Immunosuppressive Azathioprine Research Articles

MUTYH mediates the toxicity of combined DNA 6-thioguanine and UVA radiation.

The therapeutic thiopurines, including the immunosuppressant azathioprine (Aza) cause the accumulation of the UVA photosensitizer 6-thioguanine (6-TG) in the DNA of the patients' cells. DNA 6-TG and UVA are synergistically cytotoxic and their interaction causes oxidative damage. The MUTYH DNA glycosylase participates in the base excision repair of oxidized DNA bases. Using Mutyh-nullmouse fibroblasts (MEFs) we examined whether MUTYH provides protection against the lethal effects of combined DNA 6-TG/UVA. Surprisingly, Mutyh-null MEFs were more resistant than wild-type MEFs, despite accumulating higher levels of DNA 8-oxo-7,8-dihydroguanine (8-oxoG).Their enhanced 6-TG/UVA resistance reflected the absence of the MUTYH protein and MEFs expressing enzymatically-dead human variants were as sensitive as wild-type cells. Consistent with their enhanced resistance, Mutyh-null cells sustained fewer DNA strand breaks and lower levels of chromosomal damage after 6-TG/UVA. Although 6-TG/UVA treatment caused early checkpoint activation irrespective of the MUTYH status, Mutyh-null cells failed to arrest in S-phase at late time points. MUTYH-dependent toxicity was also apparent in vivo. Mutyh-/- mice survived better than wild-type during a 12-month chronicexposure to Aza/UVA treatments that significantly increased levels of skin DNA 8-oxoG. Two squamous cell skin carcinomas arose in Aza/UVA treated Mutyh-/- mice whereas similarly treated wild-type animals remained tumor-free.

DNA repair inhibition by UVA photoactivated fluoroquinolones and vemurafenib.

Cutaneous photosensitization is a common side effect of drug treatment and can be associated with an increased skin cancer risk. The immunosuppressant azathioprine, the fluoroquinolone antibiotics and vemurafenib—a BRAF inhibitor used to treat metastatic melanoma—are all recognized clinical photosensitizers. We have compared the effects of UVA radiation on cultured human cells treated with 6-thioguanine (6-TG, a DNA-embedded azathioprine surrogate), the fluoroquinolones ciprofloxacin and ofloxacin and vemurafenib. Despite widely different structures and modes of action, each of these drugs potentiated UVA cytotoxicity. UVA photoactivation of 6-TG, ciprofloxacin and ofloxacin was associated with the generation of singlet oxygen that caused extensive protein oxidation. In particular, these treatments were associated with damage to DNA repair proteins that reduced the efficiency of nucleotide excision repair. Although vemurafenib was also highly phototoxic to cultured cells, its effects were less dependent on singlet oxygen. Highly toxic combinations of vemurafenib and UVA caused little protein carbonylation but were nevertheless inhibitory to nucleotide excision repair. Thus, for three different classes of drugs, photosensitization by at least two distinct mechanisms is associated with reduced protection against potentially mutagenic and carcinogenic DNA damage.

Use of azathioprine for graft-vs-host disease is the major risk for development of secondary malignancies after haematopoietic stem cell transplantation: a nationwide population-based study.

Background:As more patients are treated by haematopoietic stem cell transplantation (HSCT), development of secondary malignancy (SM) becomes an increasingly common issue in long-term survivors.Methods:We conducted a nationwide population-based study of the Taiwanese population to analyse patients who received HSCT between January 1997 and December 2010. Standardised incidence ratios (SIRs) were used to compare the risk of SM in HSCT patients and the general population. Multivariate analysis was performed to identify independent predictors of SM.Results:Patients receiving HSCT had a significantly greater risk of developing SM (SIR 2.00; 95% confidence interval (CI) 1.45–2.69; P<0.001). Specifically, the incidence increased for cancers of the oral cavity (SIR 14.18) and oesophagus (SIR 14.75) after allogeneic HSCT. Multivariate analysis revealed an increased SIR for cancer in patients who received the immunosuppressant azathioprine. The risk of SM also increased with greater cumulative doses of azathioprine.Conclusions:This study demonstrates an increased incidence of SM in Taiwanese patients who received allogeneic HSCT, especially for cancers of the oral cavity and oesophagus. This finding is different from results in populations of Western countries. Physicians should be cautious about azathioprine use for graft-vs-host disease after HSCT.

