Azathioprine promotes intestinal epithelial cell differentiation into Paneth cells and alleviates ileal Crohn's disease severity.

Paneth cells (PCs), a subset of intestinal epithelial cells (IECs) found at the base of small intestinal crypts, play an essential role in maintaining intestinal homeostasis. Altered PCs function is associated with diverse intestinal pathologies, including ileal Crohn's disease (CD). CD patients with ileal involvement have been previously demonstrated to display impairment in PCs and decreased levels of anti-microbial peptides. Although the immunosuppressive drug Azathioprine (AZA) is widely used in CD therapy, the impact of AZA on IEC differentiation remains largely elusive. In the present study, we hypothesized that the orally administered drug AZA also exerts its effect through modulation of the intestinal epithelium and specifically via modulation of PC function. AZA-treated CD patients exhibited an ileal upregulation of AMPs on both mRNA and protein levels compared to non-AZA treated patients. Upon in vitro AZA stimulation, intestinal epithelial cell line MODE-K exhibited heightened expression levels of PC marker in concert with diminished cell proliferation but boosted mitochondrial OXPHOS activity. Moreover, differentiation of IECs, including PCs differentiation, was boosted in AZA-treated murine small intestinal organoids and was associated with decreased D-glucose consumption and decreased growth rates. Of note, AZA treatment strongly decreased Lgr5 mRNA expression as well as Ki67 positive cells. Further, AZA restored dysregulated PCs associated with mitochondrial dysfunction. AZA-dependent inhibition of IEC proliferation is accompanied by boosted mitochondria function and IEC differentiation into PC.

Complex assessment of immunosuppression effects in prevention and treatment of adhesive disease, an experiment.

The cause of all small bowel obstruction in 60-75% of cases is adhesive development. The first and main method for adhesion prevention is undoubtedly the surgical technique, but the prevention of adhesive development is still actual. We aimed to study macroscopic and microscopic peculiarities of the intestine, peritoneum, and scars of the anterolateral abdominal wall. Also, immunological blood changes were observed in rats with the experimental created adhesive disease on the background of azathioprine introduction. The experiment was conducted on 40 rats divided into 2 subgroups: 20 animals as an experimental group (EG1) and 20 as a control group (CG1). Animals from EG received azathioprine (Moshimerampreparaty named by N.A. Semashko, Russia) in a dosage of 1 mg/100g of weight once a day for the first 3 days (starting from the day of surgery). The control group did not receive any drugs. All 40 rats survived the postoperative period. Rats were removed from the experiment on the 7th day after the operation. There were significant statistical differences in most indicators between the experimental and control groups. Phagocytic index (PI) was reduced by 4.55 due to the natural reaction of the rat organism to the surgery. Indicators of EG were a slight decrease in leukocytes and lymphocytes by 0.3 and 0.9, respectively, a moderate decrease in T-lymphocytes by no more than 2.0, and a decrease in phagocytic activity by 5.8. Immunosuppression with azathioprine significantly reduced the frequency and severity of the adhesive process of the abdominal cavity. Used in the recommended dose does not significantly inhibit important indicators of immunity and does not affect wound healing processes.

The Thiopurine Tale: An Unexpected Journey.

Exactly 70 years ago [1951] mercaptopurine was discovered by Gertrude Elion as a novel treatment option for acute leukaemia. A total of three thiopurines (also thioguanine [1950] and azathioprine [1957]) were developed over time. These immunosuppressive drugs were also successfully introduced a few decades later to prevent rejection of transplanted organs and to treat several autoimmune diseases. For her discovery of thiopurines and other antimetabolite drugs, in 1988 Elion was rewarded, together with George Hitchings and James Black, with the Nobel Prize in Physiology or Medicine. Important steps have been made in recent years to unravel its metabolism, mode of action and pharmacogenetics. Today thiopurine [based] therapy remains an essential immunosuppressive approach in treating patients with inflammatory bowel disease.

Anti-angiogenic activity of azathioprine.

