Nasal Mucosal Epithelial Cells Research Articles

The effect of transplantation of nasal mucosal epithelial cell sheets after middle ear surgery in a rabbit model

Postoperative regeneration of the middle ear mucosa and pneumatization of the middle ear cavity are of great importance after middle ear surgery. This study developed a new method to transplant autologous nasal mucosal epithelial cell sheets into the damaged middle ear cavity. The aim of this study was to evaluate postoperative healing after the transplantation of the cell sheets. Rabbit nasal mucosal epithelial cell sheets were fabricated on a temperature-responsive culture dish, and transplanted into the damaged middle ear of rabbit, which was surgically created. The healing of middle ears was evaluated by histology and X-ray computed tomography after transplantation. Functional evaluation was performed by measuring the maximum middle ear total pressure reflecting a trans-mucosal gas exchange function. Two control groups were used: the normal control group and the mucosa-eliminated control group. Transplantation of cell sheets suppressed the bone hyperplasia and the narrowing of pneumatic space in the middle ear cavity compared with the mucosa-eliminated control group. The mucosal gas exchange function was also better in the cell sheet-transplanted group. Nasal mucosal epithelial cell sheet was confirmed to be useful as an effective graft material after middle ear surgery and hopefully become a novel therapy in the future.

Ocular surface reconstruction with a tissue-engineered nasal mucosal epithelial cell sheet for the treatment of severe ocular surface diseases.

Severe ocular surface diseases (OSDs) with severe dry eye can be devastating and are currently some of the most challenging eye disorders to treat. To investigate the feasibility of using an autologous tissue-engineered cultivated nasal mucosal epithelial cell sheet (CNMES) for ocular surface reconstruction, we developed a novel technique for the culture of nasal mucosal epithelial cells expanded ex vivo from biopsy-derived human nasal mucosal tissues. After the protocol, the CNMESs had 4-5 layers of stratified, well-differentiated cells, and we successfully generated cultured epithelial sheets, including numerous goblet cells. Immunohistochemistry confirmed the presence of keratins 3, 4, and 13; mucins 1, 16, and 5AC; cell junction and basement membrane assembly proteins; and stem/progenitor cell marker p75 in the CNMESs. We then transplanted the CNMESs onto the ocular surfaces of rabbits and confirmed the survival of this tissue, including the goblet cells, up to 2 weeks. The present report describes an attempt to overcome the problems of treating severe OSDs with the most severe dry eye by treating them using tissue-engineered CNMESs to supply functional goblet cells and to stabilize and reconstruct the ocular surface. The present study is a first step toward assessing the use of tissue-engineered goblet-cell transplantation of nonocular surface origin for ocular surface reconstruction.

Abstract 5356: The role of VNN3 gene in nasopharyngeal carcinoma tumorigenesis

Abstract Nasopharyngeal carcinoma (NPC) is one of the leading cause of cancer-related deaths in southeastern China and South East Asia. However, the detailed pathogenesis mechanisms of NPC remain to be elucidated. Despite novel therapies and advances in early detection, it is often diagnosed at an advanced stage and has a poor prognosis. Recent evidences showed that SRY (sex-determining region Y)-box 5 (SOX-5) plays an important role in Nasopharyngeal tumor development, but little is known about the role of its associated protein such as VNN3 gene (a pantetheinase) in controlling tumor development. SOX-5 overexpression enhances tumor formation in NPC, and has a poor prognosis. In this study, we compared the differential gene expression in NPC cell lines and normal nasal mucosal epithelial cells by cDNA microarray analysis, and observed that the RNA and protein expression levels of SOX-5 and VNN3 were dramatically increased in NPC cell lines than normal nasal mucosal epithelial cells by qRT-PCR and Western blot analysis. In order to investigate that whether VNN3 gene could alter NPC progression, we observed at first the gene expression in different NPC cell lines using qRT-PCR analysis and found that the expression of VNN3 in NPC cell line, especially in NPC-TW01 cell line was highly upregulated. Besides, we also found that the expression level of VNN3 protein was also increased in multiple NPC cell lines and NPC biopsy specimens by immunohistochemical staining and Western bloting. In this study, we also isolated the cancer stem cell (CSC) from NPC-TW01 cell line by conditioned culture medium, highly expression level of VNN3 gene was found in the NPC-TW01 stem cells (CSC). To further analysis of the functional role of VNN3 in this NPC and CSC lines, the expression of VNN3 was decreased to about 70-80% of the control in NPC and CSC after knockdown of VNN3 by VNN3 shRNA-lentiviral infection. This in turn resulted in a significant reduction of cell proliferation, migration and invasion abilities. We also found that a decreased expression level of VNN3 (Pantetheinase), which is an amidohydrolase that hydrolyzes pantetheine into pantothenic acid (vitamin B5) as well as cysteamine, and caused an increase of intracellular reactive oxygen species (ROS), and cell death through apoptosis; the weight of NPC xenograft tumors had diminished about 51% after 6 weeks, compared with shLuc control in SCID mice bearing NPC xenografts derived from these lentiviral infected NPC and CSC cell lines. Impairment of tumor cell proliferation, accompanied with apoptosis was found in the xenografts histopathologically. These novel findings of the functional role of VNN3 may have a potential implication in molecular targeted therapy for NPC. Citation Format: Shang-Yu Huang, Yi-Ying Chen, Dah-yeou Huang, Cheng-Der Wu, Chin-Tarng Lin. The role of VNN3 gene in nasopharyngeal carcinoma tumorigenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5356. doi:10.1158/1538-7445.AM2014-5356

