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Urologists and gynecologists manage most of the patients with female pelvic floor disorders. However, lack of a single focused approach among these two disciplines and other related fields may hinder advances in clinical care and research. Herein, along with describing the background of birth of the new subspeciality, we present a practical approach by which we established an integrated model of care and discovery for FPFD at our institution. With the recent approval of this subspeciality by the Accreditation Council for Graduate Medical Education (ACGME), it is plausible that other institutions would wish to initiate similar steps toward establishment of an integrated model of care for FPFD, and hence move this new subspecialty to its new frontiers.

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.

The Patient Protection and Affordable Care Act signed into law in March 2010, has led to sweeping changes to the US health care system. The ensuing pace of change in health care regulation is unparalleled and difficult for physicians to keep up with. Because of the extraordinary challenges that have arisen, the public policy committee of the Society for Surgery of the Alimentary tract conducted a symposium at their 52nd Annual Meeting in May 2011 to educate participants on the myriad of public policy changes occurring in order to best prepare them for their future. Expert speakers presented their views on policy changes affecting diverse areas including patient safety, patient experience, hospital and provider fiscal challenges, and the life of the practicing surgeon. In all areas, surgical leadership was felt to be critical to successfully navigate the new health care landscape as surgeons have a long history of providing safe, high quality, low cost care. The recognition of shared values among the diverse constituents affected by health care policy changes will best prepare surgeons to control their own destiny and successfully manage new challenges as they emerge.

Liver stem cell (LSC) transplantation is a promising alternate approach to liver transplantation for patients with end-stage liver disease. However, the precise origin of LSCs remains unclear. Herein we determine if bone marrow mesenchymal stem cells (BMSCs) isolated from rats in acute hepatic failure (AHF) possess hepatic characteristics and have differentiation potential. BMSCs were isolated from AHF and sham-operated rats, and primary hepatocytes were isolated from normal rats for comparison. The transcriptomic profile of BMSCs and primary hepatoyctes was analyzed using the Affymetrix GeneChip Rat Genome 230 2.0 Array. BMSCs isolated from AHF and normal rats were induced to differentiate into hepatocytes in vitro and the degree of hepatic differentiation was assessed using quantitative real time RT-PCR, immunohistochemistry, and biochemical assays. AHF-derived BMSCs had a significantly different gene expression profile compared to control BMSCs. Thirty-four gene/probe sets were expressed in both AHF-derived BMSCs and primary hepatocytes, but were absent in control-derived BMSCs, including 3 hepatocyte-specific genes. Forty-four genes were up-regulated more than 2-fold in AHF-derived BMSCs compared to controls, including 3 genes involved in hepatocyte metabolism and development. Furthermore, AHF-derived BMSCs expressed more hepatocyte related genes than control BMSCs. Additional experiments to validate the differentiation of AHF-derived BMSCs, compared to control-derived BMSCs, showed that several hepatocyte-specific genes and proteins [such as albumin (ALB) and alpha fetoprotein (AFP)] were expressed earlier, and at higher levels, after 1 week of differentiation. Hepatocyte-specific metabolic functions were also significantly higher in the AHF group compared to control cells. Conclusion: AHF-derived BMSCs had a hepatic transcriptional profile and expressed hepatocyte specific genes early during differentiation, and possessed greater hepatogenic potency in vitro compared to cells isolated from control animals, further confirming their potential as a stem cell-based therapy for end-stage liver disease.

Liver tissue engineering with hepatic stem cells provides a promising alternative to liver transplantation in patients with acute and chronic hepatic failure. In this study, a three-dimensional (3D) bioscaffold was introduced for differentiation of rat bone marrow mesenchymal stem cells (BMSCs) into hepatocytes. For hepatocyte differentiation, third passage BMSCs isolated from normal adult F344 rats were seeded into collagen-coated poly(lactic-co-glycolic acid) (C-PLGA) 3D scaffolds with hepatocyte differentiation medium for 3 weeks. Hepatogenesis in scaffolds was characterized by reverse transcript PCR, western blot, confocal laser scanning microscopy (CLSM), periodic acid-Schiff staining, histochemistry, and biochemical assays with hepatic-specific genes and markers. A monolayer culture system was used as a control differentiation group. The results showed that isolated cells possessed the basic features of BMSCs. Differentiated hepatocyte-like cells in C-PLGA scaffolds expressed hepatocyte-specific markers [eg, albumin (ALB), alpha-fetoprotein, cytokeratin 18, hepatocyte nuclear factor 4alpha, and cytochrome P450] at mRNA and protein levels. Most markers were expressed in C-PLGA group 1 week earlier than in the control group. Results of biocompatibility indicated that the differentiated hepatocyte-like cells grew more stably in C-PLGA scaffolds than that in controls during a 3-week differentiation period. The significantly higher metabolic functions in hepatocyte-like cells in the C-PLGA scaffold group further demonstrated the important role of the scaffold.As the phenomenon of transdifferentiation is uncommon, our successful transdifferentiation rates of BMSCs to mature hepatocytes prove the superiority of the C-PLGA scaffold in providing a suitable environment for such a differentiation. This material can possibly be used as a bioscaffold for liver tissue engineering in future clinical therapeutic applications.

