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2.
Artigo em Inglês | MEDLINE | ID: mdl-31628202

RESUMO

Epithelial ovarian cancer (OvCa) is the most lethal female reproductive tract malignancy. A major clinical hurdle in patient management and treatment is that when using current surveillance technologies 80% of patients will be clinically diagnosed as having had a complete clinical response to primary therapy. In fact, the majority of women nonetheless develop disease recurrence within 18 mo. Thus, without more accurate surveillance protocols, the diagnostic question regarding OvCa recurrence remains framed as "when" rather than "if." With this background, we describe the case of a 61-yr-old female who presented with a 3-mo history of unexplained whole-body rash, which unexpectedly led to a diagnosis of and her treatment for OvCa. The rash resolved immediately following debulking surgery. Nearly 1 yr later, however, the rash reappeared, prompting the prospect of tumor recurrence and requirement for additional chemotherapy. To investigate this possibility, we undertook a genomics-based tumor surveillance approach using a targeted 56-gene NGS panel and biobanked tumor samples to develop personalized ctDNA biomarkers. Although tumor-specific TP53 and PTEN mutations were detectable in all originally collected tumor samples, pelvic washes, and blood samples, they were not detectable in any biosample collected beyond the first month of treatment. No additional chemotherapy was given. The rash spontaneously resolved. Now, 2 yr beyond the patient's original surgery, and in the face of continued negative ctDNA findings, the patient remains with no evidence of disease. As this single case report suggests, we believe for the first time that ctDNA can provide an additional layer of information to avoid overtreatment.

4.
Elife ; 62017 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-28826481

RESUMO

Standard treatment for metastatic prostate cancer (CaP) prevents ligand-activation of androgen receptor (AR). Despite initial remission, CaP progresses while relying on AR. AR transcriptional output controls CaP behavior and is an alternative therapeutic target, but its molecular regulation is poorly understood. Here, we show that action of activated AR partitions into fractions that are controlled preferentially by different coregulators. In a 452-AR-target gene panel, each of 18 clinically relevant coregulators mediates androgen-responsiveness of 0-57% genes and acts as a coactivator or corepressor in a gene-specific manner. Selectivity in coregulator-dependent AR action is reflected in differential AR binding site composition and involvement with CaP biology and progression. Isolation of a novel transcriptional mechanism in which WDR77 unites the actions of AR and p53, the major genomic drivers of lethal CaP, to control cell cycle progression provides proof-of-principle for treatment via selective interference with AR action by exploiting AR dependence on coregulators.


Assuntos
Regulação da Expressão Gênica , Neoplasias da Próstata/patologia , Receptores Androgênicos/metabolismo , Linhagem Celular Tumoral , Humanos , Masculino , Fatores de Transcrição/metabolismo , Transcrição Genética , Proteína Supressora de Tumor p53/metabolismo
5.
J Infect Dis ; 215(3): 387-395, 2017 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-28003350

RESUMO

Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, affects 7 million people in Latin American areas of endemicity. About 30% of infected patients will develop chronic Chagas cardiomyopathy (CCC), an inflammatory cardiomyopathy characterized by hypertrophy, fibrosis, and myocarditis. Further studies are necessary to understand the molecular mechanisms of disease progression. Transcriptome analysis has been increasingly used to identify molecular changes associated with disease outcomes. We thus assessed the whole-blood transcriptome of patients with Chagas disease. Microarray analysis was performed on blood samples from 150 subjects, of whom 30 were uninfected control patients and 120 had Chagas disease (1 group had asymptomatic disease, and 2 groups had CCC with either a preserved or reduced left ventricular ejection fraction [LVEF]). Each Chagas disease group displayed distinct gene expression and functional pathway profiles. The most different expression patterns were between CCC groups with a preserved or reduced LVEF. A more stringent analysis indicated that 27 differentially expressed genes, particularly those related to natural killer (NK)/CD8+ T-cell cytotoxicity, separated the 2 groups. NK/CD8+ T-cell cytotoxicity could play a role in determining Chagas disease progression. Understanding genes associated with disease may lead to improved insight into CCC pathogenesis and the identification of prognostic factors for CCC progression.


