Christian Hochstim

The Transcription Factor NFIA Controls the Onset of Gliogenesis in the Developing Spinal Cord

The mechanisms controlling the transition from neurogenesis to gliogenesis in the vertebrate CNS are incompletely understood. We identified a family of transcription factors, called NFI genes, which are induced throughout the spinal cord ventricular zone (VZ) concomitantly with the induction of GLAST, an early marker of gliogenesis. NFIA is both necessary and sufficient for GLAST induction in the VZ. Unexpectedly, NFIA is also essential for the continued inhibition of neurogenesis in VZ progenitors. This function is mediated by the requirement of NFIA for the expression of HES5, a Notch effector. However, Notch effectors are unable to promote glial-fate specification in the absence of NFIA. Thus, NFIA links the abrogation of neurogenesis to a generic program of gliogenesis, in both astrocyte and oligodendrocyte VZ progenitors. At later stages, NFIA promotes migration and differentiation of astrocyte precursors, a function that is antagonized in oligodendrocyte precursors by Olig2.

Identification of Positionally Distinct Astrocyte Subtypes whose Identities Are Specified by a Homeodomain Code

Astrocytes constitute the most abundant cell type in the central nervous system (CNS) and play diverse functional roles, but the ontogenetic origins of this phenotypic diversity are poorly understood. We have investigated whether positional identity, a fundamental organizing principle governing the generation of neuronal subtype diversity, is also relevant to astrocyte diversification. We identified three positionally distinct subtypes of white-matter astrocytes (WMA) in the spinal cord, which can be distinguished by the combinatorial expression of Reelin and Slit1. These astrocyte subtypes derive from progenitor domains expressing the homeodomain transcription factors Pax6 and Nkx6.1, respectively. Loss- and gain-of-function experiments indicate that the positional identity of these astrocyte subtypes is controlled by Pax6 and Nkx6.1 in a combinatorial manner. Thus, positional identity is an organizing principle underlying astrocyte, as well as neuronal, subtype diversification and is controlled by a homeodomain transcriptional code whose elements are reutilized following the specification of neuronal identity earlier in development.

Biofilm detection with hematoxylin-eosin staining.

OBJECTIVE To demonstrate that hematoxylin-eosin staining can be used to detect the presence of bacterial biofilm in patients with chronic rhinosinusitis (CRS). DESIGN A prospective study. SETTING The University of Southern California University Hospital and the Department of Otolaryngology-Head and Neck Surgery, University of Southern California, Keck School of Medicine, Los Angeles. PATIENTS A total of 34 patients: 24 undergoing endoscopic sinus surgery for CRS and 10 undergoing septoplasty with or without turbinate reduction with no history of sinusitis, were enrolled in the study. MAIN OUTCOME MEASURES Contiguous sections from patient samples were examined by both hematoxylin-eosin staining and fluorescence in situ hybridization (FISH) with the bacterial-specific probe EUB338 for evidence of bacterial biofilm. RESULTS Biofilm was detected by hematoxylin-eosin staining in 15 of 24 patients with CRS and 1 of 10 control patients. In all cases, hematoxylin-eosin staining was found to be an accurate predictor of the presence or absence of biofilm using FISH as a control standard. CONCLUSION Hematoxylin-eosin staining of surgical specimens is a reliable and available method for the detection of bacterial biofilm in chronic infectious disease.

Increased radiation sensitivity of head and neck squamous cell carcinoma with sphingosine kinase 1 inhibition

Sphingosine kinase 1 (SphK1) is an important regulator of apoptosis, survival, and proliferation in cancer cells. SphK1 expression in head and neck squamous cell cancer (HNSCC) cell lines and tumor tissue was assessed, and the efficacy of SphK1 knockdown in increasing tumor radiosensitivity was evaluated in vitro and in vivo.

