Thomas Schaffner

Calpain-mediated cleavage of Atg5 switches autophagy to apoptosis.

Autophagy-related gene (Atg) 5 is a gene product required for the formation of autophagosomes. Here, we report that Atg5, in addition to the promotion of autophagy, enhances susceptibility towards apoptotic stimuli. Enforced expression of Atg5-sensitized tumour cells to anticancer drug treatment both in vitro and in vivo. In contrast, silencing the Atg5 gene with short interfering RNA (siRNA) resulted in partial resistance to chemotherapy. Apoptosis was associated with calpain-mediated Atg5 cleavage, resulting in an amino-terminal cleavage product with a relative molecular mass of 24,000 (Mr 24K). Atg5 cleavage was observed independent of the cell type and the apoptotic stimulus, suggesting that calpain activation and Atg5 cleavage are general phenomena in apoptotic cells. Truncated Atg5 translocated from the cytosol to mitochondria, associated with the anti-apoptotic molecule Bcl-xL and triggered cytochrome c release and caspase activation. Taken together, calpain-mediated Atg5 cleavage provokes apoptotic cell death, therefore, represents a molecular link between autophagy and apoptosis — a finding with potential importance for clinical anticancer therapies.

Differential Expression of Chemokines in Normal Pancreas and in Chronic Pancreatitis

BACKGROUND & AIMS Cellular infiltrates are present already in early stages of chronic pancreatitis. The mechanisms responsible for their recruitment are unknown. Hence, we determined the differential expression of chemokine genes and their cellular sources in normal and affected pancreatic tissues. METHODS Pancreatic tissues from 23 patients with chronic pancreatitis and from 4 normal controls were subjected to in situ hybridization for detecting messenger RNA (mRNA) of the chemokine genes interleukin 8, ENA-78, MIG, MCP-1, and I-309. RESULTS Normal pancreatic tissues lack cells expressing mRNA for IL-8, ENA-78, MIG, and MCP-1. In contrast, pancreatic lobuli with mild to moderate signs of tissue alterations strongly expressed MCP-1 mRNA in centroacinar ducts, endothelia, fibroblasts, macrophages, T cells, and occasionally in nerves. Interleukin 8 and ENA-78 mRNA is preferentially detected in centroacinar ducts of pancreatic lobuli with more advanced alterations. Variable numbers of pancreas-infiltrating T cells express MIG mRNA. I-309 mRNA, however, is consistently observed in normal acini and in tissue with mild to moderate signs of tissue alterations. CONCLUSIONS The observed differential expression of distinct chemokine genes in pancreatic parenchyma and infiltrates from patients with chronic pancreatitis strongly suggests an involvement of distinct chemokines in the initiation and perpetuation of disease.

The bursa of fabricius: A central organ providing for contact between the lymphoid system and intestinal content

Abstract Small inert particles, after oral or intracloacal administration to chickens, are readily taken up and deposited throughout the medullary portion of bursal follicles. Epithelial tuft cells transporting this material are distinctively equipped with lytic enzymes. No entry of living bacteria into the lymphoid tissue was observed. These findings are indicative of an antigen-driven development of the bursal system protected by local bactericidal mechanisms.

Early and late outcome of operated and non-operated acute dissection of the descending aorta.

