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1.
Viruses ; 14(10)2022 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-36298823

RESUMO

We earlier reported substantial progress in designing gp120 antagonists. Notably, we discovered that NBD-14189 is not only the most active gp120 antagonist but also shows antiviral activity against HIV-1 Reverse Transcriptase (RT). We also confirmed its binding to HIV-1 RT by X-ray crystallography. The dual inhibition is highly significant because, intriguingly, this compound bridges the dNTP and NNRTI-binding sites and inhibits the polymerase activity of isolated RT in the enzymatic assay. This novel finding is expected to lead to new avenues in designing a novel class of HIV-1 dual inhibitors. Therefore, we needed to advance this inhibitor to preclinical assessment. To this end, we report the pharmacokinetics (PK) study of NBD-14189 in rats and dogs. Subsequently, we assessed the toxicity and therapeutic efficacy in vivo in the SCID-hu Thy/Liv mouse model. The PK data indicated a favorable half-life (t1/2) and excellent oral bioavailability (%F = 61%). NBD-14189 did not show any measurable toxicity in the mice, and treatment reduced HIV replication at 300 mg/kg per day in the absence of clear evidence of protection from HIV-mediated human thymocyte depletion. The data indicated the potential of this inhibitor as an anti-HIV-1 agent and needs to be assessed in a non-human primate (NHP) model.


Assuntos
Fármacos Anti-HIV , Infecções por HIV , Soropositividade para HIV , HIV-1 , Ratos , Camundongos , Cães , Humanos , Animais , Camundongos SCID , Infecções por HIV/tratamento farmacológico , Modelos Animais de Doenças , HIV-1/fisiologia , Fármacos Anti-HIV/uso terapêutico
2.
Proc Natl Acad Sci U S A ; 118(20)2021 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-33975958

RESUMO

Genetic editing of induced pluripotent stem (iPS) cells represents a promising avenue for an HIV cure. However, certain challenges remain before bringing this approach to the clinic. Among them, in vivo engraftment of cells genetically edited in vitro needs to be achieved. In this study, CD34+ cells derived in vitro from iPS cells genetically modified to carry the CCR5Δ32 mutant alleles did not engraft in humanized immunodeficient mice. However, the CD34+ cells isolated from teratomas generated in vivo from these genetically edited iPS cells engrafted in all experiments. These CD34+ cells also gave rise to peripheral blood mononuclear cells in the mice that, when inoculated with HIV in cell culture, were resistant to HIV R5-tropic isolates. This study indicates that teratomas can provide an environment that can help evaluate the engraftment potential of CD34+ cells derived from the genetically modified iPS cells in vitro. The results further confirm the possibility of using genetically engineered iPS cells to derive engraftable hematopoietic stem cells resistant to HIV as an approach toward an HIV cure.


Assuntos
Engenharia Genética , Infecções por HIV/terapia , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Animais , Diferenciação Celular , Células Cultivadas , Feminino , Edição de Genes , Humanos , Masculino , Camundongos
3.
Cell Rep ; 31(2): 107494, 2020 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-32294445

RESUMO

Paradoxically, early host responses to infection include the upregulation of the antiphagocytic molecule, CD47. This suggests that CD47 blockade could enhance antigen presentation and subsequent immune responses. Indeed, mice treated with anti-CD47 monoclonal antibody following lymphocytic choriomeningitis virus infections show increased activation of both macrophages and dendritic cells (DCs), enhancement of the kinetics and potency of CD8+ T cell responses, and significantly improved virus control. Treatment efficacy is critically dependent on both APCs and CD8+ T cells. In preliminary results from one of two cohorts of humanized mice infected with HIV-1 for 6 weeks, CD47 blockade reduces plasma p24 levels and restores CD4+ T cell counts. The results indicate that CD47 blockade not only enhances the function of innate immune cells but also links to adaptive immune responses through improved APC function. As such, immunotherapy by CD47 blockade may have broad applicability to treat a wide range of infectious diseases.


Assuntos
Antígeno CD47/imunologia , Antígeno CD47/metabolismo , Viroses/imunologia , Imunidade Adaptativa/imunologia , Animais , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular , Feminino , Células HEK293 , Humanos , Imunidade Inata/imunologia , Imunoterapia/métodos , Coriomeningite Linfocítica/imunologia , Vírus da Coriomeningite Linfocítica/imunologia , Macrófagos/imunologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de Sinais
4.
Viruses ; 11(3)2019 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-30871222

