Chloride ion transport and overexpression of TMEM16A in a guinea-pig asthma model.

BACKGROUND: TMEM16A, a Ca-activated Cl channel, regulates various physiological functions such as mucin secretion. However, the role of TMEM16A in hyper-secretion in asthma is not fully understood. OBJECTIVE: The aim of this study is to evaluate Cl ion transport via TMEM16A and determine the localization of TMEM16A in a guinea-pig asthma model. METHODS: Guinea-pigs were sensitized with ovalbumin (OVA) i.p. on Days 1 and 8. On Day 22, we assessed OVA challenge-induced Cl ion transport in the sensitized tracheas ex vivo in an Ussing chamber, compared with the non-sensitized tracheas. We then examined the effect of T16Ainh-A01, a TMEM16A inhibitor, on the increase in Cl ion transport. The tracheal epithelium was immunostained with an anti-TMEM16A antibody. Epithelial cells from guinea-pig tracheas were cultured at the air-liquid interface in the presence of IL-13 for in vitro study. We studied the effect of TMEM16A inhibitors on Ca-dependent agonist, uridine triphosphate (UTP)-induced increases in Cl ion transport in the cultured cells. The cells were immunostained with an anti-TMEM16A antibody, an anti-MUC5AC antibody and an anti-α-tubulin antibody. RESULTS: OVA challenge induced an increase in short circuit current within 1 min in the OVA-sensitized tracheas but not in the non-sensitized tracheas, which was inhibited by pretreatment of T16Ainh-A01. Sensitized tracheas showed goblet cell metaplasia with more positive TMEM16A immunostaining, particularly in the apical portion compared with the non-sensitized tracheas. The in vitro UTP-induced increase in Cl ion transport was strongly inhibited by pretreatment with T16Ainh-A01, benzbromarone, and niflumic acid. TMEM16A was positively immunostained at the apical portion and in the MUC5AC-positive area in IL-13-induced goblet cell metaplasia. CONCLUSIONS: Antigen challenge and Ca-dependent agonist treatment increased Cl ion transport via the overexpression of TMEM16A in goblet cell metaplasia in a guinea-pig asthma model. TMEM16A inhibitors may be useful for the treatment of hyper-secretion in asthma.

Dihydrotestosterone inhibits murine hair growth via the androgen receptor.

BACKGROUND: Androgens cause regression of human hair follicles in the parietofrontal scalp, but the precise mechanisms by which they do so are unknown. Although many investigators have elucidated the effect of androgens on hair growth by using rodents and other animals, some of the evidence is conflicting. OBJECTIVES: To investigate the effect of androgens on mouse hair regrowth and hair cycle by using androgen receptor knockout (ARKO) mice. Methods We examined the effects of dihydrotestosterone (DHT) on hair regrowth by using ARKO mice and wild-type (WT) littermates, compared the hair cycles in ARKO mice and WT littermates by histology and histomorphometry, and measured hair length and thickness in ARKO mice and WT littermates. RESULTS: DHT inhibited the hair regrowth of WT mice but not that of their ARKO littermates. The anagen phase in the second hair cycle was longer in ARKO mice than in their WT littermates. The hair of ARKO mice was longer and thicker than that of their WT littermates. CONCLUSIONS: Androgens inhibit hair growth in mice, and this inhibition might be caused by androgen-androgen receptor signals.

Truncation mutations abolish chromatin-associated activities of adenomatous polyposis coli.

The adenomatous polyposis coli (APC) is a tumor suppressor whose loss of function leads to colon cancer. APC shuttles between the nucleus and cytoplasm, however its role in the nucleus remains elusive. We have found that nuclear APC specifically associates with transcriptionally active chromatin through structural elements located downstream to the region of frequent truncation mutations found in colorectal tumors. We show that a recombinant APC fragment comprising such elements associates in vivo with euchromatin and preferentially binds in vitro to acetylated histone H3. Induction of DNA double-strand breaks (DSB) stimulates accumulation of APC at the damaged DNA chromatin marked by histone H2AX and S139-phosphorylated histone H2AX. A nuclear complex containing the DNA-dependent protein kinase catalytic subunit (DNAPKcs) and APC associates with chromatin in response to DNA DSB. APC knockdown with siRNA decreased the rate of DNA DSB-induced S139 histone H2AX phosphorylation in cells expressing endogenous full-length APC, but not in colon cancer cells with its truncation mutants, whereas ectopic APC expression stimulated the H2AX phosphorylation regardless of the type of endogenous APC. Our data suggest that APC involves in the DSB DNA repair and that truncation mutations impair chromatin-associated functions of APC.

