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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.
Poster presentation: NO-sensitive guanylyl cyclases (GC) are the principal receptors for nitric oxide (NO) and convert GTP into the second messenger cGMP. We showed that GC is prone to tyrosine phosphorylation in COS1 cells overexpressing the human holoenzyme. Similar results were obtained in PC12 cells and in rat aortic tissue slices. The major phosphorylation site was mapped to position 192 in the regulatory domain of the beta1 subunit. Tyrosine phosphorylation of GC was reduced in the presence of the inhibitors PP1 and PP2 indicating that Src-like kinases are critically involved in phosphorylation. Moreover, co-immunoprecipitation experiments revealed an interaction between Src and GC. To further analyse the relevance of this posttranslational modification we generated a phospho-specific antibody raised against pTyr192. This antibody clearly distinguishes between phosphorylated and non-phosphorylated GC and may be a powerful tool to analyse the subcellular localisation of the phosphorylated enzyme.
First paragraph (this article has no abstract) Persistent stimulation of nociceptors results in sensitization of nociceptive sensory neurons, which is associated with hyperalgesia and allodynia. The release of NO and subsequent synthesis of cGMP in the spinal cord are involved in this process. cGMP-dependent protein kinase I (PKG-I) has been suggested to act as a downstream target of cGMP, but its exact role in nociception hadn't been characterized yet. To further evaluate the NO/cGMP/PKG-I pathway in nociception we assessed the effects of PKG-I inhibiton and activaton in the rat formalin assay and analyzed the nociceptive behavior of PKG-I-/- mice. Open access article.
Generation of reactive oxygen species (ROS) is increasingly recognized as an important cellular process involved in numerous physiological and pathophysiological processes. Complex I (NADH:ubiquinone oxidoreductase) is considered as one of the major sources of ROS within mitochondria. Yet, the exact site and mechanism of superoxide production by this large membrane-bound multiprotein complex has remained controversial. Here we show that isolated complex I from Yarrowia lipolytica forms superoxide at a rate of 0.15% of the rate measured for catalytic turnover. Superoxide production is not inhibited by ubiquinone analogous inhibitors. Because mutant complex I lacking a detectable iron-sulfur cluster N2 exhibited the same rate of ROS production, this terminal redox center could be excluded as a source of electrons. From the effect of different ubiquinone derivatives and pH on this side reaction of complex I we concluded that oxygen accepts electrons from FMNH or FMN semiquinone either directly or via more hydrophilic ubiquinone derivatives.
Minimal invazif endoskopik giriflimlerin kalp cerrahisinde kullanımı ancak telemanipülatör sistemlerin kullanıma girmesiyle mümkün olmufltur. Bu çalıflmada total endoskopik revaskülarizasyon için kullanılan robotik destekli telemanipülasyon sistemleri gözden geçirilip, çalıflan ve duran kalpte uygulamalar derlenmifltir. Robotik cerrahi günümüzde halen geliflme aflamasındadır. Maliyetin yüksek oluflu ve sadece seçilmifl bir hasta grubunda uygulanabiliyor olması bu yeni tekni¤in en büyük sınırlayıcı faktörleridir. Ancak teknoloji üzerine çalıflmalar ve özellikle anastomoz tekniklerinin geliflmesiyle koroner revaskülarizasyon için bir alternatif olacaktır. Henüz istenen hedeflere ulaflılmamıflsa da gelecek umut vericidir.
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.
