Early differential diagnosis of rheumatoid arthritis (RA) is an unmet medical need in the field of rheumatology. In particular, the disease is easily confounded with other diseases like seronegative arthritis (PSO), osteoarthritis (OA), lupus or microcrystalline arthritis. These patholgies are at this early stage categorized as "Undifferentiated Arthritis" (UA). About 50% of the cases will turn out to be RA. The delay in making the correct diagnosis and initiating adequate therapy is harmful for the patients. We performed high-density transcriptomic studies on synovial biopsies from patients with arthritis, and found that synovial gene expression profiles were significantly different according to the underlying disorder. Low-density arrays were designed, targeting 100 genes able to discriminate arthritis samples according to the underlying condition. Knee synovial biopsies were obtained from patients with a definite diagnosis of RA, PSO or OA (n=40), and from UA patients (n=48). Amplified and biotinylated cDNA was hybridized on the customized arrays. In samples obtained from patients with a known diagnosis, the array had a diagnostic accuracy of 73.4%, increasing up to 79.4% in samples from untreated patients. Next, we wondered whether addition of selected clinical manifestations could increase the yield of correct diagnoses. Strikingly, the accuracy of the algorithm combining transcriptomic data and 3 features (psoriasis, arthritis of the hands, rheumatoid factors) was 92.8% in patients with a known diagnosis. Finally, we performed the same analyses in samples obtained from UA patients, and obtained a 90.3% diagnostic accuracy. A diagnosis can thus be made in patients with UA based on the combination of gene expression data and selected clinical symptoms. The list of 100 genes is now patented, and a commercial version of a diagnostic kit is under development, with the perspective of offering to patients up to 2 years gain in therapy. The solution accuracy is higher than any other diagnostic solution designed so far, including the ACR/EULAR 2010 criteria for RA diagnosis. The routine solution, named RheumaKit, is a web platform (rheumakit.com) on which rheumatologists or orthopaedic surgeons can freely register, manage patients and order sampling kits. They then take a knee synovial biopsy from their patient and send it back to a pre-specified destination. There, gene expressions are extracted and uploaded to rheumakit.com. The doctors then enter the three clinical observations required for the diagnostic computation. A virtual machine is then started on the Amazon Cloud and computes the diagnostic with our predictive model. RheumaKit is readily available as a demo version and can be tested for a virtual patient. An introduction is available on youtube (http://www.youtube.com/watch?v=wLJrUtkRO_E) and a user's manual is available on Slideshare (http://www.slideshare.net/DNAlytics/rheumakit-users-guide).

Foot movements in metatherians may be analyzed in order to unravel eutherian bipedal gait. Historical footage shows thylacine feet positioned horizontally in sway. For a clearer image, we therefore analyzed archived live videotapes of walking wombats. From take-off, the lateral side of the wombat foot stays continuously lifted, causing throughout everted foot positions during sway. In the opossum, biomechanical guidance by the cardan-like ankle joint transmits shank axial rotation to foot-eversion or foot-inversion (1). Only since recently, quantitative measurements in primates e.g. man, allow extrapolating such data to bipedal gait. After take-off, just a short distinct foot eversion helps to clear the human foot from the surface, mainly during the onset of sway (2). Very recently, sophisticated technology captured foot eversion in swing just prior to touch-down in a carnivore running at full speed (3). While doing so, this eutherian quadruped shows full toe-abduction simultaneously with full toe-extension. This phenomenon might be universal, as it was described in metatherians earlier (4). References : 1. Narain, F. H. M., van Zwieten, K. J., Gervois, P., Lippens, P. L., Reyskens, A., Colla, P., Palmers, Y., Schmidt, K. P., Vandersteen, M., Biesmans, S., Robeyns, I., Op ‘t Eijnde, B., Zinkovsky, A. V., Varzin, S. A., Lamur, K. S. (2009) Human foot inversion prior to toe-off: an analysis by means of functional morphology, and comparative anatomical observation. Journal of Vibroengineering, 11 (3), 530-535. 2. Legault-Moore, D., Chester, V. L., de Vries, G. (2012) Multisegment foot kinematics during walking in younger and older adults. Journal of Clinical Medicine Research, 4 (4), 259-266. 3. Hubbard, K., Wilson, G. et al. (2012) Video : Filming the World’s Fastest Runner. National Geographic, November Issue. 4. van Zwieten, K. J., Lippens, P. L., Honinckx, M. (1991) Gripping mechanism in Didelphis includes prehensive patterns. Belgian Journal of Zoology, 121, S1, 49-50.

Introduction The secretion of adipokines by the adipose tissue plays an important role in the pathophysiology of the metabolic syndrome in human medicine. The fat cow syndrome demonstrates similar characteristics with the metabolic syndrome. However, the effect of obesity on the adipokine profile is unknown in dairy cows. We hypothesized that in dairy cows, overconditioning may lead to an altered production of adipokines: higher circulating concentrations of leptin, resistin, IL-6 and TNF-α and lower circulating concentrations of adiponectin. Materials and methods Experimental procedures were approved by the local ethical committee of the faculty of veterinary medicine (Ghent University). Starting 2 months before the expected calving date, 10 Holstein Friesian cows (selected to assure a good spread of body condition score) were followed up 2-weekly by blood sampling, assessment of body condition score (BCS) and backfat thickness (BFT). Plasma concentrations of adiponectin, leptin, resistin and IL-6 were determined using commercial ELISA kits. Plasma concentration of TNFα was determined as described by Farney et al. (2011). Data were analyzed using a mixed effect linear model and Spearman correlation coefficients in SPSS. Results The mixed effect linear model revealed no significant (P > 0,05) association between the concentration of the different adipokines and time, BCS or BFT. There was a significant negative correlation between the average IL-6 concentration and the average concentration of adiponectin, leptin and TNF-α; and a significant positive correlation between the average concentration of TNF-α and leptin. Discussion In the present study, we measured the concentration of adiponectin, leptin, resistin, IL-6 and TNF-α in plasma samples of dairy cows with different body condition score during the dry period. The concentration of the studied adipokines does not change significantly during the dry period and is not influenced by BCS or BFT. The BCS and the BFT are tools used in practice to evaluate the accumulation of subcutaneous fat. Since in human medicine, the accumulation of abdominal fat is an important risk factor for the dysregulated production of adipokines, the same might be true for dairy cows. Therefore, one should develop a practical method to assess the amount of internally stored fat in dairy cows. The negative correlation between the average IL-6 concentration and the average adiponectin concentration is in line with findings in humans and rodents. The negative correlation between the average IL-6 concentration and the average leptin and TNF-α concentrations; and the positive correlation between the average TNF-α concentration and leptin concentration are however unexpected. In dairy cows, more research is needed to evaluate the relationship between overconditioning, abdominal fat and adipokine concentration and to identify the potential role of the different adipokines in the fat cow syndrome.

Colostrum and the health status of calves cannot be separated from each other. In comparison to other species such as human infants, a calf's immunity is fully dependent on the uptake of good quality colostrum, as soon, as fresh and as much as possible after birth. Good quality colostrum is mainly defined as encompassing a high amount of antibodies. However, from literature it is known that besides the antibodies also the colostral leukocytes (CLs) are absorbed in the calf’s gut. Therefore, we hypothesized that calves receiving CLs will have an increased immunological response to vaccination at neonatal age in comparison to calves that didn't receive those CLs. To test this hypothesis, neonatal calves (n = 28) were randomly divided into one of the following three groups: COL- (calves given colostrum without CLs from a central colostrum bank), COL+ (calves given colostrum from the same colostrum bank but after addition of CLs of the calf’s own dam) or COLfresh (calves given fresh full colostrum from their own dam). All dams had been vaccinated against tetanus toxin (TT) with 1 dose of Tetapur® two and one month before the expected calving date. The COL- and COL+ calves were further divided into 3 subgroups which were vaccinated with Tetapur® at the age of 2, 5 or 10 days, respectively, whereas all COLfresh calves were vaccinated at the age of 10 days. All calves were given a booster 23 days after the first vaccination. Blood samples were taken twice a week the first two weeks after the primary vaccination and then weekly until 3 weeks after the booster immunization to evaluate cellular (3H-thymidin lymphocyte proliferation assay) immune responses against TT. Preliminary results show that COL+ and COLfresh calves react more rapidly and higher in the 3H-thymidin proliferation assay in comparison to the COL- calves.

Low-molecular weight compounds can be applied as chemical tools to study biological processes (chemical biology) and/or can be used as starting points for the development of therapeutic agents (drug discovery). The identification of these compounds requires high-throughput screening methodologies to analyze chemical libraries in screening assays. To provide research groups the ability to screen large compound collections in high-throughput fashion, VIB has established the Compound Screening Facility (CSF). The VIB-CSF has several drug-like compound collections available that amount to a total of about 74,000 compounds and a human siRNA library that targets almost 8,000 genes. State-of-the-art liquid handling systems and detection technologies are available for screening in-house or custom collections in 96- or 384-well plate format. The platform is compatible with biochemical assays, cell-based assays in a broad range of cellular systems and assays in the model plant Arabidopsis thaliana. The core activities of the VIB-CSF consist of managing the VIB screening collections, assisting researchers in assay development/automation and performing high-throughput screenings. After hit clustering and selection, the VIB-CSF team can contribute in structure-activity analysis, IC50 measurements, cytotoxicity analysis, secondary screening for hit validation and counter screening for specificity analysis.

The acquisition of water and nutrients by plant roots is a fundamental aspect of agriculture and strongly depends on root architecture. Root branching and expansion of the root system is achieved through the development of lateral roots and is to a large extent controlled by the plant hormone auxin. However, the pleiotropic effects of auxin or auxin-like molecules on root systems complicate the study of lateral root development. Here we describe a small-molecule screen in Arabidopsis thaliana that identified naxillin as what is to our knowledge the first non-auxin-like molecule that promotes root branching. By using naxillin as a chemical tool, we identified a new function for root cap–specific conversion of the auxin precursor indole-3-butyric acid into the active auxin indole-3-acetic acid and uncovered the involvement of the root cap in root branching. Delivery of an auxin precursor in peripheral tissues such as the root cap might represent an important mechanism shaping root architecture.

Purpose: Numerous drugs act as both substrates and inhibitors of hepatic transporters such that unexpected and unwanted interactions are frequently observed, including toxic concentrations of bile acids or substrate drugs in the blood or liver. Rifampicin, for example, inhibits the uptake transporter Oatp and the efflux transporter Mrp2, which may lead to hyperbilirubinemia. In the current study, hepatic transporter disturbances by rifampicin were investigated by µSPECT imaging of 99mTc-mebrofenin, a radiolabeled Oatp and Mrp2 substrate. Based on the results, a non-invasive method to visualize and quantify disturbances in hepatic transport is proposed. Methods: Wild-type FVB mice (vehicle or rifampicin-treated) were intravenously injected with 99mTc-mebrofenin (n=3 per group). Following dynamic µSPECT and short CT acquisitions, time–activity curves of liver and gallbladder+intestines were obtained and correlated with direct blood samples. Results: The TACs of livers of vehicle control mice showed a peak of 55.2 ± 8.7 % (Cmax, liver) of the injected dose after 134 ± 41 seconds (Tmax, liver), followed by a decrease to baseline within 15 minutes (900s). The AUCliver was 6120 ± 124 MBq*s. 99mTc-mebrofenin reached the gallbladder after 147 ± 26 s. At the end of the acquisition, all the activity reached the gallbladder and intestines, the AUCgallbladder+intestines was 17752 ± 688 MBq*s. In contrast, 99mTc-mebrofenin disposition was altered following rifampicin treatments. We observed a dose-dependent delayed Tmax,liver, an increased AUCliver and a lower AUCgallbladder+intestines (p values of 0.042, 0.034 and 0.001, highest dose). Emergence of radioactivity in the gallbladder (p=0.009, highest dose) occurred later and AUCblood (p= 0.006) was higher. The mean AUCblood in mice treated with the highest dose of rifampicin was 4.3-fold greater than that of their vehicle control counterparts (p=0.006). Conclusion: The current study visualizes and quantifies disturbed hepatic uptake and biliary efflux of 99mTc-mebrofenin in a non-invasive manner. This is a promising approach to test interactions between new chemical entities and hepatobiliary transporters.

Extracellular or cell free DNA has been found to exist in many biological media such as blood and saliva. To check whether cell free DNA is present in the supernatant which is normally discarded during several DNA extraction processes, such as Chelex extraction, DNA profiles of cell pellet and concentrated supernatant from 30 artificial case like samples and from 100 real forensic samples were compared. Presence of cell free DNA was shown in all investigated sample types. Moreover, in some samples additional alleles, not detected during analysis of the cell pellet, were detected, offering valuable information which would normally have been discarded together with the supernatant. The results of this study indicate that cell free DNA deserves further consideration since it has the potential to increase the DNA yield in forensic casework samples in general and in contact traces in particular.

There is a growing interest in the use of physiologically based pharmacokinetic models for the modeling and simulation of the pharmacokinetics in pediatric patients. In order to optimize the currently available models, there is a need for research on developmental and disease-specific determinants. Liver disease has been shown to influence the hepatic biotransformation in adults, but few to no data are available on the metabolic capacity of the liver in children with hepatic diseases. Because of its major role in drug metabolism, this study focused on the in vitro cytochrome P450 activity in this specific patient population. To our knowledge, this is the first study to evaluate CYP activity in children with hepatic impairment. Thirty-one patients undergoing liver transplantation for various indications (mainly biliary atresia (n=23)) were included in the study. The in vitro activity of 6 CYP isoforms, CYP1A2, 2C9, 2C19, 2D6, 2E1, and 3A4 was determined in hepatic microsomes, through the incubation of specific probe substrates. A hypervariable activity was observed for all the isoforms. Compared to an average adult activity, low activities were seen for CYP1A2, 2C19, 2E1, and 3A4. For CYP2E1 and 3A4, a positive correlation between activity and abundance was observed. Age, co-medication, and genotype were shown to be poor predictors of the CYP activity. In contrast, a negative correlation could be observed between the Pediatric End-stage Liver Disease score and the ln(activity) for all isoforms, suggesting a decrease in CYP activity with deteriorating hepatic function. Moreover, the activity of all isoforms was correlated, demonstrating a concomitant decrease of all isoforms in young patients with liver disease. The presented data may provide support in the further optimization of a disease-specific PBPK model in this specific patient population.

Bone Therapeutics is a leading international biopharmaceutical company focused on innovative cell therapy products for the treatment of bone diseases. Utilizing the Company’s unique knowledge of the bone/joint physiology and long-standing expertise in cell therapy and cell transplantation, Bone Therapeutics has created a fully integrated business with an advanced product pipeline comprising novel bone cell products, tailored in-house production methods and minimally invasive treatment techniques. Bone Therapeutics’ lead product, PREOB®, is an autologous bone cell product, currently in Phase III clinical trials for the treatment of osteonecrosis and non-union fractures. Bone Therapeutics is also developing an allogeneic bone cell therapy product, ALLOB®, which is expected to enter the clinic in 2013 and MXB, a combined cell-matrix product for the treatment of large bone defects, currently in preclinical development. All of Bone Therapeutics’ cell therapy products are manufactured to the highest GMP standards, comply with all regulations and are protected by a rich IP estate. The bone disease and reconstruction market is one of the largest healthcare markets in the world, with more than 4 million procedures requiring bone grafts performed annually in Europe and the USA alone. Bone Therapeutics is operating in areas where demand for new products is high and competition is low. Founded in 2006, Bone Therapeutics is privately held and headquartered in Gosselies (south of Brussels), Belgium. Further information is available at: www.bonetherapeutics.com

Industrial production and purification of hydrophobin HFBII have become interesting for biotechnologists in recent years. Currently, our group has developed a novel method for extraction of HFBII using hydrophobic interactions (Industrial Crops and Production 43: 372-377, 2013), although the yield of production needs to be improved. In this study, the influence of different carbon sources on growth of Trichoderma reesei while producing HFBII was investigated. The growth behavior of T. reesei in all cases was recorded using Bioscreen C. With 40 g/L lactose, the maximum amount of HFBII was obtained after 4 days equal to 36.8±1.4 mg/L. These amounts were obtained when the lactose was consumed totally and the dry weight increased to 15.0 g/L. When the rate of lactose consumption is very high, the production of HFBII starts at a low rate of around 0.12 mg/Lh. Once almost all lactose was consumed, the rate of HFBII formation became ten times higher, reaching 1.2 mg/Lh. In contrast, when glucose or galactose were added, the amount of HFBII was not significant. Carbon starvation and extracellular enzyme expression were determined as two main conditions for HFBII production. Biodegradation of protein at stationary phase due to the lack of nutrients was proved by observing the removal of the last amino acid from the structure of HFBII.

Objective: Although serotonin reuptake inhibitors (SRIs) are generally regarded as safe drugs with few side effects, data suggest that they may compromise cellular immune responses. The objective of this study was to determine if SRIs can inhibit in vivo allo-antigen induced T cell responses in a murine model of graft-versus-host disease (GvHD). In addition, we analyzed whether the observed results are due to a direct effect of SRIs on T cell proliferation and viability. Methods: The effect of fluoxetine on murine GvHD was determined in a MHC-matched, minor HA-mismatched bone marrow transplantation model (n=13 from two experiments). Clinical symptoms and survival time were registered, and blood samples were taken at week 8 after transplantation to determine donor chimerism and the presence of alloreactive T cells. In addition, we analyzed the direct effects of six different SRIs (paroxetine, fluoxetine, sertraline, citalopram, fluvoxamine and venlafaxine) on in vitro viability and proliferative responses of peripheral blood T cells from healthy volunteers (n=6). Results: In the murine GvHD model, fluoxetine significantly reduced the clinical severity of GvHD relative to vehicle-treated control animals, without interference with the reconstitution of the hematopoietic compartment. An improvement in survival rate was also noted. In vitro results revealed an anti-proliferative effect of all compounds tested and a pro-apoptotic effect of paroxetine, fluoxetine, sertraline, fluvoxamine and citalopram. Interestingly, we found that the pro-apoptotic effect was significantly stronger in activated than in naïve T cells, indicating a higher sensitivity of activated T cells to SRI-induced apoptosis. Conclusions: These results show that fluoxetine can suppress alloreactive T cell responses in murine GvHD and that this effect is due to a direct anti-proliferative and pro-apoptotic effect on T cells, a characteristic shared by all SRIs. Given their favorable and well characterized safety profile, we conclude that SRIs may hold therapeutic potential as a novel class of T cell immunosuppressants.

Hippocampal deep brain stimulation (DBS) is an experimental therapy for patients with refractory temporal lobe epilepsy. The anti-epileptogenic potential of hippocampal DBS was not previously investigated. In this experimental study, we evaluated the effect of hippocampal Poisson distributed DBS on the development of spontaneous seizures in the systemic kainic acid (KA) rat model. Rats were implanted with a quadripolar DBS/EEG-recording electrode in the right hippocampus and a bipolar EEG recording electrode in the left hippocampus. Twenty-four hours after KA induced status epilepticus (SE) , one group (n=6) was subjected to short term DBS (ST-DBS) (Poisson Distributed Stimulation (PDS), 130 Hz, 100µs, 100µA) of 1 week, a second group (n=7) was subjected to long term DBS (LT-DBS) (PDS, 130 Hz, 100µs, 100-400µA) of 10 weeks. A control group (n=9) received sham DBS (SHAM). Continuous EEG monitoring was used to evaluate seizure frequency during and after DBS. The progression in mean daily seizure frequency after SE was the same in the SHAM and ST-DBS group, and concordant with the results seen in previous long term EEG monitoring experiments in the kainic acid model. In the LT-DBS group, group analysis shows a strong seizure suppression during 10 week long hippocampal DBS, and an altered progression in seizure frequency after SE even when stimulation was stopped. Analysis of individual seizure frequency traces reveal a decreased risk for developing a progressive type of epilepsy if an animal was in the LT-DBS group. These results show that long term hippocampal DBS efficiently suppresses seizures during stimulation and consequently changes the progression in seizure frequency in a rat model for temporal lobe epilepsy. Although our results do not rule out a mere seizure suppressive effect of hippocampal DBS, these findings support the hypothesis that hippocampal DBS exerts neuromodulatory effect that can affect epileptogenesis

Standard Illumina mate-paired libraries are constructed from 3- to 5-kb DNA fragments by a blunt-end circularization. Sequencing reads that pass through the junction of the two joined ends of a 3-5-kb DNA fragment are not easy to identify and pose problems during mapping and de novo assembly. Longer read lengths increase the possibility that a read will cross the junction. To solve this problem, we developed a mate-paired protocol for use with Illumina sequencing technology that uses Cre-Lox recombination instead of blunt end circularization. In this method, a LoxP sequence is incorporated at the junction site. This sequence allows screening reads for junctions without using a reference genome. Junction reads can be trimmed or split at the junction. Moreover, the location of the LoxP sequence in the reads distinguishes mate-paired reads from spurious paired-end reads. We tested this new method by preparing and sequencing a mate-paired library with an insert size of 3 kb from Saccharomyces cerevisiae. We present an analysis of the library quality statistics and a new bio-informatics tool called DeLoxer that can be used to analyze an IlluminaCre-Lox mate-paired data set. We also demonstrate how the resulting data significantly improves a de novo assembly of the S. cerevisiae genome

In forensic casework analysis, identification of the biological matrix and the species of a forensic trace, preferably without loss of DNA, is of major importance. The biological matrices that can be encountered in a forensic context are blood (human or non-human), saliva, semen, vaginal fluid, and to a lesser extent nasal secretions, faeces and urine. All these matrices were applied on swabs and digested with trypsin in order to obtain peptides. These peptides were injected on a mass spectrometer (ESI Q-TOF) resulting in the detection of several biomarkers that were used to build a decision tree for matrix identification. Saliva and blood were characterized by the presence of alpha-amylase 1 and hemoglobin, respectively. In vaginal fluid cornulin, cornifin and/or involucrin were found as biomarkers while semenogelin, prostate specific antigen and/or acid phosphatase were characteristic proteins for semen. Uromodulin or AMBP protein imply the presence of urine, while plunc protein is present in nasal secretions. Faeces could be determined by the presence of immunoglobulins without hemoglobin. The biomarkers for the most frequently encountered biological matrices (saliva, blood, vaginal fluid and semen) were validated in blind experiments and on real forensic samples. Additionally, by means of this proteomic approach, species identification was possible. This approach has the advantage that the analysis is performed on the first “washing” step of the chelex DNA-extraction, a solution which is normally discarded, and that one single test is sufficient to determine the identity and the species of the biological matrix, while the conventional methods require cascade testing. This technique can be considered as a useful additional tool for biological matrix identification in forensic science and holds the promise of further automation.

The expression of CD70 is normally transient and restricted to activated B- and T- cells, as well as mature dendritic cells. Chronic expression of CD70 on T cells has been demonstrated in many autoimmune indications and is linked with the methylation status of the CD70 promoter. It leads to chronic T and B cell activation resulting in disease pathology. Overexpression of CD70 has also been documented in a variety of solid and hematological tumors, where it is thought to play a role in evasion of immune surveillance and tumor proliferation and survival. ARGX-110 is a defucosylated, germlined IgG1 monoclonal antibody that selectively targets and neutralizes CD70, the ligand of CD27. Detailed biochemical and functional characterization of ARGX-110 was carried out to support its clinical development for the treatment of cancer and autoimmune disease. ARGX-110 binds to human and non-human primate CD70 with picomolar affinity. It inhibits in a dose-dependent fashion CD70/CD27-induced signalling and is believed to have strong potential in re-activating patient anti-tumor immune responses. CD70 is minimally internalized by most haematological cell lines, providing a plausible rationale to focus on enhanced ADCC as a mode of therapeutic action. In cell depletion studies, binding of ARGX-110 and activation of ADCC is associated with an increase in the rate of cell lysis at a greater efficacy than a non-ADCC-enhanced IgG1. Independence of cytotoxic potency from cell surface copy number of CD70 was also demonstrated. In addition to potent ADCC, ARGX-110 has been shown to have strong CDC and ADCP activity. In a Burkitt lymphoma SCID mouse model (CD70+ Raji cells), ARGX-110 administered intraperitoneally twice weekly resulted in a dose-dependent effect on animal survival, with 0.1 mg/kg being the fully effective dose. When administered weekly with 5 doses of 30 mg/kg in cynomolgus monkeys, ARGX-110 showed no signs of toxicity. Moreover, the antibody had no effect on the number of the overall number of T, B and NK cells. ARGX-110 is predicted to have a half life in humans of around 23 days and is planned to enter a Phase I study in patients with CD70+ malignancies early in 2013.

In approximately 30% of epilepsy patients, seizures cannot be controlled with currently available antiepileptic drugs. For these patients, new treatments with novel mechanisms of action are needed. Neuropeptide systems are promising targets for the treatment of epilepsy. Several neuropeptides such as neuropeptide Y, galanin and somatostatin have already been implicated in the control of seizure activity. Many others, however, have not been investigated in this regard. Neuropeptide FF (NPFF) is a member of the RF-amide family. NPFF and its receptors (NPFF1 and NPFF2) are differentially distributed throughout the central nervous system. We have studied the effects of NPFF receptor ligands in two rat models that can be used to predict the anticonvulsant activity of potential antiepileptic drugs: the cortical stimulation model (CSM) and the amygdala kindling model. In the CSM, a 50Hz stimulus train of pulses with increasing intensity is delivered to the motor cortex and the threshold intensity for eliciting forelimb clonus is determined through behavioral observation. The threshold is a measure for cortical excitability and correlates with anticonvulsant activity in seizure models and humans. We found that intracerebroventricular (i.c.v.) administration of NPFF (2nmol/2h) (n=7) significantly increased the threshold to 136±5% of baseline [p<0.0001 compared to control]). Threshold increase could be prevented by pretreatment with the NPFF receptor antagonist RF9 (i.c.v. 6.25nmol/2.5h) (n=6). Administration of RF9 alone had no effect on the threshold (n=7). In the amygdala kindling model, electrical stimulation of the amygdala is performed to determine the threshold for eliciting an afterdischarge. Subsequent daily stimulations at or above this threshold result in limbic seizures of gradually increasing severity. The kindling model is used to screen anticonvulsant drugs. Continuous administration of NPFF (i.c.v. 2nmol/2h) for 2h prior to stimulation significantly increased the threshold for eliciting an afterdischarge compared to control (435±72 [n=10] vs. 131±23µA [n=8] [p<0.001]). However, when rats subsequently underwent daily stimulations above this threshold, repeated administration of NPFF did not significantly delay or prevent kindling acquistion. We conclude that NPFF reduces cortical and amygdala excitability, and may therefore be an interesting drug for the treatment of epilepsy. Since NPFF did not delay amygdala kindling acquisition, we conclude that it has no effect on epileptogenesis.

Crude bio-oil, produced by fast pyrolysis of lignocellulosic biomass, is a complex mixture with a wide variety of functional groups and molecular weights. The potential of bio-oil for direct substitution of fuels and chemical feedstocks is limited due to high viscosity, high water content, low heating value, instability and corrosiveness. Consequently upgrading of bio-oil by either physical or chemical methods is required to obtain a liquid product that can be used as a liquid fuel or as chemical feedstocks in various applications. Addition of polar solvents (alcohols) to bio-oil increases the stability and also the calorific value. The stability is followed by measurement of the viscosity (at 40 °C) and the water con-tent after a forced aging test at 80 °C. Addition of 5 mm% methanol to bio-oil leads to a sub-stantial decrease (60%) of the viscosity, which is due to physical dilution and a decrease of condensation and polymerisation reactions. The water content initially lowered by physical dilution, increases rapidly by esterification and acetalization reactions. A method in using bio-oil as a fuel in a compression engine or boiler is to produce an emulsion with fossil fuels. Bio-oils are not miscible with hydrocarbon fuels, but with the aid of sur-factants they can be emulsified with diesel oil. A comprehensive screening of available com-mercial surfactants was conducted to produce stable bio-oil (5 mm%) /diesel (94 mm%) emulsions. Surfactants (1 mm%) with an HLB value around 5 à 6 deliver stable emulsions. A suitable upgrading method is catalytic hydrogenation of the bio-oil, leading to a reduction in the amount of bound oxygen. Depending on the circumstances a distinction is made between hydrodeoxygenation (HDO) and hydroprocessing (HP). HDO takes place at high pressure (100-200 bar) and temperature (300-400 °C), with the intent to produce biofuels. Hydropro-cessing goes through in milder conditions (50-100 bar, 125°C

It has been previously described that when a sample’s particle size is determined using different sizing techniques, the results can differ considerably. The purpose of this study was to review several in-process techniques for particle size determination (Spatial Filtering Velocimetry, Focused Beam Reflectance Measurements, Photometric Stereo Imaging and the Eyecon® technology) and compare them to well-known and widespread offline reference methods (Laser Diffraction and sieve analysis). To start with, a theoretical explanation of the working mechanism behind each sizing technique is presented and a comparison between them is established. Secondly, six batches of granules and pellets (i.e. spherical particles) having different sizes were measured using these techniques. The obtained size distributions and related D10, D50 and D90 values were compared using the laser diffraction particle-in-liquid method as reference technique. As expected, each technique provided different size distributions with different D values. These dissimilarities were examined and explained considering the measurement principles behind each sizing technique. The particle property measured by each particle size analyzer (particle size or chord length) and how it is measured, as well as the way in which size information is derived and calculated from this measured property and how results are presented (e.g. volume or mass distributions) are essential for the interpretation of the particle size data.

Background: Peritoneal metastases are one of the major causes of death in patients diagnosed with ovarian cancer. Unfortunately, poor prognosis and late stage of discovery hamper efficient therapy of such cancer. Therefore, recent studies suggest that specialized drug delivery systems (DDSs) or implants owing long residence time in the peritoneal cavity and ability to release anti-cancer agents may serve as an alternative for the currently used Hyperthermic IntraPeritoneal Chemotherapy (HIPEC) that lacks tumor specificity and is often associated with abdominal pain. However, neither the content of the IP fluid, nor the behavior of different delivery vehicles in the IP fluid have been investigated yet. Aims: 1) to analyze the composition of the IP fluid from murine origin and humans and compare it with the composition of other biological fluids(serum, plasma,etc). 2) to study the stability and the aggregation of widely used lipoplexes in the IP fluid and serum. Methods: IP fluids were analyzed, for their protein content, pH, etc. Lipoplexes composed of DOTAP/DOPE liposomes and siRNA labeled with Cy5 were prepared. The stability of the lipoplexes and the release of siRNA in IP fluid were determined using fluorescence correlation spectroscopy (FCS) , while the aggregation of the lipoplexes was studied using single particle tracking (SPT). Additionally, the size and the surface charge of the lipoplexes following incubation for different time points with the different fluids were evaluated by dynamic light scattering (DLS). Results and Discussion: It is known that proteins can influence the stability and aggregation of nanoparticles. We found that the used serum has a much higher protein content, when compared to the protein content of IP fluid. Therefore, we hypothesized that the effect of the IP fluid on lipoplexes would most likely be less aggressive than serum. Surprisingly, when incubated with IP fluid, more fast and pronounced siRNA release was observed by FCS measurements. Furthermore, as measured by SPT, aggregation of the liposomes in the IP fluid occurred faster than in other fluids. These results were confirmed by dynamic light scattering and gel electrophoresis. Therefore, it seems that the IP fluid can induce premature siRNA release from non-viral gene delivery complexes, making them inactive. The reason for such behavior is still not fully understood. Conclusions: when designing delivery systems and implants for IP application, careful optimization of the mechanical strength, the release and the aggregation in the IP cavity should be taken into account. Keywords: Ovarian cancer, IP fluid, DDS, release, aggregation.

Introduction Since the discovery of dendritic cells (DCs) and their role in immunity, DC vaccination has become an emerging strategy in cancer immunotherapy. By ex vivo loading a patient’s DCs with antigens and injecting them as a vaccine, specific antitumor immune responses can be induced [1, 2]. As an alternative to the currently used strategies for antigen-loading of DCs, we propose mRNA-sonoporation. Sonoporation makes use of microscopic gas bubbles that respond to pressure differences created by ultrasound waves. By adding mRNA-loaded microbubbles to DCs and exposing them to ultrasound, the microbubbles locally implode, causing a dual effect; (1) the cell membranes at the implosion site are locally damaged (sonoporated) and simultaneously (2) the mRNA is released and can be internalized through the created pores. Results We previously reported on the efficient in vitro mRNA-sonoporation of primary murine DCs, with transfection efficiencies up to 27% without compromising cell viability [3]. As fully mature and functional antigen-presenting DCs are crucial for effective cancer vaccination, we investigated the maturation status and function of the DCs after mRNA sonoporation. We discovered that mRNA-sonoporation causes partial DC maturation. In order to induce a more complete phenotypic switch, we included TriMix (a mix of 3 mRNAs that modulates the DC’s functionality [4]) in the sonoporation. In vivo T cell proliferation studies demonstrated that DCs sonoporated with antigen-encoding mRNA were capable of inducing a significant proliferation of antigen-specific T cells. The number of proliferating T cells could be boosted by co-sonoporating with antigen-mRNA and TriMix. In addition, therapy experiments were performed. Mice with pre-existing OVA-expressing tumors were vaccinated with either mGFP sonoporated DCs (control), DCs sonoporated with mOVA, or DCs sonoporated with both mOVA and TriMix. In line with the T cell proliferation results, we observed a significant slowdown of tumor outgrowth in the mOVA, but especially in the mOVA-TriMix combination group, also resulting in a prolonged overall survival. Conclusions & future perspectives To conclude, mRNA sonoporation is a promising tool in DC-based cancer immunotherapy. The major advantage of this approach is its in vivo potential: we aim to use mRNA-loaded microbubbles for immediate in vivo transfection of DCs in the lymph nodes, in this way circumventing current expensive and labor-intensive ex vivo transfection procedures. Acknowledgements Heleen Dewitte is a doctoral fellow of IWT-Vlaanderen. Ine Lentacker is a postdoctoral fellow of FWO-Vlaanderen. References [1] J. Exp. Med., 137 (1973) 1142-1162 [2] Int. J. Cancer, 94 (2001) 459-473 [3] Biomaterials, 32 (2011) 9128-9135 [4] Cancer Res., 72 (2012) 1661-1671

Burkholderia cenocepacia is an opportunistic pathogen that can cause severe lung infections in cystic fibrosis patients. Infections are often difficult to treat because of its innate resistance and its capacity to form biofilms. Biofilms play an important role in the recalcitrance of infections due to their tolerance. Tolerance is an ability to survive antibiotic treatment without expressing a resistance mechanism. Although the molecular basis of tolerance is still largely unknown, toxin-antitoxin modules (TA) are thought to play a role. The toxin (T) is a protein that inhibits an important cellular function such as translation or replication and which can form an inactive complex with an antitoxin (AT). Bactericidal antibiotics kill cells by corrupting the target functions. Inhibition of these functions would prevent antibiotics from corrupting their targets and would give rise to tolerant persister cells. In the present study we wanted to investigate whether these TA modules play a role in B. cenocepacia biofilm tolerance towards different antibiotics. Using bioinformatics tools, 17 pairs of genes were identified as possible TA modules in the genome of B. cenocepacia J2315. Based on microarray data, which indicated that these genes were upregulated in biofilms treated with tobramycin, RNA of treated and untreated B. cenocepacia J2315 biofilms and planktonic cultures was extracted and analyzed by qPCR. The expression of these TA modules in treated sessile and planktonic cultures was compared to that in untreated cultures. Toxin overexpression mutants with a rhamnose inducible promoter were constructed and evaluated for growth, resistance, biofilm formation and response to antibiotics. Generally the toxins showed a higher basal expression in sessile cells than in planktonic cells, but the expression patterns were similar for most T-AT pairs. Four toxins were upregulated in all conditions tested, whereas only one toxin was systematically downregulated. Operons showing similarity with mazEF, relBE or mqsRA and five other TA modules were significantly upregulated in biofilms and planktonic cultures after treatment with tobramycin but not after treatment with ciprofloxacin. None of the operons was upregulated in both conditions after treatment with ciprofloxacin. Others were only upregulated in sessile or in planktonic cultures, which indicates that planktonic and sessile cells may have different mechanisms to regulate the relative amounts of T/AT. Overexpression of toxins had different effects on growth, biofilm formation and the number of surviving cells after treatment. For example, overexpression of RelE or MazF resulted in a prolonged lag phase and more cells surviving antibiotic treatment. These results indicate that various TA modules may play a role in tolerance and persistence but that their exact contribution depends on the mode of growth and the antibiotic used.

