HIV infection of CD4-CD8+ T-cell line derived from patients with HAM

This studies have attempted human immunodeficiency virus (HIV) infection in four CD4+ or CD8+ T-cell lines derived from HTLV-I associated myelopathy virus (HAM) patients. Not only CD4+ cell line but also CD8+ cell line could be infected with HIV and CD4+ cell line showed a higher susceptibility than CD8+ cell line on HIV infection. HIV antigen in early stage after HIV inoculation was detected by using enzyme-linked immunosorbent assay (ELISA) rather than indirect immunofluorescence assay (IFA). HTLV-I producing CD4+ and CD8+ cell lines became to express two viral antigens (HTLV-I and HIV) after HIV inoculation. The results indicated that CD4-CD8+ T-cell line from patient with HAM can be infected with HIV. So that, we have found that other epitopes except for CD4 antigen may be associated with HIV infection.     

1141180267Mechanism of HIV transmission across female primary genital epithelial cells

Our lab is utilizing human primary genital epithelial cultures to investigate the mechanism of HIV transmission across the female genital mucosa. Primary endometrial epithelial cells (ECs) grown on matrigel- coated cell inserts were used for HIV infection studies. EC's were infected with cell- free or cell- associated R5 and X4 virus strains. Infections were performed in the presence or absence of the appropriate macrophage (U937) or T-cell (Jurkat) target cell- line in basolateral compartments of cultures. Virus was quantified from supernatants using p24 ELISA. U373-CXCR4 and CCR5 magi cell lines were utilized to measure infectious virus and tropism. Sybrgreen real time PCR was used to detect viral RNA in cell lysates. Infectious HIV particles were found only in basolateral supernatants of EC's infected with X4 strains of HIV in the presence of appropriate target cells. This indicated that EC's may not be productively infected, but were only able to transmit HIV in the presence of target cells. These results were supported by the detection of higher levels of gag gene (CT-15) in X4 target cells compared to EC's (CT-25) using real-time PCR. No infectious virus was present in primary EC's infected with cell-free R5 strain, although gag gene product was detected by real time PCR in both R5 target cells as well as EC's. P24 levels in supernatants were not indicative of infectious virus. Our data indicates that R5 and X4 virus strains have differential abilities to cross the female genital mucosa to infect target cells. The presence of target cells appears to be critical for the production of infectious HIV particles under these culture conditions. These studies are providing important information regarding the ability of HIV-1 to cross the female genital mucosa.     

Mumps virus replication in human lymphoid cell lines and in peripheral blood lymphocytes: preference for T cells.

The replication of mumps virus was studied in human continuous lymphoblastoid cell lines (LCLs) with T or B characteristics and in lymphocyte subpopulations derived from peripheral blood. T-LCLs supported effective virus replication as shown by high titers of free and cell-associated virus over 1 to 4 days after infection. By immunofluorescence analysis, the majority of cells were positive for mumps virus antigens. In contrast, the B-cell lines produced low titers of infectious virus, and only a small percentage expressed viral antigens. This resistance of the B-LCLs was found with several mumps virus strains. Cultures of peripheral blood mononuclear cells also supported mumps virus replication. Very high titers of infectious virus (10(8) PFU/ml) were observed in cultures prestimulated with phytohaemagglutinin. Studies with enriched T and B cells point to the activated T lymphocyte as the major virus-producing cell.     

gp120-independent HIV infection of cells derived from the female reproductive tract, brain, and colon

The infection of CD4 cells may have significant involvement in the transmission and long-term persistency of HIV. Using HIV clones carrying the enhanced green fluorescent protein (EGFP), we infected epithelial and glioneuronal cell lines derived from the female reproductive tract, brain, colon, and intestine. HIV infection was quantified by counting EGFP-positive cells. Infection was quantified in cell lines from the female reproductive tract, brain tissue, and colon tissue (0.36%-3.15%). Virus replicated in the infected cells and the progeny virus were infectious for CD4 cells, HeLa-CD4, and CEM T lymphocytes. Furthermore, we found that infection of these epithelial and brain cell lines is independent of gp120. The results from the infection of CD4 epithelial cells suggest that HIV can traverse epithelial cell layers by infecting them through a gp120-independent mechanism. Infection of glial and neuronal cell lines suggests that HIV infection of these cells is a probable mechanism for HIV pathogenicity in the brain and a possible cause for persistent infection in patients.     