Background Pituitary tumor transforming gene (PTTG), homologous to a mammalian securin, plays a pivotal role in cell transformation, however, its biological function(s) in normal tissues is not fully understood. Because the liver is a regenerative organ, the relevant biological function of PTTG in the liver would be more feasible to understand PTTG. Also, PTTG may be involved in the liver regeneration. Materials and methods Expressions of rat hepatic PTTG messengerRNA (mRNA) and cellular immunoreactivities during the cell proliferative period of the liver regeneration both in vitro and in vivo were tested. Results PTTG expression of the rat primary hepatocyte was stimulated by HGF in a dose dependent manner, and was suppressed when hepatocyte proliferation was inhibited by transforming growth factor-β1. A positive PTTG immunoreactive co-localizing with 5-bromo-2′-deoxyuridine (BrdU) in the hepatocyte nucleus was found and there was a concurrent sister chromatin itself by the immunofluorescent labeling of PTTG with cytokeratin 18 (CK18). Discussion Since the correlation of PTTG mRNA expression, cell proliferation and immunoreactivity were observed in primary rat cultured hepatocytes, PTTG may be a novel marker of cell proliferation both in vitro and in vivo liver regeneration.

Hepatocyte transplantation and use of bioartificial liver support systems have been suggested as potential therapies for fulminant hepatic failure. Cryopreservation in liquid nitrogen is presently the major method of long-term storage of isolated hepatocytes. However, cryopreservation can result in low cell recovery and reduction in differentiated function. Several possible mechanisms of cell death during cryopreservation have been proposed. The most important mechanisms appear to be oxidative stress and apoptosis. In this study, we isolated fresh rat hepatocytes and cryopreserved them in three media: University of Wisconsin (UW) solution, an antioxidant-containing medium, and medium containing a caspase inhibitor. Viability and function of hepatocytes cryopreserved in these media were examined. Cryopreservation conditions had no effect on hepatocyte viability after thawing. However, after culture we found significant improvements in viability and function in both antioxidant- and caspase inhibitor-treated hepatocytes at 6 and 24 h.

Objective: The HepatAssist liver support system is an extracorporeal porcine hepatocyte-based bioartificial liver (BAL). The safety and efficacy of the BAL were evaluated in a prospective, randomized, controlled, multicenter trial in patients with severe acute liver failure. Summary Background Data: In experimental animals with acute liver failure, we demonstrated beneficial effects of the BAL. Similarly, Phase I trials of the BAL in acute liver failure patients yielded promising results. Methods: A total of 171 patients (86 control and 85 BAL) were enrolled. Patients with fulminant/subfulminant hepatic failure and primary nonfunction following liver transplantation were included. Data were analyzed with and without accounting for the following confounding factors: liver transplantation, time to transplant, disease etiology, disease severity, and treatment site. Results: For the entire patient population, survival at 30 days was 71% for BAL versus 62% for control (P = 0.26). After exclusion of primary nonfunction patients, survival was 73% for BAL versus 59% for control (n = 147; P = 0.12). When survival was analyzed accounting for confounding factors, in the entire patient population, there was no difference between the 2 groups (risk ratio = 0.67; P = 0.13). However, survival in fulminant/subfulminant hepatic failure patients was significantly higher in the BAL compared with the control group (risk ratio = 0.56; P = 0.048). Conclusions: This is the first prospective, randomized, controlled trial of an extracorporeal liver support system, demonstrating safety and improved survival in patients with fulminant/subfulminant hepatic failure.

