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Acute cholecystitis is a common disease, and laparoscopic surgery is the standard of care.Optimal timing of surgery for acute cholecystitis remains controversial: either early surgery shortly after hospital admission or delayed elective surgery after a conservative treatment with antibiotics.The ACDC ("Acute Cholecystitis-early laparoscopic surgery versus antibiotic therapy and Delayed elective Cholecystectomy") study is a randomized, prospective, open-label, parallel group trial. Patients were randomly assigned to receive immediate surgery within 24 hours of hospital admission (group ILC) or initial antibiotic treatment, followed by delayed laparoscopic cholecystectomy at days 7 to 45 (group DLC). For infection, all patients were treated with moxifloxacin for at least 48 hours. Primary endpoint was occurrence of predefined relevant morbidity within 75 days. Secondary endpoints were as follows: (1) 75-day morbidity using a scoring system; (2) conversion rate; (3) change of antibiotic therapy; (4) mortality; (5) costs; and (6) length of hospital stay.Morbidity rate was significantly lower in group ILC (304 patients) than in group DLC (314 patients): 11.8% versus 34.4%. Conversion rate to open surgery and mortality did not differ significantly between groups. Mean length of hospital stay (5.4 days vs 10.0 days; P0.001) and total hospital costs (€2919 vs €4262; P0.001) were significantly lower in group ILC.In this large, randomized trial, laparoscopic cholecystectomy within 24 hours of hospital admission was shown to be superior to the conservative approach concerning morbidity and costs. Therefore, we believe that immediate laparoscopic cholecystectomy should become therapy of choice for acute cholecystitis in operable patients. (NCT00447304).

We report a case of a woman with a gastrointestinal bleeding. An esophageal ulcer was detected in the endoscopy, however a histological malignancy could not be found. A computer tomography (CT) scan showed a thickness in the distal esophagus and enlarged lymph nodes, so a malignancy was highly suspected. However, the patient refused to follow the recommended clinical procedure of a surgical intervention. Four years later a carcinoma could be ruled out because of follow-up examinations.

Death from end-stage liver disease (ESLD) because of chronic hepatitis B and C has become an increasing problem in human immunodeficiency virus (HIV)-infected patients in the last years. This is mainly because of the dramatic decrease of HIV-related morbidity and mortality since the introduction of highly active antiretroviral therapy (HAART). Although the data on the outcome of liver transplantation in HIV-infected recipients with ESLD is limited, overall results seem comparable to non-HIV-infected recipients. Therefore, liver transplant centres around the world are increasingly accepting HIV-infected individuals as organ recipients. Post-transplantation control of HIV replication is achieved by continuing HAART. As in non-HIV-infected patients, hepatitis B virus recurrence is efficiently prevented by hepatitis B immunoglobulin and antiviral therapy. Re-infection of the allograft with hepatitis C virus, however, remains an important problem, and progress to allograft cirrhosis may even be more rapid than in HIV-negative patients. Interactions in drug metabolism between the HAART components and the immunosuppressive drugs are difficult to predict and require close monitoring of drug levels and dose adjustments. The complexity in this setting makes close cooperation between transplant surgeons, hepatologists, HIV-clinicians and pharmacologists mandatory. As experience on liver transplantation in HIV-infected individuals is still limited, to date results from large prospective trials addressing key issues are needed.

Incompatibility of drug solutions administered through a common line may jeopardize the safety and effectiveness of i.v. drug therapies.[1][1] We recently reported that, in the absence of dedicated preventive strategies, incompatibilities are alarmingly frequent in an intensive care setting.[2][2]

Background Prediction of prognosis after liver transplantation (OLT) remains difficult. The present study determines if standard laboratory parameters measured within the first week after OLT correlate with outcome. Patients and Methods Laboratory parameters measured within the first weak after OLT of 328 patients were grouped either graft loss or death within 90 days after (group 1: graft loss; group 2: death; group 3: neither graft loss nor death within 90 days). Results Peak AST and ALT were significantly lower in group 3 (1867 and 1252 U/L) than in group 1 (4474 and 2077 U/L) or 2 (3121 and 1865 U/L). Bilirubin was significantly lower and γ-GT significantly higher in group 3 compared to groups 1 and 2. In multivariate analysis, high AST peaks were independently associated with death or graft loss within 90 days. An increase in γ-GT and low bilirubin early after transplantation were found to be independently associated with superior outcome. Discussion Unexpectedly, a disproportionate rise in γ-GT was associated with graft and patient survival of more than 90 days. This might be explained by regeneration phenomena in the liver indicative of a well functioning graft.

Over the past 4 decades, the surgical techniques of liver transplantation (LTx) have permanently evolved and been modified. Among these, the modified piggyback (MPB) technique by Belghiti offers specific advantages. The objective of this study was to present our single-center experience with the MPB technique in 500 cases. Recipients' perioperative data were prospectively collected and evaluated. Postoperative and specific complications, stay in the intensive and intermediate care unit, and the mortality rate with cause of death were analyzed. Most recipients were classified as Child C (49.1%). For the patients who underwent LTx for the first time, alcoholic (23.9%) and viral (22.2%) cirrhosis and hepatocellular carcinoma (15.1%) were the prevalent indications. The overall median warm ischemia time, anastomosis duration, and operative time were 45, 108, and 320 minutes, respectively. The median intraoperative blood loss was 1500 mL. A venovenous bypass was never needed to maintain hemodynamic stability. Only in a few cases was temporary inferior vena cava clamping necessary. Most prominent surgical complications were hemorrhage, hematoma, and wound dehiscence. Renal failure occurred in 6.2% of patients. The overall median stay in the intensive and intermediate care unit was 14 days. The mortality rates within 30 and 90 days were 6.3% and 13.3%, respectively. No technique-related death occurred. The MPB technique by Belghiti is a feasible and simple LTx technique. The caval flow is preserved during the anhepatic phase, and this minimizes the need for venovenous bypass or portocaval shunt. This technique requires only 1 caval anastomosis, which is easy to perform with a short anhepatic phase. To minimize the risk of outflow obstruction, attention should be paid by doing a wide cavocavostomy cranially to the donor inferior vena cava in a door-lock manner. This technique can be applied in almost all patients undergoing LTx for the first time and liver retransplantation as well.

