Session - Respiratory & Critical Care Medicine: no. S-RC-6published_or_final_versio

Fung, PCW

Ho, JCM

Lam, WK

Shum, IHY

Taylor, G

Tipoe, GL

Tsang, KWT

Zheng, L

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TitlePseudomonas aeruginosa pyocyanin (PYO) down-regulatesinducible nitric oxide synthase (iNOS) expression in humanbronchusAuthor(s) Shum, IHY; Zheng, L; Taylor, G; Tipoe, GL; Lam, WK; Fung,PCW; Ho, JCM; Tsang, KWTCitationThe 5th Medical Research Conference, Department of Medicine,The University of Hong Kong, Queen Mary Hospital, Hong Kong,China, 22-23 January 2000. In Hong Kong Practitioner, 2000, v.22 n. 2 Supp 2, p. 57, abstract no. S-RC-6Issued Date 2000URL http://hdl.handle.net/10722/46988Rights Creative Commons: Attribution 3.0 Hong Kong LicenseS-RC-6PSEUDOMONAS AERUGINOSA PYOCYANIN (PYO) DOWN-REGULATES INDUCIBLE NITRIC OXIDESYNTHASE (iNOS) EXPRESSION IN HUMAN BRONCHUSIHY Shum, L Zheng, *G Taylor, +G Tipoe, WK Lam, P Fung, JCM Ho, KWT Tsang. University Departments ofMedicine, +Anatomy and * Paediatrics, The University of Hong Kong, Hong Kong SAR, and *The Royal PostgraduateMedical School, London, UK.Pseudomonas aeruginosa (PA) is a problematic but common respiratory pathogen in cystic fibrosis andbronchiectasis. Chronic infection with PA is responsible for considerable morbidity among these patients. PAproduces PYO, which is a phenazine pigment that causes slowing of ciliary beating and disruption of respiratoryepithelium. It has also been reported recently that iNOS down-regulation occurs in the airways of patientswith cystic fibrosis although the cause of this remains obscured. We have therefore investigated the effect ofPYO on iNOS expression in human bronchial organ cultures. Paired endobronchial biopsies were collectedfrom 11 otherwise healthy subjects, who underwent bronchoscopy for suspected bronchial carcinoma, forincubation in FAD medium with either additional PYO or control medium for 16h at 37°C and 5% CO2. iNOSimmunostaining was performed on 3um paraffin sections and intracellular expression of iNOS was quantifiedon the immunostained sections with computer image analysis. The mean ± SD intensity of iNOS expressionin the control and test intraepithelial, endothelial, and submucosal cells were 84.62±32.15 & 120.79±25.33(p=0.003); 193±59 & 130+63 vessesl/mm2 (p=0.04); and 169±63 & 137± 150cells/mm2 (p=0.37) respectively.Our results indicate that PA pyocyanin down-regulates iNOS expression in bronchial epithelial and submucosalvessels. Further studies should follow to evaluate the clinical and pathological implications of our findings.s-RI-1B CELL APOPTOSIS IN SYSTEMIC LUPUS ERYTHEMATOSUS Adrian Wu. Albert Chan, Stanley Chik, CS Lau.Department of Medicine, University of Hong Kong.Systemic lupus erythematosus (SLE) is a multisystem autoimmune disorder characterized by the formation ofautoantibodies against nuclear antigens. Apoptosis plays an important role in peripheral tolerance to self-antigens. Miceinheriting the Ipr and gld mutations develop autoimmune syndromes and excessive production of many of the antibodyspecificities associated with human SLE. These animals also develop nephritis, and has uncontrolled proliferation of Bcells. Recent studies revealed that the Ipr and gld genes encode the apoptosis-inducing cell surface molecules Fas andFas ligand (FasL), respectively. This study hopes to identify possible defects in apoptosis during B cell activation inpatients with SLE.We identified patients who fulfilled the 1982 ACR criteria for SLE. These patients were clinically inactive at the time ofthe study and on low-dose immunosuppression only. Age-matched healthy subjects were recruited as controls. PBMCswere separated from 40 ml of peripheral blood and B cells were isolated by negative selection using the MACS magneticcell sorting system. B cell purity after isolation ranges from 35 to 55%. Apoptosis and Fas (CD95) expression of freshlyisolated B cells was evaluated by double staining with anti-CD 19 antibody and Annexin V-FITC conjugate or anti-CD95antibody respectively, followed by FACS analysis. B cells were incubated overnight with an agonist anti-CD95 antibody.These cells were analyzed for apoptosis by FACS the following morning. Peripheral B cells were cultured in the presenceof anti-CD40 antibody, with or without IL4. Cell surface Fas expression was evaluated after 48 hours. Anti-Fas antibodywas added after 48 hours, and the cells were incubated overnight before FACS analysis for apoptosis. We have analysed15 patients and 8 controls. The percentage of freshly isolated B cells staining positive with annexin V is low (<5%), andthey express low level of Fas. There is no significant difference between patients and controls. After overnight incubation,the percentage of B cells undergoing spontaneous apoptosis ranged from 35 to 70%. The addition of anti-Fas antibodydid not significantly change the percentage of cells undergoing apoptosis in patients or controls. B cell expression of Fasincreased after 48-hour incubation with anti-CD40 antibody to the same extent in patients and controls. However, almostall B cells underwent spontaneous apoptosis in vitro after 64 hours of incubation without stimulation. The presence ofanti-CD40 and IL4 reduced the percentage of apoptotic cells to the same extent in patients and controls. The addition ofanti-Fas after 48 hours did not change the percentage of apoptotic cells. Data so far does not suggest the presence ofdefects in B cell apoptosis in patients with inactive SLE. B cells rapidly undergo apoptosis in vitro without stimulation,and anti-Fas antibody did not affect the rate of apoptosis in patients or controls. Preliminary experiments have shownthat a combination of anti-IgM, anti-CD40 and 1L-4 can stimulate B cell proliferation in vitro. We will further look intoFas-mediated apoptosis of these activated B cells from SLE patients.- 57 -

Pseudomonas aeruginosa pyocyanin (PYO) down-regulates inducible nitric oxide synthase (iNOS) expression in human bronchus

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Pseudomonas aeruginosa pyocyanin (PYO) down-regulates inducible nitric oxide synthase (iNOS) expression in human bronchus

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