Peter Schierack

Brandenburg University of Technology Cottbus - Senftenberg, Kottbus, Brandenburg, Germany

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Publications (61)176.19 Total impact

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    ABSTRACT: Mallard ducks have demonstrated to be a likely reservoir for zoonotic E. coli strains; thus, it is possible that these ducks could also act as a reservoir for other Enterobacteriaceae members. The present study was initiated to evaluate the species distribution of Enterobacteriaceae other than E. coli in 175 fresh faecal samples collected from a population of mallard ducks. Sixty-four samples displayed detectable colonies of Enterobacteriaceae (excluding E. coli), which resulted in 75 pulsed-field gel electrophoresis (PFGE) types. Seventy-five single representatives of each PFGE type were subjected to identification with API 32NE and MALDI TOF MS systems due to the practical difficulties in species differentiation of Enterobacteriaceae. Those isolated were found to be from nine genera: Buttiauxella (15 %), Citrobacter (5 %), Enterobacter (32 %), Hafnia (1 %), Leclercia (1 %), Pantoea (7 %), Raoultella (21 %), Rahnella (7 %) and Serratia (11 %). Evaluation of antimicrobial resistance phenotypes using the disc method and detection of resistance genes using the microarray method revealed that these microbes possess resistance to β-lactams, aminoglycosides, macrolides, quinolones, rifamycine, sulphonamides, streptogramins and diaminopyrimidines. In conclusion, mallard ducks harbour a variety of non-pathogenic and pathogenic Enterobacteriaceae species like Enterobacter cloacae and Enterobacter amnigenus in their intestine and could act as a reservoir of resistant Enterobacteriaceae.
    Environmental Monitoring and Assessment 03/2015; 187(3):4346. · 1.68 Impact Factor
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    ABSTRACT: We report the population structure and dynamics of one E. coli population of wild mallard ducks in their natural environment over four winter seasons, following the characterization of 100 isolates each consecutive season. Macrorestriction analysis was used to define isolates variously as multi- or one-year PFGE types. Isolates were characterized genotypically based on virulence-associated genes (VAGs), phylogenetic markers, and phenotypically based on hemolytic activity, antimicrobial resistance, adhesion to epithelial cells, microcin production, motility and carbohydrate metabolism. Only 12 out of 220 PFGE types were detectable over more than one winter, and classified as multi-year PFGE types. There was a dramatic change of PFGE types within two winter seasons. Nevertheless, the genetic pool (VAGs) and antimicrobial resistance pattern remained remarkably stable. The high diversity and dynamics of this E. coli population were also demonstrated by the occurrence of PFGE subtypes and differences between isolates of one PFGE type (based on VAGs, antimicrobial resistance, and adhesion rates). Multi- and one-year PFGE types differed in antimicrobial resistance, VAGs and adhesion. Other parameters were not prominent colonization factors. In conclusion, the high diversity, dynamics and stable genetic pool of an E. coli population seems to enable their successful colonization of host animal population overtime. This article is protected by copyright. All rights reserved.
    Environmental Microbiology 02/2015; · 6.24 Impact Factor
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    ABSTRACT: Background: Quantification cycle (Cq) and amplification efficiency (AE) are parameters mathematically extracted from raw data to characterize quantitative PCR (qPCR) reactions and quantify the copy number in a sample. Little attention has been paid to the effects of preprocessing and the use of smoothing or filtering approaches to compensate for noisy data. Existing algorithms largely are taken for granted, and it is unclear which of the various methods is most informative. We investigated the effect of smoothing and filtering algorithms on amplification curve data. Methods: We obtained published high-replicate qPCR datasets from standard block thermocyclers and other cycler platforms and statistically evaluated the impact of smoothing on Cq and AE. Results: Our results indicate that selected smoothing algorithms affect estimates of Cq and AE considerably. The commonly used moving average filter performed worst in all qPCR scenarios. The Savitzky–Golay smoother, cubic splines, and Whittaker smoother resulted overall in the least bias in our setting and exhibited low sensitivity to differences in qPCR AE, whereas other smoothers, such as running mean, introduced an AE-dependent bias. Conclusions: The selection of a smoothing algorithm is an important step in developing data analysis pipelines for real-time PCR experiments. We offer guidelines for selection of an appropriate smoothing algorithm in diagnostic qPCR applications. The findings of our study were implemented in the R packages chipPCR and qpcR as a basis for the implementation of an analytical strategy.