Azathioprine (AZA) is the main drug used in immunomodulatory therapy in post-transplant patients or with autoimmune diseases. However, no study has evaluated the AZA angiogenic response. Therefore, this study investigated the effects of AZA on the angiogenic process through macroscopic, histological, and immunohistochemical analyses in chick embryo chorioallantoic membrane (CAM). Our results showed potent anti-angiogenic activity of AZA at the higher concentrations tested in the CAM assay. The histological analysis of CAM confirmed this effect, since AZA induced a significant reduction in all parameters evaluated. In addition, immunohistochemical evaluation of CAM revealed that AZA decreased TGF-ß and VEGF levels, important cytokines involved in the angiogenic process. Therefore, the AZA anti-angiogenic effect identified in our study provides new information for the possible application of this drug in anticancer treatment.

Clinical experience with biologic treatment in resistant eosinophilic fasciitis: Case reports and review of the literature.

RATIONALE: Eosinophilic fasciitis (EF) is an uncommon connective tissue disorder characterized by limb and trunk erythema, with symmetrical thickening of the skin. Its pathogenesis is poorly understood. Treatment consists mainly of glucocorticoids. Yet, no randomized trials have evaluated therapies for this rare disease and the optimal treatment modality remains unclear. Although most patients show partial or complete response to glucocorticoids, many relapse upon drug tapering, while others either do not respond at all or fail to sustain prolonged remission. Second-line therapy for this rare disorder includes mainly methotrexate (MTX), azathioprine, cyclosporine and hydroxychloroquine. Recently, several attempts using rituximab and intravenous immunoglobulins (IVIG) have shown good clinical results. PATIENT CONCERNS: The three patients had good clinical response to glucocorticoid treatment, followed by disease flare when the drug dose was tapered. Adding methotrexate in all patients and azathioprine to patient 3 did not lead to remission. DIAGNOSES: EF was diagnosed in all patients based on clinical presentation accompanied by fascia biopsy that demonstrated eosinophilic fasciitis. INTERVENTIONS: The patients were successfully treated with rituximab or IVIG, achieving sustained remission. OUTCOMES: The three cases had good clinical response to glucocorticoid treatment, followed by disease flare when the drug dose was tapered. The patients were then successfully treated with rituximab or IVIG, achieving sustained remission. LESSONS: This review of three cases of EF supports the results of previous reports, suggesting addition of rituximab and IVIG is an effective treatment for patients with refractory disease.

Sustained clinical response to infliximab in refractory Cronkhite-Canada syndrome.

A 59-year-old man with refractory Cronkhite-Canada syndrome (CCS) had poor clinical response to high-dose intravenous steroids, azathioprine, total parenteral nutrition and best supportive care. He remained highly symptomatic with abdominal pain, diarrhoea, recurrent sepsis and profound weight loss. Infliximab induction was given as rescue therapy, with marked clinical improvement observed within 3 weeks. This allowed steroid taper. Within 12 months of infliximab therapy, he achieved complete clinical remission and returned to his baseline weight and a full oral diet. Sequential endoscopies observed significant regression of previous marked gastrointestinal polyposis, including histological remission on colonic biopsies at 3.5 and 5 years of treatment. He currently remains in remission following 6 years of combination therapy with 5 mg/kg 8 weekly infliximab and azathioprine, and there is ongoing discussion with regard to the benefits and risks of therapy de-escalation. This case demonstrates the effectiveness of infliximab in inducing and maintaining remission in refractory CCS.

Thiopurine Drugs in the Treatment of Ulcerative Colitis: Identification of a Novel Deleterious Mutation in TPMT.

Chronic inflammatory bowel disease (IBD) includes Crohn's disease and ulcerative colitis. Both are characterized by inflammation of part of the digestive tract lining. Azathioprine (AZA) is a well-known immunosuppressant that has been known for many years for its ability to provide long-term disease remission in IBDs, but has important side effects, most of which are related to a single nucleotide polymorphism in the gene for thiopurine methyltransferase (TPMT), which ensures the degradation and efficacy of AZA. Since a direct correlation between TPMT gene polymorphisms and the haematological toxicity of the AZA treatment has been widely demonstrated, TPMT genotyping has been made necessary prior to any introduction of AZA. The monitoring of thiopurine metabolites presents one of the factors that limit wide adaptation of these thiopurines in clinical practice. Thus, identifying patients with asymmetric metabolism could help clinicians provide an ideal treatment recommendation to improve response and reduce adverse effects. Here, we review the role of AZA in the treatment of IBD and discuss the usefulness of TPMT genotyping to guide clinical decision-making. In addition, we report the identification of a new molecular alteration, never described, TPMT mutation affecting the TPMT activity and responsible for deleterious side effects in a clinical case of a 20-year-old woman patient.

