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BACKGROUND AND AIMS: In otherwise near-normal appearing biopsies by routine light microscopy, next-generation pathology (NGP) detected close pairings (immune pairs; iPAIRs) between lymphocytes and antigen-presenting cells (APCs) that predicted immunosuppression weaning failure in pediatric liver transplant (LTx) recipients (Immunosuppression Withdrawal for Stable Pediatric Liver Transplant Recipients [iWITH], NCT01638559). We hypothesized that NGP-detected iPAIRs enrich for true immune synapses, as determined by nuclear shape metrics, intercellular distances, and supramolecular activation complex (SMAC) formation. APPROACH AND RESULTS: Intralobular iPAIRs (CD45 high lymphocyte-major histocompatibility complex II + APC pairs; n = 1167, training set) were identified at low resolution from multiplex immunohistochemistry-stained liver biopsy slides from several multicenter LTx immunosuppression titration clinical trials (iWITH; NCT02474199 (Donor Alloantigen Reactive Tregs (darTregs) for Calcineurin Inhibitor (CNI) Reduction (ARTEMIS); Prospective Longitudinal Study of iWITH Screen Failures Secondary to Histopathology). After excluding complex multicellular aggregates, high-resolution imaging was used to examine immune synapse formation ( n = 998). By enriching for close intranuclear lymphocyte-APC distance (mean: 0.713 µm) and lymphocyte nuclear flattening (mean ferret diameter: 2.1), SMAC formation was detected in 29% of iPAIR-engaged versus 9.5% of unpaired lymphocytes. Integration of these morphometrics enhanced NGP detection of immune synapses (ai-iSYN). Using iWITH preweaning biopsies from eligible patients ( n = 53; 18 tolerant, 35 nontolerant; testing set), ai-iSYN accurately predicted (87.3% accuracy vs. 81.4% for iPAIRs; 100% sensitivity, 75% specificity) immunosuppression weaning failure. This confirmed the presence and importance of intralobular immune synapse formation in liver allografts. Stratification of biopsy mRNA expression data by immune synapse quantity yielded the top 20 genes involved in T cell activation and immune synapse formation and stability. CONCLUSIONS: NGP-detected immune synapses (subpathological rejection) in LTx patients prior to immunosuppression reduction suggests that NGP-detected (allo)immune activity usefulness for titration of immunosuppressive therapy in various settings.
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Hurones , Linfocitos T , Humanos , Animales , Niño , Estudios Prospectivos , Estudios Longitudinales , Hígado , Células Presentadoras de Antígenos , Aloinjertos , Rechazo de Injerto/diagnóstico , Inmunosupresores/uso terapéuticoRESUMEN
BACKGROUND AND AIMS: No consensus criteria or approaches exist regarding assessment of steatosis in the setting of human donor liver suitability for transplantation. The Banff Working Group on Liver Allograft Pathology undertook a study to determine the consistency with which steatosis is assessed and reported in frozen sections of potential donor livers. APPROACH AND RESULTS: A panel of 59 pathologists from 16 countries completed a questionnaire covering criteria used to assess steatosis in donor liver biopsies, including droplet size and magnification used; subsequently, steatosis severity was assessed in 18 whole slide images of donor liver frozen sections (n = 59). Survey results (from 56/59) indicated a wide variation in definitions and approaches used to assess and report steatosis. Whole slide image assessment led to a broad range in the scores. Findings were discussed at a workshop held at the 15th Banff Conference on Allograft Pathology, September 2019. The aims of discussions were to (i) establish consensus criteria for defining "large droplet fat" (LDF) that predisposes to increased risk of initial poor graft function and (ii) develop an algorithmic approach to determine fat droplet size and the percentage of hepatocytes involved. LDF was defined as typically a single fat droplet that expands the involved hepatocyte and is larger than adjacent nonsteatotic hepatocytes. Estimating severity of steatosis involves (i) low magnification estimate of the approximate surface area of the biopsy occupied by fat, (ii) higher magnification determination of the percentage of hepatocytes within the fatty area with LDF, and (iii) final score calculation. CONCLUSIONS: The proposed guidelines herein are intended to improve standardization in steatosis assessment of donor liver biopsies. The calculated percent LDF should be provided to the surgeon.
