Search

The aim of this study was to evaluate the in vitro steroid sensitivity as a predictor of clinical response to glucocorticoids in childhood idiopathic nephrotic syndrome (INS). Seventy-four patients (median age 4.33, interquartile range [IQR] 2.82-7.23; 63.5% male) were enrolled in a prospective multicenter study: in vitro steroid inhibition of patients' peripheral blood mononuclear cell proliferation was evaluated by [methyl-3H] thymidine incorporation assay at disease onset (T0) and after 4 weeks (T4) of treatment. Steroid dependence was associated with increased in vitro sensitivity at T4 assessed both as drug concentration inducing 50% of inhibition (IC50; odds ratio [OR] = 0.48, 95% confidence interval [CI] = 0.24-0.85; P = 0.0094) and maximum inhibition at the highest drug concentration (Imax; OR = 1.13, 95% CI = 1.02-1.31; P = 0.017). IC50 > 4.4 nM and Imax < 92% at T4 were good predictors for optimal clinical response. These results suggest that this test may be useful for predicting the response to glucocorticoid therapy in pediatric INS. [ABSTRACT FROM AUTHOR]

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.

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.

Background: The long non-coding RNA (lncRNA) growth arrest-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.

The aim of this research was the identification of novel pharmacogenomic biomarkers for better understanding the complex gene regulation mechanisms underpinning glucocorticoid (GC) action in paediatric inflammatory bowel disease (IBD). This goal was achieved by evaluating high-throughput microRNA (miRNA) profiles during GC treatment, integrated with the assessment of expression changes in GC receptor (GR) heterocomplex genes. Furthermore, we tested the hypothesis that differentially expressed miRNAs could be directly regulated by GCs through investigating the presence of GC responsive elements (GREs) in their gene promoters. Ten IBD paediatric patients responding to GCs were enrolled. Peripheral blood was obtained at diagnosis (T0) and after four weeks of steroid treatment (T4). MicroRNA profiles were analyzed using next generation sequencing, and selected significantly differentially expressed miRNAs were validated by quantitative reverse transcription-polymerase chain reaction. In detail, 18 miRNAs were differentially expressed from T0 to T4, 16 of which were upregulated and 2 of which were downregulated. Out of these, three miRNAs (miR-144, miR-142, and miR-96) could putatively recognize the 3&rsquo;UTR of the GR gene and three miRNAs (miR-363, miR-96, miR-142) contained GREs sequences, thereby potentially enabling direct regulation by the GR. In conclusion, we identified miRNAs differently expressed during GC treatment and miRNAs which could be directly regulated by GCs in blood cells of young IBD patients. These results could represent a first step towards their translation as pharmacogenomic biomarkers.

Glucocorticoids (GCs) are widely employed in inflammatory, autoimmune and neoplastic diseases, and, despite the introduction of novel therapies, remain the first-line treatment for inducing remission in inflammatory bowel disease (IBD). Given the high incidence of suboptimal response, associated with a significant number of side-effects, that are particularly severe in paediatric patients, the identification of subjects that are most likely to respond poorly to GCs is extremely important. Recent evidence suggests that the long non-coding RNA (lncRNA) GAS5 could be a potential marker of GC resistance. To address this issue, we evaluated the association between the lncRNA GAS5 and the efficacy of steroids, in terms of inhibition of proliferation, in two cell lines derived from colon and ovarian cancers, to confirm the sensitivity and specificity of these lncRNAs. These cells showed a different sensitivity to GCs and revealed differential expression of GAS5 after treatment. GAS5 was up-regulated in GC-resistant cells and accumulated more in the cytoplasm compared to the nucleus in response to the drug. The functions of GAS5 were assessed by silencing, and we found that GAS5 knock-down reduced the proliferation during GC treatment. Furthermore, for the first time, we measured GAS5 levels in 19 paediatric IBD patients at diagnosis and after the first cycle of GCs, and we demonstrated an up-regulation of the lncRNA in patients with unfavourable steroid response. Our preliminary results indicate that GAS5 could be considered a novel pharmacogenomic marker useful for the personalization of GC therapy., (© 2017 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).)

