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International audience; Knowledge of the factors contributing to the development of protective immunity after vaccination with COVID-19 mRNA vaccines is fragmentary. Thus we employed high- temporal-resolution transcriptome profiling and in-depth characterization of antibody production approaches to investigate responses to COVID-19 mRNA vaccination. There were marked differences in the timing and amplitude of the responses to the priming and booster doses. Notably, two distinct interferon signatures were identified, that differed based on their temporal patterns of induction. The first signature (S1), which was preferentially induced by type I interferon, peaked at day 2 post-prime and at day 1 post-boost, and in both instances was associated with subsequent development of the antibody response. In contrast, the second interferon signature (S2) peaked at day 1 both post-prime and post- boost but was found to be potently induced only post-boost, where it coincided with a robust inflammation peak. Notably, we also observed “post-prime-like” (S1 ++ ,S2 0/+ ) and “post-boost-like” (S1 ++ ,S2 ++ ) patterns of interferon response among COVID-19 patients. A post-boost-like signature was observed in most severely ill patients at admission to the intensive care unit and was associated with a shorter hospital stay. Interestingly, severely ill patients who stayed hospitalized the longest showed a peculiar pattern of interferon induction (S1 -/0 ,S2 + ), that we did not observe following the administration of mRNA vaccines. In summary, high temporal resolution profiling revealed an elaborate array of immune responses elicited by priming and booster doses of COVID-19 mRNA vaccines. Furthermore, it contributed to the identification of distinct interferon-response phenotypes underpinning vaccine immunogenicity and the course of COVID-19 disease.

Atypical hemolytic uremic syndrome (aHUS) is an ultra-rare disease characterized by microangiopathic hemolysis, thrombocytopenia, and renal impairment and is associated with dysregulation of the alternative complement pathway on the microvascular endothelium. Outcomes have improved greatly with pharmacologic complement C5 blockade. Abnormalities in complement genes (CFH, CD46, CFI, CFB, C3, and THBD), CFH–CFHR genomic rearrangements, and anti-FH antibodies have been reported in 40–60% of cases. The penetrance of aHUS is incomplete in carriers of complement gene abnormalities; and multiple hits, including the CFH–H3 and CD46GGAAC risk haplotypes and the CFHR1*B risk allele, as well as environmental factors, contribute to disease development. Here, we investigated the determinants of penetrance of aHUS associated with CD46 genetic abnormalities. We studied 485 aHUS patients and found CD46 rare variants (RVs) in about 10%. The c.286+2T>G RV was the most prevalent (13/485) and was associated with G RV who experienced a severe form of aHUS and developed end-stage renal failure. The father and paternal uncle with the same variant in homozygosity and six heterozygous relatives are unaffected. Flow cytometry analysis showed about 50% reduction of CD46 expression on blood mononuclear cells from the heterozygous proband and over 90% reduction in cells from the proband's unaffected homozygous father and aunt. Further genetic studies did not reveal RVs in known aHUS-associated genes or common genetic modifiers that segregated with the disease. Importantly, a specific ex vivo test showed excessive complement deposition on endothelial cells exposed to sera from the proband, and also from his mother and maternal uncle, who do not carry the c.286+2T>G RV, indicating that they share a circulating defect that results in complement dysregulation on the endothelium. These results highlight the complexity of the genetics of aHUS and indicate that CD46 deficiency may not be enough to induce aHUS. We hypothesize that the proband inherited from his mother a genetic abnormality in a complement circulating factor that has not been identified yet, which synergized with the CD46 RV in predisposing him to the aHUS phenotype.