Expression and regulation of tight junction protein Occludin in nasal polyps

To evaluate the possible role of tight junction protein Occludin in nasal polyps. The expression of Claudin-1, Occludin and ZO-1 in nasal polyps (n = 20) and healthy uncinate mucosa (n = 15) were examined using immunohistochemical staining, real-time quantitative polymerase chain reaction (PCR) and Western blot analysis. The regulatory effects of proinflammatory cytokines (IFN-γ, IL-13, IL-17, TGF-β, TGF-α) on the expression of Occludin in cultured human nasal epithelial cells were investigated. The immunohistochemical results showed that Claudin-1, Occludin and ZO-1 were detected both in the nasal polyp group and the control group. The expression sites were the cell membrane and cytoplasm of nasal mucosa epithelial cells. The mean optical density of Claudin-1, Occludin and ZO-1 were 0.187 ± 0.076,0.172 ± 0.109 and 0.098 ± 0.035 respectively in the nasal polyp group and were significantly lower than those in the control group (0.312 ± 0.101, 0.220 ± 0.069 and 0.233 ± 0.093 respectively), the differences were significant (t = 9.345, t = 3.301, t = 13.323, all P < 0.01).RT-PCR results showed that the relative expression of Occludin mRNA was 0.000 117 ± 0.000 035 in the nasal polyp group and was significantly lower than that in the control group(0.000 464 ± 0.000 134), and the difference was significant (Z = -5.0, P < 0.01) . There was no statistically significant difference in the relative expression of Claudin-1 and ZO-1 mRNA between the nasal polyp group and the control group (P > 0.05) . After the cultured human nasal epithelial cells were stimulated by IL-13, IL-17, IFN-γ and other proinflammatory cytokines, the relative expression of Occludin mRNA was 0.631 ± 0.039, 0.581 ± 0.029 and 0.648 ± 0.040, respectively. Compared with the unstimulated control group, the differences were statistically significant (t = 16.299, 24.669 and 14.995 respectively, all P < 0.05).Western blot analyse showed that the relative grayscale in the above proinflammatory cytokines stimulation groups was 0.650 ± 0.061,0.482 ± 0.106 and 0.536 ± 0.109, respectively. Compared with the unstimulated control group, the differences were statistically significant (t = 9.880, 8.442 and 7.310 respectively, all P < 0.05). The reduced expression of Occludin might be involved in the pathogenesis of nasal polyps.

Fluticasone Furoate Inhibits Cytokine Secretion from Nasal Epithelial Cells and Reduces Eosinophil Survival in an in vitro Model of Eosinophilic Inflammation

