RESUMO
Aspergillus fumigatus is an environmental fungus that causes invasive aspergillosis (IA) in immunocompromised patients. Although -CC-chemokine receptor-2 (CCR2) and Ly6C-expressing inflammatory monocytes (CCR2âºMo) and their derivatives initiate adaptive pulmonary immune responses, their role in coordinating innate immune responses in the lung remain poorly defined. Using conditional and antibody-mediated cell ablation strategies, we found that CCR2âºMo and monocyte-derived dendritic cells (Mo-DCs) are essential for innate defense against inhaled conidia. By harnessing fluorescent Aspergillus reporter (FLARE) conidia that report fungal cell association and viability in vivo, we identify two mechanisms by which CCR2âºMo and Mo-DCs exert innate antifungal activity. First, CCR2âºMo and Mo-DCs condition the lung inflammatory milieu to augment neutrophil conidiacidal activity. Second, conidial uptake by CCR2âºMo temporally coincided with their differentiation into Mo-DCs, a process that resulted in direct conidial killing. Our findings illustrate both indirect and direct functions for CCR2âºMo and their derivatives in innate antifungal immunity in the lung.
Assuntos
Células Dendríticas/imunologia , Imunidade Inata/imunologia , Monócitos/imunologia , Aspergilose Pulmonar/imunologia , Animais , Aspergillus fumigatus/imunologia , Diferenciação Celular/imunologia , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/citologia , Monócitos/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Receptores CCR2/metabolismo , Esporos Fúngicos/imunologiaRESUMO
BACKGROUND: Existing highly active antiretroviral therapy (HAART) effectively controls viral replication in human immunodeficiency virus type 1 (HIV-1) infected individuals but cannot completely eradicate the infection, at least in part due to the persistence of latently infected cells. One strategy that is being actively pursued to eliminate the latent aspect of HIV-1 infection involves therapies combining latency antagonists with HAART. However, discordant pharmacokinetics between these types of drugs can potentially create sites of active viral replication within certain tissues that might be impervious to HAART. RESULTS: A preliminary reverse genetic screen indicated that the proteasome might be involved in the maintenance of the latent state. This prompted testing to determine the effects of proteasome inhibitors (PIs) on latently infected cells. Experiments demonstrated that PIs effectively activated latent HIV-1 in several model systems, including primary T cell models, thereby defining PIs as a new class of HIV-1 latency antagonists. Expanding upon experiments from previous reports, it was also confirmed that PIs inhibit viral replication. Moreover, it was possible to show that PIs act as bifunctional antagonists of HIV-1. The data indicate that PIs activate latent provirus and subsequently decrease viral titers and promote the production of defective virions from activated cells. CONCLUSIONS: These results represent a proof-of-concept that bifunctional antagonists of HIV-1 can be developed and have the capacity to ensure precise tissue overlap of anti-latency and anti-replication functions, which is of significant importance in the consideration of future drug therapies aimed at viral clearance.
Assuntos
HIV-1/efeitos dos fármacos , Inibidores de Proteassoma/farmacologia , Ativação Viral/efeitos dos fármacos , Latência Viral/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Adulto , Fármacos Anti-HIV/farmacologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/virologia , Células Cultivadas , HumanosRESUMO
Current antiretroviral therapy (ART) provides potent suppression of HIV-1 replication. However, ART does not target latent viral reservoirs, so persistent infection remains a challenge. Small molecules with pharmacological properties that allow them to reach and activate viral reservoirs could potentially be utilized to eliminate the latent arm of the infection when used in combination with ART. Here we describe a cell-based system modeling HIV-1 latency that was utilized in a high-throughput screen to identify small molecule antagonists of HIV-1 latency. A more detailed analysis is provided for one of the hit compounds, antiviral 6 (AV6), which required nuclear factor of activated T cells for early mRNA expression while exhibiting RNA-stabilizing activity. It was found that AV6 reproducibly activated latent provirus from different lymphocyte-based clonal cell lines as well as from latently infected primary resting CD4(+) T cells without causing general T cell proliferation or activation. Moreover, AV6 complemented the latency antagonist activity of a previously described histone deacetylase (HDAC) inhibitor. This is a proof of concept showing that a high-throughput screen employing a cell-based model of HIV-1 latency can be utilized to identify new classes of compounds that can be used in concert with other persistent antagonists with the aim of viral clearance.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , HIV-1/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Antirretrovirais/uso terapêutico , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/virologia , Proliferação de Células , Desenho de Fármacos , Citometria de Fluxo/métodos , Regulação Viral da Expressão Gênica , Genoma Viral , Humanos , Lentivirus/genética , Ativação Linfocitária , Integração Viral , Latência ViralRESUMO
