Extracellular vesicles secreted by human aneuploid embryos present a distinct transcriptomic profile and upregulate MUC1 transcription in decidualised endometrial stromal cells.

STUDY QUESTION: Do extracellular vesicles (EVs) secreted by aneuploid human embryos possess a unique transcriptomic profile that elicits a relevant transcriptomic response in decidualized primary endometrial stromal cells (dESCs)? SUMMARY ANSWER: Aneuploid embryo-derived EVs contain transcripts of PPM1J, LINC00561, ANKRD34C, and TMED10 with differential abundance from euploid embryo-derived EVs and induce upregulation of MUC1 transcript in dESCs. WHAT IS KNOWN ALREADY: We have previously reported that IVF embryos secrete EVs that can be internalized by ESCs, conceptualizing that successful implantation to the endometrium is facilitated by EVs. Whether these EVs may additionally serve as biomarkers of ploidy status is unknown. STUDY DESIGN SIZE DURATION: Embryos destined for biopsy for preimplantation genetic testing for aneuploidy (PGT-A) were grown under standard conditions. Spent media (30 µl) were collected from euploid (n = 175) and aneuploid (n = 140) embryos at cleavage (Days 1-3) stage and from euploid (n = 187) and aneuploid (n = 142) embryos at blastocyst (Days 3-5) stage. Media samples from n = 35 cleavage-stage embryos were pooled in order to obtain five euploid and four aneuploid pools. Similarly, media samples from blastocysts were pooled to create one euploid and one aneuploid pool. ESCs were obtained from five women undergoing diagnostic laparoscopy. PARTICIPANTS/MATERIALS SETTING METHODS: EVs were isolated from pools of media by differential centrifugation and EV-RNA sequencing was performed following a single-cell approach that circumvents RNA extraction. ESCs were decidualized (estradiol: 10 nM, progesterone: 1 µM, cAMP: 0.5 mM twice every 48 h) and incubated for 24 h with EVs (50 ng/ml). RNA sequencing was performed on ESCs. MAIN RESULTS AND THE ROLE OF CHANCE: Aneuploid cleavage stage embryos secreted EVs that were less abundant in RNA fragments originating from the genes PPM1J (log2fc = -5.13, P = 0.011), LINC00561 (log2fc = -7.87, P = 0.010), and ANKRD34C (log2fc = -7.30, P = 0.017) and more abundant in TMED10 (log2fc = 1.63, P = 0.025) compared to EVs of euploid embryos. Decidualization per se induced downregulation of MUC1 (log2fc = -0.54, P = 0.0028) in ESCs as a prerequisite for the establishment of receptive endometrium. The expression of MUC1 transcript in decidualized ESCs was significantly increased following treatment with aneuploid compared to euploid embryo-secreted EVs (log2fc = 0.85, P = 0.0201). LARGE SCALE DATA: Raw data have been uploaded to GEO (accession number GSE234338). LIMITATIONS REASONS FOR CAUTION: The findings of the study will require validation utilizing a second cohort of EV samples. WIDER IMPLICATIONS OF THE FINDINGS: The discovery that the transcriptomic profile of EVs secreted from aneuploid cleavage stage embryos differs from that of euploid embryos supports the possibility to develop a non-invasive methodology for PGT-A. The upregulation of MUC1 in dESCs following aneuploid embryo EV treatment proposes a new mechanism underlying implantation failure. STUDY FUNDING/COMPETING INTERESTS: The study was supported by a Marie Sklodowska-Curie Actions fellowship awarded to SM by the European Commission (CERVINO grant agreement ID: 79620) and by a BIRTH research grant from Theramex HQ UK Ltd. The authors have no conflicts of interest to declare.

Alterations in glutamate, arginine, and energy metabolism characterize cerebrospinal fluid and plasma metabolome of persons with HIV-associated dementia.

