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1.
Nat Commun ; 15(1): 853, 2024 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-38286990

RESUMO

The presence of CD8+ T cells in the cytoplasm of biliary epithelial cells (BEC) has been correlated with biliary damage associated with primary biliary cholangitis (PBC). Here, we characterise the mechanism of CD8+ T cell invasion into BEC. CD8+ T cells observed within BEC were large, eccentric, and expressed E-cadherin, CD103 and CD69. They were also not contained within secondary vesicles. Internalisation required cytoskeletal rearrangements which facilitated contact with BEC. Internalised CD8+ T cells were observed in both non-cirrhotic and cirrhotic diseased liver tissues but enriched in PBC patients, both during active disease and at the time of transplantation. E-cadherin expression by CD8+ T cells correlated with frequency of internalisation of these cells into BEC. E-cadherin+ CD8+ T cells formed ß-catenin-associated interactions with BEC, were larger than E-cadherin- CD8+ T cells and invaded into BEC more frequently. Overall, we unveil a distinct cell-in-cell structure process in the liver detailing the invasion of E-cadherin+ CD103+ CD69+ CD8+ T cells into BEC.


Assuntos
Ductos Biliares , Cirrose Hepática Biliar , Humanos , Ductos Biliares/metabolismo , Cirrose Hepática Biliar/patologia , Linfócitos T CD8-Positivos/metabolismo , Células Epiteliais/metabolismo , Caderinas/metabolismo
2.
Front Pediatr ; 11: 1143051, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37181429

RESUMO

The 2022 worldwide epidemic of acute hepatitis and liver failure in young children has led to a focus on unusual causes for childhood acute hepatitis. In the UK epidemic, human herpes virus subtype 6B (HHV-6B) was detected along with adenovirus subtype-41F in severely affected children, especially in those requiring liver transplantation (LT). The lifting of COVID lock-down measures has coincided with the rise in these common childhood infections with a higher than expected rate of systemic complications. The sudden exposure of young children to common childhood infections from which they were protected during the pandemic may have induced an abnormal immune mediated response potentiated by multiple pathogen exposure. Primary HHV-6 infection is one such common childhood infection. Classically known as Roseola infantum due to the appearance of a widespread erythematous rash on fever subsidence (exanthema subitem), it has a peak incidence of 6-12 months of age and almost all children will have been infected by age 2. It is the virus most frequently associated with febrile convulsions but the more serious complications of hepatitis and liver failure are rare. We report on the historic cases of three female infants who had suspected primary HHV-6B infection, acute hepatitis and rapid progression to acute liver failure (ALF) requiring LT. Appearances of their native liver were identical to those described in children in the recent hepatitis epidemic. Deteriorating clinical trajectories of recurrent graft hepatitis and rejection-like episodes followed and all three succumbed to graft failure with HHV-6B detected posthumously in their liver allografts. Our case series and the serious complications observed with the recent rise in common childhood infections is a reminder that these routinely encountered pathogens can be deadly especially in the young immunologically untrained. We advocate for HHV-6 to be screened for routinely in children with acute hepatitis and the use of effective HHV-6 anti-viral prophylaxis to prevent recurrence post-transplant.

3.
Clin Exp Immunol ; 213(2): 243-251, 2023 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-37095599

RESUMO

Post-acute cardiac sequelae, following SARS-CoV-2 infection, are well recognized as complications of COVID-19. We have previously shown the persistence of autoantibodies against antigens in skin, muscle, and heart in individuals following severe COVID-19; the most common staining on skin tissue displayed an inter-cellular cement pattern consistent with antibodies against desmosomal proteins. Desmosomes play a critical role in maintaining the structural integrity of tissues. For this reason, we analyzed desmosomal protein levels and the presence of anti-desmoglein (DSG) 1, 2, and 3 antibodies in acute and convalescent sera from patients with COVID-19 of differing clinical severity. We find increased levels of DSG2 protein in sera from acute COVID-19 patients. Furthermore, we find that DSG2 autoantibody levels are increased significantly in convalescent sera following severe COVID-19 but not in hospitalized patients recovering from influenza infection or healthy controls. Levels of autoantibody in sera from patients with severe COVID-19 were comparable to levels in patients with non-COVID-19-associated cardiac disease, potentially identifying DSG2 autoantibodies as a novel biomarker for cardiac damage. To determine if there was any association between severe COVID-19 and DSG2, we stained post-mortem cardiac tissue from patients who died from COVID-19 infection. This confirmed DSG2 protein within the intercalated discs and disruption of the intercalated disc between cardiomyocytes in patients who died from COVID-19. Our results reveal the potential for DSG2 protein and autoimmunity to DSG2 to contribute to unexpected pathologies associated with COVID-19 infection.


