Subhash Dhawan

Curcumin (Diferuloylmethane) Inhibition of Tumor Necrosis Factor (TNF)-Mediated Adhesion of Monocytes to Endothelial Cells by Suppression of Cell Surface Expression of Adhesion Molecules and of Nuclear Factor-κB Activation

Recruitment of leukocytes by endothelial cells and their subsequent migration from the vasculature into the tissue play major roles in inflammation. In the present study, we investigated the effect of curcumin, an antiinflammatory agent, on the adhesion of monocytes to human umbilical vein endothelial cells (EC). Treatment of EC with tumor necrosis factor (TNF) for 6 hr augmented the adhesion of monocytes to EC, and this adhesion was due to increased expression of intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and endothelial leukocyte adhesion molecule-1 (ELAM-1). Pretreatment of EC for 1 hr with curcumin completely blocked their adhesion to monocytes, as well as the cell surface expression of ICAM-1, VCAM-1, and ELAM-1 in EC. Although curcumin inhibited adhesion even when administered 1 hr after TNF treatment, maximum inhibition occurred when added either 1 hr before or at the same time as TNF. As the induction of various adhesion molecules by TNF requires activation of the transcription factor NF-kappaB, the effect of curcumin on the activation of this factor in the EC was also investigated. A 30-min treatment with TNF activated NF-kappaB; the activation was inhibited in a concentration-dependent manner by pretreatment with curcumin, indicating that NF-kappaB inhibition may play a role in the suppression of expression of adhesion molecules in EC. Our results demonstrate that the antiinflammatory properties of curcumin may be attributable, in part, to inhibition of leukocyte recruitment.

Emodin (3-methyl-1,6,8-trihydroxyanthraquinone) inhibits TNF-induced NF-κB activation, IκB degradation, and expression of cell surface adhesion proteins in human vascular endothelial cells

Most inflammatory agents activate nuclear transcription factor-κB (NF-κB) which results in expression of genes for cytokines, adhesion molecules, and enzymes involved in amplification and perpetuation of inflammation. Emodin (3-methyl-1,6,8-trihydroxyanthraquinone) is an active component from the roots of Polygonum cuspidatum that has been reported to exhibit antiinflammatory properties but the mechanism is not known. In the present study we investigated the effects of emodin on the activation of NF-κB in human umbelical vein endothelial cells (EC). Treatment of EC with TNF activated NF-κB; preincubation with emodin inhibited this activation in a dose- and time-dependent manner. Emodin did not chemically modify NF-κB subunits but rather inhibited degradation of IκB, an inhibitory subunit of NF-κB. Since the promoter regions of ICAM-1, VCAM-1, and ELAM-1 contain NF-κB binding sites and these adhesion molecules are involved in the attachment of leukocytes to EC, the effect of emodin on the adhesion of monocytes to EC and the expression of these adhesion molecules was also studied. Treatment of EC with TNF for 6 h increased the adhesion of monocytes to EC, which correlated with increases in cell surface expression of ICAM-1, VCAM-1 and ELAM-1. Pretreatment of EC for 1 h with emodin inhibited both monocyte-EC attachment and expression of ICAM-1, ELAM-1 and VCAM-1. These results indicate that emodin is a potent inhibitor of NF-κB activation and expression of adhesion molecules and thus could be useful in treating various inflammatory diseases.

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.

Human immunodeficiency virus-1-tat induces matrix metalloproteinase-9 in monocytes through protein tyrosine phosphatase-mediated activation of nuclear transcription factor NF-κB

We have previously shown that human immunodeficiency virus (HIV)‐1‐tat induces the production of matrix metalloproteinase‐9 (MMP‐9) in human monocytes by a mechanism that is not understood. In the present report, we demonstrate that HIV‐tat‐induced expression of MMP‐9 is blocked by inhibitors of protein tyrosine phosphatases (PTPases). PTPase inhibitors also blocked HIV‐tat‐induced nuclear transcription factor NF‐κB activation and IκBα degradation required for MMP‐9 induction. These results suggest that HIV‐tat induces MMP‐9 in human monocytes through activation of PTPase and NF‐κB.

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.

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-α, IL-1 β, 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-κB 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-1β, IL-6, IL-8, TNF-α, and TGF-β expression within 3 h of coincubation. Fixation of macrophages, U-1, or ACH2 cells with paraformaldehyde before coculture completely abrogated the induction of NF-κB 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-α Ab down-regulated the replication of HIV-1. In addition, pretreatment of macrophage-U1 or macrophage-ACH2 cocultures with the NF-κB inhibitor (E)3-[(4-methylphenyl)sulfonyl]-2-propenenitrile (BAY 11-7082) prevented the induction of cytokine expression, indicating a pivotal role of NF-κB-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.

