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Biomedical subjects

Roger J Pomerantz

Publications and source records attributed to Roger J Pomerantz.

At least 73 records · Page 4Linked to original sources
PubMed2003

Human immunodeficiency virus type 1 enters primary human brain microvascular endothelial cells by a mechanism involving cell surface proteoglycans independent of lipid rafts.

Several studies have reported a crucial role for cholesterol-enriched membrane lipid rafts and cell-associated heparan sulfate proteoglycans (HSPGs), a class of molecules that can localize in lipid rafts, in the entry of human immunodeficiency virus type 1 (HIV-1) into permissive cells. For the present study, we examined the role of these cell surface moieties in HIV-1 entry into primary human brain microvascular endothelial cells (BMVECs), which represent an important HIV-1 central nervous system-based cell reservoir and a portal for neuroinvasion. Cellular cholesterol was depleted by exposure to beta-cyclodextrins and 3-hydroxy-3-methylglutaryl (HMG)-coenzyme A reductase inhibitors (statins), the loss of cholesterol was quantitated, and disruption of membrane rafts was verified by immunofluorescence. Nevertheless, these treatments did not affect binding of several strains of HIV-1 virions to BMVECs at 4 degrees C or their infectivities at 37 degrees C. In contrast, we confirmed that cholesterol depletion and raft disruption strongly inhibited HIV-1 binding and infection of Jurkat T cells. Enzymatic digestion of cell-associated HSPGs on human BMVECs dramatically inhibited HIV-1 infection, and our data from quantitative HIV-1 DNA PCR analysis strongly suggest that cell-associated chondroitin sulfate proteoglycans greatly facilitate infective entry of HIV-1 into human BMVECs. These findings, in combination with our earlier work showing that human BMVECs lack CD4, indicate that the molecular mechanisms for HIV-1 entry into BMVECs are fundamentally different from that of viral entry into T cells, in which lipid rafts, CD4, and probably HSPGs play important roles.

Brain↗
PubMed2003

Covalently linked human immunodeficiency virus type 1 gp120/gp41 is stably anchored in rhabdovirus particles and exposes critical neutralizing epitopes.

Rabies virus (RV) vaccine strain-based vectors show significant promise as potential live-attenuated vaccines against human immunodeficiency virus type 1 (HIV-1). Here we describe a new RV construct that will also likely have applications as a live-attenuated or killed-particle immunogen. We have created a RV containing a chimeric HIV-1 Env protein, which contains introduced cysteine residues that give rise to an intermolecular disulfide bridge between gp120 and the ectodomain of gp41. This covalently linked gp140 (gp140 SOS) is fused in frame to the cytoplasmic domain of RV G glycoprotein and is efficiently incorporated into the RV virion. On the HIV-1 virion, the gp120 and gp41 moieties are noncovalently associated, which leads to extensive shedding of gp120 from virions and virus-infected cells. The ability to use HIV-1 particles as purified, inactivated immunogens has been confounded by the loss of gp120 during preparation. Additionally, monomeric gp120 and uncleaved gp160 molecules have been shown to be poor antigenic representations of virion-associated gp160. Because the gp120 and gp41 portions are covalently attached in the gp140 SOS molecule, the protein is maintained on the surface of the RV virion throughout purification. Surface immunostaining and fluorescence-activated cell sorting analysis with anti-envelope antibodies show that the gp140 SOS protein is stably expressed on the surface of infected cells and maintains CD4 binding capabilities. Furthermore, Western blot and immunoprecipitation experiments with infected-cell lysates and purified virions show that a panel of neutralizing anti-envelope antibodies efficiently recognize the gp140 SOS protein. The antigenic properties of this recombinant RV particle containing covalently attached Env, as well as the ability to present Env in a membrane-bound form, suggest that this approach could be a useful component of a HIV-1 vaccine strategy.

Animals↗
PubMed2003

In a subset of subjects on highly active antiretroviral therapy, human immunodeficiency virus type 1 RNA in plasma decays from 50 to <5 copies per milliliter, with a half-life of 6 months.

