The Interplay Between Metabolites and MicroRNAs in Aqueous Humor to Coordinate Corneal Endothelium Integrity.

Purpose: The purpose of the study was to clarify the interplay between metabolites and microRNAs (miRs) in the aqueous humor (AqH) of bullous keratopathy (BK) patients to retain human corneal endothelium (HCE) integrity. Design: Prospective, comparative, observational study. Participants: A total of 55 patients with BK and 31 patients with cataract (Cat) as control. Methods: A biostatic analysis of miRs and metabolites in the AqH, hierarchical clustering, and a least absolute shrinkage and selection operator (Lasso) analysis were employed. The miR levels in AqH of BK (n = 18) and Cat (n = 8) patients were determined using 3D-Gene human miR chips. Hierarchical clusters of metabolites detected by liquid chromatography-mass spectrometry or gas chromatography-mass spectrometry in AqH specimens from 2 disease groups, BK (total n = 55) and Cat (total n = 31), were analyzed twice to confirm the reproducibility. The analytical procedure applied for investigating the association between metabolites and miRs in AqH was the exploratory data analysis of biostatistics to avoid any kind of prejudice. This research procedure includes a heat-map, cluster analysis, feature extraction techniques by principal component analysis, and a regression analysis method by Lasso. The cellular and released miR levels were validated using reverse transcription polymerase chain reaction and mitochondria membrane potential was assessed to determine the functional features of the released miRs. Main Outcome Measures: Identification of interacting metabolites and miRs in AqH attenuating HCE degeneration. Results: The metabolites that decreased in the AqH of BK patients revealed that 3-hydroxyisobutyric acid (HIB), 2-aminobutyric acid (AB) and branched-chain amino acids, and serine were categorized into the same cluster by hierarchical clustering of metabolites. The positive association of HIB with miR-34a-5p was confirmed (P = 0.018), and the Lasso analysis identified the interplay between miR-34a-5p and HIB, between miR-24-3p and AB, and between miR-34c-5p and serine (P = 0.041, 0.027, and 0.009, respectively). 3-hydroxyisobutyric acid upregulated the cellular miR-34a expression, mitochondrial membrane potential, and release of miR-184 in dedifferentiated cultured HCE cells. Conclusions: Metabolites and miRs in AqH may synchronize in ensuring the integrity of the HCE to maintain efficient dehydration from the stroma. Financial Disclosures: Proprietary or commercial disclosure may be found after the references.

Long-term Outcomes of Early-stage Non-stomach Gastrointestinal Mucosa-associated Lymphoid Tissue Lymphoma Treated With Radiation Therapy.

BACKGROUND/AIM: Non-stomach gastrointestinal mucosa-associated lymphoid tissue (MALT) lymphoma is rare, and there are only a few reports regarding radiation therapy (RT) for non-stomach gastrointestinal MALT lymphoma. There has been no established cure and no reports on RT use with long-term follow-up. Herein, we report a retrospective long-term investigation of early-stage non-stomach gastrointestinal MALT lymphoma. Our aim was to evaluate whether RT is a valid treatment option for this disease. PATIENTS AND METHODS: We retrospectively analyzed 6 patients who were diagnosed with early-stage non-stomach gastrointestinal MALT lymphoma and received RT. The median age was 66 years (range=38-89 years). The primary tumor originated from the duodenum in 2 patients and from the rectum in 4 patients. The RT dose was 30-34 Gy in 15-20 fractions to the involved site or field, depending on the case. RESULTS: The median follow-up time was 89.5 months (range=6-170). All patients had complete remission within 3 months after RT. The 5-year overall survival and progression-free survival rates were 83.3% and 100%, respectively. During the observation period, no patient had a confirmed recurrence. One patient died of causes unrelated to cancer or treatment. There were no late toxicities by RT. CONCLUSION: Our results show good long-term local control and no late toxicities requiring treatment. Moderate-dose RT was appropriate and well tolerated for early-stage non-stomach gastrointestinal MALT lymphoma.

Repressed miR-34a Expression Dictates the Cell Fate to Corneal Endothelium Failure.