RESUMO

Although antiretroviral therapy (ART) greatly suppresses HIV replication, lymphoid tissues remain a sanctuary site where the virus may replicate. Tracking the earliest steps of HIV spread from these cellular reservoirs after drug cessation is pivotal for elucidating how infection can be prevented. In this study, we developed an in vivo model of HIV persistence in which viral replication in the lymphoid compartments of humanized mice was inhibited by the HIV reverse transcriptase inhibitor 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) to very low levels, which recapitulated ART-suppression in HIV-infected individuals. Using a combination of RNAscope in situ hybridization (ISH) and immunohistochemistry (IHC), we quantitatively investigated the distribution of HIV in the lymphoid tissues of humanized mice during active infection, EFdA suppression, and after drug cessation. The lymphoid compartments of EFdA-suppressed humanized mice harbored very rare transcription/translation-competent HIV reservoirs that enable viral rebound. Our data provided the visualization and direct measurement of the early steps of HIV reservoir expansion within anatomically intact lymphoid tissues soon after EFdA cessation and suggest a strategy to enhance therapeutic approaches aimed at eliminating the HIV reservoir.


Assuntos
Desoxiadenosinas/farmacologia , Reservatórios de Doenças/virologia , HIV-1/fisiologia , Tecido Linfoide/virologia , Inibidores da Transcriptase Reversa/farmacologia , Replicação Viral/efeitos dos fármacos , Animais , Modelos Animais de Doenças , Feminino , Infecções por HIV/tratamento farmacológico , HIV-1/efeitos dos fármacos , Humanos , Camundongos , Camundongos Transgênicos
5.
PLoS Pathog ; 14(2): e1006856, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29470552

RESUMO

HIV-1-infected cells persist indefinitely despite the use of combination antiretroviral therapy (ART), and novel therapeutic strategies to target and purge residual infected cells in individuals on ART are urgently needed. Here, we demonstrate that CD4+ T cell-associated HIV-1 RNA is often highly enriched in cells expressing CD30, and that cells expressing this marker considerably contribute to the total pool of transcriptionally active CD4+ lymphocytes in individuals on suppressive ART. Using in situ RNA hybridization studies, we show co-localization of CD30 with HIV-1 transcriptional activity in gut-associated lymphoid tissues. We also demonstrate that ex vivo treatment with brentuximab vedotin, an antibody-drug conjugate (ADC) that targets CD30, significantly reduces the total amount of HIV-1 DNA in peripheral blood mononuclear cells obtained from infected, ART-suppressed individuals. Finally, we observed that an HIV-1-infected individual, who received repeated brentuximab vedotin infusions for lymphoma, had no detectable virus in peripheral blood mononuclear cells. Overall, CD30 may be a marker of residual, transcriptionally active HIV-1 infected cells in the setting of suppressive ART. Given that CD30 is only expressed on a small number of total mononuclear cells, it is a potential therapeutic target of persistent HIV-1 infection.


Assuntos
Linfócitos T CD4-Positivos/virologia , Infecções por HIV/virologia , HIV-1/fisiologia , Antígeno Ki-1/metabolismo , Tecido Linfoide/virologia , Reto/virologia , Ativação Transcricional , Fármacos Anti-HIV/farmacologia , Terapia Antirretroviral de Alta Atividade , Biomarcadores/sangue , Biomarcadores/metabolismo , Brentuximab Vedotin , Contagem de Linfócito CD4 , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/patologia , Células Cultivadas , Estudos de Coortes , DNA Viral/sangue , DNA Viral/metabolismo , Infecções por HIV/tratamento farmacológico , Infecções por HIV/metabolismo , Infecções por HIV/patologia , HIV-1/efeitos dos fármacos , HIV-1/isolamento & purificação , HIV-1/patogenicidade , Humanos , Imunoconjugados/farmacologia , Hibridização In Situ , Antígeno Ki-1/antagonistas & inibidores , Antígeno Ki-1/sangue , Antígeno Ki-1/química , Tecido Linfoide/efeitos dos fármacos , Tecido Linfoide/metabolismo , Tecido Linfoide/patologia , RNA Viral/sangue , RNA Viral/metabolismo , Reto/efeitos dos fármacos , Reto/metabolismo , Reto/patologia , Solubilidade , Ativação Transcricional/efeitos dos fármacos , Carga Viral/efeitos dos fármacos
6.
J Histochem Cytochem ; 66(6): 427-446, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29462571

RESUMO

Persistent tissue reservoirs of HIV present a major barrier to cure. Defining subsets of infected cells in tissues is a major focus of HIV cure research. Herein, we describe a novel multiplexed in situ hybridization (ISH) (RNAscope) protocol to detect HIV-DNA (vDNA) and HIV-RNA (vRNA) in formalin-fixed paraffin-embedded (FFPE) human tissues in combination with immunofluorescence (IF) phenotyping of the infected cells. We show that multiplexed IF and ISH (mIFISH) is suitable for quantitative assessment of HIV vRNA and vDNA and that multiparameter IF phenotyping allows precise identification of the cellular source of the ISH signal. We also provide semi-quantitative data on the impact of various tissue fixatives on the detectability of vDNA and vRNA with RNAscope technology. Finally, we describe methods to quantitate the ISH signal on whole-slide digital images and validation of the quantitative ISH data with quantitative real-time PCR for vRNA. It is our hope that this approach will provide insight into the biology of HIV tissue reservoirs and to inform strategies aimed at curing HIV.


