Evaluation of resazurin phenoxazine dye as a highly sensitive cell viability potency assay for natural killer cell-derived extracellular vesicle-based cancer biotherapeutics.

Natural killer cell-derived extracellular vesicles (NK-EVs) are candidate biotherapeutics against various cancers. However, standardised potency assays are necessary for a reliable assessment of NK-EVs' cytotoxicity. This study aims to thoroughly evaluate a highly sensitive resazurin phenoxazine-based cell viability potency assay (measurement of the cellular redox metabolism) for quantifying the cytotoxicity of NK-EVs against leukaemia K562 cells (suspension model) and breast cancer MDA-MB-231 cells (adherent model) in vitro. The assay was evaluated based on common analytical parameters setforth by regulatory guidelines, including specificity, selectivity,accuracy, precision, linearity, range and stability. Our results revealed that this resazurin-based cell viability potency assay reliably and reproducibly measured a dose-response of NK-EVs' cytotoxic activity against both cancer models. The assay showed precision with 5% and 20% variation for intra-run and inter-run variability. The assay signal showed specificity and selectivity of NK-EVs against cancer target cells, as evidenced by the diminished viability of cancer cells following a 5-hour treatment with NK-EVs, without any detectable interference or background. The linearity analysis of target cancer cells revealed strong linearity for densities of 5000 K562 and 1000 MDA-MB-231 cells per test with a consistent range. Importantly, NK-EVs' dose-response for cytotoxicity showed a strong correlation (|ρ| ∼ 0.8) with the levels of known cytotoxic factors associated with the NK-EVs' corona (FasL, GNLY, GzmB, PFN and IFN-γ), thereby validating the accuracy of the assay. The assay also distinguished cytotoxicity changes in degraded NK-EVs, indicating the ability of the assay to detect the potential loss of sample integrity. Compared to other commonly reported bioassays (i.e., flow cytometry, cell counting, lactate dehydrogenase release assay, DNA-binding reporter assay and confluence assay), our results support this highly sensitive resazurin-based viability potency assay as a high-throughput and quantitative method for assessing NK-EVs' cytotoxicity against both suspension and adherent cancer models for evaluating NK-EVs' biotherapeutics.

Circulating Blood Biomarkers and Risk of Venous Thromboembolism in Cancer Patients: A Systematic Review and Meta-Analysis.

BACKGROUND: Cancer patients have an increased risk of venous thromboembolism (VTE). Currently, the availability of highly discriminatory prediction models for VTE in cancer patients is limited. The implementation of biomarkers in prediction models might lead to refined VTE risk prediction. In this systematic review and meta-analysis, we aimed to evaluate candidate biomarkers and their association with cancer-associated VTE. METHODS: We searched Medline, EMBASE, and Cochrane Central for studies that evaluated biomarkers in adult cancer patients from inception to September 2022. We included studies reporting on VTE after a cancer diagnosis with biomarker measurements performed at a defined time point. Median/mean differences (for continuous measures) and odds ratios (for dichotomous measures) with 95% confidence intervals were estimated and pooled using random-effects models. RESULTS: We included 113 studies in the systematic review. Of these, 50 studies were included in the meta-analysis. We identified two biomarkers at cancer diagnosis (factor VIII and time to peak thrombin), three biomarkers pre-chemotherapy (D-dimer, fibrinogen, and mean platelet volume), and one biomarker preoperatively (platelet count) that had significant median or mean differences. Additionally, we found that hemoglobin <100 g/L and white blood count >11 × 109/L were significantly associated with future VTE risk only when measured at cancer diagnosis. Pre-chemotherapy neutrophil-to-lymphocyte ratio ≥3 and preoperative platelet count ≥400 × 109/L were also found to be associated with future VTE risk. CONCLUSION: In conclusion, this study identified nine candidate blood biomarkers that may help in optimizing VTE prediction in cancer patients that should be further explored in future studies.

Inherited thrombophilia gene mutations and risk of venous thromboembolism in patients with cancer: A systematic review and meta-analysis.

