RESUMO
The generation of CD8(+) T-cell memory is a major aim of vaccination. While distinct subsets of CD8(+) T-cells are generated following immunization that differ in their ability to confer long-term immunity against infection, the transcriptional profiles of these subsets within endogenous vaccine-induced CD8(+) T cell responses have not been resolved. Here, we measure global transcriptional profiles of endogenous effector (TEFF), effector memory (TEM) and central memory (TCM) CD8(+) T-cells arising from immunization with three distinct prime-boost vaccine regimens. While a proportion of transcripts were uniquely regulated within distinct CD8(+) T cell populations, we observed progressive up- or down-regulation in the expression of a majority of differentially expressed transcripts when subsets were compared in the order TN>TCM>TEM>TEFF. Strikingly, when we compared global differences in gene expression between TN, TCM, TEM and TEFF cells with known transcriptional changes that result when CD8(+) T cells repetitively encounter antigen, our analysis overwhelmingly favored a model whereby cumulative antigen stimulation drives differentiation specifically from TN>TCM>TEM>TEFF and this was common to all vaccines tested. These findings provide insight into the molecular basis of immunological memory and identify potential biomarkers for characterization of vaccine-induced responses and prediction of vaccine efficacy.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Animais , Linfócitos T CD8-Positivos/citologia , Diferenciação Celular/fisiologia , Células Cultivadas , Feminino , Citometria de Fluxo , Memória Imunológica/imunologia , Memória Imunológica/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Análise de Sequência com Séries de OligonucleotídeosRESUMO
CD8 T cells play a key role in mediating protective immunity against selected pathogens after vaccination. Understanding the mechanism of this protection is dependent upon definition of the heterogeneity and complexity of cellular immune responses generated by different vaccines. Here, we identify previously unrecognized subsets of CD8 T cells based upon analysis of gene-expression patterns within single cells and show that they are differentially induced by different vaccines. Three prime-boost vector combinations encoding HIV Env stimulated antigen-specific CD8 T-cell populations of similar magnitude, phenotype, and functionality. Remarkably, however, analysis of single-cell gene-expression profiles enabled discrimination of a majority of central memory (CM) and effector memory (EM) CD8 T cells elicited by the three vaccines. Subsets of T cells could be defined based on their expression of Eomes, Cxcr3, and Ccr7, or Klrk1, Klrg1, and Ccr5 in CM and EM cells, respectively. Of CM cells elicited by DNA prime-recombinant adenoviral (rAd) boost vectors, 67% were Eomes(-) Ccr7(+) Cxcr3(-), in contrast to only 7% and 2% stimulated by rAd5-rAd5 or rAd-LCMV, respectively. Of EM cells elicited by DNA-rAd, 74% were Klrk1(-) Klrg1(-)Ccr5(-) compared with only 26% and 20% for rAd5-rAd5 or rAd5-LCMV. Definition by single-cell gene profiling of specific CM and EM CD8 T-cell subsets that are differentially induced by different gene-based vaccines will facilitate the design and evaluation of vaccines, as well as enable our understanding of mechanisms of protective immunity.
Assuntos
Vacinas contra a AIDS/farmacologia , Linfócitos T CD8-Positivos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Subpopulações de Linfócitos T/metabolismo , Vacinas contra a AIDS/imunologia , Animais , Citometria de Fluxo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/imunologia , Lectinas Tipo C , Camundongos , Análise em Microsséries , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Receptores CCR7/metabolismo , Receptores CXCR3/metabolismo , Receptores Imunológicos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas com Domínio T/metabolismoRESUMO
The functional human immune system, including T, B, and natural killer lymphocytes, is reconstituted in NOD/Shi-scid/IL-2Rgamma(null) (NOG) mice that receive hematopoietic stem cell transplants. Here, we show that these humanized mice can recapitulate key aspects of Epstein-Barr virus (EBV) infection in humans. Inoculation with approximately 1 x 10(3) TD(50) (50% transforming dose) of EBV caused B cell lymphoproliferative disorder, with histopathological findings and latent EBV gene expression remarkably similar to that in immunocompromised patients. Inoculation with a low dose of virus (Assuntos
Infecções por Vírus Epstein-Barr/imunologia
, Infecções por Vírus Epstein-Barr/patologia
, Transtornos Linfoproliferativos/virologia
, Animais
, Anticorpos Antivirais/biossíntese
, Modelos Animais de Doenças
, Humanos
, Imunidade Celular
, Transtornos Linfoproliferativos/imunologia
, Camundongos
, Camundongos Transgênicos
, Linfócitos T/citologia
, Linfócitos T/imunologia
RESUMO
