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Kruger P.,University of Oxford | Saffarzadeh M.,University Medical Center Mainz | Weber A.N.R.,University of Tübingen | Rieber N.,University of Tübingen | And 7 more authors.
PLoS Pathogens | Year: 2015

Neutrophils, the most abundant human immune cells, are rapidly recruited to sites of infection, where they fulfill their life-saving antimicrobial functions. While traditionally regarded as short-lived phagocytes, recent findings on long-term survival, neutrophil extracellular trap (NET) formation, heterogeneity and plasticity, suppressive functions, and tissue injury have expanded our understanding of their diverse role in infection and inflammation. This review summarises our current understanding of neutrophils in host-pathogen interactions and disease involvement, illustrating the versatility and plasticity of the neutrophil, moving between host defence, immune modulation, and tissue damage. © 2015 Kruger et al.


Graser Y.,Charité - Medical University of Berlin | Czaika V.,Charité - Medical University of Berlin | Ohst T.,Charité - Medical University of Berlin | Ohst T.,Labor Berlin Charite Vivantes GmbH
JDDG - Journal of the German Society of Dermatology | Year: 2012

The prevalence of onychomycosis is increasing steadily, sevenfold alone in the US within the last twenty years. An important aspect in this development is the demographic development of the human population of the industrial countries like Germany. A fast and accurate laboratory diagnosis is essential for successful treatment because 50% of the cases are misdiagnosed when relying on the clinical appearance only. The current diagnosis of dermatophytosis, based on direct microscopy and culture of the clinical specimen, is problematic given the lacking specificity of the former and the length of time needed for the latter. Molecular techniques can help to solve these problems. In recent years, a number of in-house PCR assays have been developed to identify dermatophytes directly from clinical specimens. Based on the "Mikrobiologisch- infektiologischen Qualitätsstandards (MIQ) für Nukleinsäure- Amplifikationstechniken" and the MIQE guideline (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) 11 studies are reviewed which were published between 2007 and 2010. The present article evaluates the quality of the PCR assays regarding false positive and false negative results due to contamination, PCR format, statistical analysis, and diagnostic performance of the studies. It shows that we are only at the beginning of providing high quality PCR diagnosis of dermatophytes. © Blackwell Verlag GmbH, Berlin.


Frisch E.,Charité - Medical University of Berlin | Kaup M.,Charité - Medical University of Berlin | Egerer K.,Charité - Medical University of Berlin | Weimann A.,Labor Berlin Charite Vivantes GmbH | And 3 more authors.
Electrophoresis | Year: 2011

High-mannose and hybrid-type N-glycans are present in human serum glycoproteins in low abundance but have recently been described to play an important role in immune responses. It is therefore important to find a strategy to selectively analyze their structures in the context of health and disease in order to understand their impact on disease mechanisms. We report here the characterization of high-mannose and hybrid-type N-glycans in total human serum. To this end, N-glycans were released using Endo-β-N-acetylglucosaminidase H (Endo H) and analyzed by CE-LIF and MALDI-TOF-MS. We found that the high-mannose structures Man 5-9GlcNAc 1 represented the majority of the pool. The monoglucosylated structure Glc 1Man 9GlcNAc 1 as well as four hybrid structures could be identified. Then, we compared the Endo H-released serum glycome of patients suffering from rheumatoid arthritis with healthy controls as mannose-binding lectin deficiency (MBL) and modulation of α-mannosidase activity were previously associated with this disease. Interestingly, we observed that both high-mannose and hybrid structures were fairly constant, suggesting that circulating MBL and α-mannosidase may not affect significantly the levels of serum glycoproteins carrying these glycans. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.


Hoppe B.,Labor Berlin Charite Vivantes GmbH | Hoppe B.,Charité - Medical University of Berlin
Thrombosis and Haemostasis | Year: 2014

Fibrinogen and factor XIII are two essential proteins that are involved directly in fibrin gel formation as the final step of a sequence of reactions triggered by a procoagulant stimulus. Haemostasis is the most obvious function of the resulting fibrin clot. Different variables affect the conversion of fibrinogen to fibrin as well as the mode of fibrin polymerisation and fibrin crosslinking, hereby, critically influencing the architecture of the resulting fibrin network and consequently determining its mechanical strength and resistance against fibrinolysis. Due to fibrinogen’s structure with a multitude of domains and binding motifs the fibrin gel allows for complex interactions with other coagulation factors, with profibrinolytic as well as antifibrinolyic proteins, with complement factors and with various cellular receptors. These interactions enable the fibrin network to control its own further state (i. e. expansion or degradation), to influence innate immunity, and to function as a scaffold for cell migration processes. During the whole process of fibrin gel formation biologically active peptides and protein fragments are released that additionally influence cellular processes via chemotaxis or by modulating cell-cell interactions. Thus, it is not surprising that fibrinogen and factor XIII in addition to their haemostatic function influence innate immunity as well as cell-mediated reactions like wound healing, response to tissue injury or inflammatory processes. The present review summarises current knowledge of fibrinogen’s and factor XIII’s function in coagulation and fibrinolysis giving special emphasis on their relation to inflammation control. © Schattauer 2014.