To correct overdosing of drugs requiring adjustment based on renal function in intensive-care patients. In a prospective intervention study, we estimated individual glomerular filtration rate and assessed whether medication required dose adjustment based on renal function. Senior clinicians received a structured report containing recommendations as to whether and how to adjust dosage in the individual patient (intervention). Prevalence of overdosed drugs (primary outcome), extent of overdoses, and reasons for nonacceptance of recommendations (secondary outcomes) were assessed. Of 138 screened intensive-care patients, 68 (49%) had renal impairment, and 110 (14%) of the 805 prescribed drugs required consideration of renal function. A potential overdose was found in 53/110 drugs (48%) and this rate decreased to 26/110 (24%, P

Varicella zoster virus (VZV) seronegative patients under immunosuppressive therapy are at risk for severe life-threatening varicella. A 25-year-old male patient presented with rash and hepatitis. He had been known to suffer from Crohn's disease and received immunosuppressive treatment with azathioprine. The patient showed dyspnoea, and presented with a generalized rash with vesicular lesions typical for herpesvirus infections. He was started immediately on acyclovir therapy. Varicella infection was determined in this VZV seronegative patient in rash vesicles, blood and tracheal secretions and a high VZV viral load was detected in these specimens. The causative agent was genotyped by sequencing of several genes as a variant of the European genotype E2 containing several unique single nucleotide polymorphisms. Despite all measures, there was progressive septic shock, and the patient died due to multi-organ failure. Immunocompromised adults without varicella history are at high risk to develop life-threatening complications of varicella. Antiviral therapy with acyclovir can only be successful when administered as early as possible on suspicion of varicella infection in this group of patients. The most effective method to prevent severe varicella in immunocompromised patients is active immunization prior to immunosuppressive therapy.

The aim of the present study was (1) to determine the prevalence of intensive care unit (ICU) admissions due to an adverse drug reaction (ADR), and (2) to compare affected patients with patients admitted to the ICU for the treatment of deliberate self-poisoning using medical drugs. Prospective observational cohort study. Fourteen bed medical ICU including an integrated intermediate care (IMC) section at a tertiary referral center. A total of 1,554 patients admitted on 1 January 2003 to 31 December 2003. Ninety-nine patients were admitted to the ICU with a diagnosis of ADR (6.4% of all admissions), 269 admissions (17.3%) were caused by deliberate self-poisoning. Patients admitted for treatment of ADR had a significantly higher age, a longer treatment duration in the ICU, a higher SAPS II score, and a higher 6-month mortality than those with deliberate self-poisoning. Most patients (71.7%) suffering from ADR required advanced supportive care in the ICU while the majority of patients (90.7%) with deliberate self-poisoning could be sufficiently treated in the IMC area. All diagnostic and therapeutic procedures in the ICU except mechanical ventilation were significantly more often performed in patients with ADR. This study provides further evidence that ADR is a frequent cause of admission to medical ICUs resulting in a considerable use of ICU capacities. In the present setting patients with ADR required longer and more intense medical treatment in the ICU than those with deliberate self-poisoning.

A 29-year-old male patient with chronic hepatitis C infection and interferon alpha therapy in his medical history was admitted to the hospital because of the clinical manifestation of a pulmonary renal syndrome. High titers of proteinase-3-ANCA were detected, while an infectious agent was ruled out. After diagnosis of Wegener's granulomatosis the patient received prednisolone and cyclophosphamide pulse therapy and remission developed rapidly. Chronic hepatitis C infection as well as interferon therapy are frequently associated with autoimmune disorders. We assume that the interferon therapy itself has triggered autoimmune processes resulting in Wegener's granulomatosis in our patient. Thus we recommend to search specifically for autoimmune disorders in the past medical history and if necessary to consider a screening for autoantibodies before starting an interferon therapy. An autoimmune disease should also be taken into account if new symptoms develop under an ongoing interferon alpha therapy.

Lethal sepsis occurs when an excessive inflammatory response evolves that cannot be controlled by physiologic anti-inflammatory mechanisms, such as the recently described cholinergic anti-inflammatory pathway. Here we studied whether the cholinergic anti-inflammatory pathway can be activated by pharmacologic cholinesterase inhibition in vivo.Prospective, randomized laboratory investigation that used an established murine sepsis model.Research laboratory in a university hospital.Female C57BL/6 mice.Sepsis in mice was induced by cecal ligation and puncture. Animals were treated immediately with intraperitoneal injections of nicotine (400 microg/kg), physostigmine (80 microg/kg), neostigmine (80 microg/kg), or solvent three times daily for 3 days.Treatment with physostigmine significantly reduced lethality (por = .01) as efficiently as direct stimulation of the cholinergic anti-inflammatory pathway with nicotine (por = .05). Administration of cholinesterase inhibitors significantly down-regulated the binding activity of nuclear factor-kappaB (por = .05) and significantly reduced the concentration of circulating proinflammatory cytokines tumor necrosis factor-alpha, interleukin-1beta, and interleukin-6 (por = .001), and pulmonary neutrophil invasion (por = .05). Animals treated with the peripheral cholinesterase inhibitor neostigmine showed no difference compared with physostigmine-treated animals.Our results demonstrate that cholinesterase inhibitors can be used successfully in the treatment of sepsis in a murine model and may be of interest for clinical use.