Assuntos
Cardiomiopatia Chagásica/genética , Disfunção Ventricular/genética , Linfócitos T CD8-Positivos/imunologia , Cardiomiopatia Chagásica/sangue , Cardiomiopatia Chagásica/fisiopatologia , Citotoxicidade Imunológica/genética , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Células Matadoras Naturais/imunologia , Análise em Microsséries , Pessoa de Meia-Idade , Miocárdio/patologia , Reação em Cadeia da Polimerase em Tempo Real , Disfunção Ventricular/sangue , Disfunção Ventricular/parasitologia
6.
Clin Cancer Res ; 23(6): 1552-1563, 2017 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-27649553

RESUMO

Purpose: The high fatality-to-case ratio of ovarian cancer is directly related to platinum resistance. Exportin-1 (XPO1) is a nuclear exporter that mediates nuclear export of multiple tumor suppressors. We investigated possible clinicopathologic correlations of XPO1 expression levels and evaluated the efficacy of XPO1 inhibition as a therapeutic strategy in platinum-sensitive and -resistant ovarian cancer.Experimental Design: XPO1 expression levels were analyzed to define clinicopathologic correlates using both TCGA/GEO datasets and tissue microarrays (TMA). The effect of XPO1 inhibition, using the small-molecule inhibitors KPT-185 and KPT-330 (selinexor) alone or in combination with a platinum agent on cell viability, apoptosis, and the transcriptome was tested in immortalized and patient-derived ovarian cancer cell lines (PDCL) and platinum-resistant mice (PDX). Seven patients with late-stage, recurrent, and heavily pretreated ovarian cancer were treated with an oral XPO1 inhibitor.Results: XPO1 RNA overexpression and protein nuclear localization were correlated with decreased survival and platinum resistance in ovarian cancer. Targeted XPO1 inhibition decreased cell viability and synergistically restored platinum sensitivity in both immortalized ovarian cancer cells and PDCL. The XPO1 inhibitor-mediated apoptosis occurred through both p53-dependent and p53-independent signaling pathways. Selinexor treatment, alone and in combination with platinum, markedly decreased tumor growth and prolonged survival in platinum-resistant PDX and mice. In selinexor-treated patients, tumor growth was halted in 3 of 5 patients, including one with a partial response, and was safely tolerated by all.Conclusions: Taken together, these results provide evidence that XPO1 inhibition represents a new therapeutic strategy for overcoming platinum resistance in women with ovarian cancer. Clin Cancer Res; 23(6); 1552-63. ©2016 AACR.


Assuntos
Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Carioferinas/genética , Neoplasias Ovarianas/tratamento farmacológico , Receptores Citoplasmáticos e Nucleares/genética , Acrilatos/administração & dosagem , Transporte Ativo do Núcleo Celular/genética , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Feminino , Humanos , Hidrazinas/administração & dosagem , Carioferinas/antagonistas & inibidores , Camundongos , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Platina/administração & dosagem , Platina/efeitos adversos , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Triazóis/administração & dosagem , Ensaios Antitumorais Modelo de Xenoenxerto
7.
PLoS One ; 11(2): e0148746, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26859141

