Publications by authors named "Cornelia Brendel"

56 Publications

Expression of β-1,4-galactosyltransferases during Aging in Caenorhabditis elegans.

Gerontology 2020 10;66(6):571-581. Epub 2020 Nov 10.

Institute for Biomedical Aging Research, Leopold-Franzens-Universität Innsbruck, Innsbruck, Austria.

Background: Altered plasma activity of β-1,4-galac-tosyl-transferases (B4GALTs) is a novel candidate biomarker of human aging. B4GALT1 is assumed to be largely responsible for this activity increase, but how it modulates the aging process is unclear at present.

Objectives: To determine how expression of B4GALT1 and other B4GALT enzymes changes during aging of an experimentally tractable model organism, Caenorhabditis elegans.

Methods: Targeted analysis of mRNA levels of all 3 C. elegans B4GALT family members was performed by qPCR in wild-type and in long-lived daf-2 (insulin/IGF1-like receptor)-deficient or germline-deficient animals.

Results: bre-4 (B4GALT1/2/3/4) is the only B4GALT whose expression increases during aging in wild-type worms. In addition, bre-4 levels also rise during aging in long-lived daf-2-deficient worms, but not in animals that are long-lived due to the lack of germline stem cells. On the other hand, expression of sqv-3 (B4GALT7) and of W02B12.11 (B4GALT5/6) appears decreased or constant, respectively, in all backgrounds during aging.

Conclusions: The age-dependent bre-4 mRNA increase in C. elegans parallels the age-dependent B4GALT activity increase in humans and is consistent with C. elegans being a suitable experimental organism to define potentially conserved roles of B4GALT1 during aging.
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http://dx.doi.org/10.1159/000510722DOI Listing
November 2020

The Oncoprotein SKI Acts as A Suppressor of NK Cell-Mediated Immunosurveillance in PDAC.

Cancers (Basel) 2020 Oct 3;12(10). Epub 2020 Oct 3.

Institute for Tumor Immunology, Clinic for Hematology, Oncology and Immunology, Philipps University of Marburg, Hans-Meerwein-Strasse 3, 35043 Marburg, Germany.

Drugs targeting epigenetic mechanisms such as histone deacetylase inhibitors (HDACi) suppress tumor growth. HDACi also induce the expression of ligands for the cytotoxicity receptor NKG2D rendering tumors more susceptible to natural killer (NK) cell-dependent killing. The major acetylases responsible for the expression of NKG2D ligands (NKG2D-L) are CBP and p300. The role of the oncogene and transcriptional repressor SKI, an essential part of an HDAC-recruiting co-repressor complex, which competes with CBP/p300 for binding to SMAD3 in TGFβ signaling, is unknown. Here we show that the siRNA-mediated downregulation of in the pancreatic cancer cell lines Panc-1 and Patu8988t leads to an increased target cell killing by primary NK cells. However, the higher cytotoxicity of NK cells did not correlate with the induction of NKG2D-L. Of note, the expression of NKG2D-L and consequently NK cell-dependent killing could be induced upon LBH589 (LBH, panobinostat) or valproic acid (VPA) treatment irrespective of the SKI expression level but was significantly higher in pancreatic cancer cells upon genetic ablation of . These data suggest that SKI represses the inducible expression of NKG2D-L. The combination of HDACi with NK cell-based immunotherapy is an attractive treatment option for pancreatic tumors, specifically for patients with high SKI protein levels.
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http://dx.doi.org/10.3390/cancers12102857DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7601115PMC
October 2020

Determination of CD43 and CD200 surface expression improves accuracy of B-cell lymphoma immunophenotyping.

Cytometry B Clin Cytom 2020 11 27;98(6):476-482. Epub 2020 Jul 27.

Department of Hematology, Oncology and Immunology, Philipps University Marburg, University Hospital Giessen and Marburg, Marburg, Germany.

Background: The Matutes score (MS) was proposed to differentiate chronic lymphocytic leukemia (CLL) from other B-cell non-Hodgkin lymphomas (B-NHLs). However, ambiguous immunophenotypes are common and remain a diagnostic challenge. Therefore, we evaluated the diagnostic benefit of measuring CD200 and CD43 expression together with the standard MS antigens.

Methods: 138 lymphoma patient samples and a validation cohort of 138 additive samples were classified according to the standard MS and further assigned with one or two additional points, for high CD200 and/or CD43 expression levels. The "classical" MS and the "Matutes score-extended" (MS-e) were categorized as high (4-5/6-7), intermediate (2-3/4-5), and low (0-1/0-3). Samples were reclassified into the MS-e with focus on ambiguous cases with an intermediate "classical" MS.

Results: A total of 35 of 138 (25.4%) patient samples were assigned to the intermediate MS group and confirmed by histopathological reports as CLL (14/40.0%) and B-NHLs other than CLL (21/60%). MS-e analysis identified 13 of 14 (92.9%) of CLL cases (MS-e 4-5) and 18/21 (85.7%) non-CLL cases (MS-e ≤ 3) correctly. Overall, the sensitivity of the CLL diagnosis was significantly increased by application of MS-e compared to the "classical" MS (98.8% vs. 82.7%; p = 0.0009), while specificity of both methods was almost equal (94.7% vs. 98.3%; p = 0.4795). Of note, sole measurement of CD43 and CD200 on B-cells sufficiently differentiated CLL from non-CLL with a test accuracy superior to the "classical" MS (F1 score 96.2 vs. 93.6).

Conclusion: CD200 and CD43 have a high informative value in diagnostic immunophenotyping and facilitate the separation of CLL from other B-NHLs particularly in ambiguous cases.
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http://dx.doi.org/10.1002/cyto.b.21936DOI Listing
November 2020

Aberrant CD3-Positive, CD8-Low, CD7-Negative Lymphocytes May Appear During Viral Infections and Mimic Peripheral T-Cell Lymphoma.

Diagnostics (Basel) 2020 Apr 7;10(4). Epub 2020 Apr 7.

Department of Hematology, Oncology, Immunology, Philipps-University Marburg, Baldinger Strasse, 35043 Marburg, Germany.

