Diaphragmatic hernia in a term newborn with congenital myotonic dystrophy: case report.

Background and aim Myotonic dystrophy (DM) is a genetic disorder determined by an amplified trinucleotide CTG repeat in the untranslated region of the DMPK gene on chromosome 19q13.3. The incidence of the congenital form is 1 in 47619 live births and the mortality in the neonatal period is up to 40%. Methods: We report a case of congenital DM (CDM, also designated Myotonic Dystrophy Type 1), presented with congenital right diaphragmatic hernia and cerebral bilateral ventricular dilatation, genetically diagnosed. Conclusions: Since no case of congenital diaphragmatic hernia associated with CDM is reported, the present case report could be considered of particular interest.

Safety use of high frequency oscillatory ventilation in transport of newborn infants affected by severe respiratory failure: preliminary data in central Tuscany.

BACKGROUND: Neonatal Emergency Transport Services play a fundamental role in neonatal care. Stabilization before transport of newborns suffering from severe respiratory failure is often a challenging problem and some critically ill infants may benefit from High Frequency Oscillatory Ventilation (HFOV) as rescue treatment. In these cases, transition to conventional ventilation for transport may cause a deterioration in clinical conditions. HFOV during neonatal transport has been only exceptionally used, due to technical difficulties. Since May 2018, a new neonatal transport unit is available at the Neonatal Protected Transport Service of the Meyer University Hospital in Florence, equipped with a pulmonary ventilator capable of delivering HFOV. Therefore, we conducted an analysis on patients transferred in HFOV to Neonatal Intensive Care Unit (NICU), in order to evaluate the safety and feasibility of its use during neonatal transport. METHODS: A retrospective analysis was performed reviewing medical records of the neonates transported by Meyer Children Hospital's Neonatal Transport Service between May 2018 and December 2020, and newborns treated with HFOV during ground neonatal transport were identified. Safety was assessed by the comparison of vital signs, hemogas-analysis values and pulmonary ventilator parameters, at the time of departure and upon arrival in NICU. The dose of inotropes, the main respiratory complications (air leak, dislocation or obstruction of the endotracheal tube, loss of chest vibrations) and the number of deaths and transfer failures were recorded. RESULTS: Out of the approximate 400 newborns transported during the analysis period, 9 were transported in HFOV. We did not find any statistically significant difference in vital parameters, hemogas-analytical values and pulmonary ventilator settings recorded before and after neonatal transport of the nine patients' parameters (p > 0,05). No patient required additional inotropes during transport. No transport-related deaths or significant complications occurred during transport. CONCLUSIONS: The interest of our report is in the possibility of using HFOV during inter-hospital neonatal transfer. As far as our experience has shown, HFOV appears to be safe for the transportation of newborns with severe respiratory failure. Nevertheless, further larger, prospective and multicentre studies are needed to better evaluate the safety and efficacy of HFOV during neonatal transport.

Type I sialidosis, a normosomatic lysosomal disease, in the differential diagnosis of late-onset ataxia and myoclonus: An overview.

Lysosomal storage diseases (LSDs) are rare to extremely rare monogenic disorders. Their incidence, however, has probably been underestimated owing to their complex clinical manifestations. Sialidosis is a prototypical LSD inherited as an autosomal recessive trait and caused by mutations in the NEU1 gene that result in a deficiency of alpha-N-acetyl neuraminidase 1 (NEU1). Two basic forms of this disease, type I and type II, are known. The dysmorphic type II form features LSD symptoms including congenital hydrops, dysmorphogenetic traits, hepato-splenomegaly and severe intellectual disability. The diagnosis is more challenging in the normosomatic type I forms, whose clinical findings at onset include ocular defects, ataxia and generalized myoclonus. Here we report the clinical, biochemical and molecular analysis of five patients with sialidosis type I. Two patients presented novel NEU1 mutations. One of these patients was compound heterozygous for two novel NEU1 missense mutations: c.530A>T (p.Asp177Val) and c.1010A>G (p.His337Arg), whereas a second patient was compound heterozygous for a known mutation and a novel c.839G>A (p.Arg280Gln) mutation. We discuss the impact of these new mutations on the structural properties of NEU1. We also review available clinical reports of patients with sialidosis type I, with the aim of identifying the most frequent initial clinical manifestations and achieving more focused diagnoses.

