The long non-coding RNA GAS5 contributes to the suppression of inflammatory responses by inhibiting NF-κB activity.

Introduction: Nuclear factor kappa B (NF-κB) is a key regulator of immune and inflammatory responses. Glucocorticoid drugs (GC) act through the glucocorticoid receptor (GR) as immunosuppressant also in pediatric patients inhibiting NF-κB activity. The long non-coding RNA GAS5 interacts with the GR, influencing GC activity. No data on the role of GAS5 on GR-dependent inhibition of NF-κB activity have been published. Methods: This study investigated the impact of GAS5 on NF-κB activity in HeLa cells overexpressing GAS5, both under basal conditions and during GC treatment. The study used EMSA, RNA-immunoprecipitation (RIP), Western blotting, and bioinformatic analyses to assess NF-κB DNA binding, GAS5-p65 interaction, and NF-κB signaling pathway modulation. Results: GAS5 overexpression increased NF-κB DNA binding activity in untreated cells. RNA-IP confirmed a direct interaction between GAS5 and the NF-κB subunit p65, suggesting a potential regulatory mechanism. GAS5 overexpression led to downregulation of NF-κB target genes, TNF-α, and NR3C1. GC treatment reduced NF-κB DNA binding activity in GAS5-overexpressing cells, indicating a potential synergistic effect. Furthermore, GAS5 overexpression increased IκB levels and reduced p-p65/pan-p65 levels during GC treatment. Discussion: GAS5 appears to modulate NF-κB activity in a complex manner, influencing both basal and GC-induced signaling. The interaction between GAS5, GCs, and NF-κB is multi-faceted, and further research is needed to fully elucidate the underlying mechanisms. These findings suggest that GAS5 could be a potential target for personalized therapy, particularly in pediatric patients with inflammatory conditions.

Monoclonal antibodies against pediatric ulcerative colitis: a review of clinical progress.

INTRODUCTION: In children, ulcerative colitis (UC) is often more severe and extensive than in adults and hospitalization for acute exacerbations occurs in around a quarter of subjects. There is a need for effective drugs, which could avoid or reduce the use of corticosteroids which, especially in children, are burdened by a number of severe side effects. The introduction in therapy of monoclonal antibodies has completely changed the therapeutic scenario and the prognosis of the disease. AREAS COVERED: In this review, the use of the monoclonal antibodies directed against tumor necrosis factor (TNF)α or other inflammatory targets for the treatment of pediatric UC will be discussed. A search of the literature was done using the keywords 'pediatric,' 'ulcerative colitis,' 'inflammatory bowel disease,' 'monoclonal antibodies;' 'infliximab,' 'adalimumab,' 'golimumab,' vedolizumab," 'ustekinumab' and 'risankizumab.' EXPERT OPINION: The use of monoclonal antibodies has greatly increased in recent years in pediatric UC, both in patients who did not respond to conventional therapies, and, more often, as initial therapy. Thanks to therapeutic drug monitoring and to the availability of biologics with different targets, therapy has become more targeted and personalized, with a significant improvement in response, in quality of life, and with a good safety profile.

Neuron-Derived Extracellular Vesicles miRNA Profiles Identify Children Who Experience Adverse Events after Ketamine Administration for Procedural Sedation.

Ketamine provides the highest safety profile among sedatives for procedural sedation and analgesia in the pediatric emergency setting. However, it can cause vomiting and recovery agitation. No studies have examined epigenetic factors, such as microRNAs, for predicting the occurrence of these adverse events. Neuronal-derived extracellular vesicle microRNA profiles were studied to predict the occurrence of ketamine-induced vomiting and recovery agitation in children. For this aim, a single-center prospective pharmacoepigenetic study was performed and 50 children who underwent procedural sedation with intravenous ketamine as the only sedative drug were enrolled between October 2019 and November 2022. MiRNA profiling in plasma neural-derived extracellular vesicles was analyzed through next-generation sequencing and measured before treatment with ketamine. Twenty-two patients experienced vomiting or recovery agitation. Among the 16 differentially expressed microRNAs, the upregulated miR-15a-5p and miR-484 targeted genes related to N-methyl-D-aspartate (NMDA) receptor activity, including glutamate ionotropic receptor NMDA type subunit 2A (GRIN2A). Preliminary data confirmed lower GRIN2A levels in patients who developed these events. Downregulated miR-126-3p and miR-24-3p targeted AMPA receptor-associated genes. Functional analyses of gene targets revealed the enrichment of glutamatergic and neurotrophins signaling. Recovery agitation was associated with this network. Vomiting was related to dopaminergic and cholinergic systems. Three miRNAs (miR-18a-3p, miR-484, and miR-548az-5p) were identified as predictive biomarkers (AUC 0.814; 95% CI: 0.632-0.956) for ketamine-induced vomiting and recovery agitation. MicroRNA profiles can predict the development of ketamine-induced vomiting or recovery agitation in children. This study contributes to the understanding of the mechanisms underlying ketamine-induced adverse events.

