RESUMO
Normothermic machine perfusion (NMP) enables pretransplant assessment of high-risk donor livers. The VITTAL trial demonstrated that 71% of the currently discarded organs could be transplanted with 100% 90-day patient and graft survivals. Here, we report secondary end points and 5-year outcomes of this prospective, open-label, phase 2 adaptive single-arm study. The patient and graft survivals at 60 months were 82% and 72%, respectively. Four patients lost their graft due to nonanastomotic biliary strictures, one caused by hepatic artery thrombosis in a liver donated following brain death, and 3 in elderly livers donated after circulatory death (DCD), which all clinically manifested within 6 months after transplantation. There were no late graft losses for other reasons. All the 4 patients who died during the study follow-up had functioning grafts. Nonanastomotic biliary strictures developed in donated after circulatory death livers that failed to produce bile with pH >7.65 and bicarbonate levels >25 mmol/L. Histological assessment in these livers revealed high bile duct injury scores characterized by arterial medial necrosis. The quality of life at 6 months significantly improved in all but 4 patients suffering from nonanastomotic biliary strictures. This first report of long-term outcomes of high-risk livers assessed by normothermic machine perfusion demonstrated excellent 5-year survival without adverse effects in all organs functioning beyond 1 year (ClinicalTrials.gov number NCT02740608).
Assuntos
Transplante de Fígado , Idoso , Humanos , Constrição Patológica/etiologia , Fígado/cirurgia , Transplante de Fígado/efeitos adversos , Preservação de Órgãos , Perfusão , Estudos Prospectivos , Qualidade de VidaRESUMO
Normothermic machine perfusion (NMP) allows objective assessment of donor liver transplantability. Several viability evaluation protocols have been established, consisting of parameters such as perfusate lactate clearance, pH, transaminase levels, and the production and composition of bile. The aims of this study were to assess 3 such protocols, namely, those introduced by the teams from Birmingham (BP), Cambridge (CP), and Groningen (GP), using a cohort of high-risk marginal livers that had initially been deemed unsuitable for transplantation and to introduce the concept of the viability assessment sensitivity and specificity. To demonstrate and quantify the diagnostic accuracy of these protocols, we used a composite outcome of organ use and 24-month graft survival as a surrogate endpoint. The effects of assessment modifications, including the removal of the most stringent components of the protocols, were also assessed. Of the 31 organs, 22 were transplanted after a period of NMP, of which 18 achieved the outcome of 24-month graft survival. The BP yielded 94% sensitivity and 50% specificity when predicting this outcome. The GP and CP both seemed overly conservative, with 1 and 0 organs, respectively, meeting these protocols. Modification of the GP and CP to exclude their most stringent components increased this to 11 and 8 organs, respectively, and resulted in moderate sensitivity (56% and 44%) but high specificity (92% and 100%, respectively) with respect to the composite outcome. This study shows that the normothermic assessment protocols can be useful in identifying potentially viable organs but that the balance of risk of underuse and overuse varies by protocol.
Assuntos
Transplante de Fígado , Sobrevivência de Enxerto , Humanos , Fígado , Transplante de Fígado/efeitos adversos , Transplante de Fígado/métodos , Doadores Vivos , Preservação de Órgãos/métodos , Perfusão/métodosRESUMO
There is no effective treatment for autoimmune biliary diseases. Therefore, understanding their immunopathology is crucial. The biliary epithelial cells (BEC), expressing TLR-4, are constantly exposed to gut microbes and bacterial wall LPS, and in settings of inflammation, the immune infiltrate is dense within the peribiliary region of human liver. By dual immunohistochemistry, we affirm human intrahepatic T cell infiltrate includes CCR6+CD4+ and AhR+CD4+ T cells with potential for plasticity to Th17 phenotype. Mechanistically, we demonstrate that Th1 and Th17 inflammatory cytokines and LPS enhance human primary BEC release of the CCR6 ligand CCL20 and BEC secretion of Th17-polarizing cytokines IL-6 and IL-1ß. Cell culture assays with human BEC secretome showed that secretome polarizes CD4 T cells toward a Th17 phenotype and supports the survival of Th17 cells. BEC secretome did not promote Th1 cell generation. Additionally, we give evidence for a mutually beneficial feedback of the type 17 cell infiltrate on BEC, showing that treatment with type 17 cytokines increases BEC proliferation, as monitored by Ki67 and activation of JAK2-STAT3 signaling. This study identifies human BEC as active players in determining the nature of the intrahepatic immune microenvironment. In settings of inflammation and/or infection, biliary epithelium establishes a prominent peribiliary type 17 infiltrate via recruitment and retention and enhances polarization of intrahepatic CD4 cells toward Th17 cells via type 17 cytokines, and, reciprocally, Th17 cells promote BEC proliferation for biliary regeneration. Altogether, we provide new insight into cross-talk between Th17 lymphocytes and human primary biliary epithelium in biliary regenerative pathologies.
