Eye Washing Downregulated Angiotensin-Converting Enzyme 2 in Conjunctival Tissue Samples from Smokers.

This study aimed to (1) determine whether the expression of angiotensin-converting enzyme 2 (ACE2) and transmembrane protease serine 2 is increased in tobacco smokers, which potentially increases their susceptibility to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, and (2) assess whether eye rinsing can reduce susceptibility. This prospective study included 20 eyes of 10 smokers and 18 eyes of nine healthy non-smokers (control) for reverse-transcription polymerase chain reaction. This study also included 28 eyes of 14 smokers and 16 eyes of eight healthy non-smokers (control) for enzyme-linked immunosorbent assay. Tear and impression cytology samples were collected from the right eye of each patient. The left eye was then rinsed for 30 s, and after 5 min, the tear and impression cytology samples were collected in the same manner. The expression of the ACE2 gene was significantly higher in the conjunctiva of smokers (n = 17; median 3.07 copies/ng of total RNA) than in those of non-smokers (n = 17; median 1.92 copies/ng of total RNA, p = 0.003). Further, mRNA expression and protein levels of ACE2 were weakly correlated in smokers (r = 0.49). ACE2 protein levels in Schirmer's strip samples were significantly reduced from 5051 to 3202 pg/mL after eye washing (n = 10; p = 0.001). Ocular surface cells are susceptible to SARS-CoV-2 infection. Smoking may be a risk factor for SARS-CoV-2 infection, and eye rinsing may reduce the risk of infection.

Comprehensive Analysis of the Expression and Functions of Pattern Recognition Receptors in Differentiated Cytotrophoblasts Derived from Term Human Placentas.

Pregnant women are exposed to various microbes, some of which can harm the mother and/or fetus and can lead to life-long morbidity and even death. The syncytiotrophoblast (STB) covers the placental villi and comes into direct contact with pathogens contained in the maternal blood and plays a key role in placental host defense. However, the precise mechanisms whereby the STB recognizes and responds to pathogenic microbes remain unclear. In this study, we comprehensively analyzed the expression of functional pattern recognition receptors, which are responsible for tissue defense against pathogens, in a primary STB model differentiated from highly purified human term cytotrophoblasts (CTBs). Screening for mRNA expression and multiplex cytokine/chemokine production demonstrated that differentiated CTBs (dCTBs) predominantly expressed dsRNA receptors, including TLR3, MDA5, and RIG-I. We confirmed that term human placentas also expressed TLR3. Transcriptome analysis revealed common and unique responses of dCTBs to a synthetic dsRNA (polyinosinic-polycytidylic acid) compared with human peripheral mononuclear cells. Moreover, polyinosinic-polycytidylic acid induced the release of type I and type III IFNs (IFN-ß, IFN-λ1, IFN-λ2, IFN-λ3), as well as mRNA expression of IFN-stimulated genes (IFIT1, MX1, and OAS1). dCTBs underwent apoptosis via the mitochondrial pathway in response to dsRNA stimulation. These results suggest that dsRNA receptors expressed on the STB are key players in antiviral defense in the placenta. Elucidation of the underpinnings of these defense processes can help us better understand the pathophysiology of viral infections during pregnancy.

Axillary Lymph Node Uptake on 18F-FDG PET/CT after COVID-19 Vaccination: A Direct Comparison Study with Influenza Vaccination.

Objectives: To compare vaccinated-side axillary lymph node uptake on 18F-fluorodeoxyglucose (FDG) positron emission tomography/computed tomography (PET/CT) after coronavirus disease-2019 (COVID-19) and influenza vaccination. Methods: We retrospectively analyzed 177 patients who underwent 18F-FDG PET/CT after COVID-19 or influenza vaccination. We compared the uptake of the vaccinated-side axillary lymph nodes of 109 COVID-19 vaccinated patients with those of a lot of influenza-vaccinated patients. We also compared the uptake between 66 patients who received the first COVID-19 vaccination with 43 who received the second COVID-19 vaccination. Results: 18F-FDG-avid axillary lymph nodes on the vaccinated side were significantly more frequently observed in the COVID-19 group (45%) than in the influenza group (19%) (p<0.001). When the interval between vaccination to PET/CT was within 7 days, there was no significant difference in the frequency of 18F-FDG-avid vaccinated-side axillary lymph nodes between the groups (COVID-19 group: 41% vs. influenza group: 45%, p=0.724). When the interval was over 7 days, 18F-FDG-avid lymph nodes were much more frequent in the COVID-19 group (47%) than in the influenza group (7%) (p<0.001). Comparing the first and second COVID-19 groups, 18F-FDG-avid lymph nodes were more frequent in the second vaccination group than in the first vaccination group, but the difference was not significant. Conclusion: 18F-FDG-avid vaccinated-side axillary lymph nodes were more frequently observed in the COVID-19 group than in the influenza group. In the case of the COVID-19 vaccine, a delay of 18F-FDG PET/CT examination is recommended by a longer interval from vaccination than in the influenza vaccine.

