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The immune system is impaired in myelodysplastic syndromes (MDS) and plays a role in the pathogenesis of the disease. Here we show effects of recombinant human erythropoietin (rHuEPO) on T cell (CD4 + , CD8 + and CD4 + CD25 + ) number and function in MDS patients. Healthy (20 subjects), MDS patients without rHuEPO treatment ('MDS', 13), and MDS patients treated with rHuEPO ('MDS + EPO', 17) were examined. CD4 + and CD8 + T cell numbers were reduced and increased respectively in MDS compared to healthy subjects. EPO treatment normalized these levels. CD4 + CD25 + cell numbers, lower in MDS, were normalized in MDS + EPO. In vitro activation of CD4 + and CD8 + cells with phytohemagglutinin as measured by CD69 expression, demonstrated a 7.2 fold increase in CD4 + activation vs 13.6 fold for MDS and MDS + EPO respectively (p = 0.004); and 10.2 fold (MDS) vs 18.6 fold (MDS + EPO, p < 0.003) for CD8 + T cells. Expression of the co-stimulatory marker CD28, decreased in CD4 + and CD8 + T cells in MDS, was normalized in MDS + EPO CD4 + T cells. Subgroup analysis of milder disease (WHO RA and RARS) and more advanced disease revealed no difference in CD4 + and CD8 + T cell numbers. However, the activation of these cells in the RA/RARS subgroup was impaired in EPO-untreated and enhanced in EPO-treated MDS patients. Our data suggest that EPO treatment improves immune abnormalities in MDS and may depend on disease severity. [ABSTRACT FROM AUTHOR]

Objectives: Epithelial ovarian cancer is often diagnosed at an advanced stage due to intraperitoneal spread. Modern treatment strategies have only modestly improved the survival rates of this disease. Chimeric antigen receptor (CAR) T cells enable T cells to directly bind tumor-associated antigens in an MHC-independent manner inducing tumor rejection. A major barrier is the lack of true tumor-specific antigens followed by risk of "on-target off-tumor" toxicity. Innovative approaches are therefore needed to increase the specificity of CAR-modified T cells exclusively against tumors. The aim of this study was to assess the efficacy and safety of intraperitoneal versus intravenous CAR T cell therapy. Our working hypothesis was that intraperitoneal CAR T cell therapy will demonstrate higher cytotoxic activity towards a tumor tissue while sparing non-tumor tissues. Methods: We have constructed CARs targeting HER2 (ErbB2) that are overexpressed in ovarian cancers. CARs were expressed in human lymphocytes, and their functionality toward human ovarian cancer cell lines was evaluated in-vitro and in a murine model. Results: We have found that the anti-ErbB2 CARs secreted IFN-g (600-1800pg/ml) after stimulation with OVCAR8, SKOV3, or NAR cells compared to untransduced lymphocytes CAR T cells (50pg/ml). Treatment with an intraperitoneal injection of anti-ErbB2 CAR T cells leads to remission of the tumor and increased survival compared to the intravenous route. Intraperitoneal treatment of anti-ErbB2 CARs was less toxic to non-tumor tissues compared to the intravenous treatment. Conclusions: Our results demonstrate that the intra-peritoneal approach of CAR T cells offers a safe strategy with a clinical potential for the treatment of ovarian cancer. [ABSTRACT FROM AUTHOR]

Erythropoietin (EPO) primarily regulates red blood cell formation, and EPO serum levels are increased on hypoxic stress (e.g., anemia and altitude). In addition to anemia, recent discoveries suggest new therapeutic indications for EPO, unrelated to erythropoiesis. We investigated the skeletal role of EPO using several models of overexpression (Tg6 mice) and EPO administration (intermittent/continuous, high/low doses) in adult C57Bl6 female mice. Using microcomputed tomography, histology, and serum markers, we found that EPO induced a 32%-61% trabecular bone loss caused by increased bone resorption (+60%-88% osteoclast number) and reduced bone formation rate (-19 to -74%; P < 0.05 throughout). EPO targeted the monocytic lineage by increasing the number of bonemonocytes/macrophages, preosteoclasts, and mature osteoclasts. In contrast to the attenuated bone formation in vivo, EPO treatment in vitro did not inhibit osteoblast differentiation and activity, suggesting an indirect effect of EPO on osteoblasts. However, EPO had a direct effect on preosteoclasts by stimulating osteoclastogenesis in isolated cultures (+60%) via the Jak2 and PI3K pathways. In summary, our findings demonstrate that EPO negatively regulates bone mass and thus bears significant clinical implications for the potential management of patients with endogenously or therapeutically elevated EPO levels. [ABSTRACT FROM AUTHOR]

