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  • 1
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    Hypotension-Avoidance Versus Hypertension-Avoidance Strategies in Noncardiac Surgery : An International Randomized Controlled Trial
    American College of Physicians ; 2023
    In:  Annals of Internal Medicine Vol. 176, No. 5 ( 2023-05), p. 605-614
    Details
    In: Annals of Internal Medicine, American College of Physicians, Vol. 176, No. 5 ( 2023-05), p. 605-614
    Type of Medium: Online Resource
    ISSN: 0003-4819 , 1539-3704
    URL: Article
    DOI: 10.7326/M22-3157
    RVK:
    XA 23600
    Language: English
    Publisher: American College of Physicians
    Publication Date: 2023
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  • 2
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    Generation of a multipathogen-specific T-cell product for adoptive immunotherapy based on activation-dependent expression of CD154
    American Society of Hematology ; 2011
    In:  Blood Vol. 118, No. 4 ( 2011-07-28), p. 1121-1131
    Details
    In: Blood, American Society of Hematology, Vol. 118, No. 4 ( 2011-07-28), p. 1121-1131
    Abstract: Viral and fungal infections remain a leading cause of mortality in patients after hematopoietic stem cell transplantation (HSCT). Adoptive transfer of multipathogen-specific T cells is promising in restoring immunity and thereby preventing and treating infections, but approaches are currently limited because of time-consuming and laborious procedures. Therefore, we investigated a new strategy to simultaneously select T cells specific for viral and fungal pathogens based on activation-dependent expression of CD154. Single- and multipathogen-specific T-cell lines with high specificity for adenovirus (AdV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), Candida albicans, and/or Aspergillus fumigatus could be readily generated within 14 days irrespective of the precursor frequency. The T-cell lines responded reproducibly to endogenously processed antigen and specifically proliferated upon antigenic stimulation. Although isolation based on CD154 favors enrichment of CD4+ T cells, AdV-, EBV- and CMV-specific CD8+ T cells could be expanded and demonstrated lysis of target cells. Conversely, T cell–mediated alloreactivity was almost abrogated compared with the starting fraction. This selection and/or expansion strategy may form the basis for future adoptive immunotherapy trials in patients at risk for multiple infections and may be translated to other antigens.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood-2010-12-322610
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2011
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  • 3
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    Identification of Low Frequency Multiple Novel Aspergillus Fumigatus Specific MHC Class II Epitopes and Rapid Generation of An Aspergillus-Specific T-Cell Product Based On Activation Dependent Expression of CD154.
    American Society of Hematology ; 2009
    In:  Blood Vol. 114, No. 22 ( 2009-11-20), p. 1170-1170
    Details
    In: Blood, American Society of Hematology, Vol. 114, No. 22 ( 2009-11-20), p. 1170-1170
    Abstract: Abstract 1170 Poster Board I-192 Despite new antifungal drugs, invasive aspergillosis (IA) remains a major cause of morbidity and mortality in patients undergoing myeloablative chemotherapy and allogeneic stem cell transplantation. Invasion of Aspergillus sp. in immuncompetent individuals is primarily controlled by neutrophils, phagocytes and pathogen-specific TH1 CD4+ cells. Therefore, adoptive transfer of Aspergillus-specific CD4+ T-cells could protect patients at risk from IA. Favorable candidates for such an approach are GPI-anchored antigens, which have demonstrated induction of protective adaptive immunity in murine studies. To implement Aspergillus-specific CD4+ T-cells into the clinical setting, we aimed (i) to define which GPI-anchored antigen induces TH1 response, (ii) to map several epitopes and (iii) to generate large amounts of Aspergillus-specific TH1 cells within a short period. Several recombinant proteins were either synthesized or were received by J-P Latge. Amongst all tested proteins, only CRF-1 induced repeatedly TH1 response in healthy individuals. To identify Aspergillus-specific MHC class II epitopes, we mapped peripheral blood mononuclear cells (PBMC) of healthy individuals with a custom made peptide library of CRF-1 consisting of 95 overlapping 15mer peptides. The library was divided into a complete pool, subpools of 10 and 95 single peptides. No precursor frequencies were detected in interferon-gamma (IFN-g)-ELISPOT using the complete and sub-pools. After 7 days of stimulation with subpools, 9 peptides were identified producing high amount of IFN-g in at least 3 donors with different MHC class II