Clinical efficacy of intravesical gemcitabine combined with ubenimex in patients with non-muscle-invasive bladder carcinoma after transurethral resection of bladder tumor.

Objectives: To evaluate the clinical value of intravesical gemcitabine combined with immunotherapy in patients with non-muscle-invasive bladder carcinoma (NMIBC) after transurethral resection of bladder tumor (TURBT). Methods: Eighty patients with non-muscle-invasive urothelial carcinoma treated in Baoding No.1 Hospital from November 2016 to November 2019 were randomly divided into two groups, with 40 patients in each group. Both groups underwent TURBT. After surgery, the research group was treated with intravesical chemotherapy using gemcitabine combined with ubenimex, while the control group was given 40 mg pirarubicin by intravesical instillation. Postoperative condition was evaluated by cystoscopy every three months in both groups. The recurrence six months, one year and two years after treatment, the incidence of lower urinary tract symptoms such as dysuria, hematuria and frequent urination, general adverse drug reactions such as rashes, liver function damage and gastrointestinal reaction, as well as the changes in CD3+, CD4+, CD8+ and CD4+/CD8+ T lymphocyte subsets before and after treatment were comparatively analyzed between the two groups. Results: The recurrence rate showed no statistical significance between the two groups 6 months after treatment (p=0.17), but significant differences one year (p=0.04) and two years (p=0.03) after treatment, which were significantly lower in the research group than the control group. The incidence of adverse drug reactions was 22.5% in the research group and 7.5% in the control group, without significant difference (p=0.36). The incidence of lower urinary tract symptoms was 32.5% and 55%, respectively, in the research group and the control group. The incidence of lower urinary tract symptoms in the research group was significantly lower compared with the control group, with a statistically significant difference (p=0.04). After treatment, CD3+, CD4+ and CD4+/CD8+ levels in the research group increased significantly than those in the control group, with statistically significant differences (CD3+, p=0.01; CD4+, p=0.00; CD4+/CD8+, p=0.00). Conclusions: For NMIBC patients receiving bladder-preserving surgery, intravesical gemcitabine combined with immunotherapy can reduce the recurrence rate, relieve lower urinary tract symptoms, increase the tolerance of patients to intravesical chemotherapy and significantly improve the function of T lymphocytes, without obvious increase in adverse drug reactions. Therefore, it is safe and effective, and has certain clinical value.

Insight into the Structural Variation and Sodium Storage Behavior of Polyoxometalates Encapsulated within Single-Walled Carbon Nanotubes.

The host-guest interaction can remarkably alter the physiochemical properties of composite materials. It is crucial to clarify the mechanism by revealing the influence of the host on the electronic structure of the guest molecules. Herein, we study the structural variation of polyoxometalates (POMs) after being confined in single-walled carbon nanotubes (SWNT). What we found is that in addition to the reported charge transfer from SWNT to POM, an intramolecular electron transfer within a single POM cluster can be observed in the POM@SWNT composites. Moreover, the charge density on the bridged oxygen of POMs is prominently enhanced. The structural change and electron reconfiguration of POMs upon encapsulation in SWNT significantly speed up electron and ion transport, leading to the improved electrochemical performance for sodium ions storage.

Advanced Anode Materials for Sodium-Ion Batteries: Confining Polyoxometalates in Flexible Metal-Organic Frameworks by the "Breathing Effect".

Polyoxometalates (POMs) have shown great potential in sodium-ion batteries (SIBs) due to their reversible multielectron redox property and high ionic conductivity. Currently, POM-based SIBs suffer from the irreversible trapping and sluggish transmission kinetics of Na+. Herein, a series of POMs/metal-organic frameworks (MOFs)/graphene oxide (GO) (MOFs = MIL-101, MIL-53, and MIL-88B; POM = [PMo12O40]3-, denoted as PMo12) composites are developed as SIB anode materials for the first time. Unlike MIL-101 with large pore structures, the pores in flexible MIL-53 and MIL-88B swell spontaneously upon the accommodation of PMo12. Particularly, the PMo12/MIL-88B/GO composites deliver an excellent specific capacity of 214.2 mAh g-1 for 600 cycles at 2.0 A g-1, with a high initial Coulombic efficiency (ICE) of 51.0%. The so-called "breathing effect" of flexible MOFs leads to the relatively tight confinement space for PMo12, which greatly modulates its electronic structure, affects the adsorption energy of Na+, and eventually reduces the trapping of sodium ions. Additionally, the straight and multidimensional channels in MIL-88B significantly accelerate ion diffusion, inducing favored energetic kinetics and thus generating high-rate performance.