Membranoproliferative GN (MPGN) was recently reclassified as alternative pathway complement–mediated C3 glomerulopathy (C3G) and immune complex–mediated membranoproliferative GN (IC-MPGN). However, genetic and acquired alternative pathway abnormalities are also observed in IC-MPGN. Here, we explored the presence of distinct disease entities characterized by specific pathophysiologic mechanisms. We performed unsupervised hierarchical clustering, a data-driven statistical approach, on histologic, genetic, and clinical data and data regarding serum/plasma complement parameters from 173 patients with C3G/IC-MPGN. This approach divided patients into four clusters, indicating the existence of four different pathogenetic patterns. Specifically, this analysis separated patients with fluid-phase complement activation (clusters 1–3) who had low serum C3 levels and a high prevalence of genetic and acquired alternative pathway abnormalities from patients with solid-phase complement activation (cluster 4) who had normal or mildly altered serum C3, late disease onset, and poor renal survival. In patients with fluid-phase complement activation, those in clusters 1 and 2 had massive activation of the alternative pathway, including activation of the terminal pathway, and the highest prevalence of subendothelial deposits, but those in cluster 2 had additional activation of the classic pathway and the highest prevalence of nephrotic syndrome at disease onset. Patients in cluster 3 had prevalent activation of C3 convertase and highly electron-dense intramembranous deposits. In addition, we provide a simple algorithm to assign patients with C3G/IC-MPGN to specific clusters. These distinct clusters may facilitate clarification of disease etiology, improve risk assessment for ESRD, and pave the way for personalized treatment.

Severe/medically complicated obesity in childhood, and particularly in adolescence, is a real disability that requires an intensive and continuous approach which should follow the procedures and schedule of rehabilitation medicine. Given the lack of a specific document focusing on children and adolescents, the Childhood Obesity Study Group set out to explore the available evidence for the treatment of severe or medically complicated obesity and to set standards tailored to the specific context of the Italian Health Service. Through a series of meetings and electronic communications, the writing committee (selected from members of the Study Group) selected the key issues, explored the literature and produced a draft document which was submitted to the other experts until the final synthesis was approved by the group. In brief, the following issues were involved: (1) definition and epidemiology; (2) identification of common goals designed to regain functional competence and limit the progression of metabolic and psychological complications; (3) a multi-professional team approach; (4) the care setting. This paper is an expert opinion document on the rehabilitation of severe and medically complicated obesity in children and adolescents produced by experts belonging to the Childhood Obesity Study Group of the Italian Society for Pediatric Endocrinology and Diabetology (ISPED).

Isomorphic mutation of the SBDS gene causes Shwachman-Diamond syndrome (SDS). SDS is a rare genetic bone marrow failure and cancer predisposition syndrome. SDS cells have ribosome biogenesis and their protein synthesis altered, which are two high-energy consuming cellular processes. The reported changes in reactive oxygen species production, endoplasmic reticulum stress response and reduced mitochondrial functionality suggest an energy production defect in SDS cells. In our work, we have demonstrated that SDS cells display a Complex IV activity impairment, which causes an oxidative phosphorylation metabolism defect, with a consequent decrease in ATP production. These data were confirmed by an increased glycolytic rate, which compensated for the energetic stress. Moreover, the signalling pathways involved in glycolysis activation also appeared more activated; i.e. we reported AMP-activated protein kinase hyper-phosphorylation. Notably, we also observed an increase in a mammalian target of rapamycin phosphorylation and high intracellular calcium concentration levels ([Ca2+]i), which probably represent new biochemical equilibrium modulation in SDS cells. Finally, the SDS cell response to leucine (Leu) was investigated, suggesting its possible use as a therapeutic adjuvant to be tested in clinical trials.

Fanconi's anemia (FA) patients face severe pathological consequences. Bone marrow failure, the major cause of death in FA, accounting for as much as 80-90% of FA mortality, appears to be significantly linked to excessive apoptosis of hematopoietic cells induced by oxidative stress. However, 20-25% of FA patients develop malignancies of myeloid origin. A survival strategy for bone marrow and hematopoietic cells under selective pressure evidently exists. This study reports that lymphoblastoid cell lines derived from two FA patients displayed significant resistance to oxidative stress induced by treatments with H(2) O(2) and various glutathione (GSH) inhibitors that induce production of reactive oxygen species, GSH depletion and mitochondrial membrane depolarization. Among the various GSH inhibitors employed, FA cells appear particularly resistant to menadione (5 μm) and ethacrynic acid (ETA, 50 μm), two drugs that specifically target mitochondria. Even after pre-treatment with buthionine sulfoximine, a GSH synthesis inhibitor that induces enhanced induction of reactive oxygen species, FA cells maintain significant resistance to these drugs. These data suggest that the resistance to oxidative stress and the altered mitochondrial and metabolic functionality found in the FA mutant cells used in this study may indicate the survival strategy that is adopted in FA cells undergoing transformation. The study of redox and mitochondria regulation in FA may be of assistance in diagnosis of the disease and in the care of patients.