alleles (n=6). CD4+ T-cells clones of one peptide were then established by limited dilution cloning. Restriction to DRB1*0401 was identified using LCLs and a tetramer was generated. The peptide-specific T-cell clones showed functional activity against ethanol-inactivated fungus or fungal extracts presented by dendritic cells. To generate Aspergillus-specific TH1 cell lines, we stimulated PBMC with the MHC class II DRB1*0401 restricted peptide. After 7 days of in vitro stimulation (IVS) with interleukin (IL)-2 5U/ml added every other day, tetramer staining was between 0.3 and 11% of CD4+ cells (mean 4.2%, n=5). After 14 days of IVS with 1 restimulation of autologous peptide-pulsed monocytes at a responder: stimulator ratio of 5:1 and IL-7 and IL-15 10ng/ml added after day 7, mean percentages of tetramer staining increased to 37% (range 20-57%, n=4) and absolute cell counts were duplicated. The expansion process was further optimized using IFN-g capture assay and CD154+ MicroBead Kit (Miltenyi). In both assays, PBMC were stimulated for 16 hours, separated by magnetic beads and co-cultured with irradiated autologous PBMC for 14 days using IL-2 5U/ml till day 7 and IL-7 and -15 10ng/ml thereafter. We were unable to expand specific CD4+ cells by IFN-g capture assay in 2 of 3 donors. In contrast, we found that Aspergillus-specific CD4+ cells selected by CD154+ showed comparable tetramer specificity and expansion but higher functional activity in intracellular cytokine assay and IFN-g ELISA than CD4+ cell lines generated from the same donor using the restimulation protocol. The CD4+ cell lines generated by CD154+ expression showed proliferative capacity in CFSE when restimulated with peptides and functional activity in IFN-g ELISA against fungal extracts (n=4). To generate Aspergillus-specific TH1 cell lines recognizing multiple MHC class II epitopes we stimulated PBMC with the previously identified 9 peptides of CRF-1 protein. After 14 days of expansion using CD154+ MicroBead Kit, we measured high IFN-g response towards 4 of 9 peptides (n=3). We are generating T-cell clones and will characterize their HLA-restriction. In summary, we have identified an immunodominant Aspergillus fumigatus protein including 9 MHC class II epitopes. One epitope was characterized specific for an abundant MHC class II allele. CD4+ TH1 cells specific for this epitope can be activated by dendritic cells after uptake of whole fungi or fungal extracts. Furthermore, we have established a GMP-applicable protocol for rapid generation ( 〈 14d) of CD4+ T-cell lines specific for Aspergillus fumigatus with low precursor frequency using CD154+ separation. These CD4+ T-cell lines demonstrate functional activity against peptides and fungal extracts that could be prophylactically administered to high risk patients. Disclosures: No relevant conflicts of interest to declare.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V114.22.1170.1170
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2009
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  • 4
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    First Generation Chimeric Antigen Receptor Display Functional Defects In Key Signal Pathways Upon Antigen Stimulation
    American Society of Hematology ; 2010
    In:  Blood Vol. 116, No. 21 ( 2010-11-19), p. 2088-2088
    Details
    In: Blood, American Society of Hematology, Vol. 116, No. 21 ( 2010-11-19), p. 2088-2088
    Abstract: Abstract 2088 Introduction: A promising strategy for tumor therapy is the adoptive transfer of tumor specific T cells which are endowed with chimeric antigen receptors (CAR). First generation CARs are constructed by single chain antibodies and as signal domain the ζ chain of the CD3 complex. However, clinical trials are disappointing as adoptive transferred T-cells showed only modest persistence in patients resulting in limited clinical activity. We there for hypothesized that CAR expressing T-cells in comparison to unmodified T-cells display signaling defects when stimulated via their CARs. Methods: Cytomegalovirus(CMV)pp65 MHC I restricted CD8+ T-cells were generated, isolated by tetramer selection and modified with first generation CAR targeting CD19 and purified based on their receptor expression to more than 〉 95% purity. T-cell receptor (TCR) and CAR expression were quantified by Quantibright beads. Effector function of both T-cell populations were analyzed for specific lysis, cytokine production (IFN-g, TNF-a) and proliferation (CSFE) upon target cell stimulation. Phosphorylation of Erk, Jnk, p38 and PLC-γ was measured and analyzed with CBA Flexsets from BD. All statistical analyses have been performed using the statistical software package R. Signal peak intensities have been compared using the nonparametric wilcoxon rank sum test. Results: CMV-specific MHC-I restricted TCR as well as the CARs are