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Patients with cholestatic liver disease, including those with primary biliary cholangitis, can experience symptoms of impaired cognition or brain fog. This phenomenon remains unexplained and is currently untreatable. Bile duct ligation (BDL) is an established rodent model of cholestasis. In addition to liver changes, BDL animals develop cognitive symptoms early in the disease process (before development of cirrhosis and/or liver failure). The cellular mechanisms underpinning these cognitive symptoms are poorly understood. Herein, the study explored the neurocognitive symptom manifestations, and tested potential therapies, in BDL mice, and used human neuronal cell cultures to explore translatability to humans. BDL animals exhibited short-term memory loss and showed reduced astrocyte coverage of the blood-brain barrier, destabilized hippocampal network activity, and neuronal senescence. Ursodeoxycholic acid (first-line therapy for most human cholestatic diseases) did not reverse symptomatic or mechanistic aspects. In contrast, obeticholic acid (OCA), a farnesoid X receptor agonist and second-line anti-cholestatic agent, normalized memory function, suppressed blood-brain barrier changes, prevented hippocampal network deficits, and reversed neuronal senescence. Co-culture of human neuronal cells with either BDL or human cholestatic patient serum induced cellular senescence and increased mitochondrial respiration, changes that were limited again by OCA. These findings provide new insights into the mechanism of cognitive symptoms in BDL animals, suggesting that OCA therapy or farnesoid X receptor agonism could be used to limit cholestasis-induced neuronal senescence.
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Colestasis , Memoria a Corto Plazo , Humanos , Ratones , Animales , Colestasis/tratamiento farmacológico , Ácido Quenodesoxicólico/farmacología , Conductos Biliares/cirugía , Hígado , LigaduraRESUMEN
Sepsis is a life-threatening condition characterized by uncontrolled systemic inflammation and coagulation, leading to multiorgan failure. Therapeutic options to prevent sepsis-associated immunopathology remain scarce. Here, we established a mouse model of long-lasting disease tolerance during severe sepsis, manifested by diminished immunothrombosis and organ damage in spite of a high pathogen burden. We found that both neutrophils and B cells emerged as key regulators of tissue integrity. Enduring changes in the transcriptional profile of neutrophils include upregulated Cxcr4 expression in protected, tolerant hosts. Neutrophil Cxcr4 upregulation required the presence of B cells, suggesting that B cells promoted disease tolerance by improving tissue damage control via the suppression of neutrophils' tissue-damaging properties. Finally, therapeutic administration of a Cxcr4 agonist successfully promoted tissue damage control and prevented liver damage during sepsis. Our findings highlight the importance of a critical B-cell/neutrophil interaction during sepsis and establish neutrophil Cxcr4 activation as a potential means to promote disease tolerance during sepsis.
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Infecciones Bacterianas , Sepsis , Animales , Infecciones Bacterianas/metabolismo , Modelos Animales de Enfermedad , Ratones , Insuficiencia Multiorgánica/metabolismo , Insuficiencia Multiorgánica/patología , Neutrófilos/metabolismo , Sepsis/metabolismoRESUMEN
BACKGROUND: Uncertainty exists about how best to identify primary biliary cholangitis (PBC) patients who would benefit from second-line therapy. Existing, purely clinical, ursodeoxycholic acid (UDCA) response criteria accept degrees of liver biochemistry abnormality in responding patients, emerging data, however, suggest that any degree of ongoing abnormality may, in fact, be associated with an increased risk of adverse outcomes. This cohort study explores the link between response status, the biology of high-risk disease and its implications for clinical practice. METHODS: Proteomics, exploring 19 markers previously identified as remaining elevated in PBC following UDCA therapy, were performed on 400 serum samples, from participants previously recruited to the UK-PBC Nested Cohort between 2014 and 2019. All participants had an established diagnosis of PBC and were taking therapeutic doses of UDCA for greater than 12 months. UDCA response status was assessed using Paris 1, Paris 2 and the POISE criteria, with additional analyses using normal liver blood tests stratified by bilirubin level. Statistical analysis using parametric t tests and 1-way ANOVA. FINDINGS: Disease markers were statistically significantly higher in UDCA non-responders than in responders for all the UDCA response criteria, suggesting a meaningful link between biochemical disease status and disease mechanism. For each of the criteria, however, marker levels were also statistically significantly higher in responders with ongoing liver function test abnormality compared to those who had normalised their liver biochemistry. IL-4RA, IL-18-R1, CXCL11, 9 and 10, CD163 and ACE2 were consistently elevated across all responder groups with ongoing LFT abnormality. No statistically significant differences occurred between markers in normal LFT groups stratified by bilirubin level. INTERPRETATION: This study provides evidence that any ongoing elevation in alkaline phosphatase levels in PBC after UDCA therapy is associated with some degree of ongoing disease activity. There was no difference in activity between patients with normal LFT when stratified by bilirubin. These findings suggest that if our goal is to completely control disease activity in PBC, then normalisation of alkaline phosphatase and bilirubin should be the treatment target. This would also simplify messaging around goals of therapy in PBC, benefiting both patients and clinicians. FUNDING: Funding by the UK Medical Research Council (Stratified Medicine Programme) and an independent research grant by Pfizer. The study funders played no role in the study design, data collection, data analyses, data interpretation or manuscript writing.
