Evaluation eines Enzymimmunoassays und einer Real-Time PCR für den Nachweis von Helicobacter pylori in Stuhlmaterial von Kindern

Helicobacter pylori, ein gramnegatives Stäbchenbakterium, kann durch eine Invasion der Magenschleimhaut Erkrankungen wie die Typ B-Gastritis, die gastroduodenale Ulkuserkrankung, das Magenkarzinom sowie das MALT-Lymphom hervorrufen. Etwa die Hälfte der Weltbevölkerung ist von einer Infektion betroffen und damit potentiell einem erhöhten Risiko für diese Erkrankungen ausgesetzt. Die Behandlung einer Helicobacter pylori-Infektion erfolgt durch eine Kombinationstherapie, bestehend aus zwei Antibiotika – Clarithromycin plus Metronidazol oder Clarithromycin plus Amoxicillin – und einem Protonenpumpen-Inhibitor. Therapeutische Probleme bereiten zunehmende Antibiotika-Resistenzen von Helicobacter pylori – insbesondere Resistenzen gegen Clarithromycin, die fast immer mit einem Therapieversagen assoziiert sind. Die derzeitige Goldstandard-Diagnostik mittels Ösophagogastroduodenoskopie beinhaltet deshalb neben dem Keimnachweis immer auch eine Antibiotika-Sensitivitätstestung. Ziel dieser Arbeit war es, zwei nicht-invasive Tests – einen Enzymimmunoassay (Amplified IDEIA Hp StAR; DakoCytomation) und eine Real-Time PCR (Helicobacter pylori ClariRes assay; Ingenetix) – an Stuhlmaterial von 100 symptomatischen, therapie-naiven Kindern zu testen und mit den gängigen Methoden der Helicobacter pylori-Diagnostik zu vergleichen. Der Infektionsstatus der Kinder wurde durch die Methoden Histologie, Kultur und 13C-Harnstoff-Atemtest festgelegt. Die Standard-Methoden zeigten für 54 Kinder einen negativen Helicobacter pylori-Infektionsstatus. Dieses Ergebnis wurde durch beide Stuhltests bestätigt (Spezifität jeweils 100%). Für die restlichen 46 Kinder wurde durch die Standard-Methoden ein positiver Infektionsstatus festgestellt. Der Stuhl-EIA bestätigte bei 44 dieser Kinder das positive Ergebnis und zeigte nur zwei falsch-negative Tests (Sensitivität 95,7%). Die Real-Time PCR lieferte bei 29 dieser Kinder ein positives Ergebnis (Sensitivität 69%). Für diese 29 Kinder konnte die Clarithromycin-Sensitivitätstestung des Helicobacter pylori ClariRes assay in allen Fällen das Ergebnis des E-Tests bestätigen. Es stellte sich heraus, dass der Stuhl-EIA durch seine exzellente Sensitivität und Spezifität über alle Altersgruppen ein guter Test ist, der günstig und leicht durchzuführen ist. Gerade bei Kindern unter sechs Jahren sollte er statt des ebenfalls nicht-invasiven 13C-Harnstoff-Atemtests angewendet werden. Nachteil des Stuhl-EIA bleibt jedoch eine fehlende Clarithromycin-Sensitivitätstestung. Der Helicobacter pylori ClariRes assay stellte sich zwar ebenfalls als schnelle und einfache diagnostische Methode dar, lieferte aber einige falsch-negative Ergebnisse (14 Kinder) und ist, verglichen mit den anderen nicht-invasiven Tests, relativ teuer. Daher kann dieser Test keine Alternative zur bisherigen Diagnostik darstellen und sollte lediglich als Zusatzuntersuchung für spezielle Fragestellungen eingesetzt werden. So könnte er z.B. als Bestätigungstest nach positivem Stuhl-EIA hilfreich sein, um zusätzliche Informationen über eine möglicherweise vorhandene Clarithromycin-Resistenz zu liefern. In dieser Studie wurden nur therapie-naive Kinder in die Untersuchung einbezogen. Ob diese Tests bei Kindern nach erfolgter Eradikationstherapie ähnliche Ergebnisse liefern, bedarf einer Bestätigung durch weitere Studien.The gram negative bacterium Helicobacter pylori colonizes the gastric mucosa and potentially induces chronic gastritis, peptic ulcer disease, gastric cancer and MALT lymphoma. About half of the world’s population is infected by Helicobacter pylori and therefore at higher risk for these diseases. Treatment of Helicobacter pylori comprises a combination therapy of two antibiotics (Clarithromycin plus Amoxicillin or Clarithromycin plus Metronidazole) and a proton pump inhibitor. Therapeutic problem is an increasing antibiotic resistance of Helicobacter pylori – especially it’s resistance to Clarithromycin which is strongly associated with treatment failure. Thus, “gold standard” of Helicobacter pylori diagnostics includes gastroduodenoscopy with concomitant susceptibility testing of antibiotics. Aim of this study was to evaluate an enzyme immuno assay (Amplified IDEIA Hp StAR; DakoCytomation) and a real-time PCR (Helicobacter pylori ClariRes assay; Ingenetix) for the detection of Helicobacter pylori infection in stool sam-ples from 100 symptomatic children. The results of these stool methods were directly compared with the results obtained from histological examination of gastric biopsy specimens, culturing and the 13C-urea breath test. Fecal specimens from all 54 children who were shown to be non-infective by “gold standard” tests gave true-negative results in both stool tests (specificity 100%). Of the remaining 46 children a positive Helicobacter pylori status was shown by standard methods. The stool EIA confirmed 44 of these children to be infective giving only two negative results (sensitivity 95.7%). The real-time PCR found only 29 cases to be Helicobacter pylori positive (sensitivity 63%). For these 29 cases the Helicobacter pylori ClariRes assay confirmed all results from phenotypic Clarithromycin susceptibility testing by Etest. In summary, this investigation demonstrates that the stool EIA is a very accurate and inexpensive test that can easily and quickly be performed. Especially in children under the age of six years the test should be used instead of the 13C-urea breath test. Disadvantage of the stool EIA remains a missing Clarithromycin susceptibility testing. The Helicobacter pylori ClariRes assay is also a quick and simple diagnostic method, but showed 14 false-negative results and is relatively expensive compared to the other non-invasive methods. Thus, the real-time PCR cannot replace other tests for the accurate diagnosis of Helicobacter pylori infection in children but could be used for specific questions. For instance the Helicobacter pylori ClariRes assay could help as a confirmation test after positive stool EIA to give additional information about a possible Clarithromycin resistance. In this study only therapy-naive children were included in the investigations. Thus, other studies need to evaluate these tests for children after eradication therapy