Zimmermann M.,Charité - Medical University of Berlin | Zimmermann M.,Labor Berlin Charite Vivantes GmbH | Otto C.,Charité - Medical University of Berlin | Gonzalez J.-B.,Charité - Medical University of Berlin | And 3 more authors.
International Journal of Laboratory Hematology | Year: 2013

Introduction: The Sysmex XE-5000 is a blood and body fluid analyzer able to differentiate cells into polymorphonuclear, mononuclear, and high-fluorescent cells (HFC). The identity of HFC in cerebrospinal fluid (CSF) has been uncertain; however, compatible with their high nucleic acid content, HFC could represent intrathecal tumor cells. Here, we studied the cellular origin and the diagnostic significance of HFC in CSF. Methods: Results of CSF examinations with the XE-5000 were analyzed in 65 CSF samples with and 126 CSF samples without tumor cells, as defined by manual microscopy of CSF cytospin preparations. Results: The XE-5000 detected HFC in 51 of 65 tumor cell-positive and in 33 of 126 tumor cell-negative CSF samples (sensitivity: 78.5%, specificity: 73.8%, positive likelihood ratio: 3.0, negative likelihood ratio: 0.29). The percentages of HFC and tumor cells in CSF samples correlated (r2 = 0.41, P < 0.0001). Tumor cells escaped detection by the XE-5000 especially in CSF samples with a low percentage of tumor cells. Conclusion: While this study identifies tumor cells as the predominant correlate of HFC in CSF, it suggests that measuring HFC is not an appropriate diagnostic test for intrathecal tumor cells. However, if HFC are incidentally detected in CSF, further evaluation by CSF microscopy seems mandatory. © 2013 John Wiley & Sons Ltd.


Ervens J.,Charité - Medical University of Berlin | Ghannoum M.,Case Western Reserve University | Graf B.,Charité - Medical University of Berlin | Graf B.,Labor Berlin Charite Vivantes GmbH | Schwartz S.,Charité - Medical University of Berlin
Infection | Year: 2014

A 45-year-old male with rhinocerebral mucormycosis (Rhizopus oryzae), refractory to liposomal amphotericin B and posaconazole, received isavuconazole salvage therapy. Initial isavuconazole plasma and tissue levels were 0.76-0.86 μg/mL and 1.09-1.38 μg/g. Plasma levels increased to 1.3-3.24 μg/mL with reduced comedication. Isavuconazole was well tolerated, and the patient has remained disease-free 24 months post-antifungal therapy. © 2013 Springer-Verlag.


Detert J.,Charité - Medical University of Berlin | Bastian H.,Charité - Medical University of Berlin | Listing J.,German Rheumatism Research Center | Weiss A.,German Rheumatism Research Center | And 15 more authors.
Annals of the Rheumatic Diseases | Year: 2013

Objective: To investigate the long-term effects of induction therapy with adalimumab (ADA) plus methotrexate (MTX) in comparison with placebo (PBO) plus MTX in DMARD-naïve patients with active early rheumatoid arthritis (RA). Methods: Patients with active early RA (disease duration of ≤12 months) were randomly assigned to receive 40 mg ADA subcutaneously every other week (eow) plus MTX 15 mg/week subcutaneously or PBO plus MTX subcutaneously at 15 mg/week over 24 weeks. Thereafter, all patients received MTX monotherapy up to week 48. The primary outcome was the Disease Activity Score 28 (DAS28) at week 48. Secondary outcomes included proportions of patients in remission (DAS28<2.6), ACR responses, Health Assessment Questionnaire (HAQ) score and radiographic progression. Results: 87 patients were assigned to ADA/MTX and 85 patients to PBO/MTX. At baseline, DAS28 was 6.2 ±0.8 in the ADA/MTX and 6.3±0.9 in the PBO/MTX groups. At week 24, treatment with ADA/MTX compared with PBO/MTX resulted in a greater reduction in DAS28 (3.0±1.2 vs 3.6±1.4; p=0.009) and other secondary outcomes such as DAS28 remission rate (47.9% vs 29.5%; p=0.021) and HAQ (0.49±0.6 vs 0.72±0.6; p=0.0014). At week 48, the difference in clinical outcomes between groups was not statistically significant (DAS28: 3.2±1.4 vs 3.4 ±1.6; p=0.41). Radiographic progression at week 48 was significantly greater in patients administered PBO/MTX (Sharp/van der Heijde score: ADA/MTX 2.6 vs PBO/MTX 6.4; p=0.03, Ratingen score: 1.7 vs 4.2; p=0.01). Conclusions: A greater reduction in radiographic progression after initial combination therapy with ADA and MTX was seen at week 48, even after discontinuation of ADA treatment at week 24. This sustained effect was not found at the primary endpoint (DAS28 reduction).