RESUMO

BACKGROUND: MicroRNAs (miRNAs) regulate gene expression by interfering translation or stability of target transcripts. This interplay between miRNA and their mRNA has been proposed as an important process in cancer development and progression. We have investigated molecular networks impacted by predicted mRNA targets of differentially expressed miRNAs in patients with clear cell renal cell carcinoma (ccRCC) diagnosed with or without metastasis. MATERIAL AND METHODS: miRNA and mRNA microarray expression profiles derived from primary ccRCC from patients with (16 samples) or without diagnosed metastasis (22 samples) were used to identify anti-correlated miRNA-mRNA interaction in ccRCC. For this purpose, Ingenuity pathway analysis microRNA Target Filter, which enables prioritization of experimentally validated and predicted mRNA targets was used. By applying an expression pairing tool, the analysis was focused on targets exhibiting altered expression in our analysis, finding miRNAs and their target genes with opposite or same expression. The resulting identified interactions were revalidated by RT-qPCR in another cohort of ccRCC patients. A selection of the predicted miRNA-mRNA interactions was tested by functional analyses using miRNA knockdown and overexpression experiments in renal cancer cell lines. RESULTS: Among the significantly differentially expressed miRNAs, we have identified three miRNAs (miR-146a-5p, miR-128a-3p, and miR-17-5p) that were upregulated in primary tumors from patients without metastasis and downregulated in primary tumors from patients with metastasis. We have further identified mRNA targets, which expression were inversely correlated to these 3 miRNAs, and have been previously experimentally demonstrated in cancer setting in humans. Specifically, we showed that CXCL8/IL8, UHRF1, MCM10, and CDKN3 were downregulated and targeted by miR-146a-5p. The interaction between miR-146a-5p and their targets CXCL8 and UHRF1 was validated in cell culture experiments. CONCLUSIONS: We identified novel target genes of dysregulated miRNAs, which are involved in the transition from primary RCC without metastases into tumors generating distant metastasis.


Assuntos
Carcinoma de Células Renais/genética , Neoplasias Renais/genética , MicroRNAs/fisiologia , Metástase Neoplásica/genética , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Renais/patologia , MicroRNAs/metabolismo , RNA Mensageiro/metabolismo
8.
mBio ; 7(1): e00100-16, 2016 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-26873097

RESUMO

UNLABELLED: Lyme disease is a tick-borne illness caused by the bacterium Borrelia burgdorferi, and approximately 10 to 20% of patients report persistent symptoms lasting months to years despite appropriate treatment with antibiotics. To gain insights into the molecular basis of acute Lyme disease and the ensuing development of post-treatment symptoms, we conducted a longitudinal transcriptome study of 29 Lyme disease patients (and 13 matched controls) enrolled at the time of diagnosis and followed for up to 6 months. The differential gene expression signature of Lyme disease following the acute phase of infection persisted for at least 3 weeks and had fewer than 44% differentially expressed genes (DEGs) in common with other infectious or noninfectious syndromes. Early Lyme disease prior to antibiotic therapy was characterized by marked upregulation of Toll-like receptor signaling but lack of activation of the inflammatory T-cell apoptotic and B-cell developmental pathways seen in other acute infectious syndromes. Six months after completion of therapy, Lyme disease patients were found to have 31 to 60% of their pathways in common with three different immune-mediated chronic diseases. No differential gene expression signature was observed between Lyme disease patients with resolved illness to those with persistent symptoms at 6 months post-treatment. The identification of a sustained differential gene expression signature in Lyme disease suggests that a panel of selected human host-based biomarkers may address the need for sensitive clinical diagnostics during the "window period" of infection prior to the appearance of a detectable antibody response and may also inform the development of new therapeutic targets. IMPORTANCE: Lyme disease is the most common tick-borne infection in the United States, and some patients report lingering symptoms lasting months to years despite antibiotic treatment. To better understand the role of the human host response in acute Lyme disease and the development of post-treatment symptoms, we conducted the first longitudinal gene expression (transcriptome) study of patients enrolled at the time of diagnosis and followed up for up to 6 months after treatment. Importantly, we found that the gene expression signature of early Lyme disease is distinct from that of other acute infectious diseases and persists for at least 3 weeks following infection. This study also uncovered multiple previously undescribed pathways and genes that may be useful in the future as human host biomarkers for diagnosis and that constitute potential targets for the development of new therapies.