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Hígado Graso , Trasplante de Hígado , Biopsia , Consenso , Hígado Graso/diagnóstico , Hígado Graso/patología , Humanos , Hígado/patología , Trasplante de Hígado/métodos , Donadores Vivos , Donantes de TejidosRESUMEN
BACKGROUND & AIMS: Augmenter of liver regeneration (ALR, encoded by GFER) is a widely distributed pleiotropic protein originally identified as a hepatic growth factor. However, little is known about its roles in hepatic physiology and pathology. We created mice with liver-specific deletion of ALR to study its function. METHODS: We developed mice with liver-specific deletion of ALR (ALR-L-KO) using the albumin-Cre/LoxP system. Liver tissues were collected from ALR-L-KO mice and ALR(floxed/floxed) mice (controls) and analyzed by histology, reverse-transcription polymerase chain reaction, immunohistochemistry, electron microscopy, and techniques to measure fibrosis and lipids. Liver tissues from patients with and without advanced liver disease were determined by immunoblot analysis. RESULTS: Two weeks after birth, livers of ALR-L-KO mice contained low levels of ALR and adenosine triphosphate (ATP); they had reduced mitochondrial respiratory function and increased oxidative stress, compared with livers from control mice, and had excessive steatosis, and hepatocyte apoptosis. Levels of carbamyl-palmitoyl transferase 1a and ATP synthase subunit ATP5G1 were reduced in livers of ALR-L-KO mice, indicating defects in mitochondrial fatty acid transport and ATP synthesis. Electron microscopy showed mitochondrial swelling with abnormalities in shapes and numbers of cristae. From weeks 2-4 after birth, levels of steatosis and apoptosis decreased in ALR-L-KO mice, and numbers of ALR-expressing cells increased, along with ATP levels. However, at weeks 4-8 after birth, livers became inflamed, with hepatocellular necrosis, ductular proliferation, and fibrosis; hepatocellular carcinoma developed by 1 year after birth in nearly 60% of the mice. Hepatic levels of ALR were also low in ob/ob mice and alcohol-fed mice with liver steatosis, compared with controls. Levels of ALR were lower in liver tissues from patients with advanced alcoholic liver disease and nonalcoholic steatohepatitis than in control liver tissues. CONCLUSIONS: We developed mice with liver-specific deletion of ALR, and showed that it is required for mitochondrial function and lipid homeostasis in the liver. ALR-L-KO mice provide a useful model for investigating the pathogenesis of steatohepatitis and its complications.
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Carcinoma Hepatocelular/etiología , Hígado Graso/etiología , Neoplasias Hepáticas/etiología , Regeneración Hepática/fisiología , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/fisiología , Animales , Apoptosis , Reductasas del Citocromo/fisiología , Humanos , Metabolismo de los Lípidos , Cirrosis Hepática Experimental/etiología , Ratones , Ratones Noqueados , Mitocondrias/fisiologíaRESUMEN
IL-33 administration is associated with facilitation of Th2 responses and cardioprotective properties in rodent models. However, in heart transplantation, the mechanism by which IL-33, signaling through ST2L (the membrane-bound form of ST2), promotes transplant survival is unclear. We report that IL-33 administration, while facilitating Th2 responses, also increases immunoregulatory myeloid cells and CD4(+) Foxp3(+) regulatory T cells (Tregs) in mice. IL-33 expands functional myeloid-derived suppressor cells, CD11b(+) cells that exhibit intermediate (int) levels of Gr-1 and potent T cell suppressive function. Furthermore, IL-33 administration causes an St2-dependent expansion of suppressive CD4(+) Foxp3(+) Tregs, including an ST2L(+) population. IL-33 monotherapy after fully allogeneic mouse heart transplantation resulted in significant graft prolongation associated with increased Th2-type responses and decreased systemic CD8(+) IFN-γ(+) cells. Also, despite reducing overall CD3(+) cell infiltration of the graft, IL-33 administration markedly increased intragraft Foxp3(+) cells. Whereas control graft recipients displayed increases in systemic CD11b(+) Gr-1(hi) cells, IL-33-treated recipients exhibited increased CD11b(+) Gr-1(int) cells. Enhanced ST2 expression was observed in the myocardium and endothelium of rejecting allografts, however the therapeutic effect of IL-33 required recipient St2 expression and was dependent on Tregs. These findings reveal a new immunoregulatory property of IL-33. Specifically, in addition to supporting Th2 responses, IL-33 facilitates regulatory cells, particularly functional CD4(+) Foxp3(+) Tregs that underlie IL-33-mediated cardiac allograft survival.
