Etlingera pavieana extract attenuates TNF-α induced vascular adhesion molecule expression in human endothelial cells through NF-κB and Akt/JNK pathways.

The aim of this study was to determine whether ethanol extracts of Etlingera pavieana rhizomes (EPE) can inhibit the expression of ICAM-1 and VCAM-1 in TNF-α-stimulated human vascular endothelial cells. EPE significantly reduced ICAM-1 and VCAM-1 expression in a concentration-dependent manner. EPE also suppressed phospho-IκB level and nuclear translocation of NF-κB. EPE significantly inhibited phosphorylation of JNK and c-Jun, a major component of AP-1, but had no effects on ERK and p38 MAPK pathways. Akt phosphorylation was increased in the presence of EPE, and wortmannin and SP600125 reversed the inhibitory effects of EPE on ICAM-1 and VCAM-1 expression. Furthermore, the active EPE constituents 4-methoxycinnamyl p-coumarate and trans-4-methoxycinnamaldehyde attenuated TNF-α-induced expression of ICAM-1 and VCAM-1. Taken together, our data indicate that EPE protects against vascular inflammation in endothelial cells, in part via NF-κB and Akt/JNK signalings. In future studies, E. pavieana may be developed as a therapeutic agent or dietary supplement for treating and preventing inflammatory diseases.

AMPK Alters Detrusor Contractility During Emptying in Normal Bladder and Hypertrophied Bladder with Partial Bladder Outlet Obstruction via CaMKKß.

AMP-activated protein kinase (AMPK) has been implicated in contractility changes in bladders with partial bladder outlet obstruction (PBOO), but the role of AMPK in the contractile response of normal bladder remains unclear. We investigated the phosphorylation of AMPKα and expression of the involved upstream AMPK kinases (AMPKKs) in a model of bladders with PBOO and sought to determine whether the pharmacological inhibition of these two factors affected detrusor contractility in normal bladders, using female Sprague-Dawley rats. Cystometry and Western blot analysis were performed in rats that were subjected to PBOO induction or a sham operation. Cystometry was performed in normal rats that received selective inhibitors of AMPKα and Ca2+/calmodulin-dependent protein kinase kinase (CaMKKß) (compound C and STO-609, respectively) at doses determined in the experiments. In the PBOO bladders, bladder weight and micturition pressure (MP) were higher and AMPKα phosphorylation (T172) and CaMKKß expression was significantly reduced. Compound C and STO-609 increased MP. The increased contractile response in bladders with PBOO-induced hypertrophy was related to decreased CaMKKß/AMPK signaling activity, and the pharmacological inhibition of this pathway in normal bladders increased detrusor contractility, implying a role of CaMKKß/AMPK signaling in the bladder in the regulation of detrusor contractility.

Roles of Exosome-Like Vesicles Released from Inflammatory C2C12 Myotubes: Regulation of Myocyte Differentiation and Myokine Expression.

BACKGROUND/AIMS: The complicated differentiation processes of cells in skeletal muscle against inflammation that induce muscle atrophy are not fully elucidated. Given that skeletal muscle is a secretory organ, we evaluated the effects of inflammation on myogenic signals and myokine expression, and the roles of inflammatory exosomes released by myotubes in myogenic differentiation. METHODS: Inflammation was induced by treatment of fully differentiated C2C12 myotubes with a cytokine mixture of TNF-α and INF-γ. Exosome-like vesicles (ELVs) were isolated from conditioned media of control or inflamed myotubes and incubated with myoblasts. The expression of molecular switches that contribute to myogenic differentiation, including several kinases, their downstream targets, and myokines, were evaluated using immunoblot analysis in inflamed myotubes and in myoblasts treated with ELVs. RESULTS: Inflammation activated molecular mechanisms contributing to muscle atrophy, including AMPK, p-38 MAPK and JNK, while inhibiting Akt-mediated myogenic signals. In addition, inflammation induced myostatin expression with suppression of a myostatin-counteracting myokine, decorin. Well-characterized ELVs released from inflamed myotubes induced myoblast inflammation and inhibited myogenic mechanisms while stimulating atrophic signals. CONCLUSION: Inflammation of skeletal muscle induces muscle atrophy via multiple mechanisms, including the regulation of myokines and kinases. Inflammatory ELVs are likely to contribute to inflammation-induced muscle atrophy.