Sebaceous Carcinoma in Patients Receiving Long-Term Immunosuppresive Treatment: Case Report and Literature Review

BackgroundSebaceous carcinoma (SC) is a very rare and aggressive malignant skin cancer that appears to occur with a greater frequency in the clinical setting of chronic immunosuppression; however, it is not reported in the literature as frequently as is squamous cell carcinoma (SCC). We report 2 cases of SC in organ transplant patients from clinical and histopathological points of view. MethodsA 48-year-old patient after 3 renal transplantations (1986, 1986, and 1998) was presented to the Dermatology Department in 1999 because of a papillomatous lesion along her right upper eyelid. The lesion was excised. Histopathologically, it was diagnosed as a SC. There was no lymphovascular invasion and no metastasis; therefore no other treatment was included. No symptoms of recurrent disease were present 14 years since diagnosis. An 87-year-old patient after a renal transplantation in 1989 was referred to dermatologist in 1993 because of the lesion on his right temple. The lesion was excised; histopathologically, it was diagnosed as SC. Because of metastatic disease, he had a course of radiotherapy to the right side of the neck. The immunosuppressive drugs azathioprine and cyclosporine A were reduced. The patient died of metastatic disease 1 year later (3 years since diagnosis). Both patients had very high cumulative UV exposition during their lifetimes, and many skin cancers were diagnosed, especially SCC. ResultsIt is necessary to realize that this cancer occurs more frequently in organ transplant patients, and its correct diagnosis is an essential issue because it has significantly more aggressive behavior than does SCC. In the 2 presented patients, we observed very rapid progression of disease. Despite aggressive treatment and reduction of immunosuppressive drugs, the second patient died 3 years after diagnosis. ConclusionsRegular dermatological follow-up is required in the population of organ transplant patients to identify all skin tumors in the early stage.

Treatment of autoimmune hemolytic anemias

Autoimmune hemolytic anemia (AIHA) is a relatively uncommon disorder caused by autoantibodies directed against self red blood cells. It can be idiopathic or secondary, and classified as warm, cold (cold hemagglutinin disease (CAD) and paroxysmal cold hemoglobinuria) or mixed, according to the thermal range of the autoantibody. AIHA may develop gradually, or have a fulminant onset with life-threatening anemia. The treatment of AIHA is still not evidence-based. The first-line therapy for warm AIHA are corticosteroids, which are effective in 70-85% of patients and should be slowly tapered over a time period of 6-12 months. For refractory/relapsed cases, the current sequence of second-line therapy is splenectomy (effective approx. in 2 out of 3 cases but with a presumed cure rate of up to 20%), rituximab (effective in approx. 80-90% of cases), and thereafter any of the immunosuppressive drugs (azathioprine, cyclophosphamide, cyclosporin, mycophenolate mofetil). Additional therapies are intravenous immunoglobulins, danazol, plasma-exchange, and alemtuzumab and high-dose cyclophosphamide as last resort option. As the experience with rituximab evolves, it is likely that this drug will be located at an earlier point in therapy of warm AIHA, before more toxic immunosuppressants, and in place of splenectomy in some cases. In CAD, rituximab is now recommended as first-line treatment.

