RESUMO
We and others previously reported potent antileukemia efficacy of CD123-redirected chimeric antigen receptor (CAR) T cells in preclinical human acute myeloid leukemia (AML) models at the cost of severe hematologic toxicity. This observation raises concern for potential myeloablation in patients with AML treated with CD123-redirected CAR T cells and mandates novel approaches for toxicity mitigation. We hypothesized that CAR T-cell depletion with optimal timing after AML eradication would preserve leukemia remission and allow subsequent hematopoietic stem cell transplantation. To test this hypothesis, we compared 3 CAR T-cell termination strategies: (1) transiently active anti-CD123 messenger RNA-electroporated CART (RNA-CART123); (2) T-cell ablation with alemtuzumab after treatment with lentivirally transduced anti-CD123-4-1BB-CD3ζ T cells (CART123); and (3) T-cell ablation with rituximab after treatment with CD20-coexpressing CART123 (CART123-CD20). All approaches led to rapid leukemia elimination in murine xenograft models of human AML. Subsequent antibody-mediated depletion of CART123 or CART123-CD20 did not impair leukemia remission. Time-course studies demonstrated that durable leukemia remission required CAR T-cell persistence for 4 weeks prior to ablation. Upon CAR T-cell termination, we further demonstrated successful hematopoietic engraftment with a normal human donor to model allogeneic stem cell rescue. Results from these studies will facilitate development of T-cell depletion strategies to augment the feasibility of CAR T-cell therapy for patients with AML.
Assuntos
Transplante de Células-Tronco Hematopoéticas , Imunoterapia Adotiva/métodos , Subunidade alfa de Receptor de Interleucina-3/imunologia , Leucemia Mieloide Aguda/terapia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/imunologia , Alemtuzumab , Animais , Anticorpos Monoclonais Humanizados/farmacologia , Antígenos CD20/genética , Antígenos CD20/imunologia , Complexo CD3/genética , Complexo CD3/imunologia , Feminino , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Subunidade alfa de Receptor de Interleucina-3/antagonistas & inibidores , Subunidade alfa de Receptor de Interleucina-3/genética , Lentivirus/genética , Lentivirus/metabolismo , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/imunologia , Leucemia Mieloide Aguda/patologia , Depleção Linfocítica , Masculino , Camundongos , Camundongos Endogâmicos NOD , RNA Antissenso/genética , RNA Antissenso/imunologia , RNA Mensageiro/genética , RNA Mensageiro/imunologia , Receptores de Antígenos de Linfócitos T/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Indução de Remissão , Rituximab/farmacologia , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/transplante , Transplante Heterólogo , Resultado do Tratamento , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/genética , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Although more than half of genomic loci are believed to have antisense transcription, whether antisense transcription is involved in cytokine expression has not been studied. In this study, we show that some loci of innate immunity related genes do have antisense transcripts. We investigated the effect of several antisense RNAs, including anti-4-1BBL, anti-p100, and anti-IL-1ß, on their cognate sense gene's expression in macrophages. We found that overexpression of antisense IL-1ß transcript suppressed IL-1ß expression. Anti-IL-1ß is complementary to the sequence in the 5' upstream region of the IL-1ß promoter. Its mediated inhibition of IL-1ß production occurred at the transcriptional level. Anti-IL-1ß did not alter the methylation status of the IL-1ß promoter. However, chromatin immunoprecipitation assays revealed that the anti-IL-1ß transcript can change the chromatin structure of the IL-1ß promoter by decreasing H3K4 trimethylation on the promoter, which is at least part of the mechanism underlying the reduced binding of RNA polymerase II to the IL-1ß promoter upon anti-IL-1ß expression. Our data suggest that some antisense transcripts of innate immunity-related genes play a role by regulating cytokine expression.