Preanalytical Stability of 13 Antibiotics in Biological Samples: A Crucial Factor for Therapeutic Drug Monitoring.

The stability of antibiotic preanalytical samples is a critical factor in therapeutic drug monitoring (TDM), a practice of undoubted importance for the proper therapeutic use of antibiotics, especially in complex management patients, such as pediatrics. This review aims to analyze the data in the literature regarding the preanalytical stability of some of the antibiotics for which TDM is most frequently requested. The literature regarding the preanalytical stability of amikacin, ampicillin, cefepime, ceftazidime, ciprofloxacin, daptomycin, gentamicin, levofloxacin, linezolid, meropenem, piperacillin, teicoplanin, and vancomycin in plasma, serum, whole blood, and dried blood/plasma spot samples was analyzed. Various storage temperatures (room temperature, 4 °C, -20 °C, and -80 °C) and various storage times (from 1 h up to 12 months) as well as subjecting to multiple freeze-thaw cycles were considered. The collected data showed that the non-beta-lactam antibiotics analyzed were generally stable under the normal storage conditions used in analytical laboratories. Beta-lactam antibiotics have more pronounced instability, particularly meropenem, piperacillin, cefepime, and ceftazidime. For this class of antibiotics, we suggest that storage at room temperature should be limited to a maximum of 4 h, storage at 2-8 °C should be limited to a maximum of 24 h, and storage at -20 °C should be limited to a maximum of 7 days; while, for longer storage, freezing at -80 °C is suggested.

Expression profiles of the lncRNA antisense GAS5-AS1 in colon biopsies from pediatric inflammatory bowel disease patients and its role in regulating sense transcript GAS5.

The long non-coding RNA (lncRNA) growth arrest-specific transcript 5 (GAS5) level was demonstrated as involved in pediatric inflammatory bowel disease (IBD) pathogenesis. Since its antisense transcript GAS5-AS1 has never been investigated in IBD, this study aims to detect whether GAS5-AS1 and GAS5 levels are related to IBD clinical parameters and investigate their correlation in vitro. Twenty-six IBD pediatric patients were enrolled; paired inflamed and non-inflamed intestinal biopsies were collected. We evaluated GAS5 and GAS5-AS1 levels by real-time PCR. The role of GAS5 and GAS5-AS1 was assessed in vitro by transient silencing in THP1-derived macrophages. GAS5-AS1 and GAS5 levels were associated with patients' clinical parameters; GAS5-AS1 expression was downregulated in inflamed tissues and inversely correlated with disease activity. A positive correlation between GAS5-AS1 and GAS5 levels was observed in non-inflamed biopsies. On THP1-derived macrophages, a reduced amount of both GAS5-AS1 and GAS5 was observed; accordingly, matrix metalloproteinase (MMP) 9 was increased. After GAS5-AS1 silencing, a downregulation of GAS5 was found, whereas no effect was detected on GAS5-AS1 after GAS5 silencing.    Conclusion: This study provided for the first time new insights into the potential role of GAS5-AS1 in IBD. GAS5-AS1 modulates GAS5 levels in vitro and may serve as a potential IBD diagnostic biomarker. What is Known: • GAS5 is involved in regulating intestinal MMP-2 and MMP-9 in pediatric patients with IBD; • GAS5-AS1 has never been investigated in the context of IBD; • GAS5-AS1 regulates the expression of GAS5, increasing its stability in tissues and in vitro cell models of cancer. What is New: • GAS5-AS1 correlated with GAS5 and IBD clinical parameters; • GAS5-AS1 can modulate GAS5 levels in macrophages; • GAS5-AS1 may serve as potential IBD diagnostic biomarker.

iPSCs as a groundbreaking tool for the study of adverse drug reactions: A new avenue for personalized therapy.

