RESUMEN
BACKGROUND: Reversible ischaemia/reperfusion (I/R) liver injury has been used to induce engraftment and hepatic parenchymal differentiation of exogenous beta2-microglubulin(-)/Thy1(+) bone marrow derived cells. AIM: To test the ability of this method of hepatic parenchymal repopulation, theoretically applicable to clinical practice, to correct the metabolic disorder in a rat model of congenital hyperbilirubinaemia. METHODS AND RESULTS: Analysis by confocal laser microscopy of fluorescence labelled cells and by immunohistochemistry for beta2-microglubulin, 72 hours after intraportal delivery, showed engraftment of infused cells in liver parenchyma of rats with I/R, but not in control animals with non-injured liver. Transplantation of bone marrow derived cells obtained from GFP-transgenic rats into Lewis rats resulted in the presence of up to 20% of GFP positive hepatocytes in I/R liver lobes after one month. The repopulation rate was proportional to the number of transplanted cells. Infusion of GFP negative bone marrow derived cells into GFP positive transgenic rats resulted in the appearance of GFP negative hepatocytes, suggesting that the main mechanism underlying parenchymal repopulation was differentiation rather than cell fusion. Transplantation of wild type bone marrow derived cells into hyperbilirubinaemic Gunn rats with deficient bilirubin conjugation after I/R damage resulted in 30% decrease in serum bilirubin, the appearance of bilirubin conjugates in bile, and the expression of normal UDP-glucuronyltransferase enzyme evaluated by polymerase chain reaction. CONCLUSIONS: I/R injury induced hepatic parenchymal engraftment and differentiation into hepatocyte-like cells of bone marrow derived cells. Transplantation of bone marrow derived cells from non-affected animals resulted in the partial correction of hyperbilirubinaemia in the Gunn rat.
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Trasplante de Médula Ósea/métodos , Hiperbilirrubinemia Hereditaria/terapia , Regeneración Hepática , Acondicionamiento Pretrasplante/métodos , Animales , Bilirrubina/metabolismo , Diferenciación Celular , Modelos Animales de Enfermedad , Supervivencia de Injerto , Hepatocitos/patología , Hiperbilirrubinemia Hereditaria/metabolismo , Hiperbilirrubinemia Hereditaria/patología , Circulación Hepática , Ratas , Ratas Gunn , Daño por Reperfusión/patología , Resultado del TratamientoRESUMEN
Acute liver failure (ALF) is associated with significant morbidity and mortality. Better understanding of the pathophysiology of the disease and improvements in patient management have resulted in increased survival. Liver transplantation remains the only proven therapeutic modality. Primarily because of organ donor shortage, a number of strategies have been developed in an attempt to support patients with severe ALF until either an organ becomes available for transplantation or until they recover. Liver support strategies include use of either non-biological or biological systems. Non-biological systems include plasma exchange, hemodialysis, hemofiltration, charcoal and resin hemoperfusion. These systems are able to remove toxins, but their utility is limited by their inability to provide missing liver synthetic function. Biological liver support systems include ex vivo liver perfusion and use of hepatocyte-based extracorporeal devices. Like non-biological systems, biological ones provide a means of detoxification and in addition biotransformation and biosynthetic functions. The major limitation of these systems is the lack of availability of an effective highly differentiated human hepatocyte line for clinical use. Currently clinically tested liver support systems use either porcine hepatocytes or human hepatoma cell lines. As liver support therapy evolves, there will be a role for both biological and non-biological liver support systems to treat specific forms of liver failure.
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Fallo Hepático Agudo/terapia , Hígado Artificial , HumanosRESUMEN
BACKGROUND: Serum bile acids are increased in liver failure, but the composition of the bile acid pool in this condition has not been studied in detail. This information is of interest because of dihydroxy bile acid toxicity. METHODS: We measured serum bile acids by gas chromatography-mass spectrometry in 13 patients with fulminant liver failure and five patients with acute-on-chronic liver failure. Furthermore, serum bile acids were analysed in the same patients after 6 h of treatment with a bioartificial liver, consisting of a hollow-fibre cartridge with microcarrier-attached porcine hepatocytes and a charcoal column. RESULTS: Pre-bioartificial liver serum bile acids demonstrated a high dihydroxy/trihydroxy ratio and were higher in patients with acute-on-chronic liver failure than in those with fulminant liver failure (452.8 +/- 98.6 vs. 182.1 +/- 39.7 micro mol/L; P < 0.05). Bioartificial liver treatment decreased significantly serum bile acids in patients with fulminant liver failure (-38.8%) and acute-on-chronic liver failure (-35.8%), with a decreased dihydroxy/trihydroxy ratio. In vitro, porcine hepatocytes in the bioreactor cleared most conjugated bile acid species from pooled patient plasma. CONCLUSIONS: Acute liver failure is associated with very high serum levels of toxic bile acids that could contribute to the pathogenesis of the syndrome. Bioartificial liver treatment reduces both serum bile acid concentrations and the hydrophobicity of the bile acid pool.