Background. This study analyzes the prognostic significance of serum phosphorus in patients with acute liver failure (ALF). Methods. We performed a retrospective analysis of 112 patients with ALF. Univariate and bivariate analyses based on Kaplan-Meier recovery curves and a multivariate Classification Tree Structure Survival Analysis were performed to identify independent predictors of outcome. The variables analyzed were age, gender, race, ABO blood group, etiology of liver disease, grade of encephalopathy, serum bilirubin, prothrombin time, creatinine, serum phosphorus, phosphorus administered, phosphorus binders, and hemodialysis. Results. The median follow-up time was 5 days, the median age was 28 years, and 62% of the patients were female. The patients' routcomes were as follows: 28% recovered, 52% required orthotopic liver transplantation, and 20% died. White patients showed the best prognosis (58% recovered in the first week), and Hispanics showed the worst prognosis (0.3% recovered at 1 week) (P=0.0001). Encephalopathy and bilirubin were significant predictors of recovery (P

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 α-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 α-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. HUM PATHOL 32:1371-1375. Copyright © 2001 by W.B. Saunders Company

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 TGFβ1 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 TGFβ1 were also measured in these rats. Additionally, the effects of treatment with TGF-β1, 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 TGFβ1 up-regulated c-met. When used alone, none of the cytokines affected Sp1 DNA binding activity. In contrast, a combination of IL-6 and TGFβ1 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 TGFβ1 levels may weaken the expression of HGF receptor c-met in surviving hepatocytes through suppression of Sp1 DNA binding.

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.

During the past decade, whole organ transplantation has become the primary method of treating fulminant hepatic failure and chronic liver failure due to specific genetic, hepatocellular, and anatomic defects of liver function. However, wider application of liver transplantation is restricted by shortage of organ donors, high cost, relatively high morbidity, and need for lifelong immunosuppression. As a result, investigators have attempted to develop alternative methods to treat liver insufficiency. These ranged from use of plasma exchange to utilization of detoxification columns and extracorporeal devices loaded with various liver tissue preparations. Recent advances in hepatocyte isolation and culture techniques, improved understanding of hepatocyte-matrix interactions, availability of new biomaterials, improved hollow-fiber technology, and better understanding of flow and mass transport across semipermeable membranes have resulted in the development of a new generation of liver assist devices. Some of these devices, including the one we developed, are currently being tested in the clinical setting. In this article, past experience with biological liver-assist devices is reviewed, the field's present status is examined, and our own experience with a hepatocyte-based bioartificial liver is summarized. Areas in which further research efforts are particularly needed are briefly characterized.

Despite recent advances in medical therapy, patients with fulminant hepatic failure (FHF) have a mortality rate approaching 90%. Many patients die because of failure to arrest the progression of cerebral edema. Liver transplantation has improved survival to 65% to 75%. However, there is a shortage of donors and approximately one half of the patients with FHF will die while awaiting liver transplantation. There is thus a need to develop an extracorporeal liver assist system to help keep these patients alive and neurologically intact until either an organ becomes available for transplantation or the native liver recovers from injury. Such a system could also be used during the period of functional recovery from massive liver resection or to assist patients with decompensated chronic liver disease. Over the years, various methods utilizing charcoal and resin hemoperfusion, dialysis, plasma exchange, and other methods of blood detoxification have been developed and tested, but none have gained wide acceptance. This was due to: (i) incomplete understanding of the pathophysiology of liver failure; (ii) lack of accurate methods of assessment, quantitation, and stratification of the degree of liver dysfunction; and (iii) inadequate numbers of prospective controlled clinical trials examining the effects of specific therapeutic modalities. Liver support systems utilizing liver tissue preparations were developed in the 1950s, but it was not until recently that advances in hepatocyte isolation and culture, better understanding of hepatocyte-matrix interactions, and improved hollow-fiber technology have resulted in the development of a new generation of liver assist devices. Some of these devices are currently being tested in the clinical setting. In a preliminary clinical study, we have used a porcine hepatocyte-based liver support system to treat patients with acute liver failure as well as patients with acute exacerbation of chronic liver disease. Patients in the first group, who were candidates for transplantation, were successfully bridged to a transplant with excellent survival. No obvious benefit from bioartifical liver treatments was seen in the second group. It is possible that, in this group, patients will have to be treated earlier and for longer periods of time. Prospective controlled trials will be initiated as soon as the current phase I study is concluded to determine the efficacy of this system in both patients populations. (c) 1996 John Wiley & Sons, Inc.

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.

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-β1 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-β1 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 × 108 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-β1 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.

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.

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.