Biofilms consisting of more than one microorganism (‘multispecies biofilms’) are a common problem in clinical practice and may lead to persistent infections. These biofilm-related infections are difficult to treat as cells in a biofilm are typically highly resistant against a broad range of antimicrobial agents. While increasingly being recognized as an important and relevant phenomenon, the properties of these multispecies biofilms remain poorly studied. The aim of the present study was to set up an in vitro model system to grow multispecies biofilms in a reproducible manner and to explore the influence of the presence of Candida albicans and Staphylococcus aureus on the susceptibility of Pseudomonas aeruginosa to antimicrobial agents. Biofilms of Pseudomonas aeruginosa were grown in brain heart infusion (BHI) medium in 24-well microtiterplates, alone or in the presence of Methicillin Resistant Staphylococcus aureus (MRSA) and Candida albicans. Biofilms were then exposed to antimicrobial agents for a certain time period. Quantification of the number of cells prior to and after treatment was done via plate counting on a selective growth medium : Pseudomonas aeruginosa was plated on cetrimide agar. Staphylococcus aureus was plated on tryptic soy agar supplemented with NaCl [7,5% (w/v)] and amphotericin B, while Candida albicans was plated on Sabouraud dextrose agar supplemented with nitrofurantoin and tobramycin. The approach outlined above allowed the reliable and reproducible determination of the effect of the presence of multiple species in a biofilm on susceptibility, opening the way to use this system in high-throughput screening studies. The first results of our study indicate that the presence of C. albicans and S. aureus led to an increased sensitivity of P. aeruginosa to several antimicrobial agents. This may be due to a response of P. aeruginosa and/or a direct protective effect provided by C. albicans or S. aureus. In future experiments, next generation sequencing will be used to determine the molecular basis of this phenomenon. We will also expand our investigations to other multispecies biofilms (including those formed by Burkholderia cepacia complex bacteria). Contact: Sarah Tavernier, Sarah.Tavernier@Ugent.be

Biotechnology is gaining credible importance in the textile industry moving the trend from classical textile materials to novel multifunctional textile products with unique values and functionalities. The need for novel eco-friendly and efficient textiles demands for applying biological compounds and their benefits into textile processes and products. For biological compounds to be successfully incorporated into textile materials, they must survive the extreme parameters used in textile processes. Most living biological compounds cannot survive the extreme process conditions used in melt extrusion for spinning fibers. However, some biological compounds produce spores which are dormant and tough non-productive structures that can resist extreme environmental conditions such as low and high temperature, pressure, radiation, desiccation and extreme chemical environments. This makes such biological compounds good candidates for extrusion into fibers. A fast, reliable and economically viable technique for testing the resistance of biological compounds is therefore important. In this study, we have developed a model system that can be used to quickly study the survival of biological compounds as a function of melt extrusion process parameters like temperature, pressure and residence time. Key Words: Micro-organisms, Spores, Embedding, PET, Melt extrusion, functionalized Fibers

Excellent care for people with suspected infections involves rapid diagnosis and treatment. In this modern age, the available technologies and diagnostic methods often take at least two days to produce results, which is too slow to help clinicians. The rapid detection of bacteria, fungi, mycobacteria and their differentiation from viral pathogens would likely improve treatment outcomes and health utilization resources by guiding treatment decisions at the time and place where they are taken. In addition, the scarcity of new antimicrobial agents and the dearth of new agents in the drug development pipeline limit treatment options, particularly for patients with infections caused by multidrug-resistant (MDR) organisms. By better identifying patients who would be eligible for enrolment in clinical trials, rapid diagnostics will reduce the number of patients, time for enrolment and costs of development and will hence reduce the economic burden associated with the development of novel antimicrobial compounds. The Innovative Medicine Initiative (IMI) is a public-private partnership between the European Union and the European Federation of Pharmaceutical Industries and Associations (EFPIA) aiming to improve the drug development process by supporting a more efficient discovery and development of better and safer medicines for patients. In April 2011, IMI funded the 5-year project RAPP-ID, bringing together 5 major pharmaceutical companies, 9 Academic Institutes and 5 SME’s which are jointly developing a Point-of-Care Test (POCT) for rapid detection of bacteria, mycobacteria, fungi, viruses and host biomarkers as well as to determine resistance to antimicrobial drugs. Starting from a clinicians point-of-view, novel specific probes, novel methods of sample preparation, and demonstrated ultra-high sensitive detection methods are currently combined to provide clinicians with results in hospitalized patients in less than 2 hours and in outpatients in less than 30 minutes, additionally aiming to be used and validated in near-future clinical trials as those identified in ongoing IMI initiatives. RAPP-ID aims to provide an integrated solution that addresses the technological challenges to enhance clinical decision-making and improve the quality of care and clinical outcomes for the people of Europe and worldwide.

Aromatic glycolipids are of both medical as well as pharmaceutical interest. Antimicrobial, antiviraland antiinflammatory activities have been reported (Otto, 2000, Journal of Molecular Catalysis B: Enzymatic). Moreover, they are expected to have interesting antioxidant properties when they contain phenolic groups. The alkyl chain should enhance their ability to penetrate into the cellular membrane (Nicolosi, 2002, Journal of Molecular Catalysis B: Enzymatic). The presence of a sugar unit could also be useful to target specific cells. In this study, novel aromatic glycolipids were synthesized as useful models for studying the structure–activity relationship, in particular as regards to their aromatic group.Theireffect on cell viability when an oxidative stress is induced was tested. In parallel, their interaction with cell models (liposomes) was studied through membrane fusion and permeability experiments.

In 2003 we discovered that the lens epithelium-derived growth factor (LEDGF/p75) tethers the human immunodeficiency virus 1 (HIV-1) pre-integration complex to chromatin. Knockdown and knockout studies have validated LEDGF/p75 as a critical HIV cofactor. Small molecules blocking the interaction between LEDGF/p75 and HIV integrase are a promising new class of HIV inhibitors in late preclinical development. In the mean time it became clear that LEDGF/p75 acts as general chromatin tethering protein in the cell. Next to HIV integrase and several other known cellular proteins, LEDGF/p75 also tethers the mixed lineage leukemia (MLL1) protein complex to chromatin. MLL1 is involved in normal blood cell formation and regulation of gene expression. In addition, MLL1 translocations are found in > 70% of acute leukemias in infants < 1 year of age. Acute leukemia is a cancer characterized by rapid expansion of malignant white blood cells that accumulate in the bone marrow and interfere with the production of normal blood cells. Infants diagnosed with acute leukemia harboring an MLL1 translocation have a particularly poor prognosis, with an overall survival of ± 50%, whereas children with leukemia that does not harbor a MLL translocation have an overall survival of > 80%. We previously demonstrated that expression of an LEDGF/p75 C-terminal fragment containing the integrase-binding domain, but lacking the chromatin binding domain, fused to eGFP (eGFP-LEDGF325-530) impaired HIV-1 replication. Now we transferred this knowledge from the HIV field to target MLL-fusion mediated leukemia. In this regard we explored the same strategy to selectively interfere with the leukemogenic activity of MLL-fusion proteins. We found that expression of LEDGF325-530 impaired the clonogenic growth of MLL-fusion gene transformed human and mouse hematopoietic cells, without affecting the growth of control cells immortalized by the FLT3-ITD mutant or normal lineage marker-depleted murine bone marrow cells. Expression of LEDGF325-530 was associated with downregulation of the MLL target Hoxa9 and G1-cell cycle arrest. Structure-function analysis revealed two small LEDGF/p75 peptides, LEDGF424-435 and LEDGF375-386 phenocopying these effects. Both LEDGF325-530 and the smaller active peptides were able to disrupt the LEDGF/p75-MLL interaction. Expression of LEDGF325-530 or LEDGF375-386 fragments increased the latency period to disease development in vivo in a mouse bone marrow transplant model of MLL-AF9 induced AML. We conclude that small peptides disrupting the LEDGF/p75-MLL interface have selective anti-leukemic activity providing a direct rationale for the design of small molecule inhibitors targeting this interaction. Reference: Méreau H., De Rijck J. (2013) Impairing MLL-fusion gene mediated transformation by dissecting critical interactions with the lens epithelium-derived growth factor (LEDGF/p75). Leukemia, accepted for publication.

Metal contamination in soils is a historical problem in many regions throughout the world. The use of biomass crops to phytoremediate such soils whilst simultaneously producing a valuable non-food crop, is an interesting approach which is enjoying increasing attention. In this work, the potential of different poplar and willow clones is assessed in the context of phytoremediation of metal contaminated soils. The test site (~ 7 ha) in the Campine region was planted with five poplar clones and 8 willow clones six years prior to this study. The first harvest took place in the fourth year, so this study was conducted in the second year of the second growth cycle of the short-rotation coppice. The total metal removal by the plants was calculated by multiplying the total biomass production by the metal concentration in this biomass. The best estimation of the biomass production was found to be made by a powerfunction as defined by Al Afas et al. (2008) per shoot. The estimated total biomass production after two years regrowth varies from 2.22 to 8.15 ton DM ha-1, with the willow clone Jorr giving the highest yield. Cd accumulates 2 to 3 times more in the leaves than in the wood, and for Zn this is 4 to 9 times. Loden accumulates the highest total amount of Cd (501 ± 112 g ha-1) and Zn (16.8 ± 1.7 kg ha-1). With this Loden clone it would take 88 to 133 years to remediate the soil. Due to the long remediation time however, it is a more realistic point of view to aim for maximum biomass production using highly productive clones. The concept of focusing on biomass productivity first and removal second is referred to as ‘phytoattenuation’. Further productivity increases could be observed upon application of 5% compost before planting. Maximizing biomass productivity will ensure a steady income for the farmer and will remediate the soil as a side effect. With Jorr it would take 271 to 418 years to extract enough metals from the soil to meet Flemish regulation.

Purpose The aim was to develop a fixed dose combination product via co-extrusion, thereby producing core/coat mini-tablets with a sustained drug release core and an immediate drug release coat. Materials and methods For the core several ethylcellulose(EC)-based formulations were hot melt extruded and evaluated for release profile. In view of co-extrusion a polyethylene oxide(PEO)/polyethylene glycol(PEG) coat formulation was selected to obtain immediate release. Metoprololtartrate(MPT) and hydrochlorothiazide(HCT) were incorporated as sustained and immediate release model drugs, respectively. The formulations were processed via co-extrusion and manufactured co-extrudates were evaluated for adhesive strength between core and coat. Solid state was characterized via DSC and XRD. The co-extruded mini-tablets with different sustained MPT release profiles were filled into capsules and administered to 6 dogs. Drug plasma concentrations were determined via HPLC. Stability of the co-extrudates was evaluated after 3 months at 25°C/60%RH and 40°C/75%RH. Results By adjusting the concentration of PEO as a hydrophilic component in the plasticized EC matrix from 20% to 5% or by lowering the drug load from 30% to 15% the in vitro MPT release from the core was sustained from 80 and 100% after 2 and 8h to 36, 60, 70 and 90% after 2, 8, 12 and 24h. Co-extrudates could be processed in combination with a PEO+15%PEG coat formulation releasing the 5.6% to 2.8% HCT load completely within 45min. Adhesion between the two layers was good. DSC and XRD revealed that MPT remained crystalline after extrusion, whereas HCT was dissolved in the coat. These solid state characteristics were confirmed during the stability study. The drug plasma concentration profiles confirmed the in vitro dissolution results. For MPT the in vivo results showed sustained release characteristics ranging from 4 to 11hours. Unexpected high MPT plasma concentrations were reached with this matrix formulation allowing the administered dose to be halved. The immediate in vitro HCT release from the coat was confirmed in vivo with a Tmax value of 2hours for all co-extrudates. Conclusion Fixed-dose combination mini-tablets with a matrix core offering a range of controlled release profiles and an immediate release coat were successfully developed by co-extrusion as confirmed in an in vivo dog study.

Which decision-making processes instigate agribusiness companies’ actual business policy on commercializing, or not, genetically modified (GM) crop applications in the EU? Involving multiple agribusiness stakeholders in a qualitative research design enabled us to scrutinize the essential aspects (“perspectives”) of these multifaceted processes and enabled to reflect on industry’s perceived environmental uncertainty on (non-)GM commercialization in the EU. Our stakeholder sample included stakeholders of several industry sectors in the agriculture industry: the seed supply, the feed industry, the potato industry, the food marketing and processing industries. Eleven perspectives were identified as being essential drivers for an agribusiness company’s GM policy in the current EU environment. These perspectives were correlated in an analytical framework, resulting in three dimensions that impact on agribusiness companies’ actual GM policy: (1) the company’s perceived level playing field of GM technology; (2) the company’s experienced GM marketing environment; (3) the company’s perceived stability of a (non-) GM EU-demand. This framework enables to disclose the inherent variation present in the decision-making processes of companies with a current (seemingly) similar GM policy. Despite this variation, a stable core of like-minded perspectives were very characteristic for companies within a same industry. Our insights in agribusiness companies’ motivations to commercialize, or not, GM crop (applications) in the EU is essential to understand certain (future) market dynamics of GM crops in the EU, as changes in the external EU environment will instigate different decision-making pathways in different industries. Key words Cross-industry analysis, Business perspectives, Agribusiness, GM crop policy, EU Topic Groups Business Environment, Strategic Management and organization

Center for Medical Innovation Clinical research centers (CRC’s) and local biobanks operating as network and collaborative model The Flemish government initiated the Center for Medical Innovation (CMI) in order to integrate and facilitate harmonisation between all stakeholders in Flanders. The mission of CMI is to support a faster and more efficient translation of research findings. It was conceived as a single virtual platform, linking a.o. the Flemish Clinical Research Centers (CRC - Antwerp-Brussels-Ghent-Hasselt-Leuven), and functioning as an interface with international translational biomedical research. CMI is funded with the support of the Flemish Region Clinical Research Centers (CRC) leveraging translational biomedical research The establishment of a relevant ethical and legal framework is a known challenge especially at international level but also at national, regional and even institutional level. Moreover, the implementation of existing guidelines and instruments for data and samples, such as the EU Data Protection Directive (Directive 95/46/EC), varies thereby hampering collaboration and exchange of information and samples. Therefore, the coordinators of the Flemish CRCs prepared an ethical-legal framework based on existing guidelines and instruments. An interdisciplinary expert group was established to reflect on this proposal and a stakeholder meeting was organized at national level to validate the framework. The report generated following this validation was adopted as ethical and legal framework within the CRCs and CMI and was provided to other Belgian national & regional biobanking initiatives. The report includes the definition of human bodily material, governance i.e. custodianship, consent & access for material and data, returning information …. and aims to create a coherent operational ethical & legal framework for the CMI biobanking project and related initiatives. In conclusion, this multidisciplinary research collaboration model will create a decision-supporting framework for innovation policy with economic and social benefits.

Growing global concern about diminishing oil reserves and environmental issues, associated with chemical production of surfactants and plastics, has lead to a quest for alternative technologies. Microbial production of the biological counterparts of such products has attracted a lot of attention. The major drawbacks of these ‘bioproducts’ are the relatively high production price and the structural limitations of the naturally produced molecules. Efficient microbial production is one of the first prerequisites that need to be met in order to be competitive with (semi)-synthetic products. One of the best known examples of a microbe producing high yields of biosurfactant molecules (sophorolipids) is the yeast Starmerella bombicola. The very efficient production (up until 400 g/L) of these valuable molecules has resulted in industrial production processes with this non pathogenic yeast for applications in f.e. cleaning products and cosmetics. All these factors make S. bombicola an excellent candidate to be transformed into a production host for other interesting biomolecules. However, up until a few years ago nothing was known about the genetic background of this yeast. Today the genes of the sophorolipid biochemical pathway are known and described. Furthermore, molecular techniques and expertise concerning manipulation of this industrially important yeast have been acquired. Everything is now available to proceed to further genetic engineering of S. bombicola. In this research we aimed for the production of two totally different classes of molecules by S. bombicola. The first example we investigated was the bioplastic polyhydroxyalkanoate (PHA), which is already industrially produced using other micro-organisms and can be used as an alternative for synthetic plastics. The second example can be situated amongst the glycolipid biosurfactants and is structurally related to sophoro- and cellobioselipids. We thus provided proof of concept that S. bombicola can be transformed into a platform organism for the production of tailor-made (glycolipid) biomolecules and new promising strains are now also in the pipeline. Such achievements form the necessary base for the further elaboration of the biobased economy.

Among the list of FDA approved biologic products, 46% are freeze-dried products (Constantino & Pikal, 2004) indicating that freeze-drying is the preferred way of stabilizing biopharmaceutical drug products that are not stable for long periods of time in aqueous solution, despite the disadvantages of cost and processing time. The end product quality of freeze dried products is generally assessed via off-line time consuming laboratory testing on a few randomly collected samples. Raman and Near-Infrared (NIR) spectroscopy have been recently demonstrated to be useful tools to monitor the product behavior during the entire freeze drying process (De Beer et al., 2009a,b and 2011). Till now, mainly the excipient behavior in formulations has been spectroscopicaly monitored during freeze drying. The aim of this study is to evaluate the potential of NIR spectroscopy for evaluating the virus state in a real vaccine formulation. NIR spectra of three different freeze dried vaccine formulations, only varying in terms of viruses state, were collected. The viruses were either normal (i.e., as used in commercial formulations), concentrated (using a centrifugal filter device) or stressed (by 96h storage at room temperature). Twenty samples were analyzed per formulation. NIR spectra were recorded through the bottom of the glass vial after the freeze drying process and were preprocessed using standard normal variation and second derivatives. Principal component analysis (PCA), and partial least-squares discriminant analysis (PLSDA) were applied to classify the formulations varying according their viruses state. The collected spectra were divided into two sets: a training set which has been used to build the models and a validation set used to evaluate the models. The classification of the validation set was 100% correct using the PLSDA model. As only the virus state was differing between the three different formulations, the classification could be attributed to the virus state. These results suggest that NIR spectroscopy is a tool which can be used for the fast screening of the virus state in freeze dried formulations.

Introduction Depression is the most common comorbidity in epilepsy patients. There is a growing consensus that both disorders have a common neurobiological cause. Vagus nerve stimulation (VNS) is an add-on treatment for patients with refractory epilepsy or depression. Both the mechanism of this comorbidity and the mechanism of action of VNS are poorly understood. In this study, we investigated the antidepressant potential of VNS in the kainic acid (KA) rat model for temporal lobe epilepsy (TLE). Methods Rats (n=29) were implanted with a stimulation electrode around the left vagus nerve. Status epilepticus (SE) was elicited by i.p. KA injections in half of the animals. The control group was injected with saline (SAL). Both groups were subdivided in a VNS-treated and a SHAM-treated group. Depressive behavior was evaluated 5 weeks post-SE using the saccharin preference test (SPT) and the forced swim test (FST) to assess the behavioral correlates of anhedonia and despair. VNS and SHAM groups received 2 weeks of therapeutic VNS or no treatment respectively. Hereafter the SPT and FST were performed again. Results The KA group showed a reduced saccharin preference (SP) compared to the SAL group (p<0.001). In the FST, the KA group displayed a lower immobility rate (p<0.05) than the SAL group. Two weeks of VNS significantly increased SP in the SPT and reduced immobility in the FST within the KA VNS-treated group (p<0.001 and <0.01 respectively). Conclusion KA-injected epileptic rats display signs of anhedonia but not despair compared to their non-epileptic counterparts. Two weeks of VNS produced an increase in SP in the SPT as well as a reduction in immobility in the FST within the epileptic group. As this are both measures of antidepressant activity, our findings demonstrate the antidepressant potential of VNS in an animal model for TLE.

BACKGROUND AND OBJECTIVES: MS is an autoimmune disease, characterized by chronic inflammation and demyelination in the CNS. MS patients show disturbances in plasma lipoproteins. The different phases of the disease (i.e. relapse and remission) have been associated with higher / lower levels or modifications of certain lipoproteins. Whereas a raise in HDL levels in the plasma is associated with neurologic recovery, a raise of LDL and total cholesterol levels in the blood is associated with disease worsening. Furthermore, oxidized LDL was also found to be present in activated microglia in actively demyelinating MS lesions. Plasma lipoproteins are known to influence the monocyte phenotype and disease progression in atherosclerosis by dysregulating the cholesterol metabolism of monocytes. Despite the crucial role of monocytes in MS, not much is known about the influence of lipoproteins on the phenotype of monocytes in MS. Our goal is to determine a possible relation between changes in lipoprotein levels in the blood and the cerebrospinal fluid (CSF), the oxidation status of lipoproteins, the inflammatory phenotype of monocytes and the disease status of MS patients. METHODS: Lipoprotein profiles were formed using density gradient ultracentrifugation. Monocytes were isolated form fresh human blood samples. Phenotypes of monocytes were analyzed with qPCR. RESULTS: Our preliminary data show that LDL and Lp(a) levels are raised in MS patients. Furthermore, stimulation of monocytes of MS patients with an LXR agonist induces a higher upregulation of LXR response genes as compared to monocytes of healthy individuals, indicating a disturbed cholesterol metabolism. CONCLUSIONS: Our preliminary data indicate that lipoprotein levels are altered in MS patients. Furthermore, cholesterol metabolism in monocytes of MS patients is disturbed. Further research is required to determine the exact role of the different lipoproteins on the phenotype and function of monocytes and the disease status in MS patients.

Recombinant Nanobodies (Nbs) are a unique form of monoclonal antibodies derived from camelid heavy-chain antibodies, characterized by a single antigen binding domain. Nanobodies are small (15 KDa, 2.2 nm diameter and 4 nm height), stable, user-friendly and easy to produce in various host cells including E. coli. Nbs bind their targets with nM affinities and often interact with epitopes that are less accessible to/ or less immunogenic for classical antibodies. Nbs can be easily tailored for a wide range of applications (immunodetection, immunomodulation, intracellular expression, biosensor applications, affinity purification, etc.). Based on Nanobody technology platform, the VIB/NSF has also embarked on the development of a Nanobody-based phenotypic screening platform which presents a coupled target discovery/drug discovery platform taking advantage of the Nanobody technology robustness and the power of next generation sequencing technologies. To obtain Nanobodies against your target antigen, we need about 1 mg antigen. Three months after the start of immunization, Nanobodies that score positive in ELISA will be delivered as bacterial clones or plasmids. Support and training are provided by the Nanobody Service Facility to use or to further engineer the Nanobodies according to the customers' needs.

The collaborative platform FlandersProteomics has been set up in collaboration with FlandersBio, initally between some of the leading academic centers and research institutions in Flanders, with a view to a) better coordinate R&D activities in Mass Spectrometry and Proteomics, especially regarding instruments and infrastructure, and to add weight to attempts to improve the research infrastructure in Flanders via coordinated grant applications; b) improve the visibility of the various complementary proteomics and MS research efforts in Flanders to the academic community, industry, and the public; c) to create a coordinated platform for research collaborations with Flemish biotech and industry, which have a high identifiable need for advanced proteomics and MS technologies. Some current R&D profiles of participating partners are also presented, with particular emphasis on possible applications. This initiative will also be open to other partners, including from industry and outside Flanders.

Lignocellulosic biomass hydrolysates are increasingly being used as the feedstock for industrial fermentations. These lignocellulosic hydrolysates consist of complex mixtures of different fermentable sugars, which can be released by a combination of pre-treatment methods and subsequent hydrolysis steps. However, pre-treatment also causes the release of stressors that affect the performance of the microbial production host, inhibiting efficient fermentation of sugars into ethanol. One of the most important inhibitors in lignocellulosic hydrolysate is acetic acid. This weak organic acid is typically present in lignocellulosic hydrolysate at concentrations ranging from 1-10 g/L. However, in acidic environments (pH < 4.7), an acetic acid concentration of 5 g/L already strongly affects growth and fermentation of Saccharomyces cerevisiae strains. Even more, the utilization of xylose, the second most abundant sugar in lignocellulosic hydrolysate, is already inhibited at 3 g/L acetic acid. To prevent the inhibitory effect of acetic acid on ethanol production, we set out to increase the tolerance of industrial S. cerevisiae strains towards this weak acid. To this end, we screened the in-house yeast collection for acetic acid tolerant strains, which resulted in the identification of a yeast strain that is able to ferment glucose in the presence of 9 g/L acetic acid. To be able to transfer this property to industrial yeast strains, the genetic basis of the high acetic acid tolerance must be unravelled. However, previous research indicated that acetic acid tolerance is a polygenic trait, which makes it difficult to engineer yeast strains for improved tolerance. Our lab is therefore developing a new technology to identify causative mutations that establish superior complex phenotypes. This method, called “pooled-segregant whole-genome sequencing”, utilizes the rapid development in next-generation sequencing and makes it possible to simultaneously identify multiple quantitative trait loci (QTLs) that underlie the trait of interest. This method has now been successfully employed to unravel the genetic basis of high acetic acid tolerance of the newly identified acetic acid tolerant yeast strain. A total of five QTLs have been identified, of which two are strongly linked to acetic acid tolerance. One QTL was further dissected to single gene level and after introducing the identified gene into an industrial yeast strain that is used for bio-ethanol production, improved acetic acid tolerance was observed. This proofs that the pooled-segregant whole-genome sequencing technology is a powerful method to identify novel genes for improving industrial yeast strains. Obviously, the new technology can be employed to investigate a wide range of industrially interesting properties such as high ethanol tolerance and thermotolerance.

Biofilms, groups of microorganisms that stick together on different surfaces within the human body and escape conventional antibiotic treatment, are an increasing challenge in the medical field. These biofilms are typically found on medical devices like implants. The most recent generation of implants with open porosity enables fast osseointegration, but also presents an increased risk of microbial biofilm-associated infection. Biofilm-associated infections are responsible for 15-25% of implant failure, and necessitate burdensome and costly revision surgery. The latter is estimated to represent a supplementary medical cost of €800m/year in Europe without taking into account the pain and distress of the patients, indicating that any significant reduction of this type of implant failure is highly recommended. Until now, biocidal implant coatings have been developed that are based on either the release of silver ions, which are toxic upon accumulation, or on conventional antibiotics that have poor activity against microorganisms in biofilms. Therefore, the COATIM consortium, consisting of 5 SMEs and 5 universities/research institutes, was established with support from the European Commission (Seventh Framework Program – FP7; www.coatim.eu). COATIM aims to develop the next generation of implant coatings containing novel potent proprietary antibiofilm molecules (ABMs) with inhibitory activity against microbial biofilms. In COATIM, these ABMs are grafted or deposited on small titanium implant substrates, as a model for dental and orthopaedic implants. Next, the ABM-coated implants are evaluated for in vitro and in vivo activity in resisting microbial infection without compromising osseointegration. Finally, the ABM-coating is applied on complex orthopaedic and dental implants, allowing the exploitation of the results by industry. In parallel, the antibiofilm mode of action of the ABMs is unraveled. Data regarding the activity of selected ABMs against various microorganisms as well as cytotoxicity of these ABMs against osteoblasts will be presented.

In the field of sociomicrobiology, the interspecies signaling molecule autoinducer-2 (AI-2) is commonly linked to virulence and pathogenicity. However, while the gene encoding the AI-2 synthase LuxS is present in a wide variety of species (including non-pathogenic bacteria), the AI-2 signal transduction pathway is not found in most organisms. Our previous findings showed that the luxS gene is present in all bifidobacteria investigated and that they all produce AI-2. This raised the question whether its primary role lies in signaling or in metabolism. In order to answer this question, we inactivated the luxS gene of Bifidobacterium breve UCC2003 by insertional mutagenesis and compared the transcriptome of this mutant to that of the wild type. An overexpression mutant capable of producing more AI-2 was also constructed. Furthermore, we analysed the effects of administration of wild type B. breve to Salmonella-infected C. elegans and compared these effects with those observed in nematodes receiving the insertion mutant or the overexpression mutant.We found that 27 out of 1843 genes (= 1.47%) were significantly upregulated in the insertion mutant, whereas 105 out of 1843 genes (= 5.70%) were significantly downregulated (2 fold cut-off, p≤0.05). Approximately 18% of the upregulated genes were involved in transcription, and 11% in defense mechanisms. Among the downregulated genes, 22% were involved in carbohydrate transport and metabolism, and 10% in amino acid transport and metabolism. These results suggest a metabolic role for LuxS in bifidobacteria. Our results further show that the WT and the luxS mutants have comparable colonization capacity in nematodes, but that AI-2 production seems to be essential for bifidobacteria to confer protection against infection with S. Typhimurium.

Platelet-derived growth factor C, also known as PDGF-C, is a transforming cell factor which promotes tumor growth via several mechanisms including survival and proliferation of tumor cells, tumor neovascularization, and is upregulated in cancer tumors that are resistant to chemotherapy. In this study, a series of newly hybridoma-derived monoclonal antibodies were generated by immunizing Balb/C mice with a recombinant human PDGF-C protein. Through cell fusion, sub-cloning, and screening approach, five hybridoma cell clones expressing monoclonal antibodies were identified displaying high binding affinity to human PDGF-C with a KD ranging from 1.2 - 5.0 nM and caused almost 90 % inhibition of PDGF-C binding to its receptor. The inhibitory activity of the anti-PDGF-C clones was also evaluated in a cell-based activation assay showing IC50 ranging from 4.3 to 8.9 nM. Taken together our results show that we have developed anti-PDGF-C antibodies that bind with high affinity to human PDGF-C and display in vitro inhibitory activity at nM range. Anti-tumor activity of anti-PDGF-C antibodies is currently being evaluated in a glioblastoma xenograft model in immunodeficient mice, which express both PDGF-C and PDGFR-α.

Diagnosis of cancer in early stages is of the utmost importance for a positive prognosis. During tumor development potential markers, e.g. tumor-specific proteins, peptides and metabolic waste products, are secreted into the blood stream from the angiogenic switch onwards. Proteolytic fragments of proteins generated by tumor-specific proteases are released as well. Through a holistic omics approach, we are currently identifying and merging these secreted compounds in blood. Validated cancer type specific markers can then be used in the development of a non-invasive, patient-friendly, reliable test for a highly sensitive and highly specific diagnosis.

Vitreomacular adhesion causing vitreomacular traction is a common indication for vitrectomy. Recently, ocriplasmin received approval by the FDA for nonsurgical resolution of symptomatic vitreomacular adhesion. Ocriplasmin is a truncated form of human plasmin that cleaves fibronectin, laminin and collagen, three major components of the vitreoretinal interface that play an important role in vitreomacular adhesion. Ocriplasmin rapidly inactivates due to autolytic degradation, limiting its duration of activity. Here, we show identification of a limited number of autolytic cleavage sites and mutational analysis of these and related sites to diminish the autolysis rate. Selected mutants are being tested in animal models for increased efficacy.

Purpose The ocriplasmin MIVI-TRUST program included two phase 3, multicenter, randomized, double-masked trials to determine efficacy and safety for treatment of vitreomacular traction (VMT). We sought to identify baseline features predictive of pharmacologic VMA resolution at day 28 following an injection of ocriplasmin or placebo and to determine the effect of VMA resolution on best corrected visual acuity (BCVA). Methods Patients with OCT-confirmed VMA were randomized to receive a single intravitreal injection of 125 µg ocriplasmin (n=464) or placebo (n=188). The primary end point was VMA resolution at 28 days post-injection. A multivariate regression analysis was performed to determine independent baseline anatomic and nonanatomic features that were significantly associated with pharmacologic VMA resolution at day 28. Selected secondary end points included BCVA improvement of ≥2 or ≥3 ETDRS lines. Results Pharmacologic VMA resolution at day 28 was observed in a significantly larger proportion of patients in the ocriplasmin group (26.5%) compared to placebo (10.1%; P<0.001). Independent baseline anatomic features predictive of VMA resolution included presence of a full-thickness macular hole (FTMH, equivalent to stage II) (P=0.019), VMA diameter ≤1500 µm (P<0.001), phakic lens status (P<0.001), and absence of an epiretinal membrane (P<0.001). Independent nonanatomic baseline features predictive of VMA resolution included ocriplasmin treatment group (P<0.001) and age <65 years (P<0.001). BCVA improvement of ≥2 lines at 6 months occurred in 41.5% of responders (ie, pharmacologic VMA resolution at 28 days) compared to 20.2% of nonresponders (P<0.001). Conclusions A single injection of ocriplasmin achieved higher rates of VMA resolution compared to placebo. VMA resolution resulted in greater BCVA gain at 6 months compared to no resolution. The identification of specific baseline anatomic features predictive of response is informative in the consideration of treatment options for patients with VMT.

Staphylococcus (S.) intermedius and pseudintermedius are two species responsible for skin infections in dogs and cats. Both species can rarely cause infections in humans, usually following infected animal contacts. Recently, Methicillin-Resistant S. intermedius (MRSI) and S. pseudintermedius (MRSP) have emerged as significant nosocomial pathogens in companion animals. Their rising incidence makes them an alarming problem since there is the limited therapeutic option for animals, or even for human. The aim of this study is to investigate MRSI and MRSP presence in S. intermedius and S. pseudintermedius and to compare their genotypes and phenotypes characteristics. Two hundreds S. intermedius and pseudintermedius isolated in Japan from cats and dogs were tested for methicillin resistance. Positive isolates were characterised for virulence genes, biofilm formation and antibiotics resistance. Besides, the strains were typed by SCCmec-typing, spa-typing, PFGE and MLST. Out of the 200 tested strains, 27 were methicillin-resistant. PFGE results showed that most positive strains are not closely related. However they are shared between 4 main groups according to MLST and SCCmec-typing. Most strains showed strong biofilm formation. Finally, MRSI and MRSP were resistant to nearly 80% of the antibiotics. Virulotyping and spa-typing are currently on-going. In conclusion, results obtained showed that MRSI and MRSP are present in the studied animals and don’t seem to form a homogeneous group. Phenotypic features as strong biofilm formation and high antibiotics resistance probably help bacteria to infect and persist in animals and veterinary hospital. Moreover, such strains could represent a risk for pets owners and veterinarians.

Human embryonic stem cells (hESC) are characterized by self-renewal and pluripotency. The latter comprises the ability to differentiate to cells of the mesoderm, ectoderm and endoderm. This means that hESC can differentiate into different cell types and therefore be useful for transplantations. Not only the process of differentiation has been extensively explored, but also finding ways of keeping them undifferentiated is essential for fundamental clinical research and toxicological screenings. The expression factor Oct4 is a widely known marker for the undifferentiated status of hESC. Currently, the differentiation status is most routinely measured using immunostaining, qPCR and/or flowcytometry. Although these techniques have proven their utility in analyzing hESC, they can only be used at the end of an experiment because of their destructive nature. Here, we validate a non-destructive method for examining the differentiation of hESC by using a commercially available Oct4-eGFP Knock-In hESC line. Technical use of this cell line will eventually allow fast screening to analyze the effect of addition of several factors on hESC pluripotency. In this reporter hESC line, Oct4 is coupled to an enhanced green fluorescent protein (eGFP), so the decrease in fluorescence represents a decrease in OCT4 expression. In search of a non-destructive and fast way to define the differentiation status of these cells, this fluorescence was measured by means of fluorescence microscopy (FM) by using the Axiovision 4.6.3 Software (Carl Zeiss, München, Germany). By determination of the densitometric means of the colony and the background, the signal to noise (s/n) can be compared between two conditions on daily basis without any loss of cells. Flowcytometry was used as the golden standard to confirm FM data. To validate this non-destructive method, the hESC were differentiated by using 2 µm retinoic acid (RA) and their s/n was compared with hESC grown in 4ng/ml basic fibroblast growth factor (bFGF), a well-known culture method to sustain pluripotency. Indeed, by means of FM, a significant decrease in fluorescence was observed with RA in comparison with the bFGF condition. Flowcytometry on the beginning and the end of the experiment allowed comparison between these two conditions and confirmed the FM results. Using microscopy has the additional advantage of being able to coordinately monitor the morphology and the attachment of the hESC colonies. These parameters vary independently of Oct4 expression and are mediated by different factors. However, staining with other markers such as SSEA-1, GATA4 will still be necessary to define lineage commitment. In conclusion, we show that FM on an Oct4-eGFP Knock-In reporter hESC line is a non-destructive approach that allows fast screening of hESC pluripotency. It will simultaneously monitor differentiation status as well as colony morphology and attachment.

