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The role of therapeutic drug monitoring (TDM) of valaciclovir (VA)/aciclovir (A) and valganciclovir/ganciclovir (VG/G) in critically ill patients is still a matter of debate. More data on the dose–concentration relationship might therefore be useful, especially in pediatrics where clinical practice is not adequately supported by robust PK studies. We developed and validated a new liquid chromatography-tandem mass spectrometry (LC-MS/MS) micro-method to simultaneously quantify A and G from plasma and dried plasma spots (DPS). The method was based on rapid organic extraction from DPS and separation on a reversed-phase C-18 UHPLC column after addition of deuterated internal standards. Accurate analyte quantification using SRM detection was then obtained using a Thermo Fisher Quantiva triple-quadrupole MS coupled to an Ultimate 3000 UHPLC. It was validated following international (EMA) guidelines for bioanalytical method validation and was tested on samples from pediatric patients treated with A, VG, or G for cytomegalovirus infection following solid organ or hematopoietic stem cell transplantation. Concentrations obtained from plasma and DPS were compared using Passing–Bablok and Bland–Altman statistical tests. The assay was linear over wide concentration ranges (0.01–20 mg/L) in both plasma and DPS for A and G, suitable for the expected therapeutic ranges for both Cmin and Cmax, accurate, and reproducible in the absence of matrix effects. The results obtained from plasma and DPS were comparable. Using an LC-MS/MS method allowed us to obtain a very specific, sensitive, and rapid quantification of these antiviral drugs starting from very low volumes (50 μL) of plasma samples and DPS. The stability of analytes for at least 30 days allows for cost-effective shipment and storage at room temperature. Our method is suitable for TDM and could be helpful for improving knowledge on PK/PD targets of antivirals in critically ill pediatric patients.

Dear editor,Digoxin is a cardioactive drug with a narrow therapeutic window. Severe intoxication may require urgent treatment. The clinical picture is represented by dangerous dysrhythmias and the ...

The common practice of therapeutic drug monitoring (TDM) involves the quantification of drug plasma concentrations at a specific time in a dosing window. Although TDM for antibiotics is not considered mandatory, it may represent a valid tool for clinicians in order to limit antibiotic resistance and avoid therapeutic failures. The aim of our study was to develop and validate a high-performance liquid chromatography-diode array detection method for simultaneous quantification of 10 antibiotics in plasma. This method has a fast analytical procedure that uses the same chromatographic conditions to quantify ceftazidime, ceftriaxone, meropenem, ertapenem, ciprofloxacin, tigecycline, ampicillin, levofloxacin and piperacillin, plus the β-lactamase inhibitor tazobactam. Method validation was ensured by testing selectivity, accuracy, precision, limits of detection and quantification, recovery and stability. The calibration ranges, established accordingly to the expected plasma concentration in patients, showed a coefficient of determination >0.996 for all compounds. Within- and between-days precisions reported a coefficient of variation >15%. Similarly, the accuracy evaluation reported a relative standard deviation of

Supplemental Digital Content is Available in the Text. In inflammatory arthritis, we show an imbalance of proresolving bioactive lipid mediators in dorsal root ganglia, under persistent nociceptive states, and antinociceptive effects of maresin-1 administration., Rheumatoid arthritis-associated pain is poorly managed, often persisting when joint inflammation is pharmacologically controlled. Comparably, in the mouse K/BxN serum-transfer model of inflammatory arthritis, hind paw nociceptive hypersensitivity occurs with ankle joint swelling (5 days after immunisation) persisting after swelling has resolved (25 days after immunisation). In this study, lipid mediator (LM) profiling of lumbar dorsal root ganglia (DRG), the site of sensory neuron cell bodies innervating the ankle joints, 5 days and 25 days after serum transfer demonstrated a shift in specialised proresolving LM profiles. Persistent nociception without joint swelling was associated with low concentrations of the specialised proresolving LM Maresin 1 (MaR1) and high macrophage numbers in DRG. MaR1 application to cultured DRG neurons inhibited both capsaicin-induced increase of intracellular calcium ions and release of calcitonin gene-related peptide in a dose-dependent manner. Furthermore, in peritoneal macrophages challenged with lipopolysaccharide, MaR1 reduced proinflammatory cytokine expression. Systemic MaR1 administration caused sustained reversal of nociceptive hypersensitivity and reduced inflammatory macrophage numbers in DRG. Unlike gabapentin, which was used as positive control, systemic MaR1 did not display acute antihyperalgesic action. Therefore, these data suggest that MaR1 effects observed after K/BxN serum transfer relate to modulation of macrophage recruitment, more likely than to direct actions on sensory neurons. Our study highlights that, in DRG, aberrant proresolution mechanisms play a key role in arthritis joint pain dissociated from joint swelling, opening novel approaches for rheumatoid arthritis pain treatment.