Intracranial hypertension leading to brain stem herniation is a major cause of death in fulminant hepatic failure (FHF). Mannitol, barbiturates, and hyperventilation have been used to treat brain swelling, but most patients are either refractory to medical management or cannot be treated because of concurrent medical problems or side effects. In this study, we examined whether allogeneic hepatocellular transplantation may prevent development of intracranial hypertension in pigs with experimentally induced liver failure. Of the two preparations tested—total hepatectomy (n = 47), and liver devascularization (n = 16)—only pigs with liver ischemia developed brain edema provided, however, that animals were maintained normothermic throughout the postoperative period. This model was then used in transplantation studies, in which six pigs received intrasplenic injection of allogeneic hepatocytes (2.5 × 109 cells/pig) and 3 days later acute liver failure was induced. In both models (anhepatic state, liver devascularization), pigs allowed to become hypothermic had significantly longer survival compared to those maintained normothermic. Normothermic pigs with liver ischemia had, at all time points studied, ICP greater than 20 mmHg. Pigs that received hepatocellular transplants had ICP below 15 mmHg until death; at the same time, cerebral perfusion pressure (CPP) in transplanted pigs was consistently higher than in controls (45 ± 11 mmHg vs. 16 ± 18 mmHg; p < 0.05). Spleens of transplanted pigs contained clusters of viable hepatocytes (hematoxylin-eosin, CAM 5.2). It was concluded that removal of the liver does not result in intracranial hypertension; hypothermia prolongs survival time in both anhepatic pigs and pigs with liver devascularization, and intrasplenic transplantation of allogeneic hepatocytes prevents development of intracranial hypertension in pigs with acute ischemic liver failure.

Hepatocyte transplantation is a promising alternative to orthotopic liver transplantation in experimental animal models with genetic disorders of liver metabolism and liver failure. Fetal hepatocytes have several characteristics that make them potentially suitable as donor cells. In contrast to adult hepatocytes, fetal hepatocytes are thought to be highly proliferative, which may facilitate engraftment, expansion of transplanted cell population, and gene transfer requiring active DNA synthesis. The present study was undertaken to evaluate the proliferative capacity of fetal and adult rat hepatocytes under standardized culture conditions. Fetal (20 days of gestation) and adult hepatocytes were cultured in serum-free media at low densities and treated with growth factors. Proliferation was assessed by [3H]-thymidine incorporation and cell cycle analysis by flow cytometry. In nonstimulated cells, DNA synthesis at 4 h was about x100 higher and after 10 days in culture x20 higher in fetal compared to adult hepatocytes. When epidermal growth factor (EGF) was added, maximal DNA synthesis in fetal hepatocytes was seen at 48 h, whereas in adult hepatocytes at 72 h. For adult hepatocytes, the average increase compared to untreated cells was x13.8 with EGF, x18.5 with transforming growth factor alpha (TGF-alpha), and x7.6 with hepatocyte growth factor (HGF). For fetal hepatocytes, the increase was twofold with either EGF, TGF-alpha or HGF. EGF-, TGF-alpha- and HGF-dependent DNA synthesis was inhibited by transforming growth factor beta-1 (TGF-beta1) in both fetal and adult hepatocyte cultures; this antiproliferative effect was significantly stronger in adult hepatocyte cultures. With cyclosporine, EGF-, TGF-alpha- and HGF-dependent DNA synthesis in fetal hepatocyte cultures decreased by 36-46%, whereas in adult hepatocytes by 19-27 %. These results show that in contrast to adult hepatocytes, fetal hepatocytes have high spontaneous proliferative activity independently of growth factors and are relatively resistant to the inhibitory effect of TGF-beta1. It was also found that cyclosporine suppresses proliferation of cultured fetal hepatocytes.

Background. In contrast to adult hepatocytes, fetal hepatocytes (FH) are thought to be highly proliferative, less immunogenic, and resistant to cryopreservation and ischemic injury. These qualities could enhance FH engraftment, proliferation, and gene transfer requiring active DNA synthesis, Methods. Rat FH were obtained using the nonperfusion collagenase/DNase digestion method, Free and cultured cells were studied using electron microscopy, fluorescence-activated cell sorting, and Northern analysis using alpha-fetoprotein and albumin as markers of hepatocyte lineage, DNA synthetic activity was measured in quiescent and mitogen-stimulated fetal and adult hepatocytes by [H-3]thymidine incorporation, Susceptibility of cultured FH to retrovirally mediated gene transfer was studied using an amphotropic retroviral vector carrying the Escherichia coli lac-Z gene, Nagase analbuminemic rats were used as recipients to study the effects of intraportal FH transplantation, Analysis of serum albumin was carried out by enzyme-linked immunosorbent assay. Results, In fetal liver, 87+/-2%? of the cells showed morphological and molecular features of hepatocytes. DNA synthetic activity in nonstimulated cultured FH was 10 times greater than the maximal hepatocyte growth factor-driven response in adult rat hepatocytes., A total of 5-15% FH stained positive for X-gal; results of transduction in adult hepatocyte cultures were negative. In Nagase analbuminemic rat recipients, FH produced significant amounts of albumin only when a hepatic regenerative stimulus was applied. Immunohistochemistry confirmed presence of albumin-positive hepatocytes, Conclusions, Fetal rat liver from the late gestation period is highly enriched with hepatocyte progenitors, They are highly proliferative and susceptible to retroviral transduction and can engraft and function in the adult rat liver if transplanted under a hepatic regenerative stimulus.