Background: Glucocorticoids (GCs) are largely used in different inflammatory, autoimmune and proliferative diseases. To date their mechanism of action is not completely clear and more studies are necessary, in particular to explain the great interindividual variability in clinical response. In this panorama the glucocorticoid receptor (GR) has an important role: in fact it regulates the pharmacological response thanks to the capability to interact with different molecules (DNA, RNA, ncRNA and proteins) that are known to influence its activity., Results: In this review our aim is to highlight the knowledge about the role of protein-protein, RNAprotein interactions and epigenetic modifications on the GR and the consequent response to GCs. The characteristics of these interactions with the GR and their effects on the pharmacological activity of GCs will be examined., Conclusion: This information could contribute to the prediction of individual sensitivity to steroids through the identification of new markers of GC resistance. In addition this knowledge may be used in developing new strategies for targeted therapy., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Chronic inflammation associated with autoimmune activation is characteristic of rheumatic diseases from childhood to adulthood. In recent decades, significant improvements in the treatment of these types of disease have been achieved using disease modifying anti-rheumatic drugs (DMARDs), such as methotrexate (MTX) and, more recently, using biologic inhibitors. The recent introduction of kinase inhibitors (for example, tofacitinib; Tofa) further increases the available ARDs. However, there are patients that do not respond to any treatment strategies, for whom combination therapies are proposed. The data regarding the combined action of different drugs is lacking and the knowledge of the mechanisms of ARDs and their actions upon pathogenic lymphocytes, which are hypothesized to sustain disease, is poor. An in vitro model of inflammation was developed in the current study, in which stimulated and unstimulated lymphocytes were cultured together, but tracked separately, to investigate the action of MTX and Tofa on the two populations. By analysing lymphocyte proliferation and activation, and cytokine secretion in the culture supernatants, it was established that, due to the presence of activated cells, unstimulated cells underwent a bystander activation that was modulated by the ARDs. Additionally, varying administration schedules were demonstrated to affect lymphocytes differently in vitro, either directly or via bystander activation. Furthermore, MTX and Tofa exerted different effects; while MTX showed an antiproliferative effect, Tofa marginally effected activation, although only a slight antiproliferative action, which could be potentiated by sequential treatment with MTX. Thus, it was hypothesized that these differences may be exploited in sequential therapeutic strategies, to maximize the anti‑rheumatic effect. These findings are notable and must be accounted for, as bystander‑activated cells in vivo could contribute to the spread of autoimmune activation and disease progression.

This study evaluates the association between the long noncoding RNA GAS5 levels and the anti-proliferative effect of the glucocorticoid (GC) methylprednisolone (MP) alone and in combination with rapamycin in peripheral blood mononuclear cells (PBMCs) obtained from healthy donors. The effect of MP, rapamycin, and MP plus rapamycin was determined in 17 healthy donors by labelling metabolically active cells with [methyl-3H] thymidine and the expression levels of GAS5 gene were evaluated by real-time RT-PCR TaqMan analysis. We confirmed a role for GAS5 in modulating GC response: poor responders presented higher levels of GAS5 in comparison with good responders. Interestingly, when PBMCs were treated with the combination of rapamycin plus MP, the high levels of GAS5 observed for each drug in the MP poor responders group decreased in comparison with rapamycin (P value = 0.0134) or MP alone (P value = 0.0193). GAS5 is involved in GC resistance and co-treatment of rapamycin with GCs restores GC effectiveness in poor responders through the downregulation of the long noncoding RNA. GAS5 could be considered a biomarker to personalize therapy and a novel therapeutic target useful for the development of new pharmacological approaches to restore GC sensitivity., (© 2016 John Wiley & Sons Australia, Ltd.)

Background: Thiopurine antimetabolites are important agents for the treatment of severe diseases, such as acute lymphoblastic leukemia and inflammatory bowel disease. Their pharmacological actions depend on biotransformation into active thioguanine-nucleotides; intracellular metabolism is mediated by enzymes of the salvage pathway of nucleotide synthesis and relies on polymorphic enzymes involved in thiopurines' catabolism such as thiopurine-S-methyl transferase. Given the enzymes involved in thiopurines' metabolism, it is reasonable to hypothesize that these drugs are able to induce significant oxidative stress conditions, possibly altering their pharmacological activity., Methods: A systemic search of peer-reviewed scientific literature in bibliographic databases has been carried out. Both clinical and preclinical studies as well as mechanistic studies have been included to shed light on the role of oxidative stress in thiopurines' pharmacological effects., Results: Sixty-nine papers were included in our review, allowing us to review the contribution of oxidative stress in the pharmacological action of thiopurines. Thiopurines are catabolized in the liver by xanthine oxidase, with potential production of reactive oxidative species and azathioprine is converted into mercaptopurine by a reaction with reduced glutathione, that, in some tissues, may be facilitated by glutathione- S-transferase (GST). A clear role of GSTM1 in modulating azathioprine cytotoxicity, with a close dependency on superoxide anion production, has been recently demonstrated. Interestingly, recent genome-wide association studies have shown that, for both azathioprine in inflammatory bowel disease and mercaptopurine in acute lymphoblastic leukemia, treatment effects on patients' white blood cells are related to variants of a gene, NUDT15, involved in biotransformation of oxidated nucleotides., Conclusions: Basing on previous evidences published in literature, oxidative stress may contribute to thiopurine effects in significant ways that, however, are still not completely elucidated.