Induced pluripotent stem cells (iPSCs), obtained by reprogramming different somatic cell types, represent a promising tool for the study of drug toxicities, especially in the context of personalized medicine. Indeed, these cells retain the same genetic heritage of the donor, allowing the development of personalized models. In addition, they represent a useful tool for the study of adverse drug reactions (ADRs) in special populations, such as pediatric patients, which are often poorly represented in clinical trials due to ethical issues. Particularly, iPSCs can be differentiated into any tissue of the human body, following several protocols which use different stimuli to induce specific differentiation processes. Differentiated cells also maintain the genetic heritage of the donor, and therefore are suitable for personalized pharmacological studies; moreover, iPSC-derived differentiated cells are a valuable tool for the investigation of the mechanisms underlying the physiological differentiation processes. iPSCs-derived organoids represent another important tool for the study of ADRs. Precisely, organoids are in vitro 3D models which better represent the native organ, both from a structural and a functional point of view. Moreover, in the same way as iPSC-derived 2D models, iPSC-derived organoids are appropriate personalized models since they retain the genetic heritage of the donor. In comparison to other in vitro models, iPSC-derived organoids present advantages in terms of versatility, patient-specificity, and ethical issues. This review aims to provide an updated report of the employment of iPSCs, and 2D and 3D models derived from these, for the study of ADRs. This article is categorized under: Cancer > Stem Cells and Development.

SERS spectroscopy as a tool for the study of thiopurine drug pharmacokinetics in a model of human B leukemia cells.

Thiopurine drugs are immunomodulatory antimetabolites relevant for pediatric patients characterized by dose-dependent adverse effects such as myelosuppression and hepatotoxicity, often related to inter-individual differences, involving the activity of important enzymes at the basis of their biotransformation, such as thiopurine S-methyltransferase (TPMT). Surface Enhanced Raman Scattering (SERS) spectroscopy is emerging as a bioanalytical tool and represents a valid alternative in terms of affordable costs, shorter analysis time and easier sample preparation in comparison to the most employed methods for pharmacokinetic analysis of drugs. The aim of this study is to investigate mercaptopurine and thioguanine pharmacokinetics by SERS in cell lysates of a B-lymphoblastoid cell line (NALM-6), that did (TPMT*1) or did not (MOCK) overexpress the wild-type form of TPMT as an in vitro cellular lymphocyte model to discriminate between cells with different levels of TPMT activity on the base of the amount of thioguanosine nucleotides (TGN) metabolites formed. SERS analysis of the cell lysates was carried out using SERS substrates constituted by Ag nanoparticles deposited on paper and parallel samples were used for quantification of thiopurine nucleotides with liquid chromatography-tandem mass spectrometry (LC-MS/MS). A direct SERS detection method has been set up that could be a tool to study thiopurine drug pharmacokinetics in in vitro cellular models to qualitatively discriminate between cells that do and do not overexpress the TPMT enzyme, as an alternative to other more laborious techniques. Results underlined decreased levels of TGN and increased levels of methylated metabolites when TPMT was overexpressed, both after mercaptopurine and thioguanine treatments. A strong positive correlation (Spearman's rank correlation coefficient rho = 0.96) exists between absolute quantification of TGMP (pmol/1 x 106 cells), obtained by LC-MS/MS, and SERS signal (intensity of TGN at 915 cm-1). In future studies, we aim to apply this method to investigate TPMT activity in pediatric patients' leukocytes.

Subcutaneous tocilizumab in the management of non-infectious uveitis in children: a brief report.