Flow cytometry (FC) facilitates diagnosis of peripheral T-cell non-Hodgkin lymphoma (T-NHL), but overlapping features between reactive and neoplastic T-cell proliferations often hamper a rapid assessment. One hundred forty peripheral blood samples submitted to diagnostic FC for T-cell immunophenotyping were retrospectively analyzed. A T-cell population with a conspicuous aberrant surface epitope expression pattern was observed in 18 cases and diagnostic follow up was performed. The aberrant T-cell population exhibited a low scatter profile, a CD7-negative/low, CD8-low and CD3-positive immunophenotype, and monoclonal T-cell receptor expansion. T-NHL was ruled out by follow up in all cases. Epstein-Barr virus infection was diagnosed in 12 cases, cytomegalovirus infection in three cases; one patient had been vaccinated. The irregular subpopulation disappeared spontaneously within days or weeks. We describe a novel peripheral blood T-cell subpopulation with a low light scatter and CD8-low, CD7-negative/low and CD3-positive marker expression profile, which indicates reactive T-cell expansion in patients who present with peripheral lymphadenopathy and/or B symptoms.
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http://dx.doi.org/10.3390/diagnostics10040204DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7235783PMC
April 2020

Cloning and characterization of a novel druggable fusion kinase in acute myeloid leukemia.

Haematologica 2020 08 2;105(8):e395-e398. Epub 2019 Dec 2.

Universitätsklinikum Gießen und Marburg, Campus Marburg, Klinik für Hämatologie, Onkologie und Immunologie, Philipps Universität Marburg, Marburg

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http://dx.doi.org/10.3324/haematol.2019.237818DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7395268PMC
August 2020

Correction to: Optimizing conditions for labeling of mesenchymal stromal cells (MSCs) with gold nanoparticles: a prerequisite for in vivo tracking of MSCs.

J Nanobiotechnology 2019 Sep 17;17(1):98. Epub 2019 Sep 17.

Department of Hematology, Oncology and Immunology, Philipps University Marburg, Marburg, Germany.

The authors apologized for the unfortunate error in figure during publication of the article and they also explained that some of the solid grey graphs in Fig. 5 are intentionally based on the same data. For 8 different surface makers (CD14, CD73, CD34, CD105, CD19, CD90, CD45, HA-DR) in accordance to the guidelines of the manufacturer a panel of 4 different isotype controls were used, corresponding to 4 different fluorescence channels.
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http://dx.doi.org/10.1186/s12951-019-0527-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6749620PMC
September 2019

Outcome of non-mold effective anti-fungal prophylaxis in patients at high-risk for invasive fungal infections after allogenic stem cell transplantation.

Leuk Lymphoma 2019 08 15;60(8):2056-2061. Epub 2019 Jan 15.

a Department of Hematology, Oncology and Immunology , University Hospital Giessen and Marburg , Marburg , Germany.

Patients who develop severe graft-versus-host disease (GvHD) after allogeneic stem cell transplantation (alloSCT) have a higher risk for invasive fungal infection (IFI). At our center, fluconazole prophylaxis is standard and upfront mold-effective prophylaxis performed only in patients with specific risk constellations. A total of 290 patients undergoing alloSCT between May 2002 and August 2011 were analyzed. Patients were regarded as high-risk if they suffered from acute GvHD II-IV° or extensive chronic GvHD. The 2-year incidence of an IFI after alloSCT was 8.97% (26/290) in the entire cohort and 7.78% (7/90) in the high-risk group. Mortality due to IFI was 3.85% (1/26) without including a high-risk patient. In the multivariate analysis a pre-transplant fungal infection was the only significant risk factor for developing an IFI after alloSCT (HR = 5.298;  = .001). A fluconazole prophylaxis in patients with GvHD after alloSCT is feasible in facilities with HEPA filtration and high awareness of clinical signs for IFI.
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http://dx.doi.org/10.1080/10428194.2018.1553303DOI Listing
August 2019

Comprehensive genetic diagnosis of acute myeloid leukemia by next-generation sequencing.

Haematologica 2019 02 6;104(2):277-287. Epub 2018 Sep 6.

Department of Hematology, Oncology and Immunology, Philipps-University Marburg, and University Hospital Gießen and Marburg, Marburg, Germany

Differential induction therapy of all subtypes of acute myeloid leukemia other than acute promyelocytic leukemia is impeded by the long time required to complete complex and diverse cytogenetic and molecular genetic analyses for risk stratification or targeted treatment decisions. Here, we describe a reliable, rapid and sensitive diagnostic approach that combines karyotyping and mutational screening in a single, integrated, next-generation sequencing assay. Numerical karyotyping was performed by low coverage whole genome sequencing followed by copy number variation analysis using a novel algorithm based on -generated reference karyotypes. Translocations and DNA variants were examined by targeted resequencing of fusion transcripts and mutational hotspot regions using commercially available kits and analysis pipelines. For the identification of internal tandem duplications and partial tandem duplications, we adapted previously described tools. In a validation cohort including 22 primary patients' samples, 9/9 numerically normal karyotypes were classified correctly and 30/31 (97%) copy number variations reported by classical cytogenetics and fluorescence hybridization analysis were uncovered by our next-generation sequencing karyotyping approach. Predesigned fusion and mutation panels were validated exemplarily on leukemia cell lines and a subset of patients' samples and identified all expected genomic alterations. Finally, blinded analysis of eight additional patients' samples using our comprehensive assay accurately reproduced reference results. Therefore, calculated karyotyping by low coverage whole genome sequencing enables fast and reliable detection of numerical chromosomal changes and, in combination with panel-based fusion-and mutation screening, will greatly facilitate implementation of subtype-specific induction therapies in acute myeloid leukemia.
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http://dx.doi.org/10.3324/haematol.2018.194258DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6355503PMC
February 2019

Development, Function, and Clinical Significance of Plasmacytoid Dendritic Cells in Chronic Myeloid Leukemia.

Cancer Res 2018 11 30;78(21):6223-6234. Epub 2018 Aug 30.

Department of Hematology, Oncology and Immunology, University Hospital Giessen and Marburg, Campus Marburg, Philipps University Marburg, Marburg, Germany.

Plasmacytoid dendritic cells (pDC) are the main producers of a key T-cell-stimulatory cytokine, IFNα, and critical regulators of antiviral immunity. Chronic myeloid leukemia (CML) is caused by BCR-ABL, which is an oncogenic tyrosine kinase that can be effectively inhibited with ABL-selective tyrosine kinase inhibitors (TKI). BCR-ABL-induced suppression of the transcription factor interferon regulatory factor 8 was previously proposed to block pDC development and compromise immune surveillance in CML. Here, we demonstrate that pDCs in newly diagnosed CML (CML-pDC) develop quantitatively normal and are frequently positive for the costimulatory antigen CD86. They originate from low-level -expressing precursors. CML-pDCs also retain their competence to maturate and to secrete IFN. RNA sequencing reveals a strong inflammatory gene expression signature in CML-pDCs. Patients with high CML-pDC counts at diagnosis achieve inferior rates of deep molecular remission (MR) under nilotinib, unless nilotinib therapy is combined with IFN, which strongly suppresses circulating pDC counts. Although most pDCs are -negative in MR, a substantial proportion of CML-pDCs persists under TKI treatment. This could be of relevance, because CML-pDCs elicit CD8 T cells, which protect wild-type mice from CML. Together, pDCs are identified as novel functional DC population in CML, regulating antileukemic immunity and treatment outcome in CML. CML-pDC originates from low-level BCR-ABL expressing stem cells into a functional immunogenic DC-population regulating antileukemic immunity and treatment outcome in CML. .
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http://dx.doi.org/10.1158/0008-5472.CAN-18-1477DOI Listing
November 2018

High β-1,4-Galactosyltransferase-I expression in peripheral T-lymphocytes is associated with a low risk of relapse in germ-cell cancer patients receiving high-dose chemotherapy with autologous stem cell reinfusion.