Chimeric antigen receptor (CAR)-T cells are genetically engineered T cells, directed against a tumor-associated antigen. Extracellular vesicles (EVs) derived from CAR-T cells (CAR-T EVs) may preserve CAR-T activity and overcome one of the major obstacles responsible for CAR-T cell failure in patients with solid tumors. This study aimed to compare CAR-T EVs with their parental cells and explore their cell penetration and cytotoxic activity. Anti-HER-2 CARs were stimulated with specific target cells. EVs were isolated from the cell media and characterized for their content and functions. We found that CAR-T EVs contained a mixture of small and large EVs. Stimulated anti-HER-2+ CAR-T EVs expressed lower cytokine levels compared with their parental CAR-T cells (such as interferon gamma). Higher levels of granzyme B were found in CAR-T EVs (≥20 × ) compared with EVs from unstimulated cells (p < 0.001). Anti-HER-2+ CAR-T EVs bound and penetrated specifically into HER-2 expressing target cells. Similar cytotoxic effects measured by caspase-3/7 activity were found in CAR-T cells and their derived EVs. However, while the CAR-T cells induced massive apoptosis during the first 24 h, CAR-T EVs required 60 − 90 h. In summary, CAR-T EVs provide a novel potent immunotherapy approach that may be effective against solid tumors. [ABSTRACT FROM AUTHOR]

Abstract: Proteins of the nuclear envelope have been implicated as participating in gene silencing. BAF, a DNA- and LEM domain-binding protein, has been suggested to link chromatin to the nuclear envelope. We have previously shown that LAP2β, a LEM-domain inner nuclear membrane protein, represses transcription through binding to HDAC3 and induction of histone H4 deacetylation. We now show that LAP2ζ, the smallest LAP2 family member, is also involved in regulation of transcription. We show that similar to other LEM-domain proteins LAP2ζ interacts with BAF. LAP2ζ-YFP and BAF co-localize in the cytoplasm, and overexpression of LAP2ζ leads to reduction of nucleoplasmic BAF. Mutations in the LAP2ζ-YFP LEM domain decrease its interaction with BAF retaining the nucleo-cytoplasmic distribution of BAF. Co-expression of LAP2β and LAP2ζ results in inhibition of LAP2β-induced gene silencing while overexpression of LAP2ζ alone leads to a small increase in transcriptional activity of various transcription factors. Our results suggest that LAP2ζ is a transcriptional regulator acting predominantly to inhibit LAP2β-mediated repression. LAP2ζ may function by decreasing availability of BAF. These findings could have implications in the study of nuclear lamina-associated diseases and BAF-dependent retroviral integration. [Copyright &y& Elsevier]

Recent studies have demonstrated that erythropoietin (EPO) treatment in mice results in trabecular bone loss. Here, we investigated the dose-response relationship between EPO, hemoglobin (Hgb) and bone loss and examined the reversibility of EPO-induced damage. Increasing doses of EPO over two weeks led to a dose-dependent increase in Hgb in young female mice, accompanied by a disproportionate decrease in trabecular bone mass measured by micro-CT (µCT). Namely, increasing EPO from 24 to 540 IU/week produced a modest 12% rise in Hgb (20.2 ± 1.3 mg/dL vs 22.7 ± 1.3 mg/dL), while trabecular bone volume fraction (BV/TV) in the distal femur decreased dramatically (27 ± 8.5% vs 53 ± 10.2% bone loss). To explore the long-term skeletal effects of EPO, we treated mice for two weeks (540 IU/week) and monitored bone mass changes after treatment cessation. Six weeks post-treatment, there was only a partial recovery of the trabecular microarchitecture in the femur and vertebra. EPO-induced bone loss is therefore dose-dependent and mostly irreversible at doses that offer only a minor advantage in the treatment of anemia. Because patients requiring EPO therapy are often prone to osteoporosis, our data advocate for using the lowest effective EPO dose for the shortest period of time to decrease thromboembolic complications and minimize the adverse skeletal outcome. [ABSTRACT FROM AUTHOR]