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Abstract Background The epigenetic status of patients 6-month post-COVID-19 infection remains largely unexplored. The existence of long-COVID, or post-acute sequelae of SARS-CoV-2 infection (PASC), suggests potential long-term changes. Long-COVID includes symptoms like fatigue, neurological issues, and organ-related problems, regardless of initial infection severity. The mechanisms behind long-COVID are unclear, but virus-induced epigenetic changes could play a role. Methods and results Our study explores the lasting epigenetic impacts of SARS-CoV-2 infection. We analyzed genome-wide DNA methylation patterns in an Italian cohort of 96 patients 6 months after COVID-19 exposure, comparing them to 191 healthy controls. We identified 42 CpG sites with significant methylation differences (FDR

Abstract Background A high level of PD-L1 expression is the most relevant predictive parameter for response to immune checkpoint inhibitor (CPI) therapy in urinary bladder cancer. Existing data on the relationship between PD-L1 expression and the natural course of disease are controversial and sparse. Methods To expand our understanding of the relationship between PD-L1 expression and parameters of cancer aggressiveness, PD-L1 was analyzed on tissue microarrays containing 2710 urothelial bladder carcinomas including 512 patients with follow-up data who underwent radical cystectomy and follow-up therapies in the pre-immune checkpoint inhibitor therapy era. Results Tumor cell positivity in ≥10% of cells were seen in 513 (20%) and an immune cell positivity occurred in 872 (34%) of 2566 interpretable cancers. PD-L1 positivity in tumor cells increased from pTaG2 low grade (0.9% positive) to pTaG3 high grade (4.1%; p = 0.0255) and was even higher in muscle-invasive (pT2–4) carcinomas (29.3%; p

Abstract Background Post COVID-19 syndrome is characterized by several cardiorespiratory symptoms but the origin of patients’ reported symptomatology is still unclear. Methods Consecutive post COVID-19 patients were included. Patients underwent full clinical evaluation, symptoms dedicated questionnaires, blood tests, echocardiography, thoracic computer tomography (CT), spirometry including alveolar capillary membrane diffusion (DM) and capillary volume (Vcap) assessment by combined carbon dioxide and nitric oxide lung diffusion (DLCO/DLNO) and cardiopulmonary exercise test. We measured surfactant derive protein B (immature form) as blood marker of alveolar cell function. Results We evaluated 204 consecutive post COVID-19 patients (56.5 ± 14.5 years, 89 females) 171 ± 85 days after the end of acute COVID-19 infection. We measured: forced expiratory volume (FEV1) 99 ± 17%pred, FVC 99 ± 17%pred, DLCO 82 ± 19%, DM 47.6 ± 14.8 mL/min/mmHg, Vcap 59 ± 17 mL, residual parenchymal damage at CT 7.2 ± 3.2% of lung tissue, peakVO2 84 ± 18%pred, VE/VCO2 slope 112 [102–123]%pred. Major reported symptoms were: dyspnea 45% of cases, tiredness 60% and fatigability 77%. Low FEV1, Vcap and high VE/VCO2 slope were associated with persistence of dyspnea. Tiredness was associated with high VE/VCO2 slope and low PeakVO2 and FEV1 while fatigability with high VE/VCO2 slope. SPB was fivefold higher in post COVID-19 than in normal subjects, but not associated to any of the referred symptoms. SPB was negatively associated to Vcap. Conclusions In patients with post COVID-19, cardiorespiratory symptoms are linked to VE/VCO2 slope. In these patients the alveolar cells are dysregulated as shown by the very high SPB. The Vcap is low likely due to post COVID-19 pulmonary endothelial/vasculature damage but DLCO is only minimally impaired being DM preserved.

Abstract Background About 30% of Prostate cancer (PCa) patients progress to metastatic PCa that remains largely incurable. This evidence underlines the need for the development of innovative therapies. In this direction, the potential research focus might be on long non-coding RNAs (lncRNAs) like H19, which serve critical biological functions and show significant dysregulation in cancer. Previously, we showed a transcriptional down-regulation of H19 under combined pro-tumoral estrogen and hypoxia treatment in PCa cells that, in turn, induced both E-cadherin and β4 integrin expression. H19, indeed, acts as transcriptional repressor of cell adhesion molecules affecting the PCa metastatic properties. Here, we investigated the role of H19/cell adhesion molecules circuitry on in vivo PCa experimental tumor growth and metastatic dissemination models. Methods H19 was silenced in luciferase-positive PC-3 and 22Rv1 cells and in vitro effect was evaluated by gene expression, proliferation and invasion assays before and after treatment with the histone lysine demethylase inhibitor, GSK-J4. In vivo tumor growth and metastasis dissemination, in the presence or absence of GSK-J4, were analyzed in two models of human tumor in immunodeficient mice by in vivo bioluminescent imaging and immunohistochemistry (IHC) on explanted tissues. Organotypic Slice Cultures (OSCs) from fresh PCa-explant were used as ex vivo model to test GSK-J4 effects. Results H19 silencing in both PC-3 and 22Rv1 cells increased: i) E-cadherin and β4 integrin expression as well as proliferation and invasion, ii) in vivo tumor growth, and iii) metastasis formation at bone, lung, and liver. Of note, treatment with GSK-J4 reduced lesions. In parallel, GSK-J4 efficiently induced cell death in PCa-derived OSCs. Conclusions Our findings underscore the potential of the H19/cell adhesion molecules circuitry as a targeted approach in PCa treatment. Modulating this interaction has proven effective in inhibiting tumor growth and metastasis, presenting a logical foundation for targeted therapy.