Role of epidermal growth factor receptor in maintaining airway goblet cell hyperplasia in rats sensitized to allergen.

BACKGROUND: Stimulation of epidermal growth factor receptor (EGFR) induces airway goblet cell hyperplasia, but the role of this molecule in the maintenance of this pathologic change remains uncertain. OBJECTIVE: To determine the mechanisms by which goblet cell hyperplasia is maintained in airway epithelium, we investigated EGFR-induced signalling pathways that lead to both mucin production and antiapoptosis in vitro. We also tested whether the inhibition of EGFR tyrosine kinase speeds reversal of established goblet cell hyperplasia to normal epithelial phenotype in vivo. METHODS: MUC5AC production was measured by immunoassay, and antiapoptotic responses were determined by Bcl-2 expression and terminal deoxynucleotidyl transferase-mediated dUTP-biotin Nick End Labelling staining using NCI-H292 cells. The effect of an inhibitor of EGFR tyrosine kinase (AG1478) on goblet cell hyperplasia was also determined in rats sensitized with ovalbumin (OVA). RESULTS: MUC5AC was constitutively expressed and few apoptotic cells were observed in NCI-H292 cells under non-stimulated condition. TGF-alpha increased MUC5AC and Bcl-2 expression, an effect that was prevented by inhibitors of EGFR tyrosine kinase (AG1478), MEK (PD98059), and NF-kappaB (CAPE). After the addition of TGF-alpha, AG1478 and an inhibitor of phosphatidylinositol 3 kinase/Akt (LY294002), but not PD98059, induced a marked apoptotic response, which was prevented by the caspase inhibitor Z-VAD fmk. Goblet cell hyperplasia and EGFR expression in airway epithelium were noted in the OVA-sensitized rats. Intratracheal instillation of AG1478 induced apoptosis of goblet cells, reverting the airway epithelium to normal epithelial phenotype. CONCLUSION: These findings indicate that EGFR plays an important role in the maintenance of goblet cell hyperplasia. We speculate that inhibitors of the EGFR cascade might be an effective therapy of airway remodelling.

Integrative analysis reveals 53BP1 copy loss and decreased expression in a subset of human diffuse large B-cell lymphomas.

p53-Binding protein 1 (53BP1) encodes a critical checkpoint protein that localizes to sites of DNA double-strand breaks (DSBs) and participates in DSB repair. Mice that are 53bp1 deficient or hemizygous have an increased incidence of lymphoid malignancies. However, 53BP1 abnormalities in primary human tumors have not been described. By combining high-density single nucleotide polymorphism (HD SNP) array data and gene expression profiles, we found 9 of 63 newly diagnosed human diffuse large B-cell lymphomas (DLBCLs) with single copy loss of the chromosome 15q15 region including the 53BP1 locus; these nine tumors also had significantly lower levels of 53BP1 transcripts. 53BP1 single copy loss found with the HD SNP array platform was subsequently confirmed by fluorescence in situ hybridization. These studies highlight the role of 53BP1 copy loss in primary human DLBCLs and the value of integrative analyses in detecting this genetic lesion in human tumors.

Id2 gene-targeted crosstalk between Wnt and retinoid signaling regulates proliferation in human keratinocytes.

We investigated the effect of all-trans-retinoic acid (atRA) on proliferation in several human skin cell lines and found that antiproliferative potency of atRA correlated with the endogenous activity of canonical Wnt signaling. In HaCaT keratinocytes, we found that atRA significantly suppressed the expression of Id2, a member of the inhibitor of differentiation family of transcription factors that regulate cell growth and differentiation. However, no apparent change in the expression of other Wnt targets, like c-Myc or cyclin D1, was observed. Retinoid-induced Id2 gene suppression was associated with decreased levels of histone H3 and H4 acetylation and histone H3 Lys-4 methylation, and with recruitment of the LSD1 demethylase at the Wnt-response element (WRE) (TCF/LEF-binding site), in the Id2 gene promoter. None of such changes was detected at the WRE of c-Myc and cyclin D1 gene promoters. Inhibition of Id2 by short interfering RNA (siRNA) had a similar effect on the proliferation of HaCaT cells as exposure to atRA, whereas anti-beta-catenin siRNA significantly inhibited its antiproliferative effect. These data suggest that downregulation of Id2 gene expression through transcriptional convergence between Wnt and retinoid signaling pathways underlies the antiproliferative effect of retinoids in keratinocytes, and provide evidence of gene-targeted crosstalk between signaling pathways.