Assuntos
DNA Viral/análise , Imunofluorescência/métodos , Infecções por HIV/patologia , HIV/isolamento & purificação , Hibridização In Situ/métodos , RNA Viral/análise , Carga Viral/métodos , DNA Viral/genética , HIV/genética , Infecções por HIV/virologia , Humanos , Inclusão em Parafina/métodos , RNA Viral/genética , Análise de Célula Única/métodos , Fixação de Tecidos/métodos
7.
AIDS Res Hum Retroviruses ; 34(1): 3-8, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28691499

RESUMO

The persistence of HIV infection, even after lengthy and successful combined antiretroviral therapy (cART), has precluded an effective cure. The anatomical locations and biological mechanisms through which the viral population is maintained remain unknown. Much research has focused nearly exclusively on circulating resting T cells as the predominant source of persistent HIV, a strategy with limited success in developing an effective cure strategy. In this study, we review research supporting the importance of anatomical tissues and other immune cells for HIV maintenance and expansion, including the central nervous system, lymph nodes, and macrophages. We present accumulated research that clearly demonstrates the limitations of using blood-derived cells as a proxy for tissue reservoirs and sanctuaries throughout the body. We cite recent studies that have successfully used deep-sequencing strategies to uncover the complexity of HIV infection and the ability of the virus to evolve despite undetectable plasma viral loads. Finally, we suggest new strategies and highlight the importance of tissue banks for future research.


Assuntos
Infecções por HIV/tratamento farmacológico , Carga Viral , Latência Viral , Fármacos Anti-HIV/uso terapêutico , Antirretrovirais/uso terapêutico , Terapia Antirretroviral de Alta Atividade , Sistema Nervoso Central/virologia , Infecções por HIV/líquido cefalorraquidiano , HIV-1/efeitos dos fármacos , HIV-1/fisiologia , Humanos , Linfonodos/virologia , Macrófagos/virologia , RNA Viral/sangue , Bancos de Tecidos
8.
PLoS Pathog ; 13(2): e1006202, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-28241080

RESUMO

Although invasive cytomegalovirus (CMV) disease is uncommon in the era of antiretroviral therapy (ART), asymptomatic CMV coinfection is nearly ubiquitous in HIV infected individuals. While microbial translocation and gut epithelial barrier dysfunction may promote persistent immune activation in treated HIV infection, potentially contributing to morbidity and mortality, it has been unclear whether CMV replication in individuals with no symptoms of CMV disease might play a role in this process. We hypothesized that persistent CMV replication in the intestinal epithelium of HIV/CMV-coinfected individuals impairs gut epithelial barrier function. Using a combination of state-of-the-art in situ hybridization technology (RNAscope) and immunohistochemistry, we detected CMV DNA and proteins and evidence of intestinal damage in rectosigmoid samples from CMV-positive individuals with both untreated and ART-suppressed HIV infection. Two different model systems, primary human intestinal cells differentiated in vitro to form polarized monolayers and a humanized mouse model of human gut, together demonstrated that intestinal epithelial cells are fully permissive to CMV replication. Independent of HIV, CMV disrupted tight junctions of polarized intestinal cells, significantly reducing transepithelial electrical resistance, a measure of monolayer integrity, and enhancing transepithelial permeability. The effect of CMV infection on the intestinal epithelium is mediated, at least in part, by the CMV-induced proinflammatory cytokine IL-6. Furthermore, letermovir, a novel anti-CMV drug, dampened the effects of CMV on the epithelium. Together, our data strongly suggest that CMV can disrupt epithelial junctions, leading to bacterial translocation and chronic inflammation in the gut and that CMV could serve as a target for therapeutic intervention to prevent or treat gut epithelial barrier dysfunction during HIV infection.