BACKGROUND: Tocilizumab is a humanized monoclonal antibody that acts as an IL-6 receptor antagonist. Intravenous tocilizumab is considered an option for children with anti-TNF refractory juvenile idiopathic arthritis-associated uveitis. In contrast, the potential of subcutaneous drug use with this indication is more controversial. Due to the decreased availability of intravenous tocilizumab during the COVID-19 pandemic, we started using the subcutaneous formulation of the drug in children with anti-TNF refractory uveitis. The study analyzes the serum concentration of tocilizumab and its clinical response in patients with anti-TNF refractory uveitis who started or switched to subcutaneous administration from intravenous use. METHODS: Five patients with non-infectious uveitis were treated with subcutaneous tocilizumab. Ocular inflammation was evaluated on slit lamp examination during clinical control. Serum tocilizumab concentrations were determined by ELISA. RESULTS: The mean blood concentration of tocilizumab was 61.4 µg/mL (range 2.7-137.0.), with higher values than levels recorded in adult patients with rheumatoid arthritis treated with intravenous tocilizumab. Three patients entered clinical remission. One patient developed a mild relapse and was treated with topical steroids. Only one patient did not respond to therapy. The medication was well tolerated without severe infection or other adverse events. CONCLUSION: Our results support a possible role of subcutaneous tocilizumab in anti-TNF refractory uveitis.

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.

PACSIN2 as a modulator of autophagy and mercaptopurine cytotoxicity: mechanisms in lymphoid and intestinal cells.

PACSIN2 variants are associated with gastrointestinal effects of thiopurines and thiopurine methyltransferase activity through an uncharacterized mechanism that is postulated to involve autophagy. This study aims to clarify the role of PACSIN2 in autophagy and in thiopurine cytotoxicity in leukemic and intestinal models. Higher autophagy and lower PACSIN2 levels were observed in inflamed compared with non-inflamed colon biopsies of inflammatory bowel disease pediatric patients at diagnosis. PACSIN2 was identified as an inhibitor of autophagy, putatively through inhibition of autophagosome formation by a protein-protein interaction with LC3-II, mediated by a LIR motif. Moreover, PACSIN2 resulted a modulator of mercaptopurine-induced cytotoxicity in intestinal cells, suggesting that PACSIN2-regulated autophagy levels might influence thiopurine sensitivity. However, PACSIN2 modulates cellular thiopurine methyltransferase activity via mechanisms distinct from its modulation of autophagy.

DNA methylation of the TPMT gene and azathioprine pharmacokinetics in children with very early onset inflammatory bowel disease.

BACKGROUND: Thiopurine methyltransferase (TPMT) is a crucial enzyme for azathioprine biotransformation and its activity is higher in very early onset inflammatory bowel disease (VEO-IBD) patients than in adolescents with IBD (aIBD). AIMS: The aims of this pharmacoepigenetic study were to evaluate differences in peripheral blood DNA methylation of the TPMT gene and in azathioprine pharmacokinetics in patients with VEO-IBD compared to aIBD. METHODS: The association of age with whole genome DNA methylation profile was evaluated in a pilot group of patients and confirmed by a meta-analysis on 3 cohorts of patients available on the public functional genomics data repository. Effects of candidate CpG sites in the TPMT gene were validated in a larger cohort using pyrosequencing. TPMT activity and azathioprine metabolites (TGN) were measured in patients' erythrocytes by HPLC and associated with patients' age group and TPMT DNA methylation. RESULTS: Whole genome DNA methylation pilot analysis, combined with the meta-analysis revealed cg22736354, located on TPMT downstream neighboring region, as the only statistically significant CpG whose methylation increases with age, resulting lower in VEO-IBD patients compared to aIBD (median 9.6% vs 12%, p = 0.029). Pyrosequencing confirmed lower cg22736354 methylation in VEO-IBD patients (median 4.0% vs 6.0%, p = 4.6 ×10-5). No differences in TPMT promoter methylation were found. Reduced cg22736354 methylation was associated with lower TGN concentrations (rho = 0.31, p = 0.01) in patients with VEO-IBD and aIBD. CONCLUSION: Methylation of cg22736354 in TPMT gene neighborhood is lower in patients with VEO-IBD and is associated with reduced azathioprine inactivation and increased TGN concentrations.

A Validated HPLC-Diode Array Detection Method for Therapeutic Drug Monitoring of Thiopurines in Pediatric Patients: From Bench to Bedside.

