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Determining whether the RNA isoforms from medically relevant genes have distinct functions could facilitate direct targeting of RNA isoforms for disease treatment. Here, as a step toward this goal for neurological diseases, we sequenced 12 postmortem, aged human frontal cortices (6 Alzheimer disease cases and 6 controls; 50% female) using one Oxford Nanopore PromethION flow cell per sample. We identified 1,917 medically relevant genes expressing multiple isoforms in the frontal cortex where 1,018 had multiple isoforms with different protein-coding sequences. Of these 1,018 genes, 57 are implicated in brain-related diseases including major depression, schizophrenia, Parkinson's disease and Alzheimer disease. Our study also uncovered 53 new RNA isoforms in medically relevant genes, including several where the new isoform was one of the most highly expressed for that gene. We also reported on five mitochondrially encoded, spliced RNA isoforms. We found 99 differentially expressed RNA isoforms between cases with Alzheimer disease and controls.
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Even though alternative RNA splicing was discovered nearly 50 years ago (1977), we still understand very little about most isoforms arising from a single gene, including in which tissues they are expressed and if their functions differ. Human gene annotations suggest remarkable transcriptional complexity, with approximately 252,798 distinct RNA isoform annotations from 62,710 gene bodies (Ensembl v109; 2023), emphasizing the need to understand their biological effects. For example, 256 gene bodies have ≥50 annotated isoforms and 30 have ≥100, where one protein-coding gene (MAPK10) even has 192 distinct RNA isoform annotations. Whether such isoform diversity results from biological redundancy or spurious alternative splicing (i.e., noise), or whether individual isoforms have specialized functions (even if subtle) remains a mystery for most genes. Recent studies by Aguzzoli-Heberle et al., Leung et al., and Glinos et al. demonstrated long-read RNAseq enables improved RNA isoform quantification for essentially any tissue, cell type, or biological condition (e.g., disease, development, aging, etc.), making it possible to better assess individual isoform expression and function. While each study provided important discoveries related to RNA isoform diversity, deeper exploration is needed. We sought to quantify and characterize real isoform usage across tissues (compared to annotations). We used long-read RNAseq data from 58 GTEx samples across nine tissues (three brain, two heart, muscle, lung, liver, and cultured fibroblasts) generated by Glinos et al. and found considerable isoform diversity within and across tissues. Cerebellar hemisphere was the most transcriptionally complex tissue (22,522 distinct isoforms; 3,726 unique); liver was least diverse (12,435 distinct isoforms; 1,039 unique). We highlight gene clusters exhibiting high tissue-specific isoform diversity per tissue (e.g., TPM1 expresses 19 in heart's atrial appendage). We also validated 447 of the 700 new isoforms discovered by Aguzzoli-Heberle et al. and found that 88 were expressed in all nine tissues, while 58 were specific to a single tissue. This study represents a broad survey of the RNA isoform landscape, demonstrating isoform diversity across nine tissues and emphasizes the need to better understand how individual isoforms from a single gene body contribute to human health and disease.
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BACKGROUND: Magnetic resonance imaging (MRI) is a non-invasive imaging modality which, in conjunction with biopsies, provide a qualitative assessment of tumor response to treatment. Intravenous injection of contrast agents such as fluorine (19F) nanoemulsions labels systemic macrophages, which can, then, be tracked in real time with MRI. This method can provide quantifiable insights into the behavior of tumor-associated macrophages (TAMs) in the tumor microenvironment and macrophage recruitment during therapy. METHODS: Female mice received mammary fat pad injections of murine breast or colon cancer cell lines. The mice then received an intravenous 19F nanoemulsion injection, followed by a baseline 19F MRI. For each cancer model, half of the mice then received 8 Gy of localized radiation therapy (RT), while others remained untreated. The mice were monitored for two weeks for tumor growth and 9F signal using MRI. RESULTS: Across both cohorts, the RT-treated groups presented significant tumor growth reduction or arrest, contrary to the untreated groups. Similarly, the fluorine signal in treated groups increased significantly as early as four days post therapy. The fluorine signal change correlated to tumor volumes irrespective of time. CONCLUSION: These results demonstrate the potential of 19F MRI to non-invasively track macrophages during radiation therapy and its prognostic value with regard to tumor growth.
