RBFOX2 is critical for maintaining alternative polyadenylation patterns and mitochondrial health in rat myoblasts.

RBFOX2, which has a well-established role in alternative splicing, is linked to heart diseases. However, it is unclear whether RBFOX2 has other roles in RNA processing that can influence gene expression in muscle cells, contributing to heart disease. Here, we employ both 3'-end and nanopore cDNA sequencing to reveal a previously unrecognized role for RBFOX2 in maintaining alternative polyadenylation (APA) signatures in myoblasts. RBFOX2-mediated APA modulates mRNA levels and/or isoform expression of a collection of genes, including contractile and mitochondrial genes. Depletion of RBFOX2 adversely affects mitochondrial health in myoblasts, correlating with disrupted APA of mitochondrial gene Slc25a4. Mechanistically, RBFOX2 regulation of Slc25a4 APA is mediated through consensus RBFOX2 binding motifs near the distal polyadenylation site, enforcing the use of the proximal polyadenylation site. In sum, our results unveil a role for RBFOX2 in fine-tuning expression of mitochondrial and contractile genes via APA in myoblasts relevant to heart diseases.

Longitudinal NK cell kinetics and cytotoxicity in children with neuroblastoma enrolled in a clinical phase II trial.

BACKGROUND: Natural killer (NK) cells are one of the main effector populations of immunotherapy with monoclonal antibody and cytokines, used in combination with chemotherapy to treat children with high-risk neuroblastoma on this phase II trial. However, the impact of chemoimmunotherapy on NK cell kinetics, phenotype, and function is understudied. METHODS: We prospectively examined NK cell properties from 63 children with newly diagnosed neuroblastoma enrolled in a phase II trial (NCT01857934) and correlated our findings with tumor volume reduction after 2 courses of chemoimmunotherapy. NK cell studies were conducted longitudinally during chemoimmunotherapy and autologous hematopoietic cell transplantation (autoHCT) with optional haploidentical NK cell infusion and additional immunotherapy. RESULTS: Chemoimmunotherapy led to significant NK cytopenia, but complete NK cell recovery reliably occurred by day 21 of each therapy course as well as after autoHCT. Haploidentical NK cell infusion elevated the NK cell count transiently during autoHCT. NK cell cytotoxicity increased significantly during treatment compared with diagnosis. In addition, NK cells maintained their ability to respond to cytokine stimulation in culture longitudinally. Unsupervised cluster analysis of CD56bright NK cell count and tumor volume at diagnosis and after two courses of chemoimmunotherapy identified two patient groups with distinct primary tumor sizes and therapy responses. CONCLUSION: After profound NK cytopenia due to chemoimmunotherapy, endogenously reconstituted NK cells exhibit enhanced NK cytotoxicity compared with pretherapy measurements. Our data suggest a relationship between CD56bright expression and tumor size before and after two courses of chemoimmunotherapy; however, future studies are necessary to confirm this relationship and its predictive significance. TRIAL REGISTRATION NUMBER: NCT01857934.

Acute Kidney Injury in Pediatric Patients Receiving Allogeneic Hematopoietic Cell Transplantation: Incidence, Risk Factors, and Outcomes.

Acute kidney injury (AKI) is a common adverse event after hematopoietic cell transplantation (HCT). AKI is associated with early death or chronic kidney disease among transplant survivors. However, large-scale pediatric studies based on standardized criteria are lacking. We performed a retrospective analysis of 1057 pediatric patients who received allogeneic HCT to evaluate the incidence and risk factors of AKI according to AKI Network criteria within the first 100 days of HCT. We also determined the effect of AKI on patient survival. The 100-day cumulative incidences of all stages of AKI, stage 3 AKI, and AKI requiring renal replacement therapy (RRT) were 68.2% ± 1.4%, 25.0% ± 1.3%, and 7.6% ± .8%, respectively. Overall survival at 1 year was not different between patients without AKI and those with stage 1 or 2 AKI (66.1% versus 73.4% versus 63.9%, respectively) but was significantly different between patients without AKI and patients with stage 3 AKI with or without RRT requirement (66.1% versus 47.3% versus 7.5%, respectively; P < .001). Age, year of transplantation, donor type, sinusoidal obstruction syndrome (SOS), and acute graft-versus-host disease (GVHD) were independent risk factors for stages 1 through 3 AKI. Age, donor, conditioning regimen, number of HCTs, SOS, and acute GVHD were independent risk factors for AKI requiring RRT. Our study revealed that AKI was a prevalent adverse event, and severe stage 3 AKI, which was associated with reduced survival, was common after pediatric allogeneic HCT. All patients receiving allogeneic HCT, especially those with multiple risk factors, require careful renal monitoring according to standardized criteria to minimize nephrotoxic insults.

