Therapeutic Drug Monitoring of Quinidine in Pediatric Patients with KCNT1 Genetic Variants

Quinidine (QND) is an old antimalarial drug that was used in the early 20th century as an antiarrhythmic agent. Currently, QND is receiving attention for its use in epilepsy of infancy with migrating focal seizures (EIMFS) due to potassium sodium-activated channel subfamily T member 1 (KCNT1) genetic variants. Here, we report the application of Therapeutic Drug Monitoring (TDM) in pediatric patients carrying KCNT1 genetic variants and orally treated with QND for developmental and epileptic encephalopathies (DEE). We measured plasma levels of QND and its metabolite hydroquinidine (H-QND) by using a validated method based on liquid chromatography coupled with mass spectrometry (LC-MS/MS). Three pediatric patients (median age 4.125 years, IQR 2.375-4.125) received increasing doses of QND. Cardiac toxicity was monitored at every dose change. Reduction in seizure frequency ranged from 50 to 90%. Our results show that QND is a promising drug for pediatric patients with DEE due to KCNT1 genetic variants. Although QND blood levels were significantly lower than the therapeutic range as an anti-arrhythmic drug, patients showed a significant improvement in seizure burden. These data underlie the utility of TDM for QND not only to monitor its toxic effects but also to evaluate possible drug-drug interactions

An orally administered butyrate-releasing derivative reduces neutrophil recruitment and inflammation in dextran sulphate sodium-induced murine colitis

BACKGROUND AND PURPOSE: Butyrate has shown benefits in inflammatory bowel diseases. However, it is not often administered orally because of its rancid smell and unpleasant taste. The efficacy of a more palatable butyrate-releasing derivative, N-(1-carbamoyl-2-phenylethyl) butyramide (FBA), was evaluated in a mouse model of colitis induced by dextran sodium sulphate (DSS). EXPERIMENTAL APPROACH: Male 10 week-old BALB/c mice received DSS (2.5%) in drinking water (for 5 days) followed by DSS-free water for 7 days (DSS group). Oral FBA administration (42.5 mg·kg-1 ) was started 7 days before DSS as preventive (P-FBA), or 2 days after DSS as therapeutic (T-FBA); both treatments lasted 19 days. One DSS-untreated group received only tap water (CON). KEY RESULTS: FBA treatments reduced colitis symptoms and colon damage. P-FBA and T-FBA significantly decreased polymorphonuclear cell infiltration score compared with the DSS group. FBA reversed the imbalance between pro- and anti-inflammatory cytokines (reducing inducible NOS protein expression, CCL2 and IL-6 transcripts in colon and increasing TGFβ and IL-10). Morever, P-FBA and T-FBA limited neutrophil recruitment (by expression and localization of the neutrophil granule protease Ly-6G), restored deficiency of the butyrate transporter and improved intestinal epithelial integrity, preventing tight-junction impairment (zonulin-1 and occludin). FBA, similar to its parental compound sodium butyrate, inhibited histone deacetylase-9 and restored H3 histone acetylation, exerting an anti-inflammatory effect through NF-κB inhibition and the up-regulation of PPARγ. CONCLUSIONS AND IMPLICATIONS: FBA reduces inflammatory intestinal damage in mice indicating its potential as a postbiotic derivative without the problems associated with the oral administration of sodium butyrate

Effects of a Lactobacillus paracasei B21060 based synbiotic on steatosis, insulin signaling and toll-like receptor expression in rats fed a high-fat diet.

