Inflammation and dephosphorylation of the tight junction protein occludin in an experimental model of multiple sclerosis

Multiple sclerosis (MS) is a disease of the CNS in which inflammation, demyelination and neurodegeneration contribute to its initiation and progression. A frequently employed model of MS is experimental autoimmune encephalomyelitis (EAE). Here, to gain new insights into the disease process, an analysis of proteins in extracts of lumbar spinal cord from naïve and EAE rats was undertaken. The data mainly confirm that inflammation and blood–brain barrier (BBB) breakdown are the major hallmarks of disease in this model. Given their importance in the BBB, junctional proteins were further investigated. Occludin, a protein localizing to tight junctions in brain endothelial cells, showed strikingly increased migration in EAE when analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). This increased migration was mimicked by in vitro phosphatase treatment, implying its dephosphorylation in EAE. Occludin dephosphorylation coincided with the onset of inflammation, slightly preceding visible signs of disease, and was just prior to apparent changes in BBB permeability. These findings suggest occludin is a target for signaling processes in EAE, perhaps regulating the response of the BBB to the inflammatory environment as seen in MS.     

Expression of short hairpin RNAs against the coxsackievirus B3 exerts potential antiviral effects in Cos-7 cells and in mice

Chemically synthesized small interfering RNA (siRNA) has been used as an anti-coxsackievirus B3 (CVB3) agent. Herein, we investigated whether vector-derived short hairpin RNAs (shRNA) targeting CVB3 can exert antiviral activities, prior to their further application to viral vector system for efficient in vivo administration. Employing transient transfection assays to in vivo mouse models as well as to in vitro Cos-7 cell cultures, we directly demonstrated the potential antiviral activity of shRNAs following challenges with infectious CVB3. Of the six shRNAs that we designed, three prevented cell death from CVB3 infection by suppressing viral replication and viral production in Cos-7 cells. These were shRNA 2, which targeted the capsid protein VP1, and shRNAs 4 and 5, which targeted two different regions of the RNA-dependent RNA polymerase 3D. Furthermore, shRNAs 2 and 5 also exerted strong antiviral effects in viral replication in vivo, accompanied by attenuated pancreatic tissue damage. Through this direct evaluation system we addressed the development and application of vector-derived shRNAs as an anti-CVB3 agent, revealing new target sequences.     

Lamivudine therapy for korean children with chronic hepatitis B

PURPOSE: Lamivudine is known to be very effective in suppressing hepatitis B virus replication and virus induced necroinflammation. The aim of this study was to evaluate lamivudine therapy efficacy, predictive factors, breakthrough, prevalence of YMDD mutation, and relapse rate in Korean children with chronic hepatitis B. MATERIALS AND METHODS: Between August 1999 and February 2005, 60 children on lamivudine therapy for chronic hepatitis B were enrolled. Treatment response was defined as alanine aminotransferase (ALT) normalization, and HBeAg and HBV-DNA disappearance. RESULTS: Seroconversion rates of HBeAg and HBV- DNA were 42% and 53%, respectively, and ALT normalization rate was 88%. Seroconversion rates of HBeAg (60.0%) and anti-HBe (60.0%) were higher in patients younger than 6 years. Seroconversion rate of HBV-DNA (68.4%) and normalization rate of serum ALT (94.7%) were highest in patients between 6 and 12 years. Seroconversion rates of all HBV markers were lowest in patients older than 12 years. Predicted 3 year cumulative seroconversion rates, were 70%, 68% for HBeAg, HBV-DNA, respectively. These were calculated by Kaplan-Meier method. Cox proportional hazard regression model showed that pre-treatment ALT was a positive predictive factor for seroconversion of HBeAg and HBV-DNA. Breakthrough phenomenon was noted in 6 patients, and 3 had a YMDD mutation. CONCLUSION: Lamivudine therapy had a significant effect on HBeAg seroconversion and HBV-DNA disappearance, and ALT normalization for Korean children with chronic hepatitis B.     

