1α,25-Dihydroxyvitamin D3 Supplementation during Pregnancy Is Associated with Allergic Rhinitis in the Offspring by Modulating Immunity.

BACKGROUND: Maternal supplementation with 1α,25-dihydroxyvitamin D3 (VD3) has immunologic effects on the developing fetus through multiple pathways. This study was aimed at investigating the effects of VD3 supplementation on immune dysregulation in the offspring during allergic rhinitis. METHODS: Different doses of VD3 as well as control were given to pregnant female mice. Ovalbumin (OVA) challenge and aluminum hydroxide gel in sterile saline were used to induce allergic rhinitis in offspring mice. Nasal lavage fluids (NLF) were collected, and eosinophils were counted in NLF 24 hours after the OVA challenge. Th1, Th2, Th17, and Treg subtype-relevant cytokines, including IFN-γ, IL-4, IL-10, IL-17, TGF-ß, and OVA-IgE levels from the blood and NLF of offspring mice, were detected by the enzyme-linked immunosorbent assay (ELISA) method. The Treg subtype was analyzed by flow cytometry. Treg cells were purified from offspring and were adoptively transferred to OVA-sensitized allogenic offspring mice. The outcomes were assessed in allogenic offspring. RESULTS: Our data showed that VD3 supplementation significantly decreased the number of eosinophils, basophils, and lymphocytes in the peripheral blood and NLF. The proportion of CD4+CD25+FoxP3+Tregs had a positive correlation with VD3 in a dose-dependent manner. The levels of serum IgE, IL-4, and IL-17 were decreased while the expressions of IFN-γ, IL-10, and TGF-ß were significantly enhanced in VD3 supplementation groups. Adoptive transfer CD4+CD25+FoxP3+Tregs of VD3 supplementation groups promoted Th1 and suppressed Th2 responses in the offspring during allergic rhinitis. CONCLUSION: Our findings indicated that low dose VD3 supply in pregnant mice's diet suppressed Th2 and Th17 responses in allergic rhinitis by elevating the Th1 subtype and the proportion of CD4+CD25+FoxP3+Tregs in offspring. It suggested that low dose VD3 supply may have the potential to act as a new therapeutic strategy for allergic rhinitis.

Ex vivo assay to evaluate the efficacy of drugs targeting sphingolipids in preventing SARS-CoV-2 infection of nasal epithelial cells.

This protocol enables the testing of drugs against infection of epithelial cells with SARS-CoV-2 (severe acute respiratory syndrome coronavirus-2), using pseudo-typed replication deficient vesicular stomatitis virus particles (pp-VSV) presenting the SARS-CoV-2 spike protein. After treating human volunteers with amitriptyline, an approved antidepressant and inhibitor of the acid sphingomyelinase, freshly isolated nasal epithelial cells were infected ex vivo and infection levels were quantified. This protocol offers the possibility to rapidly test the efficacy of potential drugs in the fight against COVID-19. For complete details on the use and execution of this protocol, please refer to Carpinteiro et al. (2020).

Choosing the Right Differentiation Medium to Develop Mucociliary Phenotype of Primary Nasal Epithelial Cells In Vitro.

In vitro differentiation of airway epithelium is of interest for respiratory tissue engineering and studying airway diseases. Both applications benefit from the use of primary cells to maintain a mucociliated phenotype and thus physiological functionality. Complex differentiation procedures often lack standardization and reproducibility. To alleviate these shortfalls, we compared differentiation behavior of human nasal epithelial cells in four differentiation media. Cells were differentiated at the air-liquid interface (ALI) on collagen-coated inserts. Mucociliary differentiation status after five weeks was analyzed by electron microscopy, histology and immunohistochemistry. The amount of ciliation was estimated and growth factor concentrations were evaluated using ELISA. We found that retinoic-acid-supplemented mixture of DMEM and Airway Epithelial Cell Growth Medium gave most promising results to obtain ciliated and mucus producing nasal epithelium in vitro. We discovered the balance between retinoic acid (RA), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF) and fibroblast growth factor ß (FGF-ß) to be relevant for differentiation. We could show that low VEGF, EGF and FGF-ß concentrations in medium correspond to absent ciliation in specific donors. Therefore, our results may in future facilitate donor selection and non-invasive monitoring of ALI cultures and by this contribute to improved standardization of epithelial in vitro culture.

