Beate Tiran

Multicentric Validation of Proteomic Biomarkers in Urine Specific for Diabetic Nephropathy

Background Urine proteome analysis is rapidly emerging as a tool for diagnosis and prognosis in disease states. For diagnosis of diabetic nephropathy (DN), urinary proteome analysis was successfully applied in a pilot study. The validity of the previously established proteomic biomarkers with respect to the diagnostic and prognostic potential was assessed on a separate set of patients recruited at three different European centers. In this case-control study of 148 Caucasian patients with diabetes mellitus type 2 and duration ≥5 years, cases of DN were defined as albuminuria >300 mg/d and diabetic retinopathy (n = 66). Controls were matched for gender and diabetes duration (n = 82). Methodology/Principal Findings Proteome analysis was performed blinded using high-resolution capillary electrophoresis coupled with mass spectrometry (CE-MS). Data were evaluated employing the previously developed model for DN. Upon unblinding, the model for DN showed 93.8% sensitivity and 91.4% specificity, with an AUC of 0.948 (95% CI 0.898-0.978). Of 65 previously identified peptides, 60 were significantly different between cases and controls of this study. In <10% of cases and controls classification by proteome analysis not entirely resulted in the expected clinical outcome. Analysis of patients subsequent clinical course revealed later progression to DN in some of the false positive classified DN control patients. Conclusions These data provide the first independent confirmation that profiling of the urinary proteome by CE-MS can adequately identify subjects with DN, supporting the generalizability of this approach. The data further establish urinary collagen fragments as biomarkers for diabetes-induced renal damage that may serve as earlier and more specific biomarkers than the currently used urinary albumin.

Age Dependency of Selenium and Cadmium Content in Human Liver, Kidney, and Thyroid

Selenium and cadmium concentrations were investigated in 60 autopsy tissue samples obtained from fetal life up to adulthood (defined in this study as 25-87 y of age) in Styria, a moderately industrialized region in Austria that has a low selenium supply. During the first 2 y after birth, median liver selenium concentrations were slightly lower (i.e., 1.5 nmol/g wet weight) than concentrations found in fetal life (i.e., 2.9 nmol/g) and adulthood (2.1 nmol/g). Whereas in the fetal period median selenium content in the kidney cortex (2.1 nmol/g) and the thyroid gland (1.6 nmol/g) was lower than that found in the liver, the reverse was true for adults (i.e., kidney, 5.5 nmol/g; thyroid, 4.3 nmol/g). Tissue cadmium concentrations approached 0 during gestation. Accumulation in the kidney and liver commenced immediately after birth. In the thyroid gland of adults, significantly higher concentrations of cadmium were found. Median concentrations in adults showed no statistical significant age dependency (i.e., liver, 7.6 nmol/g; kidney, 59.8 nmol/g; thyroid, 11.2 nmol/g). In summary, the data revealed very low tissue selenium concentrations and low cadmium burdens for the Styrian population that was not exposed occupationally.

Pathological events in experimental acute pancreatitis prevented by the bradykinin antagonist, Hoe 140

1 In a previous investigation, Hoe 140, a specific and potent bradykinin B2 receptor antagonist, prevented the pancreatic oedema and the hypotension observed during acute experimental pancreatitis; however, it augmented the associated rises in the serum activities of pancreatic enzymes. Therefore, we have now investigated the consequences of the pancreatic oedema for the fate of activated enzymes released into the tissue during the course of acute pancreatitis. 2 Acute oedematous pancreatitis was induced in rats, pretreated with captopril (50 μmol kg−1, i.p.), by hyperstimulation of the exocrine function of the pancreas with the cholecystokinin analogue, caerulein (4 nmol kg−1 h−1, i.v.), for up to 120 min. 3 Pancreatic oedema began to develop 10 min after the start of the caerulein infusion, reached a maximum within about 45 min, and then declined slightly. The development of the oedema parallelled the second phase of the caerulein‐induced fall in blood pressure found in earlier experiments. No further extravasation of plasma proteins occurred during the 2nd hour of the caerulein infusion. The oedema formation was completely blocked in animals pretreated with the bradykinin receptor antagonist, Hoe 140 (100 nmol kg−1, s.c.). Pretreatment with aprotinin or soy bean trypsin inhibitor did not result in a significant inhibition of the oedema. 4 The haematocrit of animals with experimental pancreatitis showed a pronounced increase which started 10 min after the start of the caerulein infusion and reached maximal values at 60 min. The changes in haematocrit showed a reduction in total blood volume of 28% due to a 48% loss of plasma. This effect was completely blocked by Hoe 140. 5 In rats with caerulein‐induced pancreatitis, there was a time‐dependent increase in the activities of amylase and lipase in blood serum as well as in the pancreas. Pretreatment with Hoe 140 greatly augmented the caerulein‐induced rise in enzyme activities in blood serum but potently attenuated it in the pancreas. The activities of trypsin in both the blood serum and the pancreas were below or near the limit of detection in all experimental groups. 6 It is concluded that the second phase of hypotension in this model of acute pancreatitis is due to the liberation of kinins which cause a massive loss of blood plasma into the pancreas and into the retroperitoneal space. Activated enzymes are trapped in the pancreas, at least in part, by the oedema of the gland. Treatment with Hoe 140 prevents the oedema formation and greatly facilitates the egress of activated enzymes from the pancreas.

