Role of double-stranded RNA and Npro of classical swine fever virus in the activation of monocyte-derived dendritic cells

Classical swine fever virus (CSFV) is a noncytopathogenic (ncp) positive-sense RNA virus that replicates in myeloid cells including macrophages and dendritic cells (DC). The virus does not induce type I interferon (IFN-alpha/beta), which in macrophages has been related to the presence of the viral Npro gene. In the present work, the role of viral double-stranded (ds)RNA and Npro in the virus-host cell interaction has been analyzed. Higher levels of detectable dsRNA were produced by a genetically engineered cytopathogenic (cp) CSFV compared with ncp CSFV, and cp CSFV induced IFN-alpha/beta in PK-15 cells. With DC, there was only a small difference in the levels of dsRNA between the cp and ncp viruses, and no IFN-alpha/beta was produced. However, the cp virus induced a higher degree of DC maturation, in terms of CD80/86 and MHC II expression. Npro deletion mutants induced an increase in DC maturation and IFN-alpha/beta production-for both ncp and cp viruses-despite reduced replication efficiency in the DC. Deletion of Npro did not influence dsRNA levels, indicating that the interference was downstream of dsRNA turnover regulation. In conclusion, the capacity of CSFV to replicate in myeloid DC, and prevent IFN-alpha/beta induction and DC maturation, requires both regulated dsRNA levels and the presence of viral Npro.     

alpha(v)beta(3) Integrin in central nervous system tumors

alpha(v)beta(3) Is an integrin specifically expressed in endothelial cells of newly forming blood vessels. Integrin-mediated angiogenesis is hypothesized to play a central role in the development and the progression of central nervous system neoplasms. Accordingly, it is considered a potential target for antiangiogenic therapy. In the current study, we compare the expression of alpha(v)beta(3) in ependymomas, oligodendrogliomas, pilocytic astrocytomas, medulloblastomas, and vestibular schwannomas (acoustic neuromas). Samples of 5 tumors of each of the 5 tumor types were harvested surgically and frozen. After the pathological diagnosis was confirmed, immunohistochemistry was performed using an anti- alpha(v)beta(3) monoclonal antibody (LM609). The expression of alpha(v)beta(3) was assessed using a 4-tiered (0-3) grading scheme reflecting the percentage of positively staining vessels. All vestibular schwannomas demonstrated strong (grade 3) alpha(v)beta(3) expression. The expression was uniformly prominent in Antoni B regions of the tumors. Of 5 ependymomas, 4 demonstrated uniformly strong alpha(v)beta(3). Oligodendrogliomas, medulloblastomas, and pilocytic astrocytomas demonstrated more variable alpha(v)beta(3). alpha(v)beta(3) may contribute significantly to angiogenesis in vestibular schwannomas and ependymomas. Despite the high vascular density of oligodendrogliomas, pilocytic astrocytomas, and medulloblastomas, these tumors had variable moderate alpha(v)beta(3) expression. This discrepancy suggests temporal and/or regional variability in the angiogenesis in these types of tumor. This study provides the first demonstration of alpha(v)beta(3) expression in vestibular schwannomas, medulloblastomas, and pilocytic astrocytomas.     

IL-15 and IL-15Rα gene deletion: Effects on T lymphocyte trafficking and the microglial and neuronal responses to facial nerve axotomy

IL-15 is a potent T cell chemoattractant, and this cytokine and its unique α subunits, IL-15Rα, can modify immune cell expression of several T cell chemokines and their receptors. Facial nerve axotomy in mice leads to T cell migration across an intact blood–brain-barrier (BBB), and under certain conditions T cells can provide neuroprotection to injured neurons in the facial motor nucleus (FMN). Although chemokines and chemoattractant cytokines are thought to be responsible for T cell migration to the injured cell bodies, data addressing this question are lacking. This study tested the hypothesis that T cell homing to the axotomized FMN would be impaired in knockout (KO) mice with the IL-15 and IL-15Rα genes deleted, and sought to determine if microglial responsiveness and motoneuron death are affected. Both IL-15KO and IL-15RαKO mice exhibited a marked reduction in CD3⁺ T cells and had fewer MHC2⁺ activated microglia in the injured FMN than their respective WT controls at day 14 post-axotomy. Although there was a relative absence of T cell recruitment into the axotomized FMN in both knockout strains, IL-15RαKO mice had five times more motoneuron death (characterized by perineuronal microglial clusters engulfing dead motoneurons) than their WT controls, whereas dead neurons in IL-15KO did not differ from their WT controls. Further studies are needed to dissect the mechanisms that underlie these observations (e.g., central vs. peripheral immune contributions).     