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Antígeno CD11b/biosíntesis , Diferenciación Celular/inmunología , Regulación hacia Abajo/inmunología , Factores de Transcripción Forkhead/biosíntesis , Supervivencia de Injerto/inmunología , Trasplante de Corazón/inmunología , Interleucinas/fisiología , Linfocitos T Reguladores/inmunología , Animales , Aterosclerosis/inmunología , Aterosclerosis/prevención & control , Células Cultivadas , Trasplante de Corazón/patología , Interleucina-33 , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Transgénicos , Células Mieloides/citología , Células Mieloides/inmunología , Células Mieloides/metabolismo , Receptores de Quimiocina/biosíntesis , Receptores de Interleucina-1/biosíntesis , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Linfocitos T Reguladores/citología , Linfocitos T Reguladores/metabolismoRESUMEN
UNLABELLED: A perfect or nearly perfect human leukocyte antigen (HLA) match has been associated with better immediate and long-term survival of diseased donor kidney transplants. However, the effect of HLA matching for hepatic allografts remains poorly defined. Using data from the National Institutes of Diabetes and Digestive and Kidney Diseases Liver Transplantation Database, we investigated the association between HLA mismatches and hepatic allograft survival, disease recurrence, and immunosuppression interactions. A, B, and DR loci were used to calculate total mismatch scores of 0 (no mismatches in any loci) to 6 (mismatches in all loci). Seven hundred ninety-nine adults (male, 55%; female, 45%) underwent 883 liver transplants. The 10-year graft survival according to total mismatch score was as follows: 0-2, 60%; 3-4, 54%; and 5-6, 57%. There was a negative effect of mismatching at the A locus on patient survival, with shorter survival for patients with 1 or 2 mismatches compared with 0 mismatches [P = 0.05, hazard ratio (HR) = 1.6]. Patients on tacrolimus with 1 or 2 mismatches at B or DR loci appeared to have increased rates of patient and graft survival compared to patients with 0 mismatches, with the appearance of a protective effect of tacrolimus (HR = 0.67). The effect of HLA mismatching was more pronounced on certain disease recurrences. DR-locus mismatch increased recurrence of autoimmune hepatitis (P = 0.01, HR = 4.2) and primary biliary cirrhosis (P = 0.04, HR = 2). Mismatch in the A locus was associated with more recurrence of hepatitis C virus (P = 0.01, HR = 1.6) and primary sclerosing cholangitis (P = 0.03, HR = 2.9). CONCLUSION: Mismatching at the A locus decreases patient survival in liver transplant recipients, and mismatching at the DR and A loci affects recurrence of autoimmune liver diseases and hepatitis C, respectively.
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Bases de Datos como Asunto , Supervivencia de Injerto/inmunología , Antígenos HLA/inmunología , Trasplante de Hígado/inmunología , Adolescente , Adulto , Anciano , Niño , Preescolar , Ciclosporina/uso terapéutico , Femenino , Estudios de Seguimiento , Antígenos HLA-A/inmunología , Antígenos HLA-DR/inmunología , Hepatitis C/inmunología , Hepatitis C/cirugía , Hepatitis Autoinmune/inmunología , Hepatitis Autoinmune/cirugía , Humanos , Inmunosupresores/uso terapéutico , Cirrosis Hepática Biliar/inmunología , Cirrosis Hepática Biliar/cirugía , Estudios Longitudinales , Masculino , Persona de Mediana Edad , National Institute of Diabetes and Digestive and Kidney Diseases (U.S.)/estadística & datos numéricos , Tacrolimus/uso terapéutico , Resultado del Tratamiento , Estados UnidosRESUMEN
Central perivenulitis (CP) in the allograft liver can be associated with portal-based acute cellular rejection and autoimmune hepatitis or can occur in isolation (isolated CP). Although several studies have demonstrated the significance of CP, the prevalence and natural history of untreated isolated CP have not been well studied. We examined 100 adult allograft liver recipients who had long-term follow-up, had routine protocol biopsies, and received no treatment for isolated CP. Isolated CP was identified in 28 (28%) patients. It occurred late at a mean of 658 days. Interestingly, patients with late isolated CP (defined as >3 months posttransplant) usually manifested only mildly to modestly elevated liver function tests. However, late isolated CP was associated with prior and subsequent allograft complications. Nearly all (94%) cases of late isolated CP occurred in patients who had early episodes of CP and/or acute cellular rejection. Of 13 patients who developed adverse outcomes in their allografts (zone 3 fibrosis in 10, de novo autoimmune hepatitis in 3, and ductopenia in 3), all experienced episodes of prior CP, and 12 (92%) had late CP; 1 patient required retransplant for chronic rejection, but all were alive within the last year. In summary, "transplant-associated" isolated CP occurs in 28% of adult patients, early CP is predictive of late CP, and late CP (often present as isolated CP) is associated with long-term liver injury in some patients.