Additive anti-allergic effects of anti-interleukin-33 and anti-Siglec-F treatments in a murine model of allergic asthma.

BACKGROUND: Anti-interleukin-33 (anti-IL-33) and anti-Siglec-F antibodies have potent anti-allergic effects on murine allergic asthma and rhinitis and induce eosinophil apoptosis. OBJECTIVE: We aimed to determine whether post-sensitization with anti-IL-33/anti-Siglec-F treatments exhibited more potent effects compared to individual treatments in a murine allergic asthma model. MATERIAL AND METHODS: Twenty-five BALB/c mice were separated into five groups (n = 5): Group A (control), Group B (ovalbumin [OVA] challenge), Group C (OVA + anti-IL-33), Group D (OVA + anti-Siglec-F), and Group E (OVA + anti-IL-33 + anti-Siglec-F). Serum total/ OVA-specific IgE, bronchoalveolar lavage (BAL) inflammatory cells and cytokines (IL-4 and IL-5), histopathological lung properties, and airway hyperreactivity were compared. RESULTS: Ovalbumin challenge induced strong immune and inflammatory responses with > 6-fold IgE level increases; 10- to 25-fold BAL eosinophil, neutrophil, and lymphocyte count increases; and > 1.5-fold IL-4 and IL-5 level increases (p < 0.05). Whereas anti-IL-33 reduced neutrophil counts, anti-Siglec-F and anti-IL-33/anti-Siglec-F reduced both eosinophil and neutrophil counts (p < 0.05). Individual treatments reduced OVA-mediated bronchiolar infiltration by 50% (p <0.05). Ovalbumin challenge increased airway hyperreactivity by 4-fold (Group B; 2000.0 ±671.8% increase in Penh) compared to controls (Group A; 445.7 ±33.5% increase in Penh) (p = 0.016). The anti-IL-33 (Group C: 1579.4 ±973.6% increase in Penh) and anti-Siglec-F (Group D: 930.4 ±236.5%) groups demonstrated significantly reduced hyperreactivity (p = 0.029). Anti-IL-33/anti-Siglec-F therapy showed synergism towards neutrophil counts, IL-5 concentrations, bronchial infiltration, and hyperreactivity (p < 0.05). CONCLUSIONS: Combination treatment with anti-IL-33/anti-Siglec-F had more potent anti-allergic effects, reducing eosinophilic infiltration through their additive effects in a murine allergic asthma model.

Exposure to hypergravity increases serum interleukin-5 and pulmonary infiltration in mice with allergic asthma.

OBJECTIVE: We evaluated the effect of acute hypergravity (HG) on the immune response in a murine model of allergic asthma. MATERIAL AND METHODS: Twenty-eight BALB/c mice were used. Group A (control group, n = 7) mice were sensitized and challenged with normal saline. Group B (control HG exposure group, n = 7) mice were sensitized, challenged with saline, and exposed to acute HG (+10 Gz) for 4 hours. Group C (asthma group, n = 7) mice were challenged with intraperitoneal and intranasal ovalbumin (OVA) to induce asthma. Group D (asthma HG exposure group, n = 7) mice were exposed to HG for 4 hours after the induction of asthma. We estimated the total and OVA-specific serum IgE, serum titers of various cytokines, and the number of eosinophils, neutrophils, and lymphocytes in bronchoalveolar lavage (BAL) fluid. Histopathology of the lung was also evaluated. RESULTS: The serum level of interleukin (IL)-5 was significantly higher in Group D (12.9 ±4.9 pg/ml) compared to that in Group C (4.7 ±6.5 pg/ml, p = 0.017). In BAL fluid, the number of neutrophils was significantly increased in Group D compared to Group C (p = 0.014). Group D demonstrated a higher infiltration of inflammatory cells (9973.8 ±1642.7 cells/mm(2)) compared to Group C (7666.3 ±586.5 cells/mm(2), p = 0.017). This tendency of increase in infiltration was not significant in non-asthmatic animals (Group A: 4488.8 ±176.1 cells/mm(2) vs. Group B: 4946.3 ±513.7 cells/mm(2), p > 0.05). CONCLUSIONS: Acute HG exacerbated the allergic response by increasing serum IL-5 levels and promoting pulmonary infiltration of inflammatory cells.

