Sensitivity of chronically HIV-1 infected HeLa cells to electrical stimulation

Use of combination anti-retroviral drug regimens including protease inhibitors dramatically decreased morbidity and mortality rates in HIV-1 infected individuals. However, such combination therapies appear to have many side effects, in addition to the emergence of resistant HIV-1 strains. Therefore, in this study we sought to elucidate novel therapeutic principles against HIV-1 infection. We examined the effects of electrical stimulation on both chronically HIV-1_LAI infected HeLa cells (P6 HeLa/HIV-1_LAI) and uninfected cells (P6 HeLa). Cells were cultured on an optically transparent electrode and application of potential at 1.0 V vs Ag/AgCl was performed over time periods ranging from 10 min to 60 min. Both P6 HeLa/HIV-1_LAI and P6 HeLa cells were progressively damaged as the duration of electrical stimulation increased. However, P6 HeLa/HIV-1_LAl cells were much more influenced by electrical stimulation than P6 HeLa cells. The difference in damage rate was most obvious at 30 min of electrical stimulation, with damaged cells accounting for about 87% and 4% of P6 HeLa/HIV-1_LAI and P6 HeLa cells, respectively. After the application of potential for 20 min, the proliferation of P6 HeLa/HIV-1_LAI cells was markedly inhibited, while the P6 HeLa cells proliferated to an extent similar to that of uninfected cells without application of potential. These results indicate that sensitivity to electrical stimulation is much higher in chronically HIV-1 infected cells than in uninfected cells. This could be considered as a useful new approach against HIV-1 infection.     

Neutrophil elastase contributes to the development of ischemia-reperfusion-induced liver injury by decreasing endothelial production of prostacyclin in rats

We previously reported that nitric oxide (NO) derived from endothelial NO synthase (NOS) increased endothelial prostacyclin (PGI(2)) production in rats subjected to hepatic ischemia-reperfusion (I/R). The present study was undertaken to determine whether neutrophil elastase (NE) decreases endothelial production of PGI(2), thereby contributing to the development of I/R-induced liver injury by decreasing hepatic tissue blood flow in rats. Hepatic tissue levels of 6-keto-PGF(1alpha), a stable metabolite of PGI(2), were transiently increased and peaked at 1 h after reperfusion, followed by a gradual decrease until 3 h after reperfusion. Sivelestat sodium hydrochloride and L-658,758, two NE inhibitors, reduced I/R-induced liver injury. These substances inhibited the decreases in hepatic tissue levels of 6-keto-PGF(1alpha) at 2 and 3 h after reperfusion but did not affect the levels at 1 h after reperfusion. These NE inhibitors significantly increased hepatic tissue blood flow from 1 to 3 h after reperfusion. Both hepatic I/R-induced increases in the accumulation of neutrophils and the microvascular permeability were inhibited by these two NE inhibitors. Protective effects induced by the two NE inhibitors were completely reversed by pretreatment with nitro-l-arginine methyl ester, an inhibitor of NOS, or indomethacin. Administration of iloprost, a stable derivative of PGI(2), produced effects similar to those induced by NE inhibitors. These observations strongly suggest that NE might play a critical role in the development of I/R-induced liver injury by decreasing endothelial production of NO and PGI(2), leading to a decrease in hepatic tissue blood flow resulting from inhibition of vasodilation and induction of activated neutrophil-induced microvascular injury.     

The ability of konjac-glucomannan to suppress spontaneously occurring dermatitis in NC/Nga mice depends upon the particle size

Konjac glucomannan (GM) is a well-known dietary fiber with various beneficial functions, which the higher viscous GM is proposed to have the stronger potencies. Application of highly viscous GM to various food industries is limited for the sake of long elapsed time required to solubilize the GM. Thus, the exploration into any physiological function of low-viscous GM remained undone for a long time. Male 4-week-old NC/Nga mice, a model of atopic dermatitis (AD), were fed for 8 weeks on diets containing 5% each of a high-viscous GM powder, ordinary konjac or highly purified GM, and a low-viscous GM powder, pulverized GM or re-granulated fine GM. Striking suppression to the aggravation of dermatitis and the increases in scratching behaviors and IgE levels was significantly observed only in mice fed on the pulverized GM diet, but not in mice fed on the other GM diets or a control diet. We concluded that the low-viscous GM possesses a novel immunoregulatory function and the intensity of the function depends upon the particle size rather than the viscosity.     