English

Aim: The use of Azathioprine (AZA) in the prevention of organ rejection during transplantation has been documented for different organs of the body. In this study, we investigated the effects of azathioprine on the integrity of the testis of Wistar rats using histological and histochemical techniques. Methods: Eighteen adult male Wistar rats with mean weight 210&plusmn;2.65 g were randomly assigned into three groups. Group I (control) received 1 ml of normal saline &ldquo;per os&rdquo; (p.o), group II animals received 10 mg/kg AZA (p.o) while group III animals received 20 mg/kg AZA (p.o). Treatment lasted for 21 days. Twenty-four hours after treatment, animals were sacrificed; their testes excised, weighed and fixed in Bouin&rsquo;s fluid for histological evaluation using Haematoxylin and Eosin while histochemical studies were carried out using Gordon and Sweets, and Masson&rsquo;s Trichrome staining techniques. Testicular homogenate was used to assay for testosterone level. Results: Treatment with Azathioprine reduced testosterone level while the histological and histochemical findings of the testicular sections revealed cyto-architectural distortions and reduced staining intensity of collagen and reticulin connective tissue fibers in AZA treated animals compared to the control group. Conclusion: Results from the study reveal that the immunosuppressive drugazathioprine when used at a concentration above 10 mg/kg disrupts the testicular integrity of adult Wistar rats. Keywords: Azathioprine, Testis, Collagen, Reticulin

Risk of myeloid neoplasms after solid organ transplantation.

Solid organ transplant recipients have elevated cancer risks, due in part to pharmacologic immunosuppression. However, little is known about risks for hematologic malignancies of myeloid origin. We linked the US Scientific Registry of Transplant Recipients with 15 population-based cancer registries to ascertain cancer occurrence among 207,859 solid organ transplants (1987–2009). Solid organ transplant recipients had significantly elevated risk for myeloid neoplasms, with standardized incidence ratios (SIRs) of 4.6 (95% confidence interval 3.8–5.6; N=101) for myelodysplastic syndromes (MDS), 2.7 (2.2–3.2; N=125) for acute myeloid leukemia (AML), 2.3 (1.6–3.2; N=36) for chronic myeloid leukemia, and 7.2 (5.4–9.3; N=57) for polycythemia vera. SIRs were highest among younger individuals and varied by time since transplantation and organ type (Poisson regression P<0.05 for all comparisons). Azathioprine for initial maintenance immunosuppression increased risk for MDS (P=0.0002) and AML (2–5 years after transplantation, P=0.0163). Overall survival following AML/MDS among transplant recipients was inferior to that of similar patients reported to US cancer registries (log-rank P<0.0001). Our novel finding of increased risks for specific myeloid neoplasms after solid organ transplantation supports a role for immune dysfunction in myeloid neoplasm etiology. The increased risks and inferior survival should heighten clinician awareness of myeloid neoplasms during follow-up of transplant recipients.

Urinary cytopathologic findings in renal allograft recipients in Sudan: Trends, outcome and challenges

Urine cytology allows frequent monitoring of the intragraft events in renal transplantation and detection of infections associated with renal transplantation as a result of immunosuppression, specifically BK polyoma virus (Decoy cells). We conducted a cross-sectional study aimed to demonstrate the urinary cytomorphological changes among renal allograft recipients. We studied urine samples from 182 renal adult allograft recipients. All grafts were obtained from live donors. All the study population was under immunosuppressive drugs (Cyclosporine, Tacrolimus or azathioprine). Fourteen samples (7.6%) were collected during the first three months following transplantation. The slides of the centrifuged precipitates were stained with Papanicolaou and Diff Quick stains. The slides were screened by a cyto-screener and examined by a consultant cytopathologist for the presence of infectious agents and benign and malignant cellular changes. Of the 182 cases, 120 (65.9%) were negative for cytological changes, 45 (24.7%) showed bacterial infection, eight (4.4%) had BK polyoma virus infection (Decoy cells), four (2.2%) had human papilloma virus (HPV) infection (Koilocytosis) (Figure 1), three (1.6%) showed candida and two (1.1%) had cytomegalovirus infection. Renal transplant recipients are at a high risk for developing infections, including opportunistic infections; this is generally attributed to the immunosuppression status and epidemiologic exposure to infections. During the first posttransplantation year, 40–50% of recipients experience at least one infection. Smears consistent with bacterial infections were the most common finding in our study; they revealed polymorphs, macrophages, necrotic debris and background of bacteria. In our study, eight (4.4%) cases were diagnosed to have BK polyoma virus cytological changes (Decoy cells). Human polyoma viruses are members of the Papova virus family, which have a double strand DNA-genome. Recently, BK virus has been recognized as a cause of severe renal allograft dysfunction and potential graft loss, which are characterized by the presence of Decoy cells that were reported to present in 10–19.7% of urine samples from renal transplant (RT) patients.