Electronic Structure Reconfiguration of Self-Supported Polyoxometalate-Based Lithium-Ion Battery Anodes for Efficient Lithium Storage.

Polyoxometalate (POM)-based materials are considered as promising candidates for lithium-ion batteries (LIBs) due to their stable and well-defined molecular structure and reversible multielectron redox properties. Currently, POM-based electrode materials suffer from high interfacial resistance and low uniformity. Herein, we reported a self-supported POM-based anode material for LIBs by electrodepositing H3PMo12O40 (PMo12) and aniline on carbon cloth (CC) for the first time. The as-prepared polyaniline (PANi)-PMo12/CC composite exhibited an excellent reversible capacity of 1092 mA h g-1 for 200 cycles at 1 A g-1. Such an outstanding performance was attributed to the rapid electron transfer and Li+ diffusion stemming from the exposure of more active sites by the self-supported structure, the strong electrostatic interaction, and electronic structure reconfiguration between the active PMo12 cluster and conductive PANi polymer. This work provides insight into the electronic structure engineering of highly efficient LIB anode materials.

Desloratadine, a Novel Antigrowth Reagent for Bladder Cancer.

Desloratadine, a potent antagonist for human histamine H1 receptor, has been revealed to exhibit antihistaminic activity and anti-inflammatory activity. However, it is not yet known whether desloratadine has any effect on the biological behaviors of tumor cells. In this study, we aimed to investigate the effects of desloratadine on cell growth and invasion in bladder cancer EJ and SW780 cells in vitro. We observed that desloratadine inhibited cell viability of EJ and SW780 cells in a dose- and time-dependent manner. Desloratadine treatment was also revealed to suppress colony-formation ability and induce cell cycle arrest at G1 phase in EJ cells. Desloratadine promoted cell apoptosis via modulating the expression of Bcl-2, Bax, cleaved caspase 3, and cleaved caspase 9 in EJ and SW780 cells. Western blot resulted showed that desloratadine also impaired the expression of autophagy-related proteins, such as Beclin 1, P62, and LC3I/II in EJ and SW780 cells; while autophagy inhibitor LY294002 reversed the effects of desloratadine on these proteins. Moreover, desloratadine remarkably attenuated cell migration and invasion. Furthermore, we illustrated that desloratadine downregulated the expression of N-cadherin, Vimentin, Snail1, and Snail2, while upregulated the expression of E-cadherin in EJ and SW780 cells in vitro. The level of interleukin 6 was reduced in desloratadine-treated cells, while upregulation of interleukin 6 significantly abolished the anticancer activity of desloratadine in cell invasion and Bcl-2, Bax, Beclin1, LC3-I/II, N-cadherin, and E-cadherin expression in EJ cells. Taken together, our data suggest a potential anticancer activity of desloratadine on cell growth and invasion for bladder cancer, which may be mediated by diminishing the epithelial-to-mesenchymal transition and interleukin 6.

miR-125b-5p/STAT3 Pathway Regulated by mTORC1 Plays a Critical Role in Promoting Cell Proliferation and Tumor Growth.

Aberrant activation of the mammalian target of rapamycin complex 1 (mTORC1) plays a critical role in tumorigenesis. However, the precise underlying mechanism is still not fully understood. Although accumulating evidence suggests that mTORC1 signaling is regulated by microRNAs (miRNAs), whether miRNAs are involved in the tumorigenesis mediated by mTORC1 dysregulation remains largely unclear. In our study, the comparison between tuberous sclerosis complex 1 (Tsc1) -/- or Tsc2-/- mouse embryonic fibroblasts (MEFs) and the control cells revealed the involvement of microRNA-125b-5p (miR-125b-5p) in the tumorigenesis driven by mTORC1 activation. Our study also showed that loss of TSC1 or TSC2 led to significant downregulation of miR-125b-5p and upregulation of signal transducer and activator of transcription 3 (STAT3) via mTORC1 activation. Overexpression of miR-125b-5p inhibited the proliferation of the cells with hyperactivated mTORC1 both in vitro and in vivo. Furthermore, we demonstrated that STAT3 is a direct target of miR-125b-5p. Depletion of STAT3 mimicked the effect of ectopic expression of miR-125b-5p, and reintroduction of STAT3 rescued the compromised cell proliferation driven by miR-125b-5p overexpression in Tsc1-/- or Tsc2-/- MEFs. We conclude that the miR-125b-5p/STAT3 pathway plays a crucial role in hyperactivated mTORC1-mediated tumorigenesis and miR-125b-5p is a potential therapeutic target.

