Clinical characteristics in Japanese patients with chronic rhinosinusitis who underwent endoscopic sinus surgery.

OBJECTIVE: There is heterogeneity in the pathophysiology of chronic rhinosinusitis (CRS). Obtaining a detailed understanding of patient profiles in specific regions can provide valuable information not only for clinical practice but also future research plans. The aim of this study was to investigate the characteristics of patients who underwent endoscopic sinus surgery (ESS) for CRS. METHODS: This retrospective, single-center study examined the features of 453 patients with CRS who underwent ESS in the Tokyo area of Japan. The study evaluated various factors in patients with CRS including sex and age, the Japanese Epidemiological Survey of Refractory Eosinophilic Chronic Rhinosinusitis (JESREC) score, the recurrence rate of CRS, comorbidities of asthma and/or allergic diseases, and IgE sensitization to 12 inhaled allergens. RESULTS: Age-related declines in the sensitization rate to inhaled allergens were observed, and the most notable age-related decrease in specific IgE antibodies was observed for house dust mites (HDM) (p = 8.3 × 10-7). Sensitization to HDM, cat dander, and various types of fungi, including Aspergillus, was frequently observed in the CRS with asthma group, with rates of 54%, 17%, and 17%, respectively. We found that 23% of the patients had recurrence. In the recurrence group, the positive rates of specific IgE antibodies for birch and cat dander were significantly higher than in the no recurrence group. Bronchial asthma was identified as an important factor for recurrence. Among male patients, the recurrence group was younger than the no-recurrence group (p = 0.0032). Severe eosinophilic CRS (ECRS) showed early recurrence after surgery, with over the half of the patients experiencing at least one recurrence within 2 years post-surgery. Among patients with ECRS, the recurrence rate for females was 1.92 times higher than for males. CONCLUSION: Our study revealed the influences of age and sex on various clinical phenotypes of CRS patients undergoing ESS. There was a high sensitization rate to cat dander in both the recurrence and asthma groups. Further research on diverse disease etiologies is necessary to improve therapeutic strategies for patients with CRS.

Internal Jugular and Subclavian Vein Thrombosis in a Case of Ovarian Cancer.

Central venous catheter insertion and cancer represent some of the important predisposing factors for deep venous thrombosis (DVT). DVT usually develops in the lower extremities, and venous thrombosis of the upper extremities is uncommon. Early diagnosis and treatment of deep venous thrombosis are of importance, because it is a precursor of complications such as pulmonary embolism and postthrombotic syndrome. A 47-year-old woman visited our department with painful swelling on the left side of her neck. Initial examination revealed swelling of the region extending from the left neck to the shoulder without any redness of the overlying skin. Laboratory tests showed a white blood cell count of 5,800/mm3 and an elevated serum C-reactive protein of 4.51 mg/dL. Computed tomography (CT) of the neck revealed a vascular filling defect in the left internal jugular vein to left subclavian vein region, with the venous lumina completely occluded with dense soft tissue. On the basis of the findings, we made the diagnosis of thrombosis of the left internal jugular and left subclavian veins. The patient was begun on treatment with oral rivaroxaban, but the left shoulder pain worsened. She was then admitted to the hospital and treated by balloon thrombectomy and thrombolytic therapy, which led to improvement of the left subclavian venous occlusion. Histopathologic examination of the removed thrombus revealed adenocarcinoma cells, indicating hematogenous dissemination of malignant cells.

Comprehensive approach to structural and functional glycomics based on chemoselective glycoblotting and sequential tag conversion.

Changes in protein glycosylation profoundly affect protein function. To understand these effects of altered protein glycosylation, we urgently need high-throughput technologies to analyze glycan expression and glycan-protein interactions. Methods are not available for amplification of glycans; therefore, highly efficient sample preparation is a major issue. Here we present a novel strategy that allows flexible and sequential incorporation of various functional tags into oligosaccharides derived from biological samples in a practical manner. When combined with a chemoselective glycoblotting platform, our analysis enables us to complete sample preparation (from serum to released, purified, methyl-esterified, and labeled glycans) in 8 h from multiple serum samples (up to 96 samples) using a 96-well microplate format and a standard de-N-glycosylation protocol that requires reductive alkylation and tryptic digestion prior to PNGase F digestion to ensure maximal de-N-glycosylation efficiency. Using this technique, we quantitatively detected more than 120 glycans on human carcinoembryonic antigens for the first time. This approach was further developed to include a streamlined method of purification, chromatographic fractionation, and immobilization onto a solid support for interaction analysis. Since our approach enables rapid, flexible, and highly efficient tag conversion, it will contribute greatly to a variety of glycomic studies.