The antiapoptotic Bcl-2 family member Bfl-1 is up-regulated in many human tumors in which nuclear factor-kappaB (NF-kappaB) is implicated and contributes significantly to tumor cell survival and chemoresistance. We previously found that NF-kappaB induces transcription of bfl-1 and that the Bfl-1 protein is also regulated by ubiquitin-mediated proteasomal degradation. However, the role that dysregulation of Bfl-1 turnover plays in cancer is not known. Here we show that ubiquitination-resistant mutants of Bfl-1 display increased stability and greatly accelerated tumor formation in a mouse model of leukemia/lymphoma. We also show that tyrosine kinase Lck is up-regulated and activated in these tumors and leads to activation of the IkappaB kinase, Akt, and extracellular signal-regulated protein kinase signaling pathways, which are key mediators in cancer. Coexpression of Bfl-1 and constitutively active Lck promoted tumor formation, whereas Lck knockdown in tumor-derived cells suppressed leukemia/lymphomagenesis. These data demonstrate that ubiquitination is a critical tumor suppression mechanism regulating Bfl-1 function and suggest that mutations in bfl-1 or in the signaling pathways that control its ubiquitination may predispose one to cancer. Furthermore, because bfl-1 is up-regulated in many human hematopoietic tumors, this finding suggests that strategies to promote Bfl-1 ubiquitination may improve therapy.
Assuntos
Predisposição Genética para Doença , Proteínas Inibidoras de Apoptose/metabolismo , Linfoma/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ubiquitina/metabolismo , Ubiquitinação , Animais , Sobrevivência Celular/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Quinase I-kappa B/genética , Quinase I-kappa B/metabolismo , Proteínas Inibidoras de Apoptose/genética , Células Jurkat , Linfoma/genética , Camundongos , Antígenos de Histocompatibilidade Menor , Mutação , NF-kappa B/genética , NF-kappa B/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Estabilidade Proteica , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas pp60(c-src)/genética , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Transdução de Sinais/genética , Ubiquitina/genéticaRESUMO
Cells have developed a mechanism to discriminate between premature termination codons (PTCs) and normal stop codons during translation, sparking vigorous research to develop drugs promoting readthrough at PTCs to treat genetic disorders caused by PTCs. It was posed that this concept could also be applied to regulated gene therapy protocols by incorporating a PTC into a therapeutic gene, so active protein would only be made after administration of a readthrough agent. The strengths of the system are highlighted here by results demonstrating: (i) background expression levels were reduced to 0.01% to 0.0005% of wild type in unselected mass populations of cells depending upon the specific stop codon utilized and its position within the gene; (ii) expression levels responded well to multiple "On" and "Off" regulation cycles in vivo in human xenograft systems; (iii) the level of induction approached three logs using aminoglycoside activators including NB54, a newly synthesized aminoglycoside with significantly reduced toxicity; and (iv) expression levels could be appreciably altered when employing different promoters in a variety of cell types. These results strongly support the contention that this system should have important clinical applications when tight control of gene expression is required.
Assuntos
Regulação da Expressão Gênica , Terapia Genética/métodos , Processamento Pós-Transcricional do RNA/genética , Transplante Heterólogo/métodos , Aminoglicosídeos/genética , Aminoglicosídeos/metabolismo , Animais , Códon sem Sentido/genética , Vetores Genéticos , Células HEK293 , Células HeLa , Humanos , Lentivirus/genética , Camundongos , Camundongos Mutantes , Modelos Animais , Células PC12 , RNA Mensageiro , RatosRESUMO
Osteopontin (OPN) is a cytokine implicated in mediating responses to certain stressors, including mechanical, oxidative, and cellular stress. However, the involvement of OPN in responding to other physical and psychological stress is largely unexplored. Our previous research revealed that OPN is critical for hind limb-unloading induced lymphoid organ atrophy through modulation of corticosteroid production. In this study, we demonstrate that OPN(-/-) mice are resistant to chronic restraint stress (CRS)-induced lymphoid (largely thymus) organ atrophy; additionally, the stress-induced up-regulation of corticosterone production is significantly reduced in OPN(-/-) mice. Underlying this observation is the fact that normal adrenocorticotropic hormone levels are substantially reduced in the OPN(-/-) mice. Our data demonstrate both that injection of OPN into OPN-deficient mice enhances the CRS-induced lymphoid organ atrophy and that injection of a specific anti-OPN mAb (2C5) into wild-type mice ameliorates the CRS-induced organ atrophy; changes in corticosterone levels were also partially reversed. These studies reveal that circulating OPN plays a significant role in the regulation of the hypothalamus-pituitary-adrenal axis hormones and that it augments CRS-induced organ atrophy.
