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Mounting evidence suggests that storage has an impact on extracellular vesicles (EVs) properties. While −80°C storage is a widespread approach, some authors proposed improved storage strategies with conflicting results. Here, we designed a systematic study to assess the impact of −80°C storage and freeze‐thaw cycles on EVs. We tested the differences among eight storage strategies and investigated the possible fusion phenomena occurring during storage. EVs were collected from human plasma and murine microglia culture by size exclusion chromatography and ultracentrifugation, respectively. The analysis included: concentration, size and zeta potential (tunable resistive pulse sensing), contaminant protein assessment; flow cytometry for the analysis of two single fluorescent‐tagged EVs populations (GFP and mCherry), mixed before preservation. We found that −80°C storage reduces EVs concentration and sample purity in a time‐dependent manner. Furthermore, it increases the particle size and size variability and modifies EVs zeta potential, with a shift of EVs in size‐charge plots. None of the tested conditions prevented the observed effects. Freeze‐thaw cycles lead to an EVs reduction after the first cycle and to a cycle‐dependent increase in particle size. With flow cytometry, after storage, we observed a significant population of double‐positive EVs (GFP+‐mCherry+). This observation may suggest the occurrence of fusion phenomena during storage. Our findings show a significant impact of storage on EVs samples in terms of particle loss, purity reduction and fusion phenomena leading to artefactual particles. Depending on downstream analyses and experimental settings, EVs should probably be processed from fresh, non‐archival, samples in majority of cases.

Background and aimsPatients infected with SARS-CoV-2 range from asymptomatic, to mild, moderate or severe disease evolution including fatal outcome. Thus, early predictors of clinical outcome are highly needed. We investigated markers of neural tissue damage as a possible early sign of multisystem involvement to assess their clinical prognostic value on survival or transfer to intensive care unit (ICU). MethodsWe collected blood from 104 patients infected with SARS-CoV-2 the day of admission to the emergency room and measured blood neurofilament light chair (NfL), glial fibrillary acidic protein (GFAP), ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), and total tau protein levels.ResultsWe found that NfL, GFAP, and tau were significantly increased in patients with fatal outcome, while NfL and UCH-L1 in those needing ICU transfer. ROC and Kaplan–Meier curves indicated that total tau levels at admission accurately predict mortality.ConclusionsBlood neural markers may provide additional prognostic value to conventional biomarkers used to predict COVID-19 outcome.

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 CD56 bright NK 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.

AimsAlterations of the exocrine pancreas have been reported in type 1 diabetes, but their contribution to the pathogenesis of the disease is poorly understood. Here, we investigated markers of exocrine pancreas dysfunction in individuals at-risk of developing type 1 diabetes.MethodsSerum P-amylase and lipase levels were assessed in samples obtained from healthy controls, patients with new onset type 1 diabetes, relatives participating to the TrialNet Pathway to Prevention who were, at blood collection, autoantibody negative or positive for a single autoantibody (low-risk individuals), and positive for multiple autoantibodies (high-risk individuals). Linear mixed models were adopted to estimate variation of pancreatic enzymes among the groups and to evaluate the influence of high-risk HLA genotypes and residual beta cell function on exocrine pancreas function.ResultsIn adults, but not children, reduced levels of P-amylase and lipase were shown in at-risk individuals, including (for P-amylase levels only) those at low-risk, and in T1Dnew. Furthermore, while high-risk HLA genotypes negatively affected P-amylase levels in autoantibody negative adult individuals, fasting C-peptide levels did not correlate with pancreatic enzyme levels.ConclusionsExocrine pancreas dysfunction precedes the onset of type 1 diabetes in adult at-risk individuals and may be unrelated to fasting C-peptide levels.