Complex raw materials such as hydrolysate, serum and yeastolate are frequently used to support or enhance the performance of bioprocesses. They bring essential nutrients or factors to cells enabling them to reach optimal growth, density and/or protein expression. However, their undefined nature raises challenges in regard to consistency of cell culture performance and compliance with regulatory agencies requirements. Good Manufacturing Practice (GMP) and FDA Quality by Design (QbD) approaches insist on extensive control of this material. However, traditional analytical methods (eg. HPLC, NIRS) lack the ability to characterize chemically complex media to the level required and in accordance with the biomanufacturing timelines and cost constraints. As a result, many companies monitor only a handful of components, failing to get the complete picture and missing information on how it will eventually affect performance in scale-up. To address these issues, we developed an NMR-based (Nuclear Magnetic Resonance) approach to troubleshoot bioprocess and predict biomanufacturing performance of defined and undefined culture media and supplement batches. This approach combines NMR technology and statistical tools to detect markers that correlate with performance data of raw material batches specific to a cell culture or fermentation process. Thus, it enables to build a custom set of predictive tools that helps with ensuring the consistent performance of a bioproduction. Moreover, it potentially allows to identify the root causes of these variations in the raw material. As a result, it helps to 1/ troubleshoot performance variability in established processes, 2/ prevent failure and performance variation in future large-scale commercial production, and 3/ supports the establishment of built-in quality and robustness in new processes when included in a QbD strategy.

The aim of this research was to monitor the solid state of materials during hot melt extrusion in every section of the extrusion barrel, including the die, using Raman spectroscopy. For die monitoring, physical mixtures (30:70 and 50:50) of Celecoxib (Selectchemie) and Eudragit® EPO (Evonik) respectively, were extruded (PTSE 12/36, Brabender® Pharma) with 3 different screw configurations and at varying temperatures (130, 140 and 150°C). Raman spectroscopy (RXN1, Kaiser Optical Systems Inc.) was used in-line and DSC (Q2000, TA Instruments) and XRPD (D2 Phaser, Bruker) were used to verify the solid state off-line. SIPAT (Siemens) was used for data collection and alignment. PCA was performed on all Raman spectra. The spectra form clusters according to solid state. Within the amorphous group, a clear difference in relation to processing temperature can be seen, although the die temperature remained constant, indicating that process history can be identified with Raman measurements. Mixtures extruded at 150°C induce a lower pressure at the measurement point than mixtures extruded at 130°C. This pressure variation is reflected in the intensity of the collected Raman spectra. Modifications in the screw design also induce pressure variations at the measurement point, due to the different shear forces produced by the configurations. During barrel monitoring, metoprolol tartrate (MPT) (Esteve Quimica) concentration (10 and 40% in Eudragit® RSPO, Evonik), extrusion temperature (100, 120 and 140°C) and screw speed (80 and 160 rpm) were varied during extrusion (Prism Eurolab 16, Thermo Scientific) to examine their influence on the polymer/drug solid state throughout the barrel. FT-IR measurements (Bruker Vertex 70 FT-IR spectrometer) were performed to confirm the observations. When extruding a formulation with a 40% MPT concentration, broadening of MPT peaks indicates melting of MPT between sections 2 and 3, caused by the first kneading zone. Decreasing the concentration to 10% shows an additional spectral difference (i.e. peak shifts indicating interactions between MPT and carrier) between sections 5 and 6, due to formation of a solid solution. At a 10% MPT load, increasing the extrusion temperature does not influence the solid state or the barrel section where the final solid state is obtained. At a drug load of 40%, the solid state of the end product is reached further down the barrel when the temperature decreases. Doubling the screw speed when processing a 10% MPT formulation does not affect the solid state of the product or the location where it is obtained. In contrast, at 40% drug load, the section where the final product is produced, is situated earlier in the barrel when applying a higher speed. These results demonstrate that Raman spectroscopy is a suitable PAT-tool for the monitoring of the polymer-drug solid state during hot-melt extrusion. The in-line collected spectra facilitate process understanding and optimisation.

Osmotic dehydration of pomegranate seeds was carried out at different temperatures (30, 40, 50°C) in a 55°Brix solution of sucrose, glucose, and mixture sucrose & glucose (50:50 wt/wt). The most significant changes of water loss and solids gain took place during the first 20 min of dewatering. During this period, seeds water loss was estimated to 46% in sucrose, 37% in glucose and 41% in mix glucose/sucrose solution. The increase of temperature favoured the increase of water loss, weight reduction, solids gain and effective diffusivity. Differential scanning calorimetry data provided complementary information on the mobility changes of water and solute in osmodehydrated pomegranate seeds. The ratio between % frozen water and % unfreezable water decreased from 5 to 0.5 during the process. That involving the presence of very tightly bound water to the sample, which is very difficult to eliminate with this process. It also appeared that glass transition temperature depends on the types of sugar. Keywords: Osmotic dehydration; water loss; solids gain; Differential scanning calorimetry; pomegranate.

Chronic wounds are a major clinical problem, affecting millions of patients globally. Although several factors can contribute to the failure of healing of these wounds, bacterial infection is considered to be one of the most important ones. The presence of bacterial biofilms contributes to the chronic nature of these wounds. Recently, quorum sensing inhibitors (QSI) have been proposed as potential anti-biofilm agents. In addition, numerous wound dressings are commercially available. However, the effect of wound care products/dressings on bacterial biofilms is often only moderate or has insufficiently been evaluated. For this reason, we screened several commercially available wound dressings and wound care products for their ability to affect Staphylococcus aureus and Staphylococcus epidermidis biofilms in an in vitro model mimicking chronically infected wounds. In addition, several experimental dressings were evaluated. These dressings were functionalized with cyclodextrins loaded with an antimicrobial compound and dressings functionalized with QSI. The anti-biofilm properties were evaluated by confocal laser scanning microscopy and culture-dependent (i.e. standard plating) and independent quantification approaches (i.e. viability screening using a fluorescently labeled reporter strain). Further, we investigated whether QSI could enhance the susceptibility of bacterial biofilms to treatment with conventional antimicrobial agents (i.e. vancomycin and clindamycin) and to treatment with commercially available and experimental wound dressings. Finally, we evaluated whether incorporation of the QSI into wound dressings could increase the efficacy of these dressings. Several thiazolidinedione derivatives and hamamellitannin displayed both biofilm inhibitory as well as biofilm eradicating activities. Our results suggest that these compounds do not interfere with the initial attachment but affect later stages of biofilm formation, matrix production and/or detachment of biofilm cells. Pre-treatment during biofilm formation or treatment of mature biofilms with these QSI increased the susceptibility of sessile S. aureus and S. epidermidis cells towards conventional antibiotics. In addition, the efficacy of commercial wound dressings was enhanced when the QSI were added together with the different dressings and when the QSI were incorporated into the dressings. Finally, several experimental wound dressings were observed to have strong biofilm inhibitory and eradicating properties. In conclusion, several potent QSI and experimental wound dressings with strong anti-biofilm properties were developed. Our data suggest that QSI may increase the success of treatment of chronically infected wounds by increasing the susceptibility of bacterial wound biofilms towards antibiotics.

Recently, we described the existence of the Cardiac Atrial appendage Stem Cell (CASC) in human hearts (1). In vitro this new cell population possesses a superior cardiomyogenic differentiation capacity as compared to the earlier described c-kit+ CSCs and the cardiosphere-derived cells. A first pilot study, in which human CASCs were transplanted in the Göttingen minipig acute infarct model, was unsuccessful since no improvement in left ventricular ejection was obtained after 2 months. These negative results are probably due to the very limited engraftment of the transplanted CASCs. Therefore, a second pilot study to improve the engraftment of CASCs in the Göttingen minipig infarct model was set-up. From the right atrial appendage of 6 Göttingen minipigs, autologous CASCs were isolated by flow sorting based on high aldehyde dehydrogenase (ALDH) expression levels. Out of these appendages, an average of 31018±15853 CASCs could be isolated. After ex vivo expansion and viral transfection with Green Fluorescent Protein (GFP), an average of 18.1±19.6x106 autologous CASCs was intramyocardially transplanted in the peri-infarct zone after the MI. Just before transplantation the cells were dissolved in 400µL of collagen-matrigel construct and distributed over 20 injection sites. After 2 weeks to 2 months, the animals were humanely sacrificed, hearts were excised and examined with immunofluorescence. This analysis showed successful engraftment of GFP+ CASCs with a distinct cardiomyogenic differentiation as shown by the co-expression of cardiac troponin T. Importantly, there were no signs of teratoma formation after this follow-up. The survival and differentiation of CASCs in this very hostile environment of inflammation and fibrosis during the acute phase after the MI proved the potency of these cells. In the near future, a larger preclinical study will investigate these promising differentiation data in combination with functional analysis to prove the real potential of the CASCs. In conclusion, the discovery of this CASC population opens new perspectives for regeneration of infarcted hearts. References: (1) Koninckx R, Daniëls A, Windmolders S, Mees U, Macianskiene R, Mubagwa K, Steels P, Jamaer L, Dubois J, Robic B, Hendrikx M, Rummens JL, Hensen K. THE CARDIAC ATRIAL APPENDAGE STEM CELL: A NEW AND PROMISING CANDIDATE FOR MYOCARDIAL REPAIR. Cardiovasc Res. 2012 Dec 19

Introduction: Epigenetics exhibit an essential role in maintaining the pluripotent state of embryonic stem cells (ESC). Amongst other regulatory mechanisms, posttranslational histone modifications (PTMs) define the differentiation potential and status, being largely governed by the Polycomb group and trithorax group proteins. These antagonizing protein complexes establish bivalent genes, where both transcription repressing and activating histone marks are present. Upon ESC differentiation, there is significant turnover of this epigenetic signature. Duncan et al. (Cell, 2008) for the first time showed the epigenetic potential of proteolytic cleavage of the histone H3 N-terminus by cathepsin L during retinoic acid induced mouse ESC differentiation. In the study presented here, we show for the first time that histone H3 clipping also occurs during human ESC differentiation. Results: Using Western blotting, N-terminal clipping of histone H3 is demonstrated during early human ESC differentiation, in both feeder (MEFs) and feeder-free (vitronectin coating) conditions. The lower MW H3 bands appear most intense during the first days after retinoic acid induced differentiation (2 µM), constituting up to 50% or more of the total H3 content, with decreasing intensity in later stages of the differentiation experiment, indicating replacement by new full-length histones. The mass difference between intact H3 and the formed H3 fragment implies a loss of about 20 amino acids, corresponding to the findings of Duncan et al where clipping takes place after alanine 21. Found in multiple independent differentiation experiments of different human ESC cell lines, histone H3 clipping may function as a general posttranslational modification, marking histones for replacement by erasing other histone modifications. Conclusions: Although the biological relevance has not been elucidated yet, the phenomenon of histone cleavage might shed new light onto existing ESC data and this modification should be taken into account when setting up epigenetic experiments on differentiating ESC in the future. From a theoretical point of view, H3 clipping and histone cleavage in general could function as a novel event to alter epigenetic signatures, hereby expanding chromatin dynamics substantially.

Promethera Biosciences® is a Belgian Biotech company, producing the cell therapy product, HepaStem, to treat serious metabolic liver disorders. This treatment is based on human heterologous liver progenitor cells isolated from healthy adult livers. An European phase I/II clinical trial is currently ongoing for the treatment of Crigler-Najjar syndrome and Urea Cycle Disorders in a pediatric setting. To further upscale the process, minimize manual operations & related-risks, as well as to reduce overall costs, Promethera has developed a fully closed system. Currently, the manufacturing process is performed in open aseptic conditions using CellBind-treated CellStacks. The next generation production process will be based on ATMI’s Xpansion 2D multiplate bioreactor technology. This multiplate bioreactor offers a similar surface treatment & area, allowing for an easy transfer from the existing multi-tray stacks process. But in addition, it offers a fully controlled environment via real-time monitoring of temperature, dissolved oxygen, pH and media flow rate, reducing in-process variations. Monitoring of cell morphology is accomplished via Ovizio’s digital holographic microscopy. To date, Promethera’s liver progenitors were successively expanded in XP10 (6120 cm2), XP50 (30600 cm2), and XP180 (110160 cm2) without change in growth rate, cumulative population doubling time, homogeneous distribution, and cell quality (identity/purity and potency). A substantial increase in cell yield was achieved. In conclusion, the Xpansion™ 2D multiplate bioreactor offers a valuable technology for large-scale production. This technology results in reduced costs and increased batch-to-batch consistency. It allows reduction of manual handling & manpower and improvement of in-process control. Using this single-use technology, Promethera is able to increase the yield of its production process by >20-fold and reduce manpower by 50%.

Within the scope of increased process efficiency and increased production, there is the intention within the pharmaceutical industry to move from traditional batch processing to continuous processing. It offers many advantages such as avoiding scale-up issues and increasing process flexibility and efficiency. The Laboratory of Pharmaceutical Process Analytical Technology (PAT) from the University of Ghent (UGent) is currently performing research on a continuous oral solid dosage manufacturing (from-powder-to-tablet) line (ConsiGma™ 25 - GEA Pharma Systems N.V., Collette™, Wommelgem, Belgium), installed in its laboratory. There is an increasing demand from the regulatory authorities towards the pharmaceutical industries to gain a comprehensive understanding of their processes together with an accurate estimation of their robustness and reliability. Instead of providing solutions to meet these demands and requirements, authorities have published guidelines establishing the philosophy to achieve these expectations. Until now, the Laboratory of Pharmaceutical PAT (UGent) has developed a major experience in the adequate implementation of process analysers in process stream allowing correct in-line assessment of critical process and product information. This study focuses on the optimisation and the validation of the most important steps in the production line of the ConsiGma™: the continuous high-shear granulator, the multicell dryer and the mill. The aim is to define the combination of process parameters leading to the product expected specifications with a very high probability, based on data collected by the process sensors. This combination is the design space. In first instance, using implemented process sensors, quantitative methods are developed and pre-validated to accurately evaluate in real-time the critical quality attributes of the continuously produced granules. Secondly, it is investigated which process parameters have the most impact on the critical quality attributes of the granules. Thirdly, the selected critical process parameters are optimized to define the design space leading to (the desired) robust product performance. Fourthly, based on the normal operating conditions, the quantitative methods developed on the process analyzers are thoroughly validated. Finally, possibilities to implement the design space in the manufacturing environment are investigated. The present study is the first step to optimize and validate the continuous granulator, dryer and mill step of the full continuous pharmaceutical from-powder-to-tablet process, meeting the new requirements of the regulatory authorities.

Recently the interest has arisen in the pharmaceutical industry to shift its manufacturing of solid dosage forms from traditional batch production towards continuous production. The main motives for this switch are avoiding scale-up issues, reducing cycle times, reducing production costs, ensuring faster product release, reducing variability, increasing flexibility and efficiency, and improving product quality. Traditionally, the end product quality is evaluated by means of off-line and time-consuming quality assessment. It is clear that these off-line tests would annul the advantages of continuous processing. The International Conference of Harmonisation (ICH) states in its Q8 ‘Pharmaceutical Development’ guideline that the manufacturer of pharmaceuticals should have an enhanced knowledge of the product performance over a range of material attributes, manufacturing process options and process parameters. This approach fits into the Process Analytical Technology (PAT) and Quality by Design (QbD) framework, as launched by the Food and Drug Administration. The present study evaluates the effect of chemical variability of raw material properties upon the quality attributes of granules, produced using a continuous from-powder-to-tablet wet granulation line (ConsiGmaTM25). The process parameters were in first instance kept constant in order to examine the differences in the end-product quality caused by the raw materials only. Afterwards an extensive study was conducted using a Design of Experiments approach. Six different grades of a pharmaceutical excipient (X) were investigated. A mixture of consisting of 30% of X, 60% of lactose and 10% of corn starch was used for granulation. The granulation liquid was distilled water. When using the same process parameters, but different Xs a shift in particle size distribution could be identified. This shift could be attributed to a different water binding capacity of the Xs. X with a lower water binding capacity resulted in more oversized granules, whereas more fines will be produced when granulating Xs with a higher water binding capacity. This phenomenon was further studied by means of a DoE, with the temperature of the granulator barrel, the powder feed addition, screw configuration, liquid addition and X grades as factors, and particle size distribution as main response. Finally, the gained knowledge on how raw material properties influence granules is used to study how the granulation process parameters need to be set in function of the starting material properties, hence ensuring the desired quality at any time.

In cystic fibrosis patients acute lung infections or exacerbations by Pseudomonas aeruginosa or Burkholderia cepacia complex are often treated with high doses of intravenously administered antibiotics, combined with the daily use of nebulized tobramycin to prevent chronic lung infections. Once chronic lung infections are established, eradication of these pathogens is very difficult. This is due to the biofilm mode of growth of pathogenic P. aeruginosa and B. cepacia complex strains. Sessile cells are resistant to most antibiotics, partly because of the latter’s limited transport into the biofilm. In the present study, we investigated whether a liposomal tobramycin formulation can deliver a high dose of antibiotics closely to the bacterial cells and increase the efficacy of the treatment. In order to determine suitable physico-chemical characteristics of the liposomal formulation, we used single particle tracking to study the transport of positively and negatively charged 200 nm fluorescent polystyrene model nanoparticles in Burkholderia cepacia complex biofilms. Negatively-charged particles penetrated into the biofilm and were then immobilized in cell clusters, while positively-charged particles hardly penetrated into the biofilm at all but instead adhered to thread-like structures in the extracellular matrix. These thread-like structures are believed to consist of DNA as they disappeared when biofilms were grown in the presence of recombinant human DNase. Positively charged particles added to biofilms grown in the presence of rhDNase showed higher mobility and some of these particles were able to penetrate deeply into the biofilm where they were immobilized in cell clusters. Our data indicate that negatively-charged particles are directed towards cell clusters, which means that negatively-charged nanomedicines could be suitable candidates to deliver antibiotics to the bacterial clusters. Positively-charged nanomedicines appear only promising when they are administered in combination with rhDNase. Currently tobramycin loaded liposomes (both positively and negatively charged) are being produced to compare the activity of drug-loaded liposomes with free tobramycin against in vitro grown Bcc biofilms.

The importance of continuous production in the pharmaceutical industry has increased significantly over the past years as there are many advantages associated with this mode of manufacturing which are all related to economic drivers for change (quality, cost and time). In the current work, twin screw granulation, being a continuous wet granulation method for particle size enlargement, was studied. The objective was to understand the effect of screw configuration, screw speed, powder feed rate, and liquid content on particle size distribution of the granules produced in a twin-screw granulator. Experiments were conducted using a high-shear twin screw granulator, being part of the ConsiGma™-1 unit (mobile R&D unit of ConsiGmaTM series, continuous tablet manufacturing systems). A premix of α-lactose monohydrate and PVP (ratio: 97.5/2.5, w/w) was granulated along with a 0.01% cochenille red aqueous solution. Using a dedicated experimental design, the effects of screw configuration (number of kneading elements: 2, 4, 6 and 12), powder feed rate (10-25 kg/h), screw speed (600 and 900 rpm) and liquid content (4.58-6.72% (w/w), based on wet mass) on the granule size (F) distribution (GSD) were evaluated. The barrel temperature was kept constant (25 °C). Granules produced were collected and dried to measure the GSD by sieve analysis (sieve size 75-2000 µm). The quality of the granules is good when in the size range of 150-1000 µm. The mass of granules in this range is therefore defined as percentage yield for the constant throughput based on powder feed rate.. Increased liquid content and powder feed rate resulted in a lower amount of fines (F <150µm). The results showed that the degree of fill inside the barrel, controlled by screw-speed and powder feed rate, had the most significant impact on the yield. Increasing the filling degree of the barrel lowered the yield values from 46-33% to 38-31% for the size range of 150-1000 µm. Furthermore, a lower amount of oversized granules (F >1000µm) (yield reduced from maximum of 49 % to 22%) could be obtained at decreased liquid content and increased screw speed. This was attributed to the high granulability of the α-lactose monohydrate and PVP mix. The effect on the granulation process (torque) and shape within the scope of the applied experimental design are under evaluation and will be provided when becoming available. Such endeavour will significantly contribute to the physical interpretation required for future mechanistic modelling of the twin-screw granulator. Keywords: continuous twin screw granulator, experimental design, particle size distribution, process yield

Promethera® HepaStem is the company’s cell therapy product to treat serious metabolic liver disorders. Currently, an European Phase I/II clinical trial is ongoing for the treatment of children suffering from Crigler-Najjar syndrome and Urea cycle disorders. The challenge is to provide a drug product easy to reconstitute at the clinical site. For this multi-country and multi-center clinical study, Promethera has developed a GMP-compliant fully closed formulation system based on Biosafe’s Sepax 2 device. This technology allows an aseptic preparation of the final product in a non-sterile environment. The underlying idea was to prepare the product in a mobile unit brought near the clinical site, to timely and consistently deliver the cells within its shelf-life. A successful infusion of HepaStem, formulated in the mobile unit, has been accomplished in the Cliniques Universitaires Saint-Luc (Bruxelles, Belgium). Future patients enrolled in this clinical trial will receive HepaStem cells formulated in the mobile unit. In order to guarantee a flexible, highly qualitative, and economically sustainable supply chain during commercialization, the phase III formulation step will be performed by reconstitution at the hospitals. This implies further fine-tuning of the current technology towards a fully automatic process with minimal manipulations, yet ensuring consistency in terms of safety, identity, purity, and therapeutic potency. This simple process will allow reconstitution to be handled by the medical team or the hospital pharmacist. The approach offers new opportunities in the field of stem cell therapy and cell-based products with a limited shelf-life. Promethera Biosciences® future development programs will implement this innovative supply delivery system.

In the last decade, advanced microscopy techniques have entered the field of drug discovery and delivery. Especially for biotechnological pharmaceutics, the development of macro and nano carriers can benefit greatly from studying their characteristics in situ using advanced fluorescence microscopy methods [1]. While choosing the optimal labeling method for visualizing the carrier or its cargo is crucial, it seldom receives attention [2]. Traditionally, the optimal fluorescent labeling, used for studying the internal processing of a drug, is determined by the need to collect as many photons possible to obtain a good signal-to-noise ratio. Although the possibility that high labeling densities alter the intracellular processing of the molecule is considered, how and at which point this interference happens is not yet studied. The aim of this study was to elucidate the effect of labeling density on the transfection efficiency of labeled pDNA. The transfection efficiency is a good measure for the conservation of biological function of a nucleic acid, since the ultimate goal is to deliver DNA in the nucleus for transcription. Different labeling densities, up to the manufacturer’s recommended density, were tested at different steps in the transfection pathway. Due to the drastic effect on expression levels for higher labeling densities, we tried to determine at which steps in the intracellular processing labeled pDNA behaves different than its non-labeled counterpart. It was found that only the cellular uptake remains unaffected, while other steps in the transfection pathway such as endosomal escape, dissociation from the carrier and transcription are severely affected. Therefore, it seems crucial to obtain a good balance between fluorescent signal and labeling density in order to reliably follow the intracellular processing of pDNA during the various steps of the transfection pathway. In conclusion, labeling of pDNA is altering the biological properties and the labeling density must be considered as an important factor when designing an experiment. The information, included in this work, should help in determining if the labeling method and density used, yields biological relevant results for the intended research question. [1] D. Vercauteren, et al., "Dynamic Colocalization Microscopy To Characterize Intracellular Trafficking of Nanomedicines," ACS Nano, vol. 10, no. 5, p. 7874–7884, 2011. [2] N. G. Walter, C.-Y. Huang, A. J. Manzo and M. A. Sobhy, "Do-it-yourself guide: how to use the modern single-molecule toolkit," Nature Methods, vol. 5, no. 6, pp. 475-489, 2008.

Today, ultrasound is used as a well-established diagnostic imaging technique, but recently it is, in combination with microbubbles, gaining increasing interest as an alternative drug delivery method. The ability to control delivery in time and space is one of its key advantages. Microbubbles consist of a gaseous core stabilized by a lipid shell. In an ultrasonic field they start oscillating, thereby creating streamings in the surrounding fluid. These effects can cause permeabilization of cell membranes. This is called sonoporation and is a very exciting feature for drug delivery of cell impermeable drugs, e.g. genes. To optimize the technique, unraveling the underlying biophysical mechanisms is inevitable. Today sonoporation is mostly studied by indirect methods, thereby missing crucial information. Therefore we developed an ultrasound and swept field confocal microscopy setup which enables us to study the sonoporation processes in real-time. The observations obtained by microscopy were verified by flow cytometry experiments using lipoplexes as model drug. For microscopy experiments, the cell membrane of BLM cells and microbubbles were fluorescently labeled by CellMask Deep Red Plasma membrane Stain®and cholesterylBODIPY FL C12 respectively. Propidium iodide, a cell impermeable dye, was used as a permeabilization marker and emits fluorescence when bound to nucleic acids. For flow cytometry experiments, PEGylated lipoplexes were coupled to microbubbles via biotin-avidin linkage. Lipoplexes were labeled with cholesterylBODIPY FL C12 to study uptake. GFP-encoding lipoplexes were used to measure transfection efficiency. The microscopy data show that pore formation occurs, which is caused by the impact of the propelled bubbles onto the cell membrane rather than fluid streamings created by the microbubbles. The bubbles can even break through the cell membrane ending up inside the cell. Next to sonoporation, bubbles can deform the cell membrane without causing pore formation. At low ultrasound intensities deformation is the main effect, while at higher pressures pore formation predominates. These observations support the data from flow cytometry experiments. At low ultrasound intensities enhanced endocytosis of the PEGylated lipoplexes occurs, although not accompanied with higher transfection levels due to a limited endosomal escape. Higher pressures lead to increased GFP expression, indicating a different mechanism than endocytosis. Our data show that the mechanism involved in ultrasound mediated delivery depends on the ultrasound parameters used. At higher pressures pore formation occurs, thereby avoiding endosomal uptake and release of nucleic acids. Pores are created by the bubbles themself rather than fluid streamings, which implies that loading of drugs onto microbubbles is beneficial. Deformation of cell membranes, observed at lower acoustic pressures, enhanced uptake via endocytosis.

Objective: to screen a dog population from Belgium, the Netherlands and Germany for the presence of disease related mutations. Mutations associated with hip dysplasia (HD), degenerative myelopathy (DM), exercise induced collapse (EIC), neuronal ceroid lipofuscinosis 4A (NCL), centronuclear myopathy (HMLR), mucopolysaccharidosis VII (MPS VII), myotonia congenital (MG), gangliosidosis (GM1) and muscular dystrophy (Duchenne type) (GRMD) were tested. Materials and Methods: blood samples (K3EDTA) from dogs of different breeds collected to study inherited joint disorders, were used for genotyping with Kompetitive Allele Specific PCR (KASP) (N=476). Allele and genotype frequencies were calculated in those breeds with at least 12 samples (N=7). When the number of samples was sufficient, Hardy-Weinberg equilibrium (HWE) was tested. Results: genetic variation was identified for 4 out of 9 diseases: mutant alleles were found in 50, 15, 3 and 2 breeds for HD, DM, EIC and NCL respectively. Additionally mutant alleles were also identified in crossbreeds for both HD and EIC. From those crossbred dogs of which parent breeds were known, no other breeds were identified than those already known from purebred dogs. For 11 breeds only the mutant allele was identified for HD. This could be due to a relatively low number of samples or the mutant allele might be normal in these specific breeds. For HD, DM, EIC and NCL the mutant allele was detected for the first time in respectively 44, 13, 2 and 1 new breeds were the disease has not been reported previously. Disease might have been missed in these breeds so far, the mutant allele can be a marker and recombination might have caused a different association or a third explanation might be that there is a breed specific influence, a modifier locus residing in certain breeds that influences the phenotype. Only for HD and EIC HWE could be tested. For HD some breeds were in HWE (German Shepherd, Golden Retriever), another (Labrador Retriever (p = 0,001)) deviated significantly. For EIC only the Labrador Retriever could be tested and our population deviated significantly from HWE (p < 0,001). Conclusion: for 5 diseases (HMLR, MPS VII, MG, GM1, GRMD), the mutant allele could not be identified in our population. For the other 4 diseases (HD, DM, EIC, NCL), prevalence of associated mutant alleles seems strongly breed dependent. Surprisingly, mutant alleles were found in a lot of breeds where the disease has not been reported (yet?) and for some breeds only mutant alleles were identified.

Purpose The aim of present study was to develop a mucoadhesive chitosan film as an ocular cyclosporine A (CyA) delivery system for treatment of dry eye disease. Methods Blank and CyA loaded chitosan films, with glycerol as plasticizer, were prepared by the solvent casting evaporation technique and dried films were stored in a desiccator containing a saturated solution of NaBr (20°C/60% RH). Hydroxypropyl-β-cyclodextrin (HP-ß-CD) was included in order to enhance ocular CyA bioavailability. The influence of 4 parameters (polymer and drug concentration, amount of plasticizer and addition of HP-ß-CD) on the properties of chitosan films was evaluated using a full factorial design. These ocular chitosan films were characterised by their thickness, swelling and mechanical properties, drug content uniformity and release kinetics. In vitro experiments were performed in order to evaluate the cytotoxicity of these films based on cell viability (MTT assay). Anti-inflammatory activity of the chitosan films developed was determined in concanavalin A activated Jurkat T cells using interleukine-2 (IL-2) secretion as biological read-out. Results Swelling of films was significant less (p<0.05) in case of films prepared with a higher amount of glycerol as plasticizer. The dispersion of CyA in the chitosan matrix resulted in an increased film thickness, altered mechanical properties and decreased water sorption. Although content uniformity was significantly influenced by the amount of glycerol (p<0.05), the variation in distribution of CyA within different regions of film was < 10% for all films prepared. The films released 9% to 19% of loaded CyA after 24 h and higher release was measured from films with the lowest amount of glycerol. The presence of HP-β-CD had no significant influence on the drug release rate. Films were found not to be cytotoxic using HaCaT epithelial cell lines. Significant suppression (p<0.05) of IL-2 secretion in concanavaline A stimulated Jurkat T cells was measured for all CyA loaded film preparations. Conclusions These results suggest that chitosan films present a promising ocular CyA delivery system. Most appropriate formulations will be selected for in vivo studies evaluating the tolerance and efficacy of the chitosan films in a mouse model of dry eye.

Introduction Despite the great therapeutic potential of RNA interference (RNAi) , the clinical progress is hampered by the lack of efficient delivery systems able to transport the small interfering RNA (siRNA) therapeutics across different barriers in a tissue specific fashion. Since the discovery of exosomes as nature’s own RNA delivery vehicle, great interest has gone towards these small (50-100nm), cell-derived membrane vesicles. A major impediment in using exosomes as nanosized delivery vehicles is finding a suitable procedure that allows efficient loading with siRNA. A method recently described in the literature (1,2) relies on electroporation and reports encapsulation efficiencies up to 25 percent. Here we evaluated the use of electroporation as a method for siRNA loading into exosomes. Material and methods Exosomes were isolated from the conditioned culture medium of HEK293 cells by using differential centrifugation. Purified exosomes were mixed with fluorescently labeled siRNA in an Optiprep™ based electroporation buffer (1) and electroporated. The formation of aggregates was analyzed by Nanoparticle Tracking Analysis (NTA). The precipitation of the siRNA was visualized by confocal microscopy and gel electrophoresis and the siRNA encapsulation efficiency was quantified by Fluorescence Fluctuation Spectroscopy (FFS). Results When electroporating siRNA in the presence of exosomes in the Optiprep™ based buffer, encapsulation efficiencies were obtained of the same magnitude as previously reported in the literature. However, repeating the same experiment without exosomes led to comparable results. Nanoparticle tracking analysis (NTA) verified the emergence of large aggregates as a result of the electroporation-induced release of metal cations (e.g. Al3+) from cuvette electrodes. Interestingly, confocal fluorescence microscopy could visualize a clear co-precipitation of fluorescently labeled siRNA in the aluminum aggregates, explaining the high aspecific siRNA encapsulation quantified with FFS. This was further confirmed by the ability to lower the observed siRNA encapsulation by adding 1mM of EDTA to the electroporation buffer (EDTA captures released aluminum cations). Precipitation of siRNA in the absence of exosomes was now lowered to approximately 3%. Complete removal of the formed aggregates was obtained by electroporation in a citrate based acidic buffer. Strikingly under these conditions no significant encapsulation of siRNA was measured neither in the absence nor in the presence of exosomes. Conclusions Altogether, our data provide conclusive evidence that electroporation-induced precipitation strongly biased the assumed encapsulation of siRNA into exosomes. Likely, electroporation may incorporate only minute amounts of siRNA into exosomal vesicles, highlighting the necessity for more efficient loading methods. (1) Alvarez-Erviti L et al. Nat Biotechnol, 2011. (2) El-Andaloussi S et al. Nat Protoc, 2012.

To support Galapagos’ oncology franchise a matrix-free high-throughput multicellular tumor spheroid platform was developed, facilitating anti-cancer drug screening and mode of action experiments. The latter is important as Galapagos is specialized in the development of small molecules against novel targets. Spheroids are microscale spherical 3D cell clusters formed by self assembly and cell growth, and reflect many important properties of solid tumors. There is experimental evidence that some marketed drugs are exclusively effective in 3D and not in 2D culture models. This is often related to the specific expression of the molecular target. Spheroids are accepted as a more relevant in vitro model to predict compound potency in vivo. The soft agar assay has emerged as the golden standard spheroid model in drug discovery. In the soft agar assay cells are embedded in an agar matrix and the compound effects on the viability of the spheroids are measured on a 96-well format. Next to the matrix-based assays (e.g. soft agar), matrix-free technologies (hanging drops, spinner flasks, micro-patterned surfaces) are also described to generate spheroids. Those technologies have disadvantages: they need special equipment, are labour intensive, or harvesting of the spheroid cells for - protein, RNA or FACS - analysis is cumbersome. Those disadvantages are circumvented by generating spheroids on non-adherent surface plates. We developed a matrix-free high-throughput multicellular tumor spheroid platform using regular polypropylene microtiter plates (PP assay). In the PP assay cells are seeded into a 96-well plate and spheroid viability was measured 4 to 7 days after compound addition. Initial evaluation of 3 cell lines (HCT-116, U87MG and T47D) showed that there is no shift in potency for a set of reference compounds between the soft agar and the PP assay. After profiling a panel of 20 cell lines in this assay, we selected the 2 most responsive cell lines (T47D and NCI-H460) to routinely evaluate compounds. In addition, 5 cell lines (NCI-H23, NCI-H441, NCI-H1437, NCI-H2210 and A549) were chosen to evaluate potency differences in the PP assay and a 2D colony formation assay. First, a reference compound inhibiting target X was tested. The A549 and NCI-H23 cell lines were selected for further analysis of a series of 24 compounds against target X. To determine the mode of action of these compounds the feasibility of Western blotting downstream of the A549 PP assay was explored. The PP assay enabled Galapagos to determine the anti-tumoral potency of compounds in an easy, inexpensive, robust and high-throughput fashion and is a valuable asset for future compound testing.

