Biomedical subjects

Subhash Dhawan

Publications and source records attributed to Subhash Dhawan.

9 recordsLinked to original sources

PubMed2006

Antibodies against a multiple-peptide conjugate comprising chemically modified human immunodeficiency virus type-1 functional Tat peptides inhibit infection.

We demonstrated recently that selective side-chain modification of functional cysteine-rich (Tat(21-40)) and arginine-rich (Tat(53-68)) domains of the HIV-1 Tat protein blocks pathogenic activities of these peptides while retaining their immunological characteristics. In the present study, we have synthesized a multiple-peptide conjugate system comprising modified Tat(21-40) and Tat(53-68) peptides (HIV-1-Tat-MPC). Immunization of mice with this highly homogeneous 10.7 kDa HIV-1-Tat-MPC synthetic construct induced an effective immune response in mice. The antibodies generated against HIV-1-Tat-MPC efficiently suppressed Tat-induced viral replication and significantly reduced HIV-associated cytopathic effects in human monocytes. These results indicate that epitope-specific antibodies directed against functional sites of Tat protein using non-pathogenic peptides inhibit HIV pathogenesis. The HIV-1-Tat-MPC, therefore, has potential for the development of a safe, effective, and economical therapeutic vaccine to reduce the progression of HIV infection.

AIDS Vaccines↗

PubMed2006

Hemin activation ameliorates HIV-1 infection via heme oxygenase-1 induction.

Hemin, a critical component of hemoglobin, is an active ingredient of a biologic therapeutic approved by the Food and Drug Administration for the treatment of acute porphyries. This report describes a biological function of this molecule in inducing host defense against HIV-1 infection via heme oxygenase-1 (HO-1) induction. Treatment of monocytes with hemin substantially inhibited HIV replication, as evident by nearly undetectable viral RNA and cell-free HIV-1 p24 protein in a dose-dependent manner. Hemin exposure of these cells before infection, at the time of infection, or after infection caused >90% reduction of HIV DNA with substantially low levels of HIV-1 p24 and HIV-associated cytopathic effects. In addition, hemin treatment significantly suppressed infection of both monocytes and T cells inoculated with R5, X4, R5X4 tropic strains, and reverse transcriptase-resistant, azidothymidine-resistant, ddC/ddI-resistant, nivirapine-resistant, and other clinical HIV isolates. Intraperitoneal administration of hemin 4 days after HIV infection reduced viral load in the serum of human PBMC-reconstituted nonobese diabetic SCID mice by >6-fold. Suppression of HIV replication in hemin-activated cells correlated with the induction of HO-1 and was attenuated by tin protoporphyrin (SnPP) IX, an inhibitor of HO-1 activity, suggesting a pivotal role of this endogenous enzyme in the regulation of HIV infection. Hemin-induced HO-1 induction in the CCR-5, CXCR-4, and CD4 coexpressing GHOST(3) cells was consistent with the inhibition of Tat-dependent activation of long terminal repeat promoter leading to reduced GFP expression. These findings suggest an important role of hemin-induced HO-1 activity as a host defense mechanism against HIV-1 infection.

Animals↗

PubMed2006

Signal amplification systems in immunoassays: implications for clinical diagnostics.

Biomarkers in physiological specimens serve as useful sensors for clinical diagnosis. Accurate detection of specific markers is crucial for the diagnosis of disease, monitoring drug therapy and patient screening. In vitro immunoassays are probably the most common, simple and relatively inexpensive serological tools used in clinical laboratories for the diagnosis and management of disease. Despite continued efforts to improve the performance of immunoassays in the past three decades, there is a need for highly sensitive assays that can detect the lowest levels of disease markers with greater accuracy. This review summarizes recent advances made towards increasing the sensitivity of immunoassays by amplifying detection signals, with implications for the development of highly sensitive diagnostic systems; it also discusses the principles of related methodologies.

Diagnosis↗

PubMed2005

Selective side-chain modification of cysteine and arginine residues blocks pathogenic activity of HIV-1-Tat functional peptides.