Thiopurine drugs are part of the therapeutic armamentarium for pediatric patients suffering from inflammatory bowel disease (IBD) and acute lymphoblastic leukemia (ALL). The therapeutic drug monitoring of these drugs, consisting of measurements of the thiopurine metabolites thioguanine nucleotides (TGN) and methylmercaptopurine nucleotides (MMPN) are used to optimize the effectiveness of treatment and prevent adverse effects. In this context, we developed and validated a high-performance liquid chromatography-diode array detection (HPLC-DAD) method for the simultaneous quantification of thiopurine metabolites according to the most recent International Council for Harmonisation (ICH) guidelines. The calibration curves were built in the clinically relevant range of concentrations for TGN of 300-12,000 nM and for MMPN of 3000-60,000 nM. The limit of detection and the lower limit of quantification were 100 and 300 nM for TGN and 900 and 3000 nM for MMPN, respectively. The percentage of inter-day accuracy and precision (CV%) varied between 85 and 104% and 1.6 and 13.8%. Stability was demonstrated for both of the metabolites for at least 50 days at -20 °C. The proposed HPLC-DAD method showed an appropriate selectivity, specificity, linearity, accuracy, precision and good applicability to samples from patients with IBD and ALL undergoing thiopurine treatment.

Patient-derived organoids for therapy personalization in inflammatory bowel diseases.

Inflammatory bowel diseases (IBDs) are chronic inflammatory disorders of the intestinal tract that have emerged as a growing problem in industrialized countries. Knowledge of IBD pathogenesis is still incomplete, and the most widely-accepted interpretation considers genetic factors, environmental stimuli, uncontrolled immune responses and altered intestinal microbiota composition as determinants of IBD, leading to dysfunction of the intestinal epithelial functions. In vitro models commonly used to study the intestinal barrier do not fully reflect the proper intestinal architecture. An important innovation is represented by organoids, 3D in vitro cell structures derived from stem cells that can self-organize into functional organ-specific structures. Organoids may be generated from induced pluripotent stem cells or adult intestinal stem cells of IBD patients and therefore retain their genetic and transcriptomic profile. These models are powerful pharmacological tools to better understand IBD pathogenesis, to study the mechanisms of action on the epithelial barrier of drugs already used in the treatment of IBD, and to evaluate novel target-directed molecules which could improve therapeutic strategies. The aim of this review is to illustrate the potential use of organoids for therapy personalization by focusing on the most significant advances in IBD research achieved through the use of adult stem cells-derived intestinal organoids.

Atomic Force Microscopy Application for the Measurement of Infliximab Concentration in Healthy Donors and Pediatric Patients with Inflammatory Bowel Disease.

The use of infliximab has completely changed the therapeutic landscape in inflammatory bowel disease. However, despite its proven efficacy to induce and maintain clinical remission, increasing evidence suggests that treatment failure may be associated with inadequate drug blood concentrations. The introduction of biosensors based on different nanostructured materials for the rapid quantification of drugs has been proposed for therapeutic drug monitoring. This study aimed to apply atomic force microscopy (AFM)-based nanoassay for the measurement of infliximab concentration in serum samples of healthy donors and pediatric IBD patients. This assay measured the height signal variation of a nanostructured gold surface covered with a self-assembled monolayer of alkanethiols. Inside this monolayer, we embedded the DNA conjugated with a tumor necrosis factor able to recognize the drug. The system was initially fine-tuned by testing known infliximab concentrations (0, 20, 30, 40, and 50 nM) in buffer and then spiking the same concentrations of infliximab into the sera of healthy donors, followed by testing pediatric IBD patients. A good correlation between height variation and drug concentration was found in the buffer in both healthy donors and pediatric IBD patients (p-value < 0.05), demonstrating the promising use of AFM nanoassay in TDM.

Extracellular Vesicles as Innovative Tools for Assessing Adverse Effects of Immunosuppressant Drugs.