In the general population, individuals with an inherited thrombophilia have a higher risk of thrombosis, but the effect of inherited thrombophilia on the risk of cancer-associated venous thromboembolism (VTE) remains controversial. Our objective was to determine the risk of VTE in cancer patients with inherited thrombophilia. We conducted a systematic review and meta-analysis of studies reporting on VTE after a cancer diagnosis in adult patients who were tested for inherited thrombophilia. In September 2022, we searched Medline, EMBASE, and Cochrane Central. Two reviewers screened the abstracts/full texts and assessed study quality using the Quality in Prognostic Studies tool. We used Mantel-Haenszel random-effects models to estimate pooled odds ratios (OR) of VTE and 95% confidence intervals (95%CI). We included 37 and 28 studies in the systematic review and meta-analysis, respectively. Most studies focused on specific cancer types and hematologic malignancies were rare. The risk of VTE was significantly higher in cancer patients with non-O (compared with O) blood types (OR: 1.56 [95% CI: 1.28-1.90]), Factor V Leiden, and Prothrombin Factor II G20210A mutations compared with wild types (OR: 2.28 [95% CI: 1.51-3.48] and 2.14 [95% CI: 1.14-4.03], respectively). Additionally, heterozygous and homozygous methylenetetrahydrofolate reductase C677T had ORs of 1.50 (95% CI: 1.00-2.24) and 1.38 (95% CI: 0.87-2.22), respectively. Among those with Plasminogen-Activator Inhibitor-1 4G/5G, Vascular Endothelial Growth Factor (VEGF) A C634G, and VEGF C2578A mutations, there was no significant association with VTE. In conclusion, this meta-analysis provided evidence that non-O blood types, Factor V Leiden, and Prothrombin Factor II G20210A mutations are important genetic risk factors for VTE in cancer patients.

A clinically relevant large-scale biomanufacturing workflow to produce natural killer cells and natural killer cell-derived extracellular vesicles for cancer immunotherapy.

Natural killer cell-derived extracellular vesicles (NK-EVs) have shown promising potential as biotherapeutics for cancer due to their unique attributes as cytotoxic nanovesicles against cancer cells and immune-modulatory activity towards immune cells. However, a biomanufacturing workflow is needed to produce clinical-grade NK-EVs for pre-clinical and clinical applications. This study established a novel biomanufacturing workflow using a closed-loop hollow-fibre bioreactor to continuously produce NK-EVs from the clinically relevant NK92-MI cell line under serum-free, Xeno-free and feeder-free conditions following GMP-compliant conditions. The NK92 cells grown in the bioreactor for three continuous production lots resulted in large quantities of both NK cell and NK-EV biotherapeutics at the end of each production lot (over 109 viable cells and 1013 EVs), while retaining their cytotoxic payload (granzyme B and perforin), pro-inflammatory cytokine (interferon-gamma) content and cytotoxicity against the human leukemic cell line K562 with limited off-target toxicity against healthy human fibroblast cells. This scalable biomanufacturing workflow has the potential to facilitate the clinical translation of adoptive NK cell-based and NK-EV-based immunotherapies for cancer with GMP considerations.

Thrombophilia gene mutations predict venous thromboembolism in ambulatory cancer patients receiving chemotherapy.

BACKGROUND: Inherited thrombophilia and cancer both independently increase the risk of venous thromboembolism (VTE). However, whether the increased VTE risk associated with inherited thrombophilia exists in cancer patients is less clear. OBJECTIVES: Our objective was to determine the influence of inherited thrombophilia on VTE and bleeding risk in moderate-to-high-risk ambulatory cancer patients receiving chemotherapy. METHODS: We conducted a post hoc analysis using blood samples from patients enrolled in the AVERT trial to determine if previously recognized thrombophilia gene mutations (prothrombin factor [F] II G20210A, FXI, fibrinogen gamma, serpin family A member 10, FV K858R, FXIII, FV Leiden [FVL], and ABO blood) were associated with VTE or bleeding during the 7-months after starting chemotherapy. Logistic regression was used to compare heterozygous and homozygous mutations (combined) to wild-type. VTE rates, bleeding rates, and risk differences for mutations stratified by prophylactic anticoagulation use were calculated. RESULTS: Of the 447 patients, there were 39 VTE and 39 bleeding events. The odds of VTE were significantly increased with FVL mutation and non-O blood type (odds ratio [OR]: 5.2; 95% CI: 1.9-14.7 and OR: 2.7; 95% CI: 1.2-6.1, respectively). The use of anticoagulation prophylaxis resulted in complete protection in FVL patients, whereas those not receiving anticoagulation had a VTE rate of 119 per 100 patient-years. Lower VTE rates were also observed in non-O blood type patients taking prophylactic anticoagulation. No other thrombophilia genes tested were significantly associated with VTE or bleeding. CONCLUSION: Our results indicate that FVL mutation and ABO blood type may be important VTE predictors in cancer patients starting chemotherapy.

The Application of an Extracellular Vesicle-Based Biosensor in Early Diagnosis and Prediction of Chemoresponsiveness in Ovarian Cancer.

