Publications by authors named "Jan Kehrmann"

25 Publications

  • Page 1 of 1

CRISPR/Cas9-mediated demethylation of FOXP3-TSDR toward Treg-characteristic programming of Jurkat T cells.

Cell Immunol 2022 Jan 20;371:104471. Epub 2021 Dec 20.

Institute of Medical Microbiology, University Hospital Essen, University of Duisburg-Essen, Germany. Electronic address:

Demethylation of FOXP3-TSDR (Treg specific demethylated region) is a hallmark of stable differentiation and suppressive function of regulatory T (Treg) cells. Previous protocols aiming at human naïve T cell differentiation failed to implement a Treg cell specific epigenetic signature. Ten-eleven translocation (TET) enzymes catalyze DNA demethylation. Plasmids towardexpression of a fusion protein encompassing nonfunctional Cas9, the catalytic domain of TET1, blue fluorescent protein, and encoding single guide RNAs (sgRNAs) targeting specific segments of the FOXP3-TSDR were engineered and transfected into Jurkat T cells. FOXP3-TSDR methylation was analyzed by deep-amplicon bisulfite sequencing while cellular Foxp3, Tbet, Gata3, and Rorgt mRNA levels were determined by real-time PCR. Overexpression of dCas9TET1 significantly decreased Jurkat cell FOXP3-TSDR methylation and increased Foxp3 mRNA expression while expressions of master transcription factor mRNAs of other major T cell lineages remained largely unaffected. dCas9-TET1 construct transfection mediated Treg programming of patients' primary T cells might be feasible.
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http://dx.doi.org/10.1016/j.cellimm.2021.104471DOI Listing
January 2022

A Pro-Inflammatory Gut Microbiome Characterizes SARS-CoV-2 Infected Patients and a Reduction in the Connectivity of an Anti-Inflammatory Bacterial Network Associates With Severe COVID-19.

Front Cell Infect Microbiol 2021 17;11:747816. Epub 2021 Nov 17.

Institute of Medical Microbiology, University Hospital Essen, University of Duisburg-Essen, Essen, Germany.

The gut microbiota contributes to maintaining human health and regulating immune responses. Severe COVID-19 illness is associated with a dysregulated pro-inflammatory immune response. The effect of SARS-CoV-2 on altering the gut microbiome and the relevance of the gut microbiome on COVID-19 severity needs to be clarified. In this prospective study, we analyzed the gut microbiome of 212 patients of a tertiary care hospital (117 patients infected with SARS-CoV-2 and 95 SARS-CoV-2 negative patients) using gene sequencing of the V3-V4 region. Inflammatory markers and immune cells were quantified from blood. The gut microbiome in SARS-CoV-2 infected patients was characterized by a lower bacterial richness and distinct differences in the gut microbiome composition, including an enrichment of the phyla Proteobacteria and Bacteroidetes and a decrease of Actinobacteria compared to SARS-CoV-2 negative patients. The relative abundance of several genera including , and was lower in SARS-CoV-2 positive patients while the abundance of and was increased. Higher pro-inflammatory blood markers and a lower CD8 T cell number characterized patients with severe COVID-19 illness. The gut microbiome of patients with severe/critical COVID-19 exhibited a lower abundance of butyrate-producing genera and and a reduction in the connectivity of a distinct network of anti-inflammatory genera that was observed in patients with mild COVID-19 illness and in SARS-CoV-2 negative patients. Dysbiosis of the gut microbiome associated with a pro-inflammatory signature may contribute to the hyperinflammatory immune response characterizing severe COVID-19 illness.
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http://dx.doi.org/10.3389/fcimb.2021.747816DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8635721PMC
December 2021

Post-ischemic protein restriction induces sustained neuroprotection, neurological recovery, brain remodeling, and gut microbiota rebalancing.

Brain Behav Immun 2022 Feb 27;100:134-144. Epub 2021 Nov 27.

Department of Neurology, University Hospital Essen, Essen, Germany; Center for Translational and Behavioral Neurosciences, University Hospital Essen, Essen, Germany. Electronic address:

Background: Moderate dietary protein restriction confers neuroprotection when applied before ischemic stroke. How a moderately protein-reduced diet influences stroke recovery when administered after stroke, is a clinically relevant question. This question has not yet been investigated.

Methods: Male C57BL6/J mice were exposed to transient intraluminal middle cerebral artery occlusion. Immediately after the stroke, mice were randomized to two normocaloric diets: a moderately protein-reduced diet containing 8% protein (PRD) or normal diet containing 20% protein (ND). Post-stroke neurological deficits were evaluated by a comprehensive test battery. Antioxidant and neuroinflammatory responses in the brain and liver were evaluated by Western blot and RTqPCR. Stroke-induced brain injury, microvascular integrity, glial responses, and neuroplasticity were assessed by immunohistochemistry. Fecal microbiota analysis was performed using 16S ribosomal RNA amplicon sequencing.

Results: We show that PRD reduces brain infarct volume after three days and enhances neurological and, specifically, motor-coordination recovery over six weeks in stroke mice. The recovery-promoting effects of PRD were associated with increased antioxidant responses and reduced neuroinflammation. Histochemical studies revealed that PRD increased long-term neuronal survival, increased peri-infarct microvascular density, reduced microglia/macrophage accumulation, increased contralesional pyramidal tract plasticity, and reduced brain atrophy. Fecal microbiota analysis showed reduced bacterial richness and diversity in ischemic mice on ND starting at 7 dpi. PRD restored bacterial richness and diversity at these time points.

