Exact test of Hardy-Weinberg equilibrium by Markov chain Monte Carlo.

The assumption of Hardy-Weinberg equilibrium (HWE) among alleles is of fundamental importance in genetic studies. There are numerous testing methods for it using genotype counts data. The exact test is used when the sample size is not large enough for asymptotic approximations. There are several numerical methods to carry out this test, such as complete enumeration, Monte Carlo and Markov chain Monte Carlo simulations. Complete enumeration is impractical in many applications, especially when the table counts are large. The Monte Carlo method is simple to use but still difficult when the table counts become large. The Markov chain Monte Carlo method, by sampling a sub-table each time, is suitable for this latter situation. Based on switches among a few (no more than four) cells, the existing Markov chain samplers are highly dependent and inefficient for large tables. Here we consider a new Markov chain sampling, in which a sub-table of user-specified size is updated at each iteration. The resulting chain is less dependent, and the sampling is flexible and efficient. The conventional test for HWE is based on a few test statistics, such as the likelihood and the chi-squared statistic. To expand the family of test statistics, we consider a class of divergence measures for the departure of HWE. Examples are given as illustrations.     

Selection of cytotoxic T lymphocytes against autologous human melanoma from lymph nodes with metastatic melanoma using repeatedin vitro sensitization

Our goal was to determine the cytotoxic activity of effector cells in lymph nodes with metastatic melanoma. Lymphocytes contained within tumor cells from metastatic lymph nodes of two patients were allowed to proliferate in recombinant IL-2 (rIL-2, 100-1,000 units/ml) after 14–21 days of culture. Each set of lymphocytes showed cytotoxicity against autologous melanoma (AM, mean 72%) at effector to target ratio of 201 and K562 cells (mean 60%) using 4-h chromium-51 release assay. Using unlabeled AM and K562, each AM could partially block the activity against K562, but K562 could not block the activity against AM. These activated lymphocytes underwentin vitro sensitization (IVS) with irradiated AM cells and rIL-2 at 2-week intervals. After repeated IVS over about 50 days, each patient's lymphocytes showed cytotoxicity against AM (mean 54%) but not K562 (mean 5%,P < 0.001). These results indicate that different cytotoxic effector cells were present in the early and late phase of lymphocyte tumor culture. Repeated IVS resulted in the selection of specific cytotoxic T lymphocytes. Cold target inhibition assay demonstrated that melanoma cells contained common and individual AM-associated antigen in addition to K562-associated antigens.This work was supported by Biomedical Research Support Grant of the University of Arizona (no. 2S07 RR05675-20), the Elsa U. Pardee Foundation Grant, partly by the Arizona Chronic Disease Research Commission and partly by CA23074 from the National Institutes of Health, Bethesda, 20892, U.S.A.Recipient of the American Cancer Society Clinical Oncology Career Award, 1987–90.     

Development and characterisation of neutralising monoclonal antibody to the SARS-coronavirus

There is a global need to elucidate protective antigens expressed by the SARS-coronavirus (SARS-CoV). Monoclonal antibody reagents that recognise specific antigens on SARS-CoV are needed urgently. In this report, the development and immunochemical characterisation of a panel of murine monoclonal antibodies (mAbs) against the SARS-CoV is presented, based upon their specificity, binding requirements, and biological activity. Initial screening by ELISA, using highly purified virus as the coating antigen, resulted in the selection of 103 mAbs to the SARS virus. Subsequent screening steps reduced this panel to seventeen IgG mAbs. A single mAb, F26G15, is specific for the nucleoprotein as seen in Western immunoblot while five other mAbs react with the Spike protein. Two of these Spike-specific mAbs demonstrate the ability to neutralise SARS-CoV in vitro while another four Western immunoblot-negative mAbs also neutralise the virus. The utility of these mAbs for diagnostic development is demonstrated. Antibody from convalescent SARS patients, but not normal human serum, is also shown to specifically compete off binding of mAbs to whole SARS-CoV. These studies highlight the importance of using standardised assays and reagents. These mAbs will be useful for the development of diagnostic tests, studies of SARS-CoV pathogenesis and vaccine development.     

Synergistic antimicrobial effect of cefotaxime and minocycline on proinflammatory cytokine levels in a murine model of Vibrio vulnificus infection.

BACKGROUND AND PURPOSE: Vibrio vulnificus causes primary bacteremia and necrotizing wound infection, leading to high morbidity and mortality in humans. This study aimed to evaluate the antimicrobial effect of cefotaxime and minocycline on proinflammatory cytokine levels in a murine model of V. vulnificus infection. METHODS: We investigated the dynamics of proinflammatory cytokines and their modulation by antimicrobial agents using a murine model of V. vulnificus infection. The change in cytokine levels was followed over a time course to identify the antimicrobial activity of the drugs against V. vulnificus. BALB/c female mice were challenged with an intraperitoneal infection using a clinical invasive isolate of Vv05191, and their cytokine levels were assayed over various time points. RESULTS: Serum levels of tumor necrosis factor-alpha, interleukin (IL)-1 beta, and IL-6 post-infection were found to be inoculum dose-dependent and positively correlated to the subsequent fatality rate in the infected mice. With an inoculum of 6.6 x 10(6) colony-forming units and intraperitoneal administration of cefotaxime, minocycline, or both, the serum and peritoneal fluid cytokine levels increased and then declined gradually. Comparison of the 3 antimicrobial regimens revealed that the magnitude of reduction in cytokine levels was greatest in mice treated with cefotaxime-minocycline combination. Moreover, the peritoneal fluid cytokine level in the combination group was significantly lower than that in the groups treated with minocycline or cefotaxime alone. CONCLUSIONS: The current results support the superiority of the combination therapy in treating invasive V. vulnificus infections.     