Lipopolysaccharide suppresses HIV-1 replication in human monocytes by protein kinase C-dependent heme oxygenase-1 induction.

LPS is an important component of the Gram‐negative bacteria cell wall. It activates monocytes and induces multiple host immune and inflammatory responses. Interestingly, in spite of inducing host‐inflammatory responses, LPS also protects monocyte‐derived macrophages from infection by HIV‐1. In this report, we have shown that LPS treatment of human monocyte‐derived macrophages markedly suppressed HIV‐1 replication, even on addition to infected cells 24 h after infection. Inhibition of HIV‐1 replication was associated with PKC‐dependent induction of HO‐1, a cytoprotective enzyme known to catabolize heme. Pretreatment with the PKC inhibitor Go 6976 not only substantially inhibited LPS‐mediated induction of HO‐1 but also attenuated LPS‐induced suppression of HIV replication. Significant reduction of HIV replication by inhibitors of JNK, NF‐κB, and PI3K was independent of a LPS‐mediated anti‐HIV effect. Specificity of HO‐1 was confirmed by substantial reversal of LPS‐induced viral replication by pretreatment of cells with SnPP IX, an inhibitor of HO‐1 enzyme activity. These results demonstrate a previously undefined function of HO‐1 as a host defense mechanism in LPS‐mediated inhibition of HIV‐1 replication.

INTERFERON-GAMMA INHIBITS HIV-INDUCED INVASIVENESS OF MONOCYTES

HIV‐infected monocytes form highly invasive network on basement membrane matrix and secrete high levels of 92‐kd metalloproteinase (MMP‐9), an enzyme that degrades basement membrane proteins. In the present study, using matrigel as a model basement membrane system, we demonstrate that treatment of human immunodeficiency virus (HIV)‐infected monocytes with interferon‐γ at 50 U/ml inhibited the ability of infected monocytes to form an invasive network on matrigel and their invasion through the matrigel matrix. These effects were associated with a significant reduction in the levels of MMP‐9 produced by HIV‐infected monocytes treated with interferon‐γ 1 day prior to infection with HIV as compared with that of untreated HIV‐infected monocytes. Monocytes treated with interferon‐γ 1 day after HIV infection showed the presence of integrated HIV sequences; however, the levels of MMP‐9 were substantially lower than those produced by monocytes inoculated with live HIV, heat‐inactivated HIV, or even the control uninfected monocytes. Exposure of monocytes to heat‐inactivated HIV did not result in increased invasiveness or high MMP‐9 production, suggesting that regulation of metalloproteinase by monocytes was independent of CD4‐gp120 interactions and required active virus infection. Furthermore, addition of interferon‐γ to monocytes on day 10 after infection inhibited MMP‐9 production by more than threefold with no significant reduction of virus replication. These results indicate that the mechanism of interferon‐γ–induced down‐regulation of MMP‐9 levels and reduced monocyte invasiveness may be mediated by a mechanism independent of antiviral activity of IFN‐γ in monocytes. Down‐regulation of MMP‐9 in HIV‐infected monocytes by interferon‐γ may play an important role in the control of HIV pathogenesis.

Immunization with a novel HIV-1-Tat multiple-peptide conjugate induces effective immune response in mice.

We report here a novel, highly immunogenic synthetic, multiple-peptide conjugate comprising functional domains Tat(21-40) and Tat(53-68) from HIV-1 group M plus Tat(9-20) from HIV-1 group O of the HIV-Tat protein (HIV-1-Tat-MPC). Vaccination of mice with HIV-1-Tat-MPC induced an effective immune response to all three functional domains. The anti-HIV-1-Tat-MPC antibodies efficiently inhibited Tat-induced viral activation in monocytes infected with HIV(Ba-L) as well as with various clinical HIV-1 isolates, and reduced Tat-mediated cytopathicity in infected cells by 60-75%. Our results indicate that anti-HIV-1-Tat-MPC antibodies inhibit viral pathogenesis, possibly by blocking functional determinants of Tat and disrupting autocrine and paracrine actions of secreted Tat protein. This epitope-specific, synthetic Tat construct may, therefore, provide a subunit AIDS vaccine candidate for inducing an effective immunoprophylaxis response to reduce progression of HIV infection.

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.