To gain insight into liver regenerationmechanisms in fulminant hepatic failure, we comparedgene expression of hepatocyte growth factor, itsreceptor c-met, c-myc, and albumin in human normal (4cases) and fulminant (14 cases) livers by reversetranscription-polymerase chain reaction. In normallivers, hepatocyte growth factor gene was not expressed,whereas c-met, c-myc and albumin genes were always expressed. In fulminant hepatic failure,hepatocyte growth factor gene was expressed in 1 of 14cases, c-met in none of 14 cases, c-myc in 10 of 14cases, and albumin in 3 of 14 cases. Byimmunofluorescence, c-met protein was revealed in normal but not infulminant hepatic failure liver tissue. Liver tissue isunlikely to account for high hepatocyte growth factorplasma levels typical for fulminant hepatic failure. Lack of its receptor (c-met)expression may explain a poor response of fulminanthepatic failure livers to exogenous hepatocyte growthfactor that normally promotes liver growth andregeneration.

Poor engraftment and consequent loss of β-cell mass could be one of the factors that are responsible for function loss after intraportal islet transplantation (Tx). Streptozotocin-diabetic rats were transplanted with syngeneic islets, which were injected into the portal vein via an indwelling catheter connected to a subcutaneous port. In Group I (n = 6), 1,000 islets were injected in a single dose into the liver. In Group II (n = 6), five doses of 200 islets were repeatedly injected over a period of 14 days, for a total of 1,000 islets. In Group III (n = 4), five decreasing doses of islets were injected over a period of 14 days, for a total of 750 islets. Nonfasting blood glucose (n-FBG) and body weight (b.wt.) were determined twice a week and an intravenous glucose tolerance test (IVGTT) was performed at 30 and 90 days. In Group I, n-FBG decreased in 2 wk from the time of first islet injection, averaging 110 ± 21.9 mg/dL at 1 mo (p < 0.05 vs. normal controls); this value was maintained throughout the 3-mo duration of the study. In Group II, n-FBG was normalized in 2 wk averaging 90.2 ± 25 mg/dL on day 12 (p = NS vs. normal controls) and 75.8 ± 14.6 mg/dL at 1 month (p = NS vs. normal controls); this value was maintained throughout the 3-mo duration of the study. In Group III, n-FBG decreased to normal values in 2 wk, averaging 77 ± 15.7 mg/dL at 1 mo (p = NS vs. normal controls), but normoglycemia was maintained for 40 days and then followed by a progressive increase. Only in Group II, KG (percent/min decline in glucose level) was not significantly different from that of normal controls (1.702 ± 0.531 at 1 mo and 1.676 ± 0.891 at 3 mo), while it was significantly lower than normal controls in both Group I and III animals. Body weight increase after Tx correlated with the number of transplanted islets and at 90 days, Group III rats showed less increase than Groups I and II (p < 0.05), while no significant differences in b.wt. were recorded between Group I and II. The findings indicate that intraportal islet Tx, injected repeatedly and in small doses, produced better metabolic effects than injection of the same total number of islets in a single dose. Copyright © 1997 Elsevier Science Inc.

A reproducible experimental animal model of fulminant hepatic failure (FHF) resembling the clinical condition is needed. We have developed such a model in the rat by combining resection of the two anterior liver lobes (68% liver mass) with ligation of the right lobes pedicle (24% liver mass), resulting in liver necrosis; the remaining two omental lobes (8% liver mass) are left intact. Adult Sprague-Dawley rats (250-300 g) were used. Survival time was determined in 60 rats. Because maintenance of body temperature at 37 degrees C shortened survival time by half, FHF rats were not warmed during the postinduction period and were allowed to gradually enter a state of mild to moderate hypothermia (29-32 degrees C). Additionally, 42 FHF rats were killed in batches of six rats each 2, 6, 12, 18, 24, 30, and 36 hours postoperatively to evaluate changes in blood chemistry (glucose, lactate, liver function tests, prothrombin time) and to assess liver regenerative response in the residual omental liver lobes (weight, protein content, incorporation of bromodeoxyuridine [BrdU], expression of proliferation cell nuclear antigen [PCNA], mitotic activity), plasma levels of hepatocyte growth factor (HGF) and transforming growth factor beta (TGF-beta1), and tissue expression of the HGF and it's receptor c-met. Rats undergoing partial hepatectomy of 68% (PH; n = 42) and a sham operation (SO; n = 42) served as controls. All SO and PH controls survived. PH rats showed only transient decreases in body temperature, signs of modest early hepatic dysfunction (hyperlactemia, hyperammonemia, prolonged PT time), and normal restitution of liver mass. All FHF rats became comatose by 24 hours postoperatively. Most animals (90%) died within 24-48 hours postoperatively (mean, 39 +/- 11 hours). Changes in blood chemistry reflected rapid development of liver failure. Plasma HGF levels were markedly elevated and at all time points were higher than in PH controls (P < .05). At the same time, expression of HGF and c-met messenger RNA in the remnant liver was delayed. Plasma TGF-beta1 levels increased early (18 hours) and remained twofold to threefold higher than that of PH and SO controls (P < .05). There was only a 20% increase in the weight of the remnant liver lobes due to swelling. No hepatocytes stained positively for BrdU and PCNA, and none showed mitotic figures. In contrast, all PH controls showed vigorous liver regeneration. In conclusion, we have developed and characterized a novel model of FHF in rats that has a number of physiological and biochemical features seen clinically in FHF, including severely impaired ability of the residual liver tissue to regenerate.

