Effects of tepoxalin, a dual inhibitor of cyclooxygenase/5-lipoxygenase, on events associated with NSAID-induced gastrointestinal inflammation

Prostaglandins and thromboxanes are products of arachidonic acid metabolism via the cyclooxygenase (CO) enzyme and are responsible for the pain and swelling common to sites of inflammation. Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit the production of these substances and are used in the treatment of inflammatory diseases such as arthritis. However, one of the major side-effects of NSAID therapy is gastric ulceration. It is possible that inhibition of prostaglandin production and a related increase in the formation of leukotrienes via the 5-lipoxygenase (5-LO) enzymatic pathway are responsible for attracting inflammatory cells, causing local sites of inflammation and producing ulceration. To determine the effects of 5-LO inhibition on this hypothesis, studies were performed in rats to evaluate the effects of tepoxalin, a dual CO/LO inhibitor on leukotriene B₄ levels in gastric mucosa and neutrophil adhesion in mesenteric venules. In rats, chronic oral administration of an NSAID, indomethacin (2 mg/kg daily over 4 days), resulted in 40% mortality, accompanied by intestinal adhesions and perforations when evaluated 24 h after the fourth dose of drug. Additionally, neutrophil adhesion was increased in the mesenteric venules and cell infiltration was evident in the mesenteric interstitium. These gastrointestinal side-effects were inhibited in a separate group of rats administered tepoxalin (20 mg/kg, p.o) 30 min prior to each daily indomethacin treatment. Further studies were performed to determine tepoxalin's effects on early events associated with NSAID-induced gastrointestinal inflammation, including neutrophil adhesion, lipid peroxide generation and LTB₄ production. Indomethacin (100 mg/kg, p.o.) produced elevated levels of LTB₄ in rat gastric mucosa 90 min after administration. Additionally, neutrophil adhesion in mesenteric venules was increased at this dose and with the administration of another NSAID, naproxen. No generation of lipid peroxides was evident in the gastric mucosa at this timepoint. Tepoxalin (up to 400 mg/kg, p.o.) did not have an effects on gastric mucosal LTB₄ generation and lipid peroxide levels. A decrease in neutrophil adhesion was observed at the highest dose. In another study, pretreatment with tepoxalin (ED₅₀=7.5 mg/kg, p.o.) or the selective 5-LO inhibitor zileuton (100 mg/kg, p.o.) prevented the increases in gastric mucosal LTB₄ levels and neutrophil adhesion induced by indomethacin (100 mg/kg, p.o.). These data suggest that LO inhibition may play a vital role in the prevention of NSAID-induced gastric inflammation, providing insight into the lack of ulcerogenicity with tepoxalin and new approaches to anti-inflammatory therapy which may prevent gastric side effects.     

Mechanisms involved in down-regulation of intestinal IgA in rats by high cocoa intake

Previous studies have shown that rat intestinal immunoglobulin A (IgA) concentration and lymphocyte composition of the intestinal immune system were influenced by a highly enriched cocoa diet. The aim of this study was to dissect the mechanisms by which a long-term high cocoa intake was capable of modifying gut secretory IgA in Wistar rats. After 7 weeks of nutritional intervention, Peyer's patches, mesenteric lymph nodes and the small intestine were excised for gene expression assessment of IgA, transforming growth factor β, C-C chemokine receptor-9 (CCR9), interleukin (IL)-6, CD40, retinoic acid receptors (RARα and RARβ), C-C chemokine ligand (CCL)-25 and CCL28 chemokines, polymeric immunoglobulin receptor and toll-like receptors (TLR) expression by real-time polymerase chain reaction. As in previous studies, secretory IgA concentration decreased in intestinal wash and fecal samples after cocoa intake. Results from the gene expression showed that cocoa intake reduced IgA and IL?6 in Peyer's patches and mesenteric lymph nodes, whereas in small intestine, cocoa decreased IgA, CCR9, CCL28, RARα and RARβ. Moreover, cocoa-fed animals presented an altered TLR expression pattern in the three compartments studied. In conclusion, a high-cocoa diet down-regulated cytokines such as IL-6, which is required for the activation of B cells to become IgA-secreting cells, chemokines and chemokine receptors, such as CCL28 and CCR9 together with RARα and RARβ, which are involved in the gut homing of IgA-secreting cells. Moreover, cocoa modified the cross-talk between microbiota and intestinal cells as was detected by an altered TLR pattern. These overall effects in the intestine may explain the intestinal IgA down-regulatory effect after the consumption of a long-term cocoa-enriched diet.     

