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Biomedical subjects

R B Lal

Publications and source records attributed to R B Lal.

At least 19 recordsLinked to original sources
PubMed1998

Analysis of a biallelic polymorphism in the tumor necrosis factor alpha promoter and HIV type 1 disease progression.

The relevance of a TNF-alpha promoter polymorphism, a G-to-A polymorphic sequence at position-308, was examined to test whether variant alleles of TNF-alpha affect susceptibility to infection with HIV-1 and progression to AIDS. Analysis of specimens from cohorts of HIV-1 positive homosexual men demonstrated that 3 of the 32 (9.4%) HIV-1-infected long-term nonprogressors (LTNPs) were homozygous for the uncommon TNF-2 allele compared with 3 of the 196 (1.5%) HIV-1-seronegative blood donors and uninfected homosexual men (p < 0.05). There was no difference in heterozygosity among HIV-1-seropositive or -seronegative groups, although some of the seropositive men heterozygous for the TNF2 genotype were also heterozygous for CCR5delta32. However, no significant association was found between TNF genotypes and time of survival, CD4 slopes, or viral loads when seroincident (n = 109) and seroprevalent cases (n = 442) from the Chicago MACS were analyzed. Functional analysis of lymphocytes from the seronegative group revealed no difference in endogenous or mitogen-induced TNF-alpha production, as well as susceptibility to in vitro HIV-1 infection between different TNF-genotype donors. These data suggest that TNF genotypes do not play a direct role in HIV-1 disease progression; however, they could potentially be part of a multigenic linkage that may be involved in delaying progression to AIDS.

Acquired Immunodeficiency Syndrome↗
PubMed1998

CCR5 coreceptor usage of non-syncytium-inducing primary HIV-1 is independent of phylogenetically distinct global HIV-1 isolates: delineation of consensus motif in the V3 domain that predicts CCR-5 usage.

The cellular tropism of human immunodeficiency virus type 1 (HIV-1) is dependent on utilization of specific chemokine co-receptor: macrophage-tropic/non-syncytium-inducing (NSI) viruses use CCR5, whereas T-cell tropic/syncytium-inducing (SI) viruses preferentially use CXCR4. We have analyzed co-receptor usage of 24 phylogenetically distinct primary HIV-1 isolates representing group M (clades A-F) and group O with known SI and NSI phenotype, using lymphocytes from donor with nonfunctional CCR5 (CCR5-/-; homozygous 32-bp deletion). While all SI isolates infected CCR5-/- lymphocytes (and hence do not require CCR5 for viral entry), all NSI isolates, regardless of clade, did not infect CCR5-/- lymphocytes. Thus, CCR5 expression is required for infection with NSI isolates and the CCR5 usage is independent of viral genotype. To localize the viral determinant involved in CCR5 binding, the V3 sequences across the clades were aligned based on the CCR5 usage. There were conserved uncharged residues at position 11 of V3 (mostly serine/glycine) and negatively charged residues at residue 25 (mostly glutamic/aspartic acid) among all isolates that used CCR5, whereas substitution with arginine or glutamine at these two positions led to usage of a co-receptor other than CCR5. This analysis led us to identify a consensus motif S/GXXXGPGXXXXXXXE/D within the V3 loop that predicts CCR5 co-receptor usage. Most isolates, with exception of one isolate, containing the conserved motif and predicted to utilize CCR5 indeed had an absolute requirement of CCR5 expression for infectibility. Site-directed mutagenesis in the infectious molecular clone further confirmed these results. Taken together, these data provide evidence that sequences within the V3 loop provide important residues that might be directly or indirectly involved in binding to a CCR5 co-receptor.

Amino Acid Sequence↗
PubMed1998

Molecular detection and isolation of human T-cell lymphotropic virus type I (HTLV-I) from patients with HAM/TSP in São Paulo, Brazil.