Conclusion: Moderate dietary protein restriction initiated post-ischemic stroke induces neurological recovery, brain remodeling, and neuroplasticity in mice by mechanisms involving antiinflammation and, in the post-acute phase, commensal gut microbiota rebalancing.
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http://dx.doi.org/10.1016/j.bbi.2021.11.016DOI Listing
February 2022

Species-Specific Interferon-Gamma Release Assay for the Diagnosis of Complex Infection.

Front Microbiol 2021 12;12:692395. Epub 2021 Jul 12.

Institute for Transfusion Medicine, University Hospital Essen, University of Duisburg-Essen, Essen, Germany.

complex (MABC) infection has a devastating impact on the course of cystic fibrosis (CF) and non-CF lung disease. Diagnosis of MABC pulmonary disease is challenging, and current diagnostic approaches lack accuracy, especially in CF. In this study, we aimed to establish an MABC-specific interferon-γ release assay to detect host immune responses to MABC and improve diagnostics of MABC infection by the detection of antigen-specific T cells. Four species-specific proteins of MABC were overexpressed in an expression system. Purified proteins were used to stimulate peripheral blood mononuclear cells of study subjects in an ELISpot assay. Interferon-γ response of 12 subjects with established diagnosis of MABC infection (10 CF and two non-CF) was compared with 35 controls (22 CF and 13 non-CF) distributed to three control groups, 17 CF subjects without NTM infection, nine subjects with NTM infection other than MABC, and nine subjects with tuberculosis. Cellular responses in the MABC group were stronger than in the control groups, especially toward the protein MAB_0405c (39 vs. 4 spots per 300,000 PBMC, = 0.004; data represent mean values) in all patients and also in the subgroup of CF subjects (39 spots vs. 1 spot, = 0.003). Receiver operating characteristic curve analysis indicated that spot numbers of at least 20 were highly predictive of MABC infection (all patients: area under curve 0.773, sensitivity 58%, and specificity 94%; CF patients: area under curve 0.818, sensitivity 60%, and specificity 100%). In conclusion, we identified MAB_0405c as a protein that may stimulate MABC-specific interferon-γ secretion and may add to the diagnosis of MABC infection in affected patients.
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http://dx.doi.org/10.3389/fmicb.2021.692395DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8312262PMC
July 2021

GPR15 Facilitates Recruitment of Regulatory T Cells to Promote Colorectal Cancer.

Cancer Res 2021 06 16;81(11):2970-2982. Epub 2021 Mar 16.

Infection Immunology, Institute of Medical Microbiology, University Hospital Essen, University of Duisburg-Essen, Essen, Germany.

Colorectal cancer is one of the most frequent malignancies worldwide. Despite considerable progress in early detection and treatment, there is still an unmet need for novel antitumor therapies, particularly in advanced colorectal cancer. Regulatory T cells (Treg) are increased in the peripheral blood and tumor tissue of patients with colorectal cancer. Recently, transient ablation of tumor-associated Tregs was shown to foster CD8 T-cell-mediated antitumoral immunity in murine colorectal cancer models. However, before considering therapies on targeting Tregs in patients with cancer, detailed knowledge of the phenotype and features of tumor-associated Tregs is indispensable. Here, we demonstrate in a murine model of inflammation-induced colorectal cancer that tumor-associated Tregs are mainly of thymic origin and equipped with a specific set of molecules strongly associated with enhanced migratory properties. Particularly, a dense infiltration of Tregs in mouse and human colorectal cancer lesions correlated with increased expression of the orphan chemoattractant receptor GPR15 on these cells. Comprehensive gene expression analysis revealed that tumor-associated GPR15 Tregs have a Th17-like phenotype, thereby producing IL17 and TNFα. Gpr15 deficiency repressed Treg infiltration in colorectal cancer, which paved the way for enhanced antitumoral CD8 T-cell immunity and reduced tumorigenesis. In conclusion, GPR15 represents a promising novel target for modifying T-cell-mediated antitumoral immunity in colorectal cancer. SIGNIFICANCE: The G protein-coupled receptor 15, an unconventional chemokine receptor, directs Tregs into the colon, thereby modifying the tumor microenvironment and promoting intestinal tumorigenesis..
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http://dx.doi.org/10.1158/0008-5472.CAN-20-2133DOI Listing
June 2021

Stool and sputum microbiome during quinolone prophylaxis of spontaneous bacterial peritonitis: an exploratory study.

Gut Pathog 2020 30;12:51. Epub 2020 Oct 30.

Present Address: Department of Internal Medicine 1, University Hospital Frankfurt, Goethe University, Theodor-Stern-Kai 7, 60590 Frankfurt am Main, Germany.

Introduction: Quinolone prophylaxis is recommended for patients with advanced cirrhosis at high risk of spontaneous bacterial peritonitis (SBP) or with prior SBP. Yet, the impact of long-term antibiotic prophylaxis on the microbiome of these patients is poorly characterized.

Methods: Patients with liver cirrhosis receiving long-term quinolone prophylaxis to prevent SBP were prospectively included and sputum and stool samples were obtained at baseline, 1, 4 and 12 weeks thereafter. Both bacterial DNA and RNA were assessed with 16S rRNA sequencing. Relative abundance, alpha and beta diversity were calculated and correlated with clinical outcome.