OBJECTIVES: HIV-associated dementia (HAD) is the most severe clinical expression of HIV-mediated neuropathology, and the processes underlying its development remain poorly understood. We aimed to exploit high-dimensional metabolic profiling to gain insights into the pathological mechanisms associated to HAD. DESIGN: In this cross-sectional study, we utilized metabolomics to profile matched cerebrospinal fluid (CSF) and plasma samples of HAD individuals ( n  = 20) compared with neurologically asymptomatic people with HIV (ASYM, n  = 20) and healthy controls (NEG, n  = 20). METHODS: Identification of plasma and CSF metabolites was performed by liquid-chromatography or gas-chromatography following a validated experimental pipeline. The resulting metabolic profiles were analyzed by machine-learning algorithms, and altered pathways were identified by comparison with KEGG pathway database. RESULTS: In CSF, HAD patients displayed an imbalance in glutamine/glutamate ratio, decreased levels of isocitrate and arginine, and increased oxidative stress when compared with ASYM or NEG. These changes were confirmed in matched plasma samples, which in addition revealed an accumulation of eicosanoids and unsaturated fatty acids in HAD individuals. Pathway analysis in both biological fluids suggested that alterations in several metabolic processes, including protein biosynthesis, glutamate and arginine metabolism, and energy metabolism, in association to a perturbed eicosanoid metabolism in plasma, may represent the metabolic signature associated to HAD. CONCLUSION: These findings show that HAD may be associated with metabolic modifications in CSF and plasma. These preliminary data may be useful to identify novel metabolic biomarkers and therapeutic targets in HIV-associated neurological impairment.

In vivo macrophage engineering reshapes the tumor microenvironment leading to eradication of liver metastases.

Liver metastases are associated with poor response to current pharmacological treatments, including immunotherapy. We describe a lentiviral vector (LV) platform to selectively engineer liver macrophages, including Kupffer cells and tumor-associated macrophages (TAMs), to deliver type I interferon (IFNα) to liver metastases. Gene-based IFNα delivery delays the growth of colorectal and pancreatic ductal adenocarcinoma liver metastases in mice. Response to IFNα is associated with TAM immune activation, enhanced MHC-II-restricted antigen presentation and reduced exhaustion of CD8+ T cells. Conversely, increased IL-10 signaling, expansion of Eomes CD4+ T cells, a cell type displaying features of type I regulatory T (Tr1) cells, and CTLA-4 expression are associated with resistance to therapy. Targeting regulatory T cell functions by combinatorial CTLA-4 immune checkpoint blockade and IFNα LV delivery expands tumor-reactive T cells, attaining complete response in most mice. These findings support a promising therapeutic strategy with feasible translation to patients with unmet medical need.

Revealing and harnessing CD39 for the treatment of colorectal cancer and liver metastases by engineered T cells.

OBJECTIVE: Colorectal tumours are often densely infiltrated by immune cells that have a role in surveillance and modulation of tumour progression but are burdened by immunosuppressive signals, which might vary from primary to metastatic stages. Here, we deployed a multidimensional approach to unravel the T-cell functional landscape in primary colorectal cancers (CRC) and liver metastases, and genome editing tools to develop CRC-specific engineered T cells. DESIGN: We paired high-dimensional flow cytometry, RNA sequencing and immunohistochemistry to describe the functional phenotype of T cells from healthy and neoplastic tissue of patients with primary and metastatic CRC and we applied lentiviral vectors (LV) and CRISPR/Cas9 genome editing technologies to develop CRC-specific cellular products. RESULTS: We found that T cells are mainly localised at the front edge and that tumor-infiltrating T cells co-express multiple inhibitory receptors, which largely differ from primary to metastatic sites. Our data highlighted CD39 as the major driver of exhaustion in both primary and metastatic colorectal tumours. We thus simultaneously redirected T-cell specificity employing a novel T-cell receptor targeting HER-2 and disrupted the endogenous TCR genes (TCR editing (TCRED)) and the CD39 encoding gene (ENTPD1), thus generating TCREDENTPD1KOHER-2-redirected lymphocytes. We showed that the absence of CD39 confers to HER-2-specific T cells a functional advantage in eliminating HER-2+ patient-derived organoids in vitro and in vivo. CONCLUSION: HER-2-specific CD39 disrupted engineered T cells are promising advanced medicinal products for primary and metastatic CRC.

Age-induced alterations of granulopoiesis generate atypical neutrophils that aggravate stroke pathology.