Extracellular Tat protein of HIV-1 activates virus replication in HIV-infected cells and induces a variety of host factors in the uninfected cells, some of which play a critical role in the progression of HIV infection. The cysteine-rich and arginine-rich basic domains represent key components of the HIV-Tat protein for pathogenic effects of the full-length Tat protein and, therefore, could be ideal candidates for the development of a therapeutic AIDS vaccine. The present study describes selective modifications of the side-chain functional groups of cysteine and arginine amino acids of these HIV-Tat peptides to minimize the pathogenic effects of these peptides while maintaining natural peptide linkages. Modification of cysteine by introducing either a methyl or t-butyl group in the free sulfhydryl group and replacing the guanidine group with a urea linkage in the side chain of arginine in the cysteine-rich and arginine-rich Tat peptide sequences completely blocked the ability of these peptides to induce HIV replication, chemokine receptor CCR-5 expression, and NF-kappaB activity in monocytes. Such modifications also inhibited angiogenesis and migration of Kaposi's sarcoma cells normally induced by Tat peptides. Such chemical modifications of the cysteine-rich and arginine-rich peptides did not affect their reactivity with antibodies against the full-length Tat protein. With an estimated 40 million HIV-positive individuals worldwide and approximately 4 million new infections emerging every year, a synthetic subunit HIV-Tat vaccine comprised of functionally inactive Tat domains could provide a safe, effective, and economical therapeutic vaccine to reduce the progression of HIV disease.

Arginine↗

PubMed2004

Mechanisms for macrophage-mediated HIV-1 induction.

Viral latency is a long-term pathogenic condition in patients infected with HIV-1. Low but sustained virus replication in chronically infected cells can be activated by stimulation with proinflammatory cytokines such as TNF-alpha, IL-1 beta, or other host factors. However, the precise mechanism by which cellular activation induces latently infected cells to produce virions has remained unclear. In the present report, we present evidence that activation of HIV-1 replication in latently infected U1 or ACH2 cells by human macrophages is mediated by a rapid nuclear localization of NF-kappaB p50/p65 dimer with concomitant increased expression of proinflammatory cytokines. Multiplexed RT-PCR amplification of mRNA isolated from cocultures of macrophages and U1 and ACH2 cells showed significant induction of IL-1beta, IL-6, IL-8, TNF-alpha, and TGF-beta expression within 3 h of coincubation. Fixation of macrophages, U-1, or ACH2 cells with paraformaldehyde before coculture completely abrogated the induction of NF-kappaB subunits and HIV-1 replication, suggesting that cooperative interaction between the two cell types is an essential process for cellular activation. Pretreatment of macrophage-U1 or macrophage-ACH2 cocultures with neutralizing anti-TNF-alpha Ab down-regulated the replication of HIV-1. In addition, pretreatment of macrophage-U1 or macrophage-ACH2 cocultures with the NF-kappaB inhibitor (E)3-[(4-methylphenyl)sulfonyl]-2-propenenitrile (BAY 11-7082) prevented the induction of cytokine expression, indicating a pivotal role of NF-kappaB-mediated signaling in the reactivation of HIV-1 in latently infected cells by macrophages. These results provide a mechanism by which macrophages induce HIV-1 replication in latently infected cells.

Cell Communication↗

PubMed2002

Temporal relationship between V1V2 variation, macrophage replication, and coreceptor adaptation during HIV-1 disease progression.

BACKGROUND: Specific mutations in VPR and V2 potentially restrict HIV-1 replication in macrophages. Such restriction could potentially limit HIV replication in long-term non-progressors (LTNP), thus accounting for low viral load and delayed progression to AIDS. OBJECTIVE: To examine whether a specific VPR phenotype (truncated versus non-truncated) correlates with disease progression and whether elongated V2 restricts viral replication in macrophages or alters viral tropism. METHODS: Sequence analysis was carried for VPR and V1-V3 env from four rapid progressors (RPs), six late progressors (LPs), and three LTNPs in cohort of HIV-1-infected homosexual men. The replication kinetics of sequential isolates was examined in primary CD4 cells and macrophages and coreceptor usage was determined by GHOST infection assays. RESULTS: No differences were found in the VPR protein from RP and LTNP isolates. Analysis of the V2 region revealed that all RPs maintained similar V2 lengths (40 aa), whereas LPs and LTNPs acquired additional amino acids (2-13 aa) in the V2 region. Coreceptor specificity revealed that RP switch from CCR5 to multiple coreceptor usage, whereas LTNPs maintained R5 viruses. Sequential isolates from each group revealed comparable replication efficiencies in both T-cells and macrophages, regardless of the V2 length or coreceptor utilization. In addition, cross-section analysis of six LTNPs from Australia revealed extended V2 with consistent usage of CCR5 coreceptor. CONCLUSION: The present results suggest that acquisition of a V2 extension over time in HIV-1-infected LPs/LTNPs appears to correlate with maintenance of CCR5 usage among LTNPs. These findings may be important for a better understanding of the host interactions and disease progression.