Epstein-Barr virus (EBV) causes EBV-associated lymphoproliferative diseases in patients with profound immune suppression. Most of these diseases are life-threatening and the prognosis of AIDS-associated lymphomas is extremely unfavorable. Polyclonal expansion of virus infected B-cell predisposes them to transformation. We investigated the possibility of nuclear factor kappa B (NF-kappaB) inhibition by dehydroxymethylepoxyquinomicin (DHMEQ) for the treatment and prevention of EBV-associated lymphoproliferative diseases. We examined the effect of DHMEQ on apoptosis induction in four EBV-transformed lymphoblastoid cell lines as well as peripheral blood mononuclear cells infected with EBV under immunosuppressed condition. DHMEQ inhibits NF-kappaB activation in EBV-transformed lymphoblastoid cell lines and induces apoptosis by activation of mitochondrial and membranous pathways. Using an in vivo NOD/SCIDgammac mouse model, we showed that DHMEQ has a potent inhibitory effect on the growth of lymphoblastoid cells. In addition, DHMEQ selectively purges EBV-infected cells expressing latent membrane protein (LMP) 1 from peripheral blood mononuclear cells and inhibits the outgrowth of lymphoblastoid cells. These results suggest that NF-kappaB is a molecular target for the treatment and prevention of EBV-associated lymphoproliferative diseases. As a potent NF-kappaB inhibitor, DHMEQ is a potential compound for applying this strategy in clinical medicine.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linfócitos B/virologia , Benzamidas/farmacologia , Cicloexanonas/farmacologia , Herpesvirus Humano 4/fisiologia , Fatores Imunológicos/farmacologia , NF-kappa B/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Sobrevivência Celular , Humanos , Transtornos Linfoproliferativos/tratamento farmacológico , Camundongos , Camundongos SCIDRESUMO
In this study, we found that the electric potential derived from the redox reaction of ultraviolet (UV)-illuminated CD4-conjugated titanium dioxide (TiO2) inactivated a wide range of high-titered primary HIV-1 isolates, regardless of virus co-receptor usage or genetic clade. In vitro incubation of HIV-1 isolates with CD4-conjugated TiO2 (CD4-TiO2) followed by UV illumination led to inhibition of viral infectivity in both H9 cells and peripheral blood mononuclear cells as well as to the complete inactivation of plasma virions from HIV-1-infected individuals. Treatment with a newly established extra-corporeal circulation system with the photocatalyst in rhesus macaques completely inactivated plasma virus in the system and effectively reduced the infectious plasma viral load. Furthermore, plasma viremia and infectious viral loads were controlled following a second therapeutic photocatalyst treatment during primary SIV(mac239) infection of macaques. Our findings suggest that this therapeutic immunophysical strategy may help control human immunodeficiency viral infection in vivo.
Assuntos
Antígenos CD4/uso terapêutico , Infecções por HIV/tratamento farmacológico , Infecções por HIV/terapia , HIV-1/efeitos dos fármacos , Vírus da Imunodeficiência Símia/efeitos dos fármacos , Titânio/uso terapêutico , Raios Ultravioleta , Animais , Antígenos CD4/química , Linhagem Celular , DNA Viral/sangue , Circulação Extracorpórea , Infecções por HIV/virologia , HIV-1/genética , HIV-1/isolamento & purificação , HIV-1/fisiologia , Células HeLa , Humanos , Leucócitos Mononucleares/virologia , Masculino , Fotoquimioterapia , Reação em Cadeia da Polimerase , Síndrome de Imunodeficiência Adquirida dos Símios/tratamento farmacológico , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/genética , Vírus da Imunodeficiência Símia/isolamento & purificação , Vírus da Imunodeficiência Símia/fisiologia , Linfócitos T/virologia , Titânio/administração & dosagem , Titânio/química , Titânio/farmacologia , Resultado do TratamentoRESUMO
Human immunodeficiency virus type 1 (HIV-1) utilizes the macromolecular machinery of the infected host cell to produce progeny virus. The discovery of cellular factors that participate in HIV-1 replication pathways has provided further insight into the molecular basis of virus-host cell interactions. Here, we report that the suppressor of cytokine signaling 1 (SOCS1) is an inducible host factor during HIV-1 infection and regulates the late stages of the HIV-1 replication pathway. SOCS1 can directly bind to the matrix and nucleocapsid regions of the HIV-1 p55 Gag polyprotein and enhance its stability and trafficking, resulting in the efficient production of HIV-1 particles via an IFN signaling-independent mechanism. The depletion of SOCS1 by siRNA reduces both the targeted trafficking and assembly of HIV-1 Gag, resulting in its accumulation as perinuclear solid aggregates that are eventually subjected to lysosomal degradation. These results together indicate that SOCS1 is a crucial host factor that regulates the intracellular dynamism of HIV-1 Gag and could therefore be a potential new therapeutic target for AIDS and its related disorders.
