Analytical evaluation of a fully automated immunoassay for faecal calprotectin in a paediatric setting

Introduction: Faecal calprotectin (FC) is a routinely used marker for identifying and monitoring children with inflammatory bowel disease (IBD). This non-invasive test is useful for screening children with gastrointestinal symptoms to avoid unnecessary invasive procedures. In this study, we validated for the first time the performance of a fully automated particle-enhanced turbidimetric immunoassay (PETIA) on the VITROS® 5600 analyzer for measurement of FC in symptomatic children and adolescents. Materials and methods: For performance validation of the PETIA (fCAL® turbo, Bühlmann Laboratories, Switzerland) on the VITROS® 5600 analyzer (Ortho Clinical Diagnostics, USA) limit of quantitation (LoQ), linearity, precision data and calibration curve stability were defined. Additionally, 95 faecal samples were measured using the PETIA, an enzyme-linked immunosorbent assay (ELISA; fCAL®, Bühlmann Laboratories, Switzerland) and a semi-quantitative lateral flow assay (Quantum Blue Reader®, Bühlmann Laboratories, Switzerland) for agreement evaluation. Results: The LoQ for calprotectin using PETIA on the VITROS® 5600 analyzer was 21 μg/g. The linearity range was 20 - 2100 μg/g and the precision study showed a total coefficient of variation (CV) between 2.3% and 8.9%. The calibration curve was stable for 4 weeks. Using the clinical samples quantifiable by PETIA, ELISA and the semi-quantitative lateral flow assay, Passing-Bablok regression analysis and Bland-Altman plots showed good agreement. Conclusions: Due to good performance characteristics and agreement with established methods, the fully automated PETIA on the routine chemistry analyzer VITROS® 5600 is a new analytical option for the rapid determination of FC

Factors associated with metabolically healthy and unhealthy obesity

Aufgrund der stetig wachsenden Zahl, ist Adipositas heutzutage eines der größten Probleme im Gesundheitswesen weltweit. Interessanterweise ist jedoch ein nicht so kleiner Anteil an adipösen Menschen metabolisch gesund ohne Zeichen von Insulinresistenz oder systemischer Inflammation (“Metaflammation”). Obwohl das Wissen über die zugrunde liegenden Mechanismen der Pathogenese von Insulinresistenz konstant wächst, sind die Faktoren, welche zwischen “gesunder” und “ungesunder” Adipositas unterscheiden, noch immer schlecht definiert. Nicht lange her, wurde gezeigt, dass Mastzellen in der Pathogenese von Adipositas eine Rolle spielen. Messung der Genexpression des Mastzell-spezifischen Marker TPSb2 in Fettgewebe von morbiden adipösen Patienten zeigten eine erhöhte Expression im Fettgewebe und diese korrelierte signifikant positiv mit der TNF-, CCL2 (= monocyte chemotactic protein 1; MCP-1), CCL5 (MCP-5) und CD68 Genexpression. Mastzell-defiziente adipöse Mäuse zeigten entsprechend eine rückläufige proinflammatorische M1 Makrophagenmarker-Genexpression im Fettgewebe. Dennoch hatte die Mastzell-Akkumulation im adipösen humanen Fettgewebe keinen systemischen Einfluss auf Insulinresistenz oder inflammatorische Mediatoren. 25-Hydroxycholesterol, produziert durch die Cholesterol 25-Hydroxylase (Ch25H), wird ebenfalls seit Kurzem eine Rolle im Lipidmetabolismus als auch in inflammatorischen Prozessen zugeschrieben. Mäuse, auf hochkalorischer Diät gesetzt, zeigten eine Reduktion von Ch25H mRNA- und Proteinlevels im Lebergewebe. Eine Literaturrecherche von publizierten humanen Datensätzen über Ch25H bestätigte niedrigere Levels in Lebern von adipösen Patienten. Adipöse Mäuse stratifiziert in eine “gesunde, insulinsensitive” und eine “erkrankte, insulinresistente” Gruppe zeigten weiters erhöhte Ch25H mRNA- und Proteinlevels in den Lebern der erkrankten Gruppe. Eine adenovirale, murine Ch25H-Überexpression bestätigte diese Ergebnisse durch eine verbesserte Glukosetoleranz und Insulinsensitvität. Gemessene Ch25H-Levels in Serum von adipösen insulinsensitiven und insulinresistenten Patienten zeigten ebenfalls signifikante unterschiedliche Werte im insulinresistenten Patientenkollektiv. Daher kann Ch25H als möglicher zukünftiger Surrogatmarker im Serum von adipösen Patienten angedacht werden, um gesunde und ungesunde Adipositas zu erkennen.Without any doubt obesity and its growing prevalence in modern societies can be ranked among the most important challenges medicine is currently facing. Counterintuitively though, one in four obese patients is metabolically healthy; they do not show any sign of insulin resistance and are not prone to metaflammation. Although the knowledge about the pathogenesis is constantly growing, factors discriminating between “healthy” and “unhealthy” obesity is still unclear. It is a well-known fact that the development of unhealthy, insulin resistant obesity is highly influenced by inflammation within white adipose tissue (WAT). Next to typical inflammatory cell types, mast cells (MCs) are described to be correlated with insulin resistance. To investigate this issue, relationship between MCs and inflammatory markers was investigated in obese patients compared to nonobese control subjects. The obese patient group showed increased MC-specific TPSb2 expression in WAT and expression levels significantly positively correlated with proinflammatory markers. Appropriately, obese mast cell-deficient mice showed decreased pro-inflammatory M1 macrophage marker gene expressions in WAT. Nevertheless, the accumulation of MCs influenced only the local environment but had no effect on systemic responses. 25-Hydroxycholesterol (25-OHC) produced by cholesterol 25-hydroxylase (Ch25H) is also recently considered to be involved in lipid metabolism as well as in inflammatory processes. Obese mice exhibited significantly lower Ch25H levels in liver tissue. Further, obese “diseased, insulin resistant” mice showed lower Ch25H levels compared to their obese “healthy, insulin sensitive” control group. A Ch25H literature search about published human data showed as well lower levels in liver tissue of obese subjects. In line, Ch25H-overexpression in mice resulted in a healthier, more insulin sensitive phenotype. When measuring Ch25H-serum levels in an obese study population, significant different Ch25H-serum levels were found in obese insulin resistant individuals. Thus, Ch25H-level in human serum samples can used to easily identify unhealthy obesity.Abweichender Titel laut Übersetzung der Verfasserin/des VerfassersArbeit an der Bibliothek noch nicht eingelangt - Daten nicht geprüftMedizinische Universität Wien, Diss., 2019(VLID)367471

