The Voronoi theory of the normal liver lobular architecture and its applicability in hepatic zonation.

The precise characterization of the lobular architecture of the liver has been subject of investigation since the earliest historical publications, but an accurate model to describe the hepatic lobular microanatomy is yet to be proposed. Our aim was to evaluate whether Voronoi diagrams can be used to describe the classic liver lobular architecture. We examined the histology of normal porcine and human livers and analyzed the geometric relationships of various microanatomic structures utilizing digital tools. The Voronoi diagram model described the organization of the hepatic classic lobules with overall accuracy nearly 90% based on known histologic landmarks. We have also designed a Voronoi-based algorithm of hepatic zonation, which also showed an overall zonal accuracy of nearly 90%. Therefore, we have presented evidence that Voronoi diagrams represent the basis of the two-dimensional organization of the normal liver and that this concept may have wide applicability in liver pathology and research.

The outcome of patients with nephrogenic systemic fibrosis after successful kidney transplantation.

Nephrogenic systemic fibrosis (NSF) is a debilitating disease in patients with severely diminished kidney function. Currently, no standard treatment exists but improvement has been reported after restoration of kidney function. We retrospectively studied 17 NSF patients with and without successful kidney transplantation (KTx) to evaluate the effects of KTx on NSF. Nine of the 11 KTx developed NSF pretransplant whereas two developed NSF immediately after KTx with delayed graft function. Two of the six dialysis patients had previous failed kidney transplants. Age and sex were well matched. All but one patient was dialysis dependent at the time of NSF. Median follow-up was 35 months for KTx patients and 9 months for dialysis patients. Kidney transplants achieved adequate renal function with median serum creatinine of 1.4 (0.9-2.8) mg/dL and a glomerular filtration rate of 42 (19-60) mL/min/1.73 m(2). NSF improved in 54.6% of the transplanted patients and 50% of the nontransplanted patients (p = 0.86). Two KTx patients had complete resolution of their symptoms whereas four had partial improvement. Improvement in the dialysis patients was all partial. Successful KTx did not insure improvement in NSF and in fact appeared to have no significant benefit over dialysis.

Survival in portopulmonary hypertension: Mayo Clinic experience categorized by treatment subgroups.

To determine the natural history of portopulmonary hypertension (POPH), a retrospective screening-right heart catheterization-survival analysis of patients was performed. We categorized patients by three treatment subgroups: (1) no therapy for pulmonary hypertension (PH) or liver transplantation (LT), (2) therapy for PH alone and (3) therapy for PH followed by LT. Seventy-four patients were identified between 1994 and 2007. Nineteen patients received no therapy for PH and no LT representing the natural history of POPH. Five-year survival was 14%, and 54% had died within 1 year of diagnosis. Five-year survival in 43 patients receiving therapy for PH but no LT was 45%, and 12% had died within 1 year of diagnosis. Twelve patients underwent LT and 5-year survival for the nine receiving therapy for PH was 67% versus 25% in the three who were not pretreated with prostacyclin therapy. The survival of untreated patients with POPH was poor. Subgroups of patients selected to medical treatment with or without LT had better long-term survival. Mortality did not correlate with baseline hemodynamic variables, type of liver disease or severity of hepatic dysfunction. Medical therapy for POPH should be considered in all patients with POPH, but the treatment effects and impact on those considered for LT still requires well-designed, prospective study before practice guidelines can be suggested.

Transfer of porcine endogenous retrovirus across hollow fiber membranes: significance to a bioartificial liver.

