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Novel triorganotin(IV) complexes of two β-lactamic antibiotics, 6-[D-(-)-β-amino-p-hydroxyphenyl-acetamido]penicillin (=amoxicillin) and 6-[D-(-)-α-aminobenzyl]penicillin (=ampicillin), have been synthesized and investigated both in solid and solution states. The complexes corresponded to the general formula R3Sn(IV)antib·H2O (R=Me, n-Bu, Ph; antib=amox=amoxicillinate or amp=ampicillinate). Structural investigations about configuration in the solid state have been carried out by interpreting experimental IR and 119Sn Mössbauer data. In particular, IR results suggested polymeric structures both for R3Sn(IV)amox·H2O and R3Sn(IV)amp·H2O. Moreover, both antibiotics appear to behave as monoanionic bidentate ligands coordinating the tin(IV) atom through ester-type carboxylate, as well as through the β-lactamic carbonyl. Evidence that in none of these compounds water molecules were involved in coordination, was provided by thermogravimetric investigations. On the basis of 119Sn Mössbauer spectroscopy it can be inferred that tin(IV) was pentacoordinate in all of the complexes in the solid state, showing an equatorial R3Sn(IV) trigonal bipyramidal (tbp) configuration. The nature of the complexes in solution state was investigated by using 1H and 13C nuclear magnetic resonance (NMR) spectroscopy, while an 119Sn spectrum was obtained for n-Bu3Sn(IV)amp·H2O. Although 1H- and 13C-NMR measurements suggested that in dimethyl sulfoxide (DMSO)-d6 solution the polymeric structure collapsed, due to a solvolysis process of the β-lactamic carbonyl bonding to the organometallic moiety, the complexes have been shown to maintain the same trigonal bipyramidal configuration at tin(IV) atom by the coordination of a DMSO molecule. Cytotoxic activity of these novel semisynthetic antibiotic derivatives has been tested towards spermatocyte chromosomes of the mussel Brachidontes pharaonis (Mollusca: Bivalvia) using two different chromosome-staining techniques such as Giemsa and CMA3. The occurrence of typical colchicinized-like (c-like) mitoses on slides obtained from animals exposed to organotin compounds, directly confirmed the high mitotic spindle-inhibiting potency of these chemicals. In addition, by comparative analysis of spermatocyte chromosomes from untreated specimens (negative controls) and specimens treated with the triorganotin(IV) complexes, structural damages such as 'achromatic lesions' and 'chromosome breakages' have been identified. © 2002 Elsevier Science Inc. All rights reserved.

Nuovi composti di organostagno(IV) [R3SnL e R¢2SnL2 (R = Me, Ph e R¢= Me, nBu, HL= Na-Boc-Ornitina)] sono stati sintetizzati e caratterizzati sia allo stato solido (FT-IR) che in soluzione (1H e 13C NMR) per poterne valutare la citotossicità in linee cellulari tumorali [1]. Na-Boc-Ornitina si comporta come un legante chelante dello stagno con il gruppo carbossilato, mentre il gruppo amminico Na-protetto è esente dalla coordinazione. I composti sono stati testati per l’attività citotossica in vitro su cellule neoplastiche umane HepG2 di epatocarcinoma ed MCF7 di cancro al seno. L’effetto dei composti, nel range da 0.5 a 25 μM, dopo 24 h di incubazione è stato valutato mediante saggio MTT. Il Ph3Sn(Boc-Orn) ha mostrato una forte azione citotossica (IC50 0.5-1.0 μM) su entrambe le linee cellulari, mentre fino alla concentrazione 2.5μM, non è apparso influenzare significativamente la crescita di cellule umane epatiche non-maligne (Chang), indicando preferenziale azione sulle tumorali, almeno a basse concentrazioni. Inoltre, misure citofluorimetriche ed al microscopio per fluorescenza di biomarkers di apoptosi, hanno chiaramente dimostrato che Ph3Sn(Boc-Orn) causa morte cellulare programmata in maniera concentrazione-dipendente. Al fine di valutare una possibile interazione tra questo complesso e il DNA cellulare sono stati effettuati studi di CD, UV-Vis e di misura della temperatura di melting su soluzioni contenti il dodecamero d(CGCAAATTTGCG) e il complesso Ph3Sn(Boc-Orn). L'osservazione di un incremento del Tm di circa 5°C, indica l’esistenza di una interazione tra complesso organometallico e sequenza oligonucleotidica. In conclusione, il nostro studio dimostra che il complesso Ph3Sn(Boc-Orn) mostra elevata attività antitumorale, superiore di circa una unità di grandezza a quella misurata nelle stesse condizioni dal cis-platino. Il complesso Ph3Sn(Boc-Orn) è risultato essere più attivo fra quelli studiati. [1] M. Gielen, Coord. Chem. Rev., 151, (1996) 41; M.A. Girasolo, S. Rubino, P. Portanova, G. Calvaruso, G.Ruisi, G.C. Stocco, J.Organomet. Chem.,695, (2010) 609.

Azathioprine is a purine antimetabolite drug commonly used as immunomodulator in the treatment of various chronic inflammatory diseases, such as inflammatory bowel disease (IBD). Azathioprine is activated in vivo after reaction with reduced glutathione (GSH) and conversion to mercaptopurine. Although this reaction may occur spontaneously, the presence of the enzyme glutathione-S-transferase (GST), in particular of isoforms GST-A1/GST-A2 and GST-M1, can increase its speed, leading to a faster activation of azathioprine to active thioguanine nucleotides. Moreover, GSTs may contribute to azathioprine effects by modulating GSH consumption, oxidative stress and apoptosis. Indeed, in young patients with IBD, deletion of GST-M1, which determines reduced enzymatic activity, was recently associated with reduced sensitivity to azathioprine and reduced production of its active metabolites. Therefore, genetic polymorphisms in genes for GSTs may be useful to predict response to azathioprine even if more in vitro and clinical validation studies are needed.

