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Inducible Nitric Oxide Synthase (iNOS) Expression and Nitric Oxide (NO) Production in Mouse Bone Marrow-Derived Mast Cells (BMMC)
S226 Abstracts
871
Inducible Nitric Oxide Synthase (iNOS) Expression and Nitric Oxide (NO) Production in Mouse Bone Marrow-Derived Mast Cells (BMMC) T. Moon, D. A. Befus; University of Alberta, Edmonton, AB, Canada. RATIONALE: There are discrepancies in the literature about whether MC express isoforms of NOS and make NO. We hypothesized that the microenvironment conditions involved in MC maturation can play an important role in whether or not MC express NOS and produce NO. METHODS: Bone marrow cells of Balb/c mice were cultured with various culture conditions, eg., IL-3, SCF 1 IL-3, or SCF 1 IL-4 for >3 weeks and we then examined NO synthesis and NOS expression after 48 h activation with IFNg in the presence or absence of IgE/Ag. To dissect the role of NOS isoforms in NO synthesis of BMMC, MC were preincubated with selective NOS inhibitors for 30 min before activation. RESULTS: In BMMC cultured with SCF 1 IL-4, but not with IL-3 or SCF 1 IL-3, we detected iNOS mRNA by RT-PCR and iNOS protein expression by western blot and confocal microscopic analysis. After stimulation with IFN-g (5ng/ml) in the presence or absence of IgE/Ag in BMMC cultured with SCF 1 IL-4, NO production was detected using the Griess assay. Confocal microscopic analysis showed that the iNOS expression induced by IFN-g colocalized in CD1171 (c-kit1) BMMC. NO production after activation with IFN-g in BMMC cultured with SCF 1 IL-4 was abrogated (>75% inhibition) by pretreatment with the iNOS specific inhibitor, 1400 W (5 mM), partially blocked (;30%) by the general NOS inhibitor, L-NMMA (5 mM), but not blocked by the eNOS selective inhibitor, LNAME (5 mM). CONCLUSIONS: MC progenitors that develop in certain permissive microenvironments such as SCF 1 IL-4 can express iNOS after receiving appropriate signals such as IFN-g, and subsequently produce NO.
872
TUESDAY
Proteins From Whole Body Of Ascaris Suum And Ascaris Lumbricoides Seem To Have Different IgE Binding Profile On Immunoblotting Of Serum From Allergy Asthmatics Patients E. A. Egea1, D. Mendoza2, S. Lozano1, G. Garavito1; 1Universidad del Norte, Barranquilla, Colombia, 2Universidad del Magdalena, Santa Marta, Colombia. RATIONALE: IgE antibody is clearly involved in both allergic reaction and host immune response against helminthic infection. The aims of this study was to identify the IgE binding profile of proteins present on extracts of whole body of the helminths A. lumbricoides (A L) and A. Suum(AS). METHODS: A total of 14 sera from allergy asthmatic patients sensitized to D. pteronnissynus, Blomia tropicalis and AS (Specific IgE) were evaluated. Electrophoretic profiles of whole body of AS and AL were determined by SDS-PAGE and the binding proteins to IgE were detected by Immunoblotting.Five sera from non-asthmatic individuals, Elisa negative for AS were considered as a negative controls. RESULTS: Coomassie stain showed 22 majority bands in the extract of AL and 20 on AS extract. A total of 13 IgE binding proteins were identified on the AS extract and 11 on the A L extract. (Ranked with size between 200-20 kDa). 11/14 sera recognized IgE binding proteins on the A L extracts. (Size between 45-31 kDa) Those bands of 43, 36 and 31 kDa showed a relevant intensity; On the AS extracts all 14 sera recognized the same 13 proteins (size between 200-60 kDa). CONCLUSIONS: This results suggest that different allergens from extracts of whole body of either AL and AS bind to its specific serum IgE and these reactions could be responsible for different IgE binding profile in both adult worms. For this reason, identification of allergens presents in A L should be performed using extracts of this helminth.