Background: In a randomized controlled trial, thalidomide has shown to be effective in refractory Crohn's disease in children. This pilot study aimed at evaluating thalidomide in refractory pediatric ulcerative colitis (UC)., Methods: Double-blind, placebo-controlled randomized clinical trial on thalidomide 1.5 to 2.5 mg/kg/day in children with active UC despite multiple immunosuppressive treatments. In an open-label extension, nonresponders to placebo received thalidomide for an additional 8 weeks; all responders were followed up for a minimum of 52 weeks., Results: Twenty-six children with refractory UC were randomized to thalidomide or placebo. Clinical remission at week 8 was achieved by significantly more children treated with thalidomide {10/12 (83.3%) versus 2/11 (18.8%); risk ratio, 4.5 (95% confidence interval [CI], 1.2-16.4); P = 0.005; number needed to treat, 1.5}. Of the nonresponders to placebo who were switched to thalidomide, 8 of 11 (72.7%) subsequently reached remission at week 8 (risk ratio, 4.0 [95% CI, 1.1-14.7]; number needed to treat, 2.45; P = 0.01). Clinical remission in the thalidomide group was 135.0 weeks (95% CI, 32-238), compared with 8.0 weeks (95% CI, 2.4-13.6) in the placebo group (P < 0.0001). Cumulative incidence of severe adverse events was 3.1 per 1000 patient-weeks. Peripheral neuropathy and amenorrhea were the most frequent adverse events., Conclusions: In this pilot randomized controlled trial on cases of UC refractory to immunosuppressive therapy, thalidomide compared with placebo resulted in improved clinical remission at 8 weeks of treatment and in longer term maintenance of remission. These findings require replication in larger clinical studies evaluating both thalidomide efficacy and safety.

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.

Mesenchymal stem cells (MSC) are cells of stromal origin which exhibit unlimited self-renewal capacity and pluripotency in vitro. It has recently been observed that MSC may also exert a profound immunosuppressive and anti-inflammatory effect both in vitro and in vivo with consequent potential use in autoimmune disorders. We present the case of a patient suffering from childhood-onset, multidrug resistant and steroid-dependent Crohn's disease who underwent systemic infusions of MSC, which led to a temporary reduction in CCR4, CCR7 and CXCR4 expression by T-cells, and a temporary decrease in switched memory B-cells, In addition, following MSC infusion, lower doses of steroids were needed to inhibit proliferation of the patient's peripheral blood mononuclear cells. Despite these changes, no significant clinical benefit was observed, and the patient required rescue therapy with infliximab and subsequent autologous hematopoietic stem cell transplantation. The results of biological and in vitro observations after MSC use and the clinical effects of infusion are discussed, and a brief description is provided of previous data on MSC-based therapy in autoimmune disorders.

Aim: To evaluate variation of the concentration of thiopurine metabolites after 5-aminosalicylate (5-ASA) interruption and the role of genetic polymorphisms of N-acetyl transferase (NAT) 1 and 2., Methods: Concentrations of thioguanine nucleotides (TGN) and methymercaptopurine nucleotides (MMPN), metabolites of thiopurines, were measured by high performance liquid chromatography in 12 young patients (3 females and 9 males, median age 16 years) with inflammatory bowel disease (6 Crohn's disease and 6 ulcerative colitis) treated with thiopurines (7 mercaptopurine and 5 azathioprine) and 5-ASA. Blood samples were collected one month before and one month after the interruption of 5-ASA. DNA was extracted and genotyping of NAT1, NAT2, inosine triphosphate pyrophosphatase (ITPA) and thiopurine methyl transferase (TPMT) genes was performed using PCR assays., Results: Median TGN concentration before 5-ASA interruption was 270 pmol/8 x 10(8) erythrocytes (range: 145-750); after the interruption of the aminosalicylate, a 35% reduction in TGN mean concentrations (absolute mean reduction 109 pmol/8 × 10(8) erythrocytes) was observed (median 221 pmol/8 × 10(8) erythrocytes, range: 96-427, P value linear mixed effects model 0.0011). Demographic and clinical covariates were not related to thiopurine metabolites concentrations. All patients were wild-type for the most relevant ITPA and TPMT variants. For NAT1 genotyping, 7 subjects presented an allele combination corresponding to fast enzymatic activity and 5 to slow activity. NAT1 genotypes corresponding to fast enzymatic activity were associated with reduced TGN concentration (P value linear mixed effects model 0.033), putatively because of increased 5-ASA inactivation and consequent reduced inhibition of thiopurine metabolism. The effect of NAT1 status on TGN seems to be persistent even after one month since the interruption of the aminosalicylate. No effect of NAT1 genotypes was shown on MMPN concentrations. NAT2 genotyping revealed that 6 patients presented a genotype corresponding to fast enzymatic activity and 6 to slow activity; NAT2 genotypes were not related to thiopurine metabolites concentration in this study., Conclusion: NAT1 genotype affects TGN levels in patients treated with thiopurines and aminosalicylates and could therefore influence the toxicity and efficacy of these drugs; however the number of patients evaluated is limited and this has to be considered a pilot study.

