Candidate biomarkers from the integration of methylation and gene expression in discordant autistic sibling pairs

While the genetics of autism spectrum disorders (ASD) has been intensively studied, resulting in the identification of over 100 putative risk genes, the epigenetics of ASD has received less attention, and results have been inconsistent across studies. We aimed to investigate the contribution of DNA methylation (DNAm) to the risk of ASD and identify candidate biomarkers arising from the interaction of epigenetic mechanisms with genotype, gene expression, and cellular proportions. We performed DNAm differential analysis using whole blood samples from 75 discordant sibling pairs of the Italian Autism Network collection and estimated their cellular composition. We studied the correlation between DNAm and gene expression accounting for the potential effects of different genotypes on DNAm. We showed that the proportion of NK cells was significantly reduced in ASD siblings suggesting an imbalance in their immune system. We identified differentially methylated regions (DMRs) involved in neurogenesis and synaptic organization. Among candidate loci for ASD, we detected a DMR mapping to CLEC11A (neighboring SHANK1) where DNAm and gene expression were significantly and negatively correlated, independently from genotype effects. As reported in previous studies, we confirmed the involvement of immune functions in the pathophysiology of ASD. Notwithstanding the complexity of the disorder, suitable biomarkers such as CLEC11A and its neighbor SHANK1 can be discovered using integrative analyses even with peripheral tissues

Defining Kawasaki disease and pediatric inflammatory multisystem syndrome-temporally associated to SARS-CoV-2 infection during SARS-CoV-2 epidemic in Italy: results from a national, multicenter survey

Background: There is mounting evidence on the existence of a Pediatric Inflammatory Multisystem Syndrome-temporally associated to SARS-CoV-2 infection (PIMS-TS), sharing similarities with Kawasaki Disease (KD). The main outcome of the study were to better characterize the clinical features and the treatment response of PIMS-TS and to explore its relationship with KD determining whether KD and PIMS are two distinct entities. Methods: The Rheumatology Study Group of the Italian Pediatric Society launched a survey to enroll patients diagnosed with KD (Kawasaki Disease Group - KDG) or KD-like (Kawacovid Group - KCG) disease between February 1st 2020, and May 31st 2020. Demographic, clinical, laboratory data, treatment information, and patients' outcome were collected in an online anonymized database (RedCAP®). Relationship between clinical presentation and SARS-CoV-2 infection was also taken into account. Moreover, clinical characteristics of KDG during SARS-CoV-2 epidemic (KDG-CoV2) were compared to Kawasaki Disease patients (KDG-Historical) seen in three different Italian tertiary pediatric hospitals (Institute for Maternal and Child Health, IRCCS "Burlo Garofolo", Trieste; AOU Meyer, Florence; IRCCS Istituto Giannina Gaslini, Genoa) from January 1st 2000 to December 31st 2019. Chi square test or exact Fisher test and non-parametric Wilcoxon Mann-Whitney test were used to study differences between two groups. Results: One-hundred-forty-nine cases were enrolled, (96 KDG and 53 KCG). KCG children were significantly older and presented more frequently from gastrointestinal and respiratory involvement. Cardiac involvement was more common in KCG, with 60,4% of patients with myocarditis. 37,8% of patients among KCG presented hypotension/non-cardiogenic shock. Coronary artery abnormalities (CAA) were more common in the KDG. The risk of ICU admission were higher in KCG. Lymphopenia, higher CRP levels, elevated ferritin and troponin-T characterized KCG. KDG received more frequently immunoglobulins (IVIG) and acetylsalicylic acid (ASA) (81,3% vs 66%; p = 0.04 and 71,9% vs 43,4%; p = 0.001 respectively) as KCG more often received glucocorticoids (56,6% vs 14,6%; p < 0.0001). SARS-CoV-2 assay more often resulted positive in KCG than in KDG (75,5% vs 20%; p < 0.0001). Short-term follow data showed minor complications. Comparing KDG with a KD-Historical Italian cohort (598 patients), no statistical difference was found in terms of clinical manifestations and laboratory data. Conclusion: Our study suggests that SARS-CoV-2 infection might determine two distinct inflammatory diseases in children: KD and PIMS-TS. Older age at onset and clinical peculiarities like the occurrence of myocarditis characterize this multi-inflammatory syndrome. Our patients had an optimal response to treatments and a good outcome, with few complications and no deaths

Interaction between influenza virus and the host cell: role of the cytoskeleton in the induction of a resistance condition to viral infection