    Clinical Chemistry 12/2014; · 7.77 Impact Factor
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    ABSTRACT: Abstract Background: Autoantibodies to exocrine-pancreatic glycoprotein 2 (anti-GP2) are Crohn's disease (CD) markers. However, CD-specific antibodies have also been found in celiac-disease (CeD) patients, in which type 1 diabetes-specific autoantibodies against endocrine pancreatic targets can be present. We investigated whether anti-GP2 are also present in CeD, a disease like CD which is also characterised by intestinal mucosal inflammation with barrier impairment. Methods: Antibodies against GP2, tissue transglutaminase (tTG), deamidated gliadin (dGD), glutamic decarboxylase (GAD), and islet antigen-2 (IA2) were tested in sera from 73 CD patients, 90 blood donors (BD), and 79 (58 de novo) CeD patients (2 consecutive sera were available from 40 patients). Results: IgA and/or IgG anti-GP2 were found in 15/79 (19.0%) CeD patients on at least one occasion, in 25/73 (34.2%) CD patients, and in 4/90 (4.4%) BD (CeD vs. CD, p=0.042; BD vs. CeD and CD, p<0.001, respectively). Amongst the 58 de novo CeD patients, anti-GP2 IgA and/or IgG were present in 11 (19.0%). Anti-GP2 IgA was significantly less prevalent in CeD compared with CD (p=0.004). Anti-GP2 IgA and IgG in CD patients demonstrated a significantly higher median level compared to patients with CeD (p<0.001, p=0.008, respectively). IgA anti-GP2 levels correlated significantly with IgA anti-tTG and anti-dGD levels in CeD Spearman's coefficient of rank correlation (ρ)=0.42, confidence interval (CI): 0.26-0.56, p<0.001; ρ=0.54, CI 0.39-0.65, p<0.001, respectively. Conclusions: The presence of anti-GP2 in CeD patients supports the notion that loss of tolerance to GP2 can probably be a manifestation of an autoinflammatory process in this intestinal disorder.
    Clinical Chemistry and Laboratory Medicine 11/2014; · 2.96 Impact Factor
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    ABSTRACT: Due to increased release or production of Shiga toxin by Enterohemorrhagic Escherichia coli (EHEC) after exposure to antimicrobial agents, the role of antimicrobial agents in EHEC mediated infections remains controversial. Probiotics are therefore rapidly gaining interest as an alternate therapeutic option. The well-known probiotic strain Escherichia coli Nissle 1917 (EcN) was tested in vitro to determine its probiotic effects on growth, Shiga toxin (Stx) gene expression, Stx amount and associated cytotoxicity on the most important EHEC strains of serotype O104:H4 and O157:H7. Following co-culture of EcN:EHEC in broth for 4 and 24 h, the probiotic effects on EHEC growth, toxin gene expression, Stx amount and cytotoxicity were determined using quantitative real time-PCR, Stx-ELISA and Vero cytotoxicity assays.Probiotic EcN strongly reduced EHEC numbers (cfu) of O104:H4 up to (68%) and O157:H7 to (72.2%) (P < 0.05) in LB broth medium whereas the non-probiotic E. coli strain MG1655 had no effect on EHEC growth. The level of stx expression was significantly down-regulated, particularly for the stx2a gene. The stx down-regulation in EcN co-culture was not due to reduced numbers of EHEC. A significant inhibition in Stx amounts and cytotoxicity were also observed in sterile supernatants of EcN:EHEC co-cultures.These findings indicate that probiotic EcN displays strong inhibitory effects on growth, Shiga toxin gene expression, amount and cytotoxicity of EHEC strains. Thus, EcN may be considered as a putative therapeutic candidate, in particular against EHEC O104:H4 and O157:H7.
