RESUMO
We have previously reported that hepatitis B virus (HBV)-specific CD8(+) cytotoxic T lymphocytes and CD4(+) helper T lymphocytes can inhibit HBV replication in the liver of HBV transgenic mice by secreting interferon (IFN)-gamma when they recognize viral antigen. To determine whether an activated innate immune system can also inhibit HBV replication, in this study we activated natural killer T (NKT) cells in the liver of HBV transgenic mice by a single injection of alpha-galactosylceramide (alpha-GalCer), a glycolipid antigen presented to Valpha14(+)NK1.1(+) T cells by the nonclassical major histocompatibility complex class I-like molecule CD1d. Within 24 h of alpha-GalCer injection, IFN-gamma and IFN-alpha/beta were detected in the liver of HBV transgenic mice and HBV replication was abolished. Both of these events were temporally associated with the rapid disappearance of NKT cells from the liver, presumably reflecting activation-induced cell death, and by the recruitment of activated NK cells into the organ. In addition, prior antibody-mediated depletion of CD4(+) and CD8(+) T cells from the mice did not diminish the ability of alpha-GalCer to trigger the disappearance of HBV from the liver, indicating that conventional T cells were not downstream mediators of this effect. Finally, the antiviral effect of alpha-GalCer was inhibited in mice that are genetically deficient for either IFN-gamma or the IFN-alpha/beta receptor, indicating that most of the antiviral activity of alpha-GalCer is mediated by these cytokines. Based on these results, we conclude that alpha-GalCer inhibits HBV replication by directly activating NKT cells and by secondarily activating NK cells to secrete antiviral cytokines in the liver. In view of these findings, we suggest that, if activated, the innate immune response, like the adaptive immune response, has the potential to control viral replication during natural HBV infection. In addition, the data suggest that therapeutic activation of NKT cells may represent a new strategy for the treatment of chronic HBV infection.
Assuntos
Antivirais/imunologia , Galactosilceramidas/imunologia , Vírus da Hepatite B/imunologia , Células Matadoras Naturais/imunologia , Ativação Linfocitária/imunologia , Replicação Viral/imunologia , Animais , Antivirais/administração & dosagem , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Relação Dose-Resposta a Droga , Feminino , Galactosilceramidas/administração & dosagem , Vírus da Hepatite B/efeitos dos fármacos , Vírus da Hepatite B/genética , Vírus da Hepatite B/fisiologia , Humanos , Células Matadoras Naturais/efeitos dos fármacos , Ativação Linfocitária/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fatores de Tempo , Replicação Viral/efeitos dos fármacosRESUMO
We have previously shown that hepatitis B virus (HBV) replication is abolished in the liver of HBV transgenic mice by inflammatory cytokines induced by HBV-specific cytotoxic T cells and during unrelated viral infections of the liver. We now report that intrahepatic HBV replication is also inhibited in mice infected by the malaria species Plasmodium yoelii 17X NL. P. yoelii infection triggers an intrahepatic inflammatory response characterized by the influx of natural killer cells, macrophages, and T cells. During this process, interferon (IFN)-gamma and IFN-alpha/beta suppress HBV gene expression and replication in the liver. Collectively, the data suggest that malaria infection might influence the course and pathogenesis of HBV infection in coinfected humans.