BACKGROUND: A porcine endogenous retrovirus (PERV) capable of infecting human cells has been identified. This study was designed to determine whether hollow fiber membranes, such as those used in a bioartificial liver, block the transfer of PERV. METHODS: Three hollow fiber cartridges (HFCs) were studied in duplicate: cellulose fibers with 70 kD nominal molecular weight cut-off (MWCO), polysulfone fibers with 400 kD MWCO, and mixed cellulose fibers with 200 nm porosity. PK15 cells (porcine kidney cell line), known to produce PERV, were grown in the intraluminal compartment of HFCs fiber cartridges. Samples of medium were collected from both intraluminal and extraluminal compartments of the HFCs fiber cartridge during 14 days of culture. Samples were screened for PERV using reverse transcription polymerase chain reaction. All positive samples were tested for PERV infectivity in human 293 cells. RESULTS: PERV was detected in all samples from the intraluminal space and all intraluminal samples seemed to infect 293 cells. All extraluminal samples from the fibers of 200 nm porosity tested positive for PERV. Detection of PERV in the extraluminal space was delayed by fibers of 400 kD MWCO and 70 kD MWCO until at least day 3 and day 7, respectively, after inoculation of PK15 cells. Positive extraluminal samples from fibers of 400 kD MWCO and 70 kD MWCO did not infect 293 cells. CONCLUSION: Pore size, membrane composition, and duration of exposure influenced the transfer of PERV across HFCs. Some HFCs decrease the risk of viral exposure to patients during bioartificial liver therapy.

Thymoglobulin induction decreases rejection in solitary pancreas transplantation.

BACKGROUND: Solitary pancreas transplants, both pancreas transplant alone (PTA) and pancreas after kidney (PAK), have higher rejection rates and lower graft survivals than simultaneous pancreas-kidney transplants (SPK). The aim of this study is to compare three different antibody induction regimens in solitary pancreas transplant recipients and to assess the role of surveillance pancreas biopsies in the management of these patients. METHODS: Solitary pancreas transplant recipients between 01/98 to 02/00 (n=29) received induction with either daclizumab (1 mg/kg on day 0, 7, 14), OKT 3 (5 mg/day x0-7), or thymoglobulin (1.5 mg/kg/day x0-10). Maintenance immunosuppression was similar for the three groups. All rejections were biopsy-proven either by surveillance/protocol or when clinically indicated. RESULTS: The 1-year graft survival was 89.3% overall and 91.7% in the thymoglobulin group. Thymoglobulin significantly decreased rejection in the first 6 months when compared with OKT3 or daclizumab (7.7 vs. 60 vs. 50%). Acute rejections were seen on surveillance biopsies in the absence of biochemical abnormalities in 40% of patients. CONCLUSIONS: Thymoglobulin induction regimen led to a low incidence of acute rejection and a high rate of graft survival in solitary pancreas transplants. In addition, surveillance biopsies were useful in the detection of early acute rejection in the absence of biochemical abnormalities.

Hepatocyte liver-assist systems--a clinical update.

Liver failure is a serious problem that affects thousands of people in the United States each year. Other than liver transplantation, a supportive therapy has been unavailable for patients with liver failure that is refractory to medical treatment. An apparent solution to this problem is a hepatocyte liver-assist system. Such a system is composed of mammalian hepatocytes loaded in a mechanical apparatus, such as a hollow fiber cartridge. During extracorporeal perfusion of the system, the hepatocytes provide metabolic function to the patient with liver failure. At least two extracorporeal hepatocyte systems have shown promise in human clinical trials of acute liver failure. In fact, one system has gained approval from the Food and Drug Administration for testing in a randomized multicenter clinical trial. In this article, key issues of clinical testing are reviewed, and major contributions and questions that remain unresolved are emphasized.

Pancreas transplantation for the prevention of diabetic nephropathy.

Diabetic nephropathy is the leading cause of kidney failure in the United States. Poor glycemic control, hypertension, and smoking have been implicated as risk factors for the development and progression of diabetic nephropathy in patients with type 1 diabetes mellitus. Improved medical therapy including angiotensin-converting enzyme inhibitors and tight glycemic control with use of intensive insulin therapy have been shown to reduce the progression of diabetic nephropathy substantially based on albumin excretion rates. Despite these improvements in medical management, many patients still experience progression from early diabetic nephropathy to end-stage renal disease. Successful pancreas transplantation leads to normal glycemic control in patients with type 1 diabetes, but historically it has generally been limited to patients with both kidney failure and diabetes. In this review of the current treatment of diabetic nephropathy, we examine the potential role of preemptive pancreas transplantation in patients with diabetic nephropathy.

Cytoprotective influence of ZVAD-fmk and glycine on gel-entrapped rat hepatocytes in a bioartificial liver.

