32 research outputs found
Charakterisierung des Pyrophosphatmetabolismus bei Pseudoxanthoma elasticum
No full textDabisch-Ruthe M. Charakterisierung des Pyrophosphatmetabolismus bei Pseudoxanthoma elasticum. Bielefeld: Universität Bielefeld; 2014.Pseudoxanthoma elasticum (PXE) ist eine autosomal rezessive Erkrankung des Bindegewebes, welche durch progressive Kalzifizierung und Fragmentierung der elastischen Fasern in der Haut, der Bruchmembran des Auges sowie der Gefäßwände charakterisiert ist. Ursächlich für PXE sind Mutationen im ATP-Bindungskassettentransporter-Protein 6 (ABCC6). Ein Zusammenhang zwischen ABCC6 und der Matrixkalzifizierung konnte bis heute nicht hergestellt werden. Insbesondere ist die Bedeutung von Pyrophosphat (PPi) als wichtigster lokaler Regulator des Kalzifizierungsprozesses und die der PPi-metabolisierenden Enzyme im Kalzi-fizierungsprozess bei PXE unklar.
Im ersten Teil dieser Arbeit wurden ausgewählte Sequenzvarianten in Genen PPi-metabolisierender Enzyme auf eine mögliche Assoziation in einer PXE-Patienten- und einer Kontrollkohorte (jeweils n = 190) untersucht. Die Einzelnukleotidvarianten c.1190-65C>A im Gen der Alkalischen Phosphatase, c.313+9G>T im Gen der Ektonukleotidpyrophosphatase 1 (ENPP1) und c.294C>T im Ankylosis-Gen kamen bei PXE signifikant häufiger vor. Für das krankheitsassoziierte Allel c.313+9T konnte eine hoch signifikante Odds Ratio von 27,96 (1,66-472,30; P=0,0008) detektiert werden, weshalb von einem starken Risikofaktor für PXE ausgegangen werden kann.
In einem zweiten Teil der Arbeit wurde der Kalzifizierungsprozess dermaler Fibroblasten von PXE-Patienten und gesunden Kontrollen unter dem Einfluss verschiedener Kalzifizierungsinduktoren untersucht. Dabei konnten erhöhte intrazelluläre Kalziumkonzentrationen in PXE-Fibroblasten detektiert werden, welche auf eine gestörte zelluläre Kalziumhomöostase hindeuten. Zum ersten Mal wurde gezeigt, dass die Regulation der kalzifizierungsinhibitorischen Proteine vom eingesetzten Kalzifizierungsinduktor abhängig ist. Des Weiteren konnte ein geeignetes Modellsystem für die Untersuchung der in vitro-Kalzifizierung bei PXE ohne den Einsatz von Phosphaten oder Kalzium zur Kalzifizierungsinduktion etabliert werden. In kalzifizierten PXE-Fibroblasten konnten erstmalig stark verminderte extrazelluläre Matrixmolekülexpressionen in Kombination mit hohen Expressionen der Matrixmetalloproteinasen 2 und 12 detektiert werden. Diese Ergebnisse zeigen, dass die pathologische Kalzifizierung eine wichtige Rolle in der PXE-Pathogenese spielt.
Der Hauptaspekt dieser Arbeit war die Untersuchung der PPi-Homöostase bei PXE. Erstmalig konnte eine Beteiligung von ABCC6 an der zellulären PPi-Homöostase gezeigt werden. Dabei korrelierte eine signifikant erhöhte Matrixkalzifizierung mit signifikant reduzierter ENPP1-Expression und -Aktivität. Verglichen mit Kontrollzellen wurden in PXE-Fibroblasten signifikant reduzierte extra- (e) und intrazelluläre (i) PPi-Konzentrationen detektiert. Die PXE-Fibroblasten kalzifizierten unter geringen PPi-Konzentrationen im Medium, während die Kontrollzellen die reduzierten ePPi-Konzentrationen mit der Generierung und dem Transport von iPPi ausgleichen und so eine Kalzifizierung verhindern konnten. Diese Ergebnisse weisen auf eine Funktionsstörung in der PPi-Bereitstellung aufgrund von PPi-Transportdefekten und reduzierter ENPP1-Aktivität in PXE-Fibroblasten hin.
