Effectiveness of pharmacological procedural sedation in children with autism spectrum disorder: A systematic review and meta-analysis.

AIM: Management of primary healthcare and routine minor procedures for children with autism spectrum disorder (ASD) can be challenging; therefore, when behavioural strategies fail, sedative medications are often employed. We evaluated the effectiveness of the current pharmacological strategies for managing children with ASD. METHODS: We performed a systematic review and meta-analysis of the current approaches for procedural sedation in children with ASD. RESULTS: Twenty studies met inclusion criteria. Dexmedetomidine, midazolam, propofol and chloral hydrate were the most efficient agents for successful procedures, while propofol had the highest number of adverse events. The most frequently used agents were dexmedetomidine and midazolam or a combination of the two, and the effectiveness of dexmedetomidine plus midazolam was superior to dexmedetomidine alone. CONCLUSION: Multiple effective drug regimens exist for procedural sedation in children with ASD. These results could support the development of specific guidelines for procedural sedation in children with ASD.

Upside-Down Preference in the Forskolin-Induced In Vitro Differentiation of 50B11 Sensory Neurons: A Morphological Investigation by Label-Free Non-Linear Microscopy.

In this study, we revealed a peculiar morphological feature of 50B11 nociceptive sensory neurons in in vitro culture related to the forskolin-induced differentiation of these cells growing upside-down on cover glass supports. Multi-photon non-linear microscopy was applied to monitor increased neurite arborization and elongation. Under live and unstained conditions, second harmonic generation (SHG) microscopy could monitor microtubule organization inside the cells while also correlating with the detection of cellular multi-photon autofluorescence, probably derived from mitochondria metabolites. Although the differentiated cells of each compartment did not differ significantly in tubulin or multi-photon autofluorescence contents, the upturned neurons were more elongated, presenting a higher length/width cellular ratio and longer neurites, indicative of differentiated cells. SHG originating from the axons' microtubules represented a proper tool to study neurons' inverted culture in live conditions without exogenous staining. This work represents the first instance of examining neuronal cell lines growing and differentiated in an upside-down orientation, allowing a possible improvement of 50B11 as a model in physiology studies of sensory neurons in peripheric nervous system disease (e.g., Fabry disease, Friedreich ataxia, Charcot-Marie-Tooth, porphyria, type 1 diabetes, Guillain-Barré syndrome in children) and analgesic drug screening.

Gel-Based Proteomic Identification of Suprabasin as a Potential New Candidate Biomarker in Endometrial Cancer.

Endometrial cancer (EC) is the most frequent gynaecologic cancer in postmenopausal women. We used 2D-DIGE and mass spectrometry to identify candidate biomarkers in endometrial cancer, analysing the serum protein contents of 10 patients versus 10 control subjects. Using gel-based proteomics, we identified 24 candidate biomarkers, considering only spots with a fold change in volume percentage ≥ 1.5 or intensity change ≤ 0.6, which were significantly different between cases and controls (p < 0.05). We used Western blotting analysis both in the serum and tissue of 43 patients for data validation. Among the identified proteins, we selected Suprabasin (SBSN), an oncogene previously associated with poor prognosis in different cancers. SBSN principal isoforms were subjected to Western blotting analysis in serum and surgery-excised tissue: both isoforms were downregulated in the tissue. However, in serum, isoform 1 was upregulated, while isoform 2 was downregulated. Data-mining on the TCGA and GTEx projects, using the GEPIA2.0 interface, indicated a diminished SBSN expression in the Uterine Corpus Endometrial Cancer (UCEC) database compared to normal tissue, confirming proteomic results. These results suggest that SBSN, specifically isoform 2, in tissue or serum, could be a potential novel biomarker in endometrial cancer.

Correction to: Photobiomodulation therapy at different wavelength impacts on retinoid acid-dependent SH-SY5Y differentiation.

After publication of this paper, the authors determined an error in the author group. The correct presentation of authors are below.

Proteomic Study Identifies Glycolytic and Inflammation Pathways Involved in Recurrent Otitis Media.