A prospective, multicenter, open-label trial of zoledronic acid in patients with hormone refractory prostate cancer

PURPOSE: The short-term safety and efficacy of zoledronic acid for the treatment of skeletal metastasis was evaluated in patients with hormone-refractory prostate cancer. PATIENTS AND METHODS: A total of 19 hormone-refractory prostate cancer patients with bone metastases were enrolled. All patients received up to six infusions of zoledronic acid (4 mg, given intravenously over 15 minutes, every 3-4 weeks). Safety was assessed by monitoring adverse events and serum creatinine levels. Efficacy was assessed by monitoring skeletal-related events, brief pain inventory score, quality of life score, type of pain medication, and analgesic score. Mean age of patients was 67.3 years (46-86 years), mean time from diagnosis of bone metastases was 27.6 months (0-117 months), and mean time from diagnosis of hormone-refractory disease was 7.5 months (0-26 months). RESULTS: There was no clinically significant change in serum creatinine levels. Eleven adverse events (musculoskeletal disorders and systemic disorders) in 8 patients were classed as having a possible relationship to study drug. Fifteen patients completed six courses of zoledronic acid infusion. There were no significant changes in the brief pain inventory composite scores, quality of life questionnaire scores or analgesic score. No new skeletal-related events developed during the treatment period. CONCLUSION: Zoledronic acid administered in this study as a 15-minute infusion demonstrated an acceptable and well-known safety profile in patients with refractory prostate cancer with bone metastases. However, prospective placebo- controlled clinical trials are required to elucidate the efficacy of zoledronic acid.     

Beneficial effects of thiazolidinediones on diabetic nephropathy in OLETF rats

PURPOSE: Diabetic nephropathy is the most serious of complications in diabetes mellitus. Thiazolidinedione (TZD) is thought to ameliorate diabetic nephropathy; however, the mechanism underlying this effect has not been elucidated. We hypothesized that the vascular endothelial growth factor (VEGF) participates in the pathogenesis of diabetic nephropathy and that TZD may be beneficial for the treatment of diabetic nephropathy because of the effect it has on VEGF. MATERIALS AND METHODS: 23 Otsuka- Long-Evans-Tokushima-Fatty (OLETF) rats and eight control Long-Evans-Tokushima-Otsuka (LETO) rats were divided into the following four groups: LETO group, control OLETF group, pioglitazone treated group (10mg/ kg/day), and rosiglitazone treated group (3mg/kg/day). RESULTS: A progressive increase in urinary protein excretion was observed in the diabetic rats. Glomerular VEGF expression in the control OLETF rats was significantly higher than in the control LETO rats. However, there was a significant reduction in both the glomerular VEGF expression and the VEGF mRNA levels after treatment with pioglitazone and rosiglitazone. The twenty-four hour urine protein levels were significantly decreased in both groups of the treated OLETF rats. CONCLUSION: These results suggest that TZD may have beneficial effects on diabetic nephropathy by reducing the VEGF expression.     

Assembly of collagen-binding peptide with collagen as a bioactive scaffold for osteogenesis in vitro and in vivo

Bioactive scaffolds inducing cell adhesion, differentiation have been premise for optimal formation of target tissue. Collagen has been employed as a tissue regenerative scaffold especially for bone regeneration and has been chemically surface-modified to present bioactivity. Herein, we show that peptide, denoted as collagen-binding motif (CBM, GLRSKSKKFRRPDIQYPDATDEDITSHM) identified from osteopontin (OPN) protein, was able to specifically bind collagen without chemical conjugation, while presenting apatite forming capability in vitro and in vivo. Collagen surface alone was not able to induce noticeable apatite nucleation however, mineralization was evident when assembled with CBM peptide, implying that the collagen-CBM assembly played a pivotal role in biomineralization. In vivo result further demonstrated that the CBM peptide in complex with material was able to induce bone formation by helping mineralization in the bone defect. Taken together, the CBM peptide herein and its assembly with collagen can be applied as an inducer of biomineralization as well as a bioactive scaffold for bone regeneration.     

HIV-1 prophylactic vaccine comprised of topical DermaVir prime and protein boost elicits cellular immune responses and controls pathogenic R5 SHIV162P3

Topical DNA vaccination (DermaVir) facilitates antigen presentation to naive T cells. DermaVir immunization in mice, using HIV-1 Env and Gag, elicited cellular immune responses. Boosting with HIV-1 gp120 Env and p41 Gag augmented Th1 cytokine levels. Intramuscular DNA administration was less efficient in priming antigen-specific cytokine production and memory T cells. In rhesus macaques, DermaVir immunization induced Gag- and Env-specific Th1 and Th2 cytokines and generation of memory T cells. Boosting of DermaVir-primed serum antibody levels was noted following gp140(SHIV89.6P)/p27(SIV) immunization. Rectal challenge with pathogenic R5-tropic SHIV162P3 resulted in control of plasma viremia (4/5 animals) that was reflected in jejunum, colon and mesenteric lymph nodes. An inverse correlation was found between Gag- and Env-specific central memory T cell responses on the day of challenge and plasma viremia at set point. Overall, the topical DermaVir/protein vaccination yields central memory T cell responses and facilitates control of pathogenic SHIV infection.