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Cirrosis Hepática Biliar , Ácido Ursodesoxicólico , Fosfatasa Alcalina , Bilirrubina , Colagogos y Coleréticos/uso terapéutico , Estudios de Cohortes , Humanos , Cirrosis Hepática Biliar/diagnóstico , Cirrosis Hepática Biliar/tratamiento farmacológico , Resultado del Tratamiento , Ácido Ursodesoxicólico/uso terapéuticoRESUMEN
BACKGROUND AND AIMS: Stratified therapy has entered clinical practice in primary biliary cholangitis (PBC), with routine use of second-line therapy in nonresponders to first-line therapy with ursodeoxycholic acid (UDCA). The mechanism for nonresponse to UDCA remains, however, unclear and we lack mechanistic serum markers. The UK-PBC study was established to explore the biological basis of UDCA nonresponse in PBC and identify markers to enhance treatment. APPROACH AND RESULTS: Discovery serum proteomics (Olink) with targeted multiplex validation were carried out in 526 subjects from the UK-PBC cohort and 97 healthy controls. In the discovery phase, untreated PBC patients (n = 68) exhibited an inflammatory proteome that is typically reduced in scale, but not resolved, with UDCA therapy (n = 416 treated patients). Nineteen proteins remained at a significant expression level (defined using stringent criteria) in UDCA-treated patients, six of them representing a tightly linked profile of chemokines (including CCL20, known to be released by biliary epithelial cells (BECs) undergoing senescence in PBC). All showed significant differential expression between UDCA responders and nonresponders in both the discovery and validation cohorts. A linear discriminant analysis, using serum levels of C-X-C motif chemokine ligand 11 and C-C motif chemokine ligand 20 as markers of responder status, indicated a high level of discrimination with an AUC of 0.91 (CI, 0.83-0.91). CONCLUSIONS: UDCA under-response in PBC is characterized by elevation of serum chemokines potentially related to cellular senescence and was previously shown to be released by BECs in PBC, suggesting a potential role in the pathogenesis of high-risk disease. These also have potential for development as biomarkers for identification of high-risk disease, and their clinical utility as biomarkers should be evaluated further in prospective studies.