Clinical depression is the third leading contributor to the global disease burden. Despite its high prevalence and considerable socioeconomic impact, very little is known about the pathophysiology of depression. Classic theories on serotonergic dysfunction and cortisol hypersecretion have been studied extensively, but fail to provide sufficient explanations for the etiology of the disease. Increasing numbers of studies now support the idea that depression is not caused by just one factor, but a range of causes including genetic and environmental contributors. Findings from preclinical and clinical studies suggest that inflammatory processes may also play a role in the etiology of depression, at least in a subset of vulnerable individuals. For instance, therapeutic stimulation of the immune system in cancer or hepatitis C patients has been found to result in depression in up to half of these patients. Systemic administration of bacterial endotoxin is commonly used to study inflammation-induced depressive-like behavior in rodents. Here we investigated immune-to-brain communication in mice by examining the effects of peripheral endotoxin administration on neuroinflammation. We used techniques ranging from transgenic bioluminescent mouse models to multiplex analysis of cytokine and chemokine levels in serum and brain. Endotoxin-induced neuroinflammation was further substantiated by quantitative immunohistochemistry of microglia specific markers. Finally we evaluated endotoxin-treated mice in a panel of behavioral paradigms to assess depression-like effects. Our results indicate that systemic immune activation induces neuroinflammation in a time-dependent manner. This neuroinflammation is accompanied by sickness behavior but not by a clearly distinguishable depressive-like behavior.

In the context of cancer therapy, many different nanoformulations have been suggested for the delivery of siRNA into tumor cells. Despite several efforts, the accumulation of nanoparticles in the tumor site after intravenous injection based on passive and active targeting has not been successful until now. An alternative strategy is the attachment of nanoparticles to cells that have tumoritropic migratory properties. The objective of this project is to create a method for decorating the surface of tumoritropic cells (e.g. cytotoxic CD8+ T lymphocytes) with nanoparticles that can be released again from the cell surface at the tumor site by an endogenous or exogenous trigger. The siRNA target that will be used is STAT3 (signal transducer and activator of transcription 3). STAT3 upregulates genes crucial for survival, proliferation, angiogenesis, metastasis and promotes the expression of immune suppressive factors. Liposomes were selected as model nanoparticles and coupled to the cell membrane of CD8+ T lymphocytes via a pyridyl dithiopropionate modified lipid (PE-PDP) incorporated into the liposomal bilayer. This linker lipid forms a disulfide bond with free thiol groups at the surface of the cells. To determine the amount of nanoparticles at the surface, liposomes are fluorescently labeled. The fluorescence per cell is measured by flow cytometry and the attached nanoparticles are visualized by confocal microscopy. These data and images demonstrate that we are able to anchor an appropriate amount of liposomes to the surface of primary CD8+ T lymphocytes without internalization. To allow triggered detachment from the surface, aspecific binding of the liposomes should be avoided. This was tested by comparing nanoparticles with and without a specific linker and by incubating the liposome-coated cells with cysteine to trigger the release of the nanoparticles. Increasing the amount PE-PDP lipids from 10 to 50 wt% leads to a high increase in specific T-cell attachment with 40-50% aspecific binding remaining at highest PE-PDP incorporation. Preliminary flow cytometry data demonstrate that after incubation with cysteine ~50% of the total amount anchored liposomes can be released again from the CD8+ T cell surface. Cell-mediated delivery of nanoparticles is a promising therapeutic strategy for the accumulation of siRNA loaded nanoparticles in the tumor site. Further optimization of the coupling process is ongoing to develop a successful reversible coupling strategy.

Currently, physicians are confronted with a low success rate when treating cystic fibrosis (CF) patients for pulmonary biofilm infections. Biofilms are communities of bacteria embedded in a self-produced extracellular matrix and are notorious for evading the immune system and their increased antibiotic resistance. Possible reasons for the failing antibiotic treatment include inactivation and limited diffusion of antibiotics in CF mucus or the biofilm matrix. While it was shown in vitro that the encapsulation of antibiotics in nanoparticles can improve their antimicrobial activity in biofilms, the highly viscoelastic mucus hampers nanoparticle transport. [1,2] In order to develop a nanocarrier with optimal transport properties in both the mucus and the biofilm matrix, we have evaluated the transport of different types of nanoparticles using confocal and single particle tracking microscopy (SPT). The transport properties of 100 nm carboxylate-, amine- and polyethylene glycol (PEG)-modified polystyrene nanospheres were evaluated in Burkholderia multivorans, Pseudomonas aeruginosa biofilms and human CF sputum. The SPT results indicated that PEGylation greatly improves nanoparticle transport both in biofilms and CF sputum. [3] Next, it was investigated whether a size limit exists for efficient penetration into denser clusters of B. multivorans biofilms using differently sized nanoparticles, including PEGylated silica nanoparticles (25, 45 and 75 nm) and PEGylated polystyrene nanospheres (100 and 200 nm). The PEGylated silica nanoparticles could penetrate into the denser clusters of the biofilm. Swept field confocal microscopy showed that the 200 nm PEGylated polystyrene particles are less mobile in denser regions of the biofilm compared to the 100 nm particles. Our data indicate that formulation of antimicrobial agents in PEGylated nanocarriers smaller than 100 nm in diameter could lead to more efficient transport into the bacterial clusters. In conclusion, SPT microscopy allows to obtain accurate measurements of nanoparticle transport in complex biological media and can therefore be a valuable new tool for the evaluation and development of nanoparticles for the treatment of biofilm-related infections. [1] C. Beaulac, et al., “In-vitro bactericidal efficacy of sub-MIC concentrations of liposome-encapsulated antibiotic against Gram-negative and Gram-positive bacteria,” Antimicrob. Chemother. 41 (1), 35-41 (1998) [2] N. Sanders, et al., “Cystic fibrosis sputum: a barrier to the transport of nanospheres,” Am. J. Respir. Crit. Care Med. 162, 1905-1910 (2000) [3] K. Forier, et al., “Transport of nanoparticles in cystic fibrosis sputum and bacterial biofilms by single-particle tracking microscopy,” Nanomedicine, DOI: 10.2217/nnm.12.129

Similar to all other major cancers, lung cancer incidence and mortality recently started to decline in the US. However, it is still the main cause of cancer-related death. Improved surgical techniques and combined therapies have increased the 1-year survival to ~45%. When detected early, the 5-year survival rate in localized (stage I or II) disease is ~50%. Unfortunately, only a minority of cancers are detected early, resulting in a 5-year survival of 17% overall. These survival rates emphasize the importance of early detection. Recently, a study showed that the use of spiral CT screening for lung cancer resulted in a 20% reduction in mortality, but a high false positive rate (96%) was observed. Complementing traditional screening methods with molecular triage markers has proven beneficial for diagnostic purposes. Epigenetic markers, i.e. aberrant DNA-methylation, are observed early during oncogenesis. Sputum of a test set of 92 patients, 40 cancer cases and 52 cancer-free controls, was evaluated as a non-invasive source to detect the presence of non-small cell lung cancer. The methylation status of 11 candidate markers (HOXA9, TAC1, DPSYL4, SOX17, HOXD1, CDO1, RASSF1, JAM3, SFRP2, GPNMB and GREM1) was tested using a quantitative methylation-specific PCR approach. Cutoffs for single markers were optimized and performance was calculated. To demonstrate clinical utility, the minimal specificity was fixed at 90%. Four genes showed adequate sensitivity (HOXA9: 53%, CDO1: 58%, RASSF1: 75% and SFRP2: 55%) as single markers. Combinations of two markers were explored using all 11 genes in search of marker complementarity. RASSF1 was present in all marker panels, with HOXA9, TAC1, SOX17, HOXD1, CDO1, SFRP2 or GREM1 as second gene, reaching sensitivities of 85%. Twelve stage I or II lung cancers in this study were used for an early detection subset analysis. Single marker assays of four genes were able to sensitively identify such cases (HOXA9: 67%, CDO1: 58%, RASSF1: 83% and SFRP2: 67%). When RASSF1 was paired with HOXA9, SFRP2 or GREM1, all early stage cases were successfully identified. This feasibility study demonstrates that aberrant DNA-methylation can be specifically detected in sputum samples of cancer patients. Panels of RASSF1 with either one of HOXA9, SFRP2 and GREM1 are particularly promising for the early detection of lung cancer, offering therapeutic advantages and potentially better clinical outcomes.

The health benefits of food products and ingredients can only be fully understood and evaluated if their digestive and fermentative profiles have been characterized under standardized protocols. Well-designed in vitro simulation technologies are essential to assess the effects of pro- and prebiotics on modulating the intestinal microbiome towards improved health. ProDigest based its activities on the validated SHIME (Simulator of the Human Intestinal Microbial Ecosystem) technology. The SHIME is internationally accepted as a representative simulation of the gastrointestinal tract (GIT) and a valuable tool for innovative product development. The SHIME is a dynamic and controlled model of the GIT that simulates in sequence the stomach and small intestine digestions, and the 3 colonic regions (ascending, transverse and descending) with its complex and stable microbial communities, which are structurally and functionally similar to the human colonic microbiota. Altough the predictive value of this model has been long validated, it does not test the ultimate effects of these food products on human intestinal epithelia and immune system, as it lacks the host biological compartment. Therefore, to evaluate the biological effect of microbiota and metabolites on the gut wall activity, we developed a number of cell culture assays in combination with SHIME experiments. Samples taken from specific colonic compartments from the SHIME are directly introduced in these cell models, and changes in biological parameters are investigated. The unique aspect of this model relates to the fact that it is not the isolated individual product which is tested, but rather the complete environmental matrix, containing both the product and its direct metabolites produced by the microbial community. This is certainly more relevant when one wants to evaluate the final health effects of the tested food product. In order to test for the specific effect of a given product, we make use of a co-culture in vitro model where human intestinal epithelial-like cells are co-cultured in the presence of immune cells. This model, by mimicking the intestinal mucosal environment, allows testing for putative beneficial effects both on the intestinal epithelia and on the immune system. Recently, we have used this technology to validate the beneficial effects of EpiCor, a yeast-fermentate dietary supplement. EpiCor has been shown to have immune-modulating properties in clinical trials and laboratory assays. However, its intestinal fate and mechanism of immune protection via the gut was largely unknown. Therefore, we have made use of our integrated in vitro models on one hand, to elucidate the product’s intestinal metabolism and effect on the gut microbiota, and on the other hand, to evaluate the effects of EpiCor-derived metabolites on the intestinal epithelia and inflammation.

There is a growing interest in photothermal therapy where damage is deliberately inflicted to malignant tissues by laser light. Gold nanoparticles are often used as enhancers of the photothermal effect since they can efficiently absorb laser light and convert it into thermal energy. When laser light is delivered as a brief but intense pulse, the gold nanoparticle becomes extremely hot and a water vapour nanobubble (VNBs) can emerge around the particle in tissue. The bubble will expand up to several hundred nm until the thermal energy from the particle is consumed, after which the bubble violently collapses. This produces shock waves that can damage neighbouring structures. In this new project, the aim is to make use of the controlled induction of VNBs to enable cytosolic delivery of nanomaterials for biomedicine, including quantum dots for bioimaging and nanomedicines for drug delivery. In order to generate VNBs, a nanosecond pulsed laser will be incorporated into a custom-built laser-based microscope system [2]. The set-up will be further modified so as to enable photothermal imaging of GNP induced VNBs. Cytosolic delivery of nanoparticles will be evaluated in ARPE-19 retinal pigment epithelium cells, A549 lung epithelial cells and C17.2 neural progenitor cells. It will be investigated if (A) cell membrane perforation or rather (B) endocytosis followed by endosomal rupture is the better strategy for cytosolic delivery of these nanomaterials; (C) if co-incubation with GNPs or rather the incorporation of the GNPs into the nanomaterials produces the best results; (D) if nanomaterials can be delivered to cells specifically through this method by using targeted GNPs in a cell co-culture. References 1. Skirtach A G, Dejugnat C, Braun D, Susha A S, Rogach A L and Sukhorukov G B 2007 Nanoparticlesdistribution control by polymers: aggregates versus nonaggregates J. Phys. Chem. C 111 555-64 2. Braeckmans K, Buyens K, Bouquet W, Vervaet C, Joye P, De Vos F, Plawinski L, Doeuvre L, Angles-Cano E, Sanders N N, Demeester J and De Smedt S C 2010 Sizing Nanomatter in Biological Fluidsby Fluorescence Single Particle Tracking Nano Letters 10 4435-42

Introduction: Recent evidence suggests that tumor growth and metastases are sustained by myeloid-derived suppressor cells (MDSC), which over-express arginase-1 (ARG) and cause T-cell suppression. We hypothesize that MDSC impair tumor cell radiosensitivity through ARG-mediated depletion of L-arginine, an essential substrate for the biosynthesis of the endogenous radiosensitizer nitric oxide (NO). Aim: We explored whether MDSC and ARG can be used as markers for tumor growth in experimental mouse models and in the clinical setting of colorectal cancer. We further examined whether low L-arginine levels are associated with an impaired generation of NO by inducible NO synthase (iNOS) and a reduced radiosensitivity of hypoxic tumor cells. Materials & methods: MDSC (CD11b+Gr-1+) and ARG levels in mouse colon CT26 and mammary 4T1 models were analyzed by flow cytometry (FCM) and RT-PCR. Mice were inoculated I.M. with either CT26 or 4T1 carcinoma cells. After 17 days, MDSC were quantified in the spleen by FCM. In addition, ARG levels were analyzed by RT-PCR and FCM. Similar analyses were performed on human blood MDSC (HLA-DRlowCD14+) derived from CRC patients. To model the hypoxic tumor microenvironment, mouse splenocytes were cultured in 1% oxygen. Activation of iNOS was induced by LPS/IFN-γ and analysed by RT-PCR, FCM and Griess assay. Radiosensitivity was analysed in a model of metabolic hypoxia by colony formation assay. Results: The growth of both CT26 and 4T1 tumors in BALB/c mice was associated with an accumulation of MDSC in the spleen from 2-4% (in naive mice) to 8 and 35% respectively. Freshly isolated splenocytes and MDSC showed low ARG levels. Contrasting, hypoxia-conditioned splenocytes from tumor-bearing mice revealed a drastic transcriptional activation of ARG (up to 525.000-fold), and ARG+ MDSC. This corresponds to the transcriptional level of ARG found in the tumor; in the tumor there is an higher percentage of ARG+ MDS. On the other hand splenocytes from tumor-free mice did not display ARG activation. The iNOS/NO-mediated radiosensitization of mammary EMT6 tumor cells was strictly dependent on L-arginine levels, with a marginal effect of 1.2-fold at 10-30 µM while approaching a 2.4-fold effect at a physiological concentration of 125 µM. MDSC levels in CRC patients were increased, as compared with healthy donors (9% versus 4%). Patients with locally advanced CRC had 16-fold higher ARG expression in blood monocytes, as compared to healthy donors. 4 weeks after pre-operative radiotherapy the ARG levels decreased by 2-fold. Conclusions: Although tumor progression is clearly associated with the expansion of MDSC, their hypoxic conditioning appears to be crucial to uncover ARG as a tumor biomarker in vivo. iNOS/NO mediated radiosensitisation of hypoxic tumor cells was drastically impaired at low L-arginine levels which could be depleted by ARG+ MDSC. In a clinical setting, we consider ARG a potential biomarker in locally advanced rectal cancer.

Introduction: The Wilms’ Tumor protein 1 (WT1) has been characterized as a nearly universal tumor-associated antigen, being overexpressed in a variety of solid and hematological malignancies. This feature, together with the limited expression of WT1 in normal tissues and its strong immunogenic potential, has rendered WT1 an attractive target for cancer immunotherapy. Dendritic cells (DCs), engineered to express WT1 by mRNA electroporation, have entered the clinical trial stage and represent promising candidates for immunotherapy. In the present study, we sought to optimize the antigenic expression and presentation of WT1 mRNA-electroporated DCs in order to enhance their immunostimulatory potential. Materials and Methods: Optimization of a WT1 cDNA-containing plasmid (wild-type WT1) vector was performed by a step-by-step approach: (1) The WT1-sequence was flanked by the HLA class II-sorting signal of DC-LAMP to enhance presentation via both HLA class I and class II molecules; (2) the nuclear localization sequence (NLS) of WT1 was deleted to promote cytoplasmic expression; (3) the WT1-DNA sequence was optimized in silico for maximizing translational efficiency; and finally (4) the complete WT1-sig-DC-LAMP fragment was cloned from the original pGEM plasmid into the pST1 plasmid expression vector (a kind gift from U. Sahin, Mainz, Germany). Results: Monocyte-derived DCs were electroporated with WT1-encoding mRNA, transcribed in vitro from the different DNA templates. Immunocytochemical analysis demonstrated that each of the aforementioned optimization strategies led to an enhanced expression of WT1 protein by DCs, expect for the codon optimization step. This could be confirmed by intracellular staining for WT1, showing maximal expression levels 4h post-electroporation and expression of WT1 protein up to 48h after electroporation with the pST1 plasmid expression vector. Moreover, deletion of NLS inhibited transport to the nucleus and enhanced cytoplasmic expression. Functional comparison was performed by coculture experiments of WT1 mRNA-electroporated DCs and a CD8+ T cell clone recognizing the HLA-A2 immunodominant WT1(126–134) epitope. Increased secretion of IFN-γ following coculture with a WT1(126–134)–specific CD8+ T cell clone proved the functional superiority of our optimized WT1-encoding mRNA construct. The superiority of this optimized construct was further demonstrated in an in vivo mouse model. Immunization with optimized WT1 mRNA could induce reduction of a WT1-expressing tumor without signs of autotoxicity by the CTLs. Conclusion: We report on new strategies to optimize the translational characteristics of WT1-encoding mRNA, resulting in an improved antigen presentation capacity of WT1 mRNA-electroporated dendritic cells that was confirmed by in vivo results. These findings may have important implications for the future design of more effective DC-based vaccines

Several advances are taking place in pharmaceutical production processes nowadays [1]. The focus is on the formulation of the Active Pharmaceutical Ingredients into final drug products. One specific point of progress in industrial applications is the change from batch-wise to continuous production processes [2-3]. The traditional batch processes rely mostly on off-line time-consuming and less efficient laboratory testing to evaluate the quality of the product [4]. Continuous production processes, relying on in-line measurements and real-time adjustment of sensitive process variables, would be a step forward towards more efficient production processes. A batch is well defined, and as such it is easy to perform a quality assurance, since a batch can simply be accepted or rejected. However, monitoring the process during continuous operation and being able to interfere during production is more economical as the loss in product is limited, and less waste is produced as such [4-5]. In this contribution the real-time in-line measurements of the continuous from-powder-to-tablet manufacturing line (ConsiGmaTM, GEA Pharma Systems) are used to follow-up and control the process. The continuous line consists of three parts: a continuous twin screw granulator (high shear), a six-segmented fluidized bed dryer system and a discharge system. A large number of variables are continuously logged and allow making a mass and energy balance over the fluidized bed dryer unit. By making of mass balance of the water content over the dryer unit the moisture content of the granules leaving the dryer can be calculated, which is based on an increase of the humidity of the air passing the dryer. Based on several datasets, it can be concluded that it is indeed possible to use the standard logged measurements to monitor the process. As such, an important conclusion is that expensive probes (e.g. NIR [6]) are not always needed to implement in the final production system, but are extremely useful in process development to support development of detailed process knowledge. [1] K. Morris et al., Advances in pharmaceutical materials and processing, Pharmaceutical Science 1 (1998) 235-245. [2] C. Vervaet, J. Remon, Continuous granulation in the pharmaceutical industry, Chem. Eng. Sci. 60 (2005) 3949-3957. [3] H. Leuenberger, New trends in the production of pharmaceutical granules: the classical batch concept and the problem of scale-up., Eur. J. Pharm. Biopharm. 52 (2001) 279-288. [4] S. Mortier et al., Mechanistic modelling of fluidized bed drying processes of wet porous granules: A review, Eur. J. Pharm. Biopharm. 79 (2011) 205-225. [5] H. Leuenberger, New trends in the production of pharmaceutical granules: batch versus continuous processing., Eur. J. Pharm. Biopharm. 52 (2001) 289-296. [6] P. Frake et al., Process control and end-point determination of a fluid bed granulation by application of near infra-red spectroscopy, Int. J. Pharm. 151 (1997) 75-80.

The advent of low cost next-generation sequencing methods is having an effect on the study of the microbial world, on the field of metagenomics and the study of uncultured bacterial diversity. Direct investigation of the vast majority of bacteria, irrespective of their cultivability and taxonomic identities, has challenged the concept of species diversity and has led to various new insights into the composition and functionalities of microbial communities. To analyze and assess the microbial diversity, most studies use open source packages or a variety of scripts. We developed a tool in the BioNumerics® (BN) software for the analysis, quantification, visualization and comparison of microbial communities starting from sequence reads, independent of the sequencing platform used. Within the graphical user interface, the concept of a metagenomics analysis workflow is used, allowing user-specific adaptations. BN makes use of the mothur (1) project. The latter software was a respons to the microbial ecology community by incorporating the functionality of numerous applications into one command-line application. BN uses the flexibility of the algorithms implemented in mothur and elaborates on these results by creating a supplementary interactive MS Windows®-based reporting service including geographical visualization tools, data mining and various charts and statistics for the interpretation and manipulation of the results. BN thus offers a single analysis environment starting from raw read sequence import, sequence trimming and cleaning, chimera removal, sequence clustering and OTU identification using diverse on-line databases to end up with visualization of the OTU abundances or the evaluation of the diversities using a plethora of indices. Performing each analysis step is facilitated by an intuitive user interface and visual feedback via flexible reporting tools. In this study, we will illustrate this new tool with publicly available genomic data sets. The integration of the metagenomics functionality enables the combination of formerly used environmental analyses with the newly obtained metagenomics results allowing comparison between the different methods. (1) Schloss PD et al. 2009. Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. AEM 75(23):7537-41.

Society is demanding a more efficient use of resources in the agricultural and fishery sector and related processing industries. Current state-of-the-art performs mainly partial valorisation often with low added-value of the end-product. Yet many of these by-products have the potential to result in end-products with higher added-value applications. The GeNeSys project (Use of By-Products as System Innovation) is an integrated ILVO project, aiming to identify innovative, if possible integrated, valorisation pathways for by-products and wastes generated in Flanders. The project approaches the problem in three different empirical research cases that is, agricultural by-products, discards from fisheries and composting. Throughout every phase of the project, stakeholder participation will be pursued. Classic science-driven innovation research often has an intrinsic methodological difficulty which hinders end-user adoption. This transdisciplinary research project aims at bundling expertise and thus involving stakeholder participation. Therefore a fourth and final research case with a methodological objective guides the others. By devising instruments and methods to achieve stakeholder participation, this case enables the development of successful, socially supported solutions for valorising agro-food and fishery by-products and wastes. In this specific case the development of an integrated valorisation pathway for agricultural by-products is targeted. A logical approach is to focus first on the evaluation of the potential of high-value applications such as cosmetics, pharmaceuticals, functional food ingredients etc. By-products of this primary valorisation process can in their turn, afterwards be used for other, lower-value applications (e.g. feed, bio-energy, composting). To develop such a bio-refinery concept, different bottlenecks have to be overcome such as stabilisation techniques, seasonal availability of the by-products and wastes, logistic aspects and the economic feasibility of the valorisation process. Therefore, a double-loop process is adopted: the project starts with an exploration cycle to select the by-product(s) with the highest valorisation potential based on scientific/technological and socio-economic criteria. Therefore the current situation in Flanders is evaluated (crop production volumes, volume of by-products, functional components, etc.) and a current state-of-the-art is formulated. Afterwards, an innovation and research cycle is conducted to elaborate further on the selected by-product(s) and its targeted valorisation process.

Background Conjugation of -N-acetylglucosamine to Ser/Thr residues is a reversible post-translational modification of many proteins, controlled by two unique enzymes: O-GlcNAc transferase (OGT) and β-N-acetyl-glucosaminidase (OGA). Hundreds of proteins are O-GlcNAc-ylated but besides biochemical details of specificity and characteristics, the physiological and pathological repercussions and significance remain largely unknown in vivo, particularly in CNS. Tau is important for microtubule stability and transport, causing synaptic defects and neurodegeneration by unknown molecular mechanisms. In Alzheimer's disease, accumulating amyloid peptides aggravate phosphorylation of Tau by activation of both GSK3 isozymes, modeled in our bigenic mice biAT and biGT mice (Terwel et al, 2008). Tau.P301L mice suffer cognitive deficits (4-6 mo), motor dysfunction (6-8 mo), loss of bodyweight, ending with precocious death (8-12 mo, mean 9.5 mo). Aggregates of Tau in brainstem correlate with upper-airway dysfunction and death (Dutschmann et al., 2010; Menuet et al, 2011a,b). Increasing O-GlcNAc-ylation by OGA inhibition was explored here in vivo in old Tau.P301L and young biAT mice. Results Chronic treatment of ageing Tau.P301L mice mitigated loss in body-weight and motor deficits. Moreover, 3-fold more treated Tau.P301L mice survived at the pre-fixed endpoint at age 9.5 months. In addition, O-GlcNAc-ase inhibition significantly improved breathing parameters of Tau.P301L mice, which pharmacologically underpins the relation of mortality to upper-airway defects. Biochemically, O-GlcNAc-ylation of brain proteins increased rapidly and stably by systemic inhibition of OGA. Conversely no biochemical evidence was obtained that protein Tau.P301L was O-GlcNAc-ylated, and was its phosphorylation only marginally affected. Similarly, chronic OGA-inhibition of young biAT mice (beginning at 3 mo) for 4 months prevented their early mortality, improved their general condition and improved their typical anxiety. But also in the biAT model, phosphorylation of protein tau was not markedly affected. Conclusion Pharmacologically increasing protein O-GlcNAc-ylation markedly improved clinical condition, and prolonged survival of ageing Tau.P301L mice with tauopathy, and young adult biAT mice with beginning combined amyloid and Tau pathology. Surprisingly, in both models the clinical benefits were not reflected by biochemical parameters of protein Tau. The pharmacological target of the OGA inhibitor is proposed to be located downstream in the pathological cascade opening a novel venue to understand - and eventually treat - neurodegeneration.

Studying the dynamic behavior of nanoparticles in biomaterials is an important aspect in many research areas. In the biomedical and pharmaceutical fields, for example, the mobility and the aggregation of molecules and nanomaterials is an important aspect in order to develop a successful therapeutic agent. Moreover, the development of nanoparticles, such as iron oxide, quantum dots and gold nanoparticles, for medical and pharmaceutical applications is increasing. To enable safe and reproducible results, accurate nanoparticle characterization in the intended biological environment is a prerequisite. Optical microscopy, and especially fluorescence microscopy, plays a key role in obtaining a better insight in the mechanism that regulate the interaction of nanoparticles with biological materials such as body fluids, tissues and cells. In recent years a novel illumination strategy, that goes under the name of Light Sheet Microscopy (LSM), has emerged as a powerful alternative to confocal scanning microscopy. LSM uses a thin sheet of light that illuminates a particular plane in the sample orthogonal to the detection pathway. Since the light sheet coincides with the focal plane of the detection objective lens, only objects within the focal plane are illuminated, while regions outside the focal plane remain dark. This results in an optical section with excellent contrast due to the absence of out-of-focus light. Compared to classic confocal microscopy, LSM has the advantage of offering high-speed imaging with superior detection sensitivity at a substantially reduced cost. Despite its capabilities, LSM has not found widespread application yet. The main drawback of this technique is the necessity of positioning two objective lens in close proximity with the sample mounted in a special sample holder, a condition that is not compatible with commercial microscope bodies. To address this problem, two types of disposable sample holders with integrated optical components for producing sheet illumination in the sample have been designed. The first design is based on a planar waveguide with an integrated microchannel to achieve an illumination plane in the center of the microchannel. The second design is based on a tilted micromirror that reflects the vertical laser illumination coming from the objective lens into a horizontal sheet of light. Exploiting the advantage of such technique, the chip will be used to demonstrate improved single particle tracking size and concentration measurement using model nanospheres. At the same time, the ability of the chip to follow the behavior of nanoparticle in living cells will be tested by imaging the post-endocytic trafficking of PEI polyplexes in cells as a function of time.

Background An emergent technique in microbiology is matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Its high sensitivity and high throughput made it a very interesting method for the identification of large numbers of microbial isolates. The fact that a given species can be identified through unique biomarkers opens up new perspectives to be able to discriminate and identify different members of a microbial community. With this, important advantages are the comparative simplicity of a given species' mass spectrum and its high reproducibility. Methods We investigated the effect of an increasing complexity on the quality of MS profiles and as a consequence the usability of MALDI-TOF for analyzing microbial communities such as present in fermented foods. In addition, an attempt was made to determine the sensitivity of the technique. For this, mixtures of increasing complexity were subjected to MALDI-TOF MS analysis. In parallel, denaturing gradient gel electrophoresis (DGGE), a commonly applied approach which has proven its usefulness studying fermented food products, was carried out. Visual and cluster analysis were performed for data interpretation. Results The complexity of MS profiles increased with growing numbers of species added to the mixture. Yet, spectral profiles remained of good quality permitting mixtures of different species composition to be compared. Mixtures only differing in one species could easily be distinguished. Despite quality profiles, visual interpretation became more difficult as complexity increased. Current software limitations could not yet give an answer to this problem. As a result, individual members could not always be retrieved from the community profile, explaining the need for specialized data interpretation software allowing more in-depth analyses. Sensitivity of MALDI-TOF MS was at least as high as for DGGE with a detection limit around 1%. Again, actual sensitivity could be higher as this observation could also be biased by the aforementioned restrictions. Conclusions Preliminary results support the application of MALDI-TOF MS for fast and high throughput analysis of food microbiota and indicate possibilities for fast-paced monitoring of for example contaminations, the effect of varying process parameters on the diversity and dynamics etc.

INTRODUCTION Despite the fact that the immune system is able to recognize and kill tumor cells, objective tumor regression is rarely observed. This can be partly attributed to the occurrence of suppressive mechanisms that counteract tumor-specific immune responses. One important immunosuppressive cell type are the myeloid-derived suppressor cells (MDSCs). Although the main site of action of MDSCs in the context of cancer is most likely situated in the tumor microenvironment, so far the study of these cells has been largely restricted to spleen-derived MDSCs. AIM In this study we compared the suppressive capacity of splenic and tumor-derived MDSCs in different subcutaneous mouse tumor models. We also investigated the expression and the role in the suppressive function of the B7 family member CD80 on these spleen- and tumor-derived cells. Finally, we investigated whether MDSCs and regulatory T cells (Treg) co-operate in the suppression of T-cell responses. METHODS EG7-OVA, LLC or MO4 cells were injected subcutaneously into C57BL/6 mice. MDSC subpopulations were sorted by FACS and cultured in the presence of anti-CD3/CD28 stimulated splenocytes after which proliferation of T-cells was evaluated. Flow cytometry was used to determine the expression of CD80 on MDSCs. To evaluate the effect of tumor-derived factors on the expression of CD80 by MDSCs we used a transwell assay and flow cytometry. Sorted Treg were also used in co-culturing experiments in order to determine whether there’s a co-operation between these Treg and MDSCs in the suppression of T-cells. RESULTS In all models, splenic granulocytic MDSCs (MDSC-G) strongly suppressed CD4+ T-cell proliferation while the suppressive effect on CD8+ T-cell proliferation was less pronounced. Not only proliferation was impaired, also the cytokine secretion by splenocytes cultured in the presence of MDSC-G was dramatically decreased. Splenic monocytic MDSCs (MDSC-M) have a lower suppressive capacity, compared to the MDSC-G, on both CD4+ and CD8+ T-cell proliferation. Both MDSC-G and MDSC-M isolated from the tumor microenvironment have a much stronger suppressive activity compared to MDSCs isolated from the spleen of tumor-bearing mice. The expression of CD80 was higher on tumor-derived MDSC-G compared to their peripheral counterparts. Direct contact with tumor cells is required for the upregulation of CD80 and CD80+ MDSCs are more suppressive than CD80- MDSCs. Co-culture of Treg and MDSCs leads to a stronger suppression of CD8+ T-cell proliferation compared to the suppression observed by Treg or MDSCs alone. CONCLUSIONS We have shown that tumor-infiltrating MDSCs possess a stronger suppressive capacity than their peripheral counterparts and that direct contact with tumor cells leads to an upregulation of CD80 on these MSDCs. The results indicate that Treg and MDSCs can co-operate in the suppression of CD8+ T-cell function. However, the nature of this co-operation is still under investigation.

In the past decade, iron oxide-based nanoparticles have been established as highly valuable tools for magnetic cell labeling. Especially in the field of magnetic resonance imaging (MRI), so-called superparamagnetic iron oxide nanoparticles (SPIOs) have been widely used as T2-contrast agents for in vivo cell imaging [1]. To stabilize this magnetic particles, they are surrounded by a coating consisting of for example (synthetic or natural e.g., dextrans or proteins) or amphiphilic molecules (e.g., fatty acids or phospholipids). When iron oxide cores are surrounded by a phospholipid bilayer, they are referred to as ‘magnetoliposomes’ (MLs) [2]. To generate these structures, a water compatible magnetic fluid is first synthesized by co-precipitation of Fe2+ and Fe3+ salts, followed by dialysis of this suspension in the presence of an excess of sonicated, small unilamellar vesicles, ultimately resulting in phospholipid-Fe3O4 complexes which can be readily captured from the solution by high gradient magnetophoresis (HGM) [3]. Examination of the architecture of the phospholipid coat reveals the presence of a typical bilayered phospholipid arrangement which provides several advantages, such as a high biocompatibility and reduced biodegradability. As the combined features of both the magnetizable colloid and the versatile lipid bilayer are joined, the resulting MLs can be successfully exploited in a great array of biological and biomedical applications, including long-term tracking by MRI, hyperthermia cancer treatment and as sustained drug delivery systems [4]. Our recent goals regarding the application of MLs include the development and validation of different types of MLs, functionalized with biological molecules (small molecules targeting receptors or transporters on the cell surface or monoclonal antibodies and potentially nanobodies), for targeted liver and beta cells and their visualization with MRI. References: [1] Shapiro et al., Proc. Nat. Acad. Sci. 101, 10901 (2004). [2] Soenen et al., Nanomedicine. 4(2), 177 (2009). [3] De Cuyper and Soenen. Methd. In Mol. Biol. 605, 97 (2010). [4] Soenen et al., Biomaterials. 32, 1748 (2011). Acknowledgments: Rita Ribeiro is an Early Stage Researcher for the EC FP7 Marie Currie ITN 'BetaTrain' project (ZL36271100-401).

Purpose Topical administration of small interfering RNA (siRNA) nanocarriers is a promising approach in the treatment of pulmonary disorders. However, an efficient siRNA delivery vehicle should still overcome the various extra- and intracellular barriers encountered following inhalation therapy. Recent work of our research group demonstrated that biodegradable cationic dextran nanogels (DEX-NGs) are promising carriers for siRNA. Upon inhalation therapy, alveolar surfactant will be one of the first interfaces that siRNA loaded DEX-NGs (siDEX-NGs) encounter. Therefore, it is of great importance to evaluate the impact of the surfactant on the gene silencing potential of siDEX-NGs. Methods DEX-NGs are engineered through inverse mini-emulsion photo-polymerization of methacrylated dextran and cationic methacrylate monomers in mineral oil. We used dynamic light scattering and fluorescence single particle tracking (fSPT) to define the size of the particles. The coating with alveolar surfactant preparations (Curosurf®) of DEX-NGs was visualized by cryogenic transmission electron microscopy. The complexation of siRNA was determined by fluorescence fluctuation spectroscopy. Flow cytometry and confocal microscopy were used to evaluate gene silencing and cellular internalization. Results siDEX-NGs mediate a marked RNAi effect in various pulmonary cell lines. However, the interaction with Curosurf® reversed the net surface charge of the particles, resulting in significantly hampered cellular uptake. Remarkably, the RNAi effect of siDEX-NGs following interaction with Curosurf® was maintained, indicating an improved intracellular siRNA distribution mediated by the surfactant. In addition, Curosurf® was able to recover the RNAi effect of inactive anionic siDEX-NGs. The impact of Curosurf® was compared with anionic synthetic liposomes (ASLs) that consist of the most abundant phospholipids in alveolar surfactant. Both markedly impede siDEX-NG cellular internalization but in contrast to Curosurf®, ASLs almost completely abrogate gene silencing induced by siDEX-NGs. An improved RNAi effect by virtue of Curosurf® coating was also demonstrated in hepatoma cells, indicating that the positive influence of alveolar surfactant on the intracellular siRNA biodistribution is not limited to cell lines of pulmonary origin. Interestingly, lipid based siRNA nanocarriers (Lipofectamine RNAiMAXTM) seem to be incompatible with alveolar surfactants. Conclusions Our data demonstrate that the interaction with natural alveolar surfactant potentiates the gene silencing activity of siDEX-NGs in different cell lines. This is in stark contrast to a cationic lipoplex formulation. Hybrid alveolar surfactant coated polymeric nanoparticles will be further pursued as a biomimetic siRNA delivery platform.