Assuntos
Produtos do Gene gag/metabolismo , Infecções por HIV/metabolismo , HIV-1/metabolismo , Proteínas Supressoras da Sinalização de Citocina/fisiologia , Replicação Viral , Síndrome da Imunodeficiência Adquirida/terapia , Linhagem Celular , Membrana Celular/metabolismo , Humanos , Células Jurkat , Microscopia Eletrônica , Microscopia Eletrônica de Transmissão , Muramidase/química , Plasmídeos/metabolismo , Processamento Pós-Transcricional do RNA , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Proteína 1 Supressora da Sinalização de Citocina , Proteínas Supressoras da Sinalização de Citocina/metabolismoRESUMO
In a previous study, we demonstrated that humanized NOD/SCID/IL2Rgamma(null) (hNOG) mice constructed with human hematopoietic stem cells (HSCs) allow efficient human immunodeficiency virus type 1 (HIV-1) infection. However, HIV-1 infection could be monitored for only 43 days in the animals due to their short life spans. By transplanting HSCs without any myeloablation methods, the mice successfully survived longer than 300 days with stable engraftment of human cells. The mice showed high viremia state for more than the 3 months examined, with systemic HIV-1 infection and gradual decrease of CD4+ T cells analogous to that in humans. These capacities of the hNOG mice are very attractive for modeling mechanisms of AIDS progression and therapeutic strategy.
Assuntos
Modelos Animais de Doenças , Infecções por HIV/virologia , HIV-1/crescimento & desenvolvimento , Transplante de Células-Tronco Hematopoéticas , Animais , Contagem de Linfócito CD4 , Feminino , Camundongos , Camundongos Knockout , Camundongos SCID , ViremiaRESUMO
Replication-defective adenovirus type 5 (Ad5) vector-based vaccines are widely known to induce strong immunity against immunodeficiency viruses. To exploit this immunogenicity while overcoming the potential problem of preexisting immunity against human adenoviruses type 5, we developed a recombinant chimeric adenovirus type 5 with type 35 fiber vector (rAd5/35). We initially produced a simian immunodeficiency virus (SIV) gag DNA plasmid (rDNA-Gag), a human immunodeficiency virus type 1 (HIV-1) 89.6 env DNA plasmid (rDNA-Env) and a recombinant Ad5/35 vector encoding the SIV gag and HIV env gene (rAd5/35-Gag and rAd5/35-Env). Prime-boost vaccination with rDNA-Gag and -Env followed by high doses of rAd5/35-Gag and -Env elicited higher levels of cellular immune responses than did rDNAs or rAd5/35s alone. When challenged with a pathogenic simian human immunodeficiency virus (SHIV), animals receiving a prime-boost regimen or rAd5/35s alone maintained a higher number of CD4(+) T cells and remarkably suppressed plasma viral RNA loads. These findings suggest the clinical promise of an rAd5/35 vector-based vaccine.
Assuntos
Vacinas contra a AIDS/imunologia , Síndrome da Imunodeficiência Adquirida/prevenção & controle , Produtos do Gene gag/imunologia , Vacinas contra a SAIDS/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/prevenção & controle , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Adenoviridae/genética , Animais , Anticorpos Antivirais/sangue , Antígenos Virais/genética , Antígenos Virais/imunologia , Contagem de Linfócito CD4 , Modelos Animais de Doenças , Produtos do Gene gag/genética , Vetores Genéticos , Haplorrinos , Humanos , RNA Viral/sangue , Vírus da Imunodeficiência Símia/genética , Vírus da Imunodeficiência Símia/imunologia , Carga Viral , Produtos do Gene env do Vírus da Imunodeficiência Humana/genéticaRESUMO
Constitutive nuclear factor kappaB (NF-kappaB) activation characterizes Hodgkin/Reed-Sternberg (H-RS) cells. Blocking constitutive NF-kappaB has been shown to be a potential strategy to treat Hodgkin lymphoma (HL). Here, for the first time we show that although constitutive NF-kappaB level of H-RS cell lines is very high, topoisomerase inhibitors further enhance NF-kappaB activation through IkappaB kinase activation in not only H-RS cell lines with wild-type IkappaBalpha, but also in those with IkappaBalpha mutations and lacking wild-type IkappaBalpha. Thus, both constitutive and inducible NF-kappaB are potential targets to treat HL. We also present the data that indicate the involvement of IkappaBbeta in NF-kappaB induction by topoisomerase inhibitors. A new NF-kappaB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ) inhibited constitutive NF-kappaB activity and induced apoptosis of H-RS cell lines. DHMEQ also inhibited the growth of H-RS cells without significant systemic toxicity in a NOD/SCID/gammac(null) (NOG) mice model. DHMEQ and topoisomerase inhibitors revealed enhancement of apoptosis of H-RS cells by blocking inducible NF-kappaB. Results of this study suggest that both constitutive and inducible NF-kappaB are molecular targets of DHMEQ in the treatment of HL. The results also indicate that IkappaBbeta is involved in NF-kappaB activation in H-RS cells and IkappaBbeta substitutes for IkappaBalpha in H-RS cells lacking wild-type IkappaBalpha.