Assuntos
Infecções por Citomegalovirus/virologia , Citomegalovirus , Infecções por HIV/virologia , Mucosa Intestinal/virologia , Replicação Viral , Animais , Coinfecção , Infecções por Citomegalovirus/imunologia , Feminino , Humanos , Imuno-Histoquímica , Hibridização In Situ , Interleucina-6/biossíntese , Interleucina-6/imunologia , Mucosa Intestinal/patologia , Masculino , Camundongos , Camundongos SCID , Permeabilidade
9.
J Virol ; 90(20): 8984-93, 2016 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-27466425

RESUMO

UNLABELLED: While combined antiretroviral therapy (cART) can result in undetectable plasma viral loads, it does not eradicate HIV infection. Furthermore, HIV-infected individuals while on cART remain at an increased risk of developing serious comorbidities, such as cancer, neurological disease, and atherosclerosis, suggesting that during cART, tissue-based HIV may contribute to such pathologies. We obtained DNA and RNA env, nef, and pol sequences using single-genome sequencing from postmortem tissues of three HIV(+) cART-treated (cART(+)) individuals with undetectable viral load and metastatic cancer at death and performed time-scaled Bayesian evolutionary analyses. We used a sensitive in situ hybridization technique to visualize HIV gag-pol mRNA transcripts in cerebellum and lymph node tissues from one patient. Tissue-associated virus evolved at similar rates in cART(+) and cART-naive (cART(-)) patients. Phylogenetic trees were characterized by two distinct features: (i) branching patterns consistent with constant viral evolution and dispersal among tissues and (ii) very recently derived clades containing both DNA and RNA sequences from multiple tissues. Rapid expansion of virus near death corresponded to wide-spread metastasis. HIV RNA(+) cells clustered in cerebellum tissue but were dispersed in lymph node tissue, mirroring the evolutionary patterns observed for that patient. Activated, infiltrating macrophages were associated with HIV RNA. Our data provide evidence that tissues serve as a sanctuary for wild-type HIV during cART and suggest the importance of macrophages as an alternative reservoir and mechanism of virus spread. IMPORTANCE: Combined antiretroviral therapy (cART) reduces plasma HIV to undetectable levels; however, removal of cART results in plasma HIV rebound, thus highlighting its inability to entirely rid the body of infection. Additionally, HIV-infected individuals on cART remain at high risk of serious diseases, which suggests a contribution from residual HIV. In this study, we isolated and sequenced HIV from postmortem tissues from three HIV(+) cART(+) individuals who died with metastatic cancer and had no detectable plasma viral load. Using high-resolution evolutionary analyses, we found that tissue-based HIV continues to replicate, evolve, and migrate among tissues during cART. Furthermore, cancer onset and metastasis coincided with increased HIV expansion, suggesting a linked mechanism. HIV-expressing cells were associated with tissue macrophages, a target of HIV infection. Our results suggest the importance of tissues, and macrophages in particular, as a target for novel anti-HIV therapies.


Assuntos
Antirretrovirais/uso terapêutico , Infecções por HIV/complicações , Infecções por HIV/virologia , HIV/isolamento & purificação , Neoplasias/complicações , Resposta Viral Sustentada , Carga Viral , Terapia Antirretroviral de Alta Atividade , Autopsia , Cerebelo/virologia , DNA Viral/genética , Variação Genética , HIV/classificação , HIV/genética , Infecções por HIV/tratamento farmacológico , Hibridização In Situ , Linfonodos/virologia , Filogenia , RNA Viral/genética , Análise de Sequência de DNA , Homologia de Sequência , Produtos do Gene env do Vírus da Imunodeficiência Humana/genética , Produtos do Gene nef do Vírus da Imunodeficiência Humana/genética , Produtos do Gene pol do Vírus da Imunodeficiência Humana/genética
10.
J Virol ; 90(20): 8968-83, 2016 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-27466426

