Monogenic diabetes clinic (MDC): 3-year experience.

AIM: In the pediatric diabetes clinic, patients with type 1 diabetes mellitus (T1D) account for more than 90% of cases, while monogenic forms represent about 6%. Many monogenic diabetes subtypes may respond to therapies other than insulin and have chronic diabetes complication prognosis that is different from T1D. With the aim of providing a better diagnostic pipeline and a tailored care for patients with monogenic diabetes, we set up a monogenic diabetes clinic (MDC). METHODS: In the first 3 years of activity 97 patients with non-autoimmune forms of hyperglycemia were referred to MDC. Genetic testing was requested for 80 patients and 68 genetic reports were available for review. RESULTS: In 58 subjects hyperglycemia was discovered beyond 1 year of age (Group 1) and in 10 before 1 year of age (Group 2). Genetic variants considered causative of hyperglycemia were identified in 25 and 6 patients of Group 1 and 2, respectively, with a pick up rate of 43.1% (25/58) for Group 1 and 60% (6/10) for Group 2 (global pick-up rate: 45.5%; 31/68). When we considered probands of Group 1 with a parental history of hyperglycemia, 58.3% (21/36) had a positive genetic test for GCK or HNF1A genes, while pick-up rate was 18.1% (4/22) in patients with mute family history for diabetes. Specific treatments for each condition were administered in most cases. CONCLUSION: We conclude that MDC may contribute to provide a better diabetes care in the pediatric setting.

Comparison of two advanced hybrid closed loop in a pediatric population with type 1 diabetes: a real-life observational study.

OBJECTIVE: The Advanced Hybrid Closed Loop (AHCL) systems have provided the potential to ameliorate glucose control in children with Type 1 Diabetes. The aim of the present work was to compare metabolic control obtained with 2 AHCL systems (Medtronic 780G system and Tandem Control IQ system) in a pediatric real-life clinical context. RESEARCH DESIGN AND METHODS: It is an observational, real-life, monocentric study; thirty one children and adolescents (M:F = 15:16, age range 7.6-18 years, mean age 13.05 ± 2.4 years, Diabetes duration > 1 year) with T1D, previously treated with Predictive Low Glucose Suspend (PLGS) systems and then upgraded to AHCL have been enrolled. CGM data of the last four weeks of "PLGS system" (PRE period) with the first four weeks of AHCL system (POST period) have been compared. RESULTS: For both AHCL systems, Medtronic 780G and Tandem Control IQ, respectively TIR at 4 weeks significantly increased, from 65.7 to 70.5% (p < 0.01) and from 64.8 to 70.1% (p < 0.01). (p < 0.01). The comparison between CGM metrics of the 2 evaluated systems doesn't show difference at baseline (last four weeks of PLGS system) and after four weeks of AHCL use. CONCLUSIONS: To our knowledge, this study is the first real-life one comparing 2 AHCL systems in a pediatric population with T1D. It shows an improvement in glucose control when upgrading to AHCL. The comparison between the two AHCL systems did not show significant differences in the analyzed CGM metrics, meaning that the algorithms currently available are equally effective in promoting glucose control.

Brain mapping across 16 autism mouse models reveals a spectrum of functional connectivity subtypes.

Autism Spectrum Disorder (ASD) is characterized by substantial, yet highly heterogeneous abnormalities in functional brain connectivity. However, the origin and significance of this phenomenon remain unclear. To unravel ASD connectopathy and relate it to underlying etiological heterogeneity, we carried out a bi-center cross-etiological investigation of fMRI-based connectivity in the mouse, in which specific ASD-relevant mutations can be isolated and modeled minimizing environmental contributions. By performing brain-wide connectivity mapping across 16 mouse mutants, we show that different ASD-associated etiologies cause a broad spectrum of connectional abnormalities in which diverse, often diverging, connectivity signatures are recognizable. Despite this heterogeneity, the identified connectivity alterations could be classified into four subtypes characterized by discrete signatures of network dysfunction. Our findings show that etiological variability is a key determinant of connectivity heterogeneity in ASD, hence reconciling conflicting findings in clinical populations. The identification of etiologically-relevant connectivity subtypes could improve diagnostic label accuracy in the non-syndromic ASD population and paves the way for personalized treatment approaches.

Reduced SNAP-25 increases PSD-95 mobility and impairs spine morphogenesis.