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Due to alternative splicing, human protein-coding genes average over eight RNA isoforms, resulting in nearly four distinct protein coding sequences per gene. Long-read RNAseq (IsoSeq) enables more accurate quantification of isoforms, shedding light on their specific roles. To assess the medical relevance of measuring RNA isoform expression, we sequenced 12 aged human frontal cortices (6 Alzheimer's disease cases and 6 controls; 50% female) using one Oxford Nanopore PromethION flow cell per sample. Our study uncovered 53 new high-confidence RNA isoforms in medically relevant genes, including several where the new isoform was one of the most highly expressed for that gene. Specific examples include WDR4 (61%; microcephaly), MYL3 (44%; hypertrophic cardiomyopathy), and MTHFS (25%; major depression, schizophrenia, bipolar disorder). Other notable genes with new high-confidence isoforms include CPLX2 (10%; schizophrenia, epilepsy) and MAOB (9%; targeted for Parkinson's disease treatment). We identified 1,917 medically relevant genes expressing multiple isoforms in human frontal cortex, where 1,018 had multiple isoforms with different protein coding sequences, demonstrating the need to better understand how individual isoforms from a single gene body are involved in human health and disease, if at all. Exactly 98 of the 1,917 genes are implicated in brain-related diseases, including Alzheimer's disease genes such as APP (Aß precursor protein; five), MAPT (tau protein; four), and BIN1 (eight). As proof of concept, we also found 99 differentially expressed RNA isoforms between Alzheimer's cases and controls, despite the genes themselves not exhibiting differential expression. Our findings highlight the significant knowledge gaps in RNA isoform diversity and their medical relevance. Deep long-read RNA sequencing will be necessary going forward to fully comprehend the medical relevance of individual isoforms for a "single" gene.
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The success of several cell-based therapies and prevalent use of magnetic resonance imaging (MRI) in the clinic has fueled the development of contrast agents for specific cell tracking applications. Safe and efficient labeling of non-phagocytic cell types such as T cells nonetheless remains challenging. We developed a one-stop shop approach where the T cell sorting agent also labels the cells which can subsequently be depicted using non-invasive MRI. We compared the MR signal effects of magnetic-assisted cell sorting microbeads (CD25) to the current preclinical gold standard, ferumoxytol. We investigated in vitro labeling efficiency of regulatory T cells (Tregs) with MRI and histopathologic confirmation. Thereafter, Tregs and T cells were labeled with CD25 microbeads in vitro and delivered via intravenous injection. Liver MRIs pre- and 24 h post-injection were performed to determine in vivo tracking feasibility. We show that CD25 microbeads exhibit T2 signal decay properties similar to other iron oxide contrast agents. CD25 microbeads are readily internalized by Tregs and can be detected by non-invasive MRI with dose dependent T2 signal suppression. Systemically injected labeled Tregs can be detected in the liver 24 h post-injection, contrary to T cell control. Our CD25 microbead-based labeling method is an effective tool for Treg tagging, yielding detectable MR signal change in cell phantoms and in vivo. This novel cellular tracking method will be key in tracking the fate of Tregs in inflammatory pathologies and solid organ transplantation.
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Meios de Contraste , Óxido Ferroso-Férrico , Microesferas , Coloração e Rotulagem , Imageamento por Ressonância Magnética/métodosRESUMO
BACKGROUND: Cerebral glucose hypometabolism is consistently observed in individuals with Alzheimer's disease (AD), as well as in young cognitively normal carriers of the Ε4 allele of Apolipoprotein E (APOE), the strongest genetic predictor of late-onset AD. While this clinical feature has been described for over two decades, the mechanism underlying these changes in cerebral glucose metabolism remains a critical knowledge gap in the field. METHODS: Here, we undertook a multi-omic approach by combining single-cell RNA sequencing (scRNAseq) and stable isotope resolved metabolomics (SIRM) to define a metabolic rewiring across astrocytes, brain tissue, mice, and human subjects expressing APOE4. RESULTS: Single-cell analysis of brain tissue from mice expressing human APOE revealed E4-associated decreases in genes related to oxidative phosphorylation, particularly in astrocytes. This shift was confirmed on a metabolic level with isotopic tracing of 13C-glucose in E4 mice and astrocytes, which showed decreased pyruvate entry into the TCA cycle and increased lactate synthesis. Metabolic phenotyping of E4 astrocytes showed elevated glycolytic activity, decreased oxygen consumption, blunted oxidative flexibility, and a lower rate of glucose oxidation in the presence of lactate. Together, these cellular findings suggest an E4-associated increase in aerobic glycolysis (i.e. the Warburg effect). To test whether this phenomenon translated to APOE4 humans, we analyzed the plasma metabolome of young and middle-aged human participants with and without the Ε4 allele, and used indirect calorimetry to measure whole body oxygen consumption and energy expenditure. In line with data from E4-expressing female mice, a subgroup analysis revealed that young female E4 carriers showed a striking decrease in energy expenditure compared to non-carriers. This decrease in energy expenditure was primarily driven by a lower rate of oxygen consumption, and was exaggerated following a dietary glucose challenge. Further, the stunted oxygen consumption was accompanied by markedly increased lactate in the plasma of E4 carriers, and a pathway analysis of the plasma metabolome suggested an increase in aerobic glycolysis. CONCLUSIONS: Together, these results suggest astrocyte, brain and system-level metabolic reprogramming in the presence of APOE4, a 'Warburg like' endophenotype that is observable in young females decades prior to clinically manifest AD.