Fatal hepatitis B virus reactivation in a chronic myeloid leukemia patient during imatinib mesylate treatment.

Reactivation of chronic hepatitis B virus (HBV) infection in patients undergoing chemotherapy is well-documented, but reactivation during imatinib mesylate treatment has not been reported. This study reports a 54-year-old man, without prior liver dysfunction but with chronic HBV infection, in whom fatal HBV reactivation occurred during treatment of chronic myeloid leukemia (CML) with imatinib mesylate. He developed fulminant hepatitis followed by marked elevation of HBV DNA polymerase, probably from the lymphocytopenic and immunosuppressive status induced by imatinib mesylate. Imatinib mesylate is widely used to treat CML patients. Although therapy with imatinib mesylate is generally well tolerated, the case presented here suggests that viral reactivation should be considered, even when using imatinib mesylate to treat CML.

The molecular basis of vitamin D-dependent rickets type I.

Vitamin D 1alpha-hydroxylase is a key enzyme for the vitamin D-calcium homeostasis. Recently, 1alpha-hydroxylase cDNA and gene were cloned. Human 1alpha-hydroxylase gene is located at chromosome 12q13.3. Several inactivating mutations in the 1alpha-hydroxylase gene were found in VDDR I patients, and it was established that 1alpha-hydroxylase gene is responsible for VDDR I. To date, various mutations spreading over all exons have been reported. The cloning of 1alpha-hydroxylase gene will further lead to the better understanding of vitamin D regulation in both normal and pathological states. In addition, 1alpha-hydroxylase knock-out mice, which is recently generated, would be a useful model animal for VDDR I.

Validity and safety of sputum induction by inhaled uridine 5'-triphosphate.

UNLABELLED: Inhalation of hypertonic saline during sputum induction causes bronchoconstriction. We studied the validity and safety of sputum induction by uridine 5'-triphosphate (UTP). Sputum was induced by a 5-min inhalation of hypertonic saline (3%) on Day 1 and UTP (5 mg/ml in 0.9% saline) on Days 8 and 15 in 16 healthy subjects and 16 patients with mild-to-moderate asthma. Inhaled UTP produced twofold greater amounts of sputum than did hypertonic saline. There were significant differences in oxygen desaturation and bronchoconstriction during the procedure between the two methods: the maximal fall in Sa(O(2)), the AUC of the Sa(O(2))-time response, and the fall in PEF were less in the subjects who received UTP than in those who received hypertonic saline. Sputum total cell and differential cell counts, with a high proportion of eosinophils in asthmatics, were similar between specimens obtained by hypertonic saline and UTP. When we compared two consecutive measurements on the UTP-induced sputum samples, the reproducibility calculated by the intraclass correlation coefficient was high for the proportion of eosinophils, neutrophils, and macrophages. Therefore, inhalation of UTP aerosols may provide an effective, relatively noninvasive, valid, and reproducible method of sputum induction for the assessment of airway inflammation in asthma. KEYWORDS: uridine triphosphate; induced sputum; airway inflammation; bronchoconstriction; asthma

Staphylococcus aureus bacteriuria and surgical site infections by methicillin-resistant Staphylococcus aureus.