RESUMO

UNLABELLED: HIV infection treatment strategies have historically defined effectiveness through measuring patient plasma HIV RNA. While combined antiretroviral therapy (cART) can reduce plasma viral load (pVL) to undetectable levels, the degree that HIV is eliminated from other anatomical sites remains unclear. We investigated the HIV DNA levels in 229 varied autopsy tissues from 20 HIV-positive (HIV(+)) cART-treated study participants with low or undetectable plasma VL and cerebrospinal fluid (CSF) VL prior to death who were enrolled in the National Neurological AIDS Bank (NNAB) longitudinal study and autopsy cohort. Extensive medical histories were obtained for each participant. Autopsy specimens, including at least six brain and nonbrain tissues per participant, were reviewed by study pathologists. HIV DNA, measured in tissues by quantitative and droplet digital PCR, was identified in 48/87 brain tissues and 82/142 nonbrain tissues at levels >200 HIV copies/million cell equivalents. No participant was found to be completely free of tissue HIV. Parallel sequencing studies from some tissues recovered intact HIV DNA and RNA. Abnormal histological findings were identified in all participants, especially in brain, spleen, lung, lymph node, liver, aorta, and kidney. All brain tissues demonstrated some degree of pathology. Ninety-five percent of participants had some degree of atherosclerosis, and 75% of participants died with cancer. This study assists in characterizing the anatomical locations of HIV, in particular, macrophage-rich tissues, such as the central nervous system (CNS) and testis. Additional studies are needed to determine if the HIV recovered from tissues promotes the pathogenesis of inflammatory diseases, such as HIV-associated neurocognitive disorders, cancer, and atherosclerosis. IMPORTANCE: It is well-known that combined antiretroviral therapy (cART) can reduce plasma HIV to undetectable levels; however, cART cannot completely clear HIV infection. An ongoing question is, "Where is HIV hiding?" A well-studied HIV reservoir is "resting" T cells, which can be isolated from blood products and succumb to cART once activated. Less-studied reservoirs are anatomical tissue samples, which have unknown cART penetration, contain a comparably diverse spectrum of potentially HIV-infected immune cells, and are important since <2% of body lymphocytes actually reside in blood. We examined 229 varied autopsy specimens from 20 HIV(+) participants who died while on cART and identified that >50% of tissues were HIV infected. Additionally, we identified considerable pathology in participants' tissues, especially in brain, spleen, lung, lymph node, liver, aorta, and kidney. This study substantiates that tissue-associated HIV is present despite cART and can inform future studies into HIV persistence.


Assuntos
Antirretrovirais/uso terapêutico , Autopsia , DNA Viral/análise , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , Carga Viral , Humanos , Estudos Longitudinais , Reação em Cadeia da Polimerase em Tempo Real
11.
J Biol Chem ; 291(19): 10332-46, 2016 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-26957545

RESUMO

HIV evades eradication because transcriptionally dormant proviral genomes persist in long-lived reservoirs of resting CD4(+) T cells and myeloid cells, which are the source of viral rebound after cessation of antiretroviral therapy. Dormant HIV genomes readily produce infectious virus upon cellular activation because host transcription factors activated specifically by cell stress and heat shock mediate full-length HIV transcription. The molecular chaperone heat shock protein 90 (Hsp90) is overexpressed during heat shock and activates inducible cellular transcription factors. Here we show that heat shock accelerates HIV transcription through induction of Hsp90 activity, which activates essential HIV-specific cellular transcription factors (NF-κB, NFAT, and STAT5), and that inhibition of Hsp90 greatly reduces gene expression mediated by these factors. More importantly, we show that Hsp90 controls virus transcription in vivo by specific Hsp90 inhibitors in clinical development, tanespimycin (17-(allylamino)-17-demethoxygeldanamycin) and AUY922, which durably prevented viral rebound in HIV-infected humanized NOD scid IL-2Rγ(-/-) bone marrow-liver-thymus mice up to 11 weeks after treatment cessation. Despite the absence of rebound viremia, we were able to recover infectious HIV from PBMC with heat shock. Replication-competent virus was detected in spleen cells from these nonviremic Hsp90 inhibitor-treated mice, indicating the presence of a tissue reservoir of persistent infection. Our novel findings provide in vivo evidence that inhibition of Hsp90 activity prevents HIV gene expression in replication-competent cellular reservoirs that would typically cause rebound in plasma viremia after antiretroviral therapy cessation. Alternating or supplementing Hsp90 inhibitors with current antiretroviral therapy regimens could conceivably suppress rebound viremia from persistent HIV reservoirs.


Assuntos
Infecções por HIV/prevenção & controle , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Latência Viral/efeitos dos fármacos , Animais , Antivirais/farmacologia , Benzoquinonas/farmacologia , Western Blotting , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Feminino , Infecções por HIV/metabolismo , Infecções por HIV/virologia , HIV-1/isolamento & purificação , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Lactamas Macrocíclicas/farmacologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID
12.
Stem Cell Reports ; 6(1): 137-49, 2016 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-26724903

RESUMO

B cells are key components of cellular and humoral immunity and, like all lymphocytes, are thought to originate and renew from hematopoietic stem cells (HSCs). However, our recent single-HSC transfer studies demonstrate that adult bone marrow HSCs do not regenerate B-1a, a subset of tissue B cells required for protection against pneumonia, influenza, and other infections. Since B-1a are regenerated by transfers of fetal liver, the question arises as to whether B-1a derive from fetal, but not adult, HSCs. Here we show that, similar to adult HSCs, fetal HSCs selectively fail to regenerate B-1a. We also show that, in humanized mice, human fetal liver regenerates tissue B cells that are phenotypically similar to murine B-1a, raising the question of whether human HSC transplantation, the mainstay of such models, is sufficient to regenerate human B-1a. Thus, our studies overtly challenge the current paradigm that HSCs give rise to all components of the immune system.