Background: Fluticasone furoate (FF) is an intranasal corticosteroid indicated for the treatment of allergic rhinitis (AR). However, the anti-inflammatory effects of FF in the nasal mucosa have yet to be investigated thoroughly. The aim of this study was to investigate the effect of FF on eosinophil survival and cytokine secretion from nasal mucosa epithelial cells. Methods: Epithelial cells obtained from nasal mucosa were stimulated with 10% fetal bovine serum (FBS) in the presence of FF (from 10^-12 to 10^-7<smlcap>M</smlcap>) for 6-24 h. Cytokine [granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-6 and IL-8] concentrations in supernatants were measured by ELISA. Peripheral blood eosinophils were incubated for 4 days with epithelial cell secretions in the presence or absence of FF (from 10^-12 to 10^-7<smlcap>M</smlcap>) and survival was assessed by Trypan blue dye exclusion. Results are expressed as medians of the minimum effective concentration and IC values. Results: FBS stimulated the secretion of GM-CSF, IL-6 and IL-8. FF significantly inhibited GM-CSF (up to 10^-10<smlcap>M</smlcap>, IC₂₅ = 12.6 p<smlcap>M</smlcap>), IL-6 (up to 10^-10<smlcap>M</smlcap>, IC₂₅ = 65.8 p<smlcap>M</smlcap>) and IL-8 (up to 10^-11<smlcap>M</smlcap>, IC₂₅ = 8.6 p<smlcap>M</smlcap>) secretion induced by FBS (n = 8). Epithelial cell secretions induced eosinophil survival from day 1 to day 4 (n = 6). This effect was significantly inhibited by FF (up to 10^-12<smlcap>M</smlcap>) at day 3 (IC₅₀ = 3.22 n<smlcap>M</smlcap>) and day 4 (IC₅₀ = 1.29 n<smlcap>M</smlcap>). Conclusions: The results obtained in this in vitro model suggest that FF may reduce upper airway eosinophilic inflammation through decreasing cytokine secretion from epithelial cells and reducing eosinophil survival.

Regional differences in the expression of innate host defense molecules in sinonasal mucosa

Nasal mucosal epithelial cells form the first line of defense by maintaining a barrier to restrict potentially harmful airborne substances and pathogens. To aid in host defense, specialized epithelial cells in submucosal glands as well as on the mucosal surface secrete mucous that helps to immobilize pathogens and other harmful substances. Beneath this mucous blanket resides an aqueous surface liquid layer, which allows proper ciliary function to clear mucous and entrapped pathogens. In concert with maintenance of a physical barrier, airway epithelium secretes dozens of antimicrobial peptides and proteins that become incorporated into the mucous and aqueous lining fluids1, 2. It has become clear that proper functioning of the epithelium is essential for suppressing the growth of pathogenic organisms and promoting healthy upper airway physiology. Dysfunction in innate immune expression of host defense molecules has been linked to many airway diseases3-8. Surprisingly, the regional distribution of important epithelial derived antimicrobial proteins secreted into upper airways has not been studied in detail. In this study, we analyzed the expression of select antimicrobial proteins that are known to be secreted into the lining fluids of the upper airways in two different anatomical sites of the sinonasal mucosa in healthy human subjects, namely the inferior region (inferior turbinate - IT), and the superior region (uncinate tissue - UT). We chose IT, as it is the proximal point of contact of inhaled air containing particulate matter and potential pathogens. Moreover, studies of CRS and other diseases of the upper airways routinely use IT tissue as a control tissue to make comparisons with nasal polyps. UT was chosen for its important role in the drainage pathways of maxillary, frontal and anterior ethmoid sinuses. We chose to study the expression of S100A7 (S100 family), hBD2 (β-Defensin family), SPLUNC1 (PLUNC family), and lactoferrin, as they represent broad families of antimicrobial proteins, which are either constitutively present or induced by inflammation triggered by pathogens and collectively have antimicrobial effects against a variety of pathogens (bacteria, fungi and viruses). Sinonasal tissue samples from IT and UT were collected from subjects undergoing surgery to correct non-inflammatory conditions, including facial anatomical defects (deformity, trauma etc.), to improve airflow and during the course of skull base surgery to remove tumors. The subjects were not diagnosed with any upper or lower airway diseases at the time of sample collection. Detailed patient characteristics are provided in table EI. The Investigational Review Board of Northwestern University approved all methods for the present study, and all patients provided informed consent. At the time of surgery, tissues and nasal epithelial scraping cells were collected and stored for further analysis. Tissue samples and epithelial cells were analyzed for mRNA expression by real time PCR. Protein expression and localization were analyzed using ELISA and immunohistochemistry, respectively. AB/PAS staining was performed to characterize the glandular differences between IT and UT. Details of the methods used can be found in the online repository and our other publications5, 7.

Abstract 3006: The CCL28 oncogene in NPC pathogenesis.