Recurrent acute otitis media (RAOM) in children is clinically defined as the occurrence of at least three episodes of acute otitis media over a course of 6 months. A further common pathological condition of interest in the context of pediatric otolaryngology is adenotonsillar hypertrophy (ATH), a common cause of obstructive sleep apnea syndrome. Aimed at unraveling the differential modulation of proteins in the two pathologies and at understanding the possible pathways involved in their onset, we analyzed the proteomic profile of the adenoids from 14 RAOM and ATH patients by using two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS). The 2-DE coupled with MS allowed us to identify 23 spots with significant (p-value < 0.05) changes in protein amount, recognizing proteins involved in neutrophil degranulation and glycolysis pathways.

MMAB, a novel candidate gene to be screened in the molecular diagnosis of Mevalonate Kinase Deficiency.

Mevalonate kinase deficiency (MKD) is an autosomal recessive inflammatory disease. Mutations in MVK gene are associated with MKD with modest genotype-phenotype correlation. In spite of recent guidelines indicating specific MVK mutations for the more severe form or the milder one, little is known about MVK variability within and between populations. The aim of this work is to provide supplementary information about MVK variability useful in the molecular diagnosis of MKD, as well as to unravel the presence of novel genes potentially involved as involved in the clinical heterogeneity of MKD phenotype. We used a population-based approach, coupled with Combined Annotation-Dependent Depletion (CADD) score, to analyze the level of genetic variability for common and putatively deleterious MVK variants. We also performed Exome screening with the Illumina Human Exome Bead Chip on 21 MKD patients to double-check our in silico findings. Haplotype block detection in different populations revealed the existence of two blocks in MVK; interestingly, the first haploblock comprises the promoter region shared with MMAB gene. Analyses of MMAB and MVK genetic variants in 21 MKD patients strengthen our observations showing a novel scenario in which the same mutations commonly associated with MKD are found coupled with different combination of MMAB rs7134594 SNP was already described as associated with HDL cholesterol level and present in the haploblock promoter region. The rs7134594 SNP is reported as an eQTL for MVK and MMAB. Hypothesizing the presence of genetic variants modulating the complex phenotypic spectrum of MKD, we suggest that future directions in screening for MKD pathogenic variants should focus both MMAB and MVK genes.

Alendronate treatment induces IL-1B expression and apoptosis in glioblastoma cell line.

Alendronate (ALD), one among the nitrogen-containing bisphosphonates (NBPs), is currently used for the treatment of many pathological conditions. Unfortunately, although generally tolerated, NBPs treatment has been associated with central nervous system (CNS) adverse outcomes, such as amnesia, hallucinations and visual disturbances. So, we analyzed the effect of ALD treatment in glial cells, the main sources of cholesterol for neurons and principal cells involved in the immunological defense of the brain. We treated a glial cell line (U87-MG) with increasing doses of ALD (0.1, 1, 10, 25, 50 µM) for 48 h, aimed at evaluating the influence of this drug treatment on IL-1B expression, NLRP3 and CASP1 expression, mitochondrial activity and apoptotic cell death. We observed that ALD treatment, at the higher concentrations, induced a significant increase of IL-1B, NLRP3, CASP1 expression, provoked apoptosis and also mitochondrial damage in U87-MG. Considering the reported CNS adverse outcomes of NBPs treatment, our results confirm ALD side-effects on glial cell model.

Lack of Prenylated Proteins, Autophagy Impairment and Apoptosis in SH-SY5Y Neuronal Cell Model of Mevalonate Kinase Deficiency.

