Advances in high-throughput proteomic approaches have provided substantial momentum to novel dise... more Advances in high-throughput proteomic approaches have provided substantial momentum to novel disease-biomarker discovery research and have augmented the quality of clinical studies. Applications based on multiplexed microsphere suspension array technology are making strong in-roads into the clinical diagnostic/prognostic practice. Conventional proteomic approaches are designed to discover a broad set of proteins that are associated with a specific medical condition. In comparison, multiplex microsphere immunoassays use quantitative measurements of selected set(s) of specific/particular molecular markers such as cytokines, chemokines, pathway signaling or disease-specific markers for detection, metabolic disorders, cancer, and infectious agents causing human, plant and animal diseases. This article provides a foundation to the multiplexed microsphere suspension array technology, with an emphasis on the improvements in the technology, data analysis approaches, and applications to tran...
The frequent occurrence of Kaposi's sarcoma (KS) in association with the acquired immune defi... more The frequent occurrence of Kaposi's sarcoma (KS) in association with the acquired immune deficiency syndrome (AIDS) could be due to the fact that the etiological agent of this tumor is the same retrovirus causing AIDS, to another oncogenic virus frequently found in AIDS patients, or to the unmasking of the tumorigenic potential of KS cells by immunosuppression. We have therefore investigated the presence of DNA sequences homologous to the AIDS retrovirus, cytomegalovirus (CMV), and hepatitis B virus in 13 KS necropsies and biopsies from AIDS patients. All KS DNA samples were negative for AIDS retrovirus or hepatitis B DNA sequences. Two DNAs from necropsies contained CMV DNA, but the data suggested the presence of replicating CMV DNA due to generalized infection. We have also studied cell cultures derived from KS skin biopsies of AIDS patients. These cultures had a short lifetime in vitro and expressed some markers of endothelial cells. The cells were not tumorigenic in nude mic...
To analyze HIV-1 genes in a nonhuman primate model for lentivirus infection and AIDS, recombinant... more To analyze HIV-1 genes in a nonhuman primate model for lentivirus infection and AIDS, recombinant SIV/HIV-1 (SHIV) clones were constructed from two HIV-1 subtype-A isolates (HIV-1 SF170 and HIV-1 Q23-17 from individuals in Africa) and two HIV-1 subtype-E isolates (HIV-1 9466 and HIV-1 CAR402 from AIDS patients in Thailand and Africa), respectively. These four SHIV clones, designated SHIV-A-170, SHIV-A-Q23, SHIV-9466.33, and SHIV-E-CAR, contain envelope (env) genes from the subtype-A orE viruses. Interestingly, SHIV-A-170, SHIV-A-Q23, and SHIV-9466.33 were restricted for replication in cultures of macaque lymphoid cells, whereas SHIV-E-CAR replicated efficiently in these cells. Additional studies to define the block to replication in macaque cells were focused on the subtype-E clone SHIV-9466.33. A SHIV intragenic env clone, containing sequence-encompassing V1/V2 regions of HIV-1 CAR402 and V3/V4/V5 regions of SHIV-9466.33, infected and replicated in macaque lymphoid cells. These results indicated that the sequence-encompassing V1/V2 region of HIV-1 9466 was responsible for the block of the SHIV-9466.33 replication in macaque cells. Analysis of viral DNA in acutely infected macaque cells revealed that SHIV-9466.33 was blocked at a step at/or before viral DNA synthesis, presumably during the process of virion entry into cells. In a fluorescence-based cell-cell fusion assay, fusion pore formation readily took place in cocultures of cells expressing the SHIV-9466.33 env glycoprotein with macaque T-lymphoid cells. Taken together, these results demonstrated that the block of SHIV-9466.33 replication in macaque cells is at an early step after fusion pore formation but before reverse transcription.
The nucleotide sequence of molecular clones of DNA from a retrovirus, ARV-2, associated with the ... more The nucleotide sequence of molecular clones of DNA from a retrovirus, ARV-2, associated with the acquired immune deficiency syndrome (AIDS) was determined. Proviral DNA of ARV-2 (9737 base pairs) has long terminal repeat structures (636 base pairs) and long open reading frames encoding gag (506 codons), pol (1003 codons), and env (863 codons) genes. Two additional open reading frames were identified. Significant amino acid homology with several other retroviruses was noted in the predicted product of gag and pol, but ARV-2 was as closely related to murine and avian retroviruses as it was to human T-cell leukemia viruses (HTLV-I and HTLV-II). By means of an SV-40 vector in transfected simian cells, the cloned gag and env genes of ARV-2 were shown to express viral proteins.