Juvenile idiopathic arthritis (JIAs) is the most common chronic rheumatic disease of childhood and is an important cause of disability. The folic acid analog methotrexate is the first choice disease-modifying anti-rheumatic drug in this disease, however, 35-45% of patients fail to respond. Molecular elements, such as variants in genes of pharmacological relevance, influencing response to methotrexate in JIA, would be important to individualize treatment strategies. Several studies have evaluated the effects of candidate genetic variants in the complex pathway of genes involved in methotrexate pharmacodynamics and pharmacokinetics, however, results are still contrasting and no definitive genetic marker of methotrexate response useful for the clinician to tailor therapy of children with JIA has been identified. Recently, genome-wide approaches have been applied, identifying new potential biological processes involved in methotrexate response in JIA such as TGF-beta signaling and calcium channels. If these genomic results are properly validated and integrated with innovative analyses comprising deep sequencing, epigenetics, and pharmacokinetics, they will greatly contribute to personalize therapy with methotrexate in children with JIA.

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.

Idiopathic nephrotic syndrome represents the most common type of primary glomerular disease in children: glucocorticoids (GCs) are the first-line therapy, even if considerable interindividual differences in their efficacy and side effects have been reported. Immunosuppressive and anti-inflammatory effects of these drugs are mainly due to the GC-mediated transcription regulation of pro- and anti-inflammatory genes. This mechanism of action is the result of a complex multistep pathway that involves the glucocorticoid receptor and several other proteins, encoded by polymorphic genes. Aim of this review is to highlight the current knowledge on genetic variants that could affect GC response, particularly focusing on children with idiopathic nephrotic syndrome.

Azathioprine is a purine antimetabolite drug commonly used to treat inflammatory bowel disease (IBD). In vivo it is active after reaction with reduced glutathione (GSH) and conversion to mercaptopurine. Although this reaction may occur spontaneously, the presence of isoforms M and A of the enzyme glutathione-S-transferase (GST) may increase its speed. Indeed, in pediatric patients with IBD, deletion of GST-M1, which determines reduced enzymatic activity, was recently associated with reduced sensitivity to azathioprine and reduced production of azathioprine active metabolites. In addition to increase the activation of azathioprine to mercaptopurine, GSTs may contribute to azathioprine effects even by modulating GSH consumption, oxidative stress and apoptosis. 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.

Objective: Immunosuppressive drugs may induce an increase of the gingival connective tissue in the extracellular matrix. The aim of this study was to assess the effectiveness of nonsurgical periodontal treatment in reducing gingival overgrowth (GO) in transplant patients taking cyclosporin A (CsA) or tacrolimus (Tcr)., Method and Materials: An observational cohort study employing 68 transplant patients with diagnosis of GO, 51 taking CsA and 17 in therapy with Tcr, was performed at the Periodontal Unit of the School of Dental Sciences (University of Trieste, Italy). Percentages of plaque index (PI), bleeding on probing (BoP), sites with probing depth (PD) > 3 mm, and hypertrophy index (HI) were registered at baseline, 90 days, 180 days, and at 1 year after nonsurgical periodontal therapy. Furthermore, HI at baseline and after 1 year was investigated by multiple linear regression., Results: Both groups have significantly improved their clinical parameters: CsA group: PIbaseline = 41.67%; PIyear = 33%; BoPbaseline = 13.88%; BoPyear = 6.94%; PD > 3 mmbaseline = 18.6%; PD > 3 mmyear = 12.96%; HIbaseline = 22%; HIyear = 10%; Tcr group: PIbaseline = 40.73%; PIyear = 38.54%; BoPbaseline = 20.78%; BoPyear = 12.5%; PD > 3 mmbaseline = 21.53%; PD > 3 mmyear = 13.19%; HIbaseline = 12%; HIyear = 6.5%. Age showed a statistical negative correlation with HI at baseline (P < .05), while PD > 3 mm was positively correlated to the baseline HI (P < .001). Only HI at baseline showed a statistically significant negative relation with HI at 1 year (P < .001)., Conclusion: After nonsurgical periodontal therapy no patients needed additional periodontal surgery. Nonsurgical periodontal treatment itself represents an efficacious therapy in transplant patients treated with CsA and Tcr.

