17种植物中蛋白质提取物的抗HIV—1活性

以化合物对ＨＩＶ－１诱导Ｃ８１６６细胞形成合胞体的抑制实验和化合物对ＨＩＶ－１感染细胞的保护实验作为初筛方法,筛选了来源７科１７种植物的４７个样品,其中８个样品经测定是核糖体失活蛋白,其余为粗提蛋白。     

检测HIV—1载量的荧光实时定量PCR技术的建立其应用

准确测定HIV－1的前病毒载量和病毒载量的技术,在感染者预后和艾滋病患者药物治疗效果的评价以及艾滋病的其它研究方面,都具有十分重要的应用价值。以定量的HIV－1 DNA和RNA为标准外参照,利用SYBR Green荧光染料和GeneAmp5700Sequence Detection System（5700系统）,建立了测定HIV－1的前病毒载量和病毒载量的荧光实时定量PCR技术,以病毒感染细胞和培养上清上材料,测定了三种化合物（AZT,GL和WT）对细胞内的前病毒载量和培养上清中的病毒载量的抑制活性,并与合胞体形成抑制方法测定化合物抗病毒活性的结果进行了比较,根据病毒载量、前病毒载量和合胞体形成计算出的三种化合物的治疗指数均依次变小,提出以荧光实时定量PCR技术测定前病毒载量,会在评价药物在体内外根除或减少存在于CD4休止或记忆T淋巴细胞中的HIV－1前病毒方面有特别的价值。     

细胞间传播在人类免疫缺陷病毒感染中的作用

理论和实验均有人类免疫缺陷病毒可能通过细胞间的机制传播。本文提出了认和体液中细胞相关性病毒启动感染的根据，分析了病毒通过形成合胞体及以粘附为基础，从一个细胞向另一转移的机制，着重强调了不形成合胞体的细胞－细胞间传播，为艾滋病的治疗提供了新的思路。     

组胺H3受体激活剂高通量筛选细胞模型的研究

摘 要 目的 建立基于报告基因的组胺H3受体（H3R）激活剂的高通量筛选模型,用此模型对收集到的中草药化合物组分进行筛选,以发现新的组胺H3R激活剂。 方法 将H3R基因质粒（H3R/pCDNA3.1-hygro）与报告基因质粒（3XCRE-LUC）按3：1的比例共转染入HEK293细胞,建立了稳定的H3R配体的报告基因筛选细胞株。激活剂与细胞表面H3R结合后,激活相应的信号通路,调节Forskolin刺激后的报告基因的表达,通过测定荧光素酶报告基因表达水平的变化,评估激活剂影响H3受体的生物活性。 结果 通过对筛选条件,如激活剂孵育时间、Forskolin终浓度、化合物溶剂的选择、溶剂DMSO终浓度等的优化,建立了可靠的筛选方法,并对多种中草药萃取物进行了筛选,找到了两种对H3R有活性的中药组分。结论 建立的细胞模型可以有效的应用于以组胺H3受体为靶点的高通量药物筛选。     

检测HIV-1载量的荧光实时定量PCR技术的建立及其应用

准确测定HIV-1的前病毒载量和病毒载量的技术,在感染者预后和艾滋病患者药物治疗效果的评价以及艾滋病的其它研究方面,都具有十分重要的应用价值。以定量的HIV-1DNA和RNA为标准外参照,利用SYBRGreen荧光染料和GeneAmp5700 Sequence Detection System(5700系统),建立了测定HIV-1的前病毒载量和病毒载量的荧光实时定量PCR技术。以病毒感染细胞和培养上清为材料,测定了三种化合物(AZT,GL和WT)对细胞内的前病毒载量和培养上清中的病毒载量的抑制活性,并与合胞体形成抑制方法测定化合物抗病毒活性的结果进行了比较。根据病毒载量、前病毒载量和合胞体形成计算出的三种化合物的治疗指数均依次变小,提出以荧光实时定量PCR技术测定前病毒载量,会在评价药物在体内外根除或减少存在于CD4休止或记忆T淋巴细胞中的HIV-1前病毒方面有特别的价值。     