Assuntos
Ductos Biliares/patologia , Comunicação Celular/fisiologia , Células Epiteliais/fisiologia , Hepatopatias/imunologia , Células Th17/fisiologia , Proliferação de Células , Células Cultivadas , Humanos , Interleucina-17/farmacologia , Lipopolissacarídeos/farmacologia , Hepatopatias/patologia , Receptores de Hidrocarboneto Arílico/fisiologia , Receptores CCR6/fisiologiaRESUMO
The increasing disparity between the number of patients listed for transplantation and the number of suitable organs has led to the increasing use of extended criteria donors (ECDs). ECDs are at increased risk of developing ischaemia reperfusion injury and greater risk of post-transplant complications. Ischaemia reperfusion injury is a major complication of organ transplantation defined as the inflammatory changes seen following the disruption and restoration of blood flow to an organ-it is a multifactorial process with the potential to cause both local and systemic organ failure. The utilisation of machine perfusion under normothermic (37 degrees Celsius) and hypothermic (4-10 degrees Celsius) has proven to be a significant advancement in organ preservation and restoration. One of the key benefits is its ability to optimise suboptimal organs for successful transplantation. This review is focused on examining ischaemia reperfusion injury and how machine perfusion ameliorates the graft's response to this.
Assuntos
Sobrevivência de Enxerto , Transplante de Fígado/métodos , Fígado/irrigação sanguínea , Preservação de Órgãos/métodos , Perfusão , Traumatismo por Reperfusão/terapia , Animais , HumanosRESUMO
Liver ischaemia-reperfusion injury (IRI) is an intrinsic part of the transplantation process and damages the parenchymal cells of the liver including hepatocytes, endothelial cells and cholangiocytes. Many biomarkers of IRI have been described over the past two decades that have attempted to quantify the extent of IRI involving different hepatic cellular compartments, with the aim to allow clinicians to predict the suitability of donor livers for transplantation. The advent of machine perfusion has added an additional layer of complexity to this field and has forced researchers to re-evaluate the utility of IRI biomarkers in different machine preservation techniques. In this review, we summarise the current understanding of liver IRI biomarkers and discuss them in the context of machine perfusion.