The aim of the study was to validate the impact of the single-nucleotide polymorphism rs2413739 (T > C) in the PACSIN2 gene on thiopurines pharmacological parameters and clinical response in an Italian cohort of pediatric patients with acute lymphoblastic leukemia (ALL) and inflammatory bowel disease (IBD). In ALL, PACSIN2 rs2413739 T allele was associated with a significant reduction of TPMT activity in erythrocytes (p = 0.0094, linear mixed-effect model, multivariate analysis considering TPMT genotype) and increased severe gastrointestinal toxicity during consolidation therapy (p = 0.049). A similar trend was present also for severe hematological toxicity during maintenance. In IBD, no significant effect of rs2413739 could be found on TPMT activity, howeverazathioprine effectiveness was reduced in patients carrying the T allele (linear mixed effect, p = 0.0058). In PBMC from healthy donors, a positive correlation between PACSIN2 and TPMT protein concentration could be detected (linear mixed effect, p = 0.045). These results support the role of PACSIN2 polymorphism on TPMT activity and mercaptopurine adverse effects in patients with ALL. Further evidence on PBMC and pediatric patients with IBD supports an association between PACSIN2 variants, TPMT activity, and thiopurines effects, even if more studies are needed since some of these effects may be tissue specific.

Purpose: Idiopathic nephrotic syndrome (INS) is the most frequent form of childhood nephrotic syndrome. Steroids represent the best therapeutic option; however, inter-individual differences in their efficacy and side effects have been reported. To date, there is no way to predict patients’ resistance and/or dependence. Alterations in the cytokine profile of INS patients might contribute to proteinuria and glomerular damage and affect drug sensitivity. Methods: The cytokine plasma levels were measured in 21 INS children at diagnosis to investigate the association among cytokines pattern and clinical response. Patients were selected on the basis of their clinical response: 7 steroid sensitive (SS), 7 dependent (SD), and 7 resistant (SR). Significant results were then analyzed in 41 additional pediatric INS patients. Results: Within the 48 cytokines analyzed, macrophage migration inhibitory factor (MIF) was a good predictor of steroid response. Indeed, SR patients showed significantly higher MIF plasma levels compared with all others (p = 0.022; OR = 4.3, 95%CI = 1.2–25.4): a cutoff concentration of MIF > 501 pg/ml significantly discriminated SR patients (sensitivity = 85.7%, specificity = 71.4%). On the contrary, SD patients showed lower MIF plasma levels compared with others (p = 0.010; OR = 0.12, 95%CI = 9.2 × 10−3–6.7 × 10−1). Significant results were confirmed in the entire cohort. Conclusions: Our comprehensive cytokine analysis indicates that assessing MIF plasma levels at diagnosis could predict response to glucocorticoids in children with INS.

Patients with ulcerative colitis (UC) using marijuana have been reported to experience symptomatic benefit. Cannabidivarin (CBDV) is a safe non-psychoactive phytocannabinoid able to activate and desensitize TRPA1, a member of the TRP channels superfamily, which plays a pivotal role in intestinal inflammation. Here, we have investigated the potential intestinal anti-inflammatory effect of CBDV in mice and in biopsies from pediatric patients with active UC. Colonic inflammation was induced in mice by dinitrobenzenesulfonic acid (DNBS). The effect of orally administered CBDV on macroscopic and microscopic damage, inflammatory parameters (i.e. myeloperoxidase activity, intestinal permeability and cytokine production) and faecal microbiota composition, was evaluated 3 days after DNBS administration. TRPA1 expression was studied by RT-PCR in inflamed colons of mice as well as in mucosal colonic biopsies of children with active UC, whose response to incubation with CBDV was also investigated. CBDV attenuates, in a TRPA1-antagonist sensitive manner, DNBS-induced signs of inflammation including neutrophil infiltration, intestinal permeability, and cytokine (i.e. IL-1β, IL-6 and the chemokine MCP-1) production. CBDV also alters the dysregulation of gut microbiota associated to colitis. Finally, CBDV lessens cytokine expression in colonic biopsies from pediatric patients with ulcerative colitis, a condition in which TRPA1 was up-regulated. Our preclinical study shows that CBDV exerts intestinal anti-inflammatory effects in mice via TRPA1, and in children with active UC. Since CBDV has a favorable safety profile in humans, it may be considered for possible clinical trials in patients with UC.

Microbial nucleic acid recognition serves as the major stimulus to an antiviral response, implying a requirement to limit the misrepresentation of self nucleic acids as non-self and the induction of autoinflammation. By systematic screening using a panel of interferon-stimulated genes we identify two siblings and a singleton variably demonstrating severe neonatal anemia, membranoproliferative glomerulonephritis, liver fibrosis, deforming arthropathy and increased anti-DNA antibodies. In both families we identify biallelic mutations in DNASE2, associated with a loss of DNase II endonuclease activity. We record increased interferon alpha protein levels using digital ELISA, enhanced interferon signaling by RNA-Seq analysis and constitutive upregulation of phosphorylated STAT1 and STAT3 in patient lymphocytes and monocytes. A hematological disease transcriptomic signature and increased numbers of erythroblasts are recorded in patient peripheral blood, suggesting that interferon might have a particular effect on hematopoiesis. These data define a type I interferonopathy due to DNase II deficiency in humans., Nucleic acid sensing is important to ensure that an innate immune response is only mounted against microbial nucleic acid. Here, the authors identify loss-of-function mutations in the DNASE2 gene that cause type I interferon-mediated autoinflammation due to enhanced systemic interferon signaling.

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.

Drug-induced pancreatitis is a gastrointestinal adverse effect concerning about 2% of drugs. The majority of cases are mild to moderate but severe episodes can also occur, leading to hospitalization or even death. Unfortunately, the mechanisms of this adverse reaction are still not clear, hindering its prevention, and the majority of data available of this potentially life-threatening adverse effect are limited to case reports leading to a probable underestimation of this event. In particular, in this editorial, special attention is given to thiopurine-induced pancreatitis (TIP), an idiosyncratic adverse reaction affecting around 5% of inflammatory bowel disease (IBD) patients taking thiopurines as immunosuppressants, with a higher incidence in the pediatric population. Validated biomarkers are not available to assist clinicians in the prevention of TIP, also because of the inaccessibility of the pancreatic tissue, which limits the possibility to perform dedicated cellular and molecular studies. In this regard, induced pluripotent stem cells (iPSCs) and the exocrine pancreatic differentiated counterpart could be a great tool to investigate the cellular and molecular mechanisms underlying the development of this undesirable event. This particular type of stem cells is obtained by reprogramming adult cells, including fibroblasts and leukocytes, with a set of transcription factors known as the Yamanaka's factors. Maintaining unaltered the donors' genetic heritage, iPSCs represent an innovative model to study the mechanisms of adverse drug reactions in individual patients' tissues not easily obtainable from human probands. Indeed, iPSCs can differentiate under adequate stimuli into almost any somatic lineage, opening a new world of opportunities for researchers. Several works are already available in the literature studying liver, central nervous system and cardiac cells derived from iPSCs and adverse drug effects. However, to our knowledge no studies have been performed on exocrine pancreas differentiated from iPSCs and drug-induced pancreatitis, so far. Hence, in this editorial we focus specifically on the description of the study of the mechanisms of TIP by using IBD patient-specific iPSCs and exocrine pancreatic differentiated cells as innovative in vitro models.

