Isolation of dengue viruses by intracerebral inoculation of mosquito larvae

The isolation of dengue viruses from clinical specimens has always posed a particularly difficult problem. The use of invertebrate cell cultures such as AP-61 and C6/36 has reduced the time required for definitive diagnosis to within a week. More recently, inoculation of adult mosquitoes has been used but it requires more than a week to reach a confirmed laboratory diagnosis. We describe a method using intracerebral inoculation of immobilized fourth instar of Toxorhynchites splendens larvae for the isolation of dengue viruses from clinical specimens which yields results within a few days following incubation at 32 degrees C.     

A phage-displayed cyclic peptide that interacts tightly with the immunodominant region of hepatitis B surface antigen.

The surface antigen (HBsAg) of hepatitis B virus (HBV) is highly conformational and generally evokes protective humoral immune response in human. A disulfide constrained random heptapeptide library displayed on the coat protein III of filamentous bacteriophage M13 was employed to select specific ligands that interact with HBsAg subtype ad. Fusion phages carrying the amino acid sequence ETGAKPH and other related sequences were isolated. The binding site of peptide ETGAKPH was located on the immunodominant region of HBsAg. An equilibrium binding assay in solution showed that the phage binds tightly to HBsAg with a relative dissociation constant (KDrel) of 2.9+/-0.9 nM. The phage bearing this peptide has the potential to be used as a diagnostic reagent and two assays for detecting HBsAg in blood samples are described.     

Serum and adipocyte resistin in polycystic ovary syndrome with insulin resistance

BACKGROUND: The aim of this study was to investigate the relationship between resistin and insulin resistance in patients with polycystic ovary syndrome (PCOS). METHODS: We compared serum resistin levels in 17 PCOS women and 10 lean, healthy, age-matched non-PCOS women and also compared levels of insulin receptor (IR), phosphatidylinositol-3 kinase (PI3-kinase), glucose transporter 4 (GLUT4) protein and resistin mRNA in adipocytes isolated from the omental adipose tissue of five of the PCOS patients and five age- and weight-matched, non-PCOS controls, to look for local defects in insulin action in PCOS. RESULTS: The PCOS group was hyperinsulinaemic and displayed an impaired insulin response in a 75 g oral glucose tolerance test and an abnormal homeostasis model insulin resistance index. Serum resistin levels were similar in PCOS patients and controls; however, resistin mRNA levels were 2-fold higher in adipocytes from PCOS patients. No correlation was found between serum resistin levels and either the BMI or testosterone levels. Western blot analysis showed that adipocyte levels of insulin receptor, PI3-kinase, and GLUT4 were respectively decreased by 56, 39.4 and 54% in PCOS patients compared with controls. CONCLUSIONS: These results suggest that overexpression of the resistin gene in adipocytes may be a local determinant factor in the pathogenesis of PCOS.     

Inflammatory cytokines reaction elicited by root-end filling materials

Cytokines are inflammation in the list of tissue reactions that cytokines control of cell and tissue growth, development, and differentiation. Root-end filling materials often contact with existing periapical tissue, and they need to be biocompatible with remnant periapical tissue. The aim of this study was to focus the effects of the root end filling materials on bone cell viability and expression of inflammatory cytokines and their role in maintaining health and stability of the restored dental tissues. Calcium hydroxide-based (Life), zinc oxide eugenol-based (Super EBA), and mineral trioxide aggregate-based (MTA) root-end filling materials were used to investigate their effect on a human osteosarcoma cell line (U2OS). The cell attachment assay was observed microscopically, and the expression of interleukin-2, -4, and -10 were quantified by enzyme-linked immunosorbent assay. Any resultant difference between the root-end filling material was analyzed statistically by one-way analysis of variance. The results showed that the best cell attachment to root-end filling material occurred with MTA. The IL-4 (0.824 +/- 0.396) and IL-10 (2.06 +/- 1.24) levels were greater for the MTA group, whereas IL-2 expression for the three kinds of root-end filling materials was similar. All materials were able to induce expression of inflammatory cytokines from cultured bone cells.     

Comparative effects of tetrandrine and berbamine on acute and relapsing experimental allergic encephalitis in Lewis rats.

The effects of the bisbenzylisoquinoline alkaloids tetrandrine and berbamine on experimental allergic encephalitis (EAE) in Lewis rats was investigated. Amelioration of the acute-phase EAE was only minimal. However, the incidence of relapsing EAE was reduced by 41 and 65% for tetrandrine and berbamine, respectively, at the non-toxic treatment schedule of 60 mg/kg administered on alternate days. These results suggest that bisbenzylisoquinoline compounds may have a potential role in the therapy of progressive multiple sclerosis.     

In vitro immunosuppressive properties of the plant alkaloid tetrandrine

Tetrandrine is an extract of the creeper Stephania tetrandra used in traditional Chinese medicine for the treatment of rheumatic diseases. It has recently been shown to retard and even reverse the lesions of silicosis in humans and rats. Data presented in this report indicate that tetrandrine has potent immunosuppressive properties. Mitogen-induced lymphoproliferative responses were markedly reduced even when tetrandrine was added after the initiation of cultures. In vitro antibody synthesis by B cells was also suppressed by tetrandrine, as was the natural killer cell-mediated lysis of K562 cells. It does not interfere with receptor-ligand binding, but does affect the inositol triphosphate second messenger system. These effects of tetrandrine were observed at nontoxic concentrations as shown by lymphocyte viability studies. These results indicate that tetrandrine possesses potent immunosuppressive properties, and may warrant further study in animal models of chronic inflammatory conditions, autoimmune diseases and transplant rejection.     

Anti-inflammatory and immunosuppressive properties of the bis-benzylisoquinolines: in vitro comparisons of tetrandrine and berbamine

Tetrandrine and berbamine are two naturally occurring analogues with a bis-benzylisoquinoline structure. Comparative in vitro studies show that tetrandrine has significantly greater suppressive effects on adherence, locomotion and 3H-deoxyglucose uptake of neutrophils, as well as the mitogen-induced lymphocyte responses and mixed lymphocyte reactions. Also, tetrandrine displayed anti-oxidant activity while berbamine did not. By contrast, berbamine demonstrated a significantly greater capacity for inhibition of NK cell cytotoxicity. These results show that tetrandrine is superior to berbamine in most aspects of anti-inflammatory and immunosuppressive activity. Since these two alkaloids differ by only one substitution in the side chain of one of the benzene rings, these findings may provide further insight into structure-activity relationships and clues to the synthesis and development of active analogues of this promising class of drugs for the treatment of chronic inflammatory diseases.     

Comparative immunopharmacology and toxicology of the bisbenzylisoquinoline alkaloids tetrandrine and berbamine.

Comparison of the bisbenzylisoquinolines tetrandrine and berbamine shows that both drugs are equipotent in terms of enhancement of antibody responses and suppression of delayed-type hypersensitivity (DTH) responses to sheep red blood cell antigens. Both compounds are also equally active when given to mice during the induction and expression phases of DTH. Using a model of experimental brucellosis in mice, it was found that both compounds did not affect antibody responses, while they caused equipotent suppression of DTH. By contrast, berbamine but not tetrandrine caused significant suppression of spleen weight. Also, berbamine caused a significantly greater enhancement of spleen colony counts of Brucella abortus than tetrandrine. Short-term toxicology studies showed no toxic effects at bioactive doses.