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La terapia farmacologica delle malattie infiammatorie croniche intestinali (MICI), ed in particolare l'uso di anticorpi monoclonali contro il fattore di necrosi tumorale alfa (TNFα), come ad esempio l’infliximab e l’adalimumab, ha completamente rivoluzionato il trattamento delle MICI, grazie alla loro capacità di indurre e mantenere la remissione clinica. Il trattamento con agenti anti-TNFα ha successo nella maggior parte dei pazienti affetti da MICI, ma una percentuale di pazienti va incontro ad una perdita di risposta durante l’induzione (10-20%) o nelle fasi più tardive del trattamento (fino al 45%) e allo sviluppo di reazioni avverse al farmaco (20%). Vi sono prove crescenti che suggeriscono che il fallimento del trattamento possa essere associato a livelli di farmaco nel sangue inadeguati, alla comparsa di anticorpi anti-farmaco e/o alla presenza di varianti genetiche. Lo studio dei meccanismi alla base di questa variabilità interindividuale diventa quindi un obiettivo importante nella pratica clinica, con lo scopo di migliorare gli outcomes clinici e ottenere una terapia personalizzata, ad hoc per il paziente. In questo contesto, questa tesi ha dimostrato: - l'utilità del point of care come alternativa ai saggi ELISA, per il monitoraggio terapeutico dell’infliximab nei pazienti pediatrici affetti da MICI; - incoraggianti risultati preliminari nell’utilizzo dell’AFM come metodica innovativa per il monitoraggio dell’infliximab nei sieri di pazienti pediatrici con MICI. È stata infatti dimostrata una buona correlazione tra la variazione di altezza e la concentrazione di infliximab permettendo così di sfruttare in futuro questa tecnica, grazie alla sua capacità di analisi multiplexing, per dosare contemporaneamente infliximab e anticorpi anti-farmaco; - una correlazione tra livelli più elevati di adalimumab durante l’induzione e la risposta clinica a lungo termine nei pazienti pediatrici affetti da MICI; - il ruolo del polimorfismo nel gene FCGR3A (rs396991) nei pazienti pediatrici con MICI: questo polimorfismo sembra influenzare la risposta all’infliximab e la suscettibilità alla produzione di anticorpi anti-farmaco. Questi dati supportano l'utilità della genotipizzazione di geni candidati per predire la risposta all’infliximab, riducendo i costi delle terapie ed aumentando l’efficacia del trattamento; - la validità del modello in vitro di linfociti T CD4+ (Jurkat) per lo studio del meccanismo d'azione dell’infliximab; - il ruolo del CD69 come marker di risposta all’infliximab. Pharmacological therapy of inflammatory bowel disease (IBD), and in particular the use of antibodies against tumor necrosis factor alpha (TNFα), such as infliximab and adalimumab, has led to a revolution in the treatment of IBD thanks to their capability to induce and maintain clinical remission. Treatment with anti-TNFα agents is successful in a majority of patients with IBD, yet it can fail in a proportion of patients leading to loss of response during the induction phase (10-20%) or over time (up to 45%) and to the development of adverse drug reactions (20%). There is increasing evidence suggesting that treatment failure may be associated with inadequate blood drug levels, the appearance of anti-drug antibodies and/or the presence of genetic variants. Studying the mechanism underlying this inter-individual variability becomes an important research goal to improve clinical practice. In this context, this thesis has demonstrated: - the utility of point of care as reliable option for real-time therapeutic drug monitoring of infliximab in children, showing a good agreement with traditional ELISA assays; - encouraging preliminary results of AFM methodology for infliximab monitoring in sera of pediatric IBD patients. A good correlation was found between signal height variation and infliximab concentration and this technique can be exploited in the future, thanks to its multiplexing capability, to dose infliximab and anti-drug antibodies at the same time; - the use of therapeutic drug monitoring to predict the efficacy of anti–TNFα agents in order to optimize treatment and minimize side effects. Early treatment modification can avoid complications: higher adalimumab levels during early treatment obtained from non–trough level serum samples predict long-term remission in children with IBD; - the role of FCGR3A SNP in pediatric IBD patients: this SNP seems to affect infliximab response and influence anti-drug antibodies production susceptibility; these data support the utility of genotyping candidate genes to predict infliximab response in children with IBD, resulting in more cost-effective and safe therapies; - the validity of Jurkat CD4+ T cells in vitro model for the study infliximab mechanism of action; - the role of CD69 as marker of infliximab response.