Abstract Background Erythropoietic protoporphyria is a rare disorder which represents an important health problem in children, causing painful photosensitivity. Little is known on the correlation between genetic profile and clinical manifestations. The standard of care for Erythropoietic protoporphyria is based on avoiding sun and using sun protections, but recent literature has suggested that cimetidine may have a role in improving sun sensitivity. Herein we report our case series describing the successful use of cimetidine and analyzing potential phenotype-genotype correlations. Case presentation This case series describes five patients presented to our Rheumatology Service complaining sun sensitivity. Blood exams and genetic analysis were consistent with the diagnosis of erythropoietic protoporphyria. Four of 5 patients received cimetidine in addition to standard therapies and the effect of treatment was evaluated by Erythropoietic Protoporphyria - Quality of Life questionnaire. Conclusions Erythropoietic protoporphyria usually manifests in early childhood after a short sun exposure. Skin manifestations are the main reason for investigations, although sometimes they can be more subtle, leading to a significant diagnostic delay. Skin diseases in children can have profound effects on their family and social relationships. A treatment with cimetidine appears to be an excellent therapeutic option in children with Erythropoietic protoporphyria.

Abstract Background Several scientific contributions have summarized the “lessons learnt” during the coronavirus disease 2019 (COVID-19) pandemic, but only a few authors have discussed what we have learnt on how to design and conduct research during a pandemic. The main intent of this study was to summarize the lessons learnt by an Italian multidisciplinary research group that developed and conducted a longitudinal study on COVID-19 patients infected during the first wave in March 2020 and followed-up for 3 years. Methods A qualitative research approach embedded into the primary CORonavirus MOnitoRing study (CORMOR) study was developed, according to the the consolidated criteria for reporting qualitative research. Multiple data collection strategies were performed: each member was invited to report the main lessons learnt according to his/her perspective and experience from the study design throughout its conduction. The narratives collected were summarized and discussed in face-to-face rounds. The narratives were then thematically analysed according to their main topic in a list that was resent to all members to check the content and their organization. The list of the final “lessons learnt” has been agreed by all members, as described in a detailed fashion. Results Several lessons were learnt while designing and conducting a longitudinal study during the COVID-19 pandemic and summarised into ten main themes: some are methodological, while others concern how to conduct research in pandemics/epidemics/infectious disease emergencies. Conclusions The multidisciplinary approach, which also included patients’ perspective, helped us to protect the consistency and quality of the research provided in pandemic times. The lesson learnt suggest that our research approach may benefit from changes in education, clinical practice and policies.

Abstract Background Serological testing of patients consulting for typical erythema migrans (EM) is not recommended in European recommendations for diagnosis of Lyme borreliosis (LB). Little is known on the level of adherence of French general practitioners to these recommendations. The objectives were to estimate the proportion of Lyme borreliosis serological test prescription in patients with erythema migrans seen in general practice consultations in France, and to study the factors associated with this prescription. Methods LB cases with an EM reported by the French general practitioners (GPs) of the Sentinelles network between January 2009 and December 2020 were included. To assess the associations with a prescription of a serological test, multilevel logistic regression models were used. Results Among the 1,831 EM cases included, a prescription for a LB serological test was requested in 24.0% of cases. This proportion decreased significantly over the study period, from 46.8% in 2009 to 15.8% in 2020. A LB serological prescription was associated with patients with no reported tick bite (Odds Ratio (OR): 1.95; 95% confidence interval [1.23–3.09]), multiple EM (OR: 3.82 [1.63–8.92]), EM of five centimeters or more (OR: 4.34 [2.33–8.08]), and GPs having diagnosed less than one EM case per year during the study period (OR: 5.28 [1.73–16.11]). Conclusions Serological testing of patients consulting for EM is not recommended in European recommendations for diagnosis of Lyme borreliosis. Therefore, the significant decrease in the rate of LB serological test for EM over the study period is encouraging. The factors identified in this study can be used to improve messaging to GPs and patients. Further efforts are needed to continue to disseminate diagnostic recommendations for LB to GPs, especially those who rarely see patients with EM.

Abstract Background The present study investigates whether epigenetic differences emerge in the heart of patients undergoing cardiac surgery for an aortic valvular replacement (AVR) or coronary artery bypass graft (CABG). An algorithm is also established to determine how the pathophysiological condition might influence the human biological cardiac age. Results Blood samples and cardiac auricles were collected from patients who underwent cardiac procedures: 94 AVR and 289 CABG. The CpGs from three independent blood-derived biological clocks were selected to design a new blood- and the first cardiac-specific clocks. Specifically, 31 CpGs from six age-related genes, ELOVL2, EDARADD, ITGA2B, ASPA, PDE4C, and FHL2, were used to construct the tissue-tailored clocks. The best-fitting variables were combined to define new cardiac- and blood-tailored clocks validated through neural network analysis and elastic regression. In addition, telomere length (TL) was measured by qPCR. These new methods revealed a similarity between chronological and biological age in the blood and heart; the average TL was significantly higher in the heart than in the blood. In addition, the cardiac clock discriminated well between AVR and CABG and was sensitive to cardiovascular risk factors such as obesity and smoking. Moreover, the cardiac-specific clock identified an AVR patient's subgroup whose accelerated bioage correlated with the altered ventricular parameters, including left ventricular diastolic and systolic volume. Conclusion This study reports on applying a method to evaluate the cardiac biological age revealing epigenetic features that separate subgroups of AVR and CABG.

Abstract Background COVID-19 had devastating effects on children’s and adolescents’ life, including neuropsychological impairment, discontinuation of social life and education. Since June 2021, antiCOVID19 vaccination has become available to adolescents in Italy up to 12 years and since December 2021 to children aged more than 5 years. The pediatric population represents a challenging target for vaccination. Aim of the study is to perform a survey among adolescents to explore factors associated with COVID 19 immunization and their perceptions about COVID-19 vaccines. Methods Italian students aged 10–17 years were invited to participate in an anonymous online survey regarding their immunization against COVID-19 and their opinion on the immunization practice through a web link to the questionnaire. The study period was March-June 2022. Statistical analysis was performed with SPSS v 21. Results In the study period, 895 students entered the survey. A total of 87.3% of respondents were immunized against SARS-CoV2. The most important predictors of being immunized against SARS-CoV2 were having both parents immunized (p