检测HIV-1载量的荧光实时定量PCR技术的建立及其应用

准确测定HIV 1的前病毒载量和病毒载量的技术 ,在感染者预后和艾滋病患者药物治疗效果的评价以及艾滋病的其它研究方面 ,都具有十分重要的应用价值。以定量的HIV 1DNA和RNA为标准外参照 ,利用SYBRGreen荧光染料和GeneAmp5 70 0SequenceDetectionSystem (5 70 0系统 ) ,建立了测定HIV 1的前病毒载量和病毒载量的荧光实时定量PCR技术。以病毒感染细胞和培养上清为材料 ,测定了三种化合物 (AZT ,GL和WT)对细胞内的前病毒载量和培养上清中的病毒载量的抑制活性 ,并与合胞体形成抑制方法测定化合物抗病毒活性的结果进行了比较。根据病毒载量、前病毒载量和合胞体形成计算出的三种化合物的治疗指数均依次变小 ,提出以荧光实时定量PCR技术测定前病毒载量 ,会在评价药物在体内外根除或减少存在于CD4休止或记忆T淋巴细胞中的HIV 1前病毒方面有特别的价值。     

评价艾滋病治疗药物鼠模型的研究进展

理想的动物模型,特别是小动物模型,对于发展和评价艾滋病的治疗药物是非常重要的。近年来,有关艾滋病动物模型的研究取得了一些进展。本文仅就评价艾滋病治疗药物中常用的鼠模型研究的进展作一综述。大动物模型研究的进展请参考有关文献［‘－’］。l鼠白血病病毒模型鼠白血病病毒（Murineleukemiavirus,MuLV）属于反转录病毒科。将它接种于敏感鼠后,可以导致牌和淋巴结增大,免疫抑制和机会性感染等症状。有人称其为鼠艾滋病（MurineAcquiredImmunodeficiencySyndrom,MAIDS）14］。近年来MuLV／MAIDS模型系统已经被应用于发…     

胃肠平滑肌运动单位SIP合胞体的生理与病理生理学意义

胃肠平滑肌层富有特殊分化的两种间质细胞,包括Cajal间质细胞(interstitial cells of Cajal,ICC)以及血小板衍生因子受体α阳性细胞(platelet-derived growth factor receptorα-positive cells,PDGFRα~+细胞)。ICC和PDGFRα~+细胞分别与平滑肌细胞(smooth muscle cells,SMC)形成缝隙连接调控平滑肌的收缩功能,因此,这三种细胞共同构成功能性的合胞体,称为SMC、ICC和PDGFRα~+细胞合胞体(SIP合胞体)。各种神经递质、体液因子、内源性生物活性分子以及药物等可以通过SIP合胞体影响胃肠运动。本文综述了SIP合胞体及其作用机制以及生理与病理生理学意义。     

用于筛选抑制丙型肝炎病毒内部核糖体进入位点活性药物的整合型细胞药物筛选模型的构建和评价

目的：构建一个能用于筛选抑制丙型肝炎病毒内部核糖体进入位点（HCV—IRES）活性药物的整合型细胞药筛模型。方法：构建携带HCV 5＇-UTR调控报告基因分泌型碱性磷酸酶（SEAP）基因的质粒pHCV5’-SEAP和用作筛选标记的pNeo^R,线性化后共转染于Huh-7细胞中,经G418筛选后得到抗性细胞克隆,进行SEAP定量检测筛选后,得到能用于筛选抑制HCV-IRES活性药物的整合型细胞药筛模型;连续培养24代,用MTT法测细胞相对活力评价该药筛模型的生长稳定性,用SEAP定量检测法评价该药筛模型的SEAP表达稳定性,用反义寡聚核苷酸抑制试验评价模型的药筛灵敏度。结果：G418筛选后得到26个抗性细胞克隆;对其中5个抗性细胞克隆进行SEAP定量检测筛选后,得到3个能表达SEAP的阳性细胞克隆;连续培养24代过程中,该药筛模型的生长稳定性大干80％,SEAP表达稳定性达95％。结论：构建的细胞模型具有良好的稳定性和药筛灵敏度,符合作为药物筛选模型的要求。     

