Biomedical subjects

James E K Hildreth

Publications and source records attributed to James E K Hildreth.

14 recordsLinked to original sources

PubMed2006

Exosomes and HIV Gag bud from endosome-like domains of the T cell plasma membrane.

Exosomes are secreted, single membrane organelles of approximately 100 nm diameter. Their biogenesis is typically thought to occur in a two-step process involving (1) outward vesicle budding at limiting membranes of endosomes (outward = away from the cytoplasm), which generates intralumenal vesicles, followed by (2) endosome-plasma membrane fusion, which releases these internal vesicles into the extracellular milieu as exosomes. In this study, we present evidence that certain cells, including Jurkat T cells, possess discrete domains of plasma membrane that are enriched for exosomal and endosomal proteins, retain the endosomal property of outward vesicle budding, and serve as sites of immediate exosome biogenesis. It has been hypothesized that retroviruses utilize the exosome biogenesis pathway for the formation of infectious particles. In support of this, we find that Jurkat T cells direct the key budding factor of HIV, HIV Gag, to these endosome-like domains of plasma membrane and secrete HIV Gag from the cell in exosomes.

Antigens, CD↗

PubMed2006

Production and characterization of a monoclonal antibody against Niemann Pick type C protein.

Niemann Pick type C is a severe, incurable disease caused, in the majority of cases, by mutations in the Niemann Pick type C protein 1 (NPC1). The pathology and biochemical changes associated with the disease have been extensively studied. However, the function of the protein is still unknown, and recent studies challenge the established concept that a defect in cholesterol and sphingolipids transport is the primary cause of this human lipidosis. Clearly defining the mechanisms by which defects in this protein lead to the disease phenotype will require further studies on the structure and function of this protein. Therefore, the development of a well-characterized monoclonal antibody (MAb) against this protein to facilitate such studies is an important goal. Here, we describe the production and characterization of such a MAb. The antibody is demonstrated to be highly specific and species cross-reactive. Function studies show that the antibody induces the NPC1 disease phenotype in cells, making it highly likely that the antibody blocks function of the NPC1 protein.

Animals↗

PubMed2006

Characterization of an anti-lymphocyte function-associated antigen-1 antibody in a simian immunodeficiency virus-pig-tailed macaque (Macaca nemestrina) model.

The simian immunodeficiency virus (SIV)/pig-tailed macaque (Macaca nemestrina) model of acquired immune deficiency syndrome (AIDS) is a powerful system in which to study cell adhesion molecules and retroviral pathogenesis in vivo. Preliminary experiments were conducted to examine the role of lymphocyte function-associated antigen 1 (LFA-1) in early SIV infection in vivo by using an LFA-1 monoclonal antibody (MHM.23) specific to human LFA-1. In vitro studies revealed that at concentrations of > or = 20 microg/ml, MHM.23 blocked LFA-1-mediated adhesion and T-cell activation (>90%) of pig-tailed macaque peripheral blood mononuclear cells (PBMCs). In addition, SIVmac239 infection of macaque cells was inhibited in a dose-dependant manner by MHM.23. Administration of MHM.23 to pig-tailed macaques inhibited LFA-1-ICAM-1-mediated activity in vivo and maintained binding on macaque cells for < or = 4 d. Our in vitro studies indicated that at an MHM.23 concentration of 20 microg/ml, macaque PBMCs were completely saturated. Our in vivo studies determined that 5 mg/kg MHM.23 intravenously every 24 h was required to maintain saturating levels and inhibit LFA-1-ICAM-1 function in pig-tailed macaques.

Acquired Immunodeficiency Syndrome↗

PubMed2005

Enhancement of cell mediated immune responses using lipid depleted lentivirus as immunogen: a novel approach for inducing recognition of new viral epitopes.