Importance of Cutaneous Vasculitis in Rheumatology

Cutaneous vasculitis may present as a significant component of many systemic vasculitic syndromes such as rheumatoid vasculitis or anti-neutrophil cytoplasmic antibody (ANCA) associated primary vasculitic syndromes like microscopic polyangiits. Cutaneous vasculitis manifested most often as palpable purpura or infiltrated erythema indicating dermal small vessel vasculitis and less commonly as nodular erythema, livedo reticularis, deep ulcers or digital gangrene suggesting subcutaneous muscular vessel vasculitis.Coexistence of dermal small vessel vasculitis and subcutaneous muscular vessel vasculitis usually indicated systemic vasculitis such as connective tissue disease (CTD)-associated vasculitis, ANCA associated vasculitis. Behcet’s disease, polyarteiritsnodosa, Cogan’s syndrome, and malignancy associated. An appropriate lesional skin biopsy is the key to obtaining the diagnosis. Treatment for cutaneous vasculitis should include devoid of the triggers (excessive standing, infection, drugs) and exclusion of vasculitis-like syndromes (psudovasculitis). In most instances, cutaneous vasculitis represents a self-limited condition and will be relieved by leg elevation, avoidance of standing and therapy with non-steroidal antiinflammatory drugs.For mild recurrent or persistent disease, colchicine and daps one are first-choice agents. Severe cutaneous and systemic disease requires more potent immunosuppression (prednisone plus azathioprine, methotrexate, cyclophosphamide).The new biologic therapies that work via cytokine blockade or lymphocyte depletion such as tissue necrosis factor-inhibitor infliximab and the anti-B cell antibody rituximab, respectively, are showing benefit in certain settings such as CTD and ANCA associated vasculitis. Rheumatologists who are familiar with the complicated clinical and histopathological spectrum of cutaneous vasculitis and correlation of systemic vasculitis as well as the technique of skin biopsy will make the diagnosis earlier in vasculitis syndromes with skin involvement.

Immunosuppression induced acute pancreatitis in renal transplant recipient – Imaging and interventional management

Acute pancreatitis is a rare but often lethal complication in post-transplant patients. We describe a case of acute pancreatitis in a male patient 11 years after renal transplantation with azathioprine for immunosuppression as possible causative agent. Laboratory and imaging diagnosis of acute pancreatitis followed by successful endoscopic interventional management using transgastric catheter drainage is presented.

The TOSCA Study of Immune Surveillance in Cerebrospinal Fluid (CSF) in Crohnʼs Disease (CD) Patients Treated with Anti-MAdCAM Monoclonal Antibody (MAb) PF-00547659: Cohort 1 Clinical Results