Assuntos
Linfócitos B/imunologia , Diferenciação Celular/imunologia , Células-Tronco Fetais/transplante , Transplante de Tecido Fetal/métodos , Transplante de Células-Tronco Hematopoéticas/métodos , Animais , Animais Recém-Nascidos , Linfócitos B/citologia , Linfócitos B/metabolismo , Diferenciação Celular/genética , Linhagem da Célula/genética , Linhagem da Célula/imunologia , Células Cultivadas , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/imunologia , Células-Tronco Embrionárias/metabolismo , Células-Tronco Fetais/imunologia , Células-Tronco Fetais/metabolismo , Citometria de Fluxo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/imunologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Fígado/citologia , Fígado/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos
13.
J Neurovirol ; 22(3): 275-81, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-26572785

RESUMO

Two innovative studies recently identified functional lymphatic structures in the meninges that may influence the development of HIV-associated neurological disorders (HAND). Until now, blood vessels were assumed to be the sole transport system by which HIV-infected monocytes entered the brain by bypassing a potentially hostile blood-brain barrier through inflammatory-mediated semi-permeability. A cascade of specific chemokine signals promote monocyte migration from blood vessels to surrounding brain tissues via a well-supported endothelium, where the cells differentiate into tissue macrophages capable of productive HIV infection. Lymphatic vessels on the other hand are more loosely organized than blood vessels. They absorb interstitial fluid from bodily tissues where HIV may persist and exchange a variety of immune cells (CD4(+) T cells, monocytes, macrophages, and dendritic cells) with surrounding tissues through discontinuous endothelial junctions. We propose that the newly discovered meningeal lymphatics are key to HIV migration among viral reservoirs and brain tissue during periods of undetectable plasma viral loads due to suppressive combinational antiretroviral therapy, thus redefining the migration process in terms of a blood-lymphatic transport system.


Assuntos
Complexo AIDS Demência/virologia , Encéfalo/virologia , HIV-1/fisiologia , Sistema Linfático/virologia , Meninges/virologia , Monócitos/virologia , Complexo AIDS Demência/imunologia , Complexo AIDS Demência/patologia , Barreira Hematoencefálica/imunologia , Barreira Hematoencefálica/virologia , Encéfalo/imunologia , Movimento Celular , Quimiocinas/biossíntese , Quimiocinas/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/virologia , Endotélio Vascular/imunologia , Endotélio Vascular/virologia , HIV-1/patogenicidade , Humanos , Sistema Linfático/imunologia , Macrófagos/imunologia , Macrófagos/virologia , Meninges/imunologia , Monócitos/imunologia , Linfócitos T/imunologia , Linfócitos T/virologia , Internalização do Vírus
14.
Antimicrob Agents Chemother ; 59(7): 4190-8, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25941222

RESUMO

Like normal cellular nucleosides, the nucleoside reverse transcriptase (RT) inhibitor (NRTI) 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) has a 3'-hydroxyl moiety, and yet EFdA is a highly potent inhibitor of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) replication with activity against a broad range of clinically important drug-resistant HIV isolates. We evaluated the anti-HIV activity of EFdA in primary human cells and in HIV-infected humanized mice. EFdA exhibited excellent potency against HIVJR-CSF in phytohemagglutinin-stimulated peripheral blood mononuclear cells (PBMCs), with a 50% inhibitory concentration of 0.25 nM and a selectivity index of 184,000; similar antiviral potency was found against 12 different HIV clinical isolates from multiple clades (A, B, C, D, and CRF01_AE). EFdA was readily absorbed after oral dosing (5 mg/kg of body weight) in both mice and the rhesus macaque, with micromolar levels of the maximum concentration of drug in serum (Cmax) attained at 30 min and 90 min, respectively. Trough levels were at or above 90% inhibitory concentration (IC90) levels in the macaque at 24 h, suggesting once-daily dosing. EFdA showed reasonable penetration of the blood-brain barrier in the rhesus macaque, with cerebrospinal fluid levels at approximately 25% of plasma levels 8 h after single oral dosing. Rhesus PBMCs isolated 24 h following a single oral dose of 5 mg/kg EFdA were refractory to SIV infection due to sufficiently high intracellular EFdA-triphosphate levels. The intracellular half-life of EFdA-triphosphate in PBMCs was determined to be >72 h following a single exposure to EFdA. Daily oral administration of EFdA at low dosage levels (1 to 10 mg/kg/day) was highly effective in protecting humanized mice from HIV infection, and 10 mg/kg/day oral EFdA completely suppressed HIV RNA to undetectable levels within 2 weeks of treatment.


