Search for human herpesvirus 6 and human cytomegalovirus in bronchoalveolar lavage from patients with human immunodeficiency virus-1 and respiratory disorders

Virus isolation and viral DNA detection by the polymerase chain reaction were used to investigate the presence of human herpesvirus 6 (HHV-6) and human cytomegalovirus (HCMV) in bronchoalveolar lavage from 34 human immunodeficiency virus-1 (HIV-1)-infected patients with respiratory disorders. The aim was to assess the presence of reactivated HHV-6 in lung tissues for a subsequent evaluation of the frequency of virus involvement in respiratory clinical manifestations in the course of HIV-1 infection. Bronchoalveolar lavage samples were tested for the presence of HCMV, as a routine investigation within a protocol monitoring opportunistic infections in symptomatic HIV-1 patients. Whereas HCMV DNA was detected by the polymerase chain reaction in 12 bronchoalveolar lavage specimens, 10 of which were also positive for virus isolation, all samples were negative for HHV-6 by both virological procedures. The HHV-6 DNA finding in bronchoalveolar lavage from an HIV-1-seronegative patient with renal carcinoma, investigated accidentally together with the bronchoalveolar lavage specimens from HIV-1 seropositive patients, stressed the HHV-6 polymerase chain reaction-negative results in the bronchoalveolar lavage samples under study. It is concluded that the lung may be a target organ for HCMV infection in HIV-1-seropositive patients affected by respiratory symptoms but that this does not seem to be the case for HHV-6. © 1996 Wiley-Liss, Inc.     

Indeterminate western blot patterns in a cohort of individuals at high risk for human immunodeficiency virus (HIV-1) exposure

Our objective was to map serial patterns of Western blot reactivity over time of a cohort of initially ELISA-negative, Western blot-indeterminate individuals from a high-risk group and to determine if these individuals were at increased risk of harboring occult HIV-1 infection. A 2-year prospective study used serial ELISA, two types of Western blot, immunologic profiles, HIV-1 culture, and analysis by polymerase chain reaction. Subjects were 20 ELISA-negative, Western blot indeterminate homosexual volunteers and 20 matched seronegative controls. Results showed that 19 of 20 study subjects completed a mean of 17.0 months of clinical and laboratory follow-up. Reactivities with p24 and/or with p55 were the two most commonly observed Western blot patterns, occurring in 70% of individuals. Specific Western blot reactivity was dependent upon the particular immunoblot preparation being used and varied considerably on a longitudinal basis. No individual pattern appeared predictive of an increased likelihood of subsequent seroconversion to HIV-1 relative to controls. By all other criteria including polymerase chain reaction analysis, samples from 17 of 19 individuals remained negative for HIV-1 at each time point. Two individuals evolved from an indeterminate to a positive Western blot and, simultaneously, from a negative to a positive polymerase chain reaction analysis, during follow-up. Our conclusions were as follows. ELISA-negative, Western blot-indeterminate individuals from a high-risk group show marked variability in immunoblot findings over time, and these patterns do not appear predictive of an increased likelihood of infection. Polymerase chain reaction analysis is clearly of value in providing confirmation of the low probability of infection in this group, although in patients who do become infected, detection by this test may not always precede diagnosis by serologic methods.     

Low T-cell responsiveness to activation via CD3/TCR is a prognostic marker for acquired immunodeficiency syndrome (AIDS) in human immunodeficiency virus-1 (HIV-1)-infected men

In a cohort of 300 HIV-1-infected homosexual men, studied longitudinally, the prognostic value of T-cell proliferative responses for development of AIDS was analyzed. In 15 persons we observed that, at seroconversion, T-cell reactivity to anti-CD3 monoclonal antibody (Mab) and phytohemagglutinin (PHA) was decreased to 20 and 60% of the normal values, respectively. After seroconversion, within 3 months, the response to anti-CD3 Mab and PHA returned to 60 and 80%, respectively, of the normal magnitude and declined thereafter. To investigate whether low T-cell reactivity correlated with progression to AIDS, groups of progressors and nonprogressors were compared. In individuals who progressed to AIDS, already 12 months before diagnosis, responses to anti-CD3 Mab were virtually absent, whereas at that time CD4+ cell numbers and reactivity to horse anti-human lymphocyte serum and PHA were still comparable to values observed for nonprogressors and the asymptomatic population as a whole. In a disease-free survival analysis, low anti-CD3 Mab reactivity predicted progression to AIDS. Our findings demonstrate that low T-cell responsiveness to anti-CD3 Mab is an early marker of progression toward AIDS in HIV-1 infected individuals.     

