Production and characterization of monoclonal antibodies raised against recombinant human granzymes A and B and showing cross reactions with the natural proteins

The human serine proteases granzymes A and B are expressed in cytotoplasmic granules of activated cytotoxic T lymphocytes and natural killer cells. Recombinant granzyme A and granzyme B proteins were produced in bacteria, purified and then used to raise specific mouse monoclonal antibodies. Seven monoclonal antibodies (mAb) were raised against granzyme A, which all recognized the same or overlapping epitopes. They reacted specifically in an immunoblot of interleukin-2 (IL-2) stimulated PBMNC with a disulfide-linked homodimer of 43 kDa consisting of 28 kDa subunits. Seven mAb against granzyme B were obtained, which could be divided into two groups, each recognizing a different epitope. On an immunoblot, all mAb reacted with a monomer of 33 kDa protein. By immunohistochemistry, these mAb could be used to detect granzymes A and B expression in activated CTL and NK cells. The availability of these mAb may facilitate studies on the role of human cytotoxic cells in various immune reactions and may contribute to a better understanding of the role of granzmes A and B in the cytotoxic response in vivo

Possible modification of BRSK1 on the risk of alkylating chemotherapy-related reduced ovarian function

STUDY QUESTION: Do genetic variations in the DNA damage response pathway modify the adverse effect of alkylating agents on ovarian function in female childhood cancer survivors (CCS)? SUMMARY ANSWER: Female CCS carrying a common BR serine/threonine kinase 1 (BRSK1) gene variant appear to be at 2.5-fold increased odds of reduced ovarian function after treatment with high doses of alkylating chemotherapy. WHAT IS KNOWN ALREADY: Female CCS show large inter-individual variability in the impact of DNA-damaging alkylating chemotherapy, given as treatment of childhood cancer, on adult ovarian function. Genetic variants in DNA repair genes affecting ovarian function might explain this variability. STUDY DESIGN, SIZE, DURATION: CCS for the discovery cohort were identified from the Dutch Childhood Oncology Group (DCOG) LATER VEVO-study, a multi-centre retrospective cohort study evaluating fertility, ovarian reserve and risk of premature menopause among adult female 5-year survivors of childhood cancer. Female 5-year CCS, diagnosed with cancer and treated with chemotherapy before the age of 25 years, and aged 18 years or older at time of study were enrolled in the current study. Results from the discovery Dutch DCOG-LATER VEVO cohort (n = 285) were validated in the pan-European PanCareLIFE (n =465) and the USA-based St. Jude Lifetime Cohort (n = 391). PARTICIPANTS/MATERIALS, SETTING, METHODS: To evaluate ovarian function, anti-Miillerian hormone (AMH) levels were assessed in both the discovery cohort and the replication cohorts. Using additive genetic models in linear and logistic regression, five genetic variants involved in DNA damage response were analysed in relation to cyclophosphamide equivalent dose (CED) score and their impact on ovarian function. Results were then examined using fixed-effect meta-analysis. MAIN RESULTS AND THE ROLE OF CHANCE: Meta-analysis across the three independent cohorts showed a significant interaction effect (P= 3.0 x 10(-4)) between rs11668344 of BRSK 1 (allele frequency = 0.34) among CCS treated with high-dose alkylating agents (CED score >= 8000 mg/m(2)), resulting in a 2.5-fold increased odds of a reduced ovarian function (lowest AMH tertile) for CCS carrying one G allele compared to CCS without this allele (odds ratio genotype AA: 2.01 vs AG: 5.00). LIMITATIONS, REASONS FOR CAUTION: While low AMH levels can also identify poor responders in assisted reproductive technology, it needs to be emphasized that AMH remains a surrogate marker of ovarian function. WIDER IMPLICATIONS OF THE FINDINGS: Further research, validating our findings and identifying additional risk contributing genetic variants, may enable individualized counselling regarding treatment-related risks and necessity of fertility preservation procedures in girls with cancer

Genetic fine mapping and genomic annotation defines causal mechanisms at type 2 diabetes susceptibility loci.

We performed fine mapping of 39 established type 2 diabetes (T2D) loci in 27,206 cases and 57,574 controls of European ancestry. We identified 49 distinct association signals at these loci, including five mapping in or near KCNQ1. 'Credible sets' of the variants most likely to drive each distinct signal mapped predominantly to noncoding sequence, implying that association with T2D is mediated through gene regulation. Credible set variants were enriched for overlap with FOXA2 chromatin immunoprecipitation binding sites in human islet and liver cells, including at MTNR1B, where fine mapping implicated rs10830963 as driving T2D association. We confirmed that the T2D risk allele for this SNP increases FOXA2-bound enhancer activity in islet- and liver-derived cells. We observed allele-specific differences in NEUROD1 binding in islet-derived cells, consistent with evidence that the T2D risk allele increases islet MTNR1B expression. Our study demonstrates how integration of genetic and genomic information can define molecular mechanisms through which variants underlying association signals exert their effects on disease

An Expanded Genome-Wide Association Study of Type 2 Diabetes in Europeans.