Valorisation is a hot item in all research branches. There is a global interest in more efficient production processes with minimization of waste and maximisation of profit. The project ‘Use of By-products as System Innovation’ (GeNeSys) aspires to close loops by valorising by-products from agriculture, horticulture and fisheries. Participatory processes will be included in order to develop instruments for successful system innovations. One of the case studies focuses on finding useful applications for discarded fish and benthos. The reform of the European Common Fisheries Policy (CFP) wants to transition fisheries into a sustainable industry that only fishes at the level of maximum sustainable yield. A discard ban is one of the controversial measures. The industry fears having to land a large amount of inferior products. In order to run a profitable business fishermen need the hold to be filled with valuable marine products. The reasons for discarding are various: fish may be damaged, undersized, quota restricted or low of value. Discarding fish can be seen as an enormous waste of resources. Making use of discards instead of throwing them back could be a big step towards a sustainable fishery. Not only would landing them give scientists more accurate catch data but it would also stimulate fishermen into using more selective catching methods. Recognizing the fact that discards can’t be completely avoided it’s important to find a valuable use for them. The diversity of discards awakens the thought that there could be equally diverse applications. Based on a self developed roadmap for scope definition we will determine the exact characteristics of the by-products. The results will show the most promising valorisation pathways. Previous research has listed numerous nutritional and non nutritional uses for marine products and derivatives. Based on literature possible valorisation products are unsaturated fatty acids, chitin, hydroxyapatite, astaxanthin, enzymes, hydrolysed proteins, fish oil, fertilizer, gelatines, vitamins, carotenoids and many more. The great challenge with the extraction of components is keeping the fish as fresh as possible. The key to success is to determine those products that are marketable. To ensure successful commercial integration of the generated innovations, this project relies heavily on participatory processes with all the stakeholders. The project has a double-loop process. Adjustments and improvements are made after the first loop. The system is upscaled during the second loop. This 4-year study wants to create innovations that will help the fishing industry cope with the discard ban. Goals of this project are minimal food losses, maximal value of the landings, and a contribution to the CFP’s vision of sustainable management of the fisheries industry.

TNF-α/cycloheximide (CHX)-induced apoptosis of the mouse intestinal epithelial cell line MODE-K, corresponds with the production of reactive oxygen species (ROS); CORM-A1 (100 µM), a carbon monoxide (CO)-releasing molecule and resveratrol (75 µM), a polyphenolic antioxidant in red wine reduced both effects (Babu et al., Curr. Pharm. Des. 2012). Activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and dysregulation of mitochondrial oxidative phosphorylation are considered to be the two major endogenous sources of ROS production in cells. We therefore investigated the involvement of mitochondrial ROS to TNF-α-induced oxidative stress in MODE-K cells, and the influence of CORM-A1 and resveratrol in this in-vitro model. Carboxy-H2DCFDA is a widely used cell-permeable indicator for total intracellular ROS. It is nonfluorescent until the acetate groups are removed by intracellular esterases. Oxidation can then occur within the cell by various active oxygen species, converting it irreversibly to the fluorescent form DCF. DHR123 has been proposed to measure mitochondrial ROS production. It concentrates in the mitochondria where it can be oxidized by mitochondrial ROS to generate the fluorescent rhodamine 123 (R123). Sytox Red Dead cell stain stains the nucleic acids of dead cells and exhibits bright red fluorescence. Mode-K cells (passage 10-35) were grown for 36h and serum starved overnight. Then, the cells were exposed to various concentrations of TNF-α/CHX for 6h; simultaneous detection of ROS production and cell death has been performed using either carboxy-H2DCFDA or DHR123 together with Sytox Red in a single experimental setup using flow cytometric analysis. For experiments involving CORM-A1 and resveratrol, the confluent cells were pretreated with the test drugs for 1 h followed by its co-treatment with TNF-α/CHX for 6h. Treatment of TNF-α/CHX increased mean fluorescence intensity (∆MFI) of both carboxy-H2DCFDA and DHR123-derived fluorescence in a concentration-dependent manner with concomitant increase in cell death as measured with Sytox Red positivity. Pretreatment with CORM-A1 significantly reduced the TNF-α/CHX-induced ∆MFI of DCF while it did not influence the ∆MFI of R123. Concomitantly, CORM-A1 reduced TNF-α/CHX-induced cell death. Pretreatment with resveratrol also reduced TNF-α/CHX-induced cell death but this was accompanied by a significant reduction of the TNF-α/CHX-induced ∆MFI of both ROS sensitive probes, implying that resveratrol reduced both total ROS and mitochondria-derived ROS. Mitochondrial ROS thus seems an important contributor to TNF-α-induced oxidative stress in intestinal epithelial cells. The anti-apoptotic effect of CORM-A1 seems to be independent of reduction of mitochondrial ROS. However, the inhibitory action of resveratrol on mitochondrial ROS production might contribute to its anti-apoptotic effect.

The current hyperpigmentation treatments are often associated with a lack of efficacy and major side-effects. We hypothesized that microRNA (miRNA) based treatments may offer an attractive alternative by specifically targeting key genes in melanogenesis. The aim of this study was to identify miRNAs interfering with the pigmentary process and to assess their functional role. MiRNA profiling was performed on mouse melanocytes (melan-a) after 3 consecutive treatments involving forskolin and ssUV irradiation. The differential expression of miRNAs was determined by a multiplex qPCR platform utilizing RT stem-loop primers and Taqman probes. Sixteen miRNAs were identified as differentially expressed in treated melan-a cells versus untreated cells. Remarkably, a 15-fold downregulation of miR-145 was detected. We decided to investigate its effect on genes involved in melanogenesis or melanosome transport by use of functional assays. Modulation of miR-145 expression in melan-a cells and human primary melanocytes resulted in a significant down- or upregulation of major pigmentation genes or genes involved in the transport of melanosomes to the cell periphery. Moreover, target prediction database analysis combined with luciferase reporter assays showed direct targeting of myosin Va by miR-145. Transient overexpression of miR-145 in human melanocytes resulted in a reduced MYO5A protein level followed by a perinuclear accumulation of melanosomes and a clearly visible depigmentation effect in vitro. In conclusion, this study established for the first time a miRNA signature associated with forskolin and ssUV treatment. The significant down- or upregulation of myosin Va together with other important pigmentation genes, after modulation of miR-145 expression, suggests a key role for miR-145 in the regulation of melanogenesis. Therefore, miR-145 is a potential therapeutic target in the treatment of skin pigmentation disorders.

Introduction: With the growing interest in epigenetics, histone proteomics has become an emerging field. Posttranslational modifications (PTMs) on histone tails play an essential role in fine-tuning gene expression programs and are of particular interest in defining embryonic stem cell (ESC) pluripotency. One such radical PTM is the N-terminal cleavage of histone H3 during retinoic acid (RA) induced differentiation of mouse ESC, which brushes away the first 21 amino acids and their PTMs (Duncan et al, Cell, 2009). In previous experiments in our lab we were able to detect for the first time a truncated form of H3 during RA induced differentiation of human ESC. Unfortunately the exact cleavage site still remains unclear. This is mainly because detection by mass spectrometry requires additional efforts. With multiple PTMs found mainly at lysine and arginine, a bottom-up approach using trypsin as a proteolytical enzyme does not generate interpretable data. Top-down mass spectrometry on the other hand has the advantage of measuring intact proteins, thereby circumventing the problem of small peptide fragments and enhancing identification of multiple PTMs at once. In this study we will focus on top-down characterization of bovine histones and purified calf H3 in order to facilitate the identification of cleaved H3 (cH3) formed during human stem cell differentiation. Results: Using an ESI-Q-TOF mass spectrometer preceded by reversed phase chromatography (RPLC), we were able to identify the four core histones present in a mixture of bovine histones using the deconvolution algorithm MaxEnt 1 (MassLynx, Waters). Yet, because of the entangled complexity of its different modifications, H3 is by far the hardest catch. Therefore we first focused on the top-down analysis of purified calf H3 to obtain greater insight into the different modification statuses and their respective retention times on the RPLC. Using these insights, we moved on to identify the cleaved form of H3 in a histone extract taken from differentiating human ESC. Conclusions: Although the biological relevance has not been elucidated yet, the study of histone cleavage will most probably shed new light onto existing ESC data. Also, from an experimental point of view, it is extremely important to realize that any approach that focuses on epigenetic modifications on the H3 N-tail, is very sensitive to this truncation event. Top-down MS detection of histones is untargeted in nature and is thus a very applicable method to study histone epigenetics.

Objectives: The fysicochemical and biomedical properties of the fluorobenzyl analogues 2-[18F]FMLP and 4-[18F]FMLP were earlier reported [1-4]. As the fluorethyl group is known to show a high stability in vivo, it was also decided to synthesize and evaluate the fluoroethyl analogues 2-[18F]FELP and 4-[18F]FELP with regard to their affinity for the LAT1 amino acid transport system which expressed in excess in tumor cells vis à vis normal tissue. Methods: The radiosynthesis of 2-[18F]FELP and 4-[18F]FELP, the stability study and the in vitro evaluation were performed in analogy with previously described methods [1,2]. Results: Both 2-[18F]FELP and 4-[18F]FELP were synthesized in a commercially available module with an overall radiochemical yield of 32 % and a radiochemical purity of more than 99 %. The new compounds were shown to be stable in 0.9% saline for at least 8 hours. The in vitro binding studies on rat rhabdomyosarcoma R1M cells over-expressing LAT1, showed that 2-[18F]FELP features a higher affinity (Km = 16 µM ) for the LAT1 than 4-[18F]FELP (Km= 94 µM) but stimulates natural amino acid efflux from the cells in a 8 times less extend than the 4-ethyl analogue. Conclusions: Both 2-[18F]FELP and 4-[18F]FELP can be efficiently synthetized in a commercially available module and show appropriate shelf stability. More important is the high affinity of 2-[18F]FELP for the LAT1 transport system that, coupled to a lower efflux stimulation renders it the properties of a blocker. These properties opens also new perspective for the therapeutic feature of the non radioactive analogue. Future research will have to prove whether these characteristics will lead to improved tumor visualization and characterization with PET and generate a potential therapeutic tool. References: [1] Kersemans K, et al (2008) J Nucl Med Biol, 35, 425–432. [2] Kersemans K, et al (2010) J Label Compd Radiopharm, 53, 58–62. [3] Kersemans K, et al (2010) J Label Compd Radiopharm, 54, 220. [4] Kersemans K, et al (2012) Int J Chem Kinet, 44, 705–711.

Cervicovaginal fluid (CVF) is composed of secretions originating from organs that are part of the female genital tract, including vagina, cervix, endometrium and ovaries. As a result, this fluid contains a wealth of information (eg. biomarkers) concerning the status of these organs. Cervix cancer is caused by one or more infections of the cervix with an oncogenic form of human papillomavirus (mostly HPV 16 and 18). Today several screening methods exist (Pap-smear, HPV genotyping, colposcopy) but each has its own disadvantages. The need for a specific and sensitive biomarker that can easily be detected is therefore high. Six post-menopausal CVF samples from healthy women and six samples from precancerous women were analyzed separately. These samples were concentrated and run over a 2D-LC-MS/MS proteomics platform (RP-C4, µCap-RP-C18, automated spotting on MALDI targets and identification by MALDI-TOF/TOF and Mascot). Quantification was performed by spectral counting. For validation by ELISA, samples from healthy and precancerous pre-menopausal women were used. After comparison of the 2x6 samples, we identified one protein that was present and absent in all CVF samples from precancerous and healthy women, respectively (‘qualitative’ biomarker). Moreover, we found four proteins that showed a marked up- or down regulation in one of the two conditions (at least 3-fold; ‘quantitative’ biomarkers). ELISA experiments on an additional 30 samples from healthy (n=17) and precancerous pre-menopausal (n=13) women confirmed the discriminative power of the above mentioned ‘qualitative’ biomarker. Further ELISA experiments on the other biomarkers are ongoing. Additionally, experiments on longitudinal samples (i.e. a time-dependent series of samples from the same individual) showing progressive infection or clearing are ongoing by using both ELISA and complete proteome analysis (iTRAQ quantification). Although further research on higher patient numbers is needed to validate our results, the ‘discovery phase’ of biomarkers for identification of the precancerous state in CVF looks promising. If such a CVF biomarker(s) could indeed be put forward, applications such as combination with current screening techniques and/or development of a self-diagnosis test could be considered.

During antimicrobial chemotherapy there is no straightforward way to follow-up on the pharmacological effect of the treatment. Therefore, based on data from large clinical trials and/or in-vitro studies, specific treatment targets are defined in terms of pharmacokinetic-pharmacodynamic (PK-PD) indices. These targets may than serve as useful intermediate endpoints to guide antimicrobial chemotherapy dosing. Based on clinical trials conducted with cipro-, gati- , grepa- and levofloxacin the use of particular AUIC targets (AUIC > 125 or AUIC > 250; depending on the intended rate of bacterial killing) are advocated. In vulnerable populations where altered pharmacokinetics are expected, like the post-bariatric surgery patients, pharmacokinetic-pharmacodynamic simulations provide a way to assess the potential influence of these expected PK alterations on drug therapy. In this study we (i) used a population approach to describe pharmacokinetic variability in a patient cohort of 12 post bariatric surgery patients and (ii) used this model in a PK-PD simulation study to assess the target attainment rate for a standard 400 mg oral dose of moxifloxacin in this population against a hypothetical Streptococcus pneumonia infection. Our analysis clearly reveals that (i) for antimicrobial dosing the “one dose fits all” paradigm does not hold and (ii) that efficient moxifloxacin dosing optimization is hampered by the discussion on which AUIC target to pursue to ensure optimal patient therapy. To our opinion our study results highlight the need for a thorough efficacy trial in post-bariatric surgery patients on one hand and further PK-PD simulations on data obtained in the healthy patient population to ascertain adequate PTA’s against Streptococcus pneumonia in this population. These conclusions are further empowered by the recent reports of increasing fluoroquinolone resistance against, amongst others, Streptococcus pneumonia, in countries with a high consumption of fluoroquinolone antibiotics (e.g. Belgium).

The adult human heart is unable to restore damage after myocardial infarction. In the last decade, stem cell therapy has emerged in an attempt to repair cardiac tissue in mainly two ways: (1) directly, by regeneration of functional myocardium and (2) indirectly, by paracrine actions enhancing tissue healing. Recently, we reported the isolation of Cardiac Atrial appendage Stem Cells (CASCs) from patients with ischemic heart disease (IHD). The isolation procedure relies on elevated activity of aldehyde dehydrogenase (ALDH), a typical stem cell feature. CASCs are a favorable candidate for cardiac regeneration, as they are ex vivo expandable without loss of differentiation potential. Alternatively, mesenchymal stem cells (MSCs) are reported to secrete paracrine factors that modify the cardiac healing process by e.g. influencing resident cardiac stem cells (CSCs). In this study, an in vitro 3D collagen assay was utilized to test whether conditioned medium from bone marrow MSCs (MSC-CM) was able to promote migration of endogenous CSCs from human cardiac tissue. Therefore, cardiac tissue fragments obtained from patients with IHD were cultured inside a native collagen type I matrix and subjected to serum-free low glucose DMEM (control) or MSC-CM. After 6 days, cells from human heart tissue were mobilized under the influence of MSC-CM, pointing out that soluble factors secreted by MSCs were responsible for this migratory response. In a next step, we wanted to investigate which particular cell type was mobilized. Therefore, mobilized cells were isolated and expanded in order to characterize them. These cells displayed stem cell properties like clonogenic potential (45,9±14,8%, n=11) and expression of pluripotency associated genes like oct-4, nanog, dppa-3, tbx-3, c-myc and klf-4. Flow cytometrically, mobilized cells were CD49c+, CD73+, CD44+, CD29+, CD105+, CD13+ and CD90+, while CD34, CD45, CD271, CD140b and CD117 could not be detected. Similar to CASCs, mobilized cells displayed high ALDH activity in culture and differentiated towards cardiomyocytes as shown by expression of troponin-T and -I after 1 week of co-culture with neonatal rat cardiomyocytes. These data indicate that mobilized cells display strong similarities with resident CSCs, more specifically with CASCs. In conclusion, these findings demonstrate that soluble factors secreted by MSCs can exert migratory actions on cells present in adult human heart tissue, including CSCs. Although the functional capacity of CASCs is currently still under investigation in a Göttingen minipig infarct model, these data could open doors towards new therapies in which paracrine factors are utilized to enhance survival, homing and differentiation capacities of transplanted/resident stem cells.

The simple antibody platform (SIMPLETM) of arGEN-X uses active immunization of outbred llamas to generate monoclonal antibodies for clinical purposes. GPCRs are considered as difficult targets for the identification of monoclonal antibodies (mAbs) since the integrity of the receptor depends on its membrane environment. In order to demonstrate the capability of the SIMPLE Antibody™ platform to generate mAbs to such a challenging target we raised mAbs against glucagon receptor (GCGR), a type B GPCR. By doing DNA immunization of 2 outbred llamas we were able to generate a strong, GCGR-specific immune response. Combinatorial Fab libraries were built by PCR-amplification of variable regions of the llama IgG1, and cloning into a phagemid vector. Phage selections on the recombinant extracellular domain of GCGR, as well as on full length GCGR, resulted in the identification of in total 10 different families of high affinity GCGR-specific clones, both lambda and kappa. The affinities of these mAbs are in the low nM range and show very good off-rate. Some of them were able to neutralize glucagon-mediated cAMP increase in GCGR-overexpressing cells with low nM potencies. With these 10 mAbs we were able to distinguish 5 different epitopes, demonstrating the high epitope coverage of the platform.

Biofilms are defined as microbial communities of cells which are attached to a surface and embedded in an extracellular polymeric matrix. Cells in these biofilms show a reduced susceptibility to most antifungal agents. Azoles have a fungistatic effect due to the inhibition of the enzyme 14α-demethylase, involved in ergosterol biosynthesis. Previous research has shown that miconazole (an imidazole) also possesses fungicidal activity. Nevertheless, 1% - 10% of sessile Candida cells are tolerant to miconazole. The aim of this study was to identify genes which are involved in fungal biofilm formation and to unravel the mechanisms of resistance of these biofilms to miconazole treatment. A screening of a Saccharomyces cerevisiae deletion mutant bank was carried out. To this end, mature biofilms were grown in 96-well microtitre plates and treated with miconazole (1 mg/ml) for 24h. The biofilm formation and susceptibility to miconazole of each mutant were examined with a resazurin-based cell viability assay. Our S. cerevisiae deletion mutant bank contains approximately 5000 mutants. 340 mutants (6.9%) showed significant differences (P < 0.01) in biofilm formation compared to the WT ; the majority (241/340 mutants) formed less biofilm. 99 mutants showed significantly increased biofilm formation. Based on the biological function of the deleted genes, peroxisomal transport and mitochondrial organization seem to play an essential role in biofilm formation. Furthermore, mutants with reduced biofilm formation were affected in genes which previously have been described to be involved in biofilm formation. 386 mutants (7.8%) showed significantly different (P < 0.01) susceptibility to miconazole compared to the WT ; 136 mutants were more susceptible and 250 mutants were more tolerant to miconazole than the WT. Analysis of the biological function of the genes deleted in these mutants revealed that genes involved in transcription, peroxisomal transport and lipid metabolism contribute to resistance to miconazole. In addition, previously described genes involved in general stress responses were also found.

Staphylococcus aureus is one of the most abundant nosocomial pathogens worldwide. It causes a variety of infections ranging from soft tissue infections to life threatening conditions such as endocarditis, resulting in approximately 18,000 deaths per year in the US. Unfortunately, these infections are hard to treat as methicillin resistant S. aureus (MRSA) strains are resistant to different classes of antibiotics. In addition, the ability to form biofilms contributes to this resistance. A possible alternative treatment is situated in the field of bacterial interference. For instance, Staphylococcus epidermidis, part of the human skin flora, produces an extracellular serine protease which inhibits S. aureus growth and biofilm formation. In the present study, we investigated the protease secretion of 114 S. epidermidis isolates recovered from endotracheal tube (ET) biofilms from mechanically ventilated patients. To this end, we performed azocasein assays in microtiter plates. 110 of the 114 (96%) S. epidermidis isolates secreted proteases and most (95%) of the isolates produced a mixture of serine, cysteine and metalloproteases. Interestingly, protease production of a particular S. epidermidis isolate was significantly higher (p < 0.05) when S. aureus was absent from that same clinical sample (i.e. the ET biofilm). When MRSA Mu50 biofilms were treated with the supernatant (SN) of protease positive S. epidermidis isolates, a significant reduction (p < 0.05) in biomass was observed. Images obtained with confocal laser scanning microscopy showed that MRSA Mu50 biofilms treated with SN of protease positive S. epidermidis strains were thinner and had a more patchy structure. In contrast, treatment of MRSA Mu50 biofilms with SN of protease negative S. epidermidis did not affect biofilm structure. Together, these data indicate that protease positive S. epidermidis strains affect S. aureus biofilm formation. Currently, we are investigating if the presence of protease positive S. epidermidis strains also affects the antibiotic susceptibility of S. aureus strains. In addition, transcriptomic experiments have been started to elucidate the S. aureus genes for which the expression is affected by protease positive S. epidermidis strains.

Objective: Multiple Sclerosis (MS) is a chronic inflammatory, autoimmune disease of the central nervous system, characterized by demyelination of neurons. Clinically Isolated Syndrome (CIS) is a possible first manifestation of MS and a subgroup of these CIS patients will develop MS. Cerebrospinal fluid (CSF) of both CIS and MS patients is characterized by the presence of oligoclonal band antibodies. However, little is known about the antigens to which these antibodies are directed. The aim of this study was to identify novel antigens for CIS and explore their prognostic potential to predict conversion to MS. Methods: To identify novel antigenic targets for CIS, a phage display technique, called serological antigen selection (SAS), was applied on CSF from 4 CIS patients, who developed MS. Two cDNA phage display libraries suitable for SAS have been constructed, which were derived from normal and MS brain, respectively. Antibody reactivity towards candidate CIS antigens was tested in CSF and serum from patients with CIS (n=123/108), MS (n=65/n=44) and other (inflammatory) neurological diseases (n=75/n=38) as well as in serum from healthy controls (n=44). Results: Using SAS, a panel of 6 novel CIS candidate antigens was identified, all derived from selections with the normal brain cDNA library. Apart from antibody reactivity in CSF from CIS patients used for the SAS technology, antibody reactivity was also detected in CSF from additional CIS patients and Relapsing-Remitting (RR) MS patients. Serum antibody reactivity was significantly increased in CIS and RR-MS as compared to healthy and neurological controls (p=0.03). For 2 antigens prognostic potential could be demonstrated, because the frequency of both CSF and serum antibody positive patients was higher in CIS patients who converted to MS as compared to CIS patients without conversion. Conclusions: A panel of 6 novel CIS antigens was identified to which antibody reactivity was primarily detected in CIS and RR-MS as compared to controls. Prognostic potential to predict conversion to MS could be demonstrated for 2 antigens. Future research is needed to study the role of these antigens in CIS and MS pathogenesis.

In the development process for new medicines, safety and efficacy are the fundamental concerns of the pharmaceutical industry. Tests for genotoxicity and carcinogenicity play a key role in this respect. Research using model organisms has provided critical breakthroughs in our understanding of life, in particular with respect to development, genetics and cell biology. The aim of this project (Neomodels) is to evaluate the suitability of flatworms as test (model) organisms to detect carcinogens, proliferation inducers or blockers, and central nervous system affecting agents. These new models are needed to fill the niches left open by the other current model organisms and in vitro assays. Flatworms are best known for their amazing regenerative capacity—even a fragment as small as 1/279th the size of the original individual has the capacity to regenerate into an entire animal. The key to the regenerative prowess of these creatures are totipotent stem cells, called neoblasts (hence NEO(blast)MODELS). Thus, flatworms offer a simplified model for studying chemically induced carcinogenesis & anti-cancer therapy within the complexity of the entire organism and during the regeneration of multiple tissues. The main goal of this project is to pre-validate these new model organisms & assays and to obtain a proof of concept of their applicability as new pre-clinical model systems. We will show data on the assay we have developed to test for the safety of compounds in vivo, on the development of a short-term cancer model and on an efficacy study.

The limitations of current rat C-peptide assays led us to develop a time-resolved fluorescence immunoassay (TR-FIA) for measurements in plasma, incubation media and tissue/cell extracts. The assay uses two monoclonal antibodies, binding to different parts of the C-peptide molecule, and allowing, respectively, capture of the peptide and its detection by europium-labeled streptavidin. It is performed on 25 µL samples for a dynamic range between 66 pM up to 3900 pM C-peptide, and displays over 95 percent recovery of added peptide in the range of 111 to 2786 pM. Its inter- and intraassay coefficients of variations are, respectively, lower than 7.6 and 4.8%. Cross-reactivities by rat insulin, and human, porcine and mouse C-peptide are negligible. The assay has been validated for in vivo and in vitro measurements of C-peptide release and cellular content. Release patterns were similar than those for insulin and occurred in equimolar concentrations for both peptides. The molar C-peptide contents in purified beta cells and isolated islets were similar to the corresponding insulin contents. This was also the case for pancreatic extracts containing protease inhibitors. Besides its analytical qualities, the in-house development and validation of assays such as the one presented in this study is also financially attractive.

When Newton saw an apple fall, he found a way of proving that the earth turns round. When we saw diseased cows, we found Hidden Markov models to get them sound (1). While studying pressures and suctions, Sir Isaac performed some deductions. While studying disease and marker concentrations, we quantified their causal associations (2). How probabilities connect, Reverend Bayes did tell. How animal infectious diseases spread, our epidemiological models foretell (3). Whether or not the bell-shaped curve is accurate, de Moivre and Gauss may hesitate. Whether or not an animal resists or tolerate, an infection our models will estimate (4). Then, if Einstein called pure mathematics the poetry of logical ideas, our model mathematics are the poetry of veterinary art. (1) Detilleux. A hidden Markov model to predict early mastitis from test-day somatic cell scores. Animal, 2011. (2) Detilleux, Reginster, Chines, Bruyère. A Bayesian path analysis to estimate causal effects of bazedoxifene acetate on incidence of vertebral fractures, either directly or through non-linear changes in bone mass density. Stat Methods Med Res, 2012. (3) Detilleux, Theron, Duprez, Redding, Bertozzi, Mainil, Hanzen. Structural equation models to estimate risk of infection and tolerance to bovine mastitis. Gen. Sel. Evol., 2013 (4) Detilleux. A mathematical model to study resistance and tolerance to infection at the animal and population levels: application to E. coli mastitis. Front Genet. (2012)

The development of fast, sensitive, and affordable technologies for the detection of DNA in clinical and environmental diagnostics has been the subject of intense research to date. Of particular interest is the accurate detection of point mutations, so-called single nucleotide polymorphisms (SNPs), in genetic material of various origins. It is well-known that these SNPs for example can play a significant role in the onset and development of many, if not all, types of cancer cystic fibrosis and antibiotic resistance for bacteria. As such their identification is vitally important for making accurate clinical treatment decisions. We therefore developed a new technology that enables fast and sensitive detection at an affordable cost (<100€ per test) that is amenable to miniaturization. This technology is called fiber optic (FO) SPR. Efforts by our research group have shown that the FO-SPR sensor can be applied in an automated setup for both immuno- and DNA based bioassays, which can rival with the ELISA golden standards. Here, we present the application of FO-SPR in real-time monitoring of solid-phase PCR and mutation screening, which is a major step forward towards cheap integrated DNA biosensors. Moreover, this concept displays some advantages over real-time PCR. While commercial qPCR provides endpoint measurements of fluorescence intensity, our biosensor permits monitoring of the reaction kinetics of every PCR cycle in real time. DNA target concentrations were detected in the femtomolar range using Au nanoparticles (NPs) as signal enhancers. Furthermore, because read-out is Au NP based, possible interference due to primer dimerization does not occur and measurements are possible even in opaque media. Additionally, FO-SPR sensors were used in combination with high-resolution sample heating to monitor the melting behaviour of DNA duplexes. This allowed not only for screening of SNPs (single nucleotide polymorphisms) in real-time but also for designating their location at the specific sequential site. The FO-SPR is therefore ideally suited for fast and sensitive genetic screening of short fragments (ranging from 30-80bp). For instance, we demonstrated that mutations in the p53 gene and several other sequences could be accurately identified in less than 20 minutes. Further developments of this technology in terms of miniaturization and integration of a clinical sample preparation will allow it to evolve towards a market-ready point of care biosensor.

Historically, in vitro enzyme kinetic experiments were conducted to have a rough idea on the intrinsic metabolic clearance of liver-derived in vitro systems without a physiologically relevant focus on this preclinical part of the R&D process. However, with the recent shift towards the use of these kinetic data for quantitative PBPK modeling & simulation, the experiments for determination of enzyme kinetic parameters should be performed, in addition to linearity assessment, with concentrations mimicking the in vivo obtained concentrations as closely as possible, ensuring ‘extrapolatibility’ from in vitro to in vivo pharmacokinetics (avoid rubbish in-rubbish out principle). This study represents the ‘bottom-up’ scaling of in vitro enzyme kinetic data of tramadol, chosen as a representative probe for multi CYP450 involvement, using the IVIVE-linked PBPK modeling and simulation platform Simcyp®. In essence, in vitro enzyme kinetic data of a test drug is mechanistically scaled to in vivo pharmacokinetics, using in vitro-in vivo extrapolation (IVIVE) principles in a physiologically-based pharmacokinetic (PBPK) modeling and simulation environment. Enzyme kinetics of tramadol were investigated in pooled human liver microsomes and recombinant CYP enzyme systems. A range of concentrations was tested in either system to determine the observed in vitro clearance vs. substrate concentration. Non-linear models that best described the kinetic data were constructed in R to extract intrinsic clearance (CLint), Km, and Vmax parameters. In turn, these are used as input for IVIVE-PBPK models in order to predict in vivo pharmacokinetics. Three different in vitro input models were investigated: model 1 is based on intrinsic clearance (CLint) values from HLM corrected for specific CYP450 contributions from a chemical inhibition assay; model 2 is based on CLint values obtained in recombinant enzyme systems; model 3 is based on CLint values from HLM corrected for specific CYP450 contribution by CLint values obtained from the recombinant enzymes. The outcome of every model was validated against tramadol in vivo pharmacokinetic data from literature. Model 3 gave the best predictions in terms of visual predictive check, median fold error (MFE) and variability fold error (VFE) on the predicted clearance. Predictions were considered as good clinical estimates, being between 0.8- and 1.25-fold of the observed clearance. The IVIVE-linked PBPK approach proved to be a strong integrative approach, combining in vitro, as well as in vivo and in silico data, to provide good clinical estimates of the in vivo PK of tramadol in adults, and gain mechanistic insight in relevant disposition covariates at hand.

Background and Aims. Rheumatoid arthritis and inflammatory bowel disease have in common that both are systemic autoimmune chronic inflammatory diseases, with a great impact on the patient’s quality of life and gives rise to important economic and social costs. Mesenchymal stem cells (MSCs) of allogeneic origin have been reported to suppress effector T-cell responses in vitro, to have therapeutic effects in some immune disorders and certain capacity to restore immune tolerance. The aim of our work was to determine the distribution, and potential anti-inflammatory and therapeutic effects of human adipose-derived MSCs (hASCs) in well-established models of rheumatoid arthritis and inflammatory bowel disease using several routes of administration: intraperitoneal (IP), intravenous (IV), endoscopic (EN) and intralymphatic (IL), an innovative route for ASC administration, developed by us. Methods. We optimized the administration of hASCs in the inguinal lymph nodes of mice and compared the distribution of Luciferase-expressing hASCs in healthy and diseased mice using IL, IV and IP routes of administration. We examined the therapeutic action of hASCs in colitis induced by administration of trinitrobenzene sulfonic acid or dextran sodium sulphate (using IP, IV, EN and IL routes), and in arthritis induced by administration of chicken collagen II (using IV and IL routes) in immunocompetent mice. Efficacy was determined by clinical signs of the disease and incorporating state of the art techniques such as microCT scanning and bioimaging. Results. Distribution of hASCs varied depending on the route of administration and the inflammatory status, and a different mobilization throughout the lymphatic system and main organs was observed depending on the experimental conditions. A correlation between localization of hASCs in the site of inflammation and therapeutic benefit was also found. The infusion of hASCs ameliorated the severity of colitis and arthritis. We report here that hASCs administered via different routes show therapeutic effect. Our data show for the first time that IL administration can also be an effective mode of ASC administration. Conclusions. Adult hASCs are attractive candidates for cell-based therapy for the treatment of inflammatory and autoimmune disorders which are associated with enormous health, social and economic burden.

Intradermal vaccination, i.e. delivery of vaccines in the dermal layer of the skin, has many advantages, including dose-sparing properties, improved immune response, improved safety and simpler logistics of delivery. Despite these advantages, devices to accurately deliver vaccines in the dermal layer of the skin are scarce. Medical scientists, product developers and economists of the University of Antwerp (UA) and Artesis University College joined forces with VOXDALE BVBA, a design and engineering SME based in Antwerp, to develop a device that allows for intradermal administration of substances, including vaccine antigens. Additionally, this device (called VAX-ID®) meets preset technological challenging requirements in terms of safety, waste management, ease of use, use by untrained medical staff, suitability for use in elderly and children and at anatomic sites other than the deltoid region, next to assembly and production requirements. The multi-disciplinary approach, combined with a distinct university-industry collaboration led to the development of a prototype device VAX-ID®. Additionally, a grant was provided by the Belgian Industrial Research & Development Fund (BiR&D). The latter provided funding for 3 master theses (Applied Economics, Nursing and Midwifery, Product Development) supporting the development of a prototype device, development of a business model and clinical evaluation of the device in healthy volunteers. A phase 1 clinical trial was performed in 102 healthy volunteers to allow evaluation of the usability and acceptability of the VAX-ID® device for injections in the deltoid region and the forearm. A questionnaire showed that the injection using saline solution was perceived as non-painful by all the vaccinees. Also, usage was optimized through a usability study with the vaccinators. A UK patent application has been filed for VAX-ID® and PCT filing is currently ongoing. Based on the developed business model and the assessment of the market potential for VAX-ID®, a UA spin-off company, Novosanis NV, has been set-up. The spin-off company will be responsible for commercialization and marketing of VAX-ID®. Additionally, further valorization opportunities, including licensing deals are currently being explored. In conclusion, after a very successful first phase this innovative intradermal injection device VAX-ID® can now be assessed for clinical immunogenicity and safety with vaccine antigen in larger cohorts of adults, children and elderly. A newly established UA spin-off company, Novosanis NV, will bring this product to the market and is looking for further valorization and licensing opportunities.