BACKGROUND/AIMS: Mevalonate Kinase Deficiency (MKD), is a hereditary disease due to mutations in mevalonate kinase gene (MVK). MKD has heterogeneous clinical phenotypes: the correlation between MVK mutations and MKD clinical phenotype is still to be fully elucidated. Deficiency of prenylated proteins has been hypothesized as possible MKD pathogenic mechanism. Based on this hypothesis and considering that neurologic impairment characterizes Mevalonic Aciduria (MA), the most severe form of MKD, we studied the effects of I268T and N301T MVK mutations on protein prenylation, autophagy and programmed cell death in SH-SY5Y neuroblastoma cell lines. METHODS: SH-SY5Y cells were transiently transfected, with the pCMV-6 plasmid containing MVK wild type and the two mutated sequences. Protein prenylation levels were evaluated using GFP-RhoA-F to assess farnesylation, and GFP-RhoA to evaluate geranylgeranylation; autophagy was measured by evaluating LC3 and p62 protein levels, while Annexin V-FITC and Propidium Iodide staining allowed apoptosis detection. RESULTS: MVK mutants' over-expression causes decreased levels of farnesylation and geranylgeranylation, and also increased LC3 lipidation in SH-SY5Y, with concomitant p62 accumulation. Treatment with bafilomycin A1 (an inhibitor of vacuolar H+-ATPase, a late autophagy inhibitor) further increase LC3-II and p62 levels, suggesting that degradation of autophagolysosome could be impaired. SH-SY5Y, with both MVK mutants, showed apoptosis increase; the presence of N301T associated with augmented cell death. CONCLUSIONS: We hypothesize that mevalonate pathway impairment causes alteration of farnesylation and geranylgeranylation proteins and alteration of the autophagic flux; these changes can induce apoptosis, possibly more relevant in the presence of N301T mutation.

Antiretroviral Treatment in HIV-1-Positive Mothers: Neurological Implications in Virus-Free Children.

Since the worldwide introduction of antiretroviral therapy (ART) in human immunodeficiency virus type 1, HIV-1-positive mothers, together with HIV-1 testing prior to pregnancy, caesarian birth and breastfeeding cessation with replacement feeding, a reduction of HIV-1 mother-to-child transmission (MTCT) has been observed in the last few years. As such, an increasing number of children are being exposed in utero to ART. Several questions have arisen concerning the neurological effects of ART exposure in utero, considering the potential effect of antiretroviral drugs on the central nervous system, a structure which is in continuous development in the fetus and characterized by great plasticity. This review aims at discussing the possible neurological impairment of children exposed to ART in utero, focusing attention on the drugs commonly used for HIV-1 MTCT prevention, clinical reports of ART neurotoxicity in children born to HIV-1-positive mothers, and neurologic effects of protease inhibitors (PIs), especially ritonavir-"boosted" lopinavir (LPV/r) in cell and animal central nervous system models evaluating the potential neurotoxic effect of ART. Finally, we present the findings of a meta-analysis to assess the effects on the neurodevelopment of children exposed to ART in utero.

HIV Protease Inhibitors Apoptotic Effect in SH-SY5Y Neuronal Cell Line.

BACKGROUND: Prophylactic treatment regimens to prevent mother-to-child HIV transmission include protease inhibitors Lopinavir and Ritonavir. Lopinavir and Ritonavir have been reported to be able to induce intracellular oxidative stress in diverse cellular models, however scarce informations are available about protease inhibitor effects of in the central nervous system (CNS). In our study we evaluated the impact of protease inhibitors on a cell neuronal model. METHODS: We treated a neuroblastoma cell line (SH-SY5Y) with increasing doses of Lopinavir and Ritonavir (0.1-1-10-25-50 µM), used alone or in combination, evaluating the impact of these drugs in terms of mitochondrial activity, with MTT cell proliferation assay; mRNA expression of heme oxygenase (HemeOH) and reactive oxygen species (ROS) levels with 2',7'-dichlorofluorescin diacetate (H2DCFDA) in order to assess oxidative stress; apoptotic cell death with flow cytometry. RESULTS: We observed that Lopinavir and Ritonavir treatment, at 25 and/or 50 µM concentrations, induced mitochondrial damage, increase of heme oxygenase RNA expression levels and ROS generation, followed by apoptosis in SH-SY5Y. CONCLUSIONS: Our in vitro model demonstrates a damaging effect of HIV protease inhibitors on the neuroblastoma cell line, thus partially mimicking the impact of these drugs on the CNS of children born to HIV positive mothers undergone to antiretroviral treatment.

Mevalonate Pathway Blockade, Mitochondrial Dysfunction and Autophagy: A Possible Link.