expressed at same density levels and T-cells show equally lysis of targets either in the time of lysis onset as well the maximal lysis. In contrast, cytokine production (IFN, TNF-a) as well as antigen driven proliferation was reduced in CAR expressing T-cells when compared to CMV-specific CD8+ T-cells upon target exposure. PLC-γ was phosphorylated within minutes after target contact by CMV-specific CD8+ T-cells whereas CAR transduced CMV-specific CD8+ T-cells showed no significant phosphorylation of PLC-γ to target cell exposure. T-cell activated via CAR's demonstrated a statistically significant reduction of maximal phosphorylation in comparison to CMV-specific T-cells for ERK, for JNK and for p38. To exclude that CAR modification of CMV-specific CD8+ T-cells may impair signaling, CAR-CMV-specific CD8+ T-cells were exposed to CMVpp65 expressing targets. Killing, cytokine production and signal intensity were restored in comparison to parental CMV-specific CD8+ T-cells. Conclusion: CAR expressing T-cells show functionally signs of split anergy by efficient target elimination but fails to produce significant levels of cytokines and do not proliferate in response to target stimulation. Split anergy is not due to reduced expression of the CAR's but due to a complete lack of phosphorylation of PLC-γ as well as reduced phosphorylation of MAP-kinases ERK, p38 and JNK. These results potentially explain why primary CAR expressing T-cells fail to show significant clinical efficacy. Analysis of adequate phosphorylation, as proposed here, may be a powerful tool to identify the most promising second generation CARs for clinical studies. Disclosures: No relevant conflicts of interest to declare.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V116.21.2088.2088
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2010
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    detail.hit.zdb_id: 80069-7
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  • 5
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    Cross-protective TH1 immunity against Aspergillus fumigatus and Candida albicans
    American Society of Hematology ; 2011
    In:  Blood Vol. 117, No. 22 ( 2011-06-02), p. 5881-5891
    Details
    In: Blood, American Society of Hematology, Vol. 117, No. 22 ( 2011-06-02), p. 5881-5891
    Abstract: T cell–mediated heterologous immunity to different pathogens is promising for the development of immunotherapeutic strategies. Aspergillus fumigatus and Candida albicans, the 2 most common fungal pathogens causing severe infections in immunocompromised patients, are controlled by CD4+ type 1 helper T (TH1) cells in humans and mice, making induction of fungus-specific CD4+ TH1 immunity an appealing strategy for antifungal therapy. We identified an immunogenic epitope of the A fumigatus cell wall glucanase Crf1 that can be presented by 3 common major histocompatibility complex class II alleles and that induces memory CD4+ TH1 cells with a diverse T-cell receptor repertoire that is cross-reactive to C albicans. In BALB/c mice, the Crf1 protein also elicits cross-protection against lethal infection with C albicans that is mediated by the same epitope as in humans. These data illustrate the existence of T cell–based cross-protection for the 2 distantly related clinically relevant fungal pathogens that may foster the development of immunotherapeutic strategies.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood-2010-12-325084
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2011
    detail.hit.zdb_id: 1468538-3
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  • 6
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    Identification and Characterization of Human Aspergillus Fumigatus-Specific Tr1-(Like) Cells
    American Society of Hematology ; 2011
    In:  Blood Vol. 118, No. 21 ( 2011-11-18), p. 181-181
    Details
    In: Blood, American Society of Hematology, Vol. 118, No. 21 ( 2011-11-18), p. 181-181
    Abstract: Abstract 181 Introduction: The ubiquitous mold Aspergillus fumigatus (A. fumigatus) induces two forms of pathogenesis: invasive aspergillosis in neutropenic patients and allergic aspergillosis in patients with chronic obstructive lung disease as well as in immunosuppressed patients. Mouse models of aspergillosis suggest that not only effector T cells (Teff) but also regulatory T cells (Treg) play a crucial role for the regulation of a protective T cell-mediated immunity to A. fumigatus. However, it is little-known about the involvement of Treg during A. fumigatus infection in humans. In order to develop new therapeutical strategies for the treatment of aspergillosis this project aims to understand the influence of regulatory T cells on A. fumigatus infection in humans. Material/Methods: A. fumigatus-specific CD4+ T cell clones were established from PBMC of healthy donors. Based on this