Abstract Background: There is limited information to compare the qualitative and semi-quantitative performance of rapid diagnostic tests (RDT) and serology for the assessment of antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Therefore, the objective of the study was (a) to compare the efficacy of SARS-CoV-2 antibody detection between RDT and laboratory serology, trying to identify appropriate semi-quantitative cut-offs for RDT in relation with quantitative serology values and to (b) evaluate diagnostic accuracy of RDT compared to the NAAT gold standard in an unselected adult population. Methods: SARS-CoV-2 antibodies were simultaneously measured with lateral flow immunochromatographic assays (LFA), the Cellex qSARS-CoV-2 IgG/IgM Rapid Test (by capillary blood), the iFlash-SARS-CoV-2 IgG/IgM chemiluminescent immunoassay (CLIA) (by venous blood) and the nucleic acid amplification test (NAAT) in samples from in- and out-patients with confirmed, suspected and negative diagnosis of coronavirus disease 2019 (COVID-19) attending Udine Hospital (Italy) (March-May 2020). Interpretation of RDT was qualitative (positive/negative) and semi-quantitative based on a chromatographic intensity scale (negative, weak positive, positive). Results: Overall, 720 paired antibody measures were performed on 858 patients. The qualitative and semiquantitative agreement analysis performed in the whole sample between LFA and CLIA provided a Kendall’s tau of 0.578 (p

Abstract Background Extracapsular proximal femur metastasis could be treated by synthesis or resection and megaprosthesis. No universal accepted guidelines are present in the literature. The aim of our study is to analyze of patients with metastases in the trochanteric region of the femur treated by a single type of intramedullary nailing or hip megaprosthesis. Methods We retrospectively reviewed all patients affected by extracapsular metastases of proximal femur. Anthropometric and anamnestic data, routine blood exams and complications were collected. VAS score and MSTS score was administered before the surgery, ad 1–6-12 months after surgery. An un-paired T test and Chi-square were used. Multiple linear regression and logistic regression was performed. Significance was set for p

Abstract Background Early start of highly active antiretroviral therapy (HAART) in perinatally HIV-1 infected children is the optimal strategy to prevent immunological and clinical deterioration. To date, according to EMA, only 35% of antiretroviral drugs are licenced in children 25%. At last check, during the off label regimen, the 80% (40/50) of patients had an undetectable VL, and 90% (45/50) of them displayed CD4 + T lymphocyte percentage > 25%. The most widely used off-label drugs were: dolutegravir/abacavir/lamivudine (16%; 8/50), emtricitbine/tenofovir disoproxil (22%; 11/50), lopinavir/ritonavir (20%; 10/50) and elvitegravir/cobicistat/emtricitabine/ tenofovir alafenamide (10%; 10/50). At logistic regression analysis, detectable VL before starting the current HAART regimen was a risk factor for receiving an off-label therapy (OR: 2.41; 95% CI 1.13–5.19; p = 0.024). Moreover, children

Abstract Background Several studies have evaluated the survivorship and clinical outcomes of proximal femoral replacement (PFR) in complex primary and revision total hip arthroplasty with severe proximal femoral bone loss; however, there remains no consensus on the overall performance of this implant. We therefore performed a systematic review of the literature in order to examine survivorship and complication rates of PFR usage. Methods A systematic review of the literature according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines was performed. A comprehensive search of PubMed, MEDLINE, EMBASE, and the Cochrane Database of Systematic Reviews was conducted for English articles using various combinations of keywords. Results In all, 18 articles met the inclusion criteria. A total of 578 PFR were implanted. The all-cause reoperation-free survivorship was 76.6%. The overall complication rate was 27.2%. Dislocation was the most common complication observed and the most frequent reason for reoperation with an incidence of 12.8 and 7.6%, respectively. Infection after PFR had an incidence of 7.6% and a reoperation rate of 6.4%. The reoperation rate for aseptic loosening of the implant was 5.9%. Overall, patients had improved outcomes as documented by postoperative hip scores. Conclusion PFR usage have a relatively high complication rate, however, it remains an efficacious treatment option in elderly patients with osteoporotic bone affected by severe proximal femoral bone loss. Modular designs have shown reduced dislocations rate and higher survivorship free from dislocation. However, PFR should only be used as salvage procedure when no other reconstruction options are available.

Abstract Recurrent respiratory infections (RRIs) are a common clinical condition in children, in fact about 25% of children under 1 year and 6% of children during the first 6 years of life have RRIs. In most cases, infections occur with mild clinical manifestations and the frequency of episodes tends to decrease over time with a complete resolution by 12 years of age. However, RRIs significantly reduce child and family quality of life and lead to significant medical and social costs. Despite the importance of this condition, there is currently no agreed definition of the term RRIs in the literature, especially concerning the frequency and type of infectious episodes to be considered. The aim of this consensus document is to propose an updated definition and provide recommendations with the intent of guiding the physician in the complex process of diagnosis, management and prevention of RRIs.

Abstract Background The debate on the best treatment strategy for atrial fibrillation (AF) has expanded following the introduction of the so-called “hybrid procedure” that combines minimally invasive epicardial ablation with endocardial catheter ablation. However, the advantage of the hybrid approach over conventional epicardial ablation remains to be established. Methods From June 2008 to December 2020, 609 surgical AF ablation procedures through a right minithoracotomy were performed at our institution. From 2008 to 2011, a unipolar radiofrequency (RF) device was used, whereas from 2011 to 2020 a bipolar RF device was used. In addition, between September 2016 and April 2017, 60 patients underwent endocardial completion of epicardial linear ablation. In 30 of these latter patients, surgical isolation of the Bachmann’s bundle (BB) was also performed. Starting from 2021, surviving patients at follow-up were asked to undergo electrocardiographic evaluation and left ventricular function assessment and to complete a questionnaire addressing quality of life and predisposing factors for recurrent AF. Results The ablation procedure was completed in all patients. Upon discharge, 30 (4.9%) patients showed recurrence of AF, whereas the remaining patients (95.1%) were in sinus rhythm. All patients in whom a hybrid approach was used either with or without BB ablation were discharged in sinus rhythm. After a mean follow-up of 74 months, 122 (20%) patients developed recurrent AF, including 19.9% in whom a unipolar RF device was used, 21% in whom a bipolar RF device was used, 23% who had undergone a hybrid procedure without BB ablation and 3.3% who had undergone a hybrid procedure with BB ablation. On multivariate analysis, reduced left ventricular ejection fraction, worsening of European Heart Rhythm Association symptom class, and cognitive impairment or depression during follow-up were found to be significantly associated with AF recurrence. Conclusions Surgical AF ablation through a right minithoracotomy is safe and may allow the creation of additional linear lesions, particularly in the BB. The placement of adjunctive linear lesions in the setting of a hybrid procedure can be more effective in reducing the risk for AF recurrence than isolated surgical ablation or hybrid ablation without the addition of further linear lesions, with no incremental risk to the patient.