Assuntos
Vírus da Hepatite B/imunologia , Hepatite B/imunologia , Interferons/imunologia , Fígado/imunologia , Malária/imunologia , Plasmodium yoelii/fisiologia , Alanina Transaminase/sangue , Animais , Northern Blotting , Citocinas/biossíntese , Citocinas/genética , Citocinas/imunologia , Hepatite B/complicações , Hepatite B/virologia , Vírus da Hepatite B/genética , Vírus da Hepatite B/fisiologia , Humanos , Interferons/biossíntese , Interferons/genética , Fígado/parasitologia , Fígado/patologia , Fígado/virologia , Macrófagos/imunologia , Malária/complicações , Malária/parasitologia , Camundongos , Camundongos Transgênicos , Linfócitos T/imunologia , Replicação ViralRESUMO
Using transgenic mice that replicate hepatitis B virus (HBV) at high levels in the liver as recipients of HBV-specific cytotoxic T lymphocytes (CTLs), we showed that the chemokines responsive to gamma-2/IFN-gamma inducible protein ([Crg2]IP-10) and monokine induced by interferon-gamma (Mig) are rapidly and strongly induced in the liver after CTL transfer. The transferred CTLs produce neither chemokine; rather, they activate (via the secretion of IFN-gamma) hepatocytes and nonparenchymal cells of the liver to produce (Crg2)IP-10 and Mig. Importantly, blocking these chemokines in vivo reduces the recruitment of host-derived lymphomononuclear cells into the liver and the severity of the liver disease without affecting the IFN-gamma-dependent antiviral potential of the CTLs. The finding that neutralization of these chemokines is associated with maintenance of antiviral effects but diminished tissue damage may be significant for the development of immunotherapeutic approaches for the treatment of chronic HBV infection.
Assuntos
Citotoxicidade Imunológica , Vírus da Hepatite B/imunologia , Hepatite B/imunologia , Interferon gama/imunologia , Monocinas/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Quimiocina CXCL10 , Hepatite B/genética , Hepatite B/patologia , Interferon gama/genética , Fígado/imunologia , Fígado/patologia , Fígado/virologia , Camundongos , Camundongos Transgênicos , Monocinas/genéticaRESUMO
Antigen presentation by major histocompatibility complex (MHC) class I molecules requires peptide supply by the transporters associated with antigen processing (TAPs), which select substrates in a species- and, in the rat, allele-specific manner. Conflicts between TAPs and MHC preferences for COOH-terminal peptide residues in rodent cells strongly reduce the efficiency of MHC class I antigen presentation. Although human TAP is relatively permissive, some peptide ligands for human histocompatibility leukocyte antigen class I molecules are known to possess very low TAP affinities; the significance of these in vitro findings for cellular antigen presentation is not known. We studied two naturally immunodominant viral epitopes presented by HLA-A2 that display very low affinities for human TAP. Low TAP affinities preclude minimal epitope access to the endoplasmic reticulum (ER) and assembly with HLA-A2 in vitro, as well as presentation by minigene-expressing cells to cytotoxic T lymphocytes. However, NH(2)-terminally but not COOH-terminally extended epitope variants with higher TAP affinities assemble in vitro and are presented to cytotoxic T lymphocytes with high efficiency. Thus, human TAP can influence epitope selection and restrict access to the ER to epitope precursors. Analysis of TAP affinities of a panel of viral epitopes suggests that TAP selection of precursors may be a common phenomenon for HLA-A2-presented epitopes. We also analyzed HLA-A2-eluted peptides from minigene-expressing cells and show that an NH(2)-terminally extended variant with low A2 binding affinity undergoes ER processing, whereas another with high affinity is presented unmodified. Therefore, the previously reported aminopeptidase activity in the ER can also act on TAP-translocated peptides.
Assuntos
Apresentação de Antígeno/imunologia , Proteínas de Transporte/imunologia , Retículo Endoplasmático/imunologia , Antígeno HLA-A2/imunologia , Complexo Principal de Histocompatibilidade/imunologia , Linfócitos T/metabolismo , Animais , Linfócitos B/metabolismo , Transporte Biológico , Testes Imunológicos de Citotoxicidade , Epitopos , Hepacivirus/imunologia , Vírus da Hepatite B/imunologia , Humanos , Camundongos , Camundongos Knockout , Peptídeos/imunologia , Precursores de ProteínasRESUMO
BACKGROUND: Human gammadelta T cells can be activated by phospho-antigens and aminobisphosphonates such as zoledronate. Because they can kill tumor cells in a major histocompatibility complex (MHC)-unrestricted manner, adoptive transfer of activated gammadelta T cells may represent a novel cancer immunotherapy. We tested whether gammadelta T cells from advanced cancer patients can be expanded by zoledronate. METHODS: Peripheral blood mononuclear cells from healthy donors and patients with advanced non-small cell lung cancer, bone metastatic breast or prostate cancer, or lung metastatic colorectal cancer, were stimulated with zoledronate (5 microM) and interleukin (IL)-2 (1000 IU/mL) for 14 days. The phenotype and function of the expanded gammadelta T-cell populations from healthy donors and cancer patients were compared. RESULTS: Gammadelta T cells from cancer patients and healthy donors responded to zoledronate equally well in terms of both phenotype and function. gammadelta T cells grew rapidly in vitro and expression of effector molecules, such as interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, perforin, granzyme B, FasL and TRAIL, increased over time. Cytotoxicity peaked on days 12-14, and proliferation continued up to 14 days, during which time>1x10(9) gammadelta T cells could be obtained from a starting sample of 45-70 mL peripheral blood. DISCUSSION: Using the agent zoledronate, already widely used in the clinic, we have established that efficient large-scale ex vivo expansion of gammadelta T cells from cancer patients is possible. These cells exert potent cytotoxicity and may be used for autologous cellular immunotherapy of cancer.