Assuntos
Doença de Lyme/genética , Transcriptoma , Adulto , Animais , Biomarcadores/sangue , Borrelia burgdorferi/fisiologia , Feminino , Perfilação da Expressão Gênica/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Doença de Lyme/diagnóstico , Doença de Lyme/tratamento farmacológico , Doença de Lyme/microbiologia , Masculino , Redes e Vias Metabólicas/genética , Pessoa de Meia-Idade , Estados Unidos
9.
Gynecol Oncol ; 134(3): 591-8, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24995581

RESUMO

OBJECTIVE: High-grade serous ovarian cancer (HGSOC) that is resistant to platinum-based chemotherapy has a particularly poor prognosis. Response to platinum has both prognostic survival value and dictates secondary treatment strategies. Using transcriptome analysis, we sought to identify differentially expressed genes/pathways based on a tumor's platinum response for discovering novel predictive biomarkers. METHODS: Seven primary HGSOC tumor samples, representing two extremes of platinum sensitivity/timing of disease recurrence, were analyzed by RNA-Seq, Ingenuity Pathways Analysis (IPA) and Upstream Regulator Analysis (URA), and used to explore differentially expressed genes and prevalent molecular and cellular processes. Progression-free and overall survival (PFS, OS) was estimated using the Kaplan-Meier method in two different sample sets including GEO and TCGA data sets. RESULTS: IPA and URA highlighted an IRF1-driven transcriptional program (P=0.0017; z-score of 3.091) in the platinum sensitive improved PFS group. QRT-PCR analysis of 31 HGSOC samples demonstrated a significant difference in PFS between low and high IRF1 expression groups (P=0.048) and between groups that were platinum sensitive versus not (P=0.016). In a larger validation data set, increased levels of IRF1 were associated with both increased PFS (P=0.043) and OS (P=0.019) and the effect on OS was independent of debulking status (optimal debulking, P=0.025; suboptimal, P=0.041). CONCLUSION: Transcriptome analysis identifies IRF1, a transcription factor that functions both in immune regulation and as a tumor suppressor, as being associated with platinum sensitivity and an independent predictor of both PFS and OS in HGSOC.


Assuntos
Antineoplásicos/uso terapêutico , Cisplatino/uso terapêutico , Cistadenocarcinoma Seroso/tratamento farmacológico , Cistadenocarcinoma Seroso/genética , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Cistadenocarcinoma Seroso/mortalidade , Intervalo Livre de Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Fator Regulador 1 de Interferon/genética , Fator Regulador 1 de Interferon/fisiologia , Pessoa de Meia-Idade , Neoplasias Ovarianas/mortalidade , Prognóstico , Taxa de Sobrevida
10.
Pac Symp Biocomput ; : 17-28, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23424108

RESUMO

Knowledge of immune system and host-pathogen pathways can inform development of targeted therapies and molecular diagnostics based on a mechanistic understanding of disease pathogenesis and the host response. We investigated the feasibility of rapid target discovery for novel broad-spectrum molecular therapeutics through comprehensive systems biology modeling and analysis of pathogen and host-response pathways and mechanisms. We developed a system to identify and prioritize candidate host targets based on strength of mechanistic evidence characterizing the role of the target in pathogenesis and tractability desiderata that include optimal delivery of new indications through potential repurposing of existing compounds or therapeutics. Empirical validation of predicted targets in cellular and mouse model systems documented an effective target prediction rate of 34%, suggesting that such computational discovery approaches should be part of target discovery efforts in operational clinical or biodefense research initiatives. We describe our target discovery methodology, technical implementation, and experimental results. Our work demonstrates the potential for in silico pathway models to enable rapid, systematic identification and prioritization of novel targets against existing or emerging biological threats, thus accelerating drug discovery and medical countermeasures research.


