Molecular and metabolic effects of local immune activation in the ruminal epithelium

Modern dairy and beef cattle efficiently produce large quantities of high value products, milk and meat respectively, for human consumption. In order to meet the energy demands of production, cattle are fed diets consisting of rapidly fermentable carbohydrates. As a result, ruminal acidosis may occur. The acidotic conditions in the ruminal fluid can result in compromised barrier function and, potentially, translocation of microbes and microbe-associated molecular patterns, (MAMP) from the lumen across the ruminal epithelium. Translocation of microbes or MAMP may lead to an interaction with the ruminal epithelial cells (REC), thus inducing a local, pro-inflammatory response. However, little is known about the capability of REC to initiate such a response as well as the effects of inflammation on the physiological functions of the ruminal epithelium. The objective of this research was to assess the inflammatory response of the ruminal epithelium and to investigate the potential effects of inflammation on nutrient uptake and metabolism using in vivo, ex vivo and primary cell culture models. In Chapter 3, ruminal papillae biopsies were collected from beef heifers following induction of subacute ruminal acidosis (SARA). The papillae were used to evaluate differential gene expression and toll-like receptor (TLR) 4 quantification. Despite an increase in ruminal fluid concentration of LPS, gene expression of inflammatory molecules and immunohistofluorescent analysis of TLR4 protein expression indicated an anti-inflammatory response 2 d following the SARA challenge. The Ussing chamber model was used in Chapter 4 to explore the effects of LPS exposure on the inflammatory response and the potential effects on butyrate flux and metabolism. Analysis of gene expression suggested that the pro-inflammatory response to LPS may have been suppressed or prevented by the epithelial barrier. In tissue exposed to LPS, butyrate flux tended to increase linearly (P = 0.063); however, production of β-hydroxybutyrate (BHB) was not affected (P = 0.21), suggesting that the impact of LPS exposure on metabolism of the ruminal epithelium was minimal. To further evaluate REC responses to LPS exposure, a cell culture model was established (Chapter 5). Using that model in Chapter 6, I evaluated the effects of dose, duration, and timing of LPS exposure on viability and gene expression of pattern recognition receptors (PRR), pro-inflammatory cytokines, chemokines, and other immunomodulatory molecules in cultured primary REC. There was no indication that LPS negatively impacted cell viability, but exposure to LPS increased TLR2 and TLR4 expression and induced a pro-inflammatory response. Results suggested that the REC response was influenced by LPS dose and duration of exposure, and that gene expression may have been regulated to prevent an excessive pro-inflammatory response and potential damage to the cells. In Chapter 7, cultured REC were exposed to LPS when grown with or without the addition of short-chain fatty acids (SCFA) to the cell culture media in order to evaluate the effects of the inflammatory response on metabolic function. Results showed that LPS exposure tended to increase glucose utilization compared to control REC (31.8 versus 28.7 ± 2.7%; P = 0.072). Analysis of gene and protein expression further indicated that nutrient transport and metabolism in the cells may have been moderately altered by the inflammatory response. Overall, the results of this research indicate that exposure of the REC to LPS activates an inflammatory response, the nature of which is dependent on the dose, duration and timing of exposure. However, the response may differ between whole tissue and cultured REC. The data suggested that although metabolism of the REC may have been altered, the changes observed were moderate, indicating that key functions of the ruminal epithelium are resilient to local inflammatory responses

Mortality and pulmonary complications in patients undergoing surgery with perioperative SARS-CoV-2 infection: an international cohort study

Background: The impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on postoperative recovery needs to be understood to inform clinical decision making during and after the COVID-19 pandemic. This study reports 30-day mortality and pulmonary complication rates in patients with perioperative SARS-CoV-2 infection. Methods: This international, multicentre, cohort study at 235 hospitals in 24 countries included all patients undergoing surgery who had SARS-CoV-2 infection confirmed within 7 days before or 30 days after surgery. The primary outcome measure was 30-day postoperative mortality and was assessed in all enrolled patients. The main secondary outcome measure was pulmonary complications, defined as pneumonia, acute respiratory distress syndrome, or unexpected postoperative ventilation. Findings: This analysis includes 1128 patients who had surgery between Jan 1 and March 31, 2020, of whom 835 (74·0%) had emergency surgery and 280 (24·8%) had elective surgery. SARS-CoV-2 infection was confirmed preoperatively in 294 (26·1%) patients. 30-day mortality was 23·8% (268 of 1128). Pulmonary complications occurred in 577 (51·2%) of 1128 patients; 30-day mortality in these patients was 38·0% (219 of 577), accounting for 81·7% (219 of 268) of all deaths. In adjusted analyses, 30-day mortality was associated with male sex (odds ratio 1·75 [95% CI 1·28–2·40], p\textless0·0001), age 70 years or older versus younger than 70 years (2·30 [1·65–3·22], p\textless0·0001), American Society of Anesthesiologists grades 3–5 versus grades 1–2 (2·35 [1·57–3·53], p\textless0·0001), malignant versus benign or obstetric diagnosis (1·55 [1·01–2·39], p=0·046), emergency versus elective surgery (1·67 [1·06–2·63], p=0·026), and major versus minor surgery (1·52 [1·01–2·31], p=0·047). Interpretation: Postoperative pulmonary complications occur in half of patients with perioperative SARS-CoV-2 infection and are associated with high mortality. Thresholds for surgery during the COVID-19 pandemic should be higher than during normal practice, particularly in men aged 70 years and older. Consideration should be given for postponing non-urgent procedures and promoting non-operative treatment to delay or avoid the need for surgery. Funding: National Institute for Health Research (NIHR), Association of Coloproctology of Great Britain and Ireland, Bowel and Cancer Research, Bowel Disease Research Foundation, Association of Upper Gastrointestinal Surgeons, British Association of Surgical Oncology, British Gynaecological Cancer Society, European Society of Coloproctology, NIHR Academy, Sarcoma UK, Vascular Society for Great Britain and Ireland, and Yorkshire Cancer Research

Claudin-4 Undergoes Age-Dependent Change in Cellular Localization on Pig Jejunal Villous Epithelial Cells, Independent of Bacterial Colonization

Newborn piglets are immunologically naïve and must receive passive immunity via colostrum within 24 hours to survive. Mechanisms by which the newborn piglet gut facilitates uptake of colostral cells, antibodies, and proteins may include FcRn and pIgR receptor-mediated endocytosis and paracellular transport between tight junctions (TJs). In the present study, FcRn gene (FCGRT) was minimally expressed in 6-week-old gut and newborn jejunum but it was expressed at significantly higher levels in the ileum of newborn piglets. pIgR was highly expressed in the jejunum and ileum of 6-week-old animals but only minimally in neonatal gut. Immunohistochemical analysis showed that Claudin-5 localized to blood vessel endothelial cells. Claudin-4 was strongly localized to the apical aspect of jejunal epithelial cells for the first 2 days of life after which it was redistributed to the lateral surface between adjacent enterocytes. Claudin-4 was localized to ileal lateral surfaces within 24 hours after birth indicating regional and temporal differences. Tissue from gnotobiotic piglets showed that commensal microbiota did not influence Claudin-4 surface localization on jejunal or ileal enterocytes. Regulation of TJs by Claudin-4 surface localization requires further investigation. Understanding the factors that regulate gut barrier maturation may yield protective strategies against infectious diseases