Purpose: Studies with natalizumab (N) showed that inhibition of the α4β7 integrin-MAdCAM interaction resulted in relevant and durable improvement in Crohn's disease (CD) activity. However, N is a non-selective agent that also inhibits the α4β1-VCAM interaction in the central nervous system (CNS), which may be the plausible cause of progressive multifocal leukoencephalopathy (PML): a debilitating, often fatal viral infection that results from impaired CNS immune surveillance. PF-00547659 is a fully human monoclonal antibody that binds to gut-specific MAdCAM with no affinity for VCAM. As MAdCAM is not found in the CNS, this strategy should be gut-selective and eliminate the risk of PML. Methods: TOSCA is an open-label, clinical trial to study the effects of PF-00547659 (PF) on CNS immune surveillance. There are two sequential cohorts: Cohort 1, to establish the baseline cellular composition of CSF prior to treatment with PF, and Cohort 2, to investigate the effects of an induction course of high-dose PF on those cell groups. We present the clinical results of induction therapy with PF on CD activity in Cohort 1. Adults with active CD (hsCRP >5 mg/mL and HBI ≧8, or active lesions on endoscopy, or evidence of active CD on imaging), who had either failure or intolerance to both an immunosuppressive drug (azathioprine, 6-mercaptopurine or methotrexate) and an anti-TNF agent, were enrolled. Subjects underwent two lumbar punctures, and then began treatment with PF, 225 mg SQ every 4 weeks. The final visit was at week 12, when responders were permitted into an open-label extension study. Non-responders continued for 6 months' follow-up. Results: Ten subjects entered -- seven without stoma, so HBI was measured. Baseline characteristics: mean (SD) of age (y) 43.4 (16.9), median (min, max) of CD duration (y) 13.5 (3.5, 23.9), and mean (SD) of HBI 8.4 (2.8). At week 8, mean (SD) HBI had declined to 2.1 (2.0), and 6/7 (85.7%) subjects met criteria for clinical remission (HBI<5), while at week 12, mean (SD) of HBI was 2.0 (1.4) and 6/7(85.7%) subjects were in remission. One subject had treatment emergent serious adverse events of parastomal enterocutaneous abscess and fatigue. Both events resolved in 9 days. No pattern of AEs or laboratory abnormalities was seen. Conclusion: No definitive efficacy or safety conclusion can be drawn from this small, open-label study. However, we observed in CD subjects with significant symptoms and failure or intolerance to all other approved agents, clinically relevant improvement of disease activity without significant safety concerns. Disclosure - Fabio Cataldi Employee, of Pfizer inc. Kenneth Gorelick Employee of Pfizer inc Sunday Rivers Employee of Pfizer inc Mina Hassan-Zahraee Employee of Pfizer inc John Cheng Employee of Pfizer inc Bo Jin Employee of Pfizer inc Jenny Yanhua.Zhang Employee of Pfizer inc William Sandborn Consultant Pfizer Walter Reinisch Consultant Pfizer Geert D'Haens Consultant Pfizer Andre Van Gossum None Richard Ransohoff None Olaf Stuve None. This research was supported by an industry grant from Pfizer Inc.

Tuberculin skin testing in inflammatory bowel disease patients from an endemic area of Brazil

ObjectiveInflammatory bowel disease (IBD) is a chronic disorder involving the gastrointestinal tract. Immunosuppressive drugs are usually prescribed to treat IBD patients, and this treatment can lead to tuberculosis reactivation. This paper aimed to analyze tuberculin skin test (TST) results in IBD patients at a reference center in Brazil. MethodsWe evaluated TST results in IBD patients using a cross-sectional study. We also analyzed the medical records of patients treated at a reference IBD outpatient unit where TST is routinely performed. ResultsWe reviewed 119 medical records of 57 (47.9%) Crohn's disease (CD), 57 (47.9%) ulcerative colitis (UC) and 5 (4.2%) indeterminate colitis (IC) patients. The mean (SD) age was 43.5 (13.7) years old. TST was positive in 24 (20.2%) of the patients. TST was positive in 16/57 (28.1%) UC and 6/57 (10.5%) CD patients (prevalence ratio [PR] 2.7). Forty-one patients (34.5%) were taking immunosuppressive drugs (azathioprine or prednisone) at the time of the TST, and six of these patients (14.6%) had positive test results. Two patients using infliximab had negative TST results. Thirty-five of the 41 patients (85.4%) on immunosuppressive treatment were anergic compared with 73.1% (57/78) of the untreated patients (PR 1.2). ConclusionsPatients with IBD have TST results similar to the general Brazilian population. Within the IBD population, CD patients have a lower frequency of TST positivity than UC patients.