Insulin resistance (IR) has been identified as crucial pathophysiological factor in the development and progression of non-alcoholic fatty liver disease (NAFLD). Although mounting evidence suggests that perturbation of gut microflora exacerbates the severity of chronic liver diseases, therapeutic approaches using synbiotic has remained overlooked. Here, we show that a synbiotic composed by Lactobacillus paracasei B21060 plus arabinogalactan and fructo-oligosaccharides lessens NAFLD progression in a rat model of high fat feeding. IR and steatosis were induced by administration of high fat diet (HFD) for 6 weeks. Steatosis and hepatic inflammation, Toll-like receptor (TLR) pattern, glucose tolerance, insulin signaling and gut permeability were studied. Liver inflammatory markers were down-regulated in rats receiving the synbiotic, along with an increased expression of nuclear peroxisome proliferator-activated receptors and expression of downstream target genes. The synbiotic improved many aspects of IR, such as fasting response, hormonal homeostasis and glycemic control. Indeed it prevented the impairment of hepatic insulin signaling, reducing the phosphorylation of insulin receptor substrate-1 in Ser 307 and down-regulating suppressor of cytokine signaling 3. Gene expression analysis revealed that in the liver the synbiotic reduced cytokines synthesis and restored the HFD-dysregulated TLR 2, 4 and 9 mRNAs toward a physiological level of expression. The synbiotic preserved gut barrier integrity and reduced the relative amount of Gram-negative Enterobacteriales and Escherichia coli in colonic mucosa. Overall, our data indicate that the L. paracasei B21060 based synbiotic is effective in reducing the severity of liver injury and IR associated with high fat intake, suggesting its possible therapeutic/preventive clinical utilization

Extensively hydrolyzed casein formula alone or with L. rhamnosus GG reduces β-lactoglobulin sensitization in mice

Background: Extensively hydrolyzed casein formula (EHCF) has been proposed for the prevention and is commonly used for the treatment of cow's milk allergy (CMA). The addition of the probiotic Lactobacillus rhamnosus GG (LGG) to EHCF may induce faster acquisition of tolerance to cow's milk. The mechanisms underlying this effect are largely unexplored. We investigated the effects of EHCF alone or in combination with LGG on β-lactoglobulin (BLG) sensitization in mice. Methods: Three-week-old C3H/HeOuJ mice were sensitized by oral administration of BLG using cholera toxin as adjuvant at weekly intervals for 5 weeks (sensitization period). Two experimental phases were conducted: (i) EHCF or EHCF+LGG given daily, starting 2 weeks before the sensitization period and then given daily for 5 weeks and (ii) EHCF or EHCF+LGG given daily for 4 weeks, starting 1 week after the sensitization period. Diet free of cow's milk protein was used as control. Acute allergic skin response, anaphylactic symptom score, body temperature, intestinal permeability, anti-BLG serum IgE, and interleukin (IL)-4, IL-5, IL-10, IL-13, IFN-γ mRNA expression were analyzed. Peptide fractions of EHCF were characterized by reversed-phase (RP)-HPLC, MALDI-TOF mass spectrometry, and nano-HPLC/ESI-MS/MS. Results: Extensively hydrolyzed casein formula administration before or after BLG-induced sensitization significantly reduced acute allergic skin reaction, anaphylactic symptom score, body temperature decrease, intestinal permeability increase, IL-4, IL-5, IL-13, and anti-BLG IgE production. EHCF increased expression of IFN-γ and IL-10. Many of these effects were significantly enhanced by LGG supplementation. The peptide panels were similar between the two study formulas and contained sequences that could have immunoregulatory activities. Conclusions: The data support dietary intervention with EHCF for CMA prevention and treatment through a favorable immunomodulatory action. The observed effects are significantly enhanced by LGG supplementation. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Lt

Hydroxytyrosol prevents metabolic impairment reducing hepatic inflammation and restoring duodenal integrity in a rat model of NAFLD.