Assuntos
Benzamidas/farmacologia , Cicloexanonas/farmacologia , Proteínas I-kappa B/metabolismo , NF-kappa B/metabolismo , Células de Reed-Sternberg/metabolismo , Animais , Apoptose , Caspases/metabolismo , Linhagem Celular , Daunorrubicina/farmacologia , Ativação Enzimática , Etoposídeo/farmacologia , Doença de Hodgkin , Humanos , Quinase I-kappa B/metabolismo , Proteínas I-kappa B/genética , Proteínas I-kappa B/fisiologia , Leucócitos Mononucleares/metabolismo , Camundongos , Mutação , Inibidor de NF-kappaB alfa , NF-kappa B/antagonistas & inibidores , Transplante de Neoplasias , Inibidores da Topoisomerase , Transplante Heterólogo , Células Tumorais CultivadasRESUMO
Critical to the development of an effective HIV/AIDS model is the production of an animal model that reproduces long-lasting active replication of HIV-1 followed by elicitation of virus-specific immune responses. In this study, we constructed humanized nonobese diabetic/severe combined immunodeficiency (NOD/SCID)/interleukin-2 receptor gamma-chain knockout (IL2Rgamma(null)) (hNOG) mice by transplanting human cord blood-derived hematopoietic stem cells that eventually developed into human B cells, T cells, and other monocytes/macrophages and 4 dendritic cells associated with the generation of lymphoid follicle-like structures in lymphoid tissues. Expressions of CXCR4 and CCR5 antigens were recognized on CD4+ cells in peripheral blood, the spleen, and bone marrow, while CCR5 was not detected on thymic CD4+ T cells. The hNOG mice showed marked, long-lasting viremia after infection with both CCR5- and CXCR4-tropic HIV-1 isolates for more than the 40 days examined, with R5 virus-infected animals showing high levels of HIV-DNA copies in the spleen and bone marrow, and X4 virus-infected animals showing high levels of HIV-DNA copies in the thymus and spleen. Furthermore, we detected both anti-HIV-1 Env gp120- and Gag p24-specific antibodies in animals showing a high rate of viral infection. Thus, the hNOG mice mirror human systemic HIV infection by developing specific antibodies, suggesting that they may have potential as an HIV/AIDS animal model for the study of HIV pathogenesis and immune responses.
Assuntos
Modelos Animais de Doenças , Anticorpos Anti-HIV/biossíntese , Infecções por HIV/imunologia , Tecido Linfoide/patologia , Viremia/imunologia , Animais , Medula Óssea/patologia , Medula Óssea/virologia , Linfócitos T CD4-Positivos/virologia , Linhagem da Célula , Transplante de Células-Tronco de Sangue do Cordão Umbilical , DNA Viral/análise , Suscetibilidade a Doenças , Feminino , Anticorpos Anti-HIV/sangue , Humanos , Subunidade gama Comum de Receptores de Interleucina/deficiência , Subunidade gama Comum de Receptores de Interleucina/genética , Subpopulações de Linfócitos/patologia , Tecido Linfoide/virologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Receptores CXCR4/análise , Receptores CXCR4/genética , Receptores CXCR5 , Receptores de Quimiocinas/análise , Receptores de Quimiocinas/genética , Baço/patologia , Baço/virologia , Timo/patologia , Timo/virologia , Transplante HeterólogoRESUMO
One of the mechanisms by which HIV infection induces the depletion of CD4+ T cells has been suggested to be impairment of T-cell development in the thymus, although there is no direct evidence that this occurs. To examine this possibility, we compared T-cell maturation in the intrathymic progenitors between macaques infected with an acute pathogenic chimeric simian-human immunodeficiency virus (SHIV), which causes profound and irreversible CD4+ T-cell depletion, and macaques infected with a less pathogenic SHIV, which causes only a transient CD4+ T-cell decline. Within 27 days post-inoculation (dpi), the two virus infections caused similar increases in plasma viral loads and similar decreases in CD4+ T-cell counts. However, in the thymus, the acute pathogenic SHIV resulted in increased thymic involution, atrophy and the depletion of immature T cells including CD4(+)CD8(+) double-positive (DP) cells, whereas the less pathogenic SHIV did not have these effects. Ex vivo differentiation of CD3(-)CD4(-)CD8(-) triple-negative (TN) intrathymic progenitors to DP cells was assessed by a monkey-mouse xenogenic fetal thymus organ culture system. Differentiation was impaired in the TN intrathymic progenitors of the acute pathogenic SHIV-infected monkeys, while differentiation was not impaired in the TN intrathymic progenitors of the less pathogenic SHIV-infected monkeys. These differences suggest that dysfunction of thymic maturation makes an important contribution to the irreversible depletion of circulating CD4+ T cells in vivo.