Progressive myoclonus epilepsy in Gaucher Disease due to a new Gly-Gly mutation causing loss of an Exonic Splicing Enhancer.

BACKGROUND: Patients with Gaucher Disease (GD) exhibit three phenotypes, including type 1 (non-neuronopathic), type 2 (acute neuronopathic), and type 3 (subacute neuronopathic). AIM: Identifying which GBA changes represent benign polymorphisms and which may result in disease-causing mutations is essential for diagnosis and genotype/phenotype correlations but is often challenging. RESULTS: Here, we describe a patient with type 3 GD, presenting with drug-resistant epilepsy, who bears a set of GBA polymorphic variants including the novel c.363A > G (Gly82Gly) synonymous mutation. In silico predictions, mRNA and functional studies revealed that the new Gly82Gly mutation causes skipping of GBA exon 4, leading to a severe reduction of the wild type GBA mRNA. This is the first report of a synonymous change causing GD through loss of an exonic splicing enhancer sequence. The synonymous mutation is in trans with the Asn188Ser missense mutation, thus making the Asn188Ser responsible for the patient's phenotype and strengthening the association of Asn188Ser with the particular neurological phenotype of type 3 GD. CONCLUSION: We strengthen the association of Asn188Ser with the type 3 GD phenotype and progressive myoclonus epilepsy. Our data confirm that in silico predictions and mRNA analysis are mandatory in discriminating pathological mutations from the background of harmless polymorphisms, especially synonymous changes.

Neuroprotective effects of topiramate and memantine in combination with hypothermia in hypoxic-ischemic brain injury in vitro and in vivo.

Hypoxic-ischemic encephalopathy (HIE) is a major cause of perinatal mortality and subsequent severe neurological sequelae. Mild hypothermia is a standard therapy for HIE, but is used only in selected Reference Centers and in neonates >1800 g. Since neuronal death following HIE occurs by a cascade of events triggered by activation of glutamate receptors, we used in vitro and in vivo models of HIE to examine whether the AMPA/kainate receptor antagonist topiramate and the NMDA receptor antagonist memantine could exert neuroprotective effects, alone or in combination with hypothermia. For the in vitro experiments, rat organotypic hippocampal slices were exposed to a 30 min duration of oxygen-glucose deprivation (OGD): treatment with topiramate (1 µM) and memantine (10-30 µM) or hypothermia (35 °C or 32 °C) significantly attenuated CA1 damage after 24 h. The combination of hypothermia with topiramate and memantine enhanced their protective effect. For the in vivo experiments, we used 7 day-old rat pups subjected to permanent left common carotid artery occlusion followed by 120 min of hypoxia. Administration of topiramate or memantine (i.p., 20 mg/kg) immediately and 2 h after hypoxia or exposure to hypothermia (32 °C for 4 h beginning 1 h after hypoxia) significantly reduced the extent of the resulting infarct. The combination of topiramate or memantine with hypothermia elicited a reduction of the infarct that was greater than that produced by drugs or hypothermia alone. Notably, memantine displayed a higher degree of neuroprotection as compared to topiramate both in vitro and in vivo and, when used alone at 20 mg/kg in vivo, produced a greater reduction in brain damage than observed using topiramate in combination with hypothermia. These results suggest that memantine may be more advantageous than topiramate as a therapeutic agent in neonates with HIE treated with hypothermia.

Safety and efficacy of topiramate in neonates with hypoxic ischemic encephalopathy treated with hypothermia (NeoNATI): a feasibility study.