Assuntos
Transplante de Fígado/métodos , Traumatismo por Reperfusão/diagnóstico , Animais , Biomarcadores/metabolismo , Endotelina-1/metabolismo , Fator 2 de Crescimento de Fibroblastos/metabolismo , Humanos , Interleucinas/metabolismo , Transplante de Fígado/efeitos adversos , Transplante de Fígado/instrumentação , MicroRNAs/metabolismo , Traumatismo por Reperfusão/etiologia , Traumatismo por Reperfusão/metabolismoRESUMO
Strategies to increase the use of steatotic donor livers are required to tackle the mortality on the transplant waiting list. We aimed to test the efficacy of pharmacological enhancement of the lipid metabolism of human livers during ex situ normothermic machine perfusion to promote defatting and improve the functional recovery of the organs. Because of steatosis, 10 livers were discarded and were allocated either to a defatting group that had the perfusate supplemented with a combination of drugs to enhance lipid metabolism or to a control group that received perfusion fluid with vehicle only. Steatosis was assessed using tissue homogenate and histological analyses. Markers for lipid oxidation and solubilization, oxidative injury, inflammation, and biliary function were evaluated by enzyme-linked immunosorbent assay, immunohistochemistry, and in-gel protein detection. Treatment reduced tissue triglycerides by 38% and macrovesicular steatosis by 40% over 6 hours. This effect was driven by increased solubility of the triglycerides (P = 0.04), and mitochondrial oxidation as assessed by increased ketogenesis (P = 0.008) and adenosine triphosphate synthesis (P = 0.01) were associated with increased levels of the enzymes acyl-coenzyme A oxidase 1, carnitine palmitoyltransferase 1A, and acetyl-coenzyme A synthetase. Concomitantly, defatted livers exhibited enhanced metabolic functional parameters such as urea production (P = 0.03), lower vascular resistance, lower release of alanine aminotransferase (P = 0.049), and higher bile production (P = 0.008) with a higher bile pH (P = 0.03). The treatment down-regulated the expression of markers for oxidative injury as well as activation of immune cells (CD14; CD11b) and reduced the release of inflammatory cytokines in the perfusate (tumor necrosis factor α; interleukin 1ß). In conclusion, pharmacological enhancement of intracellular lipid metabolism during normothermic machine perfusion decreased the lipid content of human livers within 6 hours. It also improved the intracellular metabolic support to the organs, leading to successful functional recovery and decreased expression of markers of reperfusion injury.
Assuntos
Fígado Gorduroso/patologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Transplante de Fígado , Preservação de Órgãos/métodos , Perfusão/métodos , Coleta de Tecidos e Órgãos/métodos , Aloenxertos/metabolismo , Aloenxertos/patologia , Antracenos , Butiratos/farmacologia , Colforsina/farmacologia , Fígado Gorduroso/diagnóstico , Fígado Gorduroso/metabolismo , Estudos de Viabilidade , Feminino , Humanos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Masculino , Pessoa de Meia-Idade , Nicotinamida Fosforribosiltransferase/farmacologia , Preservação de Órgãos/instrumentação , Perfusão/instrumentação , Perileno/análogos & derivados , Perileno/farmacologia , Soluções Farmacêuticas/farmacologia , Compostos de Fenilureia/farmacologia , Traumatismo por Reperfusão/diagnóstico , Traumatismo por Reperfusão/etiologia , Traumatismo por Reperfusão/prevenção & controle , Tiazóis/farmacologia , Coleta de Tecidos e Órgãos/efeitos adversosRESUMO
While majority of liver transplants worldwide continue to be performed using deceased donor organs, the demands for donor livers continues to exceed the current supply. In an attempt to maximize the number of potentially usable donor livers and expand the current donor pool, there is intense global research by various groups exploring the role of machine perfusion in the liver transplantation, particularly with respect to the machine perfusion of extended-criteria liver donors. In this review, the authors summarize the current field of machine perfusion strategies as applied to deceased donor liver transplantation and how therapeutic targeting of the liver sinusoidal endothelial cell may improve the quality of donor livers.
