Ernst Klar

Acute cholecystitis: early versus delayed cholecystectomy, a multicenter randomized trial (ACDC study, NCT00447304).

Objective:Acute cholecystitis is a common disease, and laparoscopic surgery is the standard of care. Background: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. Methods: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. Results: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; P < 0.001) and total hospital costs (&OV0556;2919 vs &OV0556;4262; P < 0.001) were significantly lower in group ILC. Conclusions: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)

Reduction in Mortality With Delayed Surgical Therapy of Severe Pancreatitis

The indications for surgery in acute pancreatitis have changed significantly in the past two decades. Medical charts of patients with acute pancreatitis treated at our institution were analyzed to assess the effects of changes in surgical treatment on patient outcomes. A total of 136 patients with radiologically defined severe pancreatitis were primarily treated or referred to our institution between 1980 and 1997. Severity of the disease (Ranson score), indications for surgical intervention, timing of surgery, and mortality rates were compared during three study periods: 1980 to 1985 (period I), 1986 to 1990 (period II), and 1991 to 1997 (period III). In period I patients underwent exploratory laparotomy if their clinical status did not improve markedly within 72 hours of admission to the hospital, whereas during period II surgery was reserved for patients who had secondary organ failure together with pancreatic necrosis seen on CT scan. During period III the aim was to operate as late as possible in the presence of pancreatic necrosis or when infected necrosis was suspected. The policy of limiting the indications for surgery resulted in a decrease in surgically treated patients from 68% to 33% (P < 0.001). Likewise, surgical intervention was performed later. In period I, 73% of operations were performed within 72 hours of admission, compared to 32% in period III (P = 0.008). The mortality rate for patients who underwent early surgery (within 72 hours) was higher than for those who underwent late surgical exploration of the abdomen (P = 0.02). Overall, the mortality rate for patients with severe pancreatitis was reduced from 39% to 12% (P = 0.003). Mortality among patients treated nonoperatively did not change significantly. The present study supports the policy of delayed surgery in severe acute pancreatitis. Early surgical intervention often results in unnecessary procedures with an increase in the number of deaths. Whenever possible, prolonged observation allows selection of patients who are likely to benefit from delayed surgery or nonoperative treatment.

Expression of the multidrug resistance proteins MRP2 and MRP3 in human hepatocellular carcinoma.

Treatment of hepatocellular carcinoma (HCC) by chemotherapy is often impeded by the intrinsic multidrug resistance (MDR) of this frequent primary cancer of the liver. The MDR phenotype can be caused by ATP‐dependent export of chemotherapeutic drugs across the plasma membrane being mediated by transporters of the MDR P‐glycoprotein family or of the multidrug resistance protein (MRP) family. To elucidate the role of MRP family members in HCC, we analyzed the expression and subcellular localization of MRP1 (ABCC1), MRP2 (ABCC2) and MRP3 (ABCC3); all 3 isoforms have been shown to confer resistance to chemotherapeutic drugs. Semiquantitative RT‐PCR demonstrated that MRP2 and MRP3 mRNA expression in HCC was at least 10‐fold higher than MRP1 mRNA expression. MRP2 immunostaining was observed in 87% (33/38) of HCC samples. MRP2 was localized in the plasma membrane in a polarized fashion, either in trabecular structures resembling the canalicular membrane or in the luminal membrane when cells had a pseudoglandular arrangement. MRP3 was detected in all samples examined (9/9) by RT‐PCR and by immunofluorescence microscopy. MRP3 was localized to the basolateral membrane of carcinoma cells. Double‐label immunofluorescence microscopy with antibodies specific for MRP2 or MRP3 indicated that carcinoma cells expressed both MRP isoforms simultaneously. When MRP1 was detected by immunofluorescence microscopy, it was localized on the intracellular membranes of carcinoma cells. Thus, plasma membrane expression of MRP2 and MRP3, but not of MRP1, can contribute to the MDR phenotype of HCC.

