The Anti-Inflammatory Drug Leflunomide Is an Agonist of the Aryl Hydrocarbon Receptor

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates the toxicity and biological activity of dioxins and related chemicals. The AhR influences a variety of processes involved in cellular growth and differentiation, and recent studies have suggested that the AhR is a potential target for immune-mediated diseases.During a screen for molecules that activate the AhR, leflunomide, an immunomodulatory drug presently used in the clinic for the treatment of rheumatoid arthritis, was identified as an AhR agonist. We aimed to determine whether any biological activity of leflunomide could be attributed to a previously unappreciated interaction with the AhR. The currently established mechanism of action of leflunomide involves its metabolism to A771726, possibly by cytochrome P450 enzymes, followed by inhibition of de novo pyrimidine biosynthesis by A771726. Our results demonstrate that leflunomide, but not its metabolite A771726, caused nuclear translocation of AhR into the nucleus and increased expression of AhR-responsive reporter genes and endogenous AhR target genes in an AhR-dependent manner. In silico Molecular Docking studies employing AhR ligand binding domain revealed favorable binding energy for leflunomide, but not for A771726. Further, leflunomide, but not A771726, inhibited in vivo epimorphic regeneration in a zebrafish model of tissue regeneration in an AhR-dependent manner. However, suppression of lymphocyte proliferation by leflunomide or A771726 was not dependent on AhR.These data reveal that leflunomide, an anti-inflammatory drug, is an agonist of the AhR. Our findings link AhR activation by leflunomide to inhibition of fin regeneration in zebrafish. Identification of alternative AhR agonists is a critical step in evaluating the AhR as a therapeutic target for the treatment of immune disorders

Multiplexed CRISPR/Cas9 Targeting of Genes Implicated in Retinal Regeneration and Degeneration

Thousands of genes have been implicated in retinal regeneration, but only a few have been shown to impact the regenerative capacity of Müller glia—an adult retinal stem cell with untapped therapeutic potential. Similarly, among nearly 300 genetic loci associated with human retinal disease, the majority remain untested in animal models. To address the large-scale nature of these problems, we are applying CRISPR/Cas9-based genome modification strategies in zebrafish to target over 300 genes implicated in retinal regeneration or degeneration. Our intent is to enable large-scale reverse genetic screens by applying a multiplexed gene disruption strategy that markedly increases the efficiency of the screening process. To facilitate large-scale phenotyping, we incorporate an automated reporter quantification-based assay to identify cellular degeneration and regeneration-deficient phenotypes in transgenic fish. Multiplexed gene targeting strategies can address mismatches in scale between “big data” bioinformatics and wet lab experimental capacities, a critical shortfall limiting comprehensive functional analyses of factors implicated in ever-expanding multiomics datasets. This report details the progress we have made to date with a multiplexed CRISPR/Cas9-based gene targeting strategy and discusses how the methodologies applied can further our understanding of the genes that predispose to retinal degenerative disease and which control the regenerative capacity of retinal Müller glia cells

Chemical and biological approaches to identify vertebrate tissue regeneration pathways

Numerous human conditions would be improved if therapies to encourage tissue regeneration were available. The goal of regenerative medicine is to encourage the body's intrinsic ability to repair and restore tissues lost by disease, injury or aging. While certain vertebrates have the inherent capacity to regenerate, mammals do not. To study tissue regeneration we developed an early life stage zebrafish model. Through comparative global mRNA expression analysis in regenerating tissues isolated from adult caudal fins, hearts and larval fins, we discovered that raldh2 is induced across all regenerating platforms and its expression is critical during early stages of regeneration. Our studies determined the role of Wnt and Fgf in larval regenerating tissue, establishing the early life stage model as a powerful platform to study regeneration. Utilizing this model we developed a rapid in vivo larval regeneration assay to identify small molecule modulators of regeneration. Our initial screening of a 2000 member FDA approved drug library identified eight glucocorticoids (GCs) that inhibited regeneration. We chose beclomethasone dipropionate (BDP) as a representative glucocorticoid receptor (GR) ligand and performed mRNA expression analysis in BDP exposed fin regenerates to identify downstream effectors of GR that are required to block tissue regeneration. Bioinformatic analysis revealed that Cripto-1 mRNA expression increased significantly following BDP exposure. We hypothesized that misexpression of Cripto-1, an Activin inhibitor, was necessary for GR ligands to block tissue regeneration. Suppression of Cripto-1 by morpholino or retinoic acid exposure restored regeneration in the presence of BDP supporting our hypothesis. Our chemical biological screen also identified 21 glucocorticoids that activated GR but did not impact regeneration. We hypothesized that differences in ligand structure induced alternate GR conformational changes and these structural differences resulted in distinct regenerative activity. Docking studies identified that ligands with large substitutions at position17 induce an energetically stable active GR confirmation that correlates with the blocking of tissue regeneration. Our research identified novel GR ligands with cortisol backbones and bulky C17 substitutions that confirmed our hypothesis. Collectively, our results demonstrate the power of the larval zebrafish regeneration model to understand the pathways that permit tissue regeneration