Assuntos
Células Endoteliais/transplante , Transplante de Fígado/métodos , Fígado/cirurgia , Preservação de Órgãos/métodos , Perfusão/métodos , Traumatismo por Reperfusão/prevenção & controle , Doadores de Tecidos/provisão & distribuição , Animais , Seleção do Doador , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Sobrevivência de Enxerto , Humanos , Fígado/metabolismo , Fígado/patologia , Transplante de Fígado/efeitos adversos , Preservação de Órgãos/efeitos adversos , Preservação de Órgãos/instrumentação , Perfusão/efeitos adversos , Perfusão/instrumentação , Traumatismo por Reperfusão/etiologia , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Fatores de Risco , Transdução de Sinais , Resultado do Tratamento , Listas de EsperaRESUMO
Long-standing research has shown that increased lipid content in donor livers is associated with inferior graft outcomes posttransplant. The global epidemic that is obesity has increased the prevalence of steatosis in organ donors, to the extent that it has become one of the main reasons for declining livers for transplantation. Consequently, it is one of the major culprits behind the discrepancy between the number of donor livers offered for transplantation and those that go on to be transplanted. Steatotic livers are characterized by poor microcirculation, depleted energy stores because of an impaired capacity for mitochondrial recovery, and a propensity for an exaggerated inflammatory response following reperfusion injury culminating in poorer graft function postoperatively. Ex situ machine perfusion, currently a novel method in graft preservation, is showing great promise in providing a tool for the recovery and reconditioning of marginal livers. Hence, reconditioning these steatotic livers using machine perfusion has the potential to increase the number of liver transplants performed. In this review, we consider the problematic issues associated with fatty livers in the realm of transplantation and discuss pharmacological and nonpharmacological options that are being developed to enhance recovery of these organs using machine perfusion and defatting strategies.
Assuntos
Gorduras/metabolismo , Fígado Gorduroso/fisiopatologia , Transplante de Fígado , Doadores Vivos/provisão & distribuição , Preservação de Órgãos/métodos , Perfusão , HumanosRESUMO
Hypothermic oxygenated perfusion (HOPE) and normothermic perfusion are seen as distinct techniques of ex situ machine perfusion of the liver. We aimed to demonstrate the feasibility of combining both techniques and whether it would improve functional parameters of donor livers into transplant standards. Ten discarded human donor livers had either 6 hours of normothermic perfusion (n = 5) or 2 hours of HOPE followed by 4 hours of normothermic perfusion (n = 5). Liver function was assessed according to our viability criteria; markers of tissue injury and hepatic metabolic activity were compared between groups. Donor characteristics were comparable. During the hypothermic perfusion phase, livers down-regulated mitochondrial respiration (oxygen uptake, P = 0.04; partial pressure of carbon dioxide perfusate, P = 0.04) and increased adenosine triphosphate levels 1.8-fold. Following normothermic perfusion, those organs achieved lower tissue expression of markers of oxidative injury (4-hydroxynonenal, P = 0.008; CD14 expression, P = 0.008) and inflammation (CD11b, P = 0.02; vascular cell adhesion molecule 1, P = 0.05) compared with livers that had normothermic perfusion alone. All livers in the combined group achieved viability criteria, whereas 40% (2/5) in the normothermic group failed (P = 0.22). In conclusion, this study suggests that a combined protocol of hypothermic oxygenated and normothermic perfusions might attenuate oxidative stress, tissue inflammation, and improve metabolic recovery of the highest-risk donor livers compared with normothermic perfusion alone.
Assuntos
Seleção do Doador/normas , Transplante de Fígado/métodos , Preservação de Órgãos/métodos , Perfusão/métodos , Aloenxertos/metabolismo , Aloenxertos/cirurgia , Biomarcadores/análise , Biomarcadores/metabolismo , Isquemia Fria/instrumentação , Isquemia Fria/métodos , Estudos de Viabilidade , Humanos , Fígado/metabolismo , Fígado/cirurgia , Testes de Função Hepática , Transplante de Fígado/normas , Preservação de Órgãos/instrumentação , Estresse Oxidativo , Perfusão/instrumentação , Isquemia Quente/instrumentação , Isquemia Quente/métodosRESUMO
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 & controleRESUMO
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/fisiologiaRESUMO
Tumour necrosis factor-like weak inducer of apoptosis (TWEAK) and its receptor fibroblast growth factor-inducible 14 (Fn14) have been associated with liver regeneration in vivo. To further investigate the role of this pathway we examined their expression in human fibrotic liver disease and the effect of pathway deficiency in a murine model of liver fibrosis. The expression of Fn14 and TWEAK in normal and diseased human and mouse liver tissue and primary human hepatic stellate cells (HSCs) were investigated by qPCR, western blotting and immunohistochemistry. In addition, the levels of Fn14 in HSCs following pro-fibrogenic and pro-inflammatory stimuli were assessed and the effects of exogenous TWEAK on HSCs proliferation and activation were studied in vitro. Carbon tetrachloride (CCl4 ) was used to induce acute and chronic liver injury in TWEAK KO mice. Elevated expression of both Fn14 and TWEAK were detected in acute and chronic human liver injury, and co-localized with markers of activated HSCs. Fn14 levels were low in quiescent HSCs but were significantly induced in activated HSCs, which could be further enhanced with the profibrogenic cytokine TGFß in vitro. Stimulation with recombinant TWEAK induced proliferation but not further HSCs activation. Fn14 gene expression was also significantly up-regulated in CCl4 models of hepatic injury whereas TWEAK KO mice showed reduced levels of liver fibrosis following chronic CCl4 injury. In conclusion, TWEAK/Fn14 interaction leads to the progression of fibrotic liver disease via direct modulation of HSCs proliferation, making it a potential therapeutic target for liver fibrosis.