Aspirin Inhibits Chlamydia pneumoniae–Induced Nuclear Factor-κB Activation, Cytokine Expression, and Bacterial Development in Human Endothelial Cells

Objective—Chlamydia pneumoniae has been associated with atherosclerosis. Infection of vascular endothelial cells with C pneumoniae increases the expression of proatherogenic cytokines mediated by nuclear factor (NF)-&kgr;B, a transcription factor. The present study was designed to test the effect of aspirin on C pneumoniae–induced NF-&kgr;B activation, interleukin expression, and bacterial development in cultured human endothelial cells. Methods and Results—Aspirin, its metabolite salicylic acid, and 2 other unrelated NF-&kgr;B inhibitors showed a strong concentration-dependent inhibitory effect on chlamydial growth, indicated by the reduction of bacterial inclusions and the titer of infectious progeny. Involvement of the transcription factor NF-&kgr;B was confirmed by electrophoretic mobility shift assay and by transfection experiments with appropriate decoy oligodeoxynucleotides. Attenuation of the C pneumoniae–induced activation of NF-&kgr;B by aspirin also reduced the secretion of interleukin-6 and interleukin-8, indicating efficient inhibition of NF-&kgr;B gene expression. Reduction of chlamydial growth was not caused by apoptosis of the host cell, as determined by monitoring characteristic chromatin condensation. Conclusions—These data provide evidence that NF-&kgr;B–mediated gene activation represents a crucial step in the developmental cycle of C pneumoniae. Aspirin exerts an anti-chlamydial effect that is due to the inhibition of C pneumoniae–induced NF-&kgr;B activation, which might account for some of the cardioprotective activity of aspirin.

Analysing cell-free plasma DNA and SLE disease activity.

Eur J Clin Invest 2011; 41 (6): 579–583

EFFECTS OF PANCREATIC ENZYME PREPARATIONS ON ERYTHROCYTE GLUTATHIONE PEROXIDASE ACTIVITIES AND PLASMA SELENIUM CONCENTRATIONS IN CYSTIC FIBROSIS

To substitute for exocrine pancreatic insufficiency, patients with cystic fibrosis (CF) take pancreatic enzymes (PE) originating from porcine pancreas. Five different pancreatic enzyme preparations used by our patients contained 0.5-1.4 microg selenium per g tablet. In patients taking PE in doses that were gradually increased to improve fat absorption during a 48-month period, the effects of PE dose on erythrocyte selenium-dependent glutathione peroxidase (SeGSH-Px) activities and plasma selenium concentrations were studied. At baseline, erythrocyte SeGSH-Px activities were significantly lower in patients (p=.01), while plasma selenium concentrations did not differ between patients and healthy subjects. When PE dose and, consequently, selenium intake from PE was increased, erythrocyte SeGSH-Px activities (p < .001) and plasma selenium concentrations (p=.02) increased. Changes in SeGSH-Px activities during the initial 8 months correlated with those in selenium intake from PE (r=0.67, p < .001). Plasma selenium concentrations plateaued at 12 months and erythrocyte SeGSH-Px activities did so at 36 months, when patients had reached SeGSH-Px activities similar to those of healthy subjects. At 48 months, patients took an average lipase dose of 17400 U x kg(-1) x d(-1) and selenium dose from PE of 0.53 microg x kg(-1) x d(-1). We conclude that selenium content of PE preparations has a significant effect on SeGSH-Px activity in patients with CF. This form of selenium supply needs to be taken into account when selenium supplements are given to patients with CF.

Involvement of tissue kallikrein but not plasma kallikrein in the development of symptoms mediated by endogenous kinins in acute pancreatitis in rats

In order to investigate the mechanism of kinin release leading to vascular symptoms in acute interstitial‐oedematous pancreatitis, the novel, selective inhibitors of tissue kallikrein, (2S,2′R)‐2‐(2′‐amino‐3′‐(4′‐chlorophenyl)propanoylamino)‐N‐(3‐guanidinopropyl)‐3‐(1‐naphthyl)propanoamide (FE999024, CH‐2856), and of plasma kallikrein, (2′S,2′′R)‐4‐(2′‐(2′′(carboxymethylamino)‐3′′‐cyclohexyl‐propanoylamino)‐3′‐phenyl‐propanoylamino)piperidine‐1‐carboxamidin (FE999026, CH‐4215), were used in experimental caerulein‐induced pancreatitis in rats. Oedema formation and plasma protein extravasation during the 2 h infusion of caerulein were inhibited in a dose‐dependent manner by i.p. pretreatment with FE999024 (7–60 μmol kg−1) while FE999026 had no effect at the same doses. Haemoconcentration and hypovolaemia associated with the pancreatic oedema formation during pancreatitis were significantly attenuated by FE999024 at a dose of 20 μmol kg−1. The reduction in circulating plasma volume was not affected by FE999026. Accumulation of amylase and lipase in the pancreas was dose‐dependently reduced by FE999024 while enzyme activities in the blood serum were increased by FE999024 at 60 μmol kg−1 indicating improved enzyme removal from the tissue. Enzyme activities in the tissue and in the blood remained unaffected by FE999026. FE999024 (20 μmol kg−1) largely inhibited increased tissue kallikrein‐like activity in the pancreas during acute pancreatitis and also strongly attenuated influx of plasma kallikrein into the tissue. FE999026 (20 μmol kg−1) significantly inhibited plasma kallikrein‐like activity in the pancreas but had no effect on tissue kallikrein‐like activity. In conclusion, vascular kinin‐mediated symptoms observed during oedematous pancreatitis in the rat are caused by the action of tissue kallikrein in the pancreas whereas an involvement of plasma kallikrein seems to be unlikely.