Assuntos
Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Interleucina-1beta/genética , RNA Antissenso/genética , Animais , Linhagem Celular , Imunoprecipitação da Cromatina , Regulação da Expressão Gênica/genética , Imunidade Inata/imunologia , Interleucina-1beta/imunologia , Lipopolissacarídeos/imunologia , Camundongos , Regiões Promotoras Genéticas/genética , Regiões Promotoras Genéticas/imunologia , RNA Antissenso/imunologia , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Previous studies have confirmed long noncoding RNA LEMD1-AS1 (LEMD1-AS1) as a functional factor in several tumors. The present work is aimed at exploring the prognostic and diagnostic values of LEMD1-AS1 in patients with epithelial ovarian cancer (EOC). We examined the expressions of LEMD1-AS1 in pan-cancer from TCGA microarray datasets and GTEx Project. The expressions of LEMD1-AS1 were detected by qRT-PCR in EOC specimens and normal ovarian specimens from 30 EOC patients. The χ 2 test was applied to compare the clinicopathological characteristics of different groups. ROC curves were established to determine the diagnostic values of LEMD1-AS1 in screening EOC tissues. The association of LEMD1-AS1 expression with clinical outcome was determined by the Kaplan-Meier methods and COX assays. A decreased expression of LEMD1-AS1 was observed in EOC tissues compared to matched normal specimens (p < 0.01). Low LEMD1-AS1 expression could be used to distinguish EOC from adjacent normal specimens. A clinical study revealed that patients with low LEMD1-AS1 expression have a shorter overall survival (p = 0.035) and progress-free interval (p = 0.041) than those with high LEMD1-AS1 expression. The Spearman correlation test revealed that LEMD1-AS1 expressions were negatively associated with the expressions of neutrophil and myeloid dendritic cell. Overall, our finding suggested that LEMD1-AS1 may have potential roles as a potential biomarker and/or a therapeutic target in EOC.
Assuntos
Carcinoma Epitelial do Ovário , Proteínas de Neoplasias , Neoplasias Ovarianas , RNA Antissenso , Carcinoma Epitelial do Ovário/diagnóstico , Carcinoma Epitelial do Ovário/genética , Carcinoma Epitelial do Ovário/imunologia , Carcinoma Epitelial do Ovário/patologia , Regulação para Baixo , Feminino , Humanos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/imunologia , Neoplasias Ovarianas/diagnóstico , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/imunologia , Neoplasias Ovarianas/patologia , Prognóstico , RNA Antissenso/genética , RNA Antissenso/imunologia , RNA Longo não Codificante/genética , RNA Longo não Codificante/imunologiaRESUMO
Both malignant pleural mesothelioma and advanced stage lung cancer are associated with a poor prognosis. Unfortunately, current treatment regimens have had only a modest effect on their progressive course. Gene therapy for thoracic malignancies represents a novel therapeutic approach and has been evaluated in a number of clinical trials over the last two decades. Using viral vectors or anti-sense RNA, strategies have included induction of apoptosis, tumor suppressor gene replacement, suicide gene expression, cytokine-based therapy, various vaccination approaches, and adoptive transfer of modified immune cells. This review will consider the clinical results, limitations, and future directions of gene therapy trials for thoracic malignancies.