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Aerobiose , Apolipoproteína E4/fisiologia , Glucose/metabolismo , Glicólise , Sintomas Prodrômicos , Adolescente , Adulto , Idoso , Doença de Alzheimer/diagnóstico , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Animais , Apolipoproteína E4/genética , Astrócitos/metabolismo , Sequência de Bases , Química Encefálica , Células Cultivadas , Diagnóstico Precoce , Metabolismo Energético , Feminino , Cromatografia Gasosa-Espectrometria de Massas , Técnicas de Introdução de Genes , Humanos , Metabolômica , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Oxirredução , Fosforilação Oxidativa , Consumo de Oxigênio/genética , Caracteres Sexuais , Análise de Célula Única , Adulto JovemRESUMO
Autotaxin (ATX) is a secreted enzyme that generates the bioactive lipid lysophosphatidic acid (LPA). We generated mice with global inducible post-natal inactivation or adipose-specific loss of the Enpp2 gene encoding ATX. The animals are phenotypically unremarkable and exhibit differences in adipocyte size and adipose tissue expression of inflammatory genes after high fat feeding without gross differences in fat distribution or body mass. Surprisingly, both models of Enpp2- deficiency exhibited marked protection from high fat diet-induced hepatic steatosis. This phenotype was not associated with differences in dietary fat absorption but may be accounted for by differences in hepatic expression of genes involved in de novo synthesis of triglycerides. These findings suggest that pharmacological inhibition of ATX might be protective against hepatic steatosis.
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Adipócitos/metabolismo , Dieta Hiperlipídica/efeitos adversos , Inflamação/metabolismo , Obesidade/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Tecido Adiposo/metabolismo , Adiposidade/fisiologia , Animais , Gorduras na Dieta/metabolismo , Lisofosfolipídeos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Triglicerídeos/metabolismoRESUMO
Research into gene therapy for heart failure has gained renewed interest as a result of improved safety and availability of adeno-associated viral vectors (AAV). While magnetic resonance imaging (MRI) is standard for functional assessment of gene therapy outcomes, quantitation of gene transfer/expression relies upon tissue biopsy, fluorescence or nuclear imaging. Imaging of gene expression through the use of genetically encoded chemical exchange saturation transfer (CEST)-MRI reporter genes could be combined with clinical cardiac MRI methods to comprehensively probe therapeutic gene expression and subsequent outcomes. The CEST-MRI reporter gene Lysine Rich Protein (LRP) was cloned into an AAV9 vector and either administered systemically via tail vein injection or directly injected into the left ventricular free wall of mice. Longitudinal in vivo CEST-MRI performed at days 15 and 45 after direct injection or at 1, 60 and 90 days after systemic injection revealed robust CEST contrast in myocardium that was later confirmed to express LRP by immunostaining. Ventricular structure and function were not impacted by expression of LRP in either study arm. The ability to quantify and link therapeutic gene expression to functional outcomes can provide rich data for further development of gene therapy for heart failure.
