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Mensen A.,Institute of Medical Immunology | Mensen A.,Experimental and Clinical Research Center | Na I.-K.,Experimental and Clinical Research Center | Hafer R.,Jena University Hospital | And 4 more authors.
Journal of Cancer Research and Clinical Oncology | Year: 2014

Purpose: Rabbit antithymocyte globulin (ATG) is commonly used before allogeneic hematopoietic stem cell transplantation (allo-HSCT) to prevent graft-versus-host disease. Studies comparing the effect of different ATG preparations and dosages on immune reconstitution and risk for Epstein–Barr virus (EBV)-mediated post-transplant lymphoproliferative disorder (PTLD) are rare.Methods: In this retrospective study, we determined T and B cell subsets by flow cytometry after allo-HSCT in children, who received ATG-Genzyme (ATG-G, n = 15), ATG-Fresenius (ATG-F, n = 25) or no-ATG treatment (n = 19). Additionally, PCR-quantified EBV-genome copy counts were correlated with incidence of PTLD.Results: We could confirm a dose-dependent impairment of CD8+ and CD4+ T cell regeneration by ATG-G, including naïve and memory CD4+ T cells. No differences were seen between the currently applied dosages of 5–10 mg/kg ATG-G and 20–60 mg/kg ATG-F. Significantly delayed T cell subset reconstitution was determined only at high dosages of 20–60 mg/kg ATG-G compared to ATG-F. B cell reconstitution was comparably impaired in ATG-G- and ATG-F-treated patients. Although the incidence of EBV reactivation was similar in both ATG groups, EBV copy counts of >104 copies/105 peripheral blood mononuclear cells and the occurrence of PTLD were only found in ATG-G-treated patients.Conclusions: We conclude that high, but importantly not currently applied low dosages of ATG-G, impair thymic T cell regeneration and memory T cell immunity to a greater extent than ATG-F in pediatric patients. In addition, our results suggest an increased risk for EBV-PTLD when treated with ATG-G. Prospective studies are warranted to compare different ATG preparations with regard to the immune reconstitution and EBV-PTLD. © 2014, Springer-Verlag Berlin Heidelberg.


Abou-El-Enein M.,Berlin Brandenburg Center for Regenerative Therapies | Abou-El-Enein M.,Charité - Medical University of Berlin | Romhild A.,Berlin Brandenburg Center for Regenerative Therapies | Kaiser D.,Berlin Brandenburg Center for Regenerative Therapies | And 6 more authors.
Cytotherapy | Year: 2013

Background aims. Advanced therapy medicinal products (ATMP) have gained considerable attention in academia due to their therapeutic potential. Good Manufacturing Practice (GMP) principles ensure the quality and sterility of manufacturing these products. We developed a model for estimating the manufacturing costs of cell therapy products and optimizing the performance of academic GMP-facilities. Methods. The 'Clean-Room Technology Assessment Technique' (CTAT) was tested prospectively in the GMP facility of BCRT, Berlin, Germany, then retrospectively in the GMP facility of the University of California-Davis, California, USA. CTAT is a two-level model: level one identifies operational (core) processes and measures their fixed costs; level two identifies production (supporting) processes and measures their variable costs. The model comprises several tools to measure and optimize performance of these processes. Manufacturing costs were itemized using adjusted micro-costing system. Results. CTAT identified GMP activities with strong correlation to the manufacturing process of cell-based products. Building best practice standards allowed for performance improvement and elimination of human errors. The model also demonstrated the unidirectional dependencies that may exist among the core GMP activities. When compared to traditional business models, the CTAT assessment resulted in a more accurate allocation of annual expenses. The estimated expenses were used to set a fee structure for both GMP facilities. A mathematical equation was also developed to provide the final product cost. Conclusions. CTAT can be a useful tool in estimating accurate costs for the ATMPs manufactured in an optimized GMP process. These estimates are useful when analyzing the cost-effectiveness of these novel interventions. © 2013, International Society for Cellular Therapy.