High proportion of PD‐1‐expressing CD4+ T cells in adipose tissue constitutes an immunomodulatory microenvironment that may support HIV persistence

We and others have demonstrated that adipose tissue is a reservoir for HIV. Evaluation of the mechanisms responsible for viral persistence may lead to ways of reducing these reservoirs. Here, we evaluated the immune characteristics of adipose tissue in HIV‐infected patients receiving antiretroviral therapy (ART) and in non‐HIV‐infected patients. We notably sought to determine whether adipose tissue's intrinsic properties and/or HIV induced alteration of the tissue environment may favour viral persistence. ART‐controlled HIV infection was associated with a difference in the CD4/CD8 T‐cell ratio and an elevated proportion of Treg cells in subcutaneous adipose tissue. No changes in Th1, Th2 and Th17 cell proportions or activation markers expression on T cell (Ki‐67, HLA‐DR) could be detected, and the percentage of CD69‐expressing resident memory CD4⁺ T cells was not affected. Overall, our results indicate that adipose‐tissue‐resident CD4⁺ T cells are not extensively activated during HIV infection. PD‐1 was expressed by a high proportion of tissue‐resident memory CD4⁺ T cells in both HIV‐infected patients and non‐HIV‐infected patients. Our findings suggest that adipose tissue's intrinsic immunomodulatory properties may limit immune activation and thus may strongly contribute to viral persistence.     

Impact of the Hayflick Limit on T cell responses to infection: lessons from aging and HIV disease

Aging and HIV disease show certain immunological similarities. In both situations, control over viral infection is diminished, and there is an increase in certain types of cancer. The immune cell type responsible for controlling viral infections and cancer is the so-called CD8 or cytotoxic T cell. In elderly persons and individuals chronically infected with HIV, there are high proportions of CD8 T cells that resemble cells that reach the end stage of replicative senescence in cell culture after repeated rounds of antigen-driven proliferation. Senescent cultures are characterized by irreversible cell cycle arrest, shortened telomeres, inability to upregulate telomerase, loss of CD28 expression, and apoptosis resistance. Strategies that retard replicative senescence may, therefore, provide novel approaches to enhancing immune function during aging and HIV disease.     

HIV/AIDS与肝炎病毒感染者CD4+、CD8+淋巴细胞数的比较

目的：探讨ＨＩＶ携带者或ＡＩＤＳ病人（简称ＨＩＶ／ＡＩＤＳ）与肝炎病毒感染者的ＣＤ４＾＋、ＣＤ８＾＋淋巴细胞变化,并进行比较。方法：用免疫磁珠法（ＤＹＮＡ ｂｅａｄｓ）检测ＣＤ４＾＋、ＣＤ８＾＋Ｔ淋巴细胞数,用双侧ｔ检验进行统计学处理。结果：全部ＨＩＶ／ＡＩＤＳ的ＣＤ４＾＋Ｔ淋巴细胞降低,且幅度大,８３．４％低于５００细胞／μｌ。在病毒性肝炎中降低的百分比和幅度均明显低于前者,只有２８．８％低于５     

CD4+ NK cells can be productively infected with HIV, leading to downregulation of CD4 expression and changes in function

NK cells mediate the innate immune response, and HIV-infected individuals demonstrate altered NK cell phenotype and function. We find that CD4+ NK cells are susceptible to HIV infection; this could account for the NK cell dysfunction seen in HIV-infected individuals. CD4+ NK cells express CXCR4 and can be infected with X4-tropic viruses and some primary R5-utilizing viral isolates. Treatment with the CXCR4 ligands AMD3100 and SDF-1α partially blocks infection with X4-tropic virus, treatment with anti-CCL Igs upregulates CCR5 surface expression and enables infection with HIV-Bal. HIV infection of NK cells results in CD4 downregulation and the production of infectious virus. HIV-infected CD4+ NK cells mediate NK cell cytotoxicity, however, HIV infection is associated with decreased chemotaxis towards IL-16. Thus, HIV infection of CD4+ NK cells could account for the NK cell dysfunction observed in HIV-infected individuals. Furthermore infected NK cells could serve as a viral reservoir of HIV in vivo.     