clone pool Treg clones were identified due to their inability to proliferate in the absence of costimulation assessed by 3[H]-TdR incorporation as well as their Ag-specific cytokine production and phenotype determined by flow cytometry. Treg function was analyzed by their ability to suppress proliferation of autologous CD4+ T cells using CFSE dilution. Results: We identified A. fumigatus-specific T cell clones that exhibited marginal detectable proliferation after restimulation with immobilized anti-CD3 mAb in the absence of costimulation. However, these T cell clones vigorously proliferated in response to restimulation with their cognate antigen. A more detailed characterization showed that these suppressor T cell clones produced high amounts of IL-10 and moderate levels of IFN-gamma upon Ag-specific restimulation and expressed low amounts of Foxp3 but not Helios, a transcription factor that had recently been linked to natural occurring Treg. Most importantly, these T cell clones suppressed Ag-specific expansion of CD4+ Teff. This effect was contact-independent since suppression of Ag-specific CD4+ T cell expansion detected in transwell experiments was comparable to cocultures that enabled cellular-contact. Furthermore, anti-CD3/CD28-induced proliferation of naïve CD4+ T cells was not reduced in the presence of culture supernatants obtained from suppressor T cell clones after their antigen-specific restimulation in the absence of DCs. Conclusions: We identified for the first time A. fumigatus-specific CD4+ T cell clones with a Tr1(-like) IL-10+IFN-gamma+Foxp3lowHelios− phenotype. These cells suppressed expansion of A. fumigatus-specific Teff in an Ag-specific manner mediated by soluble factors released from Tr1(-like) cell clones. Since these factors did not affect CD4+ T cell proliferation in the absence of DCs our data suggest, that Tr1(-like) cell clones rather negatively regulate the stimulatory capacity of DCs leading to a reduced expansion of Ag-specific CD4+ T cells. Therefore these Tr1(-like) cells might play a protective role during A. fumigatus infection in humans. Thus, adoptive transfer of A. fumigatus-specific Treg could be useful to enhance protective immunity in patients with chronic A. fumigatus infection. Disclosures: Topp: Micromet: Consultancy, Honoraria.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V118.21.181.181
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2011
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  • 7
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    BLEACH&STAIN 15-marker Multiplexed Imaging in 3,098 Human Carcinomas Reveals Six Major PD-L1–driven Immune Phenotypes with Distinct Spatial Orchestration
    American Association for Cancer Research (AACR) ; 2023
    In:  Molecular Cancer Research Vol. 21, No. 6 ( 2023-06-01), p. 605-613
    Details
    In: Molecular Cancer Research, American Association for Cancer Research (AACR), Vol. 21, No. 6 ( 2023-06-01), p. 605-613
    Abstract: Multiplex fluorescence IHC (mfIHC) approaches were yet either limited to six markers or limited to a small tissue size that hampers translational studies on large tissue microarray cohorts. Here we have developed a BLEACH & STAIN mfIHC method that enabled the simultaneous analysis of 15 biomarkers (PD-L1, PD-1, CTLA-4, panCK, CD68, CD163, CD11c, iNOS, CD3, CD8, CD4, FOXP3, CD20, Ki67, and CD31) in 3,098 tumor samples from 44 different carcinoma entities within one week. To facilitate automated immune checkpoint quantification on tumor and immune cells and study its spatial interplay an artificial intelligence–based framework incorporating 17 different deep-learning systems was established. Unsupervised clustering showed that the three PD-L1 phenotypes (PD-L1+ tumor and immune cells, PD-L1+ immune cells, PD-L1−) were either inflamed or noninflamed. In inflamed PD-L1+patients, spatial analysis revealed that an elevated level of intratumoral M2 macrophages as well as CD11c+ dendritic cell (DC) infiltration (P & lt; 0.001 each) was associated with a high CD3+ CD4± CD8± FOXP3± T-cell exclusion and a high PD-1 expression on T cells (P & lt; 0.001 each). In breast cancer, the PD-L1 fluorescence intensity on tumor cells showed a significantly higher predictive performance for overall survival (OS; AUC, 0.72, P & lt; 0.001) compared with the commonly used percentage of PD-L1+ tumor cells (AUC, 0.54). In conclusion, our deep-learning–based BLEACH & STAIN framework facilitates rapid and comprehensive assessment of more than 60 spatially orchestrated immune cell subpopulations and its prognostic relevance. Implications: The development of an easy-to-use high-throughput 15+1 multiplex fluorescence approach facilitates the in-depth understanding of the immune tumor microenvironment (TME) and enables to study the prognostic relevance of more than 130 immune cell subpopulations.