Proceedings of the National Academy of Sciences, 1995
To elucidate the functions of human immunodeficiency virus type 1 (HIV-1) genes in a nonhuman pri... more To elucidate the functions of human immunodeficiency virus type 1 (HIV-1) genes in a nonhuman primate model, we have constructed infectious recombinant viruses (chimeras) between the pathogenic molecular clone of simian immunodeficiency virus (SIV) SIVmac239 and molecular clones of HIV-1 that differ in phenotypic properties controlled by the env gene. HIV-1SF33 is a T-cell-line-tropic virus which induces syncytia, and HIV-1SF162 is a macrophage-tropic virus that does not induce syncytia. A DNA fragment encoding tat, rev, and env (gp160) of SIVmac239 has been replaced with the counterpart genetic region of HIV-1SF33 and HIV-1SF162 to derive chimeric recombinant simian/human immunodeficiency virus (SHIV) strains SHIVSF33 and SHIVSF162, respectively. In the acute infection stage, macaques inoculated with SHIVSF33 had levels of viremia similar to macaques infected with SIVmac239, whereas virus loads were 1/10th to 1/100th those in macaques infected with SHIVSF162. Of note is the relativ...
The envelope ( env ) glycoprotein of human immunodeficiency virus type 1 (HIV-1) determines sever... more The envelope ( env ) glycoprotein of human immunodeficiency virus type 1 (HIV-1) determines several viral properties (e.g., coreceptor usage, cell tropism, and cytopathicity) and is a major target of antiviral immune responses. Most investigations on env have been conducted on subtype-B viral strains, prevalent in North America and Europe. Our study aimed to analyze env genes of subtype-E viral strains, prevalent in Asia and Africa, with a nonhuman primate model for lentivirus infection and AIDS. To this end, we constructed a simian immunodeficiency virus/HIV-1 subtype-E (SHIV) recombinant clone by replacing the env ectodomain of the SHIV-33 clone with the env ectodomain from the subtype-E strain HIV-1 CAR402 , which was isolated from an individual in the Central African Republic. Virus from this recombinant clone, designated SHIV-E-CAR, replicated efficiently in macaque peripheral blood mononuclear cells. Accordingly, juvenile macaques were inoculated with cell-free SHIV-E-CAR by t...
Rhesus macaques infected with simian immunodeficiency virus (SIV) containing either a large nef d... more Rhesus macaques infected with simian immunodeficiency virus (SIV) containing either a large nef deletion (SIVmac239Δ 152 nef) or interleukin-2 in place of nef developed high virus loads and progressed to simian AIDS. Viruses recovered from both juvenile and neonatal macaques with disease produced a novel truncated Nef protein, tNef. Viruses recovered from juvenile macaques infected with serially passaged virus expressing tNef exhibited a pathogenic phenotype. These findings demonstrated strong selective pressure to restore expression of a truncated Nef protein, and this reversion was linked to increased pathogenic potential in live attenuated SIV vaccines.
Background: The primate lentiviruses, human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2... more Background: The primate lentiviruses, human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) and simian immunodeficiency virus (SIV), encode a conserved accessory gene product, Nef. In vivo, Nef is important for the maintenance of high virus loads and progression to AIDS in SIV-infected adult rhesus macaques. In tissue culture cells expressing Nef, this viral protein interacts with a cellular serine kinase, designated Nef-associated kinase. Results: This study identifies the Nef-associated kinase as a member of the p21activated kinase (PAK) family of kinases and investigates the role of this Nefassociated kinase in vivo. Mutants of Nef that do not associate with the cellular kinase are unable to activate the PAK-related kinase in infected cells. To determine the role of cellular kinase association in viral pathogenesis, macaques were infected with SIV containing point-mutations in Nef that block PAK activation. Virus recovered at early time points after inoculation with mutant virus was found to have reverted to prototype Nef function and sequence. Reversion of the kinase-negative mutant to a kinase-positive genotype in macaques infected with the mutant virus preceded the induction of high virus loads and disease progression. Conclusions: Nef associates with and activates a PAK-related kinase in lymphocytes infected in vitro. Moreover, the Nef-mediated activation of a PAKrelated kinase correlates with the induction of high virus loads and the development of AIDS in the infected host. These findings reveal that there is a strong selective pressure in vivo for the interaction between Nef and the PAKrelated kinase.