Soluble guanylyl cyclase (sGC) is the major cytosolic receptor for nitric oxide (NO) that converts GTP into the second messenger cGMP in a NO-dependent manner. Other factors controlling this key enzyme are intracellular proteins such as Hsp90 and PSD95, which bind to sGC and modulate its activity, stability, and localization. To date little is known about the effects of posttranslational modifications of sGC, although circumstantial evidence suggests that reversible phosphorylation may contribute to sGC regulation. Here we demonstrate that inhibitors of protein-tyrosine phosphatases such as pervanadate and bisperoxo(1,10-phenanthroline)oxovanadate(V) as well as reactive oxygen species such as H2O2 induce specific tyrosine phosphorylation of the β1 but not of the α1 subunit of sGC. Tyrosine phosphorylation of sGCβ1 is also inducible by pervanadate and H2O2 in intact PC12 cells, rat aortic smooth muscle cells, and in rat aortic tissues, indicating that tyrosine phosphorylation of sGC may also occur in vivo. We have mapped the major tyrosine phosphorylation site to position 192 of β1, where it forms part of a highly acidic phospho-acceptor site for Src-like kinases. In the phosphorylated state Tyr(P)-192 exposes a docking site for SH2 domains and efficiently recruits Src and Fyn to sGCβ1, thereby promoting multiple phosphorylation of the enzyme. Our results demonstrate that sGC is subject to tyrosine phosphorylation and interaction with Src-like kinases, revealing an unexpected cross-talk between the NO/cGMP and tyrosine kinase signaling pathways at the level of sGC.
Background: Osteoarthritis (OA) has a high prevalence in primary care. Conservative, guideline orientated approaches aiming at improving pain treatment and increasing physical activity, have been proven to be effective in several contexts outside the primary care setting, as for instance the Arthritis Self management Programs (ASMPs). But it remains unclear if these comprehensive evidence based approaches can improve patients' quality of life if they are provided in a primary care setting. Methods/Design: PraxArt is a cluster randomised controlled trial with GPs as the unit of randomisation. The aim of the study is to evaluate the impact of a comprehensive evidence based medical education of GPs on individual care and patients' quality of life. 75 GPs were randomised either to intervention group I or II or to a control group. Each GP will include 15 patients suffering from osteoarthritis according to the criteria of ACR. In intervention group I GPs will receive medical education and patient education leaflets including a physical exercise program. In intervention group II the same is provided, but in addition a practice nurse will be trained to monitor via monthly telephone calls adherence to GPs prescriptions and advices and ask about increasing pain and possible side effects of medication. In the control group no intervention will be applied at all. Main outcome measurement for patients' QoL is the GERMAN-AIMS2-SF questionnaire. In addition data about patients' satisfaction (using a modified EUROPEP-tool), medication, health care utilization, comorbidity, physical activity and depression (using PHQ-9) will be retrieved. Measurements (pre data collection) will take place in months I-III, starting in June 2005. Post data collection will be performed after 6 months. Discussion: Despite the high prevalence and increasing incidence, comprehensive and evidence based treatment approaches for OA in a primary care setting are neither established nor evaluated in Germany. If the evaluation of the presented approach reveals a clear benefit it is planned to provide this GP-centred interventions on a much larger scale.
The illusion of apparent motion can be induced when visual stimuli are successively presented at different locations. It has been shown in previous studies that motion-sensitive regions in extrastriate cortex are relevant for the processing of apparent motion, but it is unclear whether primary visual cortex (V1) is also involved in the representation of the illusory motion path. We investigated, in human subjects, apparent-motion-related activity in patches of V1 representing locations along the path of illusory stimulus motion using functional magnetic resonance imaging. Here we show that apparent motion caused a blood-oxygenation-level-dependent response along the V1 representations of the apparent-motion path, including regions that were not directly activated by the apparent-motion-inducing stimuli. This response was unaltered when participants had to perform an attention-demanding task that diverted their attention away from the stimulus. With a bistable motion quartet, we confirmed that the activity was related to the conscious perception of movement. Our data suggest that V1 is part of the network that represents the illusory path of apparent motion. The activation in V1 can be explained either by lateral interactions within V1 or by feedback mechanisms from higher visual areas, especially the motion-sensitive human MT/V5 complex.
Introduction: ScFv(FRP5)-ETA is a recombinant antibody toxin with binding specificity for ErbB2 (HER2). It consists of an N-terminal single-chain antibody fragment (scFv), genetically linked to truncated Pseudomonas exotoxin A (ETA). Potent antitumoral activity of scFv(FRP5)-ETA against ErbB2-overexpressing tumor cells was previously demonstrated in vitro and in animal models. Here we report the first systemic application of scFv(FRP5)-ETA in human cancer patients.
