Calpain mediates caspase-dependent apoptosis initiated by hydrogen peroxide in pancreatic acinar AR42J cells.

Several studies have shown that oxidative stress induces apoptosis in many cellular systems including pancreatic acinar cells. However, the exact molecular mechanisms leading to apoptosis remain partially understood. This study aimed to investigate the role of the cytosolic cysteine protease calpain in H2O2-induced apoptosis in pancreatic AR42J cells. Apoptosis was evaluated using flow cytometric analysis of sub-G1 DNA populations, electron-microscopic analysis, caspase-3-specific αII-spectrin breakdown, and measuring the proteolytic activities of the initiator caspase-12 and caspase-8, and the executioner caspase-3. H2O2 induced an increase in the calpain proteolytic activity immediately after starting the experiments that tended to return to a nearly normal level after 8 h and could be attributed to m-calpain. Whereas no caspase-12, caspase-8 and caspase-3 activations could be detected within the first 0.5 h, significantly increased proteolytic activities were observed after 8 h compared with the control. At the same time, the cells showed first ultrastructural hallmarks of apoptosis and a decreased viability. In addition, αII-spectrin fragmentation was identified using immunoblotting that could be attributed to both calpain and caspase-3. Calpain inhibition reduced the activities of caspase-12, caspase-8, and caspase-3 leading to a decrease in the number of apoptotic cells. Immunoblotting analyses of caspase-12 and caspase-8 indicate that calpain may be involved in the activation process of both proteases. The results suggest that H2O2-induced apoptosis of AR42J cells requires activation of m-calpain initiating the endoplasmic reticulum stress-induced caspase-12 pathway and a caspase-8-dependent pathway. The findings also suggest that calpain may be involved in the execution phase of apoptosis.

Diagnostic procedures in tularaemia with special focus on molecular and immunological techniques.

Tularaemia is a severe bacterial zoonosis caused by the highly infectious agent Francisella tularensis. It is endemic in countries of the northern hemisphere ranging from North America to Europe, Asia and Japan. Very recently, Francisella-like strains causing disease in humans were described from tropical northern Australia. In the last decade, efforts have been made to develop sensitive and specific immunological and molecular techniques for the laboratory diagnosis of tularaemia and also for the definite identification of members of the species F. tularensis and its four subspecies. Screening for the keyword 'Francisella' a Medline search over the last decade was performed and articles describing diagnostic methods for tularaemia and its causative agent were selected. Besides classical microbiological techniques (cultivation, biochemical profiling, susceptibility testing) several new immunological and molecular approaches to identify F. tularensis have been introduced employing highly specific antibodies and various polymerase chain reaction (PCR)-based methods. Whereas direct antigen detection by enzyme-linked immunosorbent assay (ELISA) or immunofluorescence might allow early presumptive diagnosis of tularaemia, these methods--like all PCR techniques--still await further evaluation. Therefore, diagnosis of tularaemia still relies mainly on the demonstration of specific antibodies in the host. ELISA and immunoblot methods started to replace the standard tube or micro-agglutination assays. However, the diagnostic value of antibody detection in the very early clinical phase of tularaemia is limited. Francisella tularensis is regarded as a 'highest priority' biological agent (category 'A' according to the CDC, Atlanta, GA, USA), thus rapid and reliable diagnosis of tularaemia is required not only for a timely onset of therapy, the handling of outbreak investigations but also for the surveillance of endemic foci. Only very recently, evaluated test kits for serological diagnosis of human tularaemia became available, while the introduction of standardized molecular techniques for detection and typing is still missing.

Calpain activation contributes to oxidative stress-induced pancreatic acinar cell injury.

Oxygen radicals have been implicated as mediators in the pathogenesis of pancreatic acinar cell necrosis. However, the sequence of events between the oxidative insult and cell damage remains unclear. In the current study, we investigated whether the Ca(2+)-regulated cytosolic cysteine protease calpain is activated by oxidative stress and contributes to oxidant-induced acinar cell damage. Isolated rat pancreatic acinar cells were exposed to hydrogen peroxide (H(2)O(2))-generated oxidative stress in the presence or absence of the Ca(2+) chelator 1,2-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester (BAPTA-AM) and different calpain inhibitors including benzyloxycarbonyl-valyl-phenylalanine methyl ester. Calpain activation was studied by fluorescence spectrophotometry and immunoblotting. Cell injury was assessed by lactate dehydrogenase (LDH) release and characterization of the cellular ultrastructure including fluorescence-labeled actin filaments. Exposure of acinar cells to H(2)O(2) provoked a time- and dose-dependent increase in calpain proteolytic activity involving the ubiquitous isoforms mu- and m-calpain. The activation of calpain reflected the time course of developing cytotoxicity as demonstrated by increased LDH release. Inhibition of oxidant-induced calpain activity by BAPTA-AM and various calpain inhibitors provoked a decline in oxidant-induced cell injury. In particular, changes in the actin filament organization characterized by an increase in the basolateral actin and by a detachment of actin from the cell membrane in the region of membrane blebs were clearly reduced. In summary, our findings suggest that acinar cell damage through oxidative stress requires activation of calpain and that the actin cytoskeleton belongs to the cellular targets of the protease. The results support the hypothesis that calpain activation may play a role in the development of acute pancreatitis.