Reverse hydroxamate-based selective TACE inhibitors

Reverse hydroxamate-based selective TACE inhibitors are described. They have potent TACE inhibitory activities and excellent selectivities against MMP-1, 2, 3, 8, 9, 13, 14, and 17. One representative compound, 18 has demonstrated an excellent oral inhibitory activity of the lipopolysaccharide (LPS)-stimulated TNF-alpha production in rats.     

IL-12 gene therapy is an effective therapeutic strategy for hepatocellular carcinoma in immunosuppressed mice

Immunosuppressive therapy for organ transplantation is essential for controlling rejection. When liver transplantation is performed as a therapy for hepatocellular carcinoma (HCC), recurrent HCC is one of the most fatal complications. In this study, we show that intratumoral murine IL-12 (mIL-12) gene therapy has the potential to be an effective treatment for malignancies under immunosuppression. C3H mice (H-2(k)), injected with FK506 (3 mg/kg) i.p., were s.c. implanted with 2.5 x 10(6) MH134 cells (H-2(k)) and we treated the established HCC with electroporation-mediated gene therapy using mIL-12 plasmid DNA. Intratumoral gene transfer of mIL-12 elevated intratumoral mIL-12, IFN-gamma, and IFN-gamma-inducible protein-10, significantly reduced the number of microvessels and inhibited the growth of HCC, compared with HCC-transferred control pCAGGS plasmid. The inhibition of tumor growth in immunosuppressed mice was comparable with that of mIL-12 gene therapy in immunocompetent mice. Intratumoral mIL-12 gene therapy enhanced lymphocytic infiltration into the tumor and elicited the MH134-specific CTL response even under FK506. The dose of FK506 was sufficient to prevent the rejection of distant allogenic skin grafts (BALB/c mice, H-2(d)) and tumors, B7-p815 (H-2(d)) used as transplants, during mIL-12 gene therapy against MH134. Ab-mediated depletion studies suggested that the inhibition of tumor growth, neovascularization, and spontaneous lung metastasis by mIL-12 was dependent almost entirely on NK cells and partially on T cells. These results suggest that intratumoral mIL-12 gene therapy is a potent effective strategy not only to treat recurrences of HCC in liver transplantation, but also to treat solid malignant tumors in immunosuppressed patients with transplanted organ.     

Inhibition of B cell death causes the development of an IgA nephropathy in (New Zealand white x C57BL/6)F(1)-bcl-2 transgenic mice

Little is known about the pathogenic mechanisms of IgA nephropathy, despite being the most prevalent form of glomerulonephritis in humans. We report in this study that in (New Zealand White (NZW) x C57BL/6)F(1) mice predisposed to autoimmune diseases, the expression of a human bcl-2 (hbcl-2) transgene in B cells promotes a CD4-dependent lupus-like syndrome characterized by IgG and IgA hypergammaglobulinemia, autoantibody production, and the development of a fatal glomerulonephritis. Histopathological analysis of glomerular lesions reveals that the glomerulonephritis observed in these animals resembles that of human IgA nephropathy. The overexpression of Bcl-2 in B cells selectively enhances systemic IgA immune responses to T-dependent Ags. Significantly, serum IgA purified from (NZW x C57BL/6)F(1)-hbcl-2 transgenic mice, but not from nontransgenic littermates, shows reduced levels of galactosylation and sialylation and an increased ability to deposit in the glomeruli, as observed in human patients with IgA nephropathy. Our results indicate that defects in the regulation of B lymphocyte survival associated with aberrant IgA glycosylation may be critically involved in the pathogenesis of IgA nephropathy, and that (NZW x C57BL/6)F(1)-hbcl-2 Tg mice provide a new experimental model for this form of glomerulonephritis.     