Tofacitinib (Tofa) is an inhibitor of Janus Kinase 3, developed for the treatment of autoimmune diseases and for the prevention of transplant rejection. Due to its selective action on proliferating cells, Tofa can offer a way to block T cell activation, without toxic effects on resting cells. However, few studies have investigated the effects of Tofa on lymphocyte activation in vitro. Our aim was to study the action of Tofa on different lymphocyte subsets after in vitro stimulation and to track the behaviour of treated cells after interruption of the treatment. Peripheral blood lymphocytes were stimulated in vitro with mitogen and treated with two concentrations of Tofa. After a first period in culture, cells were washed and further incubated for an additional time. Lymphocyte subsets, activation phenotype and proliferation were assessed at the different time frames. As expected, Tofa was able to reduce the activation and proliferation of lymphocytes in the first four days of treatment. In addition the drug led to a relative decrease of Natural Killer, B cells and CD8 T cells compared to CD4 T cells. However, treated cells were still viable after the first period in culture and begun to proliferate, strikingly, in a dose dependent manner when the drug was removed from the environment by replacing the culture medium. This novel data does not necessarily predict a similar behaviour in vivo, but can warn about the clinical use of this drug when a discontinuation of treatment with Tofa is considered for any reason.

Goals: To investigate, in young patients with inflammatory bowel disease (IBD) treated with azathioprine, the association between genetic polymorphisms of thiopurine-S-methyl-transferase (TPMT), inosine-triphosphate-pyrophosphatase (ITPA), and glutathione-S-transferases (GST), involved in azathioprine metabolism, the concentration of the main metabolites of azathioprine, thioguanine nucleotides (TGNs) and the methylated nucleotides (MMPN), and the dose of the medication., Background: Azathioprine is widely used in IBD as an immunosuppressive agent, particularly to maintain remission in patients with steroid refractory disease. Azathioprine is a prodrug and requires conversion to its active form mercaptopurine, which has no intrinsic activity, and is activated by the enzymes of the purine salvage pathway to TGNs. Polymorphisms in genes of enzymes involved in azathioprine metabolism influence the efficacy and toxicity of treatment., Study: Seventy-five young patients with IBD treated with azathioprine at least for 3 months were enrolled and genotyped for the selected genes; for these patients, TGN and MMPN metabolites were measured by high performance liquid chromatography in erythrocytes., Results: GST-M1 deletion was associated with lower TGN/dose ratio (P=0.0030), higher azathioprine dose requirement (P=0.022), and reduced response to therapy (P=0.0022). TPMT variant genotype was associated with lower MMPN concentration (P=0.0064) and increased TGN/dose ratio (P=0.0035). ITPA C94A polymorphism resulted in an increased MMPN concentration (P=0.037)., Conclusions: This study describes the effect of candidate genetic polymorphisms in TPMT, ITPA, and GST-M1 on azathioprine pharmacokinetics in IBD patients, showing, for the first time, relevant effects of GST-M1 genotype on azathioprine metabolites concentration.

In spite of the introduction in therapy of highly effective biological agents, glucocorticoids (GCs) are still employed to induce remission in moderate to severe inflammatory bowel diseases (IBD), but considerable inter-individual differences in their efficacy and side effects have been reported. The effectiveness of these drugs is indeed very variable and side effects, particularly severe in pediatric patients, are common and often unpredictable: the understanding of the complex gene regulation mediated by GCs could shed light on the causes of this variability. In this context, microRNAs (miRNAs) represent a new and promising field of research. miRNAs are small non-coding RNA molecules that suppress gene expression at post-transcriptional level, and are fine-tuning regulators of diverse biological processes, including the development and function of the immune system, apoptosis, metabolism and inflammation. Emerging data have implicated the deregulated expression of certain miRNA networks in the pathogenesis of autoimmune and inflammatory diseases, such as IBD. There is a great interest in the identification of the role of miRNAs in the modulation of pharmacological response; however, the association between miRNA and GC response in patients with IBD has not yet been evaluated in a prospective clinical study. The identification of miRNAs differently expressed as a consequence of GC treatment in comparison to diagnosis, represents an important innovative approach that could be translated into clinical practice. In this review we highlight the altered regulation of proteins involved in GC molecular mechanism by miRNAs, and their potential role as molecular markers useful for predicting in advance GC response.