Aquaporins 8 and 9 as Possible Markers for Adult Murine Lacrimal Gland Cells.

Aquaporins (AQPs) are proteins that selectively transport water across the cell membrane. Although AQPs play important roles in secretion in the lacrimal gland, the expression and localization of AQPs have not been clarified yet. In the current study, we investigated the expression pattern of AQP family members in the murine lacrimal gland during development. Lacrimal gland tissues were harvested from E13.5 and E17.5 murine embryos and from mice 8 weeks of age (adults). Corneal and conjunctival tissues from the latter served as controls. Total RNA was isolated and analyzed for the expression of AQP family members using qPCR. The localization of AQPs in the adult lacrimal gland in adult murine lacrimal glands was also analyzed. Expression of Aqp8 and Aqp9 mRNAs was detected in the adult lacrimal gland but not in the cornea, conjunctiva, or fetal lacrimal gland. AQP8 and AQP9 and α-SMA partially colocalized around the basal regions of the acinar unit. The levels of Aqp3 mRNAs and protein were much lower in the adult lacrimal gland but were readily detected in the adult cornea and conjunctiva. Our study suggests that AQP8 and AQP9 may serve as markers for adult murine lacrimal gland, ductal, and myoepithelial cells.

The FADS mouse: A novel mouse model of atopic keratoconjunctivitis.

BACKGROUND: Atopic keratoconjunctivitis (AKC) is a chronic allergic conjunctival disease. However, a mouse model of AKC to investigate the underlying mechanism of the therapeutic agents and estimate their efficacy has not been established. We recently generated mice in which Ikk2 is specifically deleted in facial skin fibroblasts and found that these mice spontaneously develop atopic dermatitis (AD)-like facial skin inflammation and scratching behaviors; thus, we named them facial AD with scratching (FADS) mice. OBJECTIVE: We sought to evaluate whether the ocular lesions that FADS mice spontaneously develop are similar to those of patients with AKC and to estimate the efficacy of topical treatments with tacrolimus and betamethasone for FADS mice by using tear periostin, a novel biomarker for allergic conjunctival disease. METHODS: FADS mice, in which Ikk2 is deleted in dermal fibroblasts, were generated by crossing female Ikk2Flox/Flox mice to male Nestincre; Ikk2Flox/+ mice. We conducted histologic analysis of the ocular lesions in FADS mice. Furthermore, we measured periostin in the tears collected from FADS mice untreated or treated with tacrolimus or betamethasone. RESULTS: The FADS mice exhibited severe blepharitis and scratch behaviors for their faces. In these mice, corneal epithelium and stroma showed hyperplasia and infiltration of eosinophils, mast cells, and TH2/TC2 cells. Periostin was significantly expressed in the lesions and tear periostin was upregulated. Betamethasone showed more suppressive effects than did tacrolimus on severe corneal lesions and increased tear periostin level. CONCLUSIONS: The FADS mouse is a novel mouse model of AKC and is useful to examine the therapeutic effects of anti-AKC agents.

Elevated levels of prostaglandin E2 in the tears of patients with severe allergic conjunctivitis and primary cultured conjunctival cells are suppressed by ketotifen and dexamethasone.

OBJECTIVE: We examined the production of prostaglandin E2 (PGE2), which is the key prostaglandin involved in inflammatory disorders of the ocular surface. Tears and conjunctival fibroblasts were evaluated in order to assess allergic inflammation and the effect of specific drugs. METHODS AND ANALYSIS: PGE2 was measured in tears from both patients and normal volunteers. Primary cultures of human conjunctival fibroblasts were incubated with interleukin (IL)-4 and tumour necrosis factor (TNF)-α with or without ketotifen fumarate or dexamethasone. The culture supernatants were removed 24 hours after exposure and the concentrations of PGE2 were quantified by ELISA. RESULTS: Significantly higher levels of PGE2 were observed in the tears of patients with severe allergic conjunctivitis than in those with post-surgical inflammation (p=0.02), and this production was reduced by eye drops. Stimulation with IL-4 and TNF-α induced the generation of PGE2 in supernatants of conjunctival fibroblasts, and this production was significantly downregulated by ketotifen fumarate or steroids. CONCLUSION: PGE2 may participate in the pathogenesis of severe ocular allergic disease, and both ketotifen fumarate and steroid reduce the production of PGE2.