Purpose: To reveal the mechanism triggering the functional disparity between degenerated and non-degenerated corneal endothelium cells in the water efflux from corneal stroma to the anterior chamber. Methods: The varied levels of the microRNA (miR)-34, miR-378, and miR-146 family in human corneal endothelium and cultured cells thereof were investigated using 3D-Gene Human miRNA Oligo Chips. Concomitantly, CD44, p53, c-Myc, matrix metalloprotease (MMP)-2 expression, and Ras homolog gene family member A (Rho A) activity was correlated to the expression intensities of these microRNAs, partly complemented with their altered expression levels with the transfection of the corresponding mimics and inhibitors. The levels of miRs were further associated with intracellular pH (pHi) and mitochondrial energy homeostasis. Results: P53-inducible miR-34a/b repressed CD44 expression, and CD44 was repressed with the elevated c-Myc. The repressed miR-34a activated the CD44 downstream factors Rho A and MMP-2. MiR-34a mimics downregulated pHi, inducing the skewing of mitochondrial respiration to oxidative phosphorylation. The oxidative stress (H2O2) induced on human corneal endothelial cells, which repressed miR-34a/b expression, may account for the impaired signaling cascade to mitochondrial metabolic homeostasis necessary for an efficient water efflux from the corneal stroma. Conclusions: The upregulated expression of CD44, through repressed miR-34a/b by reactive oxygen species and elevated c-Myc by oxidative stress, may impair mitochondrial metabolic homeostasis, leading to human corneal endothelial failure.

Potential participation of CTRP6, a complement regulator, in the pathology of age related macular degeneration.

PURPOSE: To investigate the localized expression of C1q/tumor necrosis factor related protein (CTRP) 6 in human age-related macular degeneration (AMD) retinal tissues. EXPERIMENTAL STUDY DESIGN: 4 AMD and 3 non-AMD whole eyes of Caucasian donors were used. Eyecups were excised at Eye Bank CorneaGen, Inc. METHODS: To elucidate the effects of CTRP6, C3b was measured by an enzyme-linked immunosorbent-like assay. CFB versus CTRP6 competitive binding assay was applied to clarify the inhibition by CTRP6 of C3bBb complex formation. The cornea, iris, lens, and vitreous were removed and the eyes were cut into a posterior eye-cup including the retina, choroid, and sclera. Six-µm-thick serial sections of frozen samples underwent hematoxylin-eosin (HE) staining and indirect immunohistochemical staining using primary antibodies, anti-CTRP6, -CTRP5, -CTRP10, -Complement factor H (CFH) and -Clusterin (CLU). Results The two in vitro studies confirmed that CTRP6 has an inhibitory effect on alternative pathways of complement (APC) function and that the molecular target of CTRP6 is the inhibition of the formation of C3bBb. Localized expression for CTRP6 and CFH was found in the drusen of the AMD eyes, both associated with APC inhibition, CLU associated with membrane-attack complex (MAC) inhibition, and CTRP5 associated with retinal degeneration. CONCLUSION: The localized expression of CTRP6 in the drusen of AMD eyes may open a new insight into the possible involvement of APC regulatory factors in the pathogenesis of AMD, together with the known CFH so far analyzed solely as an APC inhibitor.

Mitochondrial miRNA494-3p in extracellular vesicles participates in cellular interplay of iPS-Derived human retinal pigment epithelium with macrophages.

To explore new molecular targets for therapy in human model systems by discerning the role of extracellular vesicle (EV) microRNAs (miRs) secreted by human retinal pigment epithelium (hRPE) cells and their cellular interplay with macrophages (Mps). Human Mps differentiated from THP-1 cells stimulated by phorbol myristate acetate were co-cultured with induced pluripotent stem cell-derived differentiated hRPE (iPS-hRPE) cells in Transwell® system separated by 0.40 µm or 0.03 µm filters. EV-associated CD63+ proteins (CD63+ EV) were detected by western blotting, and secreted EVs were analyzed by Nanosight tracking. The miR profiles of the secreted EVs were determined using 3D-gene human microRNA chips (Toray Industries, Inc.). Levels of CD63+ EV were increased in co-cultures concomitantly with the increased production of EV particles (50-150 nm). The increased production of EVs was associated with higher production of MCP-1, IL-6, IL-8 from hRPE cells, and VEGF and repressed production of TNF-α from Mps and pigment epithelium-derived factor (PEDF) from RPE cells. Ultracentrifugation of semi-purified EVs increased the secretion of these pro-inflammatory cytokines and EV particles from hRPE cells, but this effect was eliminated in transwells equipped with 0.03 µm filters, whereas no repression of PEDF and TNF-α secretion occurred. 3D-gene miR analysis revealed a selective increase in secretion of miR494-3p in EVs from iPS-hRPE cells during the interplay with Mps. The miRs in EVs secreted by hRPE cells may have a critical role in the vicious inflammatory cycle, whereas repression of TNF-α and PEDF require cell-to-cell contact that is independent of EVs or exosomes. MiR494-3p may be a candidate molecular target of diagnosis and therapy for age-related macular degeneration.