Results: Overall, 35 stool and 19 sputum samples were obtained from 11 patients. Two patients died (day 9 and 12) all others were followed for 180 days. Reduction of Shannon diversity and bacterial richness was insignificant after initiation of quinolone prophylaxis (p > 0.05). Gut microbiota were significantly different between patients (p < 0.001) but non-significantly altered between the different time points before and after initiation of antibiotic prophylaxis (p > 0.05). A high relative abundance of > 20% during quinolone prophylaxis was found in three patients. Specific clinical scenarios (development of secondary infections during antibiotic prophylaxis or the detection of multidrug-resistant ) characterized these patients. Sputum microbiota were not significantly altered in individuals during prophylaxis.

Conclusion: The present exploratory study with small sample size showed that inter-individual differences in diversity of gut microbiota were high at baseline, yet quinolone prophylaxis had only a moderate impact. High relative abundances of during follow-up might indicate failure of or non-adherence to quinolone prophylaxis. However, our results may not be clinically significant given the limitations of the study and therefore future studies are needed to further investigate this phenomenon.
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http://dx.doi.org/10.1186/s13099-020-00389-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7596951PMC
October 2020

Two fatal cases of plague after consumption of raw marmot organs.

Emerg Microbes Infect 2020 Dec;9(1):1878-1880

National Center for Zoonotic Disease Ministry of Health, Ulaanbaatar, Mongolia.

Marmots are an important reservoir of and a source of human plague in Mongolia. We present two fatal cases of plague after consumption of raw marmot organs and discuss the distribution of natural foci of in Mongolia.
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http://dx.doi.org/10.1080/22221751.2020.1807412DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7473306PMC
December 2020

Molecular Profiling of Keratinocyte Skin Tumors Links Overabundance and Increased Human β-Defensin-2 Expression to Growth Promotion of Squamous Cell Carcinoma.

Cancers (Basel) 2020 Feb 26;12(3). Epub 2020 Feb 26.

Institute of Pathology, Medical University of Graz, Neue Stiftingtalstrasse 6, 8010 Graz, Austria.

The skin microbiota plays a prominent role in health and disease; however, its contribution to skin tumorigenesis is not well understood. We comparatively assessed the microbial community compositions from excision specimens of the main human non-melanoma skin cancers, actinic keratosis (AK), squamous cell carcinoma (SCC) and basal cell carcinoma (BCC). Keratinocyte skin tumors are characterized by significantly different microbial community compositions, wherein AK and SCC are more similar to each other than to BCC. Notably, in SCC, which represents the advanced tumor entity and frequently develops from AK, overabundance of Staphylococcus aureus, a known skin pathogen, was noted. Moreover, S. aureus overabundance was significantly associated with increased human β-defensin-2 (hBD-2) expression in SCC. By challenging human SCC cell lines with , a specific induction of hBD-2 expression and increased tumor cell growth was seen. Increased proliferation was also induced by directly challenging SCC cells with hBD-2. Together, our data indicate that a changed microbial community composition in SCC, specified by overabundance, might promote tumor cell growth via modulation of hBD-2 expression.
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http://dx.doi.org/10.3390/cancers12030541DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7139500PMC
February 2020

Case Report: Eye Infection: The First Human Case in Germany.

Am J Trop Med Hyg 2020 02;102(2):350-351

Department of Ophthalmology, University Hospital Essen, University Duisburg-Essen, Essen, Germany.

eye worm is a nematode transmitted by drosophilid flies not only primarily to carnivores and lagomorphs but also to humans. Only a few cases have been reported in Europe (Italy, France, and Portugal). Here, we report the first eye infection in a German patient.
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http://dx.doi.org/10.4269/ajtmh.19-0483DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7008349PMC
February 2020

Depletion of Foxp3 regulatory T cells is accompanied by an increase in the relative abundance of Firmicutes in the murine gut microbiome.

Immunology 2020 03 12;159(3):344-353. Epub 2019 Dec 12.

Institute of Medical Microbiology, University Hospital Essen, University of Duisburg-Essen, Essen, Germany.

A reciprocal interaction exists between the gut microbiota and the immune system. Regulatory T (Treg) cells are important for controlling immune responses and for maintaining the intestinal homeostasis but their precise influence on the gut microbiota is unclear. We studied the effects of Treg cell depletion on inflammation of the intestinal mucosa and analysed the gut microbiota before and after depletion of Treg cells using the DEpletion of REGulatory T cells (DEREG) mouse model. DNA was extracted from stool samples of DEREG mice and wild-type littermates at different time-points before and after diphtheria toxin application to deplete Treg cells in DEREG mice. The V3/V4 region of the 16S rRNA gene was used for studying the gut microbiota with Illumina MiSeq paired ends sequencing. Multidimensional scaling separated the majority of gut microbiota samples from late time-points after Treg cell depletion in DEREG mice from samples of early time-points before Treg cell depletion in these mice and from gut microbiota samples of wild-type mice. Treg cell depletion in DEREG mice was accompanied by an increase in the relative abundance of the phylum Firmicutes and by intestinal inflammation in DEREG mice 20 days after Treg cell depletion, indicating that Treg cells influence the gut microbiota composition. In addition, the variables cage, breeding and experiment number were associated with differences in the gut microbiota composition and these variables should be respected in murine studies.
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http://dx.doi.org/10.1111/imm.13158DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7011623PMC
March 2020

Ileostomy for steroid-resistant acute graft-versus-host disease of the gastrointestinal tract.

Ann Hematol 2019 Oct 23;98(10):2407-2419. Epub 2019 Jul 23.