Assuntos
Fármacos Anti-HIV/uso terapêutico , Compostos de Diazônio/uso terapêutico , Farneseno Álcool/análogos & derivados , Infecções por HIV/tratamento farmacológico , Inibidores da Transcriptase Reversa/uso terapêutico , Animais , Fármacos Anti-HIV/administração & dosagem , Barreira Hematoencefálica , Compostos de Diazônio/administração & dosagem , Compostos de Diazônio/farmacocinética , Farneseno Álcool/administração & dosagem , Farneseno Álcool/farmacocinética , Farneseno Álcool/uso terapêutico , Citometria de Fluxo , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , Meia-Vida , Humanos , Técnicas In Vitro , Macaca mulatta , Camundongos , Camundongos SCID , Monócitos/efeitos dos fármacos , Monócitos/virologia , RNA Viral/biossíntese , RNA Viral/efeitos dos fármacos , Inibidores da Transcriptase Reversa/administração & dosagem , Inibidores da Transcriptase Reversa/farmacocinética , Vírus da Imunodeficiência Símia , Viremia/tratamento farmacológico , Viremia/virologia
15.
Virology ; 462-463: 115-25, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24971704

RESUMO

Highly potent broadly neutralizing human monoclonal antibodies hold promise for HIV prophylaxis and treatment. We used the SCID-hu Thy/Liv and BLT humanized mouse models to study the efficacy of these antibodies, primarily PG16, against HIV-1 clades A, B, and C. PG16 targets a conserved epitope in the V1/V2 region of gp120 common to 70-80% of HIV-1 isolates from multiple clades and has extremely potent in vitro activity against HIVJR-CSF. PG16 was highly efficacious in SCID-hu mice as a single intraperitoneal administration the day before inoculation of R5-tropic HIV directly into their Thy/Liv implants and demonstrated even greater efficacy if PG16 administration was continued after Thy/Liv implant HIV inoculation. However, PG16 as monotherapy had no activity in humanized mice with established R5-tropic HIV infection. These results provide evidence of tissue penetration of the antibodies, which could aid in their ability to prevent infection if virus crosses the mucosal barrier.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Anticorpos Neutralizantes/uso terapêutico , Anticorpos Anti-HIV/uso terapêutico , Infecções por HIV/terapia , HIV-1/imunologia , Animais , Modelos Animais de Doenças , Proteína gp120 do Envelope de HIV/imunologia , Masculino , Camundongos , Camundongos SCID , Resultado do Tratamento
16.
Virology ; 436(1): 162-72, 2013 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-23200770

RESUMO

We previously showed that reduced infectivity of HIV with incompletely processed capsid-spacer protein 1 (CA-SP1) is rescued by cellular activation or increased expression of HSP90AB1, a member of the cytosolic heat shock protein 90 family. Here we show that HSP90AB1 is present in HIV virions and that HSP90AB1, but not nonfunctional mutated HSP90AB1(E42A+D88A), restores infectivity to HIV with mutations in CA that alter core stability. Further, the CA mutants were hypersensitive to pharmacological inhibition of HSP90AB1. In agreement with Roesch et al. (2012), we found that culturing HIV at 39.5°C enhanced viral infectivity up to 30-fold in human peripheral blood mononuclear cells (p=0.002) and rescued CA-mutant infectivity in nonactivated cells, concurrent with elevated expression of HSP90AB1 during hyperthermia. In sum, the transdominant effect of HSP90AB1 on CA-mutant HIV infectivity suggests a potential role for this class of cellular chaperones in HIV core stability and uncoating.


Assuntos
Proteínas do Capsídeo/genética , HIV/genética , HIV/patogenicidade , Proteínas de Choque Térmico HSP90/metabolismo , Linhagem Celular , Células HEK293 , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Temperatura Alta , Humanos , Células Jurkat , Leucócitos Mononucleares/virologia , Mutação , Linfócitos T/virologia , Proteínas do Core Viral/genética
17.
J Virol ; 86(23): 12795-805, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22973041

RESUMO

Human cytomegalovirus (HCMV) is the leading viral cause of birth defects and life-threatening lung-associated diseases in premature infants and immunocompromised children. Although the fetal lung is a major target organ of the virus, HCMV lung pathogenesis has remained unexplored, possibly as a result of extreme host range restriction. To overcome this hurdle, we generated a SCID-hu lung mouse model that closely recapitulates the discrete stages of human lung development in utero. Human fetal lung tissue was implanted into severe combined immunodeficient (CB17-scid) mice and inoculated by direct injection with the VR1814 clinical isolate of HCMV. Virus replication in the fetal lung was assessed by the quantification of infectious virus titers and HCMV genome copies and the detection of HCMV proteins by immunohistochemistry and Western blotting. We show that HCMV efficiently replicated in the lung implants during a 2-week period, forming large viral lesions. The virus productively infected alveolar epithelial and mesenchymal cells, imitating congenital infection of the fetal lung. HCMV replication triggered apoptosis near and within the viral lesions and impaired the production of surfactant proteins in the alveolar epithelium. Our findings highlight that congenital and neonatal HCMV infection can adversely impact lung development, leading to pneumonia and acute lung injury. We have successfully developed a small-animal model that closely recapitulates fetal and neonatal lung development and provides a valuable, biologically relevant tool for an understanding of the lung pathogenesis of HCMV as well as other human respiratory viruses. Additionally, this model would greatly facilitate the development and testing of new antiviral therapies for HCMV along with select human pulmonary pathogens.


