Human cytokine-induced killer cells have enhanced in vitro cytolytic activity via non-viral interleukin-2 gene transfer

Modulation of the immune system by genetically modified immunological effector cells is of potential therapeutic value in the treatment of malignancies. Interleukin-2 (IL-2) is a crucial cytokine which induces potent antitumor response. Cytokine-induced killer cells (CIK) have been described as highly efficient cytotoxic effector cells capable of lysing tumor cell targets and are capable of recognizing these cells in a non-MHC restricted fashion. Dendritic cells (DC) are the major antigen presenting cells. This study evaluated the antitumor effect of CIK cells which were non-virally transfected with IL-2 and co-cultured with pulsed and unpulsed DC. Human CIK cells generated from peripheral blood were transfected in vitro with plasmid encoding for the human IL-2. Transfection involved a combination of electrical parameters and a specific solution to deliver plasmid directly to the cell nucleus by using the Nucleofector(® )electroporation system. Nucleofection resulted in the production of IL-2 with a mean of 478.5 pg/10(6 )cells (range of 107.6–1079.3 pg /10(6 )cells/24 h) compared to mock transfected CIK cells (31 pg/10(6 )cells) (P = 0.05). After co-culturing with DC their functional ability was assessed in vitro by a cytotoxicity assay. On comparison with non-transfected CIK cells co-cultured with DCs (36.5 ± 5.3 %), transfected CIK cells co-cultured with DC had a significantly higher lytic activity of 58.5 ± 3.2% (P = 0.03) against Dan G cells, a human pancreatic carcinoma cell line

Effects of recombinant adenovirus-mediated expression of IL-2 and IL-12 in human B lymphoma cells on co-cultured PBMC

BACKGROUND: Modulation of the immune system by genetically modified lymphoma cell vaccines is of potential therapeutic value in the treatment of B cell lymphoma. However, the anti-tumor effect of any single immunogene transfer has so far been limited. Combination treatment of recombinant IL-2 and IL-12 has been reported to be synergistic for inducing anti-tumor responses in solid tumors but the potential of IL-2/IL-12 gene modified B cell lymphoma cells has not been explored yet. METHODS: Using three different human B cell lymphoma cell lines and primary samples from patients with B cell neoplasms, expression levels of the coxsackie B-adenovirus receptor (CAR) and alpha (v) integrins were analyzed by fluorescence-activated cell sorter (FACS). Adenoviral transduction efficiencies were determined by GFP expression analysis and IL-2 and IL-12 cytokine production was quantified by enzyme-linked immunosorbent (ELISA) assays. Proliferative activities of peripheral blood mononuclear cells (PBMC) stimulated with either cytokine derived from supernatants of transduced lymphoma cells were measured by cell proliferation (MTT) assays. An EuTDA cytotoxicity assay was used to compare cytotoxic activities of IL-2 and/or IL-12 stimulated PBMC against unmodified lymphoma cells. RESULTS: We found that B cell lymphoma cell lines could be transduced with much higher efficiency than primary tumor samples, which appeared to correlate with the expression of CAR. Adenoviral-expressed IL-2 and IL-12 similarly led to dose-dependent increases in proliferation rates of PBMC obtained from healthy donors. IL-2 and/or IL-12 transduced lymphoma cells were co-cultured with PBMC, which were assayed for their cytolytic activity against unmodified lymphoma cells. We found that IL-2 stimulated PBMC elicited a significant anti-tumor effect but not the combined effect of IL-2/IL-12 or IL-12 alone. CONCLUSION: This study demonstrates that the generation of recombinant adenovirus modified lymphoma cell vaccines based on lymphoma cell lines expressing IL-2 and IL-12 cytokine genes is technically feasible, induces increases in proliferation rates and cytotoxic activity of co-cultured PBMC, and warrants further development for the treatment of lymphoma patients in the future

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

Invasive fungal infections in neutropenic enterocolitis: A systematic analysis of pathogens, incidence, treatment and mortality in adult patients