We tested the hypothesis that removal of viral lipids using diisopropylether can enhance antigenicity of SIVmac251. DIPE delipidation removed cholesterol from SIVmac251 without significant loss of viral protein or RNA. Mice immunized with the same SIV preparation but boosted with delipidated SIVmac251 exhibited significantly broader and higher cellular and humoral immune responses compared to live or AT-2-inactivated virus. As little as 1microg (total protein) of delipidated virus was sufficient to induce such enhanced immune responses. Thus, solvent treated lentivirus may provide a novel strategy to generate immune responses to additional viral epitopes.

Animals↗

PubMed2004

The evolution of alloimmunity and the genesis of adaptive immunity.

Infectious agents select for host immune responses that destroy infectious nonself yet maintain tolerance to self. Here we propose that retroviruses and other host-antigen associated pathogens (HAAPs) select for the genetic, biochemical, and cell biological properties of alloimmunity, also known as the histocompatibility or tissue rejection response. This hypothesis predicts the major observations regarding histocompatibility responses, including: (i) their existence in animals as diverse as sponges and humans; (ii) extreme polymorphism and balanced allele frequencies at histocompatibility loci, including the human MHC and blood group loci; (iii) the frequency dependent selection of histocompatibility alleles; (iv) the ancient age of many alloantigenic polymorphisms; (v) the high ratio of nonsynonymous mutations to synonymous mutations at histocompatibility loci; (vi) disassortative mating based on MHC alleles; (vii) the inability to explain the existence and continuing selection of histocompatibility alleles by other more conventional biochemical and genetic paradigms; and (viii) the susceptibility of HAAPs, particularly retroviruses such as HIV (human immunodeficiency virus), to histocompatibility reactions. In addition, the hypothesis that HAAPs select the forms and molecules of alloimmunity offers simple explanations for the evolution of histocompatibility systems over time, the initial selection of hypervariable immune mechanisms, and the genesis of adaptive immunity.

Alleles↗

PubMed2003

Evidence that HIV budding in primary macrophages occurs through the exosome release pathway.

Lipid rafts are specialized regions of cell membranes enriched in cholesterol and sphingolipids that are involved in immune activation and signaling. Studies in T-cells indicate that these membrane domains serve as sites for release of human immunodeficiency virus (HIV). By budding through lipid rafts in T-cells, HIV selectively incorporates raft markers and excludes non-raft proteins. This process has been well studied in T-cells, but it is unknown whether lipid rafts serve as budding sites for HIV in macrophages. Recently, we proposed a new model of retroviral biogenesis called the Trojan exosome hypothesis (Gould, S. J., Booth, A., and Hildreth, J. E. K. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 10592-10597). This model proposes that retroviruses coopt the existing cellular machinery for exosomal release. Here, we performed the first test designed to differentiate between the lipid raft hypothesis of retroviral biogenesis and the Trojan exosome hypothesis. Using macrophages, we examined the relative abundance of several host proteins on the cell surface, in lipid rafts, and on both HIV particles and exosomes derived from these cells. Our results show significant differences in the abundance of host proteins on the cell surface and in HIV. Moreover, our data demonstrate discordance in the abundance of some proteins in lipid rafts and in HIV. Finally, our data reveal a strong concordance between the host cell protein profile of exosomes and that of HIV. These results strongly support the Trojan exosome hypothesis and its prediction that retroviral budding represents exploitation of a pre-existing cellular pathway of intercellular vesicle trafficking.

Antigens, CD↗

PubMed2003

The Trojan exosome hypothesis.

We propose that retroviruses exploit a cell-encoded pathway of intercellular vesicle traffic, exosome exchange, for both the biogenesis of retroviral particles and a low-efficiency but mechanistically important mode of infection. This Trojan exosome hypothesis reconciles current paradigms of retrovirus-directed transmission with the unique lipid composition of retroviral particles, the host cell proteins present in retroviral particles, the complex cell biology of retroviral release, and the ability of retroviruses to infect cells independently of Envelope protein-receptor interactions. An exosomal origin also predicts that retroviruses pose an unsolvable paradox for adaptive immune responses, that retroviral antigen vaccines are unlikely to provide prophylactic protection, and that alloimmunity is a central component of antiretroviral immunity. Finally, the Trojan exosome hypothesis has important implications for the fight against HIV and AIDS, including how to develop new antiretroviral therapies, assess the risk of retroviral infection, and generate effective antiretroviral vaccines.