    International Journal of Medical Microbiology 10/2014; 305(1). · 3.42 Impact Factor
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    ABSTRACT: The international consensus for the classification of antiphospholipid syndrome (APS) requires clinical and laboratory criteria to be considered at an equal level for diagnosing APS. Thus, detection of antiphospholipid antibodies (aPL) being a hallmark of APS has been the object of intensive investigation over the past 40 years. However, appropriate detection of aPL still remains a laboratory challenge due to their heterogeneity comprising autoantibodies reactive to different phospholipid-binding plasma proteins, such as beta-2 glycoprotein I (β2GPI) and prothrombin. The relevance of aPL interacting with phospholipids other than cardiolipin (CL, diphosphatidylglycerol), such as phosphatidylserine (PS), remains elusive with regard to the diagnosis of APS. Recently, the concept of aPL profiling has been introduced to assess the risk of thrombotic complications in patients with APS. New assay techniques, apart from enzyme-linked immunosorbent assays (ELISAs) recommended by the international consensus for the classification of APS, have been proposed for multiplexing of aPL testing. Line immunoassays (LIAs) employing a novel hydrophobic solid phase for the simultaneous detection of different aPL seem to be an intriguing alternative. We evaluated a novel multiplex LIA employing a hydrophobic membrane coated with different phospholipid (PL)-binding proteins or PLs. The performance characteristics of this new multiplexing assay technique demonstrated its usefulness for aPL profiling.
    Lupus 10/2014; 23(12):1262-4. · 2.48 Impact Factor
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    ABSTRACT: Anti-neutrophil cytoplasmic antibodies (ANCA) are the serological hallmark of small vessel vasculitis, so called ANCA-associated vasculitis. The international consensus requires testing by indirect immunofluorescence (IIF) on human ethanol-fixed neutrophils (ethN) as screening followed by confirmation with enzyme-linked immunosorbent assays (ELISAs). This study evaluates the combination of cell- and microbead-based digital IIF analysis of ANCA in one reaction environment by the novel multiplexing CytoBead technology for simultaneous screening and confirmatory ANCA testing. Sera of 592 individuals including 118 patients with ANCA-associated vasculitis, 133 with rheumatoid arthritis, 49 with infectious diseases, 77 with inflammatory bowel syndrome, 20 with autoimmune liver diseases, 70 with primary sclerosing cholangitis and 125 blood donors were tested for cytoplasmic ANCA (C-ANCA) and perinuclear ANCA (P-ANCA) by classical IIF and ANCA to proteinase 3 (PR3) and myeloperoxidase (MPO) by ELISA. These findings were compared to respective ANCA results determined by automated multiplex CytoBead technology using ethN and antigen-coated microbeads for microbead immunoassays. There was a good agreement for PR3- and MPO-ANCA and a very good one for P-ANCA and C-ANCA by classical and multiplex analysis (Cohen's kappa [κ] = 0.775, 0.720, 0.876, 0.820, respectively). The differences between classical testing and CytoBead analysis were not significant for PR3-ANCA, P-ANCA, and C-ANCA (p<0.05, respectively). The prevalence of confirmed positive ANCA findings by classical testing (IIF and ELISA) compared with multiplex CytoBead analysis (IIF and microbead immunoassay positive) resulted in a very good agreement (κ = 0.831) with no significant difference of both methods (p = 0.735). Automated endpoint-ANCA titer detection in one dilution demonstrated a very good agreement with classical analysis requiring dilution of samples (κ = 0.985). Multiplexing by CytoBead technology can be employed for simultaneous screening and quantitative confirmation of ANCA. This novel technique provides fast and cost-effective ANCA analysis by automated digital IIF for the first time.