BACKGROUND: Neutropenic enterocolitis is a life-threatening complication most frequently occurring after intensive chemotherapy in acute leukaemias. Gramnegative bacteria constitute the most important group of causative pathogens. Fungi have also been reported, but their practical relevance remains unclear. The guidelines do not address concrete treatment recommendations for fungal neutropenic enterocolitis. METHODS: Here, we conducted a metaanalysis to answer the questions: What are frequency and mortality of fungal neutropenic enterocolitis? Do frequencies and microbiological distribution of causative fungi support empirical antimycotic therapy? Do reported results of antimycotic therapy in documented fungal neutropenic enterocolitis help with the selection of appropriate drugs? Following a systematic search, we extracted and summarised all detail data from the complete literature. RESULTS: Among 186 articles describing patients with neutropenic enterocolitis, we found 29 reports describing 53 patients with causative fungal pathogens. We found no randomised controlled trial, no good quality cohort study and no good quality case control study on the role of antifungal treatment. The pooled frequency of fungal neutropenic enterocolitis was 6.2% calculated from all 860 reported patients and 3.4% calculated from selected representative studies only. In 94% of the patients, Candida spp. were involved. The pooled mortality rate was 81.8%. Most authors did not report or perform antifungal therapy. CONCLUSION: In patients with neutropenic enterocolitis, fungal pathogens play a relevant, but secondary role compared to bacteria. Evidence concerning therapy is very poor, but epidemiological data from this study may provide helpful clues to select empiric antifungal therapy in neutropenic enterocolitis

Anticancer Dose Adjustment for Patients with Renal and Hepatic Dysfunction: From Scientific Evidence to Clinical Application

Most anticancer agents exhibit a narrow therapeutic index, i.e., a small change in plasma concentrations can lead to a less efficacious treatment or an unacceptable degree of toxicity. This study aimed at providing health professionals with a feasible and time-saving tool to adapt the dose of anticancer agents for patients with renal or hepatic dysfunction. A guideline for anticancer agents was developed based on a literature search. An algorithm was generated to enhance the efficiency of the dose adaptation process. Finally, the dosing guideline was converted into an easy-to-use ExcelTM tool. The concept was applied to a total of 105 adult patients at the Centre for Integrated Oncology, Bonn, Germany. In total, 392 recommendations for dose adaptation were made and 320 (81.6%) recommendations were responded to by the oncologists. 98.4% of the recommendations were accepted. The algorithm simplifies the decision and screening process for high-risk patients. Moreover, it provides the possibility to quickly decide which laboratory tests are required and whether a dose adjustment for a particular anticancer drug is needed. The ExcelTM tool provides a recommended individual dose for patients with renal or hepatic dysfunction. The effectiveness of this strategy to reduce toxicity should be investigated in further studies before being adopted for routine use

Synergistic integration of histone deacetylase inhibitors apparently enhances the cytokine‐induced killer cell efficiency in multiple myeloma via the NKG2D pathway

Abstract Objectives The rapid recognition of epigenetic manipulation's potential in restricting cancer cell capabilities spurred translational initiatives, including histone deacetylase inhibitors (HDACis). Clinical trials on multiple myeloma (MM) demonstrated substantial benefits of HDACis, coupled with promising outcomes from cytokine‐induced killer cell (CIK) immunotherapy. Intriguingly, the unexplored synergy of HDACis and CIK cell immunotherapy in MM prompted our study. Methods We examined clinically relevant HDACis (panobinostat/LBH589 and romidepsin) alongside CIK cells derived from peripheral blood mononuclear cells across diverse MM cell lines (U266, RPMI8226, OPM‐2 and NCI‐H929). Utilising various in vitro methodologies, we investigated how HDACis enhance CIK cell lysis of myeloma cells through NKG2D/NKG2D ligand interactions. Results The results of our analysis indicated several key findings. (1) Enhanced cytotoxicity of CIK cells in MM cells when combined with HDACis. (2) Significant increase in apoptosis, suggesting HDACis and CIK may together enhance apoptotic effects in specific MM cell lines. (3) Elevated IFN‐γ secretion and alterations in granzyme B secretion because of the independent activity of HDACis. (4) Notably, HDACis increased the expression of MICA/B and ULBP2, crucial for inducing antitumor cytotoxicity of NKT cells. Validation through NKG2D receptor blocking in CIK cells with a purified mouse antihuman NKG2D antibody further supported our findings. Conclusions Our analyses provide sufficient evidence to consider this clinically forgotten instance (HDACis‐CIK cell combination) as a therapeutic priority for MM treatment. Furthermore, we suggest that NKG2D/NKG2D‐ligand interactions activating NK/NKT cells may contribute to enhanced myeloma cell lysis in response to HDACis treatment by CIK cells