艾滋病治疗药物-HIV抑制剂作用机制及研究进展

艾滋病（AIDS）是由人类免疫缺陷病毒（HIV）感染而引起的慢性进行性致死性传染病,又称获得性免疫缺陷综合症,目前无有效治愈的方法,严重危害着人类的健康。现今,艾滋病治疗药物主要包括逆转录酶抑制剂、蛋白酶抑制剂、进入抑制剂、整合酶抑制剂四大类化学药物和一些中草药制剂。抗HIV药物虽然不能完全治愈艾滋病,但可以控制艾滋病病情的发展,延长患者的无病生存期,提高患者的生活质量。本文就艾滋病发病机制、HIV抑制药物的抗病机制、副作用及其研究进展做一综述。     

Retroperitoneal fibromatosis and acquired immunodeficiency syndrome in macaques: epidemiologic studies

At the University of Washington Regional Primate Research Center, a simian acquired immunodeficiency syndrome (SAIDS) associated with retroperitoneal fibromatosis (RF) has been observed in 82 macaques since 1976, including 77 pigtailed macaques (Macaca nemestrina), two long-tailed macaques (M. fascicularis), one Japanese macaque (M. fuscata) and two rhesus macaques (M. mulatta). The syndrome is characterized by immunodeficiency accompanied by a fibroproliferative lesion, primarily affects young monkeys (1-3 years) and has a high case fatality rate. Based on the occurrence of RF in colony-born and non-colony-born monkeys, the minimum incubation period for natural exposure is believed to be about 9 months. The incidence of RF was 0.9% in M. nemestrina, 0.1% in M. fascicularis, 1.0% in M. fuscata and 0.4% in M. mulatta. There were no significant differences in the incidence of RF by sex or seasonality. Epidemiologic studies were focused on 42 juvenile M. nemestrina that developed RF between January 1980 and June 1983, and the results were compared with 42 age- and sex-matched controls. The incidence of RF was 5.7% in monkeys 12-24 months old and 3.4% in monkeys 24-36 months old, but less than 1.0% in age groups of under 1 year and over 3 years. No significant associations were found for housing history, parentage, generations or ancestral origins. Epidemiologic information and preliminary viral studies suggest a type D retrovirus may be the causative agent in RF and SAIDS. RF associated with SAIDS appears to be an excellent model for Kaposi's sarcoma associated with human AIDS.     

Viral etiologies of AIDS: facts and hypotheses

All the epidemiological features suggest that the acquired immunodeficiency syndrome (AIDS) is caused by a single transmissible agent and surely a virus. First, cytomegalovirus, Epstein-Barr virus and hepatitis B virus have been proposed as possible etiological agents of AIDS. A direct link between ubiquitous viruses and the occurrence of the disease has been discarded. At present time, etiological researches provide evidence that retroviruses are the best candidates for the etiology of AIDS. These agents could be directly responsible of the profound suppression of the cell-mediated immunity observed in patients with AIDS. Two human retroviruses are now proposed: human T-cell leukemia virus (HTLV) or lymphadenopathy associated virus (LAV). Moreover simian AIDS (SAIDS) occurred spontaneously at several primate centers in USA; a retrovirus partially related to Mason Pfizer monkey virus appears to be the etiologic agent of SAIDS.     