BACKGROUND: Ovarian cancer (OVCA) is the most fatal gynecological cancer with late diagnosis and plasma gelsolin (pGSN)-mediated chemoresistance representing the main obstacles to treatment success. Since there is no reliable approach to diagnosing patients at an early stage as well as predicting chemoresponsiveness, there is an urgent need to develop a diagnostic platform for such purposes. Small extracellular vesicles (sEVs) are attractive biomarkers given their potential accuracy for targeting tumor sites. METHODS: We have developed a novel biosensor which utilizes cysteine-functionalized gold nanoparticles that simultaneously bind to cisplatin (CDDP) and plasma/cell-derived EVs, affording us the advantage of predicting OVCA chemoresponsiveness, and early diagnosis using surface-enhanced Raman spectroscopy. RESULTS: We found that pGSN regulates cortactin (CTTN) content resulting in the formation of nuclear- and cytoplasmic-dense granules facilitating the secretion of sEVs carrying CDDP; a strategy used by resistant cells to survive CDDP action. The clinical utility of the biosensor was tested and subsequently revealed that the sEV/CA125 ratio outperformed CA125 and sEV individually in predicting early stage, chemoresistance, residual disease, tumor recurrence, and patient survival. CONCLUSION: These findings highlight pGSN as a potential therapeutic target and provide a potential diagnostic platform to detect OVCA earlier and predict chemoresistance; an intervention that will positively impact patient-survival outcomes.

Androgen-induced exosomal miR-379-5p release determines granulosa cell fate: cellular mechanism involved in polycystic ovaries.

Polycystic ovarian syndrome (PCOS) is a complex multi-factorial syndrome associated with androgen excess and anovulatory infertility. In the current study, we investigated the role of dihydrotestosterone-induced exosomal miR-379-5p release in determining the destiny of the developing follicles. Our hypothesis was that androgen regulates granulosa cell miR-379-5p content by facilitating its exosomal release in a follicular-stage dependent manner, a process which determines granulosa cell fate. Compared to human non-PCOS subjects, individuals with PCOS exhibit higher follicular fluid free testosterone levels, lower exosomal miR-379-5p content and granulosa cell proliferation. Androgenized rats exhibited lower granulosa cell miR-379-5p but higher phosphoinositide-dependent kinase-1 (PDK1; a miR-379-5p target) content and proliferation. Androgen reduced granulosa cell miR-379-5p content by increasing its exosomal release in preantral follicles, but not in antral follicles in vitro. Studies with an exosomal release inhibitor confirmed that androgen-induced exosomal miR-379-5p release decreased granulosa cell miR-379-5p content and proliferation. Ovarian overexpression of miR-379-5p suppressed granulosa cell proliferation, and basal and androgen-induced preantral follicle growth in vivo. These findings suggest that increased exosomal miR-379-5p release in granulosa cells is a proliferative response to androgenic stimulation specific for the preantral stage of follicle development and that dysregulation of this response at the antral stage is associated with follicular growth arrest, as observed in human PCOS.

Granulosa cell-derived miR-379-5p regulates macrophage polarization in polycystic ovarian syndrome.

Polycystic ovarian syndrome (PCOS) is associated with hyperandrogenemia and ovarian antral follicle growth arrest. We have previously demonstrated that androgen-induced exosomal release of miR-379-5p (miR379) from preantral follicle granulosa cells increases the proliferation of target cells via phosphoinositide-dependent kinase 1 (PDK1) upregulation. Androgen also increases inflammatory M1 macrophage abundance, but reduces anti-inflammatory M2 polarization in rat antral and preovulatory follicles. However, the role of small extracellular vesicles (sEVs; also known as exosomes) secretion in determining the cellular content and function of miRNAs in exosome-receiving cells is largely unknown. Our objectives were to determine: 1) the regulatory role of granulosa cells (GC)-derived exosomal miR379 on macrophage polarization and ovarian inflammation; 2) whether miR379-induced M1 polarization regulates GC proliferation; and 3) if this regulated process is follicular stage-specific. Compared with non-PCOS subjects, PCOS subjects had a higher M1/M2 ratio, supporting the concept that PCOS is an inflammatory condition. Ovarian overexpression of miR379 increased the number of M1 macrophages and the M1/M2 ratio in preantral follicles specifically. Transfection of macrophages with a miR379 mimic reduced the cellular content of PDK1 and induced M0→M1 polarization; whereas its inhibitor polarized M0→M2. Conditioned media from macrophages transfected with miR379 mimic and follicular fluid from PCOS subjects had higher galectin-3 content, a pro-inflammatory cytokine which specifically suppresses human antral follicle GC proliferation. These results indicate that miR379 inhibits M2 macrophage polarization, a condition which suppresses GC proliferation in a follicle stage-dependent manner, as exhibited in PCOS.