Three of five virally suppressed human immunodeficiency virus type I (HIV-1)-infected patients treated with highly active antiretroviral therapy and followed intensively with a supersensitive reverse transcriptase PCR assay with a lower limit of quantitation of 5 copies/ml showed statistically significant viral load decays below 50 copies/ml, with half-lives of 5 to 8 months and a mean of 6 months. This range of half-lives is consistent with the estimated half-life of the latent HIV-1 reservoir in the peripheral blood. Those patients without decay of viral load in plasma may have significant cryptic HIV-1 residual replication.

Acquired Immunodeficiency Syndrome↗
PubMed2003

Recombinant rhabdoviruses as potential vaccines for HIV-1 and other diseases.

The failure to develop vaccines to protect against important infectious diseases such as human immunodeficiency virus type I (HIV-1) or Hepatitis C virus (HCV) has increased the interest in new vaccine strategies. One of these methods is immunization with an attenuated recombinant viral vector expressing a foreign antigen, which could protect individuals from later exposure to the respective pathogen. A new method to recover a non-segmented negative-stranded RNA virus (NNSV) from cDNA was described for the first time for rabies virus (RV), a member of the rhabdovirus family. The same approach was successfully used for another rhabdovirus, vesicular stomatitis virus (VSV), and opened the possibility to use rhabdoviruses as vaccine vehicles and biomedical tools. Further research showed that the genomes of rhabdoviruses are highly flexible, easy to manipulate, and able to express large and even multiple foreign genes, and therefore are excellent vaccine candidates. In addition, it has been shown for both RV and VSV that their single surface glycoprotein G, which is responsible for attachment and fusion to the host cell, can functionally be replaced by other viral or cellular glycoproteins. This review gives an overview of the use of RV and VSV as promising new candidates in the fight against HIV-1 and other human diseases.

AIDS Vaccines↗
PubMed2002

Ethanol strongly potentiates apoptosis induced by HIV-1 proteins in primary human brain microvascular endothelial cells.

Ethanol may have significant effects on human immunodeficiency virus type I (HIV-1) pathogenesis in vivo. As such, the effects of ethanol treatment were studied on the proapoptotic potential of various HIV-1 proteins in primary isolated human brain microvascular endothelial cells (MVECs), a major cellular component of the blood-brain barrier. Low-passage primary brain MVECs were treated with recombinant HIV-1 proteins Nef, Vpr, Tat and gp120 proteins from X4, R5, and X4R5 viral strains, with and without ethanol at various relevant concentrations. The apoptotic potential of each HIV-1 protein with and without ethanol was compared with cells treated with ethanol alone or GST protein as a control, under similar conditions. Specific HIV-1 proteins induced apoptosis in primary isolated human brain MVECs, which was potentiated on treatment with 0.1 and 0.3% (v/v) ethanol. Cotreatment with ethanol and specific HIV-1 proteins showed enhanced lactate dehydrogenase release, compared with MVECs treated with ethanol alone. The presence of ethanol in in vitro culture medium also enhanced HIV-1 protein-mediated tumor necrosis factor-alpha production, compared with cells treated with ethanol alone or GST protein. Thus, these studies demonstrate ethanol's potential for inducing apoptosis of human MVECs with relevant HIV-1-specific proteins and suggest a potential synergistic effect in augmenting HIV-1 neuroinvasion and neuropathogenesis in vivo.

Apoptosis↗
PubMed2002

Potent suppression of viral infectivity by the peptides that inhibit multimerization of human immunodeficiency virus type 1 (HIV-1) Vif proteins.

Virion infectivity factor (Vif) is essential for the replication of human immunodeficiency virus type 1 (HIV-1) in vivo, but its function remains uncertain. Recently, we have shown that Vif proteins are able to form multimers, including dimers, trimers, or tetramers. Because the multimerization of Vif proteins is required for Vif function in the viral life cycle, we propose that it could be a novel target for anti-HIV-1 therapeutics. Through a phage peptide display method, we have identified a set of 12-mer peptides containing a PXP motif that binds to HIV-1 Vif protein. These proline-enriched peptides potently inhibited the Vif-Vif interaction in vitro. We have also screened a set of synthesized Vif peptides (15-mer), which covers all the amino acids of the HIV-1 Vif protein sequence, for their ability to inhibit the Vif-Vif interaction in vitro. We demonstrated that Vif-derived proline-enriched peptides that contain the (161)PPLP(164) domain are able to inhibit the Vif-Vif interaction. Conversely, the deletion of the (161)PPLP(164) domain of Vif protein will significantly impair the capability of Vif proteins to interact with each other, indicating that the (161)PPLP(164) domain plays a key role in Vif multimerization. All these results demonstrate that the proline-enriched peptides block the multimerization of Vif through interfering with the polyproline interfaces of Vif formed by (161)PPLP(164) domain. Moreover, these peptides which inhibit the Vif-Vif interaction in vitro potently inhibit HIV-1 replication in the "nonpermissive" T-cells. We propose that this study starts a novel strategy to develop structural diverse inhibitors of Vif such as peptidomimetics or small organic molecules.