Effects of vitamin E depletion/repletion on biomarkers of oxidative stress in healthy aging.

Abstract: The effects on ex vivo LDL resistance to oxidation and biomarkers of in vivo oxidative stress in response to 3‐month dietary vitamin E restriction to 25% of recommended intake and 2‐month unrestricted dietary intake and supplementation with 800 IU/d were studied in 100 healthy, nonsmoking 20‐75‐year‐old volunteers. Significant changes in vitamin E status were associated with decreases and increases, respectively, in LDL resistance to oxidation in the depletion and supplementation period and with decreases in lipid peroxidation and oxidative DNA modification in the supplementation period. Healthy aging was not associated with enhanced susceptibility to oxidation in the depletion period.

Hypoglycaemic coma due to falsely high point-of-care glucose measurements in an ICU-patient with peritoneal dialysis: a critical incidence report

Sir: Hypoglycaemia is a frequent complication in the establishment of tight glycaemic control by means of intensified insulin therapy [1]. Patients in intensive care are often not capable to communicate the presence of hypoglycaemia-associated adverse events and, thus, the reliability and accuracy of glucose measurements are especially important in this environment [2]. Therefore, every attempt should be taken to ensure that point-of-care glucose testing is reliable and technically unavoidable limitations of devices in certain patient groups are well communicated between industry and ICU personnel. In this context, we report a critical incidence due to falsely high pointof-care glucose measurements in a 68-year-old man with a history of diabetes and continuous ambulatory peritoneal dialysis, who was hospitalised for sepsis. In the morning of the first day the patient was increasingly disorientated and dyspnoeic on the ward. Results of an arterial blood gas analysis (Cobas B221, Roche Diagnostics, glucose oxidase method) showed a glucose value of 183 mg/dl. Peritoneal dialysate was exchanged but due to further deterioration of his condition he was admitted to the ICU in the afternoon. At the ICU a capillary glucose value of 496 mg/dl was attained at 19:00 using a bedside glucose meter [Accu-Chek Go, Roche Diagnostics using GDH-PQQ (glucose dehydrogenase pyrroloquinolinequinone test method)]. Despite aggressive insulin administration (*70 IU over 8 h) no accordant decline of glucose was observed (Fig. 1a). At 2:30 in the morning, the patient developed tachyarrhythmia, but this seemed not to disturb his sleep. The nurse in charge suspected hypokalemia following high-dose insulin infusion to be causal for the formation of arrhythmias, she performed an analysis for electrolytes (Cobas B221, Roche Diagnostics). In fact hypokalemia was present (3.2 mmol/l), but more surprisingly a routinely included blood glucose measurement revealed a value of 38 mg/dl, which was in major disagreement to the capillary measurement performed just before (393 mg/dl). At that time the patient was not asleep, but in a hypoglycaemic coma and regained consciousness promptly after glucose administration. On the next day blood samples of the patient were analysed for glucose with commonly used glucose-meters (Fig. 1b). The falsely high glucose values measured with certain devices

Anti-inflammatory actions of perfluorooctanoic acid and peroxisome proliferator-activated receptors (PPAR) α and γ in experimental acute pancreatitis

Perfluorooctanoic acid (PFOA) and agonists of peroxisome proliferator-activated receptors (PPAR) alpha and gamma were investigated for potential anti-inflammatory effects in cerulein-induced acute pancreatitis in rats. PFOA significantly reduced both leukocyte accumulation and prostanoid synthesis. The PPAR-alpha agonist clofibrate had no effect on leukocyte activation but significantly inhibited prostanoid synthesis whereas the PPAR-gamma agonist rosiglitazone significantly reduced leukocyte activation but did not affect synthesis of prostaglandins in the pancreas. Neither PFOA, nor clofibrate or rosiglitazone had an effect on the formation of the inflammatory edema or elevated levels of lipase activity in the blood serum. In summary, PFOA attenuates the accumulation of activated leukocytes and reduces the synthesis of prostanoids in the pancreas during cerulein-induced acute pancreatitis. An activation of PPAR-alpha causes inhibition of prostanoid synthesis while activation of PPAR-gamma inhibits leukocyte activation.