吗啡对HIV-1在MT2细胞内复制影响

目的 探讨吗啡对人类免疫缺陷病毒-1(HIV-1)在MT2细胞中复制的影响.方法 将MT2细胞按单纯随机分组的原则分为吗啡处理组、吗啡+纳洛酮处理组、纳洛酮处理组和病毒对照组,先用浓度为10-8 mol/L的纳洛酮预处理吗啡+纳洛酮处理组和纳洛酮处理组0.5h,再用10-12、10- 10和10-8 mol/L 3个浓度的吗啡处理吗啡+纳洛酮处理组和吗啡处理组24h,然后每组细胞加入HIV -1感染MT2细胞,并分别于感染的第3、4、5和6d取培养上清测定HIV -1 p24抗原表达.结果 HW-1感染MT2细胞第3、4、5、6d,10-12、10-10和10-8 mol/L 3个浓度吗啡处理组的HIV-1 p24抗原表达均高于病毒对照组,差异均有统计学意义(P＜0.01);吗啡+纳洛酮处理组、纳洛酮处理组与病毒对照组HIV-1 p24抗原表达比较,差异均无统计学意义(P＞0.05);第3、4、5、6d,3个浓度吗啡处理组的HIV-1 p24抗原表达量比对照组增加的倍数比较,差异均有统计学意义(P＜0.05);10-12、10-10和10-8 mol/L 3个浓度吗啡处理组在不同时间HIV-1 p24抗原表达量比对照组增加倍数比较,3个吗啡处理组的HIV-1 p24抗原表达量比对照组增加倍数均随着感染时间的延长而呈现递增的趋势(P＜0.05).结论 吗啡能够促进HIV-1在MT2细胞和内的复制,并且随感染时间的延长呈现递增趋势;吗啡促进HIV-1复制的作用可被阿片受体阻滞剂纳洛酮阻断.     

新生小牛血清在精制地鼠肾细胞狂犬疫苗组织培养中对肾细胞保护作用的观察

在精制地鼠肾细胞狂犬疫苗生产中 ,地鼠肾细胞在组织培养中的生长状况对狂犬病病毒的培养起着决定性作用 ,而细胞生长的质量和新生小牛血清对肾细胞的保护与促进生长作用有直接关系。为了解新生小牛血清在制备细胞悬液中对肾细胞的保护作用、对实验组和对照组细胞生长的差异及病毒液的毒力 ,笔者做了大量的实验观察和比较。1　材料与方法1.1　材料　新生小牛血清 (沈阳安迪生物高科技公司提供 ) ,浓度为 8% ;0 .2 5 %水解乳蛋白ＨａｎＫｓ生长液 ;0 .4%人血蛋白ＨａｎＫｓ -ＳＭＬ维持液 ;12～ 14ｄ龄地鼠肾细胞 ,实验组和对照组解剖取肾 ,…     

HIV-1重组病毒载体疫苗研究进展

研制有效的疫苗是预防和控制HIV-1的理想途径.重组活病毒载体疫苗能诱导广泛的细胞和体液免疫应答,并在临床试验中展现出良好的应用前景,已成为当前HIV-1疫苗研究的一个热点.此文就近年来针对HIV-1的各种重组病毒载体疫苗研究进展作了综述.     

冰毒对HIV-1在巨噬细胞内复制影响的体外实验研究

目的探讨冰毒对人类免疫缺陷病毒-1型（human immunodeficiency virus 1,HIV-1）在巨噬细胞中复制的作用。方法将巨噬细胞按单纯随机分组原则分为：（1）冰毒＋多巴胺受体D1阻滞剂（SCH23390）处理组,（2）冰毒处理组,（3）SCH23390处理组,（4）病毒对照组。先用浓度为10-5mol／L的SCH23390预处理（1）、（3）组1h,再用10μmol／L的冰毒处理（1）组24h,同时用10～mol／L、10μmol／L及2．5×10-4mol／L的冰毒处理（2）组24h,然后每组加入等量的HIV-1进行感染,于感染的第4、6、8、10d取培养上清,用酶联免疫吸附剂测定（enzymelinked immunosorbnent assay,ELISA）检测培养上清的HIV-1 p24抗原,比较各组之间抗原表达量差异。结果HIV-1感染巨噬细胞第4、6、8、10d,病毒对照组的p24抗原表达量均低于3个不同浓度冰毒处理组（均有P〈0．01）;且各浓度冰毒处理组的p24抗原表达量比对照组增加的倍数,随着感染时间的延长而具有上升的趋势,时间．效应差异有统计学意义（F=155．94,P〈0．001）;而冰毒＋SCH23390处理组、SCH23390处理组分别与病毒对照组HIV-1p24抗原表达比较,差异均无统计学意义（均有P〉0．05）。结论冰毒能够促进HIV-1在巨噬细胞内的复制,并随感染时间的延长呈递增趋势;冰毒促进HIV-1复制的作用可被多巴胺受体D1阻滞剂（SCH23390）阻断。     

