Functional differences in human aortic valve interstitial cells from patients with varying calcific aortic valve disease.

Calcific aortic valve disease (CAVD) is characterized by progressive stiffening of aortic valve (AV) tissues, inducing stenosis and insufficiency. Bicuspid aortic valve (BAV) is a common congenital defect in which the AV has two leaflets rather than three, with BAV patients developing CAVD decades years earlier than in the general population. Current treatment for CAVD remains surgical replacement with its continued durability problems, as there are no pharmaceutical therapies or other alternative treatments available. Before such therapeutic approaches can be developed, a deeper understanding of CAVD disease mechanisms is clearly required. It is known that AV interstitial cells (AVICs) maintain the AV extracellular matrix and are typically quiescent in the normal state, transitioning into an activated, myofibroblast-like state during periods of growth or disease. One proposed mechanism of CAVD is the subsequent transition of AVICs into an osteoblast-like phenotype. A sensitive indicator of AVIC phenotypic state is enhanced basal contractility (tonus), so that AVICs from diseased AV will exhibit a higher basal tonus level. The goals of the present study were thus to assess the hypothesis that different human CAVD states lead to different biophysical AVIC states. To accomplish this, we characterized AVIC basal tonus behaviors from diseased human AV tissues embedded in 3D hydrogels. Established methods were utilized to track AVIC-induced gel displacements and shape changes after the application of Cytochalasin D (an actin polymerization inhibitor) to depolymerize the AVIC stress fibers. Results indicated that human diseased AVICs from the non-calcified region of TAVs were significantly more activated than AVICs from the corresponding calcified region. In addition, AVICs from the raphe region of BAVs were more activated than from the non-raphe region. Interestingly, we observed significantly greater basal tonus levels in females compared to males. Furthermore, the overall AVIC shape changes after Cytochalasin suggested that AVICs from TAVs and BAVs develop different stress fiber architectures. These findings are the first evidence of sex-specific differences in basal tonus state in human AVICs in varying disease states. Future studies are underway to quantify stress fiber mechanical behaviors to further elucidate CAVD disease mechanisms.

Decreased serotonin transporter activity in the mitral valve contributes to progression of degenerative mitral regurgitation.

Degenerative mitral valve (MV) regurgitation (MR) is a highly prevalent heart disease that requires surgery in severe cases. Here, we show that a decrease in the activity of the serotonin transporter (SERT) accelerates MV remodeling and progression to MR. Through studies of a population of patients with MR, we show that selective serotonin reuptake inhibitor (SSRI) use and SERT promoter polymorphism 5-HTTLPR LL genotype were associated with MV surgery at younger age. Functional characterization of 122 human MV samples, in conjunction with in vivo studies in SERT-/- mice and wild-type mice treated with the SSRI fluoxetine, showed that diminished SERT activity in MV interstitial cells (MVICs) contributed to the pathophysiology of MR through enhanced serotonin receptor (HTR) signaling. SERT activity was decreased in LL MVICs partially because of diminished membrane localization of SERT. In mice, fluoxetine treatment or SERT knockdown resulted in thickened MV leaflets. Similarly, silencing of SERT in normal human MVICs led to up-regulation of transforming growth factor ß1 (TGFß1) and collagen (COL1A1) in the presence of serotonin. In addition, treatment of MVICs with fluoxetine not only directly inhibited SERT activity but also decreased SERT expression and increased HTR2B expression. Fluoxetine treatment and LL genotype were also associated with increased COL1A1 expression in the presence of serotonin in MVICs, and these effects were attenuated by HTR2B inhibition. These results suggest that assessment of both 5-HTTLPR genotype and SERT-inhibiting treatments may be useful tools to risk-stratify patients with MV disease to estimate the likelihood of rapid disease progression.

Cardiometabolic profiles in children and adults with overweight and obesity and down syndrome.