OBJECTIVE At present debate continues concerning the optimal mode of treatment for type B dissections. Controversies are mainly due to discordant results regarding survival following medical or surgical treatment. We assessed early and long-term outcome of acute dissection of the descending aorta treated by emergency aortic replacement, medical treatment or delayed surgery. METHODS Between 1980 and 1995, 225 patients were hospitalized in the medical or surgical department of our institution with the diagnosis of acute type B aortic dissection. A total of 38 patients (16.8%) underwent replacement of the descending aorta within the first week after hospital admission. Primary indications for immediate surgery were: rupturing aneurysm (n = 15), diameter of the descending aorta (n = 13), malperfusion of the thoracoabdominal aorta (n = 8) and pseudocoarctation syndrome with uncontrollable hypertension (n = 2). All other patients (n = 187) underwent primary conservative treatment on the intensive care unit, including appropriate anti-hypertensive medication. In 12 of them, surgery was denied because of age or significant concomitant diseases. RESULTS Hospital mortality after urgent or emergency surgery was 21% (8/38 patients) for the overall time period. There has been a significant decrease in hospital mortality during the last 5 year-period (12% versus 30% between 1980 and 1994). Causes of death were: cardiac failure in 3, bleeding complications in 2, postoperative mesenteric ischemia in 2 and septicemia in one patient. From the 30 operative survivors, 9 (30%) patients required further surgery on the native aorta after a mean follow-up of 48 +/- 13 months. Hospital mortality during conservative treatment was 17.6% (33/187 patients). Main causes of death were rupture in 14, thoraco-abdominal malperfusion in 13 and cardiac failure in 3 patients, whereas in 3 patients, the cause of death could not be evaluated. In this group, 9 patients had to be shifted to early surgery during the initial hospitalization because of impending rupture (n = 4), rapidly increasing diameter (n = 2) and suspicion of intestinal ischemia (n = 3). After hospital discharge, surgery for chronic dissection was performed in 47 patients, mainly because of expanding descending aortic aneurysm. Hospital mortality was 8% (4/47 patients). Actuarial survival rates after surgery during the first admission were 85 +/- 6% at 5 years and 61 +/- 8% at 10 years, versus 76 +/- 5 and 50 +/- 7% respectively, following conservative treatment (P < 0.001). CONCLUSION Nowadays, acute type B dissection can be treated surgically with a reasonable perioperative risk. Despite aggressive anti-hypertensive treatment, hospital mortality of primary conservative treatment is still high and a substantial percentage of patients requires surgery during initial hospitalization. Main causes of death in both groups are rupture and abdominal malperfusion: therefore, closed clinical and radiologic assessment of the whole thoraco-abdominal aorta is of utmost importance. Long-term results are satisfying; unlimited radiographic follow-up allows for detection of potential severe complications and for proper planning of elective reoperations when indicated.

Sodium thiosulfate prevents vascular calcifications in uremic rats

Accelerated vascular calcification is a severe complication of chronic kidney disease contributing to high morbidity and mortality in patients undergoing renal replacement therapy. Sodium thiosulfate is increasingly used for the treatment of soft tissue calcifications in calciphylaxis. Therefore, we determined whether it also prevents development of vascular calcifications in chronic kidney disease. We found that uremic rats treated by thiosulfate had no histological evidence of calcification in the aortic wall whereas almost three-fourths of untreated uremic rats showed aortic calcification. Urinary calcium excretion was elevated and the calcium content of aortic, heart, and renal tissue was significantly reduced in the thiosulfate-treated compared to non-treated animals. Sodium thiosulfate treatment transiently lowered plasma ionized calcium and induced metabolic acidosis. It also lowered bone strength in the treated animals compared to their normal controls. Hence, sodium thiosulfate prevented vascular calcifications in uremic rats, likely by enhancing acid- and/or chelation-induced urinary calcium loss. The negative impact on rat bone integrity necessitates a careful risk-benefit analysis before sodium thiosulfate can be used in individual human patients.

In vitro model for the study of necrosis and apoptosis in native cartilage.

