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Published Article / The initial section of this manuscript focus on the ultra-structure of a unique class of heat stable cell-bound lipopolysaccharides (endotoxin) produced by Gram-negative bacteria. Subsequently, this paper summarises literature on the human body's response when challenged with endotoxins present in food and further explores the influence of food manufacturing and storage practices on endotoxin production and release by bacteria commonly isolated from food. Finally, this paper presents a brief description on the methods applied by the food industry to quantify endotoxins.

No description available.

Pulmonary intravascular macrophages (PIMs) promote lung inflammation and are found in ruminants, horses, pigs, cats, and dolphins, but not in primates, rats and mice. Rabbits are used to study mechanisms of lung inflammation in humans, but disagreement exists whether rabbits have PIMs. This study examined rabbits for PIMs, and their influence on endotoxin-induced lung inflammation.<p> Rabbits were treated with gadolinium chloride (10 mg/kg intravenous: Group GC, n=6) to produce apoptosis in PIMs, or with saline (Group SAL, n=6). Rabbits were euthanized 48 hours later. Light microscopic examination of epoxy-embedded rabbit lung sections revealed mononuclear phagocytes in alveolar septa. Transmission electron microscopy confirmed PIMs with lysosomes and close attachment to capillary endothelium. Light microscopic immuno-cytochemistry using rabbit anti-macrophage antibody (RAM-11) showed staining of septal and alveolar macrophages. There was no difference in number of RAM-11 positive septal cells between SAL and GC rabbits (P=0.2).<p> Rabbits were administered intravenous E.coli 0127:B8 endotoxin (100 Ýg/kg) 48 hours after GC (GC-LPS; n=5) or SAL treatment (SAL-LPS; n=6), and euthanized 24 hours later. Rabbits in both LPS treated groups were hypocalcaemic and exhibited compensated metabolic acidosis compared to SAL rabbits. Four rabbits died in the SAL-LPS group within 24 hours of the endotoxin treatment and were replaced. None died in the GC-LPS group (Chi-square comparison for survival P=0.063). Greater numbers of septal heterophils were found in groups SAL-LPS and GC-LPS compared to SAL and GC. TNFÑ protein in serum, and IL-1Ò and IL-6 mRNA in lung tissues were increased in SAL-LPS compared to SAL and GC rabbits. Lung tissues from SAL-LPS rabbits but not in GC-LPS showed moderate inflammation, but lung wet/dry ratios were not different. Lung tissue TNFÑ, IL-1Ònand IL-6 mRNA, myeloperoxidase activity, and serum TNFÑ were reduced in GC-LPS animals compared to SAL-LPS. Immuno-electron microscopy revealed TNFÑ in PIMs in normal and LPS-treated rabbits. Lung and liver tissue TNFÑ, IL-8 and MCP-1 protein concentrations were not different between groups. GC did not appear to reduce liver inflammation. These data show that rabbits have low numbers of PIMs. GC treatment induced apoptosis in PIMs and reduced endotoxin-induced lung inflammation and mortality.