Conjunctival Injection Reduction in Patients with Atopic Keratoconjunctivitis Due to Synergic Effect of Bovine Enteric-Coated Lactoferrin in 0.1% Tacrolimus Ophthalmic Suspension.

Lactoferrin (LF), a multifunctional glycoprotein found in mammalian milk, is reported to have immunoregulatory effects. The present study aimed to evaluate whether enteric-coated LF (eLF) could improve symptoms in patients with atopic keratoconjunctivitis (AKC). This randomized double-blind placebo-controlled single-center trial comprised Japanese patients (n = 20; aged 22-60 years) with AKC. Patients treated with 0.1% tacrolimus ophthalmic suspension (TALYMUS®) were administered eLF (400 mg/d of bovine LF) or placebo tablets for 12 weeks. Conjunctival injection was examined, papillae formation in the palpebral conjunctiva was evaluated, and corneal fluorescein score, itchy sensation in end-point itching scale, and serum allergic parameters were assessed. Conjunctival injection was significantly reduced in the LF group than in the placebo group (p = 0.0017, Mann-Whitney U-test). Papillae formation in the palpebral conjunctiva showed a statistical decrease in the LF group than in the placebo group (p = 0.010, unpaired T-test). LF combined with TALYMUS® could be a promising treatment strategy to mitigate AKC.

COVID-19 pneumonia in a patient with adult T-cell leukemia-lymphoma.

Although some patients with COVID-19 develop only mild symptoms, fatal complications have been observed among those with comorbidities. As patients with cancer are immunocompromised, they are thought to have a high risk of severe illness associated with COVID-19. We report a COVID-19 patient with adult T-cell leukemia-lymphoma (ATL) who was treated using favipiravir. A 69-year-old woman with lymphoma-type ATL was treated using cyclophosphamide, doxorubicin, vincristine, prednisolone and mogamulizumab (M-CHOP) with substantial efficacy. However, in cycle 4 of M-CHOP therapy, she developed fever with mild cough. The patient was admitted to the hospital and CT revealed bilateral ground-glass opacities. SARS-CoV-2 was detected by RT-PCR and the patient was diagnosed with COVID-19. Considering severe immunosuppression caused by ATL, we initiated favipiravir therapy. Subsequently, the fever improved without antipyretics and her C-reactive protein level decreased rapidly. SARS-CoV-2 PCR tests were negative on days 17 and 18 of favipiravir therapy, and the patient was discharged without residual disease on the final CT. This is the first documented case of COVID-19 in a patient with ATL. Although severe immunosuppression caused by ATL was present, severe COVID-19 pneumonia did not develop. The immunosuppressed condition caused by hematological malignancy may not always be a risk factor for severe illness associated with COVID-19. Further accumulation of data regarding COVID-19 in patients with hematological malignancies is warranted to clarify the risk factors for severe illness, the best-in-class antiviral agent, and the optimal treatment strategy in this population.

Effects of Docosahexaenoic Acid on Chemokine Expression in Human Conjunctival Fibroblasts.

Purpose: We assessed the production of chemokines by human conjunctival fibroblasts in response to inflammation and the effects of omega (ω)-3 fatty acids on chemokine expression.Methods: Primary cultures of human conjunctival fibroblasts were incubated with interleukin-4 (IL-4) and tumor necrosis factor-alpha (TNF-α). The expression of eotaxin-1 and RANTES in response to pretreatment with docosahexaenoic acid (DHA) was investigated. Moreover, western blotting was used to evaluate the effects of DHA on the activation of nuclear factor (NF)-κB and signal transducer and activator of transcription 6 (STAT6).Results: The expression of eotaxin-1 mRNA was significantly suppressed by pretreatment with DHA with IL-4 and TNF-α costimulation. RANTES expression was similarly suppressed, but the difference was not significant. The secretion of eotaxin-1 and RANTES was significantly lower in DHA-pretreated cells than in vehicle-treated cells. Western blotting for NF-κB and STAT6 showed that these proteins were downregulated in the DHA pretreatment group compared with those in the vehicle control group.Conclusion: The results of this study suggested that DHA could have applications in the management of allergic inflammation.