Type 1 diabetes (T1D) is a chronic autoimmune disease resulting in progressive destruction of β-cells. Several factors affecting lymphocyte and antigen-presenting cells, including dendritic cells (DCs), contribute to defective maintenance of tolerance in T1D. DC-10 are a subset of human DCs involved in IL-10-mediated tolerance. A precise monitoring of DC-10 in the peripheral blood is possible thanks to the discovery of specific biomarkers. DC-10, being cells that naturally express HLA-G, may be used for the appropriate staging of the disease. By enumerating and phenotypically characterizing DC-10 in the peripheral blood of subjects at different stages of T1D development—first-degree relatives (FDRs) of T1D patients, without (Ab neg ) or with (Ab pos ) autoantibodies, T1D patients at onset, and age-matched healthy controls (HCs)—we showed that DC-10 contain a high proportion of HLA-G-expressing cells as compared with monocytes. We reported that a low frequency of DC-10 during disease development is paralleled with the increased proportion of pro-inflammatory cDC2 cells. Moreover, DC-10 number and phenotype differ from Ab neg FDRs, Ab pos FDRs, and T1D patients compared with HCs, and DC-10 from T1D patients express low levels of CD83. Finally, multiple regression analysis, considering DC-10 and HLA-G-related parameters, showed that Ab neg FDRs are more similar to subjects with autoimmunity than to HCs. This is the first demonstration that impairment in DC-10 number and phenotype, specifically CD83 expression, is associated with risk of developing T1D, suggesting a possible use of CD83 + DC-10 to stratify individuals at risk of T1D in conjunction with classical prognostic factors.

Background Alzheimer’s disease (AD) affects the global quality of life of persons who suffer from it and their caregivers, because of the behavioral and psychological consequences associated with the pathology and its caring. The Alzheimer Café (AC) is one example of approach aimed to help persons and caregivers deal with their disease. Aim This is a pilot study focusing on the efficacy of AC in relieving caregivers’ and persons’ burdens due to dementia. Methods The quality of life of both caregivers and persons who attended the AC was compared with the quality of life of those who did not. Basic and instrumental daily activities and neuropsychiatric functioning were assessed. Caregivers also answered to general well-being and caregiving burden questionnaires. The evaluation took place at the beginning of the intervention and after 1, 3, 6, 9 and 12 months. Results Caregivers who joined the AC with their persons with dementia showed to have significantly benefited in the daily care of persons with dementia, in terms of total well-being, vitality, and emotional burden. Discussion Although improvements were not observed in persons with dementia who attended the AC, significant benefits were reported by their caregivers, suggesting that the intervention may produce better management of social and economic problems and lead to better emotional support. Conclusions The AC seems to help families of AD persons to better manage the disease, and also delay the institutionalization of these persons, which is certainly an ambitious goal for an incurable disorder such as Alzheimer’s disease.

Background Type 1 Diabetes (T1D) exhibits considerable heterogeneity, impacting prediction, prevention, diagnosis, and treatment. Precision medicine aims to tailor treatments using ‘endotypes’—subtypes of disease with distinct pathophysiological mechanisms. However, proposed endotypes often lack mechanistic associations with clinical outcomes for accurately identifying T1D cases. Methods This study introduces an approach leveraging the multi-omics factor analysis (MOFA) strategy, a computational method for unsupervised integration analysis, to explore endotypes. Analyzing data from 146 new-onset children with T1D (54 females, 92 males; age range 5–18 years), including circulating immunome, transcriptome, and serum metabolic hormones, we identify 12 factors explaining variability across the three data sets. Results Here we find no associations, either direct or through clustering, between these 12 factors and clinical parameters, genetic predisposition, or disease outcome. These results suggest that a combination of clinical phenotypes might be responsible for the differences across T1D cases. Conclusions These findings challenge the assumption that T1D heterogeneity reflects diverse developmental mechanisms. These results add to the ongoing debate on endotypes and carry important implications for clinical trial design—particularly in how treatments are evaluated for their effectiveness across broad and diverse patient populations. Codazzi et al. employ multi-omics factor analysis, integrating immunome, transcriptome, and serum metabolic hormone data, to identify potential subtypes of pediatric Type 1 Diabetes. These findings reveal no significant associations, suggesting that blood-derived omics data may not suffice to identify distinct subtypes of Type 1 Diabetes. Plain Language Summary This study seeks to understand why type 1 diabetes (T1D) affects individuals differently and whether specific subtypes of the disease, known as “endotypes,” can explain this variability to inform personalized treatment strategies. We analyze data from 146 children newly diagnosed with T1D, examining immune activity, gene expression, and hormone levels. We identify 12 factors that account for some variability among patients but find no clear associations between these factors and disease outcomes or genetic risk. These findings suggest that T1D is shaped by a complex interplay of factors rather than by distinct underlying mechanisms, highlighting the need for a more refined understanding of disease heterogeneity to guide effective treatments and the design of future clinical trials.