In einem letzten Teil dieser Arbeit wurde die Eigenschaft von PPi als potentieller Kalzifizierungsinhibitor bei PXE untersucht. Eine Supplementation von PPi führte im Zellkulturmodell zu einer signifikant reduzierten Kalzifizierung und deutet auf eine mögliche Kompensation der gestörten PPi-Bereitstellung in PXE-Fibroblasten hin. Diese Ergebnisse liefern Hinweise auf eine potentielle Therapieoption für PXE-Patienten.
Zusammenfassend wurde in dieser Arbeit PPi erstmalig als Schlüsselmetabolit der PXE-Pathogenese und als Inhibitor der pathologischen Kalzifizierung bei PXE in einem neu etablierten Modellsystem, welches ohne Kalzifizierungsinduktor auskommt, beschrieben
Inactivation of Murine Norovirus on Fruit and Vegetable Surfaces by Vapor Phase Hydrogen Peroxide
No full textVapor phase hydrogen peroxide (H2O2) can be utilized to inactivate murine norovirus (MNV), a surrogate of human norovirus, on surface areas. However, vapor phase H2O2 inactivation of virus on fruits and vegetables has not been characterized. In this study, MNV was used to determine whether vaporized H2O2 inactivates virus on surfaces of various fruits and vegetables (apples, blueberries, cucumbers, and strawberries). The effect of vapor phase H2O2 decontamination was investigated with two application systems. Plaque assays were performed after virus recovery from untreated and treated fresh produce to compare the quantity of infective MNV. The Mann-Whitney U test was applied to the test results to evaluate the virus titer reductions of treated food samples, with significance set at P <= 0.05. The infective MNV populations were significantly reduced on smooth surfaces by 4.3 log PFU (apples, P < 0.00001) and 4 log PFU or below the detection limit (blueberries, P = 0.0074) by treatment with vapor phase H2O2 (60 min, maximum of 214 ppm of H2O2). Similar treatments of artificially contaminated cucumbers resulted in a virus titer reduction of 1.9 log PFU. Treatment of inoculated strawberries resulted in 0.1and 2.8-log reductions of MNV. However, MNV reduction rates on cucumbers (P = 0.3809) and strawberries (P = 0,7414) were not significant. Triangle tests and color measurements of untreated and treated apples, cucumbers, blueberries, and strawberries revealed no differences in color and consistency after H2O2 treatment. No increase of the H2O2 concentration in treated fruits and vegetables compared with untreated produce was observed. This study reveals for the first time the conditions under which vapor phase H2O2 inactivates MNV on selected fresh fruit and vegetable surfaces
Effects of Technological Processes on the Tenacity and Inactivation of Norovirus Genogroup II in Experimentally Contaminated Foods
No full textContaminated food is a significant vehicle for human norovirus transmission. The present study determined the effect of physicochemical treatments on the tenacity of infective human norovirus genogroup II in selected foods. Artificially contaminated produce was subjected to a number of processes used by the food industry for preservation and by the consumer for storage and preparation. Virus recovery was carried out by using ultrafiltration and was monitored by using bacteriophage MS2 as an internal process control. Norovirus was quantified by using monoplex one-step TaqMan real-time reverse transcription (RT)-PCR and an external standard curve based on recombinant RNA standards. An RNase pretreatment step was used to avoid false-positive PCR results caused by accessible RNA, which allowed detection of intact virus particles. Significant reductions in titers were obtained with heat treatments usually applied by consumers for food preparation (baking, cooking, roasting). Generally, processes used for preservation and storage, such as cooling, freezing, acidification (≥pH 4.5), and moderate heat treatments (pasteurization), appear to be insufficient to inactivate norovirus within a food matrix or on the surface of food. Besides data for persistence in processed food, comparable data for individual matrix-specific protective effects, recovery rates, and inhibitory effects on the PCRs were obtained in this study. The established procedure might be used for other noncultivable enteric RNA viruses that are connected to food-borne diseases. The data obtained in this study may also help optimize the process for inactivation of norovirus in food by adjusting food processing technologies and may promote the development of risk assessment systems in order to improve consumer protection
Usage of cold hydrogen peroxide vapour for inactivation of murine norovirus on fruit and vegetable surfaces.