Apoptosis plays a role in everything from early development to ageing and in a host of disease states. Studying this important process in the in vivo state is critical, to understand its varied role and to open further avenues of therapeutic intervention. The present paper presents an ex vivo bovine articular cartilage model to study apoptotic and necrotic processes following acute injury. Ex vivo bovine articular cartilage was assessed 1, 3 and 6 days following holmium : YAG laser treatment (780 mJ). Markers to visualize cell viability, caspase‐3 activity, changes in mitochondrial membrane potential and the degree of DNA fragmentation (TUNEL assay) were used alone or in various combinations. Standard histology and transmission electron microscopy (TEM) were also performed for a more comprehensive assessment. A significant progression (p < 0.05) of ethidium/caspase‐3‐positive signal depth at day 3 preceded a significant increase (p < 0.05) in TUNEL signal depth by day 6. The mitochondrial matrix marker CMXRos was shown to provide an alternative to calcein‐AM for assessing cell viability. The identification of chondrocyte apoptosis morphology by TEM was not conclusive. Nevertheless, TEM revealed that cells which were clearly necrotic also stained positively for TUNEL, thus indicating the risk of using TUNEL alone for the assessment of apoptosis. The model described here allows the rapid, spatial and temporal determination of cell viability and of apoptotic and necrotic processes in whole‐tissue specimens after acute injury, and permits study of the balance between these events. The assessment of healthy and diseased cartilage and of the effects of surgical, pharmaceutical or in vitro intervention are immediate applications of these protocols. Moreover, this model may be useful for the study of key mechanisms involved in apoptosis or for the establishment of other markers of apoptosis. Copyright

Loss of the CBX7 protein expression correlates with a more aggressive phenotype in pancreatic cancer.

Polycomb group (PcG) proteins function as multiprotein complexes and are part of a gene regulatory mechanism that determines cell fate during normal and pathogenic development. Several studies have implicated the deregulation of different PcG proteins in neoplastic progression. Pancreatic ductal adenocarcinoma is an aggressive neoplasm that follows a multistep model of progression through precursor lesions called pancreatic intraepithelial neoplasia (PanIN). Aim of this study was to investigate the role of PcG protein CBX7 in pancreatic carcinogenesis and to evaluate its possible diagnostic and prognostic significance. We analysed by immunohistochemistry the expression of CBX7 in 210 ductal pancreatic adenocarcinomas from resection specimens, combined on a tissue microarray (TMA) including additional 40 PanIN cases and 40 normal controls. The results were evaluated by using receiver operating characteristic (ROC) curve analysis for the selection of cut-off scores and correlated to the clinicopathological parameters of the tumours and the outcome of the patients. Expression of E-cadherin, a protein positively regulated by CBX7, was also assessed. A significantly differential, and progressively decreasing CBX7 protein expression was found between normal pancreatic tissue, PanINs and invasive ductal adenocarcinoma. Loss of CBX7 expression was associated with increasing malignancy grade in pancreatic adenocarcinoma, whereas the maintenance of CBX7 expression showed a trend toward a longer survival. Moreover, loss of E-cadherin expression was associated with loss of CBX7 and with a trend towards worse patient survival. These results suggest that CBX7 plays a role in pancreatic carcinogenesis and that its loss of expression correlates to a more aggressive phenotype.

Clinical significance of cell cycle- and apoptosis-related markers in biliary tract cancer: a tissue microarray-based approach revealing a distinctive immunophenotype for intrahepatic and extrahepatic cholangiocarcinomas.

Cholangiocarcinoma is the second most common malignant tumor of the liver. We analyzed, immunohistochemically, the significance of cell cycle- and apoptosis-related markers in 128 cholangiocarcinomas (42 intrahepatic, 70 extrahepatic, and 16 gallbladder carcinomas) combined in a tissue microarray. Follow-up was available for 57 patients (44.5%). In comparison with normal tissue (29 specimens), cholangiocarcinomas expressed significantly more frequently p53, bcl-2, bax, and COX-2 (P.05 <). Intrahepatic tumors were significantly more frequently bcl-2+ and p16+, whereas extrahepatic tumors were more often p53+ (P < .05). Loss of p16 expression was associated with reduced survival of patients. Our data show that p53, bcl-2, bax, and COX-2 have an important role in the pathogenesis of cholangiocarcinomas. The differential expression of p16, bcl-2, and p53 between intrahepatic and extrahepatic tumors demonstrates that there are location-related differences in the phenotype and the genetic profiles of these tumors. Moreover, p16 was identified as an important prognostic marker in cholangiocarcinomas.