Pharmacogenetics (PGx) association studies are used to discover, replicate, and validate the association between an inherited genotype and a treatment outcome. The objective of this tutorial is to provide trainees and novice PGx researchers with an overview of the major decisions that need to be made when designing and conducting a PGx association study. The first critical decision is to determine whether the objective of the study is discovery, replication, or validation. Next, the researcher must identify a patient cohort that has all of the data necessary to conduct the intended analysis. Then, the investigator must select and define the treatment outcome, or phenotype, that will be analyzed. Next, the investigator must determine what genotyping approach and genetic data will be included in the analysis. Finally, the association between the genotype and phenotype is tested using some statistical analysis methodology. This tutorial is divided into five sections; each section describes commonly used approaches and provides suggestions and resources for designing and conducting a PGx association study. Successful PGx association studies are necessary to discover and validate associations between inherited genetic variation and treatment outcomes, which enable clinical translation to improve efficacy and reduce toxicity of treatment.

To assess whether NLRP3 gene promoter methylation was able to discriminate glucocorticoid (GC)-resistant from GC-sensitive idiopathic nephrotic syndrome (INS), patients with minimal change disease (MCD) or focal segmental glomerulosclerosis (FSGS), we measured the methylation level of NLRP3 promoter in DNA from peripheral blood cells of 10 adult patients with GC-resistant FSGS already in hemodialysis and 18 patients with GC-sensitive INS (13 MCD/5 FSGS) and in 21 pediatric patients with INS with MCD/FSGS before starting any treatment. Association of NLRP3 inflammasome with GC resistance was recapitulated in vitro in monocytic cell lines (THP-1 and U937). In both adults and pediatric patients, NLRP3 promoter methylation was significantly reduced in GC-resistant compared with GC-sensitive patients. Indeed, NLRP3 methylation distinguished GC-resistant and GC-sensitive patients (area under the receiver operating characteristic curve [AUROC] 86.7% in adults, p = 0.00019, and 73.5% in children, p = 0.00097). NLRP3 knock-down augmented sensitivity to GCs in THP-1 cells, whereas NLRP3 inflammasome activation lowered GC receptor concentration, increasing GC resistance in U937 cells. Our results uncovered a new biological mechanism by which patients with INS may acquire GC resistance, that could be used in future as a novel noninvasive diagnostic tool. Study Highlights WHAT IS THE CURRENT KNOWLEDGE ON THE TOPIC? ☑ Approximately 80% of patients with idiopathic nephrotic syndrome (INS) respond to glucocorticoids, with the remaining 20% being steroid-resistant. WHAT QUESTION DID THIS STUDY ADDRESS? ☑ Whether NLRP3 gene promoter methylation was able to discriminate glucocorticoid-resistant from glucocorticoid (GC)-sensitive INS. WHAT DOES THIS STUDY ADD TO OUR KNOWLEDGE? ☑ In both adults and children, NLRP3 promoter methylation was significantly reduced in leukocytes of patients with GC-resistant compared with GC-sensitive INS. NLRP3 inflammasome activation lowered GC receptor concentration and augmented GC resistance, whereas NLRP3 knockdown increased sensitivity to GCs in cell lines representative of monocytes (U937 and THP1). HOW MIGHT THIS CHANGE CLINICAL PHARMACOLOGY OR TRANSLATIONAL SCIENCE? ☑ Our findings uncovered a new biological mechanism whereby patients with INS may develop resistance to GCs that could be used in the future as a novel noninvasive diagnostic tool.

No abstract available

Ataxia telangiectasia (AT) and Aicardi–Goutières syndrome (AGS) are inherited disorders of immunity with prevalent neurological phenotype. Available treatments are only partially effective, and the prognosis is poor. Induced pluripotent stem cells (iPSCs) are obtained by reprogramming patient somatic cells, preserving the donor individual genetic heritage and creating patient-specific disease models, useful to investigate pathogenesis and drug effects and to develop precision therapies. The aim is to investigate the cytotoxicity of a panel of immunomodulators using iPSCs of patients with AT or different forms of AGS (AGS1, AGS2, and AGS7). iPSCs were obtained by reprogramming AT and AGS patients’ cells and, as a control, the BJ normal human fibroblast line, using Sendai virus. Cytotoxic effects of two drugs proposed to treat respectively AT and AGS (dexamethasone and mepacrine) were tested by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay after 72 hours' exposure. Data were obtained also for other immunomodulatory drugs (thioguanine, mercaptopurine, thalidomide, and lenalidomide). Relative expression of genes involved in the tested drug pathways was analyzed. AGS7-derived iPSCs displayed altered viability when treated with a low dose of mepacrine and higher expression of cyclic guanosine monophosphate–adenosine monophosphate synthase, which is the main target for mepacrine action. AGS7-derived iPSCs were also more sensitive to thioguanine, while AGS2 and AT iPSCs were less sensitive to this medication than the BJ-iPSC. All iPSCs were equally sensitive to mercaptopurine and resistant to dexamethasone, thalidomide, and lenalidomide. This work establishes an innovative in vitro model that is useful to investigate the mechanisms of drugs potentially effective in AT and AGS.