饮食性缺铜、缺锌对小鼠细胞因子产生的影响

３周龄小鼠饲用６周缺铜饮食后，出现胸腺萎缩、肝脏肿大以及明显的缺铜指征，包括血清Ｃｐ活性、肝Ｃｕ含量、ＣＣＯ和Ｃｕ，Ｚｎ－ＳＯＤ活性明显下降。缺钢小鼠产生的ＴＮＦ－α、ＩＬ－１和ＩＬ－６水平明显低于正常组小鼠。小鼠饲予７周缺锌饮食后，出现明显脱毛，虽然肝Ｚｎ含量降低不明显，但ＴＮＦ－α、ＩＬ－１和ＩＬ－６活性明显降低，说明上述细胞因子是小鼠缺锌的敏感指征之一。本文结果显示正常Ｃｕ、Ｚｎ水平对ＴＮＦ－α、ＩＬ－１和ＩＬ－６产生的重要性，并提示这些细胞因子的改变可能是缺铜、缺锌引起的免疫功能损害的细胞、分子学机理之一。     

干扰素基因定向进化库的建立及初步筛选

目的构建α干扰素基因家族定向文库。方法利用D NAshuffling技术对人、鸡和猪α干扰素基因进行人工改造，结合噬菌体展示技术构建噬菌体基因改组文库，并对文库进行验证。结果构建了α干扰素基因家族定向文库，其容量为3．5×10^7pfu；测序得到了3条与猪、人α干扰素基因高度同源的序列。结论得到了1个高质量的α干扰素基因家族定向文库，为今后筛选高效的α干扰素奠定了基础。     

Influence of antibody and complement components on phagocytosis and chemiluminescence of macrophages

Macrophages are known to release reactive oxygen species (O₂^?, ¹O₂, H₂O₂, OH·) in response to various membrane stimuliHowever, our studies show that phagocytic stimulation of macrophages is not necessarily accompanied by a stimulation of the oxidative burstWhereas IgG-opsonized erythrocytes were capable to induce phagocytosis and a chemiluminescence response, both being dependent on the number of IgG bound per erythrocyte, C3b-bearing erythrocytes were well ingested but failed to induce any chemiluminescence reactionFurthermore, stimulation of macrophages, via the Fc-receptors, seems to alter their functional state in regard to the activation of a receptor, which enables them to recognize membrane lesions on the target erythrocyteThe presence of IgG and membrane lesions, e.gthe C5b-9-complex of complement, induced a marked increase in chemiluminescence compared with stimulation by IgG-bearing particles aloneThe augmented response of macrophages was at least in part due to an additional release of H₂O₂, which was not liberated in response to IgG-bearing erythrocytesThis «Alesion recognizing receptor» in the macrophage membrane could not be activated by stimulation of C3b-receptors, indicating its functional linkage to the Fc-receptors.     

Increased Mycobacterium tuberculosis growth in HIV-1-infected human macrophages: role of tumour necrosis factor-alpha

Synergism between Mycobacterium tuberculosis (M. tuberculosis) and HIV-1 infections was demonstrated in several in vitro models and clinical studies. Here, we investigated their reciprocal effects on growth in chronically HIV-1-infected promonocytic U1 cells and in acutely infected monocyte-derived macrophages (MDM). Phagocytosis of M. tuberculosis induced HIV-1 expression in U1 cells, together with increased TNF-alpha production. M. tuberculosis growth, evaluated by competitive PCR, was greater in HIV-1-infected MDM compared to uninfected cells. M. tuberculosis phagocytosis induced greater TNF-alpha and IL-10 production in HIV-1-infected MDM than in uninfected cells. In uninfected MDM, addition of TNF-alpha and IFN-gamma decreased, whereas IL-10 increased M. tuberculosis growth. On the contrary, in HIV-1-infected MDM, addition of TNF-alpha and IFN-gamma increased, whereas IL-10 has no effect on M. tuberculosis growth. TNF-alpha seems to play a pivotal role in the enhanced M. tuberculosis growth observed in HIV-1-infected MDM, being unable to exert its physiological antimycobacterial activity. Here, for the first time we demonstrated an enhanced M. tuberculosis growth in HIV-1-infected MDM, in line with the observed clinical synergism between the two infections.     