BACKGROUND: Infection with HTLV-I is etiologically linked with HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). However some patients with chronic progressive paraparesis resembling HAM/TSP have been shown to be infected with HTLV-II. OBJECTIVE: To clarify the role of each of these human retroviruses in the etiology of HAM/TSP in São Paulo, Brazil. STUDY DESIGN: A detailed serological and molecular analysis of HTLV-I/II infection was performed in a cohort of 19 patients with HAM/TSP attending a neurological clinic. RESULTS: Plasma samples analyzed for anti-HTLV-I/II antibodies using a Western blot assay, comprising HTLV-I (rgp46I)- and HTLV-II (rgp46II)-specific recombinant env epitopes, demonstrated reactivity to rgp46I and hence were typed as seropositive for HTLV-I. Presence of HTLV genomic sequences in peripheral blood mononuclear cells (PBMC) was sought after by PCR using consensus primers SK 110 and SK 111 for the pol region of HTLV proviral DNA followed by hybridization with type-specific probes--SK 112 (HTLV-I) and SK 188 (HTLV-II). Southern blots from all individuals hybridized with SK 112 but not with SK 188, further confirming HTLV-I infection. Cocultivation of PBMC from eight of these patients with activated lymphocytes from normal individuals resulted in active viral production, detected as presence of soluble p24gag antigen in culture supernatants. Investigation of risk factors for HTLV-I infection in these individuals revealed that five out of 19 patients studied (26.3%) had received blood transfusions previous to disease onset.

Adult↗
PubMed1998

Plasmodium falciparum antigen-induced human immunodeficiency virus type 1 replication is mediated through induction of tumor necrosis factor-alpha.

Because malaria-stimulated cytokine production may have deleterious effects on human immunodeficiency virus type 1 (HIV-1) replication, the effects of Plasmodium falciparum antigens on HIV-1 replication were studied. Stimulation with malarial antigens significantly enhanced HIV-1 replication of HIV-1LAV and primary HIV-1 isolates (subtype A) in CD8-depleted peripheral blood mononuclear cells from naive donors. The malarial antigen-induced activation of HIV-1 was due to cellular activation as judged by the expression of cell activation markers and proliferative responses. While malarial antigen stimulation increased expression of tumor necrosis factor (TNF-alpha) and interleukin-6 (IL-6), only monoclonal antibodies (MAbs) to TNF-alpha inhibited malarial antigen-induced HIV-1 replication, whereas MAb to IL-6 had no effect. Malarial antigen increased HIV-1 replication by increasing viral mRNA expression and by activating long terminal repeat-directed viral transcription. These data suggest that P. falciparum infection can modulate HIV-1 pathogenesis by activating lymphocytes and stimulating viral replication through the production of cytokines.

Animals↗
PubMed1998

Down-regulation of interleukin-10 expression and production is associated with spontaneous proliferation by lymphocytes from human T lymphotropic virus type II-infected persons.

Cytokines from peripheral blood mononuclear cells (PBMC) from human T lymphotropic virus (HTLV)-II-infected persons were studied to delineate the mechanism(s) of spontaneous lymphocyte proliferation (SLP). Culturing HTLV-II-infected PBMC that spontaneously proliferate (SLP+) resulted in greater mRNA expression and production of interferon-gamma, interleukin (IL)-4, and IL-5, with a concomitant decrease in IL-10, than was seen with nonproliferating (SLP ) and normal PBMC. While IL-2 mRNA expression was higher, production was lower in SLP+ PBMC than in SLP and normal PBMC, implying that the proliferating cells are utilizing IL-2. Neutralization of IL-2 resulted in partial inhibition, suggesting that other cytokines also affect SLP. Addition of recombinant IL-10 inhibited the proliferation of SLP+ PBMC. Further, blocking costimulatory signals with monoclonal antibodies against CD80/CD86 resulted in increased IL-10 production with concomitant inhibition of SLP. The mechanism(s) underlying HTLV-II-associated SLP in vitro involve increased utilization of IL-2 and down-regulation of IL-10.

Antigens, CD↗
PubMed1998

Efficient expression and rapid purification of human T-cell leukemia virus type 1 protease.

Human T-cell leukemia virus type 1 (HTLV-1) is an oncovirus that is clinically associated with adult T-cell leukemia. We report here the construction of a pET19-based expression clone containing HTLV-1 protease fused to a decahistidine-containing leader peptide. The recombinant protein is efficiently expressed in Escherichia coli, and the fusion protein can be easily purified by affinity chromatography. Active mature protease in yields in excess of 3 mg/liter of culture can then be obtained by a novel two-step refolding and autoprocessing procedure. The purified enzyme exhibited Km and Kcat, values of 0.3 mM and 0.143 sec(-1) at pH 5.3 and was inhibited by pepstatin A.

Amino Acid Sequence↗
PubMed1998

Cloning and expression of a human T-lymphotropic virus type 1 protein with reverse transcriptase activity.