Herpes simplex virus (HSV) is an infrequent cause of viral hepatitis, accounting for 1.4% [1] of all cases with acute liver failure, and is associated with a high mortality in adults. HSV 1 and 2 can both cause fulminant acute hepatitis that occurs mainly in immunocompromised patients. Symptoms may be unspecific and include fever, abdominal pain and flulike symptoms. Therapeutic options include intravenous acyclovir therapy and liver transplantation [2–6]. Nevertheless, prognosis for fulminant HSV hepatitis is poor and in a recent French report, all five patients with hepatic failure due to HSV died [1], two of them prior to transplantation, two up to 1 month after transplantation; one patient survived for one year. In contrast, a recent publication suggests that the spectrum and severity of HSV hepatitis reported in the literature over-represents fulminant presentations, and that HSV hepatitis in immunocompromised host occurs with a higher frequency and lower mortality than described so far [7]. The following report describes a kidney and pancreas graft recipient with fulminant HSV2 hepatitis, who underwent successful liver transplantation in combination with acyclovir and foscarnet therapy. Case report

Background Unquestionably viral diversity and genetic heterogeneity in hepatitis C virus (HCV) infection and other viral diseases play an essential role in viral immune escape and the development of chronicity. Despite this knowledge most vaccine approaches against HCV have excluded this important issue. Moreover the feasibility of developing an effective HCV vaccine has been questioned, mainly because prophylactic immunity against HCV cannot be achieved in chimpanzees by either vaccination or previous HCV infection, and reinfection in men has been reported, most likely due to genetic shift and immune escape. To analyse and characterize a new technique of a ‘multigenotype’- and/or ‘library’-vaccine, we established an envelope 1 (E1) plasmid vaccine against HCV and characterized humoral and cellular immune responses after vaccination in a mouse model. Material and methods Normally genetic information of one or two target proteins is cloned into a DNA-vaccine. In our approach we cloned a defined number of different genotypes and subtypes (defined vaccine, DV) or the genetic information from 20 patients (undefined) into a plasmid (library vaccine, LV). Results As expected, immunized animals showed both stronger humoral (ELISA) and cellular (T-cell proliferation, ELISPOT) immune responses against genotype 1, since the stimulating antigen was genotype 1 derived. However, not all genotype 1 immunized animals recognized this viral antigen leading to the assumption that some epitopes lost their immunogenicity through a change in the amino acid sequence. Interestingly, some of the genotype 4 and 5 immunized mice sera were able to react against E1 protein. Conclusion Most of the assays showed immune reactivity against the DV or LV vaccine demonstrating the cross-reactive potential of such a vaccination approach. This cloning and immunization strategy based on the viral heterogeneity of the virus has in our view major implications for HCV, a virus with a broad viral genetic diversity, and may become in the future in the context of DNA- or viral-based vaccination strategies a possibility to overcome viral immune escape both in the prophylactic or therapeutic setting.

Hepatitis C virus (HCV) is the causative agent of most cases of chronic liver disease, cirrhosis, and hepatocellular carcinoma (HCC) affecting more than 170 million people world-wide. Progress in elucidating the nature of HCV and the development of new therapeutic strategies is hampered fundamentally by the absence of adequate small animal models simulating natural HCV infection. The creation of conditional mouse lines with the tetracycline-controlled gene expression system holds new perspectives for simulation of wild-type HCV infection in a small animal model. Transgenic mice were established with tetracycline-inducible coexpression of HCV core or HCV open reading frame (ORF) and luciferase. In long-term induction experiments, mice were examined for immunopathological changes after expression of HCV proteins. Inducible and liver-specific expression of transgenes was detected by Western blot, immunoprecipitation, luciferase assay and in vivo imaging of bioluminescence of luciferase in genetically modified mice. Ectopic expression levels were determined quantitatively in the liver, kidney, heart and spleen of mice in the induced and non-induced state. During long-term induction an elevation of aminotransaminases (ALT) was observed only in HCV core/ORF-expressing mice, but HCV-specific immune response was not confirmed by in vitro immunological assays. The histology of liver sections provided evidence of steatosis, which was correlated with an inflammatory response. The inducible HCV-transgenic mouse lines provide further evidence of liver pathogenesis in the presence of inflammation during liver-specific expression of HCV proteins and offer new insights into the effects of temporally and spatially controlled protein expression of HCV. J. Med. Virol. 79: 1103–1112, 2007.

Biliary complications remain a substantial cause of morbidity following liver transplantation. They have been reported to occur in a rate of 10-15% of full-size transplantations and may be higher in living donor, split or reduced size liver transplantations. The most common biliary complications following liver transplantations are leaks and strictures. In both, the incidence varies with respect to type of graft and donor as well as the type of biliary anastomosis. The management of the biliary complications requires a multidisciplinary approach and has changed over the past decade, favoring endoscopic and radiological techniques. Surgical revision including retransplantation is reserved for patients in whom endoscopic and interventional modalities are unsuccessful.