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Cirrosis Hepática Biliar/tratamiento farmacológico , Ácido Ursodesoxicólico/uso terapéutico , Anciano , Sistema Biliar/citología , Sistema Biliar/metabolismo , Biomarcadores/sangre , Estudios de Casos y Controles , Quimiocinas/sangre , Células Epiteliales/metabolismo , Femenino , Humanos , Cirrosis Hepática Biliar/sangre , Cirrosis Hepática Biliar/metabolismo , Masculino , Persona de Mediana Edad , Proteoma , Insuficiencia del TratamientoRESUMEN
OBJECTIVE: TGF-ß2 (TGF-ß, transforming growth factor beta), the less-investigated sibling of TGF-ß1, is deregulated in rodent and human liver diseases. Former data from bile duct ligated and MDR2 knockout (KO) mouse models for human cholestatic liver disease suggested an involvement of TGF-ß2 in biliary-derived liver diseases. DESIGN: As we also found upregulated TGFB2 in liver tissue of patients with primary sclerosing cholangitis (PSC) and primary biliary cholangitis (PBC), we now fathomed the positive prospects of targeting TGF-ß2 in early stage biliary liver disease using the MDR2-KO mice. Specifically, the influence of TgfB2 silencing on the fibrotic and inflammatory niche was analysed on molecular, cellular and tissue levels. RESULTS: TgfB2-induced expression of fibrotic genes in cholangiocytes and hepatic stellate cellswas detected. TgfB2 expression in MDR2-KO mice was blunted using TgfB2-directed antisense oligonucleotides (AON). Upon AON treatment, reduced collagen deposition, hydroxyproline content and αSMA expression as well as induced PparG expression reflected a significant reduction of fibrogenesis without adverse effects on healthy livers. Expression analyses of fibrotic and inflammatory genes revealed AON-specific regulatory effects on Ccl3, Ccl4, Ccl5, Mki67 and Notch3 expression. Further, AON treatment of MDR2-KO mice increased tissue infiltration by F4/80-positive cells including eosinophils, whereas the number of CD45-positive inflammatory cells decreased. In line, TGFB2 and CD45 expression correlated positively in PSC/PBC patients and localised in similar areas of the diseased liver tissue. CONCLUSIONS: Taken together, our data suggest a new mechanistic explanation for amelioration of fibrogenesis by TGF-ß2 silencing and provide a direct rationale for TGF-ß2-directed drug development.
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Colangitis Esclerosante , Silenciador del Gen , Cirrosis Hepática Biliar , Cirrosis Hepática , Oligonucleótidos Antisentido , Factor de Crecimiento Transformador beta2/genética , Factor de Crecimiento Transformador beta2/metabolismo , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Animales , Colangitis Esclerosante/metabolismo , Colangitis Esclerosante/patología , Modelos Animales de Enfermedad , Descubrimiento de Drogas , Regulación de la Expresión Génica , Células Estrelladas Hepáticas/metabolismo , Humanos , Cirrosis Hepática/metabolismo , Cirrosis Hepática/patología , Cirrosis Hepática/prevención & control , Cirrosis Hepática Biliar/metabolismo , Cirrosis Hepática Biliar/patología , Ratones , Ratones Noqueados , Regulación hacia Arriba , Miembro 4 de la Subfamilia B de Casete de Unión a ATPRESUMEN
Primary biliary cholangitis (PBC) is a chronic autoimmune liver disease. Approximately 30% of patients do not respond to therapy with ursodeoxycholic acid (UDCA). Previous studies have implicated increased senescence of cholangiocytes in patients who do not respond to UDCA. This may increase the release of cytokines which drive pathogenic T cell polarization. As FXR agonists are beneficial in treating UDCA non-responsive patients, the current study was designed to model the interactions between cholangiocytes and CD4+ T cells to investigate potential immunomodulatory mechanisms of bile acid receptor agonists. Human cholangiocytes were co-cultured with CD4+ T cells to model the biliary stress response. Senescent cholangiocytes were able to polarize T cells toward a Th17 phenotype and suppressed expression of FoxP3 (P = 0.0043). Whilst FXR and TGR5 receptor agonists were unable directly to alter cholangiocyte cytokine expression, FGF19 was capable of significantly reducing IL-6 release (P = 0.044). Bile acid receptor expression was assessed in PBC patients with well-characterized responsiveness to UDCA therapy. A reduction in FXR staining was observed in both cholangiocytes and hepatocytes in PBC patients without adequate response to UDCA. Increased IL-6 expression by senescent cholangiocytes represents a potential mechanism by which biliary damage in PBC could contribute to excessive inflammation.
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In this Article, the sentence: "After 7 months of HFD, MUP-uPA mice developed HCC15, which contained numerous (usually 50-100 per tumour) non-recurrent coding mutations in pathways that are mutated in human HCC (Fig. 2d and Extended Data Fig. 6a).", should have read: "After 7 months of HFD, MUP-uPA mice developed HCC15, which contained numerous (usually 50-100 per tumour) non-recurrent mutations in pathways that are mutated in human HCC (Fig. 2d and Extended Data Fig. 6a).". This has been corrected online. In Extended Data Fig. 6a and b, which show the number of point mutations identified per sample and the mutational signatures, all sequence variants (including non-coding mutations) are shown. Fig. 2d also presents all variants compared to human mutations. In the Supplementary Information to this Amendment, we now provide the comparisons of all variants and coding variants to human mutations.
