Homologous and heterologous antibody responses to lipopolysaccharide after enterohemorrhagic Escherichia coli infection

To evaluate antibody responses against lipopolysaccharide (LPS: O157, O26, and O111) in enterohemorrhagic Escherichia coli(EHEC) infection, sera of 24 schoolchildren associated with the Morioka outbreak in 1997 and of 74 sporadic patients suspected of having EHEC infection were examined. Using a positive standard serum, quantitative evaluation of LPS antibodies by an enzyme-linked immunosorbent assay (ELISA) was established. High levels of specific IgM and IgA antibodies against homologous E. coli LPS were present in the acute period and are characteristic of EHEC. This could be used for the serological diagnosis of EHEC infection, except for early infants and the elderly. In addition to the specific homologous response, multiple antibody responses against different serotypes other than those isolated were demonstrated in many cases by qualitative analysis using Western blotting.     

Induction of hepatic injury by hepatitis C virus-specific CD8+ murine cytotoxic T lymphocytes in transgenic mice expressing the viral structural genes

In the present study, we generated killer cells specific for hepatitis C virus (HCV) structural protein by re-stimulation of immune spleen cells from H-2(d) haplotype transgenic (Tg) mice, expressing the core, E1, E2, and NS2 genes of HCV regulated by the Cre/loxP switching system. The generated killer cells were conventional CD8(+)L(d) class-I MHC molecule-restricted cytotoxic T lymphocytes (CTLs) and specific for the HCV E1 structural protein. Because the CTLs could also kill hepatocytes from the Tg mice expressing HCV structural proteins in vitro, we attempted to transfer those CTLs intravenously into interferon regulatory factor-1 (IRF-1) negative, CD8-deficient Tg mice representing the HCV structural genes on hepatocytes to examine whether the inoculated CD8(+) CTLs can eliminate hepatocytes expressing the HCV genes in vivo. We observed an elevation of serum ALT level as well as damage of the liver tissue histologically. To our knowledge, this is the first demonstration to show that HCV-specific CD8(+) CTLs specifically attack hepatocytes expressing the HCV structural proteins both in vitro and in vivo.     

Use of RNA interference-mediated gene silencing and adenoviral overexpression to elucidate the roles of AKT/protein kinase B isoforms in insulin actions

Insulin plays a central role in the regulation of glucose homeostasis in part by stimulating glucose uptake and glycogen synthesis. The serine/threonine protein kinase Akt has been proposed to mediate insulin signaling in several processes. However, it is unclear whether Akt is involved in insulin-stimulated glucose uptake and which isoforms of Akt are responsible for each insulin action. We confirmed that expression of a constitutively active Akt, using an adenoviral expression vector, promoted translocation of glucose transporter 4 (GLUT4) to plasma membrane, 2-deoxyglucose (2-DG) uptake, and glycogen synthesis in both Chinese hamster ovary cells and 3T3-L1 adipocytes. Inhibition of Akt either by adenoviral expression of a dominant negative Akt or by the introduction of synthetic 21-mer short interference RNA against Akt markedly reduced insulin-stimulated GLUT4 translocation, 2-DG uptake, and glycogen synthesis. Experiments with isoform-specific short interference RNA revealed that Akt2, and Akt1 to a lesser extent, has an essential role in insulin-stimulated GLUT4 translocation and 2-DG uptake in both cell lines, whereas Akt1 and Akt2 contribute equally to insulin-stimulated glycogen synthesis. These data suggest a prerequisite role of Akt in insulin-stimulated glucose uptake and distinct functions among Akt isoforms.     

Clonal expansion of double-positive intraepithelial lymphocytes by MHC class I-related chain A expressed in mouse small intestinal epithelium

Expression of a distant homologue MHC class I molecule, MHC class I-related chain A (MICA), has been found to be stress inducible and limited to the intestinal epithelium. This nonclassical MHC molecule is associated with various carcinomas in humans. To understand the biological consequences of MICA expression in the gut, we generated transgenic (Tg) mice (T3(b)-MICA Tg) under the control of the T3(b) promoter. The T3(b)-MICA Tg mice expressed MICA selectively in the intestine and had an increased number of TCRalphabeta CD4CD8alphaalpha, double-positive (DP) intraepithelial lymphocytes (IELs) in the small bowel. These MICA-expanded DP IELs exhibited a bias to Vbeta8.2 and overlapped motifs of the complementarity-determining region 3 region among various Tg mice. Hence, the overexpression of MICA resulted in a clonal expansion of DP IELs. Studies in model of inflammatory bowel disease showed that transgenic MICA was able to attenuate the acute colitis induced by dextran sodium sulfate administration. Therefore, this unique in vivo model will enable investigation of possible influences of stress-inducible MICA on the gut immune surveillance.     