Assuntos
Autoanticorpos , COVID-19 , Humanos , Autoanticorpos/metabolismo , Soroterapia para COVID-19 , SARS-CoV-2 , Miocárdio
4.
Inflamm Bowel Dis ; 29(9): 1409-1420, 2023 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-36378498

RESUMO

BACKGROUND: We aimed to predict response to biologics in inflammatory bowel disease (IBD) using computerized image analysis of probe confocal laser endomicroscopy (pCLE) in vivo and assess the binding of fluorescent-labeled biologics ex vivo. Additionally, we investigated genes predictive of anti-tumor necrosis factor (TNF) response. METHODS: Twenty-nine patients (15 with Crohn's disease [CD], 14 with ulcerative colitis [UC]) underwent colonoscopy with pCLE before and 12 to 14 weeks after starting anti-TNF or anti-integrin α4ß7 therapy. Biopsies were taken for fluorescein isothiocyanate-labeled infliximab and vedolizumab staining and gene expression analysis. Computer-aided quantitative image analysis of pCLE was performed. Differentially expressed genes predictive of response were determined and validated in a public cohort. RESULTS: In vivo, vessel tortuosity, crypt morphology, and fluorescein leakage predicted response in UC (area under the receiver-operating characteristic curve [AUROC], 0.93; accuracy 85%, positive predictive value [PPV] 89%; negative predictive value [NPV] 75%) and CD (AUROC, 0.79; accuracy 80%; PPV 75%; NPV 83%) patients. Ex vivo, increased binding of labeled biologic at baseline predicted response in UC (UC) (AUROC, 83%; accuracy 77%; PPV 89%; NPV 50%) but not in Crohn's disease (AUROC 58%). A total of 325 differentially expressed genes distinguished responders from nonresponders, 86 of which fell within the most enriched pathways. A panel including ACTN1, CXCL6, LAMA4, EMILIN1, CRIP2, CXCL13, and MAPKAPK2 showed good prediction of anti-TNF response (AUROC >0.7). CONCLUSIONS: Higher mucosal binding of the drug target is associated with response to therapy in UC. In vivo, mucosal and microvascular changes detected by pCLE are associated with response to biologics in inflammatory bowel disease. Anti-TNF-responsive UC patients have a less inflamed and fibrotic state pretreatment. Chemotactic pathways involving CXCL6 or CXCL13 may be novel targets for therapy in nonresponders.


Assuntos
Produtos Biológicos , Colite Ulcerativa , Doença de Crohn , Doenças Inflamatórias Intestinais , Humanos , Doença de Crohn/diagnóstico por imagem , Doença de Crohn/tratamento farmacológico , Doença de Crohn/genética , Inibidores do Fator de Necrose Tumoral/uso terapêutico , Doenças Inflamatórias Intestinais/diagnóstico por imagem , Doenças Inflamatórias Intestinais/tratamento farmacológico , Doenças Inflamatórias Intestinais/genética , Colite Ulcerativa/diagnóstico por imagem , Colite Ulcerativa/tratamento farmacológico , Colite Ulcerativa/genética , Fator de Necrose Tumoral alfa/uso terapêutico , Terapia Biológica , Produtos Biológicos/uso terapêutico , Expressão Gênica , Fluoresceínas/uso terapêutico , Lasers , Proteínas Adaptadoras de Transdução de Sinal , Proteínas com Domínio LIM
5.
Gut ; 71(7): 1399-1411, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-34548339

RESUMO

OBJECTIVE: Tissue-resident memory T cells (TRM) are vital immune sentinels that provide protective immunity. While hepatic CD8+ TRM have been well described, little is known about the location, phenotype and function of CD4+ TRM. DESIGN: We used multiparametric flow cytometry, histological assessment and novel human tissue coculture systems to interrogate the ex vivo phenotype, function and generation of the intrahepatic CD4+ T-cell compartment. We also used leukocytes isolated from human leukocyte antigen (HLA)-disparate liver allografts to assess long-term retention. RESULTS: Hepatic CD4+ T cells were delineated into three distinct populations based on CD69 expression: CD69-, CD69INT and CD69HI. CD69HICD4+ cells were identified as tissue-resident CD4+ T cells on the basis of their exclusion from the circulation, phenotypical profile (CXCR6+CD49a+S1PR1-PD-1+) and long-term persistence within the pool of donor-derived leukcoocytes in HLA-disparate liver allografts. CD69HICD4+ T cells produced robust type 1 polyfunctional cytokine responses on stimulation. Conversely, CD69INTCD4+ T cells represented a more heterogenous population containing cells with a more activated phenotype, a distinct chemokine receptor profile (CX3CR1+CXCR3+CXCR1+) and a bias towards interleukin-4 production. While CD69INTCD4+ T cells could be found in the circulation and lymph nodes, these cells also formed part of the long-term resident pool, persisting in HLA-mismatched allografts. Notably, frequencies of CD69INTCD4+ T cells correlated with necroinflammatory scores in chronic hepatitis B infection. Finally, we demonstrated that interaction with hepatic epithelia was sufficient to generate CD69INTCD4+ T cells, while additional signals from the liver microenvironment were required to generate liver-resident CD69HICD4+ T cells. CONCLUSIONS: High and intermediate CD69 expressions mark human hepatic CD4+ TRM and a novel functionally distinct recirculating population, respectively, both shaped by the liver microenvironment to achieve diverse immunosurveillance.