The Effect of Cytokine-Stimulation and Pharmacologic Intervention on PGE2 Production in Primary Human Conjunctival and Corneal Cells.

We studied the production of PGE2 by human conjunctival and corneal cells in response to inflammation, and reduction of inflammation with non-steroidal anti-inflammatory drugs. Primary cultures of human conjunctival epithelial cells, fibroblasts, corneal epithelial cells, and keratocytes were incubated with IL-4 and TNF-α. PGE2 and COX-2 levels were analyzed. Effects of anti-inflammatory and anti-immune drugs on PGE2 production were also investigated. IL-4 and TNF-α induced the generation of PGE2 and COX-2 in conjunctival and corneal cells. Epithelial PGE2 production was significantly lower than in keratocytes and fibroblasts, which was down-regulated by aspirin. IL-4 and TNF-α enhanced the inflammatory response via prostaglandin production which contributed to ocular surface inflammation. Prostaglandin production was higher in stromal cells than epithelial cells. These results suggest that the epithelial barrier disruption may contribute to ocular allergic inflammation by the PGE2 production from stromal cells. Moreover, NSAIDs were effective in suppressing PGE2 production in our experiment.

What echocardiographic indices are predictive of patent ductus arteriosus surgical closure in early preterm infants? A prospective multicenter cohort study.

BACKGROUND: No echocardiographic indices for predicting the need for preterm patent ductus arteriosus (PDA) surgery have been tested with an adequate sample size. We tested the hypothesis that some echocardiographic indices have better predictive ability for the need for PDA surgery. METHODS: We prospectively collected data from infants with gestational ages between 23 and 29 weeks at 34 Japanese neonatal intensive care units over 14 months. Data points were 1, 3, 7, and 14 days of age and, if applicable, before PDA surgery. We assessed five echocardiographic indices. Volume and dimension indices were adjusted for birth body weight (BBW). For each echocardiographic index, the worst value among all data points in nonsurgical patients or the value just before surgery in surgical patients was used. Multivariate logistic regression was applied with adjustment for clinical status. RESULTS: In total, 691 patients were analyzed, of whom 61 (8.8%) underwent surgery, as guided using the criteria in the protocol. The areas under the receiver-operating characteristic curve for PDA diameter (0.86) and PDA diameter/BBW (0.86) were the largest, followed by those of left pulmonary artery end-diastolic velocity (LPAedv) (0.80), and left atrial volume/BBW (0.80). CONCLUSIONS: Considering the measurement's easiness and independence of body size, PDA diameter and LPAedv may serve as useful indices for assessing the need for PDA surgery in early preterm infants.

Induction of human regulatory innate lymphoid cells from group 2 innate lymphoid cells by retinoic acid.

BACKGROUND: Group 2 innate lymphoid cells (ILC2s) play critical roles in induction and exacerbation of allergic airway inflammation. Thus clarification of the mechanisms that underlie regulation of ILC2 activation has received significant attention. Although innate lymphoid cells are divided into 3 major subsets that mirror helper effector T-cell subsets, counterpart subsets of regulatory T cells have not been well characterized. OBJECTIVE: We sought to determine the factors that induce regulatory innate lymphoid cells (ILCregs). METHODS: IL-10+ ILCregs induced from ILC2s by using retinoic acid (RA) were analyzed with RNA-sequencing and flow cytometry. ILCregs were evaluated in human nasal tissue from healthy subjects and patients with chronic rhinosinusitis with nasal polyps and lung tissue from house dust mite- or saline-treated mice. RESULTS: RA induced IL-10 secretion by human ILC2s but not type 2 cytokines. IL-10+ ILCregs, which were converted from ILC2s by means of RA stimulation, expressed a regulatory T cell-like signature with expression of IL-10, cytotoxic T lymphocyte-associated protein 4, and CD25, with downregulated effector type 2-related markers, such as chemoattractant receptor-homologous molecule on TH2 cells and ST2, and suppressed activation of CD4+ T cells and ILC2s. ILCregs were rarely detected in human nasal tissue from healthy subjects or lung tissue from saline-treated mice, but numbers were increased in nasal tissue from patients with chronic rhinosinusitis with nasal polyps and in lung tissue from house dust mite-treated mice. Enzymes for RA synthesis were upregulated in airway epithelial cells during type 2 inflammation in vivo and by IL-13 in vitro. CONCLUSION: We have identified a unique immune regulatory and anti-inflammatory pathway by which RA converts ILC2s to ILCregs. Interactions between airway epithelial cells and ILC2s play an important roles in the generation of ILCregs.

