Cholesterol 25-hydroxylase is a metabolic switch to constrain T cell-mediated inflammation in the skin.

Interleukin-27 (IL-27) is an immunoregulatory cytokine whose essential function is to limit immune responses. We found that the gene encoding cholesterol 25-hydroxylase (Ch25h) was induced in CD4+ T cells by IL-27, enhanced by transforming growth factor­ß (TGF-ß), and antagonized by T-bet. Ch25h catalyzes cholesterol to generate 25-hydroxycholesterol (25OHC), which was subsequently released to the cellular milieu, functioning as a modulator of T cell response. Extracellular 25OHC suppressed cholesterol biosynthesis in T cells, inhibited cell growth, and induced nutrient deprivation cell death without releasing high-mobility group box 1 (HMGB1). This growth inhibitory effect was specific to actively proliferating cells with high cholesterol demand and was reversed when extracellular cholesterol was replenished. Ch25h-expressing CD4+ T cells that received IL-27 and TGF-ß signals became refractory to 25OHC-mediated growth inhibition in vitro. Nonetheless, IL-27­treated T cells negatively affected viability of bystander cells in a paracrine manner, but only if the bystander cells were in the early phases of activation. In mouse models of skin inflammation due to autoreactive T cells or chemically induced hypersensitivity, genetic deletion of Ch25h or Il27ra led to worse outcomes. Thus, Ch25h is an immunoregulatory metabolic switch induced by IL-27 and dampens excess bystander T effector expansion in tissues through its metabolite derivative, 25OHC. This study reveals regulation of cholesterol metabolism as a modality for controlling tissue inflammation and thus represents a mechanism underlying T cell immunoregulatory functions.

A selective, high affinity 5-HT 2B receptor antagonist inhibits visceral hypersensitivity in rats.

BACKGROUND: RS-127445 is a selective, high affinity 5-HT(2B)receptor antagonist. We investigated whether 5-HT(2B)receptor antagonists can reduce colonic visceral hypersensitivity caused by restraint stress or by proximal colonic inflammation. METHODS: Visceral hypersensitivity was induced in rats by either restraint stress or injection of 2, 4, 6-trinitrobenzene sulfonic acid (TNBS) into the proximal colon. Restraint stress produced a significant increase in numbers of abdominal contractions evoked by colorectal distension (CRD), measured as a quantitative index of visceral nociception in rats. Seven days after TNBS injection, the pain threshold to CRD at the non-inflamed distal colon, that was determined as the minimum pressure required to evoke abdominal cramp, was significantly decreased. The effect of RS-127445 on visceral hypersensitivity was assessed in either naïve or TNBS-treated rats. KEY RESULTS: Oral administration of a selective, high affinity 5-HT(2B)receptor antagonist, RS-127445, significantly inhibited visceral hypersensitivity provoked by restraint stress (35 to 74% inhibition at 1 to 10 mg kg(-1)). Oral RS-127445 produced a significant suppression of TNBS-induced visceral hypersensitivity (15 to 62% inhibition at 3 to 30 mg kg(-1)), although it was without significant effect on the visceral nociceptive threshold of naïve rats. RS-127445 (1 to 30 mg kg(-1), p.o.) also dose-dependently reduced the restraint stress-induced defecation in naïve and TNBS-treated rats. CONCLUSIONS & INFERENCES: These results suggest that 5-HT(2B)receptors are involved in signaling from the colon in rats in which there is visceral hypersensitivity and that a selective 5-HT(2B)receptor antagonist could have therapeutic potential for the treatment of gut disorders characterized by visceral hypersensitivity.

Synthesis and pharmacological activities of novel bicyclic thiazoline derivatives as hepatoprotective agents II. (7-Alkoxycarbonyl-2,3,5,6-tetrahydropyrrolo[2,1-b]thiazol-3-ylidend) acetamid derivatives.

A series of exomethylenic bicyclic thiazoline derivatives (3a--i) was synthesized and evaluated for hepatoprotective activity against galactosamine-induced and monoclonal antibody-induced acute liver injuries in rats. The structure-activity relationships were investigated. Among the compounds synthesized, N-methyl-(7-isopropoxy-carbonyl-6,6-dimethyl-2,3,5,6- tetrahydropyrrolo[2,1-b]thiazol-3-ylidene)acetamide (3i) exhibited the most potent hepatoprotective activity. This compound suppressed galactosamine-induced hepatic injury at 100 mg/kg by oral administration and further prevented monoclonal antibody-induced hepatic injury at 30 mg/kg by intraperitoneal injection, as judged from the changes in serum transaminase activities.