Functional genomics analyses of differential macaque peripheral blood mononuclear cell infections by human immunodeficiency virus-1 and simian immunodeficiency virus

The pathogenicity of the primate lentiviruses, human, and simian immunodeficiency viruses, is host-specific. Previous studies indicated that the highly pathogenic human lentivirus HIV-1 has markedly reduced pathogenicity compared to the pathogenic simian lentivirus SIV in pigtail macaques (Macaca nemestrina). We therefore hypothesized that the pigtail macaque peripheral blood mononuclear cells (mPBMCs) would respond differently to infections of HIV-1 and pathogenic SIV. To elucidate the cellular responses to the infections of HIV-1 and SIV, we infected mPBMC with these two viruses. Like infections in vivo, HIV-1 and SIV demonstrated distinct replication kinetics in mPBMCs, with HIV-1 replicating at significantly lower levels. Similarly, gene expression profiling facilitated by macaque-specific oligonucleotide microarrays also revealed distinct expression patterns of genes between the HIV-1- and SIV-infected mPBMCs; in particular, genes associated with the antigen presentation, T cell receptor, ERK/MAPK signaling, Wnt/beta-catenin signaling, and natural killer cell signaling pathways were differentially regulated between these two viruses. Most interestingly, despite the lower levels of replication, HIV-1 triggered a more robust regulation of immune response genes early after infection; the converse was true in SIV-infected mPBMCs. Our results therefore suggest that macaques may be controlling the infection of HIV-1 at an early stage through coordinated regulation of host defense pathways.     

Prognostic significance of cytotoxic T cells in individuals infected with human immunodeficiency virus

Nine dual-fluorescence combinations were used to enumerate T-cell subsets in 112 human immunodeficiency virus type 1-infected patients. Two blood samples were analyzed, with a 6-month interval between the tests, in 53 of these 112 patients. The alteration in CD4 over this period of time correlated with the change in CD8 and CD8S6F1 (P<0.02 andP<0.01), irrespective of the disease stage. Two groups of patients were defined by the CD8S6F1 subset at the first normal levels. Changes in numbers of CD4, CD4CD45RA, and CD4CD29 were significantly higher in group B than in group A patients. The absolute count of CD8S6F1 could thus serve as an indicator of the ensuing depletion of the CD4 population, as well as the CD4 subsets.     

HLA class I serotypes and cytotoxic T-lymphocyte responses among human immunodeficiency virus-1-uninfected Thai volunteers immunized with ALVAC-HIV in combination with monomeric gp120 or oligomeric gp160 protein boosting

Antigen-induced cellular immunogenicity may vary between populations due to differences in human leukocyte antigen (HLA) diversity and, hence, may play a critical role in the protection afforded by vaccines. In the setting of two, phase I/II human immunodeficiency virus-1 vaccine trials of a recombinant canarypox prime, and boosting with either recombinant monomeric gp120 or oligomeric gp160, we assessed the association between specific human leukocyte antigen (HLA) class I serotypes and the presence of cytotoxic T-lymphocyte response measured by 51Cr-release assay. HLA class I serotypes A11, A24, A33, B46, and B75 were the most common, present in 10% or more of 245 individuals studied. Forty of 187 (21.4%) Thai adults who received either ALVAC-HIV with gp120 or oligomeric gp160 or ALVAC alone had a precursor cytolytic CD8 T-cell response (pCTL). HLA-B44 was positively and significantly associated with a pCTL response (odds ratio 7.6, 95% CI: 2.7-21.2), whereas B46 was negatively associated but not robust when adjusted for multiple comparisons. Responses to Env proteins accounted for the majority (nine of 11) of pCTL activity among those persons with B44. This HLA class I serotype occurred in 9.4% of participants overall (including the placebo group), less commonly than what is reported from populations of European ancestry. These results strengthen the importance of assessing HLA class I distributions in conjunction with studies of vaccines designed to elicit cellular immunity in different populations.     

Programmed cell death (apoptosis) as a mechanism of cell death in peripheral blood mononuclear cells from cats infected with feline immunodeficiency virus (FIV).

FIV is a lentivirus infection of cats which induces an immunodeficiency syndrome associated with early qualitative defects in antigen-specific T cell function and with late quantitative defects in CD4+ T lymphocytes. We have observed that peripheral blood mononuclear cells (PBMC) from FIV-infected cats have impaired survival in culture. The mechanism of this in vitro dysfunction and depletion is not known. We have proposed that inappropriate induction of programmed cell death (apoptosis) could account for these in vitro defects. Here, we report that PBMC from FIV-infected cats, with impaired T cell blastogenesis and impaired survival in vitro, undergo an active cell death upon culture, which has the morphological and biochemical characteristics of programmed cell death (PCD). Apoptosis occurred in all six asymptomatic FIV-infected cats, and in none of the nine uninfected cats, which were studied. Changes in cell morphology under both light and electron microscopy, and fragmentation of genomic DNA were characteristic for apoptosing cells. Cell death was spontaneous and occurred in the absence of any stimuli, and culture with the T cell mitogen, concanavalin A (Con A), did not significantly enhance cell death. Activation-induced cell death was inhibited, in a dose-dependent manner, by addition to the incubation medium of zinc, which has been shown to inhibit the action of endonuclease responsible for the characteristic fragmentation of DNA. Since apoptosis has recently been implicated in AIDS pathogenesis, FIV infection may prove useful to study this aspect of retroviral, in particular HIV, infection.     