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. ispartof: WORLD JOURNAL OF GASTROENTEROLOGY vol:28 issue:24 pages:2636-2653 ispartof: location:United States status: published

Background: The T-cell engager antibody blinatumomab (BlincytoTM) represents a promising rescue therapy for relapsed/refractory CD19+ acute lymphoblastic leukemia (B-ALL), although ∼20-30% of patients still do not respond to treatment. Blinatumomab creates a tight synapsis between CD3+ T-lymphocytes and leukemic CD19+ B-cells, resulting in a granzyme B (GzB)-mediated specific lysis of leukemic cells. Methods: Aim of the study was to provide evidence that variability in blinatumomab response could have a genetic basis in PAX5, one of the most often mutated genes in B-ALL, affecting the CD19 surface expression on lymphoblasts, and could be explored in vitro by means of a cytofluorimetric assay, staining both surface antigens (CD45, CD19 and CD3) and intracytoplasmic markers (7AAD, Syto16). Two human immortalized B-ALL cell lines (NALM6 and REH) were chosen for their different PAX5 and CD19 protein levels, as verified by western blot and flow cytometry, respectively. Results: In contrast to NALM6, REH cells do not express the full-length PAX5 protein and show less CD19 on the cell surface (fluorescence peak median intensity: 9155 versus 28895). Co-cultures of CD3+ T-lymphocytes from healthy donors and B-ALL cell lines were seeded at an effector-to-target cell ratio of 1:10 for simulating the condition existing in the bone marrow due to the malignant invasion of blast cells. Co-cultures were exposed in vitro to blinatumomab and the simultaneous increase in blast mortality and T-lymphocytes activation induced by the drug was observed at day +7 (both effects: P

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.

Diseases affecting the immune system, such as inflammatory bowel disease (IBD), juvenile idiopathic arthritis (JIA), and acute lymphoblastic leukemia (ALL), are pathological conditions affecting the pediatric population and are often associated with alterations in the intestinal microbiota, such as a decrease in bacterial diversity. Growing evidence suggests that gut microbiota can interfere with chemotherapeutic and immunosuppressant drugs, used in the treatment of these diseases, reducing or facilitating drug efficacy. In particular, the effect of intestinal microflora through translocation, immunomodulation, metabolism, enzymatic degradation, and reduction of bacterial diversity seems to be one of the reasons of interindividual variability in the therapeutic response. Although the extent of the role of intestinal microflora in chemotherapy and immunosuppression remains still unresolved, current evidence on bacterial compositional shifts will be taken in consideration together with clinical response to drugs for a better and personalized therapy. This review is focused on the effect of the intestinal microbiota on the efficacy of pharmacological therapy of agents used to treat IBD, JIA, and ALL.

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.

A mutualistic relationship between the composition, function and activity of the gut microbiota (GM) and the host exists, and the alteration of GM, sometimes referred as dysbiosis, is involved in various immune-mediated diseases, including inflammatory bowel disease (IBD). Accumulating evidence suggests that the GM is able to influence the efficacy of the pharmacological therapy of IBD and to predict whether individuals will respond to treatment. Additionally, the drugs used to treat IBD can modualate the microbial composition. The review aims to investigate the impact of the GM on the pharmacological therapy of IBD and vice versa. The GM resulted in an increase or decrease in therapeutic responses to treatment, but also to biotransform drugs to toxic metabolites. In particular, the baseline GM composition can help to predict if patients will respond to the IBD treatment with biologic drugs. On the other hand, drugs can affect the GM by incrementing or reducing its diversity and richness. Therefore, the relationship between the GM and drugs used in the treatment of IBD can be either beneficial or disadvantageous.

Introduction: Thiopurine methyltransferase (TPMT) catalyzes the S-methylation of thiopurines (mercaptopurine (MP) and tioguanine (TG)), chemotherapeutic agents used in the treatment of acute lymphoblastic leukemia (ALL). Polymorphisms in TPMT gene encode diminished activity enzyme, enhancing accumulation of active metabolites, and partially explaining the inter-individual differences in patients’ clinical response. Areas covered: This review gives an overview on TPMT gene and function, and discusses the pharmacogenomic implications of TPMT variants in the prevention of severe thiopurine-induced hematological toxicities and the less known implication on TG-induced sinusoidal obstruction syndrome. Additional genetic and non-genetic factors impairing TPMT activity are considered. Literature search was done in PubMed for English articles published since1990, and on PharmGKB. Expert opinion: To titrate thiopurines safely and effectively, achieve the right degree of lymphotoxic effect and avoid excessive myelosuppression, the optimal management will combine a preemptive TPMT genotyping to establish a safe initial dose with a close phenotypic monitoring of TPMT activity and/or of active metabolites during long-term treatment. Compared to current ALL protocols, replacement of TG by MP during reinduction phase in TPMT heterozygotes and novel individualized TG regimens in maintenance for TPMT wild-type subjects could be investigated to improve outcomes while avoiding risk of severe hepatotoxicity.

Implementation of pharmacogenetics (PGx) in the clinical practice of cancer therapy is one of the goals for a precision medicine. For some anticancer drugs (fluoropyrimidines and irinotecan) precise recommendations by regulatory agencies are available. However, in the future, implementation of PGx in clinical practice should also consider the anticancer drugs without available pharmacogenetic guidelines and the increasingly important role of rare PGx variants. To overcome barriers to PGx implementation we need to focus on pharmacogenetic test clinical utility by adopting a panel-based approach and sensitize both physician and patients to the therapeutic value of pharmacogenetic tests.

Introduction: Adalimumab is effective in inducing and maintaining remission in children with inflammatory bowel diseases (IBD). Therapeutic drug monitoring is an important strategy to maximize the response rates, but data on the association of serum adalimumab levels are lacking. This study aimed to assess the association of adalimumab concentrations at the end of induction and early during maintenance for long-term response.Materials and Methods: Serum samples for adalimumab level measurement were collected during routine visits between adalimumab administrations and therefore not necessarily at trough, both during the induction (week 4 ± 4) and maintenance phases (week 22 ± 4, 52 ± 4, and 82 ± 4). Adalimumab and anti-adalimumab antibodies were measured retrospectively using enzyme-linked immunosorbent assays (ELISA). Disease activity was determined by Pediatric Crohn's Disease Activity Index or Pediatric Ulcerative Colitis Activity Index.Results: Thirty-two children (median age 14.9 years) were enrolled. Sixteen, 15, 14, and 12 patients were in remission at weeks 4, 22, 52, and 82, respectively. Median adalimumab concentration was higher at all time points in patients achieving sustained clinical remission. Adalimumab levels correlated with clinical and biochemical variables. Adalimumab concentration above 13.85 and 7.54 μg/ml at weeks 4 and 22 was associated with remission at weeks 52 and 82.Conclusions: Adalimumab non-trough levels are associated with long-term response in pediatric patients with IBD.