Synthesis and pharmacological activities of novel bicyclic thiazoline derivatives as hepatoprotective agents. I. 8-Ethoxycarbonyl-5,6-dihydrothiazolo[2,3-c][1,4]thiazine derivatives.

A series of bicyclic thiazoline derivatives (4a-s) was synthesized and evaluated for hepatoprotective activity against galactosamine-induced and monoclonal antibody-induced acute liver injuries in rats. The structure-activity relationships were investigated. Among the compounds synthesized, ethyl 3-(N-methylcarbamoyl)-5,6-dihydrothiazolo[2,3-c][1,4]thiazin e-8- carboxylate (4p) exhibited remarkable hepatoprotective activity and lower toxicity. This compound suppressed galactosamine-induced hepatic injury at 100 mg/kg by gavage and further prevented monoclonal antibody-induced hepatic injury at 30 mg/kg by intraperitoneal injection, as evaluated by measuring changes in serum transaminase activities.

Species- and organ-specificity and expression of 105 kD liver cell membrane glycoprotein antigen during rat development.

Species- and organ-specificity of a rat liver cell antigen recognized by a monoclonal antibody (MoAb) from a hybridoma clone RM-1 was investigated immunohistologically and immunoelectronmicroscopically. In rats, the MoAb reacted specifically with liver cell membrane, but not with other organs tested, including the brain, thymus, heart, lung, stomach, spleen, kidney, small and large intestines, testis and muscle. Furthermore, it was found that liver from the human, monkey, mouse, rabbit, guinea pig, dog, cat, cattle and chicken showed no specific reaction with this MoAb. The results indicated that the antigen was organ- and species-specific and designated as rat liver cell-specific membranous antigen (RLSA). RLSA was expressed scarcely along cell junctions of immature hepatocytes at the 19th day of pregnancy. This increased gradually, and was expressed along all surface membranes after birth.

Antisecretory and antiulcer effects of ebselen, a seleno-organic compound, in rats.

The effects of ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-on), a metal-containing organic compound, on gastric secretion and gastric ulceration were examined in rats. Intraduodenal ebselen (30 to 300 mg/kg) significantly and dose-dependently inhibited gastric secretion in pylorus-ligated rats. Both aspirin- and water-immersion restraint stress-induced ulcers were significantly prevented by oral administration of ebselen at doses equivalent to the antisecretory doses. These results indicate that the antisecretory effect of ebselen underlies its antiulcer effect in these models.

Effect of DS-4574, a novel peptidoleukotriene antagonist with mast cell stabilizing action, on acute gastric lesions and gastric secretion in rats.

DS-4574 is a peptidoleukotriene antagonist with mast cell stabilizing activity. In the present study, we studied the effects of this compound on gastric secretion and various acute gastric lesions in rats. Intraduodenal administration of DS-4574 at doses of 5 to 10 mg/kg significantly and dose-dependently inhibited gastric acid secretion in pylorus-ligated rats, but a further increase in the dose up to 50 mg/kg did not cause any further inhibition. Shay ulceration in response to pylorus ligation was dose-dependently prevented by DS-4574 (10-25 mg/kg, i.d.). Water-immersion restraint stress- and aspirin-induced gastric ulcers were also significantly prevented in a dose-related manner by oral pretreatment with DS-4574 (10-50 mg/kg). The lower doses of DS-4574 (1-10 mg/kg, p.o.) significantly and dose-dependently protected the gastric mucosa against the necrotizing action of either absolute ethanol or concentrated hydrochloric acid, indicating that this compound possesses a potent gastroprotective activity. These antiulcer and gastric protective effects of DS-4574 were more potent than those of cimetidine used as a reference drug. These findings suggest that DS-4574 is useful for peptic ulcer therapy, as well as for the therapy of various allergic diseases, including asthma.

Protective effect of 16,16-dimethyl prostaglandin E2 on isolated rat hepatocytes against complement-mediated immune attack.