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Ácidos y Sales Biliares/sangre , Fallo Hepático/sangre , Fallo Hepático/terapia , Hígado Artificial , Adolescente , Adulto , Anciano , Femenino , Cromatografía de Gases y Espectrometría de Masas , Encefalopatía Hepática/sangre , Encefalopatía Hepática/terapia , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Recently it was shown that a population of cells in the bone marrow-expressing hematopoietic stem cell antigens could differentiate into hepatocytes. However, explicitly committed hepatocyte progenitors, which exhibit highly differentiated liver functions, immediately upon isolation, have not yet been isolated from bone marrow. After studying common antigens on blast-like cells in fetal and adult regenerating cholestatic rat livers and human regenerating and malignant livers, we hypothesized that beta-2-microglobulin-negative (beta(2)m(-)) cells might represent dedifferentiated hepatocytes and/or their progenitors. Utilizing a two-step magnetic bead cell-sorting procedure, we show that in bone marrow from rat and human, beta(2)m(-)/Thy-1(+) cells consistently express liver-specific genes and functions. After intraportal infusion into rat livers, bone marrow-derived hepatocyte stem cells (BDHSC) integrated with hepatic cell plates and differentiated into mature hepatocytes. In a culture system simulating liver regeneration and containing cholestatic serum, these cells differentiated into mature hepatocytes and metabolized ammonia into urea. This differentiation was dependent on a yet nondescript humoral signal existing in the cholestatic serum. Transmission electron microscopy and three-dimensional digital reconstruction confirmed hepatocyte ultrastructure of cultured BDHSC.
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Células Madre Hematopoyéticas/fisiología , Hepatocitos/química , Hepatocitos/trasplante , Albúminas/metabolismo , Animales , Diferenciación Celular , Células Cultivadas , Colestasis Intrahepática/metabolismo , Colestasis Intrahepática/patología , Hepatocitos/citología , Separación Inmunomagnética , Hígado/metabolismo , Regeneración Hepática , ARN Mensajero/biosíntesis , Ratas , Ratas Endogámicas Lew , Ratas Sprague-Dawley , Antígenos Thy-1/análisis , Antígenos Thy-1/inmunología , Microglobulina beta-2/análisis , Microglobulina beta-2/inmunologíaRESUMEN
Liver transplantation is the only clinically effective method of treating acute liver failure. However, wider application of this therapeutic modality is restricted primarily by shortage of donor organs. In the search for alternative methods of liver replacement therapy, investigators have focused on transplantation of normal allogeneic hepatocytes and on the development of liver support systems utilizing isolated hepatocytes. Since all human livers suitable for cell harvest are being used for transplantation, hepatocyte therapy using human tissue would require growing of cells in vitro. Unfortunately, although hepatocytes have tremendous capacity to proliferate in vivo, their ability to grow in culture is severely limited. Stromal cells from bone marrow and other blood-forming organs have been found to support hematopoiesis. In this paper, we show that bone marrow-derived stromal cells (BMSCs) enhance proliferation and support differentiation of rat hepatocytes in culture. Further, we demonstrate that in hepatocyte/BMSC co-cultures, clonal expansion of small hepatocytes (SH) is increased. Using semipermeable membrane cultures, we established that direct cell-cell contact is necessary for stimulation of cell proliferation. We also show that BMSCs which are in direct contact with hepatocytes and SH colonies express Jagged1. This suggests a potential role for Notch signaling in the observed effects. Finally, we present evidence that the expression and activity of liver specific transcription factors, CCAAT/enhancer binding proteins and liver specific key enzymes such as tryptophan 2,3-dioxygenase, are improved in hepatocyte/BMSC co-cultures. In conclusion, results of this study indicate that BMSCs could facilitate proliferation and differentiation of primary rat hepatocytes and their progenitors (SH) in vitro.