Oncoimmunology 2018;7(5):e1423169. Epub 2018 Feb 16.

Department of Hematology, Oncology and Immunology at the Philipps-University Marburg; Baldinger Strasse, Marburg, Germany.

Survival of patients with germ-cell cancer (GCC) and primary progression or relapse after cisplatin-based first-line chemotherapy is highly heterogeneous, ranging from close to zero to more than 70%. We investigated β-1,4-Galactosyltransferase-I () expression levels in peripheral lymphocytes in a cohort of 46 testicular cancer patients. enhances immune cell crosstalk via glycosylation of surface molecules. A high expression level of in T-lymphocytes, but not in monocytes, was associated with a lower risk of relapse with a hazard ratio (HR) of 0.66 (95% confidence interval (CI) of HR: 0.45-0.97; = 0.02) upon multivariate Cox regression analysis. Correspondingly, interleukin 10 (IL10), a cytokine released by cytotoxic T-cells, was likewise significantly elevated in T-lymphocytes of non-relapse GCC patients (HR: 0.3; 95% CI of HR: 0.14-0.65; = 0.002). Our data indicate that glycosylation and activation of T-lymphocytes may play a pivotal role in disease control in GCC patients with primary progressive or relapsed disease.
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http://dx.doi.org/10.1080/2162402X.2017.1423169DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5927517PMC
February 2018

Maintenance of cellular respiration indicates drug resistance in acute myeloid leukemia.

Leuk Res 2017 11 28;62:56-63. Epub 2017 Sep 28.

Klinik für Hämatologie, Onkologie und Immunologie, Universitätsklinikum Gießen und Marburg, Standort Marburg, Philipps-Universität Marburg, Baldingerstraße, 35033 Marburg, Germany.

Primary resistance to induction therapy is an unsolved clinical problem in acute myeloid leukemia (AML). Here we investigated drug resistance in AML at the level of cellular metabolism in order to identify early predictors of therapeutic response. Using extracellular flux analysis, we compared metabolic drug responses in AML cell lines sensitive or resistant to cytarabine or sorafenib after 24h of drug treatment to a small cell lung cancer (SCLC) cell line exposed to etoposide. Only drug-resistant AML cells maintained oxidative metabolism upon drug exposure while SCLC cells displayed an overall metabolic shift towards glycolysis, i.e. a Warburg effect to escape drug toxicity. Moreover, primary AML blasts displayed very low glycolytic activity, while oxygen consumption was readily detectable, indicating an essential role of oxidative pathways in the bioenergetics of AML blasts. In line with these observations, analysis of the mitochondrial membrane potential using tetramethylrhodamine ethyl ester staining and flow cytometry allowed for clear discrimination between drug sensitive and resistant AML cell line clones and primary blasts after 24h of treatment with cytarabine or sorafenib. Our data reveal a distinct metabolic phenotype of resistant AML cells and suggest that disrupting oxidative metabolism rather than glycolysis may enhance the cytotoxic effects of chemotherapy in AML.
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http://dx.doi.org/10.1016/j.leukres.2017.09.021DOI Listing
November 2017

Immunosuppressive capacity of mesenchymal stem cells correlates with metabolic activity and can be enhanced by valproic acid.

Stem Cell Res Ther 2017 04 26;8(1):100. Epub 2017 Apr 26.

Department of Hematology, Oncology and Immunology, Philipps-University Marburg, Baldingerstraße, 35043, Marburg, Germany.

Background: Mesenchymal stem cells (MSCs) have entered the clinic as an Advanced Therapy Medicinal Product and are currently evaluated in a wide range of studies for tissue regeneration or in autoimmune disorders. Various efforts have been made to standardize and optimize expansion and manufacturing processes, but until now reliable potency assays for the final MSC product are lacking. Because recent findings suggest superior therapeutic efficacy of freshly administered MSCs in comparison with frozen cells, we sought to correlate the T-cell suppressive capacity of MSCs with their metabolic activity.

Methods: Human MSCs were obtained from patients' bone fragments and were employed in coculture with peripheral blood mononuclear cells (PBMCs) in an allogeneic T-cell proliferation assay to measure immunosuppressive function. Metabolic activity of MSCs was measured in real time in terms of aerobic glycolysis quantified by the extracellular acidification rate and mitochondrial respiration quantified by the oxygen consumption rate.

Results: We show that MSC-induced suppression of T-cell proliferation was highly dependent on individual healthy donors' lymphocytes. Moreover, coculture with PBMCs increased the glycolytic and respiratory activity of MSCs considerably in a PBMC donor-dependent manner. The twofold to threefold enhancement of cell metabolism was accompanied by higher T-cell suppressive capacities of MSCs. The cryoprotectant dimethyl sulfoxide decreased metabolic and immunosuppressive performances of MSCs while valproic acid (VPA) increased their glycolytic, respiratory and T-cell suppressive capacity.

Conclusions: Functional fitness of MSCs can be determined by measuring metabolic activity and can be enhanced by exposure to VPA. Pretesting the increment of metabolic activity upon interaction of donor MSCs with patient T-cells provides a rational approach for an individualized potency assay prior to MSC therapy.
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http://dx.doi.org/10.1186/s13287-017-0553-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5406996PMC
April 2017

Optimizing conditions for labeling of mesenchymal stromal cells (MSCs) with gold nanoparticles: a prerequisite for in vivo tracking of MSCs.

J Nanobiotechnology 2017 Mar 29;15(1):24. Epub 2017 Mar 29.

Department of Hematology, Oncology and Immunology, Philipps University Marburg, Marburg, Germany.