The efficacy of 0.1% tacrolimus ophthalmic suspension in the treatment of severe atopic keratoconjunctivitis.

BACKGROUND: Severe atopic keratoconjunctivitis (AKC) is a relatively rare disease, and some cases are refractory to conventional steroid treatment. OBJECTIVE: To examine the efficacy of 0.1% tacrolimus ophthalmic suspension in treating severe AKC during a 1-year follow-up. METHODS: This was a single-center, retrospective clinical study. Sixty eyes from 30 patients with severe AKC who were treated with 0.1% tacrolimus ophthalmic suspension 4 times per day, were included. The mean age of the patients was 21.5 ± 13.7 years. The severity of objective signs was observed at baseline (before treatment), at 2 weeks, and at 1, 2, 3, 6, and 12 months after treatment initiation. Ten objective signs of palpebral conjunctiva, bulbar conjunctiva, limbus, and cornea were assessed using 4 grades (0 = normal; 1+ = mild; 2+ = moderate; 3+ = severe). Safety was assessed based on the incidence and the severity of adverse events. RESULTS: The total score of the 10 clinical signs significantly decreased from baseline 2 weeks after initiating tacrolimus eye drop treatment, except at 2 months. The mean total score of clinical signs was 13.6 ± 6.6 at the beginning of treatment, and decreased to 5.4 ± 4.8 12 months after initiation. Treatment was gradually tapered, with increasing intervals between applications. Additional medications were required to provide relief in 18 patients during follow-up. No patient discontinued treatment due to adverse drug effects. Herpes keratitis was observed in 3 cases during follow-up. However, these cases were completely controlled. CONCLUSION: The 0.1% tacrolimus ophthalmic suspension is effective for the treatment of severe AKC refractory to standard conventional treatments throughout a full year.

JESREC score and mucosal eosinophilia can predict endotypes of chronic rhinosinusitis with nasal polyps.

OBJECTIVE: Recently, JESREC score and mucosal eosinophil count have been used to diagnose eosinophilic chronic rhinosinusitis (ECRS) in Japan. However, it remains unknown whether the subtypes of CRS diagnosed by these criteria have different endotypes. In the present study, we investigated whether JESREC score and mucosal eosinophil count were appropriate for classification of CRS subgroups into endotypes. METHODS: A cross-sectional study involving 71 consecutive patients with CRS with nasal polyps (CRSwNP) and 13 control patients was performed. Nasal polyp tissues from CRSwNP patients and uncinate process tissues from control patients were collected for analysis of inflammatory cells by immunohistochemistry and measurement of cytokines and chemokines by ELISA and quantitative real-time PCR. We compared the differences between subtypes according to JESREC score and mucosal eosinophil count and investigated the subgroups with different endotypes by cluster analysis and principal component analysis. RESULTS: In the 71 CRSwNP patients, 9 patients had JESREC score <11 and mucosal eosinophil count <70/HPF (Group A), 20 patients had JESREC score ≥11 and mucosal eosinophil count <70/HPF (Group C), and 42 patients had JESREC score ≥11 and mucosal eosinophil count ≥70/high-power field (HPF) (Group D). Semiquantitative analysis of inflammatory cells showed that eosinophils, neutrophils, macrophages, mast cells, and basophils differed significantly between the subgroups. At the mRNA level, CLC, IL5, IL13, CCL11, CCL24, CCL26, POSTN, CSF3, and IL8 showed significant differences. At the protein level, eotaxin-2/CCL24, eotaxin-3/CCL26, and G-CSF had significant differences. Cluster analysis using gene expression levels in 55 CRS patients and 11 control patients revealed that the patients could be classified into five clusters. Cluster 1 (n=27) contained all patients with Group D. Cluster 2 (n=11) comprised all control patients. Cluster 3 (n=4) included mixed subtypes: one with Group A and three with Group D. Cluster 4 (n=7) and Cluster 5 (n=17) contained all patients with Groups A and C, respectively. Furthermore, the principal component analysis revealed that the subtypes had different characteristics. CONCLUSION: CRS subtypes based on JESREC score and mucosal eosinophil count showed different inflammatory patterns, and unsupervised statistical analyses supported the classification that can predict endotypes. From these results, we concluded that the classification based on JESREC score and mucosal eosinophil count was useful for predicting CRS endotypes.