Importance: Pediatric-onset Crohn disease is more aggressive than adult-onset disease, has high rates of resistance to existing drugs, and can lead to permanent impairments. Few trials have evaluated new drugs for refractory Crohn disease in children., Objective: To determine whether thalidomide is effective in inducing remission in refractory pediatric Crohn disease., Design, Setting, and Patients: Multicenter, double-blind, placebo-controlled, randomized clinical trial of 56 children with active Crohn disease despite immunosuppressive treatment, conducted August 2008-September 2012 in 6 pediatric tertiary care centers in Italy., Interventions: Thalidomide, 1.5 to 2.5 mg/kg per day, or placebo once daily for 8 weeks. In an open-label extension, nonresponders to placebo received thalidomide for an additional 8 weeks. All responders continued to receive thalidomide for an additional minimum 52 weeks., Main Outcomes and Measures: Primary outcomes were clinical remission at week 8, measured by Pediatric Crohn Disease Activity Index (PCDAI) score and reduction in PCDAI by ≥25% or ≥75% at weeks 4 and 8. Primary outcomes during the open-label follow-up were clinical remission and 75% response., Results: Twenty-eight children were randomized to thalidomide and 26 to placebo. Clinical remission was achieved by significantly more children treated with thalidomide (13/28 [46.4%] vs 3/26 [11.5%]; risk ratio [RR], 4.0 [95% CI, 1.2-12.5]; P = .01; number needed to treat [NNT], 2.86). Responses were not different at 4 weeks, but greater improvement was observed at 8 weeks in the thalidomide group (75% response, 13/28 [46.4%] vs 3/26 [11.5%]; RR, 4.0 [95% CI, 1.2-12.5]; NNT = 2.86; P = .01; and 25% response, 18/28 [64.2%] vs 8/26 [30.8%]; RR, 2.1 [95% CI, 1.1-3.9]; NNT = 2.99; P = .01). Of the nonresponders to placebo who began receiving thalidomide, 11 of 21 (52.4%) subsequently reached remission at week 8 (RR, 4.5 [95% CI, 1.4-14.1]; NNT = 2.45; P = .01). Overall, 31 of 49 children treated with thalidomide (63.3%) achieved clinical remission, and 32 of 49 (65.3%) achieved 75% response. Mean duration of clinical remission in the thalidomide group was 181.1 weeks (95% CI, 144.53-217.76) vs 6.3 weeks (95% CI, 3.51-9.15) in the placebo group (P < .001). Cumulative incidence of severe adverse events was 2.1 per 1000 patient-weeks, with peripheral neuropathy the most frequent severe adverse event., Conclusions and Relevance: In children and adolescents with refractory Crohn disease, thalidomide compared with placebo resulted in improved clinical remission at 8 weeks of treatment and longer-term maintenance of remission in an open-label follow-up. These findings require replication to definitively determine clinical utility of this treatment., Trial Registration: clinicaltrials.gov Identifier: NCT00720538.

Objectives: Etiological periodontal therapy is effective in reducing cyclosporin A-induced gingival overgrowth, but a high variability among subjects has been observed. This study aimed to evaluate the role of polymorphisms in PAI-1 and A BCB1 genes on the course of this side effect following periodontal therapy., Method and Materials: Forty-five transplant patients were subjected to nonsurgical periodontal therapy and evaluated for hypertrophy index, probing depths, bleeding, and plaque scores at baseline, and after 3 and 6 months. A BCB1 (C3435T and G2677T) and PAI-1 (4G/5G) polymorphisms were studied with polymerase chain reaction-restriction fragment length polymorphism and allele-specific polymerase chain reaction respectively., Results: All the monitored periodontal indexes decreased significantly during the six months. Modeling of hypertrophy index by linearmixed- effect models (allowing non-normal distribution of the outcome variable hypertrophy index) resulted in the selection as the most significant model, of the one comprising the independent variables: time, C 3435T genotype, and their interaction term. This model indicated that C 3435T-mutated patients had significantly higher baseline hypertrophy index values (90% Markov chain Monte C arlo empirical confidence intervals: 5.08, 30.00). The decrease in hypertrophy index values over time showed a trend toward being faster in mutated than nonmutated patients (interaction time: C 3435T nonmutated, 90% Markov chain Monte C arlo empirical confidence interval: -11.08, -0.40). When hypertrophy index values were normalized, the significance and trend were lost. No effect of the A BCB1 G2677T and PAI-1 4G/5G polymorphisms was observed., Conclusion: These preliminary results suggest that C 3435T polymorphism is a genetic factor that could influence the course of cyclosporin A-induced gingival overgrowth in transplant patients subjected to periodontal therapy.

