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 influximab 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

Transcriptomic profiling of 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, 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. Cereblon 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 mechanisms underpinning thalidomide action in pediatric IBD. Ten IBD pediatric patients clinically responsive to thalidomide were prospectively enrolled. RNA-sequencing and functional enrichment analysis was carried out on peripheral blood mononuclear cells obtained before and after treatment with thalidomide. RNA-sequencing analysis revealed 378 differentially expressed genes 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 induced by 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 altered candidate pathways observed in patients. These results represent a unique resource for enhanced understanding of thalidomide mechanism in patients with IBD, providing novel potential targets associated with drug response.Book of abstract: 4th Belgrade Bioinformatics Conference, June 19-23, 202

SVILUPPO DI UN BIOSENSORE E ANALISI GENOMICHE PER LO STUDIO DELLA RISPOSTA ALL'INFLIXIMAB IN PAZIENTI PEDIATRICI CON MALATTIE INFIAMMATORIE CRONICHE INTESTINALI

La terapia farmacologica delle malattie infiammatorie croniche intestinali (MICI), ed in particolare l'uso di anticorpi monoclonali contro il fattore di necrosi tumorale alfa (TNF\u3b1), come ad esempio l\u2019infliximab e l\u2019adalimumab, ha completamente rivoluzionato il trattamento delle MICI, grazie alla loro capacit\ue0 di indurre e mantenere la remissione clinica. Il trattamento con agenti anti-TNF\u3b1 ha successo nella maggior parte dei pazienti affetti da MICI, ma una percentuale di pazienti va incontro ad una perdita di risposta durante l\u2019induzione (10-20%) o nelle fasi pi\uf9 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 variabilit\ue0 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'utilit\ue0 del point of care come alternativa ai saggi ELISA, per il monitoraggio terapeutico dell\u2019infliximab nei pazienti pediatrici affetti da MICI; - incoraggianti risultati preliminari nell\u2019utilizzo dell\u2019AFM come metodica innovativa per il monitoraggio dell\u2019infliximab nei sieri di pazienti pediatrici con MICI. \uc8 stata infatti dimostrata una buona correlazione tra la variazione di altezza e la concentrazione di infliximab permettendo cos\uec di sfruttare in futuro questa tecnica, grazie alla sua capacit\ue0 di analisi multiplexing, per dosare contemporaneamente infliximab e anticorpi anti-farmaco; - una correlazione tra livelli pi\uf9 elevati di adalimumab durante l\u2019induzione 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\u2019infliximab e la suscettibilit\ue0 alla produzione di anticorpi anti-farmaco. Questi dati supportano l'utilit\ue0 della genotipizzazione di geni candidati per predire la risposta all\u2019infliximab, riducendo i costi delle terapie ed aumentando l\u2019efficacia del trattamento; - la validit\ue0 del modello in vitro di linfociti T CD4+ (Jurkat) per lo studio del meccanismo d'azione dell\u2019infliximab; - il ruolo del CD69 come marker di risposta all\u2019infliximab.Pharmacological therapy of inflammatory bowel disease (IBD), and in particular the use of antibodies against tumor necrosis factor alpha (TNF\u3b1), 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-TNF\u3b1 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\u2013TNF\u3b1 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\u2013trough 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

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

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

Iron Bioavailability in the Extracellular Environment Is More Relevant Than the Intracellular One in Viability and Gene Expression: A Lesson from Oligodendroglioma Cells

Oligodendroglioma (OG) is a brain tumor that contributes to <1% of brain tumor diagnoses in the pediatric population. Unfortunately, pediatric OG remains without definitive molecular characteristics to aid in diagnosis, and little is known about the tumor microenvironment. Tumor cells’ metabolism and proliferation rate are generally higher than those of healthy cells, so their iron demand is also significantly higher. This consideration underlines the great importance of iron for tumor development and progression. In this context, this study aims to evaluate the effect of iron in a cellular in vitro model of human oligodendroglioma brain tumor. Cell morphology, the effect of siderotic medium on cell growth, iron uptake, and the expression of iron-metabolism-related genes were evaluated via optic microscopy, ICP-MS, confocal microscopy, and real-time PCR, respectively. This study underlines the great importance of iron for tumor development and progression and also the possibility of reducing the available iron concentration to determine an antiproliferative effect on OG. Therefore, every attempt can be promising to defeat OG for which there are currently no long-term curative therapies

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

Abstract 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

A Validated HPLC&ndash;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&mdash;diode array detection (HPLC&ndash;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&ndash;12,000 nM and for MMPN of 3000&ndash;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 &minus;20 &deg;C. The proposed HPLC&ndash;DAD method showed an appropriate selectivity, specificity, linearity, accuracy, precision and good applicability to samples from patients with IBD and ALL undergoing thiopurine treatment

Targeting kinase-activating genetic lesions to improve therapy of pediatric acute lymphoblastic leukemia

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): t(9;22)(q34;q11.2) 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 B-immunophenotype 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 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.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): t(9;22)(q34;q11.2) 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 B-immunophenotype 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 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