Unlike most other characterized retroviruses, there is little published information on the biochemical properties of human T-lymphotropic virus type 1 (HTLV-1) reverse transcriptase (RT). Specifically, no reports of a cloned functional RT enzyme have been published. Since the RT enzyme is an essential component of the virus, our objective was to clone, express, and purify a functional RT enzyme from HTLV-1. Our approach was to clone and express a protein of approximately 60 to 65 kDa that we hypothesized would correspond to the RT region encoded by the pol reading frame. The predicted region encoding the RT enzyme comprised nucleotides 2617 to 4312 of the HTLV-1 MT-2 isolate. A putative RT gene was obtained by PCR and was ligated into various prokaryotic expression vectors. A novel cloning approach allowed us to generate a stable clone in the prokaryotic expression vector pGEX-4T-1 and produce a recombinant protein of approximately 60 to 65 kDa. The partially purified protein displays RT activity in both amplification RT (AMP-RT) assays and traditional RT assays. This is the first report of a cloned protein from HTLV-1 which displays RT activity and is the first step in the characterization of HTLV-1 RT.

Amino Acid Sequence↗
PubMed1998

Genetically divergent strains of human immunodeficiency virus type 2 use multiple coreceptors for viral entry.

Several members of the seven-transmembrane chemokine receptor family have been shown to serve, with CD4, as coreceptors for entry by human immunodeficiency virus type 1 (HIV-1). While coreceptor usage by HIV-1 primary isolates has been studied by several groups, there is only limited information available concerning coreceptor usage by primary HIV-2 isolates. In this study, we have analyzed coreceptor usage of 15 primary HIV-2 isolates, using lymphocytes from a donor with nonfunctional CCR5 (CCR5 -/-; homozygous 32-bp deletion). Based on the infections of PBMCs, seven of these primary isolates had an absolute requirement for CCR5 expression, whereas the remaining eight exhibited a broader coreceptor usage. All CCR5-requiring isolates were non-syncytium inducing, whereas isolates utilizing multiple coreceptors were syncytium inducing. Blocking experiments using known ligands for chemokine receptors provided indirect evidence for additional coreceptor utilization by primary HIV-2 isolates. Analysis of GHOST4 cell lines expressing various chemokine receptors (CCR1, CCR2b, CCR3, CCR4, CCR5, CXCR4, BONZO, and BOB) further defined specific coreceptor usage of primary HIV-2 isolates. The receptors used included CXCR4, CCR1-5, and the recently described receptors BONZO and BOB. However, the efficiency at which the coreceptors were utilized varied greatly among the various isolates. Analysis of V3 envelope sequences revealed no specific motif that correlated with coreceptor usage. Our data demonstrate that primary HIV-2 isolates are capable of using a broad range of coreceptors for productive infection in vitro. Additionally, our data suggest that expanded coreceptor usage by HIV-2 may correlate with disease progression.

Amino Acid Sequence↗
PubMed1998

Activation of human T-cell leukemia virus type 1 tax gene expression in chronically infected T cells.

Expression of human T-cell leukemia virus type 1 (HTLV-1) is regulated both by the HTLV-1 Tax transactivator and by cellular transcriptional factors binding to the viral long terminal repeat (LTR), suggesting that cellular signals may play a role in regulating viral expression. Treatment of cells chronically infected with HTLV-1, which express low levels of HTLV-1 RNAs and Tax protein, with phorbol esters (i.e., phorbol12-myristate 13- acetate [PMA]), phytohemagglutinin (PHA), sodium butyrate, or combinations of cytokines resulted in induction of HTLV- 1 gene expression. PMA or PHA treatment following cotransfection of HTLV-1 Tax expression plasmids resulted in synergistic activation of HTLV-1 LTR-directed gene expression, apparently involving tyrosine ki- nase- mediated pathways. These results suggest that cellular activation stimuli may cooperate with HTLV-1 Tax to enhance expression of integrated HTLV-1 genomes and thus may play a role in the pathogenesis of HTLV-1 disease.

Cell Line↗
PubMed1997

Transcriptional activation of the intercellular adhesion molecule 1 (CD54) gene by human T lymphotropic virus types I and II Tax is mediated through a palindromic response element.