Background: The differential diagnosis between Alzheimer's disease (AD) and dementia with Lewy bodies (DLB) is difficult. There is growing interest in the use of cerebrospinal fluid (CSF) α-synuclein as a potential biomarker for α-synucleinopathies. Studies produced conflicting results due to the examination of splicing variants of α-synuclein and absence of autopsy confirmation in research studies. Objective: To determine the potential of α-synuclein-140 as a biomarker for α-synucleinopathies. Methods: CSF α-synuclein-140 concentrations were determined by a prototype multiplex xMAP bead based assay specific for human α-synuclein-140 (Innogenetics NV, Belgium). In addition, CSF biomarkers Aβ1-42 T-tau and P-tau181P (INNOTEST®, Innogenetics NV, Belgium) were determined. The population included patients with AD (n=129), DLB (n=45) , Parkinson's Disease (PD) (n=30), PD dementia (PDD) (n=23), multiple system atrophy (MSA) (n=7), and cognitively healthy controls (HC) (n=29). A subset had autopsy-confirmed dementia diagnoses (AD: n=32; DLB: n= 13). Results: As compared to controls (111±53pg/mL) and α-synucleinopathies (104±50pg/mL), CSF α-synuclein levels were increased in AD (147±74pg/mL) (p< 0.001). These results were confirmed in the subset of patients with autopsy-confirmed AD (155[125-263] pg/mL) and DLB (93[72-129] pg/mL) patients (p=0.001). CSF α-synuclein levels were correlated with CSF T-tau (ρ=0.655; p< 0.001) and P-tau181P (ρ=0.776; p< 0.001) levels in autopsy-confirmed AD patients. Conclusion: As CSF α-synuclein levels were significantly increased in AD as compared to α-synucleinopathies and HC, α-synuclein might have value as a biomarker for differential dementia diagnosis. Probably neurodegeneration caused increased CSF levels of α-synuclein in AD given the significant correlations with CSF T-tau and P-tau181P levels.

In the diagnostic field, detection of DNA mutations is performed using many different techniques , among which PCR and sequencing are the most often used. The former has higher sensitivity, the latter is more economic and more flexible towards detection of different mutations. The use of hybridization techniques, like microarrays, is rather uncommon for mutation detection. On the one hand, this is somewhat surprising given the fact that these technologies are nowadays mature, affordable and widely used. On the other hand, for a clear quantitative interpretation of microarray data, insight and use of the physic-chemical foundations of the underlying process, which is much less known, turns out to be important. We have extensively studied the hybridization free energies in DNA which resulted in a very precise quantification of molecular interactions in DNA microarrays [1][2]. Most recently, we used this information for the detection, identification and quantification of hotspot point mutations in the K-RAS oncogene, an important genetic marker for colorectal and lung cancer diagnostics and treatment stratification. Using a dilution series of a cancer cell line and wild type samples, we have shown that the detection sensitivity is below 1% of mutant sequence. Due to the intrinsic parallel character of the microarray technology, this approach makes it possible to detect hundreds of different point mutations in a single run. [1] Hooyberghs,J., Van Hummelen,P. and Carlon,E. (2009) The effects of mismatches on hybridization in DNA microarrays: determination of nearest neighbor parameters. NUCLEIC ACIDS RESEARCH, 37. [2] Hadiwikarta,W.W., Walter,J.C., Hooyberghs,J. and Carlon, E. (2012) Probing Hybridization parameters from microarray experiments: nearest neighbor model and beyond. NUCLEIC ACIDS RESEARCH, 40.

Many neurodegenerative diseases can be characterized by a gradual modification of the cellular environment resulting in macroscopic brain changes. Groupwise analysis of large sets of magnetic resonance (MR) images, visualizing the morphology of the human brain, can provide reliable and measurable image features indicative for a specific disease and/or disease stage. Such features, called biomarkers, can contribute to early diagnosis, to the assessment of therapy response as well as to the study of the underlying causes of the neurodegenerative disease. We developed a Bayesian framework for cross-sectional studies that performs groupwise analysis of a set of brain MR images and delivers potential biomarkers. Our framework analyses a heterogeneous set of brain MR images, for instance of both normal controls as well as of patients with different neurodegenerative disease(s) (stages). The framework simultaneously performs segmentation of each image into the major tissue classes (white matter, grey matter, CSF) and automatically clusters all images of the set in homogeneous subgroups based on the image morphology extracted from the image segmentations. Along with the segmentation and clustering, a probabilistic brain atlas is constructed for each cluster, visualizing the mean morphology of the subgroup and guiding the segmentation and clustering processes. The constructed morphological subgroups can be correlated with the clinical diagnosis of each subject, while the clustering process reveals the distinctive cluster-specific image features in general and in each individual image. The framework allows taking additional clinical knowledge into account in the clustering process to support research of specific clinical questions. The framework is validated on multiple sets of brain MR images, including the simulated BrainWeb data set and sets extracted from the large international initiatives ADNI and OASIS. The experiments show that our framework provides accurate and less biased image segmentations and that it is capable of detecting the clinically relevant morphological subgroups within the heterogeneous data set as well as the corresponding cluster-specific features. Finally, it is also illustrated that our framework for combined segmentation and clustering can find distinctive image features that would not have been picked up by handling both processes separately. In conclusion, the presented method combines segmentation, clustering and atlas construction in a unified probabilistic framework such that all techniques can cooperate and become more data-driven. We provide initial evidence that our method improves the representation of the morphology of disease stages, and facilitates the detection of novel subgroups and of novel disease-specific features. Therefore, our method can become an important tool for the development of novel and refined imaging biomarkers, providing new insights in structural change and development.

In the never ending search for new and better treatments for debilitating or life threatening diseases, advanced therapeutic medicinal products (ATMPs) are the new kids on the block. ATMPs are medicines for human use that are based on gene therapy, somatic-cell therapy or tissue engineering. The first examples of such medicines are now passing through the different stages of pre-clinical and clinical development and regulatory approval. The ability to successfully manufacture ATMPs on a consistent and cost-efficient basis will ultimately be a key element to make such products regulatory approvable and available to a large number of patients. From early development onwards, decisions need to be targeted towards the end goal: delivering safe and efficacious products with consistent quality at an affordable cost Our experience can stand as a guidance for producing commercial cell therapy products as we have successfully passed key tests: ATMP approval of ChrondroCelect, cGMP approval of a pilot facility and the new large scale manufacturing site, commercial production and supply to hospitals. Based on the case study of ChondroCelect, the first centrally approved ATMP in Europe, we will discuss the various factors that drive the routine manufacturing process. Since the process set-up is directly impacting the lay-out of the cleanrooms, we will also clarify the link between product development and site lay-out, as exemplified by TiGenix’ new, state of the art, 2500 m2 cell therapy facility. In this presentation, we will illustrate the life cycle from development to routine manufacture of such innovative products, and highlight factors that need to be taken into account from development onwards.

The blends of volatile organic compounds (VOCs) emitted by plants have been reported to vary qualitatively and quantitatively depending on the plant species and its biotic stress status (e.g. herbivorous insect). As a host plant for diamondback moth (Plutella xylostella - Lepidoptera: Yponomeutidae), Arabidopsis thaliana Col-0 (A.t Col-0) was studied for the biological interactions between plant and chewer pest, was studied for the biological interactions between plant and chewer pest, focusing on VOCs emission pattern. To gain insight into this topic, the changes in volatile profiles of A.t Col-0 were compared due to feeding by P. xylostella at different densities and residence duration of on plant leaves (0, 3, 9, 20 pest larvae within 0-4 h and 4-8 h infestation, respectively). Headspace solid-phase micro-extraction coupled with gas chromatography - mass spectrometry (HS-SPME-GC/MS) analyses showed that the relative emission of several volatile compounds changed significantly for Arabidopsis plants subjected to P.xylostella infestation. Several changes in alcohol, terpenes, aldehydes and ketones concentrations occurred. The proportion of sulfur containing metabolites increased significantly according to the number of pests and increasing duration of P.xylostella infestation, dimethyl disulfide being the main compound from infested plants. Our results indicated that the release of VOCs from P. xylostella infested A.t Col-0 was likely to influence another Lepidoptera species (Pieris rapae). The elucidation of this pest and related host plant interactions may be important in understanding the volatile emission from plant – chewing insect interactions, including both biotic and abiotic ones in ongoing experiments.

Freeze drying is often the preferred drying technique to stabilize biopharmaceutical protein formulations. However, these proteins can suffer from stresses during the freeze-drying process itself which can induce changes in the protein’s three dimensional conformation. The aim of the present study was to investigate if in-line Raman spectroscopy is complementary to in-line NIR spectroscopy to visualize and evaluate the protein conformation during freeze drying. Furthermore, it was investigated whether the protein protection by cryo- and lyoprotectants can be evaluated by Raman and NIR spectroscopy during processing. The tested cryo- and lyoprotectants were sucrose and PVP because the mechanism of stabilisation is different for these two protectants. The high stability of the used model protein formulation, immunoglobulin G (IgG), obliged us to use extreme stress conditions during freeze drying. IgG1 was therefore freeze dried with different concentrations of NaSCN to obtain different protein conformations (native, partly denatured, denatured) in the freeze-dried product. During the freeze drying process, Raman and NIR spectra were recorded every process minute via NIR and Raman probes implemented in the freeze dryer chamber. The frequency of the amide A/II band (near 4850cm-1 ) in the NIR spectrum was monitored as a function of water elimination. During protein dehydration, these two features are well correlated in the absence of protein unfolding. Deviations from this correlation indicated by shifts to higher amide A/II frequencies were shown to be related to protein unfolding. Due to overwhelming water and ice signals in NIR spectra, it was impossible to monitor the protein structure during the entire freeze drying process. In-line Raman spectroscopy overcomes this problem and allows monitoring the protein structure during the entire freeze-drying process since ice is a weak Raman scatterer. It also allowed to evaluate the protein-cryoprotectant interactions during the freezing step by analyzing the amide I (1650cm-1) and amide III band (1240 cm-1). Spectral shifts and broadenings of these Raman signals reflect changes in protein conformation and intra-/intermolecular interactions. This study proves the complementarity of Raman and NIR spectroscopy for the freeze-drying process monitoring of protein formulations

Nowadays, millions of people suffer from blinding disorders severely affecting their everyday life. Current treatment consists mainly of managing the symptoms with small molecule drugs or antibodies. Thanks to recent advances in genomic screening, however, several of these disorders have been linked to genetic defects of the retina, implying gene therapy could provide a definite cure for the patients. Still, one of the major hurdles in gene therapy remains the delivery of the intact gene therapeutics to their site of action. To protect the nucleic acids from degradation and guide them to the appropriate target location, they are usually combined with carrier materials to form nano-sized complexes. Nonviral carrier materials, such a polymers and liposomal formulations, aren’t as capable as viral vectors yet, but their ease of manufacture, low immunogenicity and adaptability make them promising alternatives for gene delivery. In retinal gene therapy, the therapeutics should be delivered to the back of the eye, where the retinal target cells are situated. The eye is an attractive target organ for exploring gene therapy due to its isolation from the rest of the body by the blood-retina barrier (BRB). Unfortunately, this is also what prevents the efficient delivery of therapeutics injected in the blood stream to the ocular tissue. Instead, direct intraocular administration, such as intravitreal injection, offers an attractive alternative. Intravitreal injection allows to deliver the nanomedicines with minimal invasiveness and minimal adverse effects to the vitreous humour, the central part of the eye, thus efficiently bypassing the BRB. Evidently, once administered to the vitreous humour, nanomedicines should still be mobile to reach the back of the eye. However, the vitreous gel has previously been shown to impede gene nanomedicine mobility, being composed of a network of proteins and other charged compounds which can trap the injected therapeutics. To aid in the rational design of novel gene vectors for intravitreal injection, we have developed an assay to accurately evaluate the mobility of fluorescently labelled nanoparticles in an ex vivo model of intact vitreous humour. By using fluorescence single particle tracking microscopy, detailed information on the interactions between nanoparticles and the vitreal network could be obtained. To establish general relationships between intravitreal mobility and nanoparticle characteristics, we investigated model nanoparticles of surface characteristics and different sizes ranging from 0.1 to 1 µm. Interestingly, rather than size, electrostatic and hydrophobic interactions appear to be the key determinants for the diffusion rate of nanoparticles in vitreous humor. These findings were confirmed when our methodology was used to evaluate the suitability for intravitreal administration of gene nanomedicines based on a promising class of polymer gene delivery vectors.

NIPP1, an interactor of protein Ser/Thr phosphatase-1 (PP1), is an essential regulator of embryonic development and cell proliferation in multicellular eukaryotes. It regulates Polycomb Group (PcG) proteins, which are involved in the silencing of genes implicated in cell proliferation and differentiation. PcG proteins are important for stem cell self-renewal and proliferation capacities, whereas their deregulation often contributes to the development of cancer. Importantly, NIPP1 associates with a subset of PcG targets and is required for the repression of these genes. A complete NIPP1 knockout in mice is embryonic lethal just before onset of gastrulation. To further explore the function of NIPP1 in vivo we generated mice where NIPP1 is conditionally removed from epithelial liver cells. The absence of NIPP1 from hepatocytes and cholangiocytes was confirmed immunohistochemically. Liver-specific NIPP1 knockout (NIPP1L-KO) mice were viable and did not show an overt phenotype up to an age of 1 year. However, in the NIPP1L-KO mice an immunostaining for the cholangiocyte and progenitor cell marker cytokeratine 19 revealed an early progenitor cell response. After one year, the liver of knockout mice showed areas of obvious progenitor cell response associated with bile duct hyperplasia which, surprisingly, were strongly linked with the recurrence of NIPP1 expression. Next, we have initiated liver cancer by a single intraperitoneal injection of diethylnitrosamine (DEN) at 14 days of age, and sacrificed the mice at 10 months. In this liver cancer model, we found a significant decrease in liver cancer development in the NIPP1L-KO mice, as compared to control mice. We confirmed that NIPP1 was absent in nearly all epithelial liver cells, indicating that NIPP1 has oncogenic properties in vivo. Intriguingly, chronic injection of DENA during 25 weeks resulted in an increased sensitivity towards the development of both hepatocellular and cholangiocarcinomas in the NIPP1L-KO mice. However, this liver cancer model was associated with a normal expression of NIPP1 in most hepatocytes and cholangiocytes in the NIPP1L-KO, hinting at the replacement of damaged liver cells by NIPP1-containing cells, probably originating from progenitor cells that had escaped Cre-recombination. In conclusion, our data suggest that NIPP1 has oncogenic properties and is an attractive novel therapeutic target for liver cancer.

Purpose Since the integration of bone is crucial for the success of the implant, more and more studies are performed on animals to compare the osseointegration between implants or implant coatings. The aim of this study is to point out the benefits of automated, (bio)medical image-based quantification of the osseointegration for a reliable comparison. Methods and materials At 6, 12 and 26 weeks after implantation, 16 samples containing the implant are extracted from the sheep's tibia or femur and scanned with a micro-CT scanner, providing 48 3D images of resolution 7.5 x 7.5 x 7.5 μm3. The first step in the quantitative analysis is the detection of the cylindrical dowel in the images, which is performed by the combination of a circular Hough transform and linear curve fitting. Next, 60 volumes of interest (VOIs) are determined around the implant (5 layers of 12 segments), each having a thickness (outer boundary compared to inner boundary) of around 2 mm and a volume of 16.7 mm3. In these VOIs, the main morphometric parameters are computed by extracting the (trabecular) bone surface out of the image with an image-specific bone threshold and an edge-based isosurface algorithm. The bone volume density (BV/TV) is defined as the percentage of bone within the VOI; the bone surface density (BS/TV) as the area of bone surface per mm3; trabecular thickness (Tb.Th) as the average thickness of the trabeculae, approximated by Tb.Th = 2.(BV/TV)/(BS/TV) using a plate model; trabecular separation (Tb.Sp) as the separation of the trabeculae, approximated by Tb.Sp = 2.(1-BV/TV)/(BS/TV). The different parameters highlight different aspects of the integration. E.g., more and thinner trabeculae highly increase BS/TV, but have limited effect on the BV/TV. Results We measured average values of 39.27%, 4.94 mm-1, 0.157 mm and 0.353 mm for BV/TV, BS/TV, Tb.Th and Tb.Sp, respectively, and noticed an increased integration for implants extracted at a later stage. The computed morphometric values strongly correspond to our visual impression. Moreover, since the quantitative measurements are automated, they are more reproducible: the observer variability is reduced to 0.92%, 0.14 mm-1, 0.005 mm and 0.017 mm for BV/TV, BS/TV, Tb.Th and Tb.Sp, respectively. In addition, quantitative analyses are less time consuming and allow estimating the integration around the whole implant and not just at one place. Conclusion The use of quantitative image measurements in clinical trials has proven to be beneficial in terms of accuracy and reproducibility. Therefore, it allows to distinguish implants or implant coatings w.r.t. osseointegration at an early stage. The sequel of the study will provide more quantitative results on the comparisons.

Nanotechnology is an emerging science, which provides promising tools for novel applications in engineering, pharmaceutics and medicine. Although nanomaterials offer great opportunities for innovation and technological development, an increased human exposure implies that potential health impacts should be carefully addressed. Therefore, it is critical that the nano-bio interactions are characterized in advance in order to prevent health issues associated with nanomaterials. In the present study, 40 nm sized monodisperse polystyrene nanoparticles, stained with fluorescent dyes, are used to provide insights into the intracellular dynamics after nanoparticle uptake. By the use of live cell imaging and image correlation-based methods, the bio-interaction and mobility of these nanoparticles are explored in different in vitro models including primary dendritic cells, the human alveolar epithelial A549 cell line and the human cervical epithelial HeLa cell line. Temporal image correlation spectroscopy (TICS) and spatio-temporal image correlation spectroscopy (STICS) are performed to investigate the intracellular nanoparticle dynamics. Interactions with subcellular compartments are studied with spatio-temporal image cross-correlation spectroscopy (STICCS). Insights in nanoparticles’ intracellular fate contribute to elucidate the cellular pathways which take place after nanoparticle uptake, and their possible interference with normal cell function.

After intrapulmonary administration of gene-based vaccines, most may end-up in non-immune cells. It is known that cross-presentation of the antigen by these cells plays an important role in the establishment of the adaptive immune response. However, the cytokine signature that is associated with recognition of mRNA by PRRs of non-immune cells has not been studied in detail. In this report we show that carrier-mediated delivery of unmodified mRNA caused overexpression of TLR3 in respiratory cells. This was accompanied with a massive production of type I interferons and other immunostimulating cytokines. The recognition of mRNA by the innate immune system was associated with cell death mediated by substantial overexpression of caspase-1. The viability of murine respiratory cells was less affected by mRNA transfection, which is in line with the lower transfection efficiency, lower innate immune response and the absence of a massive caspase-1 upregulation in these cells. Recognition of the delivered mRNA by TLR3 had also a negative effect on the translation of the transgene. Finally, the cytotoxicity of the transfected mRNA was abolished when modified nucleotides were incorporated in the mRNA. Additionally, we also administered our mRNA/carrier complexes to the lungs of mice and determined the local innate immune response. Carrier-mediated delivery of unmodified mRNA activates innate immune system by TLR3 signaling. The following induction of immunostimulating cytokines and caspase-1-mediated cell death, pyroptosis, in lung epithelial cells may help the residing professional antigen presenting cells to present the encoded antigens and to create a suitable cytokine environment to obtain the appropriate immune answer. However, the induction of the innate immune response does also decrease the translation of the mRNA. Whether this will decrease the efficacy of mRNA vaccines will be dependent on the system used for mRNA delivery.

In vitro companion diagnostic devices provide information that is essential for the safe and effective use of a corresponding therapeutic product (e.g. Diabetes glucose monitor). Nowadays, their development has been encouraged by facilitating the related patent processes and supporting the reimbursements of costs coupled to their use. This is particularly beneficial for treatment of chronic illnesses (e.g. Crohn’s disease, rheumatoid arthritis, etc.) as they require life-long administration of biologicals (drugs) as well as monitoring of their levels in the patient blood for determining the next biological dose. Detected high levels of biologicals warn for possible long term overdose with adverse effects while low levels indicate a need for a higher dose or an insensitivity to the costly therapy. Maintaining the ideal blood biological levels results in less patient hospitalizations and increasing the chances of remission. Analysis of the patient blood lowest biological levels (trough levels) during patient consultation in a lab near the doctor’s office requires fast, accurate, easy and affordable diagnostic test. Over the past decades, there has been a remarkable progress in the field of biosensing technologies attempting to realize these requirements. Biosensors are analytical devices that use biological components for target recognition and subsequently convert this molecular event into a physically detectable signal. Surface plasmon resonance (SPR) biosensors are one of the most advanced label free, real time detection technologies that provides quantification, next to on activity, type or abundance of the biomolecular compound. Although there has been a constant technical improvement in biosensor devices, most of the commercially available SPR platforms are still bulky instruments with expensive optical components. Our research group has developed an innovative fiber optic (FO) SPR platform that is applicable outside the specialized research environment due to several features. First, the FO-SPR sensor consists of low-cost components with a single-use sensor tip integrated on an automated device. As such, this platform has next to multichannel performance the flexibility in combining the specific test types according to the samples. Furthermore, easy to use nanoparticle based sample preparation improves the detection limits of our biosensor, and thus its sensitivity, beyond the state of the art. Next to antibody based bioassays, FO-SPR sensor has been successfully applied in DNA-based bioassays. Thus, the DNA amplification/melting processes can be monitored in real-time, which has not been accomplished before with commercial SPR devices as they do not allow for accurate and broad temperature control. By exploiting this concept, single nucleotide polymorphisms were screened in real-time, which makes FO-SPR ideally suited for fast screening of genetic mutations for bacterial resistance (MRSA) or cancer.

Objectives It is assumed that the concentration of amyloid-β1-40 (Aβ1-40) in cerebrospinal fluid (CSF) reflects the total amount of Aβ protein in the brain and thus allows a better interpretation of inter-individual differences in Aβ quantity than the Aβ1-42 concentration. In this study, Aβ1-40 was added to the existing CSF biomarker panel of Aβ1-42, total tau (T-tau) and phosphorylated tau (P-tau181P) in order to test whether the accuracy of the differential dementia diagnosis improved. Methods The concentration of Aβ1-40 (INNOTEST® β-amyloid(1-40) prototype version, Innogenetics NV, Belgium) and the other biomarkers (INNOTEST®, Innogenetics NV, Belgium) was determined in CSF samples from 80 Alzheimer’s disease (AD) patients, 75 non-AD dementia patients and 30 controls. A large proportion of the study population had autopsy-confirmed dementia (AD: 73/80=91%; non-AD: 38/75=51%). Results The levels of Aβ1-40 were decreased in AD (10856±4745 pg/mL) and non-AD patients (10519±4491 pg/mL) when compared to controls (14760±7846 pg/mL) (p=0.002 and p=0.001). The Aβ1-42/Aβ1-40 ratio (p<0.001) was significantly decreased in AD (0.043±0.021) as compared to non-AD patients (0.064±0.027) and controls (0.053±0.023). In order to differentiate AD from non-AD patients a decision tree was constructed. The diagnostic accuracy (80%) of the decision tree that contained Aβ1-42, Aβ1-40, P-tau181P and the Aβ1-42/Aβ1-40 ratio was significantly better than the diagnostic accuracy (74%) of the decision tree without Aβ1-40 and the Aβ1-42/Aβ1-40 ratio (p<0.001). Conclusion In conclusion, adding CSF Aβ1-40 and the CSF Aβ1-42/Aβ1-40 ratio to a biomarker-based decision tree increased the diagnostic accuracy for discriminating AD from non-AD.

The atmosphere has been described as one of the last frontiers of biological exploration on Earth. Despite our intimate relationship with the air around us, the composition of microbial communities in the atmosphere is still poorly defined, and our knowledge about the functional potential of airborne microbes (both beneficial and pathogenic) is scant. Only recently, it has been discovered that airborne microbes are more than just passive inhabitants of the atmosphere: they are metabolically active and well adapted to the harsh atmospheric conditions. Studying airborne microbes has important implications for our general health, agriculture, national security and climate research since microbes act as cloud condensation and ice nuclei. It can also open new frontiers for biotechnological applications based on the enzymes of these unexplored microbes. In this project, airborne microbes are studied in relation to air pollution in the region of Antwerp (Belgium) and surroundings, to investigate whether airborne microbes can be used for biomonitoring and bioremediation purposes and whether microbial endotoxins interact with other air pollutants such as particulate matter. Potential confounding factors such as seasonality, meteorological conditions, and variability of bacterial sources are taken into account. Furthermore, low densities of airborne microbes demands sensitive analysis techniques, such as next-generation sequencing technology methods for community analysis. This approach compensates for the obvious bias and underestimation of the total microbial diversity of culture-dependent methods. Sensitive bioassays are also applied for the biomonitoring of endotoxins. Furthermore, our challenge is to introduce standardization of the recent techniques, from air collection to sample-processing methods, so as to allow for comparison between and within studies.

Objectives: Previous research has highlighted several implementations to overcome difficulties in producing [11C]methylated PET-tracers, such as the necessity for rapid, reliable and efficient automated procedures. Despite the improvements that have been proposed, the final purification and work-up steps are still subject to enhancement. We aimed at developing a downscaled HPLC purification strategy for [11C]raclopride and [11C]DASB, using analytical HPLC columns and non-toxic solvents that are suitable for IV administration. Methods: [11C]raclopride was radiosynthesized by bubbling [11C]methyl triflate, in carrier helium, for 3.5 minutes into a sealed vial containing the precursor, (S)-O-desmethylraclopride (100 µg), and an equimolar amount of sodium hydroxide (1M) in acetone (200 µL). For the radiosynthesis of [11C]DASB a similar procedure was applied: gaseous [11C]methyl triflate was bubbled into a sealed vial containing the precursor, N-desmethylDASB (50 µg), in acetone (200 µL). When the radioactivity in the reaction vial reached maximum activity levels, the crude reaction mixture was diluted with eluent and loaded directly on a 750 µL HPLC loop. Several analytical columns in combination with ethanol containing eluents were examined to optimize the chromatographic resolution. Results: Using an analytical SupelcosilTM SuplexTM pKb-100 HPLC column in combination with citrate buffer pH3 / ethanol: 75/25 (V/V) as mobile phase and a flow of 1 mL/min, [11C]raclopride eluted after merely 5 minutes while it was separated at baseline level from all other compounds. The fraction containing the product was collected within a maximum of 2 minutes and transferred over two consecutive 0.22 µm filters into a sterile vial containing 2.930 mL physiological saline and 70 µL sodium bicarbonate (84 mg/mL). For the purification of [11C]DASB, the optimum chromatographic conditions were obtained by using an AlltimaTM HP CN column along with a mixture of 25 mM phosphate buffer pH 7 / ethanol: 65/35 (V/V) as mobile phase and a flow of 1 mL/min. [11C]DASB eluted after 8.5 minutes. The fraction containing the product was collected within a maximum of 2 minutes and passed over 2 consecutive 0.22 µm filters into a sterile vial containing 5 mL physiological saline. Both tracers were directly suitable for IV administration. Conclusion The combination of aforementioned reaction conditions and the described original purification strategies for [11C]raclopride and [11C]DASB resulted in straightforward produced injectable solutions with higher specific activities and substantially reduced administration volumes. Besides the fact that this is favourable for patient administration, these findings are of great importance in the field of small animal PET studies, where the mass and the volume of the injected tracer are two limiting factors which tend to determine the maximum injected dose.

Progress in oncology drug development is limited by scarcity of in vivo models that authentically reproduce tumor growth and metastatic progression. The lack of preclinical models reliable predicting the efficacy of a drug at the time clinical trials are initiated is one of the underlying factors for the low yield rate in anticancer drug development. Moreover, since the number of potential therapeutic options increases, and the costs for (pre)clinical testing escalate, the use of model systems to help prioritize which compound to select for clinical investigation becomes increasingly important. Before entering clinical development, agents are usually tested against high-passage commercially obtained cell lines and xenografts established from these lines. In contrast to those commonly used cell line-xenograft models, patient-derived tumor xenograft (PDTX) models are established from the immediate transfer of fresh tumor tissue from patients into immunodeficient mice (F1) and reimplantation in successive generations of mice (F2, F3,…). Despite these serial passages, the intratumor heterogeneity and histologic characteristics are maintained in the PDTX and, consequently, a better retention of the morphological and molecular markers of the source tumors is reached. Besides, PDTX models represent a more complete spectrum of molecular subtypes than cell line-xenografts. Based on genomic and gene expression profiling, it is demonstrated that PDTXs maintain the majority of the key genetic profile and global pathway activity as observed in the original patient’s tumor. Although the concept of PDTX models has been in existence for decades, their value in oncology drug development is just becoming realized as individualized therapy approaches translational cancer therapy. At UZ Leuven, a PDTX Platform and complementary Biobank representing the different subsets of tumors is currently established. At this stage, endometrial, breast, colorectal, pancreatic, head and neck tumors and melanoma are included, though, in the future, the platform will be extended to other tumor types as well. Due to a close connection between the clinic and research facility, tumors are processed within max. 1h after surgery. After histologic, molecular and genetic validation, the models become available for both basic and translational research. Recent data on the establishment of endometrial PDTX models show the feasibility and the high grade of similarity with the original patient tumor (data presented in the abstract by Coenegrachts et al.). Ultimately, our PDTX platform will allow better evaluation of new agents in all different tumor types. Moreover, relevant biomarkers will be identified that can lead to better stratified patient trials in a later phase. With this approach, more information on molecular criteria for response to targeted therapies can be generated and guide the selection of the tumor type in which new agents will become tested.

MASPIT (for MAmmalian Small molecule-Protein Interaction Trap) is a three-hybrid technology that enables the identification of interactions between small organic compounds and their target proteins in living human cells [1,2]. Applying the assay in a cell array screening setup [3], large collections of proteins can be screened efficiently at high throughput. Starting from a small molecule methotrexate conjugate, a target profile can be mapped and validated in only a couple of weeks’ time. The MASPIT platform offers a number of important advantages over existing target profiling technologies. First, the assay operates in intact human cells, which is the native background of the targeted protein(s) and the context in which the future drug will operate. Second, it is an unbiased screening approach, not restricted to a limited panel of related proteins but covering a broad area of the human proteome. Third, the high throughput of the screening platform ensures rapid turnaround times and flexibility with regard to parallel screens or iterative rounds of screening compound analogs during a hit or lead optimization process. To facilitate efficient methotrexate conjugation of small molecules of interest, a versatile methotrexate reagent has been developed that enables straightforward coupling using ‘click’ chemistry (see the poster of De Clercq et al. for more details on the chemical aspects of the MASPIT technology). The screening collection currently comprises a set of 12.500 full size human proteins, and will further grow to genome-wide coverage in the near future. The technology has recently been evaluated in pilot screens for the breast cancer drug tamoxifen and the cell dedifferentiating agent reversine, successfully identifying both known and novel cellular targets. These results validate the MASPIT cell array screening platform as a powerful novel tool for in cell profiling of both on- and off-target proteins of drug or lead molecules. In a next step, the platform will be developed into a screening service towards biotech and pharma companies. References [1] Caligiuri, M. et. al., Chem. Biol. 2006, 13, 711–722. [2] Risseeuw, M.D.P. et al., ChemMedChem 2013, in press. [3] Lievens, S. et al., J. Proteome Res. 2009, 8, 877-886.

Galapagos discovers and develops small molecule and antibody therapies with novel modes-of-action. The company progresses one of the largest pipelines in biotech, covering several disease areas. Our unique Cystic Fibrosis research toolbox led to the identification and characterization of possible new drug leads. Cystic Fibrosis (CF) is a fatal genetic disease, caused by a defect in the CF Transmembrane Regulator (CFTR) gene encoding a chloride channel protein present in the apical membrane of epithelial cells, which contributes to the water homeostasis in the body. An absence or non-functional form of CFTR disturbs this water balance and causes dehydration of e.g. lung epithelium, which in turn prevents ciliary clearance of mucus in the lungs. Over time CF patients suffer from obstruction of the bronchia by mucus plugs, leading to severe inflammation and finally remodelling of the lung tissue. The majority of CF patients share a common mutation in the CFTR gene: a deletion of phenylalanine at position 508 that prevents the CFTR protein from trafficking correctly to the membrane. Moreover, the minimal amount of protein that does reach the plasma membrane is barely functional. Therefore, to offer proper treatment for F508del-CFTR patients, two hurdles need to be taken: CFTR amounts on the plasma membrane need to be increased (= correction) and the functionality of the channel needs to be improved (= potentiation). We developed a research toolbox to identify and characterize small molecules that can correct and/or potentiate F508del-CFTR. Our YFP-halide assay is based on epithelial lung cell lines that carry the F508del-CFTR phenotype. Screening of this assay delivered several hit series. More complex biology systems (pseudostratified human epithelia from CF-human primary bronchial epithelial cells) are applied to characterize hit series. A fully functional electrophysiology platform (Ussing chambers, TECC, patch-clamp) was established, as well as technologies to provide insight in the mode-of-action of the small molecules (glycosylation, cell surface expression, airway surface liquid determination). The assays in the CF research toolbox will be highlighted.

Endometrial cancer is the most frequent malignancy of the female genital tract. Treatment modalities for endometrial cancer are limited for primary advanced and recurrent disease leading to a poor overall survival of these patients. Development of new treatment strategies is therefore warranted but may be hampered by the lack of representative pre-clinical in vivo models. Patient-derived tumor xenograft (PDTX) models appear to have better retention of the morphological and molecular markers of the source tumors, despite serial passages across several generations of mice. PDTX models of several tumor types (including colorectal, breast, lung cancers) are currently widely available for oncology research, whereas data on PDTX models for endometrial cancer are lacking. In our study, we here present preliminary data on the establishment and characterization of these PDTX models for endometrial cancer. We established PDTXs of different histological subtypes of endometrial cancer within the framework of the PDTX platform at KU Leuven – UZ Leuven (abstract presented by Hermans et al.). After written informed consent of the patient and within 1h after surgery, tumor samples were implanted s.c. in NOD-SCID mice (F1 generation). When tumors have reached ~1000 mm3, tumors were retransplanted in NOD-SCID and/or nude mice. At this moment, a success rate of ~50% is reached, which is comparable to other tumor types. More in particular, 18 PDTX models, of different histological subtypes (endometrioid, serous, carcinosarcoma, undifferentiated carcinoma), were initiated, of which 7 were successful (i.e. tumor growth in at least F1 and F2 generation). Five PDTX models were categorized as ‘failed’ since no tumor growth was observed 8 months after implantation. Six other PDTX models were only recently initiated. Tumor growth rate in F1 xenografts was generally slower than in the subsequent generations (F2, F3,…). A protocol for re-implantation of cryopreserved xenografts was successfully optimized. Histological analyses showed that general tumor morphology (by H&E staining), proliferation capacity (Ki-67), hormone receptor status (ER, PR) and PTEN status remained similar in F1- F2-F3 xenografts as compared to the original patient tumor. Hotspot mutation profiling (Sequenom) showed that mutations present in the original tumor were also observed in the xenograft tumors and no additional mutations in the xenografts were identified. Whole exome sequencing and gene expression analyses are currently ongoing and/or planned in near future. Finally, we successfully evaluated the in vivo responsiveness to standard chemotherapy and a PI3K – mTOR inhibitor, BEZ-235, in one of the established models. In conclusion we here show first evidence for the establishment of PDTX models for endometrial cancer that can be used for future preclinical drug evaluation.