Pharmacological characterization of the 3D MucilAir™ nasal model.

The preclinical evaluation of nasally administered drug candidates requires screening studies based on in vitro models of the nasal mucosa. The aim of this study was to evaluate the morpho-functional characteristics of the 3D MucilAir™ nasal model with a pharmacological focus on [ATP]-binding cassette (ABC) efflux transporters. We initially performed a phenotypic characterization of the MucilAir™ model and assessed its barrier properties by immunofluorescence (IF), protein mass spectrometry and examination of histological sections. We then focused on the functional expression of the ABC transporters P-glycoprotein (P-gp), multidrug resistance associated protein (MRP)1, MRP2 and breast cancer resistance protein (BCRP) in bidirectional transport experiments. The MucilAir™ model comprises a tight, polarized, pseudo-stratified nasal epithelium composed of fully differentiated ciliated, goblet and basal cells. These ABC transporters were all expressed by the cell membranes. P-gp and BCRP were both functional and capable of actively effluxing substrates. The MucilAir™ model could consequently represent a potent tool for evaluating the interaction of nasally administered drugs with ABC transporters.

The potential of Olea europaea extracts to prevent TGFß1-induced epithelial to mesenchymal transition in human nasal respiratory epithelial cells.

BACKGROUND: One of the molecular mechanisms involved in upper airway-related diseases is epithelial-to-mesenchymal transition (EMT). Olea europaea (OE) has anti-inflammatory properties and thus, great potential to prevent EMT. This study aimed to investigate the effect of OE on EMT in primary nasal human respiratory epithelial cells (RECs). METHODS: Respiratory epithelial cells were isolated and divided into four groups: control (untreated), treated with 0.05% OE (OE group), EMT induced with 5 ng/ml of transforming growth factor beta-1 (TGFß1 group) and treated with 5 ng/ml TGFß1 + 0.05% OE (TGFß1 + OE group). The effects of OE treatment on growth kinetics, morphology and protein expression in RECs were evaluated. Immunocytochemistry analysis was performed to quantitate the total percentage of E-cadherin and vimentin expression from day 1 to day 3. RESULTS: There were no significant differences between untreated RECs and OE-treated RECs in terms of their morphology, growth kinetics and protein expression. Induction with TGFß1 caused RECs to have an elongated spindle shape, a slower proliferation rate, a higher expression of vimentin and a lower expression of E-cadherin compared with the control. Cells in the TGFß1 + OE group had similar epithelial shape to untreated group however it had no significant differences in their proliferation rate when compared to TGFß1-induced RECs. Cells treated with TGFß1 + OE showed significantly reduced expression of vimentin and increased expression of E-cadherin compared with the TGFß1 group (P < 0.05). CONCLUSION: The ability of OE to inhibit EMT in RECs was shown by TGFb1-induced EMT REC morphology, growth kinetics and protein expression markers (E-cadherin and vimentin) upon treatment with OE and TGFß1. Therefore, this study could provide insight into the therapeutic potential of OE to inhibit pathological tissue remodelling and persistent inflammation.

[Experimental study on the effect of Yiqi Wenyang Decoction on nasal mucosa infiltration of NK cells in mice with allergic rhinitis].