Interferonopathies are rare genetic conditions defined by systemic inflammatory episodes caused by innate immune system activation in the absence of pathogens. Currently, no targeted drugs are authorized for clinical use in these diseases. In this work, we studied the contribution of sulforaphane (SFN), a cruciferous-derived bioactive molecule, in the modulation of interferon-driven inflammation in an immortalized human hepatocytes (IHH) line and in two healthy volunteers, focusing on STING, a key-component player in interferon pathway, interferon signature modulation, and GSTM1 expression and genotype, which contributes to SFN metabolism and excretion. In vitro, SFN exposure reduced STING expression as well as interferon signature in the presence of the pro-inflammatory stimulus cGAMP (cGAMP 3 h vs. SFN+cGAMP 3 h p value &lt, 0.0001, cGAMP 6 h vs. SFN+cGAMP 6 h p &lt, 0.001, one way ANOVA), restoring STING expression to the level of unstimulated cells. In preliminary experiments on healthy volunteers, no appreciable variations in interferon signature were identified after SFN assumption, while only in one of them, presenting the GSTM1 wild type genotype related to reduced SFN excretion, could a downregulation of STING be recorded. This study confirmed that SFN inhibits STING-mediated inflammation and interferon-stimulated genes expression in vitro. However, only a trend towards the downregulation of STING could be reproduced in vivo. Results obtained have to be confirmed in a larger group of healthy individuals and in patients with type I interferonopathies to define if the assumption of SFN could be useful as supportive therapy.

The pathogenic role of the overactivated ABL1 tyrosine kinase (TK) pathway is well recognized in some forms of BCR-ABL1 like acute lymphoblastic leukemia (ALL); TK inhibitors represent a useful therapeutic choice in these patients who respond poorly to conventional chemotherapy. Here we report a novel peptide biosensor (PABL)-ELISA assay to investigate ABL1 activity in four immortalized leukemic cell lines with different genetic background. The PABL sequence comprises an ABL1 tyrosine (Y) phosphorylation site and a targeting sequence that increases the specificity for ABL1; additional peptides (Y-site-mutated (PABL-F) and fully-phosphorylated (PPHOSPHO-ABL) biosensors) were included in the assay. After incubation with whole cell lysates, average PABL phosphorylation was significantly increased (basal vs. PABL phosphorylation: 6.84 ± 1.46% vs. 32.44 ± 3.25%, p-value < 0.0001, two-way ANOVA, Bonferroni post-test, percentages relative to PPHOSPHO-ABL in each cell line). Cell lines expressing ABL1-chimeric proteins (K562, ALL-SIL) presented the higher TK activity on PABL; a lower signal was instead observed for NALM6 and REH (p < 0.001 and p < 0.05 vs. K562, respectively). Phosphorylation was ABL1-mediated, as demonstrated by the specific inhibition of imatinib (p < 0.001 for K562, NALM6, ALL-SIL and p < 0.01 for REH) in contrast to ruxolitinib (JAK2-inhibitor), and occurred on the ABL1 Y-site, as demonstrated by PABL-F whose phosphorylation was comparable to basal levels. In order to validate this novel PABL-ELISA assay on leukemic cells isolated from patient’s bone marrow aspirates, preliminary analysis on blasts derived from an adult affected by chronic myeloid leukaemia (BCR-ABL1 positive) and a child affected by ALL (BCR-ABL1 negative) were performed. Phosphorylation of PABL was specifically inhibited after the incubation of BCR-ABL1 positive cell lysates with imatinib, but not with ruxolitinib. While requiring further optimization and validation in leukemic blasts to be of clinical interest, the PABL-based ELISA assay provides a novel in vitro tool for screening both the aberrant ABL1 activity in BCR-ABL1 like ALL leukemic cells and their potential response to TK inhibitors.

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.

Thiopurines are immunomodulators used in the treatment of acute lymphoblastic leukemia and inflammatory bowel diseases. Adverse reactions to these agents are one of the main causes of treatment discontinuation or interruption. Myelosuppression is the most frequent adverse effect; however, approximately 5%-20% of patients develop gastrointestinal toxicity. The identification of biomarkers able to prevent and/or monitor these adverse reactions would be useful for clinicians for the proactive management of long-term thiopurine therapy. In this editorial, we discuss evidence supporting the use of PACSIN2, RAC1, and ITPA genes, in addition to TPMT and NUDT15, as possible biomarkers for thiopurine-related gastrointestinal toxicity.

Although the use of glucocorticoids (GC) is well established, the therapeutic response to these agents often shows important interindividual differences, in particular among young patients with inflammatory bowel diseases (IBD). Currently, GC resistance or dependence cannot be predicted by clinical or laboratory findings. The aim of this study was to investigate the association of gender and age with GC efficacy and with the expression of Glucocorticoid-Induced Leucine Zipper (GILZ). One hundred thirty patients (mean age at enrolment 12.6 years, 53 Crohn’s disease, 70 males) were enrolled in this retrospective study. IBD patients with active disease despite prednisone at a daily dose of up to 2 mg/kg over a period of 4 weeks were defined as steroid resistant. Patients who initially responded but relapsed upon dose reduction were considered steroid-dependent. Total RNA was extracted from biopsies of 14 patients (9 males) and the levels of GILZ mRNA were evaluated by real-time PCR. Association between clinical response to prednisone and the considered demographic variables was evaluated using logistic regression models. After 4 weeks of treatment, 112 patients were responders to prednisone and 18 were resistant; at this time-point, resistant patients were older than responders (p=0.032). After 12 weeks, 42, 71 and 12 patients were sensitive, dependent and resistant respectively; at this time-point, females were more prone than males to develop prednisone dependence vs a good response (p=0.028) while age had no effect. Age was associated with response both at 4 and 12 weeks in the subgroups of females: resistant patients were older than sensitive ones at 4 weeks (p=0.02). Likewise, at 12 weeks of therapy, dependent patients resulted older than sensitive ones (p=0.05). No association of age with prednisone response was found in males. In a subgroup of 14 patients (5 females), GILZ mRNA expression in intestinal biopsies was higher in males (p=0.0031). Patients with unfavorable response (7) presented lower GILZ expression at disease onset in comparison to the responder group (p=0.017). Older females with IBD have a higher incidence of prednisone unfavorable response and reduced intestinal expression of the GC pharmacodynamic marker GILZ.

