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898 Molecular characterization of a glycoprotein allergen of Aspergillus fumigatus
S306
Abstracts
898
Molecular Aspergillus
J ALLERGY CLIN IMMUNOL JANUARY 2000
Characterization of a Glycoprotein Allergen of Fumigatus Shailly Nigam*, P VGK Sarmaf. PC Ghosh#, Vsha P. Sarma* *Centre for Biochemical Technology. Delhi,India tSri Venkateshwara College.University of Delhi, Delhi, India *Department of Biochemistry, University of Delhi South Campus, Delhi, India Allergens and antigens of A.fumigatus are extremely complex macromolecules with multifunctional properties and some of them are proposed to be virulent factors in Aspergillus mediated disorders. A.fumigatus secretes ribonucleases, catalases. elastases and proteases with IgG and IgE binding activity with the sera of patients of aspergillosis. A number of allergens and antigens of A.fumigatus in the molecular weight range of 14-70kD exhibit specific IgG and IgE binding with the sera of aspergillosis patients. This suggests the possible presence of homologous sequential or conformational epitopes among various immunodominant allergens. Such analysis on the structural and biological functional aspects of Aspergillus antigens would further shed light on their mechanism of action in the pathogenesis of fungal diseases. Earlier work in our laboratory focussed on an l8kD allergen with respect to its cloning, expression and structure functional aspects (GenBank accession number:AF181859).With this in view, in the current study the gene encoding an immunodominant 55kD glycoprotein allergen was cloned and expressed. The gene encoding this allergen was amplified by Polymerase Chain Reaction using A.fumigatus genomic DNA of an Indian clinical strain isolated from an aspergilloma patient (Af-285). This product was then cloned in the SmaI site of pUCl8. The recombinants were selected and the fusion protein was expressed in E.coli JM 109 cells. The immunological activity ( IgG & IgE ) of the expressed fusion protein was observed using specific hyperimmune rabbit sera and also with the sera obtained from aspergillosis patients. The biological activity of the recombinant protein was examined with respect to the protease and cytotoxic activity. The cloned fragment and the PCR product have been sequenced. A synthetic peptide of this 55kD allergen has been observed to be allergenic. Further its N-terminal twenty amino acid sequence is homologous to the deduced sequence of Aspf 2 which is one of the immunodominant allergens. The identification of presence of such common sequential epitopes and their characterization at molecular level would enhance our knowledge on structural homology among these fungal aller-
899 Expression ders of Acute E. Wenzelf. Christman#.
of Secretory Leukocyte Protease Inhibitor in Disorand Chronic Lung Injury John A. Kennedy*. Sal/~ Kevin K. Brownf, Pert-o E. Petrides&
Gordon Malcolm
R. Bernard#. Kingi Cliffird
Brim1 W D. Wright*
*Amgen, Inc. Thousand Oaks, CA tNationa1 Jewish Medical and Research Center, Denver, CO $Vanderbilt University. Nashville, TN $Humboldt University, Berlin, Germany IUniversity of Alberta, Edmonton, Canada Secretory leukocyte protease inhibitor (SLPI) is a naturally occurring serine protease inhibitor produced by mucosal epithelial cells, serous cells, and bronchiolar goblet cells in human airways. SLPI exhibits broad spectrum inhibitory activity against mast cell and leukocyte serine proteases implicated in the pathology of disorders of acute and chronic lung injury. In order to assess the potential contribution of SLPI to anti-protease activity in such disorders, SLPI levels were assessed in bronchoalveolar lavage fluid (BALF) from normal volunteers and patients with severe asthma, idiopathic pulmonary fibrosis, adult respiratory distress syndrome (ARDS).