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Indoleamine 2,3-dioxygenase (IDO)-induced immunosuppression can be clinically beneficial for autoimmune diseases. Primary biliary cirrhosis (PBC) is characterized by autoimmune lesions of intrahepatic bile duct epithelial cells that may lead to irreversible cirrhosis or hepatocellular carcinoma. The present study assessed the expression and function of IDO in a cell culture model and in PBC patients. IDO expression was monitored in a human immortalized but non-malignant biliary epithelial cell (iBEC) line. Increased expression of IDO1/2 was observed in the iBECs following stimulation with interferon-γ (IFN-γ). The induction of IDO was IFN-γ-dependent, but was independent of the transforming growth factor-ß (TGF-ß) pathway. IDO enzymatic activity was observed in the supernatant of iBECs following stimulation with IFN-γ using colorimetric assays. A total of 47 serum samples from PBC patients were used to examine IDO activity by high-performance liquid chromatography, with samples from 24 healthy volunteers used as controls. Patients with PBC exhibited an increased rate of tryptophan to kynurenine conversion (P>0.01). Liver sections from patients with PBC (n=5) and those of healthy controls (n=5) were used for immunohistochemical studies. IDO expression was observed in biliary epithelial cells and in hepatocytes of PBC patients. Finally, the effect of tryptophan metabolites on human cluster of differentiation (CD) 4+ T cells in inducing polarization towards a regulatory T cell phenotype was examined. 3-Hydroxykynurenine significantly upregulated the fraction of CD4+ cells expressing forkhead box p3 (Foxp3). The results of the present study suggest a therapeutic opportunity for the management of PBC and indicate that tryptophan catabolism could serve as a potential biomarker to monitor disease progression.
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This corrects the article DOI: 10.1038/nature24302.
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The role of adaptive immunity in early cancer development is controversial. Here we show that chronic inflammation and fibrosis in humans and mice with non-alcoholic fatty liver disease is accompanied by accumulation of liver-resident immunoglobulin-A-producing (IgA+) cells. These cells also express programmed death ligand 1 (PD-L1) and interleukin-10, and directly suppress liver cytotoxic CD8+ T lymphocytes, which prevent emergence of hepatocellular carcinoma and express a limited repertoire of T-cell receptors against tumour-associated antigens. Whereas CD8+ T-cell ablation accelerates hepatocellular carcinoma, genetic or pharmacological interference with IgA+ cell generation attenuates liver carcinogenesis and induces cytotoxic T-lymphocyte-mediated regression of established hepatocellular carcinoma. These findings establish the importance of inflammation-induced suppression of cytotoxic CD8+ T-lymphocyte activation as a tumour-promoting mechanism.