Aging accounts for increased risk and dismal outcome of ischemic stroke. Here, we investigated the impact of age-related changes in the immune system on stroke. Upon experimental stroke, compared with young mice, aged mice had increased neutrophil clogging of the ischemic brain microcirculation, leading to worse no-reflow and outcomes. Aged mice showed an enhanced granulopoietic response to stroke that led to the accumulation of CD101+CD62Llo mature and CD177hiCD101loCD62Llo and CD177loCD101loCD62Lhi immature atypical neutrophils in the blood, endowed with increased oxidative stress, phagocytosis and procoagulant features. Production of CXCL3 by CD62Llo neutrophils of the aged had a key role in the development and pathogenicity of aging-associated neutrophils. Hematopoietic stem cell rejuvenation reverted aging-associated neutropoiesis and improved stroke outcome. In elderly patients with ischemic stroke, single-cell proteome profile of blood leukocytes identified CD62Llo neutrophil subsets associated with worse reperfusion and outcome. Our results unveil how stroke in aging leads to a dysregulated emergency granulopoiesis impacting neurological outcome.

Sarcopenia and Patient's Body Composition: New Morphometric Tools to Predict Clinical Outcome After Ivor Lewis Esophagectomy: a Multicenter Study.

BACKGROUND: The impact of preoperative body composition as independent predictor of prognosis for esophageal cancer patients after esophagectomy is still unclear. The aim of the study was to explore such a relationship. METHODS: This is a multicenter retrospective study from a prospectively maintained database. We enrolled consecutive patients who underwent Ivor-Lewis esophagectomy in four Italian high-volume centers from May 2014. Body composition parameters including total abdominal muscle area (TAMA), visceral fat area (VFA), and subcutaneous fat area (SFA) were determined based on CT images. Perioperative variables were systematically collected. RESULTS: After exclusions, 223 patients were enrolled and 24.2% had anastomotic leak (AL). Sixty-eight percent of patients were sarcopenic and were found to be more vulnerable in terms of postoperative 90-day mortality (p = 0.028). VFA/TAMA and VFA/SFA ratios demonstrated a linear correlation with the Clavien-Dindo classification (R = 0.311 and 0.239, respectively); patients with anastomotic leak (AL) had significantly higher VFA/TAMA (3.56 ± 1.86 vs. 2.75 ± 1.83, p = 0.003) and VFA/SFA (1.18 ± 0.68 vs. 0.87 ± 0.54, p = 0.002) ratios. No significant correlation was found between preoperative BMI and subsequent AL development (p = 0.159). Charlson comorbidity index correlated significantly with AL (p = 0.008): these patients had a significantly higher index (≥ 5). CONCLUSION: Analytical morphometric assessment represents a useful non-invasive tool for preoperative risk stratification. The concurrent association of sarcopenia and visceral obesity seems to be the best predictor of AL, far better than simple BMI evaluation, and potentially modifiable if targeted with prehabilitation programs.

Neural stem cell transplantation in patients with progressive multiple sclerosis: an open-label, phase 1 study.

Innovative pro-regenerative treatment strategies for progressive multiple sclerosis (PMS), combining neuroprotection and immunomodulation, represent an unmet need. Neural precursor cells (NPCs) transplanted in animal models of multiple sclerosis have shown preclinical efficacy by promoting neuroprotection and remyelination by releasing molecules sustaining trophic support and neural plasticity. Here we present the results of STEMS, a prospective, therapeutic exploratory, non-randomized, open-label, single-dose-finding phase 1 clinical trial ( NCT03269071 , EudraCT 2016-002020-86), performed at San Raffaele Hospital in Milan, Italy, evaluating the feasibility, safety and tolerability of intrathecally transplanted human fetal NPCs (hfNPCs) in 12 patients with PMS (with evidence of disease progression, Expanded Disability Status Scale ≥6.5, age 18-55 years, disease duration 2-20 years, without any alternative approved therapy). The safety primary outcome was reached, with no severe adverse reactions related to hfNPCs at 2-year follow-up, clearly demonstrating that hfNPC therapy in PMS is feasible, safe and tolerable. Exploratory secondary analyses showed a lower rate of brain atrophy in patients receiving the highest dosage of hfNPCs and increased cerebrospinal fluid levels of anti-inflammatory and neuroprotective molecules. Although preliminary, these results support the rationale and value of future clinical studies with the highest dose of hfNPCs in a larger cohort of patients.