The Influence of Exercise-Associated Small Extracellular Vesicles on Trophoblasts In Vitro.

Exercise induces the release of small extracellular vesicles (sEVs) into circulation that are postulated to mediate tissue cross-talk during exercise. We previously reported that pregnant individuals released greater levels of sEVs into circulation after exercise compared to matched non-pregnant controls, but their biological functions remain unknown. In this study, sEVs isolated from the plasma of healthy pregnant and non-pregnant participants after a single bout of moderate-intensity exercise were evaluated for their impact on trophoblasts in vitro. Exercise-associated sEVs were found localized within the cytoplasm of BeWo choriocarcinoma cells, used to model trophoblasts in vitro. Exposure to exercise-associated sEVs did not significantly alter BeWo cell proliferation, gene expression of angiogenic growth factors VEGF and PLGF, or the release of the hormone human chorionic gonadotropin. The results from this pilot study support that exercise-associated sEVs could interact with trophoblasts in vitro, and warrant further investigation to reveal their potential role in communicating the effects of exercise to the maternal-fetal interface.

Predicting chemoresponsiveness in epithelial ovarian cancer patients using circulating small extracellular vesicle-derived plasma gelsolin.

BACKGROUND: Resistance to chemotherapy continues to be a challenge when treating epithelial ovarian cancer (EOC), contributing to low patient survival rates. While CA125, the conventional EOC biomarker, has been useful in monitoring patients' response to therapy, there are no biomarkers used to predict treatment response prior to chemotherapy. Previous work in vitro showed that plasma gelsolin (pGSN) is highly expressed in chemoresistant EOC cell lines, where it is secreted in small extracellular vesicles (sEVs). Whether sEVs from tumour cells are secreted into the circulation of EOC patients and could be used to predict patient chemoresponsiveness is yet to be determined. This study aims to identify if sEV-pGSN in the circulation could be a predictive biomarker for chemoresistance in EOC. METHODS: Sandwich ELISA was used to measure pGSN concentrations from plasma samples of 96 EOC patients (primarily high grade serous EOC). sEVs were isolated using ExoQuick ULTRA and characterized using western blot, nanoparticle tracking analysis, and electron microscopy after which pGSN was measured from the sEVs. Patients were stratified as platinum sensitive or resistant groups based on first progression free interval (PFI) of 6 or 12 months. RESULTS: Total circulating pGSN was significantly decreased and sEV-pGSN increased in patients with a PFI ≤ 12 months (chemoresistant) compared to those with a PFI > 12 months (chemosensitive). The ratio of total pGSN to sEV-pGSN further differentiated these groups and was a strong predictive marker for chemoresistance (sensitivity: 73.91%, specificity: 72.46%). Predetermined CA125 was not different between chemosensitive and chemoresistant groups and was not predictive of chemoresponsiveness prior to treatment. When CA125 was combined with the ratio of total pGSN/sEV-pGSN, it was a significant predictor of chemoresponsiveness, but the test performance was not as robust as the total pGSN/sEV-pGSN alone. CONCLUSIONS: Total pGSN/sEV-pGSN was the best predictor of chemoresponsiveness prior to treatment, outperforming the individual biomarkers (CA125, total pGSN, and sEV-pGSN). This multianalyte predictor of chemoresponsiveness could help to inform physicians' treatment and follow up plan at the time of EOC diagnosis, thus improving patients' outcomes.

Urinary interleukin-9 in youth with type 1 diabetes mellitus.