针刺对多囊卵巢综合征大鼠卵巢TGF-α、EGFR表达的影响

目的:研究针刺对多囊卵巢综合征(PCOS)大鼠卵巢转化生长因子α(TGF-α)、表皮生长因子受体(EGFR)表达的影响,探讨针刺促排卵的作用机制。方法:24日龄雌性大鼠颈背部皮下注射脱氢表雄酮(DHEA)的油溶液制作(PCOS)模型,对照组同期皮下注射油剂。PCOS大鼠随机分为模型组和针刺组。模型组不作处理,针刺组大鼠从80日龄起针刺关元、中极、双侧三阴交、双侧子宫穴,1次/天,15min/次,连续6周。治疗结束后各组大鼠断头处死,迅速取血并分离血清,-20℃冰箱保存,待测性激素水平。摘取双侧卵巢,称重,4%多聚甲醛固定,作HE染色和免疫组织化学染色。结果:与模型组相比,针刺组卵巢湿重、卵巢TGF-α、EGFR表达及血清睾酮(T)、雌二醇(E2)水平均显著降低(P<0.01),而卵泡刺激素(FSH)、黄体生成素(LH)、孕酮(P4)水平差异无统计学意义(P>0.05)。结论:针刺能显著降低PCOS大鼠卵巢TGF-α及EGFR的表达,抑制TGF-α对卵巢和激素合成的作用,改善PCOS大鼠多囊样变和高雄激素血症,促进排卵。     

Expression of alpha-methylacyl-CoA racemase (P504s) in various malignant neoplasms and normal tissues: astudy of 761 cases

Alpha-methylacyl CoA racemase (AMACR), also known as P504S, plays an important role in peroxisomal beta-oxidation of branched-chain fatty acids. It has recently been shown that AMACR is highly expressed in prostate cancer and that it may be an important diagnostic marker for prostate carcinoma. However, little is known about expression of AMACR in normal tissues and other malignant tumors. In this study, we investigated expression of AMACR in 539 malignant tumors and 222 normal human tissues of various types by immunohistochemical analysis. mRNA levels of AMACR in normal organs and in selected tumors were assessed by real time PCR. In normal tissue, high expression of AMACR mRNA was identified in liver, kidney and salivary gland, while AMACR protein was detected in liver (hepatocytes), kidney (tubular epithelial cells), lung (only bronchial epithelial cells), and gallbladder (only mucosal epithelial cells). High expression of AMACR mRNA was found in prostate, liver, and kidney cancers but rarely in stomach and bladder cancers. A high percent of adenocarcinomas arising from these organs express AMACR, including 17 of 21 (81%) of hepatocellular carcinomas and 18 of 24 (75%) of renal cell carcinomas. In addition, carcinomas arising from tissues normally not expressing AMACR were also positive for the antigen, including 17 of 18 (94%) prostate carcinomas, 9 of 29 (31%) of urothelial carcinomas, and 4 of 15 (27%) of gastric adenocarcinomas. Two hundred and fifty cases of adenocarcinomas from lung, breast, pancreas, bile duct, adrenal gland, salivary gland, ovary, thyroid and endometrium were negative or rarely positive for AMACR. Neuroendocrine carcinomas rarely expressed AMACR. Melanomas, squamous cell carcinomas, basal cell carcinomas, soft tissue tumors (including epithelioid sarcomas and synovial sarcoma), thymomas, and germ cell tumors were negative for AMACR. Our data provide important baseline information for using AMACR in clinical practice and also are valuable in furthering understanding of the pathogenic role of AMACR in malignant neoplasms.     

Expression of TNFalpha by muscle fibers in biopsies from children with untreated juvenile dermatomyositis: association with the TNFalpha-308A allele

Juvenile dermatomyositis (JDM) is the most common pediatric inflammatory myopathy. In patients with JDM, the A --> G polymorphism in the tumor necrosis factor alpha (TNFalpha)-308 promoter region (TNFalpha-308A) is associated with prolonged disease course and increased production of TNFalpha by peripheral blood mononuclear cells (Arthritis Rheum. 43, 2368-2377, 2000). Magnetic resonance imaging directed biopsies from 21 white children with untreated JDM were evaluated for TNFalpha expression. Using monoclonal antibody to TNFalpha, fresh frozen sections were processed by the standard immunohistochemical technique. We investigated the association among the expression of TNFalpha by muscle fibers, disease activity, duration of untreated disease, and the TNFalpha-308 polymorphism. Untreated children with JDM who had the TNFalpha-308A allele had an increased number of TNFalpha stained muscle fibers than children with the TNFalpha-308G allele (P = 0.001). There was no association with disease activity or duration of untreated disease. We speculate that muscle fiber production of TNFalpha provides a microenvironment in which TNFalpha acts synergistically with other mediators to prolong muscle fiber damage.