Suppression of epithelial signal transducer and activator of transcription 1 activation by extracts of Aspergillus fumigatus.

Aspergillus fumigatus (AF) is often pathogenic in immune-deficient individuals and can cause life-threatening infections such as invasive aspergillosis. The pulmonary epithelial response to AF infection and the signaling pathways associated with it have not been completely studied. BEAS-2B cells or primary human bronchial epithelial cells were exposed to extracts of AF and challenged with IFN-ß or the Toll-like receptor 3 agonist double-stranded RNA (dsRNA). Cytokine release (B-cell activating factor of the TNF family [BAFF], IFN-γ-induced protein-10 [IP-10], etc.) was assessed. AF extract was separated into low-molecular-weight (LMW) and high-molecular-weight (HMW) fractions using ultra 4 centrifugal force filters to characterize the activity. Real-time PCR was performed with a TaqMan method, and protein estimation was performed using ELISA techniques. Western blot was performed to assess phosphorylation of signal transducer and activator of transcription 1 (STAT1). IFN-ß and dsRNA induced messenger RNA (mRNA) expression of BAFF (350- and 452-fold, respectively [n = 3]) and IP-10 (1,081- and 3,044-fold, respectively [n = 3]) in BEAS-2B cells. When cells were pretreated with AF extract for 1 hour and then stimulated with IFN-ß or dsRNA for 6 hours, induction of BAFF and IP-10 mRNA was strongly suppressed relative to levels produced by IFN-ß and dsRNA alone. When compared with control, soluble BAFF and IP-10 protein levels were maximally suppressed in dsRNA-stimulated wells treated with 1:320 wt/vol AF extract (P < 0.005). Upon molecular size fractionation, a LMW fraction of AF extract had no measurable suppressive effect on IP-10 mRNA expression. However, a HMW fraction of the AF extract significantly suppressed IP-10 expression in BEAS-2B cells that were stimulated with dsRNA or IFN-ß. When BEAS-2B cells were pretreated with AF extract and then stimulated with IFN-ß, reduced levels of pSTAT1 were observed, with maximum suppression at 4 and 6 hours. Our results show that AF extracts suppressed expression of inflammatory cytokines in association with inhibition of the IFN-ß signaling pathway and suppression of the formation of pSTAT1.

Giant axon formation in mice lacking Kell, XK, or Kell and XK: animal models of McLeod neuroacanthocytosis syndrome.

McLeod neuroacanthocytosis syndrome (MLS) is a rare X-linked multisystem disease caused by XK gene mutations and characterized by hematological and neurological abnormalities. XK, a putative membrane transporter, is expressed ubiquitously and is covalently linked to Kell, an endothelin-3-converting enzyme (ECE-3). Absence of XK results in reduction of Kell at sites where both proteins are coexpressed. To elucidate the functional roles of XK, Kell, and the XK-Kell complex associated with pathogenesis in MLS, we studied the pathology of the spinal cord, anterior roots, sciatic nerve, and skeletal muscle from knockout mouse models, using Kel(-/-), Xk(-/-), Kel(-/-)Xk(-/-), and wild-type mice aged 6 to 18 months. A striking finding was that giant axons were frequently associated with paranodal demyelination. The pathology suggests probable anterograde progression from the spinal cord to the sciatic nerve. The neuropathological abnormalities were found in all three genotypes, but were more marked in the double-knockout Kel(-/-)Xk(-/-) mice than in either Kel(-/-) or Xk(-/-) mice. Skeletal muscles from Xk(-/-) and Kel(-/-)Xk(-/-) mice showed mild abnormalities, but those from Kel(-/-) mice were similar to the wild type. The more marked neuropathological abnormalities in Kel(-/-)Xk(-/-) mice suggest a possible functional association between XK and Kell in nonerythroid tissues.

Comparative studies of macrophage-biased responses in mice to infection with Toxoplasma gondii ToxoDB #9 strains of different virulence isolated from China.