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Rechazo de Injerto/etiología , Venas Hepáticas/patología , Hepatitis Autoinmune/etiología , Cirrosis Hepática/etiología , Hepatopatías/complicaciones , Trasplante de Hígado/efectos adversos , Hígado/patología , Adolescente , Adulto , Anciano , Conductos Biliares/patología , Progresión de la Enfermedad , Femenino , Estudios de Seguimiento , Rechazo de Injerto/epidemiología , Rechazo de Injerto/patología , Hepatitis Autoinmune/epidemiología , Hepatitis Autoinmune/patología , Humanos , Hígado/irrigación sanguínea , Hígado/cirugía , Cirrosis Hepática/epidemiología , Cirrosis Hepática/patología , Hepatopatías/epidemiología , Hepatopatías/etiología , Hepatopatías/patología , Masculino , Persona de Mediana Edad , Necrosis , Prevalencia , Reoperación , Factores de Riesgo , Índice de Severidad de la Enfermedad , Factores de Tiempo , Trasplante Homólogo , Vénulas/patologíaRESUMEN
The expression of cyclooxygenase-2 (COX-2) is increased in human cholangiocarcinoma. However, the biologic function and molecular mechanisms of COX-2 in the control of cholangiocarcinoma cell growth have not been well established. This study was designed to examine the direct effect of COX-2 and its inhibitor celecoxib on the growth of human intrahepatic cholangiocarcinoma cells. Overexpression of COX-2 or treatment with prostaglandin E(2) (PGE(2)) enhanced human cholangiocarcinoma cell growth, whereas antisense depletion of COX-2 in these cells decreased PGE(2) production and inhibited growth. These findings demonstrate a direct role of COX-2-mediated PGE(2) in the growth regulation of human cholangiocarcinoma cells. Furthermore, the COX-2 inhibitor celecoxib induced a dose-dependent inhibition of cell growth, cell cycle arrest at the G(1)-S checkpoint, and induction of cyclin-dependent kinase inhibitors p21(waf1/cip1) and p27(kip1). However, the high concentration of celecoxib (50 micro M) required for inhibition of growth, the incomplete protection of celecoxib-induced inhibition of cell growth by PGE(2) or COX-2 overexpression, and the fact that overexpression or antisense depletion of COX-2 failed to alter the level of p21(waf1/cip1) and p27(kip1) indicate the existence of a COX-2-independent mechanism in celecoxib-induced inhibition of cholangiocarcinoma cell growth.
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Neoplasias de los Conductos Biliares/patología , Conductos Biliares Intrahepáticos , Proteínas de Ciclo Celular/biosíntesis , Colangiocarcinoma/patología , Ciclinas/biosíntesis , Inhibidores de la Ciclooxigenasa/farmacología , Isoenzimas/fisiología , Prostaglandina-Endoperóxido Sintasas/fisiología , Sulfonamidas/farmacología , Proteínas Supresoras de Tumor/biosíntesis , Celecoxib , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa 2 , Dinoprostona/fisiología , Humanos , Proteínas de la Membrana , PirazolesRESUMEN
BACKGROUND: Histologic evaluation of small bowel allograft biopsies is important for the diagnosis of acute rejection. However, a standard histologic schema to grade the severity of intestinal acute rejection is not currently available. The primary goal of this study was to develop a histologic grading system for the diagnosis of small bowel allograft acute rejection. METHODS: We evaluated 3268 small bowel allograft biopsies obtained from adult patients who underwent small bowel transplantation at the University of Pittsburgh Medical Center between 1990 and 1999. A histologic grading system was proposed and validated by retrospective correlation with clinical outcomes. RESULTS: Among the 3268 biopsies, 180 acute rejection episodes were diagnosed (88 indeterminate, 74 mild, 14 moderate, and 4 severe). All four histologically diagnosed, severe acute rejection episodes resulted in graft failure before resolution, despite aggressive immunosuppressive therapy. Four of the 14 moderate acute rejection episodes were associated with unfavorable clinical outcomes. In contrast, the 74 mild and 88 indeterminate acute rejection episodes were not associated with unfavorable clinical outcomes. Statistical analysis for trend revealed that grades indicating more severe acute rejection episodes were associated with a greater probability of unfavorable outcomes (P<0.01). In addition, there was good overall agreement among different pathologists regarding the diagnosis of acute rejection using the proposed schema, suggesting that this system is practical. CONCLUSIONS: This study provides a reliable predictive schema for assessment of the severity of human small bowel acute rejection.
