ORMDL3 overexpression facilitates FcεRI-mediated transcription of proinflammatory cytokines and thapsigargin-mediated PERK phosphorylation in RBL-2H3 cells.

INTRODUCTION: The chromosomal region 17q21 harbors the human orosomucoid-like 3 (ORMDL3) gene and has been linked to asthma and other inflammatory diseases. ORMDL3 is involved in the unfolded protein response (UPR), lipid metabolism, and inflammatory reactions. We investigated the effects of ORMDL3 overexpression in RBL-2H3 cells to determine the contribution of ORMDL3 to inflammatory disease development. METHODS: We generated ORMDL3 stably overexpressing RBL-2H3 cells to assess degranulation, transcriptional upregulation of interleukin-4 (IL-4), tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1), and mitogen-activated protein kinase (MAPK) phosphorylation via FcεRI. In addition, we examined the effects of ORMDL3 overexpression on thapsigargin (TG)-mediated proinflammatory cytokine transcription and UPR by monitoring MAPK, protein kinase-like endoplasmic reticulum kinase (PERK), and inositol-requiring enzyme 1 (IRE1) phosphorylation. RESULTS: Overexpression of ORMDL3 enhanced IL-4, TNF-α, and MCP-1 expression after FcεRI cross-linking, whereas the sphingosine-1-phosphate (S1P) agonist FTY720 suppressed this enhancement. There was no significant difference in degranulation and MAPK phosphorylation via FcεRI-mediated activation between vector-transfected and ORMDL3-overexpressing cells. ORMDL3 overexpression accelerated TG-mediated PERK phosphorylation, while MAPK phosphorylation and proinflammatory cytokine expression showed no significant changes in ORMDL3-overexpressing cells. CONCLUSIONS: Our findings suggest that ORMDL3 plays an important role in regulating proinflammatory cytokine expression via the S1P pathway and selectively affects the UPR pathway in mast cells.

GIP Receptor Antagonist, SKL-14959 Indicated Alteration of the Lipids Metabolism to Catabolism by the Inhibition of Plasma LPL Activity, Resulting in the Suppression of Weight Gain on Diets-Induced Obesity Mice.

INTRODUCTION: Glucose-dependent insulinotropic polypeptide (GIP) plays a crucial role in the regulation of lipid metabolism via lipoprotein lipase (LPL). GIP receptor antagonist, SKL-14959, suppressed the weight gain in the diet-induced obesity model. However, the mechanism is not unclear. Therefore, we aimed to give insight into the reason. METHODS: Mice were divided into three groups of the low-fat diet, high-fat diets mixture with or without SKL-14959 for 151 days, and were monitored body weight and food consumption through the test. Oral glucose tolerance test (OGTT) and insulin tolerance test (ITT) were also performed. After that, blood, liver, muscle and adipose tissue were collected. Blood samples were measured glycosylated hemoglobin A1c (HbA1c), glucose, insulin, GIP level and plasma LPL activity. Triacylglycerol (TG) contents of liver and muscles were also measured. Moreover, a simple correlation analysis was performed. RESULTS: SKL-14959 suppressed the body weight gain, decreased body mass index (BMI), HbA1c, and fasting glucose level, and trended to decline adipose tissues weight and TG contents compared with the vehicle, and inhibited plasma LPL activity. OGTT and ITT in the SKL-14959 group were not significantly changed relative to the vehicle. Additionally, upon treatment with SKL-14959 treatment, weight gain had weak correlation with lipase activity. Furthermore, lipase activity was associated with the fat mass and not white but red muscle TG contents and liver TG contents were not associated with lipase activity but HbA1c. IN CONCLUSION: SKL-14959 might direct lipids metabolism to catabolism by inhibition of plasma LPL activity, resulting in the suppression of weight gain on diets-induced obesity mice.

Enhanced 15-Lipoxygenase 1 Production is Related to Periostin Expression and Eosinophil Recruitment in Eosinophilic Chronic Rhinosinusitis.

