Development of glandular models from human nasal progenitor cells.

Hyperplasia/hypertrophy of submucosal glands contributes to mucus overproduction in chronic diseases of the upper and lower respiratory tracts, especially in adult and pediatric chronic rhinosinusitis. Mechanisms that lead to glandular hyperplasia/hypertrophy are markedly understudied, reflecting a lack of in vitro model systems wherein airway epithelial progenitor cells differentiate into glandular cells. In this study, we developed and compared several in vitro three-dimensional systems using human nasal epithelial basal cells (HNEBCs) cultured by different methods on two types of extracellular matrices. We demonstrate that HNEBCs cultured on Matrigel (Corning, Tewksbury, MA) form glandular acini-like structures, whereas HNEBCs embedded in a collagen type I matrix form a network of tubules. Fibroblast-conditioned medium increases tubule formation in collagen type I. In contrast, HNEBCs cocultured with fibroblasts self-aggregate into organotypic structures with tubules and acini. These observations provide morphological evidence that HNEBCs are pluripotent and retain the capacity to differentiate into structures resembling specific structural components of submucosal glands depending on the extracellular matrices and culture conditions. The resultant models should prove useful in targeting cross-talk between epithelial cells and fibroblasts to decipher molecular mechanisms and specific signals responsible for the development of glandular hyperplasia/hypertrophy, which in turn may lead to new therapeutic strategies for chronic rhinosinusitis and other inflammatory respiratory diseases characterized by glandular hyperplasia/hypertrophy.

Immunotherapy Induced Adrenal Insufficiency: An Underdiagnosed Cause of Persistent Hypotension in Cancer.

Endocrinopathies following immunotherapy have infrequently been documented in the literature. Adrenal insufficiency is a rare consequence of pembrolizumab immunotherapy, with incidence reported to be between 0.98 and 1.3%. We present the case of a 34-year-old female with triple negative breast cancer on chemotherapy who presented with generalized weakness with tachycardia, tachypnea and hypotension unresponsive to fluids. Despite initial improvement with intravenous hydrocortisone and midodrine, the patient continued to be symptomatically hypotensive following discharge and required re-admission. AM cortisol level was found to be < 0.5 ug/dl and ACTH was <1.5 pg/dL, consistent with secondary adrenal insufficiency. CT abdomen and pelvis was unremarkable for adrenal pathology. Patient had been initiated on pembrolizumab (Keytruda) 4 months prior to presentation as part of neoadjuvant chemotherapy. The patient was provided supportive treatment with discharge on fludrocortisone, prednisone, and midodrine. This case reports an unusual consequence of immune checkpoint inhibitors, in which early diagnostic testing, identification, and management is critical.

Glandular gene expression of sinus mucosa in chronic rhinosinusitis with and without cystic fibrosis.

Secretory cells in submucosal glands (SMGs) secrete antibacterial proteins and mucin glycoproteins into the apical lumen of the respiratory tract, and these are critical for innate immune mucosal integrity. Glandular hyperplasia is manifested in diseases with obstructive respiratory pathologies associated with mucous hypersecretion, and is predominant in the sinus mucosa of patients with chronic rhinosinusitis (CRS), cystic fibrosis (CF), and clinical symptoms of CRS. To gain insights into the molecular basis of SMG hyperplasia in CRS, gene expression microarray analyses were performed to identify the differences in global and specific gene expression in the sinus mucosa of control, CRS, and CRS/CF patients. A marked up-regulation of 11 glandular-associated genes in CRS and CRS/CF sinus mucosa was evident. The RNA and protein expressions of the four most highly up-regulated genes (DSG3, KRT14, PTHLH, and OTX2) were evaluated. An increased expression of DSG3, KRT14, and PTHLH was demonstrated at the mRNA and protein levels in both CRS and CRS/CF sinus mucosa, whereas the increased expression of OTX2 was evident only for CRS/CF sinus mucosa, implicating OTX2 as a CF-specific gene. Immunofluorescence analysis localized DSG3, PTHLH, and OTX2 to serous cells, and KRT14 to myoepithelial cells, in SMGs. Because glandular hyperplasia is a central histologic feature of CRS, the identification of overexpressed glandular genes in the sinus mucosa lays the groundwork for future studies of glandular hyperplasia, and may ultimately lead to the development of novel treatments for mucous hypersecretion in patients with CRS.