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Rechazo de Injerto/patología , Intestino Delgado/patología , Intestino Delgado/trasplante , Índice de Severidad de la Enfermedad , Enfermedad Aguda , Adulto , Biopsia , Femenino , Humanos , Masculino , Persona de Mediana Edad , Pronóstico , Estudios Retrospectivos , Método Simple Ciego , Trasplante HomólogoRESUMEN
Lymphocyte cultures grown from liver allograft biopsies were shown to exhibit alloreactivity towards donor cells as measured by primed lymphocyte testing (PLT). The PLT specificity was determined in assays using HLA typed panel cells and/or by inhibition testing with HLA specific monoclonal antibodies. Certain cultures exhibited PLT specificity towards class I HLA antigens of the donor, whereas others were specific for class II HLA antigens or recognized mixtures of class I and II antigens. These PLT specificity patterns were compared with clinical, histological and laboratory findings on the liver transplant patients at the time of the biopsy. Biopsies yielding class I specific PLT cells were taken generally during the earlier posttransplant period, whereas class II specific cells were grown from later biopsies. There was no significant correlation of the PLT specificity towards class I vs II antigens with the levels of total or direct bilirubin, serum glutamate oxaloacetic transaminase (SGOT), and serum glutamate pyruvate transaminase (SGPT), although a trend towards higher values was noted for biopsies presenting with a class II specific infiltrate. However, the levels of gamma glutamyl transpeptidase (GGTP) and alkaline phosphatase (AP) were significantly increased when biopsies yielded class II specific rather than class I specific PLT cells. Biopsy histology showed more damage to bile duct epithelium in association with class II PLT specificity whereas intense but often reversible infiltrates were found in biopsies yielding class I specific cells. The elevated GGTP and AP levels are probably related to the interaction of class II specific T cells with bile duct epithelium, which has been shown to express induced class II HLA antigens on their cell surface.
RESUMEN
In the steady state, hepatic antigen (Ag)-presenting cells (APC) generally dampen systemic inflammatory responses to gut-derived Ags. Our studies focus on the role of specific liver APC populations, both non-parenchymal cells (dendritic cells [DC], Kupffer cells, and hepatic stellate cells [HSC]) and parenchymal cells, in the molecular regulation of tissue damage (ischemia and reperfusion [I/R] injury) and immunity following liver transplantation. We focus on factors that either promote or overwhelm the natural tendency of the liver to suppress inflammatory/immune responses. We are also examining molecular mechanisms that regulate liver DC maturation and function and that determine their role in the control of allogeneic T-cell function and the fate of the transplanted liver. Our studies are also aimed at elucidating mechanisms by which HSC regulate DC and T-cell function. These investigations may provide new targets for therapeutic intervention in liver inflammation.
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Células Presentadoras de Antígenos/inmunología , Células Presentadoras de Antígenos/metabolismo , Trasplante de Hígado/inmunología , Hígado/inmunología , Hígado/metabolismo , Animales , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Células Dendríticas/ultraestructura , Células Estrelladas Hepáticas/inmunología , Células Estrelladas Hepáticas/metabolismo , Humanos , Tolerancia Inmunológica/inmunología , Inmunidad Innata/inmunología , Ratones , Daño por Reperfusión/inmunología , Daño por Reperfusión/patologíaRESUMEN
Studies on liver interstitial dendritic cells (DC) indicate that the maturation and function of these important antigen-presenting cells may be suppressed by continual exposure to microbial products from the gut, in particular, bacterial lipopolysaccharide. New evidence is emerging for a role of specific intracellular regulators of signal transduction and of cytokines in the hepatic microenvironment, which may contribute to a hyporesponsive state in liver DC. Analysis of signaling molecule expression within DC in liver transplant tissue is likely to uncover its relation to allograft outcome.
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Células Dendríticas/inmunología , Trasplante de Hígado/inmunología , Hígado/inmunología , Animales , Células Presentadoras de Antígenos/inmunología , Endotoxinas/inmunología , Supervivencia de Injerto/inmunología , Humanos , Tolerancia Inmunológica/inmunología , Interleucina-10/inmunología , Interleucina-12/inmunología , Ratones , Receptor Toll-Like 10/inmunología , Receptores Toll-Like/inmunología , Trasplante Homólogo/inmunología , Resultado del TratamientoRESUMEN
The unique immunologic environment of the liver, together with its anatomic location downstream of the gut, influences the maturation and function of its interstitial dendritic cell (DC) populations. These well-equipped, antigen-presenting cells play critical roles in regulation of innate and adaptive immunity. New information is emerging about the molecular regulation of liver DC maturation and function, and their tolerogenic potential, while new insight is being gained regarding interactions between liver DC and other immune effector cell populations (NK, NKT cells) in addition to T cells. During transplantation, factors that affect liver DC biology include ischemia-reperfusion injury, liver regeneration, viral infection and the actions of anti-inflammatory and immunosuppressive drugs. Herein, we review the molecular and cell biology of hepatic DC populations in relation to the regulation of alloimmune responses and liver transplant outcome.