    Type of Medium: Online Resource
    ISSN: 1541-7786 , 1557-3125
    URL: Article
    DOI: 10.1158/1541-7786.MCR-22-0593
    Language: English
    Publisher: American Association for Cancer Research (AACR)
    Publication Date: 2023
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  • 8
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    Simultaneous Inhibition of Hsp90 and Akt Disable Immune Functions of Alloreactive T Cells in a Synergistic Manner
    American Society of Hematology ; 2011
    In:  Blood Vol. 118, No. 21 ( 2011-11-18), p. 4697-4697
    Details
    In: Blood, American Society of Hematology, Vol. 118, No. 21 ( 2011-11-18), p. 4697-4697
    Abstract: Abstract 4697 Introduction: Despite advances in prevention and post transplant immuno-suppressive strategies Graft versus Host Disease (GvHD) remains a major cause of morbidity and mortality in patients undergoing allogeneic hematopoietic stem cell transplantation. Recently we have shown, that heat shock protein 90 (Hsp90) inhibitor 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (DMAG) selectively depletes alloreactive T cells in vitro. Here, we demonstrate that DMAG and isoform-specific Akt1 and 2 inhibitor Akti-1/2 interact in a synergistic manner to suppress essential immune functions of alloreactive human T cells in vitro. Methods: Human CD3+ T cells were activated by coculture for 5 days with allogeneic dendritic cells (DC) in the presence of DMAG and Akti-1/2 alone and in combination. In another setting the inhibitors were added for the last 24 hours of T cell culture. Subsequently, the effect of Hsp90, Akt and Akt/Hsp90 inhibition on proliferation, apoptosis and cell cycle of alloreactive T cells was determined. Results Here, we demonstrate that human CD3+ T cells activated in a physiological manner by coculture with allogeneic DCs have an increased expression of Akt2 isoform compared to unstimulated human T cells. Inhibition of Akt activity by isoform-specific Akt1 and 2 inhibitor Akti-1/2 decreased proliferation of alloreactive T cells in a dose-dependent manner. This effect can be attributed to modest apoptosis induction and induction of cell cycle arrest in G1-phase. Cell cycle arrest is accompanied by accumulation of cell cycle regulator proteins p21WAF/CIP1 and p27KIP1. Simultaneous inhibition of Akt and Hsp90 induced a synergistic effect on proliferation suppression of alloreactive T cells. Akti-1/2 (1 μM) caused for example 26% proliferation decrease that was reinforced to 65% and 69% when combined with 0.5 μM and 2 μM DMAG, respectively. Similar results were obtained, when inhibitors were added at the beginning of the T cell culture. To exclude that the observed effect relies on inhibitor-induced DC dysfunction, we selectively target Hsp90 and Akt2 function in the T cell population by Hsp90β- and Akt2-specific siRNA. T cells transfected with Hsp90β- or Akt2-specific siRNA showed only moderate down-regulation of Hsp90β and Akt2 expression as determined by western blot. However, siRNA-mediated knockdown resulted in a significant decrease of proliferation in alloreactive T cells compared to negative control siRNA transfected T cells. Furthermore, T cells transfected with Hsp90β- and Akt2-specific siRNA in combination demonstrated no distinct down-regulation of Akt2 or Hsp90β expression compared to T cells transfected with Akt2- or Hsp90β-specific siRNA alone, but showed significant reduction of proliferation in comparison to T cells transfected with only one specific siRNA. To identify the underlying mechanism of increased proliferation suppression of agent combination, we investigated by western blot analysis expression of Akt substrate proteins and Hsp90 client proteins which are known to regulate proliferation of T