ICPL_ESIQuant – a Powerful Freeware Tool for Handling Proteomics LCESI- MS2 Experiments

Among the MS-based quantitative methods using stable isotope labelling, the Isotope-Coded Protein Label (ICPL) technique has emerged as a powerful tool to identify and relatively quantify thousands of proteins within complex protein mixtures. The ICPL_ESIQuant 3.0 software package is one of the key components of the ICPL-ESI workflow, covering data processing steps like LC-MS feature detection, ICPL doublet/triplet/quadruplet quantification as well as a merging step of LC-MS features and Mascot search results. As unique features, the software performs isotope pattern overlap corrections and utilizes additional chemical knowledge, e.g. the physico-chemical properties of the ICPL labels, to discard false positive isotope pattern, which significantly improves the quality of the final peptide and protein results. ICPL_ESIQuant is the first freeware tool on the market, which supports both the shotgun proteomics strategy using Data Dependent Acquisition (DDA) and the directed proteomics strategy using mass inclusion lists for precursor ion selection. ICPL_ESIQuant 3.0 (32 and 64 bit versions) can be downloaded from https://sourceforge.net/projects/icplquant/ files

Targeting of the master receptor MOM19 to mitochondria

The targeting of proteins to mitochondria involves the recognition of the precursor proteins by receptors on the mitochondrial surface followed by insertion of the precursors into the outer membrane at the general insertion site GIP. Most mitochondrial proteins analyzed so far use a mitochondrial outer membrane protein of 19 kilodaltons (MOM19) as an import receptor. The gene encoding MOM19 has now been isolated. The deduced amino acid sequence predicts that MOM19 is anchored in the outer membrane by an NH2-terminal hydrophobic sequence, while the rest of the protein forms a hydrophilic domain exposed to the cytosol. MOM19 was targeted to the mitochondria via a pathway that is independent of protease-accessible surface receptors and controlled by direct assembly of the MOM19 precursor with GIP

Architecture of coatomer: Molecular characterization of delta-COP and protein interactions within the complex