sepsis, post-bone marrow transplant idiopathic pulmonary syndrome or pulmonary carcinoma. In addition, sputum samples from patients with chronic obstructive pulmonary disease (COPD) or cystic fibrosis were assessed. SLPI concentrations were quantified by enzyme immunoassay. In normal BALF, SLPI was present at 20.9 rig/ml. In comparison. the following levels were observed in patient BALF: severe asthma 52.0 nglml idiopathic pulmonary fibrosis 17.0 rig/ml ARDS 245.6 rig/ml (p= 0.003 vs. normal) sepsis 433.6 rig/ml (p= 0.0009 vs. normal) idiopathic pulmonary syndrome 43.9 rig/ml (p= 0.025 vs. normal) and pulmonary carcinoma 396.7 nglml (p= 0.004 vs. normal). In COPD sputum, the SLPI content was 794.5 rig/ml compared with I. I nglml in sputum from patients with cystic fibrosis (p=0.0003 vs. COPD). In contrast. the matrix metalloproteinase inhibitor TIMP- I was significantly elevated in all patient populations. The reduced level of SLPI in cystic fibrosis sputum may reflect the ability of Pseudomonas elastase to degrade SLPI. These results suggest that the pathophysiology of these airway disorders may be associated with differential upregulation of SLPI expression. 900
The Role of Protease Activated Receptors in Cystic Fibrosis Lung Pathology Joshua Fink*t. Andrew McWilliamf#. Geoffre? Stenwt*f *Department of Microbiology tuniversity of Western Australia *Department of Medicine Cystic fibrosis (CF) is a major human disease characterized by repeated, progressively more severe, respiratory infection and inflammation. The respiratory epithelium (RE) is known to be important in a variety of pathological conditions including CF, as it is capable of synthesising a range of pro-infhunmatory agents in response to a variety of stimuli. In this regard, it is known that endogenous and exogenous proteases are potent inducers of proinflammatory cytokines from RE. How these protease-induced effects are mediated at the cellular level, and what effect the CF genetic defect has on RE pro-inflammatory function, is unclear. To this end, we have begun to investigate the role of a novel family of protease-activated receptors (PAR) in this process. Thus far. 4 PAR have been identified and differentiated on the basis of their activation by various proteases; PAR I ,3 and 4 are activated by thrombin whereas PAR-2 and 4 are activated by trypsin or tryptase. Immunohistochemical studies. using specific antibodies, indicated the presence of PAR- I, -2 and -3, but not -4. in airway epithelial cell lines possessing the CF genetic defect (CFiTE29o-) and also in those without the defect (BEAS-2B and A549). The cell lines were then exposed to agonist peptides (range O-500 mM) consisting of the six amino acid residues corresponding to the nascent N-terminus portion of receptors 1 to -4 and their effect on IL-6 and IL-8 secretion determined by ELISA. PAR-I and -2 peptides caused the release of both cytokines from the non-CF derived cell lines. In contrast, PAR1 peptide, but not PAR-2 caused the release of cytokines from the CF-derived cell line. Stimulation with PAR-3 and -4 did not result in cytokine responses in any cell line. An additive effect for PAR-I and PAR-2 was observed for cytokine secretion in both the A549 and CF/TE29ocell lines despite the lack of any response from the CF-derived cells following treatment with PAR-2 peptide alone. Maximum cytokine release for both types of cell line was obtained with about 400 mM peptide. The cytokine response in the CF/TE29ocell line was significantly lower than that seen in the non-CF cell lines although similar levels of cytokines were obtained in both types of cell line after PMA stimulation. These data suggest that the CF defect may modulate epithelial cell pro-inflammatory functions involving PAR.
Abstracts
898
Molecular Aspergillus
J ALLERGY CLIN IMMUNOL JANUARY 2000
Characterization of a Glycoprotein Allergen of Fumigatus Shailly Nigam*, P VGK Sarmaf. PC Ghosh#, Vsha P. Sarma* *Centre for Biochemical Technology. Delhi,India tSri Venkateshwara College.University of Delhi, Delhi, India *Department of Biochemistry, University of Delhi South Campus, Delhi, India Allergens and antigens of A.fumigatus are extremely complex macromolecules with multifunctional properties and some of them are proposed to be virulent factors in Aspergillus mediated disorders. A.fumigatus secretes ribonucleases, catalases. elastases and proteases with IgG and IgE binding activity with the sera of patients of aspergillosis. A number of allergens and antigens of A.fumigatus in the molecular weight range of 14-70kD exhibit specific IgG and IgE binding with the sera of aspergillosis patients. This suggests the possible presence of homologous sequential or conformational epitopes among various immunodominant allergens. Such analysis on the structural and biological functional aspects of Aspergillus antigens would further shed light on their mechanism of action in the pathogenesis of fungal diseases. Earlier work in our laboratory focussed on an l8kD allergen with respect to its cloning, expression and structure functional aspects (GenBank accession number:AF181859).With this in view, in the current study the gene encoding an immunodominant 55kD glycoprotein allergen was cloned and expressed. The gene encoding this allergen was amplified by Polymerase Chain Reaction using A.fumigatus genomic DNA of an Indian clinical strain isolated from an aspergilloma patient (Af-285). This product was then cloned in the SmaI site of pUCl8. The recombinants were selected and the fusion protein was expressed in E.coli JM 109 cells. The immunological activity ( IgG & IgE ) of the expressed fusion protein was observed using specific hyperimmune rabbit sera and also with the sera obtained from aspergillosis patients. The biological activity of the recombinant protein was examined with respect to the protease and cytotoxic activity. The cloned fragment and the PCR product have been sequenced. A synthetic peptide of this 55kD allergen has been observed to be allergenic. Further its N-terminal twenty amino acid sequence is homologous to the deduced sequence of Aspf 2 which is one of the immunodominant allergens. The identification of presence of such common sequential epitopes and their characterization at molecular level would enhance our knowledge on structural homology among these fungal aller-