Assuntos
Terapia Genética/métodos , Neoplasias Pulmonares/terapia , Mesotelioma/terapia , Neoplasias Pleurais/terapia , Transferência Adotiva/métodos , Animais , Apoptose/genética , Apoptose/imunologia , Vacinas Anticâncer/genética , Vacinas Anticâncer/imunologia , Vacinas Anticâncer/uso terapêutico , Ensaios Clínicos como Assunto , Citocinas/genética , Citocinas/imunologia , Genes Transgênicos Suicidas/genética , Genes Transgênicos Suicidas/imunologia , Genes Supressores de Tumor , Vetores Genéticos/genética , Vetores Genéticos/imunologia , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/imunologia , Mesotelioma/diagnóstico , Mesotelioma/genética , Mesotelioma/imunologia , Neoplasias Pleurais/diagnóstico , Neoplasias Pleurais/genética , Neoplasias Pleurais/imunologia , Prognóstico , RNA Antissenso/genética , RNA Antissenso/imunologia , Vírus/genéticaRESUMO
CD39 is an ectonucleotidase that initiates conversion of extracellular nucleotides into immunosuppressive adenosine. CD39 is expressed by regulatory T (Treg)-cells, where it mediates immunosuppression, and by a subset of T-helper (Th) 17-cells, where it limits pathogenicity. CD39 is regulated via single-nucleotide-polymorphisms and upon activation of aryl-hydrocarbon-receptor and oxygen-mediated pathways. Here we report a mechanism of CD39 regulation that relies on the presence of an endogenous antisense RNA, transcribed from the 3'-end of the human CD39/ENTPD1 gene. CD39-specific antisense is increased in Treg and Th17-cells of Crohn's disease patients over controls. It largely localizes in the cell nucleus and regulates CD39 by interacting with nucleolin and heterogeneous-nuclear-ribonucleoprotein-A1. Antisense silencing results in CD39 upregulation in vitro and amelioration of disease activity in a trinitro-benzene-sulfonic-acid model of colitis in humanized NOD/scid/gamma mice. Inhibition/blockade of antisense might represent a therapeutic strategy to restore CD39 along with immunohomeostasis in Crohn's disease.
Assuntos
Antígenos CD/genética , Apirase/genética , Doença de Crohn/genética , RNA Antissenso/genética , Animais , Antígenos CD/imunologia , Apirase/imunologia , Doença de Crohn/imunologia , Feminino , Humanos , Camundongos , Camundongos Endogâmicos NOD , RNA Antissenso/imunologia , Linfócitos T Reguladores/imunologia , Células Th17/imunologiaRESUMO
Autoimmune diseases affect approximately 1 in 21 persons in the United States. Treatment often requires long-term cytotoxic therapy. How and why these deleterious diseases occur is unclear. A serendipitous finding in our laboratory using serum from patients with autoimmune vasculitis led us to develop the theory of autoantigen complementarity, a novel concept that may elucidate the etiological and pathogenetic mechanisms underlying autoimmune disease in general. The theory proposes that the inciting immunogen that elicits a cascade of immunological events is not the self-antigen (the autoantigen) or its mimic but rather a protein that is complementary in surface structure to the autoantigen; that is, a protein homologous or identical to the amino acid sequence of translated antisense RNA from the noncoding strand of the autoantigen gene. The cascade begins when this complementary protein initiates the production of antibodies that in turn elicit an anti-antibody or anti-idiotypic response. These anti-idiotypic antibodies can now react with the autoantigen. Strikingly, homology search of complementary proteins yields microbial and fungal proteins, thus indicating that invading micro-organisms can deliver the inciting immunogen. Curiously, approximately 50% of our patients transcribe the complementary protein's antisense RNA. If it transpires that these aberrant RNAs are translated, the complementary protein would be produced by the individual. Here we review published research investigating complementary proteins, anti-idiotypic immune responses, and antisense transcripts, all of which support complementary proteins as initiators of autoimmune disease. In addition, we provide possible microbial and/or fungal organisms that may incite some of the most studied autoimmune diseases. Lastly, we propose mechanisms by which cell-mediated autoimmunity can be triggered by autoantigen complementarity. Based on our data and the contributions of the researchers described in this review, identification of proteins complementary to autoantigens is likely to be informative in most autoimmune diseases. This vein of study is in the early phases; however, we expect "autoantigen complementarity" is an underlying mechanism in many autoimmune diseases.
