RESUMO
BACKGROUND: Congenital cytomegalovirus (CMV) infection is a major public health problem. Antiviral therapies administered during pregnancy might prevent vertical CMV transmission and disease in newborns, but these agents have not been evaluated in clinical trials. The guinea pig model of congenital CMV infection was therefore used to test the hypothesis that antiviral therapy, using the agent agent cyclic cidofovir (cHPMPC), could prevent congenital CMV infection. RESULTS: Pregnant outbred Hartley guinea pigs were challenged in the early-third trimester with guinea pig CMV (GPCMV) and treated with placebo, or the antiviral agent, cyclic cidofovir. To optimize detection of vertical infection, an enhanced green fluorescent protein (eGFP)-tagged virus was employed. Compared to placebo, cyclic cidofovir-treated dams and pups had reduced mortality following GPCMV challenge. The magnitude of GPCMV-induced maternal and fetal mortality in this study was reduced from 5/25 animals in the placebo group to 0/21 animals in the treatment group (p = 0.05, Fisher's exact test). By viral culture assay, antiviral therapy was found to completely prevent GPCMV transmission to the fetus. In control pups, 5/19 (26%) were culture-positive for GPCMV, compared to 0/16 of pups in the cyclic cidofovir treatment group (p < 0.05, Fisher's exact test). CONCLUSION: Antiviral therapy with cyclic cidofovir improves pregnancy outcomes in guinea pigs, and eliminates congenital CMV infection, following viral challenge in the third trimester. This study also demonstrated that an eGFP-tagged recombinant virus, with the reporter gene inserted into a dispensable region of the viral genome, retained virulence, including the potential for congenital transmission, facilitating tissue culture-based detection of congenital infection. These observations provide support for clinical trials of antivirals for reduction of congenital CMV infection.
Assuntos
Antivirais/farmacologia , Citosina/análogos & derivados , Transmissão Vertical de Doenças Infecciosas/prevenção & controle , Organofosfonatos/farmacologia , Complicações Infecciosas na Gravidez/tratamento farmacológico , Infecções por Roseolovirus/prevenção & controle , Roseolovirus/crescimento & desenvolvimento , Animais , Cidofovir , Citosina/farmacologia , Modelos Animais de Doenças , Feminino , Cobaias , Gravidez , Complicações Infecciosas na Gravidez/virologia , Resultado da Gravidez , Roseolovirus/genética , Infecções por Roseolovirus/congênito , Infecções por Roseolovirus/transmissãoRESUMO
A primary obstacle to an HIV-1 cure is long-lived viral reservoirs, which must be eliminated or greatly reduced. Cure strategies have largely focused on monitoring changes in T cell reservoirs in peripheral blood (PB), even though the lymphoid tissues (LT) are primary sites for viral persistence. To track and discriminate viral reservoirs within tissue compartments we developed a specific and sensitive next-generation in situ hybridization approach to detect vRNA, including vRNA+ cells and viral particles ("RNAscope"), vDNA+ cells ("DNAscope") and combined vRNA and vDNA with immunohistochemistry to detect and phenotype active and latently infected cells in the same tissue section. RNAscope is highly sensitive with greater speed of analysis compared to traditional in situ hybridization. The highly sensitive and specific DNAscope detected SIV/HIV vDNA+ cells, including duplexed detection of vDNA and vRNA or immunophenotypic markers in the same section. Analysis of LT samples from macaques prior to and during combination antiretroviral therapy demonstrated that B cell follicles are an important anatomical compartment for both latent and active viral persistence during treatment. These new tools should allow new insights into viral reservoir biology and evaluation of cure strategies.
