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Biomedical subjects

A T Haase

Publications and source records attributed to A T Haase.

At least 37 records · Page 2Linked to original sources
PubMed1998

Tracking members of the simian immunodeficiency virus deltaB670 quasispecies population in vivo at single-cell resolution.

Genetically distinct lentiviruses constitute a quasispecies population that can evolve in response to selective forces. To move beyond characterization of the population as a whole to the behavior of individual members, we devised an in situ hybridization approach that uses genotype-specific probes. We used probes that detect simian immunodeficiency viruses (SIV) that differ in sequence in the V1 region of the surface envelope glycoprotein (env) gene to investigate the replication and cellular tropisms of four viral variants in the tissues of infected rhesus macaques. We found that the V1 genotypic variants replicated in spatially defined patterns and to different extents at each anatomic site. The two variants that replicated most extensively in animals with AIDS were detected in both macrophages and T lymphocytes in tissues. By extension of this approach, it will be possible to investigate the role of individual lentiviruses in a quasispecies in pathogenesis and to evaluate the effects of antiviral or immunotherapeutic treatment on select members of a quasispecies.

Animals↗
PubMed1998

Human immunodeficiency virus-2 infection in baboons is an animal model for human immunodeficiency virus pathogenesis in humans.

OBJECTIVE: To assess disease progression in baboons (Papio cynocephalus) that were infected with two human immunodeficiency virus-2 (HIV-2) isolates. METHODS: Eight baboons were inoculated intravenously with either HIV-2UC2 or HIV-2UC14 and were followed for a 2- to 7-year period of observation. RESULTS: Six of 8 baboons showed lymphadenopathy and other signs of HIV-related disease, 3 of 8 baboons had an acute phase CD4+ T-cell decline, and 2 of 5 baboons infected with the HIV-2UC2 isolate progressed to an acquired immunodeficiency syndrome-like disease. Human immunodeficiency virus-2-specific pathology in lymphatic tissues included follicular lysis, vascular proliferation, and lymphoid depletion. Both neutralizing antibodies and a CD8+ T-cell antiviral response were associated with resistance to disease. CONCLUSIONS: Disease progression and the development of acquired immunodeficiency syndrome in HIV-2-infected baboons have similarities to human HIV infections.

Acquired Immunodeficiency Syndrome↗
PubMed1997

Reduction of HIV-1 in blood and lymph nodes following potent antiretroviral therapy and the virologic correlates of treatment failure.

Potent antiretroviral therapy can reduce plasma HIV RNA levels below the threshold of detection for periods of a year or more. The magnitude of HIV RNA reduction in the lymphoid tissue in patients with suppression of HIV RNA levels in plasma beyond 6 months has not been determined. We evaluated levels of HIV RNA and DNA and characterized resistance mutations in blood and inguinal lymph node biopsies obtained from 10 HIV-infected subjects who received 36-52 weeks of indinavir (IDV)/zidovudine (ZDV)/lamivudine (3TC), IDV, or ZDV/3TC. After 1 year of therapy, viral RNA levels in LN of individuals remained detectable but were log10 = 4 lower than in subjects on the triple drug regimen with interruption of therapy or in those treated with ZDV/3TC alone, who had viral loads in their lymph nodes indistinguishable from those expected for untreated patients. In all cases viral DNA remained detectable in lymph nodes and peripheral blood mononuclear cells (PBMC). When plasma virus suppression was incomplete, lymph node and PBMC cultures were positive and drug resistance developed. These studies indicate that pronounced and sustained suppression of plasma viremia by a potent antiretroviral combination is associated with low HIV RNA levels in the lymph nodes 1 year after treatment. Conversely, the persistence of even modest levels of plasma virus after 1 year of treatment reflects ongoing viral replication, the emergence of drug resistance, and the maintenance of high burdens of virus in the lymph nodes.

Anti-HIV Agents↗
PubMed1997

Integration of visna virus DNA occurs and may be necessary for productive infection.

