Control of antibody-antigen interaction using anion-induced conformational change in antigen peptide

The binding of a monoclonal antibody to an epitope peptide was controlled by the conformational change of the epitope peptide induced by anions. We synthesized peptides in which the epitope sequence DTYRYI for the monoclonal antibody AU1 is located between amphiphilic peptides (KKLL)n and (LLKK)n. In the absence of an appropriate anion, the peptide was in a random coil state and the epitope was linear. In contrast, in the presence of an appropriate anion, the peptide exhibited an anti-parallel alpha-helical structure and the epitope was subsequently 'bent'. In the presence of 41 microM triphosphate, the association constant between the antibody and the peptide was decreased by one order of magnitude in the case of n = 3 and at least three orders of magnitude in the case of n = 4 or 5. Oligo-DNAs, as anions, dissociated the antibody-peptide complex, whereas triphosphate did not. The DNA concentrations required for 50% dissociation of the antibody-peptide complex at pH 7.5 were 4x10(-8), 1x10(-7) and 6x10(-6) M for decamer, octamer and hexamer DNA, respectively.     

Dual renin gene targeting by Cre-mediated interchromosomal recombination

This study describes a new approach to targeting clustered genes. Our study began with the establishment of two lines of mice carrying different mutations in either Ren1 or Ren2. These two genes, both encoding renin, span over 40 kb in tandem on chromosome 1. Each gene was mutated by gene targeting to contain loxP sites. These two mutants and Cre transgenic mice were mated to produce offspring carrying the mutant Ren1 and Ren2 genes, as well as the Cre transgene concurrently. Initially, two mutant Ren genes were located on separate chromosomes. Southern analysis of mice from the second generation revealed that the mutant Ren1 and Ren2 were interchromosomally recombined at the loxP sites to produce a new dually mutated allele on the chromosome at the rate of 9.6% (7/73). Thus, interchromosomal recombination can be efficiently programmed by mating as designed using the Cre-loxP system.     

Membranous glomerulonephritis development with Th2-type immune deviations in MRL/lpr mice deficient for IL-27 receptor (WSX-1)

MRL/lpr mice develop spontaneous glomerulonephritis that is essentially identical with diffuse proliferative glomerulonephritis (World Health Organization class IV) in human lupus nephritis. Lupus nephritis is one of the most serious complications of systemic lupus erythematosus. Diffuse proliferative glomerulonephritis is associated with autoimmune responses dominated by Th1 cells producing high levels of IFN-gamma. The initial mounting of Th1 responses depends on the function of the WSX-1 gene, which encodes a subunit of the IL-27R with homology to IL-12R. In mice deficient for the WSX-1 gene, proper Th1 differentiation was impaired and abnormal Th2 skewing was observed during infection with some intracellular pathogens. Disruption of the WSX-1 gene dramatically changed the pathophysiology of glomerulonephritis developing in MRL/lpr mice. WSX-1-/- MRL/lpr mice developed disease resembling human membranous glomerulonephritis (World Health Organization class V) with a predominance of IgG1 in glomerular deposits, accompanied by increased IgG1 and IgE in the sera. T cells in WSX-1-/- MRL/lpr mice displayed significantly reduced IFN-gamma production along with elevated IL-4 expression. Loss of WSX-1 thus favors Th2-type autoimmune responses, suggesting that the Th1/Th2 balance may be a pivotal determinant of human lupus nephritis development.     

A novel role of hepatocyte growth factor as an immune regulator through suppressing dendritic cell function

Hepatocyte growth factor (HGF) plays an important role in many biological events such as angiogenesis, cell proliferation, anti-fibrosis and antiapoptosis. It is well known that HGF promotes tumor progression and suppresses development of fibrosis after tissue injury. In contrast, its role in immune-mediated disorders has not been fully clarified. In the present study, we examined the role of HGF in Ag-specific immune response using in vitro studies and an experimental model of allergic airway inflammation. We first confirmed that dendritic cells (DCs) expressed the receptor for HGF, c-met, which was not expressed in T cells. Treatment with HGF both in vitro and in vivo potently suppressed DC functions such as Ag-presenting capacity, thus down-regulating Ag-induced Th1- and Th2-type immune responses. Exogenous administration of the HGF expression plasmid into Ag-primed mice markedly suppressed the development of airway eosinophilia and airway hyperresponsiveness, which was induced by Ag inhalation, with suppression of the Ag-presenting capacity of DCs in the lung. HGF exhibited these immunosuppressive effects without up-regulation of IL-10 or TGF-beta. We also found that expression of endogenous HGF in the lung significantly increased following Ag sensitization and inhalation challenges. Finally, neutralization of endogenous HGF in vivo significantly increased airway eosinophilia and airway hyperresponsiveness with up-regulation of the Ag-presenting capacity of DCs in the lung. These results demonstrated a novel, significant, and possibly therapeutic role of HGF as a potent regulator in immune-mediated disorders such as asthma.     

The participation of myofibroblasts in the capsular formation of human conventional and chromophobe renal cell carcinomas

The presence of myofibroblasts has been elucidated in neoplastic capsules of various organs. In the present article, we examine the presence of myofibroblasts in the capsule of renal cell carcinoma (RCC) and discuss the origin of the myofibroblasts. Nineteen renal tumors (conventional RCC, n=17; chromophobe RCC, n=2) with evident and totally surrounded fibrous capsule were selected. Abundant myofibroblasts were immunohistochemically observed in the capsule of the RCCs. These findings were confirmed by electron and immunoelectron microscopic studies of three conventional RCCs. Type III and I collagens were predominant in the outer and inner layers of the RCC capsule, respectively. The cytoplasm of the tubular epithelial cells in the tissue surrounding the neoplastic capsule stained positively for transforming growth factor (TGF)-beta 1. In situ hybridization detected type I collagen mRNA in myofibroblasts of the capsule. Myofibroblasts may participate in the capsular formation of conventional and chromophobe RCCs through the collagen production.     

Biting reduces acute stress-induced oxidative stress in the rat hypothalamus

We investigated the inhibitory effect of para-masticatory activity, namely biting, on restraint stress-induced oxidative stress. A blood brain barrier-permeable nitroxyl spin probe, 3-methoxycarbonyl-2,2,5,5,-tetramethylpyrrolidine-1-oxyl (MC-PROXYL), was administered to rats and L-band electron spin resonance (ESR) and ESR-computerized tomography (ESR-CT) imaging were used to show that the decay rate constant of MC-PROXYL in the hypothalamus of isolated brain after 30 min of restraint stress was more rapid than in unrestrained control rats, suggesting that restraint was associated with oxidative stress. Interestingly, biting during restraint stress caused the decay rate constant of MC-PROXYL in isolated brain to approach that of the control group. These observations suggest that biting suppresses oxidative stress induced by restraint stress, and that the anti-stress effect of masticatory motor activity movements, such as biting, are important for reducing the adverse effects associated with exposure to psychological stressors.