BACKGROUND: Different from three clonal lineages of Toxoplasma gondii in North America and Europe, the genotype China 1 is predominantly prevalent in China. However, there are different virulent isolates within China 1, such as virulent TgCtwh3 and avirulent TgCtwh6, and little is known about differences in macrophage activation between them. The objective of this study focused on cytokine production, phenotype and markers of activated macrophages, and correlated signaling pathway induced by the two isolates. METHODS: Adherent peritoneal macrophages (termed Wh3-Mφ and Wh6-Mφ, respectively) harvested from infected mice were cultured for detection of Nitric Oxide and arginase activity, and activated markers on Wh3-Mφ/Wh6-Mφ were determined by flow cytometry. In in vitro experiments, the levels of IL-12p40 and TNF-α were measured using ELISA kits, and mRNA expressions of IL-12p40, TNF-α, iNOS, Arg-1 and Ym1 were assayed by real-time PCR. To confirm the activation state of NF-kB p65 in infected cells stained by IF, protein levels of iNOS, Arg-1, Ym1, nuclear NF-κB p65, and phosphorylation of STAT6/STAT3/IκBα were evaluated by Western Blotting. A one-way ANOVA test was used to compare differences among multiple groups. RESULTS: The result revealed that contrary to the virulent TgCtwh3, the less virulent TgCtwh6 isolate induced a significant increase in IL-12p40 and TNF-α. Although both isolates down-regulated CD80, CD86 and MHCII molecule expression on macrophages, TgCtwh3 promoted up-regulation of PD-L2 and CD206. Wh6-Mφ generated a high level of NO whereas Wh3-Mφ up-regulated Ym1 and arginase expression at transcriptional and protein levels. In terms of signaling pathway, TgCtwh3 induced phospho-STAT6, conversely, TgCtWh6 led to NF-κB p65 activation. CONCLUSIONS: The virulent TgCtwh3 isolate induced macrophages to polarize toward alternatively activated cells with STAT6 phosphorylation, whereas the less virulent TgCtwh6 elicited the development of classically activated macrophages with nuclear translocation of NF-κB p65. This discrepancy suggests that it is necessary to thoroughly analyze the genotype of TgCtwh3 and TgCtwh6, and to further study other effector molecules that contribute to the macrophage polarization in T. gondii.

Changes in red cell ion transport, reduced intratumoral neovascularization, and some mild motor function abnormalities accompany targeted disruption of the Mouse Kell gene (Kel).

Kell (ECE-3), a highly polymorphic blood group glycoprotein, displays more than 30 antigens that produce allo-antibodies and, on red blood cells (RBCs), is complexed through a single disulfide bond with the integral membrane protein, XK. XK is a putative membrane transporter whose absence results in a late onset form of neuromuscular abnormalities known as the McLeod syndrome. Although Kell glycoprotein is known to be an endothelin-3-converting enzyme, the full extent of its physiological function is unknown. To study the functions of Kell glycoprotein, we undertook targeted disruption of the murine Kel gene by homologous recombination. RBCs from Kel(-/-) mice lacked Kell glycoprotein, Kell/XK complex, and endothelin-3-converting enzyme activity and had reduced levels of XK. XK mRNA levels in spleen, brain, and testis were unchanged. In Kel(-/-) mice RBC Gardos channel activity was increased and the normal enhancement by endothelin-3 was blunted. Analysis of the microvessels of tumors produced from LL2 cells indicated that the central portion of tumors from wild-type mice were populated with many mature blood vessels, but that vessels in tumors from Kel(-/-) mice were fewer and smaller. The absence of Kell glycoprotein mildly affected some motor activities identified by foot splay on the drop tests. The targeted disruption of Kel in mouse enabled us to identify phenotypes that would not be easily detected in humans lacking Kell glycoprotein. In this regard, the Kell knockout mouse provides a good animal model for the study of normal and/or pathophysiological functions of Kell glycoprotein.

Nasal saline irrigation facilitates control of allergic rhinitis by topical steroid in children.

