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Moral distress (MD) in healthcare providers is widely recognized as a serious issue in critical care contexts. It has the potential to have negative impacts on both personal and professional wellbeing, the quality of care provided and staff turnover. The aim of this study was to investigate the relationship between MD and burnout among neonatal intensive care unit (NICU) healthcare professionals and identify the possible factors associated with its occurrence. Participants were asked to complete an online survey, which covered sociodemographic and professional information and included two self-report questionnaires (Italian Moral Distress Scale-Revised and Maslach Burnout Inventory). The sample comprised 115 healthcare providers (nurses and physiotherapists: 66.1%; physicians: 30.4%; healthcare assistants: 3.5%) working in four NICUs located within the province of Turin, Italy. The results revealed overall low levels of MD, with no significant differences between nurses/physiotherapists and physicians. Nurses/physiotherapists showed a statistically significant higher percentage of personal accomplishment burnout (32.9%) compared with physicians (8.6%; p = 0.012). MD was associated with the emotional exhaustion dimension of burnout. Spirituality and/or religiousness was shown to be a moderating variable. Further research is needed to deepen our understanding of the correlation between MD and burnout and the role of spirituality and/or religiousness as moderators.

Regulatory T cells (Tregs) play fundamental roles in maintaining peripheral tolerance to prevent autoimmunity and limit legitimate immune responses, a feature hijacked in tumor microenvironments in which the recruitment of Tregs often extinguishes immune surveillance through suppression of T-effector cell signaling and tumor cell killing. The pharmacological tuning of Treg activity without impacting on T conventional (Tconv) cell activity would likely be beneficial in the treatment of various human pathologies. PIP4K2A, 2B, and 2C constitute a family of lipid kinases that phosphorylate PtdIns5P to PtdIns(4,5)P2. They are involved in stress signaling, act as synthetic lethal targets in p53-null tumors, and in mice, the loss of PIP4K2C leads to late onset hyperinflammation. Accordingly, a human single nucleotide polymorphism (SNP) near the PIP4K2C gene is linkedwith susceptibility to autoimmune diseases. How PIP4Ks impact on human T cell signaling is not known. Using ex vivo human primary T cells, we found that PIP4K activity is required for Treg cell signaling and immunosuppressive activity. Genetic and pharmacological inhibition of PIP4K in Tregs reduces signaling through the PI3K, mTORC1/S6, and MAPK pathways, impairs cell proliferation, and increases activation-induced cell death while sparing Tconv. PIP4K and PI3K signaling regulate the expression of the Treg master transcriptional activator FOXP3 and the epigenetic signaling protein Ubiquitin-like containing PHD and RING finger domains 1 (UHRF1). Our studies suggest that the pharmacological inhibition of PIP4K can reprogram human Treg identity while leaving Tconv cell signaling and T-helper differentiation to largely intact potentially enhancing overall immunological activity.

The immune system is a complex network that acts to protect vertebrates from foreign microorganisms and carries out immunosurveillance to combat cancer. In order to avoid hyper-activation of the immune system leading to collateral damage tissues and organs and to prevent self-attack, the network has the intrinsic control mechanisms that negatively regulate immune responses. Central to this negative regulation are regulatory T (T-Reg) cells, which through cytokine secretion and cell interaction limit uncontrolled clonal expansion and functions of activated immune cells. Given that positive or negative manipulation of T-Regs activity could be utilised to therapeutically treat host versus graft rejection or cancer respectively, understanding how signaling pathways impact on T-Regs function should reveal potential targets with which to intervene. The phosphatidylinositol-3-kinase (PI3K) pathway controls a vast array of cellular processes and is critical in T cell activation. Here we focus on phosphoinositide 3-kinases (PI3Ks) and their ability to regulate T-Regs cell differentiation and function.