Frequent simian foamy virus infection in persons occupationally exposed to nonhuman primates

The recognition that AIDS originated as a zoonosis heightens public health concerns associated with human infection by simian retroviruses endemic in nonhuman primates (NHPs). These retroviruses include simian immunodeficiency virus (SIV), simian T-cell lymphotropic virus (STLV), simian type D retrovirus (SRV), and simian foamy virus (SFV). Although occasional infection with SIV, SRV, or SFV in persons occupationally exposed to NHPs has been reported, the characteristics and significance of these zoonotic infections are not fully defined. Surveillance for simian retroviruses at three research centers and two zoos identified no SIV, SRV, or STLV infection in 187 participants. However, 10 of 187 persons (5.3%) tested positive for SFV antibodies by Western blot (WB) analysis. Eight of the 10 were males, and 3 of the 10 worked at zoos. SFV integrase gene (int) and gag sequences were PCR amplified from the peripheral blood lymphocytes available from 9 of the 10 persons. Phylogenetic analysis showed SFV infection originating from chimpanzees (n = 8) and baboons (n = 1). SFV seropositivity for periods of 8 to 26 years (median, 22 years) was documented for six workers for whom archived serum samples were available, demonstrating long-standing SFV infection. All 10 persons reported general good health, and secondary transmission of SFV was not observed in three wives available for WB and PCR testing. Additional phylogenetic analysis of int and gag sequences provided the first direct evidence identifying the source chimpanzees of the SFV infection in two workers. This study documents more frequent infection with SFV than with other simian retroviruses in persons working with NHPs and provides important information on the natural history and species origin of these infections. Our data highlight the importance of studies to better define the public health implications of zoonotic SFV infections.     

Retroperitoneal fibromatosis and acquired immunodeficiency syndrome in macaques: clinical and immunologic studies

A simian acquired immunodeficiency syndrome (SAIDS) associated with retroperitoneal fibromatosis (RF) has been observed in several species of macaque at the Washington Regional Primate Research Center. Clinical signs were recurrent diarrhea, weight loss, mesenteric lymphadenopathy, and opportunistic infections. Most affected macaques in the later stages of illness showed marked immunodeficiency. Response of peripheral blood mononuclear cells to mitogens was impaired significantly. There was sharply depressed primary and secondary antibody response to the T-cell dependent antigen, bacteriophage phi X174. Affected monkeys did not switch from IgM to IgG antibody following a secondary immunization, as did normal macaques. Twenty-four (67%) of 36 affected animals with progressive RF or deteriorated stages of illness had hypoproteinemia and hypoalbuminemia. Quantitative serum immunoglobulins of 23 cases showed that eight (35%) had hypogammaglobulinemia, six (26%) had hypergammaglobulinemia, and the remainder (39%) were within the normal range. Opportunistic infections were predominantly bacterial pathogens. Type D retrovirus appeared to be closely associated with RF-affected macaques (12/12 or 100%). The case fatality rate (including animals sacrificed after prolonged illness) was 98%. The leading cause of death was due directly to RF lesions in 43%, to enterocolitis in 36%, septicemia in 12%, amyloidosis in 5%, and malignant lymphoma (2%). Clinical, immunologic and pathologic changes reveal an acquired immunodeficiency syndrome that has many similarities to human AIDS. SAIDS and RF may be a useful model for studying human AIDS.     

人工饲养中国猕猴中SRV、STLV和BV的流行病学调查

非人灵长类动物是十分重要的生物医学资源。由于与人类在生理生化、免疫、遗传等方面近似,猕猴是重要的非人灵长类实验动物之一。然而,猕猴作为自然宿主,易感染D型逆转录病毒(simian type D retrovirus,SRV)和T淋巴细胞白血病病毒(simian T lymphotropic virus,STLV)这两种逆转录病毒,并可能会影响AIDS猕猴动物模型等的研究结果。猴B病毒(ceropithecine herpesvirus1,BV)对猕猴及动物从业人员均有危害。云南省拥有较大规模的中国猕猴繁殖种群。基于以上原因,建立SPF级别的中国猕猴种群十分必要。该文应用PCR技术筛查了人工饲养种群中411只中国猕猴的SRV、STLV和BV感染流行情况。结果表明:SRV、STLV和BV的阳性感染率分别为19.71%(81/411)、13.38%(55/411)和23.11%(95/411)。同时比较分析了不同性别及年龄组中国猕猴的病毒感染情况。该研究将有助于建立SPF级别的中国猕猴繁殖种群。     

Role of the SH3-Ligand Domain of Simian Immunodeficiency Virus Nef in Interaction with Nef-Associated Kinase and Simian AIDS in Rhesus Macaques