Individuals with Down syndrome (DS) are at increased risk for being overweight/obese, but the associated cardiometabolic risk (CR) is not clear. Cross-sectional anthropometric and clinical laboratory data from a multi-site, international cohort of individuals with DS were analyzed to determine cardiometabolic risk by reporting observed distributions of cardiometabolic biomarkers in overweight/obese individuals with DS throughout the lifespan. Descriptive statistics and regression analyses by age categories determined the distributive percentiles for cardiometabolic biomarkers and tested for adiposity as a predictor of CR. Across seven DS clinics, data were collected on 240 patients between the ages of 3 and 63 years, with one quarter overweight and three quarters obese among children and nearly all adults being obese. In children and adults, most cardiometabolic biomarker profiles showed distributive values within normal ranges. Blood lipids were positively associated with body mass index (BMI) in children (high density lipid-cholesterol, p = 0.01; low density lipid-cholesterol, p = 0.02). Levels of hs-CRP were elevated in both children and adults, with BMI positively associated with hs-CRP in adults with DS (p = 0.04). Liver enzyme values were positively associated with BMI in children and adults. The data suggest that in contrast to the general population, in individuals with Down syndrome, being overweight and obese does not appear to confer a significantly increased risk for cardiometabolic disease by biomarker profile. Individuals with DS who are overweight/obese appear to have unique cardiometabolic profiles unrelated to adiposity, notable for increased hs-CRP and normal HA1c levels.

Safety and Long-Term Immunogenicity of BNT162b2 Vaccine in Individuals with Down Syndrome.

We aimed to evaluate the safety and immunogenicity of the BNT162b2 vaccine in young people with Down syndrome (DS), and to compare their humoral immune response with those of the healthy controls (HC). Individuals with DS and HC received the BNT162b2 vaccine. Longitudinal blood samples were collected on the day of vaccination, twenty-one days after the first dose, seven days after the second dose, and six months after the first dose. Both the local and systemic adverse events reported by participants were mild. Pain at the injection site was the most reported local adverse event, while fever was the systemic adverse event. Humoral responses showed a significant increase of anti-S and anti-S trimeric antibody (Ab) levels after both doses of vaccine in both groups. In comparison with HC, Ab levels in individuals with DS were similar at T21, but significantly lower, both in terms anti-S and anti-S trimeric, at T28 (respectively p = 0.0003 and p = 0.0001). At T180 both groups showed a significant reduction of anti-S trimeric Ab levels compared to T28 (p = 0.0004 and p < 0.0001 for DS and HC, respectively). Individuals with DS exhibit a good humoral response to the BNT162b2 vaccine; however, similarly to in HC, the immune response wanes over time.

LPHN2 inhibits vascular permeability by differential control of endothelial cell adhesion.

Dynamic modulation of endothelial cell-to-cell and cell-to-extracellular matrix (ECM) adhesion is essential for blood vessel patterning and functioning. Yet the molecular mechanisms involved in this process have not been completely deciphered. We identify the adhesion G protein-coupled receptor (ADGR) Latrophilin 2 (LPHN2) as a novel determinant of endothelial cell (EC) adhesion and barrier function. In cultured ECs, endogenous LPHN2 localizes at ECM contacts, signals through cAMP/Rap1, and inhibits focal adhesion (FA) formation and nuclear localization of YAP/TAZ transcriptional regulators, while promoting tight junction (TJ) assembly. ECs also express an endogenous LPHN2 ligand, fibronectin leucine-rich transmembrane 2 (FLRT2), that prevents ECM-elicited EC behaviors in an LPHN2-dependent manner. Vascular ECs of lphn2a knock-out zebrafish embryos become abnormally stretched, display a hyperactive YAP/TAZ pathway, and lack proper intercellular TJs. Consistently, blood vessels are hyperpermeable, and intravascularly injected cancer cells extravasate more easily in lphn2a null animals. Thus, LPHN2 ligands, such as FLRT2, may be therapeutically exploited to interfere with cancer metastatic dissemination.

Altered Responsiveness to TGFß and BMP and Increased CD45+ Cell Presence in Mitral Valves Are Unique Features of Ischemic Mitral Regurgitation.

[Figure: see text].

Management of pediatric post-infectious neurological syndromes.

