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

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

Acinetobacter baumannii Resistance to Sulbactam/Durlobactam: A Systematic Review

Infections caused by carbapenem-resistant Acinetobacter baumannii (CRAB) have limited therapeutic options. Sulbactam-durlobactam is a combination of two beta lactamase inhibitors with activity against CRAB under phase 3 clinical investigation. We performed a systematic review on in vitro studies reporting A. baumannii resistances against sulbactam/durlobactam. We considered "resistant" species to be those with MIC >= 8 mg/L. Ten studies were included in the review (9754 tested isolates). Overall, 2.3% of A. baumannii were resistant to sulbactam/durlobactam, and this percentage rose to 3.4% among CRAB subgroups and to 3.7% among colistin-resistant strains. Resistance was 100% among metallo beta-lactamase-producing strains. Overall, in 12.5% of cases, sulbactam/durlobactam resistance was associated with the production of NDM-1, in 31.7% of cases with the substitutions in the PBP3 determinants, and in the remaining cases the resistance mechanism was unknown. In conclusion, A. baumannii resistance towards sulbactam/durlobactam is limited, except for MBL-producing strains

A new proof of evidence of cysteamine quantification for therapeutic drug monitoring in patients with cystinosis

Background To date, measurement of intracellular cystine is used for the therapeutic monitoring of patients affected by cystinosis in treatment with cysteamine. Since this method is time and sample consuming, development of a faster method to quantify cysteamine would be extremely useful in order to help clinicians to adjust dosages of cysteamine and to define better the pharmacokinetic profile of this drug. The aim of the study was to develop a liquid chromatography tandem mass spectrometry method for the quantification of cysteamine in plasma samples and to test its applicability on plasma samples derived from patients with nephropathic infantile cystinosis in treatment with cysteamine. Results The percentage of accuracy of the developed method varied between 97.80 and 106.00% and CV% between 0.90 and 6.93%. There was no carry over. The calibration curves were built from 2.5 to 50 mu M. The limit of detection and the lower limit of quantification occurred at 0.25 and 1.25 mu M respectively. Cysteamine was stable up to 2 months at -20 degrees C. Concentrations of cysteamine and intracellular cystine of 4 patients were in line with data previously reported. Conclusion The proposed method showed an appropriate selectivity, specificity, linearity, sensibility, accuracy, precision and good applicability to samples

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

Role of Oxidative Stress Mediated by Glutathione-S-transferase in Thiopurines' Toxic Effects

Azathioprine (AZA), 6-mercaptopurine (6-MP), and 6-thioguanine (6-TG) are antimetabolite drugs, widely used as immunosuppressants and anticancer agents. Despite their proven efficacy, a high incidence of toxic effects in patients during standard-dose therapy is recorded. The aim of this study is to explain, from a mechanistic point of view, the clinical evidence showing a significant role of glutathione-S-transferase (GST)-M1 genotype on AZA toxicity in inflammatory bowel disease patients. To this aim, the human nontumor IHH and HCEC cell lines were chosen as predictive models of the hepatic and intestinal tissues, respectively. AZA, but not 6-MP and 6-TG, induced a concentration-dependent superoxide anion production that seemed dependent on GSH depletion. N-Acetylcysteine reduced the AZA antiproliferative effect in both cell lines, and GST-M1 overexpression increased both superoxide anion production and cytotoxicity, especially in transfected HCEC cells. In this study, an in vitro model to study thiopurines' metabolism has been set up and helped us to demonstrate, for the first time, a clear role of GST-M1 in modulating AZA cytotoxicity, with a close dependency on superoxide anion production. These results provide the molecular basis to shed light on the clinical evidence suggesting a role of GST-M1 genotype in influencing the toxic effects of AZA treatment

Long Noncoding RNA GAS5: A Novel Marker Involved in Glucocorticoid Response

Glucocorticoids (GCs) exert their effects through regulation of gene expression after activation in the cytoplasm of the glucocorticoid receptor (GR) encoded by NR3C1 gene. A negative feedback mechanism resulting in GR autoregulation has been demonstrated through the binding of the activated receptor to intragenic sequences called GRE-like elements, contained in GR gene. The long noncoding RNA growth arrest-specific transcript 5 (GAS5) interacts with the activated GR suppressing its transcriptional activity. The aim of this study was to evaluate the possible role of GAS5 and NR3C1 gene expression in the antiproliferative effect of methylprednisolone in peripheral blood mononuclear cells and to correlate the expression with individual sensitivity to GCs. Subjects being poor responders to GCs presented higher levels of GAS5 and NR3C1 in comparison with good responders. We suggest that abnormal levels of GAS5 may alter GC effectiveness, probably interfering with the mechanism of GR autoregulation

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

: The thiopurine drugs are immunomodulatory antimetabolites that are 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 over expressed, 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/1x106 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 patients' leukocytes

PACSIN2 modifies miRNAs in extracellular vesicles, modulating thiopurine response

Thiopurines, such as mercaptopurine, are antimetabolites, used in the treatment of acute lymphoblastic leukemia (ALL) and inflammatory bowel disease (IBD). PACSIN2 rs2413739 is associated with gastrointestinal toxicity in children with ALL and with drug-efficacy in IBD pediatric patients. PACSIN2 is involved in vesicular trafficking and may affect the release and content of extracellular vesicles (EVs), which mediate cell communication and whose cargo modifies phenotypes of target cells. This study evaluates mechanisms associating PACSIN2 polymorphism with interindividual variability in efficacy of thiopurines, by considering the role of PACSIN2 in sorting specific miRNA in EVs. Effects of stable PACSIN2 knock-down (KD) were evaluated in intestinal LS180 cells. MTT cytotoxicity assay was used to verify mercaptopurine-sensitivity. EVs, released by LS180 KD and MOCK control cells were isolated by ultracentrifuge and characterized by nanoparticle tracking analysis (NTA). EVs miRNA-sequencing was performed by Illumina Hi-seq 2000. EVs may alter drug cytotoxicity, therefore LS180 MOCK and KD cells were co-treated with mercaptopurine and EVs. Statistical analysis was performed using t-test and ANOVA. Mercaptopurine was more cytotoxicity in LS180 KD cells (IC50 MOCK 3.23; IC50 KD 2.18 μM). No differences were observed by NTA in release of EVs between MOCK and KD cells (t-test, p = 0.13). PACSIN2 KD altered intracellular and EVs expression of 6 and 24 miRNAs respectively. EVs released by reduced mercaptopurine cytotoxicity (about 10%) and Rac1 protein expression in KD cells (ANOVA, p < 0.001), probably because they transport different miRNAs. In conclusion, PACSIN2 KD increase mercaptopurine cytotoxicity, probably, by deregulation of miRNA expression in cells and EVs. These results will be further investigated to better explain the link between PACSIN2 and EVs, whose miRNAs could provide a new scenario in personalizing thiopurine treatment.Book of abstract: 4th Belgrade Bioinformatics Conference, June 19-23, 202

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 auto-phagy. 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 in-flammatory bowel disease pediatric patients at diagnosis. PAC-SIN2 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 cyto-toxicity 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