Abstract Background The use of Spinal Cord Stimulation (SCS) system to treat medically refractory neuropathic pain is increasing. Severe neuropathic pain can be found in giant chest wall arteriovenous malformations (AVMs), exceedingly rare and debilitating abnormalities, rarely reported during pregnancy. Case presentation We present a report of a pregnant patient with implanted Spinal Cord Stimulation (SCS) system because of painful thoracic AVM scheduled for an urgent cesarean section in which we used lumbar ultrasound (US) to rule out the possibility to damage SCS electrodes and to find a safe site to perform spinal anesthesia. Conclusions The use of lumbar US to find a safe site for a lumbar puncture in presence of SCS system in a patient affected by painful thoracic AVM makes this case a particularly unique operative challenge and offers a new possible use of ultrasound to detect a safe space in patients with SCS implant.

Abstract Background Borrelia miyamotoi is a relapsing fever Borrelia species transmitted by ticks of the Ixodes ricinus complex. Human disease caused by B. miyamotoi was first described in Russia and later in the USA and Japan. Additionally, five cases of meningoencephalitis in immunocompromised patients and one case in an apparently immunocompetent patient were described. Methods We investigated the presence of B. miyamotoi in I. ricinus nymphs and in patients suspected of human granulocytic anaplasmosis, in Alsace (France), an endemic area for I. ricinus ticks and Lyme borreliosis, using direct (PCR) and indirect diagnosis (glycerophosphoryldiester-phosphodiesterase (GlpQ) serology). Results Borrelia miyamotoi was found in 2.2% of 4354 ticks collected between 2013 and 2016. None of the 575 blood samples, collected from the patients suspected of HGA, was found positive for B. miyamotoi by PCR. Acute and late sera from 138 of these 575 patients were available. These paired sera were tested for IgM and IgG antibodies against the B. miyamotoi GlpQ antigen. A total of 14 out of 138 patients had at least one positive parameter (i.e. anti-GlpQ IgG and/or IgM). One patient seroconverted for IgG, and three had isolated IgM in the acute serum. These three patients were treated with doxycycline which could have prevented seroconversion. After reviewing clinical data and other biological tests performed, co-exposure among different microorganisms vectored by ticks or serological cross-reactivity could not be ruled out in these different cases. One patient had persistent IgG, which strongly suggests previous exposure to B. miyamotoi. Conclusions Humans can be exposed to B. miyamotoi through tick bites in Alsace. We present serological data for possible B. miyamotoi exposure or infection of patients with fever after tick bite. Future studies should determine the incidence, clinical course and burden of this emerging tick-borne disease in other parts of Western Europe.

Abstract Background Massive hemoptysis is a serious complication in Cystic Fibrosis (CF), occurring commonly in older patients. Bronchial artery embolization (BAE) can be performed to stop the bleeding. BAE is generally safe and effective, but can sometimes lead to serious complications. We report the first case of temporary unilateral diaphragmatic paralysis associated to lung consolidation following BAE in a pediatric CF female patient. This complication worsened the lung function of the patient who underwent lung transplantation after 9 months. Case presentation A 14 years old female CF patient followed by the CF center of Florence presented low-grade fever, cough increase and recurrent episodes of major hemorrhages such as to carry out a BAE. Within 24 h the patient started to complain of severe thoracic pain in the right hemithorax, increased dyspnea and fever. A computed tomographic angiography and a dynamic fluoroscopic evaluation revealed the right diaphragmatic paralysis, not present before the procedure. After 4 days the clinical condition and radiological imaging had improved with restored mobility of the right hemidiaphragm. Nine months later, she required mechanical ventilation, and subsequently the initiation of extracorporeal membrane oxygenation (ECMO) for a pulmonary exacerbation with septic shock. Lung transplantation in ECMO was performed with success. Conclusion Clinicians should be aware of the possibility of phrenic nerve injury with BAE in pediatric CF patients.

Abstract Severe asthma has a substantial epidemiological impact on children and biological treatments can be an option to take into account, as they target specific molecules and pathways involved in its pathogenesis. Modern medicine is continuously and progressively oriented towards tailored treatments designed specifically for the pathology patterns observed in individual patients and identified as endotypes with associated biomarkers. In this regard, biologic treatments in asthma are one of the best examples. Among the biological drugs currently available, omalizumab is the one with the greatest amount of data on efficacy and safety, and the one we have more real-life clinical experience with. However, mepolizumab will likely be accessible soon globally for clinical use. Moreover, research on biological drugs for the treatment of severe asthma is expanding rapidly, with some molecules currently used in adult patients that could be registered also for pediatric use and new molecules that could be available in the future. On the other hand, due to this potential abundance of therapeutic options, new criteria could become necessary to guide clinicians through an evidence-based choice between omalizumab and these new drugs. For the same reason, more data collected specifically from pediatric clinical trials are necessary. In this review we aim to analyze the factors that could help clinicians make their choice and to highlight the unmet need for a more evidence-based choice.