HIV-1B''特异性细胞毒性T细胞与疾病进展关系

目的 了解人类免疫缺陷病毒Ⅰ型(HIv-1)B'亚型特异性细胞毒性T细胞(CTL)功能与中国HIV-1感染者疾病进展关系.方法 将覆盖HIV-1B亚型Gag p17、p24和p2p7p1p6全长的54个重叠多肽作为抗原,用酶联免疫斑点实验(ELISPOT)检测58例HIV-1感染者特异性CTL对上述多肽的应答情况.结果 中国HIV-1感染者特异性CTL可识别多个HIV-1B'Gag表位,反应宽度与病毒载量显著负相关(r=-0.374,P=0.004),与CD4 T细胞绝对计数显著正相关(r=0.425,P=0.001),反应强度与病毒载量显著负相关(r=-0.285,P=0.030).长期不进展者识别 HIV-1B'Gag多肽的反应宽度显著高于无症状感染者和艾滋病患者(P=0.001,P=0.005).结论 我国HIV-1感染者体内存在识别不同HIV-1B'Gag多肽表位的特异性CTL应答,并且与疾病进展相关.     

HIV-1 AE重组型疫苗构建及免疫原性研究

目的利用AE亚型的膜蛋白,研发抗HIV-1AE重组型的疫苗并进行免疫原性评价。方法构建表达中国流行株97CNGX2F（HIV-1 AE2F）包膜蛋白gp140的重组痘苗病毒rVVT140AE。使用表达gp140的DNA疫苗和rVVT140AE用初免-加强方式免疫小鼠。免疫结束后第2周处死小鼠,分别检测特异性抗体滴度和特异性T细胞反应。结果本疫苗所活化的特异性抗体滴度在3 200和51 200之间,几何平均值为11 143;总T细胞免疫反应强度为（1 918±442）SFCs/10^6脾细胞,其中针对env1、env2、env3、env4肽池的免疫反应强度分别为（1 280±330）SFCs/10^6脾细胞,（66±16）SFCs/10^6脾细胞,（163±34）SFCs/10^6脾细胞和（409±96）SFCs/10^6脾细胞,均显著高于空载体对照组（〈10 SFCs/10^6脾细胞）。结论本实验构建的HIV-1 AE2F株包膜蛋白gp140重组痘苗病毒疫苗可作为针对我国HIV-1流行株的候选疫苗免疫原。     

实验室检测脊髓灰质炎疫苗滴度应注意的问题

Ｈｅｐ－２细胞是脊髓灰质炎（脊灰）病毒的敏感细胞之一，其敏感性受多种因素制约，本文以培养Ｈｅｐ－２细胞是否使用乳蛋白水解物及小牛血清的质量差异对脊灰糖丸疫苗滴度的影响进行了分析。结果表明：乳蛋白水解物的使用可使脊灰三价糖丸疫苗总滴度平均提高０．３６ＬｏｇＣＣＩＤ５０／粒，显著高于未使用乳蛋白水解物培养的Ｈｅｐ－２细胞。另外，小中血清的影响不容忽视，本文发现有些进口小牛血清对病毒的生长产生非特异性抑制，对脊灰病毒的抑制滴度可达１．９ＬｏｇＣＣＩＤ５０／ｍｌ以上。这提示我们。组织培养试剂的更换应格外慎重，实验室标准体系的建立也是非常必要的。     

HLA-Ⅰ等位基因对HIV-1特异性细胞毒性T细胞免疫应答的影响

目的 探讨中国人群HLA-Ⅰ等位基因对HIV-1抗原特异性CTL免疫应答的影响.方法 采用酶联免疫斑点(ELISPOT)技术,以HIV-1 P24区域的12个重叠肽段作为特异性肽段表位,刺激100例HIV-1感染者的外周血单核细胞(PBMC),检测IFN-γ分泌细胞频率;并用PCR特异性引物扩增法测定上述研究对象的HL...     