5-Fluorouracil/irinotecan induced lethal toxicity as a result of a combined pharmacogenetic syndrome: report of a case.

Combination cancer chemotherapy induced toxicity can be associated with combined pharmacogenetic syndromes. Dihydropyrimidine dehydrogenase (DPD) is the principal enzyme involved in the catabolic detoxification of 5-fluorouracil (5FU). A heterozygous G > A transition at the 5' splicing donor consensus sequence in intron 14 leading to exon 14 skipping (IVS14+1 G > A, DPYD*2A) with partial loss of enzyme activity may be partly responsible for 5FU induced toxicity, whereas irinotecan associated toxicity may in part be explained by an aberrant UGT1A1 promoter (TA)(n) genotype underlying Gilbert's syndrome with reduced liver glucuronidation activity. This report describes a 44 year old white woman who suffered from severe gastrointestinal and haematological toxicity while undergoing 5FU(24h)/folinic acid/irinotecan treatment for adenocarcinoma of the sigmoid colon. Despite appropriate supportive treatment, her condition rapidly deteriorated and led to death. Molecular analysis revealed a hitherto undescribed combined pharmacogenetic syndrome, consisting of heterozygosity for the DPD IVS14+1 G > A mutation and UGT1A1 (TA)(6/7) heterozygosity, which probably contributed to the fatal outcome in this patient.

Predicting the risk of early atherosclerotic disease development in children after repair of aortic coarctation.

AIMS: Increased cardiovascular morbidity is manifested a long time after the repair of aortic coarctation (CoA). By way of impaired flow-mediated vasodilation (FMD) and increased intima media thickness (IMT), surrogate parameters of atherosclerosis, cardiovascular risk factors (RFs) can be correlated with early vascular wall changes in children. This study investigated whether changes in arterial wall function and morphology are detectable in children after coarctation repair. METHODS AND RESULTS: We examined 28 children after successful repair of CoA vs. 30 control subjects. All children underwent identical screening, with a broad RF profile and FMD/IMT measurements. CoA-children presented significantly (P < 0.001) impaired FMD (4.87 +/- 2.6 vs. 10.2 +/- 3.1%) and higher IMT values (P < 0.001) than the controls (0.48 +/- 0.08 vs. 0.38 +/- 0.05 mm). The blood pressure during rest and exercise and the left ventricular mass were significantly elevated, but no additional RF could be identified in CoA-children. Only a remaining pressure gradient related significantly to FMD. CONCLUSION: This study documents early vascular wall changes in children after successful coarctation repair. Arterial hypertension and a resting pressure gradient are the major contributing factors to early atherosclerotic development and should be primary targets for therapy. Vascular status should be monitored regularly by FMD and IMT.

[The diagnostic validity of non-invasive pancreatic function tests--a meta-analysis]. / Die diagnostische Validität nichtinvasiver Pankreasfunktionstests -- Eine Metaanalyse.

BACKGROUND: The paper discusses the non-invasive (tubeless) pancreatic function tests used to diagnose exocrine pancreatic insufficiency (EI). Studies evaluating the diagnostic validity of these tests are integrated into a meta-analysis, provided that they comply with the following criteria: The sensitivity (Ss) of a test has to be calculated by comparing it with an invasive function test which is accepted as the gold standard of pancreatic function diagnostics. Furthermore, the test must differentiate between slight (sl), moderate (md) and severe (sv) EI. For assessment of the specificity (Sp), the control group should not contain healthy persons but rather patients with other gastrointestinal diseases and a normal pancreatic function. In the statistical evaluation, each study was weighted according to the number of persons included. RESULTS: Tests (n = sum of persons included in all analysed studies): Fecal chymotrypsin: Ss (n = 169) 54 % (sl EI), 53 % (md EI), 89 % (sv EI), Sp (n = 202) 74 %. NBT-PABA test: Ss (n = 394) 49 % (sl EI), 64 % (md EI), 72 % (sv EI), Sp (n = 218) 83 %. Pancreolauryl test: Ss (n = 320) 63 % (sl EI), 76 % (md EI), 94 % (sv EI), Sp (n = 171) 85 %. Fecal elastase-1: Ss (n = 307) 54 % (sl EI), 75 % (md EI), 95 % (sv EI), Sp (n = 347) 79 %. Additional tests discussed but not included in the meta-analysis were fecal fat, (13)C breath tests, amino acid consumption test, serum tests. CONCLUSION: None of the non-invasive pancreatic function tests is sensitive enough to diagnose reliably a slight to moderate exocrine pancreatic insufficiency.