Biochemical characterization of a new recombinant TNF receptor-hyFc fusion protein expressed in CHO cells.

The currently used Tumor Nectosis Factor (TNF)-α blockers such as infliximab, adalimumab and etanercept have Fc regions of the human IgG1 subtype have advantages in terms of in vivo half-life, however these could raise potential concerns for unwanted effector-mediated effects, such as antibody dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). To address this issue, we constructed a novel hybrid protein with decreased ADCC and CDC potentials by fusing the TNF receptor to a hybrid Fc (hyFc) containing CH2 and CH3 regions of IgG4 and highly flexible hinge regions of IgD which neither has ADCC and CDC activities. The resulting fusion protein, TNFR-hyFc, was over-expressed in CHO cells. For use as a pre-clinical material in pharmacology, PK and toxicological evaluations were carried out for biochemical characterization which was then compared with etanercept that has similarity in structure. Amino acid composition analysis and peptide mapping showed that the expressed TNFR-hyFc matched the theoretical composition derived from the DNA sequence. Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) showed that TNFR-hyFc is 2.9 kDa larger than etanercept. MALDI-TOF after removal of N-glycans by PNGase treatment showed that TNFR-hyFc is 3.9 kDa larger than etanercept. Isoelectric focusing and monosaccharide analysis showed that TNFR-hyFc is slightly more acidic than etanercept. N-terminal amino acid sequencing showed that N-terminal heterogeneity is present in both TNFR-hyFc and etanercept, although the ratios are somewhat different. Glycan analysis showed that the main glycan form is bi-antennary, similar to etanercept.

The pharmacology study of a new recombinant TNF receptor-hyFc fusion protein.

TNF-α-blocking agents such as infliximab, adalimumab and etanercept are widely used for the treatment of severe inflammatory diseases including rheumatoid arthritis and psoriasis. The currently used TNF-α blockers have Fc regions of the human IgG1 subtype, which is advantageous in terms of in vivo half-life but also raise the potential for unwanted effector-mediated effects, such as antibody dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). To address this issue, we constructed a novel hybrid protein by fusing the TNF receptor (TNFR) with a hybrid Fc (hyFc) consisting of the CH2 and CH3 regions of IgG4 and the highly flexible hinge regions of IgD which would not have ADCC and CDC activity. The resulting fusion protein, TNFR-hyFc, was over-expressed in CHO and pharmacological characteristics were evaluated in comparison with the structurally similar etanercept. TNFR-hyFc effectively neutralized TNF-α in L929 bioassay and showed a 1.5-fold higher neutralizing activity compared to etanercept. In a pharmacokinetic study in cynomolgus monkeys, TNFR-hyFc showed plasma half-life and AUC comparable to etanercept. In a mouse collagen induced arthritis model, TNFR-hyFc showed significant amelioration of arthritis compared to etanercept or vehicle control. In an LPS-induced septic shock model, TNFR-hyFc showed a similar level of protection against mortality as etanercept. These results confirm the feasibility of the TNFR-hyFc as an effective TNF-α blocker for the treatment of inflammatory diseases.

Beneficial effect of anti-interleukin-33 on the murine model of allergic inflammation of the lower airway.

OBJECTIVE: Interleukin (IL)-33, which mediates the T(h)2 allergic pathway, may play a key role in allergic airway inflammation. This study was conducted to evaluate the therapeutic potential of anti-IL-33 antibody for treatment of allergic inflammation of the lower airway in a murine model. METHODS: Twenty-four BALB/c mice were used in this study. Saline was used for sensitization and challenge of mice in Group A (control group, n = 6). Mice in Group B (ovalbumin (OVA) group, n = 6) received intraperitoneal (ip) and intranasal OVA challenge. In Group C (control IgG group, n = 6), mice received ip injection with control IgG prior to OVA challenge. Mice in Group D (anti-IL-33 group, n = 6) received an ip injection of anti-IL-33 prior to challenge. Measurements of serum total and OVA-specific IgE and the number of eosinophils, neutrophils, and lymphocytes in bronchoalveolar lavage (BAL) fluid were performed. We performed histopathologic examination to evaluate the degree of eosinophilic infiltration in lung tissue. Airway hyperreactivity was measured according to change of enhanced pause (Penh). RESULTS: A significant decrease in serum total and OVA-specific IgE and the number of eosinophils and neutrophils in BAL fluid was observed in Group D, compared with Group B or Group C (p < .05). In Group D, treatment with anti-IL-33 resulted in a significant decrease in eosinophilic infiltration in lung tissue, compared with Group B and Group C (p < .05). Degree of airway hyperreactivity, measured by Penh, showed a significant decrease in the anti-IL-33 treatment group, compared with the OVA group or the control IgG treatment group (p < .01, at 50 mg/mL of methacholine). CONCLUSIONS: Anti-IL-33 has therapeutic potential for treatment of allergic inflammation of the lower airway.