Methods: We have performed a phase I dose-finding study, with the objective to assess the maximum tolerated dose and the dose-limiting toxicity of intravenously injected scFv(FRP5)-ETA. Eighteen patients suffering from ErbB2-expressing metastatic breast cancers, prostate cancers, head and neck cancer, non small cell lung cancer, or transitional cell carcinoma were treated. Dose levels of 2, 4, 10, 12.5, and 20 μg/kg scFv(FRP5)-ETA were administered as five daily infusions each for two consecutive weeks.
Results: No hematologic, renal, and/or cardiovascular toxicities were noted in any of the patients treated. However, transient elevation of liver enzymes was observed, and considered dose limiting, in one of six patients at the maximum tolerated dose of 12.5 μg/kg, and in two of three patients at 20 μg/kg. Fifteen minutes after injection, peak concentrations of more than 100 ng/ml scFv(FRP5)-ETA were obtained at a dose of 10 μg/kg, indicating that predicted therapeutic levels of the recombinant protein can be applied without inducing toxic side effects. Induction of antibodies against scFv(FRP5)-ETA was observed 8 days after initiation of therapy in 13 patients investigated, but only in five of these patients could neutralizing activity be detected. Two patients showed stable disease and in three patients clinical signs of activity in terms of signs and symptoms were observed (all treated at doses ≥ 10 μg/kg). Disease progression occurred in 11 of the patients.
Conclusion: Our results demonstrate that systemic therapy with scFv(FRP5)-ETA can be safely administered up to a maximum tolerated dose of 12.5 μg/kg in patients with ErbB2-expressing tumors, justifying further clinical development.
Background: To investigate the occupational risk of tuberculosis (TB) infection in a low-incidence setting, data from a prospective study of patients with culture-confirmed TB conducted in Hamburg, Germany, from 1997 to 2002 were evaluated. Methods: M. tuberculosis isolates were genotyped by IS6110 RFLP analysis. Results of contact tracing and additional patient interviews were used for further epidemiological analyses. Results: Out of 848 cases included in the cluster analysis, 286 (33.7%) were classified into 76 clusters comprising 2 to 39 patients. In total, two patients in the non-cluster and eight patients in the cluster group were health-care workers. Logistic regression analysis confirmed work in the health-care sector as the strongest predictor for clustering (OR 17.9). However, only two of the eight transmission links among the eight clusters involving health-care workers had been detected previously. Overall, conventional contact tracing performed before genotyping had identified only 26 (25.2%) of the 103 contact persons with the disease among the clustered cases whose transmission links were epidemiologically verified. Conclusion: Recent transmission was found to be strongly associated with health-care work in a setting with low incidence of TB. Conventional contact tracing alone was shown to be insufficient to discover recent transmission chains. The data presented also indicate the need for establishing improved TB control strategies in health-care settings.
Human endothelial circulating progenitor cells (CPCs) can differentiate to cardiomyogenic cells during co-culture with neonatal rat cardiomyocytes. Wnt proteins induce myogenic specification and cardiac myogenesis. Here, we elucidated the effect of Wnts on differentiation of CPCs to cardiomyogenic cells. CPCs from peripheral blood mononuclear cells were isolated from healthy volunteers and co-cultured with neonatal rat cardiomyocytes. 6–10 days after co-culture, cardiac differentiation was determined by α-sarcomeric actinin staining of human lymphocyte antigen-positive cells (fluorescence-activated cell-sorting analysis) and mRNA expression of human myosin heavy chain and atrial natriuretic peptide. Supplementation of co-cultures with Wnt11-conditioned medium significantly enhanced the differentiation of CPCs to cardiomyocytes (1.7 ± 0.3-fold), whereas Wnt3A-conditioned medium showed no effect. Cell fusion was not affected by Wnt11-conditioned medium. Because Wnts inhibit glycogen synthase kinase-3β, we further determined whether the glycogen synthase kinase-3β inhibitor LiCl also enhanced cardiac differentiation of CPCs. However, LiCl (10 mm) did not affect CPC differentiation. In contrast, Wnt11-conditioned medium time-dependently activated protein kinase C (PKC). Moreover, the PKC inhibitors bisindolylmaleimide I and III significantly blocked differentiation of CPCs to cardiomyocytes. PKC activation by phorbol 12-myristate 13-acetate significantly increased CPC differentiation to a similar extent as compared with Wnt11-conditioned medium. Our data demonstrate that Wnt11, but not Wnt3A, augments cardiomyogenic differentiation of human CPCs. Wnt11 promotes cardiac differentiation via the non-canonical PKC-dependent signaling pathway.