Amino Acid Sequence↗
PubMed2002

Approaching eradication of highly active antiretroviral therapy-persistent human immunodeficiency virus type 1 reservoirs with immune activation therapy.

Highly active antiretroviral therapy (HAART) has dramatically altered the human immunodeficiency virus type 1 (HIV-1) pandemic in the developed world. Most patients treated with HAART will maintain clinically undetectable plasma virus loads with concomitant dramatic decreases in mortality and morbidity. Nevertheless, HAART does not eradicate HIV-1 infection on the basis of persistent low-level or cryptic viral replication and, of importance, latent provirus in resting CD4+ T lymphocytes. New approaches are now being developed for stimulation of "HAART-persistent" reservoirs. Immune activation therapy (IAT) has begun to be used in attempts to stimulate the HIV-1 latent reservoir. These studies and new approaches to activating latent virus in resting CD4+ T cells are reviewed and critically analyzed in the present report. Development of novel IAT may lead to long-term remission or viral eradication in the future.

Anti-HIV Agents↗
PubMed2002

Intensification and stimulation therapy for human immunodeficiency virus type 1 reservoirs in infected persons receiving virally suppressive highly active antiretroviral therapy.

Highly active antiretroviral therapy (HAART) has led to significant changes in mortality and morbidity in the human immunodeficiency virus type 1 (HIV-1) epidemic. Nevertheless, because of molecular mechanisms of viral persistence, HAART does not eradicate HIV-1. Didanosine and hydroxyurea were added to the antiretroviral regimens of 3 HIV-1-infected men who were receiving stable HAART and who had HIV-1 RNA levels <50 copies/mL at the initiation of the study protocol, as a novel intensification to attack cryptic viral replication; low-dose OKT3 was then administered, followed by a course of interleukin-2, to stimulate latent provirus. Replication-competent virus was undetectable after treatment, and plasma viral RNA was either undetectable or <5 copies/mL. In trial periods during which no antiretroviral therapy was administered, the patients developed plasma viral rebound. This translational approach combines novel intensification and stimulation therapy to deplete residual HIV-1 reservoirs. Additional experimental approaches must be developed if HIV-1 eradication is to become possible in patients receiving virally suppressive HAART.

Adult↗
PubMed2002

A trans-dominant negative HIV type 1 Rev with intact domains of NLS/NOS and NES.

The nuclear diffusion inhibitory signal (NIS) is a 15-amino acid peptide motif (10-24; EDLLKAVRLIKFLYQ) in the N-terminus of the HIV-1 Rev protein. NIS appears to be involved in maintaining the proper nucleocytoplasmic trafficking and intracellular stability of HIV-1 Rev. Deletion in NIS leads to Rev functional inactivity, and these data led to further investigation of its possible inhibitory effects on Rev function. An HIV-1 proviral rescue assay was utilized to evaluate Rev function. The association between wild-type Rev molecules, or wild-type Rev with Revd23, an NIS mutant, plus Rev-responsive element (RRE) interactions in cultured cells were also evaluated. Revd23 showed a potent trans-dominant negative phenotype, while multimerization with wild-type Rev and Revd23-RRE binding in cells were found to reveal no significant changes from wildtype. These results suggest that the potential trans-dominance mechanism of Revd23 may differ from that of a Rev construct, RevM10, with mutations in the C-terminus nuclear export signal (NES). As such, these data will be useful in the rational design of novel antiretroviral approaches targeting HIV-1 Rev.

Active Transport, Cell Nucleus↗
PubMed2002

Primary isolated human brain microvascular endothelial cells express diverse HIV/SIV-associated chemokine coreceptors and DC-SIGN and L-SIGN.