Ceruloplasmin-Deficient Mice Show Dysregulation of Lipid Metabolism in Liver and Adipose Tissue Reduced by a Protein Replacement.

Ceruloplasmin is a ferroxidase that plays a role in iron homeostasis; its deficiency fosters inter alia iron accumulation in the liver, which expresses the soluble form of the protein secreted into the bloodstream. Ceruloplasmin is also secreted by the adipose tissue, but its role in adipocytes has been poorly investigated. We hypothesized that ceruloplasmin might have a role in iron/lipid interplay. We investigated iron/lipid dysmetabolism in the liver and adipose tissue of the ceruloplasmin-deficient mouse (CpKO) model of aceruloplasminemia and evaluated the effectiveness of ceruloplasmin replacement. We found that CpKO mice were overweight, showing adipose tissue accumulation, liver iron deposition and steatosis. In the adipose tissue of CpKO mice, iron homeostasis was not altered. Conversely, the levels of adiponectin and leptin adipokines behaved opposite to the wild-type. Increased macrophage infiltration was observed in adipose tissue and liver of CpKO mice, indicating tissue inflammation. The treatment of CpKO mice with ceruloplasmin limited liver iron accumulation and steatosis without normalizing the expression of iron homeostasis-related proteins. In the CpKO mice, the protein replacement limited macrophage infiltration in both adipose and hepatic tissues reduced the level of serum triglycerides, and partially recovered adipokines levels in the adipose tissue. These results underline the link between iron and lipid dysmetabolism in ceruloplasmin-deficient mice, suggesting that ceruloplasmin in adipose tissue has an anti-inflammatory role rather than a role in iron homeostasis. Furthermore, these data also indicate that ceruloplasmin replacement therapy may be effective at a systemic level.

Neural precursor cells tune striatal connectivity through the release of IGFBPL1.

The adult brain retains over life endogenous neural stem/precursor cells (eNPCs) within the subventricular zone (SVZ). Whether or not these cells exert physiological functions is still unclear. In the present work, we provide evidence that SVZ-eNPCs tune structural, electrophysiological, and behavioural aspects of striatal function via secretion of insulin-like growth factor binding protein-like 1 (IGFBPL1). In mice, selective ablation of SVZ-eNPCs or selective abrogation of IGFBPL1 determined an impairment of striatal medium spiny neuron morphology, a higher failure rate in GABAergic transmission mediated by fast-spiking interneurons, and striatum-related behavioural dysfunctions. We also found IGFBPL1 expression in the human SVZ, foetal and induced-pluripotent stem cell-derived NPCs. Finally, we found a significant correlation between SVZ damage, reduction of striatum volume, and impairment of information processing speed in neurological patients. Our results highlight the physiological role of adult SVZ-eNPCs in supporting cognitive functions by regulating striatal neuronal activity.

Combined unsupervised and semi-automated supervised analysis of flow cytometry data reveals cellular fingerprint associated with newly diagnosed pediatric type 1 diabetes.

An unbiased and replicable profiling of type 1 diabetes (T1D)-specific circulating immunome at disease onset has yet to be identified due to experimental and patient selection limitations. Multicolor flow cytometry was performed on whole blood from a pediatric cohort of 107 patients with new-onset T1D, 85 relatives of T1D patients with 0-1 islet autoantibodies (pre-T1D_LR), 58 patients with celiac disease or autoimmune thyroiditis (CD_THY) and 76 healthy controls (HC). Unsupervised clustering of flow cytometry data, validated by a semi-automated gating strategy, confirmed previous findings showing selective increase of naïve CD4 T cells and plasmacytoid DCs, and revealed a decrease in CD56brightNK cells in T1D. Furthermore, a non-selective decrease of CD3+CD56+ regulatory T cells was observed in T1D. The frequency of naïve CD4 T cells at disease onset was associated with partial remission, while it was found unaltered in the pre-symptomatic stages of the disease. Thanks to a broad cohort of pediatric individuals and the implementation of unbiased approaches for the analysis of flow cytometry data, here we determined the circulating immune fingerprint of newly diagnosed pediatric T1D and provide a reference dataset to be exploited for validation or discovery purposes to unravel the pathogenesis of T1D.

Continuous sensing of IFNα by hepatic endothelial cells shapes a vascular antimetastatic barrier.