Quantitative glycomics of human whole serum glycoproteins based on the standardized protocol for liberating N-glycans.

Global glycomics of human whole serum glycoproteins appears to be an innovative and comprehensive approach to identify surrogate non-invasive biomarkers for various diseases. Despite the fact that quantitative glycomics is premised on highly efficient and reproducible oligosaccharide liberation from human serum glycoproteins, it should be noted that there is no validated protocol for which deglycosylation efficiency is proven to be quantitative. To establish a standard procedure to evaluate N-glycan release from whole human serum glycoproteins by peptide-N-glycosidase F (PNGase F) treatment, we determined the efficiencies of major N-glycan liberation from serum glycoproteins in the presence of reducing agents, surfactants, protease treatment, or combinations of pretreatments prior to PNGase F digestion. We show that de-N-glycosylation efficiency differed significantly depending on the condition used, indicative of the importance of a standardized protocol for the accumulation and comparison of glycomics data. Maximal de-N-glycosylation was achieved when serum was subjected to reductive alkylation in the presence of 2-hydroxyl-3-sulfopropyl dodecanoate, a surfactant used for solubilizing proteins, or related analogues, followed by tryptic digestion prior to PNGase F treatment. An optimized de-N-glycosylation protocol permitted relative and absolute quantitation of up to 34 major N-glycans present in serum glycoproteins of normal subjects for the first time. Moreover PNGase F-catalyzed de-N-glycosylation of whole serum glycoproteins was characterized kinetically, allowing accurate simulation of PNGase F-catalyzed de-N-glycosylation required for clinical glycomics using human serum samples. The results of the current study may provide a firm basis to identify new diagnostic markers based on serum glycomics analysis.

Reorganization of ZO-1 by sodium-dependent glucose transporter activation after heat stress in LLC-PK1 cells.

Heat stress (HS) induces activation of high-affinity sodium-dependent glucose transporter (SGLT1) in porcine renal LLC-PK(1) cells. In this study, we investigated the roles of SGLT1 activation in reorganization of zonula occludens-1 (ZO-1), a cytosolic tight junction (TJ) protein, after HS. HS (42 degrees C, 3 h) caused decrease in transepithelial electrical resistance (TER). Subsequent incubation at 37 degrees C for 12 h increased TER above pre-HS level. The treatment of phloridzin, a potent SGLT1 inhibitor, or the replacement of glucose with a nonmetabolizable glucose analog blocked the recovery of TER and increased the transepithelial flux of FITC-dextran (4,000 Da). Immunofluorescent staining of ZO-1 showed that HS diffused ZO-1 from cell contact to cytosolic sites. Furthermore, the fraction of ZO-1 was distributed from the Triton X-100 insoluble to the Triton X-100 soluble pool. After incubation at 37 degrees C for 12 h, cell contact and ZO-1 extractability with Triton X-100 returned to pre-HS conditions, but the recovery was completely prevented by phloridzin. Tyrosine kinases activity was increased by HS that was inhibited by phloridzin. Genistein and CGP77675, tyrosine kinases inhibitors, blocked the recovery of TER and increased the transepithelial flux of FITC-dextran. Furthermore, these inhibitors prevented the recovery of cell contact and ZO-1 extractability with Triton X-100 as same as phloridzin. These findings suggested that the activation of SGLT1 reorganized ZO-1 mediated by elevation of tyrosine kinases activity after heat injury.

Recovery from heat shock injury by activation of Na+-glucose cotransporter in renal epithelial cells.