Assuntos
Linfócitos T CD4-Positivos , Fígado , Linfócitos T CD8-Positivos , Citocinas/imunologia , Humanos , Memória Imunológica , Fígado/imunologia , Monitorização Imunológica
6.
Front Immunol ; 12: 729143, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34630404

RESUMO

B lymphocytes are multitasking cells that direct the immune response by producing pro- or anti-inflammatory cytokines, by presenting processed antigen for T cell activation and co-stimulation, and by turning into antibody-secreting cells. These functions are important to control infection in the liver but can also exacerbate tissue damage and fibrosis as part of persistent inflammation that can lead to end stage disease requiring a transplant. In transplantation, immunosuppression increases the incidence of lymphoma and often this is of B cell origin. In this review we bring together information on liver B cell biology from different liver diseases, including alcohol-related and metabolic fatty liver disease, autoimmune hepatitis, primary biliary and primary sclerosing cholangitis, viral hepatitis and, in infants, biliary atresia. We also discuss the impact of B cell depletion therapy in the liver setting. Taken together, our analysis shows that B cells are important in the pathogenesis of liver diseases and that further research is necessary to fully characterise the human liver B cell compartment.


Assuntos
Linfócitos B/imunologia , Hepatopatias/imunologia , Fígado/imunologia , Fatores Etários , Animais , Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Diferenciação Celular , Humanos , Agentes de Imunomodulação/uso terapêutico , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Hepatopatias/metabolismo , Hepatopatias/patologia , Hepatopatias/terapia , Depleção Linfocítica , Fenótipo , Rituximab/uso terapêutico , Transdução de Sinais
7.
Sci Adv ; 6(43)2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-33087346

RESUMO

The serpinopathies are among a diverse set of conformational diseases that involve the aberrant self-association of proteins into ordered aggregates. α1-Antitrypsin deficiency is the archetypal serpinopathy and results from the formation and deposition of mutant forms of α1-antitrypsin as "polymer" chains in liver tissue. No detailed structural analysis has been performed of this material. Moreover, there is little information on the relevance of well-studied artificially induced polymers to these disease-associated molecules. We have isolated polymers from the liver tissue of Z α1-antitrypsin homozygotes (E342K) who have undergone transplantation, labeled them using a Fab fragment, and performed single-particle analysis of negative-stain electron micrographs. The data show structural equivalence between heat-induced and ex vivo polymers and that the intersubunit linkage is best explained by a carboxyl-terminal domain swap between molecules of α1-antitrypsin.

8.
Front Immunol ; 11: 1226, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32714318

RESUMO

Background: Pre-clinical research with multi-potent adult progenitor cells (MAPC® cells, Multistem, Athersys Inc., Cleveland, Ohio) suggests their potential as an anti-inflammatory and immunomodulatory therapy in organ transplantation. Normothermic machine perfusion of the liver (NMP-L) has been proposed as a way of introducing therapeutic agents into the donor organ. Delivery of cellular therapy to human donor livers using this technique has not yet been described in the literature. The primary objectives of this study were to develop a technique for delivering cellular therapy to human donor livers using NMP-L and demonstrate engraftment. Methods: Six discarded human livers were perfused for 6 h at 37°C using the Liver Assist (Organ Assist, Groningen). 50 × 106 CMPTX-labeled MAPC cells were infused directly into the right lobe via the hepatic artery (HA, n = 3) or portal vein (PV, n = 3) over 20 min at different time points during the perfusion. Perfusion parameters were recorded and central and peripheral biopsies were taken at multiple time-points from both lobes and subjected to standard histological stains and confocal microscopy. Perfusate was analyzed using a 35-plex multiplex assay and proteomic analysis. Results: There was no detrimental effect on perfusion flow parameters on infusion of MAPC cells by either route. Three out of six livers met established criteria for organ viability. Confocal microscopy demonstrated engraftment of MAPC cells across vascular endothelium when perfused via the artery. 35-plex multiplex analysis of perfusate yielded 13 positive targets, 9 of which appeared to be related to the infusion of MAPC cells (including Interleukin's 1b, 4, 5, 6, 8, 10, MCP-1, GM-CSF, SDF-1a). Proteomic analysis revealed 295 unique proteins in the perfusate from time-points following the infusion of cellular therapy, many of which have strong links to MAPC cells and mesenchymal stem cells in the literature. Functional enrichment analysis demonstrated their immunomodulatory potential. Conclusion: We have demonstrated that cells can be delivered directly to the target organ, prior to host immune cell population exposure and without compromising the perfusion. Transendothelial migration occurs following arterial infusion. MAPC cells appear to secrete a host of soluble factors that would have anti-inflammatory and immunomodulatory benefits in a human model of liver transplantation.