Hepatic metastases are a poor prognostic factor of colorectal carcinoma (CRC) and new strategies to reduce the risk of liver CRC colonization are highly needed. Herein, we used mouse models of hepatic metastatization to demonstrate that the continuous infusion of therapeutic doses of interferon-alpha (IFNα) controls CRC invasion by acting on hepatic endothelial cells (HECs). Mechanistically, IFNα promoted the development of a vascular antimetastatic niche characterized by liver sinusoidal endothelial cells (LSECs) defenestration extracellular matrix and glycocalyx deposition, thus strengthening the liver vascular barrier impairing CRC trans-sinusoidal migration, without requiring a direct action on tumor cells, hepatic stellate cells, hepatocytes, or liver dendritic cells (DCs), Kupffer cells (KCs) and liver capsular macrophages (LCMs). Moreover, IFNα endowed LSECs with efficient cross-priming potential that, along with the early intravascular tumor burden reduction, supported the generation of antitumor CD8+ T cells and ultimately led to the establishment of a protective long-term memory T cell response. These findings provide a rationale for the use of continuous IFNα therapy in perioperative settings to reduce CRC metastatic spreading to the liver.

Colorectal cancer remains one of the most widespread and deadly cancers worldwide. Poor health outcomes are usually linked to diseased cells spreading from the intestine to create new tumors in the liver or other parts of the body. Treatment involves surgically removing the initial tumors in the bowel, but patient survival could be improved if, in parallel, their immune system was 'boosted' to destroy cancer cells before they can form other tumors. Interferon alpha is a small protein which helps to coordinate how the immune system recognizes and deactivates foreign agents and cancerous cells. It has recently been trialed as a colorectal cancer treatment to prevent tumors from spreading to the liver, but only with limited success. This partly because interferon-alpha is usually administered in high and pulsed doses, which cause severe side effects through the body. Instead, Tran, Ferreira, Alvarez-Moya et al. aimed to investigate whether continuously delivering lower amounts of the drug could be a better approach. This strategy was tested on mice in which colorectal cancer cells had been implanted into the wall of the large intestine. Continuous administration minimized the risk of the implanted cancer cells spreading to the liver while also creating fewer side effects. The team was able to identify an optimum delivery strategy by varying how much interferon-alpha the animals received and when. Further experiments also revealed a new mechanism by which interferon-alpha prevented the spread of colorectal cancer. Upon receiving continuous doses of the drug, a group of liver cells started to generate a physical barrier which stopped cancer cells from being able to invade the organ. The treatment also promoted long-term immune responses that targeted diseased cells while being safe for healthy tissues. If confirmed in clinical trials, these results suggest that colorectal patients undergoing tumor removal surgery may benefit from also receiving interferon-alpha through continuous delivery.

Radiomic and gEnomic approaches for the enhanced DIagnosis of clear cell REnal Cancer (REDIRECt): a translational pilot methodological study.

Background: The combination of radiomic and transcriptomic approaches for patients diagnosed with small clear-cell renal cell carcinoma (ccRCC) might improve decision making. In this pilot and methodological study, we investigate whether imaging features obtained from computed tomography (CT) may correlate with gene expression patterns in ccRCC patients. Methods: Samples from 6 patients who underwent partial nephrectomy for unilateral non-metastatic ccRCC were included in this pilot cohort. Transcriptomic analysis was conducted through RNA-sequencing on tumor samples, while radiologic features were obtained from pre-operative 4-phase contrast-enhanced CT. To evaluate the heterogeneity of the transcriptome, after a 1,000 re-sampling via bootstrapping, a first Principal Component Analyses (PCA) were fitted with all transcripts and a second ones with transcripts deriving from a list of 369 genes known to be associated with ccRCC from The Cancer Genome Atlas (TCGA). Significant pathways in each Principal Components for the 50 genes with the highest loadings absolute values were assessed with pathways enrichment analysis. In addition, Pearson's correlation coefficients among radiomic features themselves and between radiomic features and transcripts expression values were computed. Results: The transcriptomes of the analysed samples showed a high grade of heterogeneity. However, we found four radiogenomic patterns, in which the correlation between radiomic features and transcripts were statistically significant. Conclusions: We showed that radiogenomic approach is feasible, however its clinical meaning should be further investigated.