Assuntos
Corticosterona/sangue , Osteopontina/antagonistas & inibidores , Osteopontina/sangue , Estresse Psicológico/imunologia , Timo/patologia , Hormônio Adrenocorticotrópico/sangue , Animais , Anticorpos Monoclonais , Atrofia , Sistema Hipotálamo-Hipofisário/imunologia , Camundongos , Camundongos Knockout , Osteopontina/genética , Sistema Hipófise-Suprarrenal/imunologia , Restrição Física , Timo/imunologiaRESUMO
Interactions between CD40 and its ligand CD154 are involved in the progression of both cell mediated and innate immunity. These interactions are brought about by the transient expression of CD154 on activated CD4(+) T cells, which is regulated, in part, at the level of mRNA turnover. Here we have focused on analyzing the pattern of post-transcriptional regulation in mouse CD4(+) T cells in response to activation. Initial experiments identify a region of the murine CD154 mRNA that binds a polypyrimidine tract-binding protein-containing complex (mComplex I), which is activation-dependent and binds to a single CU-rich site within the 3' uTR Subsequent findings demonstrate that in vivo polyclonal activation of T cells leads to a pattern of differential CD154 mRNA stability that is directly dependent on extent of activation. Furthermore, in vitro activation of antigen-primed T cells shows that the CD154 mRNA half-life increases relative to that of unprimed cells. Importantly, this is the first report demonstrating that the regulation of CD154 in vivo is connected to an activation-induced program of mRNA decay and thus provides strong evidence for post-transcriptional mechanisms having a physiological role in regulating CD154 expression during an ongoing immune response.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Ligante de CD40/genética , Processamento Pós-Transcricional do RNA , Estabilidade de RNA , Regiões 3' não Traduzidas/genética , Regiões 3' não Traduzidas/metabolismo , Animais , Anticorpos Monoclonais/farmacologia , Antígenos/imunologia , Sequência de Bases , Antígenos CD28/imunologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Ensaio de Desvio de Mobilidade Eletroforética , Citometria de Fluxo , Humanos , Células Jurkat , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência do Ácido Nucleico , Fatores de TempoRESUMO
The employment of HIV-1-based vectors in clinical trials is controversial mainly due to the lethal nature of the virus. HIV-2 is less pathogenic in nature and therefore is likely to be safer for vector design and production. We developed HIV-2-based self-inactivating vectors in which 520 bp out of 554 bp of the viral U3 was deleted. Interestingly, high titers were obtained only when an exogenous promoter was used to drive expression of viral RNA. It was found that the vectors could target a wide range of mammalian cell types including primary neuronal cells and could yield long term expression. It is also noteworthy that the HIV-2 vectors could be effectively cross-packaged into HIV-1 core, which might provide for enhanced safety by reducing the recombination potential of the system.