The preferred therapy for acute and chronic liver insufficiency and severe heritable disorders of liver metabolism is whole-organ transplantation. However, due to the shortage of organ donors and high cost, alternative therapeutic approaches have been proposed, including transplantation of normal allogeneic hepatocytes. Recently, it has been reported that many hepatocytes transplanted into the spleen migrated to the liver. We therefore carried out a series of large-animal experiments to reexamine the intrasplenic route and to develop a method for large-scale hepatocellular transplantation in pigs.Allogeneic porcine hepatocytes were transplanted using the following routes: (1) retrograde injection of cells via the splenic vein, (2) intraarterial injection of cells, (3) direct intrasplenic injection of cells after laparotomy, (4) percutaneous intrasplenic injection of cells under laparoscopic control, (5) laparoscopic intrasplenic injection of cells. The number of cells injected varied from 2 x 10(9) to 10 x 10(9) cells.Of all the methods tested, only direct intrasplenic injection of 2 bln of cells was found to be compatible with survival. However, even with this "small" number of cells (2% original liver mass), there was a significant risk of spleen infarction, perisplenic adhesion formation, and portal vein thrombosis. The laparoscopic approach was found to be reliable, simple, and safe.Even though the spleen is considered by many authors the optimal site for hepatocellular transplantation, transplantation of cells in a number needed to support the failing liver may be associated with significant complications, morbidity, and mortality.

UNLABELLED The only clinically proven effective treatment of fulminant hepatic failure (FHF) is orthotopic liver transplant (OLT). However, many patients die before an organ becomes available. Thus, there is a need for development of an extracorporeal liver support system to "bridge" these patients either to OLT or spontaneous recovery. We developed a bioartificial liver (BAL) based on plasma perfusion through a circuit of a hollow-fiber cartridge seeded with matrix-anchored porcine hepatocytes to treat patients with severe acute liver failure. Two groups of patients were studied. Group 1 (n = 12): patients with FHF. All patients were successfully "bridged" to OLT. "Bridge" time to OLT was 21-96 hr (mean: 39.3 hr). All patients were discharged neurologically intact. Reversal of decerebration was noted in all 11 deep stage 4 coma patients. There was reduction in intracranial pressure (ICP mmHg, 18.2 +/- 2.2 to 8.5 +/- 1.2; p < 0.004) and increase in cerebral perfusion pressure (CPP mmHg, 71.1 +/- 4.0 to 84.7 +/- 2.6; p < 0.006). Laboratory values pre- and post-BAL treatment: glucose (mg/dl) 122 +/- 11 to 183 +/- 21, p < 0.002; ammonia (mumol/l) 155.6 +/- 13.2 to 121.6 +/- 9.5, p < 0.02; total bilirubin (mg/dl) 21.6 +/- 2.8 to 18.2 +/- 2.2, p < 0.001; PT (sec) 23.2 +/- 1.7 to 21.9 +/- 1.0, p < 0.3. Group II (n = 8): patients with chronic liver failure experiencing acute exacerbation. Two patients survived and later underwent OLT. Six patients (not OLT candidates) died 1-14 days after last BAL treatment. Laboratory values pre- and post-treatment: ammonia (mumol/l) 201 +/- 47 to 143 +/- 25, p < 0.06; total bilirubin (mg/dl) 22.8 +/- 5.2 to 19.5 +/- 4.4, p < 0.01; PT (sec) 22.5 +/- 2.0 to 21.8 +/- 1.1, p < 0.6. CONCLUSION our clinical experience with the BAL suggests that it may serve as "bridge" to OLT in patients with FHF primarily by reversing intracranial hypertension, but it is not a substitute for OLT in patients with end-stage liver disease who are non-transplant candidates.