Pulmonary intravascular macrophages (PIMs) promote lung inflammation and are found in ruminants, horses, pigs, cats, and dolphins, but not in primates, rats and mice. Rabbits are used to study mechanisms of lung inflammation in humans, but disagreement exists whether rabbits have PIMs. This study examined rabbits for PIMs, and their influence on endotoxin-induced lung inflammation.<p> Rabbits were treated with gadolinium chloride (10 mg/kg intravenous: Group GC, n=6) to produce apoptosis in PIMs, or with saline (Group SAL, n=6). Rabbits were euthanized 48 hours later. Light microscopic examination of epoxy-embedded rabbit lung sections revealed mononuclear phagocytes in alveolar septa. Transmission electron microscopy confirmed PIMs with lysosomes and close attachment to capillary endothelium. Light microscopic immuno-cytochemistry using rabbit anti-macrophage antibody (RAM-11) showed staining of septal and alveolar macrophages. There was no difference in number of RAM-11 positive septal cells between SAL and GC rabbits (P=0.2).<p> Rabbits were administered intravenous E.coli 0127:B8 endotoxin (100 Ýg/kg) 48 hours after GC (GC-LPS; n=5) or SAL treatment (SAL-LPS; n=6), and euthanized 24 hours later. Rabbits in both LPS treated groups were hypocalcaemic and exhibited compensated metabolic acidosis compared to SAL rabbits. Four rabbits died in the SAL-LPS group within 24 hours of the endotoxin treatment and were replaced. None died in the GC-LPS group (Chi-square comparison for survival P=0.063). Greater numbers of septal heterophils were found in groups SAL-LPS and GC-LPS compared to SAL and GC. TNFÑ protein in serum, and IL-1Ò and IL-6 mRNA in lung tissues were increased in SAL-LPS compared to SAL and GC rabbits. Lung tissues from SAL-LPS rabbits but not in GC-LPS showed moderate inflammation, but lung wet/dry ratios were not different. Lung tissue TNFÑ, IL-1Ònand IL-6 mRNA, myeloperoxidase activity, and serum TNFÑ were reduced in GC-LPS animals compared to SAL-LPS. Immuno-electron microscopy revealed TNFÑ in PIMs in normal and LPS-treated rabbits. Lung and liver tissue TNFÑ, IL-8 and MCP-1 protein concentrations were not different between groups. GC did not appear to reduce liver inflammation. These data show that rabbits have low numbers of PIMs. GC treatment induced apoptosis in PIMs and reduced endotoxin-induced lung inflammation and mortality.

The gastrointestinal epithelium normally sealed by tight junctions, which act as a structural barrier and paracellular channels. Inflammation can increase the permeability of microvasculature that result in plasma leakage. Mammalian intestinal epithelium has many goblet cells which discharge mucus in the inflammatory response. The discharging mucus functioning is as a defensive barrier and lubricant. The mucus layer is the anatomical site at which the host first encounters gut bacteria, physical damage, and chemical stimulant. The heat shock response is one of the most primitive cellular defense mechanism. A variety of stressful situations including environmental (ultraviolet radiation or heavy metals), pathological (infections or malignancies), or physiological (growth factors) stimuli induce heat shock proteins. This study investigated the effect of heat shock on endotoxin-induced plasma leakage and goblet cell mucus discharge in the small intestine of rats of Sprague-Dawley (SD) and Long-Evans (LE) strains. India ink was used as the tracer to detect leaky microvessels. The mucus secretion of the goblet cells of intestinal villi was observed with scanning electron microscopy and calculated with digital morphomertric software SimplePCI. Our results showed that endotoxin-induced plasma leakage and goblet cell discharging in the two strains increased significantly as compared to rat groups receiving saline. Numerous openings on the epithelial surface of villi resulted from compound exocytosis of mucus granules in goblet cells. Either 30 min or 1h after LPS injection, heat shock pretreatment in LE rats LPS-induced plasma leakage in the duodenum and ileum was reduced by 58-80% (P<0.01). 1 h after LPS injection in LE rats pretreated with heat shock, the number of discharging goblet cells in the ileum was reduced (P<0.05). In SD rats, heat shock inhibited LPS-induced plasma leakage in the duodenum and ileum at 1h after LPS injection by 56-68% (P<0.01), and the number of discharging goblet cells was reduced in the duodenum and ileum (P<0.05). In conclusion, heat shock could protect rat intestine from endotoxin-induced inflammation and mucus secretion.

Universiẗat, Diss., 2007--Giessen.