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Células de la Médula Ósea/citología , Hepatocitos/citología , Células del Estroma/fisiología , Animales , Bromodesoxiuridina/metabolismo , Proteínas Potenciadoras de Unión a CCAAT/biosíntesis , Proteínas Potenciadoras de Unión a CCAAT/genética , Proteínas de Unión al Calcio , Comunicación Celular , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular , División Celular , Células Cultivadas , Hepatocitos/metabolismo , Péptidos y Proteínas de Señalización Intercelular , Proteína Jagged-1 , Cinética , Masculino , Proteínas de la Membrana , Fenotipo , Biosíntesis de Proteínas , Proteínas/genética , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , Proteínas Serrate-Jagged , Células Madre/citología , Células Madre/metabolismoRESUMEN
Intracranial hypertension leading to brainstem coning is a major cause of death in fulminant hepatic failure (FHF). We have developed a bioartificial liver (BAL) utilizing plasma perfusion through a bioreactor loaded with porcine hepatocytes and a column with activated charcoal. In a Phase I clinical trial, we observed a decrease in intracranial pressure (ICP) in FHF patients. However, these patients received BAL therapy together with other measures. We therefore examined whether BAL therapy alone could prevent development of intracranial hypertension in pigs with surgically induced FHF. Pigs (40-60 kg) underwent end-to-side portacaval shunt, transection of all hepatic ligaments, and placement of slings around the hepatic artery and bile duct. After 3 days, the slings were tightened to induce liver necrosis. After 4 h, Group 1 pigs (n = 6) underwent a 6 h treatment with the BAL utilizing 10 billion cryopreserved pig hepatocytes and a charcoal column, Group 2 pigs (n = 6) with the BAL containing charcoal but no cells, and Group 3 pigs (n = 6) with the BAL containing neither cells nor charcoal. Group 1 pigs maintained a normal ICP during BAL treatment and for 14 h afterward and because of this effect they survived longer than Groups 2 and 3 animals. In contrast, Groups 2 and 3 pigs showed an early (6-8 h) rise in ICP.
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Hipertensión Intracraneal/terapia , Fallo Hepático/mortalidad , Fallo Hepático/terapia , Hígado Artificial , Análisis de Varianza , Animales , Trasplante de Células/métodos , Modelos Animales de Enfermedad , Femenino , Hepatocitos/trasplante , Hipertensión Intracraneal/complicaciones , Hipertensión Intracraneal/mortalidad , Fallo Hepático/complicaciones , Probabilidad , Sensibilidad y Especificidad , Tasa de Supervivencia , Porcinos , Resultado del TratamientoRESUMEN
BACKGROUND: Earlier we described a model of fulminant hepatic failure (FHF) in the rat where partial hepatectomy is combined with induction of right liver lobe necrosis. In FHF rats, lack of hepatocyte proliferation was associated with delayed expression of HGF and HGF receptor c-met. Since the c-met promoter region has Sp1 binding sites, we decided to examine whether in FHF rats down-regulation of c-met is associated with decreased Sp1 function and whether changes in blood HGF, IL-6, and TGFbeta1 levels might be responsible for these effects. MATERIALS AND METHODS: Induction of FHF, partial (2/3) hepatectomy (PH), and sham hepatectomy (SH) was performed in adult Sprague-Dawley rats. The levels of c-met mRNA and Sp1 DNA binding activity were studied in rat liver remnants at different time points after surgery. Blood levels of HGF, IL-6, and TGFbeta1 were also measured in these rats. Additionally, the effects of treatment with TGF-beta1, IL-6, or a combination of both on c-met expression and Sp1 DNA binding were studied in HGF-induced rat hepatocyte cultures. RESULTS: Compared to SH rats, in PH rat livers c-met was up-regulated after 6 h and Sp1 DNA binding was at or only slightly lower than levels at all time points studied. In FHF rat livers, c-met expression was markedly reduced after 2 and 6 h, moderate after 12 h, and undetectable after 24 h. At the same time, Sp1 DNA binding was detected at 2 h postinduction only. In FHF rats, blood levels of all three cytokines showed early and sustained elevation. In vitro, IL-6 had no effect on c-met expression, whereas TGFbeta1 up-regulated c-met. When used alone, none of the cytokines affected Sp1 DNA binding activity. In contrast, a combination of IL-6 and TGFbeta1 down-regulated c-met expression as well as Sp1 DNA binding activity. These effects were dependent on the IL-6 concentration used. This study suggests that following massive loss of hepatocyte mass in rats, early increase in blood IL-6 and TGFbeta1 levels may weaken the expression of HGF receptor c-met in surviving hepatocytes through suppression of Sp1 DNA binding.