Mycophenolic acid and 6-mercaptopurine both inhibit B-cell proliferation in granulomatosis with polyangiitis patients, whereas only mycophenolic acid inhibits B-cell IL-6 production.

Granulomatosis with polyangiitis (GPA) is an autoimmune disease affecting mainly small blood vessels. B-cells are important in the GPA pathogenesis as precursors of autoantibody-producing cells but likely also contribute (auto)antibody-independently. This has been underlined by the effectiveness of B-cell-depletion (with Rituximab) in inducing and maintaining disease remission. Mycophenolate-mofetil (MMF) and azathioprine (AZA) are immunosuppressive therapies frequently used in GPA-patients. Interestingly, MMF-treated GPA-patients are more prone to relapses than AZA-treated patients, while little is known about the influence of these drugs on B-cells. We investigated whether MMF or AZA treatment (or their active compounds) alters the circulating B-cell subset distribution and has differential effects on in vitro B-cell proliferation and cytokine production in GPA-patients that might underlie the different relapse rate. Circulating B-cell subset frequencies were determined in samples from AZA-treated (n = 13), MMF-treated (n = 12), untreated GPA-patients (n = 19) and matched HCs (n = 41). To determine the ex vivo effects of the active compounds of MMF and AZA, MPA and 6-MP respectively, on B-cell proliferation and cytokine production, PBMCs of untreated GPA-patients (n = 29) and matched HCs (n = 30) were cultured for 3-days in the presence of CpG-oligodeoxynucleotides (CpG) with MPA or 6-MP. After restimulation (with phorbol myristate acetate, calcium-ionophore), cytokine-positive B-cell frequencies were measured. Finally, to assess the effect of MMF or AZA treatment on in vitro B-cell proliferation and cytokine production, PBMCs of MMF-treated (n = 18), and AZA-treated patients (n = 28) and HCs (n = 41) were cultured with CpG. The memory B-cell frequency was increased in AZA- compared to MMF-treated patients, while no other subset was different. The active compounds of MMF and AZA showed in vitro that MPA decreased B-cell proliferation in GPA-patients and HCs. B-cell proliferation in MMF- and AZA-treated patients was not different. Finally, the IL-6+ B-cell frequency was decreased by MPA compared to 6-MP. No differences in IL-10+, IL-6+ or TNFα+ B-cell proportions or proliferation were found in MMF- and AZA-treated patients. Our results indicate that MMF could be superior to AZA in inhibiting B-cell cytokine production in GPA-patients. Future studies should assess the effects of these immunosuppressive drugs on other immune cells to elucidate mechanisms underlying the potential differences in relapse rates.

Long-term outcomes of patients with ulcerative proctitis: Analysis from a large referral centre cohort.

INTRODUCTION: Long-term outcomes of patients with ulcerative proctitis (UP) have been poorly investigated, since these patients are excluded from participation in randomized controlled clinical trials. OBJECTIVE: The aim of this study was to investigate the prognostic and therapeutic long-term outcomes of patients with UP. METHODS: A retrospective study of patients with UP followed at our referral centre between 1 January 1998 and 1 January 2019 was performed. Treatment success was defined as clinical response (significant improvement in UP-related symptoms) and endoscopic response (mayo endoscopic sub-score of 0 or 1) if available at last follow-up. RESULTS: From a total of 1561 patients with ulcerative colitis, 118 patients with UP were identified. A total of 36 (31%) patients were refractory to rectal and oral therapy with 5-ASA and corticosteroids, necessitating azathioprine as monotherapy in 19 (16%) patients and/or biological therapies in 33 (28%) patients. After a median follow-up of 71 months (interquartile range 29-149 months), treatment success was observed in 103/118 (87%) UP patients and in 25/36 (69%) patients with refractory UP. Clinical response rates were significantly higher for refractory UP patients treated with biologicals (23/33; 70%) compared to ones treated with azathioprine (2/19; 11%; p = 0.001). CONCLUSION: Good clinical outcomes were recorded in UP, with treatment success in 87% of patients. Nevertheless, 28% needed escalation to biologicals. Long-term outcome in patients on biologicals was superior to azathioprine.