OBJECTIVE: This study was designed to determine whether nasal saline irrigation improved the symptoms and signs of allergic rhinitis (AR) and whether nasal saline irrigation could be used as a complementary management of AR in children while less steroids were used. METHOD: 26 children with AR were divided into three groups and were given nasal saline irrigation and/or topical steroid. Symptoms and signs of AR and mucociliary clearance (MCC) were evaluated, and concentration of soluble intercellular adhesion molecule (sICAM)-1 in nasal secretion was detected. RESULTS: In AR children treated with nasal irrigation and tapered topical steroid at week 8 and week 12, a significant improvement in symptoms and signs was observed, and a significant decrease in the mean values of MCC and the mean concentrations of sICAM-1 in nasal secretions was also detected. CONCLUSION: Nasal saline irrigation can be viewed as a good adjunctive treatment option for AR. It permitted the use of less topical steroids for controlling AR in children, which will contribute to fewer side effects and less economic burden.

Expression profiles of mouse Kell, XK, and XPLAC mRNA.

Kell and XK are related because in red cells they exist as a disulfide-bonded complex. Kell is an endothelin-3-converting enzyme, and XK is predicted to be a transporter. Absence of XK, which is accompanied by reduced Kell on red cells, results in acanthocytosis and late-onset forms of central nervous system and neuromuscular abnormalities that characterize the McLeod syndrome. In this study, expression of mouse XK, XPLAC, a homolog of XK, and Kell were compared by in situ hybridization histochemistry (ISHH) and RT-PCR. ISHH showed that Kell and XK are coexpressed in erythroid tissues. ISHH detected XK, but not Kell, mRNA in testis, but RT-PCR indicated that both Kell and XK are coexpressed. XK, but not Kell, was significantly expressed in brain, spinal cord, small intestine, heart, stomach, bladder, and kidney. ISHH did not detect XK in skeletal muscle but RT-PCR did. In brain, XK was predominantly expressed in neuronal rather than in supportive cells. By contrast, XPLAC was predominantly expressed in the thymus. Coexpression of Kell and XK in erythroid tissues and the different expressions in non-erythroid tissues suggest that XK may have a complementary hematological function with Kell and a separate role in other tissues.

Mechanical stretch modulates the promoter activity of the profibrotic factor CCN2 through increased actin polymerization and NF-kappaB activation.

The connective tissue growth factor known as CCN2 is an inducible, profibrotic molecule that becomes aberrantly expressed in mechanical overload-bearing tissues. In this study, we found that CCN2 gene expression is rapidly induced in cyclically stretched bladder smooth muscle cells (SMCs) in vitro and in the detrusor muscle of a mechanically overloaded bladder in a rat model of experimental urethral obstruction. The activity of CCN2 promoter constructs, transiently transfected into cultured SMCs, was increased (up to 6-fold) by continuous cyclic stretching. Molecular analyses of the CCN2 promoter by serial construct deletions, cis-element mutagenesis, and electrophoretic mobility shift assays revealed that a highly conserved NF-kappaB binding site located within the CCN2 proximal promoter region is responsible for the activation of the promoter by stretch. Chromatin immunoprecipitation assays showed that NF-kappaB binds to the endogenous CCN2 promoter in both stretched cells and mechanically overloaded bladder tissues. Furthermore, stretch-dependent CCN2 promoter activity was significantly reduced upon inhibition of either phosphatidylinositol 3-kinase, p38 stress-activated kinase, or RhoA GTPase and was completely abolished upon inhibition of actin polymerization. Concordantly, actin polymerization was increased in either mechanically stretched cells or overloaded bladder tissues. Incubation of cultured SMCs with a cell-penetrating peptide containing the N-terminal sequence, Ac-EEED, of smooth muscle alpha-actin, altered both actin cytoskeleton organization and stretch-mediated nuclear relocation of NF-kappaB, and subsequently, it reduced CCN2 promoter activity. Thus, mechanical stretch-induced changes in actin dynamics mediate NF-kappaB activation and induce CCN2 gene expression, which probably initiates the fibrotic reactions observed in mechanical overload-associated pathologies.

Signaling transduction pathways involved in basophil adhesion and histamine release.