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Meios de Contraste , Dependovirus/genética , Técnicas de Transferência de Genes , Genes Reporter , Vetores Genéticos/genética , Coração/fisiologia , Imageamento por Ressonância Magnética/métodos , Animais , Células Cultivadas , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/terapia , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Exposure to dioxins and related persistent organic pollutants likely contributes to cardiovascular disease (CVD) risk through multiple mechanisms including the induction of chronic inflammation. Epidemiological studies have shown that leaner individuals may be more susceptible to the detrimental effects of lipophilic toxicants because they lack large adipose tissue depots that can accumulate and sequester these pollutants. This phenomenon complicates efforts to study mechanisms of pollutant-accelerated atherosclerosis in experimental animal models where high-fat feeding and adipose expansion limit the bioavailability of lipophilic pollutants. Here, we investigated whether a model dioxin-like pollutant, PCB 126, could increase inflammation and accelerate atherosclerosis in Ldlr-/- mice fed a low-fat atherogenic diet. We fed Ldlr-/- mice the Clinton/Cybulsky diet (10% kcal fat, 0.15% cholesterol) and sacrificed mice at 8, 10, or 12 weeks postPCB (2 doses of 1 µmol/kg) or vehicle gavage. To characterize this novel model, we examined the effects of PCB 126 on markers of systemic inflammation, hematological indices, fatty livers, and atherosclerotic lesion size. Mice exposed to PCB 126 exhibited significantly increased plasma inflammatory cytokine levels, increased circulating biomarkers of CVD, altered platelet, and red blood cell counts, increased accumulation of hepatic fatty acids, and accelerated atherosclerotic lesion formation in the aortic root. PCB 126 also increased circulating neutrophils, monocytes, and macrophages as determined by flow cytometry analysis. Exposure to dioxin-like PCB 126 increases inflammation and accelerates atherosclerosis in mice. This low-fat atherogenic diet may provide a useful tool to study the mechanisms linking exposure to lipophilic pollutants to increased risk of CVD.
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Aterosclerose/induzido quimicamente , Citocinas/sangue , Poluentes Ambientais/toxicidade , Lipídeos/sangue , Bifenilos Policlorados/toxicidade , Receptores de LDL/deficiência , Animais , Aorta/efeitos dos fármacos , Aorta/patologia , Aterosclerose/sangue , Aterosclerose/imunologia , Aterosclerose/patologia , Biomarcadores/sangue , Contagem de Células Sanguíneas , Peso Corporal , Dieta Aterogênica , Inflamação , Masculino , Camundongos Knockout , Receptores de LDL/genéticaRESUMO
The structurally simple glycero- and sphingo-phospholipids, lysophosphatidic acid (LPA) and sphingosine-1-phosphate, serve as important receptor-active mediators that influence blood and vascular cell function and are positioned to influence the events that contribute to the progression and complications of atherosclerosis. Growing evidence from preclinical animal models has implicated LPA, LPA receptors, and key enzymes involved in LPA metabolism in pathophysiologic events that may underlie atherosclerotic vascular disease. These observations are supported by genetic analysis in humans implicating a lipid phosphate phosphatase as a novel risk factor for coronary artery disease. In this review, we summarize current understanding of LPA production, metabolism, and signaling as may be relevant for atherosclerotic and other vascular disease.
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Aterosclerose/metabolismo , Lisofosfolipídeos/fisiologia , Fosfatidato Fosfatase/fisiologia , Diester Fosfórico Hidrolases/fisiologia , Tecido Adiposo/enzimologia , Animais , Apolipoproteínas E/deficiência , Aterosclerose/genética , Doença da Artéria Coronariana/epidemiologia , Doença da Artéria Coronariana/genética , Doença da Artéria Coronariana/prevenção & controle , Predisposição Genética para Doença , Humanos , Lisofosfolipídeos/metabolismo , Camundongos , Camundongos Knockout , Fosfatidato Fosfatase/deficiência , Fosfatidato Fosfatase/genética , Placa Aterosclerótica/metabolismo , Risco , Esfingosina/análogos & derivados , Esfingosina/metabolismoRESUMO
Syngeneic graft-versus-host disease (SGVHD), a chronic inflammatory disease, develops following irradiation, syngeneic bone marrow transplantation (BMT) and treatment with the immunosuppressive agent cyclosporine A (CsA). We have shown that TH1 and TH17 cytokine responses are increased during the development of SGVHD. The current study was designed to further investigate the involvement of TH17 immunity in SGVHD-associated colitis. IL-23 is a TH17 cytokine responsible for maintaining the effector functions of TH17 cells. The administration of anti-mouse IL-23p19 was shown to significantly reduce the clinical symptoms of primary and secondary SGVHD-associated colitis resulting in a significant reduction in both TH1 and TH17 associated cytokine expression. These results demonstrate that the TH17-associated cytokine, IL-23, may prove to be a beneficial therapeutic target in the treatment of chronic colon inflammation.