Abstract Background To best of our knowledge, this is the first quantitative ethnobotanical study with the aim of documenting the local knowledge and practices of using plants for curing diseases in the Cava de’ Tirreni area, Salerno Province, Campania Region, Italy. The present ethnobotanical field study, carried out during 2016–2017, documents the local uses of 119 plant species for medicinal, food and domestic purposes. Methods Ethnobotanical data were documented from 70 informants: field data were collected and information on the uses of plants was gathered through semi-structured and structured interviews with persons who still retain traditional ethnobotanical knowledge. Documented data were evaluated using the quantitative ethnobotanical index of use value (UV). Results Overall, the informants native of the area were interviewed and 277 use-reports have been recorded. The scientific names, local names, plant part used, preparation and administration processes are reported and compared with practices in other Southern Italian regions. In total, 101 species are documented as medicinal, 36 as food or food aromatizer, 29 for domestic and handicraft uses, 10 in veterinary medicine. More or less 64% of all species have more uses and over half of the food plants (23 species) are also used for medicinal purposes. Conclusions The comparison of the documented species and their uses with ethnobotanical literature of other Italian regions reveals that the traditional plant knowledge in this area shows strong similarities with adjacent Southern Italian areas. Some of the recorded species and administration processes however seem to be unique for the zone.

Abstract Background Glioma is the most common and primary brain tumors in adults. Despite the available multimodal therapies, glioma patients appear to have a poor prognosis. The Hedgehog (Hh) signaling is involved in tumorigenesis and emerged as a promising target for brain tumors. Glabrescione B (GlaB) has been recently identified as the first direct inhibitor of Gli1, the downstream effector of the pathway. Methods We established the overexpression of Gli1 in murine glioma cells (GL261) and GlaB effect on cell viability. We used 1H-nuclear magnetic resonance (NMR) metabolomic approach to obtain informative metabolic snapshots of GL261 cells acquired at different time points during GlaB treatment. The activation of AMP activated protein Kinase (AMPK) induced by GlaB was established by western blot. After the orthotopic GL261 cells injection in the right striatum of C57BL6 mice and the intranasal (IN) GlaB/mPEG5kDa-Cholane treatment, the tumor growth was evaluated. The High Performance Liquid Chromatography (HPLC) combined with Mass Spectrometry (MS) was used to quantify GlaB in brain extracts of treated mice. Results We found that GlaB affected the growth of murine glioma cells both in vitro and in vivo animal model. Using an untargeted 1H-NMR metabolomic approach, we found that GlaB stimulated the glycolytic metabolism in glioma, increasing lactate production. The high glycolytic rate could in part support the cytotoxic effects of GlaB, since the simultaneous blockade of lactate efflux with α-cyano-4-hydroxycinnamic acid (ACCA) affected glioma cell growth. According to the metabolomic data, we found that GlaB increased the phosphorylation of AMPK, a cellular energy sensor involved in the anabolic-to-catabolic transition. Conclusions Our results indicate that GlaB inhibits glioma cell growth and exacerbates Warburg effect, increasing lactate production. In addition, the simultaneous blockade of Gli1 and lactate efflux amplifies the anti-tumor effect in vivo, providing new potential therapeutic strategy for this brain tumor. Graphical Abstract

Abstract Background Vaccination represents one of the most effective means of preventing infections for the population and for the public health in general. Recently there has been a decline in vaccinations, also among healthcare workers (HCWs). The aim of the study is to detect the knowledge, skills, attitudes and barriers of HCWs regarding vaccinations in a tertiary children’s hospital in order to support clinical management in immunisation practices. Methods An observational study was conducted on 255 subjects over a period of 8 months. The 31-item questionnaire considered profession, level of instruction and different ages. It included questions taken from a questionnaire used for a Canadian research and one used by the Bellinzona hospital. A 4-point Likert scale and closed-ended questions were used. A confidence interval of 95%, p value ≤ 0.05, Chi-square, ANOVA and the Kruskal-Wallis test were considered. Results In the last 5 years less than one third of the sample were vaccinated against flu. 77.8% (n.130) of nurses and 45.8% (n.19) of doctors were not vaccinated (p

Abstract Volcanic islands pose several major types of natural hazards, often interconnected and concentrated in relatively small areas. The quantification of these hazards must be framed from a multi-hazard perspective whilst building on existing single-hazard analyses. Ischia is a densely inhabited volcanic island with a long eruptive history lasting more than 150 ka (last in 1302 AD) characterized by the significant asymmetric resurgence of a caldera block. Here, we review the state-of-art of the natural hazards of Ischia, aiming at building a solid base for future holistic multi-hazard quantifications. We frame our analysis in three steps: i) review of geological, historical and current activity; ii) review of available hazard models and analyses; iii) development of an interpretative framework for the interdependent hazards. The results highlight that volcanic activity has been quite intense and many volcano-related hazardous phenomena have affected the island including in very recent times, both for eruptive (phreatic or magmatic eruptions) and non-eruptive (earthquakes, landslides, and tsunamis) phenomena. The effects of some of them (e.g. tsunamis, tephra) are also relevant beyond the island territory. Quantitative hazard assessments are almost absent and should be developed in the future considering the evident interconnections between hazards. To this end, we propose a conceptual interpretative multi-hazard framework that highlights the fundamental role played by the resurgent block in controlling and connecting the different hazards, in terms of both spatial distribution of the sources and temporal clustering.