BACKGROUND: Extracellular vesicles (EVs) are a heterogeneous family of small vesicles released by donor cells and absorbed by recipient cells, which represent important mediators with fundamental roles in both physiological and pathological conditions. EVs are present in a large variety of biological fluids and have a great diagnostic and prognostic value. They have gained the interest of the scientific community due to their extreme versatility. In fact, they allow us to hypothesize new therapeutic strategies since, in addition to being cell signal mediators, they play an important role as biomarkers, drug vehicles, and potential new therapeutic agents. They are also involved in immunoregulation, have the ability to transmit resistance to a drug from one cell to a more sensitive one, and can act as drug delivery systems. OBJECTIVE: The main reciprocal interactions between EVs and immunosuppressive drugs will be presented. RESULTS: The known interactions between EVs and immunosuppressive drugs, in particular cyclosporin, glucocorticoids, rapamycin, methotrexate, cyclophosphamide, eculizumab, infliximab, certolizumab, etanercept, glatiramer acetate, and fingolimod are presented. CONCLUSION: This review provides relevant information on the links between EVs and immunosuppressive drugs with a focus on EVs' role as tools to assess the effects of immunosuppressants, suggesting innovative properties and new possible therapeutic uses.

Inflammatory Bowel Disease and Risk of Colorectal Cancer: An Overview From Pathophysiology to Pharmacological Prevention.

Increased risk of colorectal cancer (CRC) in inflammatory bowel disease (IBD) patients has been attributed to long-standing chronic inflammation, with the contribution of genetic alterations and environmental factors such as the microbiota. Moreover, accumulating data indicate that IBD-associated CRC (IBD-CRC) may initiate and develop through a pathway of tumorigenesis distinct from that of sporadic CRC. This mini-review summarizes the current knowledge of IBD-CRC, focusing on the main mechanisms underlying its pathogenesis, and on the important role of immunomodulators and biologics used to treat IBD patients in interfering with the inflammatory process involved in carcinogenesis.

Interplay of OpdP Porin and Chromosomal Carbapenemases in the Determination of Carbapenem Resistance/Susceptibility in Pseudomonas aeruginosa.

Carbapenem resistance in Pseudomonas aeruginosa strains responsible for chronic lung infections in cystic fibrosis (CF) patients is mainly due to loss of the OprD protein and, limited to meropenem and doripenem, to overexpression of efflux pumps. However, recent reports of isolates showing inconsistent genotype-phenotype combinations (e.g., susceptibility in the presence of resistance determinants and vice versa) suggest the involvement of additional factors whose role is not yet fully elucidated. Among them, the OpdP porin as an alternative route of entry for carbapenems other than OprD and the overexpression of two chromosomal carbapenemases, the Pseudomonas-derived cephalosporinase (PDC) and the PoxB oxacillinase, have recently been reconsidered and studied in specific model strains. Here, the contribution of these factors was investigated by comparing different phenotypic variants of three strains collected from the sputum of colonized CF patients. Carbapenem uptake through OpdP was investigated both at the functional level, by assessing the competition exerted by glycine-glutamate, the OpdP's natural substrate, against imipenem uptake, and at the molecular level, by comparing the expression levels of opdP genes by quantitative real-time PCR (qRT-PCR). Moreover, overexpression of the chromosomal carbapenemases in some of the isolates was also investigated by qRT-PCR. The results showed that, even if OprD inactivation remains the most important determinant of carbapenem resistance in strains infecting the CF lung, the interplay of other determinants might have a nonnegligible impact on bacterial susceptibility, being able to modify the phenotype of part of the population and consequently complicating the choice of an appropriate therapy. IMPORTANCE This study examines the interplay of multiple factors in determining a pattern of resistance or susceptibility to carbapenems in clinical isolates of Pseudomonas aeruginosa, focusing on the role of previously poorly understood determinants. In particular, the impact of carbapenem permeability through OprD and OpdP porins was analyzed, as well as the activity of the chromosomal carbapenemases AmpC and PoxB, going beyond the simple identification of resistance determinants encoded by each isolate. Indeed, analysis of the expression levels of these determinants provides a new approach to determine the contribution of each factor, both individually and in coexistence with the other factors. The study contributes to understanding some phenotype-genotype discordances closely related to the heteroresistance frequently detected in P. aeruginosa isolates responsible for pulmonary infections in cystic fibrosis patients, which complicates the choice of an appropriate patient-specific therapy.