The mevalonate pathway, crucial for cholesterol synthesis, plays a key role in multiple cellular processes. Deregulation of this pathway is also correlated with diminished protein prenylation, an important post-translational modification necessary to localize certain proteins, such as small GTPases, to membranes. Mevalonate pathway blockade has been linked to mitochondrial dysfunction: especially involving lower mitochondrial membrane potential and increased release of pro-apoptotic factors in cytosol. Furthermore a severe reduction of protein prenylation has also been associated with defective autophagy, possibly causing inflammasome activation and subsequent cell death. So, it is tempting to hypothesize a mechanism in which defective autophagy fails to remove damaged mitochondria, resulting in increased cell death. This mechanism could play a significant role in Mevalonate Kinase Deficiency, an autoinflammatory disease characterized by a defect in Mevalonate Kinase, a key enzyme of the mevalonate pathway. Patients carrying mutations in the MVK gene, encoding this enzyme, show increased inflammation and lower protein prenylation levels. This review aims at analysing the correlation between mevalonate pathway defects, mitochondrial dysfunction and defective autophagy, as well as inflammation, using Mevalonate Kinase Deficiency as a model to clarify the current pathogenetic hypothesis as the basis of the disease.

The mitochondrial-related effect of the 905 nm photobiomodulation therapy on 50B11 sensory neurons.

Photobiomodulation therapy (PBMT) is known as a complementary tool to alleviate pain sensation in patients, nevertheless, there is still a gap of knowledge on its mechanism of action, thus limiting its clinical employment. In this study, a possible molecular mechanism of the 905 nm PBMT (0.25 W/cm2 ; 3, 6, 12, and 18 J/cm2 , 5 Hz) analgesic effect was tested on 50B11 cells, by investigating its impact on mitochondria. A decrement of adenosine triphosphate was detected, moreover, an increment of total reactive oxygen species and mitochondrial superoxide anion was found after PBMT with all protocols tested. PBMT at 18 J diminished the mitochondrial membrane potential, and influenced mitochondrial respiration, decreasing the oxygen consumption rate. Finally, a decrement of extracellular signal-regulated kinase 1/2 phosphorylation was observed with the protocol using 12 J. Taken together these findings highlighted the intracellular effects, mainly correlated to mitochondrial, induced by 905 nm PBMT in sensory neurons, indicating the central role of this organelle in the cellular response to 905 nm near-infrared laser light.

Transient Receptor Potential Ankyrin 1 (TRPA1) Methylation and Chronic Pain: A Systematic Review.

BACKGROUND AND OBJECTIVE: Chronic pain represents a major global health issue in terms of psycho-physiological, therapeutic, and economic burden, not limited to adults but also to the pediatric age. Despite its great impact, its molecular mechanisms have still not been completely unraveled. Focusing on the impact of epigenetics in the pain complex trait, we assessed the association between chronic pain and the methylation pattern of TRPA1, a key gene related to pain sensitivity. METHODS: We conducted a systematic review retrieving articles from three different databases. After deduplication, 431 items were subjected to manual screening, and then 61 articles were selected and screened again. Of these, only six were maintained for meta-analysis and analyzed using specific R packages. RESULTS: Six articles were divided into two groups (group 1: comparison of mean methylation levels between healthy subjects and patients with chronic pain; group 2: correlation between mean methylation levels and pain sensation). A non-significant mean difference was obtained from the analysis of group 1 with a value of 3.97 (95% C.I. -7.79; 15.73). Analysis of group 2 showed a high level of variability between studies (correlation = 0.35, 95% C.I. -0.12; 0.82) due to their heterogeneity (I2 = 97%, p < 0.01). CONCLUSIONS: Despite the high variability observed in the different studies analyzed, our results suggest that hypermethylation and increased pain sensitivity could be connected, possibly due to the variation of TRPA1 expression.

Gene expression profiling in white blood cells reveals new insights into the molecular mechanisms of thalidomide in children with inflammatory bowel disease.