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Transferência Adotiva , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/terapia , Subunidade p19 da Interleucina-23/antagonistas & inibidores , Animais , Citocinas/biossíntese , Feminino , Mediadores da Inflamação/metabolismo , Subunidade p19 da Interleucina-23/metabolismo , Camundongos , Transplante IsogênicoRESUMO
BACKGROUND: The pathogenesis of inflammatory bowel disease (IBD) is complex and the need to identify molecular biomarkers is critical. Epithelial cells play a central role in maintaining intestinal homeostasis. We previously identified five "signature" biomarkers in colonic epithelial cells (CEC) that are predictive of disease phenotype in Crohn's disease. Here we investigate the ability of CEC biomarkers to define the mechanism and severity of intestinal inflammation. METHODS: We analyzed the expression of RelA, A20, pIgR, tumor necrosis factor (TNF), and macrophage inflammatory protein (MIP)-2 in CEC of mice with dextran sodium sulfate (DSS) acute colitis or T-cell-mediated chronic colitis. Factor analysis was used to combine the five biomarkers into two multifactorial principal components (PCs). PC scores for individual mice were correlated with disease severity. RESULTS: For both colitis models, PC1 was strongly weighted toward RelA, A20, and pIgR, and PC2 was strongly weighted toward TNF and MIP-2, while the contributions of other biomarkers varied depending on the etiology of inflammation. Disease severity was correlated with elevated PC2 scores in DSS colitis and reduced PC1 scores in T-cell transfer colitis. Downregulation of pIgR was a common feature observed in both colitis models and was associated with altered cellular localization of pIgR and failure to transport IgA. CONCLUSIONS: A multifactorial analysis of epithelial gene expression may be more informative than examining single gene responses in IBD. These results provide insight into the homeostatic and proinflammatory functions of CEC in IBD pathogenesis and suggest that biomarker analysis could be useful for evaluating therapeutic options for IBD patients.
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Biomarcadores/metabolismo , Colite/genética , Sulfato de Dextrana/toxicidade , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Perfilação da Expressão Gênica , Mucosa Intestinal/metabolismo , Doença Aguda , Animais , Doença Crônica , Colite/induzido quimicamente , Colite/patologia , Células Epiteliais/patologia , Feminino , Imunofluorescência , Proteínas de Homeodomínio , Mucosa Intestinal/patologia , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Murine syngeneic graft-versus-host disease (SGVHD) results in chronic colon and liver inflammation following syngeneic bone marrow transplantation (BMT) and treatment with the calcineurin inhibitor, cyclosporine A (CsA). SGVHD was initially thought to arise as a result of an autoreactive immune response, but more recently it has been shown that enhanced antimicrobial responses develop in SGVHD mice. Consequently, we performed studies to analyze the role of the microbiota in the development of murine SGVHD. Treatment with broad-spectrum antibiotics eliminated disease-associated inflammatory immune responses and pathology, linking the role of the microbiota and microbial-specific immunity to the development of murine SGVHD. In a broader context, these results bring into question the role that anti-microbial immune responses play in post-transplant immune pathologies that develop following allogeneic stem cell transplantation and use of calcineurin inhibitors.
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Murine syngeneic graft-versus-host disease (SGVHD) initiates colon and liver inflammation following lethal irradiation, reconstitution with syngeneic bone marrow transplantation, and therapy with the immunosuppressive agent cyclosporine A. Previous studies have demonstrated that the inducible disease is mediated by CD4(+) T cells with increased reactivity of peripheral and liver-associated lymphocytes against intestinal microbial Ags. In the current report, studies were performed to analyze the specificity of the CD4(+) T cell response of T cells isolated from diseased animals and to determine the in vivo role of the microbiota to the development of SGVHD. Increased major histocompatibility Ag (MHC) class II-restricted responsiveness of SGVHD CD4(+) T cells against microbial Ags isolated from the ceca of normal animals was observed. The enhanced proliferative response was observed in the CD62L(-) memory population of CD4(+) T cells. To determine the role of the bacterial microbiota in the development of murine SGVHD, control and CsA-treated bone marrow transplantation animals were treated with broad-spectrum antibiotics (metronidazole, ciprofloxacin) after transplantation. Cyclosporine A-treated animals that were given antibiotic therapy failed to develop clinical symptoms and pathological lesions in the target tissues characteristic of SGVHD. Furthermore, the reduction in intestinal bacteria resulted in the elimination of the enhanced antimicrobial CD4(+) T cell response and significantly reduced levels of the inflammatory cytokines, IFN-γ, IL-17, and TNF-α. The elimination of the disease-associated inflammatory immune responses and pathology by treatment with broad-spectrum antibiotics definitively links the role of the microbiota and microbial-specific immunity to the development of murine SGVHD.