AIDS Vaccines↗

PubMed2003

Involvement of macrophage mannose receptor in the binding and transmission of HIV by macrophages.

The human immunodeficiency virus (HIV) is an enveloped virus whose surface glycoprotein gp120 binds CD4 on target cell membranes to initiate infection. About half of the carbohydrates on gp120 are terminally mannosylated, a pattern common to many pathogens. We have examined the ability of macrophage mannose receptor (MMR) on primary monocyte-derived macrophages to bind HIV and facilitate its transmission to T cells. We adapted the tyramide signal amplification system for fluorescence detection of HIV bound to macrophages. Our data show that approximately 60% of the initial association of HIV with macrophages that lack expression of DC-SIGN (a dendritic cell-specific ICAM-3 receptor/HIV-1-binding protein) is MMR mediated, as evidenced by inhibition with mannan, D-mannose, EDTA, and soluble mannose-binding lectin, but not by D-galactose. This inhibition is not seen in cells that lack MMR. Macrophages are able to mediate transmission of bound HIV to co-cultured T cells, and this transmission is blocked up to 80% by inhibitors of MMR binding. Unlike virus bound to DC-SIGN, macrophage-bound HIV has a slightly lower half-life compared to free virus, with no transmission in co-culture observed beyond 24 h after virus binding to macrophages. Results obtained with endocytosis inhibitors indicate that this decrease in viral longevity is due to rapid internalization of macrophage-bound HIV. Together, these results suggest a substantial role for MMR in the binding and transmission of HIV-1 by macrophages.

Cell Adhesion Molecules↗

PubMed2003

Lipid rafts and HIV pathogenesis: virion-associated cholesterol is required for fusion and infection of susceptible cells.

We have shown that HIV budding occurs at cholesterol-rich membrane microdomains called lipid rafts (Nguyen and Hildreth, J Virol 2000;74:3264-3272). This observation prompted us to examine the role in HIV entry of cholesterol in the membrane of cells. We recently reported that host cell cholesterol is required for HIV infection (Liao et al., AIDS Res Hum Retroviruses 2001;17:1009-1019). In the present study we examined the role of virion-associated cholesterol in HIV infection by modulating the cholesterol content of virions and infected cells with 2-hydoxypropyl-beta-cyclodextrin (beta-cyclodextrin). Our results show that removal of cholesterol from the membrane of HIV-infected cells dramatically lowered virus release and that virions released from cholesterol-depleted cells are minimally infectious. Exposure of infectious HIV particles to beta-cyclodextrin resulted in a dose-dependent inactivation of the virus. In both cases, the effect was attributable to loss of cholesterol and could be reversed by replenishing cholesterol. beta-Cyclodextrin-treated, noninfectious HIV retained its ability to bind cells. Western blot, p24 core ELISA, and reverse transcription assays indicated that virions remained intact after treatment with beta-cyclodextrin at concentrations that abolished infectivity. Electron microscopy revealed that beta-cyclodextrin-treated HIV had a morphology very similar to that of untreated virus. R18 fluorescence dequenching studies showed that beta-cyclodextrin-treated HIV did not fuse to the membrane of susceptible cells. Dequenching was restored by replenishing virion-associated cholesterol. The results indicate that cholesterol in HIV particles is strictly required for fusion and infectivity. These observations in combination with those of past studies indicate beta-cyclodextrin to be an excellent candidate for use as a chemical barrier for AIDS prophylaxis.