Abstract Background Malignant spinal cord compression (MSCC) is associated withpoor prognosis and may lead to permanent paralysis, sensory loss, and sphincter dysfunction. Very limited data are available on incidence and etiology of MSCC in pediatric population. We aimed to examine etiology, clinical presentation and treatment of pediatric patient with MSCC admitted to the Santobono-Pausilipon Children’s Hospital, Naples, Italy. Methods Forty-four children under 18 yearsadmitedsince 2007 and assessed for MSCC clinical presentations, evaluation, and treatment.were retrospectively collected from our institutional pediatric oncology and neurosurgery database. Results The median age at time of MSCC diagnosis was 52 months, with a peak in young (≤3 years) patients. The leading cause of MSCC was extramedullary tumors (63.6%), in particular neuroblastoma (27.2%) followed by Ewing sarcomas (15.9%). Cord compression was the presenting feature of a new malignancy in 33 (75%) patients, and a consequence of metastatic disease progression or relapse in the remaining 11 (25%) patients. Motor deficit was the initial symptoms of spinal compression in all patients, while pain was present in about 60% of patients, followed by sphincteric deficit (43.2%). The primary tumor site was located in the neck in 3 (6.8%) patients, thorax in 16 (36.4%), cervico-thoracic region in 3 (6.8%), thoraco-lumbar region in 8 (18.2%), abdomen in 5 (11.4%), lumbar-sacral region in 7 (15.9%) and thoracic-lumbar-sacral region in 1 (2.3%). The median length of the interval between symptom onset and tumor diagnosis varied widely from 0 to 360 days in the entire population, however this interval was significantly shorter in patients with known neoplasia in comparisonto patients with new diagnosis (at relapse 7 days [interquartile range 3–10] vs at diagnosis 23 days [7–60]). Pre and post-operative spine magnetic resonance imagingwas performed in all cases, and most(95%) patients underwent neurosurgical treatment as first treatment. Severe motor deficit was associated with younger age and severe motor deficit at diagnosis was associated withworst motor outcomes at discharge from neurosurgery. Patients with progression or relapsed disease showed a worst prognosis, while the majority of patients (70.5%) were alive at 5 years after diagnosis. Conclusions The natural history of MSCC in children is associated to permanent paralysis, sensory loss, and sphincter dysfunction, thus prompt diagnosis and correct management are needed to minimize morbidity. Treatment strategies differed widely among cancer types and study groups in the absence of optimal evidence-based treatment guidelines. When the diagnosis is uncertain, surgery provides an opportunity to biopsy the lesion in addition to treating the mass.

Abstract Background Bovine surfactants are known to be clinically equivalent but it is unclear if porcine or bovine surfactants at their licensed dose should be preferred to treat respiratory distress syndrome in preterm neonates. Methods We performed a comprehensive review of biochemical and pharmacological features of surfactants to understand the biological plausibility of any clinical effect. We then performed a pragmatic meta-analysis comparing internationally marketed porcine and bovine surfactants for mortality and respiratory outcomes. Search for randomised controlled trials with no language/year restrictions and excluding “grey” literature, unpublished or non-peer reviewed reports was conducted, following Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines and the most recent methodological recommendations. Results Sixteen articles were included in the review and 14 in the meta-analysis (1491 neonates). 200 mg/kg poractant-α (a porcine surfactant) was associated with lower BPD/mortality (OR 0.632[95%CI:0.494, 0.809];p

Abstract Background Diagnosis and treatment of primary lung adenocarcinoma in children remains challenging given its rarity. Here we highlight the clinical history, pathological evaluation, genomic findings, and management of a very young patient with metastatic lung adenocarcinoma. Case presentation A 10-year-old white girl presented with brain metastases due to primary pulmonary adenocarcinoma. Next generation sequencing analysis with “Comprehensive Cancer Panel” highlighted the presence of multiple non-targetable mutations in the FLT4, UBR5, ATM, TAF1, and GUCY1A2 genes. She was treated aggressively with chemotherapy, surgery, and radiation therapy for local and distant recurrence. Eventually, therapy with nivolumab was started compassionately, and she died 23 months after diagnosis. Conclusions Extremely rare cancers in children such as lung adenocarcinoma need accurate and specific diagnosis in order to develop an optimal plan of treatment. It is also necessary to underline that “children are not little adults,” thus implying that an adult-type cancer in the pediatric population might have a different etiopathogenesis. Diagnostic confirmation and primary treatment of such rare conditions should be centralized in reference centers, collaborative networks, or both, with multidisciplinary approaches and very specific expertise.

Abstract Background Cobalamin C (cblC) defect is the most common inborn error of Vitamin B12 metabolism often causing severe neurological, renal, gastrointestinal and hematological symptoms. Onset with pulmonary hypertension (PAH) and atypical hemolytic-uremic syndrome (aHUS) is rare. Case presentation We describe the case of a 2-years old child, previously in good health, admitted to the hospital with severe respiratory symptoms, rapid worsening of clinical conditions, O2 desaturation and palmo-plantar edema. The patient showed PAH and laboratory findings compatible with aHUS. cblC defect, an inborn error of metabolism, was identified as the cause of all the symptoms described (cardiac, respiratory and renal involvement). Results of neonatal screening for inborn errors of metabolism had been negative. Administration of IM OHCbl (intramuscular hydroxocobalamin), oral betaine and symptomatic treatment with diuretics and anti-hypertensive systemic and pulmonary drugs induced dramatic improvement of both cardiac and systemic symptoms. Conclusions In this case of cblC defect the metabolic treatment completely reverted symptoms of aHUS and PAH. The course was favorable, and the prognosis is what we foresee for the future.

Abstract Background Invasive bacterial diseases (IBD) are a serious cause of hospitalization, sequelae and mortality. Albeit a low incidence, an increase in cases due to H. influenzae was registered in the past 4 years and, in the Tuscany region, an excess of cases due to N. meningitidis since 2015 is alarming. The purpose of this study is to deepen the knowledge of IBD epidemiology in Tuscany with particular attention to temporal trends. Methods Tuscan residents hospitalized for IBD from January 1st 2000 to March 18th 2016 were selected from the regional hospital discharge database based on ICD-9-CM codes. Age-specific and standardized hospitalization rates were calculated together with case-fatality rates (CFRs). A time-trend analysis was performed; whereas, prognostic factors of death were investigated through univariable and multivariable analyses. Results The average standardized hospitalization rates for invasive meningococcal diseases (IMD), invasive pneumococcal diseases and invasive diseases due to H. influenzae from 2000 to 2016 were 0.6, 1.8, and 0.2 per 100,000, respectively. The average CFRs were 10.5%, 14.5% and 11.5% respectively with higher values in the elderly. Older age was significantly associated with higher risk of death from all IBD. A significant reduction in hospitalization rates for IMD was observed after meningococcal C conjugate vaccine introduction. The Annual Percentage Change (APC) was -13.5 (95% confidence interval (CI) -22.3; -3.5) in 2005–2013 but has risen since that period. Furthermore, a significant increasing trend of invasive diseases due to H. influenzae was observed from 2005 onwards in children 1–4 years old (APC 13.3; 95% CI 0; 28.3). Conclusions This study confirms changes in the epidemiology of invasive diseases due to H. influenzae and IMD. Furthermore, attention is called to the prevention of IBD in the elderly because of the age group’s significantly higher rate of hospitalizations and deaths for all types of IBD.