Assuntos
Vetores Genéticos , HIV-2/genética , Transdução Genética , Animais , Linhagem Celular , Linhagem Celular Tumoral , Células Cultivadas , Clonagem Molecular , Deleção de Genes , Repetição Terminal Longa de HIV , Humanos , Plasmídeos/genética , Regiões Promotoras Genéticas , RatosRESUMO
Substantial progress in the development of new anti-viral drugs has taken place in recent years. Most of these new drugs belong to three groups of compounds, nucleoside analogs, thymidine kinase-dependent nucleotide analogs and specific viral enzyme inhibitors. Although these drugs revolutionized the treatment of several viral diseases, the involvement of the immune system is crucial for complete recovery and prevention of re-infection. New advances in the understanding of immune regulation mechanisms, mainly the role of cytokines, led to the development of several new immunologically-based anti-viral drugs and treatments. The most studied group of immunomodulators is the cytokines, some of which were shown to act as potent stimulators of immune responses. Other, non-cytokine immune modulators have also been successfully employed in both humans and experimental animals as anti-viral drugs of which several are currently in clinical trials. Advances in genetic engineering and transgenic mouse technologies facilitated the production of humanized as well as authentic human anti-viral monoclonal antibodies. Some of these antibodies proved to be clinically efficacious and are commercially produced as anti-viral drugs. As is often the case in anti-viral treatments, a combination of conventional and an immune-mediated anti-viral drugs or a combination therapy involving immunomodulators, therapeutic vaccines, immune intervention and even gene therapy might prove most efficacious as a treatment for a particular virus. Most of the advances made in anti-viral treatments have also been applied to the development of new vaccines. Some of the classical attenuated viruses are being replaced by recombinant attenuated viruses. Recombinant viral vaccines containing genes encoding other viral antigens and/or cytokines are being tested as new vaccines. Several chimeric viral vaccines have proven efficacious in experimental animals and are now in different phases of clinical trials. This review will encompass the major new developments in this field, including some that have not yet been subjected to human trials.
Assuntos
Adjuvantes Imunológicos/farmacologia , Antivirais/farmacologia , Vacinas Virais/imunologia , Animais , Anticorpos Monoclonais/uso terapêutico , Citocinas/fisiologia , Humanos , Vacinas de DNA/imunologia , Vacinas Sintéticas/imunologiaRESUMO
Stem cells are unique cell populations with the ability to undergo self-renewal and differentiation. These cells have been identified in a wide range of tissues and possess varied differentiation potentials. Tissue-specific stem cells have typically been thought to have limited differentiation capabilities. We show here that fibroblast-like cells isolated from mouse brain possess cross-germ layer differentiation abilities. These cells were found to express typical mesenchymal stem cell markers (CD44, CD29, and CD105) and were able to be passaged more than 50 times. When treated under defined conditions, the brain-derived cells were able to generate many different cell types including adipocytes, osteocytes, astrocytes, neurons, and even hepatocyte-like cells. The hepatocyte-like cells not only expressed liver cell-specific markers, but also exhibited the capacity for glycogen storage and low-density lipoprotein uptake. These results demonstrate the existence of cells in the brain with three-germ-layer differentiation potential.
Assuntos
Encéfalo/citologia , Diferenciação Celular/fisiologia , Linhagem da Célula/fisiologia , Células-Tronco/citologia , Animais , Antígenos de Diferenciação/metabolismo , Citometria de Fluxo , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos BALB C , Corantes de Rosanilina , Células-Tronco/fisiologiaRESUMO
INTRODUCTION: Local synthesis of interferon within B16 tumors mediates anti-tumor effects. Based on reports that stem cells are recruited to tumors, and because systemic administration of interferon causes dose-limiting undesirable side effects, we wanted to improve the anti-tumor effects of interferon while simultaneously minimizing its systemic side effects by employing mesenchymal stem cells (MSCs) as tumor-localized ectopic producers of interferon. Many vectors exist to fulfill this purpose, but their transfection efficiency and resulting expression levels vary considerably. METHODS: To follow both the recruitment to tumors and the synthesis of interferon by MSCs, we designed a bicistronic vector system that permits fluorescent visualization of vector-transfected and interferon-producing MSCs. We used Mu-IFNαA cDNA as the first cistron and the cherry fluorescent protein cDNA as the second cistron, whose translation requires the internal ribosome entry sequence (IRES) from the encephalomyocarditis virus 5' untranslated region. Observing inconsistent expression of these cistrons in various vectors and cell lines, especially compared with a control plasmid pmaxGFP, we optimized the expression of this bicistronic message by mutating pcDNA3 to facilitate exchange of the promoter and polyadenylation segments controlling both the gene of interest and the eukaryotic antibiotic resistance gene as well as the eukaryotic antibiotic resistance gene itself, and effectively compare the effects of these exchanges, creating plasmid pc3.5. RESULTS: Murine MSCs stably and ectopically expressing Mu-IFNαA inhibited the establishment of tumors in homogeneic C57/BL6 mice. Mu-IFNαA expressed from the bicistronic message is fully biologically active, but is expressed at only two-thirds of the level observed from a monocistronic message. Cap-dependent translation is threefold more efficient than IRES-driven translation in 293T, B16, and MSC cell lines. Both efficient expression and good transfection efficiency require strong expression of the gene of interest and a chimeric intron. High doses of Mu-IFNαA within tumors inhibited tumor establishment but may not inhibit tumor growth. CONCLUSIONS: Our modified vector and its derived plasmids will find use in stem cell therapeutics, gene expression, mRNA regulation, and transcription regulation. Local release of Mu-IFNαA within tumors may differently affect tumor establishment and tumor growth.