Background: Mesenchymal stromal cells (MSCs) have an inherent migratory capacity towards tumor tissue in vivo. With the future objective to quantify the tumor homing efficacy of MSCs, as first step in this direction we investigated the use of inorganic nanoparticles (NPs), in particular ca. 4 nm-sized Au NPs, for MSC labeling. Time dependent uptake efficiencies of NPs at different exposure concentrations and times were determined via inductively coupled plasma mass spectrometry (ICP-MS).

Results: The labeling efficiency of the MSCs was determined in terms of the amount of exocytosed NPs versus the amount of initially endocytosed NPs, demonstrating that at high concentrations the internalized Au NPs were exocytosed over time, leading to continuous exhaustion. While exposure to NPs did not significantly impair cell viability or expression of surface markers, even at high dose levels, MSCs were significantly affected in their proliferation and migration potential. These results demonstrate that proliferation or migration assays are more suitable to evaluate whether labeling of MSCs with certain amounts of NPs exerts distress on cells. However, despite optimized conditions the labeling efficiency varied considerably in MSC lots from different donors, indicating cell specific loading capacities for NPs. Finally, we determined the detection limits of Au NP-labeled MSCs within murine tissue employing ICP-MS and demonstrate the distribution and homing of NP labeled MSCs in vivo.

Conclusion: Although large amounts of NPs improve contrast for imaging, duration and extend of labeling needs to be adjusted carefully to avoid functional deficits in MSCs. We established an optimized labeling strategy for human MSCs with Au NPs that preserves their migratory capacity in vivo.
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http://dx.doi.org/10.1186/s12951-017-0258-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5372278PMC
March 2017

Diverse Applications of Nanomedicine.

ACS Nano 2017 03 14;11(3):2313-2381. Epub 2017 Mar 14.

CIC biomaGUNE , Paseo de Miramón 182, 20014, Donostia - San Sebastián, Spain.

The design and use of materials in the nanoscale size range for addressing medical and health-related issues continues to receive increasing interest. Research in nanomedicine spans a multitude of areas, including drug delivery, vaccine development, antibacterial, diagnosis and imaging tools, wearable devices, implants, high-throughput screening platforms, etc. using biological, nonbiological, biomimetic, or hybrid materials. Many of these developments are starting to be translated into viable clinical products. Here, we provide an overview of recent developments in nanomedicine and highlight the current challenges and upcoming opportunities for the field and translation to the clinic.
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http://dx.doi.org/10.1021/acsnano.6b06040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5371978PMC
March 2017

A rational two-step approach to KRAS mutation testing in colorectal cancer using high resolution melting analysis and pyrosequencing.

BMC Cancer 2016 08 2;16:585. Epub 2016 Aug 2.

Klinik für Hämatologie, Onkologie und Immunologie, Universitätsklinikum Gießen und Marburg, Standort Marburg, Philipps-Universität Marburg, Baldingerstraße, Marburg, Germany.

Background: KRAS mutation testing is mandatory in the management of metastatic colorectal cancer prior to treatment with anti-EGFR antibodies as patients whose tumors express mutant KRAS do not benefit from these agents. Although the U.S. Food and Drug Administration has recently approved two in-vitro diagnostics kits for determination of KRAS status, there is generally no consensus on the preferred method and new tests are continuously being developed. Most of these techniques focus on the hotspot mutations at codons 12 and 13 of the KRAS gene.

Methods: We describe a two-step approach to KRAS codon 12/13 mutation testing involving high resolution melting analysis (HRM) followed by pyrosequencing using the Therascreen KRAS Pyro kit (Qiagen) of only those samples that are not clearly identified as KRAS wildtype or mutant by HRM. First, we determined KRAS status in a panel of 61 colorectal cancer samples using both methods to compare technical performance and concordance of results. Subsequently, we evaluated practicability and costs of our concept in an independent set of 120 colorectal cancer samples in a routine diagnostic setting.

Results: HRM and pyrosequencing appeared to be equally sensitive, allowing for clear detection of mutant alleles at a mutant allele frequency ≥12.5 %. Pyrosequencing yielded more exploitable results due to lower input requirements and a lower rate of analysis failures. KRAS codon 12/13 status was called concordantly for 98.2 % (56/57) of all samples that could be successfully analysed by both methods and 100 % (19/19) of samples that were identified mutant by HRM. Reviewing the actual effort and expenses for KRAS mutation testing in our laboratory revealed, that the selective use of pyrosequencing for only those samples that could not be analysed by HRM increased the fraction of valid results from 87.5 % for HRM alone to 99.2 % (119/120) while allowing for a net reduction of operational costs of >75 % compared to pyrosequencing alone.

Conclusions: Combination of HRM and pyrosequencing in a two-step diagnostic procedure constitutes a reliable and economic analysis platform for KRAS mutation testing in colorectal cancer in a clinical setting.
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http://dx.doi.org/10.1186/s12885-016-2589-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4971616PMC
August 2016

Contact-dependent abrogation of bone marrow-derived plasmacytoid dendritic cell differentiation by murine mesenchymal stem cells.

Biochem Biophys Res Commun 2016 07 24;476(1):15-20. Epub 2016 May 24.

Institute for Clinical Immunology and Transfusion Medicine, Universities of Giessen and Marburg Lung Center (UGMLC), German Center for Lung Research (DZL), University Hospital Giessen und Marburg, Justus-Liebig-University Giessen, Germany.

Plasmacytoid dendritic cells (pDCs) are rare central regulators of antiviral immunity and unsurpassed producers of interferon-α (IFN-α). Despite their crucial role as a link between innate and adaptive immunity, little is known about the modulation of pDC differentiation by other bone marrow (BM) cells. In this study, we investigated the modulation of pDC differentiation in Flt-3 ligand (Flt3L)-supplemented BM cultures, using highly purified mesenchymal stem cells (MSCs) that were FACS-isolated from murine BM based on surface marker expression and used after in vitro expansion. Initial analysis revealed an almost complete inhibition of BM-derived pDC expansion in the presence of >2% MSC. This inhibition was cell contact-dependent and soluble factor-independent, as indicated by trans-well experiments. The abrogation of functional pDC development by MSCs was confirmed after TLR9 stimulation, revealing a complete, contact-dependent suppression of the IFN-a producing capacity of pDCs in Flt3L MSC BM co-cultures. MSC selectively inhibited pDC development in contrast to myeloid DC development, as indicated by the significantly increased numbers of myeloid DC in Flt3L-supplemented BM cultures. The absence of significant MSC-mediated inhibitory effects on myeloid DC differentiation was confirmed by additional experiments in GM-CSF/IL-4-supplemented BM cultures. In summary, we describe a novel contact-dependent immunomodulatory mechanism of MSC that targets the BM-derived expansion of functional pDCs.
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http://dx.doi.org/10.1016/j.bbrc.2016.05.108DOI Listing
July 2016

A Gain-Of-Function Mutation in the Plcg2 Gene Protects Mice from Helicobacter felis-Induced Gastric MALT Lymphoma.