Numerous studies have reported successful allotransplantation of hepatocytes. However, none have shown long-term correction of a liver-related metabolic defect. In this study, we used a method of regional hepatocyte transplantation and subsequent induction of transplanted cell proliferation by regeneration response in the transplant-bearing liver lobes. New Zealand White rabbits were used as cell donors and Watanabe heritable hyperlipidemic (WHHL) rabbits were used as cell recipients (2 x 10(8) cells/rabbit). All recipient rabbits were maintained on daily cyclosporine. Two weeks after baseline serum cholesterol determination, group I WHHL rabbits (n = 7) received an infusion of cells into the right lateral liver lobe, and a loose ligature was placed around the portal venous branch supplying the anterior lobe. After 1 week, to allow engraftment, the portal venous branch was ligated, which resulted in the atrophy of the affected liver parenchyma and induction of hyperplasia in the transplant-bearing liver tissue. Group II rabbits (n = 6) were transplanted with New Zealand White hepatocytes without portal branch ligation (PBL) and group III rabbits (n = 4) were subjected to sham transplantation (saline) and PBL. The experimental period extended to 150 days after transplantation. All WHHL rabbits transplanted with normal hepatocytes showed reduction in serum cholesterol and low-density lipoprotein (LDL) levels. Group I (PBL-stimulated) recipients demonstrated a more pronounced and sustained effect than group II animals (P < 0.05). Group III controls showed only a slight, typical for aging decrease in serum cholesterol. Group I recipient livers perfused with LDL labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethyl indocarbocyanine perchlorate (DiI) showed much higher numbers of DiI-LDL-positive hepatocytes than those of group II recipients. In conclusion, a liver regeneration stimulus enhanced the population of transplanted hepatocytes and their functional effect in a large animal model of inborn error of liver metabolism.

Ex vivo reproduction of liver microstructure using isolated hepatocytes is critical for bioartificial liver use. We have developed a method of producing matrix-induced liver cell aggregates (MILCA) using a small number of collagen-coated beads as a nidus for formation of hepatocyte aggregates. Porcine hepatocytes were obtained by EDTA/collagenase digestion. Cell viability was assessed by trypan blue exclusion and LDH release. Cytochrome P-450 activity was determined at 4 and 24 hours by measuring the formation of 7-hydroxycoumarine (7-HC) from 7-ethoxycoumarine (7-EC). At 4 hours, the viability of MILCA was 92 +/- 2%, LDH release was 100 +/- 22 U/L and 7-HC formation was 140 +/- 34 nM/g cells. At 24 hours, MILCA viability remained greater than 90%, but 7-HC formation was lower than that of parallel control monolayer hepatocyte cultures (194 +/- 43 vs 481 +/- 78 nM/g cells; p < 0.002). On transmission electron microscopy, MILCA ultrastructure resembled that of a normal liver (maintenance of cell polarity, gap junctions, bile canaliculi, intact organellae, glycogen granules). MILCA were subsequently inoculated into hollow-fiber bioreactors which were perfused for 6 hours with plasma recovered from patients with fulminant hepatic failure (n = 6; 5 x 10(9) cells/cartridge, recirculation of 350 ml of plasma at 400 ml/min). In these studies, lidocaine (20 micrograms/ml) was cleared in less than 3 hours and 7-HC production at 6 hours was 71 +/- 8 nM/g cells. Other MILCA effects noted in this system included lowering of plasma lactate, bilirubin and ammonia and increase in the level of several non-essential amino acids.

Anatomical separation of pancreatic islets in some teleost fish makes them a useful source of pancreatic endocrine tissue. Islets were harvested from tropical Tilapia fish (Oreochromis nilotica) and cultured for 24 hr at 37°C. Eight athymic nude mice were rendered diabetic by streptozotocin (STZ) and transplanted under the kidney capsule with fish islets. After transplantation (Tx), nonfasting blood glucose (n-FBG), which was in all recipients >450 mg/dl, decreased to