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Antibacterianos/uso terapêutico , Doença Enxerto-Hospedeiro/tratamento farmacológico , Doença Enxerto-Hospedeiro/imunologia , Animais , Antibacterianos/classificação , Transplante de Medula Óssea/imunologia , Transplante de Medula Óssea/patologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/microbiologia , Linfócitos T CD4-Positivos/patologia , Linhagem Celular , Proliferação de Células , Ciprofloxacina/uso terapêutico , Feminino , Doença Enxerto-Hospedeiro/patologia , Antígenos de Histocompatibilidade Classe II/imunologia , Inflamação/tratamento farmacológico , Inflamação/imunologia , Inflamação/patologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/microbiologia , Mucosa Intestinal/patologia , Metronidazol/uso terapêutico , Camundongos , Camundongos Endogâmicos C3HRESUMO
Syngeneic graft vs. host disease (SGVHD) was first described as a graft vs. host disease-like syndrome that developed in rats following syngeneic bone marrow transplantation (BMT) and cyclosporin A (CsA) treatment. SGVHD can be induced by reconstitution of lethally irradiated mice with syngeneic bone marrow cells followed by 21 days of treatment with the immunosuppressive agent CsA. Clinical symptoms of the disease appear 2-3 wk following cessation of CsA therapy, and disease-associated inflammation occurs primarily in the colon and liver. CD4(+) T cells have been shown to play an important role in the inflammatory response observed in the gut of SGVHD mice. Time-course studies revealed a significant increase in migration of CD4(+) T cells into the colon during CsA therapy, as well as significantly elevated mRNA levels of TNF-α, proinflammatory chemokines, and cell adhesion molecules in colonic tissue of CsA-treated animals compared with BMT controls, as early as day 14 post-BMT. Homing studies revealed a greater migration of labeled CD4(+) T cells into the gut of CsA-treated mice at day 21 post-BMT than control animals via CsA-induced upregulation of mucosal addressin cell adhesion molecule. This study demonstrates that, during the 21 days of immunosuppressive therapy, functional mechanisms are in place that result in increased homing of CD4(+) T effector cells to colons of CsA-treated mice.
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Transplante de Medula Óssea/fisiologia , Medula Óssea/fisiologia , Linfócitos T CD4-Positivos/fisiologia , Colo/química , Colo/efeitos dos fármacos , Ciclosporina/farmacologia , Imunossupressores/farmacologia , Condicionamento Pré-Transplante , Animais , Western Blotting , Moléculas de Adesão Celular/biossíntese , Movimento Celular/efeitos dos fármacos , Quimiocinas/biossíntese , Colo/metabolismo , Citocinas/biossíntese , Citometria de Fluxo , Imunofluorescência , Camundongos , Camundongos Endogâmicos C3H , Receptores de Retorno de Linfócitos/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Syngeneic graft-versus-host disease (SGVHD) develops following lethal irradiation, reconstitution with syngeneic bone marrow (BM) and treatment with a 21 day course of the immunosuppressive agent cyclosporine A (CsA). Clinical symptoms of SGVHD appear 2-3 weeks post-CsA with inflammation occurring in the colon and liver. Previously we have demonstrated that CD4(+) T cells and a T helper cell type 1 cytokine response (T(H)1) are involved in the development of SGVHD associated intestinal inflammation. Studies have recently discovered an additional T cell lineage that produces IL-17 and is termed T(H)17. It has been suggested that inflammatory bowel disease is a result of a T(H)17 response rather than a T(H)1 response. This study was designed to investigate T(H)17 involvement in SGVHD-associated colitis. Following induction of SGVHD, the levels of T(H)17 and T(H)1 cytokine mRNA and protein were measured in control and SGVHD animals. In vivo cytokine neutralization was performed to determine the role of the prototypic T(H)17 cytokine, IL-17, in the disease process. We found that during CsA-induced murine SGVHD there was an increase in both T(H)17 and T(H)1 mRNA and cytokines within the colons of diseased mice. The administration of an anti-mouse IL-17A mAb did not alter the course of disease. However, neutralization of IL-17A resulted in an increased production of IL-17F, a related family member, with an overlapping range of effector activities. These results demonstrate that in the pathophysiology of SGVHD, there is a redundancy in the T(H)17 effector molecules that mediate the development of SGVHD.