Acute lymphoblastic leukemia (ALL) is the most common pediatric hematological malignancy; notwithstanding the success of ALL therapy, severe adverse drugs effects represent a serious issue in pediatric oncology, because they could be both an additional life threatening condition for ALL patients per se and a reason to therapy delay or discontinuation with important fallouts on final outcome. Cancer treatment-related toxicities have generated a significant need of finding predictive pharmacogenomic markers for the a priori identification of at risk patients. In the era of precision medicine, high throughput genomic screening such as genome wide association studies (GWAS) might provide useful markers to tailor therapy intensity on patients' genetic profile. Furthermore, these findings could be useful in basic research for better understanding the mechanistic and regulatory pathways of the biological functions associated with ALL treatment toxicities. The purpose of this review is to give an overview of high throughput genomic screening of the last 10 years that had investigated the landscape of ALL treatment-associated toxicities. This article is categorized under: Cancer > Genetics/Genomics/Epigenetics.

Glucocorticoids (GCs) are commonly used as therapeutic agents for immune-mediated diseases and leukemia. However, considerable inter-individual differences in efficacy have been reported. Several reports indicate that the inhibitor of mTOR rapamycin can reverse GC resistance, but the molecular mechanism involved in this synergistic effect has not been fully defined. In this context, we explored the differential miRNA expression in a GC-resistant CCRF-CEM cell line after treatment with rapamycin alone or in co-treatment with methylprednisolone (MP). The expression analysis identified 70, 99 and 96 miRNAs that were differentially expressed after treatment with MP, rapamycin and their combination compared to non-treated controls, respectively. Two pathways were exclusively altered as a result of the co-treatment: the MAPK and ErbB pathways. We validated the only miRNA upregulated specifically by the co-treatment associated with the MAPK signaling, miR-331-3p. Looking for miR-331-3p targets, MAP2K7, an essential component of the JNK/MAPK pathway, was identified. Interestingly, MAP2K7 expression was downregulated during the co-treatment, causing a decrease in terms of JNK activity. miR-331-3p in mimic-transfected cells led to a significant decrease in MAP2K7 levels and promoted the reversion of GC resistance in vitro. Interestingly, miR-331-3p expression was also associated with GC-resistance in patient leukemia cells taken at diagnosis. The combination of rapamycin with MP restores GC effectiveness through the regulation of different miRNAs, suggesting the important role of these pharmacoepigenetic factors in GC response. Electronic supplementary material The online version of this article (10.1007/s00280-020-04122-z) contains supplementary material, which is available to authorized users.

Pharmacogenomics is one of the emerging approaches to precision medicine, tailoring drug selection and dosing to the patient’s genetic features. In recent years, several pharmacogenetic guidelines have been published by international scientific consortia, but the uptake in clinical practice is still poor. Many coordinated international efforts are ongoing in order to overcome the existing barriers to pharmacogenomic implementation. On the other hand, existing validated pharmacogenomic markers can explain only a minor part of the observed clinical variability in the therapeutic outcome. New investigational approaches are warranted, including the study of the pharmacogenomic role of the immune system genetics and of previously neglected rare genetic variants, reported to account for a large part of the inter-individual variability in drug metabolism. In this Special Issue, we collected a series of articles covering many aspects of pharmacogenomics. These include clinical implementation of pharmacogenomics in clinical practice, development of tools or infrastractures to support this process, research of new pharmacogenomics markers to increase drug efficacy and safety, and the impact of rare genetic variants in pharmacogenomics.

The marine polyether palytoxin (PLTX) is one of the most toxic natural compounds, and is involved in human poisonings after oral, inhalation, skin and/or ocular exposure. Epidemiological and molecular evidence suggest different inter-individual sensitivities to its toxic effects, possibly related to genetic-dependent differences in the expression of Na+/K+-ATPase, its molecular target. To identify Na+/K+-ATPase subunits, isoforms correlated with in vitro PLTX cytotoxic potency, sensitivity parameters (EC50: PLTX concentration reducing cell viability by 50%, Emax: maximum effect induced by the highest toxin concentration, 10&minus, 7 M) were assessed in 60 healthy donors&rsquo, monocytes by the MTT (methylthiazolyl tetrazolium) assay. Sensitivity parameters, not correlated with donors&rsquo, demographic variables (gender, age and blood group), demonstrated a high inter-individual variability (median EC50 = 2.7 &times, 10 M, interquartile range: 0.4&ndash, 13.2 &times, 10 M, median Emax = 92.0%, interquartile range: 87.5&ndash, 94.4%). Spearman&rsquo, s analysis showed significant positive correlations between the &beta, 2-encoding ATP1B2 gene expression and Emax values (rho = 0.30, p = 0.025) and between Emax and the ATP1B2/ATP1B3 expression ratio (rho = 0.38, p = 0.004), as well as a significant negative correlation between Emax and the ATP1B1/ATP1B2 expression ratio (rho = &minus, 0.30, p = 0.026). This toxicogenetic study represents the first approach to define genetic risk factors that may influence the onset of adverse effects in human PLTX poisonings, suggesting that individuals with high gene expression pattern of the Na+/K+-ATPase &beta, 2 subunit (alone or as &beta, 2/&beta, 1 and/or &beta, 3 ratio) could be highly sensitive to PLTX toxic effects.