Assuntos
Células-Tronco Adultas , Transplante de Fígado , Doadores Vivos , Transplante de Células-Tronco , Células-Tronco Adultas/citologia , Células-Tronco Adultas/metabolismo , Biomarcadores , Terapia Baseada em Transplante de Células e Tecidos , Quimiocinas/metabolismo , Terapia Combinada , Citocinas/metabolismo , Imunofluorescência , Humanos , Imuno-Histoquímica , Imunofenotipagem , Imunoterapia , Transplante de Fígado/métodos , Preservação de Órgãos/métodos , Perfusão , Proteoma , Transplante de Células-Tronco/métodos
9.
Hepatology ; 72(4): 1253-1266, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-31925805

RESUMO

BACKGROUND AND AIMS: Lifetime risk of biliary tract cancer (BTC) in primary sclerosing cholangitis (PSC) may exceed 20%, and BTC is currently the leading cause of death in patients with PSC. To open new avenues for management, we aimed to delineate clinically relevant genomic and pathological features of a large panel of PSC-associated BTC (PSC-BTC). APPROACH AND RESULTS: We analyzed formalin-fixed, paraffin-embedded tumor tissue from 186 patients with PSC-BTC from 11 centers in eight countries with all anatomical locations included. We performed tumor DNA sequencing at 42 clinically relevant genetic loci to detect mutations, translocations, and copy number variations, along with histomorphological and immunohistochemical characterization. Regardless of the anatomical localization, PSC-BTC exhibited a uniform molecular and histological characteristic similar to extrahepatic cholangiocarcinoma. We detected a high frequency of genomic alterations typical of extrahepatic cholangiocarcinoma, such as TP53 (35.5%), KRAS (28.0%), CDKN2A (14.5%), and SMAD4 (11.3%), as well as potentially druggable mutations (e.g., HER2/ERBB2). We found a high frequency of nontypical/nonductal histomorphological subtypes (55.2%) and of the usually rare BTC precursor lesion, intraductal papillary neoplasia (18.3%). CONCLUSIONS: Genomic alterations in PSC-BTC include a significant number of putative actionable therapeutic targets. Notably, PSC-BTC shows a distinct extrahepatic morpho-molecular phenotype, independent of the anatomical location of the tumor. These findings advance our understanding of PSC-associated cholangiocarcinogenesis and provide strong incentives for clinical trials to test genome-based personalized treatment strategies in PSC-BTC.


Assuntos
Neoplasias dos Ductos Biliares/genética , Colangiocarcinoma/genética , Colangite Esclerosante/complicações , Adolescente , Adulto , Idoso , Neoplasias dos Ductos Biliares/mortalidade , Neoplasias dos Ductos Biliares/patologia , Neoplasias dos Ductos Biliares/terapia , Criança , Colangiocarcinoma/mortalidade , Colangiocarcinoma/patologia , Colangiocarcinoma/terapia , Inibidor p16 de Quinase Dependente de Ciclina/genética , Feminino , Genes p53 , Genômica , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Proteínas Proto-Oncogênicas p21(ras)/genética , Adulto Jovem
10.
Cell Rep ; 29(6): 1610-1620.e4, 2019 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-31693899

RESUMO

CD4+ T cells play critical roles in directing immunity, both as T helper and as regulatory T (Treg) cells. Here, we demonstrate that hepatocytes can modulate T cell populations through engulfment of live CD4+ lymphocytes. We term this phenomenon enclysis to reflect the specific enclosure of CD4+ T cells in hepatocytes. Enclysis is selective for CD4+ but not CD8+ cells, independent of antigen-specific activation, and occurs in human hepatocytes in vitro, ex vivo, and in vivo. Intercellular adhesion molecule 1 (ICAM-1) facilitates T cell early adhesion and internalization, whereas hepatocytes form membrane lamellipodia or blebs to mediate engulfment. T cell internalization is unaffected by wortmannin and Rho kinase inhibition. Hepatocytes engulf Treg cells more efficiently than non-Treg cells, but Treg cell-containing vesicles preferentially acidify overnight. Thus, enclysis is a biological process with potential effects on immunomodulation and opens a new field for research to fully understand CD4+ T cell dynamics in liver inflammation.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Endocitose/imunologia , Endossomos/imunologia , Hepatócitos/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Linfócitos T Reguladores/imunologia , Linfócitos T CD4-Positivos/ultraestrutura , Linfócitos T CD8-Positivos/imunologia , Adesão Celular/genética , Linhagem Celular , Endocitose/genética , Endossomos/genética , Fatores de Transcrição Forkhead/metabolismo , Humanos , Tolerância Imunológica , Molécula 1 de Adesão Intercelular/genética , Fígado/imunologia , Proteínas de Membrana Lisossomal/metabolismo , Lisossomos/metabolismo , Microscopia Eletrônica de Varredura , Pinocitose , Linfócitos T Reguladores/ultraestrutura , beta Catenina/genética , beta Catenina/metabolismo
11.
Liver Transpl ; 24(10): 1453-1469, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30359490