AIMS: Interleukin-9 (IL-9) attenuates podocyte injury in experimental kidney disease, but its role in diabetic nephropathy is unknown. We sought to relate urinary IL-9 levels to the release of podocyte-derived extracellular vesicles (EVs) in youth with type 1 diabetes. We related urinary IL-9 levels to clinical variables and studied interactions between urinary IL-9, vascular endothelial growth factor (VEGF), tumor necrosis factor alpha (TNFα) and interleukin-6 (IL-6) on urinary albumin/creatinine ratio (ACR) a functional measure of podocyte injury. METHODS: We performed an analysis of urine samples and clinical data from a cohort of youth with type 1 diabetes (n = 53). Cytokines were measured using a Luminex platform (Eve Technologies), and nanoscale flow cytometry was employed to quantify urinary podocyte-derived EVs. All urinary measures were normalized to urinary creatinine. RESULTS: Mean age was 14.7 ± 1.6 years, and the mean time from diagnosis was 6.7 ± 2.9 years. Mean HbA1c was 70.3 ± 13.9 mmol/mol, mean ACR was 1.3 ± 1.9 mg/mmol, and mean eGFR was 140.3 ± 32.6 ml/min/1.73 m2. IL-9 was inversely related to podocyte EVs (r = - 0.56, p = 0.003). IL-9 was also inversely related to blood glucose, HbA1C and eGFR (r = - 0.44, p = 0.002; r = - 0.41, p = 0.003; r = - 0.49, p < 0.001, respectively) and positively correlated with systolic BP (r = 0.30, p = 0.04). There was a significant interaction between IL-9, EVs and ACR (p = 0.0143), and the relationship between IL-9 and ACR depended on VEGF (p = 0.0083), TNFα (p = 0.0231) and IL-6 levels (p = 0.0178). CONCLUSIONS: IL-9 is associated with podocyte injury in early type 1 diabetes, and there are complex interactions between urinary IL-9, inflammatory cytokines and ACR.

Extracellular vesicles in gestational diabetes mellitus: A scoping review.

Gestational diabetes mellitus (GDM) is one of the most common complications of pregnancy worldwide. Despite extensive study, the molecular mechanisms leading to GDM and associated perinatal complications are not well understood. The condition is also associated with an increased risk of future cardiometabolic disease in both mothers and their offspring. Thus, there is a pressing need for the development of effective screening tools and to identify novel molecular mechanisms responsible for the short and long-term risks associated with GDM. In this regard, extracellular vesicles (EVs) offer promise as novel biomarkers of GDM-mediated changes to both mother and fetus. The purpose of this scoping review is to provide an overview of studies examining EVs in the context of GDM. EMBASE and Ovid Medline were searched for articles published from inception to December 2020. We update current knowledge in this area and identify key knowledge gaps with recommendations for future research.

Pulmonary and Neurologic Effects of Mesenchymal Stromal Cell Extracellular Vesicles in a Multifactorial Lung Injury Model.

Rationale: Bronchopulmonary dysplasia, a chronic respiratory condition originating from preterm birth, is associated with abnormal neurodevelopment. Currently, there is an absence of effective therapies for bronchopulmonary dysplasia and its associated brain injury. In preclinical trials, mesenchymal stromal cell therapies demonstrate promise as a therapeutic alternative for bronchopulmonary dysplasia. Objectives: To investigate whether a multifactorial neonatal mouse model of lung injury perturbs neural progenitor cell function and to assess the ability of human umbilical cord-derived mesenchymal stromal cell extracellular vesicles to mitigate pulmonary and neurologic injury. Methods: Mice at Postnatal Day 7 or 8 were injected intraperitoneally with LPS and ventilated with 40% oxygen at Postnatal Day 9 or 10 for 8 hours. Treated animals received umbilical cord-mesenchymal stromal cell-derived extracellular vesicles intratracheally preceding ventilation. Lung morphology, vascularity, and inflammation were quantified. Neural progenitor cells were isolated from the subventricular zone and hippocampus and assessed for self-renewal, in vitro differentiation ability, and transcriptional profiles. Measurements and Main Results: The multifactorial lung injury model produced alveolar and vascular rarefaction mimicking bronchopulmonary dysplasia. Neural progenitor cells from lung injury mice showed reduced neurosphere and oligodendrocyte formation, as well as inflammatory transcriptional signatures. Mice treated with mesenchymal stromal cell extracellular vesicles showed significant improvement in lung architecture, vessel formation, and inflammatory modulation. In addition, we observed significantly increased in vitro neurosphere formation and altered neural progenitor cell transcriptional signatures. Conclusions: Our multifactorial lung injury model impairs neural progenitor cell function. Observed pulmonary and neurologic alterations are mitigated by intratracheal treatment with mesenchymal stromal cell-derived extracellular vesicles.

Plasma Gelsolin Confers Chemoresistance in Ovarian Cancer by Resetting the Relative Abundance and Function of Macrophage Subtypes.