16,16-Dimethyl prostaglandin E2 was examined for its ability to inhibit complement-mediated in vitro hepatocytolysis by an antigen-antibody reaction. In the presence of fresh rat serum as a source of complement, 5-min culture of isolated rat hepatocytes with a monoclonal antibody against a rat liver-specific membranous antigen resulted in a marked, significant elevation in lactate dehydrogenase leakage into the culture medium. However, with heat-inactivated rat serum, such a reaction did not occur, indicating that the hepatocytolysis induced by the antibody was attributable to the membrane damaging action of complement activated by an antigen-antibody reaction. Pretreatment of the hepatocyte with 16,16-dimethyl prostaglandin E2 significantly suppressed the cytolytic reaction induced by the antibody in a concentration-dependent manner. These results show that 16,16-dimethyl prostaglandin E2 is capable of protecting isolated rat hepatocytes against the membrane-damaging insult of activated complement.

Protection by 16,16-dimethyl prostaglandin E2 and dibutyryl cyclic AMP against complement-mediated hepatic necrosis in rats.

16,16-Dimethyl prostaglandin E2, a known cytoprotective agent, was examined for its ability to protect the liver against complement-mediated necrosis induced by an intravenous injection of a monoclonal antibody against a rat liver-specific antigen in rats. The hepatic injury induced by the antibody was characterized by (a) rapid development of numerous massive hemorrhagic foci of necrotic liver cells, (b) marked increases in serum liver enzyme activities and (c) pronounced reduction in the CH50 level, presumably as a result of complement consumption in the liver. Pretreatment with 16,16-dimethyl prostaglandin E2 at intraperitoneal doses of 20 and 100 micrograms/kg suppressed the hepatic injury, as evidenced by markedly mitigated liver-cell necrosis and much smaller increases in the serum-enzyme activities compared with the values in diseased control animals. The prostaglandin analogue failed to prevent serum complement consumption in response to the antibody injection or affect the CH50 level at the preinjury stage, indicating that neither complement inactivation nor interference with the antigen-antibody reaction was involved in the hepatic protection. The hepatoprotective doses of 16,16-dimethyl prostaglandin E2 produced a significant increase in liver cyclic AMP content in a dose-related manner. In addition, intravenous dibutyryl cyclic AMP at 3 and 10 mg/kg dose-dependently prevented histological and biochemical changes in the hepatic damage without altering the rate of reduction in serum complement activity. Like 16,16-dimethyl prostaglandin E2, dibutyryl cyclic AMP did not affect the preinjury CH50 level.(ABSTRACT TRUNCATED AT 250 WORDS)

A rat model of acute liver necrosis induced by a monoclonal antibody to liver-specific antigen and complement.

Acute massive hepatic injury was induced in rats by a monoclonal antibody against a rat liver-specific membrane antigen, and its histological characteristics were investigated. A single intravenous injection of murine ascites containing a monoclonal antibody produced numerous hemorrhagic foci of degenerated and necrotic liver cells predominantly in zones 1 (the periportal area) and 2 (the area of transition between the periportal zone and the perivenular zone) of the liver lobule within 10 min. Massive hepatocellular necroses were observed 1 hr later, but no inflammatory cell infiltration occurred in and around the necrotic foci. Immunohistological study demonstrated marked deposition of the third component of the complement system in the necrotic area. Serum complement activity was sharply decreased immediately after the injection of the antibody, suggesting that the hepatic necrosis is ascribable to a complement-mediated immune attack on the liver cell membrane induced by the antigen-antibody reaction. The hepatic necrosis in response to monoclonal-antibody injection did not progress to a chronic disease and healed almost completely, changing to scar tissues within 2 wk. Although it is not clear whether this hepatic injury has any clinical relevance, this antibody/complement model may be useful for investigating the cause and therapy of hepatic diseases such as fulminant hepatitis.

Colonization of Helicobacter pylori in the gastric mucosa of Mongolian gerbils.

Helicobacter pylori was orally inoculated into Mongolian gerbils. The organisms were able to colonize in the gastro-mucosal layer of the gerbils, especially in those gerbils which had mucosal lesions caused by indomethacin treatment. The pathological changes developed by H. pylori infection were restricted to the stomachs, and only slightly inflammatory cells were observed.

[Comparison of three methods for detecting Campylobacter pylori and measurement of human antibody titers against the whole organisms].

In order to detect Campylobacter pylori in the gastric mucosa, three different methods as 1) culture of the organisms, 2) immunostaining by monoclonal antibody against the cells, and 3) urease test were compared. In the disease group, positive % of each methods was 64, 75 and 74. However, positive % common to the three methods was only 48. The corresponding rate of culture and urease tests, and that of culture and staining methods was 81% and 60%, respectively. Therefore, it was concluded that urease test was a useful method, and that the three methods were necessary to confirm the existence of the organisms in the gastric mucosa. Total positive % of culture and staining methods in disease and control groups was 77 and 31, respectively, indicating that positive % of the disease group was much higher than that of the control group. Anti C. pylori titer was compared between culture-positive and culture-negative patients by ELISA. The titer of the former was significantly higher than that of the latter.

