发酵支原体抗HIV-1作用机制的研究

目的　研究发酵支原体抗HIV 1作用机制。方法　以发酵支原体PG18为实验菌株 ,制备支原体培养上清液和支原体菌体蛋白。用高效液相层析纯化发酵支原体PG18培养上清的蛋白 ,检测各部分逆转录酶抑制活性和核酸酶活性。用ELISA法检测发酵支原体对正常人PBMC及感染HIV 1的人PBMC分泌IL 10的影响及rhIL 10在体外对HIV 1复制的抑制作用。结果　发酵支原体PG18培养上清蛋白质相对分子质量 (Mr)为 (6 7～ 10 0 )× 10 3 和 (10～ 2 5 )× 10 3 时 ,分别具有 36 %和 34 %的RT活性抑制作用和部分核酸酶活性。发酵支原体PG18菌体蛋白可显著诱导正常人PBMC及感染HIV 1的PBMC分泌IL 10 ,且对感染HIV 1的PBMC分泌IL 10的诱导活性更强。rhIL 10可抑制HIV 1在PBMC中的复制 ,并随剂量的增加而增强。结论　发酵支原体PG18培养上清的逆转录酶活性抑制作用和菌体蛋白对PBMC分泌IL 10的促进作用为发酵支原体PG18抗HIV作用的相关机制     

Inhibition of immediate-type allergic reactions by Prunella vulgaris in a murine model.

We studied the effect of aqueous extract of Prunella vulgaris (Labiatae) (PVAE) on immediate-type allergic reactions. PVAE (0.005 to 1 g/kg) dose-dependently inhibited systemic anaphylactic shock induced by compound 48/ 80 in rats. When PVAE was given as pretreatment, at concentrations ranging from 0.005 to 1 g/kg, the serum histamine levels induced by compound 48/ 80 were reduced in a dose-dependent manner. PVAE (0.001 to 1 g/kg) inhibited the passive cutaneous anaphylaxis activated by anti-dinitrophenyl (DNP) IgE antibody dose dependently. PVAE also inhibited the histamine release induced by compound 48/80 or anti-DNP IgE from the rat peritoneal mast cells (RPMC). The level of cyclic AMP in RPMC, when PVAE was added, significantly increased, compared with that of normal control. Moreover, PVAE (0.01 and 0.1 mg/ml) had a significant inhibitory effect on anti-DNP IgE-mediated tumor necrosis factor-alpha production from RPMC. These results indicate that PVAE inhibits immediate-type allergic reactions in rats.     

In vitro infection of human epidermal Langerhans' cells with HIV-1.

Epidermal Langerhans' cells (LC) from human immunodeficiency virus type-1 (HIV-1)-infected patients harbour HIV-1 proviral DNA and RNA. In the present study, we investigated whether LC from epidermis of normal, HIV-seronegative subjects could be infected in vitro with HIV-1. Epidermal cells (EC) spontaneously detached from epidermal sheet cultures were enriched for LC (10-25% of CD1a+/CD4+ cells), deprived of contaminating T cells and then incubated with HIV-1IIIB. After 24 hr, purified LC and LC-depleted EC fractions were obtained by immunomagnetic separation. Polymerase chain reaction (PCR) analysis showed the presence of HIV-1 proviral DNA (gag) only in purified LC. In addition, LC-enriched EC, purified LC, LC-depleted EC or the non-permissive cell line, TF-1, the latter having being previously challenged with HIV-1IIIB for the same length of time as the EC, were co-cultivated with C8166 cells, and the co-cultures assessed for the presence of HIV DNA by PCR. Co-cultures of C8166 cells with purified LC or LC-enriched EC previously exposed to HIV-1IIIB exhibited a time-dependent increase in HIV proviral DNA. In contrast, PCR analysis of C8166 cells co-cultured with either LC-depleted EC or TF-1 cells gave negative results. Finally, C8166 cells co-cultured with HIV-infected LC formed syncytia, showed membrane budding and released numerous retroviral particles. The results indicate that LC from normal subjects can be infected in vitro with HIV and can transmit infection to myeloid cells. This in vitro model may help in understanding the regulation of HIV infection of LC.     

In vitro anti-HIV-1 antibody production in subjects in different stages of HIV-1 infection.

