Generation of Leukaemia-Derived Dendritic Cells (DCleu) to Improve Anti-Leukaemic Activity in AML: Selection of the Most Efficient Response Modifier Combinations.

Dendritic cells (DC) and leukaemia derived DC (DCleu) are potent stimulators of anti-leukaemic activity in acute myeloid leukaemia (AML) and can be generated from mononuclear cells in vitro following standard DC/DCleu-generating protocols. With respect to future clinical applications though, DC/DCleu-generating protocols specifically designed for application in a whole-blood-(WB)-environment must be established. Therefore, we developed ten new DC/DCleu-generating protocols (kits; Kit-A/-C/-D/-E/-F/-G/-H/-I/-K/-M) for the generation of DC/DCleu from leukaemic WB, containing calcium-ionophore, granulocyte-macrophage-colony-stimulating-factor (GM-CSF), tumour-necrosis-factor-alpha, prostaglandin-E1 (PGE1), prostaglandin-E2 (PGE2) and/or picibanil (OK-432). All protocols were evaluated regarding their performance in generating DC/DCleu using refined classification and/or ranking systems; DC/DCleu were evaluated regarding their performance in stimulating anti-leukaemic activity using a cytotoxicity fluorolysis assay. Overall, we found the new kits capable to generate (mature) DC/DCleu from leukaemic WB. Through refined classification and ranking systems, we were able to select Kit-I (GM-CSF + OK-432), -K (GM-CSF + PGE2) and -M (GM-CSF + PGE1) as the most efficient kits in generating (mature) DC/DCleu, which are further competent to stimulate immunoreactive cells to show an improved anti-leukaemic cytotoxicity as well. This great performance of Kit-I, -K and -M in mediating DC/DCleu-based anti-leukaemic immunity in a WB-environment in vitro constitutes an important and directive step for translating DC/DCleu-based immunotherapy of AML into clinical application.

Dendritic Cell-Triggered Immune Activation Goes along with Provision of (Leukemia-Specific) Integrin Beta 7-Expressing Immune Cells and Improved Antileukemic Processes.

Integrin beta 7 (ß7), a subunit of the integrin receptor, is expressed on the surface of immune cells and mediates cell-cell adhesions and interactions, e.g., antitumor or autoimmune reactions. Here, we analyzed, whether the stimulation of immune cells by dendritic cells (of leukemic derivation in AML patients or of monocyte derivation in healthy donors) leads to increased/leukemia-specific ß7 expression in immune cells after T-cell-enriched mixed lymphocyte culture-finally leading to improved antileukemic cytotoxicity. Healthy, as well as AML and MDS patients' whole blood (WB) was treated with Kit-M (granulocyte-macrophage colony-stimulating factor (GM-CSF) + prostaglandin E1 (PGE1)) or Kit-I (GM-CSF + Picibanil) in order to generate DCs (DCleu or monocyte-derived DC), which were then used as stimulator cells in MLC. To quantify antigen/leukemia-specific/antileukemic functionality, a degranulation assay (DEG), an intracellular cytokine assay (INTCYT) and a cytotoxicity fluorolysis assay (CTX) were used. (Leukemia-specific) cell subtypes were quantified via flow cytometry. The Kit treatment of WB (compared to the control) resulted in the generation of DC/DCleu, which induced increased activation of innate and adaptive cells after MLC. Kit-pretreated WB (vs. the control) led to significantly increased frequencies of ß7-expressing T-cells, degranulating and intracellular cytokine-producing ß7-expressing immune cells and, in patients' samples, increased blast lysis. Positive correlations were found between the Kit-M-mediated improvement of blast lysis (vs. the control) and frequencies of ß7-expressing T-cells. Our findings indicate that DC-based immune therapies might be able to specifically activate the immune system against blasts going along with increased frequencies of (leukemia-specific) ß7-expressing immune cells. Furthermore, ß7 might qualify as a predictor for the efficiency and the success of AML and/or MDS therapies.

PGE1-Containing Protocols Generate Mature (Leukemia-Derived) Dendritic Cells Directly from Leukemic Whole Blood.

Dendritic cells (DCs) and leukemia-derived DC (DCleu) are potent stimulators of various immunoreactive cells and they play a pivotal role in the (re-) activation of the immune system. As a potential treatment tool for patients with acute myeloid leukemia, we developed and analyzed two new PGE1-containing protocols (Pici-PGE1, Kit M) to generate DC/DCleu ex vivo from leukemic peripheral blood mononuclear cells (PBMCs) or directly from leukemic whole blood (WB) to simulate physiological conditions. Pici-PGE1 generated significantly higher amounts of DCs from leukemic and healthy PBMCs when compared to control and comparable amounts as the already established protocol Pici-PGE2. The proportions of sufficient DC-generation were even higher after DC/DCleu-generation with Pici-PGE1. With Kits, it was possible to generate DCs and DCleu directly from leukemic and healthy WB without induction of blast proliferation. The average amounts of generated DCs and DCleu-subgroups were comparable with all Kits. The PGE1 containing Kit M generated significantly higher amounts of mature DCs when compared to the PGE2-containing Kit K and increased the anti-leukemic-activity. In summary PGE1-containing protocols were suitable for generating DC/DCleu from PBMCs as well as from WB, which reliably (re-) activated immunoreactive cells, improved the overall ex vivo anti-leukemic activity, and influenced cytokine-release-profiles.

Cardiac MRI left ventricular global function index and quantitative late gadolinium enhancement in unrecognized myocardial infarction.

PURPOSE: To compare left ventricular global function index (LVGFI) and quantitative late gadolinium enhancement (LGE) in patients with unrecognized myocardial infarction (UMI), recognized myocardial infarction (RMI) and without myocardial infarction (MI). MATERIAL AND METHODS: Under waiver of the Institutional Review Board 235 patients (age 63.5±10.5years, 57 female) were retrospectively evaluated. All patients had undergone cardiac MRI at 1.5T for symptoms of CAD. 67 patients (29%) had suffered a known RMI before. Functional imaging and full-intensity late gadolinium enhancement (LGE) imaging were evaluated for LVGFI and quantitative LGE mass. RESULTS: Of 168 patients without history of RMI, 48 patients (29%) had UMI, 120 patients had no MI. LVGFI was lower in RMI patients (34±8% [range 16;52]), and UMI patients (35±8% [range 10;51]), compared to patients with no MI (38±7% [range 16;55]) respectively and similar between RMI and UMI patients. RMI patients had full-intensity LGE in 11±6% of left ventricular myocardial mass (LVMM). UMI patients had LGE in 9±5% of LVMM. RMI patients had significantly more LGE than UMI patients (p=0.0096). CONCLUSION: LGE quantification is effective to assess infarction scar size in RMI and UMI patients. LVGFI provides information on cardiac function and morphology but does not allow for a reliable differentiation between patients with and without history of MI, due small differences and wide overlap of LVGFI values for all three patient groups. This may be a reason why LVGFI is not applied in clinical routine.