The potential mechanisms of action of polyphenols in nonalcoholic fatty liver disease (NAFLD) are overlooked. Here, we evaluate the beneficial therapeutic effects of hydroxytyrosol (HT), the major metabolite of the oleuropein, in a nutritional model of insulin resistance (IR) and NAFLD by high-fat diet. Young male rats were divided into three groups receiving (1) standard diet (STD; 10.5% fat), (2) high-fat diet (HFD; 58.0% fat) and (3) HFD+HT (10 mg/kg/day by gavage). After 5 weeks, the oral glucose tolerance test was performed, and at 6th week, blood sample and tissues (liver and duodenum) were collected for following determinations. The HT-treated rats showed a marked reduction in serum AST, ALT and cholesterol and improved glucose tolerance and insulin sensitivity, reducing homeostasis model assessment index. HT significantly corrected the metabolic impairment induced by HFD, increasing hepatic peroxisome proliferator activated receptor PPAR-α and its downstream-regulated gene fibroblast growth factor 21, the phosphorylation of acetyl-CoA carboxylase and the mRNA carnitine palmitoyltransferase 1a. HT also reduced liver inflammation and nitrosative/oxidative stress decreasing the nitrosylation of proteins, reactive oxygen species production and lipid peroxidation. Moreover, HT restored intestinal barrier integrity and functions (fluorescein isothiocyanate-dextran permeability and mRNA zona occludens ZO-1). Our data demonstrate the beneficial effect of HT in the prevention of early inflammatory events responsible for the onset of IR and steatosis, reducing hepatic inflammation and nitrosative/oxidative stress and restoring glucose homeostasis and intestinal barrier integrity

THERAPEUTIC EFFICACY OF PROBIOTICS AND SHORT CHAIN FATTY ACIDS IN GASTROINTESTINAL DISEASE: EVALUATION OF METABOLIC, HORMONAL AND INFLAMMATORY PARAMETERS

In these three years we studied probiotics and postbiotics efficacy and their mechanism/s in preventing or limiting gastrointestinal diseases, such as hepatic steatosis and ulcerative colitis. On the basis of strength anatomical correlation between liver and gut, we wanted to observe if microorganisms present in gut and their postbiotic derivatives, the short chain fatty acids, were able to prevent or cure not only local intestinal disease but also to limit extraintestinal and systemic pathologies, i.e. liver steatosis and insulin-resistance. Our results showed a protective and curative effect of probiotics and short chain fatty acids in limiting the onset and the progression of these pathologies. We demonstrate several mechanisms of action for these effects: probiotics and postbiotics restore alteration in gut microflora, in particular, Lactobacillus paracasei B21060 can compete with pathogen bacteria avoiding them to attach intestinal epithelium and stimulating immune response. Both probiotics and short chain fatty acids, in particular butyrate, are able to influence directly colonic functions improving innate immune defense mechanisms. In particular, Lactobacillus paracasei B21060 and butyrate-based compounds improved not only tight-junction structure reducing intestinal permeability but also stimulated mucin and defensine production increasing antimicrobial properties of intestinal epithelial cells. Alteration of tight-junctions or mucin production together with changes in microbiota composition, can be considered as predisposing factors to development of gastrointestinal disease. Specially in western life style, bad alimentary habits taken together with smoke or alchol consumption, represent a favourable situation for the onset of these morbidity. In this context, daily assumption of probiotics or directly of their fermentation products as butyrate, could be considered a valid approach for prevention or treatment of gastrointestinal pathologies. In our models of Non-alcoholic fatty liver disease (NAFLD) and ulcerative colitis (UC), both a synbiotic based on Lactobacillus paracasei B21060 and butyrate prevented the beginning of the principal events which underlie progression of these conditions such as insulin-resistance and intestinal inflammation. The enhancement of epithelial barrier integrity by the synbiotic, through an increase in tight junction proteins expression (ZO-1 and occludin), determines a reduction of DAMPs, PAMPs, and FFA afflux to liver. These effects are related to reduction of hepatic TLR4 expression, and its accessory molecule CD14, TLR2 and TLR9 and NF-κB-activation with a decrease in inflammatory gene transcription. The reduced expression of inflammatory cytokines (TNF- α and IL-6) leads to an improvement of insulin signaling (reduction of IRS-1 phosphorylation in Ser 307 and down-regulation of SOCS3), accompanied by an increase in insulin sensitivity. The synbiotic increases PPAR-α expression in liver, contributing to the reduction of inflammation and the increase in fatty acid catabolism. Consistently, in adipose tissue the synbiotic increases PPAR-γ expression and adiponectin transcription, contributing to reduce inflammation. Finally, the therapeutic/preventive role of the synbiotic in hepatic steatosis could be related to its ability to limit inflammatory liver damage, insulin signaling impairment, and imbalance of TLR patterns induced by high fat diet. Our results support probiotics as innovative, preventive and therapeutic strategy for NAFLD, using synbiotic preparations containing selected strain with clear and demonstrated beneficial immunomodulatory effects. Among probiotics, L. paracasei B21060, can be considered a potential approach, limiting the main pathogenetic events involved in the onset of insulin-resistance and steatosis induced by high fat diet. This synbiotic, alone or in combination with other therapies, could be useful in the treatment of fatty liver in children who are hardly able to follow a program of hypocaloric diet and regular physical activity. Also sodium butyrate and its palatable chemical derivative N-(1-carbamoyl-2-phenylethyl) butyramide (FBA) were able to attenuate steatosis and liver injury with reduction of inflammatory responses via suppression of Toll-like receptors and NF-kB pathways. In a murine model of ulcerative colitis, we used dextran sodium sulphate at 2.5% since it is able to reproduce the complexity and event’s cascade typical of moderate to active colitis development. Our data demonstrated that the synbiotic-based Lactobacillus paracasei B21060 was able to prevent immune cell infiltration and to reduce tissue damage in colon, modulating inflammatory and anti-inflammatory cytokines and adipokines, and preventing tight-junction loss. We assessed also the role of inflammosome in this model and its modulation by synbiotic. Moreover the marked changes in defensin-β and mucin-1 expression demonstrated the capability of this synbiotic formulation to maintain the intestinal mucosal homeostasis regulating mucus and antimicrobial peptides secretion by colonic mucosa. In this work we clearly showed that this synbiotic formulation was able to enhance intestinal barrier function and epithelial repair capability in response to DSS induced colitis in mice, suggesting not only a potential therapeutic role for this agent in this pathology, but also the possibility that a supplement of these lactobacilli might prevent the relapse of ulcerative colitis. In the same experimental model of colitis, postbiotics such as butyrate and FBA improved gut permeability avoiding bacterial translocation and impairing immune cell recruitment. These anti-inflammatory effects were evident by reduction in neutrophil infiltration and reduced HDAC9 transcription in colonic mucosa. These events improved intestinal immune homeostasis and tolerance vs. commensal bacteria. In addition, butyrate-based compounds restored colonocyte ability to up-take butyrate after fiber digestion: this occurred with unaltered PPAR-γ levels but blocking NF-kB activation, leading to colonocyte protection from inflammatory process. Efficacy of butyrate to limit early molecular events underlying inflammatory process linked to intestinal damage, suggests its potential clinical utility as a preventive and therapeutic strategy for ulcerative colitis. Since butyrate derivative does not have the characteristic odour of rancid cheese, this derivative may represent a viable therapeutic alternative to butyrate, favouring a better compliance and a greater effectiveness