PLoS One 2016 11;11(3):e0150411. Epub 2016 Mar 11.

Department of Hematology, Oncology and Immunology, Philipps University of Marburg, and University Hospital Giessen and Marburg, Marburg, Germany.

Gastric mucosa-associated lymphoid tissue (MALT) lymphomas develop from a chronic Helicobacter infection. Phospholipase C gamma 2 (PLCG2) is important for B-cell survival and proliferation. We used BALB/c mice with a gain-of-function mutation in the Plcg2 gene (Ali5) to analyze its role in the development of gastric MALT lymphoma. Heterozygous BALB/c Plcg2Ali5/+ and wildtype (WT) mice were infected with Helicobacter felis (H. felis) and observed up to 16 months for development of gastric MALT lymphomas. In contrast to our initial hypothesis, Plcg2Ali5/+ mice developed MALT lymphomas less frequently than their WT littermates after long-term infection of 16 months. Infected Plcg2Ali5/+ mice showed downregulation of proinflammatory cytokines and decreased H. felis-specific IgG1 and IgG2a antibody responses. These results suggested a blunted immune response of Plcg2Ali5/+ mice towards H. felis infection. Intriguingly, Plcg2Ali5/+ mice harboured higher numbers of CD73 expressing regulatory T cells (Tregs), possibly responsible for impaired immune response towards Helicobacter infection. We suggest that Plcg2Ali5/+ mice may be protected from developing gastric MALT lymphomas as a result of elevated Treg numbers, reduced response to H. felis and decrease of proinflammatory cytokines.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0150411PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4788355PMC
November 2016

Prospectively defined murine mesenchymal stem cells inhibit Klebsiella pneumoniae-induced acute lung injury and improve pneumonia survival.

Respir Res 2015 Oct 6;16:123. Epub 2015 Oct 6.

Institute for Clinical Immunology and Transfusion Medicine, Universities of Giessen and Marburg Lung Center (UGMLC), Member of the German Center for Lung Research (DZL), University Hospital Giessen und Marburg, Justus-Liebig-University Giessen, Langhansstr. 7, D-35390, Giessen, Germany.

Background: Numerous studies have described the immunosuppressive capacity of mesenchymal stem cells (MSC) but these studies use mixtures of heterogeneous progenitor cells for in vitro expansion. Recently, multipotent MSC have been prospectively identified in murine bone marrow (BM) on the basis of PDFGRa(+) SCA1(+) CD45(-) TER119(-) (PαS) expression but the immunomodulatory capacity of these MSC is unknown.

Methods: We isolated PαS MSC by high-purity FACS sorting of murine BM and after in vitro expansion we analyzed the in vivo immunomodulatory activity during acute pneumonia. PαS MSC (1 × 10(6)) were applied intratracheally 4 h after acute respiratory Klebsiella pneumoniae induced infection.

Results: PαS MSC treatment resulted in significantly reduced alveolitis and protein leakage in comparison to mock-treated controls. PαS MSC-treated mice exhibited significantly reduced alveolar TNF-α and IL-12p70 expression, while IL-10 expression was unaffected. Dissection of respiratory dendritic cell (DC) subsets by multiparameter flow cytometry revealed significantly reduced lung DC infiltration and significantly reduced CD86 costimulatory expression on lung CD103(+) DC in PαS MSC-treated mice. In the post-acute phase of pneumonia, PαS MSC-treated animals exhibited significantly reduced respiratory IL-17(+) CD4(+) T cells and IFN-γ(+) CD4(+) T cells. Moreover, PαS MSC treatment significantly improved overall pneumonia survival and did not increase bacterial load.

Conclusion: In this study we demonstrated for the first time the feasibility and in vivo immunomodulatory capacity of prospectively defined MSC in pneumonia.
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http://dx.doi.org/10.1186/s12931-015-0288-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4594670PMC
October 2015

Oncogenic NRAS Primes Primary Acute Myeloid Leukemia Cells for Differentiation.

PLoS One 2015 22;10(4):e0123181. Epub 2015 Apr 22.

Department of Hematology, Oncology and Immunology, Philipps University of Marburg, and University Clinic Giessen and Marburg, Marburg, Germany.

RAS mutations are frequently found among acute myeloid leukemia patients (AML), generating a constitutively active signaling protein changing cellular proliferation, differentiation and apoptosis. We have previously shown that treatment of AML patients with high-dose cytarabine is preferentially beneficial for those harboring oncogenic RAS. On the basis of a murine AML cell culture model, we ascribed this effect to a RAS-driven, p53-dependent induction of differentiation. Hence, in this study we sought to confirm the correlation between RAS status and differentiation of primary blasts obtained from AML patients. The gene expression signature of AML blasts with oncogenic NRAS indeed corresponded to a more mature profile compared to blasts with wildtype RAS, as demonstrated by gene set enrichment analysis (GSEA) and real-time PCR analysis of myeloid ecotropic viral integration site 1 homolog (MEIS1) in a unique cohort of AML patients. In addition, in vitro cell culture experiments with established cell lines and a second set of primary AML cells showed that oncogenic NRAS mutations predisposed cells to cytarabine (AraC) driven differentiation. Taken together, our findings show that AML with inv(16) and NRAS mutation have a differentiation gene signature, supporting the notion that NRAS mutation may predispose leukemic cells to AraC induced differentiation. We therefore suggest that promotion of differentiation pathways by specific genetic alterations could explain the superior treatment outcome after therapy in some AML patient subgroups. Whether a differentiation gene expression status may generally predict for a superior treatment outcome in AML needs to be addressed in future studies.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0123181PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4406710PMC
January 2016

Sorafenib induces paradoxical phosphorylation of the extracellular signal-regulated kinase pathway in acute myeloid leukemia cells lacking FLT3-ITD mutation.

Leuk Lymphoma 2015 3;56(9):2690-8. Epub 2015 Mar 3.

a Klinik für Innere Medizin und Hämatologie, Onkologie, Immunologie, Philipps Universität Marburg und Universitätsklinikum Gießen und Marburg , Marburg , Germany.

