Comparison of the suppressive effects of soluble CR1 and C5a receptor antagonist in acute arthritis induced in rats by blocking of CD59

We investigated the effects of suppression of complement activation at C3 level and inhibition of C5a on acute synovitis in rats. Acute synovitis was induced in Wistar rats by intra-articular (i.a.) injection into one knee of 0.3 mg of MoAb 6D1 (anti-rat CD59 antibody). In the treatment groups, soluble CR1 (sCR1) or C5a receptor (C5aR) antagonist was administered intra-articularly or intravenously and effects on the course of the acute synovitis were monitored. Synovitis induced by 6D1 was characterized by joint swelling, thickening of synovial tissue, cellular infiltration and deposition of membrane attack complex (MAC) on the synovial surface. Neither inflammatory change nor MAC deposition was found in rats which received an i.a. injection of sCR1 to suppress complement activity in the joint. Intra-articular injection of sCR1 did not reduce plasma complement activity. Intravenous administration of sCR1 suppressed plasma complement activity but had no effect on the course of the arthritis and synovitis with MAC deposition was observed. Neither i.a. nor i.v. injection of C5aR antagonist had any suppressive effects on inflammatory change or MAC deposition in synovium. The data show that inflammatory change induced by 6D1 was mediated by local complement activation and was not accompanied by systemic complement activation. C5a generation was not responsible for the observed inflammation, suggesting that other complement activation products, possibly MAC, mediate the inflammatory change observed in this model of acute synovitis in rats.     

Transforming growth factor beta is protective in host resistance against Listeria monocytogenes infection in mice.

The role of transforming growth factor beta (TGF-beta) in host resistance against Listeria monocytogenes infection was studied with mice. The constitutive expression of TGF-beta 1 mRNA was observed in the spleens and livers of mice before and after infection. Injecting the mice with anti-TGF-beta 1 peptide serum resulted in diminished antilisterial resistance, whereas the administration of human platelet-derived TGF-beta 1 enhanced the resistance. Moreover, mice were protected against lethal infection when treated with TGF-beta 1. These results suggest the TGF-beta 1 might be involved in antilisterial resistance. On the other hand, injecting the mice with TGF-beta 1 resulted in a decrease in the titers of endogenous gamma interferon, tumor necrosis factor alpha, and interleukin-6, which are crucial in antilisterial resistance, in sera and in extracts of spleen and liver. Thus, a complicated mechanism might be involved in the role of TGF-beta 1 in host resistance against L. monocytogenes infection.     

The full expression of the ity phenotype in ityr mice requires C3 activation by Salmonella lipopolysaccharide.

Our previous study has shown that the rapid and sufficient activation of complement by Salmonella lipopolysaccharide occurs in genetically resistant (Ityr) A/J mice. To assess whether the level of complement activation by a virulent strain of Salmonella typhimurium regulates the level of murine natural resistance, we compared levels of serum complement activation by S. typhimurium and kinetics of serum-opsonized S. typhimurium grown in macrophages using several strains of resistant (Ityr) and susceptible (Itys) mice. Itys macrophages killed intracellular S. typhimurium to the same extent as did Ityr macrophages when the pathogen was opsonized with Ityr serum. Opsonization of S. typhimurium with Itys serum reduced intracellular killing activity in Ityr macrophages to the same level as seen with Itys macrophages. Incubation of S. typhimurium with 25% Mg2+ EGTA (5 mm MgCl2-3 mm ethylene glycol-bis (beta-aminotheyl either)-N,N,N',N'-tetraacetic acid)-chelated Ityr serum resulted in higher levels of C3 deposition onto the surface of this bacteria, C3b generation and also C3 consumption, compared with that with Mg2+ EGTA-chelated Itys serum. Opsonization of S. typhimurium with A/J serum prior to infection increased early resistance in Itys mice. Infection with a virulent strain of S. typhimurium induced the expression of interleukin-10 (IL-10) mRNA at higher levels in C57BL/6 mice than in A/J mice. However, opsonization of S. typhimurium with A/J serum decreased bacterial growth in the spleen of C57BL/6 mice to the same level as observed for A/J mice in association with decreased expression levels of IL-10 mRNA. Moreover, administration of anti-C3 antibodies reduced the resistance of A/J mice in association with a decrease in serum levels of C3. These results indicate that the high level of complement activation via the alternative pathway in Ityr serum by a virulent strain of S. typhimurium reduces the virulence of this pathogen, which may contribute to the full expression of Ity phenotype in Ityr mice.     

Methylcobalamin induces a long-lasting enhancement of the field potential in rat suprachiasmatic nucleus slices

Optic nerve stimulation has been reported to evoke a field potential (FP) in rat suprachiasmatic nucleus (SCN) slices. Methylcobalamin,δ-(5,6-dymethylbenzimidazoyl)-Co-methyl-cobamide (Me-B₁₂) enhanced this FP and the enhancement lasted more than 1 h after washing out. Maximal enhancement (143.6±9.8%) was achieved at a concentration of 10 μM. By contrast, cyanocobalamin containing CN- instead of CH₃-showed no enhancement of the amplitude in the FP. Me-B₁₂ induced enhancement of FP was strongly blocked by an N-methyl- -aspartate (NMDA) receptor antagonist, -2-amino-5-phosphonovaleric acid (APV). These results indicate that CH₃- in the Me-B₁₂ is required to modulate the FP amplitude and the NMDA receptor is involved in the long-lasting FP enhancement induced by Me-B₁₂. The present results suggest that Me-B₁₂ modifies the photic entrainment of the circadian clock in the suprachiasmatic nucleus via an activation of NMDA receptors.     