HIV-1的潜伏储藏库

高效抗逆转录病毒治疗(HAART)降低了艾滋病的发病率和死亡率,使血浆HIV-1RNA降至常规方法检测不到的水平,但用更敏感的方法仍可检测到病毒基因和低水平的病毒复制,这是因为HIV-1潜伏储藏库的存在。含有整合HIV DNA的静止性CD4 T细胞是最主要的HIV-1潜伏储藏库,是清除病毒的主要障碍。     

HIV-1B′特异性细胞毒性T细胞与疾病进展关系

目的了解人类免疫缺陷病毒I型（HIV-1）B′亚型特异性细胞毒性T细胞（CTL）功能与中国HIV-1感染者疾病进展关系。方法将覆盖HIV-1B′亚型Gag p17、p24和p2p7plp6全长的54个重叠多肽作为抗原,用酶联免疫斑点实验（ELISPOT）检测58例HIV-1感染者特异性CTL对上述多肽的应答情况。结果中国HIV-1感染者特异性CTL可识别多个HIV-1B′Gag表位,反应宽度与病毒载量显著负相关（r=-0.374,P=0.004）,与CD4＋T细胞绝对计数显著正相关（r=0.425,P=0.001）,反应强度与病毒载量显著负相关（r=-0.285,P=0.030）。长期不进展者识别HIV-1B′Gag多肽的反应宽度显著高于无症状感染者和艾滋病患者（P=0.001,P=0.005）。结论我国HIV-1感染者体内存在识别不同HIV-1B′Gag多肽表位的特异性CTL应答,并且与疾病进展相关。     

Response of adult human volunteers to oral administration of bovine and bovine/human reassortant rotaviruses

Small groups of adult volunteers, in sequence, were inoculated orally with inactivated purified bovine rotavirus of strain NCDV, with live NCDV purified or unpurified and with two different NCDV X human rotavirus reassortant viruses. One of five volunteers given 200 micrograms of ultravioletinactivated NCDV developed a virus-neutralizing (VN) and a binding antibody response detected by enzyme-linked immunosorbent assay (ELISA). Four of 10 volunteers given from 1 X 10(6) to 1 X 10(8) p.f.u. of live NCDV developed VN antibody, but nine of 10 responded when ELISA, HAI and radioimmuno-precipitation tests for serum antibody were also considered. Two different NCDV X human serotype 1 Wa strain virus reassortants, each containing Wa gene segment 9 and the serotype 1 neutralization phenotype, were administered orally in doses up to 10(6) p.f.u. The reassortants were relatively ineffective in eliciting a serum antibody response at the dosage level employed.     

Intranasal immunization with inactivated SARS-CoV (SARS-associated coronavirus) induced local and serum antibodies in mice

SARS-CoV (severe acute respiratory syndrome-associated coronavirus) strain GZ50 was partially purified and inactivated with 1:2000 formaldehyde. In cell culture the inactivated virus blocked the replication of live virus by decreasing the TCID(5.0) of the live virus 10(3.6) to 10(4.6) times. Inactivated GZ50 was used to immunize mice intranasally either alone, or after precipitation with polyethylene glycol (PEG), or with CpG, or CTB as an adjuvant. The titer of serum neutralizing antibodies was up to 1:640. In mice immunized with adjuvants or PEG precipitated GZ50, specific IgA was detected in tracheal-lung wash fluid by immunofluorescence. Though serum antibodies were detected, no anti-SARS-IgA could be detected in mice immunized only with inactivated GZ50. The roles of adjuvants in intranasal immunization with inactivated. SARS-CoV is discussed.     