We evaluated the in vitro antibody production from peripheral blood mononuclear cells (PBMC) against HIV-1 proteins in infected adults. Fifty-four HIV-1 infected patients (four recent seroconverters, 15 asymptomatics with a CD4 count higher than 500/microliters, 27 asymptomatics with a CD4 count between 200 and 500/microliters and eight symptomatic patients) were tested. PBMC were incubated in the presence or absence of 1% pokeweed mitogen (PWM) at 37 degrees C for 8 days. Western blot assay, p24 antigen ELISA and anti-p24 antibody ELISA were performed on serum and culture supernatants. Spontaneous production of anti-env antibody in culture supernatants was evidenced in all subjects. All the positive supernatants for anti-core antibodies (18/54) were derived from asymptomatic patients. PBMC from recent seroconverters and from symptomatic patients did not produce any anti-core antibody. Antibody production decreased after stimulation with PWM. The concentration of p24 antigen did not significantly increase in p24 positive supernatants following acidification (P = 0.1), suggesting that the inability to detect p24 antibody was not due to the anti-p24 antibody complexed to p24 antigen in culture supernatants. In vitro production of anti-p24 antibodies was significantly more frequent in asymptomatic subjects with high CD4+ cell counts (P = 0.02) and was absent in recent seroconverters. This last finding suggests that during the initial phases of the infection, anti-p24 antibody production may be restricted to cells residing in lymphoid organs. In addition, the lower percentage of anti-core antibody in people with low CD4+ cell counts is not merely a consequence of the binding of the antibody to an increased amount of antigen, but probably reflects an impaired production or a sequestration of producing cells in lymphoid tissue during the late stages of the infection.     

Analysis of the spontaneous in vitro anti-HIV-1 antibody secretion by peripheral blood mononuclear cells in HIV-1 infection.

We studied the spontaneous in vitro secretion of anti-HIV-1 antibodies by peripheral blood mononuclear cells (PBMC) from HIV-1-infected patients. Specific antibody production was detected in supernatants of PBMC cultures using an ELISA; HIV-1 specificity was confirmed by antigen adsorption and Western blotting. This antibody secretion was found to be an active phenomenon and was not due to a release of plasma antibodies passively adsorbed onto the cell membranes. In all positive supernatants, anti-HIV-1-secreted antibodies were directed against env-encoded antigens and many supernatants also contained antibodies to pol- and gag-encoded antigens. PBMC from all HIV-1-infected patients tested (140 adults and 18 infants) secreted anti-HIV-1 antibodies. This production was found during all the clinical stages of HIV-1 infection. Our results suggest that this spontaneous HIV-1-specific antibody secretion represents a marker of HIV-1 infection. Detection of these antibodies could be a valuable tool for early confirmation of HIV-1 infection in neonates born to HIV-1-seropositive mothers.     

Productive infection of human fetal microglia by HIV-1.

Central nervous system disease is a frequent finding in both pediatric and adult AIDS. Microglia have been shown to be the major target of HIV-1 infection in the central nervous system. However, studies in vitro concerning susceptibility of human microglia to HIV-1 infection reported conflicting results; microglia from adult brain showed productive infection by HIV-1, whereas microglia from fetal brain did not. To investigate this further and to define the possible mechanisms responsible for this difference, we prepared highly purified human microglial cell cultures from fetuses of 16 to 24 weeks' gestation and exposed them to monocytotropic (HIV-1 JR-FL and HIV-1 JR-CSF) isolates of HIV-1. Culture supernatants were examined for the presence of p24 antigen for a 4-week period after viral exposure. Concurrently, potential cytopathic effects and cellular viral antigen expression (gp41 and p24) were examined by light microscopy in combination with immunocytochemistry. The results showed that human fetal microglia can be productively infected by HIV-1 as judged by p24 antigen capture assay, syncytia formation, and gp41 and p24 immunoreactivity of infected microglia. In addition, by electron microscopy, numerous viral particles characteristic of HIV-1 were present both in the intracellular and extracellular compartments. Uninfected cultures or astrocytes overgrown in the microglial cultures did not show evidence of infection under identical experimental conditions. These data demonstrate that human fetal microglia, like their adult counterparts, are susceptible to HIV-1 infection in vitro and can support the production of virus.     

Immune defence against HIV-1 infection in HIV-1-exposed seronegative persons.