BACKGROUND: This study was designed to determine if an anti-necrotic compound, glycine, and/or an anti-apoptotic agent, ZVAD-fmk, improved the viability and function of hepatocytes in a bioartificial liver. METHODS: Isolated rat hepatocytes were entrapped in collagen gel (1.0-10.0 x 10(6) cells/mL) and cultured in serum-free medium (1:10 ratio of gel:media) supplemented with glycine alone, ZVAD-fmk alone, or glycine and ZVAD-fmk. The cytoprotective effects of glycine and ZVAD-fmk on gel-entrapped rat hepatocytes (GERH) were determined after anoxic exposure (0-20 hours). Cell functionality (measured by urea production), cell viability (quantitated by vital staining with fluorescein diacetate:ethidium bromide [FDA:EB]), and the mechanism of cell death (verified by electron microscopy and DNA fragmentation studies) were determined for each condition. RESULTS: The viability of GERH declined gradually and then stabilized 12 hours after hepatocyte isolation. The rate of urea production by GERH was directly proportional to the number of viable hepatocytes. Apoptotic death predominated at low cell density, and necrotic cell death became significant at high cell density. Hepatocyte necrosis became more significant after exposure to longer periods of anoxia (4, 8, 12, and 20 hours). ZVAD-fmk provided dose-dependent cytoprotection to GERH with an optimum benefit at a concentration of 60 mumol/L. After anoxic exposure or under high cell density culture, glycine demonstrated a maximum benefit of inhibiting necrosis at a concentration of 3 mmol/L. The beneficial effects of glycine and ZVAD-fmk were additive. CONCLUSIONS: The metabolic activity of a hepatocyte bioartificial liver may benefit from the use of cytoprotective agents such as ZVAD-fmk and glycine.

Extracorporeal application of a gel-entrapment, bioartificial liver: demonstration of drug metabolism and other biochemical functions.

Metabolic activity of a gel-entrapment, hollow fiber, bioartificial liver was evaluated in vitro and during extracorporeal hemoperfusion in an anhepatic rabbit model. The bioartificial liver contained either 100 million rat hepatocytes (n = 12), fibroblasts (n = 3), or no cells (n = 7) during hemoperfusion of anhepatic rabbits. Eight other anhepatic rabbits were studied without hemoperfusion as anhepatic controls, and three sham rabbits served as normal controls. Albumin production rates (mean +/- SEM) were similar during in vitro (17.0 +/- 2.8 micrograms/h) and extracorporeal (18.0 +/- 4.0 micrograms/h) application of the hepatocyte bioartificial liver. Exogenous glucose requirements were reduced (p < 0.01) and euglycemia was prolonged (p < 0.001) in anhepatic rabbits treated with the hepatocyte bioartificial liver. The maximum rate of glucose production by the hepatocyte bioartificial liver ranged from 50-80 micrograms/h. Plasma concentrations of aromatic amino acids, proline, alanine, and ammonia were normalized in anhepatic rabbits during hepatocyte hemoperfusion. Gel-entrapped hepatocytes in the bioartifical liver performed sulfation and glucuronidation of 4-methylumbelliferone. P450 activity was demonstrated during both in vitro and extracorporeal application of the BAL device by the formation of 3-hydroxy-lidocaine, the major metabolite of lidocaine biotransformation by gel-entrapped rat hepatocytes. In summary, a gel-entrapment, bioartificial liver performed multiple hepatocyte-specific functions without adverse side effects during extracorporeal application in an anhepatic, small animal model. With its potential for short term support of acute liver failure, scale-up of the current bioartificial liver device is indicated for further investigations in large animal, preclinical trials.

Resolution improvements in in vivo 1H NMR spectra with increased magnetic field strength.