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.
Background: Tumor development remains one of the major obstacles following organ transplantation. Immunosuppressive drugs such as cyclosporine and tacrolimus directly contribute to enhanced malignancy, whereas the influence of the novel compound mycophenolate mofetil (MMF) on tumor cell dissemination has not been explored. We therefore investigated the adhesion capacity of colon, pancreas, prostate and kidney carcinoma cell lines to endothelium, as well as their beta1 integrin expression profile before and after MMF treatment. Methods: Tumor cell adhesion to endothelial cell monolayers was evaluated in the presence of 0.1 and 1 μM MMF and compared to unstimulated controls. beta1 integrin analysis included alpha1beta1 (CD49a), alpha2beta1 (CD49b), alpha3beta1 (CD49c), alpha4beta1 (CD49d), alpha5beta1 (CD49e), and alpha6beta1 (CD49f) receptors, and was carried out by reverse transcriptase-polymerase chain reaction, confocal microscopy and flow cytometry. Results: Adhesion of the colon carcinoma cell line HT-29 was strongly reduced in the presence of 0.1 μM MMF. This effect was accompanied by down-regulation of alpha3beta1 and alpha6beta1 surface expression and of alpha3beta1 and alpha6beta1 coding mRNA. Adhesion of the prostate tumor cell line DU-145 was blocked dose-dependently by MMF. In contrast to MMF's effects on HT-29 cells, MMF dose-dependently up-regulated alpha1beta1, alpha2beta1, alpha3beta1, and alpha5beta1 on DU-145 tumor cell membranes. Conclusion: We conclude that MMF possesses distinct anti-tumoral properties, particularly in colon and prostate carcinoma cells. Adhesion blockage of HT-29 cells was due to the loss of alpha3beta1 and alpha6beta1 surface expression, which might contribute to a reduced invasive behaviour of this tumor entity. The enhancement of integrin beta1 subtypes observed in DU-145 cells possibly causes re-differentiation towards a low-invasive phenotype.
Severe acute respiratory syndrome (SARS) is a new infectious disease caused by a novel coronavirus that leads to deleterious pulmonary pathological features. Due to its high morbidity and mortality and widespread occurrence, SARS has evolved as an important respiratory disease which may be encountered everywhere in the world. The virus was identified as the causative agent of SARS due to the efforts of a WHO-led laboratory network. The potential mutability of the SARS-CoV genome may lead to new SARS outbreaks and several regions of the viral genomes open reading frames have been identified which may contribute to the severe virulence of the virus. With regard to the pathogenesis of SARS, several mechanisms involving both direct effects on target cells and indirect effects via the immune system may exist. Vaccination would offer the most attractive approach to prevent new epidemics of SARS, but the development of vaccines is difficult due to missing data on the role of immune system-virus interactions and the potential mutability of the virus. Even in a situation of no new infections, SARS remains a major health hazard, as new epidemics may arise. Therefore, further experimental and clinical research is required to control the disease.
Keywords: Severe Acute Respiratory Syndrome; SARS; coronavirus; molecular mechanisms; therapy; vaccination
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.
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.