Identification of Mimotopes with Diagnostic Potential for Trypanosoma brucei gambiense Variant Surface Glycoproteins Using Human Antibody Fractions

Background: At present, screening of the population at risk for gambiense human African trypanosomiasis (HAT) is based on detection of antibodies against native variant surface glycoproteins (VSGs) of Trypanosoma brucei (T.b.) gambiense. Drawbacks of these native VSGs include culture of infective T.b. gambiense trypanosomes in laboratory rodents, necessary for production, and the exposure of non-specific epitopes that may cause cross-reactions. We therefore aimed at identifying peptides that mimic epitopes, hence called “mimotopes,” specific to T.b. gambiense VSGs and that may replace the native proteins in antibody detection tests. Methodology/Principal Findings A Ph.D.-12 peptide phage display library was screened with polyclonal antibodies from patient sera, previously affinity purified on VSG LiTat 1.3 or LiTat 1.5. The peptide sequences were derived from the DNA sequence of the selected phages and synthesised as biotinylated peptides. Respectively, eighteen and twenty different mimotopes were identified for VSG LiTat 1.3 and LiTat 1.5, of which six and five were retained for assessment of their diagnostic performance. Based on alignment of the peptide sequences on the original protein sequence of VSG LiTat 1.3 and 1.5, three additional peptides were synthesised. We evaluated the diagnostic performance of the synthetic peptides in indirect ELISA with 102 sera from HAT patients and 102 endemic negative controls. All mimotopes had areas under the curve (AUCs) of ≥0.85, indicating their diagnostic potential. One peptide corresponding to the VSG LiTat 1.3 protein sequence also had an AUC of ≥0.85, while the peptide based on the sequence of VSG LiTat 1.5 had an AUC of only 0.79. Conclusions/Significance: We delivered the proof of principle that mimotopes for T.b. gambiense VSGs, with diagnostic potential, can be selected by phage display using polyclonal human antibodies

Focus on 16p13.3 Locus in colon cancer

Background : With one million new cases of colorectal cancer (CRC) diagnosed annually in the world, CRC is the third most commonly diagnosed cancer in the Western world. Patients with stage I-III CRC can be cured with surgery but are at risk for recurrence. Colorectal cancer is characterized by the presence of chromosomal deletions and gains. Large genomic profiling studies have however not been conducted in this disease. The number of a specific genetic aberration in a tumour sample could correlate with recurrence-free survival or overall survival, possibly leading to its use as biomarker for therapeutic decisions. At this point there are not sufficient markers for prediction of disease recurrence in colorectal cancer, which can be used in the clinic to discriminate between stage II patients who will benefit from adjuvant chemotherapy. For instance, the benefit of adjuvant chemotherapy has been most clearly demonstrated in stage III disease with an approximately 30 percent relative reduction in the risk of disease recurrence. The benefits of adjuvant chemotherapy in stage II disease are less certain, the risk for relapse is much smaller in the overall group and the specific patients at risk are hard to identify. Materials and Methods : In this study, array-comparative genomic hybridization analysis (array-CGH) was applied to study high-resolution DNA copy number alterations in 93 colon carcinoma samples. These genomic data were combined with parameters like KRAS mutation status, microsatellite status and clinicopathological characteristics. Results : Both large and small chromosomal losses and gains were identified in our sample cohort. Recurrent gains were found for chromosome 1q, 7, 8q, 13 and 20 and losses were mostly found for 1p, 4, 8p, 14, 15, 17p, 18, 21 and 22. Data analysis demonstrated that loss of chromosome 4 is linked to a worse prognosis in our patients series. Besides these alterations, two interesting small regions of overlap were identified, which could be associated with disease recurrence. Gain of the 16p13.3 locus (including the RNA binding protein, fox-1 homolog gene, RBFOX1) was linked with a worse recurrence-free survival in our patient cohort. On the other hand, loss of RBFOX1 was only found in patients without disease recurrence. Most interestingly, above mentioned characteristics were also found in stage II patients, for whom there is a high medical need for the identification of new prognostic biomarkers. Conclusions : In conclusion, copy number variation of the 16p13.3 locus seems to be an important parameter for prediction of disease recurrence in colon cancer

Identification of Peptide Mimotopes of Trypanosoma brucei gambiense Variant Surface Glycoproteins