Nonselective blocking of the sympathetic nervous system decreases detrusor overactivity in spontaneously hypertensive rats.

The involuntary dual control systems of the autonomic nervous system (ANS) in the bladder of awake spontaneously hypertensive rats (SHRs) were investigated through simultaneous registrations of intravesical and intraabdominal pressures to observe detrusor overactivity (DO) objectively as a core symptom of an overactive bladder. SHRs (n = 6) showed the features of overactive bladder syndrome during urodynamic study, especially DO during the filling phase. After injection of the nonselective sympathetic blocking agent labetalol, DO disappeared in 3 of 6 SHRs (50%). DO frequency decreased from 0.98 ± 0.22 min(-1) to 0.28 ± 0.19 min(-1) (p < 0.01), and DO pressure decreased from 3.82 ± 0.57 cm H(2)O to 1.90 ± 0.86 cm H(2)O (p < 0.05). This suggests that the DO originating from the overactive parasympathetic nervous system is attenuated by the nonselective blocking of the sympathetic nervous system. The detailed mechanism behind this result is still not known, but parasympathetic overactivity seems to require overactive sympathetic nervous system activity in a kind of balance between these two systems. These findings are consistent with recent clinical findings suggesting that patients with idiopathic overactive bladder may have ANS dysfunction, particularly a sympathetic dysfunction. The search for newer and better drugs than the current anticholinergic drugs as the mainstay for overactive bladder will be fueled by our research on these sympathetic mechanisms. Further studies of this principle are required.

Persistent detrusor overactivity in rats after relief of partial urethral obstruction.

Detrusor overactivity (DO) persists after prostatectomy in 20% to 25% of patients with benign disease. Assuming that nonvoiding contractions (NVCs) can be used as a surrogate for DO in humans, the rat model of obstruction/deobstruction may allow us to study the pathophysiology of persistent DO after deobstruction. We investigated bladder function, with a special focus on NVCs, in rats by use of a new, modified method of obstruction and deobstruction and compared these results with those obtained by use of the conventional method. Seventy female Sprague-Dawley rats underwent 1) sham operation (n = 10), 2) obstruction by a modified method (Modif-Obs; n = 12), 3) obstruction/deobstruction by the conventional method (Conv-Obs/Deobs; n = 13), or 4) obstruction/deobstruction by the modified method (Modif-Obs/Deobs; n = 35). The Modif-Obs/Deobs animals were divided into subgroups with (DO+) and without (DO-) NVCs. Two weeks after partial urethral obstruction, the animals were deobstructed, and 1 wk later cystometry was performed with recording of intravesical and intra-abdominal pressures. NVCs were shown in all groups: Modif-Obs (80%), Conv-Obs/Deobs (100%), and Modif-Obs/Deobs (40%). In the Modif-Obs/Deobs group, bladder weight and the muscle-to-collagen ratio were higher in DO+ than in DO- rats. The Modif-Obs/Deobs group showed no mortality compared with 25% mortality in the Conv-Obs/Deobs group. The modified method may be more adequate for studying persistent DO after deobstruction, because it resulted in pressure/volume- and DO-related parameters similar to those found in the clinical situation. The persistence of DO after deobstruction may partly be due to irreversible changes in the bladder caused during the period of obstruction.

Lipid emulsion reverses Levobupivacaine-induced responses in isolated rat aortic vessels.