Dendritic cells (DC) are potent antigen-presenting cells that play a critical role in the initiation of immunity to viral infections. Direct transduction of DC appears to be the major pathway in vivo responsible for induction of antigen specific immune responses. The aim of this study was to explore the vaccine potential of DC transduced with adenoviral vectors encoding the HCV nonstructural protein 3 (AdNS3) compared to DC pulsed with recombinant NS3 (rNS3). Mice (Balb/c and C57BL/6 transgenic for HLA-A2.1) were immunized with DC based vaccines. After the immunization, antigen specific immune responses including humoral responses, cytokine secretion, and IFN-gamma-producing T cell responses were analyzed. In both strains of mice inoculated with DC transduced with an adenovirus, the generated NS3 specific antibody response and IFN-gamma-secreting T cell response were stronger than that generated by rNS3-pulsed DC. Analysis of the cytokine profiles revealed that immunization with AdNS3 transduced DC shifted the antigen specific immunity towards Th1 responses. DC transduced with AdNS3 are superior to DC pulsed with rNS3 in inducing vigorous humoral and Th1-type cellular responses against NS3. The results demonstrate for the first time the immunogenic potential of genetically modified DC by a prime and boost approach in eliciting a strong NS3-specific, cell-mediated, humoral immune response in both Balb/c mice and HLA-A2.1 transgenic mice.

Our aim was to conduct a prospective study to evaluate staging accuracy of a new coil concept for endoluminal magnetic resonance imaging (MRI) on ex vivo gastric carcinomas. Twenty-eight consecutive patients referred to surgery with a clinically proven primary gastric malignancy were included. Surgical specimens were examined with a foldable and self-expanding loop coil (8-cm diameter) at 1.5 Tesla immediately after total gastrectomy. T1- and T2-weighted and opposed-phase sequences (axial, frontal sections; 3- to 4-mm slice thickness) were acquired. Investigators blinded to any patient information analyzed signal intensity of normal gastric wall, gastric tumor, and lymph nodes. Findings were compared with histopathological staging. On surgical specimens, 2–5 gastric wall layers could be visualized. All gastric tumors (26 carcinomas, two lymphomas) were identified on endoluminal MR data (100%). Overall accuracy for T staging was 75% (18/24); sensitivity to detect serosal involvement was 80% and specificity 89%. N staging correlated in 58% (14/24) with histopathology (N+ versus N−). The endoluminal coil concept is feasible and applicable for an ex vivo setting. Endoluminal MR data provided sufficient detail for gastric wall layer differentiation, and therefore, identification of T stages in gastric carcinoma is possible. Further investigations in in vivo settings should explore the potential of our coil concept for endoluminal MR imaging.

Wilson disease is a copper storage disorder caused by mutations in the ATP7B gene leading to liver cirrhosis. It has previously been shown that lentiviral vectors can govern an efficient delivery and stable expression of a transgene. The aim of this pilot study was to prove the principle of a lentiviral gene transfer in the Long-Evans cinnamon (LEC) rat, an animal model of Wilson disease.LEC rats were treated either by systemic application of lentiviral vectors or by intrasplenic transplantation of LEC-rat hepatocytes lentivirally transduced with ATP7B. The ATP7B gene expression was analyzed by RT-PCR and immunofluorescence analysis. The therapeutic effect was assessed by analysis of liver histology, serum ceruloplasmin oxidase activity, and liver copper content.Hepatic expression of the transgene was detected at different time-points post-treatment and lasted for up to 24 weeks (end of experiment). Liver copper levels were lowered in all treatment groups compared to untreated LEC rats. Twenty-four weeks after treatment, the area of the examined liver-tissue sections occupied by fibrosis was 48.3-57.9% in untreated LEC rats and 10.7-19.8% in rats treated with cell therapy. In systemically treated rats, only small fibrous septa could be observed.These data prove for the first time that lentiviral ATP7B gene transfer is feasible in Wilson disease. In our pilot study the systemic approach was more promising in ameliorating disease progression than the transplantation of lentivirally transduced hepatocytes.

Die Entscheidung fur eine individualisierte Arzneimitteltherapie muss auf der Basis von Wirksamkeit, unerwunschten Wirkungen und Patientencompliance erfolgen. Dies gilt in besonderem Mase an den Schnittstellen der Versorgung, vor allem auch beim Ubergang von der stationaren Therapie zur ambulanten Weiterbehandlung. Aspekte der Zulassung und damit der Erstattungsfahigkeit durch den Kostentrager mussen dabei ebenfalls berucksichtigt werden. Ein Patient (mannlich, 61 Jahre, Maler) zeigte 1999 nach Phenprocoumonbehandlung wegen tiefer Venenthrombose einen Transaminasenanstieg, der nach Absetzen von Phenprocoumon vollstandig reversibel war. Diagnostisch konnten phenprocoumonunabhangige Ursachen ausgeschlossen werden. Nach einer erneuten Thrombose wurde 2003 wieder Phenprocoumon verordnet. Dies fuhrte erneut zu einem Transaminasenanstieg, schlieslich zu einer cholestatischen Hepatitis und einem fulminanten Leberversagen. Es wurde eine Transplantation durchgefuhrt mit anschliesend guter Leberfunktion unter immunsuppressiver Therapie. Aufgrund einer zweifachen tiefen Beinvenenthrombose sowie intermittierenden Vorhofflimmerns war eine Antikoagulation auch uber die peripoperative Phase hinaus indiziert. Dafur wurde Enoxaparin als niedermolekulares Heparin gewahlt. Ein niedermolekulares Heparin erscheint nach Nutzen-, Risiko- und Kostenaspekten trotz der erforderlichen subkutanen Gabe am besten geeignet, um in dieser Situation auch ambulant die gerinnungshemmende Behandlung sicherzustellen. Das nicht auszuschliesende Risiko einer extrahepatischen, immunologisch vermittelten Kreuzsensitivitat spricht gegen Cumarine. Mit Ximelagatran steht seit kurzem ein Wirkstoff zur oralen Antikoagulation in fixer Dosierung mit dokumentierter Wirksamkeit zur Verfugung. Die mogliche Lebertoxizitat insbesondere bei Langzeitanwendung spricht allerdings gegen seine Anwendung bei diesem Patienten. Fur Heparinoide, Hirudine und andere Gerinnungsinhibitoren wie insbesondere Thrombozytenaggregationshemmer existieren keine ausreichenden Wirksamkeitsbelege. Die Kriterien des Bundessozialgerichts fur eine Erstattungsfahigkeit der „off-label“-Therapie eines niedermolekularen Heparins sind in diesem Fall erfullt.