Surgical site infection (SSI) remains an important cause of morbidity among hospitalized patients. We reviewed 421 patients who underwent open urological operations between January 1993 and December 1997 in our institute. Group I consisted of 259 patients who received uncontrolled antimicrobial prophylaxis (AMP) between 1993 and 1995. Group II consisted of 162 patients who received controlled AMP between 1996 and 1997. In group II, penicillins or first to second-generation cephalosporins was used and the duration of use for these agents regulated according to the wound class of each operation. The operations with clean wounds showed the lowest rate of SSI in both groups; the operations with contaminated wounds showed the highest rate of SSI (32.0% in group I and 33.3% in group II). There was no significant difference in the total rates of SSI between the two groups (P=0.216). The most frequently isolated bacterial species was methicillin-resistant Staphylococcus aureus (MRSA), isolated in 73.3% of the cases in group I and in 93.3% in group II. There was no significant difference in the incidence of MRSA isolation between the two groups (P=0.114). The controlled AMP could not lower the incidence of MRSA-induced SSIs. In SSI patients, 22.7% of group I and 35.7% in group II, had MRSA bacteriuria before operation. The prohibition of third-generation cephalosporins and shorter duration of AMP did not reduce the incidence of SSI induced by MRSA because MRSA was not the emerging microorganism but rather a resident in the urological ward. On the other hand, the total incidence of SSI did not increase after regulation of AMP. This finding suggests that older antibacterial agents can prevent infection, except those caused by resistant microorganisms such as MRSA. The effective counter-measure for the prevention of MRSA-induced SSI is needed.

Relationship of epidermal growth factor receptors to goblet cell production in human bronchi.

To determine the relationship of epidermal growth factor receptor (EGFR) expression to mucin synthesis in human airways, we examined EGFR and MUC5AC expression at both gene and protein levels using in situ hybridization and immunohistochemical analysis in human bronchi. Bronchial mucosal biopsy specimens were obtained from 12 asthmatic subjects and 11 healthy subjects. In asthmatic airways, EGFR mRNA was expressed in the airway epithelium. EGFR immunoreactivity staining patterns varied among the asthmatic airways: staining was positive mainly in goblet cells, in basal cells, or in both. In contrast, healthy airways showed little expression of EGFR mRNA; EGFR immunoreactivity was observed mainly in goblet cells. In parallel to EGFR expression, MUC5AC mRNA expression was greater in asthmatic airways; mucous glycoconjugates that stained positively with Alcian blue/PAS were also increased in asthmatic airways. Ciliated cells were negative for EGFR and MUC5AC both in asthmatic and in healthy subjects at both mRNA and protein levels. There was a significant positive correlation between EGFR immunoreactivity and the area of MUC5AC-positive staining in both asthmatics and healthy subjects. These findings suggest a sequence of events by which EGFR activation is involved in mucin expression in asthmatic airway epithelium.

IL-13 induces mucin production by stimulating epidermal growth factor receptors and by activating neutrophils.

Mucus hypersecretion contributes to the morbidity and mortality in acute asthma. Both T helper 2 (Th2) cytokines and epidermal growth factor receptor (EGFR) signaling have been implicated in allergen-induced goblet cell (GC) metaplasia. Present results show that a cascade of EGFR involving neutrophils is implicated in interleukin (IL)-13-induced mucin expression in GC. Treatment with a selective EGFR tyrosine kinase inhibitor prevented IL-13-induced GC metaplasia dose dependently and completely. Instillation of IL-13 also induced tumor necrosis factor-alpha protein expression, mainly in infiltrating neutrophils. Control airway epithelium contained few leukocytes, but intratracheal instillation of IL-13 resulted in time-dependent leukocyte recruitment by IL-13-induced IL-8-like chemoattractant expression in airway epithelium. Pretreatment with an inhibitor of leukocytes in the bone marrow (cyclophosphamide) or with a blocking antibody to IL-8 prevented both IL-13-induced leukocyte recruitment and GC metaplasia. These findings indicate that EGFR signaling is involved in IL-13-induced mucin production. They suggest a potential therapeutic role for inhibitors of the EGFR cascade in the hypersecretion that occurs in acute asthma.

Activation of epidermal growth factor receptors is responsible for mucin synthesis induced by cigarette smoke.

Mucus hypersecretion from hyperplastic airway goblet cells is a hallmark of chronic obstructive pulmonary disease (COPD). Although cigarette smoking is thought to be involved in mucus hypersecretion in COPD, the mechanism by which cigarette smoke induces mucus overproduction is unknown. Here we show that activation of epidermal growth factor receptors (EGFR) is responsible for mucin production after inhalation of cigarette smoke in airways in vitro and in vivo. In the airway epithelial cell line NCI-H292, exposure to cigarette smoke upregulated the EGFR mRNA expression and induced activation of EGFR-specific tyrosine phosphorylation, resulting in upregulation of MUC5AC mRNA and protein production, effects that were inhibited completely by selective EGFR tyrosine kinase inhibitors (BIBX1522, AG-1478) and that were decreased by antioxidants. In vivo, cigarette smoke inhalation increased MUC5AC mRNA and goblet cell production in rat airways, effects that were prevented by pretreatment with BIBX1522. These effects may explain the goblet cell hyperplasia that occurs in COPD and may provide a novel strategy for therapy in airway hypersecretory diseases.