Abstract CCL28 Gene plays a role as an oncogene in the pathogenesis of Lymphoepitheliomatous Carcinoma of Nasopharynx Nasopharyngeal Carcinoma (NPC) is one of the most common cancers among Chinese living in southern China, Hong Kong, Singapore and Taiwan. The molecular mechanisms involved in the pathogenesis of NPC still are not yet well defined. Although it was proposed that Epstein-Barr virus (EBV) is closely associated with NPC pathogenesis, but recently many studies have reported that EBV behaves more likely as a progression factor but not an initiation factor. The purpose of this research was to find out the genes associated with NPC pathogenesis. Using cDNA microarray analysis of mRNA expression between NPC cell lines and normal nasal mucosal epithelial cells, SOX5 gene expression was found significantly increased in NPC cell lines. In our previous studies of the function of SOX-5 gene in NPC, we found that chemokine (CC motif) of ligand 28 (CCL28) was also significantly increased in NPC cell lines, especially in NPC-TW03 line which is a lymphoepitheliomatous carcinoma of nasopharynx (LE-NPC). In 50 cases of Type III NPC biopsy specimens it was found that about 46% of cases revealed high expression of CCL28 protein in their tumor cells. To further identify the relationship between SOX5 and CCL28, we found that SOX5 and CCL28 gene expressions could be reciprocally regulated in NPC cells and suggested that CCL28 may be a critical factor for the formation of LE-NPC. To study the role of CCL28 in the molecular pathogenesis of LE-NPC and its functions, we constructed a stable pBIG2i-CCL28 transfected NPC cell line. We found that tumor cells could be up-regulated to express CCL28 mRNA and protein remarkably in those transfectants. This gene could promote tumor cell migration, proliferation, invasion, and attract lymphocytes especially B cells in vitro. In addition, EBV-immortalized B cells can also be attracted by CCL28. On the other hand, EBV could infect the CCL28 transfectants; furthermore, CCL28 could enhance IgA anti EBV-VCA production from the EBV infected-B cells. However, in SCID mice bearing CCL28 transfected NPC xenograft, the tumor growth and metastatic activity were slightly to moderately increased by this gene, but it has failed to attract mouse lymphocytic infiltration, probably due to the dissimilarity of CCL28 molecular structure between human and mouse species. Also, cDNA microarray analysis of CCL28 overexpressed NPC cells showed a group of oncogenes, such as IFI6 (Interferon L-inducible Protein 6, a survival protein) up-regulated and some oncosuppressor genes such as GPR68 (a G-protein-couple receptor 68) (a tumor metastasis suppressor gene) down-regulated. It is concluded that CCL28 gene may play a role to promote the formation of LE-NPC and as an oncogene in NPC pathogenesis to promote LE-NPC migration, proliferation and invasion in addition to its chemotactic property to attract B cell migration and to enhance IgA anti EBV-VCA production from the EBV+ B cells. Citation Format: Chin-Tarng Lin, Yi-Ying Chen, Cheng-Der Wu, Dah-yeou Huang, Yuan-Sung Kuo. The CCL28 oncogene in NPC pathogenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3006. doi:10.1158/1538-7445.AM2013-3006

Fluticasone Furoate Decreases Nasal Eosinophilic Inflammation by Inhibiting Both Cytokine Secretion From Nasal Mucosa Epithelial Cells Cytokine Secretion and Eosinophil Survival

Fluticasone furoate (FF) is an intranasal corticosteroids used to treat allergic rhinitis (AR). However, the antiinflammatory effects of FF in the nasal mucosa have not been deeply investigated. The objective was to investigate the effect of FF on eosinophil survival and cytokine and sICAM-1 secretion from nasal epithelial cells.

132 Characterization of 2 Epithelial Cell Air-Liquid Interface (ALI) Culture Models for Human Healthy Nasal Mucosa and Nasal Polyps