PBX1基因剪切体表达与SLE的相关研究

了解PBX1基因各种剪切体的表达在SLE患者和正常人中是否存在差异 ,探讨PBX1的表达与SLE发病的相关性。通过PCR扩增及毛细管芯片电泳 ,确证剪切体h、k、l存在于人体 ;通过实时荧光定量PCR技术 ,对剪切体h、k、l分别进行SLE患者组和正常组的mRNA表达定量比较。结果发现这 3种剪切体在患者组中的表达较正常人明显降低 ,正常人的表达是SLE的 9～ 12倍。重度患者的k、l剪切体与轻中度的病人相比表达明显降低 ,并发狼疮性肾炎的病人k剪切体的表达较无肾累及的病人显著降低。说明PBX1基因剪切体h、k、l在SLE患者中mRNA表达水平下降 ,并与SLE活动度及肾累及有关。提示机体通过PBX1的表达量的调节可能参与SLE的发病     

Effect of illumination of nitrate and phosphate removal by coimmobilized Chlorella pyrenoidosa and activated sludge

Chlorella pyrenoidosa and activated sludge were co-immobilized with simplifying modified PVA-sulfate method. Effects of light intensity and light:dark ratio on the growth of co-immobilized algae cells and removal of nitrate and phosphate were studied. The results indicate that the growth rate of co-immobilized algae cells reduces when light intensity is decreased from 4000 Lux to 1000 Lux, while optimal light:dark ratio for algal growth is 16:8. The influence of illumination on nitrate removal is so weak that the removal percentage can reach 90 approximately 100% within 12 approximately 24 h during the experimental periods in spite of changing illumination conditions. On the other hand, phosphate removal efficiency reduces when light intensity or light:dark ratio is decreased. The highest phosphate removal percentage is 99.6% under the circumstances of 4000 Lux and full-time illumination in our experiment, while the average phosphate removal is about 78%. The change of pH value in water samples is also observed. When water sample is treated by the co-immobilized system, pH value increases in light and decreases in dark. Microorganisms' physiological action is considered as the main mechanism that leads to the change of pH value.     

The ability of heat-killed Mycobacterium vaccae to stimulate a cytotoxic T-cell response to an unrelated protein is associated with a 65 kilodalton heat-shock protein

Exogenous antigens are generally presented by Class II major histocompatibility (MHC) molecules. When administered with an adjuvant, however, they are capable of inducing a CD8+ T-cell response where antigen recognition is associated with Class I MHC. Accordingly, immunization with soluble ovalbumin (OVA) alone does not activate CD8+ cytotoxic T cells (CTL) but when given in complete Freund's adjuvant (CFA), or in formulations of a number of novel adjuvants, an OVA-specific CD8+ CTL response can be detected. We show in this report that immunization with soluble OVA mixed with heat-killed Mycobacterium vaccae, but not with other common pathogenic and saprophytic mycobacteria, can activate OVA-specific CD8+ CTL. An OVA-specific CTL response is detected when mice are immunized by either the intraperitoneal or intranasal route and their spleen cells are re-stimulated in vitro. Adjuvant activity of heat-killed M. vaccae is present in M. vaccae culture filtrate, in soluble protein components of whole M. vaccae and in the 65 kDa heat-shock protein (hsp) of M. vaccae. Mycobacterium vaccae has previously been shown to have no adverse side-effects in humans. The current results suggest that M. vaccae may be useful as an adjuvant for vaccines and other immunotherapies where CD8+ CTL responses to exogenous proteins are crucial.     

Nucleolar localization of non-structural protein 3b, a protein specifically encoded by the severe acute respiratory syndrome coronavirus

The open reading frame 3 (ORF3) of the severe acute respiratory syndrome coronavirus (SARS-CoV) genome encodes a predicted 154-amino acid protein, which lacks similarities to any known protein, and is named 3b. In this study, it was shown that 3b protein was predominately localized to nucleus with EGFP tag at its N- or C-terminus. The localization patterns were similar in different transfected cells. Immuno-fluorescence assay revealed that 3b protein was co-localized well with C23 in nucleolus. C23, B23 and fibrillarin all are important nucleolar proteins, which localize in the region of the nucleolus. Co-transfection of p3b-EGFP with pC23-DsRed, pB23-DsRed and pfibrillarin-DsRed further confirmed 3b's nucleolus localization. With construction of serial truncated mutants of 3b, a region (residues 134-154 aa) responsible for nucleolar localization was determinated in 3b protein. These results provide a new insight for further functional studies of SARS-CoV 3b protein.