Assuntos
Células Estreladas do Fígado/patologia , Cirrose Hepática/etiologia , Receptores do Fator de Necrose Tumoral/metabolismo , Fatores de Necrose Tumoral/deficiência , Actinas/metabolismo , Animais , Tetracloreto de Carbono/toxicidade , Proliferação de Células , Doença Hepática Induzida por Substâncias e Drogas , Doença Hepática Crônica Induzida por Substâncias e Drogas , Citocina TWEAK , Progressão da Doença , Fibroblastos/metabolismo , Humanos , Cirrose Hepática/patologia , Masculino , Camundongos Knockout , RNA Mensageiro/metabolismo , Fatores de Transcrição SOX9/metabolismo , Receptor de TWEAK , Fatores de Necrose Tumoral/metabolismo , Fatores de Necrose Tumoral/farmacologia , Regulação para Cima/fisiologiaRESUMO
UNLABELLED: Chronic hepatitis occurs when effector lymphocytes are recruited to the liver from blood and retained in tissue to interact with target cells, such as hepatocytes or bile ducts (BDs). Vascular cell adhesion molecule 1 (VCAM-1; CD106), a member of the immunoglobulin superfamily, supports leukocyte adhesion by binding α4ß1 integrins and is critical for the recruitment of monocytes and lymphocytes during inflammation. We detected VCAM-1 on cholangiocytes in chronic liver disease (CLD) and hypothesized that biliary expression of VCAM-1 contributes to the persistence of liver inflammation. Hence, in this study, we examined whether cholangiocyte expression of VCAM-1 promotes the survival of intrahepatic α4ß1 expressing effector T cells. We examined interactions between primary human cholangiocytes and isolated intrahepatic T cells ex vivo and in vivo using the Ova-bil antigen-driven murine model of biliary inflammation. VCAM-1 was detected on BDs in CLDs (primary biliary cirrhosis, primary sclerosing cholangitis, alcoholic liver disease, and chronic hepatitis C), and human cholangiocytes expressed VCAM-1 in response to tumor necrosis factor alpha alone or in combination with CD40L or interleukin-17. Liver-derived T cells adhered to cholangiocytes in vitro by α4ß1, which resulted in signaling through nuclear factor kappa B p65, protein kinase B1, and p38 mitogen-activated protein kinase phosphorylation. This led to increased mitochondrial B-cell lymphoma 2 accumulation and decreased activation of caspase 3, causing increased cell survival. We confirmed our findings in a murine model of hepatobiliary inflammation where inhibition of VCAM-1 decreased liver inflammation by reducing lymphocyte recruitment and increasing CD8 and T helper 17 CD4 T-cell survival. CONCLUSIONS: VCAM-1 expression by cholangiocytes contributes to persistent inflammation by conferring a survival signal to α4ß1 expressing proinflammatory T lymphocytes in CLD.