Heme oxygenase-1 in hepatocytes drives insulin resistance in mice and men

Adipositas und Diabetes betreffen mehr als 1.5 Milliarden Menschen weltweit. Interessanterweise zeigen Adipositas und Diabetes nicht immer eine Koinzidenz und die entscheidenden molekularen Determinanten zwischen "gesunden" versus "ungesunden" insulinresistenten adipösen Patienten sind noch unerforscht. Dem Enzym Hämoxygenase-1 (HO-1) wird eine große Bedeutung als Schutzfaktor mit zahlreichen anti-inflammatorischen Eigenschaften zugeschrieben. Interessanterweise konnten Studien zeigen, dass die pharmakologisch erzielte systemische HO-1 Aktivierung Adipositas und Diabetes in Mäusen und Ratten bessern kann. Im Gegensatz dazu zeigte eine rezente Studie über Leber-Irs1/Irs2 Doppelknockout-Mäuse, dass erhöhte HO-1 Spiegel sich nachteilig auf die Funktion der Leberzellen auswirken. Insgesamt gesehen sind die gegenwärtig verfügbaren in vivo Daten also widersprüchlich. Überraschenderweise fand sich in unseren gesammelten Leberbiopsien von adipösen "gesunden" und "ungesunden" Patienten eine positive Korrelation zwischen HO-1 und Insulinresistenz. Dies bestätigte sich im parallelen Mausexperiment. Adipöse insulinresistente (ob-IR) Mäuse zeigten eine signifikante HO-1 Hochregulierung verglichen mit den adipösen insulinsensitiven (ob-IS) Mäusen. Um die metabolische Rolle von Hepatozyten nun genauer zu erforschen, wurde hierfür eine Hepatozyten-spezifische Knockoutmaus ("Lhoko") generiert. Unter normaler Diät zeigten die Lhoko Mäuse keine Veränderungen in der Lebermorphologie noch in Blutparametern während metabolischen Tests. Nach 16-wöchiger kalorienreicher Ernährung fanden sich keine relevanten Unterschiede im Körpergewicht noch Unterschiede in den Glukosewerten während des oralen Glukosetoleranztests. Interessanterweise waren die Lhoko Mäuse jedoch insulinsensitiver - sie hatten erniedrigte Nüchtern-Insulinspiegel, niedrigere Glukosespiegel im Insulintoleranztest und niedrigere berechnete HOMA-IR bei gleichzeitigem Hinweis auf weniger Leberschädigung. Die Applikation von Insulin direkt in die Portalvene führte bei den adipösen Lhoko Mäusen zu einem induzierten Insulinsignal. Weiters führte der Knockout von HO-1 in den Hepatozyten zu einer erhöhter mitochondrialer Atmung mit einer erhöhten Reservekapazität. Dies war gekoppelt mit erhöhten Wasserstoffperoxid (H2O2) Werten, mit einer Hochregulierung des mitochondrialen ROS Detoxifikationssystems und einer Erhöhung der PTP1B Aktivität. Das gegensätzliche Experiment - der akuten Hochregulierung von HO-1 via Adenovirus in C57BL/6J Mäusen unter normaler Diät - führte zu einer getriggerten Glukosetoleranz und Insulinresistenz sowie zu erhöhten HOMA-IR. Mit dieser Studie konnten wir zeigen, dass der Knockout von HO-1 in Hepatozyten einer Diät induzierten Insulinresistenz entgegenwirkt und dadurch auftretene Begleiterkrankungen wie Steatosis und Leberschäden verhindern werden können. Dies zeigt, dass HO-1 die Entstehung von Insulinresistenz, Leberschäden und weiteren metabolischen Erkrankungen fördert. Somit könnte HO-1 eine neue Targettherapie für metabolische Erkrankungen darstellen.Obesity and diabetes affect more than half a billion individuals worldwide. Interestingly, the two conditions do not always coincide and the molecular determinants of 'healthy' versus 'unhealthy' insulin resistant obesity remain ill-defined. In vivo studies of various designs demonstrate that systemic activation of the heme oxygenase-1 (Abraham and Kappas) by chemical means ameliorates obesity and diabetes. On the contrary, other data suggest that increased HO-1 levels are detrimental for liver function in metabolic settings important for obesity and diabetes