Use of a BJAB-derived cell line for isolation of human herpesvirus 8

Establishment of latently infected cell lines from primary effusion lymphomas (PEL) presently is the most efficient system for the propagation of clinical strains of human herpesvirus 8 (HHV-8) in culture. Here we describe a new approach to culture productively replicating HHV-8 from patient samples. A BJAB-derived B-cell line, BBF, was found to retain HHV-8 longer, to support the latent and lytic replication programs, and to produce transmissible virus. Supernatants from n-butyrate-treated peripheral blood mononuclear cells of 24 HHV-8-seropositive renal transplant recipients were used to infect BBF cells, and replicating virus was detected in cultures from 11 patients. Moreover, BBF cells infected with saliva strains showed a highly productive profile regardless of the initial viral load, which confirms that infectious HHV-8 can be present in saliva and also suggests that saliva strains may exhibit a high tropism for B lymphocytes. In conclusion, we established an in vitro system that efficiently detects HHV-8 in samples with low viral loads and that produces infectious progeny. BBF cells can be used to propagate HHV-8 from different biological samples as well as to clarify important issues related to virus-cell interactions in a context distinct from endothelial and PEL-derived cell lines.     

Histone deacetylase inhibitor chidamide promotes reactivation of latent human immunodeficiency virus by introducing histone acetylation

Highly active antiretroviral therapy can reduce the human immunodeficiency virus (HIV) viral load in the plasma to undetectable levels. However, because of the presence of latent HIV reservoirs, it is difficult to completely eradicate HIV in infected patients. Our objective was to assess the potency of chidamide, a novel histone deacetylase inhibitor recently approved for cancer treatment by the China Food and Drug Administration, to reactivate latent HIV‐1 via histone acetylation. Viral reactivities of chidamide were accessed in 2 latent HIV pseudotype virus cell reporter systems (J‐Lat Tat‐green fluorescent protein clone A72 and TZM‐bl), a latently infected full‐length HIV virus cell system (U1/HIV), and resting CD4⁺ T cells from 9 HIV‐infected patients under highly active antiretroviral therapy with undetectable viral load. Chidamide was able to increase HIV expression in each cell line, as evidenced by green fluorescent protein, luciferase activity, and p24, as well as to reactivate latent HIV‐1 in primary CD4⁺ T cells of HIV‐infected patients. Histone acetylation adjacent to the HIV promoter in A72 cells was determined by chromatin immunoprecipitation. Chidamide was able to increase histone H3 and H4 acetylation at the HIV promoter. In brief, chidamide induced the reactivation of latent HIV in pseudotype virus reporter cells, latently infected cells, and primary CD4⁺ T cells, making this compound an attractive option for future clinical trials.     

CD44 MicroBeads accelerate HIV-1 infection in T cells

Super-paramagnetic CD44 MicroBeads (Miltenyi) designed for the isolation of infectious HIV-1 from dilute or difficult biological samples dramatically enhance the infectivity of bound HIV virions, even if the original viral suspension is merely incubated with beads. Infection of the CEM T cell line with the NL4-3 virus clone or primary human CD4 T cells with X4- and R5-tropic clones and a clade C primary virus isolate all showed accelerated p24 production and larger fractions of infected target cells. Effects could be detected very early; incubation of virus with the CD44 MicroBeads promoted higher levels of viral integration within the first infection cycle. In summary, CD44 MicroBeads provide the means not only to concentrate dilute viral samples, but also to directly facilitate within days rather than weeks the in vitro expansion of patient isolates independent of coreceptor usage and the performance of HIV replication assays that require a large fraction of infected primary T cells.     

Direct detection of infectious HIV-1 in blood using a centrifugation-indicator cell assay

Plasma HIV RNA is a useful surrogate marker for predicting HIV-1 disease progression in infected individuals but provides no information regarding the infectious viral titer. Traditional assays of infectious HIV-1 are, however, time consuming, insensitive, use non-standardized reagents and are subject to selection bias introduced by prolonged cultivation. In this pilot study infectious HIV-1 was detected directly in patient plasma using the indicator line HeLa-CD4-CCR5-LTR/beta-gal in a centrifugation-culture method. Replication competent HIV-1 was identified within 2 days of tissue culture inoculation in six (26%) of 23 plasma specimens. The capability of a new cell line, MT4-CCR5-tat, to amplify plasma HIV-1 was also tested. HIV was cultivated from ten (71%) of 14 specimens using MT4-CCR5-tat cells before titering the virus with the indicator cell assay. Using these stable cell lines in refined versions of this assay it may be feasible to develop rapid, simple methods for titering infectious plasma HIV-1 and for testing the susceptibility of the virus to antiretroviral drugs.     