J ALLERGY CLIN IMMUNOL FEBRUARY 2009
873
Natriuretic Peptide Signaling Pathway Involvement In Respiratory Syncytial Virus Infection Of Lung Epithelium X. Kong1, W. Zhang1, P. K. Jena1, R. F. Lockey2, S. S. Mohapatra1; 1University of South Florida, Tampa, FL, 2James A Haley Veteran’s Hospital, Tampa, FL. RATIONALE: This study was conducted to determine the role of the atrial natriuretic peptide signaling pathway in respiratory syncytial virus (RSV) infection using lung epithelial cells and ANP receptor knockout mice as models. METHODS: ANP signaling in human lung epithelial A549 cells was prevented by downregulation of the ANP receptor, NPRA, with small interfering RNA against NPRA. Cells were exposed to RSV and viral titers were determined at 24 and 48 h after infection. The effect of blocking ANP activity in vivo was examined by comparing RSV infection of NPRA knockout mice with wild type. RSV titers in the lung, lung histopathology and cellular infiltration, and cytokine levels in bronchoalveolar lavage fluid were measured. RESULTS: Abrogation of ANP signaling with siNPRA reduced the infectivity of RSV in human epithelial cells. In mice deficient in ANP receptor, RSV titers were lower than in wild type and lung pathology and inflammatory cytokine levels were both reduced. CONCLUSIONS: Our data demonstrate a role for ANP-induced inflammation in RSV infectivity. Viral load in the lung epithelium may be reduced through local therapeutic intervention with an ANP receptor antagonist.
874
Soluble CD27 Levels in Children with Acute and Chronic Renal Failure _ T. Kendirli, A. Ikinciog ˘ ullari, M. Ekim, F. Dog˘u, F. Yalcxınkaya, S. ¨ zcxakar, D. G€ulog˘lu, B. Dog˘anay, E. Babacan; Ankara UniveY€uksel, Z. O sity School of Medicine, Ankara, Turkey. RATIONALE: CD27 is expressed on T, activated T, memory B and natural killer cells and renal tubules. Recently, the expression of CD27 and its ligand Siva was reported in rat kidneys with ischemia/reperfusion damage. METHODS: Serum soluble sCD27 levels were measured in 15 and 17 patients with Acute Renal Failire (ARF) and Chronic Renal Failure (CRF) espectively, and 25 healthy children. Serum sCD27 levels were measured by ELISA. RESULTS: Serum sCD27 level was 365 (130-1925) U/mL in ARF, 1525 (877-3260) U/mL in CRF and 75 (40-292) U/mL in the control group (p < 0.001). No correlation existed between sCD27 and white blood cell count, urea, creatinine, electrolytes, and liver enzymes in both groups. A negative correlation was found between the sCD27 and hemoglobin levels (r:-0. 55) in the CRF group. Mortality rate was 26% in ARF group. No statistically significant difference was found in sCD27 levels between surviving (365 (130-1925) IU/L) and deceased (420 (205-612) IU/L) children.There was no significant difference between dialysis modalities and sCD27 levels. CONCLUSIONS: Increased sCD27 levels in children with acute and chronic renal failure might be a marker of renal tubule apoptosis reflecting renal ischemia-perfusion damage.
871
Inducible Nitric Oxide Synthase (iNOS) Expression and Nitric Oxide (NO) Production in Mouse Bone Marrow-Derived Mast Cells (BMMC) T. Moon, D. A. Befus; University of Alberta, Edmonton, AB, Canada. RATIONALE: There are discrepancies in the literature about whether MC express isoforms of NOS and make NO. We hypothesized that the microenvironment conditions involved in MC maturation can play an important role in whether or not MC express NOS and produce NO. METHODS: Bone marrow cells of Balb/c mice were cultured with various culture conditions, eg., IL-3, SCF 1 IL-3, or SCF 1 IL-4 for >3 weeks and we then examined NO synthesis and NOS expression after 48 h activation with IFNg in the presence or absence of IgE/Ag. To dissect the role of NOS isoforms in NO synthesis of BMMC, MC were preincubated with selective NOS inhibitors for 30 min before activation. RESULTS: In BMMC cultured with SCF 1 IL-4, but not with IL-3 or SCF 1 IL-3, we detected iNOS mRNA by RT-PCR and iNOS protein expression by western blot and confocal microscopic analysis. After stimulation with IFN-g (5ng/ml) in the presence or absence of IgE/Ag in BMMC cultured with SCF 1 IL-4, NO production was detected using the Griess assay. Confocal microscopic analysis showed that the iNOS expression induced by IFN-g colocalized in CD1171 (c-kit1) BMMC. NO production after activation with IFN-g in BMMC cultured with SCF 1 IL-4 was abrogated (>75% inhibition) by pretreatment with the iNOS specific inhibitor, 1400 W (5 mM), partially blocked (;30%) by the general NOS inhibitor, L-NMMA (5 mM), but not blocked by the eNOS selective inhibitor, LNAME (5 mM). CONCLUSIONS: MC progenitors that develop in certain permissive microenvironments such as SCF 1 IL-4 can express iNOS after receiving appropriate signals such as IFN-g, and subsequently produce NO.