Insights into the Role of MicroRNAs in the Onset and Development of Diabetic Neuropathy

Diabetic neuropathy is a serious complication of chronic hyperglycemia in diabetes patients. This complication can involve both peripheral sensorimotor and autonomic nervous system. The precise nature of injury to the peripheral nerves mediated by chronic hyperglycemia is unknown; however, several mechanisms have been proposed including polyol pathway activation, enhanced glycation of proteins and lipids, increased oxidative stress, and cytokine release in the site of injury. MicroRNAs (miRNAs) are small non-coding RNAs that mediate RNA interference by post-transcriptionally modulating gene expression and protein synthesis. Therefore, they have been implicated in several developmental, physiological, and pathophysiological processes where they modulate the expression of different proteins. Recently, miRNAs gained an increasing attention also for their role as diagnostic test in many diseases due to their stability in serum and their easy detection. Furthermore, recent studies suggest that miRNAs may be involved in diabetic neuropathy although their role in the onset and the development of this complication is not fully understood. In this review, we discuss the most recent literature providing evidence for miRNAs role in diabetic neuropathy opening new pathways to improve both early diagnosis and treatment of this complication

A novel interaction between CX3CR1 and CCR2 signalling in monocytes constitutes an underlying mechanism for persistent vincristine-induced pain