development. Surprisingly, in matched liver biopsies from obese "healthy" versus "unhealthy" patients we found that HO-1 correlated positively with insulin resistance. In agreement with the human study, comparison of obese insulin resistant (ob-IR) and obese insulin sensitive (ob-IS) mice revealed significantly higher HO-1 mRNA levels in the ob-IR subgroup. To directly assess the role of HO-1 in hepatocytes, we generated hepatocyte-specific HO-1 mutant mice ("Lhoko"). Lhoko mice on normal chow exhibited unchanged liver morphology as well as serum parameters during functional metabolic testing. Lhoko mice showed no difference in high fat diet (HFD)-induced body weight gain nor changes in glucose levels during oral glucose tolerance. However, insulin tolerance, fasting insulin and HOMA-IR indicated significantly increased insulin sensitivity in the HFD-treated Lhoko mice. Additionally, liver damage parameters in Lhoko livers were lower compared to their controls. Injection of an insulin bolus directly into the portal vein of HFD-treated Lhoko mice leaded to increased activation of insulin signaling pathway. In vitro primary Lhoko hepatocytes exhibited increased basal and maximal respiratory capacity, an increase in intracellular hydrogen peroxide (H2O2), an upregulation of the mitochondrial reactive oxygen species (ROS) detoxification system, a decrease in PTP1B activity and an increase in acute insulin receptor activatability. On the contrary, acute overexpression of HO-1 via HO-1 adenovirus tail vein injection in unchallenged chow fed C57BL/6J mice triggered glucose intolerance, insulin resistance and elevated HOMA-IR. With this study we show that hepatocyte conditional HO-1 deletion in mice evokes resistance to diet-induced insulin resistance and reduces secondary disease such as steatosis and liver toxicity. This indicates that hepatocyte HO-1 exacerbates insulin resistance, steatosis, liver damage and metabolic disease. These findings identify HO-1 inhibition as a potential therapeutic strategy for metabolic disease.submitted by Elisa WallnerAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersWien, Med. Univ., Diss., 2014OeBB(VLID)171614

Hepatic Cholesterol-25-Hydroxylase Overexpression Improves Systemic Insulin Sensitivity in Mice

Obesity is a major risk factor for several diseases including diabetes, heart disease, and some forms of cancer and due to its rapidly increasing prevalence it has become one of the biggest problems medicine is facing today. All the more surprising, a substantial percentage of obese patients are metabolically healthy when classified based on insulin resistance and systemic inflammation. Oxysterols are naturally occurring molecules that play important role in various metabolic and inflammatory processes and their levels are elevated in patients suffering from obesity and diabetes. 25-Hydroxycholesterol (25-OHC) is produced in cells from cholesterol by the enzyme cholesterol 25-hydroxylase (Ch25h) and is involved in lipid metabolism, inflammatory processes, and cell proliferation. Here, we investigated the role of hepatic Ch25h in the transition from metabolically healthy obesity to insulin resistance and diabetes. Using several different experimental approaches, we demonstrated the significance of Ch25h on the border of “healthy” and “diseased” states of obesity. Adenovirus-mediated Ch25h overexpression in mice improved glucose tolerance and insulin sensitivity and lowered HOMA-IR. Our data suggest that low hepatic Ch25h levels could be considered a risk marker for unhealthy obesity.(VLID)486245

Interference of Mycoplasma Infection in a Gene Expression Study: It Was the Environment and Not the Gene▿

