RESUMO
Nowadays, there is a strong request for the treatment of chronic HBV-infection with direct acting antivirals. Furthermore, prevalent human immunodeficiency virus (HIV-1) and hepatitis B (HBV) co-infections highlight an immediate need for dual long-acting and easily administered antivirals. To this end, we modified lamivudine (3TC), a nucleoside analog inhibitor of both viruses, into a lipophilic monophosphorylated prodrug (M23TC). Prior work demonstrated that nanoformulation of M23TC (NM23TC) enhanced drug stability, controlled dissolution and improved access to sites of viral replication. The present study evaluated the efficacy of a NM23TC in HBV-infected chimeric liver humanized mice. Levels of HBV DNA and HBsAg in plasma were monitored up to 8 weeks posttreatment. A single intramuscular dose of 75 mg/kg 3TC equivalents of nanoformulated NM23TC provided sustained drug levels and suppressed HBV replication in humanized mice for 4 weeks. The results support further development of this long-acting 3TC nanoformulation for HBV treatment and prevention.
Assuntos
Lamivudina/química , Animais , Antivirais/química , Antivirais/farmacologia , Vírus da Hepatite B/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Imuno-Histoquímica , Lamivudina/farmacologia , Masculino , Camundongos , Camundongos Knockout , Ratos , Ratos Sprague-Dawley , Replicação Viral/efeitos dos fármacosRESUMO
Stable engraftment of human lymphoid tissue in NOD/scid-IL-2Rgammacnull mice after CD34+ hematopoietic stem cell reconstitution permits the evaluation of ongoing HIV-1 infection for weeks to months. We demonstrate that HIV-1-infected rodents develop virus-specific cellular immune responses. CD8+ cell depletion, 2 or 5-7 wk after viral infection, resulted in a significant increase of HIV-1 load, robust immune cell activation, and cytopathology in lymphoid tissues but preserved CD4/CD8 double-positive thymic T cell pools. Human CD8+ cells reappeared in circulation as early as 2-3 wk. These data support a role of CD8+ cells in viral surveillance and the relevance of this humanized mouse model for the studies of HIV-1 pathobiology and virus-specific immunity.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Animais , Separação Celular , Transplante de Células-Tronco de Sangue do Cordão Umbilical , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Receptores de Interleucina-2/deficiência , Receptores de Interleucina-2/genéticaRESUMO
Few rodent models of human immunodeficiency virus type one (HIV-1) infection can reflect the course of viral infection in humans. To this end, we investigated the relationships between progressive HIV-1 infection, immune compromise, and neuroinflammatory responses in NOD/scid-IL-2Rγ(c)(null) mice reconstituted with human hematopoietic CD34(+) stem cells. Human blood-borne macrophages repopulated the meninges and perivascular spaces of chimeric animals. Viral infection in lymphoid tissue led to the accelerated entry of human cells into the brain, marked neuroinflammation, and HIV-1 replication in human mononuclear phagocytes. A meningitis and less commonly an encephalitis followed cM-T807 antibody-mediated CD8(+) cell depletion. We conclude that HIV-1-infected NOD/scid-IL-2Rγ(c)(null) humanized mice can, at least in part, recapitulate lentiviral neuropathobiology. This model of neuroAIDS reflects the virological, immunological, and early disease-associated neuropathological components of human disease.
Assuntos
Complexo AIDS Demência/etiologia , Modelos Animais de Doenças , Infecções por HIV/complicações , HIV-1/fisiologia , Camundongos , Complexo AIDS Demência/patologia , Complexo AIDS Demência/virologia , Animais , Encéfalo/imunologia , Encéfalo/virologia , Células Cultivadas , Progressão da Doença , Infecções por HIV/imunologia , Infecções por HIV/patologia , Infecções por HIV/virologia , Humanos , Subunidade gama Comum de Receptores de Interleucina/genética , Meningite Viral/complicações , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos TransgênicosRESUMO
The use of appropriate extraction and amplification controls for acellular specimens is not standardized in the clinical laboratory community. Extraction controls and checks for inhibitors of amplification in cellular specimens are most often accomplished by amplification of an internal human genomic target. This approach is not feasible for acellular specimens, which may contain little or no amplifiable genomic material. Other specimen types, such as stool, frequently contain amplification inhibitors. Failure to test for these inhibitors can result in the reporting of false-negative results. The goal of this study was to evaluate the use of a T4 bacteriophage as an extraction and amplification control for acellular specimens. The T4 bacteriophage assay was evaluated for use as a control in 290 specimens, including cerebrospinal fluid, serum, and filtered stool. Extraction procedures on two automated instruments were assessed, including the Roche MagNAPure Compact (Roche Diagnostics, Indianapolis, IN) and the QIAGEN BioRobot M48 (QIAGEN, Valencia, CA), along with the manual QIAGEN extraction method. The T4 bacteriophage can be extracted reliably and reproducibly from cerebral spinal fluid, serum, and filtered stool and, therefore, is useful as both an extraction control and inhibitor check for these specimen sources.