Introduction: Our previous study (Mohades 2012) showed differences in the mean fractional anisotropy (FA) values of some language related white matter pathways between 2 groups of bilinguals (simultaneous and sequential) and the monolingual children. Our hypothesis was that the brain of these 3 groups might have different maturation processes. Thus we examined the same group after 2 years and the same fibres were studied by means of DT-MRI based fibre tracking. We investigated the effect of time gap on the FA values of these fibres after 2 years. The pathways were:1) the left arcuate fasciculus/superior longitudinal fasciculus (lAF/lSLF);2) the left inferior occipitofrontal fasciculus (lIFOF);3)the bundle arising from the anterior part of the corpus callosum (CC) projecting to the orbital lobe (AC-OL) 4) the fibres emerging from the anterior midbody (AMB) of the CC that associate with the premotor and supplementary motor cortices (AMB-PMC). Materials and Methods A 3T MR system (Philips Achieva Release 2.5) with an 8 channel SENSE head coil was used.3 groups respectively containing 11 simultaneous bilinguals, 15 sequential bilinguals and 9 monolingual children were scanned.(right-handed, healthy males and females, all previously examined). The age range was 116-165 months and the gap between 2 scans was 19-27 months. Scan parameters:SS-SEEPI,15 non-collinear diffusion gradient directions,b= 700 s/mm2,TR/TE=6484ms/60ms, FOV=224x224x120 mm3,60 oblique axial 2mm slices, scan duration=454s. Figure1 shows the bundles: IFOF (red), AF/SLF (cyan), AC-OL (green) and AMB-PMC (yellow). Mean FA of the bundles was calculated for each subject using DTIstudio (Johns Hopkins University, Baltimore, USA). Paired-samples T-test was undertaken to compare the mean FA values of 2 runs (Table1). A P-value less than 0.05 was considered significant. Repeated measures ANOVA was used to compare the Mean FA values for 3 groups and 2 runs. Correlation analysis between the FA value and Time gap between 2 runs shows the degree of association between these factors. Results Figure2 shows the mean FA values for the 4 bundles of fibres and 2 runs. Pair-wise comparison shows significant difference in the FA value of the bundles IOF and PMC over time(Table1); and Table 2 shows the degree of correlation between FA values and the time gap between two runs. Repeated measures ANOVA showed significant difference in the FA-IOF between 2 groups of bilinguals(sig=0.039) and the FA_PMC between Monolinguals and sequential bilinguals conclusion differences between the level of FA values over the time and the positive correlation with Gap shows that the characteristics of fibres change in 2 years and this changes will be affected by bilingualism in case of some pathways like IOF that is responsible for semantic processing. Further studies will help to interpret the results and find more specific relation between these factors and bilingualism

Methods that allow high throughput identification of cellular targets of small molecules are valuable assets in pharmaceutical research. They are useful in mechanism of action studies of hits identified via phenotypic screening. Alternatively, they may uncover “off-target” proteins of established drugs, which may contribute to their therapeutic efficacy. Finally, such methods also allow profiling small molecules against a series of related intracellular targets (e.g. kinases). Mammalian Small molecule-Protein Interaction Trap (MASPIT)[1] provides a new tool for swift proteome-wide screening for intracellular targets of known small molecules. This three-hybrid system is based on the JAK/STAT signaling pathway of the cytokine receptor and enables the identification of mammalian cytosolic proteins that interact with a small molecule of interest (see the poster of Lievens et al. for more details on the biology aspects and applications of the MASPIT technology). In this poster we present a scalable synthesis of a versatile methotrexate (MTX) reagent equipped with an azide ligation handle that allows rapid -selective conjugation to yield MTX fusion compounds (MFCs) appropriate for MASPIT.[2] We selected three structurally diverse pharmacologically active compounds (tamoxifen, reversine and FK506) as model baits. After acetylene functionalization of these baits (paying close attention to SAR), MFCs were synthesized via a CuAAC reaction, demonstrating the general applicability of the MTX reagent. In analytical mode, MASPIT was able to give concentration-dependent reporter signals for the established target proteins. Furthermore, we demonstrate that the sensitivity obtained with the new MTX reagent was significantly stronger than that of a previously used non-regiomeric conjugate mixture. Finally, the FK506 MFC was explored in a cellular array screen for targets of FK506. Out of a pilot collection of nearly 2000 full-length human ORF preys, FKBP12, the established target of FK506, emerged as the prey protein that gave the highest increase in reporter signal. This indicates that our newly developed synthetic strategy for the straightforward generation of MFCs is a promising asset to uncover new intracellular targets using MASPIT cellular array screening[3]. References [1] Caligiuri, M. et. al., Chem. Biol. 2006, 13, 711–722. [2] Risseeuw, M.D.P. et al., ChemMedChem 2013, in press. [3] Lievens, S. et al., J. Proteome Res. 2009, 8, 877-886.

The emergence of resistance to antibiotics presents a global and urgent medical threat. The Infectious Disease Society of America highlighted the most problematic species, the so called ESKAPE bacteria Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa, and Enterobacteriaceae ESβL producers in which there is a danger that existing therapies will soon be no longer effective. Of particular concern is the methicillin resistant Staphylococcus aureus (MRSA) which was reported to be responsible for more deaths in US hospitals than from HIV/AIDS and tuberculosis combined. Once firmly established in hospitals worldwide, MRSA has now emerged as a significant community-acquired pathogen. Community-acquired MRSA infections are increasing, and may now involve persons without risk factors predisposing them for acquisition, including children. The discovery and development of new agents with novel mechanisms of action that do show activity against the multidrug resistant bacteria is of critical importance to have therapies that keep pace with the growing problem. Galapagos has identified DNA pol IIIα as target for developing novel antibiotics. DNA pol IIIα is ubiquitous and an essential protein in both Gram positive and Gram negative bacteria and therefore a highly desirable target for antibacterial therapy. Using this novel target, Galapagos has selected a first candidate antibiotic, CAM-1, to enter drug development. CAM-1 (GLPG1492) is identified as a highly potent inhibitor of the DNA pol IIIα enzyme isolated from S. aureus (IC50 33 nM). CAM-1 was tested against more than 250 different bacterial strains and effectively killed 100% of all drug resistant S. aureus, including MRSA. In time kill experiments, CAM-1 exhibited a cidality at concentrations 2-8 fold the MIC, and rate of kill similar to ciprofloxacin. In vivo efficacy was confirmed in several murine models of infection. In the neutropenic mouse thigh infection model, CAM-1 was shown to be as effective as linezolid at similar doses. A consistently effective dose was demonstrated between 25-50 mg/kg irrespective of the route of administration. Furthermore in vivo efficacy was seen at once-a-day frequency. CAM-1 has entered preclinical development in order to allow evaluation of safety, tolerability and pharmacokinetics in a clinical setting. The novel mode of action - inhibition of DNA pol IIIα - may be used to explore a variety of novel antibiotics, targeting bacteria for which resistance to current antibiotics has emerged.

The contamination of a non-GM maize field by cross-fertilization with pollen from a neighbouring MON810 maize field was studied by sampling plants in a co-existence field test in Flanders. To accurately quantify GM percentages, an evaluation was performed of the effect of plant development stage. Sampling at different stages in the growth process of maize has indeed consequences for relative GM quantification based on haploid genome equivalents, due to the specific maize seed composition and differences in DNA extractability from different seed tissues. By sampling different maize plants and subparts at four different stages during the growth season, the evolution of weights, absolute DNA yields, DNA densities and the ratios of endosperm and embryo relative to the total maize kernel are studied in detail. During the plant growth, plant parts with potential GM genes (embryo in kernel and cob on total plant) increase in importance on weight as well as DNA concentration level, while the endosperm drops in relative importance. In order to determine the GM percentage in one kernel, the endosperm and embryo DNA content, relative to the total kernel DNA content are taken into account. Based on these influencing factors, for the different sampling stages, expected % GM maize values are calculated for a whole (field) harvest of grain maize. This GM percentage on kernel level can further be extended to other maize usages in Flanders such as CCM (corn cob mix) and silage maize respectively, by taking into account the relative part (in weight) of grains to the total cob and of the total cob to the whole maize plant, respectively. In all calculations, it is assumed that complete cross-fertilization occurred of the non-GM field by the GM pollen coming from a surrounding field. In this context a model could be drawn for predicting the expected % GM (DNA) for lower overall (field- or harvest-based) GM admixture rates.

Foamy macrophages, containing myelin degradation products, are abundantly found in active MS lesions. Recent studies have described an altered phenotype of macrophages after myelin phagocytosis. However, mechanisms by which myelin affects the phenotype of macrophages and how this phenotype influences lesion progression remain unclear. PS is a phospholipid found in myelin. As apoptotic cell clearance by macrophages via PS induces an immunosuppressive phenotype in macrophages, we investigated the impact of myelin-derived PS on the functional properties of myelin-phagocytosing macrophages. We show that PS present in myelin reduces nitric oxide production by macrophages through activation of PPARβ/δ. Furthermore, uptake of PS by macrophages, after intravenous injection of PSLs, suppresses the production of inflammatory mediators and ameliorates EAE. The protective effect of PSLs in EAE animals is also associated with a reduced immune cell infiltration into the central nervous system and decreased splenic cognate antigen specific proliferation. Interestingly, we find that PPARs are activated in foamy macrophages in active MS lesions, indicating that degraded myelin also activates PPARs in macrophages in the human brain. Collectively, our data show that myelin modulates the phenotype of macrophages via PS-induced PPAR activation, which may subsequently dampen lesion progression in demyelinating diseases such as MS. The immunoregulatory impact of naturally-occurring myelin lipids may hold promise for future MS therapeutics.

Soluble guanylate cyclase(sGC) is a main receptor of NO (nitric oxide). The NO-sGC-cGMP pathway is involved in numerous physiological activities and related pathologies, ranging from neurotransmission to smooth muscle relaxation. It is a drugable pathway with nitrates and PDE5 inhibitors such as sildenafil as the best known drugs used in clinical practice. sGC is a heterodimeric protein consisting of an α (either α1 or α2) and a beta subunit (only β1 is active), and a prosthetic heme group that confers sensitivity to NO. In oxidative stress conditions the Fe in the heme group is oxidized resulting in an NO insensitive enzyme. Stimulators (riociguat) and activators (cinaciguat) of sGC are in clinical development for conditions such as pulmonary hypertension and heart failure. sGC stimulators enhance the effect of NO and are NO-dependent, sGC activators selectively activate the oxidized and NO-resistant version of the enzyme and are NO-independent. Primary open angle glaucoma (POAG) is a leading cause of blindness worldwide. Available therapies offer incomplete protection. The molecular signaling involved in the pathogenesis of POAG remains unknown. Here, we report that mice lacking the α1 subunit of the nitric oxide (NO) receptor soluble guanylate cyclase (sGCα1-/- mice) represent a novel and translatable animal model of POAG. Female wild-type (WT) and sGCα1-/- mice were studied. Thickness of the retinal nerve fiber layer (RNFL) was measured via spectral domain optical coherence tomography (SD-OCT). Retinal ganglion cells (RGCs) and optic nerve axons were detected histochemically and counted. Morphology of the iridocorneal angle was examined by light microscopy, ultrasound biomicroscopy, and SD-OCT. Intraocular pressure (IOP) was measured serially with a TonoLab-Tonometer. Retinal arterial function was assessed via in vivo laser ophthalmoscopy. POAG cases and controls from the Glaucoma Genes and Environment (GLAUGEN) study were studied for their association with genetic variants in the sGC locus. Age-dependent thinning of the RNFL and loss of RGC’s and optic nerve axons in the context of an open iridocorneal angle was observed in sGC α1-/- but not age-matched WT mice. The optic neuropathy associated with sGC α1–deficiency was accompanied by a modest (~2mmHg) and age-dependent increase in IOP and by retinal artery dysfunction. A candidate gene association study of POAG with paracentral vision loss, a POAG subtype thought to be associated with vascular dysregulation, identified a variant in the locus containing the genes encoding the α1 and β1 subunits of sGC. Together, these results highlight the relevance of sGC (and our animal model) in the pathogenesis of POAG.

With many ailments known today, the speed of diagnosis is one of the most important factors determining an efficient therapy. This is also true for diseases that have a genetic causality. Disorders such as cystic fibrosis and phenylketonuria (PKU), involve the presence of single nucleotide polymorphisms (SNPs). State-of-the-art techniques for mutation detection, such as Denaturing Gradient Gel Electrophoresis (DGGE), sequencing, and microarrays, require a long analysis time. Moreover, sequencing and microarrays need fluorescently labeled material, and the read-out infrastructure of both techniques is expensive. We propose an alternative technique, based on Heat-Transfer Resistance (Rth) during denaturation of dsDNA. The principle relies on the morphological change dsDNA undergoes during denaturation. This influences the efficiency of heat-transfer through that DNA layer. dsDNA molecules are stiff, upright rods, allowing heat to be transferred efficiently from the sensor surface to the liquid above, through the spaces in between the dsDNA. After denaturation, the ssDNA molecules obtain a mushroom-like shape, covering and insulating a much higher percentage of the surface, and blocking heat-transfer. This switch from dsDNA to ssDNA is clearly visible when monitoring Rth during denaturation. The temperature at which the switch occurs, differs between complementary and SNP dsDNA. Previously, a sensor was developed that was able to detect SNPs in short (~10 nm) fragments of the Phenylalanine Hydroxylase (PAH) gene, increasing the risk for PKU, with impedance spectroscopy, during chemical denaturation, within seconds. More recently, the SNPs were detected in these same fragments by monitoring Rth during thermal denaturation within the same time-scale. However, in order for this technique to be translated into a fast, label-free, and simplified microarray used in routine genetic analysis, several aspects need to be optimized. First, the Rth method must be applicable with exon-size DNA fragments. When exons are present as probes on the microarray, a gene can be screened with only a few spots. So, the maximum DNA length was determined where the Rth-based technique was still sensitive to SNPs. The detection of 1222C>T in a 120-mer fragment of exon 12 was proven. Longer fragments are under investigation up to the persistence length of dsDNA, which is 150 bp (~150 nm). DNA longer than this will curve instead of remain upright, decreasing the resolution of Rth between dsDNA and ssDNA. A second aspect to ensure the suitability of Rth in a clinical application, is the ability to distinguish homozygotes from heterozygotes. This is currently under investigation. Eventually, the Rth-based DNA sensor must be able to screen all exons simultaneously. In conclusion, monitoring Rth during the denaturation of DNA is a simple, fast, and label-free method to detect DNA mutations. Optimized, the technique will be easily transferrable to analyze other genes.

Testing of new therapeutic strategies for Parkinson’s disease (PD) is currently hampered by the lack of a robust and reproducible animal model that displays the hallmark features of PD. We developed a viral vector-based rat model for PD, presenting reproducible nigrostriatal pathology and behavioral deficits in a short time period. Therefore, we stereotactically injected adeno-associated viral vectors (rAAV2/7) encoding the human A53T α-synuclein mutant into the substantia nigra of adult rats. Progressive dopaminergic dysfunction and α-synuclein aggregation were corroborated by histopathological and biochemical analysis, non-invasive imaging, microdialysis and motor behavior. We used this optimized PD rat model to study the therapeutic effect of the immunophilin ligand FK506 in vivo. Treatment with FK506 for four weeks significantly increased the survival rate of dopaminergic neurons compared to placebo controls. To understand the mechanism-of-action of FK506 in this model, we investigated α-synuclein aggregation and neuroinflammation after FK506 treatment. Our data revealed that FK506 lowered the infiltration of cytotoxic T cells and the number of Mac 1 positive macrophages, without any apparent effect on classically activated macrophages or α-synuclein aggregation. This suggests that the immunosuppressive properties of FK506 may be the predominant factor for neuroprotection in our optimized α-synuclein rat model.

In recent years, free fatty acids (FFA) have been shown to act as direct signalling molecules through activation of several G-protein coupled membrane receptors (GPCRs). FFA2 (also known as GPR43) is activated by short chain fatty acids (SCFA) such as acetate, propionate or butyrate. FFA2 is mainly expressed on immune cells (neutrophils, monocytes, B lymphocytes), enterocytes, enteroendocrine cells and adipocytes. FFA2 has been shown to play a major role in SCFA-induced neutrophil activation and migration. Studies in FFA2 knock-out mice suggest an important contribution of FFA2 in the development and control of inflammation. GLPG0974 is identified as a potent and selective antagonist of the human FFA2. In vitro, it potently inhibits acetate-induced calcium flux in HEK293 cells (IC50 11 nM) and acetate-induced human neutrophil migration (IC50 43 nM). In a human whole blood assay, GLPG0974 inhibits acetate-stimulated neutrophil activation, as evidenced by CD11b activated epitope [AE] [removed]IC50 483 nM). GLPG0974 is also active against monkey FFA2, but not against rodent or dog FFA2. It is highly selective for FFA2 over the close homologues FFA1 and FFA3, as well as other non-related GPCRs. The safety, tolerability, pharmacokinetics (PK) and pharmacodynamics (PD) of GLPG0974 have been evaluated in healthy volunteers after single (SD) and multiple administrations for 14 days (MAD). The PD was assessed by flow cytometric measurement of neutrophil activation (CD11b[AE] expression) in whole blood upon ex vivo stimulation by acetate. Single doses up to 250 mg and multiple doses up to 400 mg daily were generally safe and well-tolerated. GLPG0974 administered under fed conditions as capsules or oral solution, was slowly absorbed with a median tmax of about 3 h and eliminated with a mean apparent terminal elimination half-life of about 5.5 h. The GLPG0974 pharmacokinetics were dose proportional over the 50 to 400 mg daily dose range. No accumulation to steady state was observed. After single administration, GLPG0974 substantially inhibited acetate-stimulated neutrophil activation in whole blood, by 61%, 73% and 89% (30, 90 and 250 mg, respectively) compared to baseline. In general, the inhibitory effect peaked at 2 h post dose and was sustained up to at least 8 h post dose. The PK/PD data showed a clear relationship between drug exposure and PD effect. The MAD study corroborated the SD results: neutrophil activation was inhibited to the same extent as observed in the SD study. The inhibitory activity was sustained for at least 12 hours after dosing and maintained over the 14-day dosing period. Inhibition of neutrophil migration into the gastro-intestinal tract may prevent neutrophil-induced tissue damage as observed with inflammatory bowel diseases, such as ulcerative colitis. A Proof-of-Concept study is initiated to evaluate the safety and efficacy of GLPG0974 in patients with mild-to-moderate ulcerative colitis.

Background Causes of many hereditary disorders can be identified through detailed inspection of the patients' genome using Next Generation Sequencing technologies. However, interpretation of the vast amount of data, typically over 20.000 variants per patient, became a bottleneck for routine implementation. To help streamlining NGS data analysis through various trimming, mapping, annotation and filtering tools, centralised web-based interfaces, such as the Galaxy platform, have been developed. Although Galaxy provides many bioinformatic tools, it lacks flexibility in downstream annotation and filtering of the resulting variants. Methods & Results We built VariantDB, a versatile annotation and filtering database implemented in MySQL, with a PHP/CGI frontend. Data added from Galaxy or FTP-upload are private, but can be shared with collaborators. Automatic annotation by ANNOVAR and snpEff, includes allele frequencies (1000g/ESP6500/dbSNPv135), functional impact (RefSeq/Ensembl/UCSC) and pathogenicity predictions (SIFT/MutationTaster/PhyloP/Polyphen2/gerp++/LRT). Custom annotation sources can be added to the xml-based configuration. Family relations allow filtering for de novo or segregating variants. Results are presented in a tabular overview, with selected annotations and hyperlinks to IGV, or exported as text files. Conclusions VariantDB allows on-demand variant filtering based on the implemented annotations. The intuitive web-interface is a very powerful tool for the interpretation of NGS data. URL: www.biomina.be/app/variantdb/

With the explosion in computer power in the last decades, computing has established a firm position in research and industry. In many fields computation has established a crucial position (e.g. aerospace or automotive design) and experiment is completely abolished or shifted to the final step in the design process. In biotechnology computation has its role, but doesn’t take a central position. Although the laws describing biomolecular processes at atomic detail are completely known, the accurate computation of those complex systems demands an enormous computational power. With current state of the art hardware, efficient software and automated algorithms the computational prediction of new and interesting molecular machines becomes possible. The investment of Flanders in a state-of-the-art supercomputer greatly enables new challenges in this field. Central to our approach is thermodynamic free energy. Free energy of the system is the driving force for processes in biochemistry/biology, hence accurate prediction of free energy, and the way it changes upon mutations are the key to successful rational design of biomolecules. One way to predict the free energy is based on statistics and existing databases, which requires the existence of relevant information in such databases or prior experimentation to build a model. On the contrary the other way only relies on the well-known laws of physics and chemistry (e.g. quantum mechanics principles, laws of thermodynamics). Here we will present an ab initio approach where the free energy will be predicted based on the first principles, and which doesn’t require prior experimentation. Current applications involve the design of selective protein-protein binding and the rational design of fluorescent proteins.

The amino-terminal tail of histones and the carboxy-tail of histone H2A protrude from the nucleosome and can be modified by many different posttranslational modifications on at least 60 different residues, thereby mediating chromatin dynamics. Several reports have recently provided strong evidence for the epigenetic potential of the enzymatic clipping of the histone H3 N-tail. During an iTRAQ proteome analysis of CLL cells we came across another major histone clipping event that has received only little attention since its discovery over 35 years ago: truncation of the histone H2A C-tail at V114. In an effort to elucidate the potential biological role of this modification, we developed a sensitive and high throughput mass spectrometry AQUA approach, based on two isotopically labeled synthetic peptides. We found that H2A clipping had no clear link to the disease under investigation but instead occurred in all myeloid hematopoietic cells investigated. Further cellular and molecular fractionation lead to the identification of the responsible enzyme, until now known as the “H2A specific protease”, as Neutrophil Elastase. With the growing interest in the epigenetic potential of histone clipping we emphasize the potential functional parallel with histone H3 clipping and caution for the impact of these clipping events on the outcome of antibody-based epigenetic approaches.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by a chronic inflammation of synovial joints. Approximately one third of RA patients test negative on the current diagnostic biomarkers rheumatoid factor (RF) and antibodies directed against cyclic citrullinated peptides (ACCP): the so-called seronegative subpopulation. Moreover, in early disease stages the sensitivity of these biomarkers is considerably lower than in established RA leading to a long diagnostic delay. As inflammation early in the disease already leads to irreversible joint damage it is of utmost importance to find additional markers for seronegative and early RA patients. In a previous study 14 novel candidate biomarkers were identified (Somers et al. J Autoimmun 2011). Here we report further validation of four of these autoantibody markers: antibody reactivity was measured in RA patients, rheumatic controls and healthy subjects in order to define sensitivity and specificity for RA, both in overall RA population and in early (symptoms for maximum one year) and seronegative subpopulations. By means of peptide ELISA antibody reactivity against the four candidate biomarkers (UH-RA.1, UH-RA.9, UH-RA.14 and UH-RA.21) was measured in 127 RA patients, 97 healthy controls and 87 rheumatic controls including psoriatic arthritis, ankylosing spondylitis, osteoarthritis and Sjögren’s syndrome patients. Amongst the 127 RA patients there were 25 early RA patients, 42 seronegative patients and 7 patients with early and seronegative disease. In this study cohort, the results demonstrated a sensitivity of UH-RA.1, UH-RA.9, UH-RA.14 and UH-RA.21 of 9%, 2%, 5% and 18% with associated specificity for RA of 95%, 98%, 100% and 89%, respectively. No less than 40% of early RA patients tested positive on the four candidate biomarkers with a sensitivity of 16% for UH-RA.1 and 20% for UH-RA.21. Amongst the seronegative RA subpopulation, 29% tested positive on the biomarkers with a sensitivity of 10% for UH-RA.1, 5% for UH-RA.14 and 17% for UH-RA.21. Three out of seven patients with early and seronegative disease tested positive for UH-RA.1 while two out of seven tested positive for UH-RA.21 (one out of seven tested positive on both biomarkers). The detection of antibody reactivity against our four candidate biomarkers in 40% of early RA patients and 29% of seronegative RA patients indicate that these biomarkers can be of additional value to the current diagnostic biomarkers for RA, with most promising results for UH-RA.1 and UH-RA.21. Our biomarkers may therefore contribute to an improved and earlier diagnosis of RA. Further analyses will be conducted to study the prognostic and theranostic potential of our biomarkers.

Reversible protein phosphorylation is one of the major molecular mechanisms behind cell signaling and regulates complex protein functions. Mapping phosphosites associated with e.g. immunological disorders contributes to the development of diagnostic and therapeutic procedures. Analytical methods in phosphoproteomics however, face many obstacles due to the transient, dynamic character and low stoichiometry of protein phosphorylation. In an effort to simultaneously circumvent mass spectrometry related pitfalls in phosphorylation quantitation we evaluated an iTRAQ (Isobaric tag for relative and absolute quantitation)-based technique which focuses on the unphosphorylated peptide precursor mass. The approach is based on the phosphopeptide mass alteration that is induced by phosphatase dephosphorylation. This peptide thus ends up in the same MSMS spectrum as its unmodified equivalent. When the untreated control and the dephosphorylated peptides are labeled by a different isobaric tag, the precursor serves as the abundant template for identification, while the skewed ratio of the reporter labels indicates the presence of a phosphosite. The iTRAQ multiplex provides the opportunity to compare both the phospho-modification and the expressional differences between multiple biological states in a single experiment. Due to the limited technical variation of this method, the number of sample fractionation steps is practically unlimited. Drawing quantitative conclusions of an iTRAQ experiment on the peptide level however, puts high demands on the data quality and requires an extensive validation of the data processing. This strategy, which focuses on the unmodified precursor peptides after removing the modification in one half of each sample, might be suitable to quantify a variety of post-translational protein modifications.

Bimetra is the Clinical Research Center for Ghent University and Ghent University Hospital (UZGent). Its mission is to facilitate, stimulate and improve translational biomedical research, from bench to bedside and from bedside to community. Indeed, the development of novel treatment options and improved patient care, based on fundamental research, is often slowed down by practical, organizational and financial hurdles. Bimetra wants to tackle these via an integrative approach, constituting of 5 platforms: • biobank activities • translational data management • clinical trial management and regulation • research and valorization (R&V) • translational core facilities The aim of Bimetra as central point of contact is therefore to • integrate, facilitate and catalyze aspects of translational biomedical research • build a biobank facility and support its data management • facilitate experimental clinical research • inventorize, reinforce and expose translational competences (intra - and extra muros) • guarantee the highest standards of ethics and quality • facilitate social and economical valorization (within strategic focus domains) • create a 'translational network' by enhancing communication and stimulating collaborations between internal and external stakeholders: clinicians, researchers, UZGent, UGent, government institutions, industry and patient organizations • monitor and promote funding opportunities • provide administrative as well as scientific support for funding applications • assist in the project management and reporting of ongoing studies More specifically, Bimetra is involved in the Center for Medical Innovation and is building out a biobank facility in the context of the Flemish Biobank Initiative. To date, Bimetra's R&V platform has: • supported more than 65 research applications • explored multiple IP related projects • facilitated 5 new patent filings in 2012 • organized more than 15 sensibilization campaigns in the field of research funding • set up and followed up more than 12 questionnaires, mainly on translational topics such as Nanotech for Health specialties, orphan diseases, biobank and infrastructure inventories, … In addition, a Coordination Center for Translational Cancer Research has been embedded in Bimetra in tight collaboration with the Oncological Center of UZGent. This was done in the context of the National Cancer Plan (Nationaal Kankerplan), a long-range national strategic plan, focusing on a number of concrete initiatives in the battle against cancer. The Center is part of Bimetra's R&V platform, although close interactions with the other platforms (i.e. Bimetra Clinics, Translational Data Management, Biobank) are maintained. In summary, Bimetra wants to reinforce the leading scientific position of the UZGent and Ghent University, by creating a solid framework where clinicians and researchers can focus on translational research under optimal circumstances.

In this research, we report a successful axillary bacterial transplantation / bacteriotherapy of the axillary microbiota to treat axillary bromhidrosis, in which offensive body odour emanated from the axillae. Bromhidrosis is caused by malodour causing bacteria that thrive in the region of the armpit. At this moment, treatments are the application of topical deodorants, antibiotics, botulinum toxin or laser operations. No fundamental solution for this problem is available, however. This research focused on applying non-odour causing bacteria in the armpit to outcompete the current malodour causing bacteria. Subsequently, the problem can be tackled at the fundaments. Subjects with significant body odour were selected for treatment. Odour assessment was done by a trained odour panel. Axillary samples were analysed by means of denaturing gradient gel electrophoresis (DGGE). A first case study was performed on a monozygotic male twin who did not co-habit, in which one twin had a significant body odour and the other one did not (p<0.05). Molecular analysis showed that one twin had mostly corynebacteria, while the other twin mostly staphylococci as dominant species in the axillae. Typically, the Corynebacterium spp. are known to cause body odour. An axillary bacterial transplantation was executed from the non-odorous twin to the odorous twin. Only one axilla was treated, whilst the other was used as a reference. Immediately after treatment, hedonic values improved for the treated axilla. Three days after the treatment, the non-treated axilla improved in hedonic values as well. The remarkable results were confirmed by DGGE as the improvements in hedonic values were associated with an increase in abundance of staphylococci and a decrease in abundance of corynebacteria. Secondly, bacteriotherapy was applied on two other subjects by means of a daily application of a pure Staphylococcus strain (during three weeks). The treatment was not successful every day, however, where there was a significant difference in hedonic value between treated and not-treated axilla, an increase of staphylococci and a decrease of corynebacteria was noticed. When treatment stopped, the original axillary microbial community was again noticed. The initial results indicated that, in one case, a permanently improved axillary community was obtained, and in two case, a temporarily improvement was obtained. This treatment opens perspectives towards the development of an enhanced deodorant, in which typical non-odour causing bacteria are added, who outcompete the malodour causing bacteria.

Neutralizing the action of several cytokines with recombinant proteins corresponding to the soluble extracellular receptor chain has proven beneficial in the treatment of various autoimmune diseases in the past years. Interleukin-33 (IL-33) is a cytokine that signals through a receptor complex consisting of ST2 and IL-1RAcP, which initiates multiple pro-inflammatory signaling pathways. Blocking the IL-33/ST2 signaling axis with the soluble (extracellular) form of the ST2 receptor (sST2) has been shown to be beneficial in experimental models of asthma and arthritis. One major drawback of the use of soluble cytokine receptor proteins, however, is their relative low ligand binding affinity. Indeed, recruitment of the IL-1RAcP co-receptor upon IL-33 stimulation has been shown to significantly increase the affinity for IL-33. Here we report a new approach to interfere with IL-33 function. We have generated a fusion protein consisting of the extracellular domains of mouse ST2 and IL-1RAcP, separated by a flexible linker (referred to as IL33trap). His-tagged recombinant IL-33trap and sST2 were expressed in human HEK293T cells and purified to homogeneity from conditioned medium by metal affinity chromatography. IL-33 neutralizing activity of the purified proteins was compared in an IL-33 bioassay in which we measure IL-33 induced NF-κB reporter gene activation. Interestingly, the IL-33trap fusion protein is considerably more potent than sST2 in neutralizing mouse IL-33 activity. A similar approach was followed to produce an IL-33trap for human IL-33, and again the IL-33trap was found to be much more potent than sST2. Binding affinity studies (BIACORE) with the IL-33 trap and sST2 proteins are ongoing. Together, these studies demonstrate the improved potential of IL-33trap for therapeutic use. This will now be further characterized using various mouse models of inflammation (asthma, arthritis). In addition, a similar cytokine trap approach will be developed for therapeutic targeting of other cytokines.

ETEC related post-weaning diarrhoea causes recurrent economic losses to the porcine rearing industry. The embargo on antibiotic prophylaxis has led to a pressing need for a suitable alternative. To evaluate feed based oral prophylactic passive immunisation against ETEC, we produced antibodies in seeds of Arabidopsis thaliana. Antibodies were designed by grafting 4 variable domains of lama heavy chain antibodies (VHH) against ETEC on the Fc part of porcine IgG and IgA. Transformants producing the 4 VHH-IgG and 4 VHH-IgA antibodies from 0.2% up to 3% of seed weight were obtained. Co-transformation of the VHH-IgA constructs with porcine joining chain and secretory component led to production of assembled dimeric and secretory IgA like antibodies in seeds. In vitro analysis of the antibody producing seed extracts were all effective in aggregating ETEC and inhibiting bacterial binding to porcine gut villous enterocytes. In a piglet feed-challenge experiment, the feed containing a milled cocktail of all the VHH-IgA based antibodies (dose 20mg/ pig/ day) protected the piglets against the challenge infection; while feed with the 4 VHH-IgG producing seeds (dose 80mg/ pig/ day) failed to offer similar protection. Piglets receiving the VHH-IgA antibodies had a swift decline in shedding of ETEC, the seroconversion was significantly lower and they had a higher weight gain. Our results show the feasibility of high-level production of simplified IgA antibodies, fit for convenient oral passive immunisation. Interestingly, it draws attention to the importance of Fc and antibody structure in passive immunisation.

Superparamagnetic nanoparticles (SPMNPs) are appealing for use in biological separation strategies. Yet, their potential remains largely unexplored because thus far research has focused on either physicochemical design, synthesis of the nanosized magnetic cores or on their application in micron-sized beads. For applications in life sciences, where extra- and intracellular interactions can be exploited, the biocompatibility of SPMNPs goes beyond their chemical composition and shape, and features like their size and more importantly their surface properties are becoming more important. Here we introduce thermal decomposition to manufacture iron oxide based SPMNPs (Ø10nm). We report how different surface functionalizations can lead to different types of interactions with living cells. Cationic aminolipid-coated SPMNPs remained surprisingly strongly associated with the cell surface. In contrast, anionic dimercaptosuccinic acid-coated SPMNPs were efficiently endocytosed/internalized and accumulated in a time-dependent manner in endosomal and lysosomal populations. Based on these results, we developed a (standardized) magnetic isolation procedure to selectively isolate plasma membranes and intracellular lysosomes. Subsequent biochemical and ultrastructural analysis consolidates the high yields and purities of such distinct membrane compartments. Our methodology therefore may provide the technological breakthrough needed in the field of subcellular ‘omics’, i.e. the analysis of the biomolecular composition of defined membrane compartments of the living cell.

Unveiling the mode of action (MOA) of chemical compounds requires gaining an insight in the pathways and regulatory cascades through which the compound act. Similarly, pathway based analysis are critical to identify the molecular mechanism of complex traits. High throughput omics data generated in in-vitro studies give a snap shot of the genetic entities that are altered upon treatment. However, the interpretation of lists of altered genetic entities in terms of pathways is still cumbersome. With the increasing number of publicly available interaction data for model organisms, it is now possible to construct a comprehensive network representation of regulatory and interaction networks in model organisms. Integrating these physical networks with in-house generated functional data can help improving insight in the cellular mode of action. We present 2 network-based approaches to (i) gain insights in the mode of action of a given treatment, and to (ii) associate complex traits/responses with downstream affected genes. In the first approach, we use a network-based method to highlight protein- protein/dna interactions or substructures in a physical network that are active in the functional data. By searching for paths (consecutive interactions) in the network that connect a highly differentially expressed seed gene with other differentially expressed genes, functional inferences can be made for genes with unaltered expression behavior that link up differentially expressed genes with known function. We present a test case to unveil the molecular subnetworks involved in acid resistance in Escherichia coli. In the second approach, we use a network-based method to search for active paths in the molecular network that associate causal genes to downstream effect, but we take into account the fact that the input nodes (candidate causal genes) can be noisy. This is a typical situation when assessing the effect of genomic alterations on observed expression behavior. With the availability of complete genomes of single strains, identifying which alterations in the DNA sequence are responsible for observed changes in gene [removed]i.e., expression quantitative trait loci (eQTL) mapping) becomes increasingly important. The proposed approach can be used to identify the causal gene within an associated genomic locus (gene prioritization) and the underlying pathways that transmit signals from the locus to the affected target. We illustrate the method on an industrially relevant trait in yeast. This research is part of the SBO-project ‘Network-based approaches for the identification and mode of action determination of antibacterial agents’ (NEMOA).

Peripheral blood mononuclear cells (PBMCs) are main actors in inflammatory processes and linked to many diseases, including rheumatoid arthritis, atherosclerosis, asthma, HIV and cancer. Moreover, they seem an interesting ‘surrogate tissue’ that can be used in biomarker discovery. In order to get a good experimental design for quantitative expression studies, the knowledge of the interindividual variation is an essential part. Therefore, PBMCs were isolated from 24 healthy volunteers (15 males, 9 females, ages 63-86) with no clinical signs of inflammation. The extracted proteins were separated using the two dimensional difference in gel electrophoresis technology (2D-DIGE), and the gel images were processed with the DeCyder 2D software. Determination of the coefficient of variation (CV) of the normalized spot volume values of these proteins, reveals that the total variation of the PBMC proteome varies between 12,99% to 148,45%, with a mean value of 28%. A supplemental look at the causes of technical variation showed that the isolation of PBMCs from whole blood is the factor which influences the experimental variance the most. This isolation should be handled with extra care and an additional washing step would be beneficial. Knowing the extent of variation, we show that at least 10 independent samples per group are needed to obtain statistical powerful data. This study demonstrates the importance of considering variance of a human population for a good experimental design for future protein profiling or biomarker studies.