Proviral integration is thought to be an obligate step of the retroviral replication cycle but the lentivirus visna has been reported to replicate in sheep choroid plexus (SCP) cultures in the absence of proviral integration. Because of new evidence that visna virus has a functional integrase, we reexamined visna virus infection of SCP cultures and found that proviral integration does indeed occur in this setting. While the majority of viral DNA remains unintegrated, integrated proviruses arise early in infection and accumulate over time. The sequences of the resulting host-virus DNA junctions show that, like other retroviruses, visna loses terminal nucleotides from its DNA upon integration. However, unlike other retroviruses, in over half the host-U3 junctions analyzed only a single nucleotide was lost such that the universally conserved CA dinucleotide, two nucleotides from the end of unintegrated viral DNA, did not directly abut host sequences in the provirus. We analyzed the role of integration in visna replication by introducing a series of five mutations into the integrase gene of molecularly cloned visna virus LV1-1KS1. Each mutation abolished viral replication, suggesting that integration may be an obligatory step in replication. We also documented productive infection of SCP cultures in which cell division had been blocked by g-irradiation. The ability of visna to integrate and to replicate in nondividing cells points to the possible utility of visna-based vectors for gene transfer into differentiated cells.

Animals↗
PubMed1997

Kinetics of response in lymphoid tissues to antiretroviral therapy of HIV-1 infection.

In lymphoid tissue, where human immunodeficiency virus-type 1 (HIV-1) is produced and stored, three-drug treatment with viral protease and reverse transcriptase inhibitors markedly reduced viral burden. This was shown by in situ hybridization and computerized quantitative analysis of serial tonsil biopsies from previously untreated adults. The frequency of productive mononuclear cells (MNCs) initially diminished with a half-life of about 1 day. Surprisingly, the amount of HIV-1 RNA in virus trapped on follicular dendritic cells (FDCs) decreased almost as quickly. After 24 weeks, MNCs with very few copies of HIV-1 RNA per cell were still detectable, as was proviral DNA; however, the amount of FDC-associated virus decreased by >/=3.4 log units. Thus, 6 months of potent therapy controlled active replication and cleared >99.9 percent of virus from the secondary lymphoid tissue reservoir.

Adult↗
PubMed1997

A new approach to investigating the relationship between productive infection and cytopathicity in vivo.

We describe a novel experimental approach to analyzing virus-host relationships and potential mechanisms of cytopathicity in vivo in simian immunodeficiency virus (SIV) infections. Progressive destruction of lymphoid tissue in the course of infection by SIV or human immunodeficiency virus (HIV) accompanies the loss of CD4+ T lymphocytes and sets the stage for AIDS. Because one of the important early events in this pathological process is lysis of follicular dendritic cells (FDCs), we investigated the controversial role of productive SIV infection in the destruction of FDCs. To differentiate productive infections from the known association of virus with FDCs as immune complexes trapped on cell surfaces, we used detection of spliced viral mRNAs in cells as evidence of productive infection. We found that spliced and unspliced viral RNAs could be detected by in situ hybridization (ISH) with specific antisense oligonucleotide probes in lymphocytes and macrophages with sensitivities of fewer than ten copies of spliced viral RNA per cell. We detected only unspliced RNA in germinal centers where FDCs reside. Thus, no productive infection of these cells can be detected in vivo by this assay, and their destruction likely occurs by indirect mechanisms that have yet to be determined.

Animals↗
PubMed1997

Simian immunodeficiency virus burden in tissues and cellular compartments during clinical latency and AIDS.

In the course of human immunodeficiency virus infection or of the related simian immunodeficiency virus (SIV), progression to AIDS is associated with high virus burdens in blood. How virus burden in the bloodstream is related to virus burden in tissue reservoirs was addressed in an animal model of rhesus macaques infected with SIV. In situ hybridization and quantitative image analysis were used to quantitate virus burden. Animals who developed AIDS had high levels of virus production and storage in lymphoid tissue reservoirs and evidence of productive infection of macrophages in the nervous system. With the quantitative approach described, it should be possible to design and assess the impact of treatment and shed light on the outstanding issues in pathogenesis.

Animals↗
PubMed1997

Kaposi's sarcoma-associated herpesvirus gene expression in endothelial (spindle) tumor cells.