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Carcinoma Hepatocelular/inmunología , Tolerancia Inmunológica/inmunología , Inmunoglobulina A/inmunología , Inflamación/inmunología , Neoplasias Hepáticas/inmunología , Enfermedad del Hígado Graso no Alcohólico/complicaciones , Enfermedad del Hígado Graso no Alcohólico/inmunología , Animales , Antígeno B7-H1/metabolismo , Antígenos CD8/deficiencia , Carcinoma Hepatocelular/etiología , Carcinoma Hepatocelular/patología , Proliferación Celular , Células Clonales/citología , Células Clonales/inmunología , Progresión de la Enfermedad , Femenino , Microbioma Gastrointestinal , Humanos , Inmunoglobulina A/metabolismo , Inflamación/etiología , Inflamación/patología , Interleucina-10/metabolismo , Cirrosis Hepática/complicaciones , Cirrosis Hepática/inmunología , Cirrosis Hepática/metabolismo , Cirrosis Hepática/patología , Neoplasias Hepáticas/etiología , Neoplasias Hepáticas/patología , Activación de Linfocitos , Masculino , Ratones , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Enfermedad del Hígado Graso no Alcohólico/patología , Células Plasmáticas/inmunología , Células Plasmáticas/metabolismo , Linfocitos T Citotóxicos/citología , Linfocitos T Citotóxicos/inmunologíaRESUMEN
High-risk primary biliary cholangitis (PBC), defined by inadequate response at one year to Ursodeoxycholic acid (UDCA), is associated with disease progression and liver transplantation. Stratifying high-risk patients early would facilitate improved approaches to care. Using long-term follow-up data to define risk at presentation, 6 high-risk PBC patients and 8 low-risk patients were identified from biopsy, transplant and biochemical archival records. Formalin-fixed paraffin-embedded (FFPE) liver biopsies taken at presentation were graded (Scheuer and Nakanuma scoring) and gene expression analysed using the NanoString® nCounter PanCancer Immunity 770-gene panel. Principle component analysis (PCA) demonstrated discrete gene expression clustering between controls and high- and low-risk PBC. High-risk PBC was characterised by up-regulation of genes linked to T-cell activation and apoptosis, INF-γ signalling and leukocyte migration and down-regulation of those linked to the complement pathway. CDKN1a, up-regulated in high-risk PBC, correlated with significantly increased expression of its gene product, the senescence marker p21WAF1/Cip, by biliary epithelial cells. Our findings suggest high- and low-risk PBC are biologically different from disease outset and senescence an early feature in high-risk disease. Identification of a high-risk 'signal' early from standard FFPE tissue sections has clear clinical utility allowing for patient stratification and second-line therapeutic intervention.
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Colangitis/genética , Colangitis/patología , Perfilación de la Expresión Génica , Transcriptoma , Adulto , Anciano , Biomarcadores , Biopsia , Colangitis/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Progresión de la Enfermedad , Femenino , Regulación de la Expresión Génica , Redes Reguladoras de Genes , Humanos , Masculino , Persona de Mediana Edad , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
The diagnosis of autoimmune hepatitis (AIH) is based on a combination of biochemical, immunological and histological features and exclusion of other causes of liver disease. Typical histological features include a chronic hepatitis pattern of injury with portal inflammation and interface activity, predominance of plasma cells in the portal infiltrate, emperipolesis, and hepatocellular rosette formation. Centrilobular injury with prominent hepatocellular necrosis and mononuclear inflammation is now recognised in the histological spectrum of AIH and may represent an early stage of the disease. Liver histology plays a major role in clinical diagnostic scoring systems and is important to confirm or support the clinical diagnosis of AIH. This review focuses on the role of histopathology in AIH and highlights the contribution of histological interpretation to the diagnosis of AIH, differential diagnosis from other entities, recognition of concurrent liver disease, and identification of the so-called overlap or variant syndromes, and addresses the importance of liver biopsy in the management and prognosis of patients with AIH.
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Hepatitis Autoinmune/patología , Hígado/patología , Biopsia , Diagnóstico Diferencial , Humanos , PronósticoRESUMEN
The X-linked transcription factor FOXP3 is expressed by epithelial cells of organs including the breast, where it is considered a tumour suppressor. The chemokine receptor CXCR4 also regulates the development of breast cancer by stimulating cell migration towards CXCL12-expressing sites of metastatic spread. During activation, human T cells show reciprocal regulation of FOXP3 and CXCR4. This study was designed to examine the role FOXP3 plays in metastatic breast cancer, with a particular focus on its potential to regulate CXCR4. Human breast cancer samples showed significantly decreased FOXP3 protein expression but an increased number of CXCR4 transcripts. In comparison with normal primary breast epithelial cells, FOXP3 was down-regulated at both transcript and protein levels in the breast cancer cell lines MCF-7 and MDA-MB-231. In the invasive MDA-MB-231 cells, the remaining FOXP3 was located predominately within the cytoplasm. Following stable FOXP3 overexpression in MDA-MB-231 cells, significant decreases were observed in the expression of ErbB2/HER2, SKP2, c-MYC, and CXCR4. In contrast, an increase in p21 expression led to inhibition of cell proliferation, with a greater proportion in the G1 phase of the cell cycle suggesting the induction of senescence. Specific knockdown of FOXP3 in normal human breast epithelial cells with siRNA significantly increased ErbB2/HER2, SKP2, c-MYC, and CXCR4, and decreased p21 expression. These cells also showed a significantly increased chemotactic response towards CXCL12, consistent with a role for FOXP3 in the regulation of cell migration. Results from this study are consistent with FOXP3 functioning as an important tumour suppressor in breast cancer. Indeed, the potential functions of FOXP3 in breast epithelium can now be extended to include regulation of CXCR4 expression and response to the pro-metastatic chemokine CXCL12.