The nef gene of the human and simian immunodeficiency viruses (HIV and SIV) is dispensable for viral replication in T-cell lines; however, it is essential for high virus loads and progression to simian AIDS (SAIDS) in SIV-infected adult rhesus macaques. Nef proteins from HIV type 1 (HIV-1), HIV-2, and SIV contain a proline-Xaa-Xaa-proline (PxxP) motif. The region of Nef with this motif is similar to the Src homology region 3 (SH3) ligand domain found in many cell signaling proteins. In virus-infected lymphoid cells, Nef interacts with a cellular serine/threonine kinase, designated Nef-associated kinase (NAK). In this study, analysis of viral clones containing point mutations in the nef gene of the pathogenic clone SIVmac239 revealed that several strictly conserved residues in the PxxP region were essential for Nef-NAK interaction. The results of this analysis of Nef mutations in in vitro kinase assays indicated that the PxxP region in SIV Nef was strikingly similar to the consensus sequence for SH3 ligand domains possessing the minus orientation. To test the significance of the PxxP motif of Nef for viral pathogenesis, each proline was mutated to an alanine to produce the viral clone SIVmac239-P¹⁰⁴A/P¹⁰⁷A. This clone, expressing Nef that does not associate with NAK, was inoculated into seven juvenile rhesus macaques. In vitro kinase assays were performed on virus recovered from each animal; the ability of Nef to associate with NAK was restored in five of these animals as early as 8 weeks after infection. Analysis of nef genes from these viruses revealed patterns of genotypic reversion in the mutated PxxP motif. These revertant genotypes, which included a second-site suppressor mutation, restored the ability of Nef to interact with NAK. Additionally, the proportion of revertant viruses increased progressively during the course of infection in these animals, and two of these animals developed fatal SAIDS. Taken together, these results demonstrated that in vivo selection for the ability of SIV Nef to associate with NAK was correlated with the induction of SAIDS. Accordingly, these studies implicate a role for the conserved SH3 ligand domain for Nef function in virally induced immunodeficiency.     

Viral factors determine progression to AIDS in simian immunodeficiency virus-infected newborn rhesus macaques.

To evaluate how viral variants may affect disease progression in human pediatric AIDS, we studied the potential of three simian immunodeficiency virus (SIV) isolates to induce simian AIDS in newborn rhesus macaques. The three virus isolates were previously shown to range from pathogenic (SIVmac251 and SIVmac239) to nonpathogenic (SIVmac1A11) when inoculated intravenously into juvenile and adult rhesus macaques. Six newborn macaques inoculated with pathogenic, uncloned SIVmac251 developed persistent, high levels of cell-associated and cell-free viremia, had no detectable antiviral antibodies, and had poor weight gain; these animals all exhibited severe clinical disease and pathologic lesions diagnostic for simian AIDS and were euthanatized 10 to 26 weeks after inoculation. Two newborns inoculated with pathogenic, molecularly cloned SIVmac239 developed persistent high virus load in peripheral blood, but both animals had normal weight gain and developed antiviral antibodies. One of the SIVmac239-infected neonates exhibited pathologic lesions diagnostic for SAIDS and was euthanatized at 34 weeks after inoculation; the other SIVmac239-infected neonate remained alive and exhibited no significant clinical disease for more than 1 year after inoculation. In contrast, three newborn rhesus macaques inoculated with the nonpathogenic molecular clone, SIVmac1A11, had transient, low-level viremia, seroconverted by 10 weeks after inoculation, had normal weight gain, and remained healthy for over 1 year. These results indicate that (i) newborn rhesus macaques infected with an uncloned, virulent SIVmac isolate have a more rapid, fulminant disease course than do adults inoculated with the same virus, (ii) the most rapid disease progression is associated with lack of a detectable humoral immune response in SIV-infected infant macaques, (iii) a molecularly cloned, attenuated SIV isolate is nonpathogenic in neonatal macaques, and (iv) SIV-infected neonatal macaques exhibit patterns of infection, virus load, and disease progression similar to those observed in human immunodeficiency virus-infected children. This SIV/neonatal rhesus model of pediatric AIDS provides a rapid, sensitive model with which to compare the virulence of SIV isolates and to study the mechanisms underlying the differences in disease progression in human immunodeficiency virus-infected infants.     