Biofilm and persistent inflammation in endoscopic sinus surgery

Bacterial biofilms have been observed in many patients with chronic rhinosinusitis, but their importance is still being investigated. This study examines the association between biofilms and other clinical findings in chronic rhinosinusitis patients. Twenty-four patients with chronic rhinosinusitis who failed medical management underwent endoscopic sinus surgery (ESS). Tissue was collected from the ethmoid sinus and analyzed for the presence of biofilm by hematoxylin and eosin staining, fluorescent in situ hybridization, and confocal scanning laser microscopy. Biofilms were classified as extensive (> 50% of mucosal surface in sample) or present (< 50% of surface). The surgeon remained blinded to the biofilm status of patients until postoperative follow-up was complete. The presence of bacterial biofilm was strongly associated with persistent mucosal inflammation after ESS (53% of biofilm-positive patients vs 0% of biofilm-negative patients, P = 0.009). The amount of biofilm was not important as there was no significant difference between the extensive and present biofilm classifications with respect to inflammation. The presence of biofilm was not associated with prior ESS, allergies, eosinophils, polyps, or presence of fungal elements.

A novel orthotopic mouse model of head and neck cancer and lymph node metastasis

Prognosis of head and neck squamous cell carcinoma (HNSCC) is largely determined by the extent of lymph node (LN) metastasis at diagnosis, and this appears to be controlled by cancer cell genetics. To examine the role of these genes in LN metastasis, we created a human-in-mouse orthotopic model of HNSCC and performed comparative microarray analysis of gene expression between populations of HNSCC cell lines derived before and after serial transplantation and in vivo metastasis in mice. Microarray analysis comparing the USC-HN3-GFP, USC-HN3-GFP-G1 and USC-HN3-GFP-G2 cell lines identified overexpression of genes implicated in epithelial-to- mesenchymal transition and the formation of cancer stem cells, including CAV-1, TLR-4 (Toll-like receptor 4), MMP-7 (matrix metalloproteinase 7), ALDH1A3, OCT-4 and TRIM-29. Ingenuity Pathway Analysis confirmed upregulation of respective gene signaling pathways in the USC-HN1-GFP-G2 cell line. Patient HNSCC samples from advanced stages overexpressed ALDH1A3, CAV-1 and MMP-7. Our results show that CAV-1, TLR-4, MMP-7, ALDH1A3, OCT-4 and TRIM-29 have increased expression in HNSCC cells selected for an enhanced metastatic phenotype and suggest that these genes may have an important role in the metastatic potential of HNSCC cells. Inhibition of these genes may therefore have prognostic and therapeutic utility in HNSCC.

Relationship of eosinophils and plasma cells to biofilm in chronic rhinosinusitis.

Background This study investigates the relationship of eosinophils and plasma cells to biofilm in chronic rhinosinusitis (CRS). A prospective observational study was performed at the Keck Hospital, University of Southern California, Department of Otolaryngology, Los Angeles, CA. Methods A total of 29 patients, 20 undergoing endoscopic sinus surgery for CRS and 9 control patients undergoing septoplasty for nasal obstruction without history or evidence of CRS, were included in this study. Contiguous sinonasal mucosa sample sections were examined by hematoxylin and eosin (H&E), fluorescence in situ hybridization (FISH), and immunohistochemistry (IHC) for biofilm, microbes, eosinophil major basic protein (EMBP), and cluster designation 27 (CD27). EMBP and CD27 were used as eosinophil and plasma cell markers, respectively. Results Biofilm was visualized in 15 of 20 patients with CRS on H&E sections, confirmed by microbial presence using FISH. Biofilm was not identified in tissue samples of the nine control patients. On IHC analysis, CD27 and EMBP expression were significantly higher in patients with CRS compared with control (p < 0.05) and had greater expression in biofilm-positive patients compared with biofilm-negative patients. Nasal polyps correlated with higher expression of CD27 and EMBP, but in CRS patients without polyps CD27 and EMBP was also significantly greater in biofilm-positive specimens compared with biofilm-negative specimens. Conclusion Biofilm presence in CRS appears to correlate to host inflammatory response involving plasma cell and eosinophil recruitment.

Bacterial biofilms and increased bacterial counts are associated with airway stenosis.