BACKGROUND: Little is known about basophil with respect to the different signaling transduction pathways involved in spontaneous, cytokine or anti-IgE induced adhesion and how this compares to IgE-dependent and IgE-independent mediator secretion. The purpose of the present study was to investigate the roles of beta1 and beta2 integrins in basophil adhesion as well as hosphatidylinositol 3-kinase (PI3K), src-kinases and extracellular signal regulated kinase (ERK) 1/2 in basophil adhesion and histamine release (HR). METHODS: Basophils (purity of 10% - 50%) were preincubated with anti-CD29 or anti-CD18 blocking antibodies before used for adhesion study. Basophils were preincubated with the pharmacological inhibitors wortmannin, PP1, PD98059 before used for adhesion and HR study. Cell adherence to bovine serum albumin (BSA) or fibronectin (Fn) was monitored using cell associated histamine as a basophil marker and the histamine was measured by the glass fiber assay. RESULTS: Basophil spontaneous adhesion to Fn was inhibited by anti-CD29. Interleukin (IL)-3, granulocyte/macrophage colony stimulating factor (GM-CSF) induced adhesion to BSA was inhibited by anti-CD18. Wortmannin at 1 micromol/L and PP1 at 20 micromol/L strongly interfered with, whereas PD98059 at 50 micromol/L weakly inhibited basophil spontaneous adhesion to Fn. One micromol/L wortmannin strongly inhibited IL-3, IL-5, GM-CSF and anti-IgE induced adhesion to BSA. PP1 at 20 micromol/L partly inhibited anti-IgE induced adhesion. Fifty micromol/L PD98059 marginally inhibited IL-5, weakly inhibited anti-IgE, partly inhibited GM-CSF induced adhesion. Wortmannin, PP1 and PD98059 inhibited anti-IgE (1:100 or 1:1000) induced basophil HR in a dose dependent manner. They inhibited calcium ionophore A23187 (10 micromol/L, 5 micromol/L) induced basophil HR in a dose dependent manner, but to different extend with PP1 being the most efficient. CONCLUSIONS: Basophil spontaneous adhesion to Fn is mediated by beta1-integrins whereas cytokine induced adhesion to BSA is mediated by beta2-integrins. PI3K, src-kinases and ERK1/2 play distinct signaling roles in basophil adhesion and HR. PI3K is the key player while ERK1/2 is the weakest participant.

Endothelin-3-converting enzyme activity of the KEL1 and KEL6 phenotypes of the Kell blood group system.

The Kell blood group protein is a metalloendopeptidase that preferentially cleaves a Trp(21)-Ile(22) bond of big endothelin-3 producing bioactive endothelin-3. Kell is a polymorphic protein, and 25 different phenotypes, because of point mutations resulting in single amino acid substitutions, have been described. It was recently reported that a recombinant form of KEL1 (K, K1) phenotype, expressed in K562 and HEK293 cells, had no endothelin-3-converting activity, in contrast to the common KEL2 (k, K2) phenotype. We demonstrate that KEL1 red blood cells and also a soluble recombinant form of KEL1 protein (s-Kell KEL1) have similar enzymatic activity as the common Kell phenotype. In addition we show that KEL6 red blood cells, which are more prevalent in persons of African heritage than in Caucasians also have endothelin-3-converting enzyme activity and that the recombinant soluble form of KEL6 protein (s-Kell KEL6) has similar K(m) values as the wild-type.

Identification of two new members, XPLAC and XTES, of the XK family.

XK, a putative membrane transporter, is a component of the XK/Kell complex of the Kell blood group system. XK's substrate is unknown but absence of the protein, as occurs in the McLeod phenotype, is associated with red cell acanthocytosis and late onset central nervous system and neuromuscular abnormalities known as the McLeod syndrome. We have cloned two cDNAs, XPLAC (GenBank accession no. AY589511) and XTES (GenBank accession no. AY989815), which are closely related to XK and define them together as the XK family. XPLAC has a 2.9 kb cDNA that encodes 462 amino acids and XTES has a 1.6 kb cDNA coding 459 amino acids. The predicted molecular weights are 53.6 kDa for XPLAC and 53.4 kDa for XTES, which are similar to that of XK, which is 50.9 kDa. Unlike XK which is ubiquitously expressed XPLAC is expressed mostly in placenta and adrenal gland while XTES is exclusively expressed in primate testis. XPLAC has 37% and XTES has 31% amino acid identity with XK protein and they are predicted to have a similar topology to XK. XPLAC, like XK, has 3 exons and is located on X chromosome at q22.1, while XTES has 4 exons and is located at 22q11.1. Phylogenetic analysis shows that there are at least 5 additional vertebrate genes that are evolutionarily distantly related to the XK family. A domain with consensus sequences (ced-8 domain) for the extended family is described.

Abrogation of oxidative stress improves insulin sensitivity in the Ren-2 rat model of tissue angiotensin II overexpression.