The recent discovery of DNA sequences of a new human herpesvirus in Kaposi's sarcoma (KS) has fueled speculation that this virus might cause KS. The mere presence, however, of a virus in a complex multicellular tumor like KS could just as well be construed as evidence of a passenger agent. We sought stronger evidence linking the KS-associated herpesvirus (KSHV) to tumor formation by using in situ hybridization to investigate the specificity, constancy, and timing of KSHV gene expression in KS tumor cells. Here we document expression of a 700-nucleotide viral RNA in every KS tumor examined, from the earliest histologically recognizable stage to advanced tumors in which the vast majority of identifiable spindle tumor cells contain this transcript. Two other KSHV RNAs were also detected in a smaller fraction of the tumor cells in all but the earliest lesion. These viral RNAs were expressed to relatively low levels in this subset; because one of these RNAs encodes a major viral capsid protein, these cells may be producing KSHV. We did not find these KSHV genes expressed in a variety of other tumors and proliferative processes, but we did detect viral gene expression in prostatic tissue, supporting a possible mechanism for sexual transmission of KSHV. The close relationship between KS and KSHV gene expression is consistent with the hypothesis that KSHV is directly involved in the etiology and pathogenesis of KS.

AIDS-Related Opportunistic Infections↗
PubMed1997

The relationship between tumor necrosis factor and human immunodeficiency virus gene expression in lymphoid tissue.

In tissue culture models of chronic human immunodeficiency virus type 1 (HIV-1) infection, cytokines such as tumor necrosis factor alpha (TNF-alpha) activate viral gene expression. We sought evidence that TNF-alpha might similarly regulate viral gene expression in vivo in the major lymphoid tissue (LT) reservoir. We used in situ hybridization, quantitative image analysis, and double-label techniques to compare cytokine and HIV-1 RNA levels in sections of tonsil and lymph node tissues obtained from individuals in early and later stages of HIV-1 infection. The levels of TNF-alpha gene expression in LT from HIV-1-infected an uninfected individuals were indistinguishable, and we found no correlation between TNF-alpha gene expression in LT and the level of HIV-1 gene expression in LT. There is thus little evidence that in vivo TNF-alpha significantly influences HIV production in LT.

Gene Expression↗
PubMed1996

Suppression of HIV replication by lymphoid tissue CD8+ cells correlates with the clinical state of HIV-infected individuals.

Lymphoid tissues from asymptomatic HIV-infected individuals, as compared with symptomatic HIV-infected subjects, show limited histopathological changes and lower levels of HIV expression. In this report we correlate the control of HIV replication in lymph nodes to the non-cytolytic anti-HIV activity of lymphoid tissue CD8+ cells. Five subjects at different stages of HIV-related disease were studied and the ability of their CD8+ cells, isolated from both lymphoid tissue and peripheral blood, to inhibit HIV replication was compared. CD8+ cells from lymphoid tissue and peripheral blood of two HIV-infected long-term survivors suppressed HIV replication at a low CD8+:CD4+ cell ratio of 0.1. The CD8+ cells from the lymphoid tissue of a third asymptomatic subject suppressed HIV replication at a CD8+:CD4+ cell ratio of 0.25; the subject's peripheral blood CD8+ cells showed this antiviral response at a lower ratio of 0.05. The lymphoid tissue CD8+ cells from two AIDS patients were not able to suppress HIV replication, and the peripheral blood CD8+ cells of only one of them suppressed HIV replication. The plasma viremia, cellular HIV load as well as the extent of pathology and virus expression in the lymphoid tissue of the two long-term survivors, were reduced compared with these parameters in the three other subjects. The data suggest that the extent of anti-HIV activity by CD8+ cells from lymphoid tissue relative to peripheral blood correlates best with the clinical state measured by lymphoid tissue pathology and HIV burden in lymphoid tissues and blood. The results add further emphasis to the importance of this cellular immune response in controlling HIV pathogenesis.

Acquired Immunodeficiency Syndrome↗
PubMed1996

Quantitative image analysis of HIV-1 infection in lymphoid tissue.