Antiulcer effects of 3-[[[2-(3,4-dimethoxyphenyl)ethyl] carbamoyl]methyl]-amino-N-methylbenzamide in experimental gastric and duodenal ulcers.

Effects of 3-[[[2-(3,4-dimethoxyphenyl)ethyl]carbamoyl] methyl]-amino-N-methylbenzamide (DQ-2511), a newly synthesized compound, were evaluated using various types of experimental gastric and duodenal ulcers in rats. Pretreatment with DQ-2511, over the dose range 30-300 mg/kg p.o., resulted in a dose-related inhibition of water-immersion stress-, serotonin-, acetylsalicylic acid (ASA)-, indometacin-, ethanol-, and 2-deoxy-D-glucose(2DG) plus indometacin-induced gastric ulcers as well as cysteamine-induced duodenal ulcers. The antiulcer potencies of DQ-2511 were equal to or greater than those of H2-receptor antagonist cimetidine in these ulcer models except for ASA- and 2DG plus indometacin-induced ulcers. The rate of healing of chronic gastric ulcers induced by acetic acid was significantly accelerated by DQ-2511 (100 and 300 mg/kg p.o.) but not by the same doses of cimetidine. DQ-2511, at doses of 30 mg/kg p.o. and above, produced a significant decrease in gastric acid and pepsin output in pylorus-ligated rats. In anesthetized rats with acute gastric fistulae, 30 mg/kg i.v. of DQ-2511 significantly inhibited gastric acid secretion stimulated by 2DG, whereas it did not affect gastric hyperacidity evoked by either carbachol, histamine or pentagastrin. At effective antiulcer doses, this compound produced a sustained increase in gastric mucosal blood flow in conscious, restrained rats. Based on these observations, DQ-2511 is characterized as a new antiulcer compound with beneficial effects on both gastric aggressive and defensive factors. Furthermore, these results indicate a possible superiority of DQ2511 over cimetidine in regard to its antiulcer potency and spectrum.

Chemiluminescence and phagocytic responses of rat polymorphonuclear neutrophils to leptospires.

The interaction of leptospires with polymorphonuclear neutrophils (PMN) was examined by the luminol-dependent chemiluminescence (CL) test. Whole blood CL changed in relation to the stage of leptospiral infection both in susceptible (SUS) and resistant (RES) rats. The intensity of CL grew with an increasing number of leptospires in the blood. CL responses were observed in isolated PMN upon exposure to living leptospires. In contrast, the same bacteria, having been inactivated by formalin, did not stimulate PMN. A variation was found in the CL response by different living strains of Leptospira. The CL intensity was arranged as follows: L. illini greater than L. biflexa greater than L. interrogans avirulent strains greater than L. interrogans virulent strains. The CL response was markedly enhanced by an opsonization of leptospires. Specific opsonization was shown to increase the rate of phagocytosis of leptospires with relation to the CL response.

Gastric cytoprotection by ebselen against the injury induced by necrotizing agents in rats.

The gastric cytoprotective effect of ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one), a novel seleno-organic antioxidative agent, against the insults of necrotizing agents was investigated in rats. Either 0.6 N HCl or acidified ethanol (60% ethanol in 150 mmol/l HCl), given orally in a volume of 1 ml, produced linear hemorrhagic necroses in the gastric mucosa within 1 h. Pretreatment with ebselen at oral doses from 10 to 100 mg/kg significantly inhibited such lesion formation induced by either necrotizing agent in a dose-related manner, and the inhibition at the highest dose (100 mg/kg p.o.) was practically complete. Light microscopic analysis also confirmed that ebselen effectively prevented the formation of deep mucosal necrosis in response to the necrotizing agents. In addition, the protection by ebselen of gastric necrosis induced by either damaging agent was not affected by pretreatment of animals with indomethacin (5 mg/kg s.c.), indicating that the protective effect of this agent was not mediated by the mild irritation on the gastric mucosa. These results demonstrate that ebselen is a potent cytoprotective agent effectively preventing the gastric mucosal injury induced by necrotizing agents.

Cytoprotective action of cetraxate against HCl.ethanol-induced gastric lesion in rats.