Assuntos
Doenças Transmissíveis Emergentes/tratamento farmacológico , Descoberta de Drogas/métodos , Algoritmos , Animais , Biologia Computacional , Simulação por Computador , Descoberta de Drogas/estatística & dados numéricos , Interações Hospedeiro-Patógeno , Humanos , Bases de Conhecimento , Camundongos , Modelos Biológicos , Projetos Piloto , Biologia de Sistemas
11.
Vaccine ; 28(41): 6704-13, 2010 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-20691653

RESUMO

The use of live recombinant attenuated Salmonella vaccines (RASV) is a promising approach for controlling infections by multiple pathogens. The highly conserved extracellular domain of the influenza M2 protein (M2e) has been shown to provide broad spectrum protection against multiple influenza subtypes sharing similar M2e sequences. An M2e epitope common to a number of avian influenza subtypes was inserted into the core antigen of woodchuck hepatitis virus and expressed in two different recombinant attenuated Salmonella Typhimurium strains. One strain was attenuated via deletion of the cya and crp genes. The second strain was engineered to exhibit a programmed delayed lysis phenotype. Both strains were able to produce both monomeric fusion proteins and fully assembled core particles. Mice orally immunized with the strain exhibiting delayed lysis induced significantly greater antibody titers than the Δcya Δcrp strain and provided moderate protection against weight loss to a low level challenge with the influenza strain A/WSN/33 modified to express the M2e sequence common to avian viruses. Further studies indicated that the Salmonella expressed core antigen induced comparable antibody levels to the purified core antigen injected with an alum adjuvant and that both are able to reduce viral replication in the lungs. To our knowledge this is the first report demonstrating Salmonella-mediated delivery of influenza virus M2e protein in a mammalian host to induce a protective immune response against viral challenge.


Assuntos
Vírus da Influenza A Subtipo H7N7/imunologia , Vacinas contra Influenza/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Vacinas de Partículas Semelhantes a Vírus/imunologia , Proteínas da Matriz Viral/imunologia , Animais , Anticorpos Antivirais/sangue , Formação de Anticorpos , Antígenos Virais/imunologia , Feminino , Vírus da Hepatite B da Marmota/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/imunologia , Proteínas Recombinantes de Fusão/imunologia , Salmonella typhimurium/imunologia
12.
J Virol ; 83(3): 1379-92, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19004949

RESUMO

Hepatitis B virus (HBV) expresses two structural forms of the nucleoprotein, the intracellular nucleocapsid (hepatitis core antigen [HBcAg]) and the secreted nonparticulate form (hepatitis e antigen [HBeAg]). The aim of this study was to evaluate the ability of HBcAg- and HBeAg-specific genetic immunogens to induce HBc/HBeAg-specific CD4(+)/CD8(+) T-cell immune responses and the potential to induce liver injury in HBV-transgenic (Tg) mice. Both the HBcAg- and HBeAg-specific plasmids primed comparable immune responses. Both CD4(+) and CD8(+) T cells were important for priming/effector functions of HBc/HBeAg-specific cytotoxic T-lymphocyte (CTL) responses. However, a unique two-step immunization protocol was necessary to elicit maximal CTL priming. Genetic vaccination did not prime CTLs in HBe- or HBc/HBeAg-dbl-Tg mice but elicited a weak CTL response in HBcAg-Tg mice. When HBc/HBeAg-specific CTLs were adoptively transferred into HBc-, HBe-, and HBc/HBeAg-dbl-Tg mice, the durations of the liver injury and inflammation were significantly greater in HBeAg-Tg recipient mice than in HBcAg-Tg mice. Importantly, liver injury in HBc/HBeAg-dbl-Tg mice was similar to the injury observed in HBeAg-Tg mice. Loss of HBeAg synthesis commonly occurs during chronic HBV infection; however, the mechanism of selection of HBeAg-negative variants is unknown. The finding that hepatocytes expressing wild-type HBV (containing both HBcAg and HBeAg) are more susceptible to CTL-mediated clearance than hepatocytes expressing only HBcAg suggest that the HBeAg-negative variant may have a selective advantage over wild-type HBV within the livers of patients with chronic infection during an immune response and may represent a CTL escape mutant.