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Doença Enxerto-Hospedeiro/imunologia , Células Th17/imunologia , Animais , Camundongos , Camundongos Endogâmicos C3H , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The murine model of cyclosporine A (CsA)-induced syngeneic graft-versus-host disease (SGVHD) is a bone marrow (BM) transplantation model that develops chronic colon inflammation identical to other murine models of CD4(+) T cell-mediated colitis. Interestingly, SGVHD animals develop chronic liver lesions that are similar to the early peribiliary inflammatory stages of clinical chronic liver disease, which is frequently associated with inflammatory bowel disease (IBD). Therefore, studies were initiated to investigate the chronic liver inflammation that develops in the SGVHD model. To induce SGVHD, mice were lethally irradiated, reconstituted with syngeneic BM, and treated with CsA. All of the SGVHD animals that developed colitis also develop chronic liver inflammation. Liver samples from control and SGVHD animals were monitored for tissue pathology, RNA for inflammatory mediators, and phenotypic analysis and in vitro reactivity of the inflammatory infiltrate. Diseased animals developed lesions of intrahepatic and extrahepatic bile ducts. Elevated levels of mRNA for molecules associated with chronic liver inflammation, including mucosal cellular adhesion molecule -1, the chemokines CCL25, CCL28, CCR9, and T(H)1- and T(H)17-associated cytokines were observed in livers of SGVHD mice. CD4(+) T cells were localized to the peribiliary region of the livers of diseased animals, and an enhanced proliferative response of liver-associated mononuclear cells against colonic bacterial antigens was observed. The murine model of SGVHD colitis may be a valuable tool to study the entero-hepatic linkage between chronic colon inflammation and inflammatory liver disease.
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Transplante de Medula Óssea/efeitos adversos , Doenças do Colo/etiologia , Doença Enxerto-Hospedeiro/complicações , Hepatopatias/etiologia , Transplante Isogênico , Animais , Doença Crônica , Doenças do Colo/patologia , Ciclosporina/uso terapêutico , Modelos Animais de Doenças , Feminino , Doença Enxerto-Hospedeiro/patologia , Imunossupressores/uso terapêutico , Inflamação/etiologia , Hepatopatias/patologia , Camundongos , Camundongos Endogâmicos C3H , Receptores Toll-Like/metabolismo , Transplante Isogênico/efeitos adversosRESUMO
BACKGROUND: Syngeneic graft-versus-host disease (SGVHD) develops after lethal irradiation, reconstitution with syngeneic bone marrow (BM), and treatment with a 21-day course of the immunosuppressant cyclosporine A (CsA). Clinical symptoms of SGVHD appear 2-3 weeks after CsA treatment, with inflammation in the colon and liver. It has been demonstrated that CD4+ T cells and a T helper cell type 1 cytokine response (Th1) are involved in the development of SGVHD associated intestinal inflammation. The immune response associated with SGVHD is thought to be the result of the reconstitution of the recipient immune system with the syngeneic donor BM. However, definitive studies have not addressed this issue experimentally. METHODS: To determine the origin of the effector cells that participate in SGVHD, C3H/HeN recipient mice were lethally irradiated and transplanted with BM from normal immunocompetent mice or from immunodeficient, severe combined immune deficient, or Rag-2-/- animals. RESULTS: CsA-treated animals, but not control animals, developed inflammation characteristic of SGVHD in the colon and liver regardless of the source of the donor marrow. Furthermore, immunologically, all CsA treated animals responded similarly with increased production of inflammatory cytokines and an increase in activated CD4+ T cells in the periphery and colon relative to controls. CONCLUSION: These results demonstrate that after lethal irradiation and in the absence of donor T cells, T cells of recipient origin can expand and mediate the induction of CsA-induced SGVHD.