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Células Dendríticas/inmunología , Regulación de la Expresión Génica/inmunología , Hepatitis Viral Humana/inmunología , Inmunofenotipificación/métodos , Neoplasias Hepáticas/inmunología , Regeneración Hepática/inmunología , Trasplante de Hígado/inmunología , Linfocitos T/inmunología , Animales , Antiinflamatorios/farmacología , Células Dendríticas/efectos de los fármacos , Humanos , Inmunosupresores/farmacología , Ratones , Receptores Toll-Like/inmunología , Receptores Toll-Like/metabolismo , Resultado del TratamientoRESUMEN
A variety of human cancers show constitutive activation of signal transducer and activator of transcription-3 (Stat3) and overexpression of cyclooxygenase-2 (COX-2). This study describes a novel cross-talk between the COX-2-controlled prostaglandin E(2) (PGE(2)) and Stat3 signaling pathways that coordinately regulate human cancer cell growth. COX-2-derived PGE(2) induces interleukin-6 production through activation of EP(4) receptor and subsequent phosphorylation of gp130/Stat3 in human cholangiocarcinoma cells. In parallel, activation of COX-2/PGE(2) signaling also enhances Stat3 phosphorylation and reporter activity through EP(1) receptor-induced activation of c-Src and EGFR in these cells. Moreover, the observations that EP(1) receptor is detected in the nucleus as well as in the Stat3.DNA binding complex and that activation of EP(1) receptor in the nuclei enhances Stat3 activation depicts a previously undescribed G protein-coupled receptor in the nucleus for Stat3 activation and tumor cell growth.
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Ciclooxigenasa 2/metabolismo , Dinoprostona/metabolismo , Fosfolipasas A/metabolismo , Receptores de Prostaglandina E/metabolismo , Factor de Transcripción STAT3/metabolismo , Línea Celular Tumoral , Núcleo Celular/metabolismo , Proliferación Celular , Citosol/metabolismo , Activación Enzimática , Regulación Neoplásica de la Expresión Génica , Fosfolipasas A2 Grupo IV , Humanos , Interleucina-6/biosíntesis , Isoenzimas/metabolismo , Receptor Cross-Talk , Receptores Acoplados a Proteínas G/metabolismo , Subtipo EP1 de Receptores de Prostaglandina E , Factor de Transcripción STAT3/genética , Transducción de SeñalRESUMEN
The mechanisms for progressive fibrosis and exacerbation by steatosis in patients with chronic hepatitis C (HCV) are still unknown. We hypothesized that proliferative blockade in HCV-infected and steatotic hepatocytes results in the default activation of hepatic progenitor cells (HPC), capable of differentiating into both biliary and hepatocyte lineages, and that the resultant ductular reaction promotes portal fibrosis. To study this concept, 115 liver biopsy specimens from subjects with HCV were scored for steatosis, inflammation, and fibrosis. Biliary epithelium and HPC were decorated by cytokeratin 7 immunoperoxidase, and the replicative state of hepatocytes was assessed by p21 and Ki-67 immunohistochemistry. A ductular reaction at the portal interface was common. There was a highly significant correlation between the area of ductular reaction and fibrosis stage (r = 0.453, P < .0001), which remained independently associated after multivariate analysis. HPC numbers also correlated with fibrosis (r = 0.544, P < .0001) and the ductular area (r = 0.624, P < .0001). Moreover, steatosis correlated with greater HPC proliferation (r = 0.372, P = .0004) and ductular reaction (r = 0.374, P < .0001) but was not an obligate feature. Impaired hepatocyte replication by p21 expression was independently associated with HPC expansion (P = .002) and increased with the body mass index (P < .001) and lobular inflammation (P = .005). In conclusion, the strong correlation between portal fibrosis and a periportal ductular reaction with HPC expansion, the exacerbation by steatosis, and the associations with impaired hepatocyte replication suggest that an altered regeneration pathway drives the ductular reaction. We believe this triggers fibrosis at the portal tract interface. This may be a stereotyped response of importance in other chronic liver diseases.