Assuntos
Linfócitos T CD4-Positivos/imunologia , HIV/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Vírus da Imunodeficiência Símia/imunologia , Timo/imunologia , Timo/virologia , Animais , Contagem de Linfócito CD4 , Linfócitos T CD4-Positivos/virologia , Diferenciação Celular/imunologia , Feminino , Citometria de Fluxo , HIV/genética , Humanos , Macaca mulatta , Camundongos , Camundongos Endogâmicos C57BL , Gravidez , RNA Viral/química , RNA Viral/genética , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/genética , Vírus da Imunodeficiência Símia/patogenicidade , Células-Tronco/imunologia , Células-Tronco/virologia , Timo/citologia , Carga ViralRESUMO
It is believed likely that immune responses are responsible for controlling viral load and infection. In this study, when macaques were primed with plasmid DNA encoding SIV gag and pol genes (SIVgag/pol DNA) and then boosted with replication-deficient vaccinia virus DIs recombinant expressing the same genes (rDIsSIVgag/pol), this prime-boost regimen generated higher levels of Gag-specific CD4+ and CD8+ T cell responses than did either SIVgag/pol DNA or rDIsSIVgag/pol alone. When the macaques were i.v. challenged with pathogenic simian/HIV, the prime-boost group maintained high CD4+ T cell counts and reduced plasma viral loads up to 30 wk after viral challenge, whereas the rDIsSIVgag/pol group showed only a partial attenuation of the viral infection, and the group immunized with SIVgag/pol DNA alone showed none at all. The protection levels were better correlated with the levels of virus-specific T cell responses than the levels of neutralization Ab responses. These results demonstrate that a vaccine regimen that primes with DNA and then boosts with a replication-defective vaccinia virus DIs generates anti-SIV immunity, suggesting that it will be a promising vaccine regimen for HIV-1 vaccine development.
Assuntos
Anticorpos Antivirais/biossíntese , Produtos do Gene gag/genética , Produtos do Gene gag/imunologia , Produtos do Gene pol/genética , Produtos do Gene pol/imunologia , Imunização Secundária , Vírus da Imunodeficiência Símia/imunologia , Vacinas de DNA/imunologia , Animais , Anticorpos Antivirais/sangue , Linfócitos T CD4-Positivos/imunologia , Células Cultivadas , Feminino , Citometria de Fluxo , Vetores Genéticos , Imunidade Celular , Interferon gama/metabolismo , Cinética , Macaca fascicularis , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/prevenção & controle , Vírus da Imunodeficiência Símia/genética , Vacinas de DNA/genética , Vaccinia virusRESUMO
To develop a new recombinant BCG (rBCG) vaccine, we constructed rBCG that expresses the full-length Gag protein of simian immunodeficiency virus (rBCG-SIVGag) at a level of 0.5 ng/mg after 3 weeks of bacterial cell culture. Intradermal (i.d.) inoculation of guinea pigs with 0.1 mg of rBCG-SIVGag resulted in the induction of delayed-type hypersensitivity (DTH) responses to both purified protein derivative (PPD) of tuberculin and SIV Gag p27 protein; responses that were maintained for the duration of the 50-week study. In contrast, guinea pigs orally vaccinated with 160 mg of the same antigen exhibited a long-lasting DTH response to the SIV Gag p27 protein, but mounted no response to PPD. Proliferative responses to SIV Gag p27 and PPD antigens were detected in both i.d. and orally immunized animals; however, the levels of PPD-specific responses were significantly higher in guinea pigs immunized by the i.d. than the oral route. A significant increase in the level of PPD- and SIV Gag p27-specific IFNgamma mRNA expression was also detected in both immunization groups receiving rBCG-SIVGag. In addition, both i.d. and oral immunization with rBCG-SIVGag induced PPD- and SIV Gag p27-specific serum IgG responses. Insertion of the SIV gag gene into BCG did not appear to change the ability of rBCG-immunized animals to elicit PPD-specific immune responses. These results indicate that rBCG-SIVGag has the ability to effectively induce long-lasting, cell-mediated and humoral immunity against both viral and bacterial antigens in guinea pigs, suggesting that rBCG-Gag has the potential to elicit immunities specific not only for tuberculosis but also for HIV at human doses.