RESUMO

Increased use of high-risk allografts is critical to meet the demand for liver transplantation. We aimed to identify criteria predicting viability of organs, currently declined for clinical transplantation, using functional assessment during normothermic machine perfusion (NMP). Twelve discarded human livers were subjected to NMP following static cold storage. Livers were perfused with a packed red cell-based fluid at 37°C for 6 hours. Multilevel statistical models for repeated measures were employed to investigate the trend of perfusate blood gas profiles and vascular flow characteristics over time and the effect of lactate-clearing (LC) and non-lactate-clearing (non-LC) ability of the livers. The relationship of lactate clearance capability with bile production and histological and molecular findings were also examined. After 2 hours of perfusion, median lactate concentrations were 3.0 and 14.6 mmol/L in the LC and non-LC groups, respectively. LC livers produced more bile and maintained a stable perfusate pH and vascular flow >150 and 500 mL/minute through the hepatic artery and portal vein, respectively. Histology revealed discrepancies between subjectively discarded livers compared with objective findings. There were minimal morphological changes in the LC group, whereas non-LC livers often showed hepatocellular injury and reduced glycogen deposition. Adenosine triphosphate levels in the LC group increased compared with the non-LC livers. We propose composite viability criteria consisting of lactate clearance, pH maintenance, bile production, vascular flow patterns, and liver macroscopic appearance. These have been tested successfully in clinical transplantation. In conclusion, NMP allows an objective assessment of liver function that may reduce the risk and permit use of currently unused high-risk livers.


Assuntos
Transplante de Fígado/efeitos adversos , Preservação de Órgãos/normas , Traumatismo por Reperfusão/diagnóstico , Sobrevivência de Tecidos , Coleta de Tecidos e Órgãos/efeitos adversos , Adulto , Idoso , Estudos de Viabilidade , Feminino , Humanos , Fígado/metabolismo , Masculino , Pessoa de Meia-Idade , Modelos Biológicos , Preservação de Órgãos/métodos , Perfusão/métodos , Perfusão/normas , Prognóstico , Traumatismo por Reperfusão/etiologia , Traumatismo por Reperfusão/prevenção & controle
12.
Liver Transpl ; 24(10): 1437-1452, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30040176

RESUMO

Ischemia/reperfusion injury (IRI) is the main cause of complications following liver transplantation. Reactive oxygen species (ROS) were thought to be the main regulators of IRI. However, recent studies demonstrate that ROS activate the cytoprotective mechanism of autophagy promoting cell survival. Liver IRI initially damages the liver endothelial cells (LEC), but whether ROS-autophagy promotes cell survival in LEC during IRI is not known. Primary human LEC were isolated from human liver tissue and exposed to an in vitro model of IRI to assess the role of autophagy in LEC. The role of autophagy during liver IRI in vivo was assessed using a murine model of partial liver IRI. During IRI, ROS specifically activate autophagy-related protein (ATG) 7 promoting autophagic flux and the formation of LC3B-positive puncta around mitochondria in primary human LEC. Inhibition of ROS reduces autophagic flux in LEC during IRI inducing necrosis. In addition, small interfering RNA knockdown of ATG7 sensitized LEC to necrosis during IRI. In vivo murine livers in uninjured liver lobes demonstrate autophagy within LEC that is reduced following IRI with concomitant reduction in autophagic flux and increased cell death. In conclusion, these findings demonstrate that during liver IRI ROS-dependent autophagy promotes the survival of LEC, and therapeutic targeting of this signaling pathway may reduce liver IRI following transplantation.


Assuntos
Células Endoteliais/fisiologia , Transplante de Fígado/efeitos adversos , Mitofagia/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Traumatismo por Reperfusão/patologia , Animais , Autofagia/fisiologia , Proteína 7 Relacionada à Autofagia/genética , Proteína 7 Relacionada à Autofagia/metabolismo , Sobrevivência Celular , Modelos Animais de Doenças , Técnicas de Silenciamento de Genes , Humanos , Fígado/citologia , Fígado/cirurgia , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Cultura Primária de Células , RNA Interferente Pequeno/metabolismo , Traumatismo por Reperfusão/etiologia , Transdução de Sinais/fisiologia
13.
Front Immunol ; 9: 44, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29422896

RESUMO

Toxic substances and microbial or food-derived antigens continuously challenge the liver, which is tasked with their safe neutralization. This vital organ is also important for the removal of apoptotic immune cells during inflammation and has been previously described as a "graveyard" for dying lymphocytes. The clearance of apoptotic and necrotic cells is known as efferocytosis and is a critical liver function to maintain tissue homeostasis. Much of the research into this form of immunological control has focused on Kupffer cells, the liver-resident macrophages. However, hepatocytes (and other liver resident cells) are competent efferocytes and comprise 80% of the liver mass. Little is known regarding the mechanisms of apoptotic and necrotic cell capture by epithelia, which lack key receptors that mediate phagocytosis in macrophages. Herein, we discuss recent developments that increased our understanding of efferocytosis in tissues, with a special focus on the liver parenchyma. We discuss the impact of efferocytosis in health and in inflammation, highlighting the role of phagocytic epithelia.


