Jochen Meyburg

Liver, liver cell and stem cell transplantation for the treatment of urea cycle defects

Despite advances in pharmacological therapy of urea cycle disorders (UCDs), the overall long-term prognosis is poor, especially for neonatal manifestations. Transplantation of liver tissue or isolated cells appears suitable for transfer of the missing enzyme. Liver transplantation (LT) for UCDs has an excellent 5-year survival rate of approximately 90% and is the only way to completely cure the disease. However, major neurological damage can only be prevented if the operation is performed during the first months of life. Unfortunately, such early LTs have a substantial risk for peri- and postoperative complications, mostly caused by a relatively large liver graft. Liver cell transplantation (LCT) is less invasive than LT, but has still to be regarded as an experimental therapy with about 100 patients treated since its first use in 1993. UCDs are a model disease for LCT, because of the poor prognosis, mainly hepatic enzyme defects, and excellent outcome after LT. So far, 10 children underwent LCT for UCDs with very few technical complications and encouraging clinical results. A first prospective study on its use in severe neonatal UCDs has recently started. However, availability of hepatocytes is limited by the scarcity of donor livers; therefore the use of stem cells is under investigation. Several different cell types may be regarded as liver stem cells, and in vivo transformation into hepatocyte-like cells has been shown in animal studies. However, a clear proof of principle in animal models of human metabolic disease is still missing, which is the prerequisite for clinical application in humans.

Liver Cell Transplantation: Basic Investigations for Safe Application in Infants and Small Children

Liver cell transplantation (LCT) is a very promising method for the use in pediatric patients. It is significantly less invasive than whole organ transplantation, but has the potential to cure or at least to substantially improve severe disorders like inborn errors of metabolism or acute liver failure. Prior to a widespread use of the technique in children, some important issues regarding safety and efficacy must be addressed. We developed a mathematical model to estimate total hepatocyte counts in relation to bodyweight to make possible more appropriate dose calculations. Different liver cell suspensions were studied at different flow rates and different catheter sizes to determine mechanical damage of cells by shear forces. At moderate flow rates, no significant loss of viability was observed even at a catheter diameter of 4.2F. Addition of heparin to the cell suspension is favored, which is in contrast to previous animal experiments. Mitochondrial function of the hepatocytes was determined with the WST-1 assay and was not substantially altered by cryopreservation. We conclude that especially with the use of small catheters, human LCT should be safe and efficient even in small infants and neonates.

Monitoring of intraportal liver cell application in children.

Despite recent advances and promising results in children, liver cell transplantation (LCT) should still be regarded as an experimental therapy. Several substantial complications are known from animal studies and individual patients. However, safety data on liver cell infusion in children are scarce. We used LCT in four children of different ages (3 weeks to 11 years, 3–40 kg) and underlying diseases [acute liver failure (n = 1), urea cycle disorders (n = 2), and Crigler-Najjar syndrome (n = 1)]. Vital parameters, portal vein flow (PVF), portal vein pressure (PVP), and liver enzymes were measured every 5 min during cell application and hourly thereafter between applications. An application protocol with discontinuation rules depending on changes in PVF and PVP was developed and successfully applied. Application was feasible in all children despite the catastrophic overall condition of the patient with acute liver failure. No application-related changes in vital parameters were found, and none of the children experienced clinical signs of portal vein thrombosis, pulmonary embolism, or anaphylactic reactions. Time courses for changes in PVF, PVP, and liver enzymes were obtained. Thorough monitoring of portal vein pressure and duplex sonography according to a defined protocol is likely to increase safety of cell application in pediatric LCT.

Liver cell transplantation in children

Abstract:  With increasing shortage of donor organs and changes in organ allocation systems, there is a considerable need for alternative or supportive techniques in the field of liver transplantation. Liver cell transplantation (LCT) is such an innovative technique that is especially promising for use in children, because it is less invasive than orthotopic liver transplantation (OLT). Two main indications exist: acute liver failure (ALF) and hepatic‐based inborn errors of metabolism. Ten children with ALF underwent LCT so far. One patient recovered without OLT, five were successfully bridged to OLT, and four died. Clinical improvement like reduced hyperammonemia, improved coagulation, and regression of hepatic encephalopathy occurred in the majority of patients. For the treatment of inborn errors of metabolism, experience exists with Crigler–Najjar syndrome type 1 (CNS 1, n = 5) and urea cycle disorders (UCD, n = 6). All but two patients experienced metabolic stabilization for a mean period of 9.7 months, before the majority of children underwent OLT. Whereas transplant success in CNS 1 patients can be easily monitored by plasma bilirubin levels, it is much more difficult in UCDs, where many factors can contribute to clinical deterioration. Nevertheless, these individual therapeutic attempts of LCT yielded encouraging results, and prospective studies should be initiated.