The persistent infection of a canine thymus cell line by equine infectious anaemia virus and preliminary data on the production of viral antigens

Equine infectious anaemia virus (EIAV) was adapted to the Cf2Th cell line, a heterologous malignant line from canine thymus. A persistent infection was monitored for 100 serial passages by demonstrating the presence of virus and viral antigens at each 10th passage by electron-microscopy, immunodiffusion and immunofluorescence. Chromosome analysis of EIAV-infected cells indicated they had a karyotype resembling the control cells of similar passage history. Virus-infected cells, grown in roller cultures for 65 days without subculturing, continuously produced viral antigens into supernatant fluids which were harvested every 3-4 days. Antigen peaks were observed at approximately 12-day intervals. Immunoprecipitin lines of identity were demonstrated between ether-extracted antigens from virus-infected canine cell line and known positive EIAV antigen extracted from infected equine spleen and a commercial source. Replication of a non-oncogenic retrovirus (EIAV) resulted in the continuous release of viral antigens from a persistently infected and infinite cell line.     

Induction of MHC class I antigen expression following infection of a human esthesioneuroblastoma cell line with cytomegalovirus and human immunodeficiency virus.

Productive infections with cytomegalovirus (CMV) and human immunodeficiency virus (HIV) were established in the Tp41ON cell line derived from a human esthesioneuroblastoma. HIV antigen expression was highest in cultures coinfected with CMV and HIV. Viral infection caused increased MHC class I antigen expression while class II and CD4 antigens remained undetectable using immunofluorescence methods. Uninfected cultures showed 10% and coinfected cultures 80% class I antigen positive cells. In coinfected cultures, CMV and HIV antigens were detected in 4% and 8% of the cells, respectively. The detection of CMV antigens in some multinucleated cells suggests coinfection with both viruses in these cells, as multinucleated cells were not found in cultures infected with CMV only. The study shows that a cell line showing neuronal differentiation in vitro can be infected with CMV and HIV and that this infection increases MHC class I antigen expression.     

Dissociation of immunologic and virologic responses to highly active antiretroviral therapy

OBJECTIVE: Immunologic markers, levels of HIV DNA, and infectious HIV were compared in partial responders (PR) to HAART who had high plasma HIV RNA levels but stable or increasing levels of CD4+ peripheral blood mononuclear cells (PBMC), and patients with complete failure (CF) who had very low or decreasing levels of CD4+ PBMC and high plasma HIV RNA levels. DESIGN AND METHODS: CD4 and CD8 levels were monitored by flow cytometry. Beta2-microglobulin (beta2M) and neopterin levels were measured by quantitative enzyme immunoassays. Plasma and PBMC from 11 PR and 13 CF were analyzed for infectious HIV levels in limiting dilution cultures. Polymerase chain reaction (PCR) assays were used to quantify cellular HIV DNA and plasma HIV RNA. RESULTS: In comparison with CF, PR had little or no CD4+ cell loss, a substantial increase in CD8+ cells, significantly fewer positive plasma HIV cultures (p = .03), lower frequencies of infectious HIV in total PBMC (p = .005) and in CD4+ PBMC (p < .001), and lower frequencies of HIV DNA in CD4+ PBMC (p = .007). CONCLUSIONS: Lower levels of infectious HIV and a lower frequency of CD4+ PBMC that contain "productive" HIV DNA in PR as compared with CF may contribute to the stable or increasing CD4+ PBMC levels of the PR. However, HAART may also have effects on lymphocyte homeostasis independent of its antiviral activity.     

Concentrations of soluble Fas and soluble Fas ligand as indicators of programmed cell death among patients coinfected with Human Immunodeficiency Virus and Hepatitis C Virus

Hepatitis C Virus (HCV) and Human Immunodeficiency Virus (HIV) coinfections can affect mechanisms of programmed cell death and therefore influence acquired immunodeficiency syndrome (AIDS) development as well as the course of chronic hepatitis C. The aim of the study was to assess soluble Fas (sFas) and soluble Fas ligand (sFasL) concentrations in HIV- and HCV-coinfected patients and, moreover, to establish their relationships with HIV viral load, CD4+ T lymphocyte count, as well as liver function tests. Seventy-eight patients were included in the study, among them 30 coinfected with HIV and HCV, 10 infected only with HIV, and 38 infected only with HCV. HIV infection was confirmed by means of Western blot analysis; HIV viral load was measured by RTPCR; and CD3+, CD4+, and CD8+ T lymphocyte counts were established by means of flow cytometry. HCV infection was confirmed through HCV RNA isolation, using RT-PCR. sFas and sFasL concentrations were measured in duplicate by ELISA. The mean CD4+ T lymphocyte count decreased in HIV- and HCV-coinfected patients versus HIV-infected individuals (429 versus 279/ml). sFasL protein was detectable principally in HIV-infected individuals without HCV infection (90%), whereas in those with HCV infection it occurred only in 11% of cases. The highest sFas concentration was observed in HCV-infected patients (25.9 ng/ml) as well as in HIV- and HCV-coinfected individuals (20.3 ng/ml). This concentration was negatively proportional to sFasL prevalence. The results of our study suggest that HCV infection in HIV-positive individuals may suppress processes of programmed cell death. There was no correlation between sFas, sFasL, and HIV-1 viral load. On the other hand, sFas concentration and the presence of sFasL were related to CD4+ T lymphocyte count.     