cells. Increased effect on proliferation suppression of agent combination compared to single agent administration is accompanied by accumulation of negative cell cycle regulator p21WAF/CIP1, whereas abundance of positive cell cycle regulator PLK1 and cyclin B1 decrease. We also show that simultaneous inhibition of Hsp90 and Akt causes a slight increase of PARP cleavage suggesting an enhancement in caspase-mediated apoptosis induction in alloreactive T cells. In addition, we determined phosphorylation state of Akt substrate proteins in CD3/CD28-activated T cells by western blot analysis and observed a considerable decrease of GSK3α phosphorylation induced by combination of Akti-1/2 and DMAG compared to single agent use. In contrast, we provide data that inhibitor combination maintains viral immunity by showing that combination treatment have no effect on number of CMV-specific CD8+ T cells. Conclusion: We suggest that combinational action of Hsp90 and Akt inhibition can be defined as a new immunosuppressive strategy suited for the treatment of GVHD and other deregulated and unwanted T cell-mediated immune responses. Disclosures: Topp: Micromet: Consultancy, Honoraria.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V118.21.4697.4697
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2011
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  • 9
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    Influence of Short and Long Hyperglycemia on Cardioprotection by Remote Ischemic Preconditioning—A Translational Approach
    MDPI AG ; 2022
    In:  International Journal of Molecular Sciences Vol. 23, No. 23 ( 2022-11-22), p. 14557-
    Details
    In: International Journal of Molecular Sciences, MDPI AG, Vol. 23, No. 23 ( 2022-11-22), p. 14557-
    Abstract: The adverse impact of common diseases like diabetes mellitus and acute hyperglycemia on morbidity and mortality from myocardial infarction (MI) has been well documented over the past years of research. In the clinical setting, the relationship between blood glucose and mortality appears linear, with amplifying risk associated with increasing blood glucose levels. Further, this seems to be independent of a diagnosis of diabetes. In the experimental setting, various comorbidities seem to impact ischemic and pharmacological conditioning strategies, protecting the heart against ischemia and reperfusion injury. In this translational experimental approach from bedside to bench, we set out to determine whether acute and/or prolonged hyperglycemia have an influence on the protective effect of transferred human RIPC-plasma and, therefore, might obstruct translation into the clinical setting. Control and RIPC plasma of young healthy men were transferred to isolated hearts of young male Wistar rats in vitro. Plasma was administered before global ischemia under either short hyperglycemic (HGs Con, HGs RIPC) conditions, prolonged hyperglycemia (HGl Con, HGl RIPC), or under normoglycemia (Con, RIPC). Infarct sizes were determined by TTC staining. Control hearts showed an infarct size of 55 ± 7%. Preconditioning with transferred RIPC plasma under normoglycemia significantly reduced infarct size to 25 ± 4% (p 〈 0.05 vs. Con). Under acute hyperglycemia, control hearts showed an infarct size of 63 ± 5%. Applying RIPC plasma under short hyperglycemic conditions led to a significant infarct size reduction of 41 ± 4% (p 〈 0.05 vs. HGs Con). However, the cardioprotective effect of RIPC plasma under normoglycemia was significantly stronger compared with acute hyperglycemic conditions (RIPC vs. HGs RIPC; p 〈 0.05). Prolonged hyperglycemia (HGl RIPC) completely abolished the cardioprotective effect of RIPC plasma (infarct size 60 ± 7%; p 〈 0.05 vs. HGl Con; HGl Con 59 ± 5%).