BACKGROUND AND AIM: The prevalence of children with hypertension is increasing, especially in obese children. This study was to assess the relationship between blood pressure, indexes of adiposity, body fat distribution and insulin resistance. METHODS AND RESULTS: Sample: 1044 children (M/F: 484/560; aged 6-11years). Anthropometry and blood pressure were measured and fasting blood samples were tested for triacylglycerol, total cholesterol, HDL cholesterol, glucose, insulin and ALT. The prevalence of high blood pressure in overweight males and females was 14.3 and 6.4%, respectively (chi(2)=16.73, p

Fanconi Anemia (FA) is a rare and complex inherited blood disorder associated with bone marrow failure and malignancies. Many alterations in FA physiology appear linked to red-ox unbalance including alterations in the morphology and structure of nuclei, intermediate filaments and mitochondria, defective respiration, reduced ATP production and altered ATP/AMP ratio. These defects are consistently associated with impaired oxygen metabolism indeed treatment with antioxidants N-acetylcysteine (NAC) and resveratrol (RV) does rescue FA physiology. Due to the importance of the intracellular calcium signaling and its key function in the control of intracellular functions we were interested to study calcium homeostasis in FA. We found that FANCA cells display a dramatically low intracellular calcium concentration ([Ca(2+)]i) in resting conditions. This condition affects cellular responses to stress. The flux of Ca(2+) mobilized by H2O2 from internal stores is significantly lower in FANCA cells in comparison to controls. The low basal [Ca(2+)]i in FANCA appears to be an actively maintained process controlled by a finely tuned interplay between different intracellular Ca(2+) stores. The defects associated with the altered Ca(2+) homeostasis appear consistently overlapping those related to the unbalanced oxidative metabolism in FA cells underlining a contiguity between oxidative stress and calcium homeostasis.

Summary Introduction. Since Regione Veneto suspended compulsory vaccination for children in 2008, and because of an increasing disaffection of parents to the vaccine practice, the vaccination rates have been slowly but steadily decreasing. The aim of this study was to analyze internal and external factors of immunization reduction and to implement potential solutions of the problem. Methods. Servizio Igiene e Sanità Pubblica of ULSS 12 Veneziana (SISP – Hygiene and Public Health Service) analyzed and addressed both, the reasons of parents who do not vaccinate their children and the internal problems regarding vaccination clinics management, information to families, procedures and guidelines and, in general, the communication skills of the vaccination staff. Results. A positive trend in vaccination rates was observed, especially in Venice historical centre. Moreover the staff reported a better working atmosphere and benefit from sharing common goals and procedures, even though the workforce was reduced of about 30% in terms of equivalent unit (EU). Discussion. The continuous quality improvement method followed in this experience led to a steady increase in vaccination coverage in all territorial clinics, to a better adhesion of guidelines and standard operating procedures and to a general professional empowerment of SISP staff. The service now offered to the population is better and more efficient, since the workforce has been reduced. Future goals are to improve information about vaccinations among the population.

Background Whether microgravity might influence tumour growth and carcinogenesis is still an open issue. It is not clear also if and how normal and transformed cells are differently solicited by microgravity. The present study was designed to verify this issue. Methods Two normal, LB and HSC93, and two transformed, Jurkat and 1310, lymphoblast cell lines were used as representative for the two conditions. Two lymphoblast lines from Fanconi's anemia patients group A and C (FA-A and FA-C, respectively), along with their isogenic corrected counterparts (FA-A-cor and FA-C-cor) were also used. Cell lines were evaluated for their proliferative ability, vitality and apoptotic susceptibility upon microgravity exposure in comparison with unexposed cells. Different parameters correlated to energy metabolism, glucose consumption, mitochondrial membrane potential (MMP), intracellular ATP content, red-ox balance and ability of the cells to repair the DNA damage product 8-OHdG induced by the treatment of the cells with 20 mM KBrO3 were also evaluated. Results Transformed Jurkat and 1310 cells appear resistant to the microgravitational challenge. On the contrary normal LB and HSC93 cells display increased apoptotic susceptibility, shortage of energy storages and reduced ability to cope with oxidative stress. FA-A and FA-C cells appear resistant to microgravity exposure, analogously to transformed cells. FA corrected cells did shown intermediate sensitivity to microgravity exposure suggesting that genetic correction does not completely reverts cellular phenotype. Conclusions In the light of the reported results microgravity should be regarded as an harmful condition either when considering normal as well as transformed cells. Modeled microgravity and space-based technology are interesting tools in the biomedicine laboratory and offer an original, useful and unique approach in the study of cellular biochemistry and in the regulation of metabolic pathways.