Herpes virus hepatitis (HSV) represents a form of acute necrotizing hepatitis, which most frequently develops in immunocompromised patients. Therapeutic options include high-dose intravenous acyclovir and liver transplantation. We report the first case of recurrent HSV hepatitis after liver retransplantation, which occurred despite continuous administration of high-dose intravenous antiviral therapy. Because explant histology pointed to initial therapy response, we thought that the reason for recurrence might be due to acyclovir resistance. Most acyclovir resistance is caused by inactivating mutations in the herpes virus thymidine kinase gene. HSV infection was detected by histology and proofed by immunohistochemistry. PCR amplification of the herpes virus thymidine kinase gene was performed on histology specimens to demonstrate the course of viral infection in liver tissue. Genotypic resistance testing of the herpes virus was performed by sequencing the thymidine kinase amplicon. In serial biopsy, HSV-DNA sequences were only detectable when histology revealed herpes hepatitis. Whereas the primary explant exhibited the wild-type thymidine kinase gene, a biopsy of the second graft one month after retransplantation, which showed recurrent herpes virus hepatitis, had a single base insertion within a homopolymeric cytosine stretch. This mutation causes a frame shift leading to a premature stop codon and results in a known acyclovir-resistant herpes strain. In conclusion, we believe that testing for acyclovir-resistant herpes strains should be considered in high-risk patients in whom viral clearance is not achieved serologically to prevent fatal recurrence of disease by using antiviral drugs such as inhibitors of HSV-DNA polymerase or viral helicase primase inhibitors.

Living-donor liver transplantation (LDLT) in adults has been expanded after becoming the standard for children in many transplant centres. Advantages of LDLT include thorough donor screening, optimization of timing for transplantation and minimal cold ischaemia time. However, the risk of donor morbidity and mortality must be considered. The preoperative evaluation of the donor typically is performed in consecutive stages. Specific donor considerations in LDLT are thrombosis and embolism, hepatic mass and hepatic steatosis. After complete evaluation, only a small proportion of potential donors are satisfactory candidates. The evaluation protocol for LDLT recipients in most centres is not different from that of cadaveric transplantation. More experience and the development of specific selection and evaluation criteria will further increase the benefit for the recipient and decrease the risk of the donor.

The mechanisms of immune evasion and the role of the early immune response in chronic infection caused by hepatitis C virus (HCV) are still unclear. Here, we present evidence for a cascade of molecular events that the virus initiates to subvert the innate immune attack. The HCV core protein induced p53-dependent gene expression of TAP1 (transporter associated with antigen processing 1) and consecutive major histocompatibility complex (MHC) class I upregulation. Moreover, in p53-deficient liver cell lines, only reconstitution with wild-type p53, but not mutated p53 lacking DNA binding capacity, showed this effect. As a consequence of increased MHC class I expression, a significantly downregulated cytotoxic activity of natural killer (NK) cells against HCV core-transfected liver cells was observed, whereas lysis by HCV-specific cytotoxic T cells was not affected. These results demonstrate a way in which HCV avoids recognition by NK cells that may contribute to the establishment of a chronic infection.

Background Different parameters have been determined for prediction of treatment outcome in hepatitis c virus genotype 1 infected patients undergoing pegylated interferon, ribavirin combination therapy. Results on the importance of vitamin D levels are conflicting. In the present study, a comprehensive analysis of vitamin D levels before and during therapy together with single nucleotide polymorphisms involved in vitamin D metabolism in the context of other known treatment predictors has been performed. Methods In a well characterized prospective cohort of 398 genotype 1 infected patients treated with pegylated interferon-α and ribavirin for 24–72 weeks (INDIV-2 study) 25-OH-vitamin D levels and different single nucleotide polymorphisms were analyzed together with known biochemical parameters for a correlation with virologic treatment outcome. Results Fluctuations of more than 5 (10) ng/ml in 25-OH-vitamin D-levels have been observed in 66 (39) % of patients during the course of antiviral therapy and neither pretreatment nor under treatment 25-OH-vitamin D-levels were associated with treatment outcome. The DHCR7-TT-polymorphism within the 7-dehydrocholesterol-reductase showed a significant association (P = 0.031) to sustained viral response in univariate analysis. Among numerous further parameters analyzed we found that age (OR = 1.028, CI = 1.002–1.056, P = 0.035), cholesterol (OR = 0.983, CI = 0.975–0.991, P