Level of UV Exposure, Skin Type, and Age Are More Important than Thiopurine Use for Keratinocyte Carcinoma Development in IBD Patients.

BACKGROUND: Retrospective studies observe an increased risk of keratinocyte carcinomas (KCs) in patients with inflammatory bowel disease (IBD) on thiopurine (TP) medication. The role of traditional risk factors such as skin type and sun protection behavior has not been studied in this population. This study aimed to examine traditional KC risk factors and thiopurine use on skin cancer development in an IBD cohort. METHODS: Consecutive IBD patients were recruited from four specialist centers in Australia and New Zealand, each with varying UV exposure indices. Data pertaining to race, skin color, freckling and sun protection behavior, dose of TP therapy, and skin cancer development were elicited through a self-reported questionnaire. RESULTS: A total of 691 IBD patients were included with 62 reporting KC development. Thiopurine usage was similar among patients who developed skin cancer compared with those who did not (92% vs. 89%, p = 0.3). There was no statistically significant association between KC development and TP dose or 6-thioguanine nucleotide levels. In multivariate modeling, four factors were independently and significantly associated with KC: age over 61 years old versus less than 30 years old (OR 6.76; 95% CI 2.38-19.18), residing in Brisbane versus Christchurch (OR 3.3; 95% CI 1.6-6.8), never staying in the shade versus staying in the shade ≥ 50% of the time (OR 3.8; 95% CI 1.4-10.5), and having a skin type that never tanned versus other skin types (OR 6.9; 95% CI 2.9-16.0). CONCLUSION: Skin type, age, and sun protection behavior are more important risk factors for KC development than thiopurine medication use in this IBD population.

Pure Red Cell Aplasia Caused by Azathioprine.

BACKGROUND: Pure Red Cell Aplasia (PRCA) is a clinical entity comprising severe normochromic normocytic anemia, reticulocytopenia, erythroblastopenia in the bone marrow, with normal leukocyte and platelets count. PRCA can be classified into congenital and acquired, with the latter characterized as idiopathic or secondary to various infections, hematological malignancies, collagen vascular diseases, thymoma, and exposure to a variety of drugs and other chemical substances. METHODS: Herein, we present a female patient, who presented with PRCA due to azathioprine treatment. RESULTS: Prompt discontinuation of the drug together with red blood cells transfusions led to complete recovery in this young patient, without any addition of immunosuppressive regimen. CONCLUSION: We followed 'the wait and see practice' instead of administering immunosuppression to our patient, after careful consideration and extensive consultation with our hematologists. This 'wait and see practice' proved to be effective in the long run.

An Open-label Study With Pirfenidone on Chronic Hypersensitivity Pneumonitis.

BACKGROUND: Chronic hypersensitivity pneumonitis (cHP) represents a severe lung disease often evolving to fibrosis with the subsequent destruction of the lung parenchyma. There are no approved therapies with confirmed efficacy to deal with this disease. METHODS: We performed an open-label, proof of concept study, to evaluate the efficacy and safety of pirfenidone added to immunosuppressive drugs on the treatment of cHP. We included 22 patients assigned to two groups: Group 1, nine patients that received prednisone plus azathioprine and Group 2, thirteen patients, received prednisone plus azathioprine and pirfenidone (ClinicalTrials.gov identifier NCT02496182). There were no significant imbalances in clinically relevant baseline characteristics between two study groups. RESULTS: After 1 year of treatment, inclusion of pirfenidone was not associated with improved forced vital capacity (primary end-point). A not significant tendency to show higher improvement of diffusion capacity of the lung for carbon monoxide (DLCO) was observed in the group receiving pirfenidone (p=0.06). Likewise, a significant improvement in the total score on the SGRQ was found in the group 2 (p=0.02) without differences in other two questionnaires related to quality of life (ATAQ-IPF and EQ-5D-3L). HRCT showed a decrease of the ground glass attenuation without changes in the fibrotic lesions and without differences between both groups. CONCLUSIONS: These findings suggest that the addition of pirfenidone to the anti-inflammatory treatment in patients with chronic HP may improve the outcome with acceptable safety profile. However, prospective randomized double-blind, placebo-controlled trials in largest cohorts are needed to validate its efficacy.