Inability of human clinical strains of Helicobacter pylori to colonize the alimentary tract of germfree rodents

Several attempts were made to colonize the alimentary tract and infect germfree BALB/c mice and germfree Sprague-Dawley rats with two human isolates of Helicobacter pylori. The alimentary tracts of mice, sacrificed at intervals between 1 day and 20 weeks after oral challenge, were culture negative for H. pylori. The alimentary tract, kidney, liver, and mesenteric lymph nodes were culture negative for H. pylori 5 h after intravenous challenge. Growth of H. pylori was inhibited by homogenates of murine stomach, small intestine, liver, and mesenteric lymph nodes. Germfree rats and mice do not appear to be readily colonized or infected by human strains of H. pylori.     

Alterations in the intestinal glycocalyx and bacterial flora in response to oral indomethacin

Nonsteroidal anti-inflammatory drugs (NSAIDs), used extensively in clinical medicine, tend to cause adverse effects in the gastrointestinal tract. Earlier work has shown that oral administration of indomethacin produced oxidative damage in the small intestine and attenuation of the glycocalyx layer of the mucosa. The present study assessed, in greater detail, the alterations produced in the glycocalyx of rat small intestinal mucosa in response to indomethacin, with specific reference to surfactant-like particles (SLP) and brush border membranes (BBM). Changes in gut flora in response to the drug were also studied, as it has been shown that luminal bacteria play a role in the pathogenesis of NSAID-induced intestinal damage. The levels of sugars such as sialic acid, fucose, hexose and hexosamine were increased in SLP and decreased in the BBM following indomethacin treatment, with the effects being maximal 24h after the administration of the drug. The composition of lipids in the SLP was also found to be altered. There was a significant increase in the number of bacteria in the luminal contents of the small intestine and caecum in these animals, as compared with controls. The number of bacteria adherent to the intestinal mucosa was also significantly higher in the drug-treated group. In vitro studies revealed that there was an increased tendency for bacteria to adhere to SLP isolated from indomethacin-treated rats. These results suggest that alterations in glycosylation of SLP and BBM in response to indomethacin, along with qualitative and quantitative changes in the luminal bacterial flora, may facilitate translocation of bacteria into the mucosa. These changes may contribute to the enteropathy observed as a result of NSAID treatment.     

Increased susceptibility of small intestine to NSAID-provoked ulceration in rats with adjuvant-induced arthritis: involvement of enhanced expression of TLR4

NSAIDs damage the small intestine as well as the stomach as adverse effects. We previously reported that the gastric ulcerogenic response to NSAIDs was markedly increased in arthritic rats. The present study was designed to examine the intestinal ulcerogenic property of indomethacin in adjuvant-induced arthritic rats in comparison with normal animals. Arthritis was induced in male Dark Agouti rats by injection of Freund's complete adjuvant into the right hindfoot. Two weeks later, indomethacin was given orally and the intestine was examined for lesions at several time points after indomethacin. Indomethacin produced intestinal lesions in both normal and arthritic rats, but in the latter, the ulcerogenic response occurred much earlier and the severity was markedly enhanced. Aminoguanidine, an inhibitor of iNOS, significantly suppressed the damage, yet the efficacy differed in normal and arthritic rats, depending on the dose schedule; the effect of post-administration (6 h after) was greater than that of pre-administration (0.5 h before) in normal rats, whereas that of post-administration was less than that of pre-administration in arthritic rats. The expression of iNOS and TLR4 in the intestine was enhanced in arthritic rats as compared with normal rats. These results suggest that the intestinal ulcerogenic response to indomethacin is markedly aggravated in arthritic rats. Notably, the onset of the ulceration was much earlier in arthritic rats than normal rats. These phenomena may be accounted for by the upregulation of iNOS/NO through the increased expression of TLR4 in the small intestine of arthritic rats.     