Background: Rapid detection and therapeutic intervention for infectious and emerging diseases is ... more Background: Rapid detection and therapeutic intervention for infectious and emerging diseases is a major scientific goal in biodefense and public health. Toward this end, cytokine profiles in human blood were investigated using a human whole blood ex vivo exposure model, called WEEM. Results: Samples of whole blood from healthy volunteers were incubated with seven pathogens including Yersinia pseudotuberculosis, Yersinia enterocolitica, Bacillus anthracis, and multiple strains of Yersinia pestis, and multiplexed protein expression profiling was conducted on supernatants of these cultures with an antibody array to detect 30 cytokines simultaneously. Levels of 8 cytokines, IL-1α, IL-1β, IL-6, IL-8, IL-10, IP-10, MCP-1 and TNFα, were significantly up-regulated in plasma after bacterial exposures of 4 hours. Statistical clustering was applied to group the pathogens based on the host response protein expression profiles. The nearest phylogenetic neighbors clustered more closely than the more distant pathogens, and all seven pathogens were clearly differentiated from the unexposed control. In addition, the Y. pestis and Yersinia near neighbors were differentiated from the B. anthracis strains. Conclusions: Cluster analysis, based on host response cytokine profiles, indicates that distinct patterns of immunomodulatory proteins are induced by the different pathogen exposures and these patterns may enable further development into biomarkers for diagnosing pathogen exposure.
Background Infection with HIV-1 results in marked immunologic insults and structural damage to th... more Background Infection with HIV-1 results in marked immunologic insults and structural damage to the intestinal mucosa, including compromised barrier function. While the development of highly active antiretroviral therapy (HAART) has been a major advancement in the treatment of HIV-1 infection, the need for novel complementary interventions to help restore intestinal structural and functional integrity remains unmet. Known properties of pre-, pro-, and synbiotics suggest that they may be useful tools in achieving this goal. Methods This was a 4-week parallel, placebo-controlled, randomized pilot trial in HIV-infected women on antiretroviral therapy. A synbiotic formulation (Synbiotic 2000®) containing 4 strains of probiotic bacteria (1010 each) plus 4 nondigestible, fermentable dietary fibers (2.5 g each) was provided each day, versus a fiber-only placebo formulation. The primary outcome was bacterial translocation. Secondary outcomes included the levels of supplemented bacteria in st...
American Journal of Respiratory and Critical Care Medicine, 2006
Rationale: HIV-infected patients with pulmonary arterial hypertension have histologic manifestati... more Rationale: HIV-infected patients with pulmonary arterial hypertension have histologic manifestations that are indistinguishable from those found in patients with idiopathic pulmonary arterial hypertension. In addition, the role of pleiotropic viral proteins in the development of plexiform lesions in HIV-related pulmonary hypertension (HRPH) has not been explored. Simian immunodeficiency virus (SIV) infection of macaques has been found to closely recapitulate many of the characteristic features of HIV infection, and thus hallmarks of pulmonary arterial hypertension should also be found in this nonhuman primate model of HIV. Objectives: To determine whether pulmonary arterial lesions were present in archived SIV-infected macaque lung tissues from Johns Hopkins University and two National Primate Research Centers. Methods: Archived macaque and human lung sections were examined via immunohistochemistry for evidence of complex vascular lesions. Results: Complex plexiform-like lesions characterized by lumenal obliteration, intimal disruption, medial hypertrophy, thrombosis, and recanalized lumena were found exclusively in animals infected with SHIV-nef (a chimeric viral construct containing the HIV nef gene in an SIV backbone), but not in animals infected with SIV. The mass of cells in the lesions were factor VIII positive, and contained cells positive for muscle-specific and smooth muscle actins. Lung mononuclear cells were positive for HIV Nef, suggesting viral replication. Endothelial cells in both the SHIV-nef macaques and patients with HRPH, but not in patients with idiopathic pulmonary arterial hypertension, were also Nef positive. Conclusions: The discovery of complex vascular lesions in SHIV-nefbut not SIV-infected animals, and the presence of Nef in the vascular cells of patients with HRPH, suggest that Nef plays a key role in the development of severe pulmonary arterial disease.