Objective: To observe the effect of Yiqi Wenyang Decoction on the infiltration and activation of NK cells in nasal mucosa of mouse model with allergic rhinitis (AR), and to explore the potential mechanism for effective intervention of AR with Yiqi Wenyang Decoction. Methods: Fourty-eight mice were randomly divided into blank group, model group, low, medium and high dose of Yiqi Wenyang Decoction group and Cetirizine group, with 8 rats in each group. After modeling of AR, the model group was filled with 0.9% sodium chloride solution. Yiqi Wenyang Decoction groups of each dose were given different concentrations of Yiqi Wenyang Decoction water extract, while the Cetirizine group was given aqueous solution of Cetirizine. The behavior, morphological changes of nasal mucosa and infiltration of NK cells in nasal mucosa were observed. The levels of IL-4 and INF-γ in nasal lavage fluid were measured. Besides, the drug safety was observed by acute toxicity test. Results: In the respect of behavioral scoring, middle and high dose of Yiqi Wenyang Decoction group were superior to the model group (number of sneezing: q value was 7.189, 8.748, respectively; number of scratching nose: q value was 12.074, 14.560, respectively; all P<0.05). In middle and high dose of Yiqi Wenyang Decoction group, the infiltration of NK cells and nasal lavage fluid IL-4 levels were lower than those in model group (IOD: q value was 10.073, 12.322, respectively; IOD/Area: q value was 10.954, 14.073, respectively; IL-4: q value was 4.705, 6.801, respectively; all P<0.05). There was no significant difference in nasal lavage fluid of INF-γ among each group (Fv=1.166, P>0.05). In acute toxicity test, no obvious poisoning symptoms and death occurred in mice. Conclusion: Yiqi Wenyang Decoction can control the nasal symptom, reduce the local NK cell infiltration of nasal mucosa and inhibit the expression of the 2-type cytokines released by NK cells, which may be related with the potential mechanism of effective intervention of AR with Yiqi Wenyang Decoction.

In vitro nasal mucosa gland-like structure formation on a chip.

The emergence of microfluidic epithelial models using diverse types of cells within a physiologically relevant microenvironment has the potential to be a powerful tool for preclinical drug screening and pathophysiological studies. However, to date, few studies have reported the development of a complicated in vitro human nasal epithelial model. The aim of this study was to produce an in vitro human nasal mucosa model for reliable drug screening and clinical applications. Here, we integrated and optimized several culture conditions such as cell type, airway culture conditions, and hydrogel scaffolds into a microfluidic chip to construct an advanced in vitro human nasal mucosa model. We observed that the inducing factors for nasal gland-like structures were secreted from activated human dermal microvascular endothelial cells. Furthermore, our in vitro nasal mucosa presented different appearance and characteristics under hypoxic conditions. Morphological and functional similarities between in vivo nasal mucosa and our model indicated its utilization as a reliable research model for nasal diseases including allergic rhinitis, chronic sinusitis, and nasal polyposis.

Chloroquine Sensitizes Nasopharyngeal Carcinoma Cells but Not Nasoepithelial Cells to Irradiation by Blocking Autophagy.

BACKGROUND: Treatment of nasopharyngeal carcinoma requires the application of high dosages of radiation, leading to severe long-term complications in the majority of patients. Sensitizing tumor cells to radiation could be a means to increase the therapeutic window of radiation. Nasopharyngeal carcinoma cells display alterations in autophagy and blockade of autophagy has been shown to sensitize them against chemotherapy. METHODS: We investigated the effect of chloroquine, a known inhibitor of autophagy, on sensitization against radiation-induced apoptosis in a panel of five nasopharyngeal carcinoma cell lines and a SV40-transformed nasoepithelial cell line. Autophagy was measured by immunoblot of autophagy-related proteins, immunofluorescence of autophagosomic microvesicles and electron microscopy. Autophagy was blocked by siRNA against autophagy-related proteins 3, 5, 6 and 7 (ATG3, ATG5, ATG6 and ATG7). RESULTS: Chloroquine sensitized four out of five nasopharyngeal cancer cell lines towards radiation-induced apoptosis. The sensitizing effect was based on the blockade of autophagy as inhibition of ATG3, ATG5, ATG6 and ATG7 by specific siRNA could substitute for the effect of chloroquine. No sensitization was seen in nasoepithelial cells. CONCLUSION: Chloroquine sensitizes nasopharyngeal carcinoma cells but not nasoepithelial cells towards radiation-induced apoptosis by blocking autophagy. Further studies in a mouse-xenograft model are warranted to substantiate this effect in vivo.