Chronic Disease↗

PubMed2002

Involvement of integrin alphavbeta3 in the pathogenesis of human immunodeficiency virus type 1 infection in monocytes.

Attachment of HIV to macrophages is a critical early event in the establishment of infection. In the present study, we demonstrate the involvement of integrin alphavbeta3 (vitronectin receptor) in HIV infection of peripheral blood monocyte-derived macrophages. Culturing monocytes in the presence of M-CSF for 3 days upregulated expression of the alphav-containing integrins, alphavbeta3 and alphavbeta5. The increase in alphavbeta3 expression was accompanied by increased HIV-1 replication by monocytes. Immunoblot analysis showed that purified HIV-gp120 protein interacted with CD4 and alphavbeta3 in immunoprecipitation experiments. Neutralizing antibodies against the alphavbeta3 integrin interfered with the coprecipitation of alphavbeta3 with an anti-gp120 antibody and substantially inhibited HIV infection of monocytes. Neutralizing antibodies against alphavbeta5 or beta1 integrins did not significantly affect HIV infection. These results indicate that HIV infection of primary monocytes requires differentiation of these cells and may involve alphavbeta3 interaction with the HIV-1 envelope protein gp120 for productive infection.

Antibodies↗

PubMed2002

Design and construction of novel molecular conjugates for signal amplification (I): conjugation of multiple horseradish peroxidase molecules to immunoglobulin via primary amines on lysine peptide chains.

Immunoconjugates are widely used for indirect detection of analytes (such as antibodies or antigens) in a variety of immunoassays. However, the availability of functional groups such as primary amines or free sulfhydryls in an immunoglobulin molecule is the limiting factor for optimal conjugation and, therefore, determines the sensitivity of an assay. In the present study, an N-terminal bromoacetylated 20 amino acid peptide containing 20 lysine residues was conjugated to N-succinimidyl-S-acetylthioacetate (SATA)-modified IgG or free sulfhydryl groups on 2-mercaptoethylamine (2-MEA)-reduced IgG molecules via a thioether (S[bond]CH(2)CONH) linkage to introduce multiple reactive primary amines per IgG. These primary amines were then covalently coupled with maleimide-activated horseradish peroxidase (HRP). The poly-HRP-antibody conjugates thus generated demonstrated greater than 15-fold signal amplification upon reaction with orthophenyldiamine substrate. The poly-HRP-antibody conjugates efficiently detected human immunodeficiency virus (HIV)-1 antibodies in plasma specimens with significantly higher sensitivity than conventionally prepared HRP-antibody conjugates in an HIV-1 solid-phase enzyme immunoassay and Western blot analysis. The signal amplification techniques reported here could have the potential for development of highly sensitive immunodiagnostic assay systems.

HIV Antigens↗

PubMed2002

Design and construction of novel molecular conjugates for signal amplification (II): use of multivalent polystyrene microparticles and lysine peptide chains to generate immunoglobulin-horseradish peroxidase conjugates.

Spherical polystyrene microparticles expressing a large number of highly reactive functional groups were chemically engineered to generate antibody-enzyme conjugates as novel signal amplification systems. Chemically modified goat anti-human IgG and horseradish peroxidase (HRP) were combined in a 1:5 ratio and attached to 0.44 microm streptavidin microparticles or N-succinimidyl-S-acetylthioacetate (SATA)-activated 0.29 microm amino microparticles with highly reactive free sulfhydryl groups on their surface. The numbers of HRP molecules/microparticle were further increased by coupling HRP to primary amines on N-terminal biotinylated or bromoacetylated polypeptides containing 20 lysine residues prior to conjugation with streptavidin or sulfhydryl groups-containing microparticles. The antibody-poly-HRP immunoconjugates contained an estimated number of 10(5)HRP/streptavidin microparticle and 10(6)HRP/amino microparticle, respectively. These microparticle immunoconjugates efficiently bound to plasma anti-HIV-1 antibodies that had been captured by HIV antigens on 5 microm carboxyl magnetic microparticles and, upon reaction with orthophenyldiamine substrate, produced a detection signal with 5-8 times more sensitivity as compared to conventional HRP-conjugated goat anti-human IgG. The signal amplification technique by microparticle immunoconjugates may provide potentially novel tools for the development of highly sensitive diagnostic systems.

HIV Antibodies↗