[Clinical long-term results of H.E.L.P.-apheresis]. / Langzeitergebnisse mit der H.E.L.P.-Apherese.

The heparin-induced extracorporeal LDL-precipitation (H.E.L.P.) procedure was approved by the German Health Authorities with the requirement of performing a five year postmarketing surveillance (PMS) study to document safety and efficiency data. A postmarketing surveillance resembles a patient registry including a patient population inhomogeneous to a certain degree with respect to clinical diagnosis and stage of the disease, respectively. Compared to clinical studies a patient registry differs by the length of regular documentation due to the fact that new patients can be recruited at any time during the 5 years of registry documentation. These restrictions must be considered to correctly interpret the PMS data presented. During the study period, a total of 628 patients were recruited at 82 HELP-treatment facilities. The mean pre-treatment LDL value was 276 mg/dl and the mean HDL value 45 mg/dl corresponding to an atherogenic index of 6.1. In addition to the regular HELP treatment, most of the patients were treated by diet and statins (88.5%) or other lipid-lowering drugs. Although 90% of all patients had been treated by maximal conventional lipid-lowering drug treatment, 42.2% of all documented patients had LDL plasma values >250 mg/dl; 19% of all documented patients exerted LDL values of >400 mg/dl and were therefore classified as homozygous FH patients. The mean surveillance time was 2 years. For 243 patients documentation was stopped early due to varying reasons: 19 patients passed away during the postmarketing surveillance; in 7 cases HELP treatment was discontinued because of adverse effects, and 78 patients changed the LDL apheresis treatment device and were therefore lost for the PMS. Lacking patient compliance and refusal of further reimbursement of the treatment costs were reasons in 28 patients, and medical reasons were documented for another 111 patients for early termination of apheresis treatment. From 58 991 documented single HELP-treatment procedures, the treated plasma volume was available as a measure for treatment efficiency. The mean treated plasma volume was 2.8 l; in 83.9% of all documented treatments a minimum of >2.5 l was treated. The mean treatment interval was 9.9 days. Out of 628 patients, 158 complained of angina before regular HELP treatment. At the final examination, the majority of these patients (87%) reported marked improvement, similar to self-reported improvement of dyspnea on exertion. The incidence of cardiac events including death, myocardial infarction, angioplasty and CABG was lower in patients with LDL >250 mg/dl (12.3%) as compared to patients with LDL <250 mg/dl (17%). For all patients, the relative rate of events was 16%. One further aim of the PMS study was the documentation of adverse effects of regular HELP treatment using a questionnaire. During five years of PMS, 2734 (4.6%) adverse effects during 59 121 documented single HELP treatments in 622 patients were recorded. To define adverse effects directly related to the HELP procedure, adverse effects of the concomitant drugs administered require careful consideration. Therefore, the incidence of adverse effects directly attributable to HELP does not exceed 1-2%.

[Current therapy of hypercholesterolemia. How much statin does your patient need?]. / Aktuelle Therapie der Hypercholesterinämie. Wie viel Statin braucht Ihr Patient?

Today, statins play an important role in the treatment of hypercholesterolemia. They have two effects on the metabolism of cholesterol: firstly, they reduce the synthesis of cholesterol and secondly they stimulate the expression of LDL receptors. LDL is reduced via both of the mechanisms. Various studies (the 4S, LIPID and CARE studies) have demonstrated the efficacy of statins in secondary prevention, that is, in patients with hypercholesterolemia and CAD. In the CARE study, for example, the statins reduced the incidence of fatal and non-fatal myocardial infarctions by 24%. A number of studies show that although primary prevention is effective, long-term tolerability is still a matter of controversy. A relatively frequent, dose-dependent side effect is myopathy, which has a reported incidence of 0.1-0.5%. In combination with fibrates, the incidence increases, and cases of rhabdomyolysis, some fatal, have been described. To minimize the side effects of statin treatment, therefore, target levels--which must be derived on the basis of the results of large studies--must be established for the individual patient.