Il lavoro sperimentale oggetto di questa tesi è volto a mettere in luce l’intervento di una componente cellulare, il citoscheletro, nella regolazione dell’infezione sperimentale della linea cellulare LLC-MK2 (rene di scimmia) con stipiti di virus influenza A. Le cellule LLC-MK2 costituiscono un modello semi-permissivo per il virus influenza, nel caso si utilizzi un inoculo virale a bassa molteplicità, e, pertanto, sono funzionali a svelare meccanismi di resistenza cellulare all’infezione virale. L’obiettivo principale è stato quello di appurare l’intervento di funzioni/componenti regolatorie dei sistemi citoscheletrici dei microtubuli e dei microfilamenti in corso di infezione con stipiti di virus influenza A (H1N1) nelle cellule LLC-MK2. L’attenzione è stata inizialmente focalizzata sul ruolo svolto dai proteasomi nella regolazione dei microtubuli e sulle ripercussioni esercitate sull’infezione virale. I risultati ottenuti attestano una diminuzione di espressione di alpha-tubulina acetilata, principale componente dei microtubuli in assetto stabilizzato, a seguito dell’attivazione delle funzioni proteolitiche dei proteasomi con la sostanza IU1; viceversa, è stato appurato un aumento dell’espressione della suddetta proteina, in conseguenza dell’inibizione dei proteasomi con la sostanza MG132. Le suddette modificazioni di assetto dei microtubuli correlavano con modulazioni dell’infezione del virus influenza. Più precisamente, l’efficienza del ciclo replicativo virale dello stipite umano NWS/33 e dello stipite aviare Mallard/03 di virus influenza A aumentava in condizioni di attivazione dei proteasomi, mentre diminuiva in seguito all'inibizione dei proteasomi. Inoltre, mediante microscopia confocale, è stata dimostrata la parziale co-localizzazione dei proteasomi con la nucleoproteina virale e con componenti dei microtubuli stabili. In particolare, il segnale di co-localizzazione era di maggiore entità nelle cellule infettate, dove sono stati osservati dei rilevanti cambiamenti nella distribuzione citoplasmatica delle proteine alpha-tubulina e MAP4. Tali risultati depongono per l’intervento dei proteasomi nella modulazione dei microtubuli nelle cellule LLC-MK2 infettate con il virus influenza, tuttavia l’azione regolatoria esercitata potrebbe essere di tipo indiretto e coinvolgere dei meccanismi di segnalazione intracellulare mediati dai microtubuli. È, inoltre, stato appurato che la depolimerizzazione dei microtubuli e dei microfilamenti con sostanze chimiche induce delle modificazioni reciproche dell’assetto di tali componenti, che “interagiscono” tra loro e svolgono un’azione sinergica, quali fattori di restrizione cellulare nei confronti di fasi precoci dell’infezione virale nelle cellule LLC-MK2. L’attenzione è stata successivamente rivolta all’intervento della proteina omologa alla proteina diafano 1 di Drosophila (DIAPH1), che regola le funzioni dei microtubuli e dei microfilamenti. Nelle cellule LLC-MK2 sottoposte a silenziamento del gene che codifica per la proteina DIAPH1 ed infettate con il virus influenza A/NWS/33 è stato osservato un aumento della produttività dell’infezione virale, unitamente a modificazioni di assetto dei microtubuli, che erano contraddistinti da una diminuita espressione di alpha- e beta-tubulina ed un’estensione ad un’area citoplasmatica più ampia, e dei microfilamenti, che erano caratterizzati da una diminuita espressione dell’actina filamentosa. L’osservazione di una localizzazione subcellulare e di un livello di espressione altamente variabili della proteina DIAPH1 in corso di infezione del virus NWS/33 nelle cellule LLC-MK2, depone per funzioni regolatorie svolte dalla proteina in specifici distretti subcellulari. Ulteriori modificazioni sono state osservate a seguito della depolimerizzazione dei microtubuli e dei microfilamenti, che depongono per l’esistenza di “interazioni” tra la proteina DIAPH1 e tali componenti del citoscheletro. L’analisi in microscopia confocale ha evidenziato, nelle cellule LLC-MK2 infettate, la parziale co-localizzazione della proteina DIAPH1 con actina, alpha-tubulina acetilata, beta-tubulina e la nucleoproteina virale. In particolare, la co-localizzazione con le componenti del citoscheletro era di entità maggiore, oltre che appannaggio di differenti aree citoplasmatiche, nelle cellule LLC-MK2 infettate, rispetto a quelle non infettate. Infine, l’analisi è stata estesa a cellule dell’epitelio respiratorio umano positive per il virus influenza A, oppure per il virus respiratorio sinciziale; tali cellule derivavano da tamponi faringei e nasali di soggetti ricoverati od osservati ambulatorialmente e pervenuti presso l’Unità Operativa di Virologia dell’Azienda Ospedaliero-Universitaria di Parma per la diagnosi di laboratorio di infezione da virus dell’apparato respiratorio. In particolare, nelle cellule positive per il virus influenza A, l’espressione della proteina DIAPH1 risultava accentuata e, al contrario, in quelle positive per il virus respiratorio sinciziale, era di scarsa entità. I risultati conseguiti rendono ragione dell’elevata complessità del rapporto che intercorre tra virus influenza e cellula ospite e pongono l’accento sulla stretta correlazione con aspetti morfologico-funzionali della cellula infettata. Le osservazioni sperimentali ottenute rafforzano le conoscenze sul fatto che la stabilizzazione dei microtubuli e la polimerizzazione dei microfilamenti, alla cui regolazione partecipano i proteasomi e la formina DIAPH1, possa concorrere in maniera significativa alla modulazione dell’infezione del virus influenza A nelle cellule LLC-MK2