Liver cell transplantation in severe infantile oxalosis—a potential bridging procedure to orthotopic liver transplantation?

BACKGROUND The infantile form of primary hyperoxaluria type I (PHI) is the most devastating PH subtype leading to early end-stage renal failure and severe systemic oxalosis. Combined or sequential liver-kidney transplantation (LKTx) is the only curative option but it involves substantial risks, especially in critically ill infants. The procedure also requires resources that are simply not available to many children suffering from PHI worldwide. Less invasive and less complex therapeutic interventions allowing a better timing are clearly needed. Liver cell transplantation (LCT) may expand the narrow spectrum of auxiliary measures to buy time until LKTx for infants can be performed more safely. METHODS We performed LCT (male neonate donor) in a 15-month-old female in reduced general condition suffering from systemic oxalosis. Renal replacement therapy, initiated at the age of 3 months, was complicated by continuous haemodialysis access problems. Living donor liver transplantation was not available for this patient. Plasma oxalate (Pox) was used as the primary outcome measure. RESULTS Pox decreased from 104.3±8.4 prior to 70.0±15.0 μmol/L from Day 14 to Day 56 after LCT. A significant persistent Pox reduction (P<0.001) comparing mean levels prior to (103.8 μmol/L) and after Day 14 of LCT until LKTx (77.3 μmol/L) was seen, although a secondary increase and wider range of Pox was also observed. In parallel, the patients clinical situation markedly improved and the girl received a cadaveric LKTx 12 months after LCT. However, biopsy specimens taken from the explanted liver did not show male donor cells by amelogenin polymerase chain reaction. CONCLUSIONS With due caution, our pilot data indicate that LCT in infantile oxalosis warrants further investigation. Improvement of protocol and methodology is clearly needed in order to develop a procedure that could assist in the cure of PHI.

Use of the Middle Colic Vein for Liver Cell Transplantation in Infants and Small Children

INTRODUCTION Because it is less invasive, intraportal liver cell transplantation (LCT) is an interesting alternative to whole organ transplantation. The inferior mesenteric vein is usually chosen for portal vein access. However, anatomical variations are common in children, so we investigated catheter insertion into the middle colic vein. PATIENTS AND METHODS Three children (3 weeks to 3 years; 3 to 14 kg) underwent LCT in our center for acute liver failure or severe neonatal urea cycle disorders. Small 4.2-French Hickman lines were surgically introduced into the middle colic vein and advanced to the portal vein stem. The patients received repetitive infusions of liver cells over a period of 4-11 days. RESULTS Catheter insertion was feasible and tolerated well despite the poor clinical condition of 1 patient and the metabolic instability in the other 2 patients. Blood could be drawn from all catheters, and measurement of portal vein pressure was possible in 2 children. The patient with acute liver failure died after 11 days from complications of the underlying disease. In the other 2 children, portal vein catheters stayed patent for several months. CONCLUSIONS The middle colic vein can be recommended for placement of intraportal LCT catheters even in small and critically ill infants.

In vivo monitoring of urea cycle activity with 13C-acetate as a tracer of ureagenesis