Responses of patients with juvenile idiopathic arthritis to methotrexate: a genomic outlook.

INTRODUCTION: Juvenile idiopathic arthritis (JIA) is a chronic disease characterized by persistent joint inflammation. JIA is the most common pediatric chronic rheumatic disease and no curative therapy is currently available. Methotrexate (MTX) is an important treatment for JIA even though a high inter-individual variability in response is observed in patients. Among the factors of this variability, genetics and epigenetics might play an important role. AREAS COVERED: This review summarizes the results of pharmacogenetic and pharmacoepigenetic studies regarding MTX response in JIA. Studies considering epigenetic factors in JIA patients are still very limited, therefore this review includes also studies performed in adult patients with rheumatoid arthritis. Moreover, the relevance of biomarkers measured in blood or urine of JIA patients in relation to MTX treatment is discussed. EXPERT OPINION: Nowadays, even though many pharmacogenomics studies have been published, a specific genetic marker predictor of MTX efficacy or adverse events has not yet been identified. Encouraging results are available and great expectations rely on the study of epigenetics. Future studies are needed in order to identify genetic and epigenetic biomarkers that can be implemented in the clinical practice.

Insights into the cellular pharmacokinetics and pharmacodynamics of thiopurine antimetabolites in a model of human intestinal cells.

Thiopurines, immunomodulating drugs used in the management of different chronic autoimmune conditions and as anti-leukemic agents, may exert in some cases gastrointestinal toxicity. Moreover, since these agents are administered orally, they are absorbed across the gastrointestinal tract epithelium. On these premises, cellular and molecular events occurring in intestinal cells may be important to understand thiopurine effects. However, quantitative information on the biotransformation of thiopurines in intestinal tissues is still limited. To shed light on biotransformation processes specific of the intestinal tissue, in this study thiopurine metabolites concentrations were analyzed by an in vitro model of human healthy colon, the HCEC cell line, upon exposure to cytotoxic concentrations of azathioprine or mercaptopurine; the investigation was carried out using an innovative mass spectrometry method, that allowed the simultaneous quantification of 11 mono-, di-, and triphosphate thionucleotides. Among the 11 metabolites evaluated, TIMP, TGMP, TGDP, TGTP, MeTIMP, MeTIDP and MeTITP were detectable in HCEC cells treated with azathioprine or mercaptopurine, considering two different incubation times before the addition of the drugs (4 and 48 h). Different associations between metabolites concentrations and cytotoxicity were detected. In particular, the cytotoxicity was dependent on the TGMP, TGDP, TGTP and MeTITP concentrations after the 4 h incubation before the addition of thiopurines. This may be an indication that, to study the association between thiopurine metabolite concentrations and the cytotoxicity activity in vitro, short growth times before treatment should be used. Moreover, for the first time our findings highlight the strong correlation between cytotoxicity and thiopurine pharmacokinetics in HCEC intestinal cells in vitro suggesting that these cells could be a suitable in vitro model for studying thiopurine intestinal cytotoxicity.

Pharmacogenetic variants of infliximab response in young patients with inflammatory bowel disease.

Infliximab is commonly used in inflammatory bowel disease (IBD), however, differences in clinical response among patients are common. Several studies have considered the possibility that these differences are caused by genetic variability even if no unique marker has been yet identified in pediatric patients. We evaluated the impact of two candidate single-nucleotide polymorphisms (SNPs) rs396991 in FCGR3A and rs1800629 in TNFα genes on infliximab response in an Italian cohort of 76 pediatric patients with IBD. Results showed that patients with the variant FCGR3A allele had a reduced clinical response at the end of induction (p value = 0.004), at 22 weeks (p value = 0.001), and at 52 weeks of treatment (p value = 0.01). A significant association between the FCGR3A variant and median infliximab levels measured during maintenance therapy was also observed: patients with wild type genotype had higher infliximab levels compared to patient with variant allele. Furthermore, patients with the variant allele had a higher probability to produce antidrug antibodies (ADAs). No association was found among the TNFα SNP, clinical response, and infliximab levels. This study addressed for the first time in pediatric patients with IBD, the association of FCGR3A SNP, infliximab response, and ADA production.