Cryopreservation of human whole blood allows immunophenotyping by flow cytometry up to 30days after cell isolation.

Immunophenotyping of whole blood (WB) by flow cytometry (FC) is used clinically to assess a patient's immune status and also in biomedical research. Current protocols recommend storage of immunolabeled samples at 4°C with FC analysis to be completed within seven days. This data acquisition window can be extended to up to one year post-labeling, but this requires cryopreservation of the samples at ultra-low temperatures (≤-80°C or in liquid nitrogen). In this study we optimized a standardized cryopreservation protocol to enable preservation of immunolabeled, human WB samples at -20°C for FC and tested its effectiveness after 0, 5, 15 or 30days. Analysis of stored samples shows that this protocol effectively preserves immunolabeled WB samples and that the duration of storage has no effect on morphology, viability or frequency of WB cell subpopulations, and that the intensity of fluorescent signal from labeled extracellular markers is fully preserved for at least 15days, and up to 30days for some markers. We demonstrate that using this protocol, we are able to differentiate resting versus activated WB cells as demonstrated by detection of significantly increased expression of CD11b by myeloid cells in WB samples pretreated with LPS (100µg/mL for 12h). Finally, we show that this method allows for labeling and detection of the intracellular cytokine (IL-8) up to 30days following cryopreservation from myeloid cells, in previously labeled and cryopreserved WB samples.

Severe Hemorrhagic Shock Induces Acute Activation and Expansion of IL-8+/IL-10+ Neutrophils with Enhanced Oxidative Reactivity in Non-Human Primates.

BACKGROUND: Neutrophilic inflammation is a mediator of morbidity and mortality in response to hemorrhagic shock. Although injury-induced neutrophil margination has long been observed, the nature of neutrophils' role in the "second hit" paradigm remains to be fully elucidated. We sought to extensively characterize neutrophil phenotype and functionality in response to severe hemorrhage in non-human primates (NHPs). METHODS: NHPs (n = 8) were subjected to severe hemorrhagic shock and resuscitation. Blood was obtained at baseline (T = 0 min), end of shock (T = 60 min), end of resuscitation (T = 180 min), T = 360 min, and 24 h (T = 1440 min). Neutrophils were quantified by complete blood count and flow cytometry. IL-8 and IL-10 production was determined by intracellular flow cytometry. Oxidation of dihydrorhodamine-123 (DHR-123) was used to determine neutrophil oxidative bursts (untreated), priming (+fMLP), and burst capacity (+PMA/ionomycin) via microplate reader ex vivo. Data are reported as mean ±â€ŠSEM; statistical significance was measured using repeated measures ANOVA with Bonferroni adjustment. P < 0.05 is considered significant. RESULTS: CD45CD11bCD16 neutrophils doubled postinjury (P < 0.0001); this was due to activated IL-8/IL-10 neutrophils that increased in frequency in relation to resting IL-8IL-10 cells. At 24 h, the proportions of activated to resting neutrophils returned to baseline levels. Resuscitative measures initially decreased neutrophil oxidative output; however, oxidative bursts, priming, and burst capacity were significantly increased at 24 h (P < 0.0025, 0.0124, and 0.0118, respectively). CONCLUSION: These results demonstrate an acute expansion and phenotypic activation of circulating neutrophils postinjury followed by a return to homeostatic proportions within 24 h; paradoxically, phenotypically "resting" neutrophils at 24 h have significantly higher oxidative potential, predisposing for exaggerated inflammatory responses. These data are consistent with clinical literature and provide important functional insight into neutrophil-mediated shock pathology.