Chitin promotes antigen-specific Th2 cell-mediated murine asthma through induction of IL-33-mediated IL-1ß production by DCs.

Chitin, which is a major component of house dust mites (HDM), fungi, crustaceans, etc., can activate immune cells, suggesting that it contributes to development of allergic disorders such as asthma. Although the pathophysiological sensitization route of asthmatic patients to allergens is considered via the respiratory tract, the roles of intranasally-administered chitin in development of asthma remain unclear. After ovalbumin (OVA) challenge, development of airway inflammation was profoundly exacerbated in mice sensitized with OVA in the presence of chitin. The exacerbation was dependent on IL-33, but not IL-25, thymic stromal lymphopoietin or IL-17A. Chitin enhanced IL-33-dependent IL-1ß production by dendritic cells (DCs). Furthermore, chitin- and IL-33-stimulated DC-derived IL-1ß promoted OVA-specific Th2 cell activation, resulting in aggravation of OVA-induced airway inflammation. These findings indicate the adjuvant activity of chitin via a new mechanism and provide important clues for development of therapeutics for allergic disorders caused by HDM, fungi and crustaceans.

Distinct gene expression profiles and regulation networks of nasal polyps in eosinophilic and non-eosinophilic chronic rhinosinusitis.

BACKGROUND: Chronic rhinosinusitis with nasal polyps (CRSwNP) is known to have 2 phenotypes in East Asia. Eosinophilic CRSwNP (ECRSwNP), defined as tissue eosinophilia and easily recurrent, is distinguished from other non-eosinophilic CRSwNP (NECRSwNP) types. However, the pathogenesis of each remains unclear. METHODS: Nasal polyp tissues from ECRS (ECRSwNP) and NECRS (NECRSwNP) patients were obtained, and their comprehensive gene expression profiles were investigated by microarray analysis. Bioinformatics approaches (eg, Ingenuity Pathway Analysis [IPA]) were used to interrogate the data sets. RESULTS: Hierarchical clustering and principal component analysis (PCA) collectively showed that ECRSwNP and NECRSwNP had distinct gene expression patterns. Of note, these genes could be divided into 8 distinctive clusters having different expression patterns and functions. Upstream Regulator Analysis revealed that not only T-helper 2 (Th2) and the eosinophilia-related molecules (interleukin 4 [IL4], IL5, and colony stimulating factor 2 [CSF2]) reported so far, but also cell cycle regulators (cyclin dependent kinase inhibitor 1A [CDKNA1] and cyclin D1 [CCND1]) and a tissue fibrosis-related molecule (transforming growth factor ß [TGFß]) were identified in ECRSwNP. On the other hand, mainly interferons (IFNs) and acute inflammatory cytokines (IL1 and IL6) were predicted as upstream regulators in NECRSwNP. CONCLUSION: These results are useful for understanding the molecular basis of the mechanisms of CRSwNP and point to new targets for developing specific biomarkers and personalized therapeutic strategies for CRSwNP.

Assessment of suitable reference genes for RT-qPCR studies in chronic rhinosinusitis.

Reverse transcription-quantitative polymerase chain reaction is a valuable and reliable method for gene quantification. Target gene expression is usually quantified by normalization using reference genes (RGs), and accurate normalization is critical for producing reliable data. However, stable RGs in nasal polyps and sinonasal tissues from patients with chronic rhinosinusitis (CRS) have not been well investigated. Here, we used a two-stage study design to identify stable RGs. We assessed the stability of 15 commonly used candidate RGs using five programs-geNorm, NormFinder, BestKeeper, ΔCT, and RefFinder. Ribosomal protein lateral stalk subunit P1 (RPLP1) and ribosomal protein lateral stalk subunit P0 (RPLP0) were the two most stable RGs in the first stage of the study, and these results were validated in the second stage. The commonly used RGs ß-actin (ACTB) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were unstable according to all of the algorithms used. The findings were further validated via relative quantification of IL-5, CCL11, IFN-γ, and IL-17A using the stable and unstable RGs. The relative expression levels varied greatly according to normalization with the selected RGs. Appropriate selection of stable RGs will allow more accurate determination of target gene expression levels in patients with CRS.