CD63+ extracellular vesicles from retinal pigment epithelial cells participate in crosstalk with macrophages in the innate inflammatory axis.

The aim of the study is to clarify the participation of extracellular vesicles (EV) secreted by murine primary retinal pigment epithelial (mpRPE) cells in the cell to cell communication with macrophages (Mps), firstly described by the authors in 2016. In ocular inflammation, Mps act as sources of tumor necrosis factor-α (TNF-α), an activator of RPE cells. TNF-α stimulates the production of monocyte chemotactic protein (MCP-1) by RPE cells, thereby causing greater recruitment of Mps to the sub-RPE space. Murine RAW 264.7 Mps cells were co-cultured with C57BL/6 mouse mpRPE cells, either together or separated in transwells, vertically or horizontally connectable, with 0.40 or 0.03 µm membrane filters. The association of EV with mpRPE or RAW 264.7 was quantified by fluorescence cell sorting (FACS) using Qdot655 streptavidin-conjugated biotinylated EV. Increased levels of CD63+ EV were detected in co-cultures by western blotting or FACS analysis, in accordance with the increased production of nanoparticles (50-150 nm) detected by Nanosight tracking analysis. The gene expressions of cytokines, MCP-1, IL-6, IL-8, and VEGF in mpRPE cells and the corresponding proteins were increased in co-cultures even in transwells, vertically connected with 0.40 µm membrane filters, while the repressed TNF-α protein production was not affected. Most of the CD63+ EVs produced by mpRPE cells in co-cultures were associated with Raw264.7, but not with mpRPE cells. Semi-purified CD63+ EV secreted from mpRPE cells, increased the secretion of MCP-1, IL-6, and VEGF in co-cultures with RAW 264.7. Culture chamber separation horizontally connected with 0.03 µm membrane filters reduced this increased secretion. Collectively, mpRPE derived CD63+ EV partly participate in the sub-retinal innate inflammation. To evaluate the functional damage of RPE cells upon chronic exposure to here defined EVs will be the critical issue to uncover their roles in chronic retinal degenerative diseases.

Epigenetic regulation of the epithelial mesenchymal transition induced by synergistic action of TNF-α and TGF-ß in retinal pigment epithelial cells.

To clarify the influence of tumor necrosis factor (TNF)-α on fibrotic phenotypes induced by transforming growth factor (TGF)-ß in retinal pigment epithelial cells (RPECs) by epigenetic regulation. Human primary retinal pigment epithelial cells (RPECs including ARPE19) were used in cultures in the presence or absence of TNF-α and/or TGF-ß2. RT2 Profiler™ (Qiagen) was used for PCR Array for fibrosis and epithelial mesenchymal transition (EMT). Microarray analysis by 3D gene DNA chip was outsourced to Toray Industries Inc. Quantification of histone acetyl transferase (HAT)-related and histone deacetylase (HDAC) related gene expression were also analyzed. HDAC and HAT activity was measured using an EpiQuik HDAC and HAT Activity/Inhibition Assay Kit (Epigentek). CD44, MMP-9, HAT, and HDAC in RPECs were analyzed by western blotting. Analysis of expression of the EMT/fibrosis related CD44 and MMP-9 phenotypes induced by TNF-α+TGF-ß2 revealed four alterations in RPECs: 1) abolition of TGF-ß2-induced α-SMA by TNF-α; 2) synergy between TNF-α+TGF-ß2 for induction of CD44 and MMP-9 phenotypes 3) no inhibition of HDAC activity by either TNF-α or TGF-ß2; and 4) significant inhibition of HAT activity by either TNF-α or TGF-ß2, but no synergy. The HDAC activation through HAT inhibition by TNF-α+TGF-ß was counteracted by HDAC inhibitors, leading to the inhibition of EMT/fibrosis. EMT/fibrotic CD44 and MMP-9 phenotypes were epigenetically upregulated by concerted action of TNF-α and TGF-ß in RPECs. The intervention in epigenetic regulation may hold potential in preventing intraocular proliferative diseases.