Modeling the Outcome of Systematic TPMT Genotyping or Phenotyping Before Azathioprine Prescription: A Cost-Effectiveness Analysis.

BACKGROUND: Thiopurine S-methyltransferase (TPMT) testing, either by genotyping or phenotyping, can reduce the incidence of adverse severe myelotoxicity episodes induced by azathioprine. The comparative cost-effectiveness of TPMT genotyping and phenotyping are not known. OBJECTIVE: Our aim was to assess the cost-effectiveness of phenotyping-based dosing of TPMT activity, genotyping-based screening and no screening (reference) for patients treated with azathioprine. METHODS: A decision tree was built to compare the conventional weight-based dosing strategy with phenotyping and with genotyping using a micro-simulation model of patients with inflammatory bowel disease from the perspective of the French health care system. The time horizon was set up as 1 year. Only direct medical costs were used. Data used were obtained from previous reports, except for screening test and admission costs, which were from real cases. The main outcome was the cost-effectiveness ratios, with an effectiveness criterion of one averted severe myelotoxicity episode. RESULTS: The total expected cost of the no screening strategy was €409/patient, the total expected cost of the phenotyping strategy was €427/patient, and the total expected cost of the genotyping strategy was €476/patient. The incremental cost-effectiveness ratio was €2602/severe myelotoxicity averted in using the phenotyping strategy, and €11,244/severe myelotoxicity averted in the genotyping strategy compared to the no screening strategy. At prevalence rates of severe myelotoxicity > 1%, phenotyping dominated genotyping and conventional strategies. CONCLUSION: The phenotype-based strategy to screen for TPMT deficiency dominates (cheaper and more effective) the genotype-based screening strategy in France. Phenotype-based screening dominates no screening in populations with a prevalence of severe myelosuppression due to azathioprine of > 1%.

The immunosuppressant drug azathioprine restrains adipogenesis of muscle Fibro/Adipogenic Progenitors from dystrophic mice by affecting AKT signaling.

Fibro/Adipogenic Progenitors (FAPs) define a stem cell population playing a pro-regenerative role after muscle damage. When removed from their natural niche, FAPs readily differentiate into adipocytes or fibroblasts. This digressive differentiation potential, which is kept under tight control in the healthy muscle niche, contributes to fat and scar infiltrations in degenerative myopathies, such as in Duchenne Muscular Dystrophy (DMD). Controlling FAP differentiation by means of small molecules may contribute to delay the adverse consequences of the progressive pathological degeneration while offering, at the same time, a wider temporal window for gene therapy and cell-based strategies. In a high content phenotypic screening, we identified the immunosuppressant, azathioprine (AZA) as a negative modulator of FAP adipogenesis. We show here that AZA negatively affects the adipogenic propensity of FAPs purified from wild type and mdx mice by impairing the expression of the master adipogenic regulator, peroxisome proliferator-activated receptor γ (PPARγ). We show that this inhibition correlates with a decline in the activation of the AKT-mTOR axis, the main pathway that transduces the pro-adipogenic stimulus triggered by insulin. In addition, AZA exerts a cytostatic effect that has a negative impact on the mitotic clonal process that is required for the terminal differentiation of the preadipocyte-committed cells.

The Inflammatory Bowel Disease Drug Azathioprine Induces Autophagy via mTORC1 and the Unfolded Protein Response Sensor PERK.

BACKGROUND: Genetic studies have strongly linked autophagy to Crohn's disease (CD), and stimulating autophagy in CD patients may be therapeutically beneficial. The aim of this study was to evaluate the effect of current inflammatory bowel disease (IBD) drugs on autophagy and investigate molecular mechanisms of action and functional outcomes in relation to this cellular process. METHODS: Autophagy marker LC3 was evaluated by confocal fluorescence microscopy and flow cytometry. Drug mechanism of action was investigated by polymerase chain reaction (PCR) array with changes in signaling pathways examined by immunoblot and quantitative reverse transcription PCR (RT-qPCR). Clearance of adherent-invasive Escherichia coli (AIEC) and levels of pro-inflammatory cytokine tumor necrosis factor alpha (TNFα) were evaluated by gentamicin protection assays and RT-qPCR, respectively. The marker LC3 was analyzed in peripheral blood mononuclear cells (PBMCs) from pediatric patients by flow cytometry. RESULTS: Azathioprine induces autophagy via mechanisms involving modulation of mechanistic target of rapamycin (mTORC1) signaling and stimulation of the unfolded protein response (UPR) sensor PERK. Induction of autophagy with azathioprine correlated with the enhanced clearance of AIEC and dampened AIEC-induced increases in TNFα. Azathioprine induced significant increase in autophagosome bound LC3-II in PBMC populations ex vivo, supporting in vitro findings. In patients, the CD-associated ATG16L1 T300A single-nucleotide polymorphism did not attenuate azathioprine induction of autophagy. CONCLUSIONS: Modulation of autophagy via mTORC1 and the UPR may contribute to the therapeutic efficacy of azathioprine in IBD.