Immunosuppressive Drug Azathioprine Reduces Aneurysm Progression Through Inhibition of Rac1 and c-Jun-Terminal-N-Kinase in Endothelial Cells

In aortic aneurysms the arterial vessel wall is dilated because of destruction of its integrity, which may lead to lethal vessel rupture. Chronic infiltration of inflammatory cells into the vessel wall is fundamental to aneurysm pathology. We aim to limit aneurysm growth by inhibition of inflammation and reducing endothelial cell (EC) activation with immunosuppressive drug azathioprine (Aza). Aza and its metabolite 6-mercaptopurine have anti-inflammatory effects on leukocytes. We here demonstrate that treatment of ECs with 6-mercaptopurine inhibits cell activation as illustrated by reduced expression of interleukin-12, CCL5, CCL2, and vascular cell adhesion molecule-1 and inhibition of monocyte-EC adhesion. The underlying mechanism of 6-mercaptopurine involves suppression of GTPase Rac1 activation, resulting in reduced phosphorylation of c-Jun-terminal-N-kinase and c-Jun. Subsequently, the effect of Aza was investigated in aneurysm formation in the angiotensin II aneurysm mouse model in apolipoprotein E-deficient mice. We demonstrated that Aza decreases de novo aortic aneurysm formation from an average aneurysm severity score of 2.1 (control group) to 0.6 (Aza group), and that Aza effectively delays aorta pathology in a progression experiment, resulting in a reduced severity score from 2.8 to 1.7 in Aza-treated mice. In line with the in vitro observations, Aza-treated mice showed less c-Jun-terminal-N-kinase activation in ECs and reduced leukocyte influx in the aortic wall. The immunosuppressive drug Aza has an anti-inflammatory effect and in ECs inhibits Rac1 and c-Jun-terminal-N-kinase activation, which may explain the protective effect of Aza in aneurysm development and, most importantly for clinical implications, aneurysm severity.

AB0623 Efficacy of tacrolimus combination therapy during the maintenance phase of systemic lupus erythematosus

BackgroundCorticosteroid therapy is effective for systemic lupus erythematosus (SLE), but many adverse effects have been observed. Tacrolimus (TAC) is an immunosuppressive agent that is used after organ transplantation. TAC is...

Tautomerism and spectroscopic properties of the immunosuppressant azathioprine

The molecular structure and the relative stabilities of the four possible tautomers of the immunosuppressant azathioprine (AZA) are calculated by DFT/B3LYP method using different basis sets. The results of the energy analysis and thermodynamic treatment of the obtained data are used to predict the relative stabilities of the AZA tautomers. The effect of solvents such as DMSO and water on the stability of the AZA tautomers was studied using the polarized continuum method (PCM) at the same level of theory. The calculation predicted that, the total energies of all tautomers are decreased indicating that all tautomers are more or less stabilized by the solvent effect. The vibrational spectra of AZA are calculated using the same level of theory and the results are compared with the experimentally measured FTIR spectra. Good correlation is obtained between the experimental and calculated vibrational frequencies (R2=0.997). The electronic spectra of AZA in gas phase and in methanol as solvent are calculated using the TD-DFT method. The calculations predicted bathochromic shift in all the spectral bands in presence of solvent compared to the gas phase. Also the NMR spectra of all tautomers are calculated and the results are correlated with the experimental NMR chemical shifts where the most stable tautomer gives the best correlation coefficient (R2=0.996).