Sensitivity of CFU-GM from normal human bone marrow and leukaemic clonogenic cells (CFU-L) from blood of patients with myelogenous leukaemia to 3'-deoxy-3'-fluorothymidine in comparison to 3'-azido-3'-deoxythymidine

3'-Deoxy-3'-fluorothymidine (FT), a thymidine analogue highly effective against HIV 1 in vitro was investigated as to its in vitro effect on normal human bone marrow CFU-GM (agar colony assay) and on human peripheral myeloid leukaemic clonogenic cells (CFU-L, colony assay in methylcellulose). For comparison, 3'-azido-3'-deoxythymidine (AZT), structurally related and used in AIDS treatment, was included in the study. Both compounds inhibit the formation of clusters and colonies from bone marrow stem cells with an [IC]50 between 10(-6) and 10(-5)M. In concentrations only 5-10 times lower than the [IC]50, FT begins to stimulate cluster and colony formation. AZT and FT also inhibit the formation of clusters and colonies from CFU-L. Compared to CFU-GM, CFU-L were more sensitive to FT, and a stimulation was not seen. It is concluded that similar side effects on bone marrow could be expected for possible use of FT against AIDS as have been found for AZT and that both compounds are potential candidates for anti-leukaemic drugs.     

Rhesus rhadinovirus infection in healthy and SIV-infected macaques at Tulane National Primate Research Center

Rhesus rhadinovirus (RRV) infection was quantified in peripheral blood mononuclear cells (PBMC) from healthy and simian immunodeficiency virus (SIV)-infected rhesus macaques (Macaca mulatta) at the Tulane National Primate Research Center and in a large collection of simian acquired immunodeficiency syndrome--(SAIDS)-associated lymphomas. Quantification of RRV load was performed by real-time PCR using amplification primers specific for the RRV interleukin-6 homologue (RRV vIL-6). RRV infection was detected infrequently and at low levels in PBMC of randomly selected healthy animals. Examination of longitudinally collected PBMC from 22 SIV-infected animals throughout progression to SAIDS revealed similarly low RRV loads that sometimes increased with advancing disease. RRV infection was detected more frequently in the peripheral blood of SIV-infected animals than in healthy animals. Examination of SAIDS-associated lymphomas showed that RRV is rare within the tumor mass, likely representing infection in an occasional tumor-infiltrating cell or contaminating blood. The results indicate that RRV infection in PBMC is not predictive of, and is apparently not required for, development of lymphoma or hyperplastic lymphadenopathy in SIV-infected animals at TNPRC.     

Morphological changes of an inflammatory myopathy in rhesus monkeys with simian acquired immunodeficiency syndrome

Eleven of 25 rhesus monkeys which died of simian acquired immunodeficiency syndrome (SAIDS) caused by infection with a type D retrovirus related to Mason-Pfizer monkey virus showed evidence of muscle weakness and atrophy and had elevated levels of muscle enzymes. Biopsies of affected muscle studied with enzyme histochemistry showed the characteristic features of polymyositis. Inflammatory cells consisting of lymphocytes, macrophages, and large vacuolated bizarre-shaped cells of undetermined type were surrounding or invading muscle fibers and were present in the perivascular spaces and endomysia septa. Within the perivascular infiltrates, lymphocytes were abundant but very few macrophages were present. Other myopathic features including profound proliferation of fibrous tissue, necrosis, and phagocytosis of muscle fibers were noted to a variable degree. The retrovirus was isolated from affected muscles. The clinical and historical features of polymyositis in rhesus monkeys with SAIDS are very similar to those of human polymyositis. The polymyositis in SAIDS induced by a type D retrovirus related to Mason-Pfizer monkey virus is an excellent primate model to study the mechanism and morphological changes of viral-induced muscle damage.