Azelastine desensitization of transient receptor potential vanilloid 1: a potential mechanism explaining its therapeutic effect in nonallergic rhinitis.

BACKGROUND: Capsaicin, a prototypic transient receptor potential vanilloid 1 (TRPV1) agonist, has been shown to be more clinically effective in the treatment of nonallergic rhinitis (NAR) compared with other rhinitis subtypes. Azelastine has also been found to be clinically effective in the treatment of NAR but its mechanism(s) of action is still poorly elucidated. This study was designed to determine, using in vitro cell lines, whether topical therapies including azelastine have activity on TRPV1 ion channels similar to capsaicin. METHODS: The effects of capsaicin (1 µM), azelastine (30 µM), bepotastine (10 µM), olopatadine (10 µM), and fluticasone (200 µM) on TRPV1 channels using mice neuronal cells (Cath.a), as surrogates for submucosal sensory neurons, and human nasal epithelial cells (hNEC) were determined and compared. For azelastine, bepotastine, and capsaicin, which elicited an agonist effect on TRPV1, live cell [Ca(2+)] signaling in Cath.a cells and hNECs expressing TRPV1 were performed in the absence and presence of capsazepine at 10 µM (a TRPV1 antagonist) or using wild-type mouse embryonic fibroblasts (wtMEFs) that express TRPV1 ion channels and TRPV1 homozygous null mutant (TRPV1-/-) knockout MEF cells as controls to establish TRPV1 channel selectivity. As azelastine has previously been found clinically effective in NAR, additional experiments were performed to determine its ability to desensitize TRPV1 ion channels and its effect on regulating intracellular calcium homeostasis. RESULTS: Cath.a cells treated with azelastine, bepotastine, or capsaicin showed a significant increase in TRPV1-dependant (Ca(2+)) specific cytosolic fluorescence. Continuous treatment with azelastine or capsaicin resulted in desensitization of TRPV1 channels. In hNECs, azelastine stimulation resulted in Ca(2+) shifts from the cytosol to mitochondria and overexpression of hematopoietic cell-specific Lyn substrate 1-associated protein X1, which may thus be effective in cytosolic Ca(2+) homeostasis. CONCLUSION: Azelastine, similar to capsaicin, exhibits direct activity on TRPV1 ion channels that may represent a novel mechanistic pathway explaining its clinical efficacy in NAR.

Preparation of mPEG2000-PLA-modified Xingnaojing microemulsion and evaluation in mucosal irritation.

Xingnaojing microemulsion (XNJ-M) administered intranasally is used for stroke treatment. Methoxy poly(ethylene glycol)-poly(lactide) (mPEG-PLA) is a block copolymer with surfactant-like properties and good biodegradability and reliable biological safety. In order to investigate the possibility to lower mucosa irritation with XNJ-M, XNJ-M modified by mPEG2000-PLA (XNJ-MM) was prepared. Different ratios of mPEG2000-PLA were synthesized. The structures and properties were confirmed by 1H-NMR, IR, and DSC. The hydrophile-lipophile balance (HLB) value and critical micellar concentration of copolymers were investigated. The in situ toad palate model was adopted to investigate the ciliotoxicity of the copolymers. The XNJ-M and XNJ-MM were prepared by dropping aqueous phase method. Mucosal irritation of different Xingnaojing (XNJ) preparations was studied by behavioral observations including sneezing and scratching nose. The epithelial thickness of nasal mucosa was evaluated and the secretory protein concentration was determined. The results of in situ toad palate model demonstrated that high HLB value mPEG2000-PLA basically showed no ciliotoxicity, while EL-35 had significant dose-dependent ciliotoxicity (P<0.05). The irritating effects of mPEG2000-PLA were significantly lower than EL-35 after seven days of treatment, based on the epithelial thickness of 0.429±0.100 mm and 0.700±0.035 mm, respectively (P<0.01). The epithelial thickness with XNJ-MM was lower than XNJ-M after seven days of treatment: 0.620±0.10 mm vs. 0.809±0.153 mm, respectively (P<0.05). The mPEG2000-PLA-modified microemulsion is a promising dosage form of XNJ, based on reduced irritation on nasal mucosa.