The measurement of cerebral metabolites using highly homologous localization techniques and similar shimming methods was performed in the human brain at 1.5 and 4 T as well as in the dog and rat brain at 9.4 T. In rat brain, improved resolution was achieved by shimming all first- and second-order shim coils using a fully adiabatic FASTMAP sequence. The spectra showed a clear improvement in spectral resolution for all metabolite resonances with increased field strength. Changes in cerebral glutamine content were clearly observed at 4 T compared to 1.5 T in patients with hepatic encephalopathy. At 9.4 T, glutamine H4 at 2.46 ppm was fully resolved from glutamate H4 at 2.37 ppm, as was the potential resonance from gamma-amino-butyric acid at 2.30 ppm and N-acetyl-aspartyl-glutamate at 2.05 ppm. Singlet linewidths were found to be as low as 6 Hz (0.015 ppm) at 9.4 T, indicating a substantial decrease in ppm linewidth with field strength. Furthermore, the methylene peak of creatine was partially resolved from phosphocreatine, indicating a close to 1:1 relationship in gray matter. We conclude that increasing the magnetic field strength increases spectral resolution also for 1H NMR, which can lead to more than linear sensitivity gains.

Knotted bowel: small-bowel obstruction from coiled peritoneal shunt catheters. Report of two cases.

Knotting of a peritoneal catheter around a loop of bowel is a rare occurrence, which may lead to bowel obstruction. The incomplete removal of two ventriculoperitoneal shunts resulted in two cases of iatrogenically knotted peritoneal catheters. One patient underwent a laparotomy for relief of obstruction and the other was successfully treated by uncoiling the catheter by means of a wire passed into its lumen. A plan for management of a knotted peritoneal catheter is outlined.

Evolution of the bioartificial liver: the need for randomized clinical trials.

The pursuit of a bioartificial liver is well documented in the literature. Early techniques of artificial liver support that have undergone clinical testing included simple exchange transfusions, extracorporeal xenogeneic or allogeneic liver perfusion, cross-circulation, hemodialysis, charcoal hemoperfusion, and plasmapheresis with plasma exchange. These techniques failed because they were unable to adequately support those hepatic functions essential for survival and because they lacked a back-up therapy, such as liver transplantation, for irreversible forms of liver disease. The concept evolved that hepatic functions essential for survival would be best performed by hepatocytes in an apparatus that allowed sustained or repetitive application. The best results have been achieved with bioartificial liver technologies that employ hepatocytes as implantable systems or extracorporeal devices. Implantable bioartificial liver systems include hepatocytes that have been on coated microcarrier beads, within microencapsulated gel droplets, within biodegradable polymeric substrates, or as spheroid hepatocyte aggregates. Extracorporeal systems include hepatocytes in suspension, on flat plates, and in hollow fiber bioreactors. Several extracorporeal systems have undergone extensive animal testing and are entering the early stages of human clinical trials. Randomized trials are needed to establish the value of bioartificial liver support in the treatment of patients with acute hepatic failure or as a bridge to liver transplantation.

Pharmacokinetic analysis verifies P450 function during in vitro and in vivo application of a bioartificial liver.

Lidocaine is a sensitive substrate for evaluating liver P450 function. In this study, metabolism of lidocaine by xenogeneic hepatocytes in a hollow fiber, bioartificial liver was measured under in vitro conditions (n = 6) and in an anhepatic rabbit model. Animals in the treatment group (n = 6) received hemoperfusion by a bioartificial liver that contained 100 million rat hepatocytes. Other anhepatic rabbits received no hemoperfusion (n = 3) or a bioartificial liver with no cells (n = 3). Lidocaine clearance was 7.0 +/- 0.6 ml/min, and the half-life of lidocaine was 5.6 +/- 0.8 hr under in vitro conditions. Conversion of lidocaine to 3-hydroxy-lidocaine was confirmed in vitro and accounted for 46% of lidocaine elimination in the hepatocyte bioartificial liver. During in vivo application of the bioartificial liver, pharmacokinetic parameters of lidocaine metabolism, including drug half-life and metabolite formation, were significantly improved in anhepatic rabbits. 3-Hydroxy-lidocaine profiles verified the activity of a P450 isozyme expressed preferentially by rat hepatocytes in the bioartificial liver. We conclude that hepatic P450 activity was provided by xenogeneic hepatocytes during in vitro and in vivo applications of a bioartificial liver.

Immunoprotection of xenocytes in a hollow fiber bioartificial liver.