Chemokines have received increasing attention due to their inhibitory activities on human immunodeficiency virus type-1 (HIV-1) and simian immunodeficiency virus (SIV) replication and the potential for chemokine receptors to assist in HIV-1/SIV entry into permissive cells. Besides CD4, which is the major receptor for HIV-1 and SIV, a number of chemokine receptors including but not limited to APJ, CCR3, CXCR4, and CCR5 may be coreceptors for HIV-1/SIV, not only in peripheral blood and lymphoid tissues but also in the central nervous system (CNS). The present studies reveal the lack of CD4, but the significant expression of various chemokine receptors, APJ, CCR3, CXCR4, and CCR5, plus C-type lectins DC-SIGN and L-SIGN on isolated primary human brain microvascular endothelial cells (MVECs). As these MVECs do not express CD4, this suggests a CD4-independent HIV/SIV entry/infection of these cells, which are the major cells constituting the human blood-brain barrier. We also found that chemokines for cognate chemokine receptors individually were unable to block binding of HIV-1 to brain MVECs. These results reveal that in primary isolated brain MVECs viral attachment is mediated by a possible previously unknown receptor(s) or by cooperative activity of various receptors. Moreover, mRNA transcripts for DC-SIGN/L-SIGN, as well as DC-SIGN protein expression, suggest the capability of MVECs to attach viral particles on cell surfaces, even though polyclonal antisera for DC-SIGN did not affect viral binding to these cells. These data will assist in further understanding lentiviral entry into the CNS.

Base Sequence↗
PubMed2002

Gene therapy using a simian virus 40-derived vector inhibits the development of in vivo human immunodeficiency virus type 1 infection of severe combined immunodeficiency mice implanted with human fetal thymic and liver tissue.

To evaluate the in vivo efficacy of gene therapy for treating human immunodeficiency virus type 1 (HIV-1) infection, a novel simian virus (SV) 40-derived vector gene delivery system that efficiently transduces human leukocytes was combined with a model using severe combined immunodeficiency mice infected with HIV-1 and implanted with human fetal thymic and liver tissue (thy/liv-SCID-hu mice). The SV40-derived vector, SV(Aw), which encodes a variable fragment antibody recognizing HIV-1 integrase (IN#33),was injected into the human thymic grafts of thy/liv-SCID-hu mice and induced IN#33 expression in most of the thymocytes in the graft. After in vivo challenge with HIV-1, IN#33 expression inhibited in vivo HIV-1 infection, as evidenced by the markedly lower number of HIV-1-infected thymocytes detected in human thymic grafts injected with the SV(Aw) vector, compared with those injected with a control SV40-derived vector. Thus, these findings demonstrate the utility of this new mouse model system for assessing the in vivo efficacy of HIV-1-specific gene therapy. In addition, these data indicate that SV40-derived vectors may provide a system capable of efficient in vivo gene delivery.

Animals↗
PubMed2002

Live and killed rhabdovirus-based vectors as potential hepatitis C vaccines.

A highly attenuated, recombinant rabies virus (RV) vaccine strain-based vector was utilized as a new immunization strategy to induce humoral and cellular responses against hepatitis C (HCV) glycoprotein E2. We showed previously that RV-based vectors are able to induce strong immune responses against human immunodeficiency virus type I (HIV-1) antigens. Here we constructed and characterized three replication-competent RV-based vectors expressing either both HCV envelope proteins E1 and E2 or a modified version of E2 which lacks 85 amino acids of its carboxy terminus and contains the human CD4 transmembrane domain and the CD4 or RV glycoprotein cytoplasmic domain. All three constructs stably expressed the respective protein(s) as indicated by Western blotting and immunostaining. Moreover, surface expression of HCV E2 resulted in efficient incorporation of the HCV envelope protein regardless of the presence of the RV G cytoplasmic domain, which was described previously as a requirement for incorporation of foreign glycoproteins into RV particles. Killed and purified RV virions containing HCV E2 were highly immunogenic in mice and also proved useful as a diagnostic tool, as indicated by a specific reaction with sera from HCV-infected patients. In addition, RV vaccine vehicles were able to induce cellular responses against HCV E2. These results further suggest that recombinant RVs are potentially useful vaccine vectors against important human viral diseases.

Animals↗
PubMed2002

Residual HIV-1 disease in seminal cells of HIV-1-infected men on suppressive HAART: latency without on-going cellular infections.