PURPOSE: To investigate the feasibility of a study based on treatment with topiramate (TPM) added to moderate hypothermia in newborns with hypoxic ischemic encephalopathy (HIE). MATERIALS AND METHODS: Multicenter randomized controlled trial. Term newborns with precocious metabolic, clinical and electroencephalographic (EEG) signs of HIE were selected according to their amplified integrated EEG pattern and randomized to receive either TPM (10 mg/kg once a day for the first three days of life) plus moderate hypothermia or hypothermia alone. Safety was assessed by monitoring cardiorespiratory parameters and blood samples collected to check renal, liver, metabolic balance and TPM pharmacokinetics. Efficacy was evaluated by the combined frequency of mortality and severe neurological disability as primary outcome. Incidence of magnetic resonance injury, epilepsy, blindness, hearing loss, neurodevelopment at 18-24 months of life was assessed as secondary outcomes. RESULTS: Forty-four asphyxiated newborns were enrolled in the study. Twenty one newborns (10 with moderate and 11 with severe HIE) were allocated to hypothermia plus TPM and 23 (12 moderate and 11 severe HIE) to hypothermia. No statistically or clinically significant differences were observed for safety, primary or secondary outcomes. However, a reduction in the prevalence of epilepsy was observed in newborns co-treated with TPM. CONCLUSIONS: Results of this pilot trial suggest that administration of TPM in newborns with HIE is safe but does not reduce the combined frequency of mortality and severe neurological disability. The role of TPM co-treatment in preventing subsequent epilepsy deserves further studies.

Human Acid ß-Glucosidase Inhibition by Carbohydrate Derived Iminosugars: Towards New Pharmacological Chaperones for Gaucher Disease.

A collection of carbohydrate-derived iminosugars belonging to three structurally diversified sub-classes (polyhydroxylated pyrrolidines, piperidines, and pyrrolizidines) was evaluated for inhibition of human acid ß-glucosidase (glucocerebrosidase, GCase), the deficient enzyme in Gaucher disease. The synthesis of several new pyrrolidine analogues substituted at the nitrogen or α-carbon atom with alkyl chains of different lengths suggested an interpretation of the inhibition data and led to the discovery of two new GCase inhibitors at sub-micromolar concentration. In the piperidine iminosugar series, two N-alkylated derivatives were found to rescue the residual GCase activity in N370S/RecNcil mutated human fibroblasts (among which one up to 1.5-fold). This study provides the starting point for the identification of new compounds in the treatment of Gaucher disease.

Familial periventricular nodular heterotopia, epilepsy and Melnick-Needles Syndrome caused by a single FLNA mutation with combined gain-of-function and loss-of-function effects.

BACKGROUND: Loss-of-function mutations of the FLNA gene cause a neuronal migration disorder defined as X-linked periventricular nodular heterotopia (PNH); gain-of-function mutations are associated with a group of X-linked skeletal dysplasias designed as otopalatodigital (OPD) spectrum. We describe a family in which a woman and her three daughters exhibited a complex phenotype combining PNH, epilepsy and Melnick-Needles syndrome (MNS), a skeletal disorder assigned to the OPD spectrum. All four individuals harboured a novel non-conservative missense mutation in FLNA exon 3. METHODS: In all affected family members, we performed mutation analysis of the FLNA gene, RT-PCR, ultradeep sequencing analysis in FLNA cDNAs and western blot in lymphocyte cells to further characterise the mutation. We also assessed the effects on RT-PCR products of treatment of patients' lymphocytes with cycloheximide, a nonsense mediated mRNA decay (NMD) inhibitor. RESULTS: We identified a novel c.622G>C change in FLNA exon 3, leading to the substitution of a highly conserved aminoacid (p.Gly208Arg). Gel electrophoresis and ultradeep sequencing revealed the missense mutation as well as retention of intron 3. Cycloheximide treatment demonstrated that the aberrant mRNA transcript-retaining intron 3 is subjected to NMD. Western blot analysis confirmed reduced FLNA levels in lymphocyte cells. CONCLUSIONS: The novel c.622G>C substitution leads to two aberrant FLNA transcripts, one of which carries the missense mutation, plus a longer transcript resulting from intron 3 retention. We propose that the exceptional co-occurrence of PNH and MNS, two otherwise mutually exclusive allelic phenotypes, is the consequence of a single mutational event resulting in co-occurring gain-of-function and loss-of-function effects.