A microcarrier cell culture process for propagating rabies virus in Vero cells grown in a stirred bioreactor under fully animal component free conditions

Rabies virus strain production in Vero cells grown on Cytodex 1 in a 2 L stirred tank bioreactor and in a medium free of components of human or animal origin (VP-SFM) is described. Cell banking procedure in VP-SFM supplemented with an animal components free mixture (10%DMSO+0.1%methylcellulose) was reported and cell growth after revitalization was assessed. Vero cells exhibited growth performances (specific growth rate and cell division number) similar to that obtained in serum containing medium. To design a scalable process that is totally free of animal-derived substances, two proteases: TrypLE Select and Accutase, were assessed as an alternative to trypsin which is routinely used for cell passage. Growth performance of Vero cells grown in VP-SFM and MEM+10% fetal calf serum (FCS) over four passages and subcultivated with either TrypLE Select or Accutase was evaluated. TrypLE Select showed the best performance in terms of specific growth rate and cell division number. Kinetics of cell growth and rabies virus production (LP2061/Vero strain) were investigated in spinner flask and in a 2 L bioreactor. In spinner flask, a maximal cell density level of 1.85x10(6) cells/mL was achieved when the cells were grown in VP-SFM on 2 g/L Cytodex 1. Cell infection experiments conducted at an MOI of 0.3 and without the medium exchange step, typically needed for serum containing rabies virus production, resulted in a maximal virus titer equal to 2x10(7) (Fluorescent Focus Unit) FFU/mL. In stirred tank bioreactor, Vero cell growth in VP-SFM on 3 g/L Cytodex 1 was shown to be sensitive to the aeration mode. Sparging the culture was detrimental for cell growth, whereas cell density level was greatly enhanced when only headspace aeration was used. A cell density level of 2.6x10(6) cells/mL was obtained when the cells were grown on 3g/L Cytodex 1 and in batch culture mode. Cell infection at an MOI of 0.1 without any medium exchange, yielded a maximal rabies virus titer of 2.4x10(7) FFU/mL. Furthermore, Vero cell growth in a 2 L bioreactor using recirculation culture mode during cell proliferation step and perfusion for virus multiplication phase was investigated. In comparison to batch culture, a higher cell density level that was equal to 5x10(6) cells/mL was reached. Cell infection under conditions similar to batch culture, resulted in a maximal virus titer equal to 1.38x10(8) FFU/mL. The potency of the pooled inactivated virus harvests showed an activity of 2.58 IU/mL which was comparable to that obtained in serum supplemented medium.     

Enhanced CD8+ T cell response to HIV-1 env by combined immunization with influenza and vaccinia virus recombinants

With the aim to determine if immunization with two different live recombinant viral vectors could lead to an enhancement of the cellular immune response to HIV-1 antigens, we have characterized the CD8+ T cell response elicited against the V3 loop epitope from HIV-1 env protein in Balb/c mice immunized with either: a recombinant influenza virus (Flu-Env) expressing the V3 loop epitope from HIV-1 strain IIIB, a vaccinia virus recombinant (VV-Env) expressing the complete HIV-1-IIIB env protein, or a combination of both. The CD8+ T cell response, measured by the ELISPOT assay, in animals primed with Flu-Env and boosted with VV-Env was 5 to 6 times higher than in animals inoculated with either Flu-Env or VV-Env alone. Similar results were obtained with recombinant viruses expressing the V3 loop epitope or the complete env protein, respectively, from the MN strain of HIV-1. Our results indicate that the use of two different live vectors for priming and boosting has a synergistic effect on the immune response against HIV-1, and could represent a novel vaccination strategy against AIDS.     

Dietary nucleotides and human immune cells. II. Modulation of PBMC growth and cytokine secretion