Advances in asthma drug discovery: evaluating the potential of nasal cell sampling and beyond.

INTRODUCTION: Inhaled corticosteroid anti-inflammatory therapy is effective at controlling disease symptoms of asthma, but a subset of patients remains symptomatic despite optimal treatment, creating a clear unmet medical need. Moreover, none of the currently available drugs for asthma are really disease-modifying or curative. Although murine models of asthma, based on transgenic and knockout animals, may offer an integrated pathophysiological system for studying the characteristics of airway inflammation and hyperresponsiveness, these alterations are noteworthily different compared with those observed in asthmatic patients. Since a clear functional and inflammatory relationship between the nasal mucosa and bronchial tissue in patients suffering from asthma and allergic rhinitis has been recognized, using preclinical models based on human nasal cells sampling might support a prompt and effective anti-inflammatory drug discovery in asthma. AREAS COVERED: The authors provide a review, which discusses the potential role of nasal cell sampling and its application in advanced drug discovery for asthma. The contents range from the similarities and differences between asthma and allergic rhinitis up to artificial airway models based on sophisticated human lung-on-a-chip devices. EXPERT OPINION: Nasal cell sampling and processing have reached a great potential in asthma drug discovery. The authors believe that models of asthma, which are based on human nasal cells, can provide valuable indications of proof of pharmacological and potential therapeutic efficacy in both preclinical and early clinical settings.

Efficacy of the oral chemoattractant receptor homologous molecule on TH2 cells antagonist BI 671800 in patients with seasonal allergic rhinitis.

BACKGROUND: The inflammatory response in patients with seasonal allergic rhinitis (SAR) is partly mediated by the prostaglandin D2 receptor chemoattractant receptor homologous molecule on T(H)2 cells (CRTH2). OBJECTIVE: We sought to investigate the efficacy and safety of the oral CRTH2 antagonist BI 671800 (50, 200, and 400 mg twice daily), fluticasone propionate nasal spray (200 µg once daily), or oral montelukast (10 mg once daily) administered for 2 weeks in patients with SAR. METHODS: In this randomized, double-blind, placebo-controlled, partial-crossover study, participants aged 18 to 65 years with a positive skin prick test to Dactylis glomerata pollen were exposed to out-of-season allergen in the environmental challenge chamber for 6 hours. The primary efficacy variable was the total nasal symptom score assessed as the area under the curve (AUC)(0-6h). RESULTS: In total, 146 patients (63.7% male; mean age, 36.1 years) were randomized. The adjusted mean total nasal symptom score AUC(0-6h) was significantly reduced versus placebo with 200 mg of BI 671800 (absolute difference, -0.85; percentage difference, -17%; P = .0026), montelukast (absolute difference, -0.74; percentage difference, -15%; P = .0115), and fluticasone propionate (absolute difference, -1.64; percentage difference, -33%; P < .0001). Compared with placebo, BI 671800 significantly reduced nasal eosinophil values (P < .05 for all doses), significantly inhibited nasal inflammatory cytokine levels (IL-4 and eotaxin, P < .05; 200 mg twice daily), and induced a dose-related reduction in ex vivo prostaglandin D2-mediated eosinophil shape change. CONCLUSION: Two hundred milligrams of BI 671800 twice daily demonstrated efficacy in treating SAR symptoms induced by environmental challenge chamber allergen exposure and had a favorable safety profile.

Isolation and culture of primary human nasal epithelial cells from anesthetized nasal epithelia.