Assuntos
Produtos do Gene gag/imunologia , Mycobacterium bovis/imunologia , Vacinação/métodos , Administração Oral , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Feminino , Expressão Gênica/genética , Expressão Gênica/imunologia , Produtos do Gene gag/genética , Vetores Genéticos/genética , Cobaias , Infecções por HIV/prevenção & controle , Hipersensibilidade Tardia/imunologia , Imunidade Celular/imunologia , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Injeções Intradérmicas , Interferon gama/genética , Intestinos/citologia , Intestinos/imunologia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Ativação Linfocitária/imunologia , Linfócitos/imunologia , Linfócitos/metabolismo , Mucosa/citologia , Mucosa/imunologia , Mycobacterium bovis/genética , Testes Cutâneos , Baço/citologia , Baço/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Transgenes/genética , Transgenes/imunologia , Tuberculina/imunologia , Tuberculose/prevenção & controle , Vacinas Combinadas/imunologiaRESUMO
Adult T-cell leukemia (ATL), an aggressive malignancy of CD4+ T cells associated with human T-cell leukemia virus type I (HTLV-I) infection, carries a very poor prognosis because of the resistance of leukemic cells to any conventional regimen, including chemotherapy. We examined the effect of ritonavir, an HIV protease inhibitor, on HTLV-I-infected T-cell lines and primary ATL cells and found that it induced apoptosis and inhibited transcriptional activation of NF-kappaB in these cells. Furthermore, ritonavir inhibited expression of Bcl-xL, survivin, c-Myc, and cyclin D2, the targets of NF-kappaB. In nonobese diabetic/severe combined immunodeficient (NOD/SCID)/gammacnull (NOG) mice, ritonavir very efficiently prevented tumor growth and leukemic infiltration in various organs of NOG mice at the same dose used for treatment of patients with AIDS. Our data indicate that ritonavir has potent anti-NF-kappaB and antitumor effects and might be clinically applicable for treatment of ATL. These results would provide a new concept and novel platform for new drug development of leukemia and solid cancer as well.
Assuntos
Apoptose/efeitos dos fármacos , Inibidores da Protease de HIV/farmacologia , NF-kappa B/metabolismo , Neoplasias Experimentais , Ritonavir/farmacologia , Transcrição Gênica/efeitos dos fármacos , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Ciclina D2 , Ciclinas/metabolismo , Modelos Animais de Doenças , Feminino , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , Humanos , Proteínas Inibidoras de Apoptose , Leucemia-Linfoma de Células T do Adulto/tratamento farmacológico , Leucemia-Linfoma de Células T do Adulto/patologia , Leucemia-Linfoma de Células T do Adulto/virologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas Associadas aos Microtúbulos/metabolismo , Pessoa de Meia-Idade , NF-kappa B/antagonistas & inibidores , NF-kappa B/genética , Proteínas de Neoplasias/metabolismo , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/patologia , Neoplasias Experimentais/virologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Survivina , Proteína bcl-X/metabolismoRESUMO
Virus-specific T-cell responses can limit immunodeficiency virus type 1 (HIV-1) transmission and prevent disease progression and so could serve as the basis for an affordable, safe, and effective vaccine in humans. To assess their potential for a vaccine, we used Mycobacterium bovis bacillus Calmette-Guérin (BCG)-Tokyo and a replication-deficient vaccinia virus strain (DIs) as vectors to express full-length gag from simian immunodeficiency viruses (SIVs) (rBCG-SIVgag and rDIsSIVgag). Cynomolgus macaques were vaccinated with either rBCG-SIVgag dermally as a single modality or in combination with rDIsSIVgag intravenously. When cynomologus macaques were primed with rBCG-SIVgag and then boosted with rDIsSIVgag, high levels of gamma interferon (IFN-gamma) spot-forming cells specific for SIV Gag were induced. This combination regimen elicited effective protective immunity against mucosal challenge with pathogenic simian-human immunodeficiency virus for the 1 year the macaques were under observation. Antigen-specific intracellular IFN-gamma activity was similarly induced in each of the macaques with the priming-boosting regimen. Other groups receiving the opposite combination or the single-modality vaccines were not effectively protected. These results suggest that a recombinant M. bovis BCG-based vector may have potential as an HIV/AIDS vaccine when administered in combination with a replication-deficient vaccinia virus DIs vector in a priming-boosting strategy.