Detection of Photofrin Fluorescence From Malignant and Premalignant Lesions in the Bronchus using a Full-color Endoscopic Fluorescence Imaging System.

STUDY OBJECTIVES: To detect invisible lung cancer and to determine field of laser radiation during PDT we developed a full-color fluorescence fiberscopic system. We tested the efficacy of this system in patients with various bronchial malignancies.System design: A fiber-optic endoscope was attached to a camera box containing a color ICCD camera which can detect from 400 to 700nm fluorescence in full-color. Light of average wavelength 405 nm was selected and radiated through the light channel of the fiberscope from a 300W Xenon lamp. PATIENTS AND METHODS: We examined nine consecutive patients with bronchial malignancy admitted in our hospital to receive PDT. Sixteen lesions in these nine patients were observed with white light and excitation light and the results were compared. Histological examinations were done by taking biopsy specimens and samples for pathological and cytological examination. After the diagnosis was confirmed, 2.0 mg/kg Photofrin was injected. Forty eight hours after the administration of Photofrin, observation of the bronchial wall was made using a full-color endoscopic fluorescence imaging system just before PDT. RESULTS: Bright red fluorescence from Photofrin was Observed in 14/14 bronchial malignancies: 3 squamous cell carcinoma, 9 squamous cell carcinoma in situ, 1 metastatic breast cancer and 1 metastatic islet cell tumor. Bright red fluorescence was also detected in 2/2 squamous dysplasia. Green autofluorescence was observed in the normal part of the bronchus. CONCLUSIONS: RESULTS of the present study suggest that the full-color endoscopic fluorescence imaging system can be used to detect malignant and premalignant lesions as red fluorescence against green autofluorescence with Photofrin administration, and this system has the potential to detect absence of autofluorescence in cancerous lesions.

Influence of body fat on the onset of vecuronium induced neuromuscular blockade.

The onset time of vecuronium, a muscle relaxant, was measured after a bolus intravenous injection of 0.15 mg kg(-1) of vecuronium into 40 surgical patients aged 59-64 years. The onset time was then compared between male and female patients and the relationship between onset time and body fat (% of body weight) was analyzed. Arterial plasma concentrations of vecuronium were measured at 75, 195, and 375 sec after administration of vecuronium to 8 patients. The female patients (n = 23) showed a shorter onset time and more body fat than the male patients (n = 17). The onset time significantly decreased with increasing body fat in both groups. When only females with body fat of less than 30% (n = 10) were compared with the male group (all male patients had body fat of less than 30%), the body fat, onset time, and regression lines between the onset time and the body fat did not differ significantly. Except in the patient with the highest body fat, plasma concentrations at 195 and 375 sec significantly increased with increasing body fat. We concluded that the higher body fat in females is largely responsible for the faster onset of vecuronium action in females. A smaller distribution volume of vecuronium may also be one of the reasons for the faster onset of vecuronium in females.

Valsalva maneuver prevents guide wire trouble associated with 22-gauge safe guide.