Assuntos
Neoplasias Ósseas/terapia , Neoplasias da Mama/terapia , Carcinoma Pulmonar de Células não Pequenas/terapia , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/terapia , Citocinas/metabolismo , Citotoxicidade Imunológica/efeitos dos fármacos , Difosfonatos/farmacologia , Imidazóis/farmacologia , Imunoterapia Adotiva , Neoplasias Pulmonares/terapia , Neoplasias da Próstata/terapia , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Linfócitos T/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias Ósseas/imunologia , Neoplasias Ósseas/secundário , Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Carcinoma Pulmonar de Células não Pequenas/imunologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Células Cultivadas , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/patologia , Citocinas/genética , Feminino , Humanos , Imunofenotipagem , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Neoplasias da Próstata/imunologia , Neoplasias da Próstata/patologia , RNA/análise , Receptores de Antígenos de Linfócitos T gama-delta/genética , Linfócitos T/metabolismo , Ácido ZoledrônicoRESUMO
Development of chronic lung allograft dysfunction involves various alloimmune-independent insults including those mediated by Toll-like receptor (TLR) signaling, which is known to activate alloimmune responses. We hypothesized that TLR signaling may also contribute to the activation of fibroblasts and promoting allograft airway fibrosis. Mouse orthotopic tracheal transplants were conducted between major histocompatibility complex (MHC)-mismatched Balb/c donor and wild-type C3H or C3H-derived TLR4 mutant recipients (nonfunctional TLR4). Immunohistochemistry on day 21 showed significantly smaller alpha-smooth muscle actin (α-SMA)-positive areas in TLR4 mutant recipients than wild-type recipients (P = .01). No difference was found for CD3+ T-cell infiltration. Proliferation of alloreactive T cells derived from the recipient spleen showed no difference between TLR4 mutant and wild-type recipients in a mixed lymphocyte reaction. The effect of TLR4 signaling was examined in primary pulmonary fibroblast cultures both with lipopolysaccharide (LPS) and transforming growth factor (TGF)-ß1. Stimulation with LPS significantly increased expression of α-SMA mRNA in wild-type fibroblasts cultured with TGF-ß1 compared with the control without LPS (P = .001). Taken together, these findings suggest disruption of TLR signaling leads to reduced activation of fibroblasts without affecting T-cell infiltration and proliferation in this model. TLR4-mediated activation of fibroblasts may be a potentially important mechanism of allograft remodeling.
Assuntos
Fibroblastos/metabolismo , Disfunção Primária do Enxerto/metabolismo , Disfunção Primária do Enxerto/patologia , Receptor 4 Toll-Like/metabolismo , Traqueia/transplante , Aloenxertos/metabolismo , Aloenxertos/patologia , Aloenxertos/fisiopatologia , Animais , Fibrose/metabolismo , Fibrose/patologia , Transplante de Pulmão , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Disfunção Primária do Enxerto/fisiopatologia , Transplante HomólogoRESUMO
We have developed a bioassay for interferons (IFN) based on measuring the amounts of 2',5' oligoadenylate synthetase (2-5AS) induced in cells of the THP-1 monocyte line in response to IFN. The assay can be completed in 20 h, gives reproducible results, and is at least 50 times more sensitive to IFN-alpha than conventional cytopathic effect inhibition antiviral assays. It is, respectively, less and much less sensitive to IFN-beta and IFN-gamma. The presence of preexisting 2-5AS activity in a sample does not influence the results. We have used this assay to measure very low levels (0.1-0.5 IU/ml) of endogenously formed IFN-alpha in serum samples from patients with various diseases and also to measure the residual small amounts of IFN-alpha still present in the serum as late as 48 h after an i.m. injection of 3 million IU, which is appreciably later than in previous methods. Thus, our highly sensitive assay offers considerable advantages, not least in relation to the clinical use of IFN.