BACKGROUND: The pathological features of chronic rhinosinusitis (CRS) with nasal polyps (CRSwNP) tissues include an eosinophilic infiltration pattern (eosinophilic CRS (ECRS)) or a less eosinophilic pattern (non-ECRS). Recently, it has been suggested that 15-lipoxygenase 1 (15-LOX-1) may have significant roles in allergic disease; however, the significance of 15-LOX-1 in CRS is not well understood. The objective of this study was to demonstrate the expression of 15-LOX-1 in CRS. METHODS: The mRNA expression levels of 15-LOX-1 and periostin in nasal tissues were measured by quantitative real-time polymerase chain reaction. We also performed an immunofluorescence study of nasal tissues. Cells of the Eol-1 eosinophilic leukemic cell line were stimulated with interleukin-33 to test the induction of 15-LOX-1. RESULTS: The expression level of 15-LOX-1 mRNA in nasal polyps (NPs) was significantly higher in ECRS patients than in non-ECRS patients. The immunofluorescence study revealed that both airway epithelial cells and eosinophils in NPs expressed 15-LOX-1. A significant correlation was seen between the number of eosinophils and the mRNA expression levels of 15-LOX-1 and periostin in nasal polyps. Moreover, interleukin-33 enhanced 15-LOX-1 expression in Eol-1 cells. CONCLUSIONS: 15-LOX-1 was shown to be a significant molecule that facilitates eosinophilic inflammation in ECRS.

Suppression of SESN1 reduces cisplatin and hyperthermia resistance through increasing reactive oxygen species (ROS) in human maxillary cancer cells.

INTRODUCTION: Cisplatin is used as a standard chemotherapeutic agent for head and neck cancer treatment. However, some head and neck cancers have cisplatin resistance, leading to difficulty in treatment and poor prognosis. Overcoming cisplatin resistance remains an important strategy to improve prognoses for head and neck cancer patients. OBJECTIVE: Elucidation of the mechanisms underlying cisplatin resistance can suggest novel targets to enhance the anticancer effects of cisplatin for treating head and neck cancers. MATERIAL AND METHODS: We used a cisplatin-resistant human maxillary cancer cell line, IMC-3CR to analyse the cisplatin resistance mechanisms. Cisplatin-induced genes were analysed in IMC-3CR cells using PCR array. Among the genes with expression increased by cisplatin, we specifically examined SESN1. SESN family reportedly regenerates peroxiredoxin and suppresses oxidative DNA injury by reactive oxygen species (ROS), which can be induced by chemotherapeutic agents such as cisplatin, radiation, and hyperthermia. The function of SESN1 in cisplatin resistance and ROS generation were analysed using specific RNAi. RESULTS: Results show that SESN1 was induced by cisplatin treatment in IMC-3CR cells. Suppression of SESN1 by RNAi induced apoptosis and reduced cell viability through enhancement of ROS after cisplatin treatment. Moreover, suppression of SESN1 enhanced the cell-killing effects of hyperthermia with increased ROS, but did not affect the cell-killing effects of radiation. CONCLUSIONS: This study demonstrated the participation of SESN1 in cisplatin and hyperthermia resistance of human head and neck cancers. SESN1 is a novel molecular target to overcome cisplatin resistance and hyperthermia resistance and improve head and neck cancer treatment.

Periostin as a novel biomarker for postoperative recurrence of chronic rhinosinitis with nasal polyps.

We previously reported that chronic rhinosinusitis with nasal polyps (CRSwNP) was subdivided into four chronic rhinosinusitis (CRS) subtypes using the JESREC scoring system. We sought to identify the gene expression profile and biomarkers related with CRSwNP by RNA-sequence. RNA-sequencing was performed to identify differentially expressed genes between nasal polyps (NPs) and inferior turbinate mucosa from 6 patients with CRSwNP, and subsequently, quantitative real-time PCR was performed to verify the results. ELISA was performed to identify possible biomarkers for postoperative recurrence. In the RNA-sequencing results, periostin (POSTN) expression was the highest in NP. We focused on POSTN and investigated the protein level of POSTN by immunohistochemistry and ELISA. POSTN was diffusely expressed in moderate and severe eosinophilic CRS using immunohistochemistry, and its staining pattern was associated with the severity of the phenotype of the CRSwNP (P < 0.05). There was a significant difference between the POSTN high/low groups for postoperative recurrence when the cutoff point was set at 115.5 ng/ml (P = 0.0072). Our data suggests that the protein expression level of POSTN was associated with the severity of CRSwNP, and serum POSTN can be a novel biomarker for postoperative recurrence of CRSwNP.

