Improved filtration method to isolate pure populations of primary bovine endometrial epithelial and stromal cells for immunological studies.

OBJECTIVES: Isolation and culture of distinct primary endometrial cells are key to reliable in-vitro models to investigate the uterine immune response and optimse new disease interventions. Details on the isolation method and purity of distinct cell populations is lacking in currently available protocols leading to inconsistent results across laboratories. METHODS: Bovine endometrial tissue from non-pregnant bovine uteri were collected immediately post-mortem and separated using differential size filtering. Isolations (n = 15) yielded an average of 3.1 × 105 ± 0.7 × 105 epithelial cells and 1.88 × 106 ± 5.44 × 105 stromal fibroblasts per uterine horn. Following expansion in culture, the purity of cell populations was confirmed using morphology and positive staining for cytokeratin and vimentin which identifies epithelial and stromal fibroblast populations, respectively. Using PCR, cDNA from both cell populations was negative for CD45, a marker of immune cells. RESULTS: On challenge with a bacterial PAMP (LPS), epithelial and stromal fibroblasts showed a marked increase in the expression of the inflammatory mediators IL8, IL6, S100A8 and S100A9, with both cell populations displaying distinct expression profiles. Here we provide a detailed methodology on the culture of primary bovine endometrial epithelial and stromal cells and demonstrate these cells provide a physiologically relevant model for studies of endometrial inflammation and its regulation.

Characterisation and expression profile of the bovine cathelicidin gene repertoire in mammary tissue.

BACKGROUND: Cathelicidins comprise a major group of host-defence peptides. Conserved across a wide range of species, they have several functions related to host defence. Only one cathelicidin has been found in humans but several cathelicidin genes occur in the bovine genome. We propose that these molecules may have a protective role against mastitis. The aim of this study was to characterise the cathelicidin gene-cluster in the bovine genome and to identify sites of expression in the bovine mammary gland. RESULTS: Bioinformatic analysis of the bovine genome (BosTau7) revealed seven protein-coding cathelicidin genes, CATHL1-7, including two identical copies of CATHL4, as well as three additional putative cathelicidin genes, all clustered on the long arm of chromosome 22. Six of the seven protein-coding genes were expressed in leukocytes extracted from milk of high somatic cell count (SCC) cows. CATHL5 was expressed across several sites in the mammary gland, but did not increase in response to Staphylococcus aureus infection. CONCLUSIONS: Here, we characterise the bovine cathelicidin gene cluster and reconcile inconsistencies in the datasets of previous studies. Constitutive cathelicidin expression in the mammary gland suggests a possible role for these host defence peptides its protection.