BACKGROUND: Post-Infectious Neurological Syndromes (PINS) are heterogeneous neurological disorders with post or para-infectious onset. PINS diagnosis is complex, mainly related to the absence of any recognized guidelines and a univocal definition. AIM OF THE STUDY: To elaborate a diagnostic guide for PINS. MATERIALS AND METHODS: We retrospectively analysed patients younger than 14 years old admitted to Bambino Gesù Children's Hospital in Rome for PINS from December 2005 to March 2018. Scientific literature using PubMed as research platform was analysed: the key words "Post-Infectious Neurological Syndromes" were used. RESULTS: A polysymptomatic presentation occurred in a percentage of 88% of the children. Motor signs and visual disturbances the most observed symptoms/signs were the most detached, followed by fever, speech disturbances, sleepiness, headache and bradipsychism. Blood investigations are compatible with inflammation, as a prodromal illnesses was documented in most cases. Normal cerebral spinal fluid (CSF) characteristics has been found in the majority of the study population. Magnetic resonance imaging (MRI) was positive for demyelinating lesions. Antibiotics, acyclovir and steroids have been given as treatment. DISCUSSION: We suggest diagnostic criteria for diagnosis of PINS, considering the following parameters: neurological symptoms, timing of disease onset, blood and CSF laboratory tests, MRI imaging. CONCLUSIONS: We propose criteria to guide clinician to diagnose PINS as definitive, probable or possible. Further studies are required to validate diagnostic criteria.

Pertussis in early life: underdiagnosed, severe, and risky disease. A seven-year experience in a pediatric tertiary-care hospital.

AIM: Pertussis continues to be a common worldwide infection in pediatric and adult populations.We aimed to study epidemiological and clinical characteristics of infants and children admitted for pertussis to a tertiary-care hospital and to investigate the risk factors for pediatric intensive care unit (PICU) admission. MATERIALS AND METHODS: With a retrospective study, we analyzed all medical reports of patients admitted to Bambino Gesù Children's Hospital in Rome from January 2011 to December 2018 with a diagnosis of pertussis. RESULTS: We examined 195 patients. The majority of hospitalized children (66.15%) were <3 months of age. No mother had received pertussis containing vaccine during pregnancy. Ten cases required admission in PICU. The age at admission was lower in PICU patients with respect to ward patients (42.8 vs 240 days; p < .0007), length of hospital stay was longer in PICU group (24.7 vs 7.52 days; p < .003). Patients who needed PICU admission had greater white blood cell count at hospital admission compared with those hospitalized in the pediatric ward. One infant died and one had encephalitis. CONCLUSIONS: Pertussis is a remerging disease. In infants, it is associated with significant morbidity and mortality. In recent years, many countries have implemented different vaccination strategies and public health measures to prevent the increase in pertussis cases. Maternal vaccination has been shown to be highly protective for infants <3 months of age before they can develop their own immunity via vaccination.

Distinct retrograde microtubule motor sets drive early and late endosome transport.

Although subcellular positioning of endosomes significantly impacts on their functions, the molecular mechanisms governing the different steady-state distribution of early endosomes (EEs) and late endosomes (LEs)/lysosomes (LYs) in peripheral and perinuclear eukaryotic cell areas, respectively, are still unsolved. We unveil that such differences arise because, while LE retrograde transport depends on the dynein microtubule (MT) motor only, the one of EEs requires the cooperative antagonism of dynein and kinesin-14 KIFC1, a MT minus end-directed motor involved in cancer progression. Mechanistically, the Ser-x-Ile-Pro (SxIP) motif-mediated interaction of the endoplasmic reticulum transmembrane protein stromal interaction molecule 1 (STIM1) with the MT plus end-binding protein 1 (EB1) promotes its association with the p150Glued subunit of the dynein activator complex dynactin and the distinct location of EEs and LEs/LYs. The peripheral distribution of EEs requires their p150Glued-mediated simultaneous engagement with dynein and SxIP motif-containing KIFC1, via HOOK1 and HOOK3 adaptors, respectively. In sum, we provide evidence that distinct minus end-directed MT motor systems drive the differential transport and subcellular distribution of EEs and LEs in mammalian cells.

PNPLA3 gene polymorphism is associated with liver steatosis in children with Down syndrome.