Machine learning approaches for prediction of bipolar disorder based on biological, clinical and neuropsychological markers: A systematic review and meta-analysis.

Applying machine learning (ML) to objective markers may overcome prognosis uncertainty due to the subjective nature of the diagnosis of bipolar disorder (BD). This PRISMA-compliant meta-analysis provides new systematic evidence of the BD classification accuracy reached by different markers and ML algorithms. We focused on neuroimaging, electrophysiological techniques, peripheral biomarkers, genetic data, neuropsychological or clinical measures, and multimodal approaches. PubMed, Embase and Scopus were searched through 3rd December 2020. Meta-analyses were performed using random-effect models. Overall, 81 studies were included in this systematic review and 65 in the meta-analysis (11,336 participants, 3903 BD). The overall pooled classification accuracy was 0.77 (95%CI[0.75;0.80]). Despite subgroup analyses for diagnostic comparison group, psychiatric disorders, marker, ML algorithm, and validation procedure were not significant, linear discriminant analysis significantly outperformed support vector machine for peripheral biomarkers (p = 0.03). Sample size was inversely related to accuracy. Evidence of publication bias was detected. Ultimately, although ML reached a high accuracy in differentiating BD from other psychiatric disorders, best practices in methodology are needed for the advancement of future studies.

Disrupting N-glycan expression on tumor cells boosts chimeric antigen receptor T cell efficacy against solid malignancies.

Immunotherapy with chimeric antigen receptor (CAR)­engineered T cells showed exceptional successes in patients with refractory B cell malignancies. However, first-in-human studies in solid tumors revealed unique hurdles contributing to poor demonstration of efficacy. Understanding the determinants of tumor recognition by CAR T cells should translate into the design of strategies that can overcome resistance. Here, we show that multiple carcinomas express extracellular N-glycans, whose abundance negatively correlates with CAR T cell killing. By knocking out mannoside acetyl-glucosaminyltransferase 5 (MGAT5) in pancreatic adenocarcinoma (PAC), we showed that N-glycans protect tumors from CAR T cell killing by interfering with proper immunological synapse formation and reducing transcriptional activation, cytokine production, and cytotoxicity. To overcome this barrier, we exploited the high metabolic demand of tumors to safely inhibit N-glycans synthesis with the glucose/mannose analog 2-deoxy-d-glucose (2DG). Treatment with 2DG disrupts the N-glycan cover on tumor cells and results in enhanced CAR T cell activity in different xenograft mouse models of PAC. Moreover, 2DG treatment interferes with the PD-1­PD-L1 axis and results in a reduced exhaustion profile of tumor-infiltrating CAR T cells in vivo. The combined 2DG and CAR T cell therapy was successful against multiple carcinomas besides PAC, including those arising from the lung, ovary, and bladder, and with different clinically relevant CAR specificities, such as CD44v6 and CEA. Overall, our results indicate that tumor N-glycosylation regulates the quality and magnitude of CAR T cell responses, paving the way for the rational design of improved therapies against solid malignancies.

Circulating CXCL10 and IL-6 in solid organ donors after brain death predict graft outcomes.

We tested the hypothesis that circulating CXCL10 and IL-6 in donor after brain death provide independent additional predictors of graft outcome. From January 1, 2010 to June 30, 2012 all donors after brain death managed by the NITp (n = 1100) were prospectively included in this study. CXCL10 and IL-6 were measured on serum collected for the crossmatch at the beginning of the observation period. Graft outcome in recipients who received kidney (n = 1325, follow-up 4.9 years), liver (n = 815, follow-up 4.3 years) and heart (n = 272, follow-up 5 years) was evaluated. Both CXCL-10 and IL-6 showed increased concentration in donors after brain death. The intensive care unit stay, the hemodynamic instability, the cause of death, the presence of risk factors for cardiovascular disease and the presence of ongoing infection resulted as significant determinants of IL-6 and CXCL10 donor concentrations. Both cytokines resulted as independent predictors of Immediate Graft Function. Donor IL-6 or CXCL10 were associated with graft failure after liver transplant, and acted as predictors of recipient survival after kidney, liver and heart transplantation. Serum donor IL-6 and CXCL10 concentration can provide independent incremental prediction of graft outcome among recipients followed according to standard clinical practice.