Retroviral envelope glycoproteins interact with cell receptors and are targets for antiviral immu... more Retroviral envelope glycoproteins interact with cell receptors and are targets for antiviral immune responses in infected hosts. Macaque simian immunodeficiency virus (SIVmac) is a T-lymphocytopathic lentivirus which causes an AIDS-like disease in rhesus macaques. The envelope gene of SIVmac encodes a precursor glycoprotein (gp160) which is cleaved into an external domain (gp130) and a transmembrane domain (gp32). To investigate the functional and immunological properties of the SIV external envelope glycoprotein, we have used genetically engineered mammalian cells to produce recombinant gp130 (rgp130). The rgp130 has the appropriate molecular weight, is glycosylated, and has native conformation as determined by binding to the cell receptor for SIV, the CD4 antigen. Rhesus macaques immunized with purified rgp130 formulated in muramyl dipeptide adjuvant generated high titers of antienvelope antibodies. Antibodies from these macaques were tested for in vitro virus neutralization; very low or undetectable levels of neutralization were observed. In contrast, neutralizing antibodies were readily detected in sera from goats immunized with rgp130. With respect to cell-mediated immunity, proliferative responses to rgp130 were demonstrated in peripheral blood monocyte cells (PBMC) from macaques immunized with the recombinant glycoprotein as well as in PBMC from SIV-infected animals. These results show that rgp130 is functional and immunogenic; the potential of rgp130 for protective immunization remains to be determined.
The human and simian immunodeficiency virus (HIV-1 and SIVmac) transmembrane proteins contain unu... more The human and simian immunodeficiency virus (HIV-1 and SIVmac) transmembrane proteins contain unusually long intracytoplasmic domains (ICD-TM). These domains are suggested to play a role in envelope fusogenicity, interaction with the viral matrix protein during assembly, viral infectivity, binding of intracellular calmodulin, disruption of membranes, and induction of apoptosis. Here we describe a novel mutant virus, SIVmac-M4, containing multiple mutations in the coding region for the ICD-TM of pathogenic molecular clone SIVmac239. Parental SIVmac239-Nef+ produces high-level persistent viremia and simian AIDS in both juvenile and newborn rhesus macaques. The ICD-TM region of SIVmac-M4 contains three stop codons, a +1 frameshift, and mutation of three highly conserved, charged residues in the conserved C-terminal alpha-helix referred to as lentivirus lytic peptide 1 (LLP-1). Overlapping reading frames for tat , rev , and nef are not affected by these changes. In this study, four juve...
One significant aspect of the acquired immune deficiency syndrome (AIDS)' epidemic is its as... more One significant aspect of the acquired immune deficiency syndrome (AIDS)' epidemic is its association with an increased incidence of neoplasia, primarily Kaposi's sarcoma and malignant lymphoma (1). The occurrence of these neoplasms appears to be intimately connected with the condition of impaired immunity present in AIDS, because similar tumors are known to be associated with other types of immunodeficiency, including congenital, drug-induced, and age-related immune deficiency syndromes (1-3). Therefore, studies of the pathogenesis of these neoplasms may provide important information concerning the biology of AIDS and the relationship between immune surveillance and tumor development in general. In particular, the study of neoplasia in patients with AIDS represents a rare opportunity to follow the development of a human tumor from the onset of a strongly predisposing condition, i.e., AIDS, to its biological and clinical appearance. Based on these considerations, we recent...
A safe, replication-defective viral vector that can induce mucosal and systemic immune responses ... more A safe, replication-defective viral vector that can induce mucosal and systemic immune responses and confer protection against many infectious pathogens, such as human immunodeficiency virus type 1 (HIV-1), may be an ideal vaccine platform. Accordingly, we have generated and tested alphavirus replicon particles encoding HIV-1 Gag from Sindbis virus (SIN-Gag) and Venezuelan equine encephalitis virus (VEE-Gag), as well as chimeras between the two (VEE/SIN-Gag). Following intramuscular (i.m.), intranasal (i.n.), or intravaginal (IVAG) immunization with VEE/SIN-Gag and an IVAG challenge with vaccinia virus encoding HIV Gag (VV-Gag), a larger number of Gag-specific CD8+ intracellular gamma interferon-expressing cells (iIFNEC) were detected in iliac lymph nodes (ILN), which drain the vaginal/uterine mucosa (VUM), than were observed after immunizations with SIN-Gag. Moreover, a single i.n. or IVAG immunization with VEE/SIN-Gag induced a larger number of cells expressing HIV Gag in ILN, and...