Assuntos
Células Epiteliais/metabolismo , Células de Kupffer/imunologia , Fígado/metabolismo , Tecido Parenquimatoso/metabolismo , Fagocitose/imunologia , Apoptose/imunologia , Epitélio/metabolismo , Hepatócitos/metabolismo , Humanos , Fígado/citologia , Necrose/imunologia , Tecido Parenquimatoso/citologia
14.
Methods Mol Biol ; 1697: 43-56, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-28560513

RESUMO

Sphingosine-1-phosphate (S1P) and the enzyme primarily responsible for its production, sphingosine kinase-1 (SphK-1), are dysregulated in multiple human diseases including cancer, multiple sclerosis (MS), diabetes, neurological diseases, fibrosis, and certain pathologies associated with impaired angiogenesis such as age-related macular degeneration (AMD). Antibody-based techniques to identify and localize S1P and SphK-1 within cells and tissue specimens represent a powerful tool, not only to understand biological role of these molecules but also to validate these unique in-class targets in multiple state diseases. Consequently, the potential applications of these molecules for therapy and diagnostic purposes are currently under investigation. Here, we describe a new improved technique, Agitated Low Temperature Epitope Retrieval (ALTER) for staining procedures, to identify expression of S1P and SphK-1 in human frozen tissue samples. The challenges encountered in the process of localization in tissue samples of lipid molecules such as S1P are discussed.


Assuntos
Fígado/metabolismo , Lisofosfolipídeos/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/análise , Esfingosina/análogos & derivados , Linhagem Celular , Secções Congeladas , Humanos , Imuno-Histoquímica , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Esfingosina/metabolismo
15.
J Hepatol ; 66(5): 919-929, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28126468

RESUMO

BACKGROUND & AIMS: Chronic hepatitis C is a global health problem with an estimated 170 million hepatitis C virus (HCV) infected individuals at risk of progressive liver disease and hepatocellular carcinoma (HCC). Autotaxin (ATX, gene name: ENPP2) is a phospholipase with diverse roles in the physiological and pathological processes including inflammation and oncogenesis. Clinical studies have reported increased ATX expression in chronic hepatitis C, however, the pathways regulating ATX and its role in the viral life cycle are not well understood. METHODS: In vitro hepatocyte and ex vivo liver culture systems along with chimeric humanized liver mice and HCC tissue enabled us to assess the interplay between ATX and the HCV life cycle. RESULTS: HCV infection increased hepatocellular ATX RNA and protein expression. HCV infection stabilizes hypoxia inducible factors (HIFs) and we investigated a role for these transcription factors to regulate ATX. In vitro studies show that low oxygen increases hepatocellular ATX expression and transcriptome analysis showed a positive correlation between ATX mRNA levels and hypoxia gene score in HCC tumour tissue associated with HCV and other aetiologies. Importantly, inhibiting ATX-lysophosphatidic acid (LPA) signalling reduced HCV replication, demonstrating a positive role for this phospholipase in the viral life cycle. LPA activates phosphoinositide-3-kinase that stabilizes HIF-1α and inhibiting the HIF signalling pathway abrogates the pro-viral activity of LPA. CONCLUSIONS: Our data support a model where HCV infection increases ATX expression which supports viral replication and HCC progression. LAY SUMMARY: Chronic hepatitis C is a global health problem with infected individuals at risk of developing liver disease that can progress to hepatocellular carcinoma. Autotaxin generates the biologically active lipid lysophosphatidic acid that has been reported to play a tumorigenic role in a wide number of cancers. In this study we show that hepatitis C virus infection increases autotaxin expression via hypoxia inducible transcription factor and provides an environment in the liver that promotes fibrosis and liver injury. Importantly, we show a new role for lysophosphatidic acid in positively regulating hepatitis C virus replication.


Assuntos
Hepacivirus/fisiologia , Diester Fosfórico Hidrolases/fisiologia , Receptores de Ácidos Lisofosfatídicos/fisiologia , Replicação Viral , Animais , Linhagem Celular , Hepatite C Crônica/complicações , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Neoplasias Hepáticas/etiologia , Camundongos , Diester Fosfórico Hidrolases/genética , Regiões Promotoras Genéticas , RNA Mensageiro/análise , Transdução de Sinais
16.
World J Gastroenterol ; 23(48): 8443-8451, 2017 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-29358854

RESUMO

Ischaemia-reperfusion injury (IRI) is the leading cause of injury seen in the liver following transplantation. IRI also causes injury following liver surgery and haemodynamic shock. The first cells within the liver to be injured by IRI are the liver sinusoidal endothelial cells (LSEC). Recent evidence suggests that LSEC co-ordinate and regulates the livers response to a variety of injuries. It is becoming increasingly apparent that the cyto-protective cellular process of autophagy is a key regulator of IRI. In particular LSEC autophagy may be an essential gatekeeper to the development of IRI. The recent availability of liver perfusion devices has allowed for the therapeutic targeting of autophagy to reduce IRI. In particular normothermic machine liver perfusion (NMP-L) allow the delivery of pharmacological agents to donor livers whilst maintaining physiological temperature and hepatic flow rates. In this review we summarise the current understanding of endothelial autophagy and how this may be manipulated during NMP-L to reduce liver IRI.