Ovarian cancer (OVCA) is the most lethal gynaecological cancer with a 5-year survival rate less than 50%. Despite new therapeutic strategies, such as immune checkpoint blockers (ICBs), tumor recurrence and drug resistance remain key obstacles in achieving long-term therapeutic success. Therefore, there is an urgent need to understand the cellular mechanisms of immune dysregulation in chemoresistant OVCA in order to harness the host's immune system to improve survival. The over-expression of plasma gelsolin (pGSN) mRNA is associated with a poorer prognosis in OVCA patients; however, its immuno-modulatory role has not been elucidated. In this study, for the first time, we report pGSN as an inhibitor of M1 macrophage anti-tumor functions in OVCA chemoresistance. Increased epithelial pGSN expression was associated with the loss of chemoresponsiveness and poor survival. While patients with increased M1 macrophage infiltration exhibited better survival due to nitric-oxide-induced ROS accumulation in OVCA cells, cohorts with poor survival had a higher infiltration of M2 macrophages. Interestingly, increased epithelial pGSN expression was significantly associated with the reduced survival benefits of infiltrated M1 macrophages, through apoptosis via increased caspase-3 activation and reduced production of iNOS and TNFα. Additionally, epithelial pGSN expression was an independent prognostic marker in predicting progression-free survival. These findings support our hypothesis that pGSN is a modulator of inflammation and confers chemoresistance in OVCA, in part by resetting the relative abundance and function of macrophage subtypes in the ovarian tumor microenvironment. Our findings raise the possibility that pGSN may be a potential therapeutic target for immune-mediated chemoresistance in OVCA.

Urinary extracellular vesicles: A position paper by the Urine Task Force of the International Society for Extracellular Vesicles.

Urine is commonly used for clinical diagnosis and biomedical research. The discovery of extracellular vesicles (EV) in urine opened a new fast-growing scientific field. In the last decade urinary extracellular vesicles (uEVs) were shown to mirror molecular processes as well as physiological and pathological conditions in kidney, urothelial and prostate tissue. Therefore, several methods to isolate and characterize uEVs have been developed. However, methodological aspects of EV separation and analysis, including normalization of results, need further optimization and standardization to foster scientific advances in uEV research and a subsequent successful translation into clinical practice. This position paper is written by the Urine Task Force of the Rigor and Standardization Subcommittee of ISEV consisting of nephrologists, urologists, cardiologists and biologists with active experience in uEV research. Our aim is to present the state of the art and identify challenges and gaps in current uEV-based analyses for clinical applications. Finally, recommendations for improved rigor, reproducibility and interoperability in uEV research are provided in order to facilitate advances in the field.

MicroRNA in Human Acute Kidney Injury: A Systematic Review Protocol.

BACKGROUND: Acute kidney injury (AKI) is a common complication of hospitalization with high morbidity and mortality for which no effective treatments exist and for which current diagnostic tools have limitations for earlier identification. MicroRNAs (miRNAs) are small non-coding RNAs that have been implicated in the pathogenesis of AKI, and some miRNAs have shown promise as therapeutic tools in animal models of AKI. However, less is known about the role of miRNAs in human AKI. OBJECTIVE: To evaluate the role of miRNAs in human subjects with AKI. DESIGN: Systematic review and meta-analysis. MEASUREMENTS: Quantification of miRNA levels from human blood, urine, or kidney biopsy samples, and measures of renal function as defined in the study protocol. METHODS: A comprehensive search strategy for Ovid MEDLINE All, Embase, Web of Science, and CENTRAL will be developed to identify investigational studies that evaluated the relationship between miRNA levels and human AKI. Primary outcomes will include measurements of kidney function and miRNA levels. Study screening, review and data extraction will be performed independently by 2 reviewers. Study quality and certainty of evidence will be assessed with validated tools. A narrative synthesis will be included and the possibility for meta-analysis will be assessed according to characteristics of clinical and statistical heterogeneity between studies. LIMITATIONS: These include (1) lack of randomized trials of miRNAs for the prevention or treatment of human AKI, (2) quality of included studies, and (3) sources of clinical and statistical heterogeneity that may affect strength and reproducibility of results. CONCLUSION: Previous studies of miRNAs in different animal models of AKI have generated strong interest on their use for the prevention and treatment of human AKI. This systematic review will characterize the most promising miRNAs for human research and will identify methodological constraints from miRNA research in human AKI to help inform the design of future studies. SYSTEMATIC REVIEW REGISTRATION: PROSPERO CRD42020201253.