Impairment of synaptic function can lead to neuropsychiatric disorders collectively referred to as synaptopathies. The SNARE protein SNAP-25 is implicated in several brain pathologies and, indeed, brain areas of psychiatric patients often display reduced SNAP-25 expression. It has been recently found that acute downregulation of SNAP-25 in brain slices impairs long-term potentiation; however, the processes through which this occurs are still poorly defined. We show that in vivo acute downregulation of SNAP-25 in CA1 hippocampal region affects spine number. Consistently, hippocampal neurons from SNAP-25 heterozygous mice show reduced densities of dendritic spines and defective PSD-95 dynamics. Finally, we show that, in brain, SNAP-25 is part of a molecular complex including PSD-95 and p140Cap, with p140Cap being capable to bind to both SNAP-25 and PSD-95. These data demonstrate an unexpected role of SNAP-25 in controlling PSD-95 clustering and open the possibility that genetic reductions of the protein levels - as occurring in schizophrenia - may contribute to the pathology through an effect on postsynaptic function and plasticity.

Association between SNAP-25 gene polymorphisms and cognition in autism: functional consequences and potential therapeutic strategies.

Synaptosomal-associated protein of 25 kDa (SNAP-25) is involved in different neuropsychiatric disorders, including schizophrenia and attention-deficit/hyperactivity disorder. Consistently, SNAP-25 polymorphisms in humans are associated with hyperactivity and/or with low cognitive scores. We analysed five SNAP-25 gene polymorphisms (rs363050, rs363039, rs363043, rs3746544 and rs1051312) in 46 autistic children trying to correlate them with Childhood Autism Rating Scale and electroencephalogram (EEG) abnormalities. The functional effects of rs363050 single-nucleotide polymorphism (SNP) on the gene transcriptional activity, by means of the luciferase reporter gene, were evaluated. To investigate the functional consequences that SNAP-25 reduction may have in children, the behaviour and EEG of SNAP-25(+/-) adolescent mice (SNAP-25(+/+)) were studied. Significant association of SNAP-25 polymorphism with decreasing cognitive scores was observed. Analysis of transcriptional activity revealed that SNP rs363050 encompasses a regulatory element, leading to protein expression decrease. Reduction of SNAP-25 levels in adolescent mice was associated with hyperactivity, cognitive and social impairment and an abnormal EEG, characterized by the occurrence of frequent spikes. Both EEG abnormalities and behavioural deficits were rescued by repeated exposure for 21 days to sodium salt valproate (VLP). A partial recovery of SNAP-25 expression content in SNAP-25(+/-) hippocampi was also observed by means of western blotting. A reduced expression of SNAP-25 is responsible for the cognitive deficits in children affected by autism spectrum disorders, as presumably occurring in the presence of rs363050(G) allele, and for behavioural and EEG alterations in adolescent mice. VLP treatment could result in novel therapeutic strategies.

Testing Aß toxicity on primary CNS cultures using drug-screening microfluidic chips.

Open microscale cultures of primary central nervous system (CNS) cells have been implemented in microfluidic chips that can expose the cells to physiological fluidic shear stress conditions. Cells in the chips were exposed to differently aggregated forms of beta-amyloid (Aß), i.e. conditions mimicking an Alzheimer's Disease environment, and treated with CNS drugs in order to assess the contribution of glial cells during pharmacological treatments. FTY720, a drug approved for the treatment of Multiple Sclerosis, was found to play a marked neuroprotective role in neuronal cultures as well as in microglia-enriched neuronal cultures, preventing neurodegeneration after cell exposure to neurotoxic oligomers of Aß.

Microglia convert aggregated amyloid-ß into neurotoxic forms through the shedding of microvesicles.

Alzheimer's disease (AD) is characterized by extracellular amyloid-ß (Aß) deposition, which activates microglia, induces neuroinflammation and drives neurodegeneration. Recent evidence indicates that soluble pre-fibrillar Aß species, rather than insoluble fibrils, are the most toxic forms of Aß. Preventing soluble Aß formation represents, therefore, a major goal in AD. We investigated whether microvesicles (MVs) released extracellularly by reactive microglia may contribute to AD degeneration. We found that production of myeloid MVs, likely of microglial origin, is strikingly high in AD patients and in subjects with mild cognitive impairment and that AD MVs are toxic for cultured neurons. The mechanism responsible for MV neurotoxicity was defined in vitro using MVs produced by primary microglia. We demonstrated that neurotoxicity of MVs results from (i) the capability of MV lipids to promote formation of soluble Aß species from extracellular insoluble aggregates and (ii) from the presence of neurotoxic Aß forms trafficked to MVs after Aß internalization into microglia. MV neurotoxicity was neutralized by the Aß-interacting protein PrP and anti-Aß antibodies, which prevented binding to neurons of neurotoxic soluble Aß species. This study identifies microglia-derived MVs as a novel mechanism by which microglia participate in AD degeneration, and suggest new therapeutic strategies for the treatment of the disease.