Summary Objective Aiming to delineate novel neuro-immune mechanisms for NGF/TrkA signalling in osteoarthritis (OA) pain, we evaluated inflammatory changes in the knee joints following injection of monoiodoacetate (MIA) in mice carrying a TrkA receptor mutation (P782S; TrkA KI mice). Method In behavioural studies we monitored mechanical hypersensitivity following intra-articular MIA and oral prostaglandin D 2 (PGD 2 ) synthase inhibitor treatments. In immunohistochemical studies we quantified joint mast cell numbers, calcitonin gene-related peptide expression in synovia and dorsal root ganglia, spinal cord neuron activation and microgliosis. We quantified joint leukocyte infiltration by flow cytometry analysis, and PGD 2 generation and cyclooxygenase-2 (COX-2) expression in mast cell lines by ELISA and Western blot. Results In TrkA KI mice we observed rapid development of mechanical hypersensitivity and amplification of dorsal horn neurons and microglia activation 7 days after MIA. In TrkA KI knee joints we detected significant leukocyte infiltration and mast cells located in the vicinity of synovial nociceptive fibres. We demonstrated that mast cells exposure to NGF results in up-regulation of COX-2 and increase of PGD 2 production. Finally, we observed that a PGD 2 synthase inhibitor prevented MIA-mechanical hypersensitivity in TrkA KI, at doses which were ineffective in wild type (WT) mice. Conclusion Using the TrkA KI mouse model, we delineated a novel neuro-immune pathway and suggest that NGF-induced production of PGD 2 in joint mast cells is critical for referred mechanical hypersensitivity in OA, probably through the activation of PGD 2 receptor 1 in nociceptors: TrkA blockade in mast cells constitutes a potential target for OA pain.

Background and purpose Butyrate has shown benefits in inflammatory bowel diseases (IBD). However, its oral administration is infrequent due to 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 5d) followed by DSS-free water for 7d (DSS group). Oral FBA administration (42.5 mg∙kg-1) started 7d before DSS as preventive (P-FBA), or 2d after DSS as therapeutic (T-FBA), and both treatments lasted at 19d. One DSS-untreated group received only tap water (CON) for totally 4 groups. Key results FBA treatments reduced colitis symptoms and colon damage. P-FBA and T-FBA significantly decreased polymorphonuclear cell infiltration score compared to the DSS group. FBA revert the imbalance between pro- and anti-inflammatory cytokines (reducing inducible NOS protein expression, chemokine (C-C motif) ligand 2 and IL-6 transcripts in colon and increasing TGF-β and IL-10). Morever, P-FBA and T-FBA limit neutrophil recruitment (by expression and localization of the neutrophil granule protease Ly-6G), restore deficiency of butyrate transporter and improve intestinal epithelial integrity, preventing tight-junction impairment (zonulin-1 and occludin). FBA, such as its parental compound sodium butyrate, inhibits histone deacetylase-9 and restores H3 histone acetylation, exerting an anti-inflammatory activity through NF-κB inhibition and PPAR-γ up-regulation. Conclusions and implications FBA reduces inflammatory intestinal damage in mice indicating its potential as post-biotic derivative, to overcome the limits of sodium butyrate's oral administration. This article is protected by copyright. All rights reserved.