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Conductos Biliares Intrahepáticos/patología , Hígado Graso/patología , Hepatitis C Crónica/patología , Hígado/patología , Células Madre/patología , Adulto , Recuento de Células , División Celular , Femenino , Fibrosis , Genotipo , Hepacivirus/genética , Hepatitis C Crónica/virología , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Transforming growth factor-beta (TGF-beta) potently inhibits the growth of human epithelial cells. However, neoplastic epithelial cells become resistant to TGF-beta-mediated mitoinhibition, and the mechanisms for this alteration during tumorigenesis are not fully understood. This study was designed to determine whether there is an association between the cytosolic phospholipase A2alpha (cPLA2alpha)-controlled eicosanoid metabolism and the growth response to TGF-beta in human liver cancer cells. TGF-beta treatment induced simultaneous Smad-mediated gene transcription and phosphorylation of cPLA2alpha. Whereas Smad activation inhibited tumor cell growth, phosphorylation of cPLA2 alpha promoted growth and counteracted Smad-mediated mitoinhibition. TGF-beta1 failed to prevent the growth of cells with high basal expression of cPLA2alpha, but inhibition of cPLA2 alpha, cyclooxygenase-2 (COX-2), or EP1 receptor restored mitoinhibition by TGF-beta1 in these cells. These results suggest that resistance of tumor cells to TGF-beta-mediated mitoinhibition involves activation of cPLA2alpha/COX-2/EP1 signaling. Furthermore, the TGF-beta1-induced Smad transcriptional activity and mitoinhibition were blocked by overexpression of cPLA2alpha or peroxisome proliferator-activated receptor-gamma (PPAR-gamma) but enhanced by depletion of cPLA2alpha or PPAR-gamma. These findings, along with the observations that cPLA2alpha activates PPAR-gamma and that PPAR-gamma binds Smad3, illustrate novel cPLA2alpha/COX-2/EP1 and cPLA2alpha/PPAR-gamma/Smad signaling pathways that counteract the mitoinhibition by TGF-beta in human cancer cells.
Asunto(s)
Citosol/enzimología , Dinoprostona/metabolismo , Neoplasias Hepáticas/metabolismo , PPAR gamma/metabolismo , Fosfolipasas A/metabolismo , Transducción de Señal , Factor de Crecimiento Transformador beta/metabolismo , División Celular , Línea Celular , Línea Celular Tumoral , Ciclooxigenasa 2 , Proteínas de Unión al ADN/metabolismo , Relación Dosis-Respuesta a Droga , Eicosanoides/metabolismo , Activación Enzimática , Genes Reporteros , Fosfolipasas A2 Grupo IV , Humanos , Immunoblotting , Isoenzimas/metabolismo , Hígado/metabolismo , Luciferasas/metabolismo , Proteínas de la Membrana , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Modelos Biológicos , Oligonucleótidos Antisentido/química , Fosforilación , Plásmidos/metabolismo , Prostaglandina-Endoperóxido Sintasas/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Receptores de Prostaglandina E/metabolismo , Subtipo EP1 de Receptores de Prostaglandina E , Proteína Smad2 , Factores de Tiempo , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Transfección , Factor de Crecimiento Transformador beta1 , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Cyclooxygenase-2 (COX-2)-controlled prostaglandin (PG) metabolism recently has been implicated in the pathogenesis of hepatocellular carcinoma (HCC). However, the biologic role and molecular mechanism of COX-2-mediated PGs in the control of liver cancer growth have not been established. This study was designed to examine the direct effect of COX-2 and its inhibitor celecoxib on the growth control of liver cancer cells. Human HCC cell lines Hep3B and HepG2 transfected with COX-2 expression vector showed increased cell growth and enhanced phosphorylation of serine/threonine protein kinase B (Akt). The level of COX-2 expression and Akt phosphorylation is correlated positively in cultured HCC cells and human liver cancer tissues. Inhibition of Akt activation by phosphatidylinositol 3-kinase (PI3-kinase) inhibitor LY294002 significantly decreased the viability of Hep3B and HepG2 cells (P <.01). These results reveal a novel role of Akt activation in COX-2-induced HCC cell survival. Furthermore, HCC cells treated with the COX-2 inhibitor celecoxib showed significant reduction of Akt phosphorylation and marked morphologic and biochemical characteristics of apoptosis. Overexpression of COX-2 or addition of exogenous PGE(2) partially prevented celecoxib-induced apoptosis (P <.01). In conclusion, our results suggest the involvement of COX-2-dependent and -independent mechanisms in celecoxib-mediated HCC cell apoptosis.