Background: In the current COVID-19 pandemic caused by the SARS-Coronavirus-2 (SARS-CoV-2) it is important to understand if breast milk may be a vehicle of infection. The possible transmission of the virus via breastfeeding is still largely unexplored. Methods: This is a prospective collaborative observational study where samples of human milk from 12 breastfeeding mothers positive for SARS-Coronavirus 2 were collected. Search for the viral RNA in breast milk samples was performed by real-time PCR methodology. All the newborns underwent a clinical follow up for the first month of life or until the finding of two sequential negative swabs. Findings: In eleven cases the search for SARS-CoV-2 RNA in milk samples resulted negative and in one case it was positive. Eleven of the twelve newborns were exclusively breastfed in the first month of life and closely monitored. Clinical outcome was uneventful. Four newborns tested positive for SARS-CoV-2 and were all detected in the first 48 hours of life, after the onset of maternal symptoms. Also the clinical course of these 4 infants, including the one who received mother’s milk positive for SARS-CoV-2, was uneventful, and all of them became SARS-CoV-2 negative within 6 weeks of life. Interpretations: Our study supports the view that SARS-CoV-2 positive mothers do not expose their newborns to additional risk of infection by breastfeeding. This is an important message that should be given to mothers and healthcare providers in this moment of uncertainty due to the COVID-19 pandemic. Clearly, the recommended hygiene measures for direct breastfeeding, as well as for milk expression when a mother and her infant need to be separated, must be carefully followed. Funding: This Study was supported by University of Turin (grant RILO2019 to EB and DL). Declaration of Interests: The authors declare no conflict of interest. Ethics Approval Statement: The study was approved by the local Ethical Committee (protocol number 0039684), and informed consent to participate in the study was obtained from each mother.

The immune system is a complex network that acts to protect vertebrates from foreign microorganisms and carries out immunosurveillance to combat cancer. In order to avoid hyper-activation of the immune system leading to collateral damage tissues and organs and to prevent self-attack, the network has the intrinsic control mechanisms that negatively regulate immune responses. Central to this negative regulation are regulatory T (T-Reg) cells, which through cytokine secretion and cell interaction limit uncontrolled clonal expansion and functions of activated immune cells. Given that positive or negative manipulation of T-Regs activity could be utilised to therapeutically treat host versus graft rejection or cancer respectively, understanding how signaling pathways impact on T-Regs function should reveal potential targets with which to intervene. The phosphatidylinositol-3-kinase (PI3K) pathway controls a vast array of cellular processes and is critical in T cell activation. Here we focus on phosphoinositide 3-kinases (PI3Ks) and their ability to regulate T-Regs cell differentiation and function.

Background: The adult mammalian heart retains residual regenerative capability via endogenous cardiac progenitor cell (CPC) activation and cardiomyocyte proliferation. We previously reported the paracrine cardioprotective capacity of human amniotic fluid-derived stem cells (hAFS) following ischemia or cardiotoxicity. Here we analyse the potential of hAFS secretome fractions for cardiac regeneration and future clinical translation.Methods: hAFS were isolated from amniotic fluid leftover samples from prenatal screening. hAFS conditioned medium (hAFS-CM) was obtained following hypoxic preconditioning. Anti-apoptotic, angiogenic and proliferative effects were evaluated on rodent neonatal cardiomyocytes (r/mNVCM), human endothelial colony forming cells (hECFC) and human CPC. Mice undergoing myocardial infarction (MI) were treated with hAFS-CM, hAFS-extracellular vesicles (hAFS-EV), or EV-depleted hAFS-CM(hAFS-DM) by single intra-myocardial administration and evaluated in the short and long term.Results: hAFS-CM improved mNVCM survival under oxidative and hypoxic damage, induced Ca2+-dependent angiogenesis in hECFC and triggered hCPC and rNVCM proliferation. hAFS-CM treatment after MI counteracted scarring, supported cardiac function, angiogenesis and cardiomyocyte cell cycle progression in the long term. hAFS-DM had no effect. hAFS-CM and hAFS-EV equally induced epicardium WT1+CPC reactivation. Although no CPC cardiovascular differentiation was observed, our data suggests contribution to local angiogenesis by paracrine modulation. hAFS-EV alone were able to recapitulate all the beneficial effects exerted by hAFS-CM, except for stimulation of vessel formation.Conclusions: hAFS-CM and hAFS-EV can improve cardiac repair and trigger cardiac regeneration via paracrine modulation of endogenous mechanisms. While both formulations are effective in sustaining myocardial renewal, hAFS-CM retains higher pro-angiogenic potential, while hAFS-EV particularly enhances cardiac function. (c) 2019 Elsevier B.V. All rights reserved.