To evaluate the role of renin-angiotensin system (RAS)-mediated oxidative stress in insulin resistance (IR), we compared the effects of the angiotensin II (ANG II) receptor blocker (ARB) valsartan and a superoxide dismutase (SOD) mimetic, tempol, on whole body glucose tolerance and soleus muscle insulin-stimulated glucose uptake in transgenic hypertensive TG(mREN-2)27 (Ren-2) rats. Ren-2 rats and Sprague-Dawley (SD) controls were given valsartan (30 mg/kg) or tempol (1 mmol/l) in their drinking water for 21 days. IR was measured by glucose tolerance testing (1 g/kg glucose ip). IR index (AUC(glucose) x AUC(insulin)) was significantly higher in the Ren-2 animals compared with SD controls (30.5 +/- 7.0 x 10(6) arbitrary units in Ren-2 vs. 10.2 +/- 2.4 x 10(6) in SD, P < 0.01). Both valsartan and tempol treatment normalized Ren-2 IR index. Compared with SD controls (100%), there was a significant increase in superoxide anion production (measured by lucigenin-enhanced chemiluminescence) in soleus muscles of Ren-2 rats (133 +/- 15%). However, superoxide production was reduced in both valsartan- and tempol-treated (85 +/- 22% and 59 +/- 12%, respectively) Ren-2 rats. Insulin (INS)-mediated 2-deoxyglucose (2-DG) uptake (%SD basal levels) was substantially lower in Ren-2 rat soleus muscle compared with SD (Ren-2 + INS = 110 +/- 3% vs. SD + INS = 206 +/- 12%, P < 0.05). However, Ren-2 rats treated with valsartan or tempol exhibited a significant increase in insulin-mediated 2-DG uptake compared with untreated transgenic animals. Improvements in skeletal muscle insulin-dependent glucose uptake and whole body IR in rats overexpressing ANG II by ARB or SOD mimetic indicate that oxidative stress plays an important role in ANG II-mediated insulin resistance.

Innate immunity of the sinonasal cavity: expression of messenger RNA for complement cascade components and toll-like receptors.

OBJECTIVE: To study the expression of important elements of the innate immune responses in human sinonasal tissue to elucidate its potential role in mucosal inflammation. DESIGN: We studied human sinonasal tissue from patients with chronic rhinosinusitis and an immortalized epithelial cell line to detect the expression of innate immune effectors and the responses of these cells to stimulation with compounds associated with pathogenic organisms. PATIENTS: Nine individuals undergoing endoscopic sinus surgery for chronic rhinosinusitis. MAIN OUTCOME MEASURES: Expression of complement components and toll-like receptors. RESULTS: We found detectable levels of messenger RNA for all toll-like receptors in human sinonasal tissue and in the BEAS-2B epithelial cell line. Expression of several components of the alternate pathway of complement (factors B, H, and I and properdin) was constitutively present in unstimulated BEAS-2B cells and was readily detectable in human sinonasal tissue. Stimulation of BEAS-2B cells with the toll-like receptor 3 ligand double-stranded RNA resulted in increased expression of messenger RNA for factors B and H but not for properdin or factor I. CONCLUSIONS: Toll-like receptors and the alternate pathway of complement are important components of innate immunity that are expressed in human sinonasal epithelium in vivo and in cultured airway epithelial cells in vitro. The expression of some of these components can be significantly induced by stimulation via toll-like receptors, and epithelial expression of components of innate immunity may play a role in inflammation in chronic rhinosinusitis.

Activation of airway epithelial cells by toll-like receptor agonists.

Toll-like receptors (TLR) play an important role in pathogen recognition and innate immunity. We investigated the presence and function of TLRs in the BEAS-2B airway epithelial cell line and primary bronchial epithelial cells. Standard real-time reverse transcriptase-polymerase chain reaction (RT-PCR) analysis and Taqman RT-PCR revealed that BEAS-2B cells express mRNA for TLR1-10. Several TLR ligands were tested for their ability to activate gene expression in BEAS-2B cells using limited microarray analyses focusing on genes of the chemokine and chemokine receptor family, cytokines, and signaling pathways. While the TLR3 ligand double-stranded RNA was the most effective epithelial activator, clear responses to flagellin, lipopolysaccharide, CpG, peptidoglycan, and zymosan were also observed. RT-PCR and/or enzyme-linked immunosorbent assay were used to confirm results obtained with microarrays for five of the induced genes: interleukin-8, serum amyloid A, TLR3, macrophage inflammatory protein-3alpha, and granulocyte-macrophage colony-stimulating factor. Stimulation of epithelial cells with double-stranded RNA induced levels of interleukin-8 exceeding 20 ng/ml and levels of serum amyloid A exceeding 80 ng/ml. Double-stranded RNA, lipopolysaccharide, zymosan A, and flagellin also induced expression of macrophage inflammatory protein-3alpha and granulocyte-macrophage colony-stimulating factor, which may facilitate immature dendritic cell migration and maturation. These results suggest that airway epithelial cells express several TLRs and that they are functionally active. Epithelial expression of TLRs may be of importance in inflammation and immunity in the airways in response to inhaled pathogens.