A new, hollow fiber bioartificial liver (BAL) was tested in an anhepatic rabbit model to assess the hollow fiber membrane as an immunologic barrier. The extracorporeal BAL contained rat hepatocytes (xenocytes) entrapped in collagen gel inside the lumen of hollow fibers with 100 kd nominal molecular weight cut-off. Blood from the anhepatic rabbit flowed in the extracapillary compartment. After 4 h of BAL hemoperfusion, the hepatocyte gels were tested for evidence of rabbit immunoglobulin and complement. Samples of the gels were stained with fluorescein isothiocyanate (FITC)-labeled antibodies directed against rabbit IgM, rabbit IgG (Fc and F[ab]2), and rabbit complement (C3) and studied by immunofluorescence microscopy. Enzyme-linked immunosorbent assay and Western blotting were used, respectively, to quantify and identify proteins inside the BAL and in the rabbit blood. These studies suggest that very little rabbit IgG (primarily fragments of IgG) and no IgM or C3 crossed the hollow fibers during hemoperfusion. Rat albumin steadily accumulated in the rabbit blood during hemoperfusion, which indicated membrane permeability to molecules of that size and stable hepatocyte function by the BAL. The authors conclude that hollow fibers with 100 kd nominal molecular weight cut-off provide immunoprotection for xenocytes in an extracorporeal BAL.

Preliminary experience with a cuffed ePTFE graft for hemodialysis vascular access.

The purpose of this study was to determine graft patency and blood flow rates in recipients of a new cuffed ePTFE graft (Venaflo graft) used for hemodialysis access. A pilot study was conducted with 12 (7 men, 5 women) consecutive patients (age range, 36-76 yr; mean, 65 yr). All patients were recipients of a new cuffed PTFE graft placed for hemodialysis access. Seven were high risk because of a prior history of clotted hemodialysis accesses (1-6; mean, 3.3). Blood flow rates were determined by ultrasound dilution technique at 3 month intervals. One year and 2 year overall graft patency rates were 90.9% and 68.2%, respectively. One graft (high risk, six prior grafts) was lost to thrombosis in the first year; two grafts (one high risk, four prior grafts) were lost to thrombosis in the second year of follow-up. No graft thrombosis resulted from stenosis at the graft-vein anastomosis. Blood flow rates ranged from 550 to 2,110 ml/min (mean, 1,086 ml/min; n = 8) when first measured 3 months after graft placement. Similar flow rates were observed at 12 months (mean, 1,043 ml/min; n = 7) and 24 months (mean, 1,014 ml/min; n = 4) in grafts available for comparison. Dialysis flow rates in excess of 350 ml/min were possible with all patent grafts. A cuffed ePTFE graft provided stable blood flow and satisfactory graft patency during 2 years of follow-up, even in high risk patients with a prior history of vascular access thrombosis.

Staining with fluorescein diacetate correlates with hepatocyte function.

To establish the importance of fluorescein diacetate (FDA) as a viability stain for cultured hepatocytes, we hypothesized that FDA staining would correlate positively with hepatocyte viability and function. Mixtures of live and dead cells were stained with FDA and scanned by flow cytometry. A close correlation was observed between the live cell fraction and percent viability as determined by FDA staining (R2 = 0.962). Hepatocytes were also sorted into low fluorescence and high fluorescence groups. Both albumin production and lidocaine metabolism (P-450 activity) were significantly increased in the high fluorescence group compared to the low fluorescence group. An automated, fluorescence-activated assay was useful for rapid assessment of hepatocyte viability. In addition, the intensity of green fluorescence following staining with FDA correlated well with two specific measures of hepatocyte function.

Impact of molecular adsorbent recirculating system therapy on tacrolimus elimination: a case report.

BACKGROUND: We report a unique case which quantifies the effect of molecular adsorbent recirculating system (MARS [Gambro, Sweden]) therapy on blood concentrations of tacrolimus in a patient treated for refractory pruritus associated with recurrent hepatitis C of the liver allograft. Tacrolimus is a low-molecular-weight, highly protein-bound drug with the potential to be removed during MARS therapy. CASE REPORT: Results of therapeutic drug monitoring revealed extracorporeal tacrolimus elimination accounted for only 0.3% of total drug removal during the session. CONCLUSIONS: Although no explanation can be offered as to why MARS contributed so little to overall tacrolimus elimination, the data clearly show minimal impact of MARS on tacrolimus blood level.