Optimizing the molecular diagnosis of GALNS: novel methods to define and characterize Morquio-A syndrome-associated mutations.

Morquio A syndrome (MPS IVA) is a systemic lysosomal storage disorder caused by the deficiency of N-acetylgalactosamine-6-sulfatase (GALNS), encoded by the GALNS gene. We studied 37 MPS IV A patients and defined genotype-phenotype correlations based on clinical data, biochemical assays, molecular analyses, and in silico structural analyses of associated mutations. We found that standard sequencing procedures, albeit identifying 14 novel small GALNS genetic lesions, failed to characterize the second disease-causing mutation in the 16% of the patients' cohort. To address this drawback and uncover potential gross GALNS rearrangements, we developed molecular procedures (CNV [copy-number variation] assays, QF-PCRs [quantitative fluorescent-PCRs]), endorsed by CGH-arrays. Using this approach, we characterized two new large deletions and their corresponding breakpoints. Both deletions were heterozygous and included the first exon of the PIEZO1 gene, which is associated with dehydrated hereditary stomatocitosis, an autosomal-dominant syndrome. In addition, we characterized the new GALNS intronic lesion c.245-11C>G causing m-RNA defects, although identified outside the GT/AG splice pair. We estimated the occurrence of the disease in the Italian population to be approximately 1:300,000 live births and defined a molecular testing algorithm designed to help diagnosing MPS IVA and foreseeing disease progression.

Sudden unexpected fatal encephalopathy in adults with OTC gene mutations-Clues for early diagnosis and timely treatment.

BACKGROUND: X-linked Ornithine Transcarbamylase deficiency (OTCD) is often unrecognized in adults, as clinical manifestations are non-specific, often episodic and unmasked by precipitants, and laboratory findings can be normal outside the acute phase. It may thus be associated with significant mortality if not promptly recognized and treated. The aim of this study was to provide clues for recognition of OTCD in adults and analyze the environmental factors that, interacting with OTC gene mutations, might have triggered acute clinical manifestations. METHODS: We carried out a clinical, biochemical and molecular study on five unrelated adult patients (one female and four males) with late onset OTCD, who presented to the Emergency Department (ED) with initial fatal encephalopathy. The molecular study consisted of OTC gene sequencing in the probands and family members and in silico characterization of the newly detected mutations. RESULTS: We identified two new, c.119G>T (p.Arg40Leu) and c.314G>A (p.Gly105Glu), and three known OTC mutations. Both new mutations were predicted to cause a structural destabilization, correlating with late onset OTCD. We also identified, among the family members, 8 heterozygous females and 2 hemizygous asymptomatic males. Patients' histories revealed potential environmental triggering factors, including steroid treatment, chemotherapy, diet changes and hormone therapy for in vitro fertilization. CONCLUSIONS: This report raises awareness of the ED medical staff in considering OTCD in the differential diagnosis of sudden neurological and behavioural disorders associated with hyperammonemia at any age and in both genders. It also widens the knowledge about combined effect of genetic and environmental factors in determining the phenotypic expression of OTCD.

Medium-chain acyl-CoA deficiency: outlines from newborn screening, in silico predictions, and molecular studies.