Polyphosphoinositides (PPIns) are a family of seven lipid messengers that regulate a vast array of signalling pathways to control cell proliferation, migration, survival and differentiation. PPIns are differentially present in various sub-cellular compartments and, through the recruitment and regulation of specific proteins, are key regulators of compartment identity and function. Phosphoinositides and the enzymes that synthesise and degrade them are also present in the nuclear membrane and in nuclear membraneless compartments such as nuclear speckles. Here we discuss how PPIns in the nucleus are modulated in response to external cues and how they function to control downstream signalling. Finally we suggest a role for nuclear PPIns in liquid phase separations that are involved in the formation of membraneless compartments within the nucleus.

Objective: To investigate whether exposure to inhibitors of gastric acidity, such as H2 blockers or proton pump inhibitors, can independently increase the risk of infections in very low birth weight (VLBW) preterm infants in the neonatal intensive care unit. Study design: This is a secondary analysis of prospectively collected data from a multicenter, randomized controlled trial of bovine lactoferrin (BLF) supplementation (with or without the probiotic Lactobacillus rhamnosus GG) vs placebo in prevention of late-onset sepsis (LOS) and necrotizing enterocolitis (NEC) in preterm infants. Inhibitors of gastric acidity were used at the recommended dosages/schedules based on the clinical judgment of attending physicians. The distribution of days of inhibitors of gastric acidity exposure between infants with and without LOS/NEC was assessed. The mutually adjusted effects of birth weight, gestational age, duration of inhibitors of gastric acidity treatment, and exposure to BLF were controlled through multivariable logistic regression. Interaction between inhibitors of gastric acidity and BLF was tested; the effects of any day of inhibitors of gastric acidity exposure were then computed for BLF-treated vs -untreated infants. Results: Two hundred thirty-five of 743 infants underwent treatment with inhibitors of gastric acidity, and 86 LOS episodes occurred. After multivariate analysis, exposure to inhibitors of gastric acidity remained significantly and independently associated with LOS (OR, 1.03; 95% CI, 1.008-1.067; P = .01); each day of inhibitors of gastric acidity exposure conferred an additional 3.7% odds of developing LOS. Risk was significant for Gram-negative (P < .001) and fungal (P = .001) pathogens, but not for Gram-positive pathogens (P = .97). On the test for interaction, 1 additional day of exposure to inhibitors of gastric acidity conferred an additional 7.7% risk for LOS (P = .003) in BLF-untreated infants, compared with 1.2% (P = .58) in BLF-treated infants. Conclusion: Exposure to inhibitors of gastric acidity is significantly associated with the occurrence of LOS in preterm VLBW infants. Concomitant administration of BLF counteracts this selective disadvantage. Trial registration: isrctn.org: ISRCTN53107700.

Mutations in ribosomal proteins RPS19, RPS24 and RPS17 have been reported in Diamond-Blackfan Anemia (DBA), an autosomal dominant disease characterised by pure red cell aplasia. DBA is the prototype of ribosomapathies: a protein synthesis defect in a tissue with a high cellular turnover is considered the cause of the erythroid progenitor failure. We have created the Diamond-Blackfan Anemia mutation database to curate and record DBA gene mutations, together with their functional consequences and clinical phenotypes. This locus-specific resource is open to future submissions and is available online (http://www.dbagenes.unito.it). It is founded on the Leiden Open (source) Variation Database (LOVD) system and includes data from sequence and structure analysis tools, genomic database resources and published reports. It lists all identified variants and background genomic information. Phenotypic data are accessed by selecting a particular mutation. The database includes 219 unique variants of which 86 are disease-causing mutations. The database will be supplemented with other DBA genes as soon as they are reported and their mutations are identified and it should be of assistance to clinicians and investigators involved in DBA research and care.

Growing evidence suggests a possible relationship between migraine and thrombotic risk factors. The aim of this study was to analyze the possible relationship between migraine and acquired and genetic thrombophilia in a young population. We compared 16 migrainous adolescents, 12 children with tension-type headache, and controls in terms of frequencies of prothrombotic polymorphisms (factor V Leiden, C677T mutation of 5,10 methylenetetrahydrofolate reductase, G20210A mutation of prothrombin), platelet aggregability, anticoagulant antibodies, blood lipid pattern, serum folate and vitamin B12 levels, homocysteinemia, coagulation parameters, and family history for migraine and precocious thrombotic events. This study confirms the link between migraine and increased platelet responsiveness. Overall, 62.5% of migrainous patients carried at least three thrombophilic factors. Our preliminary data suggest that, in order to assess prevention strategies, it could be appropriate to perform a complete thrombophilia screening in young patients suffering from migraine and with a family history of thrombosis.