OBJECTIVE: The immune system is dependent on purines and pyrimidines as building blocks for DNA and RNA synthesis to enable rapid cell proliferation and protein synthesis. Emerging evidence suggests that dietary nucleotides optimize immune function. We investigated whether growth and function of human immune cells were affected by an exogenous source of nucleotides during specific antigen challenge. METHODS: Peripheral blood mononuclear cells from healthy individuals (n = 10) were stimulated with influenza virus antigen and either DNA sodium from fish soft roe (DNA), RNA from bakers yeast (Saccharomyces cerevisiae) (RNA), 2' deoxyadenosine 5'-monophosphate sodium (dAMP), 2' deoxycytidine 5'-monophosphate sodium (dCMP), 2' deoxyguanosine 5'-monophosphate sodium (dGMP), 2' deoxyuridine 5'-monophosphate sodium (dUMP) or thymidine sodium (TMP). Growth effects were ascertained by measuring the amount of tritium-labeled thymidine, incorporated into cell DNA. Cell function was measured by detection of IFN-gamma, TNF-alpha and IL-10 production. RESULTS: Specific nucleotide derivatives alone did not affect the growth of healthy peripheral blood mononuclear cells. However, the nucleotide derivatives influenced immune cell growth and cytokine secretion when cocultured with specific antigen. DNA, RNA, dAMP, dCMP and dUMP increased influenza virus antigen induced immune cell proliferation. In contrast dGMP and TMP inhibited the antigen-induced growth response. RNA and dAMP cocultured with virus antigen significantly increased peripheral blood mononuclear cell secretion of IFN-gamma, IL-10 and TNF-alpha. DNA increased virus antigen-induced immune cell secretion of IFN-gamma only, whereas dUMP significantly increased secretion of IL-10 only. dGMP completely inhibited virus-triggered IFN-gamma secretion, whereas TMP did not change the virus induced secretion pattern of measured cytokines. CONCLUSION: Nucleotide derivatives affect growth and function of specific virus antigen-stimulated human immune cells in vitro.     

Alkyl-polyacrylate esters are strong mucosal adjuvants

Synthetic polymers were examined for their potency to enhance mucosal immune responses to inactivated antigens. Aqueous solutions of polyacrylic acid with a MW of 450 kDa (p[AA]) or an butyl–ester thereof with 16% esterification (Butyl16–p[AA]) plus antigen were administered twice intranasally in mice with a 2 week interval. The frequency of IgA-antibody secreting cells (ASCs) in lung cell suspensions was determined 1 week after the second immunisation. Both polymers significantly enhanced the IgA response against inactivated Newcastle disease virus (iNDV), inactivated influenza virus strain MRC-11 (iMRC-11), haemagglutinin/neuraminidase subunits of influenza virus strain A/Texas (HA/NA) and bovine serum albumin (BSA). Butyl16–p(AA) was significantly more effective than non-derivatised p(AA), cholera toxin B subunit (CTB) or liposomes. The factor of increase in IgA-ASCs varied from <10- to >100-fold and depended on the type of antigen, the dose of antigen and the adjuvant. Extremely high responses of about 10,000 IgA-ASCs per million lung cells were detected after immunisation with 5 μg HA/NA plus 50 μg Butyl16–p(AA). Intranasal immunisation with Butyl16–p(AA) resulted in high IgA responses, not only in the lungs, but also in the spleen and in high IgG responses in these organs. We concluded that alkyl–esters of polyacrylate are an interesting, novel category of mucosal adjuvants.     

Serologic characterization and sero-epidemiologic studies on acute hemorrhagic conjunctivitis (AHC) virus.

Serologic and sero-epidemiologic characteristics of AHC virus infection were studied by neutralization test (NT). Four-fold or greater virus neutralizing (VN) antibody response was demonstrated to the Japanese isolate of AHC virus (the J 670/71 strain) in 77.3% and 66.7% of paired sera from clinical AHC patients in Japan (1971-1973) and Tunisia (1973). The four patients from Indonesia studied in 1972 showed similar antibody response. Cross-neutralization tests of AHC virus isolated in Japan (1971), Taiwan (1971), Hong Kong (1971), Thailand (1972), Indonesia (1972), Singapore (1972), Morocco (1971) and England (1971) with three kinds of antisera prepared against Japanese, Hong Kong and Moroccan AHC virus isolates indicated their antigenic identity. However, isolates from Sinapore in 1970 (Singapore 70 virus) were not neutralized with the AHC virus antisera mentioned above: Singapore 70 virus constitutes another antigenic type, to which, however, no VN antibody rise was found in paired patients' sera from Japan, Tunisia and Indonesia. Thus, no serologic evidence supporting an etiologic role of this virus group in the development of AHC was found. Although cross-tests using monospecific antisera suggested some cross-relation between AHC and both echovirus type 4 (E4) and coxsackie A (CA), type 19, no serologic relationship between AHC and these viruses was found. Sera from healthy individuals collected before and after AHC outbreaks were tested for VN antibody against AHC virus in Japan and two epidemic foci, Ghana and Indonesia. Before the epidemic, 80 to 90% of the people lacked antibody in the three countries, but 39.7% and 45.2% of inhabitants posessed VN antibody of 1:8 or over in Ghana and Indonesia after the outbreak. In Japan, however, only a slight increase was found in VN antibody prevalence afterwards. Serologic study showed that 41.5% of horse sera were VN positive at dilutions of 1:8 or more; many cattle sera also had a low VN titer but few cynomologus monkey sera had VN activity.     