RESUMO
The induction of chemokines by interferons might represent a link between innate and adaptive immunity. Whether these induced chemokines might be useful by themselves to induce an immune response is not known. We hypothesized that the interferon-inducible chemokine CXCL10 could stimulate dendritic cells (DC) to mature and cross-present exogenous antigen to T cells, resulting in a Th1-type immune response. We found that injecting mice with CXCL10 together with ovalbumin (OVA) as a test antigen was sufficient to produce functional OVA-specific T cells in 7 of 10 mice. Further, using only CXCL10 and a peptide antigen derived from vaccinia virus, we were able to induce peptide-specific cytotoxic T cells in 4 of 4 mice tested. These cytotoxic T cells protected 9 of 10 mice from subsequent infectious challenge with vaccinia virus. Unlike traditional adjuvants, no side effects were observed in any of the injected mice. We conclude that CXCL10 co-administration with a variety of antigens may represent a unique strategy of inducing a protective T cell response to a number of pathogens that merits further study.
Assuntos
Quimiocinas CXC/imunologia , Animais , Quimiocina CXCL10 , Citotoxicidade Imunológica , Camundongos , Camundongos Endogâmicos C57BL , Ovalbumina/imunologia , Úlcera Cutânea/prevenção & controle , Linfócitos T/imunologia , Linfócitos T Citotóxicos/imunologia , Vacínia/prevenção & controle , Vaccinia virus/imunologia , Proteínas Virais/imunologiaRESUMO
Dendritic cells (DCs), because they orchestrate the immune response to microbes, represent an ideal target for pathogens attempting to evade the immune system. We hypothesized that interactions between human immunodeficiency virus (HIV) and DCs lead to the development of a semimature state, in which DCs migrate to lymph nodes but induce tolerance in T cells, rather than immunity. We found that lymph nodes from untreated HIV-infected subjects contained an abundance of semimature DCs, the disappearance of which correlated with the initiation of highly active antiretroviral therapy (HAART). Such lymph nodes also contained an abundance of T cells that had a regulatory phenotype and that persisted after HAART. Lymph node DCs from untreated HIV-infected subjects cultured with normal allogeneic T cells induced these T cells to adopt the phenotype of regulatory T cells, an ability that was lost after HAART. We conclude that HIV infection correlates with the presence of semimature DCs that stimulate T cell tolerance rather than immunity. These regulatory T cells may contribute to the lack of effective HIV immune responses.
Assuntos
Fármacos Anti-HIV/uso terapêutico , Células Dendríticas/imunologia , Infecções por HIV/imunologia , HIV-1/fisiologia , Linfócitos T Reguladores/imunologia , Terapia Antirretroviral de Alta Atividade , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD4-Positivos/virologia , Células Cultivadas , Células Dendríticas/metabolismo , Células Dendríticas/patologia , Infecções por HIV/tratamento farmacológico , Humanos , Leucócitos Mononucleares/virologia , Linfonodos/patologia , Linfonodos/virologiaRESUMO
Previous studies have suggested that G protein coupling, phospholipase C activation, phosphoinositide hydrolysis, and protein kinase C activation may be required for alpha(1B)-adrenergic receptor regulation, particularly for their endocytosis into intracellular vesicles. Accordingly, the internalization and down-regulation properties of mutated receptors with defects in G protein coupling and second messenger generation were investigated. The Delta12 and Delta5 receptors, previously shown to be defective in G protein coupling, exhibited greater agonist-induced losses of cell surface accessibility assessed by radioligand binding to intact cells on ice than for the wild-type receptor; however, these receptors were completely defective in endocytosis into intracellular vesicles assessed by sucrose density gradient centrifugation. These receptors also did not undergo down-regulation with long-term agonist exposure as did the wild-type receptor; instead, a prominent up-regulation was observed. The Y348A receptor, previously shown to be defective in phosphoinositide hydrolysis and endocytosis was also defective in down-regulation but did not exhibit significant up-regulation. In contrast, a receptor construct with amino acid residues 246 to 261 deleted (Delta[246-261]) was also defective in stimulation of phosphoinositide hydrolysis but exhibited internalization and down-regulation properties essentially identical to those for the wild-type receptor. Together, these results suggest that stimulation of phosphoinositide hydrolysis by alpha(1B)-adrenergic receptors is not required for their endocytosis or down-regulation but that similar and overlapping receptor structural domains are involved in mediating these processes.