We describe the onset of cataract in early infancy in a family with hereditary hyperferritinaemia-cataract syndrome. The two probands presented with isolated hyperferritinaemia and had developed cataracts at the age of 18 months. Two members of their family with high ferritin levels (1270–1450 µg/l) had suffered from cataract since childhood. The mutation responsible was a 32G→C change in the lateral bulge of the stem structure of the iron responsive element of the L-ferritin subunit gene. Mutations at this level cause particularly high ferritin levels, whereas the age of cataract onset and its severity are controversial subjects. In our family, early ophthalmic examination ruled out the possibility that cataract was due to age-related persistence of high ferritin levels in the lens and suggested that other factors may modulate the phenotype. Conclusion: cataract may appear early in hereditary hyperferritinaemia-cataract syndrome and this syndrome should be suspected and ferritin levels measured in all cases of cataract in children, even when the onset is in early infancy.

Background Diamond-Blackfan anaemia (DBA) is a rare inherited red cell hypoplasia characterised by a defect in the maturation of erythroid progenitors and in some cases associated with malformations. Patients have an increased risk of solid tumors. Mutations have been found in several ribosomal protein (RP) genes, i.e RPS19, RPS24, RPS17, RPL5, RPL11, RPL35A. Studies in haematopoietic progenitors from patients show that haplo-insufficiency of an RP impairs rRNA processing and ribosome biogenesis. DBA lymphocytes show reduced protein synthesis and fibroblasts display abnormal rRNA processing and impaired proliferation. Results To evaluate the involvement of non-haematopoietic tissues in DBA, we have analysed global gene expression in fibroblasts from DBA patients compared to healthy controls. Microarray expression profiling using Affymetrix GeneChip Human Genome U133A 2.0 Arrays revealed that 421 genes are differentially expressed in DBA patient fibroblasts. These genes include a large cluster of ribosomal proteins and factors involved in protein synthesis and amino acid metabolism, as well as genes associated to cell death, cancer and tissue development. Conclusion This analysis reports for the first time an abnormal gene expression profile in a non-haematopoietic cell type in DBA. These data support the hypothesis that DBA may be due to a defect in general or specific protein synthesis.

Diamond-Blackfan anemia (DBA) is an inherited disease characterized by pure erythroid aplasia. Thirty percent (30%) of patients display malformations, especially of the hands, face, heart, and urogenital tract. DBA has an autosomal dominant pattern of inheritance. De novo mutations are common and familial cases display wide clinical heterogeneity. Twenty-five percent (25%) of patients carry a mutation in the ribosomal protein (RP) S19 gene, whereas mutations in RPS24, RPS17, RPL35A, RPL11, and RPL5 are rare. These genes encode for structural proteins of the ribosome. A link between ribosomal functions and erythroid aplasia is apparent in DBA, but its etiology is not clear. Most authors agree that a defect in protein synthesis in a rapidly proliferating tissue, such as the erythroid bone marrow, may explain the defective erythropoiesis. A total of 77 RPS19 mutations have been described. Most are whole gene deletions, translocations, or truncating mutations (nonsense or frameshift), suggesting that haploinsufficiency is the basis of DBA pathology. A total of 22 missense mutations have also been described and several works have provided in vitro functional data for the mutant proteins. This review looks at the data on all these mutations, proposes a functional classification, and describes six new mutations. It is shown that patients with RPS19 mutations display a poorer response to steroids and a worse long-term prognosis compared to other DBA patients.