Noutsias M.,Charité - Medical University of Berlin | Rohde M.,Charité - Medical University of Berlin | Gldner K.,Institute of Medical Immunology | Block A.,Charité - Medical University of Berlin | And 9 more authors.
European Journal of Heart Failure | Year: 2011

AimsTo quantify and functionally characterize the intramyocardial T-cells in endomyocardial biopsies (EMBs) from patients presenting with acute myocarditis (AMC) and dilated cardiomyopathy (DCM).Methods and resultsExpression of genes characterizing Th1 [interferon (IFN)γ, Tbet-1, Eomesodermin, interleukin (IL)-27], Th2 (IL-4, IL-5, GATA3), Th17 (IL-17), regulatory [regulatory T-cells (Treg); FoxP3, TGFβ, IL-10], anergic (GRAIL), and cytotoxic T-cells (CTLs: Perforin, Granulysin, Granzyme A), as well as of functional T-cell receptor Vbeta (TRBV) families were investigated in EMBs from AMC patients (n 58) and DCM patients (n 34) by pre-amplified real-time reverse transcription-polymerase chain reaction. These data were compared with EMBs from n 19 controls. Expression of CD3d, CD3z, and TRBC (T-cell receptor beta constant region) were associated with the immunohistological diagnosis of inflammatory cardiomyopathy (DCMi). In EMBs from DCM patients with increased CD3d expression, significantly increased markers of Th1 (IFNγ, T-bet, Eomesodermin), regulatory T-cells (Treg; FoxP3, TGFβ), and cytotoxic T-cells (CTLs: Perforin, Granulysin, Granzyme A) were present, while no differential polarization of T-cells was found in EMBs form AMC patients. A differential dominance of distinct functional TRBV families was associated with different cardiotropic viruses: TRBV 11 and 24 with Parvovirus B19; TRBV4, 10 and 28 with human herpes virus type 6; and TRBV14 for Coxsackie virus, respectively.ConclusionsThe T-cell infiltrates in human DCMi are characterized by differential expression of functional T-cell markers indicating Th1, Treg, and CTLs, while no major role could be confirmed for Th17. The virus-associated differential TRBV dominance suggests an antiviral specificity of virus-induced T-cell responses in human DCMi. © 2011 The Author.


Mahrenholz C.C.,Institute of Medical Immunology | Abfalter I.G.,Johannes Kepler University | Bodenhofer U.,Johannes Kepler University | Volkmer R.,Institute of Medical Immunology | Hochreiter S.,Johannes Kepler University
Molecular and Cellular Proteomics | Year: 2011

Understanding the relationship between protein sequence and structure is one of the great challenges in biology. In the case of the ubiquitous coiled-coil motif, structure and occurrence have been described in extensive detail, but there is a lack of insight into the rules that govern oligomerization, i.e. how many α-helices form a given coiled coil. To shed new light on the formation of two- and three-stranded coiled coils, we developed a machine learning approach to identify rules in the form of weighted amino acid patterns. These rules form the basis of our classification tool, PrOCoil, which also visualizes the contribution of each individual amino acid to the overall oligomeric tendency of a given coiled-coil sequence. We discovered that sequence positions previously thought irrelevant to direct coiled-coil interaction have an undeniable impact on stoichiometry. Our rules also demystify the oligomerization behavior of the yeast transcription factor GCN4, which can now be described as a hybrid - part dimer and part trimer - with both theoretical and experimental justification. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.


Na I.-K.,Charité - Medical University of Berlin | Na I.-K.,Institute of Medical Immunology | Na I.-K.,Experimental and Clinical Research Center | Wittenbecher F.,Charité - Medical University of Berlin | And 12 more authors.
Haematologica | Year: 2013

Rabbit antithymocyte globulin-Genzyme™ is used to prevent graft-versus-host disease after allogeneic hematopoietic stem cell transplantation. Common disadvantages of treatment are infectious complications. The effects of rabbit antithymocyte globulin-Genzyme™ on thymic function have not been well-studied. Multicolor flow cytometry was used to analyze the kinetics of conventional and regulatory T cells in adult patients treated (n=12) or not treated (n=8) with rabbit antithymocyte globulin-Genzyme™ during the first 6 months after allogeneic hematopoietic stem cell transplantation. Patients treated with rabbit antithymocyte globulin-Genzyme™ had almost undetectable levels of recent thymic emigrants (CD45RA+CD31+) of both conventional and regulatory CD4 T cells throughout the 6 months after allogeneic hematopoietic stem cell transplantation whereas CD4+CD45RAmemory T cells were less affected, but their levels were also significantly lower than in patients not treated with rabbit antithymocyte globulin-Genzyme™. In vitro, rabbit antithymocyte globulin-Genzyme™ induced apoptosis and cytolysis of human thymocytes, and its cytotoxic effects were greater than those of rabbit antithymocyte globulin-FreseniusTM. Rabbit antithymocyte globulin-Genzyme™ in combination with a conditioning regimen strongly impairs thymic recovery of both conventional and regulatory CD4+ T cells. The sustained depletion of conventional and regulatory CD4+ T cells carries a high risk of both infections and graft-versus-host disease. Our data indicate that patients treated with rabbit antithymocyte globulin-Genzyme™ could benefit from thymus-protective therapies and that trials comparing this product with other rabbit antithymocyte globulin preparations or lymphocyte-depleting compounds would be informative. ©2013 Ferrata Storti Foundation.