What Is Already Known About This Subject: In vitro lymphocyte steroid sensitivity has been suggested as a useful tool to predict in vivo response to glucocorticoid treatment in different inflammatory chronic diseases. A correlation between genetic polymorphisms and clinical response to glucocorticoids has been demonstrated in these patients., What This Study Adds: The BclI polymorphism in the glucocorticoid receptor (NR3C1) gene is associated with higher methylprednisolone potency in vitro. The combined evaluation of the in vitro sensitivity to methylprednisolone and BclI polymorphism could represent an aid for physicians to adjust therapy a priori. AIM To evaluate the association between the in vitro sensitivity of peripheral blood mononuclear cells (PBMCs) to methylprednisolone (MP) and the presence of genetic polymorphisms involved in glucocorticoid (GC) response., Methods: In vitro MP inhibition of the proliferation of lymphocytes stimulated with concanavalin A was determined. Non linear regression of dose-response data was performed computing the MP concentration required to reduce proliferation to 50% (IC(50) ). The maximum inhibition achievable at the highest MP concentration (I(max) ) was also calculated. Moreover, the Taqman technique was used to analyze the BclI polymorphism in the NR3C1 gene and the Leu155His polymorphism in the NALP1 gene., Results: A significant association between the BclI mutated genotype and an increased in vitro sensitivity to GCs was observed., Conclusions: The a priori evaluation of the BclI polymorphism, associated with a lymphocyte proliferation assay, could represent a useful diagnostic tool for the optimization of steroid treatment., (© 2011 The Authors. British Journal of Clinical Pharmacology © 2011 The British Pharmacological Society.)

Pediatric inflammatory and autoimmune diseases are a wide array of systemic or organ-specific conditions, characterized by an exaggerated immune reactivity, which generally occurs in immunogenetically predisposed children. Among the most important ones, in terms of their diffusion and morbidity in the population worldwide, pediatric inflammatory bowel disease (IBD) and juvenile rheumatoid arthritis (JRA) have to be considered. The aim of personalized therapy is to give to each patient the most appropriate drug and dose regimen, in order to maximize treatment response and reduce the risk of adverse events. In general, several therapeutic options exist for pediatric inflammatory and autoimmune conditions, therefore the perspective of pharmacological tools that allow identification of patients with increased risk of treatment issues related to a particular medication, in terms of lack of efficacy or increased probability of adverse events, is particularly desirable and promising. The present review will be focused on the personalized therapy approaches already available or in development for pediatric patients with IBD or JRA, comprising pharmacokinetic, pharmacodynamic and pharmacogenetic assays.

Lymphocytes proliferation after antigen-driven activation leads to an increase in cell count, which could last some week, until apoptosis mechanisms allow the homeostatic control of the system. During the first days of this stimulation, activated lymphocytes display high resistance to apoptosis and to most immunosuppressive drugs. According to the literature, few compounds have been described to kill recently activated cells, by inhibiting metabolic processes fundamental to proliferation. The aim of our work was to evaluate comparatively these different compounds, in order to identify the best strategy to kill cells that have undergone proliferation, while sparing the repertoire of resting cells. After preliminary experiments, 3-HAA and bortezomib were selected as the most suitable compounds for our purposes. The possible synergic effect of 3-HAA with bortezomib or with manganese ions was also assessed. 3-HAA was confirmed to be the most reliable pharmacologic approach to inhibit proliferation with acceptable toxicity on resting cells. While in the case of PHA stimulation 3-HAA led to death of most lymphocytes, only a minor percentage of cells were killed after allo-stimulation, suggesting that the effect is proportional to the percentage of stimulated lymphocytes. Manganese ions further enhanced this effect, while results with bortezomib seemed to be less consistent. These results deserve further investigations to develop new procedures for targeting activated cells with pharmacological approaches., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Resistance to glucocorticoids often occurs in patients with severe inflammatory disorders. Occasionally, this resistance could be overcome by switching to a different glucocorticoid, but the mechanisms of this selectivity are not clear. We studied this condition in three patients with severe inflammatory disorders, who responded satisfactorily to betamethasone, but could not be switched to equipotent doses of methylprednisolone or prednisone. While betamethasone displayed similar activity on lymphocyte proliferation in cells obtained from the three patients and controls, higher concentrations of methylprednisolone were needed to inhibit proliferation in patients' cells. In a competition study, the concentration of methylprednisolone that inhibited 50% of specific [(3)H]dexamethasone binding was increased in patients' lymphocytes. Higher Rhodamine-123 efflux was demonstrated in CD4 T cells from two patients, suggesting that an increased activity of membrane transporters could be responsible for the selective response to different glucocorticoids, even if P-glycoprotein and MRP1 expression was not increased., (2009 Elsevier Inc. All rights reserved.)