Gain-of-function mutations in the RAS and FLT3 genes are frequently found in cells of acute myeloid leukemia (AML), leading to constitutive activation of signaling pathways that regulate fundamental cellular processes, and are therefore attractive targets for AML therapy. The multi-targeted kinase inhibitor sorafenib is efficacious in AML with FLT3-internal tandem duplication (ITD), but resistance to therapy is an important clinical problem. It is unclear whether AML lacking FLT3-ITD responds to sorafenib. Using AML cell lines, we have shown that a low concentration of sorafenib induces opposing effects depending on the oncogenic background. In FLT3-ITD positive cells sorafenib blocks Erk activity and cell proliferation, and triggers apoptosis. However, in cells lacking FLT3-ITD, sorafenib paradoxically activates Erk2, and stimulates cellular proliferation and metabolic activity. Thus, depending on the genetic context, sorafenib is a beneficial inhibitor or paradoxical activator of mitogenic signaling pathways in AML. These results harbor important consequences in planning clinical trials in AML.
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http://dx.doi.org/10.3109/10428194.2014.1003055DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4732463PMC
August 2016

A rare ancestral HLA-DRB1(∗)15:01̃DQB1(∗)02:01 haplotype and its reversion in the same Western European family.

Hum Immunol 2015 Mar 27;76(2-3):124-8. Epub 2015 Jan 27.

Diagnostic Center, Institute of Transfusion Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

The HLA-DR and -DQ loci are close neighbors on chromosome 6 that are highly linked. Many common associations between HLA-DR and DQ-alleles are known, normally transmitted as HLA-DR̃DQ haplotypes from one generation to another. Reports of very recent genetic rearrangements between HLA-DR and -DQ are rarely found in the literature. In Europeans haplotypes containing DRB1(∗)15:01, DQB1(∗)02:01, and DQA1(∗)05:01 have not been reported before. We report the finding of the rare HLA haplotype A(∗)24:02̃C(∗)07:02̃B(∗)07:02̃MICA(∗)008:01̃DRB5(∗)01:01̃DRB1(∗)15:01̃DQA1(∗)05:01̃DQB1(∗)02:01̃DPB1(∗)04:01 in a German stem cell donor with East Frisian ancestry. Our observation suggests a rare ancestral recombination between the DR and DQ loci. In order to investigate this haplotype, we typed 50/74 members of the family encompassing four generations for HLA classes I and II by serological and molecular methods. The rare haplotype was identified in 12 heterozygous carriers. Furthermore, we identified and further characterized a putative crossing over event resulting in its reversion to a common haplotype.
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http://dx.doi.org/10.1016/j.humimm.2015.01.016DOI Listing
March 2015

Antithetical NFATc1-Sox2 and p53-miR200 signaling networks govern pancreatic cancer cell plasticity.

EMBO J 2015 Feb 13;34(4):517-30. Epub 2015 Jan 13.

Department of Gastroenterology II, University Medical Center Goettingen, Goettingen, Germany

In adaptation to oncogenic signals, pancreatic ductal adenocarcinoma (PDAC) cells undergo epithelial-mesenchymal transition (EMT), a process combining tumor cell dedifferentiation with acquisition of stemness features. However, the mechanisms linking oncogene-induced signaling pathways with EMT and stemness remain largely elusive. Here, we uncover the inflammation-induced transcription factor NFATc1 as a central regulator of pancreatic cancer cell plasticity. In particular, we show that NFATc1 drives EMT reprogramming and maintains pancreatic cancer cells in a stem cell-like state through Sox2-dependent transcription of EMT and stemness factors. Intriguingly, NFATc1-Sox2 complex-mediated PDAC dedifferentiation and progression is opposed by antithetical p53-miR200c signaling, and inactivation of the tumor suppressor pathway is essential for tumor dedifferentiation and dissemination both in genetically engineered mouse models (GEMM) and human PDAC. Based on these findings, we propose the existence of a hierarchical signaling network regulating PDAC cell plasticity and suggest that the molecular decision between epithelial cell preservation and conversion into a dedifferentiated cancer stem cell-like phenotype depends on opposing levels of p53 and NFATc1 signaling activities.
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http://dx.doi.org/10.15252/embj.201489574DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4331005PMC
February 2015

Characterization of the MeCP2R168X knockin mouse model for Rett syndrome.

PLoS One 2014 26;9(12):e115444. Epub 2014 Dec 26.

University Medical Center Göttingen, Department of Child and Adolescent Health - Division of Neuropediatrics, Göttingen, Germany.

Rett syndrome, one of the most common causes of mental retardation in females, is caused by mutations in the X chromosomal gene MECP2. Mice deficient for MeCP2 recapitulate some of the symptoms seen in patients with Rett syndrome. It has been shown that reactivation of silent MECP2 alleles can reverse some of the symptoms in these mice. We have generated a knockin mouse model for translational research that carries the most common nonsense mutation in Rett syndrome, R168X. In this article we describe the phenotype of this mouse model. In male MeCP2(R168X) mice life span was reduced to 12-14 weeks and bodyweight was significantly lower than in wild type littermates. First symptoms including tremor, hind limb clasping and inactivity occurred at age 27 days. At age 6 weeks nest building, rotarod, open-field and elevated plus maze experiments showed impaired motor performance, reduced activity and decreased anxiety-like behavior. Plethysmography at the same time showed apneas and irregular breathing with reduced frequency. Female MeCP2R168X mice showed no significant abnormalities except decreased performance on the rotarod at age 9 months. In conclusion we show that the male MeCP2(R168X) mice have a phenotype similar to that seen in MECP2 knockout mouse models and are therefore well suited for translational research. The female mice, however, have a much milder and less constant phenotype making such research with this mouse model more challenging.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0115444PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4277341PMC
August 2015

Immunosuppressive capabilities of mesenchymal stromal cells are maintained under hypoxic growth conditions and after gamma irradiation.

Cytotherapy 2015 Feb 21;17(2):152-62. Epub 2014 Nov 21.

Department of Hematology, Oncology and Immunology, Philipps University Marburg, Marburg, Germany. Electronic address:

Background Aims: The discovery of regenerative and immunosuppressive capacities of mesenchymal stromal cells (MSCs) raises hope for patients with tissue-damaging or severe, treatment-refractory autoimmune disorders. We previously presented a method to expand human MSCs in a bioreactor under standardized Good Manufacturing Practice conditions. Now we characterized the impact of critical treatment conditions on MSCs with respect to immunosuppressive capabilities and proliferation.

Methods: MSC proliferation and survival after γ irradiation were determined by 5-carboxyfluorescein diacetate N-succinimidyl ester and annexinV/4',6-diamidino-2-phenylindole (DAPI) staining, respectively. T-cell proliferation assays were used to assess the effect of γ irradiation, passaging, cryopreservation, post-thaw equilibration time and hypoxia on T-cell suppressive capacities of MSCs. Quantitative polymerase chain reaction and β-galactosidase staining served as tools to investigate differences between immunosuppressive and non-immunosuppressive MSCs.