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.

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. Electronic supplementary material The online version of this article (10.1186/s12974-018-1116-6) contains supplementary material, which is available to authorized users.

Non-dioxin-like (NDL) polychlorinated biphenyls (PCBs) are persistent organic pollutants, associated with a range of adverse health effects, including interference with the immune system. In this study, we investigate the capability of NDL-PCBs 101, 153, and 180, 3 of the 6 NDL-PCBs defined as indicators, to impair the immune response in lipopolysaccharide (LPS)-activated J774A.1 and primary murine macrophages. Our results clearly demonstrate that the exposure of J774A.1 and primary macrophages to NDL-PCB 153 or 180 or all NDL-PCBs mixtures causes a significant reduction in LPS-induced cytokine/chemokine synthesis, such as tumor necrosis factor-α and interleukin-6, together with monocyte chemoattractant protein-1, involved in cell recruitment. Moreover, PCBs were found to suppress LPS-stimulated NO production, and to reduce cyclooxygenase-2 and inducible nitric oxide synthase expression in J774A.1 and primary macrophages. At mechanistic level, PCBs significantly counteract the LPS-driven toll-like receptor (TLR) 4 and CD14 upregulation, therefore inhibiting downstream nuclear factor-κB (NF-κB) activation in J774A.1. Furthermore, PCBs determine a significant loss of macrophage endocytic capacity, a prerequisite for efficient antigen presentation. Taken together, these data indicate that NDL-PCBs reduce macrophage responsiveness, particularly when they are combined at concentrations per se inactive, impairing the capability to orchestrate a proper immune response to an infectious stimulus, disrupting TLR4/NF-κB pathway.

Following peripheral axon injury, dysregulation of non-coding microRNAs (miRs) occurs in dorsal root ganglia (DRG) sensory neurons. Here we show that DRG neuron cell bodies release extracellular vesicles, including exosomes containing miRs, upon activity. We demonstrate that miR-21-5p is released in the exosomal fraction of cultured DRG following capsaicin activation of TRPV1 receptors. Pure sensory neuron-derived exosomes released by capsaicin are readily phagocytosed by macrophages in which an increase in miR-21-5p expression promotes a pro-inflammatory phenotype. After nerve injury in mice, miR-21-5p is upregulated in DRG neurons and both intrathecal delivery of a miR-21-5p antagomir and conditional deletion of miR-21 in sensory neurons reduce neuropathic hypersensitivity as well as the extent of inflammatory macrophage recruitment in the DRG. We suggest that upregulation and release of miR-21 contribute to sensory neuron–macrophage communication after damage to the peripheral nerve., Exosomes are known to contain microRNAs (miRs). Here the authors show that dorsal root ganglion neurons release exosomes containing miR-21-5p, which contributes to inflammatory cell recruitment following peripheral nerve injury.

Hypertension is an important risk factor for kidney failure and renal events in the general population. Palmitoylethanolamide (PEA) is a member of the fatty acid ethanolamine family with profound analgesic and anti-inflammatory effects, resulting from its ability to activate peroxisome proliferator activated receptor (PPAR)α. A role for this nuclear receptor has been addressed in cardiovascular system and PPARα ligands have been shown to protect against inflammatory damage especially resulting from angiotensin II hypertension. In this study, we demonstrated that PEA significantly reduced blood pressure in spontaneously hypertensive rats (SHR) and limited kidney damage secondary to high perfusion pressure. To investigate the mechanisms involved in PEA effect, we found that PEA reduced cytochrome P450 (CYP) hydroxylase CYP4A, epoxygenase CYP2C23 and soluble epoxide hydrolase enzyme expression in the kidney, accompanied by a reduction of 20-hydroxyeicosatetraenoic acid excretion in the urine. Moreover, it markedly reduced kidney oxidative and nitrosative stress accompanied by decreased expression of renal NAD(P)H oxidase and inducible nitric oxide synthase and increased expression of Cu/Zn superoxide dismutase, in the kidney of SHR. Moreover, angiotensin II receptor (AT) evaluation revealed a decrease in AT1 receptor expression and a restoration of AT2 receptor level in the kidney from PEA-treated SHR. Consistently, angiotensin converting enzyme expression was reduced, implying a decrease in angiotensin II synthesis. These results indicate that PEA treatment lowers blood pressure and can protect against hypertensive renal injury by increasing the antioxidant defense and anti-inflammatory response and modulating renin-angiotensin system.