Representações sociais dos juízes da infância e juventude na aplicação da privação de liberdade a adolescentes autores de ato infracional

Este artigo refere-se a pesquisa realizada em 2010 em municípios de Minas Gerais e cuja proposta era analisar se e em que medida as práticas jurí­dicas no campo do direito infantojuvenil ainda se respaldam nas concepções tutelares-repressivas da doutrina da “situação irregular”, que deveria ter sido suplantada após a publicação do Estatuto da Criança e do Adolescente. Foi realizada análise de sentenças judiciais, sendo possível verificar que a argumentação dos juízes é permeada por repre­sentações sociais tradicionais e estereotipadas em relação ao adolescente, à família, aos papéis de gê­nero e à privação de liberdade. The article Social Representations of the Juvenile Court Judges in the imposition of Freedom Restric­tion on Teenage Offenders refers to a research con­ducted in 2010 in some towns of Minas Gerais state with the aim of analyzing whether and to what extent practices in the field of infant-juvenile law are still based on repressive-guardianship notions from the doctrine of “irregular status”, which should have been supplanted upon the publication of the Child and Teenager Act (ECA). Court decisions were reviewed and it was found that the judges’ argumentation was pervaded by traditional and stereotypical representations in relation to the youth, the family, gender roles and the restriction of freedom. Keywords: irregular status, full-time protection, juvenile offender, restriction of freedom, social representations</strong

Age-Related Effects on the Spectrum of Cerebral Visual Impairment in Children With Cerebral Palsy

Background: Cerebral Visual Impairment (CVI) is a very common finding in children affected by Cerebral Palsy (CP). In this paper we studied the characteristics of CVI of a large group of children with CP and CVI, describing their neurovisual profiles according to three different age subgroups (subgroup 1: infants 6 months-2 years; subgroup 2: pre-school age 3-5 years; subgroup 3: school age ≥ 6 years). Methods: We enrolled 180 subjects (104 males, mean age 66 ± 42.6 months; range 6-192 months) with CP and CVI for the study. We carried out a demographic and clinical data collection, neurological examination, developmental or cognitive assessment, and a video-recorded visual function assessment including an evaluation of ophthalmological characteristics, oculomotor functions, and basic visual functions. In school-aged children, we also performed an evaluation of their cognitive-visual profiles. Results: There were signs of CVI in all the three subgroups. Subgroup 1 (62 children) and subgroup 2 (50 children) were different for fixation (p = 0.02), visual acuity (p = 0.03) and contrast sensitivity (p < 0.01), being more frequently impaired in younger children. Comparing subgroup 2 with subgroup 3 (68 children), the older children presented more frequently myopia (p = 0.02) while the younger ones esotropia (p = 0.02) and alteration in smooth pursuit (p = 0.03) and saccades (p < 0.01). Furthermore, fixation, smooth pursuit, visual acuity, contrast sensitivity and visual filed (p < 0.01) were more frequently impaired in younger children (subgroup 1) compared to the older ones. Multiple correspondence analysis (MCA) confirmed the different neurovisual profiles according to age: younger children with CP showed more signs of CVI compared to the older ones. 34 out of 68 children belonging to subgroup 3 underwent the cognitive visual evaluation; an impairment of cognitive visual skills was detected in 21 subjects. Conclusion: Younger children with CP showed more signs of CVI compared to the older ones, likely for the physiological maturation of visual system and mechanisms of neuroplasticity. In this direction, we suggest an early neurovisual evaluation to detect any weak visual functions

Expanding the spectrum of clinical severity of <scp>AICA</scp> ‐ribosiduria: Report of two siblings with mild phenotype caused by a novel pathogenic variant in <scp>ATIC</scp> gene