Discovery of a long-acting glucagon-like peptide-1 analog with enhanced aggregation propensity.

In the course of our search for new GLP-1 analogs, we screened a number of [Ser8]-GLP-1 analogs using the C-terminal helix 3 of the albumin binding domain 3 of protein G from bacterial Streptococcal G strain 148 (G148-ABD3) as appendage. Our efforts led to the discovery of [Ser8]-GLP-1 (7-35)-GVKALIDEILAA-NH2, peptide 6, as a long-acting GLP-1 analog with enhanced self-associated aggregation. Peptide 6 showed enhanced stability in rat and human plasma and an extended half-life of 5.4 h with good bioavailability in rats and subsequently prolonged therapeutic effects in diabetic mice. Analytical ultracentrifugation and TLC suggest that 6 remains oligomeric in the circulation, which accounts for its extended in vivo half-life. The present work shows the possible enhancement of medium-sized oligopeptides aggregation propensity and highlights the potential advantages of peptide aggregates for long-acting peptide drugs.

Toll-like receptor ligands induce cytokine and chemokine production in human inner ear endolymphatic sac fibroblasts.

OBJECTIVE: Against recent reports concerning cytokine or chemokine in mouse or rat inner ear cells, it is almost unknown whether human inner ear cells would produce cytokine or chemokine. We have for the first time established the human inner-ear-derived fibroblasts from endolymphatic sac. METHODS: The expression levels of Toll-like receptors (TLRs) in human endolymphatic sac fibroblasts, and the effect on cytokine or chemokine production of the TLR ligands have been examined. To demonstrate the intracellular pathways involved in the regulation of cytokine-production, we used specific inhibitors of c-Jun N-terminal kinase (JNK), extracellular signal-related kinase (ERK), p38 mitogen-activated protein kinase (p38 MAPK)-signaling and N-acetyl-l-cysteine (NAC). RESULTS: TLR 2, 3, 4 and 9 were highly expressed in human endolymphatic sac fibroblasts. The TLR 3 ligand, polyinosinic-polycytidylic acid (poly(I:C)) significantly enhanced the secretion of thymic stromal lymphopoietin (TSLP), B lymphocyte stimulator (BLyS), IFNγ-inducible protein 10 (IP-10), and macrophage inflammatory protein 1 alpha (MIP-1α) from the cells. The inhibitor of JNK strongly reduced the poly(I:C)-induced TSLP-production. The antioxidant drug, NAC also reduced the TSLP-production in fibroblasts stimulated with poly(I:C). CONCLUSION: Our findings suggest human inner-ear-endolymphatic sac derived fibroblasts can produce the cytokine and chemokine in response to TLR ligands and play a certain role during the initiation of an immune response.

Gustatory Dysfunction and Decreased Number of Fungiform Taste Buds in Patients With Chronic Otitis Media With Cholesteatoma.

OBJECTIVE: To compare the number of fungiform taste buds among patients with chronic otitis media (COM), those with pars flaccida retraction type cholesteatoma, and those with pars tensa retraction type cholesteatoma in combination with gustatory function. METHODS: Thirty-seven patients with COM, 22 patients with pars flaccida retraction type cholesteatoma, and 17 patients with pars tensa retraction type cholesteatoma were included. An average of 10 fungiform papillae (FP) per patient in the midlateral region of the tongue were observed by confocal laser scanning microscopy in vivo, and the average number of taste buds were counted. Just before the observation of FP, electrogustometry (EGM) was performed to evaluate gustatory function. RESULTS: A significant decrease of the average number of fungiform taste buds and significant elevation of EGM thresholds were clarified in the pars tensa retraction type cholesteatoma group but not in the COM or pars flaccida type cholesteatoma group. CONCLUSION: It was suggested that some neurotoxic cytokines produced by cholesteatoma tissue might affect the CTN morphology, resulting in a decreased number of fungiform taste buds and elevation of EGM threshold in patients with pars tensa retraction type cholesteatoma.

Structural analysis of the outermost hair surface using TOF-SIMS with gas cluster ion beam sputtering.