HLA-A,B,C and DR antigen frequencies in acquired immunodeficiency syndrome (AIDS) patients with opportunistic infections

During the past three years, an epidemic of acquired immunodeficiency syndromes (AIDS) involving the presence of specific forms of cancer (notably Kaposi's sarcoma) and infection (e.g., pneumocystis carinii) ordinarily seen only in severely immunosuppressed hosts has occurred among active homosexuals, Haitian immigrants, drug users, and hemophiliacs in large cities in the United States and elsewhere. An as yet unidentified viral agent is presumably the cause of the initial immunodeficiency and host genetic factors may influence the subsequent development of different clinical symptoms in different patients. We have previously reported that the HLA antigens DR5 and DR2 are associated with susceptibility to Kaposi's sarcoma (KS) in different Caucasian subpopulations. We now have also noted that AIDS patients with opportunistic infections have a normal frequency of DR2 and DR5 and a significantly increased frequency of DR3 and that the ultimate clinical expression of AIDS in patients with unexplained lymphadenopathy may depend upon genetic factors associated with these particular DR types.     

Detection of Hepatitis C virus RNA in peripheral blood mononuclear cells of patients with abnormal alanine transaminase in Ahvaz

Purpose: Hepatitis C virus (HCV) is an important agent for chronic and acute hepatitis. Occult hepatitis C remains a major health problem worldwide. Patients with chronic occult HCV may progress to cirrhosis and hepatocellular carcinoma. The aim of this study was to determine prevalence of occult hepatitis C by IS-PCR-ISH (in situ PCR in situ hybridisation) in the patients with abnormal ALT. Materials and Methods: The blood samples were taken from 53 patients including 17 females (32.1%) and 36 (67.9%) males who had abnormal alanine transaminase (ALT) for more than 1 year. The mean ALT and aspartate transaminase (AST) level were 41.02 ± 9.3 and 24.17 ± 7.3, respectively. The patients’ age were between 4 and 70-years old with mean age 38 ± 13. All the patients were negative for HCV antibody, HCV RNA and HBs Ag. The peripheral blood mononuclear cells (PBMC) were separated with ficoll gradient from each blood sample, then the cells were fixed on slides by cold acetone and followed by IS-PCR-ISH for HCV RNA detection. Results: Seventeen (32%) patients including 6 (11.3%) females and 11 (20.7%) males showed positive results for HCV RNA by in situ-PCR in situ hybridisation. Ten (18.8%) positive cases were between 20 and 40-years old and 6 (11.3%) positive patients were between 40 and 60 years old. Ten (19.6%) patients who were positive for IS-PCR-ISH also had positive anti-HBc IgG and 7 (13.2%) patients were negative for HBc-IgG. Conclusion: In the present study high rate of 32% occult hepatitis C were found among the patients with elevated ALT.     

Perinatal infection by human immunodeficiency virus type 1 (HIV-1): relationship between proviral copy number in vivo, viral properties in vitro, and clinical outcome.

Human immunodeficiency virus type 1 (HIV-1) isolates from 25 perinatally HIV-1 infected children were classified according to their capacity to replicate in vitro as rapid (R), intermediate (S/R) and slow (S) variants. R-type viruses replicated on peripheral blood mononuclear cells (PBMCs) and grew better in T-lymphoid cells, even though 9 out of 12 isolates also maintained tropism for monocytoid cells. The S/R-type isolates replicated efficiently after several days of culture, while the S-type viruses displayed only a low and transient replication activity; however, both S/R- and S-type isolates exerted viral transactivation activity in an indicator monocytoid cell line. Replication patterns in vitro were significantly associated in vivo with the number of HIV-1 copies in PBMCs as determined by polymerase chain reaction: in children with R-type isolates, the number of HIV-1 proviral DNA molecules/10(5) PBMCs ranged from 62 to 571, and in children with S/R and S isolates the range was 5-43. Seven children had severe symptomatic HIV-1 infection, and in all an R-type virus was identified; 18 children had no or only mild symptoms, and among these, S-, S/R-, and R-type isolates were found in 5, 8, and 5 cases, respectively. Besides demonstrating HIV-1 variability in perinatal infection, these findings suggest that R-type virus might be a prerequisite for disease progression.     