Outcome of Radiation Therapy for Locally Advanced Vulvar Carcinoma: Analysis of Inguinal Lymph Node.

BACKGROUND/AIM: The aim of this study was to define the outcome of radiation therapy for vulvar carcinoma, and to investigate the effectiveness of therapeutic and prophylactic inguinal lymph node (ILN) irradiation. Because reports about the treatment of ILN were limited. PATIENTS AND METHODS: Thirty consecutive vulvar carcinoma patients were treated using external beam radiation therapy (EBRT) for definitive disease (n=25) or postoperatively (n=5). Twenty-four (80%) had squamous cell carcinoma (SCC). Tumor stages (2002 UICC) ranged from 0 to IVB, with no distant metastases. RESULTS: The median total prescribed dose for primary tumor was 64.8 Gy. The 2-year overall survival rate was 25.3%. The outcome was significantly better in patients with ILNs<30 mm (p=0.005) and patients receiving prescribed doses >60 Gy (p=0.002). CONCLUSIONS: ILN diameters ≤30 mm and prescribed doses over 60 Gy were associated with ILN control in patients with vulvar carcinoma.

Outcomes of treatment for localized prostate cancer in a single institution: comparison of radical prostatectomy and radiation therapy by propensity score matching analysis.

OBJECTIVES: To compare the outcomes of radical prostatectomy (RP), intensity-modulated radiation therapy (IMRT), and low-dose-rate brachytherapy (BT) using propensity score matching analysis in patients with clinically localized prostate cancer. METHODS: A group of 2273 patients with clinically localized prostate cancer between January 2004 and December 2015 at the Yokohama City University hospital were identified. The records of 1817 of these patients, who were followed up for a minimum of 2 years, were reviewed; 462 were treated with RP, 319 with IMRT, and 1036 with BT. The patients were categorized according to the National Comprehensive Cancer Network risk classification criteria, and biochemical outcomes and overall survival rates were examined. Biochemical failure for RP was defined as prostate-specific antigen (PSA) levels > 0.2 ng/ml, and for IMRT and BT as nadir PSA level + 2 ng/ml. Propensity scores were calculated using multivariable logistic regression based on covariates, including the patient's age, preoperative PSA, Gleason score, number of positive cores, and clinical T stage. RESULTS: Median follow-up was 77 months for the RP, 54 months for IMRT, and 66 months for BT patients. After the propensity scores were adjusted, a total of 372 (186 each) and 598 (299 each) patients were categorized into RP vs IMRT and RP vs BT groups, respectively. Kaplan-Meier analysis did not show any statistically significant differences in terms of overall survival rate between these groups (RP vs IMRT: p = 0.220; RP vs BT: p = 0.429). IMRT was associated with improved biochemical failure-free survival compared to RP in all risk groups (high-risk: p < 0.001; intermediate-risk: p = 0.009; low-risk: p = 0.001), whereas significant differences were observed only in the intermediate-risk group (p = 0.003) within the RP vs BT group. CONCLUSION: The results of our propensity score analysis of mid-term localized prostate cancer treatment outcomes demonstrated no significant differences in the overall survival rate. Despite the difference in biochemical failure definition between surgery and radiotherapeutic approaches, the results of this study demonstrate improved biochemical control favoring IMRT and BT as compared to RP.

Hypofractionated radiotherapy in children with diffuse intrinsic pontine glioma.