[Teaching in the operative room: the benefit of daysurgery on surgical trainees]. / Attività didattica in sala operatoria: utilità della day-surgery nell'iter formativo dello specializzando in chirurgia.

AIM: Aim of the study was to evaluate the operative time and the incidence of post-operative complications in a group of patients undergoing Lichtenstein inguinal hernia repair performed either by surgical residents or senior surgeons in a day-surgery setting. PATIENTS AND METHODS: The study population consisted of 198 patients: group I (n=102), in which the operator was a senior surgeon, group II (n=96), in which the operator was a resident supervised by a senior surgeon. We recorded the duration of the operation and the complications following the procedure, and statistically compared them between group I and II. RESULTS: Our analysis showed that there was a statistically significant difference between the two groups only for the mean operative time, being shorter in group I (62 vs 82 min, p>0.05), while no significant difference was found for the incidence of complications. CONCLUSION: In conclusion, the day-surgery setting allows a high quality training of young surgeons, based on performing minor surgical procedures such has inguinal hernia repair. This training allows a step by step supervised learning process that does not jeopardize the efficacy of the treatment as well as the patient safety. The major cost due to the increase in operative time should be considered as an investment in young surgeons education.

Engineering injured spinal cord with bone marrow-derived stem cells and hydrogel-based matrices: a glance at the state of the art.

Concerning the broad topic of neural tissue engineering, we present a review relating to the state of the art in spinal cord injury repair strategies. Particular attention is given to spinal cord damage causes and effects, in neural and mesenchymal stem cell therapeutic approaches, in the use of hydrogels as cell carriers and in the mathematical modeling of involved phenomena. High importance is given to multidisciplinary strategies applied to spinal cord repair, since new research frontiers are believed to be now on 3D gel/cells and neuroprotective agent constructs for neural reconstruction purposes.

The effects of calcitriol and nicotinamide on residual pancreatic beta-cell function in patients with recent-onset Type 1 diabetes (IMDIAB XI).

BACKGROUND: A number of recent studies underline the importance of vitamin D in the pathogenesis of Type 1 diabetes (T1D). AIMS: The aim of this study was to investigate whether supplementation with the active form of vitamin D (calcitriol) in subjects with recent-onset T1D protects residual pancreatic beta-cell function and improves glycaemic control (HbA(1c) and insulin requirement). METHODS: In this open-label randomized trial, 70 subjects with recent-onset T1D, mean age 13.6 years +/- 7.6 sd were randomized to calcitriol (0.25 microg on alternate days) or nicotinamide (25 mg/kg daily) and followed up for 1 year. Intensive insulin therapy was implemented with three daily injections of regular insulin + NPH insulin at bedtime. RESULTS: No significant differences were observed between calcitriol and nicotinamide groups in respect of baseline/stimulated C-peptide or HbA1c 1 year after diagnosis, but the insulin dose at 3 and 6 months was significantly reduced in the calcitriol group. CONCLUSIONS: At the dosage used, calcitriol has a modest effect on residual pancreatic beta-cell function and only temporarily reduces the insulin dose.

A two year observational study of nicotinamide and intensive insulin therapy in patients with recent onset type 1 diabetes mellitus.

BACKGROUND AND AIMS: A number of trials have evaluated residual beta-cell function in patients with recent onset type 1 diabetes mellitus (DM1) treated with nicotinamide in addition to intensive insulin therapy (IIT). In most studies, only a slight decline of C-peptide secretion was observed 12 months after diagnosis; however, no data is available on C-peptide secretion and metabolic control in patients continuing nicotinamide and IIT for up to 2 years after diagnosis. PATIENTS AND METHODS: We retrospectively analysed data from 25 patients (mean age 14.7 years +/- 5 SD) with DM1 in whom nicotinamide at a dose of 25 mg/kg b. wt. was added from diagnosis (< 4 weeks) to IIT (three injections of regular insulin at meals + one NPH at bed time) and continued for up to 2 years after diagnosis. Data were also analysed from patients (n = 27) in whom IIT was introduced at diagnosis and who were similarly followed for 2 years. Baseline C-peptide as well as insulin dose and HbA1c levels were evaluated at 12 and 24 months after diagnosis. RESULTS: In the course of the follow-up, patients on nicotinamide + IIT or IIT alone did not significantly differ in terms of C-peptide secretion (values at 24 months in the two groups were 0.19 +/- 0.24 nM vs 0.19 +/- 0.13 nM, respectively). Insulin requirement (0.6 +/- 0.3 U/kg/day vs 0.7 +/- 0.2 U/kg/day at 24 months, respectively) did not differ between the two groups. However, HbA1c was significantly lower 2 years after diagnosis in patients treated with nicotinamide + IIT (6.09 +/- 0.9% vs 6.98 +/- 0.9%, respectively, p < 0.01). No adverse effects were observed in patients receiving nicotinamide for 2 years. CONCLUSION: Implementation of IIT with the addition of nicotinamide at diagnosis continued for 2 years improves metabolic control as assessed by HbA1c. In both nicotinamide and control patients, no decline in C-peptide was detected 2 years after diagnosis, indicating that IIT preserves C-peptide secretion. We conclude that nicotinamide + IIT at diagnosis of DM1 prolonged for up to 2 years can be recommended, but longer follow-up is required to determine whether nicotinamide should be continued beyond this period.