HIV-mediated immunodepression: in vitro inhibition of T-lymphocyte proliferative response by ultraviolet-inactivated virus

In order to assess whether the human retrovirus HIV, like other animal retroviruses, is endowed with intrinsic immunosuppressive activity, we studied the effects of noninfectious, uv-irradiated virus on in vitro lymphocyte function. uvHIV preparations inhibited T-cell proliferation to mitogens and alloantigens, as well as mitogen-driven IL-2 production. The inhibitory effect, which was not exerted by uv-irradiated HTLV-I, was apparently not due to a decrease in cell viability and was likely associated with thermoresistant viral component(s). The suppression proved to be selective for T-cell responses, while sparing other lymphocyte functions, such as the B-cell proliferative response to a selective B-cell mitogen. The inhibitory effect of uvHIV was not counteracted by a substantial reduction in the number of monocytes or by indomethacin. Moreover, IL-1 production by monocytes was not affected upon virus incubation. On the other hand, the proliferative response of both CD4+ and CD8+ T-cell clones was inhibited by uvHIV, suggesting that T cells represent the actual target for the inhibitory effect. Although a sizeable decrease in IL-2 production was observed following uvHIV incubation, exogenous IL-2 was not capable of reversing the virus-induced suppression of the proliferation. The possibility that the immunosuppressive activity of noninfectious HIV contributes to the T-cell defect in infected patients by mechanisms other than the cytopathic effect on CD4+ T lymphocytes is discussed.     

Relation between HIV-2 proviral load and CD4+ lymphocyte count differs in monotypic and dual HIV infections

OBJECTIVE: To explore and compare the relations between proviral DNA load and CD4+ lymphocyte counts in both HIV-2 monotypic and HIV dual infection. STUDY DESIGN/METHODS: In Dakar, Senegal, where the HIV-1 and HIV-2 epidemics overlap, serum and peripheral blood mononuclear cell (PBMC) DNA samples were collected from registered female sex workers and hospitalized patients. Sera were evaluated for reactivity to antigens of HIV-1 and HIV-2 by immunoblot; dual reactivity was confirmed with recombinant envelope peptides for HIV-1 and HIV-2. These samples were then subjected to HIV-1 and HIV-2 proviral DNA polymerase chain reaction (PCR). To evaluate the HIV-2 cellular proviral DNA loads, a quantitative competitive PCR (QC-PCR) was developed using nested primers to amplify the gag region of HIV-2. This assay used an internal competitor generated by inserting 25 bp in the first-round PCR target sequence. T-lymphocyte subset counts were estimated by flow cytometry for both HIV-2 monotypic and dually infected persons. RESULTS: 35 HIV-2-infected and 33 dually seroreactive samples were evaluated in this study. The CD4+ lymphocyte counts were similar in both groups, with mean values of 449 +/- 390 cells/mm3 for the HIV-2 monotypic infected persons and 476 +/- 308 cells/mm3 among the dually infected persons. However, the median proviral loads differed significantly, with those in the HIV-2 group ranging from 63.2 to 669.8 copies/10(5) CD4+ cells and demonstrating an inverse correlation with CD4+ lymphocyte count. The HIV dually infected persons showed less variation in viral load, ranging from 9.9 to 43.3 copies/10(5) CD4+ cells. Among the HIV dually infected persons, low HIV-2 proviral load was correlated with low CD4+ lymphocyte counts. CONCLUSIONS: The HIV-2 proviral loads in HIV dually infected persons were significantly lower than those in HIV-2 monotypically infected individuals (P < .0001), despite comparable CD4+ lymphocyte counts. These results suggest that different HIV-2 proviral dynamics prevail in HIV dual infection.