Personalized cancer treatment involves targeting the genomic aberrations driving cancer development while reducing drug toxicity due to altered drug metabolism encoded by the patient’s genome. Despite the many innovations in cancer research there remain many unanswered questions about the efficacy of personalized treatment and its applicability in clinical practice. With the current state of the art of massively parallel sequencing (MPS) technologies it is economic, scientific and technical not possible to use exome or whole genome sequencing of cancer cells to obtain efficiently genetic mutations underlying cancer etiology and progression. Targeted MPS approaches however, targeted a selected set of genes, exons or mutations have been shown to enable MPS based personalized cancer treatment. Multiplicom’s MASTR (Multiplex Amplification of Specific Targets for Resequencing) assays enable highly efficient and low cost assays for personalized cancer treatment. MASTR assays employ a simple two-step PCR protocol enabling specific amplification of the regions of interest followed by the incorporation of molecular barcodes in each amplified product to unambiguously link each read to the sample it originated from. This simple and straightforward two-step protocol enables direct compatibility with all commercially available, bench top massively parallel sequencing (MPS) platforms for high throughput and cost-effective sequencing. Multiplicom is commercializing several germline based diagnostic tests for cancer diagnosis (breast and colon cancer) and congenital diseases (CFTR, DMD, Marfan syndrome,..) and recently started developing MASTR based cancer panels for MPS based detection of somatic mutations. These panels target important cancer therapy related genes linked to many cancers, including melanoma, lung, colorectal, gastrointestinal, prostate, breast and ovarian cancer. Certain cancer panels are targeted against specific exons for detection of mutation hotspots e.g. GIST panel comprising exons 9, 11, 13, 14, 15, 16 and 17 of c-KIT and exons 8, 10, 12, 14 and 18 of PDGFRA as well as an EGFR assay amplifying exons 18 to 21. Other cancer panels are developed to amplify the complete coding region of cancer related genes: the TP53 gene panel; panel containing BRAF, NRAS, KRAS and a panel containing EGFR, ERBB2, PTEN, PIK3CA and PIK3R1. The ability of these assays to use minimal amounts of precious FFPE material will unlock a wealth of genomic information for many tumor types and will be instrumental to develop personalized treatment in the clinic. Several clinical verification studies are ongoing for these assays of which results will be presented.

Imagine what would happen if all the medical data in the world were combined. Researchers and doctors wouldn’t lose precious time. Patients’ lives would improve radically. At ONTOFORCE, we want to turn this vision into reality. So we developed a unique application that makes medical insights from across the globe accessible from one single gateway. It’s fast, effective and simple to bridge the data gap and build smart links to save lives. Linked Data as a set of best practices has become popular as a means to exchange structured information on the web. An increasing number of consumer applications are now relying on the availability of LinkedData for providing data-driven analytics services. There are currently hundreds of datasets, both open and private, in the Linked Data cloud, which allow very agile information reuse. However, quality among datasets varies - very few of these LinkedData services are entirely reliable regarding uptime, completeness, performance and ease of use. First area of interest is to improve the performance, benchmarking and reliability of data sources exposed. This will include devising and implementing metrics for evaluating availability through benchmarking, measuring performance and improving fitness for consumption. Second area to solve the challenges posed by “Big Data” from the need to optimize data access and data security, to the statistical and machine learning techniques necessary to explore and disqover the knowledge concealed in many increasingly complex datasets in target use cases in healthcare and life sciences (e.g. ChemBL, Uniprot, GO, WIkiPathways, Diseasome, Sider…) and clinical trials or patient-group contributed information (Clinical Research Results, PatientsLikeMe, LinkedCT…). The platform allows distributed joins applying federated querying and smart caching techniques aiming for optimal federation of 5-20billion data points. And last but not least, the method whereby information is presented to researchers in this linked data space needs far more attention than ever. ONTOFORCE therefor considers an optimized user experience enabling knowledge discovery as one of the key aspects of its application. For many years most of the knowledge discovery tools are perceived as expert tools and have a too high threshold for the common user. User experience experts are working together with the end-users from the beginning of the development using several UX techniques such as eye-tracking … We’re committed to deliver a groundbreaking application that will make drug discovery far more effective. Because so many people need personalized medicines today – not tomorrow.

Eukaryotic cells have to handle a continuous flow of anomalous proteins. These proteins can occur as waste products during de novo synthesis (so-called misfolded proteins), or are caused by cellular stress or disease-causing mutations. Accumulation of these aberrant proteins can induce inappropriate interactions with other cellular components or can lead to potentially toxic (pathological) protein mass. In order to maintain the integrity of the eukaryotic proteome, which is essential for cell viability, eukaryotic cells have developed several sophisticated tools that monitor protein quality control. The ERAD system, i.e. the endoplasmic reticulum (ER) associated degradation system, is a conserved pathway which controls de novo protein synthesis and protein folding, and translocates misfolded proteins from the lumen of the ER to the cytosol where they become labeled with ubiquitin prior to recognition and degradation by the proteasome. A crucial step in the protein degradatin pathway is the selective recognition of target proteins. This step is accomplished by F-box proteins. The F-box protein family is the largest protein superfamily known, the total number of members ranging from 20 in yeast and 70 in humans to more than 700 proteins in plants. Substrate binding of F-box proteins relies mostly on protein-protein interactions. However, studies in mammals have documented the existence of a group of F-box proteins (Fbs proteins) that have a sugar-binding domain which is involved in the recognition and binding of glycoproteins that fail to achieve proper folding or assembly. Recognition is achieved upon binding of the Fbs protein with a typical sugar chain that is left on the misfolded glycoprotein. After binding with the Fbs protein, the glycoprotein is labeled with ubiquitin and degraded by the proteasome. Plants also rely on the ERAD system to control the abundance of key regulatory proteins. However, degradation of folding-defective glycoproteins remains poorly understood. We are studying a novel family of plant-specific F-box proteins that highly resemble the mammalian sugar-binding F-box proteins and therefore are suggested to be functional analogues playing a central role in protein quality control in plants.

Background: Biobanks collect, process and store tissue, linked to clinical, demographic or other data, to be distributed and used for research purposes. In absence of evidence based protocols, blood, tissue and their derivatives are processed and stored in as many different ways as there are biobanks. However, various pre-analytical as well as storage factors affect the post-storage quality of these samples, but the precise nature and impact of these factors on research results has not been studied in detail. Furthermore, hospital integrated biobanks often only obtain limited volumes of tissue or blood from the clinic for storage and subsequent research. This eliminates the option to divide, process and store samples according to the golden standard needed for each potential analyte. Therefore, at the University Biobank Limburg, we are continuously searching for the optimal pre-analytical protocols which allow the broadest range of downstream molecular applications, without introducing a processing bias. Here we validated the best storage type of white blood cells (WBC) which allows the broadest range of downstream molecular applications. To this end, we assessed the quality of DNA and RNA extracted after short and middle-term storage of frozen buffy coat (BC), erythrocyte lysed white blood cell (WBC) pellets and WBC suspensions and determined the impact on analytical outcome. Methods: Peripheral blood of healthy volunteers was fractionated into BC, WBC pellets and/or WBC suspensions (n=20). These were snap-frozen in LN2 and stored at -80°C for 2 months (short-term) or 2 years (middle term). Subsequently, DNA and RNA was extracted and their yield, integrity and real-time PCR performance was analysed. C-fos and IL-8 mRNA expression levels were used to determine the effect of analyte quality on post-storage research results. Results: DNA yield, purity, integrity and PCR performance were not affected upon short or middle-term storage in different WBC storage types. In contrast, RNA integrity and PCR performance were already severely affected in short-term stored WBC suspensions, although comparable RNA yields were obtained between storage types. Moreover, middle-term storage as BC also resulted in degraded RNA compared to WBC pellets, which still yielded high quality RNA after 2 years of storage. Analysis of c-fos and IL-8 mRNA levels revealed aberrantly high levels in WBC suspensions compared to WBC pellets, despite using the same amount of RNA input, identifying storage form as a major factor influencing post-storage results. Conclusions: Qualitative DNA and RNA can be obtained from short-term stored BCs. However, storage of WBC as pellet is superior to storage as BC when demanding RNA applications are envisioned. Studies to validate the pellet as optimal long-term WBC storage type are ongoing. Researchers need to be aware that the storage form of their source material may directly bias their research results.

In double-muscled Belgian Blue (BB) cattle, caesarean section is necessary in more than 95% of parturitions due to a disproportion between the pelvic size of the dam and the size of the calf. To reduce this number by selection, it should be helpful to be able to accurately estimate the growth of the pelvis and its size at the time of calving. Overall body growth and pelvic growth were determined for 69 double-muscled Belgian Blue heifers in 3 herds in Flanders. Heifers were measured starting at 10 months of age and the measurements were repeated monthly until they were sold or slaughtered, with an average of 30 measurements/animal. Overall body size was assessed by measuring wither height (WH). Internal pelvic height (PH) and pelvic width (PW) were measured by a Rice pelvimeter (Lane Manufacturing, Colorado, USA) and pelvic area (PA) was calculated. Fifty seven animals calved at least once during this research, all by caesarean section. At 10 months of age, the average wither height was 103.0 ± 3.36 cm. Up to 24 months of age, a rapid overall body growth was noticed, with a mean increase in wither height of 1.3 ± 0.31 cm/month. After first parturition, average growth rate was 0.5 ± 0.25 cm/month further decreasing to 0.2 ± 0.20 cm/month after 36 months. Internal pelvic area at 10 months of age was 102.8 ± 19.61 cm², with PH=12.0 ± 0.90 and PW=8.51 ± 1.18 cm. The PA increased mainly during the first 24 months (9.4 ± 2.41 cm²/month) and between 24 and 36 months of life (4.4 ± 4.16 cm²/month). Nevertheless, the pelvis was still growing at 56 months of age. Pelvic area near the age of first calving (24 months) was significantly correlated with pelvic area before first insemination, with the best correlation at 12 months of age (r=0.62, P<0.001). We can state that the pelvis of a BB cow keeps growing up to 5 years of age, even when the animal has reached a mature body size. As a result, heifers in which caesarean section was necessary at first parturition may still be able to calve naturally later on in life. As overall body size is not a good indicator of pelvic size, internal pelvic measurements are necessary. Measuring heifers before first insemination could be used as a starting point for selection, as the prebreeding pelvic area is an indicator of the precalving pelvic area.

In view of the expected climate change, grass breeders are interested in forage grasses performing better under limiting conditions of water. Lolium perenne (Lp) is considered as an optimal species for grassland agriculture as it provides high yields of nutritious forage. However, these species appear to lack tolerance against abiotic stress. Festuca species appear to be more tolerant to prolonged drought periods. Drought tolerance in both grasses will be studied by means of a multidisciplinary approach (a combination of genetics, genomics, transcriptomics and phenomics). Primarily, ± 600 genotypes of Festuca arundinacea Schreb. and Lolium perenne L. selected from the UGent (Festuca) and ILVO (Lolium) breeding gene pool will be screened for drought tolerance and regrowth after a drought period. For this purpose, plants will be grown under a mobile rainout shelter. Most tolerant and susceptible genotypes of each species will be selected for a more profound study at the phenotypic level. RNAseq will provide genes that are differentially expressed when drought is induced. Extreme parents will be crossed and the offspring will both be phenotyped and genotyped. RT-qPCR will then be used to evaluate the effect of induced drought conditions on the expression of candidate genes in the entire population. A genetical genomics approach must allow to point out most interesting (e)QTL for drought tolerance on a genetic map of both grass species.

We are currently working on the encapsulation and growth of different cell types in silica hydrogel. The silica sol is produced at the Turku University of Applied Sciences in Finland by acid catalyzed hydrolysis of tetra-ethyl-ortho-silicate (TEOS). To prepare gel from the sol, we add cell culture medium in a ratio of 1/1 sol/medium. For cell encapsulation, the silica sol is mixed with medium containing different amounts of cells. Cell viability and potential proliferation is analyzed by measuring metabolic activity of the encapsulated cells. Metabolic activity results in reduction of the redox indicator resazurin to resofurin which can be detected by absorption or fluorescence. Preliminary results indicate that none of the tested cell types survive in the gel longer than 28 hours. This is probably due to a lack of sufficient large pores in the silica network. Therefore, we want to create pores in the silica hydrogel by using fast degrading (bio)polymers or other molecules as porogens. When we choose the right porogen, it will dissolve in the aqueous environment or it will be degraded by the encapsulated cells. In this way, the cells will gradually get more space to stretch out and even divide. The additives serve different purposes: immediate pore formation for cell encapsulation and improved contact with cell culture medium (in vitro) or body fluids (in vivo), enhancing cell attachment on the hydrogel surface and expanding the space for growing cells by slower degrading polymers. The latter is important in vitro, because silica has low solubility in water. In vivo silica hydrogel dissolves fast, as living tissue is not a static system. The porogens can be applied in different morphologies: in dissolved form, in suspension, as fibers or as solid particles. After degradation or dissolution of the porogen, the gel should have sufficient porosity for attachment and division of different cell types. Possible porogens are dextrin, PEG, PEO or PLA. For each porogen, the effect on gel characteristics will have to be measured, including gelation time, viscosity and elasticity. In addition, methods will be developed to control the dissolution or degradation rate. The pore size will be measured using scanning electron microscopy. After adaptation of the hydrogel, viability of encapsulated cells will again be measured. The results obtained in the silica hydrogel system will be compared to the porous cross-linked gelatin hydrogel developed by the Polymer Chemistry and Biomaterials Group in Ghent. These porous gelatin scaffolds are produced by cryogenic treatment of chemically cross-linked gelatin hydrogel, followed by removal of ice crystals through lyophylization. By varying the cryogenic parameters and gelatin concentration, the size and morphology of the pores in these hydrogels can be controlled.

Background. Few well-designed randomized controlled trials (RCT) regarding the impact of community pharmacist interventions on pharmacotherapeutic monitoring of patients with Chronic Obstructive Pulmonary Disease (COPD) have been conducted. We assessed the effectiveness of a pharmaceutical care program for patients with COPD. Methods. The PHARMACOP-trial is a single-blind 3-month RCT, conducted in 170 community pharmacies in Belgium, enrolling patients prescribed daily COPD medication, aged ≥50 years, and with a smoking history ≥10 pack-years. A computer-generated randomization sequence allocated patients to intervention, receiving protocol-defined pharmacist care, or control group, receiving usual pharmacist care (1:1 ratio, stratified by center). Interventions, focusing on inhalation technique and adherence to maintenance therapy, were carried out at start of the trial and at one month follow-up. Primary outcomes were inhalation technique and medication adherence. Secondary outcomes were exacerbation rate, dyspnea, COPD specific and generic health status and smoking behavior. Clinicaltrials.gov Identifier: NCT01260389. Findings. From December 2010 to April 2011, 734 patients were enrolled. 17 (4.7%) of control (n=363) and 25 (6.7%) of intervention group patients (n=371) were lost to follow-up. At the end of the trial, inhalation score (Mean estimated difference [∆],13.5%; 95% Confidence Interval [CI], 10.8-16.1; P<.0001) and medication adherence (∆, 8.51%; 95%CI, 4.63-12.4; P<.0001) were significantly higher in the intervention group compared to the control group. In the intervention group, a significantly lower hospitalization rate was observed (9 vs 35; Rate Ratio, 0.28; 95%CI, 0.12-0.64; P=.003). There were no significant between-group differences in emergency department visits (P=.20), dyspnea (P=.97), COPD-specific or generic health status (P=.83 and P=.19) or smoking (P=.76) Conclusion. Pragmatic pharmacist care programs improve the pharmacotherapeutic regime in patients with COPD and could reduce hospitalization rates.

The Leuven Viral Vector Core (LVVC) is a full-service facility, housed at Campus Sint-Rafaël, KU Leuven, producing high-quality, serum-free HIV-derived lentiviral (LV) vectors and adeno-associated viral (AAV) vectors on a weekly basis for internal and external research programs. LV vectors are an excellent tool to transduce difficult-to-transfect cell lines and to generate stable cell lines since they stably integrate into the host cell genome. AAV vectors outperform LV vectors for many applications in animal models. The portfolio of the LVVC covers a wide range of molecular biology tools, ranging from basic overexpression over classic short hairpin RNA (shRNA) and second generation microRNA-based shRNA knockdown technology to inducible and regulatable systems, all combined with selection markers in multicistronic expression constructs. In addition, the LVVC develops non-invasive molecular imaging vectors to follow up gene expression using reporter genes for bioluminescence imaging, fluorescence imaging and positron emission tomography (PET). This extensive portfolio enables to support a wide range of research applications. Furthermore, we have optimised vector-based techniques to generate somatic transgenic animals (i.e. mice and rats) as well as locoregional transgenic models (after stereotactic injection in rodent brain). All these viral vector-based assays and models can be applied in target validation and drug screening projects. The LVVC provides state-of-the-art biotechnology services via bilateral contracts.

Background: The University Biobank Limburg (UBiLim) is a unique collaboration of the Jessa Hospital, the Hospital East Limburg and Hasselt University. It was founded to support the growing need of biobank facilities for the translational research activities of the Limburg Clinical Research Program (LCRP) and other parties. UBiLim collects, processes and stores high quality human tissue and coupled data, and makes these available for translational and clinical research, to accelerate their impact on healthcare. UBiLim is built on the expertise of the Clinical Biobank of the Jessa Hospital, which it obtained through participation in the European FP6 project “Cancer Control using Population-based Registries and Biobanks (CCPRB)”. As a response to several biobank funding initiatives in 2008, the three partners agreed to join their activities in a collective biobank facility, named UBiLim. Financial support was obtained from Limburg Sterk Merk. Methods: A centralised storage facility with decentralised depots was set up, holding organised storage capacity at -40°C, -80°C and in liquid nitrogen vapour, all continuously monitored at temperature and equipment alarm level. Procedures for processing and storage of a plethora of sample types, suitable for state- of-the-art applications were validated and implemented. An organisational structure was established, consisting of an operational group, a scientific advisory board and a steering committee. A procedural framework was designed to request logistic and processing support or access to samples. All activities are integrated in a Quality Management System ISO15189 and based on (inter)national biobank guidelines. Finally, we provide know-how, expertise and counselling in practical study design as in sample processing and storage. Results: UBiLim is an economical biobanking approach, because of its centralised facility at one partner with depots offering first-line activities at the others’ and the shared IT solutions, compared to each partner building its own bank. Since our foundation in 2008, 12 sample collections were incorporated, resulting in an approximate total of 45000 samples, of which about half derive from LCRP projects. Four hundred samples were requested for analysis by 4 different research groups. This has already resulted in 2 peer-reviewed articles, 4 published abstracts, 5 oral presentations and 5 posters (including one poster price). Currently, 3 new sample collections are in the consulting phase and will probably start collecting spring 2013. Two historical collections are being considered for transfer. Conclusion: UBiLim is a unique, cost-efficient biobank thanks to three independent partners cooperating in one common facility. This set-up also stimulates the regional collaboration. UBiLim clearly supports and facilitates LCRP activities and has developed into an important tool for scientists. As such, UBiLim contributes indirectly but successfully to translational research.

Alzheimer’s disease (AD) is the leading neurodegenerative disorder in the world, but up until today no treatment has been proven effective against AD. Moreover, diagnosing AD has been challenging and can only be confirmed after a post-mortem brain autopsy. Therefore it is important to develop AD biomarkers, which detect AD at an early stage and discriminate AD from other dementias. Although increased cerebral spinal fluid (CSF) Aβ has been associated with an increased risk of AD development, it is not sufficiently accurate for the prediction of AD and requires a relatively invasive lumbar puncture. Alternatively, amyloid depositions and functional perturbations can be identified with Positron Emission Tomography (PET). However, this imaging technique involves the injection of radioactive tracers that emits possibly dangerous ionizing radiation. Magnetic Resonance Imaging (MRI), on the other hand offers safe and non-invasive possibilities to evaluate structural and functional defects. So far, MRI has been used primarily to assess brain atrophy in late stage AD. In order to realize the full potential of multiple MRI modalities, our research focusses on the implementation of state-of-the-art MRI techniques, examining selected aspects of AD in dedicated animal models. In our recent research we have used APPSwe/PS1L166P transgenic mice that mimic the cerebral amyloid pathology of AD. These mice start to develop Aβ-plaques from the age of 2 months, which cover the whole cerebrum from the age of 8 months onwards. Using Diffusion Kurtosis Imaging (DKI), increased microstructural complexity in the cortical areas of APPSwe/PS1L166P mice was found, suggesting that amyloid deposits augment tissue’s complexity. Additionally, we observed significantly higher magnetic transfer ratios (MTR) in the cortical regions of APPSwe/PS1L166P mice. This finding could be related with the high amyloid load present in the APPSwe/PS1L166P mice, supporting the DKI data. We cannot rule out, however, that inflammatory processes contribute to the diffusion changes. Besides the structural changes, we also found deterioration of the Functional Connectivity (FC) between the retrosplenial cortex and the hippocampus in APPSwe/PS1L166P mice. These FC alterations suggest impaired synaptic function, due to the presence of (soluble) amyloid. Moreover, altered FC might be related to the compromised spatial memory observed in these mice and AD patients. AD patients also show Aβ deposition in the cerebral vasculature, which has a profound influence on vascular integrity and function. Using susceptibility weighted imaging (SWI) we have found vascular abnormalities in different animal models of AD. In conclusion, our findings endorse the use of multimodal MRI in AD research, in preclinical research to search for new potential drug targets and to assist drug evaluation, and in the clinic to support neurological exams in order to improve diagnostic accuracy.

A crucial aspect of clinical trial management is the recruitment of patients conforming to the clinical trial protocol. A timely selection of eligible candidate patients brings considerable benefits to both hospitals (becoming and retaining the status of preferred study site) and study sponsors (reduced risk for patient accrual failures). Despite those advantages, many clinical trial selection procedures still consist of a manual inspection of electronic health records. This prolongs the recruitment process and induces the possibility of missing eligible patients (up to 60% according to several studies). Therefore, a platform to aid in the automated selection of eligible patients was developed at the Antwerp University Hospital. The platform consists of: (1) a web-based study editor allowing clinicians or study nurses to enter the inclusion and exclusion criteria of a clinical trial which is saved in a machine-readable representation, (2) a patient evaluation component which has access to data sources at a specific site so that patient eligibility can be determined, and (3) a web-based evaluation result viewer that provides an overview of patient eligibility for a study and an individual patient. The data sources used by the patient evaluation component consist of both structured information, including demographics, labs and diagnostic codes, as well as unstructured information in the form of clinical free text. Structured information is stored in I2B2, a clinical data warehouse, and unstructured information is stored in SOLR, an open-source Lucene-based platform for full-text searching and indexing. While textual data in clinical notes is the richest source of information, but also the most difficult one to extract structured information from. Therefore, we have developed techniques to combine complex criteria and text searches into compound and clinically interpretable concepts. These concepts enable the mapping of inclusion and exclusion criteria to keyword queries or lab test filters for an optimal retrieval of relevant information. In a case study to assess the formalization capabilities of this platform, we evaluated 4 clinical trials (2 oncology, 2 neurology). An average of 36.5 criteria per study are present, of which 17% are consent-, willingness or ability-based. Of the remaining criteria, 72% can be formally represented in our platform and 58% of these criteria requires extraction of information from clinical free text.

The BioMiMedics consortium is a cross-border collaboration in the Maas-Rijn Euregion that aims at the development of new and modification of existing polymers which are preferentially biodegradable. These new polymers are envisioned with applications ranging from new wound dressing materials to 3D scaffolds for tissue engineering and controlled drug delivery. Expertise of research groups in Maastricht (UM), Aachen (RWTH, INB), Liège (ULg: CERM, CEIB) and Hasselt (UH: IMO-IMOMEC, BIOMED) is combined to successfully achieve this goal. An important step during the development of the newly synthetized products is their in vitro assessment. Here, we present the use of fluorescent labels to facilitate the in vitro and in vivo monitoring of these new polymers, including their distribution, dynamics and fate in cells. Considerable attention was devoted to confirm that the fluorophores remain attached to the polymer after internalization. Techniques that are used include fluorescence correlation spectroscopy and spatio-temporal image correlation spectroscopy. This research is part of the Interreg EMR IV-A project BioMIMedics (www.biomimedics.org), and is co-financed by the European Union, local governments, research institutes and SMEs. The European Commission, investing in your future.

DNA replication errors that persist as mismatch mutations make up the molecular fingerprint of mismatch repair (MMR) deficient tumors and convey them with resistance to standard therapy. Using whole-genome and -exome sequencing, we here characterize the unique somatic substitution and insertion/deletion (indel) patterns underlying MMR-deficient tumors. In particular, we show that mutation frequencies are >30-fold increased relative to MMR-proficient tumors: substitutions mainly consist of single basepair transitions and more frequently located near indels. G:C>A:T transitions mostly occured in the context of a CG dinucleotide. Indels are largely confined to homopolymers and mostly consisted of 1 or 2 bp frameshifts. Since exonic indels result in loss-of-function frameshift mutations, indels were less frequent in the exome due to negative selection. However, a small fraction of exonic indels was positively selected and occurred as hotspot mutations, preferentially affecting the ‘DNA double-strand break (DSB) repair by homologous recombination (HR)’ pathway (on average, each MMR-deficient tumor contained 3.0±0.5 loss-of-funtion mutations in this pathway). We confirmed that the DSB repair by HR is functionally impaired in MMR-deficient tumors by immunofluorescent staining of nuclear RAD51 foci in primary cultures of MMR-deficient tumors in the presence of the PARP1 inhibitor olaparib. Furthermore, MMR-deficient cultures exhibited a dose-dependent reduction in proliferation under olaparib, whereas MMR-proficient cells did not respond (P<0.001 by repeated measurement). Sequencing of MMR-deficient tumors thus identified a synthetic lethal interaction between the major DNA repair pathways, offering novel treatment modalities for MMR-deficient tumors. Finally, high-throughput profiling of hotspot mutations also more accurately detected microsatellite instability (MSI) in several cancer types compared to standard diagnostic methods. The extended Bethesda panel, which consists of 8 microsatellite and 2 homopolymer markers, is currently used to detect MSI and has only limited sensitivity. We demonstrate that high-throughput profiling of 56 hotspot mutations outperforms Bethesda panel.

Mortality risk assessment for critically ill patients is an essential task in intensive care units (ICUs) with respect to preventive treatment and medication schemes (Strand and Flaantien, 2008). Accurate and reliable patient risk assessments are a prerequisite for clinicians and physicians to evaluate quality of care and treatment, to gain fast insights into patients’ circumstances, and to make improved clinical decisions. Tremendous amounts of heterogeneous data available at ICUs, have created big computational challenges for the development of predictive models. Over the last two decades, various mortality risk prediction systems have been proposed for critically ill patients, demonstrating good prognostic performances on their own specific patient data. The objective of this study is to develop an improved computational model for critically ill patients’ mortality risk prediction that is based on a state-of-the-art machine learning technique called Random Forests. Data were collected from the Patient Database Management System (PDMS) of the Antwerp University Hospital (UZA). All patients admitted to the ICU from May 14, 2007 to June 18, 2012 were considered for inclusion into this study. In total, there were 11,412 patient admissions included into the study cohort. We developed a novel Random Forest based model and performed a number of cross-validation experiments with different subsets of variables on the study cohort. The performances were compared against those of the SAPS3 model (Metnitz et al., 2005 and Moreno et al., 2005), which is a recently published ICU mortality risk prediction system. The experimental results demonstrate that the RF-based model outperforms this SAPS3 model. Using only variables used in the SAPS3 model, the RF-based model yields an Area Under the ROC (AUC) of 88.73%, which is significantly better than that of the SAPS3 model (p-value 2.75e-23). The AUC further increases to 91% when relevant information extracted from additional lab tests was incorporated into the model.

Many drugs exert their therapeutic activities by modulating multiple targets. Moreover, these pleiotropic effects are often associated with undesired off-target effects. Hence knowledge about the molecular pathways targeted by a (candidate) drug, including the specific drug-target interactions, is of great importance for a more efficient development of new drugs, in particular to predict side effects and explore the possibilities for drug repositioning. For many (candidate) drugs, the targets are, however, largely unknown and difficult to find among the thousands of gene products. Gene expression technologies such as microarrays and next generation sequencing enable us to observe the effect of drug treatment on the activity of all genes simultaneously and thus form the perfect starting point for drug mode of action prediction. Hence we are developing an easy-to-use analysis suite for functional characterization of drugs based on expression changes in cells or tissues after treatment. Our software will provide all necessary tools for gaining new insights into the biological effects of a drug by integrating (1) data import and preprocessing of the most common Affymetrix arrays and RNA-Seq platforms; (2) data quality assessment; (3) prediction of a drug’s targets; (4) prediction of pathways involved in a drug’s mode of effect; (5) identification of overlap with disease genes enabling side effect prediction and drug repurposing; and (6) result visualization and reporting. Drug target prediction is performed by means of an in-house developed algorithm for neighborhood analysis, integrating the expression data with functional protein association information. To test our pipeline we will apply it to some publicly available gene expression data for treatment with drugs exhibiting a well-defined mode of action.

Transmission of viruses by raw materials presents a considerable risk for production of biological products. As part of continuous improvement efforts to eliminate this risk, Genzyme has initiated several programs to evaluate different viral inactivation methods for their cell culture processes. Different technologies like gamma and ultra-violet C irradiation and nano filtration were proposed as potential methods for viral inactivation of cell culture media. Each technology has been shown to be effective for a specific range of virus types and a combination of methods would be necessary to cover a wider range, including both single-stranded and double stranded DNA viruses, and both enveloped and non-enveloped viruses. Although these methods have been known to possess excellent virus inactivation capabilities, they can damage or remove media components. The effect of the use a treated media on the cell growth, productivity and product quality cannot be predicted due to the fact that these methods can possibly cause degradation on small molecules and proteins or remove important growth factors which can be detrimental to mammalian systems. Comprehensive assessments and small scale studies are required to evaluate the impact on cell culture performance and product quality attributes of new and established legacy processes.

Genzyme uses mammalian cell culture for the production of a recombinant protein which is used as an enzyme replacement therapy. During bioreactor cell cultivation, DO probes are used to control the oxygen sparge rate to maintain the Dissolved oxygen at a fixed set point. Historically variable oxygen flow rates - unrelated to viable cell counts - during seed bioreactor runs are observed to maintain the Dissolved Oxygen at set-point. To improve oxygen sparge flow rate performance in the seed bioreactors, alternative sparge elements were selected for further evaluation. Investigation of sparge elements with different lengths, different pore sizes and porous metal media grade by high speed imaging tests were performed. Results of these experiments showed comparability of a 1 inch, 2 micron pore sized sparge element with the current 10 inch sparge elements. The alternative sparge element is easy to install on the sparger line. Further, the small size of the element prevents scratching the inner surface of the reactors during installation in the seed bioreactor due to the decreased length of the sparge lines. The 1 inch, 2 µm pore sized alternative sparge element was used in consecutive seed bioreactor runs for further investigating the influence of using this sparge element on the seed bioreactor process performance. Both seed bioreactor runs were operated according to the current production records with the only difference that the alternative sparger was installed for supplying oxygen to the seed reactor. The obtained oxygen sparge flow rates to control the DO at set-point during seed bioreactor runs with the alternative sparge element were comparable to historical runs and DO was controlled within a narrow range. Further, cell culture performance was comparable to the historical runs. Also, no increased bioburden or endotoxin results were found which indicates that the SIP cycles of the bioreactor vessels were performed successfully. Further steps are ongoing for the final implementation of this alternative sparge element in the seed bioreactors.

The MultiStem® product is an adult adherent stem cell product derived from bone marrow which has significant clinical exposure. MultiStem cells are currently in phase II clinical studies for treatment of ischemic stroke and ulcerative colitis, with Phase I studies completed in acute myocardial infarction and for GvHD prophylaxis in allogeneic hematopoietic stem cell transplantation. In addition, the cells are being evaluated in preclinical studies for the treatment of a range of other indications such as conditions involving ischemic injury, neurological injury or disease and conditions involving inflammation or immune system function. ReGenesys has made significant progress in the development of novel MultiStem culture methods for future clinical application. On the one hand, we have completed a xenobiotic-free platform to isolate, expand and cryopreserve MultiStem cells, while on the other hand we have optimized MultiStem expansion on a GMP-grade 3D hollow fiber bioreactor (Quantum, TerumoBCT) up to clinical dose scale. An indispensable part of the development and manufacturing of an advanced cell therapy product is quality control and cell comparability testing such as screening for growth, marker expression, immunosuppression, multipotent differentiation, transcriptome analysis and chromosome stability (SNP arrays), which are conducted on each newly produced batch of cells. The regulatory legislation concerning the quality requirements of these types of products is still under development and new, more stringent guidelines are expected to be released in the near future. ReGenesys therefore aims to be first in class by designing a high level cell comparability testing pipeline and employs miRNA screens and Next Generation Sequencing-based DNA methylation and RNA expression in order to characterize the molecular phenotype and mechanism of therapeutic activity of the product. Extensive screens have been carried out to identify (epigenetic) markers to characterize the MultiStem phenotype and better distinguish it from competing products (e.g. MSC). Based on these results, we have implemented new QC assays and demonstrate that these assays are an improvement to standard assays, e.g. flow cytometry or differentiation assays, to discriminate between MultiStem cells and MSC. The assays are being used to establish the MultiStem identity after modifications of the manufacturing process and have shown that this identity is retained after xenobiotic-free expansion, or after expansion on the 3D bioreactor. Currently, we are integrating the xenobiotic-free cell expansion and 3D bioreactor technology and take advantage of the novel assays we have developed for cell comparability testing. Here, we present results of xenobiotic-free and 3D bioreactor-expanded cells in comparison with standard cultured MultiStem cells and MSC.

ReGenesys BVBA is a biotech company focused on the research, development and commercialization of stem cell-based therapies and technologies. Multipotent Adult Progenitor Cells (MAPC) are bone marrow-derived non-hematopoietic adherent stem cells with a large expansion capacity and a remarkable biological plasticity. MAPC have the potential to home and integrate into damaged tissues and provide immunomodulatory and angiogenic effects by cell-cell contact and paracrine regulation. Therefore, there is growing interest in the use of MAPC as cellular therapy. The MultiStem® therapeutic product, based on MAPC technology, is an allogeneic “off the shelf" stem cell product. Like mesenchymal stem cells (MSC), allogeneic MultiStem cells are non-immunogenic and exert immunosuppressive effects in vitro. On the other hand, MultiStem cells show alterations in phenotype,differences ingene and protein expressionand have an extensive proliferation capacity as compared to MSC. MultiStem cells are currently being used in several clinical trials like acute myocardial infarct (AMI, Phase II), graft versus host disease (GVHD, Phase I), ischemic stroke (Phase I/II) and inflammatory bowel disease (ulcerative colitis, Phase II). One clinical MultiStem dose requires 100 to 200 million cells. Such large numbers of cells needed for clinical applications makes standard cell culture conditions expensive and labor intensive with the need for higher personnel resources. Besides this, the standard expansion is an open process which also signifies a higher risk of contamination. We have optimized a cell expansion system for large scale cell culture. TerumoBCT (formerly CaridianBCT) developed the Quantum Cell Expansion System for ex vivo expansion of stem cells using a bioreactor. The functionally closed automated culture system is comprised of a synthetic hollow-fiber bioreactor connected to sterile closed-loop, computer-controlled media and gas exchangers. We have tested this system to optimize the complete workflow of MultiStem culture, including stem cell isolation from bone marrow and expansion up to the scale of clinical doses. We demonstrate that MultiStem cells could be obtained on the Quantum without prior clone selection in culture dishes. Furthermore, we could reach a harvest yield of up to 1 billion cells after a 6 day culture starting from 10 million cells. A full quality control was performed showing that characteristics of MultiStem cells harvested from the Quantum were retained compared to stem cells generated according to standard cell culture conditions on cell culture plastic. In conclusion, we show that this system can provide clinical doses of cells with a strong reduction of human labor without the need of GMP-grade clean rooms.