2-Hydroxypropyl-beta-cyclodextrin↗

PubMed2003

Cholesterol depletion of human immunodeficiency virus type 1 and simian immunodeficiency virus with beta-cyclodextrin inactivates and permeabilizes the virions: evidence for virion-associated lipid rafts.

Recent evidence suggests that human immunodeficiency virus type 1 (HIV-1) particles assemble and bud selectively through areas in the plasma membrane of cells that are highly enriched with glycosylphosphatidylinositol-anchored proteins and cholesterol, called lipid rafts. Since cholesterol is required to maintain lipid raft structure and function, we proposed that virion-associated cholesterol removal with the compound 2-hydroxy-propyl-beta-cyclodextrin (beta-CD) might be disruptive to HIV-1 and simian immunodeficiency virus (SIV). We examined the effect of beta-CD on the structure and infectivity of cell-free virions. We found that beta-CD inactivated HIV-1 and SIV in a dose-dependent manner and permeabilized the viral membranes, resulting in the loss of mature Gag proteins (capsid, matrix, nucleocapsid, p1, and p6) without loss of the envelope glycoproteins. SIV also lost reverse transcriptase (RT), integrase (IN), and viral RNA. IN appeared to be only slightly diminished in HIV-1, and viral RNA, RT, matrix, and nucleocapsid proteins were retained in HIV-1 but to a much lesser degree. Host proteins located internally in the virus (actin, moesin, and ezrin) and membrane-associated host proteins (major histocompatibility complex classes I and II) remained associated with the treated virions. Electron microscopy revealed that under conditions that permeabilized the viruses, holes were present in the viral membranes and the viral core structure was perturbed. These data provide evidence that an intact viral membrane is required to maintain mature virion core integrity. Since the viruses were not fixed before beta-CD treatment and intact virion particles were recovered, the data suggest that virions may possess a protein scaffold that can maintain overall structure despite disruptions in membrane integrity.

2-Hydroxypropyl-beta-cyclodextrin↗

PubMed2003

Barrier-to-autointegration factor BAF binds p55 Gag and matrix and is a host component of human immunodeficiency virus type 1 virions.

Barrier-to-autointegration factor (BAF) is a conserved human chromatin protein exploited by retroviruses. Previous investigators showed that BAF binds double-stranded DNA nonspecifically and is a host component of preintegration complexes (PICs) isolated from cells infected with human immunodeficiency virus type 1 (HIV-1) or Moloney murine leukemia virus. BAF protects PIC structure and stimulates the integration of salt-stripped PICs into target DNA in vitro. PICs are thought to acquire BAF from the cytoplasm during infection. However, we identified two human tissues (of 16 tested) in which BAF mRNA was not detected: thymus and peripheral blood leukocytes, which are enriched in CD4(+) T lymphocytes and macrophage precursors, respectively. BAF protein was detected in activated but not resting CD4(+) T lymphocytes; thus, if BAF were essential for PIC function, we hypothesized that virions might "bring their own BAF." Supporting this model, BAF copurified with HIV-1 virions that were digested with subtilisin to remove microvesicle contaminants, and BAF was present in approximately zero to three copies per virion. In three independent assays, BAF bound directly to both p55 Gag (the structural precursor of HIV-1 virions) and its cleaved product, matrix. Using lysates from cells overexpressing Gag, endogenous BAF and Gag were coimmunoprecipitated by antibodies against Gag. Purified recombinant BAF had low micromolar affinities (1.1 to 1.4 micro M) for recombinant Gag and matrix. We conclude that BAF is present at low levels in incoming virions, in addition to being acquired from the cytoplasm of newly infected cells. We further conclude that BAF might contribute to the assembly or activity of HIV-1 PICs through direct binding to matrix, as well as DNA.

CD4-Positive T-Lymphocytes↗

PubMed2002

Sustained antibody-dependent cell-mediated cytotoxicity (ADCC) in SIV-infected macaques correlates with delayed progression to AIDS.