No full textComparison of three multiplex PCR assays for the detection of respiratory viral infections: evaluation of xTAG respiratory virus panel fast assay, RespiFinder 19 assay and RespiFinder SMART 22 assay
No full textAbstract Background A broad spectrum of pathogens is causative for respiratory tract infections, but symptoms are mostly similar. Therefore, the identification of the causative viruses and bacteria is only feasible using multiplex PCR or several monoplex PCR tests in parallel. Methods The analytical sensitivity of three multiplex PCR assays, RespiFinder-19, RespiFinder-SMART-22 and xTAG-Respiratory-Virus-Panel-Fast-Assay (RVP), were compared to monoplex real-time PCR with quantified standardized control material. All assays include the most common respiratory pathogens. Results To compare the analytical sensitivity of the multiplex assays, samples were inoculated with 13 different quantified viruses in the range of 101 to 105 copies/ml. Concordant results were received for rhinovirus, whereas the RVP detected influenzavirus, RSV and hMPV more frequently in low concentrations. The RespiFinder-19 and the RespiFinder-SMART-22 showed a higher analytical sensitivity for adenoviruses and coronaviruses, whereas the RVP was incapable to detect adenovirus and coronavirus in concentrations of 104 copies/ml. The RespiFinder-19 and RespiFinder-SMART-22A did not detect influenzaviruses (104 copies/ml) and RSV (103 copies/ml). The detection of all 13 viruses in one sample was only achieved using monoplex PCR. To analyze possible competitive amplification reactions between the different viruses, samples were further inoculated with only 4 different viruses in one sample. Compared to the detection of 13 viruses in parallel, only a few differences were found. The incidence of respiratory viruses was compared in tracheal secretion (TS) samples (n = 100) of mechanically ventilated patients in winter (n = 50) and summer (n = 50). In winter, respiratory viruses were detected in 32 TS samples (64%) by RespiFinder-19, whereas the detection rate with RVP was only 22%. The most frequent viruses were adenovirus (32%) and PIV-2 (20%). Multiple infections were detected in 16 TS samples (32%) by RespiFinder-19. Fewer infections were found in summer (RespiFinder-19: 20%; RVP: 6%). All positive results were verified using monoplex PCR. Conclusions Multiplex PCR tests have a broad spectrum of pathogens to test at a time. Analysis of multiple inoculated samples revealed a different focus of the detected virus types by the three assays. Analysis of clinical samples showed a high concordance of detected viruses by the RespiFinder-19 compared to monoplex tests.</p
Late sampling for automated culture to extend the platelet shelf life to 5 days in Germany
No full textBACKGROUND
Bacterial contamination of platelet concentrates (PCs) is still a major challenge in transfusion medicine. Different methodologic concepts and screening strategies have been developed and investigated concerning their usability. We evaluated the feasibility of BacT/ALERT automated culture (BacT/A, bioMérieux) with late sampling after 3 days at the earliest.
STUDY DESIGN AND METHODS
Twenty-four bacterial strains isolated from PCs and six relevant strains from reference stocks were spiked into apheresis-derived PCs (10-60 colony-forming units [CFU]/bag). Sampling was performed after 3 days, and bacterial detection was investigated using the two detection methods (BacT/A and BactiFlow [BF], bioMérieux). The maximum time-to-result of BacT/A was set to less than 12 hours.
RESULTS
All medium- or high-pathogenic strains are capable of proliferating to high titers, and 100% of contaminated samples were detected by BF and BacT/A (6 to ≤12 h incubation); lower detection rates of BacT/A were obtained within 6 hours of incubation (≤6 h: 76.2-93.4%). The majority of low-pathogenic isolates are also capable of growing in PCs (89.7%), showing a detection rate of 74.3% for BF versus 54.3% for BacT/A (6 to ≤12 h incubation). BacT/A failed to detect bacteria within 6 hours of incubation. Certainly, a small number of strains did not grow under PC storage conditions and were detectable by BacT/A only with increased detection times.