Introduction: Natural and synthetic glucocorticoids are widely employed in different diseases, among which are hematological and solid tumors. Their use is however associated with a number of serious side effects and by the occurrence of resistance. With the aim of separating their gene transactivating effect, more linked to side effects, from transrepressive properties, associated with therapeutic efficacy, a number of selective glucocorticoid modulators have been identified. Areas covered: This review summarizes the patent applications from 2014 to present in the field of selective glucocorticoid receptor modulators employed in cancer therapy. Only few patents have been identified, that concern the identification of new molecules or the method of use of already patented compounds. In addition, a discussion of the mechanism of action of these compounds is included. Expert opinion: Only a very limited number of patents have been applied that concern selective glucocorticoid receptor modulators and their use in cancer. Biological information is scarce for most of these patents; more research is necessary in this field in particular concerning clinical data in order to understand whether it is actually possible to improve the efficacy and therapeutic index of these compounds in cancer therapy.

Extracellular nicotinamide phosphoribosyltransferase (eNAMPT) is increased in inflammatory bowel disease (IBD) patients, and its serum levels correlate with a worse prognosis. In the present manuscript, we show that eNAMPT serum levels are increased in IBD patients that fail to respond to anti-TNFα therapy (infliximab or adalimumab) and that its levels drop in patients that are responsive to these therapies, with values comparable with healthy subjects. Furthermore, eNAMPT administration in dinitrobenzene sulfonic acid (DNBS)-treated mice exacerbates the symptoms of colitis, suggesting a causative role of this protein in IBD. To determine the druggability of this cytokine, we developed a novel monoclonal antibody (C269) that neutralizes in vitro the cytokine-like action of eNAMPT and that reduces its serum levels in rodents. Of note, this newly generated antibody is able to significantly reduce acute and chronic colitis in both DNBS- and dextran sulfate sodium (DSS)-induced colitis. Importantly, C269 ameliorates the symptoms by reducing pro-inflammatory cytokines. Specifically, in the lamina propria, a reduced number of inflammatory monocytes, neutrophils, Th1, and cytotoxic T lymphocytes are found upon C269 treatment. Our data demonstrate that eNAMPT participates in IBD and, more importantly, that eNAMPT-neutralizing antibodies are endowed with a therapeutic potential in IBD. KEY MESSAGES: What are the new findings? Higher serum eNAMPT levels in IBD patients might decrease response to anti-TNF therapy. The cytokine-like activity of eNAMPT may be neutralized with a monoclonal antibody. Neutralization of eNAMPT ameliorates acute and chronic experimental colitis. Neutralization of eNAMPT limits the expression of IBD inflammatory signature. Neutralization of eNAMPT impairs immune cell infiltration in lamina propria.

Background Intestinal inflammation and epithelial injury are the leading actors of inflammatory bowel disease (IBD), causing an excessive pro-inflammatory cytokines expression. Tristetraprolin (TTP), an mRNA binding protein, plays a role in regulating the inflammatory factors, recognizing specific sequences on the 3' untranslated region of cytokine mRNAs. TTP activity depends on its phosphorylation state: the unphosphorylated TTP degrades pro-inflammatory cytokine mRNAs; on the contrary, the phosphorylated TTP fails to destabilize mRNAs furthering their expression. The phospho-TTP forms a complex with the chaperone protein 14-3-3. This binding could be one of the factors that promote intestinal inflammation as a cause of disease progression. Aim To assess if TTP phosphorylation has a role in paediatric IBD. Methods The study was carried out on a cohort of paediatric IBD patients. For each patient enrolled, a specimen of inflamed and non-inflamed colonic mucosa was collected. Furthermore, the experiments were conducted on macrophages differentiated from blood samples of the same patients. Macrophages from healthy donors' blood were used as controls. Co-immunoprecipitation assay and immunoblotting analyses were performed to observe the formation of the phospho-TTP/14-3-3 complex. In the same samples TNF-α expression was also evaluated as major factor of the pro-inflammatory activity. Results In this work we studied indirectly the phosphorylation of TTP through the binding with the chaperone protein 14-3-3. In inflamed and non-inflamed colon mucosa of IBD paediatric patients immunoblot assay demonstrated a higher expression of the TTP in inflamed samples respect to the non-inflamed; the co-immunoprecipitated 14-3-3 protein showed the same trend of expression. In the TNF-α gene expression analysis higher levels of the cytokine in inflamed tissues compared to controls were evident. The same experiments were conducted on macrophages from IBD paediatric patients and healthy controls. The immunoblot results demonstrated a high expression of both TTP and co-immunoprecipitated 14-4-3 protein in IBD-derived macrophages in comparison to healthy donors. TNF-α protein levels from macrophages lysates showed the same trend of expression in favour of IBD paediatric patients compared to healthy controls. Conclusion In this work, for the first time, we describe a relation between phospho-TTP/14-3-3 complex and IBD. Indeed, a higher expression of TTP/14-3-3 was recorded in IBD samples in comparison to controls.

Introduction: Medical treatment of pediatric inflammatory bowel diseases (IBD) has been greatly changed by the introduction of a number of biologic agents that are able to target various players of the immune response. In particular, monoclonal antibodies against the pro-inflammatory cytokine TNF-alpha (TNF) such as infliximab, adalimumab, and golimumab are now in the clinics both in induction and maintenance therapy, and several efforts are currently ongoing to optimize the use of these drugs in children. Areas covered: This review focuses on therapeutic drug monitoring (TDM) of anti-TNF levels and antidrug antibodies (ADAs), in IBD children. A revision of the analytical assays used for assessing anti-TNF plasma levels is also provided. Expert opinion: Although there is a consensus across studies that higher anti-TNF trough levels are associated with a better clinical outcome, and that early anti-TNF serum measurements could be predictive of long-term response, it is still not clear what the best predictive time of sampling is and what the ideal target drug plasma concentration to achieve. Indeed, there are a number of published studies, particularly in pediatric cohorts, limited by the population size analyzed and more prospective large studies are needed to examine the value of these predictive markers.

Although the introduction of antibiotics in medicine has resulted in one of the most successful events and in a major breakthrough to reduce morbidity and mortality caused by infectious disease, response to these agents is not always predictable, leading to differences in their efficacy, and sometimes to the occurrence of adverse effects. Genetic variability, resulting in differences in the pharmacokinetics and pharmacodynamics of antibiotics, is often involved in the variable response, of particular importance are polymorphisms in genes encoding for drug metabolizing enzymes and membrane transporters. In addition, variations in the human leukocyte antigen (HLA) class I and class II genes have been associated with different immune mediated reactions induced by antibiotics. In recent years, the importance of pharmacogenetics in the personalization of therapies has been recognized in various clinical fields, although not clearly in the context of antibiotic therapy. In this review, we make an overview of antibiotic pharmacogenomics and of its potential role in optimizing drug therapy and reducing adverse reactions.

