Knockdown of MVK does not lead to changes in NALP3 expression or activation

Mutations in the Mevalonate Kinase gene (MVK) are causes of a rare autoinflammatory disease: Mevalonate Kinase Deficiency and its more acute manifestation, Mevalonic Aciduria. The latter is characterized, among other features, by neuroinflammation, developmental delay and ataxia, due to failed cerebellar development or neuronal death through chronic inflammation. Pathogenesis of neuroinflammation in Mevalonate Kinase Deficiency and Mevalonic Aciduria has not yet been completely clarified, however different research groups have been suggesting the inflammasome complex as the key factor in the disease development. A strategy to mimic this disease is blocking the mevalonate pathway, using HMG-CoA reductase inhibitors (Statins), while knock-out mice for Mevalonate Kinase are non-vital and their hemyzygous (i.e only one copy of gene preserved) littermate display almost no pathological features

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

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

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

Copy number variation, gene expression and histological localization of human beta-defensin 2 in patients with adeno-tonsillar hypertrophy

Both bacterial infections and innate oral immunity response participate in development of adeno-tonsillar hypertrophy (ATH). ATH can lead to obstructive sleep apnea. We investigated the beta-defensin 2 (hBD-2) encoding gene, DEFB4, by analyzing the copy number variations (CNVs) of the defensin gene cluster in patients with ATH and by correlating CNV with DEFB4 gene expression. We enrolled 79 patients with ATH, 21 of whom presented with only adenoid hypertrophy, while 58 exhibited hypertrophy of both adenoid and tonsil. CNVs of the defensin gene cluster, DEFB4 mRNA, and hBD-2 protein expression were assessed. Also, beta-defensin 2 was localized histologically using immunohistochemistry. The distribution of defensin gene cluster CNV was similar among the 79 subjects. DEFB4 expression analysis exhibited considerable inter-individual variability, but with neither specific differences among subjects nor correlation with the CNV number. Immunohistochemistry enabled localization of hBD-2 in the tonsil and adenoid epithelium. No differences in localization between the two ATH presentations were found. Inducible antimicrobial defensin peptides exhibited great inter-individual variability in terms of both CNV and gene expression, but no correlation with presentation of ATH was found

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

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. (Figure presented.).This work was supported by the Italian Ministry of Health, through the contribution given to the Institute for Maternal and Child Health IRCCS Burlo Garofolo, Trieste, Italy (RC 15/17 and RC 29/23)

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-Barre syndrome in children) and analgesic drug screening

Mast Cells in Peritoneal Fluid From Women With Endometriosis and Their Possible Role in Modulating Sperm Function

Endometriosis is a local pelvic inflammatory process, frequently associated with infertility, with altered function of immune-related cells in the peritoneal environment. Mast cells are known to be key players of the immune system and have been recently involved in endometriosis and in infertility, with their mediators directly suppressing sperm motility. In this study, we evaluated the mast cell population and their mediators in the peritoneal fluid of infertile patients with endometriosis and their impact on human sperm motility. Peritoneal fluids, collected by laparoscopy from 11 infertile patients with endometriosis and 9 fertile controls were evaluated for the presence of mast cells, tryptase levels and their effect on sperm motility. Furthermore, an in vitro model of mast cells-sperm interaction in peritoneal fluid was set up, using LAD2 cell line as a mast cell model, and analyzed from a functional as well as a morphological point of view. Mast cell peritoneal fluid population and its main mediator, tryptase, is more represented in endometriosis confirming an involvement of these cells in this disease. Anyway it appears unlikely that tryptase enriched peritoneal fluid, which fails to inhibit sperm motility, could contribute to endometriosis associated infertility. Despite of this, sperm interaction with the mast cell surface (LAD2) induced a significantly mast cell-degranulation response in the peritoneal fluid from endometriosis which could directly modulate sperm function other than motility. This evidence lead us to suppose that there is, between these elements, an interrelationship which deserves further studies

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.This research was funded by I.R.C.C.S. “B. G.”, grant number RC 02/20 and RC 15/17

Transcriptomic profiling of 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, 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. Cereblon 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 mechanisms underpinning thalidomide action in pediatric IBD. Ten IBD pediatric patients clinically responsive to thalidomide were prospectively enrolled. RNA-sequencing and functional enrichment analysis was carried out on peripheral blood mononuclear cells obtained before and after treatment with thalidomide. RNA-sequencing analysis revealed 378 differentially expressed genes 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 induced by 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 altered candidate pathways observed in patients. These results represent a unique resource for enhanced understanding of thalidomide mechanism in patients with IBD, providing novel potential targets associated with drug response.Book of abstract: 4th Belgrade Bioinformatics Conference, June 19-23, 202