Tracking human immunodeficiency virus-type 1 (HIV-1) infection at the cellular level in tissue reservoirs provides opportunities to better understand the pathogenesis of infection and to rationally design and monitor therapy. A quantitative technique was developed to determine viral burden in two important cellular compartments in lymphoid tissues. Image analysis and in situ hybridization were combined to show that in the presymptomatic stages of infection there is a large, relatively stable pool of virions on the surfaces of follicular dendritic cells and a smaller pool of productively infected cells. Despite evidence of constraints on HIV-1 replication in the infected cell population in lymphoid tissues, estimates of the numbers of these cells and the virus they could produce are consistent with the quantities of virus that have been detected in the bloodstream. The cellular sources of virus production and storage in lymphoid tissues can now be studied with this approach over the course of infection and treatment.

Adult↗
PubMed1996

RNA splice site utilization by simian immunodeficiency viruses derived from sooty mangabey monkeys.

Alternative splicing of the full-length, primary transcript into numerous subgenomic mRNAs is one way that lentiviral gene expression is regulated. Because the behaviors of different viral isolates might reflect in part differences in splicing, we Investigated the patterns of splice site utilization by simian immunodeficiency viruses (SIVs)-originally isolated from sooty mangabey monkeys (Cercocebus atys) We used reverse transcription-polymerase chain reaction (RT-PCR), molecular cloning, and DNA sequencing approaches to characterize SIVdeltaB670, a pathogenic and neurovirulent isolate, and SIVsmmH4, a related molecular clone. The majority of randomly selected SIVdeltaB670 and SIVsmmH4 partial cDNAs contained tat, rev, nef, and long terminal repeat (LTR) intron splice donor and acceptor sites positioned as expected based on the proviral sequence of SIVsmmH4. Nearly all (87%) of the partial cDNAs analyzed contained a spliced LTR intron. A greater number of partial cDNAs derived from SIVdeltaB670-infected cells contained putative alternatively spliced introns In comparison to SIVsmmH4, including two previously undocumented splice junctions involving the LTR intron splice donor. These data provide the first comprehensive analysis of splice site utilization by an isolate of SIV in comparison to a related molecular clone and the first characterization of SIVsmm splice site utilization.

Amino Acid Sequence↗
PubMed1996

Human T-lymphotropic virus type I-associated myelopathy and tax gene expression in CD4+ T lymphocytes.

Infection by human T-lymphotropic virus type I (HTLV-I) is associated with adult T-cell leukemia and a slowly progressive disease of the central nervous system (CNS), HTLV-I-associated myelopathy/tropical spastic paraparesis, characterized pathologically by inflammation and white matter degeneration in the spinal cord. One of the explanations for the tissue destruction is that HTLV-I infects cells in the CNS, or HTLV-I-infected CD4+ T lymphocytes enter the CNS, and this drives local expansion of virus-specific CD8+ cytotoxic T lymphocytes, which along with cytokines cause the pathological changes. Because both in the circulation and in the cerebrospinal fluid, CD8+ cytotoxic T lymphocytes are primarily reactive to the product of the HTLV-I tax gene, we sought evidence of expression of this gene within cells in the inflammatory lesions. After using double-label in situ hybridization techniques, we now report definitive localization of HTLV-I tax gene expression in CD4+ T lymphocytes in areas of inflammation and white matter destruction. These findings lend support to a hypothetical scheme of neuropathogenesis in which HTLV-I tax gene expression provokes and sustains an immunopathological process that progressively destroys myelin and axons in the spinal cord.

Adult↗
PubMed1996

Outpatient biopsies of the palatine tonsil: access to lymphoid tissue for assessment of human immunodeficiency virus RNA titers.