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.

It has been suggested a role of fatty acid ethanolamides in control of feeding behavior. Among these, palmitoylethanolamide (PEA) has not been directly implicated in appetite regulation and weight gain. The aim of this study was to investigate the effect of PEA on food intake and body weight and the interaction between PEA and hypothalamic leptin signaling in ovariectomized rats. Ovariectomy produced hyperphagia and increased weight gain, making it an useful model of mild obesity. Ovariectomized rats were treated with PEA (30 mg/kg sc) for 5 weeks. Then, blood was collected, and hypothalamus and adipose tissue were removed for histological, cellular, and molecular measurements. We showed that PEA caused a reduction of food intake, body weight, and fat mass. The mechanisms underlying PEA effects involved an improvement in hypothalamic leptin signaling, through a raise in signal transducer and activator of transcription 3 phosphorylation. We also reported that PEA reduced AMP-activated protein kinase-α phosphorylation and modulated transcription of anorectic and orexigenic neuropeptides in the hypothalamus. Moreover, PEA increased AMP-activated protein kinase-α phosphorylation and carnitine palmitoyltransferase 1 transcription in adipose tissue, suggesting an increase in ATP-producing catabolic pathway. PEA also polarized adipose tissue macrophages to M2 lean phenotype, associated to a reduction of inflammatory cytokines/adipokines. To demonstrate the direct effect of PEA on leptin sensitivity without interference of adiposity loss, we obtained consistent data in PEA-treated sham-operated animals and in vitro in SH-SY5Y neuroblastoma cell line. Therefore, our data provide a rationale for the therapeutic use of PEA in obese postmenopausal woman.

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).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·kgFBA 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.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.This article is part of a themed section on Principles of Pharmacological Research of Nutraceuticals. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.11/issuetoc.

Palmitoylethanolamide (PEA), a peroxisome proliferator-activated receptor-α agonist, has been demonstrated to reduce blood pressure and kidney damage secondary to hypertension in spontaneously hypertensive rat (SHR). Currently, no information is available concerning the putative effect of PEA on modulating vascular tone. Here, we investigate the mechanisms underpinning PEA blood pressure lowering effect, exploring the contribution of epoxyeicosatrienoic acids, CYP-dependent arachidonic acid metabolites, as endothelium-derived hyperpolarizing factors (EDHF), and renin angiotensin system (RAS) modulation. To achieve this aim SHR and Wistar-Kyoto rats were treated with PEA (30 mg/kg/day) for five weeks. Functional evaluations on mesenteric bed were performed to analyze EDHF-mediated vasodilation. Moreover, mesenteric bed and carotid were harvested to measure CYP2C23 and CYP2J2, the key isoenzymes in the formation of epoxyeicosatrienoic acids, and the soluble epoxide hydrolase, which is responsible for their degradation in the corresponding diols. Effect of PEA on RAS modulation was investigated by analyzing angiotensin converting enzyme and angiotensin receptor 1 expression. Here, we showed that EDHF-mediated dilation in response to acetylcholine was increased in mesenteric beds of PEA-treated SHR. Western blot analysis revealed that the increase in CYP2C23 and CYP2J2 observed in SHR was significantly attenuated in mesenteric beds of PEA-treated SHR, but unchanged in the carotids. Interestingly, in both vascular tissues, PEA significantly decreased the soluble epoxide hydrolase protein level, accompanied by a reduced serum concentration of its metabolite 14-15 dihydroxyeicosatrienoic acid, implying a reduction in epoxyeicosatrienoic acid hydrolisis. Moreover, PEA treatment down-regulated angiotensin receptor 1 and angiotensin converting enzyme expression, indicating a reduction in angiotensin II-mediated effects. Consistently, a damping of the activation of angiotensin receptor 1 underlying pathways in mesenteric beds was shown in basal conditions in PEA-treated SHR. In conclusion, our data demonstrate the involvement of epoxyeicosatrienoic acids and renin angiotensin system in the blood pressure lowering effect of PEA.