BackgroundPrimary human airway epithelial cells, when submerged in culture, undergo a dedifferentiation with loss of many features of the in vivo airway epithelium. However, when cultured in an air-liquid interface (ALI), cells develop a well-differentiated, polarized, and pseudostratified epithelium. The aim of the current study was to characterize the mucociliary differentiation of human nasal mucosa and polyp epithelial cells cultured using an ALI system.MethodsNasal mucosa (NM, n = 3) and nasal polyps (NP, n = 3) were obtained from patients undergoing nasal corrective surgery and endoscopic sinus surgery, respectively. Epithelial cells were obtained from the explant method, and differentiated in ALI culture during 28 days. Cultures were studied at different time points (0, 7, 14, 21, and 28 days): tissue ultrastructure by scanning electron microscopy (SEM) and transmission electron microscopy (TEM); mucous (MUC5AC, MUC5B) and serous (lactoferrin) cell secretion by ELISA; and cytokeratin 18 (epithelial marker), β-tubulin IV (cilia marker), MUC5AC (goblet cell marker), and p63 (basal cell marker) expression by immunocytochemistry.ResultsIn both NM and NP ALI cultures and at days 14 and 28, a pseudostratified epithelium with ciliated, mucus-secreting and basal cells was observed, and expression of cytokeratin 18, b-tubulin IV, MUC5AC and p63 was detected. In NP cultures, both MUC5AC (day 14: 2.2 ± 0.1-folds; day 28: 3.6-fold ± 0.7-fold) and MUC5B (day 14: 3.2-fold ± 0.6-fold; day 28: 3.1-fold ± 1-fold) increased over time compared to day 0 (P < 0.05). In NM cultures, only MUC5B (day 14: 3.9-fold ± 0.9-fold; day 28: 3.4-fold ± 0.4-fold; P < 0.05) but not MUC5AC increased over time compared to day 0 (P < 0.05). Secretion of lactoferrin was present but showed no changes over time in either NM or NP ALI cultures.ConclusionsEpithelial cell ALI cultures provide a well-differentiated human nasal mucosa and polyp tissues that may be used as an in vitro model to study mucin regulation, inflammatory mechanisms of upper airways, and their regulation by antiinflammatory drugs.

Foot-and-Mouth Disease Virus Replicates Only Transiently in Well-Differentiated Porcine Nasal Epithelial Cells

Three-dimensional (3D) porcine nasal mucosal and tracheal mucosal epithelial cell cultures were developed to analyze foot-and-mouth disease virus (FMDV) interactions with mucosal epithelial cells. The cells in these cultures differentiated and polarized until they closely resemble the epithelial layers seen in vivo. FMDV infected these cultures predominantly from the apical side, primarily by binding to integrin alphav beta6, in an Arg-Gly-Asp (RGD)-dependent manner. However, FMDV replicated only transiently without any visible cytopathic effect (CPE), and infectious progeny virus could be recovered only from the apical side. The infection induced the production of beta interferon (IFN-beta) and the IFN-inducible gene Mx1 mRNA, which coincided with the disappearance of viral RNA and progeny virus. The induction of IFN-beta mRNA correlated with the antiviral activity of the supernatants from both the apical and basolateral compartments. IFN-alpha mRNA was constitutively expressed in nasal mucosal epithelial cells in vitro and in vivo. In addition, FMDV infection induced interleukin 8 (IL-8) protein, granulocyte-macrophage colony-stimulating factor (GM-CSF), and RANTES mRNA in the infected epithelial cells, suggesting that it plays an important role in modulating the immune response.

A Morphologic and Immunohistochemical Study of Nasal Mucosa in Leatherworkers

The association between intestinal-type sinonasal adenocarcinoma and the occupational exposure to leather dusts has been widely documented, but the identification of precursor lesions in exposed workers has remained controversial. The purpose of this study was to investigate the histological changes and modifications in the phenotype of epithelial cells in nasal mucosa of leather workers. Biopsy specimens of the mucosa of the middle turbinate were obtained from 139 subjects who had been employed in leather factories for 10-48 years (median, 29 years). Tissue fragments were routinely processed for histological examination and immunostainings for cytokeratin 20, CDX-2, and MUC-2 were performed. Regarding the surface epithelium, the most common histopathological finding was the presence of squamous metaplasia (64.7%), which was associated with mild to moderate dysplasia in 37 cases (41.1%), and goblet cell hyperplasia was identified in 30 biopsy specimens (21.6%). Positivity for MUC-2 was detected in goblet cells of 20 of the 30 samples with goblet cell hyperplasia (66.6%), whereas no immunostaining was observed for cytokeratin 20 and CDX-2. Presence of goblet cell hyperplasia was significantly associated with longer occupational exposure in leather tanning activities (p = 0.03). None of the alterations observed (squamous metaplasia, dysplasia, or goblet cell hyperplasia) showed correlation with smoking habits. Similarly, there was no correlation between squamous metaplasia with or without dysplasia and type and duration of occupational exposure. Our data identify goblet cell hyperplasia as possible work-related alterations of nasal mucosa in leather workers. Additional investigations are needed to clarify the significance of these findings in the development of sinonasal intestinal-type carcinoma.

