Human TJP1 blocking peptide

Seven multiparous Holstein cows with a ruminal fistula were used to investigate the changes in rumen microbiota, gene expression of the ruminal epithelium, and blood biomarkers of metabolism and inflammation during the transition period. Samples of ruminal digesta, biopsies of ruminal epithelium, and blood were obtained during -14 through 28 d in milk (DIM). A total of 35 genes associated with metabolism, transport, inflammation, and signaling were evaluated by quantitative reverse transcription-PCR. Among metabolic-related genes, expression of RMGCS2 increased gradually from -14 to a peak at 28 DIM, underscoring its central role in epithelial ketogenesis. The decrease of glucose and the increase of nonesterified fatty acids and beta-hydroxybutyrate in the blood after calving confirmed the state of negative energy balance. Similarly, increases in bilirubin and decreases in albumin concentrations after calving were indicative of alterations in liver function and inflammation. Despite those systemic signs, lower postpartal expression of TLR2, TLR4, CD45, and NFKB1 indicated the absence of inflammation within the epithelium. Alternatively, these could reflect an adaptation to react against inducers of the immune system arising in the rumen (e.g., bacterial endotoxins). The downregulation of RXRA, INSR, and RPS6KB1 between -14 and 10 DIM indicated a possible increase in insulin resistance. However, the upregulation of IRS1 during the same time frame could serve to restore sensitivity to insulin of the epithelium as a way to preserve its proliferative capacity. The upregulation of TGFB1 from -14 and 10 DIM coupled with upregulation of both EGFR and EREG from 10 to 28 DIM indicated the existence of 2 waves of epithelial proliferation. However, the downregulation of TGFBR1 from 14 through 28 DIM indicated some degree of cell proliferation arrest. The downregulation of OCLN and TJP1 from -14 to 10 DIM indicated a loss of tight-junction integrity. The gradual upregulation of membrane transporters MCT1 and UTB to peak levels at 28 DIM reflected the higher intake and fermentability of the lactation diet. In addition, those changes in the diet after calving resulted in an increase of butyrate and a decrease of ruminal pH and acetate, which partly explain the increase of Anaerovibrio lipolytica, Prevotella bryantii, and Megasphaera elsdenii and the decrease of fibrolytic bacteria (Fibrobacter succinogenes, Butyrivibrio proteoclasticus). Overall, these multitier changes revealed important features associated with the transition into lactation. Alterations in ruminal epithelium gene expression could be driven by nutrient intake induced changes in microbes; microbial metabolism; and the systemic metabolic, hormonal, and immune changes. Understanding causes and mechanisms driving the interaction among ruminal bacteria and host immunometabolic responses merits further study.

Background The complexity of the neurovascular unit (NVU) poses a challenge in the investigations of drug transport across the blood-brain barrier (BBB) and the function of the brain capillary endothelium. Several in vitro models of the brain capillary endothelium have been developed. In vitro culture of primary endothelial cells has, however, been reported to alter the expression levels of various brain endothelial proteins. Only a limited number of studies have addressed this in detail. The aim of the present study was to investigate mRNA levels of selected BBB transporters and markers in in vitro models of the BBB based on bovine primary endothelial cells and compare these to the levels estimated in freshly isolated bovine brain capillaries. Methods Brain capillaries were isolated from bovine cerebral cortex grey matter. Capillaries were seeded in culture flasks and endothelial cells were obtained using a brief trypsinization. They were seeded onto permeable supports and cultured in mono-, non-contact- or contact co-culture with/without primary rat astrocytes. mRNA-expression levels of the selected BBB markers and transporters were evaluated using qPCR and monolayer integrity of resulting monolayers was evaluated by measuring the transendothelial electrical resistance (TEER). Results The capillary mRNA transcript profile indicated low expression of ABCC1 and CLDN1. The mRNA expression levels of TPA, OCLN, ABCB1, SLC2A1, SLC16A1 and SLC7A5 were significantly decreased in all culture configurations compared to freshly isolated bovine brain capillaries. ALP, VWF, ABCC1 and ABCC4 were upregulated during culture, while the mRNA expression levels of F11R, TJP1, CLDN5, CLDN1 and ABCG2 were found to be unaltered. The mRNA expression levels of VWF, ALP, ABCB1 and ABCC1 were affected by the presence of rat astrocytes. Conclusion The endothelial mRNA transcript profile in bovine capillaries obtained in this study correlated nicely with profiles reported in mice and humans. Cultured endothelial cells drastically downregulated the mRNA expression of the investigated SLC transporters but maintained expression of efflux transporter and junctional protein mRNA, implying that the bovine in vitro BBB models may serve well to investigate basic barrier biology and in vivo permeation of passively permeating compounds and efflux transporter substrates but may be less well suited for investigations of SLC-mediated transport.