Assuntos
Apoptose , Ductos Biliares/química , Hepatite/etiologia , Linfócitos T/fisiologia , Molécula 1 de Adesão de Célula Vascular/fisiologia , Adesão Celular , Células Cultivadas , Humanos , Integrina alfa4beta1/fisiologia , NF-kappa B/fisiologia , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/análise , Proteínas Proto-Oncogênicas c-akt/fisiologia , Linfócitos T/citologia , Molécula 1 de Adesão de Célula Vascular/análise , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologiaAssuntos
Metabolismo dos Lipídeos , Transplante de Fígado , Fígado Gorduroso , Humanos , Fígado , Preservação de Órgãos , PerfusãoRESUMO
Extended duration of normothermic machine perfusion (NMP) provides opportunities to resuscitate suboptimal donor livers. This intervention requires adequate oxygen delivery typically provided by a blood-based perfusion solution. Methaemoglobin (MetHb) results from the oxidation of iron within haemoglobin and represents a serious problem in perfusions lasting > 24 h. We explored the effects of anti-oxidant, N-acetylcysteine (NAC) on the accumulation of methaemoglobin. NMP was performed on nine human donor livers declined for transplantation: three were perfused without NAC (no-NAC group), and six organs perfused with an initial NAC bolus, followed by continuous infusion (NAC group), with hourly methaemoglobin perfusate measurements. In-vitro experiments examined the impact of NAC (3 mg) on red cells (30 ml) in the absence of liver tissue. The no-NAC group sustained perfusions for an average of 96 (range 87-102) h, universally developing methaemoglobinaemia (≥ 2%) observed after an average of 45 h, with subsequent steep rise. The NAC group was perfused for an average of 148 (range 90-184) h. Only 2 livers developed methaemoglobinaemia (peak MetHb of 6%), with an average onset of 116.5 h. Addition of NAC efficiently limits formation and accumulation of methaemoglobin during NMP, and allows the significant extension of perfusion duration.
Assuntos
Transplante de Fígado , Metemoglobinemia , Humanos , Transplante de Fígado/métodos , Acetilcisteína/farmacologia , Preservação de Órgãos/métodos , Metemoglobina , Fígado , Perfusão/métodosRESUMO
BACKGROUND & AIMS: Hepatitis C virus (HCV) causes progressive liver disease and is a major risk factor for the development of hepatocellular carcinoma (HCC). However, the role of infection in HCC pathogenesis is poorly understood. We investigated the effect(s) of HCV infection and viral glycoprotein expression on hepatoma biology to gain insights into the development of HCV associated HCC. METHODS: We assessed the effect(s) of HCV and viral glycoprotein expression on hepatoma polarity, migration and invasion. RESULTS: HCV glycoproteins perturb tight and adherens junction protein expression, and increase hepatoma migration and expression of epithelial to mesenchymal transition markers Snail and Twist via stabilizing hypoxia inducible factor-1α (HIF-1α). HIF-1α regulates many genes involved in tumor growth and metastasis, including vascular endothelial growth factor (VEGF) and transforming growth factor-beta (TGF-ß). Neutralization of both growth factors shows different roles for VEGF and TGFß in regulating hepatoma polarity and migration, respectively. Importantly, we confirmed these observations in virus infected hepatoma and primary human hepatocytes. Inhibition of HIF-1α reversed the effect(s) of infection and glycoprotein expression on hepatoma permeability and migration and significantly reduced HCV replication, demonstrating a dual role for HIF-1α in the cellular processes that are deregulated in many human cancers and in the viral life cycle. CONCLUSIONS: These data provide new insights into the cancer-promoting effects of HCV infection on HCC migration and offer new approaches for treatment.