Advances in high-throughput proteomic approaches have provided substantial momentum to novel dise... more Advances in high-throughput proteomic approaches have provided substantial momentum to novel disease-biomarker discovery research and have augmented the quality of clinical studies. Applications based on multiplexed microsphere suspension array technology are making strong in-roads into the clinical diagnostic/prognostic practice. Conventional proteomic approaches are designed to discover a broad set of proteins that are associated with a specific medical condition. In comparison, multiplex microsphere immunoassays use quantitative measurements of selected set(s) of specific/particular molecular markers such as cytokines, chemokines, pathway signaling or disease-specific markers for detection, metabolic disorders, cancer, and infectious agents causing human, plant and animal diseases. This article provides a foundation to the multiplexed microsphere suspension array technology, with an emphasis on the improvements in the technology, data analysis approaches, and applications to tran...
The frequent occurrence of Kaposi's sarcoma (KS) in association with the acquired immune defi... more The frequent occurrence of Kaposi's sarcoma (KS) in association with the acquired immune deficiency syndrome (AIDS) could be due to the fact that the etiological agent of this tumor is the same retrovirus causing AIDS, to another oncogenic virus frequently found in AIDS patients, or to the unmasking of the tumorigenic potential of KS cells by immunosuppression. We have therefore investigated the presence of DNA sequences homologous to the AIDS retrovirus, cytomegalovirus (CMV), and hepatitis B virus in 13 KS necropsies and biopsies from AIDS patients. All KS DNA samples were negative for AIDS retrovirus or hepatitis B DNA sequences. Two DNAs from necropsies contained CMV DNA, but the data suggested the presence of replicating CMV DNA due to generalized infection. We have also studied cell cultures derived from KS skin biopsies of AIDS patients. These cultures had a short lifetime in vitro and expressed some markers of endothelial cells. The cells were not tumorigenic in nude mic...
To analyze HIV-1 genes in a nonhuman primate model for lentivirus infection and AIDS, recombinant... more To analyze HIV-1 genes in a nonhuman primate model for lentivirus infection and AIDS, recombinant SIV/HIV-1 (SHIV) clones were constructed from two HIV-1 subtype-A isolates (HIV-1 SF170 and HIV-1 Q23-17 from individuals in Africa) and two HIV-1 subtype-E isolates (HIV-1 9466 and HIV-1 CAR402 from AIDS patients in Thailand and Africa), respectively. These four SHIV clones, designated SHIV-A-170, SHIV-A-Q23, SHIV-9466.33, and SHIV-E-CAR, contain envelope (env) genes from the subtype-A orE viruses. Interestingly, SHIV-A-170, SHIV-A-Q23, and SHIV-9466.33 were restricted for replication in cultures of macaque lymphoid cells, whereas SHIV-E-CAR replicated efficiently in these cells. Additional studies to define the block to replication in macaque cells were focused on the subtype-E clone SHIV-9466.33. A SHIV intragenic env clone, containing sequence-encompassing V1/V2 regions of HIV-1 CAR402 and V3/V4/V5 regions of SHIV-9466.33, infected and replicated in macaque lymphoid cells. These results indicated that the sequence-encompassing V1/V2 region of HIV-1 9466 was responsible for the block of the SHIV-9466.33 replication in macaque cells. Analysis of viral DNA in acutely infected macaque cells revealed that SHIV-9466.33 was blocked at a step at/or before viral DNA synthesis, presumably during the process of virion entry into cells. In a fluorescence-based cell-cell fusion assay, fusion pore formation readily took place in cocultures of cells expressing the SHIV-9466.33 env glycoprotein with macaque T-lymphoid cells. Taken together, these results demonstrated that the block of SHIV-9466.33 replication in macaque cells is at an early step after fusion pore formation but before reverse transcription.
The nucleotide sequence of molecular clones of DNA from a retrovirus, ARV-2, associated with the ... more The nucleotide sequence of molecular clones of DNA from a retrovirus, ARV-2, associated with the acquired immune deficiency syndrome (AIDS) was determined. Proviral DNA of ARV-2 (9737 base pairs) has long terminal repeat structures (636 base pairs) and long open reading frames encoding gag (506 codons), pol (1003 codons), and env (863 codons) genes. Two additional open reading frames were identified. Significant amino acid homology with several other retroviruses was noted in the predicted product of gag and pol, but ARV-2 was as closely related to murine and avian retroviruses as it was to human T-cell leukemia viruses (HTLV-I and HTLV-II). By means of an SV-40 vector in transfected simian cells, the cloned gag and env genes of ARV-2 were shown to express viral proteins.