Sawitzki B.,Institute of Medical Immunology | Sawitzki B.,Berlin Brandenburg Center for Regenerative Therapies | Reinke P.,Berlin Brandenburg Center for Regenerative Therapies | Pascher A.,Charité - Medical University of Berlin | And 2 more authors.
Current Opinion in Organ Transplantation | Year: 2010

Purpose of Review: In order to avoid toxic side effects by long-term immunosuppressive treatment, transplant research therefore focuses on new strategies to either induce tolerance or allow partial or complete immunosuppressive weaning whenever possible. This can be only safely achieved when guided by biomarkers reflecting the individual immune reactivity. Here we summarize the recent efforts to identify biomarkers and functional assays which allow an individualized minimization or complete weaning of immunosuppression in stable or 'operational' tolerant transplant patients, respectively. Recent Findings: Data obtained by two main collaborative networks, 'RISET' and 'Immune Tolerance Network' have provided a better characterization of operational tolerant kidney patients. In both studies an increased numbers of B cells and a B-cell-associated peripheral gene signature were discovered. Long-term observation of tolerant liver transplant patients undergoing immunosuppressive minimization highlight the importance of surveillance or protocol biopsies. Additionally, functional assays such as IFN-γ ELISPOT or urine markers have been shown to predict long-term graft outcome. Summary: With the recent findings we have gained a better understanding of operational tolerant patients and have identified biomarkers and assays which will be very helpful when guiding partial or complete immunosuppressive minimization. For the future, collaborative efforts are needed to design and perform prospective multicenter trials to validate the identified biomarkers across different laboratories and laboratory platforms. © 2010 Wolters Kluwer Health | Lippincott Williams & Wilkins.


Appel H.,Charité - Medical University of Berlin | Wu P.,Charité - Medical University of Berlin | Scheer R.,Charité - Medical University of Berlin | Kedor C.,Charité - Medical University of Berlin | And 5 more authors.
Journal of Rheumatology | Year: 2011

Objective. Regulatory T cells are characterized by expression of the transcription factor FoxP3 and are thought to be involved in the pathogenesis of autoimmune diseases. We determined the frequency and phenotypic characteristics of CD4+FoxP3+ T cells in the blood and synovial fluid (SF) of patients with inflammatory joint diseases. Methods. SF from 10 patients with ankylosing spondylitis (AS), 20 patients with other spondyloarthritides or with peripheral arthritis (pSpA), and 12 patients with rheumatoid arthritis (RA), and peripheral blood (PB) from 22 patients with AS, 19 with pSpA, 15 with RA, and 12 healthy controls were stained for CD4, FoxP3, CD25, and CD127 and different effector cytokines and then analyzed by flow cytometry. Methylation pattern of the Treg-specific demethylated region (TSDR) was determined after bisulfite treatment by quantitative polymerase chain reaction. Results. In all groups of patients we observed higher frequencies of Foxp3+ cells/CD4+ T cells within SF compared to PB. The frequency of synovial Foxp3+ cells/CD4+ T cells was significantly higher in patients with pSpA (18.79% ± 6.41%) compared to patients with AS (9.69% ± 4.11%) and patients with RA (5.95% ± 2.21%). CD4+FoxP3+ T cells were CD25+ and CD127- and lacked effector cytokine production in any of the different patient groups. The majority of the CD4+CD25+CD127- T cells showed demethylation of the TSDR within the Foxp3 locus, confirming its regulatory phenotype. Conclusion. Our data show accumulation of Foxp3+ T cells within inflamed joints. These Foxp3+ T cells are mainly of stable T regulatory phenotype. The high frequency of Foxp3+ T cells in pSpA might contribute to the spontaneous resolution and remitting course of arthritis in pSpA as compared to the more persistent joint inflammation in RA. The Journal of Rheumatology Copyright © 2011. All rights reserved.


