Rationale Idiopathic subglottic stenosis (iSGS) is a rare and devastating extrathoracic obstruction involving the lower laryngeal and upper tracheal airway. It arises without known antecedent injury or associated disease process. Persistent mucosal inflammation and a localized fibrotic response are hallmarks of the disease. Despite the initial clinical description of iSGS more than 40 year ago, there have been no substantive investigations into the pathogenesis of this enigmatic and progressive airway obstruction. Objectives In these studies, we present the initial characterization of the molecular pathogenesis underlying the fibrosing phenotype of iSGS. Methods Utilizing 20 human iSGS and healthy control specimens we applied histologic, immunohistochemical, molecular and immunologic techniques. Main Results We demonstrate significant activation of the canonical IL-23/IL-17A pathway in the tracheal mucosa of iSGS patients, as well as identify γδ T cells as the primary cellular source of IL-17A. Conclusions Our results suggest that aberrant mucosal immune activation is a component in of the pathogenesis of iSGS. Most critically, our work offers new targets for future therapeutic intervention. Level of Evidence NA

Rationale Idiopathic subglottic stenosis (iSGS) is an unexplained obstruction involving the lower laryngeal and upper tracheal airway. Persistent mucosal inflammation is a hallmark of the disease. Epithelial microbiota dysbiosis is found in other chronic inflammatory mucosal diseases; however, the relationship between tracheal microbiota composition and iSGS is unknown. Objectives Given the critical role for host defense at mucosal barriers, we analyzed tissue specimens from iSGS patients for the presence of microbial pathogens. Methods Utilizing 20 human iSGS, 20 intubation-related tracheal stenosis (iLTS) and 10 healthy control specimens we applied molecular, immunohistochemical, electron microscopic, immunologic and Sanger™ sequencing techniques. Main Results With unbiased culture-independent nucleic acid, protein, and immunologic approaches, we demonstrate that Mycobacterium species are uniquely associated with iSGS. Phylogenetic analysis of the mycobacterial virulence factor rpoB suggests that rather than Mycobacterium Tuberculosis (Mtb), a variant member of the Mycobacterium Tuberculosis Complex (MtbC), or a closely related novel mycobacterium is present in iSGS specimens. Conclusions These studies identify a novel pathogenic role for established large airway bacteria, and provide new targets for future therapeutic intervention. Level of Evidence NA.

Objective. To characterize the phenotype and function of fibroblasts derived from airway scar in idiopathic subglottic stenosis (iSGS) and to explore scar fibroblast response to interleukin 17A (IL-17A). Study Design. Basic science. Setting. Laboratory. Subjects and Methods. Primary fibroblast cell lines from iSGS subjects, idiopathic pulmonary fibrosis subjects, and normal control airways were utilized for analysis. Protein, molecular, and flow cytometric techniques were applied in vitro to assess the phenotype and functional response of disease fibroblasts to IL-17A. Results. Mechanistically, IL-17A drives iSGS scar fibroblast proliferation (P < .01), synergizes with transforming growth factor ß1 to promote extracellular matrix production (collagen and fibronectin; P = .04), and directly stimulates scar fibroblasts to produce chemokines (chemokine ligand 2) and cytokines (IL-6 and granulocyte-macrophage colony-stimulating factor) critical to the recruitment and differentiation of myeloid cells (P < .01). Glucocorticoids abrogated IL-17A-dependent iSGS scar fibroblast production of granulocyte-macrophage colony-stimulating factor (P = .02). Conclusion. IL-17A directly drives iSGS scar fibroblast proliferation, synergizes with transforming growth factor ß1 to promote extracellular matrix production, and amplifies local inflammatory signaling. Glucocorticoids appear to partially abrogate fibroblast-dependent inflammatory signaling. These results offer mechanistic support for future translational study of clinical reagents for manipulation of the IL-17A pathway in iSGS patients.

Temporal bone mucormycosis is a rare and morbid infectious disease, though its outcomes appear to be different for patients who present with isolated temporal bone disease as compared to those individuals who develop temporal bone mucormycosis secondary to a rhinologic source.

e22101 Background: GTE and Epigallocatechin 3-gallate (EGCG), the most abundant polyphenol in GTE, have been shown to exert inhibitory effects on carcinogenesis; modulatory effects on tumor proliferation and differentiation; and immunomodulatory effects on tumor immunity in different pre-clinical models. These pluripotent effects suggest that GTE may have clinical activity that could be exploited for treatment of chemo-insensitive but immunologically responsive tumors. Thus, the present study investigated the effects of EGCG on the proliferation and immunologic sensitivity of human renal cell cancer (RCC) and malignant melanoma (MM) cell lines. Methods: Human RCC (769-P) and MM (A375) cell lines were tested following incubation in media ± 21.8μM EGCG, a pharmacologically-achievable concentration produced by 8, 200mg capsules GTE daily. Tumor proliferation was assessed by MTS assay; lytic sensitivity to IL2-activated human peripheral blood lymphocytes (IL2PBL) by 51Chromium release assay; and gene expression by quantitative RT-PCR assay. Results: EGCG produced significant inhibition of proliferation of both RCC and MM cells (61.5% and 67.3% of media control values respectively; p<0.01 by 2-tailed, paired t test). EGCG also caused significantly increased expression (≥ 2 times (×) media control values) of pro-apoptotic genes in both RCC: BCL2L1 (5.4×); BCL2L10 (7.9×); BIK (2.9×); Caspase 5 (6.3×); and Caspase 14 (6.3×) and MM cells: Caspase8 (2X) and Card6 (2.5X). Unfortunately, EGCG pre-treatment also decreased the sensitivity of RCC (43% of control) and MM (53% of control) to IL2PBL-mediated lysis (p<0.01 respectively). This occured in association with significant upregulation of Fas ligand mRNA in RCC (6.3×) and TRAF2 mRNA in MM (33 ×). Conclusions: The results of this pre-clinical study demonstrate that EGCG exerts significant antiproliferative effects against human RCC and MM cells in vitro in association with increased expression of different pro-apoptotic genes. Unfortunately, EGCG-treated cells also demonstrated reduced sensitivity to lysis by IL2-activated human lymphocytes. Taken together, the results suggest that GTE may have activity in vivo in patients against chemoresistant RCC or MM but should not be used in conjunction with IL2 therapy. No significant financial relationships to disclose.