    PLoS ONE 09/2014; 9(9):e107743. · 3.53 Impact Factor
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    ABSTRACT: Microbead-based technologies represent elegant and versatile approaches for highly parallelized quantitative multiparameter assays. They also form the basis of various techniques for detection and quantification of nucleic acids and proteins. Nucleic acid-based methods include hybridization assays, solid-phase PCR, sequencing, and trapping assays. Microbead assays have been improved in the past decades and are now important tools in routine and point-of-care diagnostics as well as in life science. Its advances include low costs, low workload, high speed and high-throughput automation. The potential of microbead-based assays therefore is apparent, and commercial applications can be found in the detection and discrimination of single nucleotide polymorphism, of pathogens, and in trapping assays. This review provides an overview on microbead-based platforms for biosensing with a main focus on nucleic acid detection (including amplification strategies and on selected probe systems using fluorescent labeling). Specific sections cover chemical properties of microbeads, the coupling of targets onto solid surfaces, microbead probe systems (mainly oligonucleotide probes), microbead detection schemes (with subsections on suspension arrays, microfluidic devices, and immobilized microbeads), quantification of nucleic acids, PCR in solution and the detection of amplicons, and methods for solid-phase amplification. We discuss selected trends such as microbead-coupled amplification, heterogeneous and homogenous DNA hybridization assays, real-time assays, melting curve analysis, and digital microbead assays. We finally discuss the relevance and trends of the methods in terms of high-level multiplexed analysis and their potential in diagnosis and personalized medicine. Contains 211 references.
    Microchimica Acta 08/2014; · 3.72 Impact Factor
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    ABSTRACT: Dear Editor, With interest we read the paper by Juste et al1 proposing the amount of zymogen-granule membrane glycoprotein 2 (GP2) on the surface of intestinal bacteria as a Crohn's disease (CD) marker. Indeed, a decreased GP2 level was found on microbes in patients with CD as compared to those of healthy controls. GP2 is a homologue to the urinary Tamm–Horsefall protein demonstrating an antimicrobial function by binding type 1-fimbriated uropathogenic Escherichia coli (UPEC). Likewise, GP2 seems to interact with intestinal bacteria as a specific receptor of bacterial type-1 fimbriae (FimH) on intestinal microfold cells that are partaking in immune responses against such microbes.2 GP2 is overexpressed in the inflamed intestine of patients with CD and has an immunomodulating role in innate and acquired immune responses.3 ,4 Interestingly, GP2 was identified as autoantigen of pancreatic antibodies in CD.4 Altogether, these findings indicate two major GP2 sources (pancreatic/intestinal) and support a role for GP2 in the interaction between the immune system and intestinal microbiota.3 Thus, loss of tolerance to GP2 could play a role in CD's pathophysiology supposed to be exacerbated by preceding intestinal infections. In general, the findings by Juste et al1 may be explained by a lower pancreatic GP2 secretion, an impaired GP2 binding to bacteria, or by a higher prevalence of bacteria with poor or no GP2 binding in patients with CD.
    Gut 07/2014; 64(3). · 13.32 Impact Factor
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    ABSTRACT: The edition of this Special Issue was commenced in 2013 at the occasion of the 60th anniversary of the elucidation of DNA structure. This milestone has completely changed biological and medical sciences and, more recently, has triggered the development of sophisticated instrumentation. The discovery of the polymerase chain reaction (PCR) has further promoted this trend and now allows the analysis of a DNA sequences even at a level of a single molecule. Similarly, various miniaturized chip-based approaches have been introduced in the past 10 years. The transition from a laboratory scale to a microscale implies many advantages. These include, in particular, reduced sample volumes, reduced costs, shorter assay times, faster heating/cooling rates, higher throughput, and the integration of processing module cascades. The chip approach enabled, in particular, the development of sophisticated PCR assays. These include single-well and multi-well continuous flow PCR devices, stationary chamber-based PCR chips, and planar microbead-based chips. Most of these systems have common components such as miniaturized reaction chambers, heating/cooling units, and an analyzer/detector unit. Some of the devices evolved from the end-point quantification to the multiplex real-time monitoring of amplification reactions and high-resolution analysis of melting curves. These technologies contributed to breakthroughs that were envisioned years before. In particular, PCR in microfluidics and isothermal amplification have radically changed medical diagnostics and life sciences in general. Any development in field is based on the efforts of experts from highly different fields, examples being molecular