Diamond-Blackfan anemia (DBA,#MIM105650) is a rare congenital pure red cell aplasia characterized by nor-mochromic macrocytic anemia, reticulocytopenia, and normocellular bone marrow with a selective deficiency of erythroid precursors. Defects in the RPS19 gene on chromosome 19q13.2 are the main

Diamond Blackfan anemia (DBA, MIM#205900) is a rare, pure red blood cell aplasia of childhood due to an intrinsic defect in erythropoietic progenitors. About 40% of cases show malformations. Anemia is corrected by steroid treatment in half of the patients; non-responders need chronic transfusions or stem cell transplantation. Defects in the RPS19 gene, encoding the ribosomal protein (RP) S19, are the main known cause of DBA and account for more than 25% of patients. Mutations in other RPs (RPS24, RPS17, RPL35a) have been reported in a minority of patients (Gazda et al., 2006; Cmejla et al., 2007; Farrar et al., 2008) and show that DBA is a disorder of ribosome synthesis. Recently, Gadza et al. (2007) reported the involvement of two new genes (RPL5, RPL11) encoding for RPs of the large subunit in a considerable percentage of DBA patients without mutations in RPS19 or RPS24 (10% and 6,5%, respectively). Here we present the results of RPL11 mutation screening in Italian DBA patients without RPS19 or RPS24 mutations. Preliminary data of RPL5 analysis are also reported. The human RPL11 gene includes 6 exons and spans 4622 bp on chromosome 1. We identified 12 heterozygous mutations in 80 analyzed probands (15%), 10 of which were deletions of 1–47 nucleotides causing frameshift and a premature termination; another deletion (12nt) resulted in a loss of 4 aminoacids. One acceptor splice site defect (intron 5) was also detected. RPL11 mutations were spread in all exons except for exon 1. The human RPL5 gene spans 9888 bp on chromosome 1, and includes 8 exons. We identified one heterozygous mutation in RPL5 in 14 analyzed patients (7%); it is a single nucleotide deletion in exon 5 causing frameshift and a premature termination. None of these sequence changes were found on the NCBI SNP dalabase (http://www.ncbi.nlm.nih.gov/SNP/). Our mutations are expected to alter the genetic information drastically and to cause haploinsufficency. Depletion of RPs compromises ribosome biogenesis and disturbs rRNA processing at different levels (Robledo et al. 2008). Interestingly, all mutated patients (11/13) showed one or more somatic malformations; specifically, thumb anomalies and growth retardation were present in 7/11 (64%) and 6/11 (54%) patients, respectively. The erythrocyte adenosine deaminase activity was performed in 6/13 mutated patients; all of them showed an increased activity. The majority of RPL11 mutated patients were transfusion-dependent at last follow-up (58%,7/12); the RPL5 mutated patient was in clinical remission after steroid treatment. No one of the mutated patients has so far developed neoplasia. Our data show that RPL11 mutations are more frequent than expected (Gazda et al. 2007). They seem to confer a relevant association with a malformation phenotype as compared to RPS19 mutations.

The autoimmune lymphoproliferative syndrome (ALPS) is an inherited disorder of lymphocyte apoptosis caused by defects in the Fas apoptotic pathway. It is characterized by the association of chronic lymphoid accumulation and autoimmune manifestations. In a subgroup of patients the disease progresses through antibody deficiency and its clinical and laboratory features overlap those of common variable immunodeficiency (CVID). Some CVID cases are associated with mutations in gene TNFRSF13B, encoding TACI. Aim of this study was to determine whether TNFRSF13B mutations are also associated with ALPS. Methods. Genomic DNA from 31 ALPS patients was extracted from PBMCs after informed consent. Exons and intron-exon boundaries of TNFRSF13B gene were amplified by PCR and sequenced on an ABI PRISM 310 Genetic Analyzer. Results. A T>A transition at nt 275, that determined the non-conservative substitution of Isoleucine with Asparagine (I92N), was found in exon 3 from one patient. The mutation involved the second extracellular cysteine rich domain, which is critical for BAFF binding. It was not found in 100 control chromosomes. The patient showed a classical ALPS phenotype with lymphadenopathy, massive splenomegaly that required splenectomy, hepatomegaly and immune cytopenias (neutropenia and immune thrombocytopenic purpura); he had alopecia and high ANA levels. Serum immunoglobulins were normal (IgG titers at the lower level of the range); lymphocytes showed defective Fas-induced apoptosis. Discussion. TACI mutations might contribute to the ALPS phenotype in some cases. Our data confirm the relationships between ALPS and CVID and provides a possible molecular explanation for overlapping phenotypes.