Department of Bone Marrow Transplantation, West-German Cancer Center, University Hospital Essen, Hufelandstrasse, 55 45122, Essen, Germany.

Steroid-resistant acute graft-versus-host disease (GVHD) of the gastrointestinal tract associates with important morbidity and mortality. While high-dose steroids are the established first-line therapy in GVHD, no second-line therapy is generally accepted. In this analysis of 65 consecutive patients with severe, steroid-resistant, intestinal GVHD (92% stage 4), additional ileostomy surgery significantly reduced overall mortality (hazard ratio 0.54; 95% confidence interval, 0.36-0.81; p = 0.003) compared to conventional GVHD therapy. Median overall survival was 16 months in the ileostomy cohort compared to 4 months in the conventional therapy cohort. In the ileostomy cohort, both infectious- and GVHD-associated mortality were reduced (40% versus 77%). Significantly declined fecal volumes (p = 0.001) after surgery provide evidence of intestinal adaptation following ileostomy. Correlative studies indicated ileostomy-induced immune-modulation with a > 50% decrease of activated T cells (p = 0.04) and an increase in regulatory T cells. The observed alterations of the patients' gut microbiota may also contribute to ileostomy's therapeutic effect. These data show that ileostomy induced significant clinical responses in patients with steroid-resistant GVHD along with a reduction of pro-inflammatory immune cells and changes of the intestinal microbiota. Ileostomy is a treatment option for steroid-resistant acute GVHD of the gastrointestinal tract that needs further validation in a prospective clinical trial.
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http://dx.doi.org/10.1007/s00277-019-03754-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7101733PMC
October 2019

B-type natriuretic peptides for the prediction of cardiovascular events and mortality in patients living with HIV: Results from the HIV-HEART study.

Int J Cardiol 2019 Apr 23;281:127-132. Epub 2019 Jan 23.

Clinic of Dermatology, Department of Venerology, University Hospital Essen, Essen, Germany.

Aims: B-type natriuretic peptide (BNP) has been suggested to improve risk prediction of cardiovascular (CV) events and mortality. We aimed to evaluate the value of BNP to predict the composite primary endpoint of CV events and mortality alongside traditional and HIV specific risk factors in a HIV-infected population.

Methods: In this prospective multicenter HIV-HEART study we followed 808 HIV-positive subjects in the German Ruhr area for a median follow up of 120 (IQR:113-129) months since 2004. Association of BNP with the composite primary endpoint was assessed using Cox regression adjusting for traditional cardiovascular and HIV specific risk factors.

Results: At baseline, median BNP was 10.3 (IQR 5.4-18.9) pg/ml. The composite endpoint occurred in 158 (19.6%) patients. Subjects with high BNP levels showed significantly increased frequencies of CV events and death (22% for BNP ≤5 pg/ml, 30% for BNP >5 up to ≤20 pg ml, 38% for BNP >20 up to ≤35 pg ml, 59% for BNP >35 up to ≤100 pg ml and 86% for BNP >100 pg/ml, p-value < 0.01). In the fully adjusted model that included traditional CV risks as well as HIV specific factors, after a log transformation, doubling of BNP was significantly associated with increased risk for the composite endpoint (HR:1.16 (95%CI 1.01-1.33); p = 0.031). Comparing BNP of <5 pg/ml to BNP > 100 pg/ml, HR in the fully adjusted model was 3.25 (95%CI 1.50-7.08; p < 0.001).

Conclusions: Increased BNP is associated with significant excess of incident CV events and mortality in HIV-infected patients. BNP is a valuable marker to improve the prediction of CV events and mortality.
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http://dx.doi.org/10.1016/j.ijcard.2019.01.066DOI Listing
April 2019

Gut Microbiota in Human Immunodeficiency Virus-Infected Individuals Linked to Coronary Heart Disease.

J Infect Dis 2019 01;219(3):497-508

Clinic for Dermatology and Venerology, University of Duisburg-Essen.

Background: Human immunodeficiency virus (HIV) infection is an independent risk factor for coronary heart disease (CHD) and is associated with perturbation of the gut microbiota.

Methods: We analyzed gut microbiota in 30 HIV-infected individuals with CHD (CHD+) and 30 without CHD (CHD-) of the HIV-HEART study group.

Results: Gut microbiota linked to CHD was associated with lower α-diversity. Despite insignificant differences in β-diversity, co-occurrence networks of bacterial genera clearly diverged between CHD+ and CHD- individuals. Multidimensional scaling separated HIV-infected individuals into 2 microbiome clusters, dominated by the genus Prevotella or Bacteroides. The relative abundance of 49 other genera was significantly different between both clusters. The Prevotella-rich cluster was largely composed of men who have sex with men (MSM) (97%), whereas the Bacteroides-rich cluster comprised both MSM (45%) and heterosexual individuals (55%). MSM of the Bacteroides-rich cluster were characterized by reduced α-diversity, advanced immunological HIV stage, longer antiretroviral therapy with more ART regimens, and longer use of protease inhibitors, compared with Prevotella-rich MSM.

Conclusions: Community structures of gut microbiota rather than individual species might facilitate risk assessment of CHD in HIV-infected individuals. Sexual behavior appears to be an important factor affecting gut microbiota β-diversity and should be considered in future studies.
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http://dx.doi.org/10.1093/infdis/jiy524DOI Listing
January 2019

Diagnostic performance of blood culture bottles for vitreous culture compared to conventional microbiological cultures in patients with suspected endophthalmitis.

