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Im vorliegenden Fall wird von einer Fehldiagnose auf der Grundlage eines falsch-reaktiven Anti-HCV-Tests und eines falsch-reaktiven HCV-Nukleinsäureamplifikationstests (NAT) berichtet, die bei einem 58-jährigen chirurgischen Oberarzt im Rahmen einer arbeitsmedizinischen Vorsorgeuntersuchung im krankenhauseigenen Labor gestellt wurde und zu einem knapp zweimonatigen Berufsverbot führte. Basis dieser Fehldiagnose war ein wiederholt schwach reaktiver HCV-Antikörper-ELISA, der mit einem Nukleinsäureamplifikationstest, der ebenfalls schwach positiv ausfiel, überprüft wurde. Ein Antikörperbestätigungs- bzw. Ergänzungstest (Immunoblot) wurde nicht durchgeführt. Die Fehldiagnose ist jedoch nicht durch einen Testfehler, sondern durch ein Missverständnis entstanden, indem beim Kliniker zwei Laborindizien zu einem Beweis aufsummiert wurden.
Ein Krankenhauslabor und ein Einsendelabor, das mehrere Krankenhäuser versorgt, haben prospektiv 3.907 Blutkulturflaschen für das BACTEC™ 9000–System (BDDiagnostics, Heidelberg, Germany) untersucht. Dabei wurden 1.888 aerobe Flaschen, 1.880 anaerobe Flaschen und 139 pädiatrische Blutkulturflaschen verarbeitet. Es wurden der Zeitpunkt der Beimpfung und der Zeitpunkt des Einlesens der Kulturen in das Gera ̈t dokumentiert. Neben den Medientypen und dem Blutvolumen wurden folgende Daten erhoben: Die Zeit vom Einlesen in das Gerät bis zum positiven Signal (Detektionszeit), die Identifizierung des Erregers bis zur Species, die Antibiotikatherapie und die Wiederfindungsrate verglichen mit der terminalen Subkultur. Die mittlere Transportdauer betrug 21,4 h, die mittlere Detektionszeit 21,5 h. 27 Flaschen waren falsch negativ und sechs Flaschen falsch positiv. Bei sieben der falschnegativen Flaschen hatte die Partnerflasche ein positives Signal gegeben (Staphylococcus aureus, Enterobactercloacae, Enterococcus faecalis, Candida albicans, Burkholderia cepacia, zwei Pseudomonas aeruginosa-Stämme). Davon waren vier Isolate strikte Aerobier, die nichtin der anaeroben Flasche wuchsen, fünf Patienten standen unter Antibiotikatherapie und eine Flasche hatte eine Transportzeit)48 h und ist in dieser Gruppe ebenfalls aufgeführt. 15/27 falsch negative Flaschen hatten eine Transportzeit)48 h, 11 Patienten bekamen in dieser Gruppe eine Antibiotikatherapie. 6/27 falsch negative Flaschen hatten eine Transportzeit-48 h, davon wurden zwei Patienten antibiotisch behandelt. Einmal handelte es sich um C. glabrata, die nicht in der anaeroben Flaschewuchs. Der klinisch relevante Anteil der falsch negativen Blutkulturen (Isolat nicht in der Begleitflasche nachgewiesen), der innerhalb von 48 h in das BACTEC™ 9000-Gerät eingelesen wurde, betrug 0,15%.
Die HIV-1-Resistenztestung wird ein immer bedeutenderer Bestandteil des Monitorings der antiretroviralen Therapie und erfolgt in der Regel mittels Genotypisierung. Zur Zeit sind zwei Systeme kommerziell erhältlich und obwohl diese technisch nicht zu den einfach durchführbaren Methoden gehören, haben sie doch einen hohen Grad an Qualität erreicht. Modifikationen der Standardprotokolle sind für bestimmte Fragestellungen durchaus von Vorteil. Obwohl beide Systeme auf Entscheidungsregeln basierende Resistenz-Reports beinhalten, braucht es das zusätzliche Wissen und die Erfahrung des Anwenders, um die detektierten Mutationsmuster in klinisch brauchbare Resultate überführen zu können. Beide der hier detailliert beschriebenen Systeme haben ihre Vor- und Nachteile. Die Entscheidung für das eine oder andere System muss aufgrund der individuellen Bedürfnisse getroffen werden. Microarray-Systemen könnte der Markt der Zukunft gehören.