Assuntos
2',5'-Oligoadenilato Sintetase/metabolismo , Interferons/sangue , Monócitos/metabolismo , Bioensaio , Linhagem Celular , Citocinas/farmacologia , Indução Enzimática , Humanos , Indutores de Interferon/farmacologia , Interferon-alfa/sangue , Interferon gama/sangue , Monócitos/efeitos dos fármacos , Sensibilidade e Especificidade , Viroses/sangueRESUMO
We investigated the responses of peripheral blood mononuclear cells (PBMCs) to hepatitis C virus core protein in ten patients with chronic hepatitis C during interferon (IFN)-beta treatment to determine if the modulation of the immune reaction to hepatitis C virus by IFN treatment is associated with the viral clearance. Interleukin-2, interleukin-4, interleukin-10, and interferon-gamma in the supernatant of the patients' PBMC co-cultured with the HCV core antigen-presenting autologous PBMCs were measured by ELISA. Serum levels of soluble CD (sCD) 8 and sCD30 in these patients were also measured by ELISA. The production of interleukin-2 and interferon-gamma by PBMCs of sustained responders (SRs) increased after IFN-treatment, although it did not reach a significant level. Interleukin-10 was detected only in non-responders (NRs) at 0 and 4 weeks after the start of IFN treatment. Serum sCD8 level increased significantly in SR with IFN treatment. A close correlation between the serum sCD8 levels and interferon-gamma levels in the supernatant at week 8 was observed in SR. These results suggest that IFN treatment potentiates the cellular immune reaction against HCV core protein more efficiently in SR than in NR.
RESUMO
BACKGROUND: Aberrant epithelial repair is a crucial event in the airway remodeling that characterizes obliterative bronchiolitis (OB) in transplanted lungs. Recent data from experiments using epithelial cell lines and human airway tissues from lung transplant recipients suggest that epithelial to mesenchymal transition (EMT) plays an important role in OB. The aim of this study was to clarify whether EMT is involved in airway remodeling in an animal model. METHODS: We performed orthotopic tracheal transplantation from BALB/c to C57BL/6 mice with from BALC/c to BALB/c mouse grafts as controls. Five allogeneic and 3 syngeneic recipients were humanely killed at predetermined postoperative days 2-12 as well as 14 and 21. Histology was evaluated using hematoxylin-eosin (H&E) staining. We studied the expression of specific markers, including E-cadherin, an epithelial marker; α-smooth muscle actin (SMA), and S100A4, mesenchymal markers, and zinc finger E-box-binding homeobox 1 (ZEB1), an EMT-related transcription factor. RESULTS: Histologic assessment of serial H&E stains of allogeneic grafts showed remarkable pseudostratified respiratory epithelium with subepithelial inflammatory cell infiltration, as well as denuded and flattened epithelium and subepithelial fibrosis. The dynamic epithelial changes occurred earlier than the subepithelial fibrosis. Immunohistochemical evaluation indicated the emergence of α-SMA- positive epithelial cells that were most prominent on day 7. The expression of E-cadherin was attenuated in α-SMA-positive epithelial cells. S100A4 was also expressed in epithelial cells. A few days before the intraepithelial expression of α-SMA, ZEB1 emerged in the nuclei of epithelial cells. CONCLUSIONS: We observed expression of an EMT-related transcription factor and mesenchymal markers along with the attenuation of epithelial marker expression in epithelial cells, several days before prominent subepithelial fibrosis formation, results that suggest epithelial cells to play an important fibrosis role in airway remodeling during epithelial to mesenchymal transition.