Eur J Clin Microbiol Infect Dis 2018 May 9;37(5):889-895. Epub 2018 Jan 9.

Institute of Medical Microbiology, University Hospital Essen, University of Duisburg-Essen, Hufelandstr. 55, 45122, Essen, Germany.

The purpose of this investigation was to evaluate the performance of blood culture bottles in comparison to conventional microbiological culture techniques in detecting causative microorganisms of endophthalmitis and to determine their anti-infective susceptibility profiles. All consecutive cases with clinically suspected endophthalmitis in a university-based ophthalmology department between January 2009 and December 2016 were analysed in this retrospective comparative case series. Samples from 247 patients with suspected endophthalmitis underwent microbiological diagnostic work-up. All three culture methods were performed from 140 vitreous specimens. Vitreous fluid specimens were inoculated in blood culture bottles, aerobic and anaerobic broth solutions, and on solid media. Anti-infective susceptibility profiles were evaluated by semi-automated methods and/or gradient diffusion methods. Microorganisms were grown in 82 of 140 specimens for which all methods were performed (59%). Microorganisms were more frequently grown from blood culture bottles (55%) compared to broth solution (45%, p = 0.007) and solid media (33%, p < 0.0001). Considerable differences in the performance among culture media were detected for fungal pathogens. All grown fungi were detected by blood culture bottles (11 of 11, 100%). Broth solution recovered 64% and solid media 46% of grown fungi. No Gram-positive bacterium was resistant to vancomycin and all Gram-negative pathogens except for one isolate were susceptible to third-generation cephalosporins. In suspected endophthalmitis patients, blood culture bottles have a higher overall pathogen detection rate from vitreous fluid compared to conventional microbiological media, especially for fungi. The initial intravitreal antibiotic therapy with vancomycin plus third-generation cephalosporins appears to be an appropriate treatment approach for bacterial endophthalmitis.
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http://dx.doi.org/10.1007/s10096-017-3182-6DOI Listing
May 2018

Comparison of clinical and immunological findings in gnotobiotic piglets infected with O104:H4 outbreak strain and EHEC O157:H7.

Gut Pathog 2017 25;9:30. Epub 2017 May 25.

University Clinic for Swine, Department for Farm Animals and Veterinary Public Health, University of Veterinary Medicine Vienna, Veterinärplatz 1, 1220 Vienna, Austria.

Background: Shiga toxin (Stx) producing () (STEC) is the most frequent cause of diarrhoea-positive haemolytic uraemic syndrome (D + HUS) in humans. In 2011, a huge outbreak with an STEC O104:H4 strain in Germany highlighted the limited possibilities for causative treatment of this syndrome. The responsible STEC strain was found to combine Stx production with adherence mechanisms normally found in enteroaggregative (EAEC). Pathotypes of evolve and can exhibit different adhesion mechanisms. It has been shown previously that neonatal gnotobiotic piglets are susceptible for infection with STEC, such as STEC O157:H7 as well as for EAEC, which are considered to be the phylogenetic origin of O104:H4. This study was designed to characterise the host response to infection with the STEC O104:H4 outbreak strain in comparison to an STEC O157:H7 isolate by evaluating clinical parameters (scoring) and markers of organ dysfunction (biochemistry), as well as immunological (flow cytometry, assessment of cytokines/chemokines and acute phase proteins) and histological alterations (light- and electron microscopy) in a gnotobiotic piglet model of haemolytic uraemic syndrome.

Results: We observed severe clinical symptoms, such as diarrhoea, dehydration and neurological disorders as well as attaching-and-effacing lesions (A/E) in the colon in STEC O157:H7 infected piglets. In contrast, STEC O104:H4 challenged animals exhibited only mild clinical symptoms including diarrhoea and dehydration and HUS-specific/severe histopathological, haematological and biochemical alterations were only inconsistently presented by individual piglets. A specific adherence phenotype of STEC O104:H4 could not be observed. Flow cytometric analyses of lymphocytes derived from infected animals revealed an increase of natural killer cells (NK cells) during the course of infection revealing a potential role of this subset in the anti-bacterial activity in STEC disease.

Conclusions: Unexpectedly, O104:H4 infection caused only mild symptoms and minor changes in histology and blood parameters in piglets. Outcome of the infection trial does not reflect   O104:H4 associated human disease as observed during the outbreak in 2011. The potential role of cells of the innate immune system for STEC related disease pathogenesis should be further elucidated.
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http://dx.doi.org/10.1186/s13099-017-0179-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5445466PMC
May 2017

GenoType NTM-DR for Identifying Mycobacterium abscessus Subspecies and Determining Molecular Resistance.

J Clin Microbiol 2016 06 30;54(6):1653-1655. Epub 2016 Mar 30.

Institute of Medical Microbiology, University Hospital Essen, University Duisburg-Essen, Essen, Germany.

We studied the performance of a new line probe assay for identifying the subspecies and determining the macrolide and aminoglycoside resistance levels of 50 Mycobacterium abscessus isolates. Agreement of GenoType NTM-DR results with sequencing and phenotypic resistance results was 92% for subspecies identification and 98% for determining molecular and phenotypic resistance.
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http://dx.doi.org/10.1128/JCM.00147-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4879307PMC
June 2016

Plasmodium yoelii infection of BALB/c mice results in expansion rather than induction of CD4(+) Foxp3(+) regulatory T cells.

Immunology 2016 06 23;148(2):197-205. Epub 2016 Mar 23.