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Fallo Hepático Agudo/fisiopatología , Regeneración Hepática/genética , Proteínas Proto-Oncogénicas c-met/genética , Animales , División Celular/fisiología , Células Cultivadas , Regulación de la Expresión Génica/fisiología , Factor de Crecimiento de Hepatocito/sangre , Hepatocitos/citología , Interleucina-6/sangre , Masculino , ARN Mensajero/análisis , Ratas , Ratas Sprague-Dawley , Factor de Transcripción Sp1/metabolismo , Factor de Crecimiento Transformador beta/sangre , Factor de Crecimiento Transformador beta1RESUMEN
Orthotopic liver transplantation is the only definitive therapy for patients with fulminant hepatic failure (FHF). However, due to shortage of organs, a large number of patients die before a liver can be procured for transplantation. In FHF the need for a liver is particularly urgent because of rapid deterioration in the patients' condition with the onset of cerebral edema and intracranial hypertension leading to irreversible brain damage. It is thus necessary to develop an extracorporeal liver support system to help maintain patients alive and neurologically intact until an organ becomes available for transplantation. Multiple attempts have been made, ranging from the use of plasma exchange to utilization of charcoal columns and extracorporeal devices loaded with liver tissue to develop liver support systems for treating patients with acute severe liver failure. None of these systems has achieved wide clinical use, and FHF due to multiple causes continues to be associated with significant morbidity and mortality. In this paper, the authors review the history of extracorporeal liver support for acute liver failure and discuss their experience with a hollow fiber bioartificial liver support system utilizing porcine hepatocytes in the treatment of patients with acute liver failure.
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Fallo Hepático/cirugía , Hígado Artificial , Enfermedad Aguda , Animales , Diseño de Equipo , Circulación Extracorporea , Hemofiltración , Hepatocitos , Humanos , Fallo Hepático/fisiopatología , PlasmaféresisRESUMEN
We studied the explanted livers from 12 patients with fulminant hepatic failure who were treated with a bioartificial liver and subsequently underwent orthotopic liver transplantation and from 18 patients who underwent orthotopic liver transplantation without previous treatment. Ten normal livers were used as controls. In addition to morphologic evaluation, an immunohistochemical analysis was performed with the monoclonal antibodies for alpha-smooth muscle actin and proliferation marker Ki-67. The expression of these markers was graded semiquantitatively from 0 to 3+ in a blinded fashion. The zonal distribution of activated hepatic stellate cells was also evaluated. In all cases, the hepatic stellate cells were activated and expressed alpha-smooth muscle actin. In all patients with submassive or massive liver cell necrosis, the distribution of activated hepatic stellate cells was predominantly in zone 1 of the acinus (periportal area). In contrast, in cases with early nodular regeneration and no significant fibrosis, the activated hepatic stellate cells were distributed throughout the liver parenchyma, involving zones 2 and 3 of the acinus. Expression of the proliferation marker Ki-67 was graded 3+ in all patients treated with the bioartificial liver who had orthotopic liver transplantation and 2+ in patients who underwent orthotopic liver transplantation only.
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Macrófagos del Hígado/patología , Fallo Hepático/terapia , Hígado Artificial , Hígado/patología , Actinas/biosíntesis , Adolescente , Adulto , Anciano , Femenino , Humanos , Antígeno Ki-67/biosíntesis , Macrófagos del Hígado/metabolismo , Hígado/metabolismo , Fallo Hepático/metabolismo , Fallo Hepático/patología , Regeneración Hepática/fisiología , Trasplante de Hígado , Masculino , Persona de Mediana EdadRESUMEN
Fulminant hepatic failure is a devastating disease that, despite recent therapeutic advances, continues to be associated with high morbidity and mortality. Orthotopic liver transplantation has emerged as the sole modality of treatment that significantly improves survival. However, the critical shortage of donors precludes timely transplantation for all patients. Consequently, almost half of all patients with fulminant hepatic failure die before a graft becomes available. This has generated interest in developing a system that would support patients until either native liver regeneration occurs or an optimal donor liver can be found. Investigators have used biological, artificial and bioartificial techniques in an attempt to improve survival in liver failure. This article reviews the history, the current state of the art and future directions of artificial liver support.