Background: Although gut microbiota perturbation is recognized as a main contributing factor to the pathogenesis of inflammatory bowel disease, synbiotic therapies, as prevention or treatment, have remained overlooked. Objective: To verify whether Lactobacillus paracasei B21060‐based synbiotic therapy could prevent or repair colon damage in a mouse model of colitis, we performed treatments before and after colitis induction. Methods: The experimental study lasted 19 d. Experimental colitis was induced in BALB/c mice by dextran sodium sulfate (DSS, 2.5%) in drinking water (days 7‐12) followed by DSS-free water (days 13‐19) (DSS group). L. paracasei B21060 (2.5 3 10 7 bacteria/10g body weight) was orally administered 7db efore DSS synbiotic as preventive treatment (P-SYN) group] or 2 d after DSS [synbiotic as therapeutic treatment (T-SYN) group] until day 19. Another group was not treated with DSS or synbiotic and was given tap water (control group), for a total of 4 groups. Results: Compared with the DSS group, both synbiotic-treated groups had significantly less pronounced weight loss and colon damage. Consistently, mRNA levels of chemokine (C‐C motif) ligand 5 in the colon were reduced in both P-SYN and T-SYN mice compared with the DSS group (51%, P < 0.05 and 72%, P < 0.001, respectively). In the P-SYN and T-SYN groups, neutrophil elastase transcription was also reduced (51%, P < 0.01 and 59%, P < 0.001, respectively). Accordingly, oxidative/nitrosative stress was lower in P-SYN and T-SYN mice than inthe DSS group. In P-SYN and T-SYN mice, colonic gene expression of tumor necrosis factor (47%, P< 0.01 and 61%, P< 0.001, respectively) and prostaglandin-endoperoxide synthase 2 (45%, P < 0.01 and 35%, P < 0.05, respectively) was lower, whereas interleukin 10 mRNA was doubled compared with the DSS group (both P < 0.5). Remarkably, epithelial barrier integrity (zonulin and occludin) and gut protection (b-defensin and mucin expression) were completely restored in P-SYN and T-SYN mice. Conclusions: Our data highlight the beneficial effects of this synbiotic formulation in acutely colitic mice, suggesting that it may have therapeutic and possibly preventive efficacy in human colitis. J Nutr doi: 10.3945/jn.114.205989.

Non-dioxin-like polychlorinated biphenyls (NDL-PCBs) are highly lipophilic environmental contaminants that accumulate in lipid-rich tissues, such as adipose tissue. Here, we reported the effects induced by PCBs 101, 153 and 180, three of the six NDL-PCBs defined as indicators, on mature 3T3-L1 adipocytes. We observed an increase in lipid content, in leptin gene expression and a reduction of leptin receptor expression and signaling, when cells were exposed to PCBs, alone or in combination. These modifications were consistent with the occurrence of "leptin-resistance" in adipose tissue, a typical metabolic alteration related to obesity. Therefore, we investigated how PCBs affect the expression of pivotal proteins involved in the signaling of leptin receptor. We evaluated the PCB effect on the intracellular pathway JAK/STAT, determining the phosphorylation of STAT3, a downstream activator of the transcription of leptin gene targets, and the expression of SOCS3 and PTP1B, two important regulators of leptin resistance. In particular, PCBs 153 and 180 or all PCB combinations induced a significant reduction in pSTAT3/STAT3 ratio and an increase in PTP1B and SOCS3, evidencing an additive effect. The impairment of leptin signaling was associated with the reduction of AMPK/ACC pathway activation, leading to the increase in lipid content. These pollutants were also able to increase the transcription of inflammatory cytokines (IL-6 and TNFα). It is worthy to note that the PCB concentrations used are comparable to levels detectable in human adipose tissue. Our data strongly support the hypothesis that NDL-PCBs may interfere with the lipid metabolism contributing to the development of obesity and related diseases.