Thalidomide has emerged as an effective immunomodulator in the treatment of pediatric patients with inflammatory bowel disease (IBD) refractory to standard therapies. Cereblon (CRBN), a component of E3 protein ligase complex that mediates ubiquitination and proteasomal degradation of target proteins, has been identified as the primary target of thalidomide. CRBN plays a crucial role in thalidomide teratogenicity, however it is unclear whether it is also involved in the therapeutic effects in IBD patients. This study aimed at identifying the molecular mechanisms underpinning thalidomide action in pediatric IBD. In this study, ten IBD pediatric patients responsive to thalidomide were prospectively enrolled. RNA-sequencing (RNA-seq) analysis and functional enrichment analysis were carried out on peripheral blood mononuclear cells (PBMC) obtained before and after twelve weeks of treatment with thalidomide. RNA-seq analysis revealed 378 differentially expressed genes before and after treatment with thalidomide. The most deregulated pathways were cytosolic calcium ion concentration, cAMP-mediated signaling, eicosanoid signaling and inhibition of matrix metalloproteinases. Neuronal signaling mechanisms such as CREB signaling in neurons and axonal guidance signaling also emerged. Connectivity Map analysis revealed that thalidomide gene expression changes were similar to those exposed to MLN4924, an inhibitor of NEDD8 activating enzyme, suggesting that thalidomide exerts its immunomodulatory effects by acting on the ubiquitin-proteasome pathway. In vitro experiments on cell lines confirmed the effect of thalidomide on candidate altered pathways observed in patients. These results represent a unique resource for enhanced understanding of thalidomide mechanism in pediatric patients with IBD, providing novel potential targets associated with drug response.

Responses to sulfated steroids of female mouse vomeronasal sensory neurons.

The rodent vomeronasal organ plays an important role in many social behaviors. Using the calcium imaging technique with the dye fluo-4 we measured intracellular calcium concentration changes induced by the application of sulfated steroids to neurons isolated from the vomeronasal organ of female mice. We found that a mix of 10 sulfated steroids from the androgen, estrogen, pregnanolone, and glucocorticoid families induced a calcium response in 71% of neurons. Moreover, 31% of the neurons responded to a mix composed of 3 glucocorticoid-derived compounds, and 28% responded to a mix composed of 3 pregnanolone-derived compounds. Immunohistochemistry showed that neurons responding to sulfated steroids expressed phosphodiesterase 4A, a marker specific for apical neurons expressing V1R receptors. None of the neuron that responded to 1 mix responded also to the other, indicating a specificity of the responses. Some neurons responded to more than 1 individual component of the glucocorticoid-derived mix tested at high concentration, suggesting that these neurons are broadly tuned, although they still displayed strong specificity, remaining unresponsive to high concentrations of the ineffective compounds.

Ferric Carboxymaltose in the Treatment of Iron-Deficiency Anaemia in Paediatric Patients with Anastomotic Ulcers.

OBJECTIVES: The aim of this paper is to describe a case series of paediatric patients affected by anastomotic ulcers (AU), a late complication of bowel resection in infancy, focusing on the treatment of iron-deficiency anaemia (IDA) with ferric carboxymaltose (FC). METHODS: Patients with a diagnosis of AU, treated at the Paediatric Department of the Institute for Maternal and Child Health IRCCS "Burlo Garofolo" from February 2012 to December 2020 were included. Haemoglobin (Hb) values, IDA related symptoms, the need for blood transfusions, for oral or intravenous (iv) iron supplementation and for surgical resections were compared before and after treatment with FC. Adverse effects of FC were recorded. RESULTS: Ten patients with an established diagnosis of AU were identified; eight (8 out of 10) received at least one administration of FC. Lower and higher Hb values increased significantly after treatment (4.9 g/dL vs. 8.2 g/dL, p = 0.0003; 9.9 g/dL vs. 13.5 g/dL, p = 0.0008 respectively), with a significant reduction of the need for blood transfusions (p = 0.0051) and for oral and iv iron supplementation. While receiving standard therapies, seven patients (7 out of 8) complained of asthenia; this symptom resolved in all cases after FC administration. Before FC treatment, two patients (2 out of 8) required surgical resection of AU, with a recurrence of anaemia after a few weeks; after at least one FC infusion, no children needed further bowel resection for IDA. FC caused mild asymptomatic hypophosphatemia in one case. CONCLUSION: FC appears to be effective and safe in the paediatric population for the treatment of IDA resulting from AU.

Photobiomodulation for Lowering Pain after Tonsillectomy: Low Efficacy and a Possible Unexpected Adverse Effect.

