Chitosan nanoparticles as antigen vehicles to induce effective tumor specific T cell responses.

Cancer vaccinations sensitize the immune system to recognize tumor-specific antigens de novo or boosting preexisting immune responses. Dendritic cells (DCs) are regarded as the most potent antigen presenting cells (APCs) for induction of (cancer) antigen-specific CD8+ T cell responses. Chitosan nanoparticles (CNPs) used as delivery vehicle have been shown to improve anti-tumor responses. This study aimed at exploring the potential of CNPs as antigen delivery system by assessing activation and expansion of antigen-specific CD8+ T cells by DCs and subsequent T cell-mediated lysis of pancreatic ductal adenocarcinoma (PDAC) cells. As model antigen the ovalbumin-derived peptide SIINFEKL was chosen. Using imaging cytometry, intracellular uptake of FITC-labelled CNPs of three different sizes and qualities (90/10, 90/20 and 90/50) was demonstrated in DCs and in pro- and anti-inflammatory macrophages to different extents. While larger particles (90/50) impaired survival of all APCs, small CNPs (90/10) were not toxic for DCs. Internalization of SIINFEKL-loaded but not empty 90/10-CNPs promoted a pro-inflammatory phenotype of DCs indicated by elevated expression of pro-inflammatory cytokines. Treatment of murine DC2.4 cells with SIINFEKL-loaded 90/10-CNPs led to a marked MHC-related presentation of SIINFEKL and enabled DC2.4 cells to potently activate SIINFEKL-specific CD8+ OT-1 T cells finally leading to effective lysis of the PDAC cell line Panc-OVA. Overall, our study supports the suitability of CNPs as antigen vehicle to induce potent anti-tumor immune responses by activation and expansion of tumor antigen-specific CD8+ T cells.

A genome-wide association study for clinical mastitis in the dual-purpose German Black Pied cattle breed.

The dual-purpose German Black Pied Cattle (DSN) has become an endangered breed of approximately 2,550 registered cows in Germany. The breed is genetically related to Holstein-Friesian cattle because the old DSN breed contributed to the selection of the modern Holstein dairy cow. In dairy farms, breeders aim to improve animal health and well-being by reducing the number of mastitis cases, which would also reduce milk losses and treatment costs. On the genomic level, no markers associated with clinical mastitis have been reported in DSN. Therefore, we performed a genome-wide association study on 1,062 DSN cows using a univariate linear mixed model that included a relatedness matrix to correct for population stratification. Although the statistical power was limited by the small population size, 3 markers were significantly associated, and 2 additional markers showed a suggestive association with clinical mastitis. Those markers accounted for 1 to 3% of the variance of clinical mastitis in the examined DSN population. One marker was found in the intragenic region of NEURL1 on BTA26, and the other 4 markers in intergenic regions on BTA3, BTA6, and BTA9. Further analyses identified 23 positional candidate genes. Among them is BMPR1B, which has been previously associated with clinical mastitis in other dairy cattle breeds. The markers presented here can be used for selection for mastitis-resistant animals in the endangered DSN population, and can broadly contribute to a better understanding of mastitis determinants in dairy cattle breeds.

DNA Sequence Variants and Protein Haplotypes of Casein Genes in German Black Pied Cattle (DSN).

Casein proteins were repeatedly examined for protein polymorphisms and frequencies in diverse cattle breeds. The occurrence of casein variants in Holstein Friesian, the leading dairy breed worldwide, is well known. The frequencies of different casein variants in Holstein are likely affected by selection for high milk yield. Compared to Holstein, only little is known about casein variants and their frequencies in German Black Pied cattle ("Deutsches Schwarzbuntes Niederungsrind," DSN). The DSN population was a main genetic contributor to the current high-yielding Holstein population. The goal of this study was to investigate casein (protein) variants and casein haplotypes in DSN based on the DNA sequence level and to compare these with data from Holstein and other breeds. In the investigated DSN population, we found no variation in the alpha-casein genes CSN1S1 and CSN1S2 and detected only the CSN1S1*B and CSN1S2*A protein variants. For CSN2 and CSN3 genes, non-synonymous single nucleotide polymorphisms leading to three different ß and κ protein variants were found, respectively. For ß-casein protein variants A 1 , A 2 , and I were detected, with CSN2*A 1 (82.7%) showing the highest frequency. For κ-casein protein variants A, B, and E were detected in DSN, with the highest frequency of CSN3*A (83.3%). Accordingly, the casein protein haplotype CSN1S1*B-CSN2*A 1 -CSN1S2*A-CSN3*A (order of genes on BTA6) is the most frequent haplotype in DSN cattle.