Assuntos
Vacina BCG/administração & dosagem , Vetores Genéticos , Esquemas de Imunização , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Vacina Antivariólica/administração & dosagem , Vacinação , Animais , Vacina BCG/genética , Células Cultivadas , Produtos do Gene gag/genética , Produtos do Gene gag/imunologia , Imunização Secundária , Interferon gama/imunologia , Leucócitos Mononucleares/imunologia , Macaca fascicularis , Masculino , Vacinas contra a SAIDS/administração & dosagem , Vacinas contra a SAIDS/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/prevenção & controle , Vírus da Imunodeficiência Símia/imunologia , Vacina Antivariólica/genética , Especificidade da Espécie , Vacinas Sintéticas/administração & dosagemRESUMO
As there are very few reproducible animal models without conditioning available for the study of human B-cell-type Hodgkin's lymphoma (HL), we investigated the ability of HL cells to induce tumors using novel NOD/SCID/gammac(null) (NOG) mice. Four human Epstein-Barr virus-negative cell lines (KM-H2 and L428 originated from B cells, L540 and HDLM2 originated from T cells) were inoculated either subcutaneously in the postauricular region or intravenously in the tail of unmanipulated NOG mice. All cell lines successfully engrafted and produced tumors with infiltration of cells in various organs of all mice. Tumor cells had classical histomorphology as well as expression patterns of the tumor marker CD30, which is a cell surface antigen expressed on HL. Tumor progression in mice inoculated with B-cell-type, but not T-cell-type, HL cells correlated with an elevation in serum human interleukin-6 levels. Tumor cells from the mice also retained strong nuclear factor (NF)-kappaB DNA binding activity, and the induced NF-kappaB components were indistinguishable from those cultured in vitro. The reproducible growth behavior and preservation of characteristic features of both B-cell-type and T-cell-type HL in the mice suggest that this new xenotransplant model can provide a unique opportunity to understand and investigate the mechanism of pathogenesis and malignant cell growth, and to develop novel anticancer therapies.
Assuntos
Doença de Hodgkin/patologia , Antígeno Ki-1/genética , NF-kappa B/genética , Animais , Divisão Celular , Linhagem Celular Tumoral , Humanos , Interleucina-6/sangue , Células Jurkat , Metástase Linfática , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Metástase Neoplásica , Transplante Heterólogo/métodosRESUMO
Multiple myeloma (MM) is a fatal lymphoid malignancy that is incurable with conventional modalities of chemotherapy. Strong and constitutive activation of nuclear factor kappa B (NF-kappaB) is a common characteristic of MM cells. In our study we successfully target NF-kappaB with a novel NF-kappaB inhibitor dehydroxymethylepoxyquinomycin (DHMEQ). DHMEQ completely abrogates constitutive NF-kappaB activity and induces apoptosis of MM cells, whereas control peripheral blood mononuclear cells (PBMC) are resistant to NF-kappaB inhibition and apoptosis by DHMEQ treatment. DHMEQ inhibition of NF-kappaB triggers activation of caspases 8 and 9, as well as G0/G1 cell cycle arrest accompanied by downregulation of antiapoptotic genes Bcl-XL and c-FLIP and cell cycle progression gene cyclins D1 and D2. DHMEQ-mediated inhibition of vascular endothelial growth factor (VEGF) production in MM cells raises the possibility that DHMEQ abrogates the autocrine VEGF loop and enhances its antitumor effects by inhibiting neovascularization in the bone marrow. Using an in vivo NOD/SCID/gammac(null) (NOG) mice model, we show that DHMEQ has a potent inhibitory effect on the growth of MM cells. Compared to other compounds having the potential to inhibit NF-kappaB, DHMEQ is a unique compound that blocks the translocation of NF-kappaB p65 into the nucleus and selectively targets NF-kappaB activated in tumor cells. Therefore, our study presents a new molecular target therapy in MM.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Benzamidas/farmacologia , Cicloexanonas/farmacologia , Mieloma Múltiplo/tratamento farmacológico , NF-kappa B/antagonistas & inibidores , Animais , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD , Proteínas de Ligação ao Cálcio , Caspase 3 , Caspase 8 , Caspase 9 , Caspases/metabolismo , Linhagem Celular Tumoral , Ciclina D , Ciclina D1/metabolismo , Ciclinas/metabolismo , Modelos Animais de Doenças , Regulação para Baixo , Ativação Enzimática/efeitos dos fármacos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Glicoproteínas de Membrana , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Mieloma Múltiplo/metabolismo , Proteínas do Tecido Nervoso , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Sinaptotagmina I , Sinaptotagminas , Translocação Genética/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Proteína bcl-XRESUMO
To clarify hematological abnormalities following infection with human immunodeficiency virus (HIV), we examined the hematopoietic capability of bone marrow by using cynomolgus monkeys infected with pathogenic simian/human immunodeficiency virus (SHIV) strain C2/1, an animal model of HIV infection. The relationship between the progress of the infection and the CD4/CD8 ratio of T lymphocytes or the amount of SHIV C2/1 viral load in the peripheral blood was also investigated. A colony assay was performed to assess the hematopoietic capability of bone marrow stem cells during the early and advanced phases of the infection. Colonies of granulocytes-macrophages (GM) were examined by PCR for the presence of the SIVmac239 gag region to reveal direct viral infection. There was a remarkable decrease in the CFU-GM growth on days 1 and 3 postinoculation, followed by recovery on day 56. During the more advanced stage, the CFU-GM growth decreased again. There was minimal evidence of direct viral infection of pooled cultured CFU-GM despite the continuously low CD4/CD8 ratios. These results indicate that the decrease in colony formation by bone marrow stem cells is reversible and fluctuates with the advance of the disease. This decrease was not due to direct viral infection of CFU-GM. Our data may support the concept that, in the early phase, production of inhibitory factors or deficiency of a stimulatory cytokine is responsible for some of the bone marrow defects described in the SHIV C2/1 model.