The protective effect of cetraxate, an antiulcer and antigastritis agent, on HCl.ethanol-induced gastric lesions was investigated in rats. Oral administration of 1 ml of HCl.ethanol (60% ethanol in 150 mM HCl) induced within 1 hr linear hemorrhagic necrosis in the gastric mucosa. Either oral or intraperitoneal treatment with cetraxate (30-300 mg/kg) significantly inhibited such macroscopic gastric lesions in a dose-related manner, and the inhibition at the oral highest dose (300 mg/kg) was practically complete. Histological analysis also confirmed that cetraxate effectively prevented deep mucosal necrosis, but showed that it was without protective effect on the surface epithelial disruption and submucosal edema in response to HCl.ethanol. The antilesion activity of cetraxate was of statistically significance for at least 3 hr after a single injection, and it was hardly affected by the removal of the gastric contents just prior to application of the necrotizing agent. However, subcutaneous treatment of rats with indomethacin (5 mg/kg) resulted in a partial but significant attenuation in the protection afforded by cetraxate, suggesting that dual mechanisms related and unrelated to endogenous prostaglandins may be involved in its protective activity. The results demonstrate that cetraxate is a potent cytoprotective agent effectively preventing the formation of gastric mucosal necrosis induced by HCl.ethanol.

Biological activities of leptospiral lipopolysaccharide.

Lipopolysaccharide extracted with phenol-water from Leptospira interrogans serovar copenhageni strain Shibaura (L-LPS) showed various biological activities. In lethality for mice, L-LPS was active (LD 50, 3.4 mg/mouse) but about 12 times less potent than Escherichia coli LPS (E-LPS) per weight basis. L-LPS had pyrogenicity for rabbits, and the fever curves showed no evidence of the classical biphasic fever produced by E-LPS. In the bone marrow of mice, L-LPS caused hemorrhages and necrosis but less severe than those caused by E-LPS. Histopathologically, fresh hemorrhages were found in the intestine, spleen, lung and the other organs at 24 h after inoculation of L-LPS. Necrosis was also found in these organs and was particularly severe in mice inoculated with more than 2 mgL-LPS. Liver necrosis was found at 7th day after inoculation of L-LPS but not after inoculation of E-LPS. L-LPS had adjuvant activity just like E-LPS. L-LPS enhanced non-specific resistance to Salmonella infection and activated mouse peritoneal macrophages to kill these organisms. L-LPS was positive in limulus test just like E-LPS. These results demonstrated similarities of L-LPS and E-LPS. Some toxic effects of L-LPS were less than those of E-LPS, but some effects of L-LPS were more than those of E-LPS. L-LPS was antigenically active and the specificity was serogroup-associated. L-LPS was composed of carbohydrate (54%), lipid (12%), protein (5%). Arabinose, xylose and rhamnose were major sugars as detected by gas chromatography. 2-keto-deoxyoctanate (KDO) was not detectable.

Phagocytosis as a defense mechanism against infection with leptospiras.

The role of macrophages in host defense was studied in vivo and in vitro. The intravenous administration of silica, an agent reported to selectively inactivate macrophages, increased the sensitivity to leptospiral infection and inhibited bacterial clearance. Active immunization with killed organisms or with leptospiral lipopolysaccharide (L-LPS), and passive immunization with a monoclonal antibody showed powerful protective effects against infection in mice. The effect of immunization decreased in silica-treated mice. These findings were supported by electron microscopic examination and observation of killing by macrophages in vitro.

Changes in duodenal mucosal blood flow and mucus glycoprotein content during cysteamine-induced duodenal ulceration in rats.

The effects of cysteamine on duodenal mucosal blood flow and duodenal glycoprotein were studied during the development of duodenal ulceration in conscious rats. Cysteamine at an ulcerogenic dose (300 mg/kg s.c.) produced a remarkable decrease of duodenal mucosal blood flow which preceded the appearance of duodenal ulcers. The reduced blood flow was followed by a significant decrease of tissue levels of glycoprotein which also occurred prior to the time when duodenal injury had reached a maximum. Cysteamine was without significant effect on the rate of incorporation of 3H-glucosamine into duodenal glycoprotein at concentrations up to 10(-3) M. These results suggest that the reduction in duodenal mucosal blood flow in response to cysteamine could possibly contribute to a decrease of duodenal glycoprotein and that both may be at least in part responsible for the incidence of duodeno-ulcerogenecity.