We have recently conducted a series of experiments to characterize the pattern of reaction of human natural antibodies (NA) with individual pig liver cells. Pooled normal human serum (PHS) was incubated with cultured pig hepatocytes (HEP), aortic endothelial cells (AEC), and portal endothelial cells (PEC), and the reaction of NA to different cell types was measured by antibody-mediated cytotoxic (MTT assay), antibody binding (ELISA), and flow cytometric analysis. The human NA displayed a differential pattern of binding with hepatocytes exhibiting a more limited expression of xenoantigen expression than either aortic or portal endothelial cells. These differences in reaction patterns were also noted for Western blot analysis of individual cell membrane extracts. Preincubation of the pig cells with anti-pig MHC antibodies did not inhibit the binding of human IgM natural antibodies to the pig cells. Comparison of the pattern of NA absorption following the use of bioartificial liver support in patients with acute hepatic failure demonstrated limited ability of pig hepatocytes to absorb substantial amounts of NA. These studies indicate that pig hepatocytes are less vulnerable to NA cytotoxicity than pig vascular endothelial cells and that pig vascular endothelial cells express xenoantigens that are unique and not found on hepatocytes.

Background: Severe liver failure is associated with high mortality. Orthotopic liver transplantation (OLT) is the only effective therapeutic modality; there is a need for a ‘bridge’ system to support patients until an organ becomes available. Methods: A bioartificial liver (BAL) was used to treat 10 patients with severe liver failure. A plasmapheresis system was used to pump patient plasma through a module with porcine hepatocytes. Each treatment lasted 6-7 h. Results: All patients tolerated the procedures(s) well. Eight patients underwent OLT following BAL treatment(s). There were two late deaths after recovery from liver failure. Five patients with increased intracranial pressure (ICP) and decerebration had ICP normalization, increased cerebral perfusion pressure and full neurologic recovery after OLT. There was improvement in the level of encephalopathy and a significant decrease in serum ammonia after BAL treatment(s). Conclusions: BAL treatment is safe and beneficial and can be successfully used as a...

OBJECTIVE: To test the safety and efficacy of a bioartificial liver support system in patients with severe acute liver failure. SUMMARY BACKGROUND DATA: The authors developed a bioartificial liver using porcine hepatocytes. The system was tested in vitro and shown to have differentiated liver functions (cytochrome P450 activity, synthesis of liver-specific proteins, bilirubin synthesis, and conjugation). When tested in vivo in experimental animals with liver failure, it gave substantial metabolic and hemodynamic support. METHODS: Seven patients with severe acute liver failure received a double lumen catheter in the saphenous vein; blood was removed, plasma was separated and perfused through a cartridge containing 4 to 6 x 10(9) porcine hepatocytes, and plasma and blood cells were reconstituted and reinfused. Each treatment lasted 6 to 7 hours. RESULTS: All patients tolerated the procedure(s) well, with neurologic improvement, decreased intracranial pressure (23.0 +/- 2.3 to 7.8 +/- 1.7 mm Hg; p < 0.005) associated with an increase in cerebral perfusion pressure, decreased plasma ammonia (163.3 +/- 21.3 to 112.2 +/- 9.8 microMoles/L; p < 0.01), and increased encephalopathy index (0.60 +/- 0.17 to 1.24 +/- 0.22; p < 0.03). All patients survived, had a liver transplant, and were discharged from the hospital. CONCLUSIONS: This bioartificial liver is safe and serves as an effective "bridge" to liver transplant in some patients.

Despite recent advances in medical supportive therapy, patients with severe fulminant hepatic failure (FHF) have mortality rate approaching 90%. Investigators have attempted to improve survival by using various extracorporeal liver support systems loaded with sorbents and liver tissue preparations. None of them succeeded in gaining clinical acceptance and orthotopic liver transplantation (OLT) remains a primary therapeutic option for patients with FHF. In this study, authors discuss the systems which utilize isolated hepatocytes. Most of these devices were tested in vitro and in animals with chemically and surgically induced liver failure. In some studies, signficant levels of detoxification and liver functions were achieved. The authors describe their own hepatocyte-based artificial liver (BAL). It is based on plasma perfusion through a hollow-fiber module seeded with matrix-anchored porcine hepatocytes. The BAL was used 14 times to treat 9 patients with acute liver failure. On 10 occasions, a charcoal column was included in the plasma circuit. Each treatment lasted 7 +/- 1 h. All procedures were tolerated well and 8 patients (including 6 patients with FHF) underwent OLT. Five patients with increased intracranial pressure (ICP) and evidence of decerebration had normalization of ICP and enjoyed full neurologic recovery after OLT. Laboratory data showed evidence for bilirubin conjugation, decrease in blood ammonia, maintenance of low lactic acid levels, and increase in the ration between the branched chain and aromatic amino acids. No allergic reactions to xenogeneic hepatocytes were observed. The authors conclude that BAL treatment with porcine hepatocytes appears to be safe and can help maintain patients alive and neurologically intact until a liver becomes available for transplantation. (c) 1994 John Wiley & Sons, Inc.