Assuntos
Autofagia/fisiologia , Células Endoteliais/fisiologia , Transplante de Fígado/efeitos adversos , Fígado/fisiopatologia , Traumatismo por Reperfusão/fisiopatologia , Autofagia/efeitos dos fármacos , Humanos , Fígado/irrigação sanguínea , Fígado/citologia , Fígado/efeitos dos fármacos , Preservação de Órgãos/efeitos adversos , Preservação de Órgãos/métodos , Perfusão/efeitos adversos , Perfusão/métodos , Substâncias Protetoras/farmacologia , Substâncias Protetoras/uso terapêutico , Traumatismo por Reperfusão/etiologia , Traumatismo por Reperfusão/prevenção & controle , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Temperatura
17.
J Autoimmun ; 77: 45-54, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27784538

RESUMO

Primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC) are immune-mediated biliary diseases that demonstrate prominent and restricted genetic association with human leukocyte antigen (HLA) alleles. In PBC, anti-mitochondrial antibodies (AMA) are specific and used as diagnostic biomarkers. PSC-relevant auto-antibodies remain controversial despite a distinct HLA association that mirrors archetypical auto-antigen driven disorders. Herein, we compared antibody-secreting B cells (ASCs) in PSC and PBC liver explants to determine if liver-infiltrating ASCs represent an opportune and novel source of disease-relevant auto-antibodies. Using enzymatic digestion and mechanical disruption, liver mononuclear cells (LIMCs) were isolated from fresh PSC and PBC explants and plasmablast (CD19+CD27+CD38hiCD138-) and plasma cell (CD19+CD27+CD38hiCD138+) ASCs were enumerated by flow cytometry. We observed 45-fold fewer plasma cells in PSC explants (n = 9) compared to PBC samples (n = 5, p < 0.01) and 10-fold fewer IgA-, IgG- and IgM-positive ASCs (p < 0.05). Liver-infiltrating ASCs from PSC and PBC explants were functional and produced similar concentrations of IgA, IgG and IgM following 2 weeks of culture. Antibody production by PBC ASCs (n = 3) was disease-specific as AMA to pyruvate dehydrogenase complex E2 subunit (PDC-E2) was detected by immunostaining, immunoblotting and ELISA. Antibody profiling of PSC supernatants (n = 9) using full-length recombinant human protein arrays (Cambridge Protein Arrays) revealed reactivities to nucleolar protein 3 (5/9) and hematopoietic cell-specific Lyn substrate 1 (3/9). Array analysis of PBC supernatants (n = 3) detected reactivities to PDC-E2 and hexokinase 1 (3/3). In conclusion, we detected unique frequencies of liver-infiltrating ASCs in PSC and PBC and in so doing, highlight a feasible approach for understanding disease-relevant antibodies in PSC.


Assuntos
Autoanticorpos/imunologia , Linfócitos B/imunologia , Colangite Esclerosante/diagnóstico , Colangite Esclerosante/imunologia , Cirrose Hepática Biliar/diagnóstico , Cirrose Hepática Biliar/imunologia , Fenótipo , Adolescente , Adulto , Idoso , Formação de Anticorpos/imunologia , Antígenos CD20/metabolismo , Autoanticorpos/sangue , Autoimunidade , Linfócitos B/patologia , Biomarcadores , Colangite Esclerosante/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Imunofluorescência , Humanos , Imunofenotipagem , Fígado/imunologia , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática Biliar/metabolismo , Contagem de Linfócitos , Masculino , Pessoa de Meia-Idade , Plasmócitos/imunologia , Plasmócitos/metabolismo , Adulto Jovem
18.
J Hepatol ; 66(1): 28-38, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27531641

RESUMO

BACKGROUND & AIMS: The high replication and mutation rate of hepatitis C virus (HCV) results in a heterogeneous population of viral sequences in vivo. HCV replicates in the liver and infected hepatocytes occur as foci surrounded by uninfected cells that may promote compartmentalization of viral variants. Given recent reports showing interferon stimulated gene (ISG) expression in chronic hepatitis C, we hypothesized that local interferon responses may limit HCV replication and evolution. METHODS: To investigate the spatial influence of liver architecture on viral replication we measured HCV RNA and ISG mRNA from each of the 8 Couinaud segments of the liver from 21 patients undergoing liver transplant. RESULTS: HCV RNA and ISG mRNA levels were comparable across all sites from an individual liver but showed up to 500-fold difference between patients. Importantly, there was no association between ISG and HCV RNA expression across all sites in the liver or plasma. Deep sequencing of HCV RNA isolated from the 8 hepatic sites from two subjects showed a similar distribution of viral quasispecies across the liver and uniform sequence diversity. Single genome amplification of HCV E1E2-envelope clones from 6 selected patients at 2 hepatic sites supported these data and showed no evidence for HCV compartmentalization. CONCLUSIONS: We found no differences between the hepatic and plasma viral quasispecies in all patients sampled. We conclude that in end-stage liver disease HCV RNA levels and the genetic pool of HCV envelope sequences are indistinguishable between distant sites in the liver and plasma, arguing against viral compartmentalization. LAY SUMMARY: HCV is an RNA virus that exists as a quasispecies of closely related genomes that are under continuous selection by host innate and adaptive immune responses and antiviral drug therapy. The primary site of HCV replication is the liver and yet our understanding of the spatial distribution of viral variants within the liver is limited. High resolution sequencing of HCV and monitoring of innate immune responses at multiple sites across the liver identified a uniform pattern of diversity and argues against viral compartmentalization.