Comparative studies on starvation - and indomethacin - induced ulcerations in albino rats.

Experimental models of chronic and acute peptic ulcerations were produced in the albino rats by means of prolonged starvation and indomethacin administration. In the case of acute indomethacin-induced peptic ulceration, the effects of anticholinergic drugs on the ulcers produced were also studied. Starving the rats for a period of seven days produced gastric ulceration in all the rats used while indomethacin produced gastric ulceration within five hours in all the rats used. Severe ulceration of the degree found in human peptic ulcer disease was produced only by chronic starvation. Anticholinergic drugs ameliorated indomethacin-induced gastric ulceration, partly at least, by reducing intra-gastric acidity.     

Establishment of tolerance to commensal bacteria requires a complex microbiota and is accompanied by decreased intestinal chemokine expression

Intricate regulation of tolerance to the intestinal commensal microbiota acquired at birth is critical. We hypothesized that epithelial cell tolerance toward early gram-positive and gram-negative colonizing bacteria is established immediately after birth, as has previously been shown for endotoxin. Gene expression in the intestine of mouse pups born to dams that were either colonized with a conventional microbiota or monocolonized (Lactobacillus acidophilus or Eschericia coli) or germ free was examined on day 1 and day 6 after birth. Intestinal epithelial cells from all groups of pups were stimulated ex vivo with L. acidophilus and E. coli to assess tolerance establishment. Intestine from pups exposed to a conventional microbiota displayed lower expression of Ccl2, Ccl3, Cxcl1, Cxcl2, and Tslp than germ-free mice, whereas genes encoding proteins in Toll-like receptor signaling pathways and cytokines were upregulated. When comparing pups on day 1 and day 6 after birth, a specific change in gene expression pattern was evident in all groups of mice. Tolerance to ex vivo stimulation with E. coli was only established in conventional animals. Colonization of the intestine was reflected in the spleen displaying downregulation of Cxcl2 compared with germ-free animals on day 1 after birth. Colonization reduced the expression of genes involved in antigen presentation in the intestine-draining mesenteric lymph nodes, but not in the popliteal lymph nodes, as evidenced by gene expression on day 23 after birth. We propose that microbial detection systems in the intestine are upregulated by colonization with a diverse microbiota, whereas expression of proinflammatory chemokines is reduced to avoid excess recruitment of immune cells to the maturing intestine.     

Reutilization by maternal and embryonal cells of nuclear materials from H3-thymidine labeled lymphocytes introduced into the lumen of intestine of rats

Summary H³-thymidine labeled lymphocytes from thymus and lymph nodes of donor rats were washed and injected in to the intestine of recipient rats on the 11th and 19th day of gestation; subsequent labeling of maternal and embryonal cells was studied autoradiographically 24 hours after injection. In 12-day embryos, numerous stem cells or hemocytoblasts were labeled frequently intensely. In 20-day embryos, stem cells or hemocytoblasts scattered throughout the liver were often labeled. In other fetal tissues at this stage, cells in thymus, spleen, mesenteric lymph node and intestine were labeled but scarcely and weakly. In mothers, labeling in lymphoid tissues was scarce but definite, in thymus, mesenteric lymph node and spleen. These results suggest that nuclear materials from lymphocytes emigrated into the intestinal canal of the mother could be reutilized by maternal and embryonal cells.     