A hair cuticle, which consists of flat overlapping scales that surround the hair fiber, protects inner tissues against external stimuli. The outermost surface of the cuticle is covered with a thin membrane containing proteins and lipids called the epicuticle. In a previous study, the authors conducted a depth profile analysis of a hair cuticle's amino acid composition to characterize its multilayer structure. Time-of-flight secondary ion mass spectrometry with a bismuth primary ion source was used in combination with the C60 sputtering technique for the analysis. It was confirmed that the lipids and cysteine-rich layer exist on the outermost cuticle surface, which is considered to be the epicuticle, though the detailed structure of the epicuticle has not been clarified. In this study, depth profile analysis of the cuticle surface was conducted using the argon gas cluster ion beam (Ar-GCIB) sputtering technique, in order to characterize the structure of the epicuticle. The shallow depth profile of the cuticle surface was investigated using an Ar-GCIB impact energy of 5 keV. Compared to the other amino acid peaks rich in the epicuticle, the decay of 18-methyleicosanic acid (18-MEA) thiolate peak was the fastest. This result suggests that the outermost surface of the hair is rich in 18-MEA. In conclusion, our results indicate that the outermost surfaces of cuticles have a multilayer (lipid and protein layers), which is consistent with the previously proposed structure.

[Clinical Outcome of Laparoscopic Partial Nephrectomy].

The purpose of this study was to evaluate the safety and feasibility of laparoscopic partial nephrectomy with a small renal tumor. Between September 2004 and October 2014, 69 patients who underwent laparoscopic partial nephrectomy in Kansai Rosai Hospial were examined. The mean patient age was 60.3 years, and the mean tumor size was 24.5 mm. The mean estimated blood loss was 111 cc. The mean cold ischemic time was 59.7 minutes, and the mean warm ischemic time was 31.3 minutes. There were 5 complications : intraoperative ureteral injury, blood transfusion, postoperative perinephric hematoma, portsite bleeding, urinary fistula, respectively. All of the cases were cured with non-surgical treatment except ureteral injury which was repaired intraoperatively. The postoperative eGFR loss was 11%. At present, no patients have developed local recurrence or distant metastasis. The initial outcome of laparoscopic partial nephrectomy in our hospital was satisfactory in terms of safety, renal function and cancer control.

Comparison of regeneration of the chorda tympani nerve and gustatory function recovery after severing the nerve between pediatric and adult patients.

OBJECTIVE: To compare the incidence of regeneration of the chorda tympani nerve (CTN) and taste function recovery between pediatric and adult patients after severing the CTN. STUDY DESIGN: Retrospective study. SETTING: University hospital. PATIENTS: Consecutive 31 pediatric patients and 61 adult patients who underwent staged tympanoplasty (86 patients) or reoperation (6 patients) because of chronic otitis media with (89 patients) or without cholesteatoma (3 patients) and whose CTN was severed during primary surgery were included. INTERVENTION: Therapeutic. MAIN OUTCOME MEASURES: The severed nerves were readapted or approximated on the temporalis muscle fascia used to reconstruct the eardrum. During secondary surgery, we tried to detect regenerated CTN. Preoperative and postoperative gustatory function was assessed using electrogustometry. RESULTS: Regenerated CTN was detected during secondary surgery in 58.1% (18/31) of pediatric patients and 31.1% (19/61) of adults (p < 0.05). However, the number of patients with preoperatively elevated electrogustometry threshold was greater in adult patients. When the subjects were limited to those with preoperatively normal taste function, there was no significant difference between the 2 groups. Among patients with CTN regeneration, the incidence of taste function recovery was higher (12/18; 66.7%) in children than that (6/19; 31.6%) in adults (p < 0.05). However, there was no significant difference when the subjects were limited to those with preoperatively normal taste function. CONCLUSION: Chronic inflammation may not severely affect the CTN in pediatric patients, resulting in higher incidences of CTN regeneration and gustatory function recovery after severing the nerve, compared with those in adult patients.

Interleukin-19 downregulates interleukin-4-induced eotaxin production in human nasal fibroblasts.