CONCLUSIONS
Late sampling after 3 days at the earliest, combined with reduced BacT/A incubation following the negative-to-date concept, offer an alternative opportunity to extend the shelf life of PCs from 4 to 5 days in Germany. The sensitivity of BacT/A with late sampling is nearly comparable to BF; the time-to-result is considerably longer
Application of cold nebulized hydrogen peroxide for inactivation of murine norovirus, bacteria and bacteria spores on surfaces in food production.
No full textPyrophosphates as a major inhibitor of matrix calcification in Pseudoxanthoma elasticum
No full textBackground
Pseudoxanthoma elasticum (PXE) is a rare hereditary disorder characterized by late onset and progressive calcification of elastic fibers in skin, eyes and the cardiovascular system, exemplifying a model for conditions characterized by soft tissue calcification.
Objective
The aim of our study was to characterize cellular inorganic pyrophosphate (PPi) homeostasis in PXE.
Methods
Gene expression of PPi metabolizing enzymes was determined by quantitative real-time PCR after incubation up to 21 days with or without addition of Na2HPO4. Extracellular and cytosolic PPi concentrations were measured by enzyme-linked bioluminescence assay. ALP and ENPP1 activity was determined spectrophotometrically. We further established a human cell culture model suitable for investigating PXE and related disorders without addition of artificial calcification triggers.
Results
Independently of the experimental conditions, PXE fibroblasts revealed a higher degree of matrix calcification. We observed that matrix calcification was associated with altered gene expression of PPi metabolizing enzymes in PXE fibroblasts. In this context, PXE fibroblasts exhibited significantly higher expression of ALP and OPN and reduced mRNA expression and activity of ENPP1. Here, for the first time cytosolic and extracellular PPi levels were shown to be strongly reduced in PXE fibroblasts. We further showed that PPi concentration in bovine and human sera additives had a strong impact on matrix calcification. In a last experimental line, we demonstrated that addition of PPi analogs reduced matrix calcification of PXE fibroblasts most likely by reducing ALP and OPN mRNA expression, restoring ENPP1 activity and subsequently elevating PPi concentrations.
Conclusion
The results of our study along with recent findings point to the essential role of PPi as the central regulatory metabolites preventing matrix calcification in PXE. But what remains to be determined is the underlying molecular mechanism leading to depletion of PPi in PXE. We further suggest that supplementation of PPi analogs might counteract pathological calcification in PXE and related disorders
Variants in genes encoding pyrophosphate metabolizing enzymes are associated with Pseudoxanthoma elasticum
No full textObjectives
Pseudoxanthoma elasticum (PXE) is a rare hereditary disorder characterized by progressive calcification and fragmentation of elastic fibers. Because of the great clinical variability between PXE patients the involvement of modifier genes was recently suggested. Therefore, we investigated the association of single nucleotide variants (SNVs) in selected candidate genes known to regulate cellular pyrophosphate metabolism.
Design and methods
We used RLFP analyses to evaluate the distribution of SNVs in alkaline phosphatase (ALP), ectonucleotide pyrophosphatase 1 (ENPP1) and ankylosis (ANKH) in DNA samples from 190 German PXE patients and 190 age- and sex-matched healthy controls. Statistical analyses were performed using Fisher exact test and Bonferroni correction.
Results
The screening revealed three different SNVs in three genes, which were associated with PXE. The SNV c.1190-65C > A (rs1780329, minor allele frequency (MAF) patients: 0.17; controls: 0.11; P = 0.04) in the ALP gene was significantly more frequent in PXE patients. Furthermore, PXE was highly associated with ANKH p.A98A genotype TT (P = 0.0012), although the MAF was not different between patients and controls. After correction for multiple testing according to the Bonferroni method, one SNV in the ENPP1 gene (c.313 + 9G > T, rs7773477) remained significantly associated with PXE with significantly higher MAF values in the patient cohort (MAF: 0.04 vs. 0.00; P = 0.0024) and a high association with PXE susceptibility (OR 27.96).
Conclusion
Polymorphisms in ALP, ENPP1 and ANKH are important genetic risk factors contributing to PXE
Collected Material for Scrapbook 1995-1996
No full textPhotocopy of a newspaper article from The San Antonio Informer titled "Author Ruthe Winegarten, educators honored with reception." Includes photographs and handwritten notes