biology, surface chemistry, microfluidics, and of course engineering. Systems are becoming smaller and more widely applicable, for example to diagnostics, environmental monitoring, and life sciences. This Special Issue gives a representative selection of the progress made in this field and in technologies related to affinity biosensors in a wider sense. The Issue includes reviews and original papers. Many of them are based on presentations given at the 7th Senftenberg Innovationsforum on Multiparameter Diagnostics in April 2013 which was organized by Brandenburg University of Technology Cottbus - Senftenberg (Germany). We have mainly included manuscripts on new analytical techniques, on end-user applications, new instrumentation, sensors and materials, with a main focus on the analysis of nucleic acids. The first part (4 reviews) gives an overview on current technologies such as extraction, amplification and detection of DNA in microfluidic chip-based and microbead-based assays. This includes recent developments in solid-phase enzymatic assays and the application in multiparametric diagnostics. The second part (Chip-based technologies for amplification, detection and analyses of nucleic acids) mainly focuses on end-point and real-time PCR techniques. Articles cover topics ranging from fundamental research to commercial applications. Part 3 (Application of chip-based technologies for amplification, detection and analyses) covers aspects of reference gene selection, quantification of gene expression, PCR in microfluidics, microarray technology in PCR, conventional and isothermal amplification methods as well as fluorescent detection. The final part (Chip-based technologies for affinity sensing) focuses on novel techniques for use in affinity sensing which have a wide application potential including analysis of nucleic acids. We are confident that this Special Issue represents a timely overview on this field and will fuel novel ideas for microanalytical systems. Last, but not least, we would like to thank the authors of this Special Issue for their excellent articles. We also are grateful to the reviewers for their numerous constructive comments on these manuscripts. We truly hope that readers will enjoy reading the result of this collective effort. V. M. Mirsky, S. Rödiger, P. Schierack, D. Roggenbuck (Editors) Brandenburg University of Technology, Cottbus – Senftenberg (Germany)
    Microchimica Acta 04/2014; · 3.43 Impact Factor
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    ABSTRACT: Autoantibodies are well-established biomarkers of autoimmune rheumatic diseases. Their detection in the routine clinical laboratory provides key information for early and differential diagnosis, prognosis, and monitoring of disease-activity. However, the currently existing variety of laboratory tests and methods as well as different diagnostic and clinical algorithms of autoimmune testing is the reason for lack of reproducibility and harmonization across immunological laboratories. Novel technical developments in the area of digital analysis of immunofluorescent images paved the way for the improvement of intra- and interlaboratory standardizationof test results. Additionally, digital immunofluorescence analysis enables the simultaneous multiparametric combination of cell- or tissue-based indirect immunofluorescence screening tests with confirmatory testing employing microparticles covered with purified autoantigens in one reaction environment.
    Medical Immunology 03/2014; 16(3):221-226.
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    ABSTRACT: Enteropathogenic Escherichia coli (EPEC) are recognized as important intestinal pathogens that frequently cause acute and persistent diarrhea in humans and animals. The use of probiotic bacteria to prevent diarrhea is gaining increasing interest. The probiotic E. coli strain Nissle 1917 (EcN) is known to be effective in the treatment of several gastro-intestinal disorders. While both in vitro and in vivo studies have described strong inhibitory effects of EcN on enteropathogenic bacteria including pathogenic E. coli, the underlying molecular mechanisms remain largely unknown. In this study, we examined the inhibitory effect of EcN on infections of porcine intestinal epithelial cells with atypical enteropathogenic E. coli (aEPEC) with respect to single infection steps including adhesion, microcolony formation and attaching and effacing phenotype. We show that EcN drastically reduced the infection efficiencies of aEPEC by inhibiting bacterial adhesion and growth of microcolonies, but not the attaching and effacing of adherent bacteria. The inhibitory effect correlated with EcN adhesion capacities and was predominantly mediated by F1C fimbriae, but also by H1 flagellae, which served as bridges between EcN cells. Furthermore, EcN seemed to interfere with the initial adhesion of aEPEC to host cells by secretion of inhibitory components. These components do not appear to be specific for EcN but we propose that the strong adhesion capacities enable EcN to secrete sufficient local concentrations of these inhibitory factor. The results of this study are consistent with a mode of action whereby EcN inhibits secretion of virulence associated proteins of EPEC, but not their expression.