: 5-Amino-4-imidazolecarboxamide-ribosiduria (AICA-ribosiduria) is an extremely rare inborn error of purine biosynthesis metabolism caused by pathogenic variants in ATIC gene that encodes a protein catalyzing the last steps of the de novo purine biosynthesis. To date, only six cases have been reported presenting a severe phenotype characterized by coarse facies and variable dysmorphic features, intrauterine and postnatal growth retardation, severe and early neurodevelopment delay, profound congenital visual deficit, scoliosis and, less frequently, epilepsy, aortic coarctation, chronic hepatic cytolysis, nephrocalcinosis and mild genitalia malformation. In this article, we report two new cases of AICA-ribosiduria carrying new pathogenic variants in ATIC (c.421C>T;p.Arg141Ter and c.1753A>G p.Thr585Ala) associated to a milder phenotype compared to previously reported patients. Particularly, the children showed few dysmorphic features (bulging forehead, depressed nasal bridge, and flat nasal tip), postnatal growth impairment, psychomotor delay since the second year of life, reduction of visual acuity (from mild impairment to low vision from the age of 5 years and to partial blindness from the age of 7 years) and mild hepatic dysfunctions. Scoliosis as well as epilepsy, renal involvement, or genitalia malformation were not detected. According to literature data, we found an abnormal accumulation of intermediates of de novo purine biosynthesis in the urine of both siblings. This report expands the spectrum of phenotypic severity associated to ATIC biallelic pathogenic variants and prompts the need to investigate ultra-rare causes of metabolic disorders such as AICA-ribosiduria in subjects with early neurological and sensory involvement of uncertain etiology

Evaluation of the assay for hepatitis B virus (HBV) surface antigen quantification in the laboratory diagnosis of HBV infection

EVALUATION OF THE ASSAY FOR HEPATITIS B VIRUS (HBV) SURFACE ANTIGEN QUANTIFICATION IN THE LABORATORY DIAGNOSIS OF HBV INFECTION De Conto Flora, Fazzi Alessandra, Medici Maria Cristina, Arcangeletti Maria Cristina, Pinardi Federica, Ferraglia Francesca, Chezzi Carlo, Calderaro Adriana Unit of Microbiology and Virology, Department of Clinical and Experimental Medicine, University of Parma, Parma, Italy Background: Hepatitis B virus infection is a global public health problem, affecting around 2 billion people worldwide. HBV infection is a leading cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. The quantitative measurement of hepatitis B surface antigen (HBsAg) monitors the progress of chronic hepatitis B, and its rapid decline may be a predictor of the efficacy of antiviral therapy. Although the measurement of serum HBV DNA is the gold standard method for viral load evaluation, the assay is expensive and time consuming, while HBsAg quantification (qHBsAg) is rapid and cost-effective. The aim of this study is to compare the results of qHBsAg and HBV DNA determination, referred to subjects with chronic hepatitis B. Material/methods: During the 2013-2015 period, 371 plasma or serum samples of subjects attending the University Hospital of Parma (Northern Italy) were subjected to qHBsAg, by means of the ARCHITECT HBsAg assay (Abbott, Wiesbaden, Germany), with a specificity and sensitivity of 99.87% and 99.52%, respectively, as reported by the manufacturer. Of these subjects, 333 (89,8%) were subjected to HBV DNA quantification, by means of the COBAS AmpliPrep/COBAS TaqMan HBV version 2.0 assay (Roche, Mannheim, Germany). Results: Of the 371 individuals analysed, 224 (60.4%) were males (median age of 53 ± 15.3 years) and 147 (39.6%) females (median age of 52 ± 15.3 years); 235 (63.3%) were Italians and 136 (36.7%) foreigners. The subjects aged ≥61 years (30.7%) prevailed, and 359 (96.8%) were positive for qHBsAg. The comparison of the mean HBsAg level of the 359 positive subjects with those of different derived subpopulations evidenced higher HBsAg levels for HBeAg-positive subjects (3.1%; P<0.0001), subjects infected with genotype D of HBV (3.6%), and females (39%). Conversely, males (61%) and human immunodeficiency virus type 1 (HIV 1) co-infected subjects (3.6%) showed lower HBsAg levels. Moreover, HBsAg levels decreased with age, with significant differences for subjects aged ≤30 (P<0.0001) and ≥51 (P<0.05) years. The accordance among the results of qHBsAg and HBV DNA determination was of 69.7% (232/333 samples) and the qHBsAg assay sensitivity of 99.6% (227/228 samples). Conclusion: This study assesses that many factors may influence HBsAg levels, such as sex, age, HIV co-infection, HBeAg status, and HBV genotype. Although the sensitivity of qHBsAg assay is high, the discrete accordance with HBV DNA determination envisages that HBsAg measurement cannot be a reliable substitute, but a complementary assay, which allows better chronic HBV infection monitoring