Proceedings of the National Academy of Sciences, 1995
To elucidate the functions of human immunodeficiency virus type 1 (HIV-1) genes in a nonhuman pri... more To elucidate the functions of human immunodeficiency virus type 1 (HIV-1) genes in a nonhuman primate model, we have constructed infectious recombinant viruses (chimeras) between the pathogenic molecular clone of simian immunodeficiency virus (SIV) SIVmac239 and molecular clones of HIV-1 that differ in phenotypic properties controlled by the env gene. HIV-1SF33 is a T-cell-line-tropic virus which induces syncytia, and HIV-1SF162 is a macrophage-tropic virus that does not induce syncytia. A DNA fragment encoding tat, rev, and env (gp160) of SIVmac239 has been replaced with the counterpart genetic region of HIV-1SF33 and HIV-1SF162 to derive chimeric recombinant simian/human immunodeficiency virus (SHIV) strains SHIVSF33 and SHIVSF162, respectively. In the acute infection stage, macaques inoculated with SHIVSF33 had levels of viremia similar to macaques infected with SIVmac239, whereas virus loads were 1/10th to 1/100th those in macaques infected with SHIVSF162. Of note is the relativ...
The envelope ( env ) glycoprotein of human immunodeficiency virus type 1 (HIV-1) determines sever... more The envelope ( env ) glycoprotein of human immunodeficiency virus type 1 (HIV-1) determines several viral properties (e.g., coreceptor usage, cell tropism, and cytopathicity) and is a major target of antiviral immune responses. Most investigations on env have been conducted on subtype-B viral strains, prevalent in North America and Europe. Our study aimed to analyze env genes of subtype-E viral strains, prevalent in Asia and Africa, with a nonhuman primate model for lentivirus infection and AIDS. To this end, we constructed a simian immunodeficiency virus/HIV-1 subtype-E (SHIV) recombinant clone by replacing the env ectodomain of the SHIV-33 clone with the env ectodomain from the subtype-E strain HIV-1 CAR402 , which was isolated from an individual in the Central African Republic. Virus from this recombinant clone, designated SHIV-E-CAR, replicated efficiently in macaque peripheral blood mononuclear cells. Accordingly, juvenile macaques were inoculated with cell-free SHIV-E-CAR by t...
Rhesus macaques infected with simian immunodeficiency virus (SIV) containing either a large nef d... more Rhesus macaques infected with simian immunodeficiency virus (SIV) containing either a large nef deletion (SIVmac239Δ 152 nef) or interleukin-2 in place of nef developed high virus loads and progressed to simian AIDS. Viruses recovered from both juvenile and neonatal macaques with disease produced a novel truncated Nef protein, tNef. Viruses recovered from juvenile macaques infected with serially passaged virus expressing tNef exhibited a pathogenic phenotype. These findings demonstrated strong selective pressure to restore expression of a truncated Nef protein, and this reversion was linked to increased pathogenic potential in live attenuated SIV vaccines.
Background: The primate lentiviruses, human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2... more Background: The primate lentiviruses, human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) and simian immunodeficiency virus (SIV), encode a conserved accessory gene product, Nef. In vivo, Nef is important for the maintenance of high virus loads and progression to AIDS in SIV-infected adult rhesus macaques. In tissue culture cells expressing Nef, this viral protein interacts with a cellular serine kinase, designated Nef-associated kinase. Results: This study identifies the Nef-associated kinase as a member of the p21activated kinase (PAK) family of kinases and investigates the role of this Nefassociated kinase in vivo. Mutants of Nef that do not associate with the cellular kinase are unable to activate the PAK-related kinase in infected cells. To determine the role of cellular kinase association in viral pathogenesis, macaques were infected with SIV containing point-mutations in Nef that block PAK activation. Virus recovered at early time points after inoculation with mutant virus was found to have reverted to prototype Nef function and sequence. Reversion of the kinase-negative mutant to a kinase-positive genotype in macaques infected with the mutant virus preceded the induction of high virus loads and disease progression. Conclusions: Nef associates with and activates a PAK-related kinase in lymphocytes infected in vitro. Moreover, the Nef-mediated activation of a PAKrelated kinase correlates with the induction of high virus loads and the development of AIDS in the infected host. These findings reveal that there is a strong selective pressure in vivo for the interaction between Nef and the PAKrelated kinase.
