Gene expression profiling in pbMEC - in search of molecular biomarkers to predict immunoglobulin production in bovine milk.

BACKGROUND: Optimization of the immunoglobulin (Ig) yield in bovine milk used as therapeutic immune milk or whey for the prevention of Clostridium difficile-associated diarrhea in humans is of great importance to improve the economic efficiency of production. Individual dairy cows have diverse immune responses upon vaccination, resulting in a variable Ig yield in blood and milk. Therefore, it is advisable to pre-select cows with the best ability to produce and secrete high yields of specific Igs. RESULTS: The gene expression profile of pbMEC (primary bovine mammary epithelial cells), challenged with the gram-positive, non-mastitis, pathogen Clostridium difficile showed distinct and significant differences in the gene expression of effector molecules of the innate immune system. A number of genes were identified that could possibly serve as molecular biomarkers to differentiate high responder cows from low responder cows. These identified genes play key roles in the promotion of innate immunity. CONCLUSION: Using a gene expression profiling approach, we showed that upon others, especially the gene expression of the pro-inflammatory cytokines was altered between the high and low responder cows. Those genes are indicated as potential molecular biomarkers in the pre-selection of cows that are able to secrete high immunoglobulin yields in milk.

Effect of zearalenone on reproductive parameters and expression of selected testicular genes in mice.

We tested the effect of two different concentrations (150µg/l and 0.15µg/l) of mycotoxin zearalenone (ZEA) on the reproductive parameters and expression of testicular genes in male mice. In adult males, no reduction of body or reproductive organ weight was observed, and the seminiferous tubules were morphologically normal with ongoing spermatogenesis. However, we found decreased sperm concentration, increase of morphologically abnormal spermatozoa and increased binding of apoptotic marker annexin V. This study was also focused on the evaluation of gene expression profiles of 28 genes playing important roles during the processes occurring in the testicular tissue. We detected changes in the expression of genes important for proper spermatogenesis. Surprisingly, we observed a stronger effect after exposure to the lower dose of ZEA.

Association of obesity susceptibility gene variants with metabolic syndrome and related traits in 1,443 Czech adolescents.

Genome-wide association studies have revealed several gene variants associated with obesity; however, only a few studies have further investigated their association with metabolic syndrome. We performed a study of eleven variants in/near genes TMEM18, SH2B1, KCTD15, PCSK1, BDNF, SEC16B, MC4R, and FTO in Czech adolescents and analysed their association with obesity, metabolic syndrome and related traits. Genotyping was performed in 1,443 adolescents aged 13.0-17.9 years. Anthropometric parameters, biochemical parameters and blood pressure were assessed. Metabolic syndrome was defined according to the International Diabetes Federation. The FTO rs9939609 variant was associated with overweight/obesity (OR 1.40, 95% CI 1.21-1.63, P < 0.001). The minor allele of TMEM18 rs7561317 was related to underweight (OR 1.78, 95% CI 1.14-2.79, P = 0.015). BDNF rs925946 and MC4R rs17782313 were associated with metabolic syndrome (OR 1.53, 95% CI 1.14-2.04, P = 0.005; 1.51, 95% CI 1.12-2.04, P = 0.009). The PCSK1 rs6235 variant was negatively related to increased blood glucose (OR 0.69, 95% CI 0.49-0.97, P = 0.040). In conclusion, the FTO variant was associated with overweight/obesity in Czech adolescents. Moreover, MC4R and BDNF variants increased the risk of metabolic syndrome, probably through their effect on abdominal obesity. The PCSK1 variant may have a protective role in the development of type 2 diabetes.

Microfluidic high-throughput RT-qPCR measurements of the immune response of primary bovine mammary epithelial cells cultured from milk to mastitis pathogens.

Bovine mastitis, the inflammation of the udder, is a major problem for the dairy industry and for the welfare of the animals. To better understand this disease, and to implement two special techniques for studying mammary gland immunity in vitro, we measured the innate immune response of primary bovine mammary epithelial cells (pbMEC) from six Brown Swiss cows after stimulation with the heat-inactivated mastitis pathogens, Escherichia coli 1303 and Staphylococcus aureus 1027. The cells were extracted and cultivated from milk instead of udder tissue, which is usually done. The advantages of this technique are non-invasiveness and less contamination by fibroblasts. For the first time, pbMEC gene expression (GE) was measured with a microfluidic high-throughput real-time reverse transcription-quantitative PCR platform, the BioMark HD™ system from Fluidigm. In addition to the physiological analysis, the precision and suitability of this method was evaluated in a large data set. The mean coefficient of variance (± s.e.) between repeated chips was 4.3 ± 0.4% for highly expressed and 3.3 ± 0.4% for lowly expressed genes. Quantitative PCR (qPCR) replicate deviations were smaller than the cell culture replicate deviations, indicating that biological and cell culture differences could be distinguished from the background noise. Twenty-two genes (complement system, chemokines, inflammatory cytokines, antimicrobial peptides, acute phase response and toll-like receptor signalling) were differentially expressed (P < 0.05) with E. coli. The most upregulated gene was the acute phase protein serum amyloid A3 with 618-time fold. S. aureus slightly induced CCL5, IL10, TLR4 and S100A12 expression and failed to elicit a distinct overall innate immune response. We showed that, with this milk-derived pbMEC culture and the high-throughput qPCR technique, it is possible to obtain similar results in pbMEC expression as with conventional PCR and with satisfactory precision so that it can be applied in future GE studies in pbMEC.