Altered conditioned fear behavior in glutamate decarboxylase 65 null mutant mice

We investigated the involvement of the 65 kDa isoform of glutamic acid decarboxylase (GAD65) and GAD65-mediated γ-aminobutyric acid (GABA) synthesis in the formation and expression of Pavlovian fear memory. To this end, behavioral, endocrine and autonomic parameters were examined during conditioned fear retrieval of mice with targeted ablation of the GAD65 gene (GAD65^–/– mice). These mutant mice were found to display specific fear behavior (freezing, escape), as well as autonomic (increased defecation) and endocrine activation (increased plasma corticosterone) during fear memory retrieval. However, freezing was reduced and flight and escape behavior were increased in GAD65^–/– mice compared to their wild type and heterozygous littermates, while corticosterone levels and defecation rates did not differ between genotypes. Active defensive behavior of GAD65^–/– mice was observed during both auditory cued and contextual retrieval of fear memory, as well as immediately after conditioning. These data indicate a selectively altered behavioral fear response in GAD65^–/– mice, most likely due to deficits in threat estimation or the elicitation of appropriate conditioned fear behavior, and suggest that GAD65 is a genetic determinant of conditioned fear behavior. GAD65^–/– mice provide a valuable tool to further dissect the GABAergic mechanisms involved in fear and anxiety and to model GABA-related neurological and psychiatric disorders.     

Environmental Estrogen-Like Chemicals and Hydroxyl Radicals Induced by MPTP in the Striatum: A Review

Oxygen free radical formation has been implicated in lesions caused by the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and iron. Although MPTP produces a parkinsonian syndrome after its conversion to 1-methyl-4-phenylpyridine (MPP⁺) by type B monoamine oxidase (MAO) in the brain, the etiology of this disease remains obscure. This review focuses on the role of an environmental neurotoxin chemically related to MPP⁺-induced free radical generation in the pathogenesis of Parkinson's disease. Environmental-like chemicals, such as para-nonylphenol or bisphenol A, significantly stimulated hydroxyl radical (OH) formation in the striatum. Allopurinol, a xanthine oxidase inhibitor, prevents para-nonylphenol and MPP⁺-induced OH generation. Tamoxifen, a synthetic nonsteroidal antiestrogen, suppressed the OH generation via dopamine efflux induced by MPP⁺. These results confirm that free radical production might make a major contribution at certain stages in the progression of the injury. Such findings may be useful in elucidating the actual mechanism of free radical formation in the pathogenesis of neurodegenerative brain disorders, including Parkinson's disease and traumatic brain injuries.     

Highly potent inhibitors of TNF-alpha production. Part II: metabolic stabilization of a newly found chemical lead and conformational analysis of an active diastereoisomer

Design and synthesis of metabolically stabilized inhibitors of TNF-alpha production, which could be new drug candidates, are reported. Conformational analysis of an active diastereoisomer was performed based on biological evaluations of the conformationally fixed indane derivatives 17 and 18. Structure-activity relationships (SARs) based on biological evaluations of the optically active derivatives are also discussed. Full details including chemistry are reported.     

Highly potent inhibitors of TNF-alpha production. Part 1: Discovery of chemical leads

The discovery of 2-acylamino-2-phenylethyl disodium phosphates and as structurally novel inhibitors of TNF-alpha production is reported. Structure-activity relationships (SARs) are also discussed.     

Immunohistochemical detection of human gastrointestinal glutathione peroxidase in normal tissues and cultured cells with novel mouse monoclonal antibodies.

This is the first report to describe the successful detection of human gastrointestinal glutathione peroxidase in normal tissues by Western blotting and immunohistochemical staining techniques. Four hybridoma clones producing monoclonal antibodies (MAbs) against the human gastrointestinal glutathione peroxidase were established from mice immunized with a gastrointestinal glutathione peroxidase-derived peptide. The MAbs did not crossreact with other members of the glutathione peroxidase family, be it cellular glutathione peroxidase, phospholipid hydroperoxide glutathione peroxidase, or extracellular glutathione peroxidase. Although the MAbs were found to react with a 24-kD protein in a Western blotting assay using gastric carcinoma cell extracts as antigen, they did not react with a B-lymphoblastoid cell extract. Immunohistochemical staining showed gastrointestinal glutathione peroxidase localized in the cytoplasm and in the nucleus of gastric carcinoma cells. Moreover, gastrointestinal glutathione peroxidase was detected in tissue extracts of human stomach, small intestine, large intestine, liver, and gallbladder by Western blotting, and its localization was immunohistochemically confirmed in the mucosal epithelia of the basal area of gastric pits and intestinal crypts.     

Human T-cell receptor TCRAV,TCRBV, and TCRAJ sequences newly found in T-cell clones reactive with allogeneic HLA class II antigens

Correspondence to: F. Obata.