Novel non-viral method for transfection of primary leukemia cells and cell lines

BACKGROUND: Tumor cells such as leukemia and lymphoma cells are possible targets for gene therapy. However, previously leukemia and lymphoma cells have been demonstrated to be resistant to most of non-viral gene transfer methods. METHODS: The aim of this study was to analyze various methods for transfection of primary leukemia cells and leukemia cell lines and to improve the efficiency of gene delivery. Here, we evaluated a novel electroporation based technique called nucleofection. This novel technique uses a combination of special electrical parameters and specific solutions to deliver the DNA directly to the cell nucleus under mild conditions. RESULTS: Using this technique for gene transfer up to 75% of primary cells derived from three acute myeloid leukemia (AML) patients and K562 cells were transfected with the green flourescent protein (GFP) reporter gene with low cytotoxicity. In addition, 49(+/- 9.7%) of HL60 leukemia cells showed expression of GFP. CONCLUSION: The non-viral transfection method described here may have an impact on the use of primary leukemia cells and leukemia cell lines in cancer gene therapy

Pulsen mit Blastenzelllysat oder Blasten-Gesamt-RNA richtet die durch dendritische Zellen vermittelte Aktivität von Zytokin-induzierten Killerzellen gegen allogene akute myeloische Zellen

Immunotherapeutic strategies may be a treatment option in patients with refractory acute myelogenous leukemia (AML) or, in cases of complete remission after conventional therapy regimens, may help to reduce disease recurrence or delay time to progression. Evidence suggests a key role of dendritic cells (DCs) in cancer immunotherapy due to their capacity to present tumour antigens to effector cells. We generated cytokine-induced killer (CIK) cells from healthy donors and examined their responses in vitro in an LDH release assay against three cell lines and allogeneic HLA non-matched blasts from three patients with de novo AML after coincubation with autologous peripheral blood monocyte-derived DCs. Although DCs were unable to enhance CIK cell effects against all three cell lines tested, the cytotoxic activity against the patients’ AML cells increased after coculture with mature DCs, which was significant in two of three patients. However, neither prior pulsing of the DCs with blast cell lysates nor with leukemic cell-derived total RNA further enhanced the lytic capacity of the CIK cells. On the contrary, pulsing reduced or even reversed the cytotoxic activity of the effector cells. This decrease of allogeneic cytotoxicity led us to conclude that monocyte-derived DCs may be useful in autologous or allogeneic vaccine strategies for the treatment of AML or in priming donor lymphocytes in vitro, but unfractionated antigens as pulsing agents may have inhibitory effects on T cell efficiency and their employment in immunotherapeutic strategies for AML seems questionable.Immuntherapeutische Strategien können eine Behandlungsoption bei Patienten mit refraktärer akuter myeloischer Leukämie (AML) sein oder in den Fällen einer kompletten Remission nach konventionellen Therapieformen helfen, das Wiederauftreten der Krankheit zu verhindern oder die Zeit bis zur Progression zu verlängern. Es gibt Hinweise darauf, dass dendritische Zellen (DCs) eine zentrale Rolle in der Krebs-Immuntherapie spielen aufgrund ihrer Fähigkeit, tumorantigene Effektor-Zellen zu präsentieren. Wir stellten Zytokin-induzierte Killer (CIK)-Zellen von gesunden Spendern her und untersuchten deren Reaktionen in vitro in einem Laktatdehydrogenase (LDH)-Assay gegen Zelllinien und allogene HLA nicht übereinstimmende Blasten von drei Patienten mit de novo AML nach Koinkubation mit autologen aus dem peripheren Blut abgeleiteten DCs. Obwohl DCs die CIK Zellen Wirksamkeit gegen alle drei getesteten Zelllinien nicht verbessern konnten, wurde die zytotoxische Aktivität gegen die Patienten-AML-Zellen nach Kokultur mit reifen DCs in zwei von drei Patienten signifikant erhöht. Doch weder ein Pulsen der DCs mit blastären Zelllysaten noch mit aus leukämischen Zellen gewonnener Gesamt-RNA konnten die lytische Kapazität der CIK-Zellen weiter verbessern. Im Gegenteil, gepulste DCs reduzierten sogar die zytotoxische Aktivität der Effektorzellen. Dieser Rückgang der allogenen Zytotoxizität führte uns zu dem Schluss, dass von Monozyten abgeleitete DCs nützlich sein könnten in autologen oder allogenen Impfstrategien zur Behandlung von AML. Unfraktionierte Antigene zum Pulsen von DC können dagegen hemmende Wirkung auf T-Zellen haben

SCORE2-diabetes : 10-year cardiovascular risk estimation in type 2 diabetes in Europe

Aims To develop and validate a recalibrated prediction model (SCORE2-Diabetes) to estimate the 10-year risk of cardiovascular disease (CVD) in individuals with type 2 diabetes in Europe. Methods and results SCORE2-Diabetes was developed by extending SCORE2 algorithms using individual-participant data from four large-scale datasets comprising 229 460 participants (43 706 CVD events) with type 2 diabetes and without previous CVD. Sex-specific competing risk-adjusted models were used including conventional risk factors (i.e. age, smoking, systolic blood pressure, total, and HDL-cholesterol), as well as diabetes-related variables (i.e. age at diabetes diagnosis, glycated haemoglobin [HbA1c] and creatinine-based estimated glomerular filtration rate [eGFR]). Models were recalibrated to CVD incidence in four European risk regions. External validation included 217 036 further individuals (38 602 CVD events), and showed good discrimination, and improvement over SCORE2 (C-index change from 0.009 to 0.031). Regional calibration was satisfactory. SCORE2-Diabetes risk predictions varied several-fold, depending on individuals' levels of diabetes-related factors. For example, in the moderate-risk region, the estimated 10-year CVD risk was 11% for a 60-year-old man, non-smoker, with type 2 diabetes, average conventional risk factors, HbA1c of 50 mmol/mol, eGFR of 90 mL/min/1.73 m(2), and age at diabetes diagnosis of 60 years. By contrast, the estimated risk was 17% in a similar man, with HbA1c of 70 mmol/mol, eGFR of 60 mL/min/1.73 m(2), and age at diabetes diagnosis of 50 years. For a woman with the same characteristics, the risk was 8% and 13%, respectively. Conclusion SCORE2-Diabetes, a new algorithm developed, calibrated, and validated to predict 10-year risk of CVD in individuals with type 2 diabetes, enhances identification of individuals at higher risk of developing CVD across Europe