Abstract Background Fever phobia is still a major issue in paediatrics. We report knowledge of a sample of Italian paediatricians performed six years after the release of the Italian guidelines for the management of fever in children (IFG). Methods A questionnaire, developed following the IFG recommendations and previously administered to 300 paediatricians in 2012, was proposed to all the paediatricians attending the 2015 National Congress of Practice Paediatrics, held in Florence, Italy. Changes in answers over time were analyzed. Results 70.2% (562/800) paediatricians returned the questionnaire. The recommended site and device for body temperature measurement in children > 1 year was correctly chosen by 89.3% of participants (vs. 80.7% of 2012 participants; P

Abstract Background Sarcoidosis in pediatric age is uncommon and challenging diagnosis, because manifestations can be significantly variable and non-specific since it is a multisystem disease, and virtually any organ system may be involved. Case presentation In this report, we describe the case of a 12-year-old girl presenting with fatigue and weight loss, with a painless hepato-splenomegaly without additional clinical signs on physical examination. In our patient, once we had ruled out infections, malignancies and granulomatous diseases of childhood, we made diagnosis of sarcoidosis, finding suggestive histological features in two different tissues (liver and lymph nodes) with lung involvement. Conclusions Our case points out that pediatricians should consider sarcoidosis in the differential diagnosis in case of systemic symptoms, even in absence of other specific clinical clues, because they represent the most common clinical manifestations on presentation in children, in order to refer promptly the young patient to specialist evaluation.

Abstract Background Lyme borreliosis in humans results in a range of clinical manifestations, thought to be partly due to differences in the pathogenicity of the infecting strain. This study compared European human clinical strains of Borreliella afzelii (previously named Borrelia afzelii) using multilocus sequence typing (MLST) to determine their spatial distribution across Europe and to establish whether there are associations between B. afzelii genotypes and specific clinical manifestations of Lyme borreliosis. For this purpose, typing was performed on 63 strains, and data on a further 245 strains were accessed from the literature. Results All 308 strains were categorized into 149 sequence types (STs), 27 of which are described here for the first time. Phylogenetic and goeBURST analyses showed short evolutionary distances between strains. Although the main STs differed among the countries with the largest number of strains of interest (Germany, the Netherlands, France and Slovenia), the B. afzelii clinical strains were less genetically structured than those previously observed in the European tick population. Two STs were found significantly more frequently in strains associated with clinical manifestations involving erythema migrans, whereas another ST was found significantly more frequently in strains associated with disseminated manifestations, especially neuroborreliosis. Conclusions The MLST profiles showed low genetic differentiation between B. afzelii strains isolated from patients with Lyme borreliosis in Europe. Also, clinical data analysis suggests the existence of lineages with differential dissemination properties in humans.

Abstract Background Gender-based medicine is an innovative branch of biomedical research and represents a new perspective for the future of health research. Many studies have been published on gender medicine in adults but very few data regarding children are available. Literature search and results A literature search covering articles published between 1stJuly, 2006 and 1st February, 2017 and concerning children only was conducted using multiple keywords and standardized terminology in Pubmed database. The search was limited to English-language publications. All relevant articles on endocrines, neurological, psychiatric, gastrointestinal, immunological, oncological, rheumatic, pneumological disorders, infectious diseases and analgesia were evaluated and pertinent articles were included in this review. Most of the available studies on gender disparity in childhood are about endocrine and neuro-psychiatric disorders, while there are few data in other areas of medicine. Conclusions Even if several studies on pediatric gender differences can be found on literature, few of them move forwards to analyze the reasons of the observed diversity. No data on pharmacokinetic and pharmacodynamic differences between boys and girls can be found. Hence, more efforts should be directed to investigate these topics in childhood.

Abstract Background Invasive micropapillary carcinoma of the breast (IMPC) is a histological tumor variant that occurs with low frequency characterized by an inside-out formation of tumor clusters with a pseudopapillary arrangement. IMPC is an aggressive tumor with poor clinical outcome. In addition, this histological subtype usually expresses human epidermal growth factor receptor 2 (HER2) which also correlates with a more aggressive tumor. In this work we studied the clinical significance of IMPC in HER2-positive breast cancer patients treated with adjuvant trastuzumab. We also analyzed mucin 4 (MUC4) expression as a novel biomarker to identify IMPC. Methods We retrospectively studied 86 HER2-positive breast cancer patients treated with trastuzumab and chemotherapy in the adjuvant setting. We explored the association of the IMPC component with clinicopathological parameters at diagnosis and its prognostic value. We compared MUC4 expression in IMPC with respect to other histological breast cancer subtypes by immunohistochemistry. Results IMPC, either as a pure entity or associated with invasive ductal carcinoma (IDC), was present in 18.6% of HER2-positive cases. It was positively correlated with estrogen receptor expression and tumor size and inversely correlated with patient’s age. Disease-free survival was significantly lower in patients with IMPC (hazard ratio = 2.6; 95%, confidence interval 1.1–6.1, P = 0.0340). MUC4, a glycoprotein associated with metastasis, was strongly expressed in all IMPC cases tested. IMPC appeared as the histological breast cancer subtype with the highest MUC4 expression compared to IDC, lobular and mucinous carcinoma. Conclusion In HER2-positive breast cancer, the presence of IMPC should be carefully examined. As it is often not informed, because it is relatively difficult to identify or altogether overlooked, we propose MUC4 expression as a useful biomarker to highlight IMPC presence. Patients with MUC4-positive tumors with IMPC component should be more frequently monitored and/or receive additional therapies.