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Fallo Hepático/terapia , Hígado Artificial , Animales , Historia del Siglo XX , Humanos , Hígado Artificial/historiaRESUMEN
In fulminant hepatic failure, survival is not possible without recovery of sufficient hepatocyte mass. Remarkably, only a few studies exist that provide insight into the mechanisms that control proliferation of residual hepatocytes after extensive hepatocyte loss. In this regard, the role of growth-regulatory factors, including pro-inflammatory cytokines such as interleukin-6 (IL-6), is not well understood. In the present study we show that in rats with critically low (10%) hepatocyte mass, whether with or without ongoing liver cell necrosis, inhibition of liver regeneration is associated with early and sustained increase in blood IL-6 levels. Under these conditions, the signal transducer and activator of transcription (Stat3) DNA binding activity was lowered at the time of G1/S cell-cycle transition. We further demonstrate that the protein inhibitor of activated Stat3 (PIAS3) and the suppressor of cytokine signaling (SOCS-1) were up-regulated early after induction of liver failure (6-12 h). In vitro, IL-6 induced PIAS3 expression in HGF stimulated rat hepatocytes. These findings suggest that after massive hepatocyte loss, an early and rapid rise in blood IL-6 levels may weaken the hepatic regenerative response through up-regulation of Stat3 inhibitors PIAS3 and SOCS-1.
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Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/metabolismo , Fallo Hepático Agudo/metabolismo , Fallo Hepático Agudo/patología , Regeneración Hepática , Proteínas Proto-Oncogénicas , Proteínas Represoras , Transactivadores/antagonistas & inhibidores , Transactivadores/metabolismo , Animales , Antígenos CD/metabolismo , Proteínas Portadoras/genética , División Celular/efectos de los fármacos , Células Cultivadas , Receptor gp130 de Citocinas , ADN/biosíntesis , ADN/genética , ADN/metabolismo , Hepatectomía , Factor de Crecimiento de Hepatocito/farmacología , Interleucina-6/sangre , Interleucina-6/farmacología , Janus Quinasa 2 , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Hígado/cirugía , Fallo Hepático Agudo/cirugía , Regeneración Hepática/efectos de los fármacos , Masculino , Glicoproteínas de Membrana/metabolismo , FN-kappa B/metabolismo , Necrosis , Unión Proteica , Proteínas Tirosina Quinasas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores de Interleucina-6/metabolismo , Factor de Transcripción STAT3 , Proteína 1 Supresora de la Señalización de Citocinas , Proteínas Supresoras de la Señalización de Citocinas , Factor de Transcripción AP-1/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Mechanisms that control function and repair of the injured liver remain unclear. We hypothesized that after liver injury, elevated blood TGF-beta1 levels may reflect an adaptive response to help maintain differentiated functions in surviving hepatocytes affected by excessive amounts of HGF. We thus studied the effect of HGF, EGF, TGF-beta1, HGF + TGF-beta1, or EGF + TGF-beta1 on the expression of liver-enriched transcription factors and genes which remain under their regulatory activity. The peak [3H]thymidine uptake induced by 20 ng/ml of either HGF or EGF was seen after 72 h; however, DNA binding of C/EBP and HNF1 decreased already after 6 h (electrophoretic mobility shift assay). Addition of TGF-beta1 antagonized these effects. Also at the mRNA level, TGF-beta1 counteracted at one point or another the decrease in C/EBPalpha, C/EBPbeta, HNF1beta, and HNF4 expression; HNF1alpha and COUP-TF showed similar responses and, additionally, were downregulated by TGF-beta1 at 24 h (Northern blot). Albumin and apolipoprotein B mRNA levels were decreased after 24-h treatment with HGF, whereas addition of TGF-beta1 increased their levels. The same pattern was found with EGF, but not until 48 h. PEPCK mRNA was dramatically lowered with either EGF or HGF, and TGF-beta1 did not counteract these effects. Id-1 was expressed only in cultures treated for 24 and 48 h with both the mitogen (EGF, HGF) and TGF-beta1 and in those treated for 48 h with TGF-beta1 alone.