Assuntos
Infecções por Citomegalovirus/congênito , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Alvéolos Pulmonares/virologia , Proteínas Associadas a Surfactantes Pulmonares/metabolismo , Replicação Viral/fisiologia , Animais , Apoptose/fisiologia , Western Blotting , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Masculino , Camundongos , Camundongos SCID , Alvéolos Pulmonares/citologia , Estatísticas não Paramétricas , Proteínas Virais/isolamento & purificação
19.
Antimicrob Agents Chemother ; 56(4): 2162-5, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22252805

RESUMO

PC-1505 is a C34 peptide derived from the heptad repeat 2 region of HIV-1 gp41 conjugated to human serum albumin for sustained in vivo activity. One single preexposure dose of PC-1505 reduced viral RNA in HIV-1-infected SCID-hu Thy/Liv mice by 3.3 log10 and protected T cells from virus-mediated depletion. In contrast, a single preexposure dose of Truvada reduced viral RNA by only 0.8 log10 and was substantially less effective in preventing T cell depletion.


Assuntos
Inibidores da Fusão de HIV/farmacologia , Infecções por HIV/prevenção & controle , Peptídeos/farmacologia , Animais , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Relação Dose-Resposta a Droga , Combinação de Medicamentos , Combinação Emtricitabina e Fumarato de Tenofovir Desoproxila , Genes MHC Classe I/genética , Proteína do Núcleo p24 do HIV/sangue , Inibidores da Fusão de HIV/química , Infecções por HIV/virologia , Camundongos , Camundongos SCID , Compostos Organofosforados/farmacologia , Peptídeos/química , RNA Viral/sangue , Timócitos/efeitos dos fármacos , Timócitos/metabolismo , Timócitos/virologia
20.
J Virol ; 86(6): 3327-36, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22238321

RESUMO

The development of small animal models for the study of HIV transmission is important for evaluation of HIV prophylaxis and disease pathogenesis. In humanized bone marrow-liver-thymus (BLT) mice, hematopoiesis is reconstituted by implantation of human fetal liver and thymus tissue (Thy/Liv) plus intravenous injection of autologous liver-derived hematopoietic stem progenitor cells (HSPC). This results in reconstitution of human leukocytes in the mouse peripheral blood, lymphoid organs, and mucosal sites. NOD-scid interleukin-2 receptor-negative (IL-2Rγ(-/-)) (NSG)-BLT mice were inoculated intravaginally with HIV and were monitored for plasma viremia by a branched DNA assay 4 weeks later. T-cell activation was determined by expression of CD38 and HLA-DR on human CD4(+) and CD8(+) T cells in mouse peripheral blood at the time of inoculation and 4 weeks later. Additional BLT mice were treated with human alpha interferon 2b (IFN-α2b) (intron A) and assessed for T-cell activation. Productive HIV infection in BLT mice was associated with T-cell activation (increases in CD38 mean fluorescence intensity and both the frequency and absolute number of CD38(+) HLA-DR(+) T cells) that correlated strongly with plasma viral load and was most pronounced in the CD8(+) T-cell compartment. This T-cell activation phenotype was recapitulated in NSG-BLT mice treated with intron A. HIV susceptibility correlated with the number of HSPC injected, yet a number of mice receiving the Thy/Liv implant alone, with no HSPC injection, were also susceptible to intravaginal HIV. These results are consistent with studies linking T-cell activation to progressive disease in humans and lend support for the use of NSG-BLT mice in studies of HIV pathogenesis.


Assuntos
Infecções por HIV/imunologia , HIV-1/fisiologia , Interferon-alfa/imunologia , Ativação Linfocitária , Linfócitos T/imunologia , Animais , Modelos Animais de Doenças , Feminino , Infecções por HIV/virologia , HIV-1/imunologia , Humanos , Interferon alfa-2 , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Receptores de Interleucina-2/genética , Receptores de Interleucina-2/imunologia , Proteínas Recombinantes/imunologia , Linfócitos T/fisiologia
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