Objectives - To evaluate the effects of equine sCD14 and monoclonal antibodies (mAbs) to equine CD14 on LPS-induced TNF° expression of equine peripheral blood mononuclear cells (PBMCs). To determine serum concentrations of soluble (sCD14) in a population of horses with gastrointestinal diseases or other illnesses likely to result in endotoxemia; and identify relationships with clinical data. Animals - Part 1; 10 healthy horses. Part 2; 55 clinical cases and 23 healthy control horses. Procedure - Part 1; PBMCs were incubated with Escherichia coli LPS, CD14 mAb, sCD14, CD14 mAb plus E coli LPS or sCD14 plus E coli LPS. Supernatants were collected at 6 hours and assayed for tumor necrosis factor ° (TNF°) activity. Part 2; Serum sCD14 was measured at admission and then at 24 and 48 hours after admission using a bead-based multiplex assay. Results - Part 1; Pre-incubation with CD14 mAb did not inhibit LPS-induced TNF° protein production in isolated equine monocytes. Use of sCD14 inhibited LPS-induced TNF° protein production in isolated monocytes in a concentration-dependent manner. Part 2; Serum concentration of sCD14 was positively related to duration of clinical signs (P = 0.007), respiratory rate (P=0.04) and band neutrophil count (P = 0.0002). There was no correlation between serum concentration of sCD14 and heart rate, temperature, hematocrit, lactate, white blood cell count, fibrinogen, creatinine, urea nitrogen, glucose and anion gap values. Serum sCD14 did not correlate with outcome at any time point for clinical cases. / Master of Science

Obese individuals present with an increased inflammatory tone as compared to healthy, normal-weight individuals, which is associated with insulin resistance. One factor hypothesized to contribute to increased inflammation in obese and diabetic states is elevated blood endotoxin levels, also known as metabolic endotoxemia. In healthy rodents (non-obese and insulin sensitive), there is evidence that blood endotoxin levels fluctuate over the course of the day with elevations in the post-prandial state that return to baseline levels in the post-absorptive state. High-fat feeding in these animals altered these fluctuations causing endotoxin levels to remain high throughout the day. The effects of alterations in endotoxin levels on glucose metabolism are not understood. The goal of this study was to determine the effects of short-term and long-term increases in endotoxin of a low magnitude on insulin signaling in a human primary cell line as well as the effects of short-term endotoxin treatments on glucose homeostasis in a C57/Bl6 mouse model. First, we tested the hypothesis in cell culture that short-term low-dose endotoxin treatments would enhance insulin-signaling and glycogen synthesis while long-term treatments would have inhibitory effects. Under our second hypothesis, we examined whether short-term low-dose treatments of endotoxin would contribute to improvements in glucose tolerance in a mouse model. In contrast to our first hypothesis, short-term endotoxin treatments did not improve insulin signaling or glycogen synthesis although long-term treatments did contribute to decreases in glycogen synthesis. Interestingly, short-term endotoxin treatments resulted in significant improvements in glucose clearance in the mouse model; this is believed to be partly attributed to LPS inhibiting gluconeogenesis. Future studies are necessary to understand the mechanisms responsible for altered glucose metabolism in response to low magnitude changes in LPS levels. / Ph. D.

Endotoxemia is responsible for severe illness in horses. Individuals can become unresponsive to the endotoxin molecule after an initial exposure; this phenomenon has been called developing a state of "endotoxin tolerance" (ET). ET has been induced in horses in vivo; however, cytokine expression associated with ET has not been investigated. The purpose of this study was to develop and validate a method for inducing ET in equine peripheral blood mononuclear cells (PBMCs) in vitro, and to describe the cytokine profile which is associated with the ET. Blood was collected from 6 healthy horses and PBMCs were isolated. ET was induced by culturing cells with three concentrations of endotoxin given to induce ET, and evaluated after a second dose of endotoxin given to challenge the cells. The relative mRNA expression of IL-10 and IL-12 was measured by use of quantitative PCR. ET was induced in all cells (n=6) exposed to the 2-step endotoxin challenge. In PBMCs treated with 1.0 ng/ml of endotoxin followed by challenge with 10 ng/ml of endotoxin, the relative mRNA expression of IL-10 in tolerized cells was not different from positive control cells. In contrast, the relative mRNA expression of IL-12 in tolerized cells was decreased by 15-fold after the second endotoxin challenge compared with positive control cells. This experiment demonstrated a reliable method for the ex vivo induction of ET in equine PBMCs. A marked suppression of IL-12 production is associated with ET. The production of IL-10 was not altered in ET in our model. / Master of Science