Medium-chain acyl-CoA dehydrogenase deficiency (MCADD) is a disorder of fatty acid oxidation characterized by hypoglycemic crisis under fasting or during stress conditions, leading to lethargy, seizures, brain damage, or even death. Biochemical acylcarnitines data obtained through newborn screening by liquid chromatography-tandem mass spectrometry (LC-MS/MS) were confirmed by molecular analysis of the medium-chain acyl-CoA dehydrogenase (ACADM) gene. Out of 324.000 newborns screened, we identified 14 MCADD patients, in whom, by molecular analysis, we found a new nonsense c.823G>T (p.Gly275∗) and two new missense mutations: c.253G>C (p.Gly85Arg) and c.356T>A (p.Val119Asp). Bioinformatics predictions based on both phylogenetic conservation and functional/structural software were used to characterize the new identified variants. Our findings confirm the rising incidence of MCADD whose existence is increasingly recognized due to the efficacy of an expanded newborn screening panel by LC-MS/MS making possible early specific therapies that can prevent possible crises in at-risk infants. We noticed that the "common" p.Lys329Glu mutation only accounted for 32% of the defective alleles, while, in clinically diagnosed patients, this mutation accounted for 90% of defective alleles. Unclassified variants (UVs or VUSs) are especially critical when considering screening programs. The functional and pathogenic characterization of genetic variants presented here is required to predict their medical consequences in newborns.

Galactosialidosis: review and analysis of CTSA gene mutations.

BACKGROUND: Mutations in the CTSA gene, that encodes the protective protein/cathepsin A or PPCA, lead to the secondary deficiency of ß-galactosidase (GLB1) and neuraminidase 1 (NEU1), causing the lysosomal storage disorder galactosialidosis (GS). Few clinical cases of GS have been reported in the literature, the majority of them belonging to the juvenile/adult group of patients. METHODS: The correct nomenclature of mutations for this gene is discussed through the analysis of the three PPCA/CTSA isoforms available in the GenBank database. Phenotype-genotype correlation has been assessed by computational analysis and review of previously reported single amino acid substitutions. RESULTS: We report the clinical and mutational analyses of four cases with the rare infantile form of GS. We identified three novel nucleotide changes, two of them resulting in the missense mutations, c.347A>G (p.His116Arg), c.775T>C (p.Cys259Arg), and the third, c.1216C>T, resulting in the p.Gln406* stop codon, a type of mutation identified for the first time in GS. An Italian founder effect of the c.114delG mutation can be suggested according to the origin of the only three patients carrying this mutation reported here and in the literature. CONCLUSIONS: In early reports mutations nomenclature was selected according to all CTSA isoforms (three different isoforms), thus generating a lot of confusion. In order to assist physicians in the interpretation of detected mutations, we mark the correct nomenclature for CTSA mutations. The complexity of pathology caused by the multifunctions of CTSA, and the very low numbers of mutations (only 23 overall) in relation to the length of the CTSA gene are discussed.In addition, the in silico functional predictions of all reported missense mutations allowed us to closely predict the early infantile, late infantile and juvenile phenotypes, also disclosing different degrees of severity in the juvenile phenotype.

New clinical and molecular insights on Barth syndrome.

BACKGROUND: Barth syndrome (BS) is an X-linked infantile-onset cardioskeletal disease characterized by cardiomyopathy, hypotonia, growth delay, neutropenia and 3-methylglutaconic aciduria. It is caused by mutations in the TAZ gene encoding tafazzin, a protein involved in the metabolism of cardiolipin, a mitochondrial-specific phospholipid involved in mitochondrial energy production. METHODS: Clinical, biochemical and molecular characterization of a group of six male patients suspected of having BS. Three patients presented early with severe metabolic decompensation including respiratory distress, oxygen desaturation and cardiomyopathy and died within the first year of life. The remaining three patients had cardiomyopathy, hypotonia and growth delay and are still alive. Cardiomyopathy was detected during pregnancy through a routine check-up in one patient. All patients exhibited 3-methylglutaconic aciduria and neutropenia, when tested and five of them also had lactic acidosis. RESULTS: We confirmed the diagnosis of BS with sequence analysis of the TAZ gene, and found five new mutations, c.641A>G p.His214Arg, c.284dupG (p.Thr96Aspfs*37), c.678_691del14 (p.Tyr227Trpfs*79), g.8009_16445del8437 and g.[9777_9814del38; 9911-?_14402del] and the known nonsense mutation c.367C>T (p.Arg123Term). The two gross rearrangements ablated TAZ exons 6 to 11 and probably originated by non-allelic homologous recombination and by Serial Replication Slippage (SRS), respectively. The identification of the breakpoints boundaries of the gross deletions allowed the direct detection of heterozygosity in carrier females. CONCLUSIONS: Lactic acidosis associated with 3-methylglutaconic aciduria is highly suggestive of BS, whilst the severity of the metabolic decompensation at disease onset should be considered for prognostic purposes. Mutation analysis of the TAZ gene is necessary for confirming the clinical and biochemical diagnosis in probands in order to identify heterozygous carriers and supporting prenatal diagnosis and genetic counseling.