Die 1990 eingeführten ersten kommerziellen HCV-Antikörper-Screening Tests wurden im Laufe der Jahre bezüglich ihrer Sensitivität und Spezifität erheblich verbessert. Inzwischen sind auch standardisierte Verfahren zum qualitativen und quantitativen HCV-RNA-Nachweis verfügbar, die Dank der Einführung eines internationalen Standards miteinander vergleichbar sind. Aber auch mittels Antigen-ELISA ist es möglich, die im Patientenblut zirkulierende Virusmenge zu quantifizieren. Einer der Hauptübertragungswege – Bluttransfusion und Blutprodukte – der HCV-Infektion wurde durch die Verbesserung der virologischen Diagnostik nahezu eliminiert. Inzwischen sind i. v.-Drogenabhängige die Hauptrisikogruppe für eine HCV-Infektion. Bislang nur zu Forschungszwecken etablierte Methoden zur Messung der zellulären Immunität oder auch die Messung neutralisierender Antikörper könnten zum Beispiel im Rahmen einer Impfstoffentwicklung an Bedeutung gewinnen.
Highly sensitive qualitative and quantitative automatednucleic acid amplification tests (NATs) that are commercially available for the detection of hepatitis B virus (HBV)infection have been developed only in the last few years.The potential indications for HBV NATs are: follow-up ofchronic hepatitis B, therapy and antiviral resistance monitoring, determination of infectivity and transmission risk,detection of occult (HBsAg-negative and HBV DNA-positive) infection and mutant virus which may escape serologic diagnosis, blood donor screening, and resolution ofunusual or discordant serologic constellations. Although NATs are now widely implemented in the routine diagnosis of clinical laboratories, there are several importantissues which need to be further investigated. Standardisation of NATs used for the monitoring of antiviral therapyand follow-up of chronic infection is still lacking, and theclinical significance of HBV DNA levels needs to be clarified. The influence of genetic variability in terms of genotype variation has been poorly investigated so far.Although there are highly sensitive automated NATs forblood donor screening available, their implementation is still subject to discussion and certain countries rejectedHBV DNA testing for blood donation for reasons of poor cost-effectiveness.
Calreticulin is a Ca2+ -binding chaperone that resides in the lumen of the endoplasmic reticulum and is involved in the regulation of intracellular Ca2+ homeostasis and in the folding of newly synthesized glycoproteins. In this study, we have used site-specific mutagenesis to map amino acid residues that are critical in calreticulin function. We have focused on two cysteine residues (Cys(88) and Cys(120)), which form a disulfide bridge in the N-terminal domain of calreticulin, on a tryptophan residue located in the carbohydrate binding site (Trp(302)), and on certain residues located at the tip of the "hairpin-like" P-domain of the protein (Glu(238), Glu(239), Asp(241), Glu(243), and Trp(244)). Calreticulin mutants were expressed in crt(-/-) fibroblasts, and bradykinin-dependent Ca2+ release was measured as a marker of calreticulin function. Bradykinin-dependent Ca2+ release from the endoplasmic reticulum was rescued by wild-type calreticulin and by the Glu(238), Glu(239), Asp(241), and Glu(243) mutants. The Cys(88) and Cys(120) mutants rescued the calreticulin-deficient phenotype only partially ( approximately 40%), and the Trp(244) and Trp(302) mutants did not rescue it at all. We identified four amino acid residues (Glu(239), Asp(241), Glu(243), and Trp(244)) at the hairpin tip of the P-domain that are critical in the formation of a complex between ERp57 and calreticulin. Although the Glu(239), Asp(241), and Glu(243) mutants did not bind ERp57 efficiently, they fully restored bradykinin-dependent Ca2+ release in crt(-/-) cells. This indicates that binding of ERp57 to calreticulin may not be critical for the chaperone function of calreticulin with respect to the bradykinin receptor.
NAD(P)H oxidase, the main source of reactive oxygen species in vascular cells, is known to be regulated by redox processes and thiols. However, the nature of thiol-dependent regulation has not been established. Protein disulfide isomerase (PDI) is a dithiol/disulfide oxidoreductase chaperone of the thioredoxin superfamily involved in protein processing and translocation. We postulated that PDI regulates NAD(P)H oxidase activity of rabbit aortic smooth muscle cells (VSMCs). Western blotting confirmed robust PDI expression and shift to membrane fraction after incubation with angiotensin II (AII, 100 nm, 6 h). In VSMC membrane fraction, PDI antagonism with bacitracin, scrambled RNase, or neutralizing antibody led to 26-83% inhibition (p < 0.05) of oxidase activity. AII incubation led to significant increase in oxidase activity, accompanied by a 6-fold increase in PDI refolding isomerase activity. AII-induced NAD(P)H oxidase activation was inhibited by 57-71% with antisense oligonucleotide against PDI (PDIasODN). Dihydroethidium fluorescence showed decreased superoxide generation due to PDIasODN. Confocal microscopy showed co-localization between PDI and the oxidase subunits p22(phox), Nox1, and Nox4. Co-immunoprecipitation assays supported spatial association between PDI and oxidase subunits p22(phox), Nox1, and Nox4 in VSMCs. Moreover, in HEK293 cells transfected with green fluorescent protein constructs for Nox1, Nox2, and Nox4, each of these subunits co-immunoprecipitated with PDI. Akt phosphorylation, a known downstream pathway of AII-driven oxidase activation, was significantly reduced by PDIasODN. These results suggest that PDI closely associates with NAD(P)H oxidase and acts as a novel redox-sensitive regulatory protein of such enzyme complex, potentially affecting subunit traffic/assembling.