DKK3 Overexpression Increases the Malignant Properties of Head and Neck Squamous Cell Carcinoma Cells.

DKK3, a member of the dickkopf Wnt signaling pathway inhibitor family, is believed to be a tumor suppressor because of its reduced expression in cancer cells. However, our previous studies have revealed that DKK3 expression is predominantly observed in head and neck/oral squamous cell carcinoma (HNSCC/OSCC). Interestingly, HNSCC/OSCC patients with DKK3 expression showed a high rate of metastasis and poorer survival, and siRNA-mediated knockdown of DKK3 in HNSCC-derived cancer cell lines resulted in reduced cellular migration and invasion. From these data, it was hypothesized that DKK3 might exert an oncogenic function specific to HNSCC. In the present research, the DKK3 overexpression model was established, and its influences were investigated, together with molecular mechanism studies. The DKK3 expression profile in cancer cell lines was investigated, including HNSCC/OSCC, esophageal, gastric, colorectal, pancreatic, prostatic, and lung cancers. DKK3 overexpression was performed in HNSCC-derived cells by transfection of expression plasmid. The effects of DKK3 overexpression were assessed on cellular proliferation, migration, invasion, and in vivo tumor growth. The molecular mechanism of DKK3 overexpression was investigated by Western blotting and microarray analysis. DKK3 overexpression significantly elevated cellular proliferation, migration, and invasion, as well as increased mRNA expression of cyclin D1 and c-myc. However, reporter assays did not show TCF/LEF activation, suggesting that the increased malignant property of cancer cells was not driven by the Wnt/ß-catenin pathway. For the investigation of the pathways/molecules in DKK3-mediated signals, the Western blot analyses revealed that phosphorylation of Akt (S473) and c-Jun (Ser63) was elevated. The application of a PI3K kinase inhibitor, LY294002, on HSC-3 DKK3 cells significantly decreased tumor cell proliferation, migration, and invasion. From these results, we demonstrated that DKK3 might contribute to cellular proliferation, invasion, migration, and tumor cell survival in HNSCC cells through a mechanism other than the canonical Wnt signaling pathway, which might be attributed to PI3K-Akt signaling.

A Rho-associated coiled-coil containing kinases (ROCK) inhibitor, Y-27632, enhances adhesion, viability and differentiation of human term placenta-derived trophoblasts in vitro.

Although human term placenta-derived primary cytotrophoblasts (pCTBs) represent a good human syncytiotrophoblast (STB) model, in vitro culture of pCTBs is not always easily accomplished. Y-27632, a specific inhibitor of Rho-associated coiled-coil containing kinases (ROCK), reportedly prevented apoptosis and improved cell-to-substrate adhesion and culture stability of dissociated cultured human embryonic stem cells and human corneal endothelial cells. The Rho kinase pathway regulates various kinds of cell behavior, some of which are involved in pCTB adhesion and differentiation. In this study, we examined Y-27632's potential for enhancing pCTB adhesion, viability and differentiation. pCTBs were isolated from term, uncomplicated placentas by trypsin-DNase I-Dispase II treatment and purified by HLA class I-positive cell depletion. Purified pCTBs were cultured on uncoated plates in the presence of epidermal growth factor (10 ng/ml) and various concentrations of Y-27632. pCTB adhesion to the plates was evaluated by phase-contrast imaging, viability was measured by WST-8 assay, and differentiation was evaluated by immunofluorescence staining, expression of fusogenic genes and hCG-ß production. Ras-related C3 botulinum toxin substrate 1 (Rac1; one of the effector proteins of the Rho family) and protein kinase A (PKA) involvement was evaluated by using their specific inhibitors, NSC-23766 and H-89. We found that Y-27632 treatment significantly enhanced pCTB adhesion to plates, viability, cell-to-cell fusion and hCG-ß production, but showed no effects on pCTB proliferation or apoptosis. Furthermore, NSC-23766 and H-89 each blocked the effects of Y-27632, suggesting that Y-27632 significantly enhanced pCTB differentiation via Rac1 and PKA activation. Our findings suggest that Rac1 and PKA may be interactively involved in CTB differentiation, and addition of Y-27632 to cultures may be an effective method for creating a stable culture model for studying CTB and STB biology in vitro.