Sudden unexpected infant death (SUDI) in a newborn due to medium chain acyl CoA dehydrogenase (MCAD) deficiency with an unusual severe genotype.

Medium chain acyl CoA dehydrogenase deficiency (MCAD) is the most common inborn error of fatty acid oxidation. This condition may lead to cellular energy shortage and cause severe clinical events such as hypoketotic hypoglycemia, Reye syndrome and sudden death. MCAD deficiency usually presents around three to six months of life, following catabolic stress as intercurrent infections or prolonged fasting, whilst neonatal-onset of the disease is quite rare. We report the case of an apparently healthy newborn who suddenly died at the third day of life, in which the diagnosis of MCAD deficiency was possible through peri-mortem blood-spot acylcarnitine analysis that showed very high concentrations of octanoylcarnitine. Genetic analysis at the ACADM locus confirmed the biochemical findings by demonstrating the presence in homozygosity of the frame-shift c.244dup1 (p.Trp82LeufsX23) mutation, a severe genotype that may explain the unusual and very early fatal outcome in this newborn. This report confirms that inborn errors of fatty acid oxidation represent one of the genetic causes of sudden unexpected deaths in infancy (SUDI) and underlines the importance to include systematically specific metabolic screening in any neonatal unexpected death.

Safety and efficacy of topiramate in neonates with hypoxic ischemic encephalopathy treated with hypothermia (NeoNATI).

BACKGROUND: Despite progresses in neonatal care, the mortality and the incidence of neuro-motor disability after perinatal asphyxia have failed to show substantial improvements. In countries with a high level of perinatal care, the incidence of asphyxia responsible for moderate or severe encephalopathy is still 2-3 per 1000 term newborns. Recent trials have demonstrated that moderate hypothermia, started within 6 hours after birth and protracted for 72 hours, can significantly improve survival and reduce neurologic impairment in neonates with hypoxic-ischemic encephalopathy. It is not currently known whether neuroprotective drugs can further improve the beneficial effects of hypothermia. Topiramate has been proven to reduce brain injury in animal models of neonatal hypoxic ischemic encephalopathy. However, the association of mild hypothermia and topiramate treatment has never been studied in human newborns. The objective of this research project is to evaluate, through a multicenter randomized controlled trial, whether the efficacy of moderate hypothermia can be increased by concomitant topiramate treatment. METHODS/DESIGN: Term newborns (gestational age ≥ 36 weeks and birth weight ≥ 1800 g) with precocious metabolic, clinical and electroencephalographic (EEG) signs of hypoxic-ischemic encephalopathy will be randomized, according to their EEG pattern, to receive topiramate added to standard treatment with moderate hypothermia or standard treatment alone. Topiramate will be administered at 10 mg/kg once a day for the first 3 days of life. Topiramate concentrations will be measured on serial dried blood spots. 64 participants will be recruited in the study. To evaluate the safety of topiramate administration, cardiac and respiratory parameters will be continuously monitored. Blood samplings will be performed to check renal, liver and metabolic balance. To evaluate the efficacy of topiramate, the neurologic outcome of enrolled newborns will be evaluated by serial neurologic and neuroradiologic examinations. Visual function will be evaluated by means of behavioural standardized tests. DISCUSSION: This pilot study will explore the possible therapeutic role of topiramate in combination with moderate hypothermia. Any favourable results of this research might open new perspectives about the reduction of cerebral damage in asphyxiated newborns.