BACKGROUND: The goal of this in vitro study was to investigate the effects of lipid emulsion (LE) on local anesthetic levobupivacaine-induced responses in isolated rat aorta and to determine whether the effect of LE is related to the lipid solubility of local anesthetics. METHODS: Isolated rat aortic rings were suspended for isometric tension recording. The effects of LE were determined during levobupivacaine-, ropivacaine-, and mepivacaine-induced responses. Endothelial nitric oxide synthase and caveolin-1 phosphorylation was measured in human umbilical vein endothelial cells treated with levobupivacaine alone and with the addition of LE. RESULTS: Levobupivacaine produced vasoconstriction at lower, and vasodilation at higher, concentrations, and both were significantly reversed by treatment with LE. Levobupivacaine and ropivacaine inhibited the high potassium chloride-mediated contraction, which was restored by LE. The magnitude of LE-mediated reversal was greater with levobupivacaine treatment than with ropivacaine, whereas this reversal was not observed in mepivacaine-induced responses. In LE-pretreated rings, low-dose levobupivacaine- and ropivacaine-induced contraction was attenuated, whereas low-dose mepivacaine-induced contraction was not significantly altered. Treatment with LE also inhibited the phosphorylation of endothelial nitric oxide synthase induced by levobupivacaine in human umbilical vein endothelial cells. CONCLUSIONS: These results indicate that reversal of levobupivacaine-induced vasodilation by LE is mediated mainly through the attenuation of levobupivacaine-mediated inhibition of L-type calcium channel-dependent contraction and, in part, by inhibition of levobupivacaine-induced nitric oxide release. LE-mediated reversal of responses induced by local anesthetics may be related to their lipid solubility.

Hypoxia-Induced Suppression of Antiapoptotic Bcl-2 Expression in Human Bladder Tumor Cells Is Regulated by Caveolin-1-Dependent Adenosine Monophosphate-Activated Protein Kinase Activity.

PURPOSE: Adenosine monophosphate-activated protein kinase (AMPK) is thought to inhibit cell proliferation or promote cell death, but the details remain unclear. In this study, we propose that AMPK inhibits the expression of anti-apoptotic B-cell lymphoma 2 (Bcl-2) by relying on the hypoxia-inducible factor 1 alpha (HIF-1α)-induced caveolin-1 (Cav-1) expression pathway in noninvasive human bladder tumor (RT4) cells. METHODS: In cells exposed to a hypoxic environment (0.5% oxygen), the levels of expression and phospho-activity of the relevant signaling enzymes were examined via Western blots and reverse transcription-polymerase chain reaction. Cell proliferation was assessed using a Cell Counting Kit-8 assay. RESULTS: The level of expression of Cav-1 was very low or undetectable in RT4 cells. Hypoxia was associated with significantly decreased cell growth, along with marked induction of HIF-1α and Cav-1 expression; additionally, it suppressed the expression of the antiapoptotic marker Bcl-2 while leaving AMPK activity unchanged. Under hypoxic conditions, HIF-1α acts as a transcription factor for Cav-1 mRNA gene expression. The cell growth and Bcl-2 expression suppressed under hypoxia were reversed along with decreases in the induced HIF-1α and Cav-1 levels by AMPK activation with metformin (1mM) or phenformin (0.1mM). In addition, pretreatment with AMPK small interfering RNA not only increased the hypoxia-induced expression of HIF-1α and Cav-1, but also reversed the suppression of Bcl-2 expression. These results suggest that HIF-1α and Cav-1 expression in hypoxic environments is regulated by basal AMPK activity; therefore, the inhibition of Bcl-2 expression cannot be expected when AMPK activity is suppressed, even if Cav-1 expression is elevated. CONCLUSION: For the first time, we find that AMPK activation can regulate HIF-1α induction as well as HIF-1α-induced Cav1 expression, and the hypoxia-induced inhibitory effect on the antiapoptotic pathway in RT4 cells is due to Cav-1-dependent AMPK activity.

Three-Dimensional Reconstruction of Neurovascular Network in Whole Mount Preparations and Thick-Cut Transverse Sections of Mouse Urinary Bladder.