BACKGROUND & AIMS: Nonalcoholic fatty liver disease (NAFLD) is the most common form of chronic liver disease. Insulin resistance (IR) appears to be critical in its pathogenesis. We evaluated the effects of sodium butyrate (butyrate) and its synthetic derivative N-(1-carbamoyl-2-phenyl-ethyl) butyramide (FBA) in a rat model of insulin resistance and steatosis induced by high-fat diet (HFD). METHODS: After weaning, young male Sprague-Dawley rats were divided into 4 groups receiving different diets for 6 weeks: 1. control group (standard diet); 2. HFD; 3. HFD plus butyrate (20 mg/kg/die) and 4. HFD plus FBA (42.5 mg/Kg/die, the equimolecular dose of butyrate). Liver tissues of the rats were analyzed by Western blot and real-time PCR. Insulin resistance, liver inflammation and Toll-like pattern modifications were determined. RESULTS: Evaluation of these two preparations of butyrate showed a reduction of liver steatosis and inflammation in HFD fed animals. The compounds showed a similar potency in the normalisation of several variables, such as transaminases, homeostasis model assessment for insulin resistance index, and glucose tolerance. Both treatments significantly reduced hepatic TNF-α expression and restored GLUTs and PPARs, either in liver or adipose tissue. Finally, FBA showed a higher potency in reducing pro-inflammatory parameters in the liver, via suppression of Toll-like receptors and NF-κB activation. CONCLUSIONS: Our results demonstrated a protective effect of butyrate in limiting molecular events underlying the onset of IR and NAFLD, suggesting a potential clinical relevance for this substance. In particular, its derivative, FBA, could represent an alternative therapeutic option to sodium butyrate, sharing a comparable efficacy, but a better palatability and compliance.

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.

This paper is dedicated to the memory of our wonderful colleague Professor Alfredo Colonna, who passed away the same day of its acceptance. Fatty liver accumulation, inflammatory process and insulin resistance appear to be crucial in non-alcoholic fatty liver disease (NAFLD), nevertheless emerging findings pointed an important role also for iron overload. Here, we investigate the molecular mechanisms of hepatic iron metabolism in the onset of steatosis to understand whether its impairment could be an early event of liver inflammatory injury. Rats were fed with control diet or high fat diet (HFD) for 5 or 8 weeks, after which liver morphology, serum lipid profile, transaminases levels and hepatic iron content (HIC), were evaluated. In liver of HFD fed animals an increased time-dependent activity of iron regulatory protein 1 (IRP1) was evidenced, associated with the increase in transferrin receptor-1 (TfR1) expression and ferritin down-regulation. Moreover, ferroportin (FPN-1), the main protein involved in iron export, was down-regulated accordingly with hepcidin increase. These findings were indicative of an increased iron content into hepatocytes, which leads to an increase of harmful free-iron also related to the reduction of hepatic ferritin content. The progressive inflammatory damage was evidenced by the increase of hepatic TNF-α, IL-6 and leptin, in parallel to increased iron content and oxidative stress. The major finding that emerged of this study is the impairment of iron homeostasis in the ongoing and sustaining of liver steatosis, suggesting a strong link between iron metabolism unbalance, inflammatory damage and progression of disease.