Institute of Medical Microbiology, University Hospital Essen, University Duisburg-Essen, Essen, Germany.

Recently, we demonstrated elevated numbers of CD4(+) Foxp3(+) regulatory T (Treg) cells in Plasmodium yoelii-infected mice contributing to the regulation of anti-malarial immune response. However, it remains unclear whether this increase in Treg cells is due to thymus-derived Treg cell expansion or induction of Treg cells in the periphery. Here, we show that the frequency of Foxp3(+) Treg cells expressing neuropilin-1 (Nrp-1) decreased at early time-points during P. yoelii infection, whereas percentages of Helios(+) Foxp3(+) Treg cells remained unchanged. Both Foxp3(+) Nrp-1(+) and Foxp3(+) Nrp-1(-) Treg cells from P. yoelii-infected mice exhibited a similar T-cell receptor Vβ chain usage and methylation pattern in the Treg-specific demethylation region within the foxp3 locus. Strikingly, we did not observe induction of Foxp3 expression in Foxp3(-) T cells adoptively transferred to P. yoelii-infected mice. Hence, our results suggest that P. yoelii infection triggered expansion of naturally occurring Treg cells rather than de novo induction of Foxp3(+) Treg cells.
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http://dx.doi.org/10.1111/imm.12602DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4863568PMC
June 2016

Principal component analysis of MALDI TOF MS mass spectra separates M. abscessus (sensu stricto) from M. massiliense isolates.

BMC Microbiol 2016 Mar 1;16:24. Epub 2016 Mar 1.

Institute of Medical Microbiology, University Hospital Essen, University Duisburg-Essen, Essen, Germany.

Background: The discrimination of the members of the Mycobacterium abscessus complex is of clinical interest because one of the subspecies, M. massiliense, exhibits higher rates of response to antibiotic treatment for lung infection than do the other members of that complex. M. abscessus complex contains three subspecies that are laborious to identify; therefore, a routine diagnostic tool would be worthwhile.

Results: We used principal component analysis, hierarchical cluster analysis, and single-peak analysis to examine peak lists derived from matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) mass spectra of 50 clinical M. abscessus complex isolates, including 28 M. abscessus (sensu stricto), 19 M. massiliense, and 3 M. bolletii isolates grown in mycobacterium growth indicator tube liquid medium and prepared with a bead-based protocol. Principal component analysis but not hierarchical cluster analysis separated M. abscessus (sensu stricto) isolates and M. massiliense isolates into two clusters. Furthermore, single-peak analysis displayed 4 discriminating peaks that separated M. abscessus (sensu stricto) from M. massiliense isolates. M. bolletii isolates did not exhibit specific peaks but resembled the M. abscessus (sensu stricto) peak profile and also grouped within this principal component analysis cluster. Principal component analysis of all peak lists with the exclusion of the four discriminating peaks again separated M. abscessus (sensu stricto) from M. massiliense isolates, thus relativizing the importance of these peaks for subspecies identification.

Conclusions: Principal component analysis of peak lists derived from MALDI TOF mass spectra is a robust and convenient method of discriminating M. massiliense isolates from the other members of the M. abscessus complex.
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http://dx.doi.org/10.1186/s12866-016-0636-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4772520PMC
March 2016

Performance of Vitek MS in identifying nontuberculous mycobacteria from MGIT liquid medium and Lowenstein-Jensen solid medium.

Diagn Microbiol Infect Dis 2016 Jan 9;84(1):43-47. Epub 2015 Oct 9.

Institute of Medical Microbiology, University Hospital Essen, University Duisburg-Essen, Essen, Germany.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry is a fast and inexpensive method for bacterial identification. The aim of this study was to analyze the performance of Vitek MS in identifying 160 nontuberculous mycobacterial isolates of 24 species from Lowenstein-Jensen solid medium and BACTEC MGIT 960 liquid medium using a bead-based method. The system correctly identified 76.9% of the isolates (123 of 160) cultivated on solid medium and 76.9% (123 of 160) of positive liquid cultures. None of the isolates included in the study was misidentified. Although the overall performance of Vitek MS with the SARAMIS 4.12 database was comparable in identifying mycobacterial species grown on solid medium and in liquid medium, the identification rate varied notably between the various species analyzed, which currently limits the utility for identification in routine diagnostics for some species.
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http://dx.doi.org/10.1016/j.diagmicrobio.2015.10.007DOI Listing
January 2016

Relevance of Foxp3⁺ regulatory T cells for early and late phases of murine sepsis.

Immunology 2015 Sep 2;146(1):144-56. Epub 2015 Jul 2.

Institute of Medical Microbiology, University Hospital Essen, University of Duisburg-Essen, Essen, Germany.