Assuntos
Bacteriófago T4/isolamento & purificação , Bioensaio/métodos , Líquido Cefalorraquidiano/química , DNA/isolamento & purificação , Fezes/química , Bacteriófago T4/genética , DNA Viral/análise , DNA Viral/genética , DNA Viral/isolamento & purificação , Humanos , Técnicas de Amplificação de Ácido Nucleico , Plasmídeos , Padrões de ReferênciaRESUMO
Human immunodeficiency virus type 1 (HIV-1) infection impairs liver function, and liver diseases have become a leading cause of morbidity in infected patients. The immunopathology of liver damage caused by HIV-1 remains unclear. We used chimeric mice dually reconstituted with a human immune system and hepatocytes to address the relevance of the model to pathobiology questions related to human hepatocyte survival in the presence of systemic infection. TK-NOG males were transplanted with mismatched human hematopoietic stem/progenitor cells and hepatocytes, human albumin concentration and the presence of human immune cells in blood were monitored for hepatocytes and immune reconstitution, and mice were infected with HIV-1. HIV-1-infected animals showed a decline in human albumin concentration with a significant reduction in percentage of human hepatocytes compared to uninfected mice. The decrease in human albumin levels correlated with a decline in CD4+ cells in the liver and with an increase in HIV-1 viral load. HIV-1 infection elicited proinflammatory response in the immunological milieu of the liver in HIV-infected mice compared to uninfected animals, as determined by upregulation of IL23, CXCL10 and multiple toll-like receptor expression. The inflammatory reaction associated with HIV-1 infection in vivo could contribute to the depletion and dysfunction of hepatocytes. The dual reconstituted TK-NOG mouse model is a feasible platform to investigate hepatocyte-related HIV-1 immunopathogenesis.This article has an associated First Person interview with the first author of the paper.
Assuntos
Transplante de Coração/métodos , Síndrome do Coração Esquerdo Hipoplásico/cirurgia , Transplante de Fígado/métodos , Adolescente , Transfusão de Sangue , Feminino , Técnica de Fontan , Herpesvirus Humano 4 , Teste de Histocompatibilidade , Humanos , Síndrome do Coração Esquerdo Hipoplásico/complicações , Cirrose Hepática/complicações , Cirrose Hepática/cirurgia , Masculino , Período Pós-Operatório , Resultado do Tratamento , Adulto JovemRESUMO
Previously, we reported the evaluation of several polyplex-based gene delivery systems with respect to their effectiveness, toxicity, and cell type dependence in vitro. One system, P123-g-PEI(2K), a cationic graft block copolymer, is of particular interest as it has been demonstrated to successfully deliver genetic material to murine liver following systemic delivery [Nguyen, H. K., Lemieux, P., Vinogradov, S. V., Gebhart, C. L., Guerin, N., Paradis, G., Bronich, T. K., Alakhov, V. Y., and Kabanov, A. V. (2000) Evaluation of Polyether-Polyethyleneimine Graft Copolymers as Gene Transfer Agents. Gene Ther. 7, 126-138 (1)]. The P123-g-PEI(2K) system requires nonmodified Pluronic P123 as an excipient to stabilize the dispersion. The purpose of the current work was to more closely characterize this system, to assess the role of each component of the system to the overall transfection process. We evaluated particle size, stability, and resistance to nuclease degradation. In addition, cellular uptake and localization of plasmid, as well as transgene expression, were evaluated following in vitro transfection of prostate cancer cells (PC-3) with various individual components of the system. Nonmodified Pluronic alone did not significantly enhance DNA uptake, transgene expression, or DNase protection. Therefore, we conclude that nonmodified Pluronic acted primarily by optimizing the size of the polyplex. Furthermore, though this system displays several characteristics thought desirable of a nonviral gene delivery system, these studies did discriminate a potential limitation of this system for in vivo applications, namely, the insufficient level of protection of plasmid DNA from nuclease degradation. This may limit the effective dose delivered, as well as limiting the effective circulation time. These studies provide vital information that will guide modification of this system to enhance the current in vivo profile.