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Hígado/citología , Hígado/fisiología , Factor de Crecimiento Transformador beta/farmacología , Factor de Crecimiento Transformador beta/fisiología , Animales , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Células Cultivadas , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Masculino , Mitógenos/farmacología , Ratas , Ratas Sprague-DawleyRESUMEN
Patients with acetaminophen-induced fulminant hepatic failure (FHF) who meet the King's College Hospital criteria have a high mortality risk (>90%) if they do not undergo liver transplantation. We have developed a treatment strategy for these patients based on the use of an extracorporeal bioartificial liver (BAL) support system. In this study, we report the results of the clinical application of BAL support in patients with acetaminophen-induced FHF. All patients were admitted to a dedicated surgical intensive care unit. They were evaluated for urgent liver transplantation and received the standard medical measures, including N-acetylcysteine administration and intracranial pressure monitoring. Moreover, they underwent daily 6-hour BAL treatments. Eight patients were treated. Three patients were bridged to liver transplantation, and five patients recovered without a transplant. All patients experienced neurological and metabolic improvement after treatments with the BAL support system. The BAL support system seems to improve the outcome of high-risk patients with acetaminophen-induced FHF, even in the absence of liver transplantation. Avoiding liver transplantation is particularly important in an era of organ shortage and high cost of transplants.
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Encefalopatía Hepática/terapia , Hígado Artificial , Acetaminofén/envenenamiento , Adolescente , Adulto , Analgésicos no Narcóticos/envenenamiento , Femenino , Escala de Coma de Glasgow , Encefalopatía Hepática/inducido químicamente , Humanos , Masculino , Persona de Mediana Edad , Resultado del TratamientoRESUMEN
Early studies showed that compensatory liver growth after anterior portal branch ligation (aPBL) may restore normoglycemia in streptozotocin (STZ)-diabetic rats, in which a subtherapeutic islet mass was previously transplanted into the liver. We hypothesized that this effect could be related to islet regeneration at the graft site. This study was designed to characterize the proliferative response of the intraportally transplanted islets, shortly after aPBL. Male Wistar-Furth rats were used as syngeneic islet donors and/or recipients. STZ-diabetic rats were divided in four groups: groups 1 and 2 underwent selective 250-islet transplantation (Tx) into the posterior liver lobes, followed by aPBL 10 days later; rats were killed 24 h (n = 9) and 48 h (n = 10) after aPBL, respectively; groups 3 and 4 underwent selective 250-islet Tx into the posterior liver lobes, followed by sham aPBL 10 days later; rats were killed 24 h (n = 3) and 48 h (n = 3) after aPBL, respectively. Two hours before killing, all animals were injected with 5'-bromo-2'-deoxyuridine (BrdU; 50 mg/kg, i.v.). Liver sections were immunostained for insulin and BrdU, and both hepatocyte and islet cell labeling index (LI) were calculated. Islet cell LI was 2.30+/-1.18% in group 1, 2.23+/-1.00% in group 2, 0.43+/-0.29% in group 3, and 0.39+/-0.21% in group 4 (group 1 vs. group 3: p<0.02; group 2 vs. group 4: p<0.01). Hepatocyte LI was 2.50+/-2.14% in group 1, 15.0+/-7.6% in group 2, 0.12 +/-0.04 in group 3, and 0.11+/-0.03% in group 4, respectively (group 1 vs. group 2: p<0.02; group 1 vs. group 3: p<0.001; group 2 vs. group 4: p<0.001). Our study showed that intraportally transplanted islets undergo a concurrent proliferative response after aPBL, although with a lower extent and a different timing when compared with the liver-cell response.
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Diabetes Mellitus Experimental/patología , Trasplante de Islotes Pancreáticos , Islotes Pancreáticos/citología , Hígado/citología , Páncreas/citología , Animales , Conductos Biliares/citología , Glucemia , Bromodesoxiuridina , División Celular , Diabetes Mellitus Experimental/sangre , Diabetes Mellitus Experimental/terapia , Ligadura , Regeneración Hepática , Masculino , Vena Porta , Ratas , Ratas Endogámicas WFRESUMEN
BACKGROUND: We earlier described a model of fulminant hepatic failure (FHF) in the rat where partial hepatectomy is combined with induction of right liver lobe necrosis. In FHF rats, lack of regeneration of the residual liver was associated with delayed expression of HGF and HGF receptor c-met and elevated blood HGF and TGF-beta1 levels. We then found that intrasplenic hepatocyte transplantation prolonged survival in FHF rats and triggered hepatocyte proliferation in the native liver. The latter effect was associated with accelerated expression of HGF and c-met mRNA in the liver and lowering of blood HGF and TGF-beta1 levels. In the present study we show that in FHF rats, treatment with a bioartificial liver (BAL) had similar effects. MATERIALS AND METHODS: FHF was induced in inbred Lewis rats and after 4 h, Group 1 rats were subjected to a 4-h whole blood perfusion through the BAL loaded with 3 x 10(8) microcarrier-attached syngeneic hepatocytes, whereas Group 2 control rats were treated with the BAL containing microcarriers only. RESULTS: Compared to sham-BAL-treated rats, the test rats lived longer (28 +/- 5 vs 17 +/- 2 h; P = 0.0005), had better coagulation parameters, maintained higher body core temperature, and showed decreased plasma TGF-beta1 levels. In addition, their liver remnants were HGF positive and showed increased DNA binding of transcription factors engaged in the modulation of hepatocyte proliferation (e.g., STAT3) and liver-specific gene expression (e.g., HNF1, HNF4, C/EBP). CONCLUSIONS: This study demonstrates that hepatocyte-based extracorporeal support not only can provide metabolic support by increasing the available functional liver mass but also is capable of modifying humoral and molecular mechanisms which are responsible for proliferation and organ-specific functions of residual hepatocytes.