Copyright © 2011 by The Rockefeller University Press.Coatomer is a cytosolic protein complex that forms the coat of COP I-coated transport vesicles. In our attempt to analyze the physical and functional interactions between its seven subunits (coat proteins, [COPs] alpha-zeta), we engaged in a program to clone and characterize the individual coatomer subunits. We have now cloned, sequenced, and overexpressed bovine alpha-COP, the 135-kD subunit of coatomer as well as delta-COP, the 57-kD subunit and have identified a yeast homolog of delta-COP by cDNA sequence comparison and by NH2-terminal peptide sequencing. delta-COP shows homologies to subunits of the clathrin adaptor complexes AP1 and AP2. We show that in Golgi-enriched membrane fractions, the protein is predominantly found in COP I-coated transport vesicles and in the budding regions of the Golgi membranes. A knock-out of the delta-COP gene in yeast is lethal. Immunoprecipitation, as well as analysis exploiting the two-hybrid system in a complete COP screen, showed physical interactions between alpha- and epsilon-COPs and between beta- and delta-COPs. Moreover, the two-hybrid system indicates interactions between gamma- and zeta-COPs as well as between alpha- and beta' COPs. We propose that these interactions reflect in vivo associations of those subunits and thus play a functional role in the assembly of coatomer and/or serve to maintain the molecular architecture of the complex.This work was supported by The Deutsche Forschungsgemeinschaft (SFB 352), the Human Frontier Science Program, and the Swiss National Science Foundation No. 31-43366.95

The monoclonal antibody E587 recognizes growing (new and regenerating) retinal axons in the goldfish retinotectal pathway

E587 is a new monoclonal antibody against a 200 kDa cell-surface glycoprotein in the fish retinotectal pathway. The E587 antigen probably belongs to the class of cell adhesion molecules, and more specifically, to the family of L1-like molecules. The immunopurified protein is recognized by the antibody against the HNK1/L2 sugar epitope (associated with most cell adhesion molecules) and by a polyclonal antiserum against chick G4, which is related to the cell adhesion molecule L1 in mouse. Moreover the NH2-terminal sequence of E587 shows similarity with L1 and Ng-CAM. The E587 immunostaining pattern in the fish retinotectal pathway suggests that the E587 antigen is a growth- associated molecule on fish retinal axons. In fish embryos, all retinal axons are labeled. In adult fish, however, only the young axons from newly added ganglion cells carry E587 staining. After optic nerve transection (ONS) and retinal axonal regeneration, all axons reexpress the E587 antigen into their terminal processes in the tectal retinorecipient layers. The reexpression of the E587 antigen is temporally regulated, and E587 immunoreactivity declines by 7 months and disappears at 12 months after ONS. We hypothesize that the E587 antigen may mediate axon-axon associations. In its restricted appearance on young axons in normal adult fish, it may contribute to the selective fasciculation of the newest axons with young axons and thus participate in the creation of the age-related fiber organization in the fish optic nerve

A horse and a zebra: an atypical clinical picture including Guillain-Barré syndrome, recurrent fever and mesenteric lymphadenopathy caused by two concomitant infections

Background While Campylobacter jejuni represents the most common cause of bacterial gastroenteritis, Yersinia pseudotuberculosis infections are very rarely diagnosed in adults. Case We report on a previously healthy patient who presented several times at our hospital with fever, Guillain-Barré syndrome, recurrent abdominal symptoms and distinct mesenteric lymphadenopathy, respectively. This complicated and diagnostically challenging course of disease was caused by a C. jejuni and Y. pseudotuberculosis coinfection. Antibiotic treatment with doxycycline was effective. Conclusion Broad serology testing was crucial to discover that two concomitant infections were causing the symptoms. This case demonstrates that when a clinical picture is not fully explained by one known infection, another infection with the same underlying risk factor has to be considered, hence “a horse and a zebra”

The cDNA sequences of cytochrome c oxidase subunit VIa from carp and rainbow trout suggest the absence of isoforms in fishes1The sequence data in this paper have been submitted to the GenBank data library under the accession numbers: BankIt88656 U83980 (trout) and BankIt88644 U83907 (carp).1

AbstractThe cDNAs of subunit VIa of cytochrome c oxidase from rainbow trout liver and carp heart are presented, revealing 82% identity of their deduced amino acid sequences. The two cDNAs are evolutionary equally distant from the livertype (VIaL) and heart-type (VIaH) of mammalian subunit VIa. The data suggest that in ectotherm fishes no isoforms of subunit VIa occur, and that the postulated tissue-specific mechanism of thermogenesis in mammals, based on interaction of ATP with subunit VIaH (Frank, V. and Kadenbach, B. (1996) FEBS Lett. 382, 121–124), is absent.© 1997 Elsevier Science B.V. All rights reserved