Rare individuals who are repeatedly exposed to HIV-1 through unprotected sexual contact fail to acquire HIV-1 infection. These persons represent a unique study population to evaluate mechanisms by which HIV-1 replication is either prevented or controlled. We followed longitudinally a group of healthy HIV-1 seronegative persons each reporting repeated high-risk sexual activities with their HIV-1-infected partner at enrollment. The volunteers were primarily (90%) male homosexuals, maintaining high risk activities with their known infected partner (45%) or multiple other partners (61%). We evaluated the quantity and specificity of HIV-1-specific T cells in 31 exposed seronegatives (ES) using a IFN-gamma ELISPOT assay to enumerate T cells recognizing epitopes within HIV-1 Env, Gag, Pol and Nef. PBMC from only three of the 31 volunteers demonstrated ex vivo HIV-1-specific IFN-gamma secretion, in contrast to nearly 30% exhibiting cytolytic responses in previous studies. These findings suggest that if T cell responses in ES are induced by HIV-1 exposure, the frequency is at low levels in most of them, and below the level of detection using the ELISPOT assay. Alternative approaches to improve the sensitivity of detection may include use of dendritic cells as antigen-presenting cells in the ex vivo assay and more careful definition of the risk behavior and extent of HIV-1 exposure in conjunction with the evaluation of T cell responses.     

Salivary sIgA response in HIV-1 infection.

Local and systemic production of total and HIV-1 specific IgA was determined in whole saliva and serum from 45 HIV-1-infected individuals and 15 healthy controls. The antigenic domains important for sIgA and IgG binding, respectively, was investigated with epitope mapping using synthetic peptides of HIV-1 proteins. Decreased levels of total sIgA in saliva were found among patients with low CD4+ cell counts in advanced stages of acquired immunodeficiency. HIV-1 specific IgA response, predominantly directed to the envelope proteins, was found in saliva and serum also at later stages of disease. Analyses using peptide enzyme-linked immunosorbent assays (ELISA) showed that the sIgA antibody response in saliva was mainly directed to the V4 region (aa 385-409) and a more C-terminal part of the V3-region (aa 325-344) compared with the IgG response, which predominantly was directed to a more central part of the V3 loop (aa 308-325). A similar picture was seen for immunoglobulins of the two isotypes derived from serum. We have in this study shown IgA epitope-specific immune response within HIV-1 gp160, indicating that antibodies of IgA isotype may recognize somewhat different antigenic domains compared to IgG antibodies.     

HIV-1 infection of in vitro cultured human monocytes: early events and influence of anti HIV-1 antibodies

Summary To characterize the role of the humoral immune response on HIV-1 infection of monocytes and macrophages (Ms) we examined the susceptibility of in vitro cultured monocyte/Ms to various HIV-1 isolates and the influence of heterologous and particularly autologous anti HIV-1 sera on this infection. Depending on the period of in vitro cultivation and the virus isolate used different patterns of susceptibility were detected. One week old monocyte/Ms were highly susceptible to HIV-1 infection, in contrast to monocyte/Ms cultured 4 weeks. The infection by virus isolated immediately after seroconversion lead to persistent infection with high level of antigen production in contrast to infection by homologous virus isolated later. MAb against the V3-IIIB loop and sCD4 inhibited the infection of monocyte/Ms in a dose dependent manner, indicating that infection requires binding to CD4 and that post binding events may be common to the infection of lymphocytes. Anti HIV-1 sera showed neutralizing activity against heterologous and even autologous escape virus. This finding, together with the observation that monocytes and Ms are infected in vivo, suggests that protection against HIV-1 infection of monocytes and Ms in vivo may not be obtainable by the humoral immune response alone.     

HLA-associated susceptibility to HIV-1 infection.

We studied HLA antigen distribution of 50 heterosexual partners of HIV+ drug abusers with more than 1 year of sexual exposure to HIV, 36 children born to seropositive mothers and 61 haemophiliac patients exposed to presumably infectious clotting factor concentrates. B52 and B44 antigens were associated with HIV resistance while B51 was associated with HIV susceptibility. Forty-nine HIV+ drug abusers, spouses of heterosexual partners studied and 25 HIV+ mothers of the children were also typed. DR11 phenotype was associated with infectiousness of HIV+ subjects. Our data suggest that the HLA region controls susceptibility to infection with HIV and infectiousness of HIV+ subjects in different risk groups.     

In vitro inhibition of Cryptosporidium parvum infection by human monoclonal antibodies.