Objectives Most airway stenoses are acquired secondary to the use of prolonged endotracheal intubation. Antibiotics have been shown to decrease local inflammation and granulation tissue formation in the trachea. However, antibiotic therapy is not 100% effective in preventing or treating granulation tissue formation. Development of bacterial biofilms may explain this finding. This study evaluates the difference between tracheal stenotic segments and normal trachea in terms of (1) presence of bacterial biofilms, (2) quantitative bacterial counts, and (3) inflammatory markers. Study Design Cross-sectional study. Setting Tertiary care academic medical center. Subjects A total of 12 patients were included in the study. Tissue from stenotic segments from 6 patients with airway stenosis undergoing open airway procedures were compared with tracheal tissue from 6 patients without airway stenosis undergoing tracheostomy. Methods Scanning electron microscopy for biofilm detection, quantitative polymerase chain reaction for quantitative analysis of bacterial count, and immunohistochemistry were performed for inflammatory markers transforming growth factor β1 (TGF-β1) and SMAD3. Results Compared with the patients without airway stenosis, patients in the airway stenosis group showed presence of bacterial biofilms, a significantly higher expression of 16S rRNA gene copies per microgram of tissue (187.5 vs 7.33, P = .01), and higher expression of TGF-β1 (91% vs 8%, P < .001) and SMAD3 (83.5% vs 17.8%, P < .001). Conclusion Bacterial biofilms, increased bacterial counts, and higher expression of TGF-β1 and SMAD3 are associated with airway stenosis.

Establishing Proximal and Distal Regional Identities in Murine and Human Tissue-Engineered Lung and Trachea.

The cellular and molecular mechanisms that underpin regeneration of the human lung are unknown, and the study of lung repair has been impeded by the necessity for reductionist models that may exclude key components. We hypothesized that multicellular epithelial and mesenchymal cell clusters or lung organoid units (LuOU) could be transplanted to recapitulate proximal and distal cellular structures of the native lung and airways. Transplantation of LuOU resulted in the growth of tissue-engineered lung (TELu) that contained the necessary cell types consistent with native adult lung tissue and demonstrated proliferative cells at 2 and 4 weeks. This technique recapitulated important elements of both mouse and human lungs featuring key components of both the proximal and distal lung regions. When LuOU were generated from whole lung, TELu contained key epithelial and mesenchymal cell types, and the origin of the cells was traced from both ActinGFP and SPCGFP donors to indicate that the cells in TELu were derived from the transplanted LuOU. Alveolar epithelial type 2 cells (AEC2s), club cells, ciliated cells marked by beta-tubulin IV, alveolar epithelial type I cells, Sox-2-positive proximal airway progenitors, p63-positive basal cells, and CGRP-positive pulmonary neuroendocrine cells were identified in the TELu. The mesenchymal components of peribronchial smooth muscle and nerve were identified with a CD31-positive donor endothelial cell contribution to TELu vasculature. TELu successfully grew from postnatal tissues from whole murine and human lung, distal murine lung, as well as murine and human trachea. These data support a model of postnatal lung regeneration containing the diverse cell types present in the entirety of the respiratory tract.

Geographic health disparities in the Los Angeles pediatric esophageal foreign body population

OBJECTIVE To assess geographical sociodemographic differences in the pediatric esophageal foreign body population of Los Angeles. METHODS We retrospectively reviewed the medical records of 128 consecutive pediatric patients at Childrens Hospital Los Angeles (CHLA) from 2014 to 2017 with a diagnosis of a retained foreign body in the esophagus removed by rigid or flexible esophagoscopy. Sociodemographic information including zip code of residence was extracted and analyzed with Chi-square, Fishers exact test, and multivariable logistic regression. RESULTS The average age of patients with a retained esophageal foreign body in this study was 2.5 years old, 52.3% were male, 91.4% had no past medical history, 53.1% were Hispanic, 82.0% had public health insurance, and 63.3% were transfers from an outside hospital. The most common foreign body removed was a coin. There were no significant differences in gender, race, type of health insurance, or income between patients that lived within 10 miles of CHLA versus farther than 10 miles. On multivariable analysis, zip codes with a high volume of esophageal foreign bodies were more likely to be lower income neighborhoods. Gender, race, type of health insurance, and distance from CHLA were not risk factors for zip codes with a high volume of esophageal foreign bodies. CONCLUSION Geographic areas in the greater Los Angeles community with a high volume of retained esophageal foreign bodies requiring endoscopic removal at our institution are associated with lower income neighborhoods. Further studies should be performed to better understand health disparities within the U.S. pediatric esophageal foreign body population.