CONTEXTE: L'insuffisance rénale aiguë (IRA) est une complication fréquente des hospitalisations avec morbidité et mortalité élevées. Il n'existe aucun traitement efficace contre l'IRA et les outils diagnostiques actuels qui permettent son dépistage précoce comportent des limites. Les microARN (miARN) sont de petits ARN non codants ayant été impliqués dans la pathogenèse de l'IRA; certains d'entre eux se sont révélés prometteurs comme outils thérapeutiques dans les modèles animaux de l'IRA. Le rôle des miARN dans l'IRA chez l'humain est cependant moins connu. OBJECTIF: Évaluer le rôle des miARN chez les sujets humains atteints d'IRA. TYPE D'ÉTUDE: Examen systématique et méta-analyze. MESURES: La quantification des taux de miARN chez l'humain à partir d'échantillons de sang, d'urine ou de biopsie rénale, et mesure de la fonction rénale telle que définie dans le protocole de l'étude. MÉTHODOLOGIE: Une stratégie de recherche exhaustive des bases de données Ovid MEDLINE All, Embase, Web of Science et CENTRAL sera élaborée afin de répertorier les études expérimentales ayant évalué la relation entre les taux de miARN et l'IRA chez l'humain. Les principaux critères d'évaluation comprendront la mesure de la fonction rénale et des taux de miARN. Deux examinateurs procéderont de façon indépendante à la sélection des études, à leur examen et à l'extraction des données. La qualité des études et la robustesse des données seront évaluées à l'aide d'outils validés. Une synthèse descriptive sera incluse et la possibilité d'une méta-analyze sera évaluée en fonction des caractéristiques de l'hétérogénéité clinique et statistique entre les études. LIMITES: Les limites de l'étude concernent notamment (i) le manque d'essais randomisés examinant les miARN pour la prévention ou le traitement de l'IRA humaine; (ii) la qualité des études incluses; et (iii) les sources d'hétérogénéité clinique et statistique susceptibles d'affecter la robustesse et la reproductibilité des résultats. CONCLUSION: Des études antérieures sur les miARN dans différents modèles animaux de l'IRA ont suscité un vif intérêt pour leur utilization dans la prévention et le traitement de l'IRA chez l'humain. Cet examen systématique caractérisera les miARN les plus prometteurs pour la recherche sur l'IRA humaine et définira les contraintes méthodologiques de telles études, ce qui aidera à orienter la conception des études futures.

Can placental growth factors explain birthweight variation in offspring of women with type 1 diabetes?

AIMS/HYPOTHESIS: Maternal hyperglycaemia alone does not explain the incidence of large offspring amongst women with type 1 diabetes. The objective of the study was to determine if there is an association between placental function, as measured by angiogenic factors, and offspring birthweight z score in women with type 1 diabetes. METHODS: This cohort study included samples from 157 Continuous Glucose Monitoring in Pregnant Women with Type 1 Diabetes (CONCEPTT) trial participants. Correlations were estimated between birthweight z score and placental growth factor (PlGF) and soluble fms-like tyrosine kinase (sFlt-1) levels measured at baseline and at 24 and 34 weeks of gestation. Linear regression was used to assess the relationship between birthweight z score and placental health, as measured by PlGF and sFlt-1/PlGF ratio, stratified by glycaemic status (continuous glucose monitoring and HbA1c measures) and adjusted for potential confounders of maternal BMI, smoking and weight gain. Higher PlGF levels and lower sFlt-1/PlGF ratios represent healthy placentas, while lower PlGF levels and higher sFlt-1/PlGF ratios represent unhealthy placentas. RESULTS: Among CONCEPTT participants, the slopes relating PlGF levels to birthweight z scores differed according to maternal glycaemia at 34 weeks of gestation (p = 0.003). With optimal maternal glycaemia (HbA1c < 48 mmol/mol [6.5%]/ or continuous glucose monitoring time above range ≤ 30%), birthweight z scores were reduced towards zero (normal weight) with increasing PlGF values (representing a healthy placenta), and increased with decreasing PlGF values. With suboptimal glycaemic status (HbA1c ≥ 48 mmol/mol [6.5%] or time above range > 30%), increasing PlGF values were associated with heavier infants. Those with a healthy placenta (PlGF > 100) and suboptimal glycaemic control had a higher mean z score (2.45) than those with an unhealthy placenta (mean z score = 1.86). Similar relationships were seen when using sFlt-1/PlGF ratio as a marker for a healthy vs unhealthy placenta. CONCLUSIONS/INTERPRETATION: In women with type 1 diabetes, infant birthweight is influenced by both glycaemic status and placental function. In women with suboptimal glycaemia, infant birthweight was heavier when placentas were healthy. Suboptimal placental function should be considered in the setting of suboptimal glycaemia and apparently 'normal' birthweight.

Prospective meta-analysis protocol on randomised trials of renin-angiotensin system inhibitors in patients with COVID-19: an initiative of the International Society of Hypertension.