Hypothermia for neonatal hypoxic-ischemic encephalopathy: may an early amplitude-integrated EEG improve the selection of candidates for cooling?

OBJECTIVE: To report our experience in the selection of newborns candidate to therapeutic hypothermia. METHODS: Retrospective study involving 47 newborns suffering from perinatal asphyxia from January 2008 to September 2011. RESULTS: Thirty-five of 47 newborns admitted to our hospital fulfilled metabolic and neurological criteria for recruitment and were cooled. aEEG was carried out in 26 of them and resulted always abnormal. In three of the 12 newborns with only metabolic criteria, aEEG was moderately abnormal. They were cooled and their outcome (evaluated by General Movements and Griffiths Mental Development Scales for children aged 0-2 years) is good. Three additional newborns who only met the metabolic criterion reached our hospital after the therapeutic window for hypothermia and exhibited seizures; their outcome is poor. CONCLUSIONS: In our experience, the inclusion of aEEG in the entry criteria would not have precluded newborns with neurological criteria from cooling. On the contrary, without an early aEEG, we would have excluded from hypothermia infants with moderate hypoxic-ischemic encephalopathy without precocious neurological signs who exhibited only the metabolic criterion, but with abnormal aEEG. If further studies will confirm that early aEEG might identify newborns suitable for cooling even in the absence of clinical signs, a revision of the entry criteria should be considered.

Strategies for reducing the incidence of skin complications in newborns treated with whole-body hypothermia.

OBJECTIVE: To present the results of a strategy designed to reduce the incidence of skin complications in newborns with hypoxic-ischemic encephalopathy treated with moderate whole-body hypothermia. DESIGN: Retrospective study. SETTING: Neonatal Intensive Care Unit (NICU). PATIENTS: Thirty-nine neonates cooled in the considered period. INTERVENTION: Starting from January 2008, for neonates treated with moderate whole-body hypothermia (33.5 °C), the cooling system was set in "automatic servo-controlled mode (ACM)", where the temperature of the circulating water could vary between 4 °C and 42 °C. Starting from January 2009, cooling blankets were used in another type of automatic mode, the "gradient variable mode (GVM)", where the circulating water was maintained at a specific pre-set gradient towards the patient's body temperature, and a specific nursing protocol (NP) was adopted. MEASUREMENTS AND MAIN RESULTS: Two of the eleven newborns treated with the "ACM" exhibited skin complications compatible with subcutaneous fat necrosis (SFN). None of the twenty-eight newborns treated with the "GVM" exhibited skin complications. A comparison of the biochemical and hematological data between these two groups revealed that newborns treated after the adopting of a NP and the "GVM" showed lower serum protein C and calcium levels, and higher platelet levels. CONCLUSIONS: Our data suggest that newborns undergoing therapeutic cooling may benefit from a specific NP and correct cooling unit setting. Should further studies confirm our data, this nursing approach could be easily adopted.

Oxidative state and IL-6 production in intestinal myofibroblasts of Crohn's disease patients.

BACKGROUND: Intestinal subepithelial myofibroblasts (ISEMFs) produce inflammatory cytokines in response to certain stimuli. In the intestine of patients with Crohn's disease (CD), cytokine synthesis is modified and an increased number of myofibroblasts has been observed. The intracellular redox state influences cytokine production and oxidative stress is present in the intestinal mucosa of CD patients. METHODS: This study was performed in ISEMFs isolated from the colon of patients with active CD and in a myofibroblast cell line derived from human colonic mucosa: 18Co cells. Cellular glutathione (GSH) levels were modulated by treatment with buthionine sulfoximine, an inhibitor of GSH synthesis, or N-acetylcysteine, a GSH precursor. GSH and oxidized glutathione (GSSG) levels were measured by high-performance liquid chromatography (HPLC) methods. Interleukin (IL)-6 production was detected by enzyme-linked immunosorbent assay (ELISA). RESULTS: ISEMFs of CD patients exhibited an increased oxidative state due to a decrease in the GSH/GSSG ratio, which is related to an increase in basal IL-6 production or is stimulated by tumor necrosis factor alpha (TNFα) or bacterial products. This relationship was also confirmed in 18Co cells. Phosphorylation and activation of ERK1/2 and p38 MAPK, which are signaling factors involved in the IL-6 synthesis, were also increased when there is oxidative stress in ISEMFs. CONCLUSIONS: This study shows for the first time in ISEMFs of CD patients an increased production of IL-6 synthesis related to the decrease in the GSH/GSSH ratio, suggesting redox regulation with the involvement of specific kinase activation. The present data shed light on the pathogenesis of inflammatory chronic processes and relapses that occur in this pathology.