Assuntos
Células-Tronco Mesenquimais/metabolismo , Plasmídeos/metabolismo , Regiões 5' não Traduzidas , Animais , Linhagem Celular Tumoral , Vírus da Encefalomiocardite/genética , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Células HEK293 , Humanos , Interferon-alfa/genética , Interferon-alfa/metabolismo , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias/prevenção & controle , Plasmídeos/genética , Regiões Promotoras Genéticas , TransfecçãoRESUMO
Selective immunoglobulin A deficiency (sIgAD) is the most common immunodeficiency in humans. Auto-reactive antibodies to human immunoglobulin A (IgA) are found in the serum of 20-40% of individuals with sIgAD. It is unknown whether these antibodies play a role in the pathogenesis of this immunodeficiency and although the prevailing thought is that they are secondary to the onset of sIgAD, there is very little, if any, support for this notion. Here, we propose that anti-IgA antibodies are in fact responsible for the removal of IgA from serum, and that the inducing antigen is most probably a xenogeneic IgA. This hypothesis is based on data obtained from an sIgAD patient in whom changes in dietary consumption of beef and/or bovine dairy products resulted in changes in anti-IgA levels in the serum. To test the hypothesis, the presence of anti-bovine IgA antibodies was tested by a highly specific enzyme-linked immunosorbent assay in serum samples from IgA-deficient and control individuals. All 13 sIgAD individuals with anti-IgA antibodies had a higher titer against bovine IgA than against human IgA. Of 23 control individuals, a surprisingly high proportion (65%) was also found to have IgG anti-bovine IgA antibodies. These results support the hypothesis that the anti-human IgA antibodies found in IgA-deficient individuals are originally produced against bovine IgA. These antibodies are found in many normal individuals, but only in cases where they cross react with endogenous human IgA, sIgAD may develop.
Assuntos
Anticorpos Anti-Idiotípicos/imunologia , Bovinos/imunologia , Deficiência de IgA/imunologia , Imunoglobulina A/imunologia , Animais , Anticorpos Anti-Idiotípicos/sangue , Reações Cruzadas/imunologia , Ensaio de Imunoadsorção Enzimática , Humanos , Deficiência de IgA/sangue , Imunoglobulina A/sangue , Imunoglobulina G/sangue , Imunoglobulina G/imunologiaRESUMO
A significant difference in the recombination rates between human immunodeficiency virus type 1 (HIV-1) and the gammaretroviruses was previously reported, with the former being 10 to 100 times more recombinogenic. It is possible that preferential copackaging of homodimers in the case of gammaretroviruses, like murine leukemia virus (MLV), led to the underestimation of their rates of recombination. To reexamine the recombination rates for MLV, experiments were performed to control for nonrandom copackaging of viral RNA, and it was found that MLV and HIV-1 exhibit similar crossover rates. The implications for control of proviral ploidy and preferential recombination during minus-strand DNA synthesis are discussed.
Assuntos
Troca Genética/genética , HIV-1/genética , Vírus da Leucemia Murina de Moloney/genética , Ploidias , RNA Viral/genética , Montagem de Vírus/genética , Animais , Células HeLa , Humanos , Camundongos , Células NIH 3T3RESUMO
Human immunodeficiency virus type 1 (HIV-1), the causative agent of AIDS in humans, exhibits a very high rate of recombination. Bearing in mind the significant epidemiological and clinical contrast between HIV-2 and HIV-1 as well as the critical role that recombination plays in viral evolution, we examined the nature of HIV-2 recombination. Towards this end, a strategy was devised to measure the rate of crossover of HIV-2 by evaluating recombinant progeny produced exclusively by heterodimeric virions. The results showed that HIV-2 exhibits a crossover rate similar to that of HIV-1 and murine leukemia virus, indicating that the extremely high rate of crossover is a common retroviral feature.