Assuntos
Carcinoma Hepatocelular/fisiopatologia , Movimento Celular/fisiologia , Hepacivirus/fisiologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Neoplasias Hepáticas/fisiopatologia , Replicação Viral/fisiologia , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Polaridade Celular/fisiologia , Progressão da Doença , Glicoproteínas/fisiologia , Hepatite C/patologia , Hepatite C/fisiopatologia , Humanos , Neoplasias Hepáticas/patologia , Junções Íntimas/fisiologia , Fator de Crescimento Transformador beta/fisiologia , Fator A de Crescimento do Endotélio Vascular/fisiologiaRESUMO
Background: Ex situ donor liver machine perfusion is a promising tool to assess organ viability prior to transplantation and platform to investigate novel therapeutic interventions. However, the wide variability in donor and graft characteristics between individual donor livers limits the comparability of results. We investigated the hypothesis that the development of a split liver ex situ machine perfusion protocol provides the ideal comparative controls in the investigation of machine perfusion techniques and therapeutic interventions, thus leading to more comparable results. Methods: Four discarded human donor livers were surgically split following identification and separation of right and left inflow and outflow vessels. Each lobe, on separate perfusion machines, was subjected to normothermic perfusion using an artificial hemoglobin-based oxygen carrier solution for 6 h. Metabolic parameters as well as hepatic artery and portal vein perfusion parameters monitored. Results: Trends in hepatic artery and portal vein flows showed a general increase in both lobes throughout each perfusion experiment, even when normalized for tissue weight. Progressive decreases in perfusate lactate and glucose levels exhibited comparable trends in between lobes. Conclusion: Our results demonstrate comparability between right and left lobes when simultaneously subjected to normothermic machine perfusion. In the pre-clinical setting, this model provides the ideal comparative controls in the investigation of therapeutic interventions.
RESUMO
Increasing evidence shows that reactive oxygen species (ROS) may be critical mediators of liver damage during the relative hypoxia of ischemia/reperfusion injury (IRI) associated with transplant surgery or of the tissue microenvironment created as a result of chronic hepatic inflammation or infection. Much work has been focused on Kupffer cells or liver resident macrophages with respect to the generation of ROS during IRI. However, little is known about the contribution of endogenous hepatocyte ROS production or its potential impact on the parenchymal cell death associated with IRI and chronic hepatic inflammation. For the first time, we show that human hepatocytes isolated from nondiseased liver tissue and human hepatocytes isolated from diseased liver tissue exhibit marked differences in ROS production in response to hypoxia/reoxygenation (H-R). Furthermore, several different antioxidants are able to abrogate hepatocyte ROS-induced cell death during hypoxia and H-R. These data provide clear evidence that endogenous ROS production by mitochondria and nicotinamide adenine dinucleotide phosphate oxidase drives human hepatocyte apoptosis and necrosis during hypoxia and H-R and may therefore play an important role in any hepatic diseases characterized by a relatively hypoxic liver microenvironment. In conclusion, these data strongly suggest that hepatocytes and hepatocyte-derived ROS are active participants driving hepatic inflammation. These novel findings highlight important functional/metabolic differences between hepatocytes isolated from normal donor livers, hepatocytes isolated from normal resected tissue obtained during surgery for malignant neoplasms, and hepatocytes isolated from livers with end-stage disease. Furthermore, the targeting of hepatocyte ROS generation with antioxidants may offer therapeutic potential for the adjunctive treatment of IRI and chronic inflammatory liver diseases.
Assuntos
Hepatócitos/metabolismo , Hipóxia/metabolismo , Fígado/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Acetilcisteína/farmacologia , Anexina A5/análise , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Feminino , Citometria de Fluxo , Hepatócitos/patologia , Humanos , Hipóxia/patologia , Inflamação/tratamento farmacológico , Células de Kupffer/metabolismo , Células de Kupffer/patologia , Fígado/patologia , Hepatopatias/tratamento farmacológico , Hepatopatias/metabolismo , Hepatopatias/patologia , Masculino , Mitocôndrias/metabolismo , Modelos Biológicos , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/metabolismo , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Adulto JovemRESUMO
[This corrects the article DOI: 10.1371/journal.pone.0224066.].
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.