Background: Rapid detection and therapeutic intervention for infectious and emerging diseases is ... more Background: Rapid detection and therapeutic intervention for infectious and emerging diseases is a major scientific goal in biodefense and public health. Toward this end, cytokine profiles in human blood were investigated using a human whole blood ex vivo exposure model, called WEEM. Results: Samples of whole blood from healthy volunteers were incubated with seven pathogens including Yersinia pseudotuberculosis, Yersinia enterocolitica, Bacillus anthracis, and multiple strains of Yersinia pestis, and multiplexed protein expression profiling was conducted on supernatants of these cultures with an antibody array to detect 30 cytokines simultaneously. Levels of 8 cytokines, IL-1α, IL-1β, IL-6, IL-8, IL-10, IP-10, MCP-1 and TNFα, were significantly up-regulated in plasma after bacterial exposures of 4 hours. Statistical clustering was applied to group the pathogens based on the host response protein expression profiles. The nearest phylogenetic neighbors clustered more closely than the more distant pathogens, and all seven pathogens were clearly differentiated from the unexposed control. In addition, the Y. pestis and Yersinia near neighbors were differentiated from the B. anthracis strains. Conclusions: Cluster analysis, based on host response cytokine profiles, indicates that distinct patterns of immunomodulatory proteins are induced by the different pathogen exposures and these patterns may enable further development into biomarkers for diagnosing pathogen exposure.
Background Infection with HIV-1 results in marked immunologic insults and structural damage to th... more Background Infection with HIV-1 results in marked immunologic insults and structural damage to the intestinal mucosa, including compromised barrier function. While the development of highly active antiretroviral therapy (HAART) has been a major advancement in the treatment of HIV-1 infection, the need for novel complementary interventions to help restore intestinal structural and functional integrity remains unmet. Known properties of pre-, pro-, and synbiotics suggest that they may be useful tools in achieving this goal. Methods This was a 4-week parallel, placebo-controlled, randomized pilot trial in HIV-infected women on antiretroviral therapy. A synbiotic formulation (Synbiotic 2000®) containing 4 strains of probiotic bacteria (1010 each) plus 4 nondigestible, fermentable dietary fibers (2.5 g each) was provided each day, versus a fiber-only placebo formulation. The primary outcome was bacterial translocation. Secondary outcomes included the levels of supplemented bacteria in st...
American Journal of Respiratory and Critical Care Medicine, 2006
Rationale: HIV-infected patients with pulmonary arterial hypertension have histologic manifestati... more Rationale: HIV-infected patients with pulmonary arterial hypertension have histologic manifestations that are indistinguishable from those found in patients with idiopathic pulmonary arterial hypertension. In addition, the role of pleiotropic viral proteins in the development of plexiform lesions in HIV-related pulmonary hypertension (HRPH) has not been explored. Simian immunodeficiency virus (SIV) infection of macaques has been found to closely recapitulate many of the characteristic features of HIV infection, and thus hallmarks of pulmonary arterial hypertension should also be found in this nonhuman primate model of HIV. Objectives: To determine whether pulmonary arterial lesions were present in archived SIV-infected macaque lung tissues from Johns Hopkins University and two National Primate Research Centers. Methods: Archived macaque and human lung sections were examined via immunohistochemistry for evidence of complex vascular lesions. Results: Complex plexiform-like lesions characterized by lumenal obliteration, intimal disruption, medial hypertrophy, thrombosis, and recanalized lumena were found exclusively in animals infected with SHIV-nef (a chimeric viral construct containing the HIV nef gene in an SIV backbone), but not in animals infected with SIV. The mass of cells in the lesions were factor VIII positive, and contained cells positive for muscle-specific and smooth muscle actins. Lung mononuclear cells were positive for HIV Nef, suggesting viral replication. Endothelial cells in both the SHIV-nef macaques and patients with HRPH, but not in patients with idiopathic pulmonary arterial hypertension, were also Nef positive. Conclusions: The discovery of complex vascular lesions in SHIV-nefbut not SIV-infected animals, and the presence of Nef in the vascular cells of patients with HRPH, suggest that Nef plays a key role in the development of severe pulmonary arterial disease.