In vitro infection of T cells with human T lymphotropic virus types I and II (HTLV-I and HTLV-II) resulted in constitutive expression of ICAM-1. Higher levels of ICAM-1 mRNA were expressed in HTLV-transformed cell lines (MT-2, MoT, C8166) when compared with uninfected T cell lines (A301). We demonstrate that this activation is conferred through a site on the ICAM-1 promoter that is activated in trans by the Tax protein of HTLV-I and HTLV-II. Enhanced promoter activity was detected when the ICAM-1 construct (-1162/+1) was transfected into HTLV-I-infected (MT-2), HTLV-II-infected (MoT, AI 1050), or an HTLV-I Tax-only-expressing (C8166) cell line as compared to the uninfected T cell line (A3.01). Cotransfection of the uninfected T cell line A3.01 with the ICAM construct along with Tax-I and Tax-II expression plasmid also resulted in increased promoter activity. Furthermore, experiments with deletion constructs of the ICAM-1 promoter region indicated that a region between -88 and -53 bp relative to the transcription start site is sufficient for Tax-inducible CAT expression. This segment includes an 11-bp palindromic segment (TTTCCGGGAAA) that has homology with the IFN-gamma and IL-6 response element. An 11-bp segment containing this regulatory region proved to be sufficient to confer Tax-I and Tax-II inducibility on a heterologous promoter (TK-CAT). Taken together these findings indicate that constitutive expression of ICAM-1 by HTLV-infected cells is influenced by the viral trans-activator protein Tax. This increased expression of ICAM-1 in response to the Tax protein may play an important role in the lymphoproliferation associated with HTLV infection.

Cell Line↗
PubMed1997

Laboratory characterization of human T cell lymphotropic virus types 1 (HTLV-1) and 2 (HTLV-2) infections in blood donors from Sao Paulo, Brazil.

Serologic screening for human T cell lymphotropic virus types 1/2 (HTLV-1/2) infection in blood donors has been recently introduced in Brazil. Analysis of 351,639 blood donations in Sao Paulo from January 1992 to October 1993 identified 1,063 positive (0.30%) and 2,238 indeterminate (0.63%) samples based on serologic confirmation using a 21e Western blot. A detailed analysis (serologic, molecular, and virologic), based on a laboratory diagnostic algorithm for characterization of HTLV-1 and HTLV-2 infections was undertaken in 50 seropositive or seroindeterminate blood donors. Modified serologic assays (2.3 Western blot that incorporate type-specific recombinant peptides) performed in 29 HTLV-1/2 positive and 21 HTLV-1/2 indeterminate donors with the 21e Western blot identified 25 as infected with HTLV-1, four with HTLV-2, five with untypable HTLV-1/2, 15 as HTLV-1/2 indeterminate, and one as seronegative. Polymerase chain reaction (PCR) analysis using DNA amplification of proviral pol and tax sequences from peripheral blood mononuclear cells confirmed HTLV-1 and HTLV-2 infections in all 2.3 Western blot seropositive donors; of the five serologically untypable donors, three were confirmed to be HTLV-1 positive, one HTLV-2 positive, and one negative by PCR. All of the seroindeterminate donors were also negative by PCR. Furthermore, HTLV-1 could be isolated in cocultures from 10 of 18 infected donors. Cell lines developed from two HTLV-1-infected donors were of T cell phenotype (CD2+, CD3+), exhibiting surface markers of activated CD4 cells (CD4+ CD25+ HLA-DR+). Thus, we provide evidence for the high seroprevalence of HTLV infection in blood donor population in Sao Paulo, Brazil compared with North American donors and propose a comprehensive serologic and genotypic diagnostic algorithm for HTLV-infected donors that has strong implications for counseling of these individuals.

Antigens, CD↗
PubMed1997

Low prevalence of HTLV-II in Mayan Indians in the Yucatan Peninsula, Mexico.

Infection with HTLV-II is endemic in Amerindians, with prevalence ranging from 0.89% - 33%. To determine the prevalence of HTLV-II among indigenous Mayans in the Yucatan Peninsula of Mexico, 440 indigenous Mayans were recruited, all native to and residents of one of six Mayan communities in the Yucatan Peninsula, (Xohuayan n=144, Yaxachen n=101, Kanxoc n=84, Xocen n=40, Nabalan n=46 and X'calot n=25) between May, 1992 and June, 1993. All of the above are pre-Hispanic settlements located in tropical forest with no immigrations for over 50 years. Of the 440 indigenous Mayans, only one woman from the X'calot tribe (0.23%) was shown to be infected with HTLV-II. A high percentage of indeterminate results was found (22/439, 5%), three of which were accounted for by the husband and two children of the positive female case. PCR analysis followed by specific restriction digestion demonstrated the virus to be of the HTLV-IIb subtype, similar to that described in the Guaymi Indians from Panama. The presence of HTLV-II in the Mayan ethnos, and in other Amerindian populations supports the idea that HTLV-II is an ancestral virus in America and that it has been sustained in "closed" communities.