The autologous intestinal microflora in the postresuscitation period after acute blood loss in rats

In this work the data obtained in the study of intestinal microbiocenosis in rats on the third day of the postresuscitation period after acute blood loss are presented. The quantitative and qualitative shifts of microflora at different biotopes, such as the wall of the small intestine, the parietal microflora of Peyer's patches, the contents and wall of the large intestine, have been characterized. Some specific features of dysbiotic changes have been revealed in comparison with the shifts of intestinal microflora in cases of dysbacteriosis caused by other reasons. The translocation of different microorganisms, including Bacterium bifidum and lactobacilli, into mesenteric lymph nodes, the liver, the spleen and the blood has been observed.     

Atropine-induced gastrointestinal cytoprotection dependences to the intact of vagal nerve against indomethacin-induced gastrointestinal mucosal and microvascular damage in rats

The non-steroidal antiinflammatory drugs, such as an indomethacin (IND), cause mucosal ulceration and increase the mucosal vascular permeability in the gastrointestinal (GI) tract. Some exogenous agents, e.g. the atropine, can protect the GI mucosa against these ulcerogenic effects. The gastrointestinal functions and mucosal protection, however, are regulated by the vagal nerve. The aims of this study was to examine the dependence of atropine-induced GI cytoprotection to the vagal innervation against the development of IND-caused ulcers and microvascular damage in the mucosa of stomach and small intestine in rats. METHODS: the observations were carried out on CFY-strain rats. The mucosal damage was produced by subcutaneous administration of IND in a 20 mg/kg dose 24 h prior to the killing of animals at the same time as the start of atropine-application, which was given in a small dose (0.1 mg/kg) every 5 h. The subdiaphragmatic bilateral surgical vagotomy was done 24 h before the experiment. The vascular permeability, indicated by the microvascular endothel damage, was measured by the appearance and concentration of intravenously administered Evans blue into the GI mucosa. The number and severity of mucosal lesions and the Evans blue content of mucosa were determined in the stomach and small intestine. RESULTS: (1) The IND caused mucosal ulcers and Evans blue extravasation into the mucosa of the stomach and small intestine. (2) The IND-induced mucosal ulceration and vascular permeability significantly decreased after atropine-administration in the same parts of GI tract. (3) The extent of cytoprotective effect of atropine against the IND was decreased after bilateral surgical vagotomy. CONCLUSIONS: (1) The IND causes microvascular endothel damage in the stomach and small intestinal. (2) The atropine has a cytoprotective effect in the stomach and small intestine against the aggressive effects of IND without decrease of gastric acid secretion. (3) The intact vagal nerve is necessary to the function of cytoprotective mechanisms of atropine against the IND.     

Post-Mortem Examination in the Diagnosis of Johne’s Disease in Goats

Post-mortem examinations play an important role in Johne’s disease programmes in Norway. The results of such examinations of samples of viscera from 2997 goats carried out during the 5-year period 1972–1976 are given. The investigations show that the demonstration of macroscopical changes in mesenteric lymph nodes and small intestine has only limited value as a guideline in the post-mortem diagnosis of Johne’s disease in goats. Often macroscopical changes were not seen or they were non-specific. Caseous and/or calcified foci in mesenteric lymph nodes in infected animals were demonstrated quite often whilst observed intestinal changes were strikingly few. Corrugation of the mucosa was rare. However, in sections of macroscopically unchanged intestine marked epithelioid cell infiltrations and abundant acid-fast bacilli were not uncommon. In sporadic cases productive inflammation with tubercle formation was seen in lymph nodes in infected animals. Bacteriological culture was by far the most reliable post-mortem diagnostic method. By this method 92% of the infected goats were detected. The corresponding figures for histological examination and microscopy were 54% and 47%, respectively.     

Mast cells are critical for protection against peptic ulcers induced by the NSAID piroxicam

Many commonly used non-steroidal anti-inflammatory drugs (NSAIDs) also cause gastrointestinal toxicity, including the development of life-threatening peptic ulcers. We report that mast cell-deficient mice have an extremely high incidence of severe peptic ulceration when exposed to the NSAID piroxicam. This enhanced ulcer susceptibility can be reversed by reconstitution with mast cells. Furthermore, wild type mice treated with diphenhydramine hydrochloride, a commonly used antihistamine that blocks histamine H1 receptors, develop a similarly high incidence of peptic ulcers following piroxicam exposure. The protective effect of mast cells is independent of TNF, blockade of H2 receptors, or acid secretion. These data indicate a critical role for mast cells and the histamine that they produce in prevention and/or repair of piroxicam-induced gastric mucosal injury. Additional studies will be required to determine whether this represents a NSAID class effect that can be exploited to develop novel therapeutic strategies to limit the incidence of NSAID-induced side effects in humans.     