Hepatitis C Virus (HCV) NS3 protease is an attractive target for antiviral agent development because it is required for viral replication. Because a stable cell culture system or small animal model to study HCV replication is not readily available, we constructed an in vitro model allowing the investigation of NS3 transcription, translation, and protease function. Sequences encoding for full length HCV genomes were cloned and transfected into HuH-7 human hepatocellular carcinoma cells to analyze NS3 transcription/translation. A plasmid pHCV ORF I luc that expresses the complete HCV coding region upstream of a luciferase reporter gene was designed to enable quantification of translated HCV proteins. Additionally, NS3 protease function was assessed by direct coexpression of NS3 and NS5 in HuH 7 cells, and the subsequent measurement of cleavage products. We found that antisense oligodeoxynucleotides (AS-ODN) interfered with NS3 translation in a dose dependent fashion; AS-ODN 5 cotransfection directed against NS3 sequences significantly inhibited protease activity as measured by cleaved NS5A levels. Finally, cleaved NS5A levels served as anindex of protease activity and Chymostatin, a protease inhibitor, almost completely blocked NS3 enzymatic activity. This cell culture system is useful in the assessment of potential antiviral agents on HCV NS3 expression and function.

Background & Aims: α-Fetoprotein (AFP) is a tumor-associated protein that is frequently expressed at high levels in hepatocellular carcinoma (HCC). The aim of the study was to characterize self-reactive cytotoxic T lymphocytes (CTLs) directed against murine AFP (mAFP) after DNA-based immunization in mice. Methods: To study CTL responses, mAFP-expressing recombinant vaccinia viruses were generated. An HCC tumor model was established in C57L/J mice by injection of syngeneic endogenously mAFP-expressing Hepa1-6 cells. Results: Gene gun and intramuscular coimmunizations of DNA expression vectors encoding mAFP with plasmids encoding murine interleukin (IL)-12, granulocyte-macrophage colony–stimulating factor, or IL-18 induced weak CTL activity against mAFP in different mouse strains. Some mice developed anti-mAFP antibody responses, suggesting breaking of immunologic ignorance. No hepatocyte damage was detectable despite low-level endogenous hepatic mAFP expression. Therapeutic immunizations of mice bearing mAFP-expressing murine HCCs induced partial regression of tumors. A significant survival benefit was observed in mice immunized with mAFP expression vector DNA but not in untreated mice or in mice immunized with mock/cytokine plasmid DNA. Conclusions: The data show that AFP may be used as a potential self tumor antigen to induce CTL and CD4 + T cell–mediated regression of AFP-expressing HCC by DNA-based immunization. GASTROENTEROLOGY 2000;119:1104-1112

Background: The combination of chronic hepatitis C virus (HCV) and ethanol may increase viral replication, impair cellular immunity, and result in severe and progressive liver disease. Because HCV nonstructural proteins play a major role in viral elimination, we examined the cellular and humoral immune responses after genetic immunization against NS5 in a chronic ethanol mouse model. Methods: Mice were fed an ethanol or isocaloric pair-fed control liquid diet and were immunized with HCV NS5-expression plasmid. Results: The humoral and cellular arms of the immune system were significantly impaired in ethanol-fed mice. Abstinence partially reversed the inhibitory effects on antibody levels and the CD4+ proliferative immune response but did not restore the CD8+ cytotoxic T-cell response to this HCV nonstructural protein. Furthermore, we determined whether murine interleukin-2 coadministration with the NS5 expression plasmid would reverse the inhibitory effects of chronic ethanol consumption; again, partial restoration was observed for B-cell and CD4+ T-cell activity, but not for cytotoxic T cells. Conclusions: These results suggest that the high rate of chronic HCV infection in alcoholics may be due to ethanol's effects on antiviral immune responses.

Hepatitis C Virus (HCV) is the major causative agent of chronic hepatitis. Since it has been difficult to obtain full-length cDNA clones of HCV including the 3' untranslated region (UTR) that give rise to replication competent virus, we generated the 3'UTR by a modified protocol of total chemical synthesis (TCS) with overlap-extension-PCR using four long oligodeoxynucleotides. A synthetic cDNA fragment of about 340 nucleotides (nt) in length was generated, subcloned and sequenced. This approach represents a rapid and easy alternative to RT-PCR from infectious serum and may be a highly valuable method to generate partial cDNA clones of HCV and other viruses including defined variants.

Most liver transplant recipients become reinfected with hepatitis C virus after OLT followed by allograft dysfunction, transplant cirrhosis and graft failure in a significant proportion of patients. Both in the pre-emptive prophylactic setting and in the treatment setting sustained virological response rates are poor compared to the precirrhotic hepatitis state. Patients with significant hepatitis should be always treated before developing cirrhosis or even with early cirrhosis. After transplantation pegylated interferon in combination with ribavirin is the most successful treatment opportunity to date, however the best time point and treatment duration as well as doses for pegylated interferons and rebavirin have to be defined.

Sickle cell trait (SCT) is an usually asymptomatic hemoglobinopathy. Cases of sudden excertional deaths in individuals with SCT have been described. We here report an exceptional case of excessive rhabdomyolysis and acute renal failure triggered by a sauna visit in a 29 year-old African American with SCT.