Analysis of SNAP-25 immunoreactivity in hippocampal inhibitory neurons during development in culture and in situ.

Synaptosomal associated protein of 25 kDa (SNAP-25) is a component of the soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment protein receptor (SNARE) complex which plays a central role in synaptic vesicle exocytosis. We have previously demonstrated that adult rat hippocampal GABAergic synapses, both in culture and in brain, are virtually devoid of SNAP-25 immunoreactivity and are less sensitive to the action of botulinum toxin type A, which cleaves this SNARE protein [Neuron 41 (2004) 599]. In the present study, we extend our findings to the adult mouse hippocampus and we also provide demonstration that hippocampal inhibitory synapses lacking SNAP-25 labeling belong to parvalbumin-, calretinin- and cholecystokinin-positive interneurons. A partial colocalization between SNAP-25 and glutamic acid decarboxylase is instead detectable in developing mouse hippocampus at P0 and, at a lesser extent, at P5. In rat embryonic hippocampal cultures at early developmental stages, SNAP-25 immunoreactivity is detectable in a percentage of GABAergic neurons, which progressively reduces with time in culture. Consistent with the presence of the substrate, botulinum toxin type A is partially effective in inhibiting synaptic vesicle recycling in immature GABAergic neurons. Since SNAP-25, beside its role as a SNARE protein, is involved in additional processes, such as neurite outgrowth and regulation of calcium dynamics, the presence of higher levels of the protein at specific stages of neuronal differentiation may have implications for the construction and for the functional properties of brain circuits.

A randomized trial of nicotinamide and vitamin E in children with recent onset type 1 diabetes (IMDIAB IX).

OBJECTIVE: Various adjuvant therapies have been introduced along with intensive insulin therapy in patients with recent onset type 1 diabetes. Nicotinamide (NA), administered at diagnosis of the disease, can have beneficial effects on the clinical remission rate, improve metabolic control and preserve or slightly increase beta-cell function, probably by reducing toxicity due to free oxygen radicals. Vitamin E, a known antioxidant, inhibits lipid peroxidation; this can lead to protection of islet beta cells from the combined effects of interleukin 1, tumor necrosis factor and gamma interferon. The aim of the present study was to investigate whether the addition of vitamin E to NA could improve metabolic control and the residual beta-cell function, as measured by C-peptide secretion, in children and adolescents with recent onset type 1 diabetes; patients were followed-up for 2 years after diagnosis. PATIENTS AND STUDY DESIGN: Recent onset type 1 diabetes patients (n=64, mean age 8.8 years) were recruited by participating centres of the IMDIAB group. Thirty-two patients were randomized to NA (25 mg/kg body weight) plus vitamin E (15 mg/kg body weight); 32 patients acted as controls and received NA only at the same dose as above. Intensive insulin therapy was applied to both treatment groups. RESULTS: There were three drop outs during the 2-year follow-up period. Overall, patients assigned to the NA+vitamin E group or the NA group did not significantly differ in terms of glycated hemoglobin (HbA1c) levels, insulin requirement or baseline C-peptide secretion. Patients diagnosed at an age of less than 9 years showed significantly reduced C-peptide levels compared with those aged over 9 years at diagnosis and at the 2-year follow-up but there were no differences between the NA and NA+vitamin E treated groups. However at 6 months, patients over 9 years of age treated with NA+vitamin E showed significantly higher C-peptide compared with the NA group (P<0.003). In both age groups and in the different treatment groups, C-peptide levels found at diagnosis were preserved 2 years later. CONCLUSIONS: The use of NA alone, or in combination with vitamin E, along with intensive insulin therapy is able to preserve baseline C-peptide secretion for up to 2 years after diagnosis. This finding is of particular interest for pre-pubertal children with type 1 diabetes and has never been reported before.