BACKGROUND: Interleukin-19 (IL-19), a member of the IL-10 family, is characterized as the cytokine suppressing the release and function of several proinflammatory cytokines. For regulation of local reaction in allergic rhinitis (AR), IL-19 might play an especially important role. METHODS: We examined effects of IL-19 on IL-4-induced eotaxin production by human nasal fibroblasts. Early receptor-mediated events (expression of the suppressors of cytokine signaling (SOCS) and phosphorylation of signal transducer and activator of transcription 6 [STAT6]) by IL-19 was examined. Knockdown methods by RNAi were administered to investigate the involvement of those signal transductions. RESULTS: Pretreatment with IL-19 downregulates IL-4-induced eotaxin production, but not interferon-γ(IFN-γ)-induced RANTES. Pretreatment with IL-19 suppressed the IL-4-induced STAT6 phosphorylation. The IL-19 induced SOCS-1, but not SOCS-3 or SOCS-5. The SOCS-1 knockdown by RNAi diminished pretreatment with IL-19-induced down-regulation of eotaxin production. CONCLUSIONS: These results suggest that IL-19 down-regulates IL-4-induced eotaxin production via SOCS-1 in human nasal fibroblasts. In non-hematopoietic cells in AR, IL-19 might be an immunosuppressive factor.

Discovery, optimization, and pharmacological characterization of novel heteroaroylphenylureas antagonists of C-C chemokine ligand 2 function.

Through the application of TRAP (target-related affinity profiling), we identified a novel class of heteroaroylphenylureas that inhibit human CCL2-induced chemotaxis of monocytes/macrophages both in vitro and in vivo. This inhibition was concentration-dependent and selective with regard to other chemokines. The compounds, however, did not antagonize the binding of (125)I-labeled CCL2 to the CCR2 receptor nor did they block CCR2-mediated signal transduction responses such as calcium mobilization. Optimization of early leads for potency and pharmacokinetic parameters resulted in the identification of 17, a potent inhibitor of chemotaxis (IC(50) = 80 nM) with excellent oral bioavailability in rats (F = 60%). Compound 17 reduced swelling and joint destruction in two rat models of rheumatoid arthritis and delayed disease onset and produced near complete resolution of symptoms in a mouse model of multiple sclerosis.

Class IA phosphatidylinositol 3-kinase in pancreatic ß cells controls insulin secretion by multiple mechanisms.

Type 2 diabetes is characterized by insulin resistance and pancreatic ß cell dysfunction, the latter possibly caused by a defect in insulin signaling in ß cells. Inhibition of class IA phosphatidylinositol 3-kinase (PI3K), using a mouse model lacking the pik3r1 gene specifically in ß cells and the pik3r2 gene systemically (ßDKO mouse), results in glucose intolerance and reduced insulin secretion in response to glucose. ß cells of ßDKO mice had defective exocytosis machinery due to decreased expression of soluble N-ethylmaleimide attachment protein receptor (SNARE) complex proteins and loss of cell-cell synchronization in terms of Ca(2+) influx. These defects were normalized by expression of a constitutively active form of Akt in the islets of ßDKO mice, preserving insulin secretion in response to glucose. The class IA PI3K pathway in ß cells in vivo is important in the regulation of insulin secretion and may be a therapeutic target for type 2 diabetes.

LL5beta directs the translocation of filamin A and SHIP2 to sites of phosphatidylinositol 3,4,5-triphosphate (PtdIns(3,4,5)P3) accumulation, and PtdIns(3,4,5)P3 localization is mutually modified by co-recruited SHIP2.

Phosphatidylinositol 3,4,5-triphosphate (PtdIns(3,4,5)P(3)) accumulates at the leading edge of migrating cells and works, at least partially, as both a compass to indicate directionality and a hub for subsequent intracellular events. However, how PtdIns(3,4,5)P(3) regulates the migratory machinery has not been fully elucidated. Here, we demonstrate a novel mechanism for efficient lamellipodium formation that depends on PtdIns(3,4,5)P(3) and the reciprocal regulation of PtdIns(3,4,5)P(3) itself. LL5beta, whose subcellular localization is directed by membrane PtdIns(3,4,5)P(3), recruits the actin-cross-linking protein Filamin A to the plasma membrane, where PtdIns(3,4,5)P(3) accumulates, with the Filamin A-binding Src homology 2 domain-containing inositol polyphosphate 5-phosphatase 2 (SHIP2). A large and dynamic lamellipodium was formed in the presence of Filamin A and LL5beta by the application of epidermal growth factor. Conversely, depletion of either Filamin A or LL5beta or the overexpression of either an F-actin-cross-linking mutant of Filamin A or a mutant of LL5beta without its PtdIns(3,4,5)P(3)-interacting region inhibited such events in COS-7 cells. Because F-actin initially polymerizes near the plasma membrane, it is likely that membrane-recruited Filamin A efficiently cross-links newly polymerized F-actin, leading to enhanced lamellipodium formation at the site of PtdIns(3,4,5)P(3) accumulation. Moreover, we demonstrate that co-recruited SHIP2 dephosphorylates PtdIns(3,4,5)P(3) at the same location.