872
TUESDAY
Proteins From Whole Body Of Ascaris Suum And Ascaris Lumbricoides Seem To Have Different IgE Binding Profile On Immunoblotting Of Serum From Allergy Asthmatics Patients E. A. Egea1, D. Mendoza2, S. Lozano1, G. Garavito1; 1Universidad del Norte, Barranquilla, Colombia, 2Universidad del Magdalena, Santa Marta, Colombia. RATIONALE: IgE antibody is clearly involved in both allergic reaction and host immune response against helminthic infection. The aims of this study was to identify the IgE binding profile of proteins present on extracts of whole body of the helminths A. lumbricoides (A L) and A. Suum(AS). METHODS: A total of 14 sera from allergy asthmatic patients sensitized to D. pteronnissynus, Blomia tropicalis and AS (Specific IgE) were evaluated. Electrophoretic profiles of whole body of AS and AL were determined by SDS-PAGE and the binding proteins to IgE were detected by Immunoblotting.Five sera from non-asthmatic individuals, Elisa negative for AS were considered as a negative controls. RESULTS: Coomassie stain showed 22 majority bands in the extract of AL and 20 on AS extract. A total of 13 IgE binding proteins were identified on the AS extract and 11 on the A L extract. (Ranked with size between 200-20 kDa). 11/14 sera recognized IgE binding proteins on the A L extracts. (Size between 45-31 kDa) Those bands of 43, 36 and 31 kDa showed a relevant intensity; On the AS extracts all 14 sera recognized the same 13 proteins (size between 200-60 kDa). CONCLUSIONS: This results suggest that different allergens from extracts of whole body of either AL and AS bind to its specific serum IgE and these reactions could be responsible for different IgE binding profile in both adult worms. For this reason, identification of allergens presents in A L should be performed using extracts of this helminth.
J ALLERGY CLIN IMMUNOL FEBRUARY 2009
873
Natriuretic Peptide Signaling Pathway Involvement In Respiratory Syncytial Virus Infection Of Lung Epithelium X. Kong1, W. Zhang1, P. K. Jena1, R. F. Lockey2, S. S. Mohapatra1; 1University of South Florida, Tampa, FL, 2James A Haley Veteran’s Hospital, Tampa, FL. RATIONALE: This study was conducted to determine the role of the atrial natriuretic peptide signaling pathway in respiratory syncytial virus (RSV) infection using lung epithelial cells and ANP receptor knockout mice as models. METHODS: ANP signaling in human lung epithelial A549 cells was prevented by downregulation of the ANP receptor, NPRA, with small interfering RNA against NPRA. Cells were exposed to RSV and viral titers were determined at 24 and 48 h after infection. The effect of blocking ANP activity in vivo was examined by comparing RSV infection of NPRA knockout mice with wild type. RSV titers in the lung, lung histopathology and cellular infiltration, and cytokine levels in bronchoalveolar lavage fluid were measured. RESULTS: Abrogation of ANP signaling with siNPRA reduced the infectivity of RSV in human epithelial cells. In mice deficient in ANP receptor, RSV titers were lower than in wild type and lung pathology and inflammatory cytokine levels were both reduced. CONCLUSIONS: Our data demonstrate a role for ANP-induced inflammation in RSV infectivity. Viral load in the lung epithelium may be reduced through local therapeutic intervention with an ANP receptor antagonist.
874
Soluble CD27 Levels in Children with Acute and Chronic Renal Failure _ T. Kendirli, A. Ikinciog ˘ ullari, M. Ekim, F. Dog˘u, F. Yalcxınkaya, S. ¨ zcxakar, D. G€ulog˘lu, B. Dog˘anay, E. Babacan; Ankara UniveY€uksel, Z. O sity School of Medicine, Ankara, Turkey. RATIONALE: CD27 is expressed on T, activated T, memory B and natural killer cells and renal tubules. Recently, the expression of CD27 and its ligand Siva was reported in rat kidneys with ischemia/reperfusion damage. METHODS: Serum soluble sCD27 levels were measured in 15 and 17 patients with Acute Renal Failire (ARF) and Chronic Renal Failure (CRF) espectively, and 25 healthy children. Serum sCD27 levels were measured by ELISA. RESULTS: Serum sCD27 level was 365 (130-1925) U/mL in ARF, 1525 (877-3260) U/mL in CRF and 75 (40-292) U/mL in the control group (p < 0.001). No correlation existed between sCD27 and white blood cell count, urea, creatinine, electrolytes, and liver enzymes in both groups. A negative correlation was found between the sCD27 and hemoglobin levels (r:-0. 55) in the CRF group. Mortality rate was 26% in ARF group. No statistically significant difference was found in sCD27 levels between surviving (365 (130-1925) IU/L) and deceased (420 (205-612) IU/L) children.There was no significant difference between dialysis modalities and sCD27 levels. CONCLUSIONS: Increased sCD27 levels in children with acute and chronic renal failure might be a marker of renal tubule apoptosis reflecting renal ischemia-perfusion damage.