Assuntos
Autoantígenos/imunologia , Doenças Autoimunes/imunologia , Animais , Humanos , Idiótipos de Imunoglobulinas/imunologia , Linfócitos/imunologia , RNA Antissenso/imunologiaAssuntos
Terapia Genética/métodos , Neoplasias Pulmonares/terapia , Mesotelioma/terapia , Neoplasias Pleurais/terapia , Transferência Adotiva/métodos , Animais , Apoptose/genética , Apoptose/imunologia , Vacinas Anticâncer/genética , Vacinas Anticâncer/imunologia , Vacinas Anticâncer/uso terapêutico , Ensaios Clínicos como Assunto , Citocinas/genética , Citocinas/imunologia , Genes Transgênicos Suicidas/genética , Genes Transgênicos Suicidas/imunologia , Genes Supressores de Tumor , Vetores Genéticos/genética , Vetores Genéticos/imunologia , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/imunologia , Mesotelioma/diagnóstico , Mesotelioma/genética , Mesotelioma/imunologia , Neoplasias Pleurais/diagnóstico , Neoplasias Pleurais/genética , Neoplasias Pleurais/imunologia , Prognóstico , RNA Antissenso/genética , RNA Antissenso/imunologia , Vírus/genéticaRESUMO
In this study we report the detection of autoantibodies to the nucleolar U3- and Th(7-2) ribonucleoprotein (RNP) particles in sera from patients with connective tissue diseases. The method described employs radioactively labelled antisense U3- and Th RNA which are hybridized to immunoprecipitated U3- or Th RNA from a HeLa cell extract. Of the 66 sera that were screened with this method seven sera (11%) precipitated only Th RNP, 16 sera (24%) precipitated only U3 RNP and 4 sera (6%) precipitated both U3- and Th RNP. Both anti-U3 RNP and anti-Th RNP activity appeared to be mostly associated with scleroderma or scleroderma-associated diseases. Using this method we also showed that some of the Th RNP particles in a cell extract are associated with the La autoantigen. We conclude that for the identification of immunoprecipitated RNAs this method is very sensitive and provides unambiguous data.
Assuntos
Anticorpos Antinucleares/análise , Endorribonucleases/imunologia , RNA Antissenso/imunologia , Ribonucleoproteínas Nucleares Pequenas/imunologia , Escleroderma Sistêmico/imunologia , Autoantígenos/imunologia , Sequência de Bases , Northern Blotting , Endorribonucleases/genética , Células HeLa , Humanos , Lúpus Eritematoso Sistêmico/imunologia , Dados de Sequência Molecular , Região Organizadora do Nucléolo/imunologia , Testes de Precipitina , RNA/imunologia , Ribonucleoproteínas Nucleares Pequenas/genéticaRESUMO
BACKGROUND: Intercellular adhesion molecule-1 (ICAM-1) is strongly induced under inflammatory conditions associated with allograft rejection, thereby promoting leukocyte recruitment and activation at the site of inflammation. Enhancement of ICAM-1 expression can also be the result of viral infection, in particular human cytomegalovirus (CMV), a frequent source of complications in the transplant recipient. In vitro studies have shown that CMV infection of endothelial cells (EC) results in the direct enhancement of ICAM-1 expression and consequent leukocyte adhesion/activation suggesting mechanisms by which CMV exacerbates graft vascular disease. Although treatment of EC with ICAM-1-specific antisense oligonucleotides has been shown to attenuate ICAM-1 induction under simulated inflammatory conditions (i.e., TNF-alpha), no studies have addressed their effectiveness on virally-induced ICAM-1 expression. RESULTS: In the current investigation, we show that the progressive increase in endothelial ICAM-1 protein expression that follows inoculation with CMV correlates with a progressive accumulation of ICAM-1 mRNA. Furthermore, we demonstrate that treatment of EC with a partially 2'-O-methoxyethyl modified ICAM-1-specific antisense oligonucleotide before viral inoculation significantly reduces CMV-associated induction of ICAM-1 protein and mRNA expression. Finally, we show that antisense-mediated attenuation in ICAM-1 expression results in a significant reduction of T lymphocyte adhesion to CMV-infected EC monolayers, an interaction that has been implicated in allogeneic T lymphocyte activation, in viral transmission to transiently adherent leukocytes and subsequent hematogenous dissemination. CONCLUSIONS: These findings demonstrate for the first time that antisense oligonucleotides can effectively reverse virally-induced host cellular protein expression, specifically ICAM-1, as well as consequent T lymphocytes adhesion, thus broadening the potential clinical utility of antisense oligonucleotides.