CONCLUSION: Using a local anesthetic agent before obtaining nasal biopsies by nasal brushing makes the sampling procedure smooth, avoids lacrimation, nasal itching/irritation, and/or sneezing and provides enough viable cells to establish primary cultures. OBJECTIVES: To examine the use of local anesthesia to avoid the irritation experienced by the subject when nasal biopsies are obtained by nasal brushing in order to culture viable nasal epithelial cells. METHODS: Nasal epithelial cells were collected from the mid-part of the inferior turbinate of healthy volunteers by brushing with interdental brushes, after spraying a topical anesthetic on the nasal mucosa. Immunocytochemistry was performed to assess the purity of epithelial cells. RESULTS: Cell samples ranging from 1.16 × 10(5) to 3.06 × 10(5) cells/per sample were obtained. Of 11 samples, 7 formed confluent cultures, while the remaining 4 samples showed only patches of epithelial cells. Neither fungal nor bacterial contamination posed a problem. Immunocytochemistry of the cytospin slides confirmed the presence of epithelial cells in the cultures. No adverse effects were experienced by the volunteers.

Anti-allergic effects of So-Cheong-Ryong-Tang, a traditional Korean herbal medicine, in an allergic rhinitis mouse model.

The herbal medicine, So-Cheong-Ryong-Tang (SCRT) has been empirically used for the treatment of allergic rhinitis for hundreds of years; however, its in vivo effects on allergic rhinitis have been rarely elucidated. We aimed to evaluate the anti-allergic effects of SCRT in an allergic rhinitis mouse model and to examine the underlying mechanism(s) of its anti-allergic effects. BALB/c mice were sensitized with ovalbumin (OVA) and alum and then challenged intranasally with OVA. SCRT (1 g/kg) was given to the treatment group, and multiple parameters of allergic responses were evaluated to determine the effects of SCRT on allergic rhinitis. SCRT reduced allergic symptoms, such as rubbing and sneezing, and eosinophil infiltration into the nasal mucosa. It also suppressed serum total IgE, OVA-specific IgE, and OVA-specific IgG1 levels and increased OVA-specific IgG2a level. SCRT significantly reduced expression of the Th2 cytokine, IL-4; however, the expression of IL-5, IFN-γ, and IL-10 was unchanged in the nasal mucosa of the treatment group (by real-time RT-PCR). In splenocyte culture, levels of both IL-4 and IL-5 decreased, and IFN-γ level increased in the treatment group; however, levels of IL-10 and TGF-ß were unaffected by administration of SCRT. This study shows that SCRT induced anti-allergic effects by decreasing, locally and systemically, the Th2 cytokine IL-4, isotype switching to IgE, and eosinophilic infiltration into the nasal mucosa in an allergic rhinitis mouse model. Thus, SCRT might be considered a potential therapeutic agent in treating allergic rhinitis.

Hop water extract inhibits double-stranded RNA-induced thymic stromal lymphopoietin release from human nasal epithelial cells.

BACKGROUND: Thymic stromal lymphopoietin (TSLP) acts as a master switch for allergic inflammation and plays a key role in allergic diseases, including allergic rhinitis. Double-stranded RNA (dsRNA) recognized by Toll-like receptor 3 (TLR3) strongly activates TSLP release from human nasal epithelial cells (HNECs). Hop (Humulus lupulus L.) extracts have been shown to have potent pharmacologic effects on inflammation. METHODS: To investigate whether a hop water extract (HWE) prevents TSLP release from HNECs, human telomerase reverse transcriptase (hTERT)-transfected HNECs, used as a model of normal HNECs, were pretreated with HWE before treatment with the TLR3 ligand Polyinosine-polycytidylic acid (poly[I:C]). RESULTS: In the hTERT-transfected HNECs, treatment with HWE significantly reduced poly(I:C)-induced production and release of TSLP in a dose-dependent manner, as well as dexamethasone. Treatment with the protein kinase C (PKC) inhibitor GF109203X and NF-κB inhibitor IMD-0354 also reduced poly(I:C)-induced TSLP release from hTERT-transfected HNECs. Treatment with HWE efficiently prevented up-regulation of PKC activity by 12-O-tetradecanoyl phorbol-13-acetate but not NF-κB activity induced by IL-1ß in hTERT-transfected HNECs. CONCLUSION: Our results clearly indicated that HWE inhibited dsRNA-induced production and release of TSLP via a PKC signal pathway in HNECs and it may have potent preventive effects against allergic rhinitis.