Chronic blockade of glutamate receptors enhances presynaptic release and downregulates the interaction between synaptophysin-synaptobrevin-vesicle-associated membrane protein 2.

During development of neuronal circuits, presynaptic and postsynaptic functions are adjusted in concert, to optimize interneuronal signaling. We have investigated whether activation of glutamate receptors affects presynaptic function during synapse formation, when constitutive synaptic vesicle recycling is downregulated. Using primary cultures of hippocampal neurons as a model system, we have found that chronic exposure to both NMDA and non-NMDA glutamate receptor blockers during synaptogenesis produces an increase in miniature EPSC (mEPSC) frequency, with no significant changes in mEPSC amplitude or in the number of synapses. Enhanced synaptic vesicle recycling, selectively in glutamatergic nerve terminals, was confirmed by the increased uptake of antibodies directed against the lumenal domain of synaptotagmin. No increased uptake was detected in neuronal cultures grown in the chronic presence of TTX, speaking against an indirect effect caused by decreased electrical activity. Enhanced mEPSC frequency correlated with a reduction of synaptophysin-synaptobrevin-vesicle-associated membrane protein 2 (VAMP2) complexes detectable by immunoprecipitation. Intracellular perfusion with a peptide that inhibits the binding of synaptophysin to synaptobrevin-VAMP2 induced a remarkable increase of mEPSC frequency in control but not in glutamate receptor blocker-treated neurons. These findings suggest that activation of glutamate receptors plays a role in the downregulation of the basal rate of synaptic vesicle recycling that accompanies synapse formation. They also suggest that one of the mechanisms through which this downregulation is achieved is an increased interaction of synaptophysin with synaptobrevin-VAMP2.

Evidence of a role for cyclic ADP-ribose in calcium signalling and neurotransmitter release in cultured astrocytes.

Astrocytes possess different, efficient ways to generate complex changes in intracellular calcium concentrations, which allow them to communicate with each other and to interact with adjacent neuronal cells. Here we show that cultured hippocampal astrocytes coexpress the ectoenzyme CD38, directly involved in the metabolism of the calcium mobilizer cyclic ADP-ribose, and the NAD+ transporter connexin 43. We also demonstrate that hippocampal astrocytes can release NAD+ and respond to extracellular NAD+ or cyclic ADP-ribose with intracellular calcium increases, suggesting the existence of an autocrine cyclic ADP-ribose-mediated signalling. Cyclic ADP-ribose-induced calcium changes are in turn responsible for an increased glutamate and GABA release, this effect being completely inhibited by the cyclic ADP-ribose specific antagonist 8-NH2-cADPR. Furthermore, addition of NAD+ to astrocyte-neuron co-cultures results in a delayed intracellular calcium transient in neuronal cells, which is strongly but not completely inhibited by glutamate receptor blockers. These data indicate that an astrocyte-to-neuron calcium signalling can be triggered by the CD38/cADPR system, which, through the activation of intracellular calcium responses in astrocytes, is in turn responsible for the increased release of neuromodulators from glial cells.

ATP mediates calcium signaling between astrocytes and microglial cells: modulation by IFN-gamma.

Calcium-mediated intercellular communication is a mechanism by which astrocytes communicate with each other and modulate the activity of adjacent cells, including neurons and oligodendrocytes. We have investigated whether microglia, the immune effector cells involved in several diseases of the CNS, are actively involved in this communication network. To address this issue, we analyzed calcium dynamics in fura-2-loaded cocultures of astrocytes and microglia under physiological conditions and in the presence of the inflammatory cytokine IFN-gamma. The intracellular calcium increases in astrocytes, occurring spontaneously or as a result of mechanical or bradykinin stimulation, induced the release of ATP, which, in turn, was responsible for triggering a delayed calcium response in microglial cells. Repeated stimulations of microglial cells by astrocyte-released ATP activated P2X(7) purinergic receptor on microglial cells and greatly increased membrane permeability, eventually leading to microglial apoptosis. IFN-gamma increased ATP release and potentiated the P2X(7)-mediated cytolytic effect. This is the first study showing that ATP mediates a form of calcium signaling between astrocytes and microglia. This mechanism of intercellular communication may be involved in controlling the number and function of microglial cells under pathophysiologic CNS conditions.