Colorectal cancer is the third most frequent cancer in industrial nations. Therapeutic strategies to treat metastatic disease and prevent recurrence are needed. Anti-tumor immunity can be induced by dendritic cells. Dendritic cells can be expanded by the fms-like tyrosine kinase 3 ligand (Flt3L) in vivo. The aim of this study was to develop an adenoviral-based immune-gene therapy of colorectal cancer with Flt3L in a BALB/c mouse model.A new Flt3L-encoding adenoviral vector (pAdFlt3L) was administered in two approaches in a CT26 colon cancer model in female BALB/c mice. In the therapeutic approach, pAdFlt3L was injected into the tail vein or directly into subcutaneous CT26 colon carcinoma tumors in BALB/c mice. In the vaccination protocol, mice were vaccinated with CT26 cell lysate and pAdFlt3L subcutaneous prior to subcutaneous application of vital CT26 cells.Application of pAdFlt3L led to high levels of Flt3L in vitro and in vivo. Significant expansion of dendritic cells after application of pAdFlt3L in vivo was confirmed by the use of CD11c and CD11b surface markers in immunohistochemistry and flow cytometry (p = 0.019). In the therapeutic approach, neither intravenous nor intratumoral treatments with pAdFlt3L lead to regression of CT26 tumors. In the vaccination protocol, vaccination completely prevented tumor growth and resulted in superior survival compared to control mice (p0.001).Our results demonstrate that immunostimulatory therapy with pAdFlt3L is effective to prevent tumor development through vaccination and may represent a therapeutic tool to prevent metastatic disease.

Primary pig hepatocytes form a tissuelike structure in an in vitro culture model that has provision for three-dimensional cell orientation, cell aggregation, decentralized cell perfusion with low metabolite gradients, integral oxygenation, and nonparenchymal cell coculture. Scanning electron microscopy (SEM) has shown that hepatocytes spontaneously form aggregates in a three-dimensional structure between and on the surface of artificial capillaries. Transmission electron microscopy (TEM) has shown that after 7 weeks of in vitro perfusion, the cell ultrastructure remains similar to that of the parenchyma in vivo. Golgi complexes, active membrane processes, reorganization of cell junctions, and bile canaliculi-like intercellular spaces were demonstrated.

Summary Little is known about the ability of hepatitis C virus (HCV) to alter early innate immune responses in infected patients. Previous studies have shown that natural killer (NK) cells are functionally impaired after interaction of recombinant HCV glycoprotein E2 with the co-stimulatory CD81 molecule in vitro; however, the functional consequences of a prolonged contact of NK cells with HCV particles have remained unclear. We have examined the phenotypes of purified, interleukin-2-activated NK cells from healthy donors and HCV genotype 1b patients after culture for 5 days with HCV pseudoparticles (HCVpp) and serum samples containing HCV genotype 1b. NK cells from healthy donors and chronic HCV patients were found to up-regulate receptors associated with activation (NKp46, NKp44, NKp30, NKG2D), while NK receptors from the killer cell immunoglobulin-like receptor family (KIR/CD158), predominantly having an inhibitory function, were significantly down-modulated after culture in the presence of HCV particles compared with control cultures of NK cells. HCV-infected sera and HCVpp elicited significantly higher secretion of the NK effector lymphokines interferon-γ and tumour necrosis factor-α. Furthermore, HCV stimulated the cytotoxic potential of NK cells from normal donors and patients. The enhanced activation of NK cells after prolonged culture with HCVpp or HCV-containing sera for 5 days suggests that these innate effector cells may play an important role in viral control during early phases of HCV infection.

AIM: To explore dendritic cells (DCs) multiple functions in immune modulation. METHODS: We used bone-marrow derived dendritic cells from BALB/c mice pulsed with pseudo particles from the hepatitis C virus to vaccinate naive BALB/c mice. Hepatitis C virus (HCV) pseudo particles consist of the genotype 1b derived envelope proteins E1 and E2, covering a non-HCV core structure. Thus, not a single epitope, but the whole “viral surface” induces immunogenicity. For vaccination, mature and activated DC were injected subcutaneously twice. RESULTS: Humoral and cellular immune responses measured by enzyme-linked immunosorbent assay and interferon-gamma enzyme-linked immunosorbent spot test showed antibody production as well as T-cells directed against HCV. Furthermore, T-cell responses confirmed two highly immunogenic regions in E1 and E2 outside the hypervariable region 1. CONCLUSION: Our results indicate dendritic cells as a promising vaccination model for HCV infection that should be evaluated further.

Eradication of chronic Hepatitis B virus (HBV) infection, marked by HBs seroconversion, is very rarely achieved by treatment with nucleoside and nucleotide analogs. Therapeutic cell based approaches, like interferon therapy, have a higher chance of seroconversion. Dendritic cells (DC) are key players in the cellular immune response and have been shown to play an important role in controlling HBV infection. In this study, the potential of ex vivo activated DC to induce specific immune responses against HBV was examined. DC derived from bone-marrow of BALB/c or C56BL/6 mice were pulsed with HBV subviral particles (HBVsvp), derived from the HepG2.2.15 cell line. HepG2.2.15 produces subviral particles consisting of the HBc and HBs proteins. Thus, the entire "viral surface" is presented to DC to induce an immune reaction. In vitro pulsation with HBVsvp successfully activated bone-marrow derived DC, demonstrated by FACS analysis showing increased MHCII, CD 86 and CCR-7. Immunization of mice, via subcutaneous injection of the activated DC, induced HBV specific immune reactions which were measured by ELISA, ELISPOT and T-cell proliferation analysis. Vaccination with ex vivo activated DC may be a promising tool for therapeutic or prophylactic approaches against the Hepatitis B virus.