Anti-TNF- therapy for renal amyloid as a complication of Crohn's disease

### Learning Point for Clinicians Renal amyloid is a rare complication of Crohn’s disease (CD). Increased mortality is associated with increased serum amyloid A (SAA) protein concentration and reducing SAA improves survival. Anti-TNF-α may treat both renal amyloid and CD. NSAID use may precipitate a CD flare. Collagenous colitis is associated with CD and amyloidosis. A 35-year-old butcher was admitted from the renal outpatient clinic for investigation and management of worsening peripheral oedema. He gave a past history of Crohn’s disease (CD). This had been asymptomatic for many months and so the patient had weaned himself of immunosuppression (azathioprine) 4 months earlier. On admission, serum albumin was <4 g/l (normal range 30–45) with proteinuria of ∼20 g/d (0–30 mg/d). Serum creatinine was normal—41 µmol/l (40–125); C-reactive protein (CRP) was 23 mg/dl (0–5). Following a diagnosis of severe nephrotic syndrome, a renal biopsy was performed. This demonstrated AA amyloid (Figure 1A). Upper and lower gastrointestinal endoscopies showed no active inflammation. The patient was optimized on standard therapy for his nephrotic syndrome—maximal doses of an angiotensin converting enzyme inhibitor and angiotensin receptor blocker alongside …

Tu1148 Mechanisms of Loss of Response to Adalimumab in Crohn's Disease

of this study was to assess the degree of correlation of serum ADA and ATA levels with indices of serologic and endoscopic mucosal inflammation in patients with inflammatory bowel disease (IBD). Methods: We designed a cross-sectional study including patients 18 years or older with CD or UC on treatment with ADA. Predictive variables included were demographics, disease phenotype, concurrent use of immunosuppressive drugs (mercaptopurine, azathioprine or methotrexate) and/or steroids, and random ATA/ADA levels (measured using a validated non-radiolabeled homogeneous mobility shift assay by Prometheus Laboratories [San Diego, CA]). The primary outcomes were the presence of macroscopic mucosal inflammation (MMI) during endoscopic exam and C-reactive protein (CRP) level. Results: 66 patients were included; 59 had CD. 62 (94%) and 18 (27%) of IBD patients had detectable ADA and ATA, respectively. A minimum ADA cutpoint of 5 μg/mL best predicted elevation of CRP levels (ROC: 0.71). The mean ADA level was significantly higher in patients with undetectable ATA that in those with ATA (12.5 vs 5.7 μg/mL respectively, p=0.001). Mean ADA levels were higher in patients with mucosal healing than in those patients with MMI (13.3 vs 8.5 μg/mL, respectively, p=0.02). The mean serum CRP level was significantly higher in those patients with detectable ATA than those lacking ATA (12 vs 2.1 mg/dL respectively, p=0.002). Detectable ATA was associated with ADA ,5 μg/ mL (OR 8.6 [95%CI:2.3-31.8;p,0.001], MMI (OR 3.8 [95%CI:1.1-13.2;p=0.03], need for steroids (OR 3.7 [95%CI:1.1-12.9;p=0.03], and previous infliximab use (OR 3.9 [95%CI:1.015.2;p=0.04]. When compared to those patients prescribed ADA monotherapy, patients combining ADA therapy with an immunomodulator had a higher mean level of ADA (9 vs 14 ug/mL, respectively, p=0.026). Conclusions: Among patients with IBD, serum ADA and ATA levels correlate well with both serologic and endoscopic inflammatory activity. An ADA level ≥5 μg/mL correlates well with lower CRP, and combination therapy with immunosuppressive drugs improves serologic levels of ADA. Further work establishing the role of measuring ADA and ATA levels in clinical care are warranted.

The immunosuppressive drug azathioprine inhibits biosynthesis of the bacterial signal molecule cyclic-di-GMP by interfering with intracellular nucleotide pool availability