INTRODUCTION: Whether ACE inhibitors (ACEi) or angiotensin II receptor blocker (ARB) therapy should be continued, initiated or ceased in patients with COVID-19 is uncertain. Given the widespread use of ACEi/ARBs worldwide, guidance on the use of these drugs is urgently needed. This prospective meta-analysis aims to pool data from randomised controlled trials (RCTs) to assess the safety and efficacy of ACEi/ARB therapy in adults infected with SARS-CoV-2. METHODS AND ANALYSIS: RCTs will be eligible if they compare patients with COVID-19 randomised to ACEi/ARB continuation or commencement versuss no ACEi/ARB therapy; study duration ≥14 days; recruitment completed between March 2020 and May 2021. The primary outcome will be all-cause mortality at ≤30 days. Secondary outcomes will include mechanical ventilation, admission to intensive care or cardiovascular events at short-term follow-up (≤30 days) and all-cause mortality at longer-term follow-up (>1 month). Prespecified subgroup analyses will assess the effect of sex; age; comorbidities; smoking status; ethnicity; country of origin on all-cause mortality. A search of ClinicalTrials.gov has been performed, which will be followed by a formal search of trial registers, preprint servers, MEDLINE, EMBASE and Cochrane Central Register of Controlled Trials to identify RCTs that meet inclusion criteria. To date, a search of ClinicalTrials.gov identified 21 potentially eligible trials for this meta-analysis. We will request trial investigators/sponsors to contribute standardised grouped tabular outcome data. ETHICS AND DISSEMINATION: Ethics approval and informed consent will be the responsibility of the individual RCTs. Dissemination of results will occur by peer-reviewed publication. The results of our analysis can inform public health policy and clinical decision making regarding ACEi/ARB use in patients with COVID-19 on a global scale.

Preclinical Studies of MSC-Derived Extracellular Vesicles to Treat or Prevent Graft Versus Host Disease: a Systematic Review of the Literature.

INTRODUCTION: Treating and preventing graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplant (HCT) remains a significant challenge. The use of mesenchymal stromal cell-derived extracellular vesicles (MSC-EVs) appears promising and a systematic review of preclinical studies is needed to accelerate the design of translational studies. METHODS: We identified 4 eligible studies from a systematic review performed on December 1, 2018. In brief, eligible studies included the treatment or prevention of GVHD in animal models and the use of MSC-EVs. Study design and outcome data were extracted and reporting was evaluated using the SYRCLE tool to identify potential bias. RESULTS: Two studies assessed the efficacy of MSC-EVs in treatment of GVHD and 2 studies address prevention. Mice treated with MSC-EVs showed improved median survival, GVHD clinical scores and histology scores as compared to untreated mice with GVHD. Prophylactic treatment with MSC-EVs attenuated GVHD severity and improved median survival as compared to no treatment or saline. CONCLUSION: Our systematic review provides important insight regarding the potential of MSC-EVs to treat or prevent GVHD. Although few studies were identified, improved survival and attenuated histologic findings of GVHD were observed in mice after MSC-EV administration for the treatment and prevention of GVHD. Dosing of EVs and route of administration remain inconsistent, however, and scalability of EV isolation for clinical studies remains a challenge. Standardized outcome reporting is needed to pool results for metanalysis. Graphical abstract.

Plasma Gelsolin Inhibits CD8+ T-cell Function and Regulates Glutathione Production to Confer Chemoresistance in Ovarian Cancer.

Although initial treatment of ovarian cancer is successful, tumors typically relapse and become resistant to treatment. Because of poor infiltration of effector T cells, patients are mostly unresponsive to immunotherapy. Plasma gelsolin (pGSN) is transported by exosomes (small extracellular vesicle, sEV) and plays a key role in ovarian cancer chemoresistance, yet little is known about its role in immunosurveillance. Here, we report the immunomodulatory roles of sEV-pGSN in ovarian cancer chemoresistance. In chemosensitive conditions, secretion of sEV-pGSN was low, allowing for optimal CD8+ T-cell function. This resulted in increased T-cell secretion of IFNγ, which reduced intracellular glutathione (GSH) production and sensitized chemosensitive cells to cis-diaminedichloroplatinum (CDDP)-induced apoptosis. In chemoresistant conditions, increased secretion of sEV-pGSN by ovarian cancer cells induced apoptosis in CD8+ T cells. IFNγ secretion was therefore reduced, resulting in high GSH production and resistance to CDDP-induced death in ovarian cancer cells. These findings support our hypothesis that sEV-pGSN attenuates immunosurveillance and regulates GSH biosynthesis, a phenomenon that contributes to chemoresistance in ovarian cancer. SIGNIFICANCE: These findings provide new insight into pGSN-mediated immune cell dysfunction in ovarian cancer chemoresistance and demonstrate how this dysfunction can be exploited to enhance immunotherapy.