Low molecular weight dextran sulfate prevents complement activation and delays hyperacute rejection in pig-to-human xenotransplantation models.

Abstract: Dextran sulfate of 5000 molecular weight (DXS 5000) is known to block complement activation as well as the intrinsic coagulation cascade by potentiation of C1 inhibitor. The effect of DXS 5000 on hyperacute rejection (HAR) was tested in pig‐to‐human xenotransplantation models. For in vitro testing, a cytotoxicity assay was used with the pig kidney cell line PK15 as target cells and fresh, undiluted human serum as antibody and complement source. Ex vivo pig lung perfusion was chosen to assess DXS 5000 in a physiologic model. Pig lungs were perfused with fresh, citrate‐anticoagulated whole human blood to which 1 or 2 mg/ml DXS 5000 were added; the lungs were ventilated and the blood de‐oxygenated. Pulmonary vascular resistance (PVR) and blood oxygenation (ΔpO2) were monitored throughout the experiment. Autologous pig blood and human blood without DXS 5000 served as controls. In the PK15 assay DXS 5000 led to a complete, dose‐dependent inhibition of human serum cytotoxicity with an average IC50 of 43 ± 18 µg/ml (n = 8). Pig lungs perfused with untreated human blood (n = 2) underwent HAR within 105 ± 64 min, characterized by increased PVR, decrease of ΔpO2, and generalized edema. Microscopically, capillary bleeding as well as deposition of human antibodies, complement and fibrin could be observed. Addition of DXS 5000 (n = 4) prolonged lung survival to 170 ± 14 min for 1 mg/ml and 250 ± 42 min for 2 mg/ml, and PVR values as well as edema formation were comparable to control lungs that were perfused with autologous pig blood (n = 2). Activation of complement (activation products in serum, deposition on lung tissue) and the coagulation system (fibrin monomers) were significantly diminished as compared to human blood without DXS 5000. Binding of anti‐Gal antibodies was not influenced, and in vitro experiments showed no evidence of complement depletion by DXS 5000. In conclusion, DXS 5000 is an efficient complement inhibitor in pig‐to‐human xenotransplantation models and therefore a candidate for complement‐inhibitory/anti‐inflammatory therapy – either alone or in combination with other substances – and warrants further investigation.

Proteomic Analysis in Aortic Media of Patients With Marfan Syndrome Reveals Increased Activity of Calpain 2 in Aortic Aneurysms

Background— Marfan syndrome (MFS) is a heritable disorder of connective tissue, affecting principally skeletal, ocular, and cardiovascular systems. The most life-threatening manifestations are aortic aneurysm and dissection. We investigated changes in the proteome of aortic media in patients with and without MFS to gain insight into molecular mechanisms leading to aortic dilatation. Methods and Results— Aortic samples were collected from 46 patients. Twenty-two patients suffered from MFS, 9 patients had bicuspid aortic valve, and 15 patients without connective tissue disorder served as controls. Aortic media was isolated and its proteome was analyzed in 12 patients with the use of 2-dimensional difference gel electrophoresis and mass spectrometry. We found higher amounts of filamin A C-terminal fragment, calponin 1, vinculin, microfibril-associated glycoprotein 4, and myosin-10 heavy chain in aortic media of MFS aneurysm samples than in controls. Regulation of filamin A C-terminal fragmentation was validated in all patient samples by immunoblotting. Cleavage of filamin A and the calpain substrate spectrin was increased in the MFS and bicuspid aortic valve groups. Extent of cleavage correlated positively with calpain 2 expression and negatively with the expression of its endogenous inhibitor calpastatin. Conclusions— Our observation demonstrates for the first time upregulation of the C-terminal fragment of filamin A in dilated aortic media of MFS and bicuspid aortic valve patients. In addition, our results present evidence that the cleavage of filamin A is highly likely the result of the protease calpain. Increased calpain activity might explain, at least in part, histological alterations in dilated aorta.