In Gram-negative bacteria, production of the signal molecule c-di-GMP by diguanylate cyclases (DGCs) is a key trigger for biofilm formation, which, in turn, is often required for the development of chronic bacterial infections. Thus, DGCs represent interesting targets for new chemotherapeutic drugs with anti-biofilm activity. We searched for inhibitors of the WspR protein, a Pseudomonas aeruginosa DGC involved in biofilm formation and production of virulence factors, using a set of microbiological assays developed in an Escherichia coli strain expressing the wspR gene. We found that azathioprine, an immunosuppressive drug used in the treatment of Crohn's disease, was able to inhibit WspR-dependent c-di-GMP biosynthesis in bacterial cells. However, in vitro enzymatic assays ruled out direct inhibition of WspR DGC activity either by azathioprine or by its metabolic derivative 2-amino-6-mercapto-purine riboside. Azathioprine is an inhibitor of 5-aminoimidazole-4-carboxamide ribotide (AICAR) transformylase, an enzyme involved in purine biosynthesis, which suggests that inhibition of c-di-GMP biosynthesis by azathioprine may be due to perturbation of intracellular nucleotide pools. Consistent with this hypothesis, WspR activity is abolished in an E. coli purH mutant strain, unable to produce AICAR transformylase. Despite its effect on WspR, azathioprine failed to prevent biofilm formation by P. aeruginosa; however, it affected production of extracellular structures in E. coli clinical isolates, suggesting efficient inhibition of c-di-GMP biosynthesis in this bacterium. Our results indicate that azathioprine can prevent biofilm formation in E. coli through inhibition of c-di-GMP biosynthesis and suggest that such inhibition might contribute to its anti-inflammatory activity in Crohn's disease.

Case Study and Review of Autoimmune Hepatitis

Autoimmune hepatitis (AIH) is a rare autoimmune disorder causing chronic liver inflammation. The disorder, sometimes called lupoid hepatitis, is not well understood because its clinical presentation is highly variable. The liver becomes inflamed due to T-cell-mediated activation of B cells that produce autoantibodies directed against liver antigens. This article details the case of a 28-year-old female patient who was diagnosed with AIH type I almost a decade ago; her therapeutic regimen may serve as a useful model for the disorder. An extensive interview was conducted with the patient. Herein, a timeline of her diagnosis is discussed, and her laboratory results are presented. Currently, she is living a fairly healthy life despite her liver disease. Her most recent liver function test results were completely normal, which is unusual with AIH. The patient was prescribed the immunosuppressive drug azathioprine to treat her AIH shortly after diagnosis. She also began following a vegan diet with unrefined sugar along with regular sleep and exercise. Currently, the patient is taking no azathioprine due to consecutively normal liver function test results over the past few years. Her continued health improvement could be credited to her strict vegan diet.

Increased incidence of squamous cell carcinoma of the skin after long‐term treatment with azathioprine in patients with auto‐immune inflammatory rheumatic diseases

Auto-immune inflammatory rheumatic diseases (AIRD) are often successfully treated with the immunosuppressant azathioprine for years. Treatment with azathioprine has been proven to increase the risk of non-melanoma skin cancer (NMSC) in transplant patients and possibly in patients with inflammatory bowel disease as well. Little is known about the risk of NMSC in AIRD patients treated with azathioprine. The aim of this study is to determine the incidence of NMSC in patients with AIRD treated with azathioprine for at least 1 year, as compared with the general Dutch population. Data were extracted from a historical cohort of patients with AIRD in a tertiary care centre. We compared the incidence to an age-matched control population and analysed risk factors for NMSC with univariate logistic regression. Fifty-nine patients were analysed. No patients were diagnosed with basal cell carcinoma and four patients with a single squamous cell carcinoma (SCC). Patients with SCC had a higher cumulative dose of azathioprine (≥ 500 g: OR 30.0 [95% CI 2.6-345.1]) and longer treatment duration (≥ 11 years: OR 13.5 [95% CI 1.3-143.6]). The risk of SCC compared with the general Dutch population was increased (standardized incidence ratio of 16.0 [95% CI 0.3-31.7]). In this cohort of patients with AIRD treated with azathioprine for at least 1 year, the risk of SCC was increased, as compared with the general population. An individual cumulative dose of at least 500 g azathioprine and a treatment duration of at least 11 years were quantified as risk factors.