Dietary nucleotides and human immune cells. II. Modulation of PBMC growth and cytokine secretion

OBJECTIVE: The immune system is dependent on purines and pyrimidines as building blocks for DNA and RNA synthesis to enable rapid cell proliferation and protein synthesis. Emerging evidence suggests that dietary nucleotides optimize immune function. We investigated whether growth and function of human immune cells were affected by an exogenous source of nucleotides during specific antigen challenge. METHODS: Peripheral blood mononuclear cells from healthy individuals (n = 10) were stimulated with influenza virus antigen and DNA-Na+ from fish soft roe, RNA from bakers yeast (Saccharomyces cerevisiae), 2'deoxyadenosine 5'-monophosphate sodium, 2'deoxycytidine 5'-monophosphate sodium, 2'deoxyguanosine 5'-monophosphate sodium, or 2'deoxyuridine 5'-monophosphate disodium. Growth effects were ascertained by measuring the amount of tritium-labeled Thymidine 5'-monophosphate sodium incorporated into cell DNA. Cell function was measured by detection of interferon-gamma (IFN-gamma), tumor necrosis factor-alpha, and interleukin-10 production. RESULTS: Specific nucleotide derivatives alone did not affect the growth of healthy peripheral blood mononuclear cells. However, the nucleotide derivatives influenced immune cell growth and cytokine secretion when cocultured with specific antigen. DNA, RNA, deoxyadenosine monophosphate, deoxycytidine monophosphate, and deoxyuridine monophosphate increased influenza virus antigen-induced immune cell proliferation. In contrast, deoxyadenosine monophosphate and thymosine monophosphate inhibited the antigen-induced growth response. RNA and deoxyadenosine monophosphate cocultured with virus antigen significantly increased peripheral blood mononuclear cell secretion of IFN-gamma, interleukin-10, and tumor necrosis factor-alpha. DNA increased virus antigen-induced immune cell secretion of IFN-gamma only, whereas deoxyuridine monophosphate significantly increased secretion of interleukin-10 only. Deoxyguanosine monophosphate completely inhibited virus-triggered IFN-gamma secretion, whereas thymosine monophosphate did not change the secretion pattern of measured cytokines. CONCLUSION: Nucleotide derivatives affect growth and function of specific virus antigen-stimulated human immune cells in vitro.     

Modulating gene expression using DNA vaccines with different 3'-UTRs influences antibody titer,seroconversion and cytokine profiles

To determine if modulating the amount of foreign antigen produced by a DNA vaccine can influence the overall intensity and cytokine polarization of the ensuing immune response, three different plasmids, each encoding the hepatitis B (HB) surface antigen, were constructed. In each construct, HBs gene expression was driven by the cytomegalovirus immediate early promoter, but differed in the 3'-untranslated regions (3'-UTR) containing the polyadenylation sequence. These 3'-UTR sequences were derived from either the hepatitis B virus (HBVpA), bovine growth hormone (BGHpA), or rabbit beta-globin (betapA). BALB/c mice were immunized intramuscularly with equimolar amounts of each plasmid and blood was collected bi-weekly. Following immunization, total IgG titers correlated with in vitro antigen production levels (from transfected CHO cells), as evidenced by the following response pattern: HBVpA>BGHpA>betapA. All groups demonstrated a heavy bias toward a Th1 immune response, as evidenced by high serum IgG2a/IgG1 ratios and the predominance of IFN-gamma over IL-4 secretion from cultured splenocytes. In addition, the HBVpA construct resulted in a seroconversion rate of 100%, in comparison to 40-50% in the BGHpA, and 0% in the betapA group. Surprisingly, splenocytes isolated from mice immunized with the betapA construct secreted the highest levels of IFN-gamma. Taken together, these findings suggest that altering the level of gene expression not only affects the overall titer and seroconversion rates of vaccinated animals, but also may play a role in modulating cytokine profiles.