Assuntos
Infecções por Citomegalovirus/imunologia , Citomegalovirus , Endotélio Vascular/imunologia , Molécula 1 de Adesão Intercelular/imunologia , RNA Antissenso/imunologia , Linfócitos T/imunologia , Adesão Celular , Endotélio Vascular/patologia , Endotélio Vascular/virologia , Regulação da Expressão Gênica/imunologia , Rejeição de Enxerto , Humanos , Molécula 1 de Adesão Intercelular/biossíntese , Molécula 1 de Adesão Intercelular/genética , Transplante de Órgãos , RNA Antissenso/genética , RNA Antissenso/farmacologia , RNA Mensageiro/biossíntese , Linfócitos T/patologia , Imunologia de TransplantesRESUMO
Glycoprotein gp43 is an immunodominant diagnostic antigen for paracoccidioidomycosis caused by Paracoccidioides brasiliensis. It is abundantly secreted in isolates such as Pb339. It is structurally related to beta-1,3-exoglucanases, however inactive. Its function in fungal biology is unknown, but it elicits humoral, innate and protective cellular immune responses; it binds to extracellular matrix-associated proteins. In this study we applied an antisense RNA (aRNA) technology and Agrobacterium tumefaciens-mediated transformation to generate mitotically stable PbGP43 mutants (PbGP43 aRNA) derived from wild type Pb339 to study its role in P. brasiliensis biology and during infection. Control PbEV was transformed with empty vector. Growth curve, cell vitality and morphology of PbGP43 aRNA mutants were indistinguishable from those of controls. PbGP43 expression was reduced 80-85% in mutants 1 and 2, as determined by real time PCR, correlating with a massive decrease in gp43 expression. This was shown by immunoblotting of culture supernatants revealed with anti-gp43 mouse monoclonal and rabbit polyclonal antibodies, and also by affinity-ligand assays of extracellular molecules with laminin and fibronectin. In vitro, there was significantly increased TNF-α production and reduced yeast recovery when PbGP43 aRNA1 was exposed to IFN-γ-stimulated macrophages, suggesting reduced binding/uptake and/or increased killing. In vivo, fungal burden in lungs of BALB/c mice infected with silenced mutant was negligible and associated with decreased lung ΙΛ-10 and IL-6. Therefore, our results correlated low gp43 expression with lower pathogenicity in mice, but that will be definitely proven when PbGP43 knockouts become available. This is the first study of gp43 using genetically modified P. brasiliensis.