COMPARISON OF DNA RENEWAL IN GERM-FREE AND CONVENTIONAL MICE USING [125I]IODODEOXYURIDINE AND [3H]THYMIDINE

Germ-free (GF) and conventional (CV) C3H mice received a single injection of 1 μCi [³H]thymidine and 3 μCi [¹²⁵I]iododeoxyuridine to provide simultaneous labeling of DNA with the two precursors. Thymus, spleen, mesenteric lymph nodes, bone marrow (femora), small intestine, colon and skin were examined for total organ activity and rate of DNA renewal 1–8 days after injection. Precursor incorporation, assayed on day 1, was lower in the thymus, mesenteric lymph nodes and femora (and, to a lesser extent, in the spleen and colon) of GF mice as compared to CV animals. The opposite was observed in the small intestine and skin, i.e. total organ activity was higher in GF animals. Differences in precursor incorporation were partly due to differences in organ weights between the two groups of mice. In comparison to CV animals, DNA renewal rates were diminished in the mesenteric lymph nodes, bone marrow, colon (following a 3-day plateau) and spleen of GF mice. Little, if any, difference was observed between the two groups with respect to the rate of DNA turnover in the thymus and skin. Radioactivity of the small intestine remained constant for 2 days. Thereafter intestinal activity in GF mice declined at an initial slow rate between days 2 and 5 followed by a rapid decrease between days 5 and 8. In CV mice the first phase of activity loss was short with the rapid decline in intestinal activity beginning on day 3. From the slopes of the regression lines, the percentage thymidine reutilization was estimated. Reutilization varied from 0 to 63% in the various organs examined, with the greatest difference between GF and CV mice occurring in the mesenteric lymph nodes.     

Similarity and difference of the response of Peyer's patches and mesenteric lymph nodes of rats to polychemotherapy]

The structure of the Peyer's patches and mesenteric lymph nodes of rats at different periods after polychemotherapy was investigated by light microscopy. After the use of antitumor drugs the number of the blasts and mytoses in the lymphoid follicles with the mesenteric lymph node bright centres and in the Peyer's patch follicles lowered, that along with the decrease of the size of the mantle zone in the lymph node follicles and the decrease of the area of the bright centres in the follicles of the lympoid formations in the intestinal wall was evident of the proliferation inhibition and B cells differentiation in the lymphoid organs. After the polychemotherapy the size of the germinative centres of the Payer's patch follicles decreased while the size of the mantle zone remained unchanged. The size of the mantle zone in the follicles of the mesenteric lymph nodes decreased while the size of the germinative centres did not change. The different responses of the lymphoid organs could be associated with some remote location of the lymph nodes with respect to the antigen source (damaged epithelium and intestinal lumen contents).     

Structure of the mesenteric lymph nodes in fetuses and offspring of white rats under the effect of indomethacin on the mother-fetus system

Mesenteric lymph nodes in fetuses and offspring of white rats have been studied during various age periods after indomethacin injection in doses 2.5 mg/kg and 1 mg/kg daily from the 18th up to 21st day of pregnancy. Dose-dependent retardation in formation of main structures of the mesenteric lymph nodes, decreasing amount of the lymphoid type cells have been revealed. Retardation in the capsule, sinuses and reticular fibers of the node takes place, as well as decrease in lympho- and plasmocytopoiesis. It is more pronounced after indomethacin in dose 2.5 mg/kg. Simultaneously, formation of the cerebral substance and appearance of lymphoid nodules and their germinative centers are delayed. After the drug effect (2.5 mg/kg), up to the age of 2 weeks the amount of tissue basophils and eosinophilic granulocytes decreases. When the dose of the drug is 1 mg/kg decreasing amount of these cells is substituted for their compensatory increase on the 2d-3d week. The data obtained demonstrate a decreasing function of the lymph nodes, that depends on the dose of indomethacin.     