    Infection and immunity 02/2014; · 4.16 Impact Factor
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    ABSTRACT: Bei Verdacht auf Vorliegen einer systemischen Autoimmunerkrankung wird für die serologische Routinediagnostik ein Zwei-Stufen-Verfahren empfohlen. Zuerst werden Autoantikörpern (AAK) mittels sensitiver zellbasierter indirekter Immunfluoreszenz (IIF)-Teste bestimmt. Ein positives Ergebnis muss aufgrund der Möglichkeit von falsch-positiven Ergebnissen mit einem weiteren, spezifischen Test bestätigt werden. Dieses sukzessive Vorgehen ist notwendig, da zurzeit keine Assaytechnik die notwendigen Anforderungen an ein einstufiges Verfahren hinsichtlich Sensitivität und Spezifität erfüllt. Im Sinne einer effektiven AAK-Diagnostik kann heute schon eine simultane Bestimmung von mehreren AAK mittels multiparametrischer Bestätigungstests die Diagnosefindung im Vergleich zu konventionellen, monoparametrischen Tests wesentlich verkürzen. Jedoch erlauben die verfügbaren multiparametrischen AAK-Nachweismethoden nicht die Kombination von Screening- und Bestätigungstesten. Deshalb wurde basierend auf der digitalen Fluoreszenz mit der hier vorgestellten CytoBead Technologie ein neuer Ansatz entwickelt. Ziel war die Kombination der empfohlenen Stufendiagnostik bestehend aus sensitivem Screening und spezifischer Bestätigungsdiagnostik in einer Reaktionsumgebung und darüber hinaus die Möglichkeit der Adaption auf die serologische Diagnostik mehrerer Autoimmunerkrankungen. Durch a) die Nutzung von Standardglasobjektträgern, b) die Kombination von nativen zellulären oder Gewebesubstraten mit antigenbeladenen fluoreszierenden Mikropartikeln (Beads) in einer Reaktionsumgebung, c) die Möglichkeit der manuellen und automatischen Auswertung mittels IIF und d) die Erhebung von quantitativen Fluoreszenzmessergebnissen konnten die Nachteile der bisher bestehenden Testsysteme überwunden werden. Das neue Prinzip ist auf verschiedene multiparametrische AAK-Nachweise wie zum Beispiel die Bestimmung von antinukleären Antikörpern und AAK gegen entsprechende nukleäre und zytoplasmatische autoantigene Zielstrukturen anwendbar. Damit wurde weiterhin die Basis für die simultane AAK-Multiparameterbestimmung für die Serologie der Zöliakie und von ANCA-assoziierten systemischen Vaskulitiden geschaffen.
    Laboratoriums Medizin 01/2014; 38(6):309-317.
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    ABSTRACT: Heterogeneous nuclear ribonucleoproteins (hnRNPs) are potent autoantigenic targets in systemic autoimmune rheumatic diseases (SARD). Loss of tolerance to the RA33 complex consisting of hnRNP A2 and its alternatively spliced variants B1 and B2 has been the interest of rheumatologists. A novel ELISA for the detection of anti-hnRNP B1 autoantibodies has been developed to investigate the prevalence thereof in 397 patients with SARD, including patients with rheumatoid arthritis (RA), spondyloarthropathy (SPA), juvenile chronic arthritis, systemic lupus erythematosus (SLE), systemic sclerosis (SSc), and Sjögren's syndrome (SS), in comparison to 174 controls. Anti-hnRNP B1 autoantibodies were significantly more prevalent in patients with SARD than controls (47/397, 11.8% versus 2/174, 1.1%; P < 0.001). In particular, anti-hnRNP B1 were found more frequently in the disease cohorts than in the controls and were present in 24/165 (14.5%) patients with RA, 6/58 (10.3%) SPA, 11/65 (16.9%) SSc, and 4/50 (8.0%) SLE. In RA patients, anti-hnRNP B1 autoantibodies correlated significantly with C-reactive protein levels and erythrocyte sedimentation rate, while in patients with SSc it was associated with features of arterial wall stiffness and presence of hypertension. Anti-hnRNP B1 autoantibodies occur in SARD and seem to be correlated with distinct clinical characteristics in patients with RA and SSc.