BACKGROUND AND AIMS: We previously demonstrated that children with Down syndrome (DS) exhibited a greater risk of steatosis than the general pediatric population. This trend was independent of obese phenotype, thus suggesting a role of genetic predisposition. Therefore, we investigated the prevalence of non-alcoholic fatty liver disease (NAFLD) and metabolic syndrome (MetS) in function of genetic susceptibility and adipocytokine levels in children with DS. METHODS AND RESULTS: A total of 84 Caucasian children with DS (age range 5-17 years), were included in this study. For all children, we collected data on anthropometric and biochemical parameters, and liver ultrasound (US). We also measured adipocytokines circulating levels and specific polymorphisms closed to NAFLD. We found a prevalence of 64.3% of liver steatosis at US, with a severe steatosis of about 4% in children with DS. The presence of steatosis in children with DS was associated with the presence of patatin-like phospholipase domain-containing 3 (PNPLA3) rs738409 variant, which also correlated with interleukin (IL)-6 levels. Moreover, we found that the 52.4% had a waist circumference > 90th percentile, 21.4% were hypertensive, 7.14% had hyperglycemia, 9.5% had hypertriglyceridemia, and 17.9% showed high-density lipoprotein cholesterol ≤ 40 mg/dl. Finally, the IL-6 and adiponectin levels correlated with steatosis, and several adipocytokines correlated with single MetS traits in children with DS. CONCLUSION: The present study explores for the first time potential pathomechanisms connecting pediatric NAFLD and MetS in DS. We found that the PNPLA3 variant is associated with steatosis, but not with MetS, in children with DS.

Effects of Pidotimod on recurrent respiratory infections in children with Down syndrome: a retrospective Italian study.

BACKGROUND: Children with Down syndrome (DS) show a high susceptibility to recurrent infections (RI), caused by immune defects and abnormalities of the airways. Our goal was to investigate the effects of Pidotimod on RI prevention in children with DS, comparing immune and clinical parameters before (T0) and after (T1) the treatment with Pidotimod. METHODS: The study was conducted at the Down syndrome outpatient Center of Bambino Gesù Children's Hospital, in Rome. We reviewed the medical records of all children with a positive history for RI and who received oral prophylaxis of Pidotimod from September 2016 to February 2017. RESULTS: Thirty-three children met the inclusion criteria (males: 51.5%; average age: 6 years ±SD: 3). We found a significant decrease in the number of children with upper respiratory infections (82% at T0 vs 24% at T1; p = 0,0001) and with lower respiratory infections (36% at T0 vs 9% at T1; p = 0.003) after treatment with Pidotimod. We also demonstrated a significant decrease in the number of children hospitalized for respiratory infections (18% at T0 vs 3% at T1; p = 0.03). We measured T and B cells in the peripheral blood and B cell function in vitro at T0 and T1. We found that the response to CpG improved at T1. A significant increase of B cell frequency (p = 0.0009), B cell proliferation (p = 0.0278) and IgM secretion (p = 0.0478) were observed in children with DS after treatment. CONCLUSIONS: Our results provided evidence that Pidotimod may be able to prevent RI in children with Down syndrome.

Complications and risk factors for severe outcome in children with measles.

OBJECTIVE AND DESIGN: Risk factors for severe measles are poorly investigated in high-income countries. The Italian Society for Paediatric Infectious Diseases conducted a retrospective study in children hospitalised for measles from January 2016 to August 2017 to investigate the risk factors for severe outcome defined by the presence of long-lasting sequelae, need of intensive care or death. RESULTS: Nineteen hospitals enrolled 249 children (median age 14.5 months): 207 (83%) children developed a complication and 3 (1%) died. Neutropaenia was more commonly reported in children with B3-genotype compared with other genotypes (29.5% vs 7.7%, p=0.01). Pancreatitis (adjusted OR [aOR] 9.19, p=0.01) and encephalitis (aOR 7.02, p=0.04) were related to severe outcome in multivariable analysis, as well as C reactive protein (CRP) (aOR 1.1, p=0.028), the increase of which predicted severe outcome (area under the receiver operating characteristic curve 0.67, 95% CI 0.52 to 0.82). CRP values >2 mg/dL were related to higher risk of complications (OR 2.0, 95% CI 1.15 to 3.7, p=0.01) or severe outcome (OR 4.13, 95% CI 1.43 to 11.8, p<0.01). CONCLUSION: The risk of severe outcome in measles is independent of age and underlying conditions, but is related to the development of organ complications and may be predicted by CRP value.

Scurvy: still a threat in the well-fed first world?