Giant pulmonary hamartoma: A rare entity

Hamartoma is one of the common benign tumor of lung. These lesions represent an abnormal proliferation and mixing of the normal components of the lung tissue. Most hamartomas are asymptomatic. We report a rare case of giant pulmonary hamartoma (12 cm in diameter) occupied almost whole of left hemithorax. Computed tomography guided fine-needle aspiration cytology revealed a benign cartilaginous neoplasm, suggestive of chondroid hamartoma. Tru-cut biopsy of the tumor confirmed the diagnosis. Complete resection of the tumor was performed. Patient was on a regular follow-up postoperatively for 6 months without any clinical and radiological relapse

Dissecting aneurysm of arch and descending thoracic aorta presenting as a left sided hemorrhagic pleural effusion

The most common cause of massive hemorrhagic effusion is malignancy. Herein we present a case of dissecting aneurysm of descending thoracic aorta presenting initially with shortness of breath due to left sided massive pleural effusion. Effusion was hemorrhagic in nature with high hematocrit value. CT scan of thorax with CT angiogram was done and that revealed the diagnosis

Diagnostic Yield of Fiber Optic Bronchoscopy and CECT Thorax in Patients of Haemoptysis with Normal Chest X-Ray

Introduction: Contrast enhanced computed tomography (CECT) thorax & Fibre Optic Bronchoscopy (FOB) are main investigations in patients of hemoptysis with normal chest X-ray. But there are limited data on their diagnostic yield especially from South East Asia. Methods: An observational study on 60 patients with hemoptysis with normal chest X -ray was conducted from April 2012 to April 2014. CECT thorax and FOB were done in all of them. Results: Out of 60 patients 38 (63.3%) were male with even age distribution. Yield of CECT was 40% (24 patients) and main diagnosis were bronchiectesis (25%) and tuberculosis (11.6%). Diagnostic yield of FOB was 47% (28 patients) with predominant diagnosis of tuberculosis (18.33%), bronchitis (18.33%) and bronchogenic carcinoma (6.66%). Overall diagnostic yield was 76.6% (46 cases). Bronchogenic carcinoma cases were all male smokers and 3 of them were more than 40 years old. Conclusion: CECT thorax and FOB are complimentary to each other in evaluation of hemoptysis with normal chest x-ray. Bronchogenic carcinoma and tuberculosis cases require specific treatment and they were diagnosed in confirm way only by FOB. So, FOB have advantage over CT scan, especially among patients with increased risk of bronchogenic carcinoma (male-smokers with age > 40 years)

Glucocorticoid receptor-dependent induction of () inhibits zebrafish caudal fin regeneration.

We previously used a chemical genetics approach with the larval zebrafish to identify small molecule inhibitors of tissue regeneration. This led to the discovery that glucocorticoids (GC) block early stages of tissue regeneration by the inappropriate activation of the glucocorticoid receptor (GR). We performed a microarray analysis to identify the changes in gene expression associated with beclomethasone dipropionate (BDP) exposure during epimorphic fin regeneration. Oncofetal cripto-1 showed > eight-fold increased expression in BDP-treated regenerates. We hypothesized that the mis-expression of cripto-1 was essential for BDP to block regeneration. Expression of cripto-1 was not elevated in GR morphants in the presence of BDP indicating that cripto-1 induction was GR-dependent. Partial translational suppression of Cripto-1 in the presence of BDP restored tissue regeneration. Retinoic acid exposure prevented increased cripto-1 expression and permitted regeneration in the presence of BDP. We demonstrated that BDP exposure increased cripto-1 expression in mouse embryonic stem cells and that regulation of cripto-1 by GCs is conserved in mammals

Glucocorticoid receptor-dependent induction of () inhibits zebrafish caudal fin regeneration.

We previously used a chemical genetics approach with the larval zebrafish to identify small molecule inhibitors of tissue regeneration. This led to the discovery that glucocorticoids (GC) block early stages of tissue regeneration by the inappropriate activation of the glucocorticoid receptor (GR). We performed a microarray analysis to identify the changes in gene expression associated with beclomethasone dipropionate (BDP) exposure during epimorphic fin regeneration. Oncofetal cripto-1 showed > eight-fold increased expression in BDP-treated regenerates. We hypothesized that the mis-expression of cripto-1 was essential for BDP to block regeneration. Expression of cripto-1 was not elevated in GR morphants in the presence of BDP indicating that cripto-1 induction was GR-dependent. Partial translational suppression of Cripto-1 in the presence of BDP restored tissue regeneration. Retinoic acid exposure prevented increased cripto-1 expression and permitted regeneration in the presence of BDP. We demonstrated that BDP exposure increased cripto-1 expression in mouse embryonic stem cells and that regulation of cripto-1 by GCs is conserved in mammals