BACKGROUND The hepatic urea cycle is the main metabolic pathway for detoxification of ammonia. Inborn errors of urea cycle function present with severe hyperammonemia and a high case fatality rate. Long-term prognosis depends on the residual activity of the defective enzyme. A reliable method to estimate urea cycle activity in-vivo does not exist yet. The aim of this study was to evaluate a practical method to quantify (13)C-urea production as a marker for urea cycle function in healthy subjects, patients with confirmed urea cycle defect (UCD) and asymptomatic carriers of UCD mutations. METHODS (13)C-labeled sodium acetate was applied orally in a single dose to 47 subjects (10 healthy subjects, 28 symptomatic patients, 9 asymptomatic carriers). RESULTS The oral (13)C-ureagenesis assay is a safe method. While healthy subjects and asymptomatic carriers did not differ with regards to kinetic variables for urea cycle flux, symptomatic patients had lower (13)C-plasma urea levels. Although the (13)C-ureagenesis assay revealed no significant differences between individual urea cycle enzyme defects, it reflected the heterogeneity between different clinical subgroups, including male neonatal onset ornithine carbamoyltransferase deficiency. Applying the (13)C-urea area under the curve can differentiate between severe from more mildly affected neonates. Late onset patients differ significantly from neonates, carriers and healthy subjects. CONCLUSION This study evaluated the oral (13)C-ureagenesis assay as a sensitive in-vivo measure for ureagenesis capacity. The assay has the potential to become a reliable tool to differentiate UCD patient subgroups, follow changes in ureagenesis capacity and could be helpful in monitoring novel therapies of UCD.

Clinical symptoms, biochemical studies and therapeutic approaches in a sibship with a new congenital tubulopathy.

Abstract We report on two siblings suffering from a new congenital tubulopathy. Following normal pregnancies not complicated by polyhydramnios, severe renal losses of potassium, chloride, sodium and magnesium occurred in the first weeks after birth. Calcium metabolism was not affected. The distal tubular chloride reabsorption was considerably decreased in the two siblings (0.25 and 0.28, respectively). Secondary hyperaldosteronism, activation of the kallikrein-kinin system and elevated urinary prostaglandin excretion were observed. The effects of indomethacin, spironolactone and captopril on symptoms, electrolyte wasting, activation of renin-angiotensin-aldosterone and kallikrein-kinin system and prostaglandin synthesis were studied. In spite of persisting elevation of prostaglandin synthesis, captopril decreased electrolyte wasting, polyuria and hyperaldosteronism most effectively. Conclusion We delineate an apparently new disorder characterized by a postnatal onset, an extremely decreased chloride reabsorption with extensive hyperchloriduria and hypermagnesiuria in the presence of normal calcium metabolism. The disorder can be distinguished from other tubulopathies with hypokalaemic alkalosis.

Pediatric liver transplantation. Introduction of a program for southern Germany in Heidelberg

Abstract:  Pediatric liver transplantation in Germany is exclusively performed in four centers in the north of Germany. We report our experience with the implementation of a new pediatric liver transplantation program in Heidelberg for children living in the south of Germany. In a pilot phase, we have transplanted eight children with various transplantation techniques including reduced size, left lateral split (segments 2 and 3), and full left split (segments 1–4). All transplantations were successful. No vascular complications occurred. The immunosuppressive regimen we used was cyclosporine A and methylprednisolone. The rate of acute rejection was three of eight patients. No patient required a retransplantation. One patient died due to a severe fungal sepsis he had acquired prior to transplantation. We conclude that in a multidisciplinary approach with careful patient selection a new program for pediatric liver transplantation can be successfully established.

ARFI shear-wave elastography with simulation of acute urinary tract obstruction in an ex vivo porcine kidney model

METHODS A total of 20 heparinized pig kidneys were investigated at 10 intrapelvic hydrostatic pressure steps (0-90 mmHg). SWE (ARFI; Virtual TouchTM IQ, Siemens) measurements were taken at three different measuring regions and in two measuring sequences using a linear ultrasonography probe (9L4, Siemens). Median values of 10 shear-wave speed (SWS) measurements were calculated for each pressure step. Logarithmic transformed median SWS values were analyzed in a linear mixed model. RESULTS SWS increased significantly with increasing intrapelvic pressure. Median SWS for all kidneys in both measuring sequences and all measuring regions was 1.47 m/s (interquartile range [IQR], 0.38 m/s) at 0 mmHg, 1.94 m/s (IQR, 0.42 m/s) at 30 mmHg, 2.07 m/s (IQR, 0.43 m/s) at 60 mmHg, 2.24 m/s (IQR, 0.49 m/s) at 90 mmHg. The correlation between pelvic pressure increase and median SWS values for the central parenchyma was significantly higher compared with the peripheral parenchyma. CONCLUSION Acutely increased renal pelvic pressure correlates with increasing SWS values in ARFI elastography in an ex vivo porcine kidney model.