We report three cases of scurvy in previously healthy children referred to us for leg pain and refusal to walk. All children had no significant medical history, symptoms had started months before and subtly advanced. Two of them presented with gingival hyperplasia and petechiae, another one reported night sweats and gingival bleeding in the past few weeks. Two had vitamin D deficiency, and all had microcytic anaemia (in one case requiring transfusional support). A nutritional screening revealed low or undetectable levels of ascorbic acid. This, along with the clinical and radiological findings, led to a diagnosis of scurvy. Vitamin C supplementation was started with rapid improvement of the children's clinical condition. Scurvy is a rare disease in the 'first world', but there are anecdotal reports of scurvy in children without any of the known risk factors for this condition. In our cases, a selective diet was the only risk factor.

Bartonella henselae in Italy: a rare seasonal infection.

BACKGROUND: Symptomatic Bartonella henselae infection is considered rare in Europe. Cat fleas transmit the microorganism between cats, but their role in transmission of B. henselae to humans has not been defined. The aim of our study was to perform a retrospective study of detected cases at our Hospital. METHODS: We retrospectively analyzed data of all children showing lymphadenopathy and a 4-fold increase in specific IgM for B. henselae over the period from June 2010 to May 2015. We therefore examined clinical data, laboratory exams in order to achieve a description of the expression of Bartonella infection in our series: age, geographical area of origin, symptoms, laboratory exams, the seat of the swelling lymph nodes with ultrasound description, and data on biopsy of lymph node when performed. RESULTS: We could identify a total of 7 patients (4 females, range of age: mean age 8.75±2.87 SD): three cases in 2011 and 1 case per year in 2010, 2012, 2013 and 2014 with an average distance between one case and the sequent of 246.16±214.54 days. All patients came from small towns with no preference between the inland and coastal areas. The infection was characterized only by lymphadenopathy with nonspecific alterations at blood tests and with no history of cat scratch. CONCLUSIONS: By our experience, Bartonella infection presents as a seasonal disease with increased incidence in autumn, with peaks in October, and a decrease after spring. In conclusion, infection with B. henselae is an issue to keep in consideration in all cases of lymphadenopathy, especially in children coming from small towns even without a declared cat scratch.

Active prophylaxis for respiratory syncytial virus: current knowledge and future perspectives.

Respiratory syncytial virus (RSV) is the most common respiratory pathogen in infants and young children but represents also an important cause of morbidity in adults, particularly in the elderly and immunocompromised persons. Despite its global impact on human health, no effective treatment is available except for supportive care and no safe vaccine has been licensed yet. Vaccine development has been hindered by several factors including vaccine enhanced disease associated with formalin-inactivated RSV vaccine, ethical concerns and lack of consensus concerning the most appropriate target antigen. In this review, we analyze history of RSV vaccine and current approaches for preventing RSV including live-attenuated, vector-based, subunit, nucleic acid-based, particle-based vaccines and we debate about concerns on target population, correlates of protection and obstacles that are slowing the progress toward a successful RSV vaccination strategy.

A rationally designed NRP1-independent superagonist SEMA3A mutant is an effective anticancer agent.

Vascular normalizing strategies, aimed at ameliorating blood vessel perfusion and lessening tissue hypoxia, are treatments that may improve the outcome of cancer patients. Secreted class 3 semaphorins (SEMA3), which are thought to directly bind neuropilin (NRP) co-receptors that, in turn, associate with and elicit plexin (PLXN) receptor signaling, are effective normalizing agents of the cancer vasculature. Yet, SEMA3A was also reported to trigger adverse side effects via NRP1. We rationally designed and generated a safe, parenterally deliverable, and NRP1-independent SEMA3A point mutant isoform that, unlike its wild-type counterpart, binds PLXNA4 with nanomolar affinity and has much greater biochemical and biological activities in cultured endothelial cells. In vivo, when parenterally administered in mouse models of pancreatic cancer, the NRP1-independent SEMA3A point mutant successfully normalized the vasculature, inhibited tumor growth, curbed metastatic dissemination, and effectively improved the supply and anticancer activity of chemotherapy. Mutant SEMA3A also inhibited retinal neovascularization in a mouse model of age-related macular degeneration. In summary, mutant SEMA3A is a vascular normalizing agent that can be exploited to treat cancer and, potentially, other diseases characterized by pathological angiogenesis.

Nonalcoholic Fatty Liver Disease in Italian Children with Down Syndrome: Prevalence and Correlation with Obesity-Related Features.