IntroductionAim of this study was to investigate the pancreatic exocrine function in patients with type 1 diabetes (T1D) by multiple non-invasive tests.Research design and methodsThe study is a single-center, cross-sectional study of pancreatic exocrine function in adult patients with new-onset or long-standing T1D and healthy controls.ResultsHealthy controls, new-onset T1D, and long-standing T1D were similar for age at the time of the study, gender and body mass index (BMI) categories. Age of onset of T1D patients with long-standing disease was younger than that of patients with new-onset T1D (p<0.001). As expected, the three groups differed for C-peptide and hemoglobin A1c (HbA1c) levels. Lipase activity measured by 13C-mixed triglyceride breath test was reduced progressively, although not significantly, from controls to recent-onset T1D and long-standing T1D participants. Fecal elastase-1 was significantly lower in participants with T1D, either new onset or long standing. Pancreatic amylase, lipase, retinol binding protein and prealbumin were significantly different across the groups, with a significant trend toward lower values in long-standing T1D and intermediate values in new-onset T1D, while no differences were observed for total amylase. The markers of impaired exocrine function tests (fecal elastase-1, serum pancreatic amylase and lipase) and of nutritional status (retinol binding protein and prealbumin levels) correlated with the reduction of fasting and urinary C-peptide.ConclusionsOur results confirm that exocrine pancreatic impairment is a feature of T1D, with low fecal elastase-1, serum pancreatic amylase and lipase as specific markers, associated with reduced levels of nutritional indexes. Moreover, the evidence of more advanced insufficiency in long-standing disease reflects the chronic nature of this process, and its correlation with the residual β-cell function suggests parallel pathways for the impairment of the endocrine and exocrine pancreatic function.

Background Here, we isolated, expanded and functionally characterized regulatory T cells (Tregs) from patients with end stage kidney and liver disease, waiting for kidney/liver transplantation (KT/LT), with the aim to establish a suitable method to obtain large numbers of immunomodulatory cells for adoptive immunotherapy post-transplantation. Methods We first established a preclinical protocol for expansion/isolation of Tregs from peripheral blood of LT/KT patients. We then scaled up and optimized such protocol according to good manufacturing practice (GMP) to obtain high numbers of purified Tregs which were phenotypically and functionally characterized in vitro and in vivo in a xenogeneic acute graft-versus-host disease (aGVHD) mouse model. Specifically, immunodepressed mice (NOD-SCID-gamma KO mice) received human effector T cells with or without GMP-produced Tregs to prevent the onset of xenogeneic GVHD. Results Our small scale Treg isolation/expansion protocol generated functional Tregs. Interestingly, cryopreservation/thawing did not impair phenotype/function and DNA methylation pattern of FOXP3 gene of the expanded Tregs. Fully functional Tregs were also isolated/expanded from KT and LT patients according to GMP. In the mouse model, GMP Tregs from LT or KT patient proved to be safe and show a trend toward reduced lethality of acute GVHD. Conclusions These data demonstrate that expanded/thawed GMP-Tregs from patients with end-stage organ disease are fully functional in vitro. Moreover, their infusion is safe and results in a trend toward reduced lethality of acute GVHD in vivo, further supporting Tregs-based adoptive immunotherapy in solid organ transplantation.

Transient partial remission, a period of low insulin requirement experienced by most patients soon after diagnosis, has been associated with mechanisms of immune regulation. A better understanding of such natural mechanisms of immune regulation might identify new targets for immunotherapies that reverse type 1 diabetes (T1D). In this study, using Cox model multivariate analysis, we validated our previous findings that patients with the highest frequency of CD4 + CD25 + CD127 hi (127-hi) cells at diagnosis experience the longest partial remission, and we showed that the 127-hi cell population is a mix of Th1- and Th2-type cells, with a significant bias toward antiinflammatory Th2-type cells. In addition, we extended these findings to show that patients with the highest frequency of 127-hi cells at diagnosis were significantly more likely to maintain β cell function. Moreover, in patients treated with alefacept in the T1DAL clinical trial, the probability of responding favorably to the antiinflammatory drug was significantly higher in those with a higher frequency of 127-hi cells at diagnosis than those with a lower 127-hi cell frequency. These data are consistent with the hypothesis that 127-hi cells maintain an antiinflammatory environment that is permissive for partial remission, β cell survival, and response to antiinflammatory immunotherapy.