UNLABELLED: The Safe guide is a central venous puncture needle that serves as both a pilot needle and as an introducer. A guide wire can be inserted into a vein through the side port at the hub of the 22-gauge Safe guides needle initially inserted as a pilot needle. However, guide wire insertion may fail due to kinking or locking at the side port. Increasing airway pressure to 20 cm H2O by squeezing a respiratory bag during insertion of the guide wire together with venous puncture was attempted to determine if would decrease guide wire trouble. SUBJECTS AND METHODS: A total of 120 patients scheduled for central venous catheterization by right internal jugular puncture were divided into two groups. Patients in group-A (n = 60) were catheterized by the conventional method and those in group-B (n = 60) were catheterized by applying the Valsalva maneuver. Three observations were made: 1) Frequency of cases in which blood back-flow occurred during withdrawal only and not upon advancement of the puncture needle. 2) Frequency of cases in which kinking and/or locking of the guide wire occurred at the hub during its insertion. And 3) the occurrence of complications. RESULTS: 1) The patency of the vein was preserved and blood back-flow was obtained during advancement of the puncture needle in all cases in which the Valsalva maneuver was applied. 2) The incidence of kinking and/or locking during insertion of the guide wire decreased from 16.7% to 3.4% by applying positive airway pressure during the Valsalva maneuver. And 3) complications were negligible. Additionally, the application of the Valsalva maneuver allowed successful guide wire insertion in 6 out of 9 cases (67%) in group-A, in which the initial attempt using the conventional method had failed. CONCLUSION: The application of positive airway pressure using the Valsalva maneuver may prevent the guide wire trouble associated with the 22-gauge Safe guide.

Analysis of genetic polymorphism in NQO1, GST-M1, GST-T1, and CYP3A4 in 469 Japanese patients with therapy-related leukemia/ myelodysplastic syndrome and de novo acute myeloid leukemia.

Several genetic polymorphisms in metabolic activation or detoxification enzymes have been associated with susceptibility to therapy-related leukemia and myelodysplastic leukemia (TRLIMDS). We analyzed gene polymorphisms of NAD(P)H:quinone oxidoreductase (NQOl), glutathione S-tranferase (GST)-MI and -TI, and CYP3A4, the enzymes of which are capable of metabolizing anticancer drugs, in 58 patients with TRL/MDS and in 411 patients with de novo acute myeloid leukemia (AML). Homozygous Ser/Ser genotype of NQOl at codon 187, causing loss of function, was more frequent in the patients with TRLIMDS (14 of 58, 24.1%; OR = 2.62) than in those with de novo AML (64 of 411, 15.6%), and control (16 of 150, 10.6%; P = 0.002). Allelic frequencies of NQOJ were different between TRL/ MDS and de novo AML (P = 0.01). In GST-MJ and -Ti, the incidence of homologous deletion was similar among the three groups. The polymorphism of the 5' promoter region of CYP3A4 was not found in persons of Japanese ethnicity. These results suggest that the NQOJ polymorphism is significantly associated with the genetic risk of TRLIMDS.

Relation of epidermal growth factor receptor expression to goblet cell hyperplasia in nasal polyps.

BACKGROUND: Because the epidermal growth factor receptor (EGFR) system regulates mucin production in airway epithelium, we hypothesized a role for this system in mucus hypersecretion that occurs in nasal polyposis. OBJECTIVE: We examined the relationship between goblet cell hyperplasia, EGFR expression, and inflammatory mediators produced by eosinophils and neutrophils in nasal polyp tissues. METHODS: Nasal polyp tissue samples from 8 patients and nasal turbinate biopsy specimens from 6 normal control subjects were examined for alcian blue/PAS staining, mucin MUC5AC (MUC5AC), and EGFR immunoreactivity and EGFR gene expression (in situ hybridization). We also examined the role of eosinophils and neutrophils in goblet cell hyperplasia. RESULTS: In control nasal mucosa alcian blue/periodic acid-Schiff- and MUC5AC-stained areas were 18.40% +/- 1.31% and 21.89% +/- 1.43%, respectively. In polyps the alcian blue/periodic acid-Schiff- and MUC5AC-stained areas were 51.30% +/- 5.85% and 52.07% +/- 6.58%, which was significantly larger than that found in control subjects (each comparison, P <.01). Four of 6 control specimens expressed EGFR messenger RNA and protein weakly in the epithelium. In polyps 4 of 8 specimens expressed EGFR gene and EGFR protein strongly; the EGFR-stained area was greater in hyperplastic than in pseudostratified epithelium. TNF-alpha immunoreactivity, expressed in eosinophils, was increased in EGFR-positive polyps compared with EGFR-negative polyps, suggesting a role for TNF-alpha in EGFR expression. Neutrophils were increased in the epithelium of EGFR-positive compared with EGFR-negative polyps, suggesting a role for these cells in mucin expression and in goblet cell degranulation. CONCLUSION: These data suggest a role for EGFR cascade in the regulation of goblet cell mucins in nasal polyps. Proof of concept will require clinical studies using selective EGFR inhibitors.