Moxifloxacin, the newest fourth-generation fluoroquinolone, has a broad spectrum of antibacterial activity covering both Gram-positive and Gram-negative aerobic and anaerobic bacteria and is therefore very well suited for the treatment of biliary tract infections. The present study aimed to determine the penetration of moxifloxacin into gallbladder tissue to evaluate its antibiotic potential in this indication.Hospitalized patients with acute cholecystitis received a single, 1 h infusion of 400 mg of moxifloxacin before cholecystectomy. Serum and gallbladder wall tissue samples were collected during surgery, and the moxifloxacin concentrations were measured by HPLC.Sixteen patients (eight men and eight women) were included between January 2007 and April 2008. The time between start of infusion and gallbladder removal ranged from 50 min to 21 h 10 min. The serum concentration at the time of cholecystectomy was between 0.39 and 4.37 mg/L, and the tissue concentration between 1.73 and 17.08 mg/kg. The tissue-to-serum concentration ratio ranged from 1.72 to 6.33.The results show that moxifloxacin penetrates well into gallbladder tissue and is therefore a therapeutic option for biliary tract infection. The highest concentrations in serum and gallbladder tissue were measured shortly after the end of a 1 h infusion. As perioperative prophylaxis, moxifloxacin should therefore be administered 30-60 min before the first surgical incision.

Background In patients presenting with acute liver failure (ALF) prediction of prognosis is vital to determine the need of transplantation. Based on the evidence that plasma disappearance rate of indocyanine green (ICG-PDR) correlates with liver cell function, we evaluated the ability of ICG-PDR measured by pulse dye densitometry to predict outcome in patients with acute liver failure. Methods Prospectively markers of hepatocellular injury, synthesis and excretion, including ICG-PDR were measured daily until liver transplantation, death, discharge from intensive care unit, or up to 7 days in 25 patients with acute liver failure. Receiver operating curve (ROC) analysis was performed to assess the value of ICG-PDR to predict outcome in ALF. Results The 25 patients analyzed included 18 that recovered spontaneously and 7 that underwent liver transplantation (n = 6) or died (n = 1). Causes of ALF included viral hepatitis (n = 4), toxic liver injury (n = 15), ischemic liver injury (n = 2), and cryptogenic liver failure (n = 4). King's college criteria were fulfilled in 85.7% of patients not recovering spontaneously and in 16.7% of patients recovering spontaneously. The mean ICG-PDR measured on day 1 in patients recovering spontaneously was 12.0 ± 7.8%/min and in patients not recovering spontaneously 4.3 ± 2.0%/min (P = 0.002). By ROC analysis the sensitivity and specificity of an ICG-PDR value ≤ 6.3%/min on study day 1 were 85.7% and 88.9%, respectively, for predicting a non spontaneous outcome in ALF. Conclusion ICG-PDR allows early and sensitive bedside assessment of liver dysfunction in ALF. Measurement of ICG-PDR might be helpful in predicting the outcome in acute liver failure. Trial registration Clinicaltrials.gov, NCT 00245310

We investigated the effect of written drug information for senior clinicians on the incidence of drug-drug interactions (DDIs) and DDI-related adverse events in intensive care patients.A prospective controlled intervention cohort study was conducted in a medical intensive and intermediate care unit in a university hospital. From 1,062 consecutive intensive care patients, those 265 (control: 136, intervention: 129) withor =8 concurrently prescribed drugs were included in the study (to include high-risk patients with polypharmacy). The DDI information for senior clinicians during an intervention period of 3 months was based on a computerised clinical decision support system (CDSS) containing information on risk and management of 9,453 drug combinations.The number of patients with at least one DDI at the end of the respective study phase decreased by 18% (relative risk reduction) from 90 (66%) patients in controls to 70 (54%) in the intervention group (p = 0.02). The relative risk of a patient suffering from at least one DDI-related adverse event decreased by 43% from 60 (44%) patients in controls to 32 (25%) in the intervention group (p0.01). Among these events, the incidence of QT(C) prolongation was reduced by 64% from 15 (11%) patients in the control group to 5 (4%) in the intervention group (p = 0.04), and the incidence of hypokalemia by 80% from 14 (10%) to 2 (2%, p0.01).Written drug information based on a CDSS considerably decreased DDIs and DDI-related adverse events in routine practice.

Over the past 4 decades, the surgical techniques of liver transplantation (LTx) have permanently evolved and been modified. Among these, the modified piggyback (MPB) technique by Belghiti offers specific advantages. The objective of this study was to present our single-center experience with the MPB technique in 500 cases. Recipients' perioperative data were prospectively collected and evaluated. Postoperative and specific complications, stay in the intensive and intermediate care unit, and the mortality rate with cause of death were analyzed. Most recipients were classified as Child C (49.1%). For the patients who underwent LTx for the first time, alcoholic (23.9%) and viral (22.2%) cirrhosis and hepatocellular carcinoma (15.1%) were the prevalent indications. The overall median warm ischemia time, anastomosis duration, and operative time were 45, 108, and 320 minutes, respectively. The median intraoperative blood loss was 1500 mL. A venovenous bypass was never needed to maintain hemodynamic stability. Only in a few cases was temporary inferior vena cava clamping necessary. Most prominent surgical complications were hemorrhage, hematoma, and wound dehiscence. Renal failure occurred in 6.2% of patients. The overall median stay in the intensive and intermediate care unit was 14 days. The mortality rates within 30 and 90 days were 6.3% and 13.3%, respectively. No technique-related death occurred. The MPB technique by Belghiti is a feasible and simple LTx technique. The caval flow is preserved during the anhepatic phase, and this minimizes the need for venovenous bypass or portocaval shunt. This technique requires only 1 caval anastomosis, which is easy to perform with a short anhepatic phase. To minimize the risk of outflow obstruction, attention should be paid by doing a wide cavocavostomy cranially to the donor inferior vena cava in a door-lock manner. This technique can be applied in almost all patients undergoing LTx for the first time and liver retransplantation as well.