Assuntos
Transição Epitelial-Mesenquimal , Traqueia/transplante , Transplante Homólogo , Animais , Feminino , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos BALB C , Traqueia/citologia , Traqueia/metabolismoRESUMO
OBJECTIVE: Surgical results have shown the superiority of human heart valve and vascular allografts over artificial prostheses when used for the treatment of infectious cardiovascular diseases. However, the mechanism of infection resistance in these allografts has not been determined. In this study the contribution of the inflammatory response after allogeneic transplantation to the antimicrobial mechanism was assessed, focusing on the induction of indoleamine 2,3-dioxygenase, a tryptophan-metabolizing enzyme. METHODS: Aortic transplantation was performed with inbred rats, and aortic allografts, isografts, and control grafts were obtained for the following analyses. The extent of inflammatory-related and indoleamine 2,3-dioxygenase gene expression was measured by means of quantitative reverse transcriptase-polymerase chain reaction, and tryptophan metabolite production in the graft was measured by means of liquid chromatographic/tandem mass spectrometric analysis. The bacteriostatic effect of each graft and tryptophan metabolites was determined by using the methicillin-resistant Staphylococcus aureus proliferation assay. RESULTS: The inflammatory response, including interferon gamma, tumor necrosis factor alpha, and indoleamine 2,3-dioxygenase gene expression, was significant in the allografts but minimal in the isografts and control grafts. Methicillin-resistant S. aureus proliferation was remarkably suppressed when cultured with the allografts but not with the control grafts. Among tryptophan metabolites, the bacteriostatic effect against methicillin-resistant S. aureus was remarkable with 3-hydroxykynurenine, with a minimum inhibitory concentration of 32 mg/L. The 3-hydroxykynurenine level in the allografts was 9-fold greater than that in the control grafts. CONCLUSION: The bacteriostatic effect of the allografts was acquired by inducing indoleamine 2,3-dioxygenase, which resulted in local production of 3-hydroxykynurenine as an antimicrobial agent. This is the first report to document a mechanism of the allograft's infection-resistant property against methicillin-resistant S. aureus growth.
Assuntos
Antibacterianos/farmacologia , Prótese Vascular/microbiologia , Indolamina-Pirrol 2,3,-Dioxigenase/farmacologia , Resistência a Meticilina/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/isolamento & purificação , Animais , Aorta/transplante , Citocinas/metabolismo , Endocardite/microbiologia , Endocardite/prevenção & controle , Cinurenina/análogos & derivados , Cinurenina/biossíntese , Ratos , Ratos Endogâmicos Lew , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/prevenção & controle , Transplante HomólogoRESUMO
In contrast to wild-type mouse mammary tumor virus (MMTV), the MMTV mutants with specific deletions in the U3 region of their long terminal repeats cause T-cell lymphomas. In 30% of T-cell lymphomas arising in BALB/c mice infected with MLA-MMTV, a leukemogenic MMTV mutant, we have found that MMTV proviruses were integrated into a short region of the Notch1 genome, so that truncated Notch1 transcripts encoding the transmembrane and the cytoplasmic domains of Notch1 protein could be expressed. Thus, Notch1 is a major target of provirus insertional mutagenesis in these T-cell lymphomas.
Assuntos
Linfoma de Células T/virologia , Vírus do Tumor Mamário do Camundongo/genética , Proteínas de Membrana/genética , Mutagênese Insercional , Provírus/genética , Receptores de Superfície Celular , Fatores de Transcrição , Animais , Sequência de Bases , Rearranjo Gênico , Linfoma de Células T/genética , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , RNA Mensageiro/análise , RNA Mensageiro/química , Receptor Notch1 , Sequências Repetidas TerminaisRESUMO
We have previously reported that intrahepatic NK T cells activated by alpha-galactosylceramide inhibit hepatitis B virus replication noncytopathically in the liver of transgenic mice. This effect is mediated by antiviral cytokines directly produced by activated NK T cells and/or by other cytokine-producing inflammatory cells that are recruited into the liver. In this study, we demonstrated that IFN-gamma produced by activated NK T cells induced parenchymal and nonparenchymal cells of the liver to produce high levels of CXC chemokine ligands 9 and 10, which mediated the intrahepatic recruitment of lymphomononuclear inflammatory cells. Recruitment of these cells was not necessary for the antiviral activity, indicating that direct activation of the intrahepatic resident NK T cell is sufficient to control viral replication in this model.