Adolescent↗
PubMed1996

Twenty-five years of HTLV type II follow-up with a possible case of tropical spastic paraparesis in the Kayapo, a Brazilian Indian tribe.

A longitudinal study, spanning 25 years and great demographic and cultural change, found a persistently high prevalence of human T-lymphotropic virus type II (HTLV-II) in the Xikrin Kayapo Indians of Brazil. More than 10% of the children continue to develop immune reactions to the virus in infancy, a sharp increase in seroprevalence occurs between ages 15 and 30 years, and prevalence in older woman still approaches 100%. This suggests that the major modes of transmission (breast milk and sexual activity) have not changed. The demonstration of stable maintenance of HTLV-II in one ethnic group makes migration theories of its dispersal more plausible. However, the infection may not be a negligible burden on population survival: at least 1 of 62 persons followed until age 40 years died of possible tropical spastic paraparesis (TSP).

Adolescent↗
PubMed1996

Costimulatory effects of T cell proliferation during infection with human T lymphotropic virus types I and II are mediated through CD80 and CD86 ligands.

The modulation of expression of CD80 and CD86 on T cells following infection with human T lymphotropic virus (HTLV)-I/II and its functional importance in T-T cell interactions was examined. Infection with HTLV-I/II leads to constitutive expression of CD80 and CD86, concomitant to down-modulation of CD28 on T cells. The CD80/CD86+ HTLV-infected T cells stimulated proliferation of allogeneic and autologous resting T cells, which could be specifically blocked by a soluble CTLA-4Ig chimeric protein, anti-CD80 or anti-CD86, but not by anti-CD54. It was necessary to inhibit interaction with both ligands (CD80 and CD86) to optimally block HTLV-mediated proliferation of allogeneic and autologous resting T cells. Simultaneous addition of anti-CD8O and anti-CD86 Abs also inhibited production of IFN-gamma, TNF-alpha, and IL-4, with no effect on IL-10 production, for both allo- and autologous T cell proliferation. Further, there was a direct correlation between the spontaneous proliferation of lymphocytes from patients infected with HTLV-II and expression of CD80, which could be blocked by simultaneous addition of anti-CD80 and anti-CD86. Taken together, these results suggest that HTLV-infected CD80/CD86+ T cells serve as APCs, leading to a sustained proliferation of T cells, and that both ligands participate in allostimulation, autologous proliferation, as well as spontaneous proliferation of HTLV-II-infected PBMC.

Adult↗
PubMed1996

Endemicity and phylogeny of the human T cell lymphotropic virus type II subtype A from the Kayapo Indians of Brazil: evidence for limited regional dissemination.

Long terminal repeat (LTR)-based restriction fragment length polymorphism (RFLP) analysis of human T cell lymphotropic virus type II (HTLV-II) from 17 seropositive Kayapo Indians from Brazil showed that all 17 samples contained a unique HTLV-IIa subtype (A-II). Additional RFLP screening demonstrated the presence of this subtype in two of three Brazilian blood donors and a Mexican prostitute and her child. In contrast, 129 samples from blood donors and intravenous drug users (IDUs) from the United States, two Pueblo Indian samples, five samples from Norwegian IDUs, and two samples from blood donors from Denmark were all found to be a different HTLV-IIa subtype (A-III). Phylogenetic analysis of two Kayapo and one Mexican LTR sequences showed that they cluster with a subtype A-II sequence from a Brazilian blood donor and with sequences from two prostitutes from Ghana and Cameroon. These results demonstrate that infection with the A-II subtype is endemic among the Kayapo Amerindians, has disseminated to non-Indian populations in Brazil, and is also present in Mexico. Furthermore, the A-II subtype does not appear to represent an origin for the HTLV-IIa infection in urban areas of the United States and Europe. This study provides evidence that HTLV-IIa may be a Paleo-Indian subtype as previously suggested for HTLV-IIb.

Base Sequence↗