Localization of inflammatory mediators in pediatric sinus mucosa.

OBJECTIVES: Microarray analyses of sinus mucosa in pediatric patients with chronic rhinosinusitis (CRS) have recently demonstrated increased messenger RNA expression of the inflammatory chemokines CXCL5 and CXCL13 and of the innate immune mediators ß-defensin 1 (DEFB1), serum amyloid A2 (SAA2), and serpin B4. The objectives of this study were to determine whether these gene products were expressed at the protein level in pediatric sinus mucosa and to determine their localization. DESIGN: Immunohistochemical analysis was used to identify protein expression and cellular localization of CXCL5, CXCL13, DEFB1, SAA2, and serpin B4. Coimmunofluorescence staining of inflammatory cells was performed to further evaluate expression of CXCL5 and CXCL13. SETTING: Pediatric tertiary care facility. PATIENTS: Fifteen children with CRS who underwent endoscopic sinus surgery and 8 children who underwent craniofacial or neurosurgical procedures for abnormalities other than sinusitis. MAIN OUTCOME MEASURES: Protein expression and cellular localization of CXCL5, CXCL13, DEFB1, SAA2, and serpin B4 in pediatric sinus mucosa. RESULTS: Ciliated and basal cells in the pseudostratified epithelium stained positively for the 5 mediators examined in both cohorts. Except for serpin B4, goblet cells did not stain for any mediators in either cohort. Glandular cells stained positively for all 5 mediators in both cohorts. Coimmunofluorescence staining of inflammatory cells showed that CXCL13 was expressed in macrophages, T and B cells but not in neutrophils. CXCL5 was detected only in T cells. CONCLUSIONS: CXCL5, CXCL13, DEFB1, SAA2, and serpin B4 were expressed at the protein level in the sinus mucosa of controls and pediatric patients with CRS and exhibited cell-specific localization. These mediators, not typically associated with pediatric CRS, may be involved in the inflammatory response and mucus hypersecretion seen in pediatric CRS.

Histologic characteristics and mucin immunohistochemistry of cystic fibrosis sinus mucosa.

OBJECTIVES: To evaluate the histologic characteristics of paranasal sinus mucosa of a disease control population and children with chronic rhinosinusitis and cystic fibrosis (CRS/CF) (1) to determine whether goblet cell (GC) hyperplasia and/or submucosal gland (SMG) hyperplasia occur in pediatric CRS/CF and (2) to compare expression and localization of MUC5AC and MUC5B mucins in the sinus mucosa of both cohorts. DESIGN: Histologic and morphometric analyses of paranasal sinus mucosa were used to quantify the number of GCs and mucin-expressing cells. Digital imaging was used to evaluate the SMG area. Immunohistochemistry was performed to identify the cellular localization of MUC5AC and MUC5B mucins, and confocal microscopy was used to determine whether MUC5AC and MUC5B mucins were expressed in the same secretory cells. SETTING: Children's National Medical Center, Washington, DC. PARTICIPANTS: Twenty-one children with CRS/CF who underwent endoscopic sinus surgical procedures and 18 children who underwent craniofacial resection or neurosurgical procedures for abnormalities other than sinusitis. RESULTS: A statistically significant increased area (4.4-fold) of SMGs was detected in the sinus mucosa of patients with CRS/CF compared with the controls (P = .02). Neither GC hyperplasia nor increased expression of MUC5AC was observed in the CRS/CF group. MUC5AC was expressed only in a subpopulation of GCs in both cohorts, and MUC5B was expressed in a subpopulation of GCs as well as in SMGs. There was a positive trend toward increased glandular MUC5B expression in the CRS/CF cohort. Colocalization of MUC5AC and MUC5B expression was observed in a subset of GCs. CONCLUSIONS: Significant SMG hyperplasia and a trend toward increased glandular MUC5B expression exist in children with CRS/CF. This suggests that SMG hyperplasia and glandular MUC5B mucin contribute to mucus overproduction in the sinus mucosa of this population.