The role of Foxp3(+) regulatory T (Treg) cells in the course of the early hyper-inflammatory and subsequent hypo-inflammatory phases of sepsis is ambiguous. Whereas Nrp1 expression has been reported to discriminate natural Treg cells from induced Treg cells, the Treg cell stability depends on the methylation status of foxp3-TSDR. To specifically evaluate the role of Foxp3(+) Treg cells in the early and late phases of sepsis, we induced sepsis by caecal ligation and puncture and subsequent Pseudomonas aeruginosa lung infection in a DEREG (DEpletion of REGulatory T cells) mouse model. We found an increase of Foxp3(+) Treg cells to all CD4(+) T cells during murine sepsis. Using a new methylation-sensitive quantitative RT-PCR method and deep amplicon sequencing, we demonstrated that natural (Nrp1(+) Foxp3(+) ) Treg cells and most induced (Nrp1(-) Foxp3(+) ) Treg cells are stable and exhibit unmethylated foxp3-TSDR, and that both Treg populations are functionally suppressive in healthy and septic mice. DEREG mice depleted of Foxp3(+) Treg cells exhibit higher disease scores, mortality rates and interleukin-6 expression levels than do non-depleted DEREG mice in early-phase sepsis, a finding indicating that Foxp3(+) Treg cells limit the hyper-inflammatory response and accelerate recovery. Treg cell depletion before secondary infection with P. aeruginosa 1 week after caecal ligation and puncture does not influence cytokine levels or the course of secondary infection. However, a moderate Treg cell recurrence, which we observed in DEREG mice during secondary infection, may interfere with these results. In summary, Treg cells contribute to a positive outcome after early-phase sepsis, but the data do not support a significant role of Treg cells in immune paralysis during late-phase sepsis.
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http://dx.doi.org/10.1111/imm.12490DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4552509PMC
September 2015

Effects of green tea compound epigallocatechin-3-gallate against Stenotrophomonas maltophilia infection and biofilm.

PLoS One 2014 1;9(4):e92876. Epub 2014 Apr 1.

Institute of Medical Microbiology, University Hospital Essen, University of Duisburg-Essen, Essen, Germany.

We investigated the in vitro and in vivo activities of epigallocatechin-3-gallate (EGCg), a green tea component, against Stenotrophomonas maltophilia (Sm) isolates from cystic fibrosis (CF) patients. In vitro effects of EGCg and the antibiotic colistin (COL) on growth inhibition, survival, and also against young and mature biofilms of S. maltophilia were determined. Qualitative and quantitative changes on the biofilms were assessed by confocal laser scanning microscopy (CLSM). Further, in vivo effects of nebulized EGCg in C57BL/6 and Cftr mutant mice during acute Sm lung infection were evaluated. Subinhibitory concentrations of EGCg significantly reduced not only biofilm formation, but also the quantity of viable cells in young and mature biofilms. CLSM showed that EGCg-exposed biofilms exhibited either a change in total biofilm biovolume or an increase of the fraction of dead cells contained within the biofilm in a dose depended manner. Sm infected wild-type and Cftr mutant mice treated with 1,024 mg/L EGCg by inhalation exhibited significantly lower bacterial counts than those undergoing no treatment or treated with COL. EGCg displayed promising inhibitory and anti-biofilm properties against CF Sm isolates in vitro and significantly reduced Sm bacterial counts in an acute infection model with wild type and CF mice. This natural compound may represent a novel therapeutic agent against Sm infection in CF.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0092876PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3972220PMC
December 2015

Impact of 5-aza-2'-deoxycytidine and epigallocatechin-3-gallate for induction of human regulatory T cells.

Immunology 2014 Jul;142(3):384-95

Institute of Medical Microbiology, University Hospital Essen, University of Duisburg-Essen, Essen, Germany.

The epigenetic regulation of transcription factor genes is critical for T-cell lineage specification. A specific methylation pattern within a conserved region of the lineage specifying transcription factor gene FOXP3, the Treg-specific demethylated region (TSDR), is restricted to regulatory T (Treg) cells and is required for stable expression of FOXP3 and suppressive function. We analysed the impact of hypomethylating agents 5-aza-2'-deoxycytidine and epigallocatechin-3-gallate on human CD4(+)  CD25(-) T cells for generating demethylation within FOXP3-TSDR and inducing functional Treg cells. Gene expression, including lineage-specifying transcription factors of the major T-cell lineages and their leading cytokines, functional properties and global transcriptome changes were analysed. The FOXP3-TSDR methylation pattern was determined by using deep amplicon bisulphite sequencing. 5-aza-2'-deoxycytidine induced FOXP3-TSDR hypomethylation and expression of the Treg-cell-specific genes FOXP3 and LRRC32. Proliferation of 5-aza-2'-deoxycytidine-treated cells was reduced, but the cells did not show suppressive function. Hypomethylation was not restricted to FOXP3-TSDR and expression of master transcription factors and leading cytokines of T helper type 1 and type 17 cells were induced. Epigallocatechin-3-gallate induced global DNA hypomethylation to a lesser extent than 5-aza-2'-deoxycitidine, but no relevant hypomethylation within FOXP3-TSDR or expression of Treg-cell-specific genes. Neither of the DNA methyltransferase inhibitors induced fully functional human Treg cells. 5-aza-2'-deoxycitidine-treated cells resembled Treg cells, but they did not suppress proliferation of responder cells, which is an essential capability to be used for Treg cell transfer therapy. Using a recently developed targeted demethylation technology might be a more promising approach for the generation of functional Treg cells.
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http://dx.doi.org/10.1111/imm.12261DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4080954PMC
July 2014

Quantification of regulatory T cells in septic patients by real-time PCR-based methylation assay and flow cytometry.

PLoS One 2012 27;7(11):e49962. Epub 2012 Nov 27.

Institute of Medical Microbiology, University Hospital Essen, University of Duisburg-Essen, Essen, Germany.

