Effects of opsonization and gamma interferon on growth of Brucella melitensis 16M in mouse peritoneal macrophages in vitro

Entry of opsonized pathogens into phagocytes may benefit or, paradoxically, harm the host. Opsonization may trigger antimicrobial mechanisms such as reactive oxygen or nitric oxide (NO) production but may also provide a safe haven for intracellular replication. Brucellae are natural intramacrophage pathogens of rodents, ruminants, dogs, marine mammals, and humans. We evaluated the role of opsonins in Brucella-macrophage interactions by challenging cultured murine peritoneal macrophages with Brucella melitensis 16M treated with complement- and/or antibody-rich serum. Mouse serum rich in antibody against Brucella lipopolysaccharide (LPS) (aLPS) and human complement-rich serum (HCS) each enhanced the macrophage uptake of brucellae. Combinations of suboptimal levels of aLPS (0. 01%) and HCS (2%) synergistically enhanced uptake. The intracellular fate of ingested bacteria was evaluated with an optimal concentration of gentamicin (2 microg/ml) to control extracellular growth but not kill intracellular bacteria. Bacteria opsonized with aLPS and/or HCS grew equally well inside macrophages in the absence of gamma interferon (IFN-gamma). Macrophage activation with IFN-gamma inhibited replication of both opsonized and nonopsonized brucellae but was less effective in inhibiting replication of nonopsonized bacteria. IFN-gamma treatment of macrophages with opsonized or nonopsonized bacteria enhanced NO production, which was blocked by N(G)-monomethyl L-arginine (MMLA), an NO synthesis inhibitor. MMLA also partially blocked IFN-gamma-mediated bacterial growth inhibition. These studies suggest that primary murine macrophages have limited ability to control infection with B. melitensis, even when activated by IFN-gamma in the presence of highly opsonic concentrations of antibody and complement. Additional cellular immune responses, e.g., those mediated by cytotoxic T cells, may play more important roles in the control of murine brucellosis.     

Involvement of protein kinase c in responses of rat dorsal horn neurons to mechanical stimuli and periaqueductal gray descending inhibition

 The effects of a protein kinase C (PKC) activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), on the activity and periaqueductal gray (PAG)-induced inhibition of rat dorsal horn neurons of the lumbar spinal cord were tested. A microdialysis fiber was placed through the dorsal horn for the purpose of local application of pharmacological agents. Extracellular single-unit recordings from dorsal horn neurons were made near the microdialysis fiber. TPA was tested on nociceptive dorsal horn cells. There was a significant increase in the background activity and responses to ”brush”, with no changes in responses to pressure and pinch stimuli. TPA also significantly blocked the PAG-induced inhibition of responses to brush, press, and pinch. These effects were eliminated by coadministration of the PKC inhibitor NPC-15437. The solvent, which contained dimethyl sulfoxide, was also tested for its effect on the responses to peripheral mechanical stimuli and PAG-induced inhibition of the dorsal horn neurons. There were no significant changes. This experiment suggests that activation of the PKC second messenger system might increase the activity of dorsal horn neurons and their responses to peripheral stimuli; in addition, the phorbol ester attenuated the PAG-induced descending inhibition of the dorsal horn neuron activity. Received: 15 May 1996 / Accepted: 14 November 1996     

Denaturing high performance liquid chromatography: high throughput mutation screening in familial hypertrophic cardiomyopathy and SNP genotyping in motor neurone disease

AIMS: To evaluate the usefulness of denaturing high performance liquid chromatography (DHPLC) as a high throughput tool in: (1) DNA mutation detection in familial hypertrophic cardiomyopathy (FHC), and (2) single nucleotide polymorphism (SNP) discovery and validation in sporadic motor neurone disease (MND). METHODS: The coding sequence and intron-exon boundaries of the cardiac beta myosin heavy chain gene (MYH7) were screened by DHPLC for mutation identification in 150 unrelated patients diagnosed with FHC. One hundred and forty patients with sporadic MND were genotyped for the A67T SNP in the poliovirus receptor gene. All DHPLC positive signals were confirmed by conventional methods. RESULTS: Mutation screening of MYH7 covered 10 kb with a total of 5700 amplicons, and more than 6750 DHPLC injections were completed within 35 days. The causative mutation was identified in 14% of FHC cases, including seven novel missense mutations (L227V, E328G, K351E, V411I, M435T, E894G, and E927K). Genotyping of the A67T SNP was performed at two different temperatures both in MND cases and 280 controls. This coding SNP was found more frequently in MND cases (13.6%) than in controls (6.8%). Furthermore, 19 and two SNPs were identified in MYH7 and the poliovirus receptor gene, respectively, during DHPLC screening. CONCLUSIONS: DHPLC is a high throughput, sensitive, specific, and robust platform for the detection of DNA variants, such as disease causing mutations or SNPs. It enables rapid and accurate screening of large genomic regions.     

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.     

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.