Results: γ irradiation of MSCs abrogated their proliferation while vitality and T-cell inhibitory capacity were preserved. Passaging and long cryopreservation time decreased the T-cell suppressive function of MSCs, and postthaw equilibration time of 5 days restored this capability. Hypoxic culture markedly increased MSC proliferation without affecting their T-cell-suppressive capacity and phenotype. Furthermore, T-cell suppressive MSCs showed higher CXCL12 expression and less β-galactosidase staining than non-suppressive MSCs.

Discussion: We demonstrate that γ irradiation is an effective strategy to abrogate MSC proliferation without impairing the cells' immunosuppressive function. Hypoxia significantly enhanced MSC expansion, allowing for transplantation of MSCs with low passage number. In summary, our optimized MSC expansion protocol successfully addressed the issues of safety and preservation of immunosuppressive MSC function after ex vivo expansion for therapeutic purposes.
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http://dx.doi.org/10.1016/j.jcyt.2014.10.004DOI Listing
February 2015

The inverse agonist DG172 triggers a PPARβ/δ-independent myeloid lineage shift and promotes GM-CSF/IL-4-induced dendritic cell differentiation.

Mol Pharmacol 2015 Feb 14;87(2):162-73. Epub 2014 Nov 14.

Institute of Molecular Biology and Tumor Research (IMT), Center for Tumor Biology and Immunology (ZTI), Philipps University, Marburg, Germany (S.L., F.F., W.M., S.M.-B., R.M.); Institute of Pharmaceutical Chemistry, Center for Tumor Biology and Immunology (ZTI), Philipps University, Marburg, Germany (F.S., W.E.D.); and Clinic for Hematology, Oncology and Immunology (G.G., C.B.); Center for Tumor Biology and Immunology (ZTI), Philipps University, Marburg, Germany

The stilbene derivative (Z)-2-(2-bromophenyl)-3-{[4-(1-methylpiperazine)amino]phenyl}acrylonitrile (DG172) was developed as a highly selective inhibitory peroxisome proliferator-activated receptor (PPAR)β/δ ligand. Here, we describe a novel PPARβ/δ-independent, yet highly specific, effect of DG172 on the differentiation of bone marrow cells (BMCs). DG172 strongly augmented granulocyte-macrophage-colony-stimulating factor (GM-CSF)-induced differentiation of primary BMCs from Ppard null mice into two specific populations, characterized as mature (CD11c(hi)MHCII(hi)) and immature (CD11c(hi)MHCII(lo)) dendritic cells (DCs). IL-4 synergized with DG172 to shift the differentiation from MHCII(lo) cells to mature DCs in vitro. The promotion of DC differentiation occurred at the expense of differentiation to granulocytic Gr1(+)Ly6B(+) cells. In agreement with these findings, transcriptome analyses showed a strong DG172-mediated repression of genes encoding neutrophilic markers in both differentiating wild-type and Ppard null cells, while macrophage/DC marker genes were up-regulated. DG172 also inhibited the expression of transcription factors driving granulocytic differentiation (Cebpe, Gfi1, and Klf5), and increased the levels of transcription factors promoting macrophage/DC differentiation (Irf4, Irf8, Spib, and Spic). DG172 exerted these effects only at an early stage of BMC differentiation induced by GM-CSF, did not affect macrophage-colony-stimulating factor-triggered differentiation to macrophages and had no detectable PPARβ/δ-independent effect on other cell types tested. Structure-function analyses demonstrated that the 4-methylpiperazine moiety in DG172 is required for its effect on DC differentiation, but is dispensable for PPARβ/δ binding. Based on these data we developed a new compound, (Z)-2-(4-chlorophenyl)-3-[4-(4-methylpiperazine-1-yl)phenyl]acrylonitrile (DG228), which enhances DC differentiation in the absence of significant PPARβ/δ binding.
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http://dx.doi.org/10.1124/mol.114.094672DOI Listing
February 2015

A cisplatin-resistant head and neck cancer cell line with cytoplasmic p53(mut) exhibits ATP-binding cassette transporter upregulation and high glutathione levels.

J Cancer Res Clin Oncol 2014 Oct 10;140(10):1689-704. Epub 2014 Jun 10.

Department of Otolaryngology, Head and Neck Surgery, University Hospital Giessen and Marburg, Campus Marburg, Baldingerstrasse, 35033, Marburg, Germany.

Purpose: Head and neck squamous cell carcinoma (HNSCC) cell lines with cytoplasmically sequestered mutant p53 (p53(mut_c)) are frequently more resistant to cisplatin (CDDP) than cells with mutant but nuclear p53 (p53(mut_n)). The aim of the study was to identify underlying mechanisms implicated in CDDP resistance of HNSCC cells carrying cytoplasmic p53(mut).

Methods: Microarray analysis, quantitative reverse transcription polymerase chain reaction, Western blot analysis and immunocytochemistry were used to identify and evaluate candidate genes involved in CDDP resistance of p53(mut_c) cells. RNAi knockdown or treatment with inhibitors together with flow cytometry-based methods was used for functional assessment of the identified candidate genes. Cellular metabolic activity was assessed with the XTT assay, and the redox capacity of cells was evaluated by measuring cellular glutathione (GSH) levels.

Results: Upregulation of ABCC2 and ABCG2 transporters was observed in CDDP-resistant p53(mut_c) HNSCC cells. Furthermore, p53(mut_c) cells exhibited a pronounced side population that could be suppressed by RNAi knockdown of ABCG2 as well as treatment with the ATP-binding-cassette transporter inhibitors imatinib, MK571 and tariquidar. Metabolic activity and cellular GSH levels were higher in CDDP-resistant p53(mut_c) cells, consistent with a higher capacity to fend off cytotoxic oxidative effects such as those caused by CDDP treatment. Finally, ABCC2/G2 inhibition of HNSCC cells with MK571 markedly enhanced CDDP sensitivity of HNSCC cells.

Conclusions: The observations in this study point to a major role of p53(mut_c) in conferring a stem cell like phenotype to HNSCC cells that is associated with ABCC2/G2 overexpression, high GSH and metabolic activity levels as well as CDDP resistance.
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http://dx.doi.org/10.1007/s00432-014-1727-yDOI Listing
October 2014

Monitoring of acute myeloid leukemia patients after allogeneic stem cell transplantation employing semi-automated CD34+ donor cell chimerism analysis.

Ann Hematol 2014 Feb 19;93(2):279-85. Epub 2013 Dec 19.

Klinik für Hämatologie, Onkologie und Immunologie, University Hospital Giessen and Marburg, Campus Marburg, Baldingerstraße, 35043, Marburg, Germany.