Assuntos
Quimiotaxia de Leucócito , Vírus da Hepatite B/fisiologia , Hepatite B/imunologia , Peptídeos e Proteínas de Sinalização Intercelular , Células Matadoras Naturais/imunologia , Subpopulações de Linfócitos T/imunologia , Replicação Viral/imunologia , Animais , Antivirais/farmacologia , Quimiocina CXCL10 , Quimiocina CXCL9 , Quimiocinas CXC/genética , Quimiocinas CXC/fisiologia , Galactosilceramidase/farmacologia , Hepatite B/genética , Hepatite B/virologia , Vírus da Hepatite B/genética , Vírus da Hepatite B/crescimento & desenvolvimento , Interferon gama/fisiologia , Cinética , Fígado/imunologia , Ativação Linfocitária , Camundongos , Camundongos Transgênicos , RNA Mensageiro/biossíntese , Receptores CXCR3 , Receptores de Quimiocinas/biossíntese , Receptores de Quimiocinas/genéticaRESUMO
Despite the high degree of homology (91%) between the nucleotide sequences of the Friend-mink cell focus-forming (MCF) and the Moloney murine leukemia virus (MuLV) genomic long terminal repeats (LTRs), the pathogenicities determined by the LTR sequences of the two viruses are quite different. Friend-MCF MuLV is an erythroid leukemia virus, and Moloney MuLV is a lymphoid leukemia virus. To map the LTR sequences responsible for the different disease specificities, we constructed nine viruses with LTRs recombinant between the Friend-MCF and Moloney MuLVs. Analysis of the leukemia induced with the recombinant viruses showed that a 195-base-pair nucleotide sequence, including a 75-base-pair nucleotide Moloney enhancer, is responsible for the tissue-specific leukemogenicity of Moloney MuLV. However, not only the enhancer but also its downstream sequences appear to be necessary. The Moloney virus enhancer and its downstream sequence exerted a dominant effect over that of the Friend-MCF virus, but the enhancer sequence alone did not. The results that three of the nine recombinant viruses induced both erythroid and lymphoid leukemias supported the hypothesis that multiple viral genetic determinants control both the ability to cause leukemia and the type of leukemia induced.
Assuntos
Vírus da Leucemia Murina de Friend/genética , Vírus da Leucemia Murina/genética , Leucemia Eritroblástica Aguda/genética , Leucemia Experimental/genética , Leucemia Linfoide/genética , Vírus Indutores de Focos em Células do Vison/genética , Vírus da Leucemia Murina de Moloney/genética , Sequências Repetitivas de Ácido Nucleico , Animais , Sequência de Bases , DNA Recombinante/fisiologia , DNA Viral/fisiologia , Elementos Facilitadores Genéticos , Vírus da Leucemia Murina de Friend/patogenicidade , Regulação da Expressão Gênica , Genes Virais , Camundongos , Vírus Indutores de Focos em Células do Vison/patogenicidade , Vírus da Leucemia Murina de Moloney/patogenicidade , Especificidade de Órgãos , Recombinação Genética , Homologia de Sequência do Ácido NucleicoRESUMO
The murine AIDS (MAIDS) virus has a unique sequence in the gag p12 region, which could be responsible for MAIDS development. RNA preparations from the spleens of normal uninfected C57BL/6 mice contain a transcript hybridizing with this sequence. Levels of the transcript in the kidney of C57BL/6 mice were higher than in the spleen, liver or thymus. Although BALB/c, NFS, DBA/2 and SL murine strains also contained genomic sequences hybridizing with the MAIDS virus-specific probe, no transcript hybridizing with the probe was detected in these strains of mice. The cDNAs carrying the transcript expressed in C57BL/6 mice were molecularly cloned. The complete nucleotide sequence of the clone indicates that the transcript is one of the endogenous murine leukaemia virus-related sequences containing large deletions from the R and U5 regions of the 5' long terminal repeat (LTR) to gag p15, from the C-terminal region of pol p40 (integrase) to the N-terminal region of env p15E, and many short deletions in the 3' LTR U3 region. The nucleotide sequence in the gag p12 region of the transcript was closely similar to that of the MAIDS virus, but the amino acid sequence was less similar because of frameshifting, even when translated. As the MAIDS virus was isolated from C57BL/6 mice with radiation-induced leukaemia, this transcript may be the progenitor of the MAIDS virus. To determine whether the gag p12 region of the transcript contains a functional sequence, a recombinant virus was generated by replacing the gag p12 region of a replication-competent BM5eco virus with that of the endogenous transcript. The recombinant virus was replication-competent, and the p12 region of the transcript retained the functional sequence present in the BM5eco virus.