899 Expression ders of Acute E. Wenzelf. Christman#.
of Secretory Leukocyte Protease Inhibitor in Disorand Chronic Lung Injury John A. Kennedy*. Sal/~ Kevin K. Brownf, Pert-o E. Petrides&
Gordon Malcolm
R. Bernard#. Kingi Cliffird
Brim1 W D. Wright*
*Amgen, Inc. Thousand Oaks, CA tNationa1 Jewish Medical and Research Center, Denver, CO $Vanderbilt University. Nashville, TN $Humboldt University, Berlin, Germany IUniversity of Alberta, Edmonton, Canada Secretory leukocyte protease inhibitor (SLPI) is a naturally occurring serine protease inhibitor produced by mucosal epithelial cells, serous cells, and bronchiolar goblet cells in human airways. SLPI exhibits broad spectrum inhibitory activity against mast cell and leukocyte serine proteases implicated in the pathology of disorders of acute and chronic lung injury. In order to assess the potential contribution of SLPI to anti-protease activity in such disorders, SLPI levels were assessed in bronchoalveolar lavage fluid (BALF) from normal volunteers and patients with severe asthma, idiopathic pulmonary fibrosis, adult respiratory distress syndrome (ARDS).
sepsis, post-bone marrow transplant idiopathic pulmonary syndrome or pulmonary carcinoma. In addition, sputum samples from patients with chronic obstructive pulmonary disease (COPD) or cystic fibrosis were assessed. SLPI concentrations were quantified by enzyme immunoassay. In normal BALF, SLPI was present at 20.9 rig/ml. In comparison. the following levels were observed in patient BALF: severe asthma 52.0 nglml idiopathic pulmonary fibrosis 17.0 rig/ml ARDS 245.6 rig/ml (p= 0.003 vs. normal) sepsis 433.6 rig/ml (p= 0.0009 vs. normal) idiopathic pulmonary syndrome 43.9 rig/ml (p= 0.025 vs. normal) and pulmonary carcinoma 396.7 nglml (p= 0.004 vs. normal). In COPD sputum, the SLPI content was 794.5 rig/ml compared with I. I nglml in sputum from patients with cystic fibrosis (p=0.0003 vs. COPD). In contrast. the matrix metalloproteinase inhibitor TIMP- I was significantly elevated in all patient populations. The reduced level of SLPI in cystic fibrosis sputum may reflect the ability of Pseudomonas elastase to degrade SLPI. These results suggest that the pathophysiology of these airway disorders may be associated with differential upregulation of SLPI expression. 900
The Role of Protease Activated Receptors in Cystic Fibrosis Lung Pathology Joshua Fink*t. Andrew McWilliamf#. Geoffre? Stenwt*f *Department of Microbiology tuniversity of Western Australia *Department of Medicine Cystic fibrosis (CF) is a major human disease characterized by repeated, progressively more severe, respiratory infection and inflammation. The respiratory epithelium (RE) is known to be important in a variety of pathological conditions including CF, as it is capable of synthesising a range of pro-infhunmatory agents in response to a variety of stimuli. In this regard, it is known that endogenous and exogenous proteases are potent inducers of proinflammatory cytokines from RE. How these protease-induced effects are mediated at the cellular level, and what effect the CF genetic defect has on RE pro-inflammatory function, is unclear. To this end, we have begun to investigate the role of a novel family of protease-activated receptors (PAR) in this process. Thus far. 4 PAR have been identified and differentiated on the basis of their activation by various proteases; PAR I ,3 and 4 are activated by thrombin whereas PAR-2 and 4 are activated by trypsin or tryptase. Immunohistochemical studies. using specific antibodies, indicated the presence of PAR- I, -2 and -3, but not -4. in airway epithelial cell lines possessing the CF genetic defect (CFiTE29o-) and also in those without the defect (BEAS-2B and A549). The cell lines were then exposed to agonist peptides (range O-500 mM) consisting of the six amino acid residues corresponding to the nascent N-terminus portion of receptors 1 to -4 and their effect on IL-6 and IL-8 secretion determined by ELISA. PAR-I and -2 peptides caused the release of both cytokines from the non-CF derived cell lines. In contrast, PAR1 peptide, but not PAR-2 caused the release of cytokines from the CF-derived cell line. Stimulation with PAR-3 and -4 did not result in cytokine responses in any cell line. An additive effect for PAR-I and PAR-2 was observed for cytokine secretion in both the A549 and CF/TE29ocell lines despite the lack of any response from the CF-derived cells following treatment with PAR-2 peptide alone. Maximum cytokine release for both types of cell line was obtained with about 400 mM peptide. The cytokine response in the CF/TE29ocell line was significantly lower than that seen in the non-CF cell lines although similar levels of cytokines were obtained in both types of cell line after PMA stimulation. These data suggest that the CF defect may modulate epithelial cell pro-inflammatory functions involving PAR.