BACKGROUND: Overall survival (OS) of patients with diffuse intrinsic pontine glioma (DIPG) is poor, with radiation therapy (RT) the only intervention that transiently delays tumor progression. Hypofractionated RT and re-irradiation at first progression have gained popularity in improving the quality of life of such patients. METHODS: We performed a retrospective review of children with DIPG treated at Kanagawa Children's Medical Center from 2000 to 2018. RESULTS: A total of 24 cases were reviewed. Median age at diagnosis was 6.3 years (1.6-14.0). Twenty patients received RT only once. Thirteen patients received conventionally fractionated RT, and seven patients received hypofractionated RT as up-front RT. Severe toxicities were not observed in patients who received hypofractionated RT. Median OS and time to progression were similar between conventionally fractionated and hypofractionated RT groups.(9.7 [95% confidence interval(CI): 7.1-11.2] versus 11.0[95% CI: 5.2-13.6] months, P = 0.60; 4.2[95% CI: 1.8-8.3] versus 7.1 [95% CI:4.5-8.7] months, P = 0.38). Four patients received re-irradiation at first progression and all patients showed transient neurological improvement and survival more than a year after diagnosis. A 4-year-old boy was re-irradiated 5-and-a-half months after the first re-irradiation; following transient neurological improvement. He survived a further 5 months. CONCLUSION: Hypofractionated RT for children with newly diagnosed DIPG is well tolerated and feasible from the viewpoint of reducing a patient's burden of treatment. Re-irradiation at first progression is suggested to be beneficial.

Outcome of postoperative radiation therapy for cholangiocarcinoma and analysis of dose-volume histogram of remnant liver.

The aim of this study was to analyze dose-volume histogram (DVH) of the remnant liver for postoperative cholangiocarcinoma (CCA) patients, to find toxicity rates, and to confirm efficacy of postoperative radiation therapy (RT).Thirty-two postoperative CCA patients received partial liver resection and postoperative RT with curative intent. The "liver reduction rate" was calculated by contouring liver volume at computed tomography (CT) just before the surgery and at CT for planning the RT. To evaluate late toxicity, the radiation-induced hepatic toxicity (RIHT) was determined by the common terminology criteria for adverse events toxicity grade of bilirubin, aspartate transaminase, alanine transaminase, alkaline phosphatase, and albumin, and was defined from 3 months after RT until liver metastasis was revealed. The radiation-induced liver disease (RILD) was also evaluated.Tumor stages were distributed as follows: I: 1, II: 8, IIIA: 1, IIIB: 6, IIIC: 14, IVA: 2. Median prescribed total dose was 50 Gy. Median follow-up time was 27 months. Two-year overall survival (OS): 72.4%, disease-free survival: 47.7%, local control: 65.3%, and the median survival time was 40 months. The median "liver reduction rate" was 21%. The OS had statistically significant difference in nodal status (P = .032) and "liver reduction rate" >30% (P = .016). In the association between the ≥grade 2 RIHT and DVH, there were significantly differences in V30 and V40 (P = .041, P = .034), respectively. The grade ≥2 RIHT rates differ also significantly by sex (P = .008). Two patients (6.2%) were suspected of RILD.We suggest that RT for remnant liver should be considered the liver V30, V40 to prevent radiation-induced liver dysfunction.

Acute and late toxicities in localized prostate cancer patients treated with low-dose 125I brachytherapy (110 Gy) in combination with external beam radiation therapy versus brachytherapy alone (160 Gy).

PURPOSE: The aim of this analysis was to compare acute and late toxicities between low-dose-rate brachytherapy (LDR-BT) (110 Gy) in combination with 45 Gy in 25 fractions external beam radiation therapy (EBRT) and LDR-BT (160 Gy) alone for localized prostate cancer. MATERIAL AND METHODS: One hundred five consecutive patients with localized prostate cancer treated from May 2014 to May 2017 were included in this retrospective analysis. Sixty patients received combination therapy and 45 patients received BT monotherapy. The LDR-BT procedure was performed using 125I seeds. RESULTS: The median follow-up time was 28 months in both groups. Three-year effect rates were overall survival: 100% in both groups. The biochemical failure rate was 2.3% in the combination group and 0% in the monotherapy group (p = 0.373). No patients died during the study period. In both groups, almost all the patients experienced acute urethritis. There was a significant difference between the combination therapy group (8.3%) and BT monotherapy group (11.1%) in late genitourinary (GU) toxicities ≥ grade 2 (p = 0.035). Only 2 patients (3.3%) in the combination therapy group developed late ≥ grade 2 rectal hemorrhage. There were no significant differences between two groups in hematuria ≥ grade 2 (p = 0.068) or rectal hemorrhage ≥ grade 2 (p = 0.206). CONCLUSIONS: To our knowledge, this is the first report to compare the GU and gastrointestinal toxicities between the combination therapy and BT monotherapy (160 Gy) for localized prostate cancer. Unexpectedly, there were more late GU toxicities (except for hematuria) in the BT monotherapy group.