Inhibition of IL-8 Production by Green Tea Polyphenols in Human Nasal Fibroblasts and A549 Epithelial Cells

The attraction of leukocytes to tissues is essential in order for inflammation and the host response to infection to occur. Airway inflammation is a very common cause illness with a substantial impact on health care. Neutrophils play an essential role in the host defense and in inflammation, but the latter may trigger and sustain the pathogenesis of a range of acute and chronic diseases. Infiltration of neutrophils occurs as a response to chemoattractant molecules by resident tissue cells. The recruitment of neutrophils in airway inflammation may account for the generation of IL-8. To evaluate the effectiveness of green tea polyphenols in the modulation of airway inflammation through the blocking of neutrophil chemokine production, nasal mucosal fibroblasts and A549 bronchial epithelial cells were analyzed for the production of IL-8. Both nasal mucosal fibroblasts and bronchial epithelial cells produced significant amounts of IL-8 through stimulation of IL-1beta. Tea polyphenols were very effective in the inhibition of IL-8 production. Among the polyphenols tested, EGCG and ECG showed strong inhibitory activity in dose-dependent manners. EGC and EC showed moderate inhibition at 48 h culture, whereas (-)catechin was not effective. Production of IL-8 after stimulation by proinflammatory cytokines in both nasal fibroblasts and bronchial epithelial cells was significantly blocked by pretreatment with green tea polyphenols.

Expression of TLR2 and TLR4 Messenger RNA in the Epithelial Cells of the Nasal Airway

Epithelium of nasal mucosa is the first line of defense against invading pathogens. This study investigated the expression of Toll-like receptor (TLR) 2 and TLR4 in epithelial cells of nasal mucosa and understood the role of TLRs in the innate immunity of nasal mucosa. Human nasal epithelial cells were obtained by scraping the middle one-third of inferior turbinates from 30 patients with chronic rhinosinusitis and 20 healthy adult volunteers. The epithelial cells are made into smears. In situ hybridization was performed for TLR2 and TLR4 messenger RNA (mRNA). TLR2 and TLR4 mRNA were expressed in the nasal epithelial cells. The expression of the two genes was significantly higher in the chronic rhinosinusitis group than in the normal control (TLR2, t = 8.605, p < 0.0005; TLR4, t = 9.050, p < 0.0005). This study is the first to establish the presence of both TLR2 and TLR4 mRNA on epithelial cells of nasal mucosa, and their expression can be up-regulated in infectious conditions. These results show that TLR2 and TLR4 may play a important role in local host defense of nasal mucosa.

Characterization of a neuraminidase-deficient influenza a virus as a potential gene delivery vector and a live vaccine.

We recently identified a packaging signal in the neuraminidase (NA) viral RNA (vRNA) segment of an influenza A virus, allowing us to produce a mutant virus [GFP(NA)-Flu] that lacks most of the NA open reading frame but contains instead the gene encoding green fluorescent protein (GFP). To exploit the expanding knowledge of vRNA packaging signals to establish influenza virus vectors for the expression of foreign genes, we studied the replicative properties of this virus in cell culture and mice. Compared to wild-type virus, GFP(NA)-Flu was highly attenuated in normal cultured cells but was able to grow to a titer of >10(6) PFU/ml in a mutant cell line expressing reduced levels of sialic acid on the cell surface. GFP expression from this virus was stable even after five passages in the latter cells. In intranasally infected mice, GFP was detected in the epithelial cells of nasal mucosa, bronchioles, and alveoli for up to 4 days postinfection. We attribute the attenuated growth of GFP(NA)-Flu to virion aggregation at the surface of bronchiolar epithelia. In studies to test the potential of this mutant as a live attenuated influenza vaccine, all mice vaccinated with >/==" BORDER="0">10(5) PFU of GFP(NA)-Flu survived when challenged with lethal doses of the parent virus. These results suggest that influenza virus could be a useful vector for expressing foreign genes and that a sialidase-deficient virus may offer an alternative to the live influenza vaccines recently approved for human use.

