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Mensen A.,Institute of Medical Immunology | Mensen A.,Experimental and Clinical Research Center | Ochs C.,Institute of Medical Immunology | Stroux A.,Institute for Biometry and Clinical Epidemiology | And 13 more authors.
Journal of Translational Medicine | Year: 2013

Background: After hematopoietic stem cell transplantation (HSCT) T- and B-cell reconstitution from primary lymphoid organs are a prerequisite for an effective early lymphocyte reconstitution and a long-term survival for adult patients suffering from acute leukemia. Here, we asked whether quantification of T cell receptor excision circle, (TREC) and kappa-deleting recombination excision circle (KREC) before and within six month after allogeneic HSCT could be used to measure the thymic and bone marrow outputs in such patients.Methods: We used a duplex real time PCR assay to quantify the absolute copy counts of TREC and KREC, and correlated the data with absolute cell counts of CD3+CD4+ T-cell and CD19+ B-cell subsets determined by flow cytometry, respectively.Results: By comparing two recently proposed naïve T cell subsets, CD31+ naive and CD31- naive T cells, we found a better correlation for the CD31+ subset with TREC level post alloHSCT, in line with the assumption that it contained T cells recently derived from the thymus, indicating that TREC levels reflected real thymic de novo production. Transitional as well as naïve B cells highly correlated with KREC levels, which suggested an association of KREC levels with ongoing bone marrow B cell output. CD45RO+ memory T cells and CD27+ memory B cells were significantly less correlated with TREC and KREC recovery, respectively.Conclusion: We conclude that simultaneous TREC/ KREC quantification is as a suitable and practicable method to monitor thymic and bone marrow output post alloHSCT in adult patients diagnosed with acute leukemia. © 2013 Mensen et al.; licensee BioMed Central Ltd.

Schmidt-Hieber M.,Hematology | Schwarck S.,Hematology | Stroux A.,Institute for Biometry and Clinical Epidemiology | Ganepola S.,Hematology | And 4 more authors.
International Journal of Hematology | Year: 2010

We analyzed cytomegalovirus (CMV) infection risk factors and immune reconstitution kinetics in 89 patients after allogeneic stem cell transplantation (allo-SCT). The use of alemtuzumab for in vivo T cell depletion (TCD) had, besides the donor/recipient CMV serostatus, the strongest influence on the CMV infection risk in univariate and multivariate analyses. In comparison to without use of in vivo TCD, the CMV infection risk [hazard ratio (HR)] was 4.82-fold after TCD with alemtuzumab, but only 1.40-fold after TCD with antithymocyte globulin (ATG). Alemtuzumab strongly depressed CD4+ and CD8+ T cell reconstitution, whereas ATG only delayed CD4+ T cell reconstitution. Considering the reconstitution kinetics of CD4 + and CD8+ T cells, CMV-specific CD8+ T cells, NK cells and the IgG concentration, only a low day +60 NK cell count (161 versus >161/μl) was significantly associated with CMV infection development (HR 2.92, p = 0.034). CMV-specific CD8+ T cells were detected in 57% of patients with a CMV-seropositive donor, but in none of the patients with a CMV-seronegative donor on day +30 (p = 0.01). Our data indicate that the type of in vivo TCD (alemtuzumab or ATG) differentially influences both the CMV infection risk and CD4+/CD8+ T cell reconstitution kinetics in patients after allo-SCT. © 2010 The Japanese Society of Hematology.

Lindner D.,University of Hamburg | Stroux A.,Institute for Biometry and Clinical Epidemiology | Scheibenbogen C.,Charite - Medical University of Berlin | Klingel K.,University of Tubingen | And 2 more authors.
Journal of Molecular and Cellular Cardiology | Year: 2014