During sepsis, a relative increase of regulatory T (Treg) cells has been reported. Its persistence is associated with lymphocyte anergy, immunoparalysis and a poor prognosis. Currently, an exact quantification of human Treg cells based on protein expression of marker molecules is ambiguous, as these molecules are expressed also by activated non-regulatory T cells. Furthermore, no firm criteria for flow cytometer gate settings exist so far. Recently, a specific DNA methylation pattern within FOXP3-TSDR has been reported that allows distinguishing Treg and non-regulatory T cells, independent of their activation status. Using this epigenetic marker, we established a single-tube real-time PCR based methylation assay (QAMA) for relative quantification of Treg cells. Validation was performed on defined ratios of methylated and unmethylated target sequence and on mixtures of Treg and non-regulatory T cells. DNA-methylation was measured in CD4(+) T cells isolated from blood samples of 30 septic patients and 30 healthy subjects and compared with results of Treg cell quantification by flow cytometry based on CD4(+) CD25(hi)CD127(low) measurement. In septic patients both methods showed an increased ratio of Treg cells to all CD4(+) T cells. In healthy individuals, the results obtained by both methods were clearly positively correlated. However, the correlation between both methods in septic patients was only weak. We showed that quantification of Treg cells by QAMA detects CD4(+) T cells with unmethylated FOXP3-TSDR, hidden in the CD25(med/low) fraction of flow cytometry. Given that unmethylated FOXP3-TSDR is the most specific feature of Treg cells to date, our assay precisely quantifies Treg cells, as it additionally detects those committed Treg cells, hidden in the CD25(med/low) fraction of CD4(+) cells. Furthermore, QAMA is a reliable method, which is easier to standardize among laboratories and can thus improve reproducibility of Treg cell quantification.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0049962PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3507919PMC
June 2013

FOXP3 Expression in GARP-Transduced Helper T Cells Is Not Associated with FOXP3 TSDR Demethylation.

Transfus Med Hemother 2011 Oct 12;38(5):287-291. Epub 2011 Sep 12.

Institut für Medizinische Mikrobiologie, Braunschweig, Germany.

AIM: Glycoprotein A repetitions predominant (GARP or LRRC32) represents a human regulatory CD4+ CD25(hi) FOXP3+ T (T(reg)) cell-specific receptor that controls FOXP3. Ectopic expression of GARP in helper T (T(h)) cells has been shown to be sufficient for the induction of FOXP3 and generation of a stable regulatory phenotype. Since expression of FOXP3 in Treg cells is epigenetically controlled by a conserved motif, the so-called T(reg)-specific demethylated region (TSDR), we asked whether GARP-mediated upregulation of FOXP3 in Th cells is similarly accompanied by demethylation of the TSDR. METHODS: DNA methylation of the FOXP3 TSDR was analyzed by direct sequencing of polymerase chain reaction (PCR) products from bisulfite-treated genomic DNA. RESULTS: Although GARP-transduced T(h) cells exhibit constitutive FOXP3 expression and a regulatory phenotype, the FOXP3 TSDR is completely methylated as in naive T(h) cells. GARP-mediated FOXP3 upregulation in T(h) cells is not associated with T(reg)-specific demethylation of the FOXP3 TSDR. CONCLUSION: Although GARP-engineered T(h) cells exhibit stable FOXP3 expression and a phenotypic reprogramming towards T(reg) cells in vitro, these cells do not completely mimic the epigenotype of natural T(reg) cells. Thus, concepts based on the genetic modification of T(h) cells as cellular therapies to treat autoimmune diseases or to control transplantation tolerance should be critically tested before any clinical application.
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http://dx.doi.org/10.1159/000331499DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3364046PMC
October 2011

TNF-alpha induces tyrosine phosphorylation and recruitment of the Src homology protein-tyrosine phosphatase 2 to the gp130 signal-transducing subunit of the IL-6 receptor complex.

J Immunol 2003 Jul;171(1):257-66

Klinik für Gastroenterologie, Hepatologie und Infektiologie, Medizinische Klinik der Heinrich Heine Universität, Düsseldorf, Germany.

Recently, it has been demonstrated that TNF-alpha and LPS induce the expression of suppressor of cytokine signaling 3 (SOCS3) and inhibit IL-6-induced STAT3 activation in macrophages. Inhibitor studies suggested that both induction of SOCS3 and inhibition of IL-6-induced STAT3 activation depend on the activation of p38 mitogen-activated protein kinase. Since recruitment of the tyrosine phosphatase Src homology protein tyrosine phosphatase 2 (SHP2) to the signal-transducing receptor subunit gp130 attenuates IL-6-mediated STAT-activation, we were interested in whether TNF-alpha also induces the association of SHP2 to the gp130 receptor subunit. In this study we demonstrate that stimulation of macrophages and fibroblast cell lines with TNF-alpha causes the recruitment of SHP2 to the gp130 signal-transducing subunit and leads to tyrosine phosphorylation of SHP2 and gp130. In this context the cytoplasmic SHP2/SOCS3 recruitment site of gp130 tyrosine 759 is shown to be important for the inhibitory effects of TNF-alpha, since mutation of this residue completely restores IL-6-stimulated activation of STAT3 and, consequently, of a STAT3-dependent promoter. In this respect murine fibroblasts lacking exon 3 of SHP2 are not sensitive to TNF-alpha, indicating that functional SHP2 and its recruitment to gp130 are key events in inhibition of IL-6-dependent STAT activation by TNF-alpha. Furthermore, activation of p38 mitogen-activated protein kinase is shown to be essential for the inhibitory effect of TNF-alpha on IL-6 signaling and TNF-alpha-dependent recruitment of SHP2 to gp130.
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http://dx.doi.org/10.4049/jimmunol.171.1.257DOI Listing
July 2003
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