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ADN/metabolismo , Encefalopatía Hepática/sangre , Encefalopatía Hepática/terapia , Hígado Artificial , Factores de Transcripción/sangre , Animales , Electroforesis en Gel de Poliacrilamida , Circulación Extracorporea , Encefalopatía Hepática/metabolismo , Encefalopatía Hepática/mortalidad , Factor de Crecimiento de Hepatocito/sangre , Factor de Crecimiento de Hepatocito/metabolismo , Masculino , Proteínas Proto-Oncogénicas c-met/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas Lew , Ribonucleasas/metabolismo , Tasa de Supervivencia , Factores de Transcripción/metabolismo , Factor de Crecimiento Transformador beta/sangreRESUMEN
During orthotopic liver transplantation (OLT) for fulminant hepatic failure (FHF), some patients develop cerebral injury secondary to intracranial hypertension. We monitored intracranial pressure (ICP) and cerebral perfusion pressure (CPP) before and during OLT in 12 FHF patients undergoing transplantation. All four patients who had normal ICP preoperatively maintained normal ICP/CPP throughout OLT. During OLT, four of the eight patients with pretransplant intracranial hypertension had six episodes of ICP increase. These episodes of intracranial hypertension occurred during failing liver dissection (n=3) and graft reperfusion (n=3). At the end of the anhepatic phase, the ICP was lower than the preoperative ICP in all patients, and was below 15 mmHg in all but one patient. These data suggest that in FHF patients who develop intracranial hypertension before OLT, dissection of the native liver and graft reperfusion are associated with a risk of brain injury resulting from intracranial hypertension and cerebral hypoperfusion.
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Encefalopatía Hepática/terapia , Hipertensión Intracraneal/etiología , Trasplante de Hígado/efectos adversos , Adulto , Edema Encefálico/etiología , Niño , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
BACKGROUND: There are limited experimental data on the nature of the humoral response elicited in humans against pig antigens. In this study, we have examined the xenoantibody (XAb) response in eight patients with acute liver failure exposed to pig hepatocytes after treatment with the bioartificial liver (BAL). METHODS: Patients' plasma samples obtained before and after BAL treatment were tested for IgM and IgG XAbs, IgG subclasses, and XAb cytotoxicity, using enzyme-linked immunosorbent assay and flow-cytometric assays. The characterization of pig aortic endothelial cell (PAEC) surface xenoantigens was analyzed by immunoprecipitation. RESULTS: We observed by day 10, a strong anti-pig IgG and IgM XAb response in patients undergoing two or more BAL treatments, with a significant increase in all the IgG subclasses; in contrast, XAb titers did not change if the patients received only one BAL treatment. The majority of the XAbs produced to porcine antigens were primarily specific for the alphaGal epitope. Both IgG and IgM XAbs were cytotoxic to PAECs, and the cytotoxic activity of IgG was associated with high levels of IgG1 and IgG3 subclasses, known to be efficient on complement activation. The characterization of porcine surface antigens demonstrated that IgM human XAbs, before and after BAL exposure, recognized xenoantigens on PAECs with similar molecular weights, suggesting that the same population of XAbs were present in the patients before and after exposure to pig antigens. CONCLUSIONS: Repetitive exposure of humans to porcine antigens after BAL treatment, results in a strong IgG and IgM XAb responses that are primarily directed against the alphaGal epitope. These XAbs are cytotoxic to PAECs and the IgG toxicity correlates with high IgG1 and IgG3 levels. Our data also suggest that no new XAb specificity emerges after porcine exposure.