Assuntos
Antígenos de Fungos/genética , Antígenos de Fungos/imunologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/imunologia , Glicoproteínas/genética , Glicoproteínas/imunologia , Paracoccidioides/genética , Paracoccidioides/imunologia , Fatores de Virulência/genética , Fatores de Virulência/imunologia , Animais , Antígenos de Fungos/metabolismo , Proteínas Fúngicas/metabolismo , Expressão Gênica/genética , Expressão Gênica/imunologia , Vetores Genéticos/genética , Vetores Genéticos/imunologia , Vetores Genéticos/metabolismo , Glicoproteínas/metabolismo , Interleucina-6/genética , Interleucina-6/imunologia , Interleucina-6/metabolismo , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Mutação/genética , Mutação/imunologia , Paracoccidioides/metabolismo , Paracoccidioidomicose/genética , Paracoccidioidomicose/imunologia , Paracoccidioidomicose/metabolismo , Paracoccidioidomicose/microbiologia , RNA Antissenso/genética , RNA Antissenso/imunologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Fatores de Virulência/metabolismoAssuntos
Oligopeptídeos/imunologia , RNA Antissenso/imunologia , RNA Bacteriano/imunologia , Vacinas Antiestafilocócicas/imunologia , Staphylococcus aureus/imunologia , Staphylococcus aureus/patogenicidade , Vacinas Sintéticas/imunologia , Animais , Humanos , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/prevenção & controle , VacinaçãoRESUMO
Retroviruses pack multiple genes into relatively small genomes by encoding several genes in the same genomic region with overlapping reading frames. Both sense and antisense HIV-1 transcripts contain open reading frames for known functional proteins as well as numerous alternative reading frames (ARFs). At least some ARFs have the potential to encode proteins of unknown function, and their antigenic properties can be considered as cryptic epitopes (CEs). To examine the extent of active immune response to virally encoded CEs, we analyzed human leukocyte antigen class I-associated polymorphisms in HIV-1 gag, pol, and nef genes from a large cohort of South Africans with chronic infection. In all, 391 CEs and 168 conventional epitopes were predicted, with the majority (307; 79%) of CEs derived from antisense transcripts. In further evaluation of CD8 T cell responses to a subset of the predicted CEs in patients with primary or chronic infection, both sense- and antisense-encoded CEs were immunogenic at both stages of infection. In addition, CEs often mutated during the first year of infection, which was consistent with immune selection for escape variants. These findings indicate that the HIV-1 genome might encode and deploy a large potential repertoire of unconventional epitopes to enhance vaccine-induced antiviral immunity.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Epitopos de Linfócito T/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , RNA Antissenso/imunologia , RNA Viral/imunologia , Transcrição Gênica/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular , Doença Crônica , Estudos de Coortes , Epitopos de Linfócito T/biossíntese , Epitopos de Linfócito T/genética , Evolução Molecular , Feminino , Produtos do Gene gag/genética , Produtos do Gene gag/imunologia , Produtos do Gene gag/metabolismo , Genes MHC Classe I/genética , Genes MHC Classe I/imunologia , Infecções por HIV/genética , HIV-1/genética , HIV-1/metabolismo , Humanos , Masculino , Polimorfismo Genético , RNA Antissenso/genética , RNA Antissenso/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , África do Sul , Produtos do Gene nef do Vírus da Imunodeficiência Humana/genética , Produtos do Gene nef do Vírus da Imunodeficiência Humana/imunologia , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismo , Produtos do Gene pol do Vírus da Imunodeficiência Humana/genética , Produtos do Gene pol do Vírus da Imunodeficiência Humana/imunologia , Produtos do Gene pol do Vírus da Imunodeficiência Humana/metabolismoRESUMO
Pollen of many grasses, trees, and weeds are the source of inhalant allergic proteins while various other plant products are allergenic only upon their ingestion as a food source. Allergenic proteins of pollen are exposed to human immune system after their rapid release from pollen upon coming in contact with moist surface of nasal mucosa. The advent of molecular cloning and ability to genetically transform plants now offer unprecedented opportunities to produce hypoallergenic plants by targeted switching off allergen production. Gene silencing strategies that operate at post-transcriptional level are highly suitable for blocking allergen production. We have demonstrated the concept of allergen gene silencing through antisense approach by producing ryegrass plants that do not produce major allergen in its pollen. Our results show the potential of antisense approach in reducing the allergenic potential of plants. Such a strategy can have a general applicability for production of transgenic plants depleted of both inhaled and ingested allergens. In addition, such an approach could also help in elucidating the in vivo function of allergen(s) in plants and contribution of an allergen to overall allergenic potential of an allergen source.