Desloratadine inhibits GM-CSF secretion from nasal mucosa and polyps and eosinophil survival induced by epithelial cells

Rationale Epithelial cells release cytokines such as GM-CSF that contribute to eosinophil inflammation in allergic rhinitis and nasal polyposis. The aim of this study was to investigate the effect of desloratadine on both GM-CSF secretion and eosinophil survival. Methods Epithelial cells from nasal mucosa (NM) and polyps (NP) were cultured and stimulated with 10% fetal bovine serum (FBS) with added desloratadine for 24 hr. GM-CSF was measured in culture supernatants (ECM) by ELISA. Peripheral blood eosinophils were cultured with ECM and desloratadine and eosinophil survival assessed by Trypan blue dye exclusion. Results are expressed as mean ± SEM of GM-CSF secretion (pg/ml) or eosinophil survival (%). Results FBS increased GM-CSF secretion (p<0.01) in epithelial cell cultures from both NM (414 ± 106, n=9) and NP (256 ± 142, n=9), compared to control media (NM=9 ± 3; NP=20 ± 11). GM-CSF-induced secretion was significantly inhibited (p<0.05) by desloratadine in both NM (up to −77%) and NP (up to −69%). Desloratadine also showed an inhibitory effect on eosinophil survival induced by ECM from both NM and NP. At 72 hr, 10-5 M desloratadine inhibited (p<0.05) eosinophil survival induced by ECM from NM (15 ± 8%, n=6) and NP (12 ± 8%, n=8) compared to ECM alone (NM=33 ± 9%; NP=43 ± 10%). Conclusions Desloratadine inhibits both GM-CSF secretion from both nasal mucosa and nasal polyp epithelial cells and eosinophil survival induced by epithelial cell secretions, suggesting that this H1 antagonist may have an antiinflammatory effect on eosinophil inflammation.

Expression of the glucocorticoid receptor alpha and beta isoforms in human nasal mucosa and polyp epithelial cells

The lower sensitivity of the inflamed nasal mucosa to glucocorticoids might be related to an increased expression of the glucocorticoid receptor (GR) beta isoform. We investigated GR α and GR β mRNA expression in epithelial cells from nasal mucosa and nasal polyps. GR α mRNA was at least 1000 times more expressed than GR β mRNA in both tissues. GR β expression (mean± SEM of 10 3 cDNA copies/μg of total RNA) was higher in nasal polyps (1.15±0.19; n=27; P<0.01) than in nasal mucosa (0.62±0.10; n=32). Nasal polyps with>3% of inflammatory cells had higher GR β levels (1.40±0.29; n=16) than both nasal mucosa ( P<0.01) and polyps with ≤3% of inflammatory cells (0.80±0.18; n=11; P<0.05). No difference in GR β expression was found between nasal mucosa and polyps with ≤ 3% of inflammatory cells. GR β expression correlated with the inflammatory cell number, especially with mast cells ( r=0.50, P<0.0001). There was no difference in GR α mRNA expression between nasal mucosa and nasal polyps. In summary, GR α is far more expressed than GR β in both tissues. The increased expression of GR β may be related to the presence of inflammatory cells.

Effect of budesonide and nedocromil sodium on IL-6 and IL-8 release from human nasal mucosa and polyp epithelial cells

We investigated the effect of budesonide and nedocromil sodium on the secretion of IL-6 and IL-8 by cultured epithelial cells from healthy nasal mucosa and nasal polyps. Human epithelial cell conditioned media was generated with fetal calf serum (FCS) in the presence or absence of budesonide and/or nedocromil sodium. Budesonide inhibited FCS-induced IL-6 and IL-8 release in a dose-dependent manner. The IC25(25% inhibitory concentration) of budesonide on IL-6 release was higher in nasal polyp than in nasal mucosa epithelial cells (34 n Mvs. 200 p M). The IC25of budesonide on IL-8 release was higher in nasal mucosa than in nasal polyps (145 p Mvs. 4 pM ). Nedocromil sodium caused a dose-related inhibitory effect on IL-8 release from nasal mucosa (IC25, 207 nM ), while it only had a significant effect in nasal polyps at 10−5M. Nedocromil sodium had no effect on IL-6 release. The inhibitory effect of budesonide was higher than that of nedocromil sodium on both nasal polyps and nasal mucosa. Budesonide and nedocromil sodium may exert their anti-inflammatory action in the respiratory mucosa by modulating the secretion of IL-6 and IL-8. The different effect of budesonide and nedocromil sodium on IL-6 and IL-8 release may be explained by differences in the mechanisms which regulate the upregulation of these cytokines in inflammatory responses.