PURPOSE: Proper functional and structural integrity of nervous and vascular system in urinary bladder plays an important role in normal bladder function and the disruption of these structures is known to be related to lower urinary tract symptoms. Here, we present an immunohistochemical staining method that delineates neurovascular structures in the mouse urinary bladder by using immunohistochemical staining with three-dimensional reconstruction. MATERIALS AND METHODS: The urinary bladder was harvested from 8-week-old C57BL/6 male mouse. Lamina propria and detrusor muscle layer were dissected for whole mount staining, and thick-cut (60-µm) sections were prepared for full-thickness bladder staining. Immunofluorescent staining of bladder tissue was performed with antibodies against CD31 (an endothelial cell marker), smooth muscle α-actin (a smooth muscle cell marker), NG2 (a pericyte marker), and ßIII-tubulin (a neuronal marker). We reconstructed three-dimensional images of bladder neurovascular system from stacks of two-dimensional images. RESULTS: Three-dimensional images obtained from thick-cut sections clearly provided good anatomic information about neurovascular structures in the three layers of bladder, such as urothelium, lamina propria, and detrusor muscle layer. Whole mount images of lamina propria and detrusor muscle layer also clearly delineated spatial relationship between nervous and vascular systems. The microvessel density was higher in the lamina propria than in the detrusor muscle layer. Nerve fibers were evenly innervated into the lamina propria and detrusor muscle. CONCLUSIONS: This study provides comprehensive insight into three-dimensional neurovascular structures of mouse urinary bladder. Our technique may constitute a standard tool to evaluate pathologic changes in a variety of urinary bladder diseases.

Mitotic catastrophe induced by overexpression of budding yeast Rad2p.

Mitotic catastrophe provokes endopolyploidy, giant cell formation and, eventually, delayed cell death. Mitotic catastrophe is induced by defective cell cycle checkpoints and by some anticancer drugs, ionizing radiation and microtubule-destabilizing agents. RAD2 is a yeast homologue of XPG, which is a human endonuclease involved in nucleotide excision repair. Here we show that Rad2p overexpression alone, in the absence of extrinsic DNA damage, causes cell growth arrest and mitotic catastrophe. Interestingly, Rad2p-induced cell growth arrest is not caused by the catalytic activity of Rad2p but rather by its C-terminal region. Cells growth-arrested by Rad2p induction do not show apoptotic phenotypes and deletion of YCA1, a yeast caspase homologue, does not affect cell growth arrest by Rad2p induction. However, Rad2p-induced cell growth arrest is released by rad9 deletion but is not affected by downstream DNA damage checkpoint genes. These observations suggest that RAD2 has a function in coordinating cell cycle regulation and damaged DNA repair.

Increased expression of nucleophosmin is associated with the pathophysiology of chronic rhinosinusitis with nasal polyposis.

OBJECTIVE: Nucleophosmin (NPM1) has been suggested to be involved in the pathophysiologic mechanism of inflammatory disorders. We measured the expression level of NPM1 in nasal polyp (NP) tissues of patients with chronic rhinosinusitis with nasal polyposis (CRSwNP). We also assessed the correlation between NPM1 expression and other parameters such as eosinophilic infiltration, inflammatory cytokines, and clinical indicators such as Lund-Mackay computed tomography (CT) score. METHODS: Thirty patients with CRSwNP were included. We performed pre-operative CT scan to determine Lund-Mackay CT scores. During endoscopic sinus surgery, we harvested NP tissues from patients with CRSwNP. We performed Sirius red staining to evaluate eosinophilia and conducted immunohistochemical staining for NPM1 and real-time PCR for cytokines including interleukin (IL)-5, IL-17A, and IL-32. RESULTS: The mRNA expression of NPM1 was significantly up-regulated in eosinophilic NP tissues (RQ 0.58 ± 0.06), compared to non-eosinophilic NP tissues (RQ 0.38 ± 0.08, p < 0.05). In the epithelium of NP tissue, a significant positive correlation was observed between eosinophilic infiltration and NPM1 expression. The expression of NPM1 was significantly correlated with that of IL-5 (r = 0.6229, p = 0.0004), IL-17A (r = 0.5971, p = 0.001), and IL-32 (r = -0.5985, p = 0.0068). There was no significant correlation between the mRNA expression of NPM1 and the Lund-Mackay CT score (Spearman r = -0.2563, p = 0.1879). CONCLUSION: Expression of NPM1 was significantly increased in eosinophilic NP tissues from patients with CRSwNP. We observed an association between NPM1 expression and various pro-inflammatory cytokines such as IL-5, IL-17, and IL-32 and eosinophilic infiltration, which is thought to contribute to the pathophysiology of NP.