Assuntos
Alérgenos/imunologia , Técnicas de Transferência de Genes , Plantas Geneticamente Modificadas/imunologia , Plantas/imunologia , Proteínas Recombinantes/imunologia , Alérgenos/genética , Plantas/genética , RNA Antissenso/genética , RNA Antissenso/imunologia , Proteínas Recombinantes/genéticaRESUMO
CIITA (class II transactivator) is a coactivator essential for transcription of MHC class II genes. In this study, a construct with a mutated CIITA gene with N-terminal domains depleted was constructed. This mutated CIITA (mCIITA) was able to repress DR and DQ expression in 45.0-60.0% of the mCIITA-transfected clones of swine endothelial cell line PIEC and in B cell line L23, as well as in human cell lines HeLa and Raji. Similarly, 30.0-46.7% of swine cell clones containing the human CIITA antisense RNA also failed to express DR molecules. However, the persistence of the DR repression on the cell lines is quite different. Transfection with mCIITA was persistent for at least 120 days, while with the CIITA antisense RNA, persistence existed for only 35-45 days. To explore the underlying mechanism, Raji cells were transfected with pUHD10-3-mCIITA, a mCIITA-containing, doxycycline-dependent plasmid. The intensity of DR repression is correlated quite well with the efficiencies of the mCIITA expression within the cells in a doxycycline dose-dependent manner. This implicates a competition between the mCIITA and its endogenous full-length counterpart. In addition, we were able to show that purified human CD4 T cells did not respond to the mCIITA-transfected PIECs in xenogeneic mixed lymphocyte endothelial reaction (MLER). The stimulating indices (SI) were only 1.0-1.5, compared with 15.2-18.2 for those transfected with empty vector or an initiation codon-depleted mCIITA that is dysfunctional for protein translation. The results we obtained, especially those for persistent suppression of class II genes, show promise for the possible development of mCIITA-transgenic swine for organ/tissue xenotransplantation.
Assuntos
Antígenos HLA-DR/imunologia , Proteínas Nucleares/genética , RNA Antissenso/metabolismo , Transativadores/genética , Animais , Regulação para Baixo , Antígenos HLA-DR/genética , Antígenos HLA-DR/metabolismo , Humanos , Mutação , Proteínas Nucleares/imunologia , Proteínas Nucleares/metabolismo , RNA Antissenso/imunologia , Análise de Sequência de Proteína , Análise de Sequência de RNA , Suínos/imunologia , Linfócitos T/imunologia , Transativadores/imunologia , Transativadores/metabolismo , TransfecçãoRESUMO
Alterations of the immunoendocrine circuit along the hypothalamic-pituitary-adrenocortical (HPA) axis in various autoimmune diseases have recently been observed, suggesting a modulatory role of this feedback regulation in the pathogenesis of autoimmune diseases. Susceptibility to experimental autoimmune encephalomyelitis (EAE) may be influenced by variations in the production of endogenous glucocorticoid hormones (GC). The adrenocortical response is central to recovery from EAE in the Lewis rat, as reflected by increased severity of the disease in adrenalectomized animals. The key role of GC in modifying the induction and progression of EAE is also emphasized by a reversal of corticoid-mediated effects through treatment with glucocorticoid receptor (GR) antagonists. We studied the relationship between defective GR function and susceptibility to EAE in transgenic (Tg) mice expressing GR antisense RNA. EAE was induced with the encephalitogenic myelin oligodendrocyte peptide (pMOG 36-50) in wild type (Wt) and transgenic (Tg) female mice bearing GR antisense RNA. pMOG 36-50 induced typical EAE in Wt mice but not in Tg mice. Histological examination of brains and spinal cords of Wt mice showed the presence of inflammation and/or demyelination, whereas in Tg mice neither were present. Although the mechanisms underlying the resistance of Tg mice to EAE induction are not yet clarified, compensatory changes at different levels of the HPA-immune axis in response to the potent immunogenic challenge are likely to participate in the observed effects. This work underlies the plasticity of the HPA-immune axis and suggests that pharmacological manipulation of neuroendocrine-immune networks may be a therapy of multiple sclerosis.