Inhibition of p38 MAPK or immunoproteasome overcomes resistance of chronic lymphocytic leukemia cells to Bcl-2 antagonist venetoclax.

Chronic lymphocytic leukemia (CLL) is a hematological neoplasm of CD19-positive mature-appearing B lymphocytes. Despite the clinical success of targeted therapies in CLL, the development of resistance diminishes their therapeutic activity. This is also true for the Bcl-2 antagonist venetoclax. We investigated the molecular mechanisms that drive venetoclax resistance in CLL, with a clear focus to provide new strategies to successfully combat it. Activation of CLL cells with IFNγ, PMA/ionomycin, and sCD40L diminished the cytotoxicity of venetoclax. We demonstrated that the metabolic activity of cells treated with 1 nM venetoclax alone was 48% of untreated cells, and was higher for cells co-treated with IFNγ (110%), PMA/ionomycin (78%), and sCD40L (62%). As of molecular mechanism, we showed that PMA/ionomycin and sCD40L triggered translocation of NFκB in primary CLL cells, while IFNγ activated p38 MAPK, suppressed spontaneous and venetoclax-induced apoptosis and induced formation of the immunoproteasome. Inhibition of immunoproteasome with ONX-0914 suppressed activity of immunoproteasome and synergized with venetoclax against primary CLL cells. On the other hand, inhibition of p38 MAPK abolished cytoprotective effects of IFNγ. We demonstrated that venetoclax-resistant (MEC-1 VER) cells overexpressed p38 MAPK and p-Bcl-2 (Ser70), and underexpressed Mcl-1, Bax, and Bak. Inhibition of p38 MAPK or immunoproteasome triggered apoptosis in CLL cells and overcame the resistance to venetoclax of MEC-1 VER cells and venetoclax-insensitive primary CLL cells. In conclusion, the p38 MAPK pathway and immunoproteasome represent novel targets to combat venetoclax resistance in CLL.

Mitochondrial Dysfunction Associates With Acute T Lymphocytopenia and Impaired Functionality in COVID-19 Patients.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection results in rapid T lymphocytopenia and functional impairment of T cells. The underlying mechanism, however, remains incompletely understood. In this study, we focused on characterizing the phenotype and kinetics of T-cell subsets with mitochondrial dysfunction (MD) by multicolor flow cytometry and investigating the association between MD and T-cell functionality. While 73.9% of study subjects displayed clinical lymphocytopenia upon hospital admission, a significant reduction of CD4 or CD8 T-cell frequency was found in all asymptomatic, symptomatic, and convalescent cases. CD4 and CD8 T cells with increased MD were found in both asymptomatic and symptomatic patients within the first week of symptom onset. Lower proportion of memory CD8 T cell with MD was found in severe patients than in mild ones at the stage of disease progression. Critically, the frequency of T cells with MD in symptomatic patients was preferentially associated with CD4 T-cell loss and CD8 T-cell hyperactivation, respectively. Patients bearing effector memory CD4 and CD8 T cells with the phenotype of high MD exhibited poorer T-cell responses upon either phorbol 12-myristate-13-acetate (PMA)/ionomycin or SARS-CoV-2 peptide stimulation than those with low MD. Our findings demonstrated an MD-associated mechanism underlying SARS-CoV-2-induced T lymphocytopenia and functional impairment during the acute phase of infection.

Health of children born through artificial oocyte activation: a pilot study.

Artificial oocyte activation (AOA) has shown to improve fertility in severe male infertility following intracytoplasmic sperm insemination (ICSI). However, the effect of AOA on the health status of children has not been studied. This pilot historical cohort study aims to evaluate physical and mental health of 79 and 89 children from 275 and 406 couples undergoing ICSI-AOA using ionomycin and conventional ICSI, respectively. The outcomes assessed were clinical pregnancy, abortion, type of delivery, and health of children (major birth defect, mental and behavior status). No significant differences were observed between the ICSI-AOA and the ICSI groups for these parameters, and the rate of major birth defects were not significantly different between the 2 groups. In this study, AOA has not imposed a greater risk on physical and mental health of children born through AOA, but for such a solid conclusion, further trails with higher number of cases are required and conclusions drawn are limited to this study.

Identification of the antibiotic ionomycin as an unexpected peroxisome proliferator-activated receptor γ (PPARγ) ligand with a unique binding mode and effective glucose-lowering activity in a mouse model of diabetes.