Direct effect of dexmedetomidine on rat isolated aorta involves endothelial nitric oxide synthesis and activation of the lipoxygenase pathway.

1. The aims of the present in vitro study were to examine the roles of pathways associated with arachidonic acid metabolism in dexmedetomidine-induced contraction and to determine which endothelium-derived vasodilators are involved in the endothelium-dependent attenuation of vasoconstriction elicited by dexmedetomidine. 2. Dexmedetomidine (10(-9)-10(-6) mol/L) concentration-response curves were constructed in: (i) aortic rings with no drug pretreatment; (ii) endothelium-denuded aortic rings pretreated with either 2 x 10(-5) mol/L quinacrine dihydrochloride, 10(-5) mol/L nordihydroguaiaretic acid (NDGA), 3 x 10(-5) mol/L indomethacin or 10(-5) mol/L fluconazole; and (iii) endothelium-intact aortic rings pretreated with either 5 x 10(-5) mol/L N(G)-nitro-l-arginine methyl ester (l-NAME), 10(-5) mol/L fluconazole, 10(-5) mol/L indomethacin, 10(-5) mol/L glibenclamide, 5 x 10(-3) mol/L tetraethylammonium or 5 x 10(-5) mol/L l-NAME plus rauwolscine (10(-5), 10(-6) mol/L). The production of nitric oxide (NO) metabolites was determined in human umbilical vein endothelial cells treated with dexmedetomidine. 3. Quinacrine dihydrochloride, NDGA and indomethacin attenuated the dexmedetomidine-induced contraction of endothelium-denuded rings. Dexmedetomidine (10(-7)-10(-6) mol/L)-induced contractions of endothelium-denuded rings were enhanced compared with those of endothelium-intact rings, as were dexmedetomidine-induced contractions of endothelium-intact rings pretreated with l-NAME or tetraethylammonium. Rauwolscine attenuated dexmedetomidine-induced contractions in endothelium-intact rings pretreated with l-NAME. Dexmedetomidine (10(-6) mol/L) was found to activate NO production. 4. Taken together, the results indicate that dexmedetomidine-induced contraction of aortic rings involves activation of the lipoxygenase and cyclo-oxygenase pathways and is attenuated by increased NO production following stimulation of endothelial alpha(2)-adrenoceptors by dexmedetomidine.

Bupivacaine-induced contraction is attenuated by endothelial nitric oxide release modulated by activation of both stimulatory and inhibitory phosphorylation (Ser1177 and Thr495) of endothelial nitric oxide synthase.

This study examined the mechanism associated with the endothelium-dependent attenuation of vasoconstriction induced by bupivacaine (BPV), with a particular focus on the upstream cellular signaling pathway of endothelial nitric oxide synthase (eNOS) phosphorylation induced by BPV in human umbilical vein endothelial cells (HUVECs). BPV concentration-response curves were investigated in the isolated rat aorta. The effects of Nω-nitro-L-arginine methyl ester (L-NAME), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), methylene blue, calmidazolium, the Src kinase inhibitor 4-amino-3-(4-chlorophenyl)-1-(t-butyl)-1H-pyrazolo[3,4-d]pyrimidine, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) and the combination of L-arginine and L-NAME on BPV-induced contraction in endothelium-intact aorta preparations were examined. The effects of BPV alone and in combination with PP2 on the phosphorylation of eNOS (at Ser1177 or Thr495), caveolin-1 and Src kinase were examined in HUVECs. BPV-induced contraction was lower in endothelium-intact aortae than in endothelium-denuded aortae. L-NAME, ODQ, methylene blue and calmidazolium increased BPV-induced contraction in endothelium-intact aortae, whereas PP2 alone and combined treatment with L-arginine and L-NAME inhibited BPV-induced contraction. Low-concentration BPV (30 µM) induced both stimulatory (Ser1177) and inhibitory (Thr495) phosphorylation of eNOS in HUVECs. However, high-concentration BPV (150 µM) induced only stimulatory (Ser1177) eNOS phosphorylation. Additionally, phosphorylation of Src kinase, caveolin-1 and inhibitory eNOS (Thr495) induced by low-concentration BPV was inhibited by PP2. These results suggest that contraction induced by low-concentration BPV is attenuated by endothelial nitric oxide release, which is modulated both stimulatory (Ser1177) and inhibitory eNOS phosphorylation (Thr495). BPV-induced phosphorylation of eNOS (Thr495) is indirectly mediated by an upstream cellular signaling pathway involving Src kinase (Tyr416) and caveolin-1 (Tyr14).