Decreased insulin secretion and accumulation of triglyceride in beta cells overexpressing a dominant-negative form of AMP-activated protein kinase.

Adenosine 5' -monophosphate-activated protein kinase (AMPK) has been implicated in the regulation of energy metabolism, although its role in the pancreatic beta cells remains unclear. In the present, we have overexpressed a dominant negative form of AMPKalpha1 subunit (Asp57Ala) tagged with c-myc epitope (AMPKalpha1-DN) in INS-1D cells with an adenoviral vector. After 48 h of adenoviral infection, overexpression of AMPKalpha1-DN in INS-1D cells was confirmed by Western blot analysis with anti-c-myc antibody. Phosphorylation of the Thr172 in AMPKalpha1/alpha2 subunit was progressively decreased in parallel with increasing number of adenoviral titers. Glucose-stimulated insulin secretion in response to 30 mmol/L glucose was decreased in INS-1D cells overexpressing AMPKalpha1-DN as compared to control cells infected with adeno- LacZ vector. Neither cellular insulin content nor insulin mRNA level was changed between the two groups. Phosphorylation of acetyl-CoA carboxylase (ACC), a down-stream substrate of AMPK, was decreased, indicating that ACC activity was increased, due to the decreased AMPK activity. In fact, intracellular triglyceride content was increased as compared to control cells. The beta-oxidation of palmitate was decreased at 30 mmol/L glucose. Insulin secretion in response to potassium chloride or glibenclamide was also decreased as compared to control cells. In conclusion, suppression of AMPK activity in beta-cells inhibited insulin secretion in response to glucose, potassium chloride or glibenclamide without altering insulin content. Accumulation of triglyceride subsequent to the activation of ACC by suppression of AMPK activity, was suggested to be, at least in part, responsible for the impaired insulin secretion through so-called lipotoxicity mechanism.

Lipopolysaccharides increase the amount of CXCR4, and modulate the morphology and invasive activity of oral cancer cells in a CXCL12-dependent manner.

Recently, it has been reported that bacterial infections play an important role in the development of cancers of the upper aero digestive tract. To examine the influence of bacterial infections on oral cancer, human oral carcinoma T3M-1 cells were treated with lipopolysaccharide (LPS) for 24 h as a model of infection. The LPS treatment increased the mRNA expression of CXCR4 and invasiveness in T3M-1 cells stimulated with CXCL12. The Rho family of small guanosine triphosphatases regulates the dynamics of the actin cytoskeleton that underlie cellular functions such as cell shape changes, migration and polarity. In T3M-1 cells treated with LPS and stimulated with CXCL12, Rac and Cdc42 were activated and caused an increase in the development of filopodia. The present findings suggest that bacterial infections enhance the invasiveness of T3M-1 cells via CXCL12/CXCR4 interaction and Cdc42-activation. Furthermore, filopodia are critical to this process.

[A case of ectopic ureterocele in a male adult found during examination of a traumatic injury].

We report a case of ectopic ureterocele in a male adult found during examination of a traumatic injury. A 26-year-old man sustained a blow to his left back during a football game and was admitted to the hospital with a complaint of abdominal pain. The computed tomographic scan showed a huge cystic mass in the retroperitoneum. A cystoscope revealed a large bulge from the left ureteral orifice to the bladder neck and another ureteral orifice distal to the bulge. Retrograde pyelography revealed an ectopic ureterocele showing a complete duplication, a lateral deviation of the left ureter and a bladder deviation to the right. An ectopic ureteral orifice was opening in the posterior urethra. An ureteral catheter inserted into this orifice revealed a dilated left ureter from the upper half of the kidney. Because the function of left upper of kidney was maintained, a transurethral incision was performed. Vesicoureteral reflux remained, but the postoperative course was uneventful. We discuss 11 cases of ectopic ureterocele in male adults including this case.