The control of human African trypanosomiasis or sleeping sickness, a deadly disease in sub-Saharan Africa, mainly depends on a correct diagnosis and treatment. The aim of our study was to identify mimotopic peptides (mimotopes) that may replace the native proteins in antibody detection tests for sleeping sickness and hereby improve the diagnostic sensitivity and specificity. We selected peptide expressing phages from the PhD.-12 and PhD.-C7C phage display libraries with mouse monoclonal antibodies specific to variant surface glycoprotein (VSG) LiTat 1.3 or LiTat 1.5 of Trypanosoma brucei gambiense. The peptide coding genes of the selected phages were sequenced and the corresponding peptides were synthesised. Several of the synthetic peptides were confirmed as mimotopes for VSG LiTat 1.3 or LiTat 1.5 since they were able to inhibit the binding of their homologous monoclonal to the corresponding VSG. These peptides were biotinylated and their diagnostic potential was assessed with human sera. We successfully demonstrated that human sleeping sickness sera recognise some of the mimotopes of VSG LiTat 1.3 and LiTat 1.5, indicating the diagnostic potential of such peptides

(Photo-)crosslinkable gelatin derivatives for biofabrication applications

Over the recent decades gelatin has proven to be very suitable as an extracellular matrix mimic for bio-fabrication and tissue engineering applications. However, gelatin is prone to dissolution at typical cell culture conditions and is therefore often chemically modified to introduce (photo-)crosslinkable functionalities. These modifications allow to tune the material properties of gelatin, making it suitable for a wide range of biofabrication techniques both as a bioink and as a biomaterial ink (component). The present review provides a non-exhaustive overview of the different reported gelatin modification strategies to yield crosslinkable materials that can be used to form hydrogels suitable for biofabrication applications. The different crosslinking chemistries are discussed and classified according to their mechanism including chain-growth and step-growth polymerization. The step-growth polymerization mechanisms are further classified based on the specific chemistry including different (photo-)click chemistries and reversible systems. The benefits and drawbacks of each chemistry are also briefly discussed. Furthermore, focus is placed on different biofabrication strategies using either inkjet, deposition or light-based additive manufacturing techniques, and the applications of the obtained 3D constructs

Characterisation of genome-wide copy number aberrations in colon cancer

Introduction : Despite improved screening programs and modern treatments, colorectal cancer (CRC) still remains the third most common cancer in men and women and the second leading cause of cancer-related death. The development of CRC is characterised by several genetic and epigenetic alterations. There is still a high rate of recurrence and metastases with a remaining need for identification of molecular biomarkers in order to predict the evolution of the disease. Aim : Our aim was to characterise copy number alterations in different subgroups of colon cancer patients. Methods : Genetic alterations were analysed using Array Comparative Genomic Hybridisation (array-CGH) in 100 resection tissues of colon tumours. The tumour DNA was isolated with the Qiagen DNeasy Blood and Tissue kit. Megapool reference DNA was used as normal control. Tumour DNA was labelled with the fluorescent dye Cy3 and the normal control was labelled with Cy5. After labelling the DNA, it was hybridised to a SurePrintG3 Human CGH Microarray, 4 × 180 K. The scanned images were analysed with an in-house developed software program, arrayCGHbase. Results : In the studied patient population, 3-year survival and recurrence of disease were correlated with the stage of colon cancer but not with tumour localisation. Preliminary results showed a significant correlation (p < 0,001) between the number of genetic alterations and overall survival of the patients (n = 100). The most prevalent copy number alterations in CRC as described in literature, were confirmed in our samples, for instance gain of chromosomes 7, 13 and 20 and losses of chromosomes 4 and 18. Moreover, we found alterations residing in a number of new interesting chromosome regions. Future analyses will have to reveal the clinical relevance in colon. cancer. Conclusion : The number of copy number alterations is associated with the overall survival of colon cancer patients. The new findings will be further investigated in depth. The link between KRAS and MSI status and the copy number alterations in the tumours will also be investigated

Recombinant antigens expressed in Pichia pastoris for the diagnosis of sleeping sickness caused by Trypanosoma brucei gambiense

Screening tests for gambiense sleeping sickness, such as the CATT/T. b. gambiense and a recently developed lateral flow tests, are hitherto based on native variant surface glycoproteins (VSGs), namely LiTat 1.3 and LiTat 1.5, purified from highly virulent trypanosome strains grown in rodents.We have expressed SUMO (small ubiquitin-like modifier) fusion proteins of the immunogenic N-terminal part of these antigens in the yeast Pichia pastoris. The secreted recombinant proteins were affinity purified with yields up to 10 mg per liter cell culture.The diagnostic potential of each separate antigen and a mixture of both antigens was confirmed in ELISA on sera from 88 HAT patients and 74 endemic non-HAT controls. Replacement of native antigens in the screening tests for sleeping sickness by recombinant proteins will eliminate both the infection risk for the laboratory staff during antigen production and the need for laboratory animals. Upscaling production of recombinant antigens, e.g. in biofermentors, is straightforward thus leading to improved standardisation of antigen production and reduced production costs, which on their turn will increase the availability and affordability of the diagnostic tests needed for the elimination of gambiense HAT