Korber M.K.,Charité - Medical University of Berlin | Langer E.,Labor Berlin Charite Vivantes GmbH | Langer E.,Charité - Medical University of Berlin | Ziemer S.,Charité - Medical University of Berlin | And 3 more authors.
Clinical and Applied Thrombosis/Hemostasis | Year: 2014

Background: Rivaroxaban (Xarelto, Bayer HealthCare, Leverkusen, Germany) is a new oral anticoagulant drug. Anticoagulants may cause bleeding, thereby requiring reliable monitoring and efficient therapy. We investigated thromboelastometry versus routine coagulation tests to measure prophylactic and therapeutic concentrations of rivaroxaban and their reversal with prothrombin complex concentrate (PCC) and activated recombinant factor VII (rFVIIa) in vitro. Methods: Rivaroxaban was solubilized, and PCC and rFVIIa were added in 2 concentrations to the rivaroxaban-spiked blood samples, and thromboelastometry and measurements were performed. Results: Rivaroxaban increased tissue factor-activated clotting time (CTExTEM) dose dependently. Activated partial prothrombin time (aPTT), prothrombin time ratio (PTR), and prothrombin time (PT) were changed significantly in both concentrations. Reversal with PCC in both dosages caused no significant change in the measured parameters. For prophylactic rivaroxaban dosage, rFVIIa changed the PT significantly but not CTExTEM, aPTT, and PTR. For therapeutic rivaroxaban dosage, the CTExTEMwas significantly reduced. The other parameters remained unaffected. Conclusions: Thromboelastometry can detect rivaroxaban effects. In vitro rFVIIa seems highly effective for reversal in contrast to PCC. Copyright © The Author(s) 2013.


Rubelt M.S.,Charité - Medical University of Berlin | Amasheh S.,Charité - Medical University of Berlin | Grobosch T.,Labor Berlin Charite Vivantes GmbH | Stein C.,Charité - Medical University of Berlin
PLoS ONE | Year: 2012

Analysis of in vitro samples with high salt concentrations represents a major challenge for fast and specific quantification with liquid chromatography-tandem mass spectrometry (LC-MS/MS). To investigate the intestinal permeability of opioids in vitro employing the Ussing chamber technique, we developed and validated a fast, sensitive and selective method based on LC-MS/MS for the determination of loperamide in HEPES-buffered Ringer's solution. Chromatographic separation was achieved with an Atlantis dC18 column, 2.1 mm×20 mm, 3μm particle size and a gradient consisting of methanol/0.1% formic acid and ammonium acetate. The flow rate was 0.7 ml/min, and the total run time was 3min. For quantification, two mass transitions for loperamide and a deuterated internal standard (methadone-d3) were used. The lower limit of loperamide quantification was 0.2 ng/ml. This new LC-MS/MS method can be used for the detection of loperamide in any experimental setup using HEPES-buffered Ringer's solution as a matrix compound. © 2012 Rubelt et al.


Mut L.,TU Berlin | Grobosch T.,Labor Berlin Charite Vivantes GmbH | Binscheck-Domass T.,Labor Berlin Charite Vivantes GmbH | Frenzel W.,TU Berlin
Biomedical Chromatography | Year: 2016

A multi-analyte screening method for the quantification of 50 acidic/neutral drugs in human plasma based on on-line solid-phase extraction (SPE)-HPLC with photodiode array detection (DAD) was developed, validated and applied for clinical investigation. Acetone and methanol for protein precipitation, three different SPE materials (two electro-neutral, one strong anion-exchange, one weak cation-exchange) for on-line extraction, five HPLC-columns [one C18 (GeminiNX), two phenyl-hexyl (Gemini C6-Phenyl, Kinetex Phenyl-Hexyl) and two pentafluorophenyl (LunaPFP(2), KinetexPFP)] for analytical separation were tested. For sample pre-treatment, acetone in the ratio 1:2 (plasma:acetone) showed a better baseline and fewer matrix peaks in the chromatogram than methanol. Only the strong anion-exchanger SPE cartridge (StrataX-A, pH 6) allowed the extraction of salicylic acid. Analytical separation was carried out on a Gemini C6-Phenyl column (150 × 4.6 mm, 3 μm) using gradient elution with acetonitrile-water 90:10 (v/v) and phosphate buffer (pH 2.3). Linear calibration curves with correlation coefficients r ≥ 0.9950/0.9910 were obtained for 46/four analytes. Additionally, this method allows the quantification of 23 analytes for therapeutic drug monitoring. Limits of quantitation ranged from 0.1 (amobarbital) to 23 mg/L (salicylic acid). Inter-/intra-day precisions of quality control samples (low/high) were better than 13% and accuracy (bias) ranged from -14 to 10%. A computer-assisted database was created for automated detection of 223 analytes of toxicological interests. Four cases of multi-drug intoxications are presented. © 2016 John Wiley & Sons, Ltd.

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