Korean medical students' attitudes toward academic misconduct: a cross-sectional multicenter study.

PURPOSE: This study investigated medical students' attitudes toward academic misconduct that occurs in the learning environment during the pre-clinical and clinical periods. METHODS: Third-year medical students from seven medical schools were invited to participate in this study. A total of 337 of the 557 (60.5%) students completed an inventory assessing their attitudes toward academic misconduct. The inventory covered seven factors: scientific misconduct (eight items), irresponsibility in class (six items), disrespectful behavior in patient care (five items), dishonesty in clerkship tasks (four items), free riding on group assignments (four items), irresponsibility during clerkship (two items), and cheating on examinations (one item). RESULTS: Medical students showed a strict attitude toward academic misconduct such as cheating on examinations and disrespectful behavior in patient care, but they showed a less rigorous attitude toward dishonesty in clerkship tasks and irresponsibility in class. There was no difference in students' attitudes toward unprofessional behaviors by gender. The graduate medical school students showed a stricter attitude toward some factors of academic misconduct than the medical college students. This difference was significant for irresponsibility in class, disrespectful behavior in patient care, and free riding on group assignments. CONCLUSION: This study indicates a critical vulnerability in medical students' professionalism toward academic integrity and responsibility. Further study evidence is needed to confirm whether this professionalism lapse is confined only to this population or is pervasive in other medical schools as well.

Expression of death receptor 4 induces caspase-independent cell death in MMS-treated yeast.

DR4, a tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor, is a key element in the extrinsic pathway of TRAIL/TRAIL receptor-related apoptosis that exerts a preferential toxic effect against tumor cells. However, TRAIL and DR4 are expressed in various normal cells, and recent studies indicate that DR4 has a number of non-apoptotic functions. In this study, we evaluated the effects of human DR4 expression in yeast to determine the function of DR4 in normal cells. The expression of DR4 in yeast caused G1 arrest, which resulted in transient growth inhibition. Moreover, treatment of DR4-expressing yeast with a DNA damaging agent, MMS, elicited drastic, and sustained cell growth inhibition accompanied with massive apoptotic cell death. Further analysis revealed that cell death in the presence of DNA damage and DR4 expression was not dependent on the yeast caspase, YCA1. Taken together, these results indicate that DR4 triggers caspase-independent programmed cell death during the response of normal cells to DNA damage.

Associations between prenatal lead exposure and birth outcomes: Modification by sex and GSTM1/GSTT1 polymorphism.

Maternal lead exposure is associated with poor birth outcomes. However, modifying effects of polymorphism in glutathione S-transferases (GST) gene and infant sex remain unexplored. Our aim was to evaluate whether associations between prenatal lead and birth outcomes differed by maternal GST genes and infant sex. Prospective data of 782 mother-child pairs from Mothers and Children's Environmental Health (MOCEH) study were used. The genotyping of GST-mu 1 (GSTM1) and theta-1 (GSTT1) polymorphisms was carried out using polymerase chain reaction. Multivariable linear regression was used to examine whether the association between blood lead (BPb) level and birth outcomes (birthweight, length, and head circumference) varied by maternal GST genes and sex. We did not find a statistically significant association between prenatal BPb levels and birth outcomes; in stratified analyses, the association between higher BPb level during early pregnancy and lower birthweight (ß=-224 per square root increase in BPb; 95% confidence interval (CI): -426, -21; false discovery rate p=0.036) was significant in males of mothers with GSTM1 null. Results were similar for head circumference model (ß=-0.78 per square root increase in BPb; 95% CI: -1.69, 0.14, p=0.095), but the level of significance was borderline. Head circumference model showed a significant three-way interaction among BPb during early pregnancy, GSTM1, and sex (p=0.046). For combined analysis with GSTM1 and GSTT1, GSTM1 null and GSTT1 present group showed a significant inverse association of BPb with birthweight and head circumference in males. Our findings of the most evident effects of BPb on the reduced birthweight and head circumference in male born to the mother with GSTM1 null may suggest a biological interaction among lead, GST genes and sex in detoxification process during fetal development.