Postoperative Chemotherapy May Not Be Necessary for Patients With ypN0-Category After Neoadjuvant Chemoradiotherapy of Rectal Cancer

PurposeAfter neoadjuvant radiochemotherapy and surgery, there is no general agreement about whether postoperative chemotherapy is necessary. With the help of clinical and pathohistologic data, prognostic factors were determined as a basis for the decision to spare a patient additional chemotherapy or to urgently recommend it.ResultsNinety-five patients treated with neoadjuvant 5-fluorouracil-based radiochemotherapy (November 4, 1997 and June 15, 2004) without distant metastases and an R0 (microscopically complete) resection were evaluated. Adjuvant chemotherapy (5-fluorouracil or 5-fluorouracil/folinic acid) was given to 65 of 95 patients (68.4 percent). The disease-free survival rate after 36 months was chosen as the target parameter (median follow-up, 36 months).MethodsThe five-year survival rate for all patients was 80.3 ± 5.6 percent; the five-year disease-free survival was 78.1 ± 5.1 percent; the five-year local control rate was 94.2 ± 5.1 percent. In the univariate and multivariate analysis of the disease-free survival, the pathohistologic lymph node status after radiochemotherapy (ypN) was the only significant prognostic parameter. Disease-free survival (36 months) for patients without lymph node metastases (ypN0) was excellent, independent of whether they had received postoperative chemotherapy (n = 43; 87.5 ± 6.0 percent) or not (n = 29; 87.7 ± 6.7 percent). Patients with ypN2 status have, despite chemotherapy, a poor disease-free survival at 30 ± 17.6 percent after 36 months.ConclusionsThese retrospective data suggest that, for some patients, postoperative chemotherapy can be spared. For patients with ypN2 status, an intensification of the postoperative chemotherapy should be considered. Further evaluation in prospective studies is urgently recommended.

Transformation of the microvascular system during multistage tumorigenesis

Simian virus SV40 large T Antigen expression in the islets of Langerhans of transgenic mice results in β‐cell hyperproliferation, onset of new blood vessel formation and the development of highly vascularized solid tumors. Angiogenesis in the RIPTag mouse model, as well as in human cancer, is a hallmark of multistage tumorigenesis and precedes the development of solid tumors. In our study, intravital microscopy was used to monitor changes in the blood vessel phenotype, microcirculation and leukocyte adhesion during the progression from normal islets to angiogenic islets and solid tumors. In RIP1‐Tag5 mice, an aberrant microangioarchitecture becomes apparent in early stages during spontaneous tumor development. Notably, the transition from normal to angiogenic islets is characterized by an increase in vessel diameter rather than vessel numbers. Thus, dilatation of existing vessels precedes vessel sprouting. Once initiated, neovascularization in angiogenic islets results in loss of vessel hierarchy and differentiation. Solid insulinomas display a higher vessel density and even more dramatic vessel heterogeneity as revealed by local “hot spots” of neovascularization and irregular vessel diameters. Strikingly, profound changes in the microangioarchitecture are already observed in early angiogenic islets suggesting that key features of the angiogenic vasculature are established prior to the expansion of tumor mass. Moreover, adhesion of leukocytes was found to be dramatically decreased in both angiogenic islets and solid tumors and correlates with morphological alterations of the vasculature. Thus, vessel transformation and reduced leukocyte‐endothelium interactions are not exclusively features of solid tumors but represent early events during tumorigenesis.

