A major concern in therapy of acute liver failure is protection of hepatocytes to prevent apoptosis and maintain liver function. Small interfering RNA (siRNA) is a powerful tool to silence gene expression in mammalian cells. To evaluate the therapeutic efficacy of siRNA in vivo we used different mouse models of acute liver failure. We directed 21-nt siRNAs against caspase 8, which is a key enzyme in death receptor-mediated apoptosis. Systemic application of caspase 8 siRNA results in inhibition of caspase 8 gene expression in the liver, thereby preventing Fas (CD95)-mediated apoptosis. Protection of hepatocytes by caspase 8 siRNA significantly attenuated acute liver damage induced by agonistic Fas (CD95) antibody (Jo2) or by adenovirus expressing Fas ligand (AdFasL). However, in a clinical situation the siRNAs most likely would be applied after the onset of acute liver failure. Therefore we injected caspase 8 siRNA at a time point during AdFasL-and adenovirus wild type (Adwt)-mediated liver failure with already elevated liver transaminases. Improvement of survival due to RNA interference was significant even when caspase 8 siRNA was applied during ongoing acute liver failure. In addition, it is of particular interest that caspase 8 siRNA treatment was successful not only in acute liver failure mediated by specific Fas agonistic agents (Jo2 and AdFasL) but also in acute liver failure mediated by Adwt, which is an animal model reflecting multiple molecular mechanisms involved in human acute viral hepatitis. Consequently, our data raise hope for future successful application of siRNA in patients with acute liver failure.

Combined coagulation factor VII (FVII) and factor X (FX) deficiency (combined FVII/FX deficiency) belongs to the group of bleeding disorders in which both factors show reduced plasma activity. It may arise from coincidental inheritance of separate coagulation factor deficiencies or a common cause as large deletions comprising both gene loci. The F7 and F10 genes are located on the long arm of chromosome 13. Here, we describe 10 cases with combined FVII/FX deficiency representing both genetic mechanisms of occurrence. Genetic analyses included direct sequencing of the F7 and F10 genes and MLPA (multiplex ligation-dependent probe amplification) for detection of heterozygous large deletions. In four patients, the combined deficiency was due to a large deletion within the terminal end of chromosome 13. In the remaining six cases the deficiency resulted from coincidental inheritance of different genetic alterations affecting both genes independently. In most cases, the genetic defects were heterozygous, presenting with prolonged PT, normal aPTT and mild or no bleeding symptoms. Only in one case compound heterozygous mutations were detected in the F10, resulting in prolonged aPTT and a more severe bleeding phenotype. To avoid a misdiagnosis of combined FVII/FX deficiency, analyses of single factor activities have to be performed in all cases with prolonged PT even if aPTT is normal. Genetic analyses are substantial for correct prediction of an inheritance pattern and a proper genetic counselling.

Allogeneic hematopoietic stem cell transplantation (HSCT) offers the possibility of cure for sickle cell disease (SCD) patients. Unfortunately, the probability of finding an HLA-matched donor for SCD patients is low. HSCT from HLA-haploidentical donors using reduced intensity conditioning, unmanipulated bone marrow and post-transplantation cyclophosphamide (ptCy) has resulted in negligible toxicity but high rates of graft rejection. We hypothesized that combining ptCy with a myeloablative reduced toxicity conditioning including serotherapy to increase immune ablation would allow for better engraftment. In a pilot approach, we treated three patients with SCD (5, 8, and 20 years old) lacking a matched donor. All patients had severe disease-related complications despite standard treatment. They received unmanipulated bone marrow from parental HLA-haploidentical donors. Conditioning consisted of alemtuzumab 0.2 mg/kg/day on days -9 and -8, fludarabine 30 mg/m/day on days -7 to -3, treosulfan 14 g/m/day on days -7 to -5, thiotepa 2 × 5 mg/kg/day on day -4, and cyclophosphamide 14.5 mg/kg/day on days -3 and -2. GVHD prophylaxis was performed using cyclophosphamide 2 × 50 mg/kg on days +3 and +4 and mycophenolate mofetil, tacrolimus from day +5. After a follow-up of 11, 14, and 30 months, all three patients are alive and well, off immunosuppression, and without symptoms of SCD. One patient experienced mild skin GVHD grade I, none showed chronic GVHD. Asymptomatic CMV reactivation was seen in two patients. HLA-haploidentical HSCT can extend the donor pool for patients with SCD. Whether intensification of the conditioning regimen and intensive immunosuppression leads to improvement in engraftment rates while still allowing a favorable toxicity profile deserves further investigation.

Inhibition of NFB enhances the susceptibility of cancer to TRAIL-mediated apoptosis and is suggested as a strategy for cancer therapy. Because the role of NFB in TRAIL-mediated apoptosis of hepatocytes is unknown, we investigated the influence of NFB-inhibition in death ligand-mediated apoptosis in hepatitis. Adenoviral hepatitis resulted in upregulation of NFB-activity, which could be inhibited by expression of IB␣-superrepressor. We treated mice after the onset of adenoviral hepatitis with adenoviruses expressing FasL (AdFasL), TRAIL (AdTRAIL), or GFP (AdGFP). In contrast to apoptosis induced by AdFasL, NFB inhibition strongly enhanced AdTRAIL-mediated apoptosis of hepatocytes. Expression of IB␣ inhibits adenoviral infection-mediated overexpression of bcl-xl, providing a molecular mechanism for TRAIL sensitization. In agreement with this hypothesis, downregulation of bcl-xl by siRNA enhanced susceptibility of hepatocytes to TRAIL, but not to FasL-mediated apoptosis, resulting in TRAIL-mediated severe liver damage after AdTRAIL application. Our data demonstrate that inhibition of NFB in adenoviral hepatitis strongly sensitizes hepatocytes to TRAIL-mediated apoptosis. Bcl-xl, in contrast to bcl-2 and c-FLIP, is strongly upregulated after viral infection and represents an essential NFB-dependent survival factor against TRAIL-mediated apoptosis. In conclusion, inhibition of NFB or bcl-xl during TRAIL therapy may harbor a risk of liver damage in patients with viral hepatitis. (HEPATOLOGY 2005;41:280-288.)

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Haemorrhagic and thrombotic events occur in both children and adults. The underlying causes are congenital or acquired disorders. In contrast to haemorrhagic disorders, inherited thrombotic disorders nearly exclusively in association with additional external risk factors lead to thrombotic events predominantly during the newborn period and adolescence. It is necessary to be aware of age-specific properties of coagulation in order to correctly interpret clinical and laboratory findings and to provide optimal care for children with haemorrhagic and thrombotic complications.

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