Assuntos
Troca Genética/genética , Evolução Molecular , HIV-2/genética , Provírus/genética , Vírion/genética , Vetores Genéticos/genética , Modelos GenéticosRESUMO
Human immunodeficiency virus type 1 (HIV-1) is not eliminated from patients even after years of antiretroviral therapy, apparently due to the presence of latently infected cells. Here we describe the development of a cell-based system of latency that can be used for high-throughput screening aimed at novel drug discovery to eradicate HIV-1 infection.
Assuntos
Fármacos Anti-HIV/farmacologia , Infecções por HIV/fisiopatologia , HIV-1/efeitos dos fármacos , Latência Viral/fisiologia , Terapia Antirretroviral de Alta Atividade , HIV-1/fisiologia , Humanos , Modelos Biológicos , Replicação Viral/efeitos dos fármacosRESUMO
The sequence of events that leads to the development of a T-dependent B-cell response has been studied for many years and much attention has focused on the importance of these interactions for the B cell and the antibody response. While a role for B cells in T-cell memory has been demonstrated, the cellular and molecular mechanisms underlying this are unclear and the importance for T cells to migrate into follicles and interact with B cells has received relatively little attention. These interactions between T and B lymphocytes are facilitated by a co-ordinated series of migration events within lymph nodes and here we propose that this migration and these events are essential for the terminal differentiation of all CD4+ T cells and without it tolerance may result.
Assuntos
Linfócitos B/imunologia , Linfonodos/imunologia , Linfócitos T/imunologia , Animais , Formação de Anticorpos/imunologia , Linfócitos T CD4-Positivos/imunologia , Diferenciação Celular/imunologia , Movimento Celular/imunologia , Humanos , Tolerância Imunológica/imunologia , Imunidade Celular/imunologia , Memória Imunológica/imunologia , Camundongos , Modelos ImunológicosRESUMO
Experimental autoimmune encephalomyelitis (EAE), an animal model for human multiple sclerosis (MS), is a typical CD4(+) T-cell-mediated autoimmune disease of the central nervous system (CNS) characterized by perivascular inflammation culminating in focal demyelinations. Like MS, EAE induced by proteolipid protein (PLP) usually follows the form of a relapsing-remitting disease. We have previously described an immunotherapy model in which infusion of autologous B cells expressing the PLP encephalitogenic determinant induced PLP-specific unresponsiveness and protected mice from induction of EAE. Here we show that the same treatment when initiated after disease onset, which resembles the clinical situation presented in MS, completely protects all treated animals from further relapses. We also show that protected animals were unresponsive to PLP as measured by delayed-type hypersensitivity (DTH). This represents a novel immunotherapeutic approach that can be exploited to develop treatments for human MS and other T-cell-mediated autoimmune diseases.
Assuntos
Autoantígenos/imunologia , Linfócitos B/imunologia , Encefalomielite Autoimune Experimental/imunologia , Transfusão de Linfócitos , Animais , Encefalomielite Autoimune Experimental/prevenção & controle , Feminino , Hipersensibilidade Tardia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos EndogâmicosRESUMO
The regulatory elements governing the process of lymphopoiesis from pluripotential stem cells to mature lymphocytes are not well understood. In this study we found that in bone marrow chimeras made by reconstituting lethally irradiated normal mice with bone marrow taken from genetically B-cell-deficient animals (microMT.B6 --> F1) the B-cell compartment is reconstituted with host-derived B cells. Similarly, in animals reconstituted with bone marrow taken from mice with genetic deficiencies in the development of T cells (TCR-/- --> F1) or both B and T cells (RAG-/- --> F1), the missing lymphocyte lineage(s) was specifically reconstituted from host-derived cells. In all chimeras, all other blood lineages were generated from donor-derived stem cells. Control chimeras (B6 --> F1) had only donor-derived hematopoietic cells as expected. The reconstituted, host-derived lymphoid compartments contained normal functional cell populations as determined by the presence of T cells expressing all 16 common TCR Vbeta families, and the presence of all antibody isotypes in the serum. Reconstituted TCR-/- --> F1 chimeras were also able to mount T-cell proliferative responses to foreign antigens equal to those of control animals. This observation would seem to suggest that during lymphopoietic reconstitution, missing lymphoid lineages can dictate their own reconstitution.