Although several in vitro lines of evidence support the potential power of antibody-dependent cell-mediated cytotoxicity (ADCC) in controlling HIV infection, the role of ADCC in the pathogenesis of HIV infection in vivo remains uncertain. There are few studies to date that longitudinally determine the plasma ADCC activity in HIV-infected subjects. We sought to establish an SIV/macaque model to perform such a longitudinal study. In the rhesus macaque cohort studied here, three of five macaques (designated Group 1) maintained higher plasma ADCC activity for at least 1 year after inoculation with SIV/17E-Br. The ADCC activity of the two remaining macaques (Group 2) fell 12 weeks after inoculation. There were also differences in longitudinal measurements of anti-SIV envelope IgG titers and CD4 counts. Group 1 macaques maintained higher antienvelope IgG titers and higher CD4(+) T cell numbers as late as 60 weeks postinoculation, while Group 2 macaques had significantly lower titers at 1 year postinoculation and lower CD4(+) T cell counts by 30 weeks postinoculation. Our study shows a correlation between humoral response, ADCC activity, and disease progression (as measured by CD4(+) T cell counts). In these animals, ADCC activity is associated with delayed progression to AIDS. Further studies are underway to determine if ADCC is a protective immune response in SIV infection or if ADCC is a marker of intact cellular and humoral immune responses.

Animals↗

PubMed2002

Oligomers of glycamino acid.

Glycamino acids, a family of sugar amino acids, are derivatives of C-glycosides that possesses a carboxyl group at the C-1 position and an amino group replacing one of the hydroxyl groups at either the C-2, 3, 4, or 6 position. We have prepared a series of glucose-type glycamino acids as monomeric building blocks: these are derivatives of 2-NH(2)-Glc-beta-CO(2)H 1, 3-NH(2)-Glc-beta-CO(2)H 2, 4-NH(2)-Glc-beta-CO(2)H 3, and 6-NH(2)-Glc-beta-CO(2)H 4 and constructed four types of homo-oligomers, beta(1-->2)-linked I, beta(1-->3)-linked II, beta(1-->4)-linked III, and beta(1-->6)-linked IV, employing the well-established N-Boc and BOP strategy. CD and NMR spectral studies of these oligomers suggested that only the beta(1-->2)-linked homo-oligomer possessed a helical structure that seems to be predetermined by the linkage position. Homo-oligomers with beta(1-->2)-linkages I and beta(1-->6)-linkages IV were also subjected to O-sulfation, and these O-sulfated oligomers were found to be able, in a linkage-specific manner, to effectively inhibit L-selectin-mediated cell adhesion, HIV infection, and heparanase activity without the anticoagulant activity associated with naturally occurring sulfated polysaccharides such as heparin.

Amino Acids↗

PubMed2002

Vaginal transmission of cell-associated HIV-1 in the mouse is blocked by a topical, membrane-modifying agent.

Because both HIV-1 virions and HIV-infected cells are present in the semen and cervical mucus of infected individuals, HIV-1 prevention strategies must consider both cell-free and cell-associated virus. Antibodies that target HIV-1 virions have been shown to prevent vaginal transmission of cell-free virus in macaques, but since cell-associated transmission has not been reliably demonstrated in this model system, no strategies to prevent such transmission have been tested. We have employed a mouse model in which SCID mice carry human peripheral blood leukocytes (HuPBLs). In these mice, vaginal transmission of cell-associated, but not cell-free, HIV-1 transmission occurs, mediated by transepithelial migration of HIV-infected cells. Topical application of beta-cyclodextrin (beta-CD), a cholesterol-sequestering agent that interferes with cell migration and budding of virus from lipid rafts, blocks transmission of cell-associated HIV-1. The HuPBL-SCID model of vaginal HIV-1 transmission should prove useful for investigating cell-associated HIV-1 transmucosal HIV-1 transmission, as well as for screening reagents for their potential efficacy in preventing sexual HIV-1 transmission.

2-Hydroxypropyl-beta-cyclodextrin↗