Assuntos
Antígenos de Hepatite/genética , Hepatite B Crônica/imunologia , Vírus da Hepatite E/imunologia , Mutação , Transferência Adotiva , Animais , Linhagem Celular Tumoral , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Linfócitos T Citotóxicos/imunologia
13.
Vaccine ; 25(9): 1593-606, 2007 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-17178179

RESUMO

The hepatitis B core antigen (HBcAg) has been proposed as a useful particulate carrier platform for poorly immunogenic peptidic and carbohydrate B cell epitopes. However, biochemical and immunologic impediments have plagued this technology. Specifically, the "assembly" problem characterized by the low yield of unstable hybrid particles resulting from the insertion of foreign sequences and the "pre-existing immunity" problem due to the fact that the HBcAg is derived from a human pathogen have limited the development of this carrier technology. As a means of addressing the "pre-existing immunity" problem we have used the core proteins from the rodent hepdnaviruses. A number of advantages to the use of the rodent hepadnaviral core proteins as opposed to the HBcAg for vaccine design were defined including: equal or superior immunogenicity at the T and B cell levels; the use of the rodent core proteins does not compromise the anti-HBc diagnostic assay; the efficacy of the rodent core proteins as vaccine carriers will not be limited by pre-existing anti-HBc antibodies that are present in previously and currently HBV-infected persons; and the HBcAg-specific tolerance present in HBV chronic carriers can be circumvented by the use of the rodent core proteins.


Assuntos
Antígenos do Núcleo do Vírus da Hepatite B/imunologia , Vírus da Hepatite B da Marmota/metabolismo , Vacinas Antimaláricas/imunologia , Malária Falciparum/prevenção & controle , Proteínas de Protozoários/imunologia , Animais , Anticorpos Antiprotozoários/sangue , Epitopos de Linfócito T , Vetores Genéticos , Antígenos do Núcleo do Vírus da Hepatite B/genética , Antígenos do Núcleo do Vírus da Hepatite B/metabolismo , Antígenos E da Hepatite B/genética , Humanos , Malária Falciparum/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Plasmodium falciparum/imunologia , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Linfócitos T/imunologia , Vacinas Sintéticas/imunologia
14.
J Virol ; 79(21): 13641-55, 2005 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-16227284

RESUMO

The hepatitis B virus core protein (HBcAg) is a uniquely immunogenic particulate antigen and as such has been used as a vaccine carrier platform. The use of other hepadnavirus core proteins as vaccine carriers has not been explored. To determine whether the rodent hepadnavirus core proteins derived from the woodchuck (WHcAg), ground squirrel (GScAg), and arctic squirrel (AScAg) viruses possess immunogen characteristics similar to those of HBcAg, comparative antigenicity and immunogenicity studies were performed. The results indicate that (i) the rodent core proteins are equal in immunogenicity to or more immunogenic than HBcAg at the B-cell and T-cell levels; (ii) major histocompatibility complex (MHC) genes influence the immune response to the rodent core proteins (however, nonresponder haplotypes were not identified); (iii) WHcAg can behave as a T-cell-independent antigen in athymic mice; (iv) the rodent core proteins are not significantly cross-reactive with the HBcAg at the antibody level (however, the nonparticulate "eAgs" do appear to be cross-reactive); (v) the rodent core proteins are only partially cross-reactive with HBcAg at the CD4+ T-cell level, depending on MHC haplotype; and (vi) the rodent core proteins are competent to function as vaccine carrier platforms for heterologous, B-cell epitopes. These results have implications for the selection of an optimal hepadnavirus core protein for vaccine design, especially in view of the "preexisting" immunity problem that is inherent in the use of HBcAg for human vaccine development.