Background: The long non-coding RNA (lncRNA) growth arrest&ndash, specific transcript 5 (GAS5) seems to be involved in the regulation of mediators of tissue injury, in particular matrix metalloproteinases (MMPs), implicated in the pathogenesis of inflammatory bowel disease (IBD). We investigated the role of GAS5 in regulating MMP2 and MMP9 expression in pediatric patients with IBD and in vitro. Methods: In total, 25 IBD patients were enrolled: For each patient paired inflamed and non-inflamed biopsies were collected. RNA was extracted and GAS5, MMP2, and MMP9 were quantified by TaqMan assay. The expression of GAS5 and MMPs was also determined in the human monocytic THP1 cells differentiated into macrophages and stimulated with lipopolysaccharide (LPS). The function of GAS5 was assessed by overexpressing the lncRNA and evaluating the MMPs levels. Results: Real-time PCR results demonstrated a downregulation of GAS5 and an upregulation of both MMPs in inflamed tissues. In vitro data confirmed the trend observed in patients for the three genes: The stimulation with LPS promoted a downregulation of GAS5 while an increase of MMPs was observed. Overexpression experiments showed that higher levels of GAS5 lead to a decrease of both enzymes. Conclusion: These results provide new information about the role of GAS5 in IBD: The lncRNA could mediate tissue damage by modulating the expression of MMPs.

Adverse drug reactions (ADRs) are major clinical problems, particularly in special populations such as pediatric patients. Indeed, ADRs may be caused by a plethora of different drugs leading, in some cases, to hospitalization, disability or even death. In addition, pediatric patients may respond differently to drugs with respect to adults and may be prone to developing different kinds of ADRs, leading, in some cases, to more severe consequences. To improve the comprehension, and thus the prevention, of ADRs, the set-up of sensitive and personalized assays is urgently needed. Important progress is represented by the possibility of setting up groundbreaking patient-specific assays. This goal has been powerfully achieved using induced pluripotent stem cells (iPSCs). Due to their genetic and physiological species-specific differences and their ability to be differentiated ideally into all tissues of the human body, this model may be accurate in predicting drug toxicity, especially when this toxicity is related to individual genetic differences. This review is an up-to-date summary of the employment of iPSCs as a model to study ADRs, with particular attention to drugs used in the pediatric field. We especially focused on the intestinal, hepatic, pancreatic, renal, cardiac, and neuronal levels, also discussing progress in organoids creation. The latter are three-dimensional in vitro culture systems derived from pluripotent or adult stem cells simulating the architecture and functionality of native organs such as the intestine, liver, pancreas, kidney, heart, and brain. Based on the existing knowledge, these models are powerful and promising tools in multiple clinical applications including toxicity screening, disease modeling, personalized and regenerative medicine.

OBJECTIVES Therapeutic drug monitoring is becoming increasingly important in clinical decision-making in children with inflammatory bowel disease (IBD). However, enzyme-linked immunosorbent assay (ELISA) assays do not allow results to be provided in real-time. We sought to compare 2 point-of-care (POC) devices for quantification of serum infliximab concentration with 2 validated ELISA assays in children with IBD. METHODS We studied 32 serum samples from 19 children with IBD treated with infliximab. Serum samples were collected immediately before drug infusion (trough level). Infliximab was measured using 2 POC infliximab assays, Quantum Blue (POC IFX/QB) and Rida Quick (POC IFX/RQ), and 2 ELISA assays: Lisa-Tracker (used as primary reference), and Promonitor (used as second control). Intraclass correlation coefficient (ICC) was assessed for quantitative comparison. Qualitative analysis was also performed to evaluate whether POC assays would correctly classify infliximab serum according to a target window (between 3 and 7 μg/mL). RESULTS ICC was 0.82 and 0.87 for POC IFX/QB and POC IFX/RQ with the primary reference ELISA assay, respectively; ICC between the 2 ELISA assays was 0.87. Classification of results according to therapeutic intervals showed good agreement between pairs of assays, with kappa of 0.67 and 0.80 for POC IFX/QB and POC IFX/RQ, respectively, with reference ELISA, and 0.81 between the 2 ELISAs. Accuracy of POC assays was better for drug levels

The contribution of candidate genetic variants involved in azathioprine biotransformation on azathioprine efficacy and pharmacokinetics in 111 young patients with inflammatory bowel disease was evaluated. Azathioprine doses, metabolites thioguanine-nucleotides (TGN) and methylmercaptopurine-nucleotides (MMPN) and clinical effects were assessed after at least 3 months of therapy. Clinical efficacy was defined as disease activity score below 10. Candidate genetic variants (TPMT rs1142345, rs1800460, rs1800462, GSTA1 rs3957357, GSTM1, and GSTT1 deletion) were determined by polymerase chain reaction (PCR) assays and pyrosequencing. Statistical analysis was performed using linear mixed effects models for the association between the candidate variants and the pharmacological variables (azathioprine doses and metabolites). Azathioprine metabolites were measured in 257 samples (median 2 per patient, inter-quartile range IQR 1-3). Clinical efficacy at the first evaluation available resulted better in ulcerative colitis than in Crohn&rsquo, s disease patients (88.0% versus 52.5% responders, p = 0.0003, linear mixed effect model, LME). TGN concentration and the ratio TGN/dose at the first evaluation were significantly higher in responder. TPMT rs1142345 variant (4.8% of patients) was associated with increased TGN (LME p = 0.0042), TGN/dose ratio (LME p &lt, 0.0001), decreased azathioprine dose (LME p = 0.0087), and MMPN (LME p = 0.0011). GSTM1 deletion (58.1% of patients) was associated with a 18.5% decrease in TGN/dose ratio and 30% decrease in clinical efficacy. GSTA1 variant (12.8% of patients) showed a trend (p = 0.049, LME) for an association with decreased clinical efficacy, however, no significant effect on azathioprine pharmacokinetics could be detected. In conclusion, GSTs variants are associated with azathioprine efficacy and pharmacokinetics.