Purpose: The aim of this pilot study was to assess the association between polymorphisms in genes that encode for proteins involved in the pharmacokinetics/pharmacodynamics of glucocorticoids and the occurrence of respiratory distress syndrome (RDS) in preterm infants born to mothers treated with a complete course of betamethasone., Methods: Sixty-two preterm infants were enrolled. The C1236T, G2677T, and C3435T polymorphisms in the ABCB1 gene, BclI, N363S and ER22/23EK in the NR3C1 gene, I105V in the GST-P1 gene and GST-M1 and GST-T1 deletions were analyzed, and their association with the occurrence of RDS was assessed., Results: In univariate analysis, the heterozygous and homozygous presence of the I105V variant in the GST-P1 gene seemed to confer protection against the occurrence of RDS (P = 0.032), while no association for all other polymorphisms was observed. In multivariate analysis, selection from the reference model of independent variables based on AIC (Akaike information criteria) maintained three variables in the model: gestation, C3435T, and GST-P1 genotype., Conclusions: Polymorphisms of the GST-P1 gene may influence the effect of antenatal steroids on the newborn lung.

A case of severe hypersensitivity syndrome, triggered by carbamazepine in the presence of a concomitant active human herpes virus (HHV) 6 and 7 infection is described. To further understand the molecular mechanism of this adverse reaction, analyses of the genetic variants of human leukocyte antigen (HLA) and of the epoxide hydrolase gene (EPHX1), previously associated with carbamazepine hypersensitivity, were performed. A lymphocyte transformation test (LTT) was conducted in order to detect drug-specific lymphocytes. In the hypersensitive patient, 2 genetic factors previously associated with intolerance to carbamazepine were detected: the allele HLA-A*3101 and homozygosity for the variant allele of SNP rs1051740 in EPHX1. Drug-specific lymphocytes could be detected by LTT when the HHV was active (positive PCR for viral DNA and increased anti-HHV 6 IgG titer), but not when it was no longer active. In conclusion, we document a case of severe carbamazepine hypersensitivity triggered by viral reactivation in a patient presenting the interaction of 2 unfavorable genetic factors., (Copyright 2009 S. Karger AG, Basel.)

Background: P-glycoprotein (Pgp), encoded by the ATP-binding cassette B1 (ABCB1) gene, is an efflux transporter located on the luminal side of intestinal epithelial cells, which protects the gut from endogenous and exogenous toxins. The association of two ABCB1 polymorphisms with the occurrence of colon cancer and long-term prognosis was evaluated in a selected patient population. The expression of Pgp in neoplastic and normal intestinal mucosa was also studied., Patients and Methods: Archival material from 51 patients, in Dukes stage B2 or C, treated for 6 months with 5-fluorouracil plus leucovorin was retrieved. The G2677T and C3435T polymorphisms were studied and immunohistochemical analysis of the tumor and adjacent normal tissue was performed., Results: The distribution of wild-type and polymorphic genotypes was similar in the patients and controls and in the patients who relapsed and those who remained event-free for 5 years. Cox proportional hazard model indicated an increased probability of relapse for older patients (p = 0.042) and C stage tumors (p = 0.030). Pgp expression was significantly lower in cancer tissue compared to normal mucosa (p < 0.001) and was related to grading, being lower in poorly-differentiated tumors (p < 0.05); however, no relationship was seen between Pgp expression, genotype and long-term prognosis., Conclusion: G2677T and C3435T polymorphisms are not associated with colon cancer risk and prognosis in a selected patient population.

ABSTRACT The transport of doxorubicin and rhodamine 123, substrates of ABC transporters, was evaluated in the BeWo stabilized trophoblast cell line. Both compounds were taken up by BeWo cells, but their intracellular concentrations were highly dependent on temperature, and significantly reduced at 4 degrees C. The P-glycoprotein inhibitors verapamil and PSC833 did not modify the intracellular concentrations of the two substrates, suggesting therefore that, in these cells, the activity of P-glycoprotein is not important. MK571, which inhibits MRPs, was on the contrary effective in increasing rhodamine 123 intracellular concentrations. The efflux of both fluorescent substrates was extremely slow, and slightly reduced by MK571. Finally, a polarized transport of doxorubicin from basal to apical side was evident, although only during the first 60 min of incubation, and was reduced by P-glycoprotein, MRP, and BCRP inhibitors. No MDR1 expression was revealed at the mRNA and protein levels; on the contrary, MRP1 and BCRP were expressed in these cells. In BeWo cells the activity of ABC transporters, and in particular of P-glycoprotein, seems to be extremely limited.