Transcriptional dynamics of induced pluripotent stem cell differentiation into ß cells reveals full endodermal commitment and homology with human islets.

BACKGROUND AIMS: Induced pluripotent stem cells (iPSCs) have the capacity to generate ß cells in vitro, but the differentiation is incomplete and generates a variable percentage of off-target cells. Single-cell RNA sequencing offers the possibility of characterizing the transcriptional dynamics throughout differentiation and determining the identity of the final differentiation product. METHODS: Single-cell transcriptomics data were obtained from four stages across differentiation of iPSCs into ß cells and from human donor islets. RESULTS: Clustering analysis revealed that iPSCs undertake a full endoderm commitment, and the obtained endocrine pancreatic cells have high homology with mature islets. The iPSC-derived ß cells were devoid of pluripotent residual cells, and the differentiation was pancreas-specific, as it did not generate ectodermal or mesodermal cells. Pseudotime trajectory identified a dichotomic endocrine/non-endocrine cell fate and distinct subgroups in the endocrine branch. CONCLUSIONS: Future efforts to produce ß cells from iPSCs must aim not only to improve the resulting endocrine cell but also to avoid differentiation into non-pancreatic endoderm cells.

Chest CT in the emergency department for suspected COVID-19 pneumonia.

PURPOSE: In overwhelmed emergency departments (EDs) facing COVID-19 outbreak, a swift diagnosis is imperative. CT role was widely debated for its limited specificity. Here we report the diagnostic role of CT in two EDs in Lombardy, epicenter of Italian outbreak. MATERIAL AND METHODS: Admitting chest CT from 142 consecutive patients with suspected COVID-19 were retrospectively analyzed. CT scans were classified in "highly likely," "likely," and "unlikely" COVID-19 pneumonia according to the presence of typical, indeterminate, and atypical findings, or "negative" in the absence of findings, or "alternative diagnosis" when a different diagnosis was found. Nasopharyngeal swab results, turnaround time, and time to positive results were collected. CT diagnostic performances were assessed considering RT-PCR as reference standard. RESULTS: Most of cases (96/142, 68%) were classified as "highly likely" COVID-19 pneumonia. Ten (7%) and seven (5%) patients were classified as "likely" and "unlikely" COVID-19 pneumonia, respectively. In 21 (15%) patients a differential diagnosis was provided, including typical pneumonia, pulmonary edema, neoplasia, and pulmonary embolism. CT was negative in 8/142 (6%) patients. Mean turnaround time for the first COVID-19 RT-PCR was 30 ± 13 h. CT diagnostic accuracy in respect of the first test swab was 79% and increased to 91.5% after repeated swabs and/or BAL, for 18 false-negative first swab. CT performance was good with 76% specificity, 99% sensitivity, 90% positive predictive value and 97% negative predictive value. CONCLUSION: Chest CT was useful to streamline patients' triage while waiting for RT-PCR in the ED, supporting the clinical suspicion of COVID-19 or providing alternative diagnosis.

Patrolling Monocytes Are Recruited and Activated by Diabetes to Protect Retinal Microvessels.

In diabetes there is a long latency between the onset of hyperglycemia and the appearance of structural microangiopathy. Because Ly6Clow patrolling monocytes (PMo) behave as housekeepers of the vasculature, we tested whether PMo protect microvessels against diabetes. We found that in wild-type mice, diabetes reduced PMo in the general circulation but increased by fourfold the absolute number of PMo adherent to retinal vessels (leukostasis). Conversely, in diabetic NR4A1-/- mice, a model of absence of PMo, there was no increase in leukostasis, and at 6 months of diabetes, the number of retinal acellular capillaries almost doubled compared with diabetic wild-type mice. Circulating PMo showed gene expression changes indicative of enhanced migratory, vasculoprotective, and housekeeping activities, as well as profound suppression of genes related to inflammation and apoptosis. Promigratory CXCR4 was no longer upregulated at longer duration when retinal acellular capillaries begin to increase. Thus, after a short diabetes duration, PMo are the cells preferentially recruited to the retinal vessels and protect vessels from diabetic damage. These observations support the need for reinterpretation of the functional meaning of leukostasis in diabetes and document within the natural history of diabetic retinopathy processes of protection and repair that can provide novel paradigms for prevention.