Radiation Therapy for Patients with Bone Metastasis from Uterine Cervical Cancer: Its Role and Optimal Radiation Regimen for Palliative Care.

AIM: To determine the role of radiation therapy for patients with bone metastasis from uterine cervical cancer and identify an optimal radiation regimen. PATIENTS AND METHODS: A total of 20 patients with bone metastases from uterine cervical cancer received radiation therapy to the pelvis. The median total dose of 60.2 Gy in the 2 Gy per fraction-equivalent dose (EQD2) was delivered to cervical tumors of all patients. Thirteen patients underwent chemotherapy during and/or following radiation therapy. RESULTS: In 18 of 20 patients, the primary tumors disappeared or were markedly reduced after radiation therapy. In all but one of 17 patients with pelvic pain and bleeding, these symptoms disappeared or were remarkably relieved. However, three patients had primary tumor progression at 7, 9, and 15 months after irradiation with total doses of 56.8, 58.4, and 68.3 Gy in EQD2, respectively. Two of these patients had relapses of bleeding and pain. The primary progression-free rate considering all patients was 69% at 1 year and 34% at 2 years. The corresponding overall survival rates were 34% at 1 year, and 8% at 2 years, with an estimated median survival time of 7 months. The number of metastatic bone sites (p=0.027) and administration of chemotherapy (p<0.001) were significant prognostic factors for survival. CONCLUSION: Radiation therapy is effective for relief of pelvic symptoms in patients with bone metastasis from uterine cervical cancer. For patients who are expected to survive for more than 1 year, almost curative-dose irradiation to primary tumors is recommended.

Radiation therapy for stage IVA uterine cervical cancer: treatment outcomes including prognostic factors and risk of vesicovaginal and rectovaginal fistulas.

PURPOSE: To evaluate the safety and efficacy of radiation therapy for stage IVA uterine cervical cancer and to identify an optimal radiation regimen. RESULTS: Seventeen of the 28 patients developed recurrence after radiation therapy (local recurrence in 10 and distant metastasis in 12). The local control and distant metastasis-free rates at 3 years in all patients were 61% and 49%, respectively. Fourteen patients died after radiation therapy, and all but 2 died of tumor progression. The disease-free, cause-specific, and overall survival rates at 3 years in all patients were 32%, 49%, and 45%, respectively, and the estimated median survival time was 32 months. Tumor size (P = 0.007) and involvement in the lower third of vagina (P = 0.006) were significant prognostic factors for local control. Older age (P = 0.018) and performance status (P = 0.020) were significant prognostic factors for distant metastasis. The presence of hydronephrosis was the sole significant prognostic factor for survival (P = 0.026). Only 2 patients developed grade 3 late toxicities (vesicovaginal fistula and radiation proctitis, respectively). MATERIALS AND METHODS: Twenty-eight patients with stage IVA uterine cervical cancer received radiation therapy. All patients initially received external pelvic irradiation at a median dose of 50.4 Gy in 28 fractions. Twenty patients also received high-dose-rate intracavitary brachytherapy at a median dose of 22 Gy in 4 fractions. These fraction sizes were lower than conventional sizes. The total median dose for all 28 patients was 68.7 Gy. CONCLUSIONS: Radiation therapy is safe and effective for treatment of stage IVA uterine cervical cancer. The reduced radiation dose per fraction may contribute to the prevention of vesicovaginal fistula formation.

The Ingenious Interactions Between Macrophages and Functionally Plastic Retinal Pigment Epithelium Cells.