[Possible value of positive PSA levels in malignant and nonmalignant breast diseases]. / Mögliche Bedeutung eines positiven PSA-Nachweises bei Patientinnen mit benignen und malignen Erkrankungen der Brust.

Measurement of serum levels of PSA is widely used as a screening tool for prostate cancer. PSA has been shown to be associated with malignancies of many other organs than prostate, including the female breast. Therefore, PSA is not prostate-specific. PSA serum levels in females increase with excess of androgens. Variable PSA expression was observed in membranes of adipocytes of fat tissue and in the endothelium of small vessels in female and male breast. There is increasing evidence that androgens play a significant role in the development and progression of breast cancer. 5alpha-reductase is an enzyme that is expressed in androgen-dependent tissues, including the female breast, catalyzing the reduction of testosterone to its more bioactive form, dihydrotestosterone, which then transactivates a number of genes. One of these genes encodes for PSA, a favorable prognostic factor in breast cancer. Interactions of PSA and sex hormones in physiological processes and in prostatic and mammary cancer have been reported. The possible influence of PSA on breast cancer growth and progression and even its physiological functions are still under controversial debate. There are some findings which might indicate similarities in the influence of steroid hormones on the development of prostate and breast malignancies, perhaps a unique hormone-dependent molecular pathway for both types of cancer.

Phenol sulphotransferase SULT1A1 polymorphism in prostate cancer: lack of association.

Preliminary evidence suggests that genetic polymorphisms in certain enzymes involved in xenobiotic metabolism and chemical defense could modify a susceptibility to prostate cancer. In the present study, two recently described phenol sulphotransferase SULT1A1 alleles (SULT1A1*1, SULT1A1*2) were investigated using a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) approach. Genotyping was performed on DNA isolated from white blood cells from 134 patients with prostate cancer and 184 healthy control subjects. Both the prostate cancer patients and the controls demonstrated similar frequencies of the variant allele SULT1A1*2 (35.1% vs 39.1%). Homozygosity for the variant allele was slightly less frequent in cancer patients than controls (12.7% vs 17.4%). Our study does not support the hypothesis that the phenol sulphotransferase variant allele SULT1A1*2 with a G/A transition at nucleotide 638 is a risk modifier for prostate cancer in the Caucasian population.

Diagnostic value of urinary alanine aminopeptidase and N-acetyl-beta-D-glucosaminidase in comparison to alpha 1-microglobulin as a marker in evaluating tubular dysfunction in glomerulonephritis patients.

To estimate the diagnostic value of tubular parameters, the urinary alanine aminopeptidase (AAP), N-acetyl-beta-D-glucosaminidase (NAG) and the alpha(1)-microglobulin (a1M) of 150 patients with histologically proven glomerulonephritis (GN) were determined. In addition, the reabsorption rate of the proximal tubule and the fractional excretion of sodium, the free water clearance and the renal function were assessed by inulin and p-aminohippurate (PAH) clearance. Compared to healthy controls, urinary AAP, NAG and a1M were found significantly elevated in GN patients. Morphological tubular changes were confirmed by significant differences in urinary laboratory parameters. In patients with tubular atrophy, the diagnostic sensitivity and specificity were calculated as follows: AAP (0.94/0.35), NAG (0.75/0.59) and a1M (0.73/0.52). In patients showing tubular protein droplets, the values were 0.90/0.17 for AAP, 0.78/0.76 for NAG and 0.84/0.74 for a1M and in patients with interstitial fibrosis, the values were AAP (0.95/0.35), NAG (0.75/0. 46) and a1M (68/0.38). Urinary AAP, NAG and a1M reflect histologically proven tubulus alteration in GN, although in most cases, the renal function is still intact. AAP indicates very early tubular impairment and, in some cases, AAP is elevated although NAG and a1M are still within normal ranges. We suggest that the enzyme activities are useful in the diagnostics of early stages of the disease.

Inositol 1,4,5-trisphosphate formation, cytoplasmic calcium dynamics, and alpha-amylase secretion of pancreatic acini isolated from aged and chronically alcohol-fed rats.