Non-dioxin-like polychlorinated biphenyls (PCBs) are stable and lipophilic chemicals that persist in the environment and tend to bioaccumulate in the food chains. In the present study, we have investigated the effect of PCBs 101, 153, and 180 on macrophage J774A.1 by assessing cell viability and apoptotic cell death. We have combined morphological techniques and biochemical ones to establish the relevance of apoptosis in macrophage cell death induced by PCBs, alone or in combination. Treatment with the examined PCBs caused the loss of cell viability and accelerated apoptosis in a concentration-dependent manner. Moreover, a synergistic effect on cell death and apoptosis was evidenced for all PCBs at concentrations which were inactive alone. The apoptosis induced by PCBs involved the increase of caspase-3 activity. Also, Bcl-2 and Bax proteins were assessed to elucidate the apoptosis machinery induced in macrophage cultures by PCBs. Our results indicate that the increase in PCB-induced apoptosis correlates with a reduction in the expression of antiapoptotic Bcl-2 and an increase in the expression of proapoptotic Bax. Interestingly, concentrations of PCBs inactive by themselves induce apoptosis when PCBs are combined. In conclusion, our findings suggest that, although less toxic than dioxin like congeners, the examined non-dioxin-like PCBs are equally dangerous as immunotoxic pollutants, also considering their presence as mixtures at higher levels than dioxin-like PCBs in biotic and abiotic matrices. © 2011 Elsevier Ireland Ltd.

Conflicting evidences suggest a role for breast milk as pro- tective factor against food allergy. The major short chain fatty acids, butyrate produced by gut microbiota exerts positive effect on immune system. We aimed to see whether butyrate concen- tration in human milk is able to prevent food allergy in an animal model of cow milk allergy. Mature breast milk butyrate concentration from 40 healthy women (aged 21–42 yrs) was assessed by gas chromatography. 4- Week-old female C3H/HeOuJ mice were sensitized by oral route with -lactoglobulin (BLG, 20 mg) plus cholera toxin (CT, 10 g) as an adjuvant in the presence or absence of butyrate. Acute aller- gic skin response,anaphylactic symptom score, body temperature, intestinal permeability, anti-BLG IgE, IL-4 and IL-10 production were assessed soon after oral challenge. Mean butyrate concen- tration in breast milk was 0.75 mM (SD ± 0.15). Based on this concentration a daily dose of 30 mg/kg body weight was calculated. The same butyrate concentration was able to significantly prevent acute allergic skin response, anaphylactic symptom score, body temperature decrease, intestinal permeability increase, anti-BLG IgE, IL-4 and IL-10 production in CMA animal model (p < .05). Our data suggest a pivotal role for butyrate as an effective human milk component in food allergy prevention.

Rosita Aitoro1,∗, Margherita Di Costanzo1, Raffaele Simeoli2, Antonio Calignano2, Claudio Pirozzi2, Maria Pina Mollica3, Giovanna Trinchese3, Rita Nocerino1, Francesco Amato1, Riccardo Troncone1, Rosaria Meli2, Roberto Berni Canani1 1 Dipartimento di Scienze Mediche Traslazionali, Universita degli Studi di Napoli “Federico II”, Napoli, Italy 2 Dipartimento di Farmacia, Universita degli Studi di Napoli “Federico II”, Napoli, Italy 3 Dipartimento di Biologia, Universita degli Studi di Napoli “Federico II”, Napoli, Italy

and seeds (5.1%), fruits and vegetables (3.1%), fish (1.8%), peanuts (1.3%), crustaceans (0.7%), other specified foods in 6.2%. 31.3% of cases received a final diagnosis of food-induced anaphylaxis but the particular food was not indicated. Conclusions: Significant increase in admission for food-induced anaphylaxis occurred in Italian children between the period 2001–2005. Similar data have been recently reported in other Western countries. Our data suggest the importance of more research to investigate the causative factors and the necessity to improve the healthcare service for this condition.