Impact of microcirculatory flow pattern changes on the development of acute edematous and necrotizing pancreatitis in rabbit pancreas

Impairment of pancreatic microcirculation has often been advocated as one pathogenic mechanism in necrotizing pancreatitis. In contrast, data on pancreatic capillary perfusion in edematous pancreatitis are scarce. It was the aim of this experimental study to compare changes in pancreatic microcirculation in edematous and necrotizing pancreatitis. Twelve rabbits were allocated to two groups. Two different models of acute pancreatitis were used. Edematous pancreatitis was elicited by intravenous administration of cerulein (25 µg/kg/hr) (N=6). Necrotizing pancreatitis of the biliary type was induced by pressure-controlled intraductal infusion of a mixture of taurocholate, trypsin, and blood (N=6). Pancreatic microcirculation was quantified by means of intravital microscopy assessing functional capillary density, blood cell velocity, and distribution of the plasma marker FITC-dextran 70. Systemic hemodynamics were maintained at baseline values by fluid administration. Regardless of edema or necrosis, pronounced extravasation of FITC-dextran was recorded in the early stage of pancreatitis. In cerulein-induced pancreatitis, hyperemia developed as indicated by an increase in blood cell velocity in the presence of homogeneous capillary perfusion. In contrast, a progressive reduction of the number of perfused capillaries was detected in necrotizing pancreatitis. In conclusion, pancreatic microvascular perfusion may be regarded as an important pathogenetic factor for the determination of acute pancreatitis.

Intravenous contrast medium aggravates the impairment of pancreatic microcirculation in necrotizing pancreatitis in the rat.

BackgroundPrevious reports demonstrated that radiographic contrast medium, as used in contrast-enhanced computed tomography, increases acinar necrosis and mortality in experimental pancreatitis. The authors studed the possibility that these changes may be related to an additional impairment of pancreatic microcirulation. MethodsFifty Wistar rats had acute pancreatitis induced by intraductal glycodeoxycholic acid (10 mmol/L for 10 min) and intravenous cerulein (5 μg/kg/hr for 6 hrs). After rehydration (16 mL/kg), pancreatic capillary perfusion was quantified by means of intravital microscopy at baseline before intravenous infusion of contrast medium (n = 25) or saline (n = 25), and 30 and 60 minutes thereafter. In addition to total capillary flow, capillaries were categorized as high-or low-flow (> or <1.6nL/min). ResultsPancreatic capillary flow did not change in either high- or low-flow capillaries after saline infusion. However, contrast medium infusion induced a significant decrease of total capillary flow (p < 0.001). Analysis according to the relative flow rate revealed that this was primarity because of a significant additional reduction of perfusion in low-flow capillaries (p < 0.0001). Furthermore, complete capillary stasis was observed in 15.9 ± 3.4% after contrast medium as compared with 3.2 ± 1.2% after saline infusion (p < 0.006). ConclusionRadiographic contrast medium aggravates the impairment of pancreatic microcirculation in experimental necrotizing pancreatitis.

Primary human hepatocytes – a valuable tool for investigation of apoptosis and hepatitis B virus infection

BACKGROUND/AIMS Apoptosis is a key event in the pathophysiology of many liver diseases. Primary human hepatocytes (PHH) provide a useful model to study physiological and pathophysiological processes in the liver. Our aim was to optimize PHH cultures to allow studies on induction of apoptosis and of hepatitis B virus (HBV) infection. METHODS PHH were isolated from human liver tissue by two-step collagenase perfusion. PHH and hepatoma cells were treated with different apoptosis-inducing agents in parallel. PHH cultures were infected with wild type HBV and transduced with HBV genomes using adenoviral vectors. RESULTS PHH were successfully isolated from 40 different tissue samples with high viability and purity. Perfusion time and seeding density turned out to be critical parameters for optimal cell yield and culture conditions, respectively. Serum addition to the medium reduced viability of PHH. PHH allowed reproducible studies of CD95-dependent and -independent apoptosis. Sensitivity towards CD95-mediated apoptosis was markedly higher than in hepatoma cells. PHH could efficiently be infected with HBV, but infection did neither induce apoptosis nor prevent CD95-induced cell death. CONCLUSIONS Our data show that PHH provide an excellent tool for the investigation of apoptosis induced by agents like death receptor-ligands and hepatotropic viruses.