BACKGROUND: HIV-1-infected men on suppressive highly active antiretroviral therapy (HAART) have a reduction of viral replication in vivo, but HIV-1 RNA is still detectable by certain ultrasensitive reverse transcriptase-PCR assays in blood plasma. Replication-competent virus can also be isolated from both peripheral blood mononuclear cells (PBMC) and seminal cells of these patients. Despite HAART, on-going in vivo infection of HIV-1-seropositive patients' PBMC was demonstrated by the detection of episomal HIV-1 moieties, known as HIV-1 two-long terminal repeat (2-LTR) DNA circles. METHODS: The present study analyzes whether new cellular infections occur in vivo in seminal cells of HIV-1-infected men on suppressive HAART. PBMC and seminal cells were isolated from a cohort of HIV-1-seropositive men taking suppressive HAART (< 50 copies HIV RNA/ml blood plasma). Viral growth assays were performed in vitro, as well as semi-quantitative PCR to detect HIV-1 2-LTR circular DNA in PBMC and seminal mononuclear cells. RESULTS: Viral growth in vitro was demonstrated in 16 out of 28 (57%) patients' PBMC, and in five patients' seminal cells (18%). Although 18 patients' PBMC were positive for HIV-1 2-LTR DNA circles, importantly, 2-LTR circular DNA was not detected in any semen sample, even when replication-competent HIV-1 virus had been recovered from a patient's seminal cells by viral co-culture assays. CONCLUSIONS: The current study suggests that in HIV-1-infected men treated with suppressive HAART, new cellular infections occur in PBMC, but that new infections do not take place in seminal cells in vivo. Thus, these findings suggest that mainly latent HIV-1 occurs in seminal cells of men on suppressive HAART, which may be a compartment-specific mechanism of residual HIV-1 disease.

Antiretroviral Therapy, Highly Active↗
PubMed2002

Combination genetic therapy to inhibit HIV-1.

Compared with single agents, combination antilentiviral pharmacotherapy targets multiple HIV-1 functions simultaneously, maximizing efficacy and decreasing chances of escape mutations. Combination genetic therapy could theoretically enhance efficacy similarly, but delivery of even single genes to high percentages of hematopoietic cells or their derivatives has proven problematic. Because of their high efficiency of gene delivery, we tested recombinant SV40-derived vectors (rSV40s) for this purpose. We made six rSV40s, each carrying a different transgene that targeted a different lentiviral function. We tested the ability of these constructs, individually and in double and triple combinations, to protect SupT1 human T lymphoma cells from HIV-1 challenge. Single chain antibodies (SFv) against CXCR4 and against HIV-1 reverse transcriptase (RT) and integrase (IN) were used, as were polymeric TAR decoys (PolyTAR) and a dominant-negative mutant of HIV-1 Rev (RevM10). Immunostaining showed that virtually all doubly treated cells expressed both transgenes. All transgenes individually protected from HIV-1 but, except for anti-CXCR4 SFv, their effectiveness diminished as challenge doses increased from 40 through 2500 tissue culture infectious dose(50) (TCID(50))/10(6) cells. However, all combinations of transgenes protected target cells better than individual transgenes, even from the highest challenge doses. Thus, combination gene therapies may inhibit HIV-1 better than single agents, and rSV40s may facilitate delivery of multigene therapeutics.

Genetic Therapy↗
PubMed2002

HIV-1 reservoirs.

In most infected individuals, HIV-1 replicates high levels throughout the duration of infection, including the clinically quiescent phase of disease. The level of this active viral replication correlates directly with disease progression and survival. The advent of combination therapeutics for HIV-1 (i.e., highly active antiretroviral therapy [HAART]) has led to dramatic reductions in viral replication in vivo and morbidity and mortality, at least in the developed world.

Anti-HIV Agents↗
PubMed2002

New methods for the detection of HIV.

In the past few years, several strides have been made in the ability to detect the presence of HIV-1 and HIV-2. This article discusses recent advances in serologic testing, including routine ELISA and Western blot tests, rapid HIV tests, home collection kits, and HIV tests using nonserum samples. The clinical application of nucleic acid-based tests also is discussed. Finally, appropriate use of these tests in both acute HIV-1 infection and in infants is reviewed.

HIV Infections↗