Invasion of human respiratory epithelial cells by Bordetella pertussis: possible role for a filamentous hemagglutinin Arg-Gly-Asp sequence and alpha5beta1 integrin

Bordetella pertussis, the agent of whooping cough, is capable of invading human respiratory epithelial cells. In this study, we investigated the mechanisms by which B. pertussis invades the human lung epithelial cell line A549 and normal human bronchial epithelial (NHBE) cells. In vitro adhesion and invasion assays using both cell types with a virulent B. pertussis strain and its isogenic mutants revealed profound defects in a mutant deficient in filamentous hemagglutinin (FHA) expression. In addition, a mutant in which an FHA Arg-Gly-Asp (RGD) site had been changed to Arg-Ala-Asp had significantly diminished invasiveness, although its adhesiveness was comparable to that of the parental strain. Furthermore, a synthetic RGD-containing hexapeptide inhibited invasion of both cell types by the virulent strain. These results demonstrate that an RGD sequence of FHA is involved in B. pertussis invasion of epithelial cells in vitro. Monoclonal antibodies directed against human alpha5beta1 integrin, but not other integrins, blocked invasion, indicating that this integrin is involved in B. pertussis invasion. Taken together, these findings suggest that B. pertussis FHA may promote invasion of human respiratory epithelial cells through the interaction of its RGD sequence with host cell alpha5beta1 integrin.     

Clinical heterogeneity of neonatal intrahepatic cholestasis caused by citrin deficiency: case reports from 16 patients

A deficiency of citrin, which is encoded by the SLC25A13 gene, causes both adult-onset type II citrullinemia (CTLN2) and neonatal intrahepatic cholestasis (NICCD). We analyzed 16 patients with NICCD to clarify the clinical features of the disease. Severe intrahepatic cholestasis with fatty liver was the most common symptom, but the accompanying clinical features were variable, namely; suspected cases of neonatal hepatitis or biliary atresia, positive results from newborn screening, tyrosinemia, failure to thrive, hemolytic anemia, bleeding tendencies and ketotic hypoglycemia. Laboratory data showed elevated serum bile acid levels, hypoproteinemia, low levels of vitamin K-dependent coagulation factors, and hypergalactosemia. Hypercitrullinemia was detected in 11 out of 15 patients examined. Most of the patients were given a lactose-free and/or medium chain triglycerides-enriched formula and lipid-soluble vitamins. The prognosis of the 16 patients is going fairy well at present, but we should observe these patients carefully to see if they manifest any symptom of CTLN2 in the future.     

Colchicine-induced apoptosis and anti-Fas localization in rat-incisor ameloblasts

Apoptosis of ameloblasts were examined by the TdT-mediated dUTP-biotin nick end-labelling method and electron microscopy 8 h after injection of colchicine. The results showed that extensive apoptosis occurred in ameloblasts of secretion to maturation zones. To determine the possible involvement of stimulators in ameloblast apoptosis, Fas, Fas ligand, tumor-necrosis-factor α, and tumor-necrosis-factor receptor 1 were examined utilizing immunohistochemistry and Western blotting analysis. Only Fas was consistently detected in the secretion, transition and maturation ameloblasts and overlying enamel organ epithelia. These results suggest that ameloblasts could undergo apoptosis by colchicine and that one of the ameloblast apoptosis mediators would be the Fas receptor.     

Alagille's syndrome. A case with a hamartomatous nodule of the liver

A male case of Alagille's syndrome associated with a hamartomatous nodule of the liver is described. The patient developed jaundice soon after birth, and was diagnosed as the syndrome with signs such as paucity of the intrahepatic bile ducts, pulmonary stenosis and embryotoxon in the cornea at 15 years of age. The liver was examined in recurrent biopsies and other tests. However, no evidence of liver cirrhosis was confirmed until his 15th year. The patient died of hepatic dysfunction when he was 17 years old. At autopsy, a large hamartomatous nodule was found in the liver showing biliary cirrhosis. Morphology of the nodule resembled that of focal nodular hyperplasia. Abnormalities of the large vessels were noted around the liver. Vascular abnormalities were also seen in the mass. The relation of these vascular abnormalities to etiological background of the syndrome and occurrence of the nodular lesion is discussed.     

Intracellular pH measurement in frog muscle by means of 31P-nuclear magnetic resonance.

The 31P-NMR technique was used for the monitoring of intracellular pH and studying its heterogeneity in the femoral biceps muscle of Rana catesbiana under anaerobic conditions. The value of intracellular pH of fresh muscle calculated from the chemical shift of intracellular inorganic phosphate (P1) was 7.3 on average and the line width of P1 was about 0.2 ppm. As the line width determined by the relaxation mechanism was 0.099 ppm, the P1 signal in fresh muscle was concluded to consist of overlapped narrow components, which indicated the heterogeneity of muscular pH (about 0.2 pH unit). Living muscle showed gradual acidification due to glycolysis and the decrease in heterogeneity. When glycolysis was suppressed by iodoacetic acid, slight alkalization due to the breakdown of creatine phosphate was observed. When the Lohmann reaction was suppressed by 2, 4-dinitro-1-fluorobenzene, rapid acidification accompanied by the appearance of a new acidic component was observed with the onset of ATP decrease. This new component was not detected in the muscle pretreated with glycerol to disrupt the transverse tubules. Therefore, it is likely that this new acidic component originates in the intracellular compartment, and not in the cellular difference.