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Neoplasias de la Mama/patología , Quimiocina CXCL12/genética , Factores de Transcripción Forkhead/genética , Regulación Neoplásica de la Expresión Génica , Receptores CXCR4/genética , Mama/metabolismo , Mama/patología , Neoplasias de la Mama/metabolismo , Ciclo Celular , Línea Celular Tumoral , Proliferación Celular , Quimiocina CXCL12/metabolismo , Quimiotaxis , Regulación hacia Abajo , Células Epiteliales/metabolismo , Células Epiteliales/patología , Femenino , Factores de Transcripción Forkhead/metabolismo , Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Invasividad Neoplásica , Metástasis de la Neoplasia , ARN Interferente Pequeño , Receptores CXCR4/metabolismoRESUMEN
BACKGROUND & AIMS: Neutrophils are important immune effectors required for sterile and non-sterile inflammatory responses. However, neutrophils are associated with pathology in drug-induced liver injury, acute alcoholic liver disease, and ischemia-reperfusion injury. An understanding of the complex mechanisms that control neutrophil recruitment to the injured liver is desirable for developing strategies aimed at limiting neutrophil-mediated cellular damage. METHODS: Wt, tlr2(-/-), tlr4(-/-), and s100a9(-/-) mice were administered CCl4 either acutely (8, 24, 48, or 72 h) or chronically (8 weeks) and livers investigated by histological (IHC for neutrophils, fibrogenesis, proliferation, and chemotactic proteins) or molecular approaches (qRT-PCR for neutrophil chemoattractant chemokines and cytokines as well as pro-fibrogenic genes). RESULTS: Mice lacking TLR2 or S100A9 failed to recruit neutrophils to the injured liver and had a defective hepatic induction of the neutrophil chemokine CXCL-2. Hierarchy between TLR2 and S100A9 proved to be complex. While induction of S100A9 was dependent on TLR2 in isolated neutrophils, there was a more complicated two-way signalling cross-talk between TLR2 and S100A9 in whole liver. However, wound-healing and regenerative responses of the liver were unaffected in these genetic backgrounds as well as in wild type mice, in which neutrophils were depleted by infusion of Ly-6G antibody. CONCLUSIONS: We have identified TLR2 and S100A8/S100A9 as key regulators of hepatic CXCL-2 expression and neutrophil recruitment. This novel TLR2-S100A9-CXCL-2 pathway may be of use in development of new strategies for selectively manipulating neutrophils in liver disease without impairing normal wound healing and regenerative responses.
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Calgranulina B/inmunología , Enfermedad Hepática Crónica Inducida por Sustancias y Drogas/inmunología , Enfermedad Hepática Inducida por Sustancias y Drogas/inmunología , Quimiocina CXCL2/inmunología , Infiltración Neutrófila/inmunología , Receptor Toll-Like 2/inmunología , Animales , Calgranulina A/inmunología , Calgranulina B/genética , Tetracloruro de Carbono/toxicidad , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Enfermedad Hepática Crónica Inducida por Sustancias y Drogas/patología , Quimiocina CXCL2/biosíntesis , Modelos Animales de Enfermedad , Humanos , Complejo de Antígeno L1 de Leucocito/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Transducción de Señal/inmunología , Receptor Toll-Like 2/deficiencia , Receptor Toll-Like 2/genética , Receptor Toll-Like 4/deficiencia , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/inmunología , Cicatrización de Heridas/inmunologíaRESUMEN
Liver transplant biopsies are routinely archived following formalin fixation and paraffin embedding and may provide an additional source of diagnostic information following transcriptomic biomarker analysis. This study was designed to compare gene transcription between resting and stressed biliary cells in culture, these cells after fixation and embedding and archival liver transplant biopsy tissue. The transcription of p21/WAF1 and transforming growth factor (TGF)-ß1 showed similar changes in the fresh and embedded liver cells. However, the expression of TGF-ß2 was markedly different between the fresh and embedded samples, suggesting that fixation can produce sequence-specific artefacts. Sufficient quantities of pure RNA were recovered from all the liver transplant biopsies to allow complementary DNA production. Measurement of the transcription of all three genes showed variability between the cases. Although the results for individual transcripts should be interpreted with care, these data do suggest the feasibility of performing a larger biomarker discovery studies using archival tissue.