We show that short-term exposure to doxycycline, as used in tetracycline-inducible gene expression models, protects cells from stress-induced death in cultures infected with Mycoplasma arginini. Coinciding with the expected maximum level of gene activity, antimicrobial effects of tetracyclines might be mistaken for antiapoptotic properties of the expressed gene product

Identification of optical density ratios in subretinal fluid as a clinically relevant biomarker in exudative macular disease

PURPOSE: To investigate the potential role of optical density ratios (ODRs) obtained from subretinal fluid analysis in exudative macular disease and to identify the predictive role of ODRs under therapy in comparison to conventional morphometric measurements (CMMs). METHODS: Fifteen patients with neovascular age-related macular degeneration (nAMD) and 15 with acute central serous chorioretinopathy (CSC) were included in this prospective comparative and interventional case series. High-definition optical coherence tomography (SD-OCT) was performed according to a standardized protocol. nAMD patients received a standard treatment consisting of three monthly doses of intravitreous ranibizumab. Best corrected visual acuity (BCVA) was assessed at baseline (BSL) and weeks 2, 4, and 12. SD-OCT parameters were compared between CSC and nAMD at baseline. Predictive factors for functional recovery under ranibizumab treatment were identified in patients with nAMD. RESULTS: ODR showed highly significant differences between CSC and nAMD, whereas it was not possible to differentiate between these diseases on the basis of CMM. During follow-up, CMM correlated with BCVA at BSL only, whereas ODR showed a significant correlation with BCVA at week 4 and 12 during antiangiogenic therapy. CONCLUSIONS: Results suggest that CMM may correlate with BCVA at BSL, but has limited predictive value regarding recovery of visual function. Most interesting, ODR correlated with BCVA under therapy and was the only parameter that was pathognomic for nAMD in contrast to CSC in this study. ODR may reflect the status of the blood-retina barrier and may be used for pathophysiologic differentiation and prognostic purposes in exudative macular disease

Fractional heat shock protein 27 urine excretion as a short-term predictor in acute exacerbation of chronic obstructive pulmonary disease

Background: Chronic obstructive pulmonary disease (COPD) is a major cause of morbidity and mortality and is characterized by episodes of acute exacerbations. Finding a systemic biomarker that reliably predicts outcome after an acute exacerbation remains a major challenge. Heat shock protein 27 (HSP27) has been previously studied in COPD, however, urine excretion trajectory and prognostic value after an exacerbation is unknown. Methods: In this retrospective post hoc analysis of a prospective study that included 253 COPD patients who were hospitalized for acute exacerbation, 207 patients were analyzed. Urine and serum were sampled at admission, discharge, and 180 days after dischargeurine excretion trajectory was analyzed and correlated with clinicopathological and survival data. Results: HSP27 urine excretion increased after an exacerbation episode [1.8% admission, 1.8% discharge, 2.3% 180 days after discharge (P=0.091)]. In severely ill patients (GOLD IV) this course was even more distinct [1.6% admission, 2.1% discharge, 2.8% 180 days after discharge (P=0.007)]. Furthermore, fractional HSP27 urine excretion at discharge was increased in GOLD IV patients (P=0.031). In Kaplan-Meier and univariable Cox proportional hazard models patients with HSP27 urine excretion below 0.845% showed significantly worse survival at 30, 90 and 180 days after discharge. In a multivariable Cox proportional hazard model including established COPD outcome parameters fractional HSP27 urine excretion remained a significant predictor of survival at 30 and 90 days after discharge. Comparing this model to our already published model that includes HSP27 serum concentration we could show that fractional HSP27 urine excretion performs better in short-term survival. Conclusions: Our findings provide novel information about fractional HSP27 urine excretion trajectory in acute exacerbation of COPD. Fractional HSP27 urine excretion may be significantly reduced during an episode of acute exacerbation in COPD patients and may be used as a predictor of short-term all-cause mortality

Increased trefoil factor 2 levels in patients with chronic kidney disease.

In chronically damaged tissue, trefoil factor family (TFF) peptides ensure epithelial protection and restitution. In chronic kidney disease (CKD), TFF1 and TFF2 are reported to be upregulated. Especially in the early phase, CKD is associated with silently ongoing renal damage and inflammation. Moreover, many patients are diagnosed late during disease progression. We therefore sought to investigate the potential of TFF2 as biomarker for CKD. We followed 118 patients suffering from predialysis CKD and 23 healthy volunteers. TFF2 concentrations were measured using ELISA. Our results showed, that median TFF2 serum levels were significantly higher in patients with later CKD stages as compared to healthy controls (p 0.75). In conclusion, urine and serum TFF2 levels of CKD patients show a different profile dependent on CKD stages. Whereas TFF2 urine levels continuously decreased with disease progression, TFF2 serum concentrations progressively increased from the early to later CKD stages, indicating changes in renal function and offering the potential to examine the course of CKD