Exposure of cells or organs to sublethal physical or chemical stresses induces disruption of cellular structures and functions. Here, we examined whether Na(+)-glucose cotransporter (SGLT1) is involved in the recovery from heat shock (HS) injury in porcine renal epithelial LLC-PK(1) cells. Recovery from HS (42 degrees C for 3 h, then 37 degrees C for 12 h) increased SGLT1 activity, assessed by [14C]alpha-methyl glucopyranoside uptake, and a maximal transport rate (V(max)) from 2.4 to 5.9 nmol/mg protein/30 min, but did not alter an apparent affinity constant (K(m)). Protein distribution of SGLT1 in apical membrane fraction was also increased after recovery from HS without changing in total membrane fraction. Membrane integrity assessed by calcein accumulation was decreased by HS, and then returned to basal level. This recovery was inhibited by phloridzin, a potent SGLT1 inhibitor, and nonmetabolizable glucose analogues. Anti-transforming growth factor-beta 1 (TGF-beta 1) antibody inhibited both elevation of SGLT1 distribution in apical membrane and recovery of calcein accumulation induced by HS. Taken together, HS increases in the number of SGLT1 protein in apical membrane mediated via TGF-beta 1 signaling pathway. The increase of glucose uptake is necessary to repair plasma membrane integrity.

Up-regulation of sodium-dependent glucose transporter by interaction with heat shock protein 70.

Heat shock stress induces some heat shock proteins, including Hsp70, and activates sodium-dependent glucose transport in porcine renal LLC-PK(1) cells, but its mechanisms have not been described in detail. We investigated whether sodium-dependent glucose transporter (SGLT1) interacts with Hsp70 to increase SGLT1 activity. Heat shock stress increased SGLT1 activity without changing SGLT1 expression. The increase of SGLT1 activity was completely inhibited by an anti-transforming growth factor-beta1 (TGF-beta1) antibody. Instead of heat shock stress, TGF-beta1 increased SGLT1 activity dose- and time-dependently without changing SGLT1 expression. We found that the amount of Hsp70 immunoprecipitated from TGF-beta1-treated cells with an anti-SGLT1 antibody was higher than that of the control cells. Transfection of an anti-Hsp70 antibody into the cells inhibited the increase of SGLT1 activity. With confocal laser microscopy, both SGLT1 and Hsp70 was localized near the apical membrane in the TGF-beta1-treated cells, and an anti-Hsp70 antibody disturbed this localization. Furthermore, we clarified that an anti-Hsp70 antibody inhibited interaction of SGLT1 with Hsp70 in vitro. These results suggest that Hsp70 forms a complex with SGLT1 and increases the expression level of SGLT1 in the apical membrane, resulting in up-regulation of glucose uptake.

Phosphatidylinositol 3-kinase mediates inhibitory effect of angiotensin II on sodium/glucose cotransporter in renal epithelial cells.

Effects of angiotensin II (ANGII) on regulation of sodium/glucose cotransporter (SGLT1) activity were investigated in LLC-PK(1) cells, renal proximal epithelial cell line. ANGII inhibited alpha-[14C] methyl-D-glucopyranoside (AMG) uptake into LLC-PK(1) cells in a dose-dependent manner. This inhibition was based on a decrease in maximal transport rate (Vmax) of AMG from 2.20 nmol/mg protein/15 min to 1.19 nmol/mg protein/15 min, although apparent affinity constant (Km) did not alter. In western blot analysis, protein level of SGLT1 in brush border membrane (BBM) was decreased by ANGII, although total SGLT1 was not altered. In the aspect of intracellular signal transduction, ANGII blocked the formation of cAMP. Pertussis toxin, an inactivator of Gi protein that control intracellular cAMP level, completely prevented the decrease of AMG uptake caused by ANGII. 8-Br-cAMP, a cell membrane permeable cAMP analogue, increased AMG uptake and protein level of SGLT1 in BBM. Both wortmannin and LY294002 that are phosphatidylinositol (PI) 3-kinase inhibitors, inhibited the SGLT1 activity, and also attenuated the effect of 8-Br-cAMP on SGLT1 activity. Those inhibitors prevented the 8-Br-cAMP-induced expression of SGLT1 in plasma membrane. We conclude that ANGII plays an important role in post-translational regulation in SGLT1. Inhibition of SGLT1 translocation is suggested to be caused by inactivation of protein kinase A and decrease of PI 3-kinase activity.