R1 resection in pancreatic cancer has significant impact on long-term outcome in standardized pathology modified for routine use

BACKGROUND The quality of a histopathologic workup after oncologic resection of pancreatic malignancies has changed the central role of surgery substantially for radical tumor clearance over the past years. The development of standardized protocols for pathologic workup increased the rate of R1 resections from around 20% up to 80%. In the present study, we investigated the incidence of R1 and its impact on survival after oncologic pancreatic resections using a standardized pathologic routine protocol. PATIENTS AND METHODS We performed 265 pancreatic resections from September 2003 to September 2010. Among 128 patients with malignant neoplasms, histology revealed ductal pancreatic adenocarcinoma in 97, ampullary cancer in 10, and distal bile duct cancer in 21 patients. Resected specimens were analyzed according to this improved standardized pathology protocol introduced in 2000. Follow-up data on overall and cancer-related survival, presence and site of tumor recurrence, and chemotherapy were obtained from 120 patients. RESULTS Pancreatic resection comprised a pylorus-preserving or classical pancreaticoduodenectomy in 112, a distal pancreatectomy in 8, and a total pancreatectomy in 7 patients. In the overall series, 56 (44%) were classified R1 resections and 68 (43%) R0 resections, 3 patients with R2 resections were excluded, leaving 125 patients for analysis. In pancreatic adenocarcinoma, the rate of R1 was 51% (48/94). R1 resection involved most frequently the circumferential margin in 86% (48/125) of the total group and in 92% (44/48) in pancreatic cancer. Follow-up was performed after a median of 17 months (range, 1-85) postoperatively. Cancer-related death rate in R0 and R1-resected patients was 60% and 83% (P < .02) in all cancers (n = 117) and 66% and 80% in patients with pancreatic adenocarcinoma (n = 88). Median tumor-related survival in R0 and R1 resections was 22 (range, 4-85) vs 14 months (range, 2-48) in all cancers (P < .002), and 19 (range, 4-85) vs 14 months (range, 2-48) in pancreatic adenocarcinoma (P < .04). Kaplan-Meier survival analysis revealed a survival benefit after R0 resection in both all cancers (P = .002) and pancreatic adenocarcinoma (P < .02). The pattern of tumor recurrence had a greater rate of regional metastases in the R1 group (P < .05). CONCLUSION Our 51% rate of R1 resections in ductal pancreatic carcinoma indicates a high quality standard of pathologic evaluation. The vast majority of R1 margins are located at the retroperitoneal dissection surface. Standardization of histopathologic analysis has a clinically relevant impact on survival after oncologic resection of pancreatic cancer and can be achieved by less extensive protocols.

Therapeutic effect of isovolemic hemodilution with dextran 60 on the impairment of pancreatic microcirculation in acute biliary pancreatitis.

Dextran of different molecular weight (Dx 40, Dx 60/70) has often been evaluated as adjunct treatment of experimental acute pancreatitis. A beneficial effect has been documented by a decrease in its lethality. However, the mechanism of action is poorly understood. A specific effect on the pancreatic microcirculation generally has not been documented and differentiation from unspecific improvement of pancreatic blood flow due to volume expansion has been difficult. This investigation was designed to quantify the effect of dextran on the impairment of pancreatic microcirculation during acute biliary pancreatitis by means of intravital microscopy. Dextran 60 (Dx 60, molecular weight 60,000) was chosen in light of the increase in vascular permeability in the early stage of pancreatitis as demonstrated previously in the same model. Isovolemic hemodilution, i.e., exchange of whole blood for Dx 60 was used as a mode of administration to achieve instantaneous onset of therapy without changes in intravascular volume. In the control group a progressive reduction of pancreatic capillary perfusion commenced 30 minutes after induction of acute pancreatitis, resulting in cessation of nutritive tissue perfusion after 3 hours. In the animals subjected to hemodilution, stabilization of the pancreatic microcirculation was accomplished throughout the observation period of 6 hours. Because volume-related effects could be excluded by the protocol and by monitoring central venous pressure and hematocrit, a specific effect of hemodilution with DX 60 on the pancreatic microcirculation is indicated by our results.