METHODS: Three-month-old female Wistar rats were fed with 20% alcohol in their drinking fluid over 6-17 mo using an interrupted feeding regimen. At different times, pancreatic acini were isolated by mild collagenase digestion. The concentrations of inositol-1,4,5-trisphosphate (1,4,5-IP3) were determined by a specific radioreceptor assay, before and at different times after stimulation with varying concentrations of CCK-8. CCK-induced dynamics of cytoplasmic calcium ([Ca2+]c) was investigated in acinar cells by confocal laser raster microscopy. Acinar alpha-amylase (Aml) secretion was measured as enzyme activity in the medium compared to the total activity in the suspension. RESULTS: In 12-13-mo-old rats, the CCK-stimulated 1,4,5-IP3 formation in acini was found to be decreased compared to young rats (age 4 mo). In rats of the same age fed with ethanol from the age of 3 mo on, 1,4,5-IP3 concentrations in acini were higher and reached values comparable to those in young rats. Correspondingly, the CCK-induced [Ca2+]c dynamics in acini isolated from 9-mo-old rats was impaired compared to that of young rats but normal in aged, chronically alcohol-fed rats. Aml secretion under CCK stimulation, however, which was decreased in aged rats, was additionally impaired after alcohol feeding. CONCLUSION: Chronic alcohol feeding modifies 1,4,5-IP3 formation, the [Ca2+]c dynamics of, and the Aml secretion of rat pancreatic acini in response to CCK stimulation. Obviously, the age-related impairment of 1,4,5-IP3 formation and [Ca2+]c dynamics is improved. In contrast, the decrease in Aml secretion of acini isolated from aged rats is more pronounced after long-term alcohol-feeding.

Severe hypophosphatemia during hematopoietic reconstitution after allogeneic peripheral blood stem cell transplantation.

A patient suffering from acute myeloid leukemia (FAB M5a) received a PBSC allograft from a matched, related donor. On day 13 after transplantation severe hypophosphatemia (0.21 mmol/l) was first noted which persisted irrespective of intravenous phosphate administration, and within 2 days reached concentrations below 0.13 mmol/l. After repeated phosphate substitution serum phosphate returned to 1.40 mmol/l on day 17. Phosphate in urine, and calcium in serum were recorded as unchanged throughout. Clinical signs and symptoms due to severe hypophosphatemia were not observed except for paresthesia in the lower extremities. The precipitous fall in serum phosphate coincided with hematopoietic reconstitution as reflected by a steep rise in leukocyte count from 0.08 x 109/l on day 10 to 5. 94 x 109/l on day 15 after transplantation. Thus, isolated hypophosphatemia was likely the result of excessive cellular phosphate uptake during hematopoietic reconstitution. Electrolyte monitoring after PBSCT should include serum phosphate to identify the hypophosphatemia associated with hematopoietic recovery.

Acute effect of H.E.L.P. treatment on radical scavenging enzyme activities, total glutathione concentrations in granulocytes, and selenium in plasma.

BACKGROUND: It has been suggested that granulocytes are activated on artificial surfaces such as dialyzer membranes or by plasma separation procedures resulting in the generation of free radicals. We reported recently that free radical scavenging enzyme (FRSE) activities of red blood cells obtained from patients undergoing hemodialysis and LDL-apheresis (LA) do not reflect an acute oxidative stress. However, because mature red cells are free of DNA and RNA, enzymes cannot be regulated on the gene level. In contrast, granulocytes are nucleated cells in which genes can be regulated, e. g. by redox sensitive transcription factors activated by extracellular oxidative stress. Therefore, granulocyte FRSE may better reflect acute oxidative stress caused by extracorporeal treatment. MATERIALS AND METHODS: Hyperlipidemic patients (n = 18) with coronary heart disease (CHD) were treated with the Heparin-induced-Extracorporeal-LDL-Precipitation (H.E.L.P.) system. Glutathione peroxidase (GSH-Px), glutathione reductase (GSSG-R), superoxide dismutase (SOD) activities, and total glutathione were determined in granulocytes before and immediately after a single LA treatment. Selenium (Se) concentrations were assessed in plasma. RESULTS: As a result of the H.E.L.P. treatment GSSG-R activity was significantly induced (+ 20%) and the GSH concentration increased (+ 41%) in granulocytes. GSH-Px activity in granulocytes (- 19%) and Se in plasma (- 27%) were significantly reduced whereas SOD activity in granulocytes was not affected by the H.E.L.P. procedure. CONCLUSION: These results show that the defence against oxygen radicals in granulocytes is affected but not severely compromised in patients undergoing regular H.E.L.P-LDL-apheresis treatment, which points to the safety of this system with respect to oxidative stress.