To characterize type 2 diabetes (T2D)-associated variation across the allele frequency spectrum, we conducted a meta-analysis of genome-wide association data from 26,676 T2D case and 132,532 control subjects of European ancestry after imputation using the 1000 Genomes multiethnic reference panel. Promising association signals were followed up in additional data sets (of 14,545 or 7,397 T2D case and 38,994 or 71,604 control subjects). We identified 13 novel T2D-associated loci (P < 5 × 10(-8)), including variants near the GLP2R, GIP, and HLA-DQA1 genes. Our analysis brought the total number of independent T2D associations to 128 distinct signals at 113 loci. Despite substantially increased sample size and more complete coverage of low-frequency variation, all novel associations were driven by common single nucleotide variants. Credible sets of potentially causal variants were generally larger than those based on imputation with earlier reference panels, consistent with resolution of causal signals to common risk haplotypes. Stratification of T2D-associated loci based on T2D-related quantitative trait associations revealed tissue-specific enrichment of regulatory annotations in pancreatic islet enhancers for loci influencing insulin secretion and in adipocytes, monocytes, and hepatocytes for insulin action-associated loci. These findings highlight the predominant role played by common variants of modest effect and the diversity of biological mechanisms influencing T2D pathophysiology.Please refer to the manuscript or visit the publisher's website for funding infomation

Development of poly(trimethylene carbonate) based implant devices and their application in oral and maxillofacial surgery

In dit proefschrift wordt de ontwikkeling en toepassing van membranen en composiet implantaten op basis van het polymeer poly(trimethyleen carbonaat) (PTMC) voor gebruik in de Mond-, Kaak- en Aangezichtschirurgie beschreven. In de inleiding van het proefschrift (hoofdstuk 1) wordt kort de evolutie van biomaterialen door de eeuwen heen tot vandaag de dag beschreven. De keuze van materialen is geëvolueerd van metalen en natuurlijke materialen naar polymere kunststoffen en resorbeerbare polymeren (kunststoffen). Allemaal met één doel: de functie van en verloren gegane delen van het lichaam te herstellen. Tegenwoordig is de belangstelling verschoven naar het gebruik van bioactieve (bio)materialen welke in staat zijn om de ontbrekende weefsels te regenereren. Bij voorkeur zijn deze materialen resorbeerbaar tijdens en na afronding van hun functie. Een probleem met resorbeerbare materialen kan echter zijn, dat hun afbraakproducten zelf de omliggende weefsels beschadigen. Dit is ongewenst en moet worden vermeden. Zo is bij het gebruik van biologisch afbreekbare polymeren voor (bot)weefselregeneratie doeleinden het van groot belang dat de afbraakproducten zelf geen negatief effect op het bot en geregenereerd botweefsel hebben. Daarom lijken polymeren die afbreken zonder de vorming van schadelijke afbraakproducten het meest geschikt. Momenteel zijn verschillende biologisch afbreekbaar polymere materialen beschikbaar voor gebruik in bot- en weefselregeneratie toepassingen, echter één materiaal in het bijzonder lijkt erg interessant en heeft meer aandacht gekregen de laatste decennia: poly (trimethyleencarbonaat) (PTMC). Het doel van dit promotieonderzoek is om implantaten op basis van PTMC te ontwikkelen en te testen voor gebruik in de Mond-, Kaak- en Aangezichtschirurgie. Dit proefschrift bestaat uit 2 delen. Het eerste deel beschrijft de ontwikkeling van resorbeerbare barrière membranen voor gebruik in guided bone regeneration (GBR) (geleide bot regeneratie) procedures voorafgaande aan het plaatsen van tandheelkundige implantaten. Het tweede deel beschrijft de ontwikkeling van resorbeerbare osteoinductieve (botinducerende) composiet implantaten voor gebruik in de reconstructie van orbitabodem fracturen. In het eerste deel van dit proefschrift, te weten hoofdstuk 2 en 3 wordt de geschiktheid van PTMC membranen in de functie van barrière membraan in GBR geëvalueerd. Hoofdstuk 2 beschrijft het effect van de nieuw ontwikkelde membranen op de botregeneratie in een studie bij ratten. In hoofdstuk 3 wordt het gedrag van het omliggende weefsel op de afbraak- en reactieproducten van de membranen geëvalueerd. Het tweede deel van dit proefschrift beschrijft de ontwikkeling en toepassing van osteoinductieve composiet materialen op basis van PTMC voor gebruik in de reconstructie van orbitabodemfracturen

Artificial bone implants, or bone grafts, of polymeric composites with bone forming properties

The present invention relates to methods for providing polymeric composites with bone forming, such as with osteogenic and/or osteoinductive and/or osteoconductive, properties. The present invention further relates to polymeric composites provided by the present method and the use of thereof for bone implants, or grafts, specifically the use thereof for orbital floor reconstruction. Specifically, the present invention relates to methods for providing a composite with bone forming properties, the method comprises the steps of: a) providing a liquid, or liquefied, polymeric composition of homopolymers or copolymers of 1,3-trimethylene carbonate (TMC); b) adding to said liquid, or liquefied, polymeric composition one or more agents with osteogenic and/or osteoinductive and/or osteoconductive properties thereby providing a dispersion of said agents in said liquid or liquefied polymeric composition; and c) crosslinking the product obtained, thereby providing a composite with bone forming properties