Assuntos
Doença Hepática Terminal , Hepacivirus , Hepatite C Crônica , Interferons/farmacologia , Fígado , Replicação Viral/efeitos dos fármacos , Adulto , Antivirais/farmacologia , Compartimento Celular/efeitos dos fármacos , Doença Hepática Terminal/etiologia , Doença Hepática Terminal/metabolismo , Doença Hepática Terminal/virologia , Feminino , Hepacivirus/efeitos dos fármacos , Hepacivirus/genética , Hepacivirus/fisiologia , Hepatite C Crônica/complicações , Hepatite C Crônica/virologia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Fígado/patologia , Fígado/virologia , Masculino , Pessoa de Meia-Idade , RNA Viral/análise , Análise de Sequência de RNA/métodos
19.
Gut ; 65(7): 1175-85, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-26078290

RESUMO

INTRODUCTION: CD248 (endosialin) is a stromal cell marker expressed on fibroblasts and pericytes. During liver injury, myofibroblasts are the main source of fibrotic matrix. OBJECTIVE: To determine the role of CD248 in the development of liver fibrosis in the rodent and human setting. DESIGN: CD248 expression was studied by immunostaining and quantitative PCR in both normal and diseased human and murine liver tissue and isolated hepatic stellate cells (HSCs). Hepatic fibrosis was induced in CD248(-/-) and wild-type controls with carbon tetrachloride (CCl4) treatment. RESULTS: Expression of CD248 was seen in normal liver of humans and mice but was significantly increased in liver injury using both immunostaining and gene expression assays. CD248 was co-expressed with a range of fibroblast/HSC markers including desmin, vimentin and α-smooth muscle actin (α-SMA) in murine and human liver sections. CD248 expression was restricted to isolated primary murine and human HSC. Collagen deposition and α-SMA expression, but not inflammation and neoangiogenesis, was reduced in CD248(-/-) mice compared with wild-type mice after CCl4 treatment. Isolated HSC from wild-type and CD248(-/-) mice expressed platelet-derived growth factor receptor α (PDGFR-α) and PDGFR-ß at similar levels. As expected, PDGF-BB stimulation induced proliferation of wild-type HSC, whereas CD248(-/-) HSC did not demonstrate a proliferative response to PDGF-BB. Abrogated PDGF signalling in CD248(-/-) HSC was confirmed by significantly reduced c-fos expression in CD248(-/-) HSC compared with wild-type HSC. CONCLUSIONS: Our data show that deletion of CD248 reduces susceptibility to liver fibrosis via an effect on PDGF signalling, making it an attractive clinical target for the treatment of liver injury.


Assuntos
Antígenos CD/genética , Antígenos CD/metabolismo , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Células Estreladas do Fígado/fisiologia , Cirrose Hepática/metabolismo , Fígado/patologia , Fator de Crescimento Derivado de Plaquetas/metabolismo , Actinas/análise , Indutores da Angiogênese/farmacologia , Animais , Antígenos CD/análise , Antígenos de Neoplasias/análise , Becaplermina , Tetracloreto de Carbono , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Células Cultivadas , Doença Crônica , Colágeno/metabolismo , Desmina/análise , Fibrose , Expressão Gênica , Células Estreladas do Fígado/química , Humanos , Inflamação/genética , Fígado/química , Cirrose Hepática/induzido quimicamente , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neovascularização Patológica/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-sis/farmacologia , RNA Mensageiro/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de Sinais/genética , Fator de Crescimento Transformador beta/genética , Vimentina/análise
20.
Sci Rep ; 5: 15040, 2015 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-26463421

RESUMO

Biodegradable elastomers are a popular choice for tissue engineering scaffolds, particularly in mechanically challenging settings (e.g. the skin). As the optimal rate of scaffold degradation depends on the tissue type to be regenerated, next-generation scaffolds must demonstrate tuneable degradation patterns. Previous investigations mainly focussed on the integration of more or less hydrolysable components to modulate degradation rates. In this study, however, the objective was to develop and synthesize a family of novel biodegradable polyurethanes (PUs) based on a poly(ε-caprolactone urea)urethane backbone integrating polyhedral oligomeric silsesquioxane (POSS-PCLU) with varying amounts of hard segments (24%, 28% and 33% (w/v)) in order to investigate the influence of hard segment chemistry on the degradation rate and profile. PUs lacking POSS nanoparticles served to prove the important function of POSS in maintaining the mechanical structures of the PU scaffolds before, during and after degradation. Mechanical testing of degraded samples revealed hard segment-dependent modulation of the materials' viscoelastic properties, which was attributable to (i) degradation-induced changes in the PU crystallinity and (ii) either the presence or absence of POSS. In conclusion, this study presents a facile method of controlling degradation profiles of PU scaffolds used in tissue engineering applications.


Assuntos
Implantes Absorvíveis , Elastômeros/química , Nanoconjugados/química , Compostos de Organossilício/química , Poliésteres/química , Alicerces Teciduais , Módulo de Elasticidade , Análise de Falha de Equipamento , Cinética , Teste de Materiais , Nanoconjugados/ultraestrutura , Tamanho da Partícula , Desenho de Prótese , Resistência à Tração , Engenharia Tecidual/instrumentação , Uretana/química , Viscosidade
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