Rapid,sensitive, specific,and quantitative detection of human T-cell leukemia virus type 1 sequence in peripheral blood mononuclear cells by an improved polymerase chain reaction method with nested primers

Improving on the nested double polymerase chain reaction (PCR) described previously, we have developed a new two-step PCR (TS-PCR) method for detecting more specifically the human T-cell leukemia virus type 1 (HTLV-1) proviral sequences in peripheral blood mononuclear cells (PBMC). In our TS-PCR method, the point of modification is to use optimal concentrations of primers in the first amplification step in the range of 0.01–0.025 µM. This increases sensitivity and specificity enough to detect from 1 to 10⁵ copies of template DNA without radioisotopes. This method is rapid because of completion in 1 day and is also applicable for quantitative detection of clinical specimens. The data show that the quantitative detection of HTLV-1 proviral sequences by this method correlates with the anti-HTLV-1 antibody titers from serologic analysis of seropositive healthy carriers. Moreover, the TS-PCR method using each specific primer was also attempted for successful detection of other viral genomes; therefore, the principle of this method is widely suitable for routine detection of genomes in the basic and clinical microbiological fields.     

Patterns of antibody reactivity to selected human immunodeficiency virus type 1 (HIV-1) gp160 epitopes in infected individuals grouped according to CD4+ cell levels

We examined sera from 160 HIV-infected individuals for antibodies reactive to HIV-1 gp160 epitopes defined by seven synthetic peptides. Seropositive individuals were placed into three groups based upon levels of circulating CD4+ cells. These groups consisted of individuals with (1) more than 400 CD4+ cells, (2) 200–400 CD4+ cells, and (3) fewer than 200 CD4+ cells/mm³. The percentage of sera containing antibodies reactive with two immunodominant gp160 epitopes (a.a. 304–321 and 600–611) was unchanged between groups, regardless of CD4 cell numbers. The percentage of sera containing antibodies reactive with weakly immunogenic gp160 epitopes, such as those defined by peptides 425–448 and 846–860, declined in the groups as CD4 values decreased. Our results suggest that the patterns of antibody reactivity to gp160 epitopes change as CD4 levels decline. A narrowing of the humoral immune response to epitopes on the envelope of HIV-1 appears to occur with disease progression.     

Detection of human herpesvirus 8 DNA sequences in peripheral blood mononuclear cells of children

Human herpesvirus 8 (HHV-8) DNA sequences were examined in peripheral blood mononuclear cell (PBMC) DNA samples of 56 children, 15 healthy adults, and 10 renal transplant patients by the polymerase chain reaction (PCR). The PCR amplification was carried out using the published KS330₂₃₃ primer pairs to amplify HHV-8 DNA sequences. The PCR-amplified products were confirmed by Southern blot hybridization with radiolabeled 233 bp HHV-8 DNA fragment, which was cloned and sequenced from the PCR-amplified product of Kaposi's sarcoma tissue. Six PCR-amplified product of four children and two renal transplant patients were cloned and sequenced. HHV-8 DNA sequences were detected in 36 of 56 (64%) normal children, in 12 of 15 (80%) healthy adults, and in all 10 renal transplant patients by Southern blot hybridization of PCR-amplified products. Six PCR-amplified products were confirmed by sequencing. These results suggest that HHV-8 infection is prevalent in the Japanese population with infection occurring in early childhood. J. Med. Virol. 53:81–84, 1997. © 1997 Wiley-Liss, Inc.     

RASSF1基因转录本A和C的表达与卵巢癌关系的研究

目的探讨RASSF1基因转录本A和C在多个卵巢癌细胞系和卵巢癌组织中所起的作用。方法应用逆转录聚合酶链反应(RT PCR)和激光捕获显微切割技术检测3个卵巢癌细胞系和80例人原发卵巢上皮性恶性肿瘤组织中RASSF1A和RASSF1CmRNA的表达。结果RASSF1A mRNA在卵巢癌SK OV3细胞中表达缺失;RASSF1A和RASSF1C在人卵巢癌组织中的表达率分别为40.0%(32/80)和91.3%(73/80)。RASSF1AmRNA的表达在浆液性癌、黏液性癌和内膜样癌组织中分别是41.2%(20/48),38.1%(8/21)和36.4%(4/11),P>0.05;临床Ⅰ期和Ⅱ期分别为71.4%和75.0%(10/14,9/12),明显高于临床Ⅲ期和Ⅳ期(26.7%、12/45和14.1%,1/9),P<0.05;高和中分化组分别为58.6%和50.0%(17/29,10/20),明显高于低分化组(16.1%,5/31),P<0.05。结论卵巢癌细胞和人原发卵巢癌组织中存在RASSF1AmRNA表达的缺失,RASSF1A的表达与卵巢癌的临床分期和组织学分级有关,可能作为一种新的抑癌基因在卵巢癌的发生和发展过程中起重要作用,RASSF1AmRNA的缺失提示预后不良。