Hyperthermia suppresses the cytotoxicity of NK cells via down-regulation of perforin/granzyme B expression

Hyperthermia, which is used as an adjunctive therapy for cancer, is known to modulate the activity of natural killer (NK) cells in vitro, but its effect in vivo is unclear. In the present study, we used a whole body hyperthermia (WBH) device heated by infrared rays to evaluate the effect of WBH on mice models. We demonstrate here that wild type C57BL/6J mice exposed to 42 degrees C for 60min had reduced NK cell cytolytic activity against YAC-1 target cells as determined by cytolytic assay. This result was confirmed using Rag-2 knockout mice, which possess functional NK but not cytolytic T or NK-T cells. Moreover, WBH decreased the mRNA expression of perforin and granzyme B in spleens of mice. But the expression of TNF cytokines (Fas ligand and TRAIL) was unchanged. These data suggest that the suppression of NK cell activity induced by WBH could be mediated through the perforin/granzyme pathway.     

Normalizing renal reducing ability prevents adriamycin-induced proteinuria

Reactive oxygen species play an important role in adriamycin (ADR) nephropathy. We showed by in vivo electron paramagnetic resonance (EPR) that renal reducing ability (RRA) declined on the 7th day after ADR administration. Proteinuria appeared after the decline in RRA. The aim of this study was to prove by in vivo EPR whether the decline in RRA is altered by scavengers such as dimethyl sulfoxide (DMSO) and dimethylthiourea (DMTU) and that it is this change which is responsible for the proteinuria in ADR nephropathy. By showing that DMSO and DMTU ameliorate the RRA, we demonstrate that the decline in RRA is related to ADR-induced proteinuria.     

Therapeutic effect of adenoviral-mediated hepatocyte growth factor gene administration on TNBS-induced colitis in mice

Inflammatory bowel disease is incurable and relapsing disease. In order to clarify the effect of HGF gene therapy for inflammatory bowel disease, the adenoviral-mediated HGF gene was intrarectally administered into TNBS-colitis-induced Balb/c mice. Adenoviral-mediated gene delivery targetted its expression mainly to intestinal epithelial cells. Mucosal damage of HGF-treated intestine was significantly improved, and compared with LacZ-treated and saline administered mice (P<0.05, each). The mice treated with intrarectal administration of pAxCAHGF showed an increased average of body weight in comparison with that of pAxCALacZ-treated and saline-treated mice (P<0.05, each). The PCNA-positive cells in pAxCALacZ-treated mice were 44.7+/-4.9%, 51.7+/-6.6%, and 53.9+/-4.5% at 10, 15, and 21 days after TNBS administration, however those in pAxCAHGF-treated mice were increased to 74.3+/-5.1%, 67.1+/-2.6%, and 69.2+/-4.6% (P<0.05, each). The TUNEL-positive cells in pAxCALacZ-treated mice were 13.3+/-5.2%, 11.5+/-2.1%, and 7.2+/-5.2%, respectively. However, those in pAxCAHGF-treated mice at 10, 15, and 21 days were significantly decreased to 5.4+/-1.8%, 3.8+/-1.3%, and 5.7+/-2.8% (P<0.05, respectively). Expression of ERK1/2 was stronger in pAxCAHGF mice than in pAxCALacZ. These data suggest that adenoviral-mediated HGF gene therapy via an intrarectal route is a promising therapy for inflammatory bowel disease.     

Glyceraldehyde-3-phosphate dehydrogenase in the extracellular space inhibits cell spreading

The occurrence and the novel function of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the extracellular space were studied. The extracellular GAPDH with the same molecular mass as the intracellular GAPDH was detected in the conditioned medium of mammalian cultured cell lines such as COS-7, HEK293, MCF-7, HepG2, PC-12, and Neuro-2a cells. Western blot analysis represented the occurrence of GAPDH, but not alpha-tubulin (an intracellular marker protein), in the conditioned medium of COS-7 cells. Furthermore, GAPDH was found in rat serum. These results indicate that GAPDH was secreted outside of the cells. Addition of GAPDH to the cultured medium of COS-7, HEK293, and HepG2 cells allowed cells to undergo morphological changes. In COS-7 cells, the extracellular GAPDH inhibited cell spreading without influencing the cell growth. Western blot and immunofluorescent microscopy analyses revealed that the extracellular GAPDH bound to COS-7 cells in time- and dose-dependent manners. However, a mutant substituting Ser for Cys at position 151 of GAPDH resulted in no binding to the cells, no decreased cell-spreading efficiency and no cell morphological changes. These results indicate that the Cys151 was involved in the binding of GAPDH to cells and the GAPDH-inhibited cell spreading.