Experimentally induced accumulation of Foxp3⁺ T cells in upper airway allergy.

BACKGROUND: It has been suggested that Foxp3(+) regulatory T (Treg) cells inhibit allergic inflammation in humans by suppressing the activation of allergen-specific effector T cells. Whether this occurs at the site of allergen exposure has not been determined. OBJECTIVE: To determine the occurrence of Foxp3(+) Treg cells in the nasal mucosa of allergic rhinitis (AR) patients and non-allergic controls after a nasal allergen challenge. METHODS: Pollen-allergic patients (n=18) and non-allergic volunteers (n=7) were challenged locally with pollen extract or placebo for 7 days outside the pollen season. Mucosal biopsies were obtained from the inferior turbinate on days 0, 1 and 7 and subjected to multi-colour immunofluorescence and blood was drawn for eosinophil counts on days 0, 2, 5 and 7. RESULTS: Only AR patients receiving pollen extract experienced typical allergic symptoms and demonstrated increased levels of eosinophils in peripheral blood and nasal mucosa. In allergic patients, a transient early increase (day 1) in CD3(+) T cells was observed in the nasal mucosa, followed by a significant increase of Foxp3(high) T cells at day 7. No changes were found in the control group. The majority of Foxp3(high) cells co-expressed CTLA-4, CD25 and CD4, and a substantial fraction expressed the proliferation marker Ki67. CONCLUSION AND CLINICAL RELEVANCE: Experimentally induced inflammation in AR patients leads to an early inflammatory response followed by accumulation of Foxp3(high) T cells in the nasal mucosa. Our findings are similar to that observed in allergic airways of experimental mice, which suggest that Treg cells are operative in allergic upper airway inflammation. It should be explored whether Treg cells accumulating in the nasal mucosa could be targets for therapeutic intervention.

The innate osteogenic potential of the maxillary sinus (Schneiderian) membrane: an ectopic tissue transplant model simulating sinus lifting.

Maxillary sinus membrane lifting is a common procedure aimed at increasing the volume of the maxillary sinus osseous floor prior to inserting dental implants. Clinical observations of bone formation in sinus lifting procedures without grafting bone substitutes were observed, but the biological nature of bone regeneration in sinus lifting procedures is unclear. This study tested whether this osteogenic activity relies on inherent osteogenic capacity residing in the sinus membrane by simulating the in vivo clinical condition of sinus lifting in an animal model. Maxillary sinus membrane cells were cultured in alpha-MEM medium containing osteogenic supplements (ascorbic acid, dexamethasone). Cultured cells revealed alkaline phosphatase activity and mRNA expression of osteogenic markers (alkaline phosphatase, bone sialoprotein, osteocalcin and osteonectin) verifying the osteogenic potential of the cells. Fresh tissue samples demonstrated positive alkaline phosphatase enzyme activity situated along the membrane-bone interface periosteum-like layer. To simulate the in vivo clinical conditions, the membranes were folded to form a pocket-like structure and were transplanted subcutaneously in immunodeficient mice for 8 weeks. New bone formation was observed in the transplants indicating the innate osteogenic potential within the maxillary Schneiderian sinus membrane and its possible contribution to bone regeneration in sinus lifting procedures.

The role of CCL22/macrophage-derived chemokine in allergic rhinitis.