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Perfilación de la Expresión Génica/métodos , Expresión Génica , Hígado/metabolismo , Formaldehído , Humanos , Hígado/patología , Trasplante de Hígado , Adhesión en Parafina , Fijación del Tejido/métodosRESUMEN
Activated T cells infiltrate a renal allograft during rejection and can respond to TGF-ß within the tubules, causing local differentiation and expression of the αE(CD103)ß7 integrin. This study was performed to examine the expression of latent TGF-ß within renal allograft tissues and to define a mechanism by which T cells can activate and respond to this latent factor. Rejecting renal allograft biopsy tissues showed increased expression of the latent TGF-ß complex, which was localized around the tubules by a mechanism that might involve interaction with heparan sulfate in the basement membrane. A cultured renal TEC line also expressed the latent complex, but these cells did not respond to this form of TGF-ß by pSmad 3. However, coculture of these cells with activated T cells induced the expression of CD103, suggesting that T cells can activate and respond to the latent TGF-ß associated with TEC. Although activated T cells expressed little cell-surface TSP-1, this was increased by culture with fibronectin or fibronectin-expressing renal TEC. Blockade of TSP-1 using LSKL peptides reduced the potential of activated T cells to differentiate in response to latent TGF-ß. This study suggests that penetration of renal tubules by activated T cells leads to increased expression of T cell-surface TSP-1, allowing activation of latent TGF-ß sequestered on heparan sulfate within the microenvironment. This mechanism may be important for localized phenotypic maturation of T cells that have infiltrated the kidney during allograft rejection.
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Rechazo de Injerto/genética , Trasplante de Riñón/patología , Túbulos Renales/patología , Linfocitos T/patología , Factor de Crecimiento Transformador beta/genética , Adulto , Anciano , Antígenos CD/genética , Antígenos CD/inmunología , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/inmunología , Comunicación Celular/efectos de los fármacos , Diferenciación Celular , Línea Celular Transformada , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Rechazo de Injerto/inmunología , Rechazo de Injerto/metabolismo , Rechazo de Injerto/patología , Heparitina Sulfato/inmunología , Heparitina Sulfato/metabolismo , Humanos , Cadenas alfa de Integrinas/genética , Cadenas alfa de Integrinas/inmunología , Trasplante de Riñón/inmunología , Túbulos Renales/inmunología , Túbulos Renales/metabolismo , Activación de Linfocitos/efectos de los fármacos , Masculino , Persona de Mediana Edad , Unión Proteica , Transducción de Señal/efectos de los fármacos , Proteína smad3/genética , Proteína smad3/inmunología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Factor de Crecimiento Transformador beta/inmunología , Factor de Crecimiento Transformador beta/farmacología , Trasplante HomólogoRESUMEN
Loss of bile duct epithelium is characteristic of early chronic rejection following liver transplantation. Recent studies have suggested that intrahepatic biliary epithelial cells can transform into myofibroblasts. This study examines the induction and molecular regulation of this transition during allograft rejection. Immortalized human cholangiocytes were stimulated with either transforming growth factor beta1 (TGFbeta1) or a T cell line, and they were examined for morphological, proteomic, and functional features. Posttransplant liver biopsy sections were also examined. Treatment of cholangiocytes with TGFbeta1 or TGFbeta-presenting T cells induced a bipolar morphology, reduced expression of E-cadherin and zona occludens 1 (ZO-1), and increased vimentin, fibronectin, matrix metalloproteinase 2 (MMP-2), MMP-9, and S100 calcium binding protein A4 (S100A4); treated cells invaded a model basement membrane. Chemokines induced T cell penetration of 3-dimensional, cultured bile duct-like structures and bile ducts in liver biopsy sections. A spatial association was observed between duct-infiltrating T cells and cholangiocyte expression of mesenchymal markers, including S100A4. Inhibition of S100A4 expression in vitro blocked TGFbeta1-mediated loss of E-cadherin and ZO-1 but did not reduce induction of fibronectin, MMP-2, or MMP-9. This study demonstrates the potential for T cells to induce an intrahepatic biliary epithelial-to-mesenchymal cell transition during chronic rejection. Furthermore, S100A4 expression by cholangiocytes was identified as a crucial regulator of this transition.