OBJECTIVES: Our objective was to assess the feasibility of using tonsillar lymphoid biopsy specimens obtained on an outpatient basis to quantitate a patient's lymphoid human immunodeficiency virus (HIV) RNA titers. DESIGN: A pilot cohort study was performed. PATIENTS: We evaluated ten HIV-seropositive patients who ranged in age from 26 to 48 years and had CD4+ cell counts ranging from 110 to 833 at enrollment. MAIN OUTCOME MEASURES: The main outcome measures were tolerance and safety of outpatient tonsil biopsies and quantitation of HIV RNA titers in tonsillar lymphoid biopsy specimens, plasma, and peripheral blood mononuclear cells determined by a new method of HIV RNA signal amplification with branched DNA probes. RESULTS: Outpatient tonsil biopsies were well tolerated and were performed without complications. Nine of 10 tonsil biopsies from the HIV-seropositive patients examined were positive for significant concentrations of HIV RNA, ranging from 106 to 101 HIV RNA equivalents per gram of tissue. All of the HIV RNA-positive tonsillar lymphoid specimens had HIV RNA titers that were 101 to 104 times greater than those recovered from plasma (per milliliter) of the same patient obtained at the time of biopsy. CONCLUSIONS: Sufficient tonsillar tissue can be obtained in an outpatient clinic setting to quantitate lymphoid HIV titers by the new branched-DNA signal amplification method with relative ease and without complication. The biopsy method described here affords ready access to the lymphoreticular system, which may help to advance our understanding of the pathogenesis of myriad immune diseases without the need for excisional node biopsies.

Adult↗
PubMed1995

Single cell transcript analysis of human immunodeficiency virus gene expression in the transition from latent to productive infection.

In the lymph nodes of individuals infected with human immunodeficiency virus (HIV), there is evidence that points to three kinds of virus-cell relationships. Virions may be associated with CD4+ lymphocytes that are actively producing virus or may be bound at the surfaces of follicular dendritic cells like other antigens. HIV is also harbored in CD4+ lymphocytes and monocytes/macrophages in a latent form as transcriptionally silenced provirus. To ultimately investigate in vivo these and other HIV-cell interactions that play such critical roles in the persistence of virus, immune dysregulation, and depletion, we have developed an in situ hybridization method that discriminates multiply spliced from singly or unspliced viral transcripts. In this report we describe the method and the results obtained with it in an analysis of the switch from latent to productive infection of chronically infected T lymphocytes in culture. We found with this single-cell technique that there are two subpopulations in the culture, a minor one of productively infected cells and a major one of latently infected cells in which only low levels of viral transcripts terminated close to the 5' end of the viral genome were detected. Shortly after activation of viral gene expression with phorbol ester, transcripts encoding Tat and Rev increase in abundancy in individual latently infected cells and this is followed by increases in and cytoplasmic export of singly or unspliced mRNAs encoding structural proteins. These studies provide insights into the regulation of HIV gene expression from a single-cell perspective and, from that perspective, transcript profiles of productively infected cells as a frame of reference for defining HIV-cell relationships in individual cells in tissue sections.

Gene Expression Regulation, Viral↗
PubMed1995

Sequence analysis of human T cell lymphotropic virus type I (HTLV-I) Env genes amplified from central nervous system tissues of patients with HTLV-I-associated myelopathy or leukemia.

Human T cell lymphotropic virus type I (HTLV-I) is a retrovirus that has been linked to HTLV-I-associated myelopathy (HAM)/tropical spastic paraparesis (TSP), a chronic or inflammatory neurological disease with some resemblance to multiple sclerosis. We used the polymerase chain reaction to amplify viral env genes in foci of inflammation and demyelination in the nervous system to adduce additional evidence of the association of HTLV-I with the neuropathological changes in HAM/TSP, and document in this report such an association. We also sought evidence of a distinct viral species in the lesions by amplifying, cloning and sequencing the env genes from tissues sections in which there were pathological changes. We did not find changes in the env gene that correlated with HTLV-I-associated neurological disease vs adult T cell leukemia or with the nervous system vs peripheral blood and lymphoid organs. We did, however, find evidence of extensive mutation and possibly deletions in the env gene in HTLV-I-associated neurological disease. We interpret these findings of increased genetic diversity as a reflection of higher rates of viral replication in HTLV-I-associated myelopathy that support a model of pathogenesis in which increased viral replication activates immune cells that subsequently enter the nervous system and cause injury by immunopathological mechanisms.

Adult↗