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.

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.

INTRODUCTION Inflammatory bowel disease is a chronic inflammation of the gut whose pathogenesis is still unclear. Although no curative therapy is currently available, a number of drugs are used in induction and maintenance therapy; however, for most of these drugs, a high inter-individual variability in response is observed. Among the factors of this variability, genetics plays an important role. Areas covered: This review summarizes the results of pharmacogenetic studies, considering the most important drugs used and in particular aminosalycilates, glucocorticoids, thiopurines, monoclonal antibodies and thalidomide. Most studies used a candidate gene approach, even if significant breakthroughs have been obtained recently from applying genome-wide studies. When available, also investigations considering epigenetics and pharmacogenetic dosing guidelines have been included. Expert opinion: Only for thiopurines, genetic markers identified as predictors of efficacy or adverse events have allowed the development of dosing guidelines. For the other drugs, encouraging results are available and great expectations rely on the study of epigenetics and integration with pharmacokinetic information, especially useful for biologics. However, to improve therapy of IBD patients with these drugs, for implementation in the clinics of pharmacogenetics, informatic clinical decision support systems and training about pharmacogenetics of health providers are needed.

Acute lymphoblastic leukemia (ALL) is the most common hematologic malignancy in children, characterized by an abnormal proliferation of immature lymphoid cells. Thanks to risk-adapted combination chemotherapy treatments currently used, survival at 5 years has reached 90%. ALL is a heterogeneous disease from a genetic point of view: patients’ lymphoblasts may harbor in fact several chromosomal alterations, some of which have prognostic and therapeutic value. Of particular importance is the translocation t(9;22)(q34;q11.2) that leads to the formation of the BCR-ABL1 fusion gene, encoding a constitutively active chimeric tyrosine kinase (TK): BCR-ABL1 that is present in ~3% of pediatric ALL patients with B-immunophenotype and is associated with a poor outcome. This type of ALL is potentially treatable with specific TK inhibitors, such as imatinib. Recent studies have demonstrated the existence of a subset of BCR-ABL1 like leukemias (~10-15% of Bimmunophenotype ALL), whose blast cells have a gene expression profile similar to that of BCR-ABL1 despite the absence of t(9;22)(q34;q11.2). The precise pathogenesis of BCR-ABL1 like ALL is still to be defined, but they are mainly characterized by the activation of constitutive signal transduction pathways due to chimeric TKs different from BCR-ABL1. BCR-ABL1 like ALL patients represent a group with unfavorable outcome and are not identified by current risk criteria. In this review, we will discuss the design of targeted therapy for patients with BCR-ABL1 like ALL, which could consider TK inhibitors, and discuss innovative approaches suitable to identify the presence of patient’s specific chimeric TK fusion genes, such as targeted locus amplification or proteomic biosensors.

International audience; Glucocorticoid receptor (GCR) transactivation reporter gene assays were used as an initial high-throughput screening on a diversified library of 1200 compounds for their evaluation as GCR antagonists. A class of imidazo[2,1-b]benzothiazole and imidazo[2,1-b]benzoimidazole derivatives were identified for their ability to modulate GCR transactivation and anti-inflammatory transrepression effects utilizing GCR and NF-κB specific reporter gene assays. Modeling studies on the crystallographic structure of the GCR ligand binding domain provided three new analogues bearing the tetrahydroimidazo[2,1-b]benzothiazole scaffold able to antagonize the GCR in the presence of dexamethasone (DEX) and also defined their putative binding into the GCR structure. Both mRNA level measures of GCR itself and its target gene GILZ, on cells treated with the new analogues, showed a GCR transactivation inhibition, thus suggesting a potential allosteric inhibition of the GCR.