Asunto(s)
Conductos Biliares/patología , Células Epiteliales/patología , Fibroblastos/patología , Rechazo de Injerto/patología , Trasplante de Hígado , Linfocitos T/patología , Conductos Biliares/inmunología , Biopsia , Cadherinas/metabolismo , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Línea Celular Transformada , Enfermedad Crónica , Colágeno , Combinación de Medicamentos , Células Epiteliales/inmunología , Células Epiteliales/metabolismo , Fibroblastos/inmunología , Técnicas de Silenciamiento del Gen , Rechazo de Injerto/inmunología , Humanos , Inmunohistoquímica , Inmunofenotipificación , Laminina , Proteoglicanos , Proteína de Unión al Calcio S100A4 , Proteínas S100/genética , Linfocitos T/inmunología , Factor de Crecimiento Transformador beta1/farmacología , Trasplante HomólogoRESUMEN
DNA damage during transplantation can activate poly-adenosine diphosphate ribose polymerase (PARP) resulting in the generation of polymers of adenosine diphosphate-ribose (PAR). Excessive linkage of PAR to nuclear proteins can induce cell death, thereby limiting the function of transplanted organs. This study uses a rat model of brain death to determine the profile of PARP activation and whether mechanisms that lead to cell death can be ameliorated by appropriate donor resuscitation. The expression of PAR-linked nuclear proteins within cardiac myocytes was greatly increased after the induction of donor brain death. Importantly, infusion of noradrenaline or vasopressin to normalize the chronic hypotension produced by brain death reduced the expression of PAR to a level below baseline. These data suggest that chronic hypotension after donor brain death has the potential to limit cardiac function through the activation of PARP; however, this early cause of graft damage can be mitigated by appropriate donor resuscitation.
Asunto(s)
Muerte Encefálica/metabolismo , Miocardio/enzimología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Animales , Daño Encefálico Crónico/etiología , Núcleo Celular/enzimología , Daño del ADN , Modelos Animales de Enfermedad , Activación Enzimática , Cinética , Miocitos Cardíacos/enzimología , Ratas , Trasplante/efectos adversosRESUMEN
The chemokine receptor CCR2 binds four pro-inflammatory monocyte chemoattractant proteins, designated MCP1/CCL2, MCP2/CCL8, MCP3/CCL7 and MCP4/CCL13. This study demonstrates the important biology of this receptor during the response to the chemokine milieu. Competitive chemotaxis and calcium flux assays were performed utilising mixtures of chemokines to assess a hierarchal arrangement of chemokine prepotency; these demonstrated that the MCP2-CCR2 interaction is able to supersede signals generated by RANTES, another pro-inflammatory chemokine, or the homeostatic chemokine SDF1. These observations were validated using three physiologically relevant monocytic cell lines. Having identified the importance of CCR2, experiments were then performed to examine the signal transduction processes coupled to this receptor. G protein coupling was initially examined; Cholera toxin reduced the chemotactic response to MCP2 (p<0.001), whilst the response to the other MCP chemokines remained normal. The response to MCP2 was uniquely inhibited by elevated concentrations of cAMP and, unlike MCP1, 3 and 4 (p<0.05), MCP2 failed to inhibit adenylate cyclase. Expression of dominant negative H-ras demonstrated that each MCP chemokine required active ras in order to elicit ERK activation and a chemotactic response. Unlike MCP1, MCP2 failed to induce nuclear translocation of activated ERK1 or subsequent induction of c-Myc expression. Akt activation also showed ligand-specific differences, with MCP2 producing a delayed response compared to the other MCP chemokines. Together these data highlight the importance of CCR2 and suggest that it is a powerful tool for fine tuning the immune response.