Assuntos
Medula Óssea/patologia , Infecções por HIV/fisiopatologia , Células-Tronco Hematopoéticas/patologia , Síndrome de Imunodeficiência Adquirida dos Símios/fisiopatologia , Animais , Relação CD4-CD8 , Feminino , HIV-1/patogenicidade , Hematopoese , Humanos , Macaca , Masculino , RNA Viral/sangue , Vírus da Imunodeficiência Símia/patogenicidade , Carga ViralRESUMO
To evaluate immunity induced by a novel DNA prime-boost regimen, we constructed a DNA plasmid encoding the gag and pol genes from simian immunodeficiency virus (SIV) (SIVgag/pol DNA), in addition to a replication-deficient vaccinia virus strain DIs recombinant expressing SIV gag and pol genes (rDIsSIVgag/pol). In mice, priming with SIVgag/pol DNA, followed by rDIsSIVgag/pol induced an SIV-specific lymphoproliferative response that was mediated by a CD4+-T-lymphocyte subset. Immunization with either vaccine alone was insufficient to induce high levels of proliferation or Th1 responses in the animals. The prime-boost regimen also induced SIV Gag-specific cellular responses based on gamma interferon secretion, as well as cytotoxic-T-lymphocyte responses. Thus, the regimen of DNA priming and recombinant DIs boosting induced Th1-type cell-mediated immunity, which was associated with resistance to viral challenge with wild-type vaccinia virus expressing SIVgag/pol, suggesting that this new regimen may hold promise as a safe and effective vaccine against human immunodeficiency virus type 1.
Assuntos
Vacinas contra a SAIDS/administração & dosagem , Vírus da Imunodeficiência Símia/imunologia , Vacinas de DNA/administração & dosagem , Vaccinia virus/imunologia , Vacinas contra a AIDS/genética , Animais , Linfócitos T CD4-Positivos/imunologia , Feminino , Genes gag , Genes pol , Vetores Genéticos , HIV-1/imunologia , Humanos , Imunização Secundária , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Recombinação Genética , Vacinas contra a SAIDS/genética , Vírus da Imunodeficiência Símia/genética , Células Th1/imunologia , Vacinas de DNA/genética , Vaccinia virus/genéticaRESUMO
Clinically and biologically relevant animal models are indispensable to evaluate both the pathophysiology and strategies for diagnosis and treatment of multiple myeloma (MM). We examined the tumorigenicity of MM cell lines KMM-1 and U-266 in an in vivo cell proliferation model using NOD/SCID/gammacnull (NOG) mice. Two cell lines were inoculated either subcutaneously (s.c.) in the post-auricular region or intravenously (i.v.) in the tail of NOG mice. The KMM-1 cell line produced a progressively growing large tumor with infiltration of the cells expressing human lambda-chain in various organs of all NOG mice, while the U-266 cell line failed to do so. Tumor cells grown in NOG mice maintained the original histomorphology, as well as expression patterns of tumor markers human lambda Ig light chain and VEGF. Tumor progression in mice also correlated with elevation of serum human soluble IL-6R and gp130. Tumor cells sustained a strong NF-kappaB activity in vivo and induced NF-kappaB components were indistinguishable from those in cells cultured in vitro. The rapid and efficient engraftment of the MM cell line in NOG mice suggests that this is a very useful animal model which could provide a novel system in which to clarify the mechanism of growth of cancer cells, as well as to develop new therapeutic regimens against MM.