Excessive accumulation of the extracellular matrix is a hallmark of many inflammatory and fibrotic diseases, including those of the kidney. This study addresses the question whether NO, in addition to inhibiting the expression of MMP-9, a prominent metalloprotease expressed by mesangial cells, additionally modulates expression of its endogenous inhibitor TIMP-1. We demonstrate that exogenous NO has no modulatory effect on the extracellular TIMP-1 content but strongly amplifies the early increase in cytokine-induced TIMP-1 mRNA and protein levels. We examined whether transforming growth factor beta (TGFbeta), a potent profibrotic cytokine, is involved in the regulation of NO-dependent TIMP-1 expression. Experiments utilizing a pan-specific neutralizing TGFbeta antibody demonstrate that the NO-induced amplification of TIMP-1 is mediated by extracellular TGFbeta. Mechanistically, NO causes a rapid increase in Smad-2 phosphorylation, which is abrogated by the addition of neutralizing TGFbeta antisera. Similarly, the NO-dependent increase in Smad-2 phosphorylation is prevented in the presence of an inhibitor of TGFbeta-RI kinase, indicating that the NO-dependent activation of Smad-2 occurs via the TGFbeta-type I receptor. Furthermore, activation of the Smad signaling cascade by NO is corroborated by the NO-dependent increase in nuclear Smad-4 level and is paralleled by increased DNA binding of Smad-2/3 containing complexes to a TIMP-1-specific Smad-binding element (SBE). Reporter gene assays revealed that NO activates a 0.6-kb TIMP-1 gene promoter fragment as well as a TGFbeta-inducible and SBE-driven control promoter. Chromatin immunoprecipitation analysis also demonstrated DNA binding activity of Smad-3 and Smad-4 proteins to the TIMP-1-specific SBE. Finally, by enzyme-linked immunosorbent assay, we demonstrated that NO causes a rapid increase in TGFbeta(1) levels in cell supernatants. Together, these experiments demonstrate that NO by induction of the Smad signaling pathway modulates TIMP-1 expression.
The tumor necrosis factor family member Fas ligand (FasL) induces apoptosis in Fas receptor-expressing target cells and is an important cytotoxic effector molecule used by CTL- and NK-cells. In these hematopoietic cells, newly synthesized FasL is stored in specialized secretory lysosomes and only delivered to the cell surface upon activation and target cell recognition. FasL contains an 80-amino acid-long cytoplasmic tail, which includes a proline-rich domain as a bona fide Src homology 3 domain-binding site. This proline-rich domain has been implicated in FasL sorting to secretory lysosomes, and it may also be important for reverse signaling via FasL, which has been described to influence T-cell activation. Here we report the identification of the Src homology 3 domain-containing adaptor protein PSTPIP as a FasL-interacting partner, which binds to the proline-rich domain. PSTPIP co-expression leads to an increased intracellular localization of Fas ligand, thereby regulating extracellular availability and cytotoxic activity of the molecule. In addition, we demonstrate recruitment of the tyrosine phosphatase PTP-PEST by PSTPIP into FasL·PSTPIP·PTP-PEST complexes which may contribute to FasL reverse signaling.
Mechanical stress is known to modulate fundamental events such as cell life and death. Mechanical stretch in particular has been identified as a positive regulator of proliferation in skin keratinocytes and other cell systems. In the present study it was investigated whether antiapoptotic signaling is also stimulated by mechanical stretch. It was demonstrated that mechanical stretch rapidly induced the phosphorylation of the proto-oncogene protein kinase B (PKB)/Akt at both phosphorylation sites (serine 473/threonine 308) in different epithelial cells (HaCaT, A-431, and human embryonic kidney-293). Blocking of phosphoinositide 3-OH kinase by selective inhibitors (LY-294002 and wortmannin) abrogated the stretch-induced PKB/Akt phosphorylation. Furthermore mechanical stretch stimulated phosphorylation of epidermal growth factor receptor (EGFR) and the formation of EGFR membrane clusters. Functional blocking of EGFR phosphorylation by either selective inhibitors (AG1478 and PD168393) or dominant-negative expression suppressed stretch-induced PKB/Akt phosphorylation. Finally, the angiotensin II type 1 receptor (AT1-R) was shown to induce positive transactivation of EGFR in response to cell stretch. These findings define a novel signaling pathway of mechanical stretch, namely the activation of PKB/Akt by transactivation of EGFR via angiotensin II type 1 receptor. Evidence is provided that stretch-induced activation of PKB/Akt protects cells against induced apoptosis.