Yoichi Kurebayashi

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.

Pharmacological characteristics of the hind paw weight bearing difference induced by chronic constriction injury of the sciatic nerve in rats

AIMS We examined the possible involvement of spontaneous on-going pain in the rat chronic constriction injury (CCI) model of neuropathic pain. MAIN METHODS The development of weight bearing deficit, as an index of spontaneous on-going pain, was investigated in comparison to that of mechanical allodynia in CCI rats. We also examined the effects of morphine and a gabapentin analogue (1S, 3R)-3-methyl-gabapentin (3-M-gabapentin) on both the CCI-induced weight bearing deficit and mechanical allodynia. KEY FINDINGS Rats with CCI demonstrated a significant reduction in weight bearing of the injured limb with a peak at a week post-operation, which was followed by a gradual recovery for over 7 weeks. The time course of development and recovery of CCI-induced weight bearing deficit appeared to follow that of foot deformation of the affected hind limb. CCI also evoked mechanical allodynia that was fully developed on a week post-operation, but showed no recovery for at least 8 weeks. 3-M-gabapentin significantly inhibited CCI-induced mechanical allodynia, but not weight bearing deficit, at 100 mg/kg p.o. Likewise, morphine was without significant effect on CCI-induced weight bearing deficit at the dose (3 mg/kg, s.c.) that could almost completely inhibit mechanical allodynia, whereas it inhibited both mechanical allodynia and weight bearing deficit at 6 mg/kg, s.c. SIGNIFICANCE The present findings suggest that CCI-induced weight bearing deficit is not a consequence of mechanical allodynia, but is attributable to spontaneous on-going pain. The rat CCI model of neuropathic pain thus represents both spontaneous on-going pain and mechanical allodynia.

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

Background  RS‐127445 is a selective, high affinity 5‐HT2Breceptor antagonist. We investigated whether 5‐HT2Breceptor antagonists can reduce colonic visceral hypersensitivity caused by restraint stress or by proximal colonic inflammation.

Abolishment of TNBS-induced visceral hypersensitivity in mast cell deficient rats

Mucosal mast cells are implicated in visceral hypersensitivity associated with irritable bowel syndrome (IBS). In this study, we investigated the role of mast cells in the development of visceral hypersensitivity by using mast cell deficient (Ws/Ws) rats and their control (W+/W+). In W+/W+ rats, an injection of 2,4,6-trinitrobenzene sulfonic acid (TNBS) into the proximal colon produced a significant decrease in pain threshold of the distal colon. Severe mucosal necrosis and inflammatory cell infiltration with concomitant increase in tissue myeloperoxidase activity were observed in the proximal colon that was directly insulted by TNBS, whereas neither necrosis nor increased myeloperoxidase activity occurred in the distal colon, indicating that TNBS-induced hypersensitivity is not caused by the local tissue damage or inflammation in the region of the gut where distention stimuli were applied. On the other hand, TNBS failed to elicit visceral hypersensitivity in Ws/Ws rats. This finding indicates that mast cells are essential for development of TNBS-induced visceral hypersensitivity in rats. Since the severity of TNBS-induced proximal colon injury and MPO activity was not affected by mast cell deficiency, it is unlikely that abolishment of visceral hypersensitivity in mast cell deficient rats was a result of altered development of the primary injury in the proximal colon. There was no difference between sham-operated Ws/Ws and W+/W+ rats in colonic pain threshold to distention stimuli, indicating that mast cells play no modulatory roles in normal colonic nociception. The present results support the view that mucosal mast cells play key roles in the pathogenesis of IBS.

Effect of a New α2δ Ligand PD-217014 on Visceral Hypersensitivity Induced by 2,4,6-Trinitrobenzene Sulfonic Acid in Rats

PD-217014, a new GABA analog (1α,3α,5α-3-aminomethyl-bicyclo[3.2.0]heptane-3-acetic acid), inhibited [3H]-gabapentin binding to α2δ subunit of voltage-gated calcium channels in a concentration-dependent manner (Ki = 18 nmol/l). Oral treatment with PD-217014 significantly inhibited the visceral hypersensitivity induced by an intra-colonic injection of trinitrobenzene sulfonic acid in rats. The anti-hyperalgesic effect of PD-217014 increased in a dose-dependent manner, and reached a plateau level at 60 mg/kg p.o. The visceral analgesia produced by PD-217014 at 30 and 60 mg/kg p.o. correlated with blood concentrations within 4 h after dosing, and maximal efficacy was obtained 2 h after dosing when the maximal blood concentration was achieved at either dose. These results indicate that PD-217014 is a potent α2δ ligand and possesses visceral analgesic activity.

Pregabalin inhibits accelerated defecation and decreased colonic nociceptive threshold in sensitized rats

Pregabalin, a ligand of alpha(2)delta subunits of voltage-gated calcium channels, reduces visceral hypersensitivity associated with irritable bowel syndrome. However, effects of pregabalin on bowel function are not well described. We investigated the effects of pregabalin on bowel dysfunction and colonic nociceptive threshold in sensitized rats. Increased fecal pellet output was evoked by non-ulcerogenic stress. Decreased colonic nociceptive threshold was induced in separate rats by administration of 2,4,6-trinitrobenzene sulfonic acid (TNBS) into the lumen of the proximal colon. Fecal pellet output was significantly increased during 2h restraint stress. Oral pregabalin (10-100mg/kg, p.o.) inhibited this increased fecal output dose-dependently, but did not change fecal output in naïve rats. The response threshold to distension of the non-inflamed distal colon was significantly decreased seven days after TNBS administration. An anti-hyperalgesic effect of pregabalin (30-100mg/kg, p.o.) that opposed the decreased colonic nociceptive threshold in TNBS-sensitized rats was observed, but nociceptive thresholds were not changed in naïve rats. Moreover, pregabalin was more potent in reducing disturbed defecation compared with reduction in nociceptive threshold to distension in TNBS-sensitized rats. This is the first report that pregabalin modulates stress-induced defecation in rats. These data indicate that pregabalin can ameliorate both altered defecation and decreases in colonic nociceptive threshold, suggesting that pregabalin might warrant investigation for the treatment of irritable bowel syndrome.

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.

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.