Assuntos
Clonagem Molecular , Vírus da Leucemia Murina/genética , RNA Mensageiro/genética , RNA Viral/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Sondas de DNA , Deleção de Genes , Genes gag , Rim/microbiologia , Vírus da Leucemia Murina/metabolismo , Fígado/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , RNA Mensageiro/análise , RNA Mensageiro/fisiologia , RNA Viral/análise , DNA Polimerase Dirigida por RNA/metabolismo , Mapeamento por Restrição , Análise de Sequência de DNA , Baço/microbiologia , Timo/microbiologia , TransfecçãoRESUMO
Endogenous feline leukemia RD114 virus genome rendered capable of infecting mouse cells by phenotypic mixing with an ecotropic murine leukemia virus (MuLV) exhibited the Fv-1 restriction pattern of the ecotropic murine virus. However, RD114 genomes phenotypically mixed with ecotropic MuLV showed one-hit dose-response kinetics, even when titrated with murine cells with the restricted Fv-1 phenotype.
Assuntos
Vírus AKR da Leucemia Murina/fisiologia , Genes Virais/fisiologia , Vírus da Leucemia Felina/fisiologia , Vírus da Leucemia Murina/fisiologia , Replicação Viral/fisiologia , Vírus AKR da Leucemia Murina/genética , Animais , Células Cultivadas , Genes Virais/genética , Vírus da Leucemia Felina/genética , Vírus da Leucemia Felina/patogenicidade , Camundongos , Camundongos Endogâmicos BALB C , Vison , Fenótipo , Virologia/métodos , Replicação Viral/genéticaRESUMO
Whether the foci on S+L- mink cells transformed by the superinfection of MuLV were induced by the secondary infection of M-MSV pseudotype generated in the cells or by the secondary infection of MuLV has remained unresolved since the requirement of secondary infection was first reported (A. Ishimoto, J. Virol. 36, 18-21, 1980). Here, we show that infection with ecotropic MuLV of S+L- mink cells transfected with the receptor gene for ecotropic MuLV induced transformed foci resembling those induced by xenotropic and amphotropic viruses. This observation and our previous results (A. Ishimoto, J. Virol. 36, 18-21, 1980) that clonal lines of S+L- mink cells chronically infected with ecotropic MuLV are morphologically indistinguishable from normal S+L- mink cells suggest that the focus formation of S+L- mink cells by superinfection with MuLV is not due to secondary spread of helper virus which transactivates the expression of the v-mos oncogene by the MuLV, but is due to the secondary infection of the defective M-MSV genome. A new S+L- mink cell line with a receptor gene for ecotropic MuLV, designated ID cells, provided a new method for titrating the ecotropic MuLV that develop few XC foci and to simultaneously detect viruses in various host ranges.