Mesalamine and azathioprine modulate junctional complexes and restore epithelial barrier function in intestinal inflammation.

Disruption of mucosal structure and barrier function contribute to the pathogenesis of inflammatory bowel disease (IBD). Efficacy of therapy in IBD is based on endoscopic mucosal healing, which occurs by a dynamic interplay of epithelial cell regeneration, migration and differentiation. Both mesalamine (5-ASA) and azathioprine (AZTP) promote this process through mechanisms not clearly understood. We examined molecular pathways implicated in epithelial barrier function that were altered by 5-ASA and AZTP. Paracellular permeability induced by inflammatory mediators was mitigated by both compounds through restoration of cellular anchoring complexes. 5-ASA and AZTP induced rearrangement and membranous localization of junctional proteins and modulated genes involved in tight junctions. Intestinal organoids from wildtype-mice treated with TNF-α and IL-10- deficient-mice displayed impaired epithelial barrier with loss of membranous E-cadherin and reduced Desmoglein-2 expression. These effects were counteracted by 5-ASA and AZTP. Unlike AZTP that exhibited antiproliferative effects, 5-ASA promoted wound healing in colon epithelial cells. Both affected cellular senescence, cell cycle distribution and restricted cells in G1 or S phase without inducing apoptosis. This study provides mechanistic evidence that molecular actions of 5-ASA and AZTP on intestinal epithelia are fundamental in the resolution of barrier dysfunction.

Effects of azathioprine and its metabolites on inflammatory cytokines in human nasal polyp organ cultures.

BACKGROUND: Oral steroids are recommended for the treatment of nasal polyps (NPs), but prolonged use is avoided because of side effects. Topical steroids can also control NPs without significant complications; however, the response to this is partially successful, and additional therapies are needed to treat glucocorticoid-resistant NPs. Azathioprine (AZA) and its first metabolite 6-mercaptopurine (6-MP) are important immunosuppressants used for the therapy of various diseases. The aim of this study was to investigate the effects of AZA and 6-MP on inflammatory cytokines in organ-cultured NPs. METHODS: NP explants were cultured using an air-liquid interface method. Cultures were maintained in the absence and presence of steroid, AZA, and 6-MP for 72 hours. Elaboration of cytokines tumor necrosis factor alpha (TNF-α), interleukin (IL)-2, IL-4, IL-5, and IL-13 into the supernatant was quantitated using the enzyme-linked immunosorbent assay (ELISA). The messenger RNA (mRNA) expression levels of TNF-α, IL-2, IL-4, IL-5, and IL-13 in cultured mucosa were measured using real-time polymerase chain reaction. Hematoxylin and eosin staining of cultured mucosa was performed to observe inflammatory cells. Immunohistochemistry was done to evaluate the distribution pattern of inflammatory cytokines in NP explants. RESULTS: On histologic examination, less inflammatory cell infiltration was found in NPs treated by steroid, AZA, and 6-MP than in control, but there was no statistical significance (p = 0.218). On immunohistochemistry, IL-13 showed a steady falling tendency in submucosal glands by steroid, AZA, and 6-MP. Expression of TNF-α, IL-2, IL-4, IL-5, and IL-13 mRNA in the NPs treated by steroid, AZA, and 6-MP were significantly lower than those of the control (p < 0.001 for all). By ELISA, IL-2 and IL-13 were significantly lower with topical steroid, AZA, and 6-MP treatment (p = 0.012 and p < 0.001). CONCLUSION: Topical AZT decreases inflammatory cytokines on human NP explants and this could have future therapeutic implications for NPs.