OBJECTIVE: To assess the prevalence of overweight/obesity in a cohort of Italian children with Down syndrome (DS) and to investigate the correlation of both obesity and DS with nonalcoholic fatty liver disease (NAFLD). STUDY DESIGN: We enrolled 280 children with DS (age range 5-18 years), who were referred to the DS outpatient clinic of the Bambino Gesù Children's Hospital in Rome. For all children, we collected the clinical history and measured anthropometric variables. Eighty-four of 280 children with DS were selected to undergo liver ultrasound scanning to evaluate the presence of NAFLD. RESULTS: Italian children with DS exhibited a prevalence of 19.64% for overweight and 12.14% for obesity. The prevalence of NAFLD in nonobese (45%) and overweight/obese (82%) children with DS is greater than in the European pediatric nonobese (5.7%) or obese population (33%). Moreover, the severity of liver brightness on ultrasound scan correlated positively with body mass index, triglycerides, low-density lipoprotein-cholesterol, and leptin levels and negatively with adiponectin. CONCLUSIONS: We demonstrated that, independently from the obese phenotype, children with DS display a greater risk to develop NAFLD than the general pediatric population.

Hospital-acquired rotavirus and norovirus acute gastroenteritis in a pediatric unit, in 2014-2015.

The occurrence of hospital-acquired acute gastroenteritis (AGE) is a major concern for public health. RotavirusA (RVA) and norovirus (NoV) are common causes of viral AGE in the pediatric population, and their role in nosocomial infections has been proven, remaining poorly investigated. To investigate RVA and NoV in hospital-acquired AGE, 55 stool samples from children with nosocomial AGE were collected between May 2014 and May 2015. To evaluate virus spreading routes, 51 environmental swabs were collected from staff and patients' rooms. Stools were tested for both RVA and NoV RNA by reverse-transcription-PCR. Nucleotide sequencing and phylogenetic analysis were performed to characterize the viruses. Forty-seven of 55 cases analyzed resulted positive for RVA. The predominant genotype was G4P[8] (18/55) followed by G1P[8] (14/55). Mixed RVA infections were also detected (7/55). Twenty-two samples were positive for NoV, and GII.4 was revealed to be the predominant genotype. Seventeen samples were positive for both RVA and NoV. This study aimed to evaluate the burden of norovirus and rotavirus nosocomial AGE, contributing to identify the environment source of infections and to activate effective strategies for intervention. The reduction in nosocomial AGE cases is an important aspect, considered the worsened disease course in transplant, cancer, and intensive care unit inpatients.

TRPM8 inhibits endothelial cell migration via a non-channel function by trapping the small GTPase Rap1.

Endothelial cell adhesion and migration are critical steps of the angiogenic process, whose dysfunction is associated with tumor growth and metastasis. The TRPM8 channel has recently been proposed to play a protective role in prostate cancer by impairing cell motility. However, the mechanisms by which it could influence vascular behavior are unknown. Here, we reveal a novel non-channel function for TRPM8 that unexpectedly acts as a Rap1 GTPase inhibitor, thereby inhibiting endothelial cell motility, independently of pore function. TRPM8 retains Rap1 intracellularly through direct protein-protein interaction, thus preventing its cytoplasm-plasma membrane trafficking. In turn, this mechanism impairs the activation of a major inside-out signaling pathway that triggers the conformational activation of integrin and, consequently, cell adhesion, migration, in vitro endothelial tube formation, and spheroid sprouting. Our results bring to light a novel, pore-independent molecular mechanism by which endogenous TRPM8 expression inhibits Rap1 GTPase and thus plays a critical role in the behavior of vascular endothelial cells by inhibiting migration.

[Vaccination in childhood: how to protect the "unprotectable"?] / Le vaccinazioni dell'infanzia: proteggere gli "improteggibili".

The failure of immunization coverage is the primary reason for the transmission and the spread of the diseases in young infants not eligible for vaccination because of age and in immunocompromised individuals. In both these categories measles, pertussis and varicella could be devastating. Pertussis, measles and varicella in the first year of life are responsible to the ED admission, the hospitalization and, exceptionally, the death. The only way to protect very young infants and immunocompromised individuals is to obtain the adequate coverage in all the population.