Cryptosporidium parvum infection of the small epithelial intestine causes unremitting diarrhea and malabsorption that can lead to chronic and sometimes fatal illness in patients with AIDS. The illness may be ameliorated by passive oral immunoglobulin therapy. The objective of this study was to produce anti-Cryptosporidium human monoclonal antibodies for evaluation as potential therapy. All human monoclonal cell lines that produced C. parvum antibodies were originally generated from the peripheral blood lymphocytes of a human immunodeficiency virus-seronegative woman. She had recovered from C. parvum infection and had a high specific antibody titer. Hybridization of these lymphocytes with a tumor cell line was accomplished by hypo-osmolar electrofusion. Twelve clones were identified by enzyme-linked immunosorbent assay (ELISA) as secreting anti-Cryptosporidium antibodies after the initial hybridization. From the 12 positive clones, two high antibody-secreting clones, 17A and 17B, were maintained in long-term culture. A second hybridization produced two other human monoclonal cell lines, EC5 and BB2. Human monoclonal antibody from the first two cell lines bound to C. parvum sporozoites and oocysts by immunofluorescence. The ability of human monoclonal antibodies to inhibit C. parvum infection in vitro was assessed by using a human enterocyte cell line, HT29.74. The antibodies of the four different human hybridomas inhibited infection by 35 to 68% (P < 0.05) compared to a control irrelevant human monoclonal antibody derived in a similar fashion. Human monoclonal antibodies are candidate molecules for immunotherapy of C. parvum infection.     

Immune modulatory effects of Prunella vulgaris L. on monocytes/macrophages

Prunella vulgaris L. (Labiatae), a popular Western and Chinese herbal medicine, has long been associated with anti-viral and anti-bacterial effects. While its anti-viral effects are attributed mainly to the inhibition of virus replication, the biological mechanisms of its anti-bacterial effects remain unknown. As a biological response modifier (BRM), the polysaccharides isolated from P. vulgaris have been shown to up-regulate the immune responses of monocytes/macrophages. However, the immune stimulatory effects seem to contradict its well-known anti-inflammatory properties. We hypothesized that the anti-microbial effects exhibited by the polysaccharides isolated from P. vulgaris encompass both anti-inflammatory and immune stimulatory effects. One of the polysaccharide fractions PV2IV markedly stimulated the production of superoxide and nitrite representing nitric oxide from murine macrophage RAW264.7 and brain macrophage BV2 cells. The amount of nitrite and superoxide produced after PV2IV stimulation was as high as that stimulated by bacterial endotoxin lipopolysaccharide (LPS) in a dose-dependent manner. In addition, PV2IV also increased cellular protein levels of inducible nitric oxide synthase (iNOS) and mRNA for tumor necrosis factor-alpha (TNFalpha). Similar to the effects of a high dose of LPS, the fraction PV2 could trigger activation-induced cell death (AICD) by stimulating caspase-3 activity and reduction of MTT uptake in monocytes/macrophages. These results may help our understanding of the molecular mechanism of P. vulgaris, which exhibited both immune stimulatory and anti-inflammatory effects against microbial invasion.     

Antibody-dependent enhancement of HIV-1 infection in human term syncytiotrophoblast cells cultured in vitro

We examined if Fc receptor-mediated antibody-dependent enhancement (FcR-ADE) or complement-mediated antibody-dependent enhancement (C′-ADE) of virus infection can contribute to increasing replication of HIV-1 in human syncytiotrophoblast (ST) cells. Here we report that both FcR-ADE and C′-ADE may result in enhanced virus release from HIV-1-infected ST cells. We show that FcR-ADF of HIV-1 infection in ST cells is mediated by FcRIII and other FcR(s) belonging to undetermined Fc classes and does not require CD4 receptors, whereas C'-ADE uses both CD4 and CR2-like receptors. FcR-ADE: seems to be more efficient in enhancing HIV-I replication than C′-ADE. While FcR-ADE leads to increased internalization of HIV-1. C′-ADE does not result in enhanced endocytosis of the virus. In addition, antibodies mediating FcR-ADE arc reactive with the gp120 viral envelope antigen, whereas antibodies involved in C′-ADE react with the viral transmembrane glycoprotein gp41. Data suggest that both FcR-ADH and C′-ADE may contribute lo the spread of HIV-1 from mother to the fetus.