Study of the favorable culture condition to produce granulocyte. Macrophage colony stimulating factor and interleukin-6 by human nasal epithelial cells

Allergic rhinitis and nasal polyposis are upper airway inflammatory conditions characterized by increased numbers of eosinophils and metachromatic cells in the epithelial layer of the nasal mucosa. However, the mechanism by which these cells accumulate still remains obscure in many respects. It is suggested that the granulocyte-macrophage colony stimulating factor (GM-CSF) plays an important role in the nasal mucosa, since the supernatant of cultured epithelial cells induce differentiation of peripheral mononuclear cells into metachromatic cells and eosinophils, and this activity was inhibited by the anti-GM-CSF antibody. In addition, the presence of several cytokines in the supernatant of nasal epithelial cell cultures has been reported. These observations suggest the possibility that GM-CSF and cytokines play an important role in the allergic and inflammatory reactions of the nasal mucosa. In the present study epithelial cells of nasal mucosa were cultured under different conditions to find out the optimal conditions of production for GM-CSF and interleukin-6 (IL-6). When the concentration of fetal calf serum (FCS) in the culture medium was varied, the number of cultured epithelial cells and the concentration of GM-CSF and IL-6 in the supernatant showed an FCS concentration-dependent increase. When nasal mucosal epithelial cells were cultured in 15% FCS, production of both GM-CSF and IL-6 was increased with increasing duration of incubation. There was no significant difference in production of GM-CSF or IL-6 between the patients with allergic rhinitis and those with nasal polyps. These results suggest that neither allergic rhinitis and nor nasal polyposis are associated with the ability of epithelial cells to produce cytokines, but the interplay of various factors in the epithelial layer, as well as of diseases, affects their ability to produce cytokines. When the condition of epithelial cell propagation was rendered favorable or unfavorable by varying the concentration of FCS in the culture medium, using the same culture system, production of GM-CSF and IL-6 was increased in a dose-dependent manner (%) favoring epithelial cell growth. These observations indicate that epithelial cells produce GM-CSF and IL-6 only when conditions are favorable for their proliferation is required.

Electron Spin Resonance Spin Trapping Assay and Immunohistochemical Localization of Superoxide Dismutases in the Rat Nasal Mucosa

Lai M-T, Ohmichi T, Ogawa T, Nishizaki K, Masuda Y. Electron spin resonance spin trapping assay and immunohistochemical localization of superoxide dismutases in the rat nasal mucosa. Acta Otolaryngol (Stockh) 1997; 117: 437-446.The immunohistochemical method and electron spin resonance (ESR) spin trapping assay were employed to detect the localization and biochemical activity of superoxide dismutases (SODs) in the rat nasal mucosa. Manganese SOD and copper-zinc SOD were immunohistochemically illustrated to be richly expressed in the epithelial cells and the subepithelial glands of nasal mucosa. The olfactory vesicles also showed positive immunostaining for manganese SOD and copper-zinc SOD. ESR spin trapping assay revealed that SOD activity in the mucosa of olfactory areas was significantly higher than in the mucosa of respiratory areas; however, the ratio of SOD activity in the mitochondrial fraction to SOD activity in the cytosolic fraction was similar, approximating 17 : 83 in the mucosa of both the olfactory and respiratory areas. The predominant localization of SODs in epithelial cells of nasal mucosa suggests the importance of mucosal epithelium in protecting nasal mucosa against cytotoxic superoxide (O2-) radicals. Epithelial goblet cells and the connective tissue of lamina propria, which showed no positive immunostaining for SODs, are considered to be vulnerable to oxidative insults implicated in the generation of O2- radicals. The higher SODs activity in the mucosa of olfactory areas implies that there is a different requirement of SOD in mucosa of the respiratory and olfactory areas on scavenging microenvironmental O2-radicals.

New insights into the mechanisms of immunotherapy

Allergen-specific immunotherapy retains a place in treatment in patients with allergic rhinitis who fail to respond to conventional treatment with antihistamines and topical corticosteroids. Studies on mechanisms of immunotherapy have previously focussed on changes in serum antibodies, including blunting of seasonal rises in specific IgE and increase in "blocking" specific IgG antibodies. Immunotherapy in patients with rhinitis has also been shown to inhibit effector cells with a decrease in nasal mucosal eosinophils and epithelial mast cells. Recent evidence suggests that these events may be orchestrated by an effect of immunotherapy on T lymphocytes with alteration from a predominant "Th2" response (interleukin 4 and 5) in favour of an additional "Th1" response (gamma interferon) which may decrease tissue eosinophilia and local IgE production. Novel therapeutic approaches to allergic diseases might include use of topical gamma interferon, immunosuppressive agents, anti-CD4 antibodies or strategies directed specifically against IL-4 or IL-5.