Determination of donor chimerism profiles in blood or bone marrow from patients with allogeneic stem cell transplantation (SCT) is useful for monitoring engraftment or predicting relapse, when specific molecular markers are lacking. CD34+ donor cell chimerism (DCC) analysis in peripheral blood samples from CD34+ acute myeloid leukemia (AML) and myleodysplastic syndrome (MDS) patients proved to be a highly sensitive diagnostic tool that is useful to detect imminent relapse significantly earlier compared to total white blood cell donor cell chimerism monitoring. However, flow-cytometric enrichment of CD34+ cells requires high efforts to human resources and equipment. We present a novel semi-automated CD34+ DCC analysis procedure-employing a magnetic cell-enrichment device, DNA extraction, and short tandem repeat profiling-without the need for flow-cytometric cell sorting. Monitoring 85 patients with AML and MDS over a period of 4 years 24 relapses were detected. Semi-automated peripheral blood CD34+ DCC was diminished below 80 % in all cases of systemic relapse. Significant decrease of the CD34+ DCC value was detected 29-42 days before overt cytological relapse. Our method provides a rapid and sensitive tool for monitoring AML and MDS patients after allogeneic SCT with regard to engraftment and early detection of relapse. Here, we propose a novel semi-automated procedure for CD34+ DCC analysis after allogeneic SCT that is simple, reliable, and therefore applicable in all hematologic laboratories.
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http://dx.doi.org/10.1007/s00277-013-1961-4DOI Listing
February 2014

Methotrexate treatment of FraX fibroblasts results in FMR1 transcription but not in detectable FMR1 protein levels.

J Neurodev Disord 2013 Sep 10;5(1):23. Epub 2013 Sep 10.

Department of Pediatrics and Pediatric Neurology, Georg August University, Robert-Koch-Strasse 40, 37075 Göttingen, Germany.

Background: Fragile X syndrome is caused by the loss of FMRP expression due to methylation of the FMR1 promoter. Treatment of fragile X syndrome patients' lymphoblastoid cells with 5-azadeoxycytidine results in demethylation of the promoter and reactivation of the gene. The aim of the study was to analyze if methotrexate, an agent which also reduces DNA methylation but with less toxicity than 5-azadeoxycytidine, has therapeutic potential in fragile X syndrome.

Methods: Fibroblasts of fragile X syndrome patients were treated with methotrexate in concentrations ranging from 1 to 4 μg/ml for up to 14 days. FMR1 and FMRP expression were analyzed by quantitative PCR and western blotting.

Results: FMR1 mRNA was detected and levels correlated positively with methotrexate concentrations and time of treatment, but western blotting did not show detectable FMRP levels.

Conclusions: We show that it is possible to reactivate FMR1 transcription in fibroblasts of fragile X syndrome patients by treatment with methotrexate. However, we were not able to show FMRP expression, possibly due to the reduced translation efficacy caused by the triplet repeat extension. Unless FMR1 reactivation is more effective in vivo our results indicate that methotrexate has no role in the treatment of fragile X syndrome.
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http://dx.doi.org/10.1186/1866-1955-5-23DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3846751PMC
September 2013

Good manufacturing practice-compliant animal-free expansion of human bone marrow derived mesenchymal stroma cells in a closed hollow-fiber-based bioreactor.

Biochem Biophys Res Commun 2013 Jan 9;430(1):325-30. Epub 2012 Nov 9.

Dept. of Hematology, Oncology and Immunology, Philipps University Marburg and Universitätsklinikum Giessen und Marburg, Germany.

Mesenchymal stroma cells (MSC) are increasingly recognized for various applications of cell-based therapies such as regenerative medicine or immunomodulatory treatment strategies. Standardized large-scale expansions of MSC under good manufacturing practice (GMP)-compliant conditions avoiding animal derived components are mandatory for further evaluation of these novel therapeutic approaches in clinical trials. We applied a novel automated hollow fiber cell expansion system (CES) for in vitro expansion of human bone marrow derived MSC employing a GMP-compliant culture medium with human platelet lysate (HPL). Between 8 and 32 ml primary bone marrow aspirate were loaded into the hollow fiber CES and cultured for 15-27 days. 2-58 million MSC were harvested after primary culture. Further GMP-compliant cultivation of second passage MSC for 13 days led to further 10-20-fold enrichment. Viability, surface antigen expression, differentiation capacity and immunosuppressive function of MSC cultured in the hollow fiber CES were in line with standard criteria for MSC definition. We conclude that MSC can be enriched from primary bone marrow aspirate in a GMP-conform manner within a closed hollow fiber bioreactor and maintain their T lymphocyte inhibitory capacity. Standardized and reliable conditions for large scale MSC expansion pave the way for safe applications in humans in different therapeutic approaches.
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http://dx.doi.org/10.1016/j.bbrc.2012.11.001DOI Listing
January 2013

Transcription factor IRF4 determines germinal center formation through follicular T-helper cell differentiation.

Proc Natl Acad Sci U S A 2012 May 2;109(22):8664-9. Epub 2012 May 2.

Institut für Medizinische Mikrobiologie und Krankenhaushygiene, Universität Marburg, 35043 Marburg, Germany.

Follicular T-helper (T(FH)) cells cooperate with GL7(+)CD95(+) germinal center (GC) B cells to induce antibody maturation. Herein, we identify the transcription factor IRF4 as a T-cell intrinsic precondition for T(FH) cell differentiation and GC formation. After immunization with protein or infection with the protozoon Leishmania major, draining lymph nodes (LNs) of IFN-regulatory factor-4 (Irf4(-/-)) mice lacked GCs and GC B cells despite developing normal initial hyperplasia. GCs were also absent in Peyer's patches of naive Irf4(-/-) mice. Accordingly, CD4(+) T cells within the LNs and Peyer's patches failed to express the T(FH) key transcription factor B-cell lymphoma-6 and other T(FH)-related molecules. During chronic leishmaniasis, the draining Irf4(-/-) LNs disappeared because of massive cell death. Adoptive transfer of WT CD4(+) T cells or few L. major primed WT T(FH) cells reconstituted GC formation, GC B-cell differentiation, and LN cell survival. In support of a T-cell intrinsic IRF4 activity, Irf4(-/-) T(FH) cell differentiation was not rescued by close neighborhood to transferred WT T(FH) cells. Together with its known B lineage-specific roles during plasma cell maturation and class switch, our study places IRF4 in the center of antibody production toward T-cell-dependent antigens.
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http://dx.doi.org/10.1073/pnas.1205834109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3365194PMC
May 2012