    Type of Medium: Online Resource
    ISSN: 1422-0067
    URL: Article
    DOI: 10.3390/ijms232314557
    Language: English
    Publisher: MDPI AG
    Publication Date: 2022
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  • 10
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    Generation of a Multiple Pathogen-Specific T Cell Product for Adoptive Immunotherapy Based on Activation-Dependent Expression of CD154
    American Society of Hematology ; 2010
    In:  Blood Vol. 116, No. 21 ( 2010-11-19), p. 2326-2326
    Details
    In: Blood, American Society of Hematology, Vol. 116, No. 21 ( 2010-11-19), p. 2326-2326
    Abstract: Abstract 2326 In patients undergoing hematopoietic stem cell transplantation (HSCT) infectious complications are frequent causing substantial morbidity and mortality. Adoptive T cell therapy specific for single pathogens has previously shown to efficiently control viral and fungal infections but approaches targeting multiple pathogens are limited to T cells generated with EBV transformed B cells that are genetically modified expressing multiple viral antigens. Infections are often experienced by different viral and fungal pathogens such as cytomegalovirus (CMV), Epstein-Barr virus (EBV), adenovirus (AdV), Aspergillus fumigatus (AF) and Candida albicans (CA) that show a wide spectrum of memory T cell frequencies. Those with a low precursor frequency are not suitable for selection methods based on the secretion of cytokines such as IFN-g. As CMV seropositivity among HSCT donors may range only between 30–40% and immunity to the other pathogens can be detected simultaneously in more than 85% of HSCT donors we focused on the generation of a multi-specific T cell product for EBV, AdV, AF and CA for easy transfer under current regulatory requirements. We aimed to develop a simple protocol which (i) is able to enrich T cells specific for pathogens with low precursor frequency and (ii) allows simultaneous expansion of multiple pathogen-specific T cells in a single culture. We determined if CD154, which is transiently expressed on antigen stimulated CD4+ but also to a lesser extend on CD8+ T cells, would be a potential candidate for selection of pathogen-specific T cells. For stimulation we used peptide pools for AdV hexon protein, EBV latent membrane protein 2 (LMP2) and CA mannose protein 65 (MP65) as well as one AF immune dominant epitope derived from the Crf1 protein. To select and expand antigen-specific T cells, we stimulated PBMC for 16 hours, separated them by CD154+ MicroBead Kit (Miltenyi) and co-cultured them with irradiated autologous PBMC with IL-2, IL-7 and IL-15 for 14 days. The isolated cells were on average 0.62% of the starting fraction and could be expanded 20- to 145-fold. The median frequency of AdV-specific T cells increased from day 1 to day 14 87-fold from 30 to 2620 spot forming counts (SFC)/2×105 cells, for EBV 229-fold from 15 to 3430 SFC/2×105 cells and for CA 960-fold from 3 to 2400 SFC/2×105 cells assessed by IFN-γ ELISPOT. AF-specific T cells that were undetectable in PBMC increased to a median of 2260 SFC/2×105 cells. Although isolation of CD154+ cells favors enrichment of CD4+ T cells, a low fraction of virus-specific CD8+ T cells were simultaneously expanded. Next, we tested the efficacy of the CD154-based enrichment for the generation of multi pathogen-specific T cell lines reactive to all 4 pathogens. Selection and expansion was comparable, there was however a notable shift in the frequencies of T cells specific for different antigens in multi pathogen-specific cultures compared to single lines. The median increase of AdV-and CA- specific T cell lines was comparable (2345 SFC/2×105 and 3205 SFC/2×105 cells) but the frequencies for EBV (575 SFC/2×105 cells) as well as for AF (465 SFC/2×105 cells) were diminished in multi-specific lines. Nevertheless, lysis of LCL pulsed with LMP2 or AdV peptide pools was efficient with 72% and 36% by single and 30% and 45% by multi-specific T cell lines (at an E:T ratio of 20:1) as assessed by 51Cr-release assay. The single and multi pathogen-specific T cell lines generated by peptides responded to endogenously processed antigens and were able to specifically proliferate upon antigen stimulation. In contrast, T cell-mediated allo-reactivity was almost abrogated when compared to the starting population. In conclusion, we established a simple expansion protocol for selection, expansion and enrichment of allo-depleted single and multiple pathogen-specific CD4+ and CD8+ T cells specific for AdV, EBV, AF and CA that may further expand if the T cells are stimulated by their native antigen in vivo. This expansion protocol may form the basis for adoptive immunotherapy trials in HSCT recipients at risk for multiple infectious complications. This study has been supported by a grant of BayImmunet. Disclosures: No relevant conflicts of interest to declare.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V116.21.2326.2326
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2010
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
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