Retroviral envelope glycoproteins interact with cell receptors and are targets for antiviral immu... more Retroviral envelope glycoproteins interact with cell receptors and are targets for antiviral immune responses in infected hosts. Macaque simian immunodeficiency virus (SIVmac) is a T-lymphocytopathic lentivirus which causes an AIDS-like disease in rhesus macaques. The envelope gene of SIVmac encodes a precursor glycoprotein (gp160) which is cleaved into an external domain (gp130) and a transmembrane domain (gp32). To investigate the functional and immunological properties of the SIV external envelope glycoprotein, we have used genetically engineered mammalian cells to produce recombinant gp130 (rgp130). The rgp130 has the appropriate molecular weight, is glycosylated, and has native conformation as determined by binding to the cell receptor for SIV, the CD4 antigen. Rhesus macaques immunized with purified rgp130 formulated in muramyl dipeptide adjuvant generated high titers of antienvelope antibodies. Antibodies from these macaques were tested for in vitro virus neutralization; very low or undetectable levels of neutralization were observed. In contrast, neutralizing antibodies were readily detected in sera from goats immunized with rgp130. With respect to cell-mediated immunity, proliferative responses to rgp130 were demonstrated in peripheral blood monocyte cells (PBMC) from macaques immunized with the recombinant glycoprotein as well as in PBMC from SIV-infected animals. These results show that rgp130 is functional and immunogenic; the potential of rgp130 for protective immunization remains to be determined.
The human and simian immunodeficiency virus (HIV-1 and SIVmac) transmembrane proteins contain unu... more The human and simian immunodeficiency virus (HIV-1 and SIVmac) transmembrane proteins contain unusually long intracytoplasmic domains (ICD-TM). These domains are suggested to play a role in envelope fusogenicity, interaction with the viral matrix protein during assembly, viral infectivity, binding of intracellular calmodulin, disruption of membranes, and induction of apoptosis. Here we describe a novel mutant virus, SIVmac-M4, containing multiple mutations in the coding region for the ICD-TM of pathogenic molecular clone SIVmac239. Parental SIVmac239-Nef+ produces high-level persistent viremia and simian AIDS in both juvenile and newborn rhesus macaques. The ICD-TM region of SIVmac-M4 contains three stop codons, a +1 frameshift, and mutation of three highly conserved, charged residues in the conserved C-terminal alpha-helix referred to as lentivirus lytic peptide 1 (LLP-1). Overlapping reading frames for tat , rev , and nef are not affected by these changes. In this study, four juve...
One significant aspect of the acquired immune deficiency syndrome (AIDS)' epidemic is its as... more One significant aspect of the acquired immune deficiency syndrome (AIDS)' epidemic is its association with an increased incidence of neoplasia, primarily Kaposi's sarcoma and malignant lymphoma (1). The occurrence of these neoplasms appears to be intimately connected with the condition of impaired immunity present in AIDS, because similar tumors are known to be associated with other types of immunodeficiency, including congenital, drug-induced, and age-related immune deficiency syndromes (1-3). Therefore, studies of the pathogenesis of these neoplasms may provide important information concerning the biology of AIDS and the relationship between immune surveillance and tumor development in general. In particular, the study of neoplasia in patients with AIDS represents a rare opportunity to follow the development of a human tumor from the onset of a strongly predisposing condition, i.e., AIDS, to its biological and clinical appearance. Based on these considerations, we recent...
A safe, replication-defective viral vector that can induce mucosal and systemic immune responses ... more A safe, replication-defective viral vector that can induce mucosal and systemic immune responses and confer protection against many infectious pathogens, such as human immunodeficiency virus type 1 (HIV-1), may be an ideal vaccine platform. Accordingly, we have generated and tested alphavirus replicon particles encoding HIV-1 Gag from Sindbis virus (SIN-Gag) and Venezuelan equine encephalitis virus (VEE-Gag), as well as chimeras between the two (VEE/SIN-Gag). Following intramuscular (i.m.), intranasal (i.n.), or intravaginal (IVAG) immunization with VEE/SIN-Gag and an IVAG challenge with vaccinia virus encoding HIV Gag (VV-Gag), a larger number of Gag-specific CD8+ intracellular gamma interferon-expressing cells (iIFNEC) were detected in iliac lymph nodes (ILN), which drain the vaginal/uterine mucosa (VUM), than were observed after immunizations with SIN-Gag. Moreover, a single i.n. or IVAG immunization with VEE/SIN-Gag induced a larger number of cells expressing HIV Gag in ILN, and...
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Papers by Paul Luciw