Angiotensin II regulation of the Na+ pump involves the phosphatidylinositol-3 kinase and p42/44 mitogen-activated protein kinase signaling pathways in vascular smooth muscle cells.

This investigation used primary cultured rat vascular smooth muscle cells to examine angiotensin II (Ang II) regulation of Na(+), K(+)-ATPase (Na(+) pump) activity, and Na(+) pump alpha(1)- and beta(1)-subunit gene transcription. This regulation was mediated through both phosphatidylinositol-3 kinase (PI3K) and p42/44 mitogen-activated protein kinase (p42/44(MAPK)) signaling pathways. Both acute (10 min) and prolonged (24 h) treatment with Ang II stimulated Na(+) pump activity. Also, prolonged exposure to Ang II (24 h) increased promoter transcription of the Na(+) pump alpha(1)- and beta(1)-subunits. Furthermore, PI3K activities because well because p42/44(MAPK) phosphorylation were increased within 10 min after Ang II treatment. To determine whether these stimulatory activities of Ang II are acting through Ang II receptors 1 and/or 2 (AT(1), AT(2)), cells were pretreated with either AT(1) receptor blocker losartan or the AT(2) receptor blocker PD 123,319. Indeed, these treatments prevented the stimulatory effect of Ang II on Na(+) pump activity at both acute and 24-h time points. Furthermore, the Ang II-stimulated alpha(1)-subunit promoter transcription was inhibited by losartan but not by the AT(2) receptor blocker. These results indicate that Ang II acts through both the AT(1) and AT(2) receptor to up-regulate Na(+) pump activity; however, Ang II regulates alpha(1)-gene transcription through AT(1) but not AT(2) receptors. It was also observed that the Ang II-stimulated beta(1)-subunit gene transcription is not mediated through either AT(1) or AT(2) receptors. To examine whether the Na(+)/H(+) exchanger is involved in Ang II-stimulated Na(+) pump activity, cells were pretreated with amiloride, a specific inhibitor of the Na(+)/H(+) exchanger. This pretreatment prevented 24 h, but not acute, Ang II-stimulated Na(+) pump activity. The 24-h Ang II-stimulated alpha(1)-subunit promoter transcription was also inhibited by amiloride. This suggests that the prolonged effect of Ang II on Na(+) pump activity is dependent on increased Na(+)/H(+) exchange. Because Ang II treatment for 10 min increased PI3K activity because well because p42/44(MAPK) phosphorylation, studies were performed to determine the involvement of PI3K and p42/44(MAPK) signaling pathways in both Ang II-stimulated Na(+) pump activity and alpha(1)- and beta(1)-gene transcription. Cells were pretreated with either the PI3K inhibitor wortmannin or the p42/44(MAPK) inhibitor PD 98059. Ang II-stimulated PI3K or p42/44(MAPK) activity was inhibited by these pretreatments. Furthermore, pretreatment of cells with the PI3K inhibitors wortmannin and LY29404 or the MAPK inhibitors U0126 and PD 98059 were all observed to inhibit Ang II-stimulated Na(+) pump activity. To more specifically determine the role of PI3K in Ang II-regulation of alpha(1)-and beta(1)-gene transcription, cells were cotransfected with a dominant-negative p85 construct. Cotransfection with dominant-negative p85 reduced Ang II-stimulated alpha(1)-but not beta(1)-gene transcription in vascular smooth muscle cells. These results indicate that Ang II acts through PI3K/p42/44(MAPK) signaling pathways to up-regulate Na(+) pump activity and alpha(1)-gene transcription and that Ang II-regulated beta(1)-gene transcription is not mediated through either PI3K or p42/44 (MAPK) signaling pathways.