Assuntos
Infecções por Hepadnaviridae/imunologia , Hepadnaviridae/imunologia , Anticorpos Anti-Hepatite/sangue , Proteínas do Core Viral/imunologia , Vacinas contra Hepatite Viral/imunologia , Sequência de Aminoácidos , Animais , Linfócitos T CD4-Positivos/imunologia , Reações Cruzadas , Epitopos de Linfócito B/imunologia , Infecções por Hepadnaviridae/sangue , Anticorpos Anti-Hepatite/imunologia , Injeções Intraperitoneais , Complexo Principal de Histocompatibilidade/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Nus , Dados de Sequência Molecular , Alinhamento de Sequência , Especificidade da Espécie , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/imunologia , Proteínas do Core Viral/genética , Vacinas contra Hepatite Viral/administração & dosagem
15.
J Virol ; 79(21): 13656-66, 2005 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-16227285

RESUMO

The particulate hepatitis core protein (HBcAg) represents an efficient carrier platform with many of the characteristics uniquely required for the delivery of weak immunogens to the immune system. Although the HBcAg is highly immunogenic, the existing HBcAg-based platform technology has a number of theoretical and practical limitations, most notably the "preexisting immunity" and "assembly" problems. To address the assembly problem, we have developed the core protein from the woodchuck hepadnavirus (WHcAg) as a new particulate carrier platform system. WHcAg appears to tolerate insertions of foreign epitopes at a greater number of positions than HBcAg. For example, both within the external loop region and outside the loop region a total of 17 insertion sites were identified on WHcAg. Importantly, the identification of an expanded number of insertion sites was dependent on additional modifications to the C terminus that appear to stabilize the various internal insertions. Indeed, 21 separate C-terminal modifications have been generated that can be used in combination with the 17 insertion sites to ensure efficient hybrid WHcAg particle assembly. This combinatorial technology is also dependent on the sequence of the heterologous insert. Therefore, the three variables of insert position, C terminus, and epitope sequence are relevant in the design of hybrid WHcAg particles for vaccine purposes.


Assuntos
Desenho de Drogas , Vírus da Hepatite B da Marmota/química , Proteínas do Core Viral/genética , Sequência de Aminoácidos , Animais , Especificidade de Anticorpos , Epitopos/genética , Epitopos/imunologia , Feminino , Imunização , Camundongos , Dados de Sequência Molecular , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Proteínas do Core Viral/imunologia
16.
J Virol ; 79(5): 3016-27, 2005 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15709022

RESUMO

The function of the hepatitis B virus (HBV) precore or HBeAg is largely unknown because it is not required for viral assembly, infection, or replication. However, the HBeAg does appear to play a role in viral persistence. It has been suggested that the HBeAg may promote HBV chronicity by functioning as an immunoregulatory protein. As a model of chronic HBeAg exposure and to examine the tolerogenic potential of the HBV precore and core (HBcAg) proteins, HBc/HBeAg-transgenic (Tg) mice crossed with T cell receptor (TCR)-Tg mice expressing receptors for the HBc/HBeAgs (i.e., TCR-antigen double-Tg pairs) were produced. This study revealed three phenotypes of HBe/HBcAg-specific T-cell tolerance: (i) profound T-cell tolerance most likely mediated by clonal deletion, (ii) T-cell clonal ignorance, and (iii) nondeletional T-cell tolerance mediated by clonal anergy and dependent on the structure, location, and concentration of the tolerogen. The secreted HBeAg is significantly more efficient than the intracellular HBcAg at eliciting T-cell tolerance. The split T-cell tolerance between the HBeAg and the HBcAg and the clonal heterogeneity of HBc/HBeAg-specific T-cell tolerance may have significant implications for natural HBV infection and especially for precore-negative chronic hepatitis.