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Anticuerpos Heterófilos/inmunología , Fallo Hepático/inmunología , Fallo Hepático/cirugía , Hígado Artificial , Hígado/citología , Animales , Formación de Anticuerpos/fisiología , Citotoxicidad Celular Dependiente de Anticuerpos/fisiología , Antígenos Heterófilos/inmunología , Aorta/inmunología , Endotelio Vascular/inmunología , Epítopos/inmunología , Diseño de Equipo , Humanos , Inmunoglobulina G/análisis , Isotipos de Inmunoglobulinas/análisis , Inmunoglobulina M/inmunología , Hígado/inmunología , Hígado/fisiología , Porcinos/inmunologíaRESUMEN
We have identified a novel human malignancy-associated gene (MAG) expressed in various malignant tumors including glioblastomas and hepatocellular carcinomas (HCCs) and in tumor preexisting conditions such as hepatitis C virus- and hepatitis B virus-induced liver cirrhosis. The expression of MAG was characterized using reverse transcription-PCR (RT-PCR), rapid amplification of cDNA ends PCR, RNA dot blotting, RNase protection assay, and Northern blot analysis. Rapid amplification of cDNA ends PCR yielded a 536-bp MAG fragment in HCC, macroregenerative liver nodules with dysplasia, and liver cirrhosis but not in normal liver or placenta. By RT-PCR, MAG expression was not found in 12 different normal tissues but found in 46 of 51 (90%) premalignant and malignant tissues of various sites. Embryonic liver and brain were positive for MAG expression together with tumors from the same organs, but the corresponding normal adult tissues were negative. By RNase protection assay, MAG mRNA was expressed in the HepG2 liver tumor cell line and in an ovarian carcinoma but not in normal liver. The estimated transcript size from Northern blot analysis was 8.8 kb. This novel gene may play a role in the progression of premalignant conditions and in the development of HCC and other cancers.
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Proteínas de Neoplasias/genética , Neoplasias/genética , Lesiones Precancerosas/genética , Adulto , Secuencia de Aminoácidos , Secuencia de Bases , Encéfalo/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Embrión de Mamíferos , Femenino , Glioblastoma/genética , Glioblastoma/patología , Hepatitis B/genética , Hepatitis B/patología , Hepatitis C/genética , Hepatitis C/patología , Humanos , Hígado/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Masculino , Datos de Secuencia Molecular , Proteínas de Neoplasias/química , Neoplasias/patología , Reacción en Cadena de la Polimerasa , Lesiones Precancerosas/patología , Embarazo , Valores de Referencia , Estudios Retrospectivos , Factores de Riesgo , Transcripción Genética , Células Tumorales CultivadasRESUMEN
To examine whether hepatocytes transplanted in the spleen can function as an ectopic liver, we performed hepatocyte transplantation in rats that were rendered anhepatic. Total hepatectomy was performed by using a novel single-stage technique. Following hepatectomy, Group 1 rats (n = 16) were monitored until death to determine survival time without prior intervention. Group 2 anhepatic rats (n = 20) were sacrificed at various times to measure blood hepatocyte growth factor (HGF) and transforming growth factor beta1 (TGF-beta1) levels. Group 3 (n = 16) rats received intrasplenic injection of isolated hepatocytes (2.5 x 10(7) cells/rat) followed by total hepatectomy after 3 days. Group 4 (n = 12) sham-transplanted rats received intrasplenic saline infusion, and after 3 days they were rendered anhepatic. Group 2, 3, and 4 rats were maintained on daily Cyclosporine A (10 mg/kg; intramuscularly). Group 1 anhepatic rats survived for 22.4 +/- 5.2 hours (standard deviation). The anhepatic state was associated with a progressive and statistically significant rise in blood HGF and TGF-beta1 levels. Rats that received hepatocyte transplantation before total hepatectomy had a significantly longer survival time than sham-transplanted anhepatic controls (34.1 +/- 8.5 vs. 15.5 +/- 4.8 hrs, P < .01). Additionally, at 12 hours post-hepatectomy, transplanted rats had significantly lower blood ammonia, prothrombin time, international normalized ratio, and TGF-beta1 levels when compared with sham-transplanted controls. In conclusion, intrasplenic transplantation of allogeneic hepatocytes prolonged survival, improved blood chemistry, and lowered blood TGF-beta1 levels in rats rendered anhepatic.