Wehrens E.J.,University Utrecht | Mijnheer G.,University Utrecht | Duurland C.L.,University Utrecht | Klein M.,University Utrecht | And 9 more authors.
Blood | Year: 2011

During the last decade research has focused on the application of FOXP3 + regulatory T cells (Tregs) in the treatment of autoimmune disease. However, thorough functional characterization of these cells in patients with chronic autoimmune disease, especially at the site of inflammation, is still missing. Here we studied Treg function in patients with juvenile idiopathic arthritis (JIA) and observed that Tregs from the peripheral blood as well as the inflamed joints are fully functional. Nevertheless, Treg-mediated suppression of cell proliferation and cytokine production by effector cells from the site of inflammation was severely impaired, because of resistance to suppression. This resistance to suppression was not caused by a memory phenotype of effector T cells or activation status of antigen presenting cells. Instead, activation of protein kinase B (PKB)/c-akt was enhanced in inflammatory effector cells, at least partially in response to TNFα and IL-6, and inhibition of this kinase restored responsiveness to suppression. We are the first to show that PKB/c-akt hyperactivation causes resistance of effector cells to suppression in human autoimmune disease. Furthermore, these findings suggest that for a Treg enhancing strategy to be successful in the treatment of autoimmune inflammation, resistance because of PKB/c-akt hyperactivation should be targeted as well. © 2011 by The American Society of Hematology.


Busse A.,Charite CBF | Letsch A.,Charite CBF | Scheibenbogen C.,Institute of Medical Immunology | Nonnenmacher A.,Charite CBF | And 3 more authors.
Journal of Translational Medicine | Year: 2010

Background: Efficacy of cancer vaccines may be limited due to immune escape mechanisms like loss or mutation of target antigens. Here, we analyzed 10 HLA-A2 positive patients with acute myeloid leukemia (AML) for loss or mutations of the WT1 epitope or epitope flanking sequences that may abolish proper T cell recognition or epitope presentation.Methods: All patients had been enrolled in a WT1 peptide phase II vaccination trial (NCT00153582) and ultimately progressed despite induction of a WT1 specific T cell response. Blood and bone marrow samples prior to vaccination and during progression were analyzed for mRNA expression level of WT1. Base exchanges within the epitope sequence or flanking regions (10 amino acids N- and C-terminal of the epitope) were assessed with melting point analysis and sequencing. HLA class I expression and WT1 protein expression was analyzed by flow cytometry.Results: Only in one patient, downregulation of WT1 mRNA by 1 log and loss of WT1 detection on protein level at time of disease progression was observed. No mutation leading to a base exchange within the epitope sequence or epitope flanking sequences could be detected in any patient. Further, no loss of HLA class I expression on leukemic blasts was observed.Conclusion: Defects in antigen presentation caused by loss or mutation of WT1 or downregulation of HLA molecules are not the major basis for escape from the immune response induced by WT1 peptide vaccination. © 2010 Busse et al; licensee BioMed Central Ltd.


PubMed | In Vivo Cellular and Molecular Imaging Center, Institute of Medical Immunology and Vrije Universiteit Brussel
Type: Journal Article | Journal: Molecular imaging and biology : MIB : the official publication of the Academy of Molecular Imaging | Year: 2016

Kupffer cells (KCs), the liver resident macrophages, are important mediators of tissue homeostasis and pathogen clearance. However, depending on the inflammatory stimuli, KCs have been involved in divergent hepato-protective or hepato-destructive immune responses. The versatility of KCs in response to environmental triggers, in combination with the specific biomarkers they express, make these macrophages attractive in vivo targets for non-invasive monitoring of liver inflammation or pathogenicity. This study aims to determine whether V-set and Ig domain-containing 4 (Vsig4) and C-type lectin domain family (Clec) 4, member F (Clec4F) can be used as imaging biomarkers for non-invasive monitoring of KCs during distinct liver inflammation models.Flow cytometry (FACS), immuno-histochemistry (IHC), and single-photon emission computed tomography (SPECT) with Tc-99m labeled anti-Vsig4 or anti-Clec4F nanobodies (Nbs) was performed to evaluate in mice KC dynamics in concanavalin A (ConA)-induced hepatitis and in non-alcoholic steatohepatitis induced via methionine choline deficiency (MCD).In homeostatic mice, Nbs targeting Clec4F were found to accumulate and co-localize with Vsig4-targeting Nbs only in the liver. Upon induction of acute hepatitis using ConA, down-regulation of the in vivo Nb imaging signal was observed, reflecting reduction in KC numbers as confirmed by FACS and IHC. On the other hand, induction of steatohepatitis resulted in higher signals in the liver corresponding to higher density of KCs. The Nb-imaging signals returned to normal levels after resolution of the investigated liver diseases.Anti-Clec4F and anti-Vsig4 Nbs targeting KCs as molecular imaging biomarkers could allow non-invasive monitoring/staging of liver pathogenesis.

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