Asunto(s)
Carcinoma Hepatocelular/enzimología , Carcinoma Hepatocelular/patología , Isoenzimas/metabolismo , Neoplasias Hepáticas/enzimología , Neoplasias Hepáticas/patología , Prostaglandina-Endoperóxido Sintasas/metabolismo , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas/metabolismo , Antineoplásicos/farmacología , Apoptosis , Celecoxib , División Celular/efectos de los fármacos , Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa/farmacología , Dinoprostona/farmacología , Humanos , Isoenzimas/farmacología , Proteínas de la Membrana , Fosforilación , Prostaglandina-Endoperóxido Sintasas/farmacología , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt , Pirazoles , Sulfonamidas/farmacología , Células Tumorales CultivadasRESUMEN
In an effort to understand the role of key eicosanoid-forming enzymes in the activation of peroxisome proliferator-activated receptor (PPAR), this study was designed to evaluate the possible contributions of cytosolic phospholipase A(2) (cPLA(2)) and group IIA secretory phospholipase A(2) (sPLA(2)) in the regulation of PPAR-mediated gene transcription in a human hepatoma cell line (HepG2). The HepG2 cells express both PPAR-alpha and -gamma but not PPAR-beta. Overexpression of cPLA(2), but not group IIA sPLA(2) in the HepG2 cells, caused a significantly increased PPAR-alpha/gamma-mediated reporter activity. Antisense inhibition of cPLA(2) resulted in a significantly decreased PPAR-alpha/gamma activity. The PPAR-alpha/gamma-induced gene transcription in the HepG2 cells was inhibited by the cPLA(2) inhibitors methyl arachidonyl fluorophosphonate and arachidonyltrifluoromethyl ketone, but not by the sPLA(2) inhibitor LY311727. The expression of PPAR-alpha-mediated endogenous gene apolipoprotein A-II was increased in cells with overexpression of cPLA(2), decreased in cells with antisense inhibition of cPLA(2), but unaltered in cells with overexpression of group IIA sPLA(2). The above results demonstrated an important role of cPLA(2), but not group IIA sPLA(2) in the control of PPAR activation. The cPLA(2)-mediated PPAR activation was likely mediated by arachidonic acid and prostaglandin E(2). This study reveals a novel intracellular function of cPLA(2) in PPAR activation in HepG2 cells. The cPLA(2) thus may represent a potential therapeutic target for the control of PPAR-related liver and metabolic disorders such as obesity, lipid metabolic disorders, diabetes mellitus, and atherosclerosis.
Asunto(s)
Carcinoma Hepatocelular/genética , Citosol/enzimología , Neoplasias Hepáticas/genética , Fosfolipasas A/fisiología , Receptores Citoplasmáticos y Nucleares/fisiología , Factores de Transcripción/fisiología , Transcripción Genética , Apolipoproteína A-II/genética , Ácido Araquidónico/metabolismo , Western Blotting , Cromatografía Líquida de Alta Presión , ADN sin Sentido/genética , Dinoprostona/farmacología , Inhibidores Enzimáticos/farmacología , Expresión Génica , Humanos , Fosfolipasas A/antagonistas & inhibidores , Fosfolipasas A/genética , Plásmidos/genética , Isoformas de Proteínas/genética , Receptores Citoplasmáticos y Nucleares/genética , Proteínas Recombinantes de Fusión , Factores de Transcripción/genética , Transfección , Tritio , Células Tumorales CultivadasRESUMEN
Ligands of peroxisome proliferator-activated receptor-gamma (PPARgamma) induce differentiation and growth inhibition in several human cancers. However, the role of PPARgamma ligands in the growth control of human cholangiocarcinoma cells remains unknown. This study was designed to investigate the biological functions and molecular mechanisms of PPARgamma ligands in the growth regulation of human cholangiocarcinoma cells. Western blot analysis showed that PPARgamma is expressed in all of the three human cholangiocarcinoma cell lines used in this study (SG231, CC-LP-1, and HuCCT1). Transient transfection assays using a peroxisome proliferator response element (PPRE) reporter construct showed that the PPARgamma expressed in human cholangiocarcinoma cells is functional as a transcription activator. Exposure of SG231, CC-LP-1, and HuCCT1 cells to PPARgamma ligands 15-deoxy-delta12, 14-prostaglandin J(2) (15d-PGJ(2)) and troglitazone for 24 to 96 hours resulted in a dose-dependent inhibition of cell growth. Flow cytometry analysis showed that 15d-PGJ(2) and troglitazone-induced cell cycle arrest at the G2/M checkpoint. Consistent with these findings, both 15d-PGJ(2) and troglitazone significantly inhibited the G2/M cyclin-dependent kinase (CDK) Cdc2 activity. Furthermore, cells treated with 15d-PGJ(2) and troglitazone showed elevated expression of p53 and two p53-controlled downstream genes, GADD45 and p21(WAF1/Cip1). Dominant negative inhibition of p53 in SG231 cells significantly blocked the 15d-PGJ(2) and troglitazone-induced growth inhibition, G2/M arrest, and GADD45/p21 induction. 15d-PGJ(2) and troglitazone failed to directly inhibit Cdc2 activity in a cell-free system in spite of direct association between GADD45 and PPARgamma proteins. In conclusion, these results show a novel p53-dependent mechanism in the PPARgamma ligand-mediated inhibition of cholangiocarcinoma growth and suggest a potential therapeutic role of PPARgamma ligands in the treatment of human cholangiocarcinoma.