    Research Journal of Immunology 01/2014; 2014:516593.
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    ABSTRACT: Abstract Crohn's disease (CrD) and ulcerative colitis (UC) are the main inflammatory bowel diseases (IBD). IBD-specific humoral markers of autoimmunity in the form of autoantibodies have been reported first in the late 1950s by demonstrating the occurrence of autoimmunity in UC, while humoral autoimmunity in CrD can be traced back to the 1970s. Ever since, the pathophysiological role of autoimmune responses in IBDs has remained poorly understood. Notwithstanding, autoreactive responses play a major role in inflammation leading to overt IBD. In CrD, approximately 40% of patients and <20% of patients with UC demonstrate loss of tolerance to antigens of the exocrine pancreas. Glycoprotein 2 (GP2) has been identified as a major autoantigenic target of the so-called pancreatic antibodies. The previously unsolved contradiction of pancreatic autoreactivity and intestinal inflammation in IBD was elucidated by demonstrating the expression of GP2 at the site thereof. Intriguingly, GP2 has been reported to be a receptor on microfold cells of intestinal Peyer's patches, which are believed to represent the origin of CrD inflammation. The development of immunoassays for the detection of antibodies to GP2 has paved the way to investigate the association of such antibodies with the clinical phenotype in CrD. Given the recently discovered immunomodulating role of GP2 in innate and adaptive intestinal immunity, this association can shed further light on the pathophysiology of IBD. In this context, the association of anti-GP2 autoantibodies as novel CrD-specific markers with the clinical phenotype in CrD will be discussed in this review.
    Clinical Chemistry and Laboratory Medicine 11/2013; · 2.96 Impact Factor
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    ABSTRACT: Different strategies of colonization or infection by E. coli result in formation of certain adhesion patterns which help also in classifying intestinal E. coli into pathotypes. Little is known about adhesion patterns and host- and tissue adaption of commensal E. coli and about E. coli originating in clinically healthy hosts carrying pathotype-specific virulence-associated genes. Adhesion pattern of E. coli (n = 282) from humans and from 18 animal species were verified on intestinal human Caco-2 and porcine IPEC-J2 cells and, furthermore, for comparison on human urinary bladder 5637, porcine kidney PK-15 epithelial and HEp-2 cells. The analysis was carried out on 150,000 images of adhesion assays.Adhesion patterns were very diverse; 88 isolates were completely non-adherent, whereas 194 adhered to at least one cell line with the dominant adhesion patterns "diffusely distributed" and "microcolony formation". Adhesion patterns "chains" and "clumps" were also visible. Chain formation was mediated by the presence of epithelial cells. Clump formation was very specific on only the 5637 cell line. All enteropathogenic (eae+) E. coli (EPEC; n = 14) were able to form microcolonies which was cell line specific for each isolate. Most EPEC formed microcolonies on intestinal IPEC-J2 and Caco-2 but several also on urinary tract cells. Shigatoxin-producing (stx+) E. coli (n = 10) showed no specific adhesion patterns. E. coli isolates were highly diverse. Commensal and pathogenic isolates can adhere in various forms, including diffuse distribution, microcolonies, chains and clumps. Microcolony formation seems to be a global adhesion strategy also for commensal E. coli.