PURPOSE: The purpose of this study was to clarify the interactions between macrophages (MPs) and RPE cells in coculture systems to investigate the functional plasticity of RPE cells. METHODS: Adherent peritoneal cells or murine MP cell line Raw 264.7 was cocultured with primary RPE cells taken from C57BL/6 mice, with or without lipopolysaccharide (LPS) or TNF-α stimulation. The cytokine levels of the culture supernatants (CSs) were then analyzed with the Bio-Plex murine 23-Plex Panel Assay Kit (Bio-Rad Laboratories). Monocyte chemoattractant protein-1 (MCP-1), IL-6, VEGF, and TNF-α in CS were further quantified by ELISA. The expression profiles, in cocultures, of complement-associated genes, TNF-α, and angiogenesis-associated genes were analyzed by quantitative real-time PCR. RESULTS: The production of MCP-1, IL-6, and VEGF was synergistically elevated when primary MPs or RAW264.7 cells and RPE cells were cocultured compared with those derived from sole cultures of MPs and RPE cells. The synergistic effect was confirmed without direct cell contact and was more prominent in the presence of LPS or TNF-α. TNF-α production by MPs was suppressed by RPE cells. Coculture of RPE cells with RAW264.7 cells increased the gene expression of C3, CFB, and VEGF genes, whereas it reduced those of complement regulatory factors CFH, CD59, clusterin, and TNF-α and antiangiogenic pigment epithelium-derived factor (PEDF). CONCLUSIONS: Our findings indicate the presence of ingenious interactions between MPs and RPE cells that forces the inflammation and complement activation in the vicinity of RPE cells under pathologic conditions.

The relationship between pollen count levels and prevalence of Japanese cedar pollinosis in Northeast Japan.

BACKGROUND: The prevalence of Japanese cedar (JC) pollinosis in Japanese children is increasing. However, few studies have reported the relationship between pollen count levels and the prevalence of pollinosis. To evaluate the relationship between JC pollen count levels and the prevalence of pollinosis in children, we investigated the sensitization and development of symptoms for JC pollen in two areas of Akita in northeast Japan with contrasting levels of exposure to JC pollen. METHODS: The study population consisted of 339 elementary school students (10-11 years of age) from the coastal and mountainous areas of Akita in 2005-2006. A questionnaire about symptoms of allergic rhinitis was filled out by the students' parents. A blood sample was taken to determine specific IgE antibodies against five common aeroallergens. RESULTS: The mean pollen count in the mountainous areas was two times higher than that in the coastal areas in 1996-2006. The prevalence rates of nasal allergy symptoms and sensitization for mites were almost the same in both areas. On the other hand, the rates of nasal allergy symptoms and sensitization for JC pollen were significantly higher in the mountainous areas than in the coastal areas. The rate of the development of symptoms among children sensitized for JC pollen was almost the same in both areas. CONCLUSIONS: These results suggest that pollen count levels may correlate with the rate of sensitization for JC pollinosis, but may not affect the rate of onset among sensitized children in northeast Japan.

A randomized control trail of stepwise treatment with fluticasone propionate nasal spray and fexofenadine hydrochloride tablet for seasonal allergic rhinitis.

BACKGROUND: In Japan, oral antihistamines are frequently used as the initial treatment for seasonal allergic rhinitis (SAR), and intranasal steroids are added when nasal symptoms worsen. This study aimed to evaluate whether starting treatment with fluticasone propionate nasal spray (FP) from the beginning of pollinosis symptoms and adding fexofenadine hydrochloride tablet (FEX) when SAR is aggravated could achieve improved amelioration of nasal symptoms throughout the pollen season in comparison with a treatment that involves starting with FEX and later adding FP. METHODS: In this pragmatic, randomized, open-label, parallel-group trial, 51 Japanese cedar pollinosis patients (age, 16-85 years) were randomly divided and administered FP 100 mcg twice daily as an initial drug with FEX 60 mg twice daily as an additional drug and the same treatment in the reverse order. Nasal symptoms were evaluated in a daily dairy using a 4-point scale. The primary outcome was area under curve of the line representing the daily total nasal symptom score in the pollen season on a graph. RESULTS: Initial treatment with FP was significantly (P = 0.0015) more effective than initial treatment with FEX in improving the primary outcome. The average daily total nasal symptom score in the initial treatment with FP group was better than that in the initial treatment with FEX group throughout the pollen season. CONCLUSIONS: Initiating treatment with FP and adding FEX might lead to improved outcomes for nasal symptoms in comparison with the same drugs administered in the reverse order.