GM1 gangliosidosis and Morquio B disease: an update on genetic alterations and clinical findings.

GM1 gangliosidosis and Morquio B syndrome, both arising from beta-galactosidase (GLB1) deficiency, are very rare lysosomal storage diseases with an incidence of about 1:100,000-1:200,000 live births worldwide. Here we report the beta-galactosidase gene (GLB1) mutation analysis of 21 unrelated GM1 gangliosidosis patients, and of 4 Morquio B patients, of whom two are brothers. Clinical features of the patients were collected and compared with those in literature. In silico analyses were performed by standard alignments tools and by an improved version of GLB1 three-dimensional models. The analysed cohort includes remarkable cases. One patient with GM1 gangliosidosis had a triple X syndrome. One patient with juvenile GM1 gangliosidosis was homozygous for a mutation previously identified in Morquio type B. A patient with infantile GM1 gangliosidosis carried a complex GLB1 allele harbouring two genetic variants leading to p.R68W and p.R109W amino acid changes, in trans with the known p.R148C mutation. Molecular analysis showed 27 mutations, 9 of which are new: 5 missense, 3 microdeletions and a nonsense mutation. We also identified four new genetic variants with a predicted polymorphic nature that was further investigated by in silico analyses. Three-dimensional structural analysis of GLB1 homology models including the new missense mutations and the p.R68W and p.R109W amino acid changes showed that all the amino acid replacements affected the resulting protein structures in different ways, from changes in polarity to folding alterations. Genetic and clinical associations led us to undertake a critical review of the classifications of late-onset GM1 gangliosidosis and Morquio B disease.

Redox regulation of ERK1/2 activation induced by sphingosine 1-phosphate in fibroblasts: involvement of NADPH oxidase and platelet-derived growth factor receptor.

BACKGROUND: Sphingosine 1-phosphate (S1P) is a sphingolipid metabolite synthesized after stimulation with growth factors or cytokines. S1P extracellular effects are mediated through specific Gi-protein coupled receptors (GPCRs). Recently, we demonstrated in NIH3T3 fibroblasts stimulated by platelet-derived growth factor (PDGF) or S1P the NADPH oxidase activation and the H(2)O(2) intracellular level increase trough the Gi protein involvement. METHODS: NIH3T3 fibroblast cell cultures were used. Western blot and quantitative analyses by Chemidoc-Quantity-One software were performed. H(2)O(2) level was assayed by fluorescence spectrophotometric analysis, and cell proliferation by counted manually or ELISA kit. RESULTS: This study demonstrates, in NIH 3T3 fibroblasts, a novel redox regulated mechanism of S1P-induced activation of ERK 1/2 related to NADPH oxidase activity and intracellular H(2)O(2) level increase with PDGF receptor tyrosine kinase involvement through a transactivation mechanism. This event is mediated by S1P(1) and S1P(3) receptors by Gi proteins and can contribute to S1P mitogenic signaling. CONCLUSION: These results can be related to mechanisms of cross-talk previously identified between receptor tyrosine kinase, including PDGFreceptor, and several GPCR ligands. GENERAL SIGNIFICANCE: The redox-sensitive ERK1/2 and PDGFr tyrosine kinase activity could be targets for therapies in diseases in which deregulation of intracellular oxidative status and the consequent alteration of S1P and/or PDGF signaling pathway are involved.