Assuntos
Antígenos do Núcleo do Vírus da Hepatite B/imunologia , Antígenos E da Hepatite B/imunologia , Vírus da Hepatite B/imunologia , Tolerância Imunológica , Animais , Animais Recém-Nascidos , Feminino , Anticorpos Anti-Hepatite B/biossíntese , Antígenos do Núcleo do Vírus da Hepatite B/genética , Antígenos E da Hepatite B/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fenótipo , Gravidez , Receptores de Antígenos de Linfócitos T/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Linfócitos T/imunologia
17.
Proc Natl Acad Sci U S A ; 101(41): 14913-8, 2004 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-15469922

RESUMO

A unique characteristic of the hepatitis B virus is the production of a secreted form (precore or HBeAg) of the structural nucleocapsid (core or HBcAg). By using T cell receptor (TCR) transgenic (Tg) and TCR x HBc/HBeAg double- and triple-Tg pairs, we demonstrate that HBeAg elicits T cell tolerance, whereas HBcAg is nontolerogenic in this system. In fact, TCR x HBc double-Tg mice spontaneously seroconvert to IgG anti-HBc positivity at an early age. However, the presence of HBeAg in the serum of TCR x HBc x HBe triple-Tg mice prevents anti-HBc seroconversion. HBeAg mediates its immunoregulatory effect by eliciting tolerance in HBc/HBeAg-specific T cells. The results suggest that hepadnaviruses have retained a secretory form of the nucleoprotein because it functions as a T cell tolerogen and regulates the immune response to the intracellular nucleocapsid. This HBeAg-mediated immune regulation may predispose to chronicity during perinatal infections and prevent severe liver injury during adult infections.


Assuntos
Antígenos da Hepatite B/imunologia , Antígenos do Núcleo do Vírus da Hepatite B/imunologia , Vírus da Hepatite B/imunologia , Proteínas do Core Viral/imunologia , Transferência Adotiva , Animais , Anergia Clonal , Citocinas/análise , Antígenos E da Hepatite B/sangue , Fígado/virologia , Camundongos , Camundongos Transgênicos , Precursores de Proteínas/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Proteínas Recombinantes/imunologia , Baço/imunologia , Baço/transplante , Linfócitos T/imunologia
18.
Virology ; 304(1): 97-104, 2002 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-12490407

RESUMO

Proper function of the Rev regulatory system is essential for the replication of lentiviruses, including feline immunodeficiency virus (FIV) and human immunodeficiency virus type 1 (HIV-1). Specifically, Rev affects the overall stability of viral mRNAs that encode necessary structural and enzymatic proteins. In turn, the eukaryotic initiation factor (eIF-5A) is indispensable for Rev function and is the only known protein whose biologically active form requires the unique amino acid, hypusine. Because 1,8-diaminooctane blocks the formation of hypusine by disrupting the cellular enzyme, deoxyhypusine synthase, thereby preventing activation of eIF-5A, we investigated the effects of 1,8-diaminooctane on posttranscriptional regulation. These are the first results to demonstrate that diaminooctane significantly reduced viral replication in a dose-dependent manner, even under conditions of contact inhibition, diminishing the compound's effect on cell proliferation. Similarly, the addition of increased concentrations of diaminooctane caused a reduction in the expression of a Rev-dependent CAT system without affecting a Rev-independent CAT system. At the RNA level, exposure of chronically infected CrFK cells to increasing concentrations of diaminooctane substantially decreased the levels of unspliced and singly spliced viral mRNAs and increased the relative amounts of multiply spliced transcripts in the cytoplasm. The findings of this study are the first demonstration that FIV, similar to HIV-1, requires eIF-5A for efficient Rev function and that small molecule intervention can indirectly target this lentivirus regulatory system.


Assuntos
Diaminas/farmacologia , Produtos do Gene rev/metabolismo , Vírus da Imunodeficiência Felina/efeitos dos fármacos , Animais , Gatos , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Produtos do Gene rev/efeitos dos fármacos , Produtos do Gene rev/fisiologia , Vírus da Imunodeficiência Felina/metabolismo , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Replicação Viral/efeitos dos fármacos
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