    Gut Pathogens 11/2013; 5(1):31. · 2.07 Impact Factor
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    ABSTRACT: Feed supplementation with the probiotic Enterococcus (E.) faecium to piglets has been found to reduce pathogenic gut microorganisms. As Escherichia (E.) coli is among the most important pathogens in pig production, we performed a comprehensive analyses to gain further insight on the influence of E. faecium NCIMB 10415 on porcine intestinal E. coli.A total of 1,436 E. coli were isolated from three intestinal habitats (mucosa, digesta, feces) of probiotic-supplemented and non-supplemented (control) piglets. E. coli were characterized via Pulsed-field gel electrophoresis (PFGE) for clonal analysis. The high diversity of E. coli was reflected by 168 clones. Multi-locus sequence typing (MLST) was used to determine the phylogenetic backgrounds which revealed 79 sequence types (STs). Pathotypes of E. coli were further defined using multiplex-PCR for virulence-associated genes.While these analyses discerned only few significant differences in the E. coli population between the feeding groups, analyses distinguishing clones that were uniquely isolated either in the probiotic, the control or in both groups revealed clear effects at the habitat level. Interestingly, ExPEC-typical clones adhering to the mucosa were significantly reduced in the probiotic group.Our data show a minor influence of E. faecium on the overall population of E. coli in healthy piglets. In contrast, this probiotic has a profound effect on mucosa-adherent E. coli. This finding further substantiates a specific effect of E. faecium strain NCIMB 10415 in piglets against pathogenic E. coli in the intestine. In addition, these data question the relevance of data based on sampling fecal E. coli only.
    Applied and Environmental Microbiology 10/2013; · 3.95 Impact Factor
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    ABSTRACT: Analysis of phosphorylated histone protein H2AX (γH2AX) foci is currently the most sensitive method to detect DNA double-strand breaks (DSB). This protein modification has the potential to become an individual biomarker of cellular stress, especially in the diagnosis and monitoring of neoplastic diseases. To make γH2AX foci analysis available as a routine screening method, different software approaches for automated immunofluorescence pattern evaluation have recently been developed. In this study, we used novel pattern recognition algorithms on the AKLIDES(®) platform to automatically analyze immunofluorescence images of γH2AX foci and compared the results with visual assessments. Dose- and time-dependent γH2AX foci formation was investigated in human peripheral blood mononuclear cells (PBMCs) treated with the chemotherapeutic drug etoposide (ETP). Moreover, the AKLIDES system was used to analyze the impact of different immunomodulatory reagents on γH2AX foci formation in PBMCs. Apart from γH2AX foci counting the use of novel pattern recognition algorithms allowed the measurement of their fluorescence intensity and size, as well as the analysis of overlapping γH2AX foci. The comparison of automated and manual foci quantification showed overall a good correlation. After ETP exposure, a clear dose-dependent increase of γH2AX foci formation was evident using the AKLIDES as well as Western blot analysis. Kinetic experiments on PBMCs incubated with 5 μM ETP demonstrated a peak in γH2AX foci formation after 4 to 8 h, while a removal of ETP resulted in a strong reduction of γH2AX foci after 1 to 4 h. In summary, this study demonstrated that the AKLIDES system can be used as an efficient automatic screening tool for γH2AX foci analysis by providing new evaluation features and facilitating the identification of drugs which induce or modulate DNA damage. © 2013 International Society for Advancement of Cytometry.
    Cytometry Part A 09/2013; · 3.71 Impact Factor

Publication Stats

694 Citations
176.19 Total Impact Points

Institutions

  • 2011–2015
    • Brandenburg University of Technology Cottbus - Senftenberg
      • Faculty of Natural Sciences (Faculty 6)
      Kottbus, Brandenburg, Germany
  • 2011–2013
    • Fachhochschule der Wirtschaft
      Paderborn, North Rhine-Westphalia, Germany
  • 2003–2012
    • Freie Universität Berlin
      • • Institute of Microbiology and Epizootics
      • • Institute of Chemistry and Biochemistry
      Berlín, Berlin, Germany
  • 2009
    • Bundesamt für Verbraucherschutz
      Brunswyck, Lower Saxony, Germany
  • 2006
    • Bundesinstitut für Risikobewertung
      Berlín, Berlin, Germany