Peroxisome proliferator-activated receptor-γ is expressed in eosinophils in nasal polyps.

BACKGROUND: Peroxisome proliferator-activated receptor-γ (PPARγ) is a member of the nuclear receptors, which regulate fatty acid metabolites. One of the natural ligands for PPARγ is 15-deoxy-Δ(12,14)-prostaglandin J(2) (15d-PGJ(2)), a major metabolite of prostaglandin D(2) (PGD(2)). Recently, PPARγ has been shown to play an important role in anti-inflammatory reactions, including within-airway allergic diseases, in a mouse model. Our aim was to clarify the expression and localization of PPARγ and PGD(2) synthase, which produces ligands of PPARγ, in nasal polyps by immunohistochemical analysis. METHODS: Nasal polyps of chronic rhinosinusitis patients (6 asthmatic patients and 6 nonasthmatic patients) were obtained during surgery. May-Grünwald-Giemsa staining was performed to evaluate the eosinophil infiltration of the polyps. To identify the cells expressing PPARγ protein and PGD(2) synthase, double immunostaining was performed using anti-PPARγ antibody, monoclonal antileukocyte antibodies, and PGD(2) synthase antibody. RESULTS: The number of eosinophils and the number of PPARγ-positive cells in the nasal polyps of the asthmatic patients were significantly higher than those in the nonasthmatic patients. PPARγ was expressed on eosinophils and T cells of the infiltrating cells in the nasal polyps. PGD(2) synthase was also expressed on PPARγ-positive cells. CONCLUSION: PPARγ is involved in nasal polyposis pathogenesis, acting on eosinophils and T cells.

Peroxisome proliferator-activated receptor gamma negatively regulates allergic rhinitis in mice.

BACKGROUND: Peroxisome proliferator-activated receptor gamma(PPAR-gamma) has been shown to play an important role in the control of inflammatory responses acting on macrophages, mast cells, T cells, and eosinophils. The present study was aimed at investigating the effects of PPAR-gamma agonist on nasal symptoms and eosinophil accumulations in nasal mucosa by using a murine allergic rhinitis model. Furthermore, we examined the expression of PPAR-gamma in the nasal mucosa in mice. METHODS: BALB/c mice were sensitized and challenged intranasally with ovalbumin. Ciglitazone, a PPAR-gamma agonist, was administered orally 6 hours before each nasal challenge. RESULTS: Administration of PPAR-gamma agonist significantly decreased the number of nasal rubs, nasal histamine responsiveness, serum IgE, IL-5 production from the spleen, and eosinophilic infiltration in the nasal mucosa. Furthermore, PPAR-gamma was expressed in eosinophils and epithelial cells in the nasal mucosa by immunohistochemistry. CONCLUSIONS: PPAR-gamma was expressed in eosinophils and epithelial cells in the nasal mucosa. Also, the oral administration of ciglitazone is effective in upper airway allergic inflammation in mice.

PACAP facilitate the nerve regeneration factors in the facial nerve injury.

Pituitary adenylate cyclase-activating polypeptide (PACAP) has been reported as a strong neurotrophic factor in the various sites of nervous system. The facial nerve injury is one of the common problems in patients at the Otolaryngology since the nerve damage could occur easily due to the anatomical characteristics. Once it happens, the regeneration is little observed and functional recovery is poor. Thus, we investigated that PACAP might have some influence for regeneration after the facial nerve transaction in the guinea pig. PACAP treatment accelerated time for the appearance of compound muscle action potentials (CMAP) after the nerve transaction (first appeared at 1 versus 2 weeks in control) and shortened the latency at 4 weeks. The number of myelinated fibers increased at 4 weeks. Histochemical demonstration of GAP-43, a growth cone protein, was observed at the injury area at 2-4 days. PACAP increased the level of glial cell line-derived neurotrophic factor (GDNF), a neurotrophin, in facial target muscles at 1 day-4 weeks. These data indicated that PACAP promotes the regeneration factors and increases the possibility of functional recovery following the facial nerve injury.