AIMS/HYPOTHESIS: Existing thiazolidinedione (TZD) drugs for diabetes have severe side effects. The aim of this study is to develop alternative peroxisome proliferator-activated receptor γ (PPARγ) ligands that retain the benefits in improving insulin resistance but with reduced side effects. METHODS: We used AlphaScreen assay to screen for new PPARγ ligands from compound libraries. In vitro biochemical binding affinity assay and in vivo cell-based reporter assay were used to validate ionomycin as a partial ligand of PPARγ. A mouse model of diabetes was used to assess the effects of ionomycin in improving insulin sensitivity. Crystal structure of PPARγ complexed with ionomycin revealed the unique binding mode of ionomycin, which elucidated the molecular mechanisms allowing the discrimination of ionomycin from TZDs. RESULTS: We found that the antibiotic ionomycin is a novel modulating ligand for PPARγ. Both the transactivation and binding activity of PPARγ by ionomycin can be blocked by PPARγ specific antagonist GW9662. Ionomycin interacts with the PPARγ ligand-binding domain in a unique binding mode with properties and epitopes distinct from those of TZD drugs. Ionomycin treatment effectively improved hyperglycaemia and insulin resistance, but had reduced side effects compared with TZDs in the mouse model of diabetes. In addition, ionomycin effectively blocked the phosphorylation of PPARγ at Ser273 by cyclin-dependent kinase 5 both in vitro and in vivo. CONCLUSIONS/INTERPRETATION: Our studies suggest that ionomycin may represent a unique template for designing novel PPARγ ligands with advantages over current TZD drugs.

Successful pregnancy after oocyte activation by a calcium ionophore for a patient with recurrent intracytoplasmic sperm injection failure, with an assessment of oocyte activation and sperm centrosomal function using bovine eggs.

OBJECTIVE: To report a successful pregnancy after intracytoplasmic sperm injection (ICSI) with artificial oocyte activation (AOA) on a patient whose fertilization rate after ICSI was extremely low; and to report on cytologic analyses of the fertilization failure (FF) eggs after ICSI and a biologic assessment of the sperm of this patient. DESIGN: Case report with an assessment of gamete function. SETTING: University hospital and an experimental laboratory. PATIENT(S): A couple with severe oligoasthenozoospermia, whose seventh attempt at ICSI ended in the failure. INTERVENTION(S): Cytologic analyses of FF eggs after ICSI, AOA after ICSI, and analyses of human sperm oocyte activation ability and centrosomal function. RESULT(S): Fertilization arrest after ICSI was observed in FF eggs at various stages of fertilization. After artificial oocyte activation by exposure to ionomycin, clinical pregnancy was confirmed, and a healthy baby was born. As assessed by heterologous ICSI of human sperm into bovine oocytes, there was no significant difference in the oocyte activation rates between the patient's and control sperm, but the sperm centrosomal function was low in the patient's sperm (48.5% vs. 69.6%). CONCLUSION(S): We report a successful pregnancy after ICSI with AOA using a calcium ionophore, after critical cytologic analyses of the FF eggs. Furthermore, sperm centrosomal function was low, indicating that sperm's ability to process the events of fertilization after the oocyte activation was poor in this patient.

Artificial oocyte activation in severe teratozoospermia undergoing intracytoplasmic sperm injection.

OBJECTIVE: To evaluate efficiency of ionomycin on fertilization and cleavage rates, embryo development, and pregnancy rate after intracytoplasmic sperm injection (ICSI) in teratozoospermic patients. DESIGN: Laboratory study. SETTING: Isfahan fertility and infertility Center, and Royan institute, Tehran, Iran. PATIENT(S): Eighty-seven couples with male factor etiology (severe teratozoospermia) undergoing ICSI. INTERVENTION(S): Ionomycin for artificial oocyte activation. MAIN OUTCOME MEASURE(S): After oocyte collection, the oocytes were randomly divided into two groups: control and artificial oocyte activation (AOA). The injected oocytes in the control group were cultured in G1. The remaining oocytes were chemically activated by exposure to 10 muM ionomycin for 10 minutes. Around 16 to 18 hours after ICSI, fertilization was assessed by the presence of pronuclei. The percentage of cleavage and high-quality embryos were calculated 48 and 72 hours after ICSI. Clinical pregnancy also was determined based on ultrasound observation of a fetal heart beat. RESULT(S): There are significant differences in the mean of fertilization and cleavage rates 72 hours after ICSI between AOA and control groups. In patients who had no fertilization in the control group and all the embryos for transfer derived from the AOA group, two pregnancies were recorded. In the patients who had poor fertilization rate (1%-33%) in the control group (14.30%), there was a significant increase in mean fertilization rate (58.31%) because of AOA. The results of the present study also reveal that the samples had motile sperm, whereas in the chemically activated group this difference was not significant. CONCLUSION (S): It can be concluded that in cases with severe teratozoospermia AOA may improve fertilization and cleavage rates, which in turn, affect the implantation and pregnancy rate.