Intestinal inflammation in TNBS sensitized rats as a model of chronic inflammatory bowel disease

An enteritis, based on a delayed-type hypersensitivity reaction, was induced in TNBS (2,4,4-trinitrobenzenesulphonic acid) sensitized rats by multiple intrajejunal challenge with TNBS via an implanted catheter. This treatment induced chronic inflammation of the distal small intestine characterized by intense hyperaemia, oedema and gut wall thickening as assessed by macroscopic scoring and weighing a defined part of the dissected intestine. Histologically, the inflammatory response included mucosal and submucosal cell infiltration by lymphocytes and histiocytes, transmural granulomatous inflammation with multinucleated cells and activated mesenteric lymph nodes. Ex vivo stimulated release of the inflammatory mediator LTB(4) in the dissected part of the intestine was increased following TNBS treatment. Drug treatment with sulphasalazine or 5-aminosalicylic acid improved the enteritis score and attenuated TNBS induced oedema formation and LTB(4) production. The applicability and relevance of this new model are discussed with respect to drug development and basic research of inflammatory bowel diseases.     

The origin of IgA-containing cells in intestinal lymph of sheep

The origin of IgA-containing cells in sheep intestinal lymph was determined by antigenically stimulating a mesenteric lymph node and by studying afferent intestinal lymph. Since antigenic stimulation of the node resulted almost exclusively in the appearance of IgG1 antibody-containing cells, it was proposed that IgA-containing cells are normally produced either in the Peyer's patches or lamina propria of the intestine. These conclusions were supported by studying lymph obtained by cannulation of a lymphatic duct afferent to the mesenteric lymph nodes. Study of the cells in afferent lymph revealed the presence of a significant population of IgA-containing blast cells. This was convincing evidence that the IgA-containing cells normally found in intestinal lymph originate from sites in the intestine.     

Systemic bacterial invasion induced by sleep deprivation

Profound sleep disruption in humans is generally believed to cause health impairments. Through comparative research, specific physical effects and underlying mechanisms altered by sleep deprivation are being elucidated. Studies of sleep-deprived animals previously have shown a progressive, chronic negative energy balance and gradual deterioration of health, which culminate in fatal bloodstream infection without an infectious focus. The present study investigated the conditions antecedent to advanced morbidity in sleep-deprived rats by determining the time course and distribution of live microorganisms in body tissues that are normally sterile. The tissues cultured for microbial growth included the blood, four major organs, six regional lymph nodes, the intestine, and the skin. The principal finding was early infection of the mesenteric lymph nodes by bacteria presumably translocated from the intestine and bacterial migration to and transient infection of extraintestinal sites. Presence of pathogenic microorganisms and their toxins in tissues constitutes a septic burden and chronic antigenic challenge for the host. Bacterial translocation and pathogenic sequelae provide mechanisms by which sleep deprivation appears to adversely affect health.     

An outbreak of Yersinia pseudotuberculosis infection in a small indoor breeding colony of red-bellied (Saguinus labiatus) tamarins

A spontaneous outbreak of yersiniosis caused by Yersinia pseudotuberculosis serotype IIB occurred in a small indoor breeding colony of red-bellied tamarins (Saguinus labiatus) during the winter of 1981. Of 35 monkeys at risk 6 died of an acute or subacute infection over a period of 23 days. Clinical signs were anorexia, weakness, listlessness and depression. The disease was characterized by focal necrosis of the liver, spleen, mesenteric lymph nodes, ulcerative enteritis, and the presence of colonies of Gram-negative bacilli in the lesions. Y. pseudotuberculosis was isolated from the liver, spleen, mesenteric lymph nodes and kidney but not from the blood, lung or intestine. Contaminated food was believed to be the source of infection.