Therapeutic targets of broad relevance are likely located in pathogenic pathways common to disorders of various etiologies. Screening for targets of this type revealed CCN genes to be consistently upregulated in multiple cardiomyopathies. We developed RNA interference (RNAi) to silence CCN2 and found this single-target approach to block multiple proinflammatory and profibrotic pathways in activated primary cardiac fibroblasts (PCFBs). The RNAi-strategy was developed in murine PCFBs and then investigated in "individual" human PCFBs grown from human endomyocardial biopsies (EMBs). Screening of short hairpin RNA (shRNA) sequences for high silencing efficacy and specificity yielded RNAi adenovectors silencing CCN2 in murine or human PCFBs, respectively. Comparison of RNAi with CCN2-modulating microRNA (miR) vectors expressing miR-30c or miR-133b showed higher efficacy of RNAi. In murine PCFBs, CCN2 silencing resulted in strongly reduced expression of stretch-induced chemokines (Ccl2, Ccl7, Ccl8), matrix metalloproteinases (MMP2, MMP9), extracellular matrix (Col3a1), and a cell-to-cell contact protein (Cx43), suggesting multiple signal pathways to be linked to CCN2. Immune cell chemotaxis towards CCN2-depleted PCFBs was significantly reduced. We demonstrate here that this RNAi strategy is technically applicable to "individual" human PCFBs, too, but that these display individually strikingly different responses to CCN2 depletion. Either genomically encoded factors or stable epigenetic modification may explain different responses between individual PCFBs. The new RNAi approach addresses a key regulator protein induced in cardiomyopathies. Investigation of this and other molecular therapies in individual human PCBFs may help to dissect differential pathogenic processes between otherwise similar disease entities and individuals. © 2013 Elsevier Ltd.

Stein L.,Private Practice | Hechler D.,Private Practice | Jessen A.B.,Private Practice | Neumann K.,Institute for Biometry and Clinical Epidemiology | And 2 more authors.
International Journal of STD and AIDS | Year: 2012

Physical activity has been recommended based on beneficial effects described in HIV-infected patients. However, such guidelines do not take into account actual sport behaviours and general attitudes towards physical activity. To evaluate actual sport activity and attitudes towards sport in HIV-infected versus non-infected individuals we conducted an anonymous questionnaire investigating the prevalence, as well as possible changes, in sports engagement and the overall attitude to physical activity. A total of 283 patients of a general care facility specialized in the treatment of HIV/AIDS in Berlin, Germany, participated; 124 were HIV infected and 159 were non-infected, mostly men who have sex with men (MSM) (88%), with a median age of 35 years. The HIV-infected participants had a median CD4+ count of 554 cells/μL and 48.8% of them were using antiretroviral therapy (ART) at the time of survey. The proportion of patients actually performing physical activity was significantly lower (P = 0.028) within the HIV-infected group (61.3%) than within the non-infected group (74.2%). This difference remained significant after accounting for possible confounders such as age, gender, injecting drug use and sexual preferences. Previously reported sport activity prevalence was similar in both groups on leaving school. From our data we could not identify an association between the time of HIV diagnosis and changes in sports activity. In conclusion, fewer HIV-infected individuals report physical activity than non-infected individuals. Sociodemographic studies to evaluate potential differences in sports behaviour are required in order to inform exercise guidelines for HIV-infected patients.

Ochsenreither S.,Charite Campus Benjamin Franklin | Fusi A.,Charite Campus Benjamin Franklin | Geikowski A.,Charite Campus Benjamin Franklin | Stather D.,Charite Campus Benjamin Franklin | And 4 more authors.
Cancer Immunology, Immunotherapy | Year: 2012

Background: Clinically effective T-cell responses can be elicited by single peptide vaccination with Wilms' tumor 1 (WT1) epitope 126-134 in patients with acute myeloid leukemia (AML). We recently showed that a predominant T-cell receptor (TCR) β chain was associated with vaccine-induced complete remission in an AML patient (patient 1). In this study, we address the question of whether this predominant clone or the accompanying Vβ11 restriction could be found in other AML patients vaccinated with the same WT1 peptide. Materials and methods: For assessment of Vβ usage, cytotoxic T lymphocytes (CTLs) from four vaccinated patients were divided into specific and non-specific by epitope-specific enrichment. Vβ families were quantified in both fractions using reverse transcribed quantitative PCR. Vβ11-positive 'complementary determining region 3' (CDR3) sequences were amplified from these samples, from bone marrow samples of 17 other vaccination patients, and from peripheral blood of six healthy controls, cloned and sequenced. Results: We observed a clear bias towards Vβ11 usage of the WT1-specific CTL populations in all four patients. The predominant CDR3β amino acid (AA) sequence of patient 1 was detected in two other patients. CDR3β loops with closely related AA sequences were only found in patient 1. There were no CDR3β AA sequences with side chains of identical chemical properties detected in any patient. Conclusion We provide the first data addressing TCR Vβ chain usage in WT1-specific T-cell populations after HLA A*0201-restricted single peptide vaccination. We demonstrate both shared Vβ restriction and the sharing of a TCR β transcript with proven clinical impact in one patient. © Springer-Verlag 2011.

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