Abstract Background The incidence of gemcitabine-induced hemolytic uremic syndrome has already been described in adults. Several approaches have been employed in the treatment of gemcitabine-induced hemolytic uremic syndrome with different outcomes. One of the most promising agents is eculizumab, which is a monoclonal antibody directed against C5 complement protein. Case presentation We reported the case of a 3-year-old white boy with medulloblastoma who underwent high-dose chemotherapy and craniospinal irradiation. Afterwards he started maintenance chemotherapy with gemcitabine and oxaliplatin. After five courses he presented a progressive clinical worsening, which resulted in a systemic thrombotic microangiopathy. Initially he was treated with rituximab without clinical improvement. Therefore he started therapy with repeated cycles of eculizumab. After seven infusions he showed a gradual improvement and finally a complete remission of gemcitabine-induced hemolytic uremic syndrome. Conclusions Eculizumab prevents serious complement-mediated vascular damage for chemotherapy-induced thrombotic microangiopathy in pediatric cases.

Abstract Background The basophil activation test (BAT), has been proposed as a possible assay for the diagnosis of immediate-type allergy to beta-lactams (BLs). The aim of this study was to assess the utility of BAT in the diagnosis of amoxicillin (AMX) or AMX-clavulanate (AMX-C) IgE-mediated hypersensitivity in children and adults. Material and methods Eighteen children and 21 adults, with clinical history of immediate reactions to AMX or AMX-C, were referred to Anna Meyer Children’s Hospital and San Giovanni di Dio Hospital, respectively. They underwent in vivo tests (skin prick test and intradermal test). Moreover, BAT with AMX or AMX-C was performed within 6 months from the reaction. Results In the pediatric group, the concordance between the skin tests (ST) and BAT results was 83.3%. Upon comparing the symptom grades and ST results to the BAT results, we found that the reaction severity and ST positivity did not correlate with BAT results in children. In the adult group, the concordance between the ST and BAT results was 61.9%. Upon comparing patients with severe reactions and patients with mild reactions in terms of BAT results, we found a BAT sensitivity of 38.5% and a specificity of 100%. When comparing the symptom grades to the BAT results, we found that no patients with mild symptoms had a positive BAT result, whereas 38.5% of patients with severe symptoms had a positive BAT result. Conclusions BAT does not seem to be a useful tool to increase the sensitivity of an allergy work-up to diagnose immediate hypersensitivity to AMX or AMX-C.

Abstract Background Nontuberculous mycobacteria (NTM) are pathogens that commonly affect the paediatric population and its most frequent manifestation is a cervicofacial lymphadenopathy. With the improvement of technologies, new species have been recently identified. Case presentation We report the first case of NMT lymphadenitis in a child caused by Mycobacterium marseillense, a newly described species belonging to Mycobacterium avium complex. Conclusions Improving the identification of these newly discovered mycobacteria, further information will be available about their clinical involvement and their best treatment.

Abstract Background Borrelia mayonii is currently the latest species belonging to the Borrelia burgdorferi (sensu lato) complex to be discovered. Interestingly it is involved in human pathology causing a high fever. We looked for its presence in post- tick bite febrile patients as well as in Ixodes ricinus ticks in an endemic area of France. Results After ensuring that our molecular technics correctly detected B. mayonii, 575 patients and 3,122 Ixodes ricinus nymphs were tested. Neither B. mayonii nor another species of the B. burgdorferi (s.l.) complex previously not reported in Europe has been identified. Conclusions For now, B. mayonii seems to be an epiphenomenon. However, its discovery broadens the etiology of post-Ixodes bite febrile syndromes.

Abstract Background Guidelines for the management of fever in children have been recently published, however “fever phobia” is still spreading. To provide information which may sustain educational interventions tailored to our population we investigated the parental and medical knowledge and management of fever in preschool children. Methods A questionnaire was administered to a convenient sample of Italian parents and paediatricians. The questionnaire elicited information about definition and cause of fever, concerns about fever, method of temperature measurement, and treatment modalities. Results Overall, 388 parents and 480 paediatricians were interviewed. All the parents believed that fever could cause at least one harmful effect and 89.9% (n = 349) believed that, if left untreated, it can cause brain damage or seizures. Parents used multiple resources to obtain information about fever but 67.8% (n = 264) considered paediatricians as their primary resource. Several wrong behaviours were found in the same proportions among parents and paediatricians: 78.5% of paediatricians (n = 377) and 77.8% of parents (n = 302) used physical method to reduce fever (P = 0.867); 27.0% of paediatricians (n = 103) and 21.4% (n = 83) of parents declared to alternate ibuprofen and acetaminophen (P = 0.953). Differently, 73.1% (n = 351) of paediatricians preferred oral to rectal administration of antipyretics compared to 48.7% (n = 190) of parents (P Conclusions Paediatricians’ attitudes greatly influence the parental behaviours and beliefs. Implementation of educational programs regarding the management of the febrile child are needed in our setting.

Abstract Background Cardiac tuberculosis is rare and usually manifests as tuberculous pericarditis. Involvement of other part of the heart is unusual and descriptions in the pediatric literature are confined to few case reports regarding mainly myocardial tuberculosis. Case presentation We describe a case of pulmonary miliary tuberculosis associated with intracardiac left atrial tuberculoma in an immunocompetent eleven-month-old infant successfully treated with surgery and antituberculous therapy. Conclusion Although unusual, involvement of endocardium in disseminated tuberculosis should be kept in mind.

Abstract Streptococcal pharyngitis is a very common pathology in paediatric age all over the world. Nevertheless there isn't a joint agreement on the management of this condition. Some authors recommend to perform a microbiological investigation in suspected bacterial cases in order to treat the confirmed cases with antibiotics so to prevent suppurative complications and acute rheumatic fever. Differently, other authors consider pharyngitis, even streptococcal one, a benign, self-limiting disease. Consequently they wouldn't routinely perform microbiological tests and, pointing to a judicious use of antibiotics, they would reserve antimicrobial treatment to well-selected cases. It has been calculated that the number of patients needed to treat to prevent one complication after upper respiratory tract infections (including sore throat), was over 4000. Even the use of the Centor score, in order to evaluate the risk of streptococcal infection, is under debate and the interpretation of the test results may vary considerably. Penicillin is considered all over the world as first line treatment, but oral amoxicillin is also accepted and, due to its better palatability, can be a suitable option. Macrolides should be reserved to the rare cases of proved allergy to β-lactams. Cephalosporins can be used in patients allergic to penicillin (with the exception of type I hypersensibility) and have been also proposed to treat the relapses.