Abstract Background A dose-limiting side effect of chemotherapeutic agents such as vincristine (VCR) is neuropathic pain, which is poorly managed at present. Chemokine-mediated immune cell/neuron communication in preclinical VCR-induced pain forms an intriguing basis for the development of analgesics. In a murine VCR model, CX3CR1 receptor-mediated signalling in monocytes/macrophages in the sciatic nerve orchestrates the development of mechanical hypersensitivity (allodynia). CX3CR1-deficient mice however still develop allodynia, albeit delayed; thus, additional underlying mechanisms emerge as VCR accumulates. Whilst both patrolling and inflammatory monocytes express CX3CR1, only inflammatory monocytes express CCR2 receptors. We therefore assessed the role of CCR2 in monocytes in later stages of VCR-induced allodynia. Methods Mechanically evoked hypersensitivity was assessed in VCR-treated CCR2- or CX3CR1-deficient mice. In CX3CR1-deficient mice, the CCR2 antagonist, RS-102895, was also administered. Immunohistochemistry and Western blot analysis were employed to determine monocyte/macrophage infiltration into the sciatic nerve as well as neuronal activation in lumbar DRG, whilst flow cytometry was used to characterise monocytes in CX3CR1-deficient mice. In addition, THP-1 cells were used to assess CX3CR1-CCR2 receptor interactions in vitro, with Western blot analysis and ELISA being used to assess expression of CCR2 and proinflammatory cytokines. Results We show that CCR2 signalling plays a mechanistic role in allodynia that develops in CX3CR1-deficient mice with increasing VCR exposure. Indeed, the CCR2 antagonist, RS-102895, proves ineffective in mice possessing functional CX3CR1 receptors but reduces VCR-induced allodynia in CX3CR1-deficient mice, in which CCR2 + monocytes are elevated by VCR. We suggest that a novel interaction between CX3CR1 and CCR2 receptors in monocytes accounts for the therapeutic effect of RS-102895 in CX3CR1-deficient mice. Indeed, we observe that CCR2, along with its ligand, CCL2, is elevated in the sciatic nerve in CX3CR1-deficient mice, whilst in THP-1 cells (human monocytes), downregulating CX3CR1 upregulates CCR2 expression via p38 MAP kinase signalling. We also show that the CX3CR1-CCR2 interaction in vitro regulates the release of pronociceptive cytokines TNF-α and IL1β. Conclusions Our data suggests that CCL2/CCR2 signalling plays a crucial role in VCR-induced allodynia in CX3CR1-deficient mice, which arises as a result of an interaction between CX3CR1 and CCR2 in monocytes

Reduced thermal sensitivity and increased opioidergic tone in the TASTPM mouse model of Alzheimer's disease

Individuals with Alzheimer's disease (AD) are in susceptible patient groups in which pain is an important clinical issue that is often underdiagnosed. However, it is unclear whether decreased pain complaints in patients with AD result from elevated pain tolerance or an impaired ability to communicate sensations. Here, we explored if AD-related pathology is present in key regions of the pain pathway and assessed whether nociceptive thresholds to acute noxious stimulation are altered in the double-mutant APPswe × PS1.M146V (TASTPM) transgenic mouse model of AD. TASTPM mice exhibited an age-dependant cognitive deficit at the age of 6 months, but not at 4 months, a deficit that was accompanied by amyloid plaques in the cortex, hippocampus, and thalamus. In the spinal cord, β-amyloid (APP/Aβ) immunoreactivity was observed in dorsal and ventral horn neurons, and the expression of vesicular glutamate transporter 2 (VGLUT2) was significantly reduced, while the expression of the inhibitory peptides enkephalins was increased in TASTPM dorsal horn, consistent with an increased inhibitory tone. TASTPM mice displayed reduced sensitivity to acute noxious heat, which was reversed by naloxone, an opioid antagonist. This study suggests that increased inhibition and decreased excitation in the spinal cord may be responsible for the reduced thermal sensitivity associated with AD-related pathology