Dendritic cells (DCs) are considered to be the most powerful antigen-presenting cells (APCs). DCs are thought to be associated with Th1 or Th2 polarization and with polarization-induced disease such as atopic dermatitis, asthma and allergic rhinitis, but its mechanism is not well known. In this study, we analyzed the mRNA expression of DCs between birch pollen allergic rhinitis and healthy controls by using cDNA array. We found that the expressions of CCL22/macrophage-derived chemokine (MDC) differed significantly. We also revealed that CCL22/MDC production was higher in patients than in healthy donors. By chemotaxis assay, CCL22/MDC can enhance the migration of patient's T cells rather than those of healthy controls. Surface marker analysis of migrated cells revealed that the most of migrated cells expressed CCR4, which were considered to be Th2 cells. Furthermore, CD1a(+) CD83(+) cells located in the nasal mucosa expressed CCL22/MDC in vivo. To the best of our knowledge, this is the first report clearly indicating the role of CCL22/MDC in allergic rhinitis.

Comprehensive interaction of dicalcin with annexins in frog olfactory and respiratory cilia.

Dicalcin (renamed from p26olf) is a dimer form of S100 proteins found in frog olfactory epithelium. S100 proteins form a group of EF-hand Ca(2+)-binding proteins, and are known to interact with many kinds of target protein to modify their activities. To determine the role of dicalcin in the olfactory epithelium, we identified its binding proteins. Several proteins in frog olfactory epithelium were found to bind to dicalcin in a Ca(2+)-dependent manner. Among them, 38 kDa and 35 kDa proteins were most abundant. Our analysis showed that these were a mixture of annexin A1, annexin A2 and annexin A5. Immunohistochemical analysis showed that dicalcin and all of these three subtypes of annexin colocalize in the olfactory cilia. Dicalcin was found to be present in a quantity almost sufficient to bind all of these annexins. Colocalization of dicalcin and the three subtypes of annexin was also observed in the frog respiratory cilia. Dicalcin facilitated Ca(2+)-dependent liposome aggregation caused by annexin A1 or annexin A2, and this facilitation was additive when both annexin A1 and annexin A2 were present. In this facilitation effect, the effective Ca(2+) concentrations were different between annexin A1 and annexin A2, and therefore the dicalcin-annexin system in frog olfactory and respiratory cilia can cover a wide range of Ca(2+) concentrations. These results suggested that this system is associated with abnormal increases in the Ca(2+) concentration in the olfactory and other motile cilia.

Effects of pollen and nasal glucocorticoid on FOXP3+, GATA-3+ and T-bet+ cells in allergic rhinitis.

BACKGROUND: T-regulatory cells (Treg) affect the balance of T(H)2 and T(H)1 cells. Treg, T(H)2 and T(H)1 cells are regulated by the FOXP3, GATA-3 and T-bet transcription factors respectively. Our aim was to determine the number of FOXP3(+), GATA-3(+) and T-bet(+) cells in nasal mucosa in symptom-free allergic rhinitis (AR) patients vs healthy controls, as well as the effects of natural pollen exposure and concomitant nasal glucocorticoid treatment on these cells. METHODS: Nasal biopsies were taken from healthy controls and patients with grass-pollen AR preseason. The AR patients were randomized to receive treatment with either fluticasone propionate (FP) or a placebo, and additional biopsies were taken during the pollen season. FOXP3(+), GATA-3(+) and T-bet(+) cells in nasal mucosa were quantified by immunohistochemistry. RESULTS: The number of FOXP3(+) and GATA-3(+) cells, but not T-bet(+) cells, was significantly higher in AR patients vs controls preseason. The number of FOXP3(+) cells remained unchanged in the former group after the pollen season but decreased significantly in the nasal mucosa as a result of FP treatment. The pollen season substantially increased the number of GATA-3(+) cells, which was inhibited by FP. The number of T-bet(+) cells was not affected by pollen or FP. CONCLUSION: These data suggest that nasal glucocorticoids attenuate the allergic inflammation partly by reducing the number of T(H)2 cells, but not by means of local upregulation of Treg cells. The local relationship between T(H)1 and T(H)2 cells as well as between Treg and T(H)2 is maintained by nasal glucocorticoid treatment.