Synergistic antitumor effect of ionomycin and cisplatin against renal cell carcinoma in vitro and in vivo.

Objectives. To characterize the synergistic antitumor effects of the calcium ionophore, ionomycin, and of cisplatin against human renal cell carcinoma cell line, ACHN, both in vitro and in vivo.Methods. The in vitro growth rate of ACHN after exposure to these compounds was measured, using the MTT assay. The apoptotic features in ACHN were evaluated by DNA ladder analysis and flow cytometric analysis. Bcl-2 and Bax expression levels in ACHN after treatment were examined by Western blot. The synergistic antitumor effects of ionomycin and cisplatin against the growth of established ACHN tumors in athymic nude mice were then tested.Results. The in vitro growth rate of ACHN was suppressed more by ionomycin and cisplatin in combination than by either alone. DNA ladder and fragmentation were more obvious when the cells were incubated with ionomycin and cisplatin together than with either reagent alone. Ionomycin treatment increased the expression level of Bax protein, whereas Bcl-2 expression was not influenced. Although an intraperitoneal injection of cisplatin or an intratumoral injection of ionomycin against subcutaneous ACHN tumors somewhat reduced tumorigenicity in nude mice, the effect was significantly enhanced by a combination of these drugs.Conclusions. The synergistic antitumor effects suggest that ionomycin-based therapy could be a novel therapeutic strategy with which to treat advanced renal cell carcinoma.

Dissociation between intracellular calcium elevation and development of human oocytes treated with calcium ionophore.

OBJECTIVE: To develop an acceptable model system to study calcium activation of human oocytes. DESIGN: Study of oocyte development and intracellular calcium [Ca]i dynamics of activated oocytes. SETTING: Research center affiliated with infertility service. MAIN OUTCOME MEASURE: Morphologic evidence of meiotic maturation and cell division under high-power Hoffman optics with an inverted microscope. Meiotic maturation was determined by the number of polar bodies or the presence of a pronucleus, and cell division was determined by evidence of a cleavage furrow or presence of blastomeres. To monitor the effect of calcium ionophore on [Ca]i levels, oocytes were incubated with fura-2 (2 microM) for 30 minutes and [Ca]i was determined by rationing the emission fluorescence (510-nm long-pass filter) during simultaneous excitation at 340 and 380 nm with a microspectrofluorimeter. RESULT(S): All oocytes loaded with fura-2 and then exposed to ionophore exhibited an isolated elevation of [Ca]i, followed by prompt return to baseline levels. None of the oocytes showed signs of cleavage or of meiotic maturation after treatment with calcium ionophore. CONCLUSION(S): Human oocytes activated with calcium ionophore A23187 or ionomycin exhibited elevated [Ca]i but remained resistant to subsequent meiotic maturation and cleavage. Our results differ from some reports of parthenogenetic activation of human oocytes. These differences may result from different activation protocols or culture conditions. Because none of the 126 oocytes cleaved after the activation protocols used in these experiments, this approach should provide an ethically acceptable model system to study calcium dynamics in human oocytes.

Adoptive transfer of bryostatin 1-activated T cells provides long-term protection from tumour metastases.

Treatment of human cancer with tumour-specific T lymphocytes is limited by the frequent unavailability of autologous tumour to stimulate T-cell growth and by the toxicity associated with high-dose interleukin-2 (IL-2) treatment. In the present study we demonstrate that Bryostatin 1 (B) plus ionomycin (I) can substitute for tumour antigen and activate tumour-bearing hosts' T-cells which provide long-term protection against tumour challenge after adoptive transfer. Lymphocytes obtained from the popliteal lymph nodes (DLN) draining an MCA-105 footpad sarcoma were stimulated with B/I, and then cultured for 7 days with 20 U ml-1 IL-2. This in vitro stimulation protocol consistently expanded cell numbers greater than 20-fold during 7 days. Mice given B/I-stimulated draining lymph node (DLN) cells were protected from specific i.v. tumour challenge for at least 15 weeks after adoptive transfer, even in the absence of IL-2 treatment. Tumour immunity conferred by B/I-activated DLN cells was systemic and independent of host T-cells. However, resistance to tumour challenge was lost when either CD4+ or CD8+ T-cells were depleted in vivo. These studies indicate that DLN cells activated with bryostatin 1 and ionomycin persist long-term in vivo as functional memory cells after adoptive transfer.