Assuntos
Transformação Celular Viral/fisiologia , Vírus Indutores de Focos em Células do Vison/fisiologia , Vison/microbiologia , Vírus do Sarcoma Murino de Moloney/fisiologia , Animais , Células CultivadasRESUMO
The defective murine AIDS (MAIDS) virus has unique sequences in its p15gag and p12gag regions. To clarify whether these sequences are responsible for the development of MAIDS, we constructed recombinant viruses by replacing various regions of the gag gene of the nonpathogenic replication-competent LP-BM5 ecotropic virus with those of the MAIDS virus. Recombinants containing both unique sequences of the MAIDS virus were replication defective and induced MAIDS. However, a recombinant containing either the p15gag or p12gag region of the MAIDS virus was also replication defective but nonpathogenic in mice. A recombinant virus containing only the p30gag region of the MAIDS virus was replication competent and nonpathogenic. These results indicate that the p15gag and p12gag regions of the MAIDS virus do not function like those of replication-competent viruses and that both of the unique sequences in the p15gag and p12gag regions are required to develop MAIDS.
Assuntos
Produtos do Gene gag/fisiologia , Vírus da Leucemia Murina/patogenicidade , Síndrome de Imunodeficiência Adquirida Murina/microbiologia , Animais , Sequência de Bases , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão , Replicação ViralRESUMO
Mouse mammary tumor virus (MMTV) is a slowly transforming retrovirus associated primarily with the induction of mammary tumors. It is widely accepted that T-cell lymphomas of various mouse strains are associated with extra proviruses of MMTV. These extra proviruses showed site-specific rearrangements in the U3 region of long terminal repeats (LTRs), consisting of about 400 nucleotide deletions and occasional substitution resulting in unique tandem repeats. However, the question of whether these mutant MMTVs cause lymphomas has not been experimentally resolved. Here we present distinct evidence that they do. We constructed chimeric MMTVs by replacing the LTR of the recently constructed pathogenic MMTV provirus clone with rearranged LTRs of MMTV proviruses obtained from two DBA/2 mouse lymphoma cell lines, MLA and DL-8, and inoculated them into BALB/c mice. These mice developed lymphomas, but no mammary tumors, 4 to 11 months postinoculation, whereas the original pathogenic MMTV clone alone induced mammary tumors. These results showed that the tissue specificity of MMTV tumorigenesis is determined by the LTR structures.
Assuntos
Rearranjo Gênico/genética , Linfoma de Células T/microbiologia , Vírus do Tumor Mamário do Camundongo/genética , Sequências Repetitivas de Ácido Nucleico/genética , Animais , Sequência de Bases , Linfoma de Células T/genética , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Provírus/genética , RNARESUMO
To examine whether the resistance allele of the Fv-4 gene (the Fv-4r gene) is a dominant inhibitory-product-encoding gene which an be used to prevent the development of murine AIDS (MAIDS), bone marrow cells from BALB/c-Fv-4wr mice were transplanted into BALB/c mice and C57BL/6 mice infected with MAIDS virus. Almost all of the virus-infected BALB/c and C57BL/6 mice developed MAIDS within 4 months and died 2 or 3 months later. However, when the virus-infected mice were subjected to cobalt irradiation and then given an intravenous injection of 10(7) BALB/c-Fv-4wr mouse bone marrow cells, the recipient mice survived much longer than the untreated mice, which suggests that the Fv-4 gene is a dominant inhibitory gene that is potentially useful in gene therapy of MAIDS.
Assuntos
Transplante de Medula Óssea , Genes Dominantes/genética , Terapia Genética/métodos , Imunidade Inata/genética , Síndrome de Imunodeficiência Adquirida Murina/terapia , Animais , Animais Recém-Nascidos , Citometria de Fluxo , Antígenos H-2/análise , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Síndrome de Imunodeficiência Adquirida Murina/mortalidade , Baço/imunologia , Análise de SobrevidaRESUMO
The Duplan strain of murine leukemia virus induces murine AIDS in C57BL/6 mice. When spleen cells from C57BL/6 mice infected with the virus were transplanted into nude mice, subcutaneous solid tumors at the transplanted sites were formed and splenomegaly and lymphadenopathy were induced. These transplantable cells were Thy-1- CD4+ alpha-beta T-cell receptor-positive T cells and integrated with the pathogenic defective viral genome. These results indicate that neoplastic cells of T-cell lineage were induced by infecting C57BL/6 mice with murine AIDS virus.