BACKGROUND: Tonsillectomy is one of the most common surgical procedures performed in children as a treatment for obstructive sleep apnea due to tonsil hypertrophy or highly recurrent tonsillitis. Odynophagia, associated with food refusal for the first few days, is a common post-operative complaint. Available drugs for pain management, while efficacious, present some drawbacks, and a novel strategy would be welcome. Photobiomodulation (PBMT), in this context, can represent a possible choice, together with pharmacological therapy. The aim of this study was to evaluate PBMT effects compared to standard pain therapy on nociceptive sensation at different time points and administration of painkiller. METHODS: A registered, controlled, randomized, double-blind clinical trial was performed. Twenty-two patients were recruited and divided into laser-treated (T) or untreated (UT) groups, based on random assignment. In T group, immediately after tonsillectomy, performed with cold dissection technique, laser light was applied to the surgery site (using a Cube 4 from Eltech K-Laser s.r.l., Treviso, Italy), and then hemostasis was performed using bismuth subgallate paste. In C group, the same procedure was performed, except that laser light was switched off. The primary outcome was the difference in pain scores between subject receiving photobiomodulation (PBMT) and subjects receiving standard care after 24 h; the secondary outcomes were pain scores at awakening and at 48 h together with distress (delirium) at awakening. RESULTS: Two patients from the T group experienced a post-surgery bleeding, and one of them required revision of the hemostasis under general anesthesia. A preliminary analysis of pain sensation reported by the patients or caregivers did not show differences between treated and untreated subjects. CONCLUSION: These data suggest that PBMT could increase post-surgical bleeding.

In vitro effects of photobiomodulation therapy on 50B11 sensory neurons: evaluation of cell metabolism, oxidative stress, mitochondrial membrane potential (MMP), and capsaicin-induced calcium flow.

The analgesic properties of photobiomodulation therapy (PBMT) have been raising increasing interest in the clinical community due to the positive effects observed on patients, nevertheless the mechanistic basis of its action on peripheral sensory neurons remains still elusive. In this study, the effect of near-infrared (NIR) PBMT at 800 and 970 nm of wavelength was investigated on the 50B11 immortalized nociceptive sensory neuronal cell line by evaluating capsaicin-induced calcium flow and different markers correlated to mitochondria, that is, ATP, reactive oxygen species (ROS), and mitochondrial membrane potential (MMP). Calcium peak stimulated by capsaicin, the ligand of TRPV1 channel, was decreased in neurons pre-irradiated with the combination of the two wavelengths. Furthermore, delivering the 800 and 970 nm separately an increment of ATP, as well as MMP hyperpolarization were detected; notably, the 800 nm wavelength also increased ROS and O2- levels. Our findings, obtained on an in vitro model of nociception, show the positive effect of PBMT on two potential photo-targets of NIR light, namely the TRPV1 channel and the mitochondria.

Early introduction oral immunotherapy for IgE-mediated cow's milk allergy: A follow-up study confirms this approach as safe and appealing to parents.

INTRODUCTION: Early introduction oral immunotherapy (E-OIT) in the first year of life can be a safe treatment for infants with cow's milk allergy (CMA). Once the protocol is completed, doubts remain whether children achieve tolerance or remain desensitized. According to current guidelines, this is determined by an avoidance period followed by a re-exposure to the food allergen during an in-hospital oral food challenge (OFC). In real life, this approach can be complicated, time-consuming, and anxiety-provoking for parents. We assessed the long-term safety of E-OIT for CMA in a cohort of children who switched to an unrestricted diet without testing the achievement of tolerance at the end of the OIT protocol. MATERIALS AND METHODS: We performed a descriptive analysis of the clinical follow-up of a cohort of children diagnosed with IgE-mediated CMA and undergoing E-OIT protocol in their first year of life. In a previous publication, the same cohort of patients had been studied to assess the feasibility of E-OIT for CMA. In the present study, we reported the results of a telephone survey, carried out through a questionnaire to their families enquiring about milk consumption and other ongoing atopic conditions of children. RESULTS: After an average of 4 years from the start of E-OIT, 62/73 patients (85% of the historical cohort) participated in the survey. Among them, all 56 patients who had previously successfully completed the protocol reported an unrestricted cow's milk intake. Ninety-three percent of these children did not experience any further allergic reactions, while the remaining 7% described only mild and transitory reactions until the 6-month period after the end of the protocol. CONCLUSIONS: This study confirmed the long-term safety of E-OIT for CMA and challenged the paradigm of the need for allergen food withdrawal to discern between desensitization and tolerance. It could be a starting point for planning future trials on this issue.