This article provides current guidelines on the treatment and prevention of childhood obesity. Since factors involved in obesity change with age, the therapeutic approach in pre-school children will be different from pupils and adolescents. The treatment will also be modulated on the basis of weight excess, weight gain velocity and complications. The main goal of the treatment should be to encourage the child and his family to have healthy lifestyle. Families who are not ready for change might benefit from counselling to improve motivation before starting treatment. A detailed alimentary and behavioural history is the start point of the treatment. The strategy of the intervention is to induce changes at three levels: 1) attitudes of parents; 2) physical activity; 3) energy intake. The treatment of the adolescents should take into account the pubertal changes and the psychological aspects of this peculiar period of life. Obesity is a chronic disease and its treatment needs long-life follow-up. The long-term results of the obesity treatment are often disappointing and we have to consider consistent prevention programs for better results.

Objectives: (a) to explore the relationship between waist circumference and certain cardiovascular risk factors in a group of girls; and (b) to assess the clinical relevance of waist circumference in identifying girls with higher cardiovascular risk across puberty. Subjects and methods: One-hundred and fifty-five overweight or obese girls aged 5–16 y were recruited. Overweight and obesity were defined on the basis of BMI, according to Cole. Results: Waist circumference was significantly correlated with plasma insulin (r=0.43; P

Isomorphic mutation of SBDS gene is the cause of Shwachman-Diamond syndrome (SDS). SDS is a rare genetic bone marrow failure and cancer predisposition syndrome. SDS cells have altered ribosome biogenesis and protein synthesis, two high-energy consuming cellular processes. The reported increment in reactive oxygen species production, endoplasmic reticulum stress response and reduced mitochondrial functionality suggest a defect in the energy production in SDS cells. In this study, we analyzed the energetic metabolism in SDS cells and find that the oxygen consumption is impaired when it is induced by pyruvate/malate or succinate. This induces poor ATP production and AMP accumulation with a consequent alteration in the ATP/AMP ratio. Also respiratory chain activity was impaired because of faulty function of the complex IV; this defect is not dependent from impaired protein synthesis despite ribosome biogenesis and transduction defects in SDS. In fact, COX5A and Cox2, two subunits of Complex IV encoded respectively by a nuclear and mitochondrial gene, were expressed at normal levels. Impaired function of complex IV could be due to an increment of cytoplasmic calcium concentration that inhibits complex IV activity. Energetic stress induces changes in cellular metabolism, stimulating or inhibiting a network of molecules involved in regulation of energetic balance, such as AMPK and mTOR. In SDS cells as consequence of energetic stress, AMPK is hyper activated and the glycolytic pathway stimulated. Surprisingly, we found that also the AKT/mTOR pathway is aberrantly hyper activated since both these proteins are hyper-phosphorylated. We can speculate that hyper activation of mTOR is a way through which SDS cells support the energy defect and protein synthesis. All these defects were recovered when the SDS cells were complemented with SDS gene. Finally, leucine is an essential amino acid that induces cell proliferation and protein synthesis, restored OXPHOS and ATP synthesis, reduced the cytoplasmic calcium concentration and the AMPK and AKT/mTOR activity, and improved in vitro erythropoiesis from SDS individuals pointing to leucine as potential tool helpful to sustain deranged energetic metabolism and erythropoiesis in SDS patients. In conclusion, we report for the first time that SDS cells suffer of energetic stress and severe respiratory defect that is related to faulty SBSD protein. These defects are compensated by an enhanced activation of AMPK, glycolysis and mTOR/Akt pathways, which appear to adequately support protein synthesis. A pivotal role in the maintenance of this altered metabolism could be played by altered calcium homeostasis. Noteworthy biochemical defects might be largely corrected by leucine which also favourably affects in vitro erythropoiesis thus pointing to biochemical defects as important determinant for impaired hematopoiesis od SDS. Disclosures Dufour: Pfizer: Consultancy.