Assuntos
Transplante de Medula Óssea , Células-Tronco Hematopoéticas/citologia , Leucopoese , Linfócitos/citologia , Células-Tronco Pluripotentes/transplante , Animais , Linfócitos B/citologia , Linhagem da Célula , Feminino , Masculino , Camundongos , Camundongos Knockout , Células-Tronco Pluripotentes/citologia , Linfócitos T/citologia , Quimeras de Transplante , Irradiação Corporal TotalRESUMO
Multiple sclerosis is an inflammatory, demyelinating disease of the CNS. Whereas oligodendrocytes have been considered the primary neural cell type most affected, recent evidence indicates that axonal and neuronal degeneration also occurs in both multiple sclerosis and experimental autoimmune encephalomyelitis (EAE), an animal model reproducing many features of multiple sclerosis. The molecular mechanisms underlying neuronal deficits in multiple sclerosis and EAE remain elusive. To address this issue, we have analysed the expression of genes encoding proteins that play critical roles in ion homeostasis, exocytosis, mitochondrial function and impulse conduction in the Lewis rat lumbar spinal cord during the clinical course of acute EAE. Transcript and protein levels of plasma membrane Ca(2+) ATPase 2 (PMCA2), an essential ion pump expressed exclusively in grey matter and involved in Ca(2+) extrusion, synapsin IIa and syntaxin 1B, important regulators of vesicular exocytosis, were dramatically decreased coincident with the onset of clinical symptoms. In contrast, changes in the expression of several other ion pumps, vesicular proteins, mitochondrial enzymes and sodium channels occurred at more advanced disease stages. Moreover, exposure of spinal cord slice cultures to kainic acid significantly reduced PMCA2 mRNA levels. Taken together, our findings suggest that glutamate, which recently has been implicated in EAE pathogenesis, suppresses neuronal PMCA2 expression leading to Ca(2+) dyshomeostasis at initial clinical phases. Consequently, perturbations in Ca(2+) balance and neurotransmitter exocytosis may partially underlie aberrant neuronal function and communication at onset of symptoms. Altered mitochondrial function and impulse conduction may exacerbate neurological deficits at subsequent disease stages.
Assuntos
ATPases Transportadoras de Cálcio/genética , Encefalomielite Autoimune Experimental/genética , Regulação da Expressão Gênica , Doença Aguda , Animais , Cálcio/metabolismo , ATPases Transportadoras de Cálcio/metabolismo , Proteínas de Transporte de Cátions , Técnicas de Cultura , Modelos Animais de Doenças , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Encefalomielite Autoimune Experimental/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Hibridização In Situ , Ácido Caínico/farmacologia , Esclerose Múltipla/genética , Esclerose Múltipla/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos/métodos , ATPases Transportadoras de Cálcio da Membrana Plasmática , RNA Mensageiro/genética , Ratos , Ratos Endogâmicos Lew , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Canais de Sódio/metabolismo , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismoRESUMO
HIV-1 evolves rapidly, which is thought to result from one or more error-prone steps in the virus life cycle. Because HIV-1 reverse transcriptase (RT) does not possess 3'- to 5'-exonucleolytic proofreading activity and because RT has been shown to be error-prone in cell free systems, it should be an important contributor to the high rate of HIV-1 mutation. However, because RNA polymerase II (pol II) synthesizes viral RNA, it might also contribute significantly to HIV-1 mutagenesis. To assess the relative contributions of RT and RNA pol II to HIV-1 mutagenesis, a system was established to study the rate and nature of mutations in HIV-1 long terminal repeats (LTRs). Owing to the unique nature of replication at the ends of the viral genome, mutational analysis of retroviral LTRs provides an opportunity to evaluate the relative contribution of HIV-1 RT and RNA pol II to viral mutagenesis. Mutational analysis was performed on both LTRs of 215 proviruses, restricted to a single cycle of replication, employing single-stranded conformational polymorphism and DNA sequencing allowing direct identification of mutations in the absence of selection and within autologous viral sequences. A total of 21 independent mutations was identified. Ten mutations were observed in both LTRs, which could have been introduced by either RT or RNA pol II, whereas the other eleven mutations were only present in a single LTR and could only have been introduced by RT. This provides the first direct evidence that HIV-1 RT contributes significantly to HIV-1 mutagenesis and is likely to be the primary engine for HIV-1 mutagenesis. Moreover, mutations were observed at the U3-R border, but the nature of the mutations and their frequency differed from experiments performed using cell-free systems suggesting that other viral and/or cellular factors contribute to fidelity at the ends of the viral genome.