Immunological and biochemical characterization of recombinant group ...

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Novozymes has recombinantly expressed the cysteine-protease Der p 1 from the house dust mite (HDM) D. pteronyssinus, and hypoallergenic variants thereof.
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Double-Stranded RNA-Induced CC Chemokine Synthesis in Human Bronchial Smooth Muscle Cells and Toll-Like Receptor 3 K. Niimi1,2, K. Asano1,2, Y. Shiraishi1,2, M. Wakaki1,2, T. Nakajima1,2, J. Kagyo1,2, Y. Suzuki1, T. Shiomi1, T. Oguma1, K. Sayama1, K. Yamaguchi1, A. Ishizaka1,2; 1Division of Pulmonary Medicine, Department of Medicine, Keio University School of Medicine, Tokyo, JAPAN, 2PfizerKeio Research Laboratories, Shinanomachi Research Park, Keio University School of Medicine, Tokyo, JAPAN. RATIONALE: Respiratory viral infection frequently causes acute exacerbation of symptoms and eosinophilic airway inflammation in patients with asthma. We examined the effect of double-stranded RNA (dsRNA), which is produced during viral replication, on the synthesis and release of eosinophilic CC chemokines such as eotaxin and RANTES in human bronchial smooth muscle cells. METHODS: Primary cultures of human bronchial smooth muscle cells (BSMC) and immortalized human bronchial epithelial cells (BEAS2B) were stimulated with dsRNA, poly I:C. The synthesis and release of eosinophilic CC chemokines was analyzed by real-time RT-PCR and ELISA. TLR3 expression on BSMC was determined with RT-PCR and flowcytometric analysis using anti-TLR3 antibody (clone 3.7). RESULTS: Stimulation with dsRNA increased the production of RANTES, but not eotaxin, in BEAS2B cells. In contrast, BSMC released significant amount of both eotaxin and RANTES in response to dsRNA in a dose-dependent manner, and eotaxin mRNA expression and protein release was further augmented in the presence of interleukin 4. RT-PCR and flowcytometric analysis confirmed the expression of TLR3 on BSMC, but dsRNA-induced release of eotaxin could not blocked by an anti-TLR3 neutralizing antibody. CONCLUSION: DsRNA induces the synthesis and release of eosinophilic chemokines such as eotaxin and RANTES in BSMC. A receptor for dsRNA, TLR3, is expressed on BSMC, but its role on the dsRNA-induced chemokine synthesis is unclear.

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Oligonucleotide Costimulation of Mouse CD4+ T Cell Proliferation Is Dependent on the Toll-Like Receptor Adaptor Molecule MyD88 D. F. LaRosa, A. E. Gelman, L. A. Turka; University of Pennsylvania School of Medicine, Philadelphia, PA. RATIONALE: Recent evidence suggests activated T cells express Tolllike receptors (TLRs), and pathogen-associated molecular patterns (PAMPs) may have direct effects on adaptive immunity. We examined the proliferative responses of mouse MyD88 -/- and wild-type CD4+ T cells to phosphorothioate oligonucleotides (sODN). METHODS: Highly purified CD4+ T cells from MyD88-/- and wild-type mice were labeled with CFSE and stimulated with anti-CD3 plus antiCD28 antibodies in the presence or absence of CpG containing sODN (sODN 1668, sODN 1826) and non-CpG containing sODNs (sODN 1720, sODN 1826C). Proliferation was measured by CSFE dilution at 72 hours post-stimulation. CD4+ T cells were also assayed for TLR-9 expression by Western blotting. RESULTS: TLR-9 expression was observed on naïve and anti-CD3 plus anti-CD28 antibody stimulated CD4+ T cells suggesting that T cells are capable of responding to TLR-9 ligands. Costimulation of CD4+ T cells by sODNs required both CD3 and CD28 ligation. Wild-type CD4+ T cell costimulation was augmented by CpG sODN 1668 and 1826 but was not dependent on the CpG motif since we also observed equivalent induced costimulation to the non-CpG ODNs 1720 and 1826C. We did not observe sODN induced costimulation in MyD88-/- CD4+ T cells irrespective of the presence of the CpG motif. CONCLUSIONS: sODN costimulation of CD4+ T cells requires CD3, CD28, and MyD88 indicating that TLR and T cell receptor signaling pathways synergize in T cells to promote adaptive immunity. Funding: NIH

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J ALLERGY CLIN IMMUNOL FEBRUARY 2005

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Interactions Between Mast Cells and Dendritic Cells

Z. Allakhverdi1, S. Mecheri2, C. Demeure2, G. Delespesse1; 1NotreDame Hospital, CHUM Research Center, Montreal, PQ, CANADA, 2Institut Pasteur, Paris, FRANCE. RATIONALE: Mast cells (MCs) are often found in close physical contact with dendritic cells (DCs) in the skin and the mucosal surfaces, the preferential sites of pathogen entry and antigen (Ag) exposure, and may be directly or indirectly activated in IgE-independent manner by certain microbes or soluble molecules, leading to a local inflammation. We have hypothesized that the ability of certain Ags to directly activate MCs affects the migration/maturation of the DCs capturing that Ag and thereby regulates the nature of the subsequent primary T cell response. METHODS: DCs were cultured in the presence or absence of culture supernatants of IgE/anti-IgE-stimulated MCs for 48 h and the cell surface marker expression was examined by FACS and their cytokine production by ELISA. T-cell priming activity of these DCs was examined in co-cultures with cord blood-derived naive CD4+ T cells. RESULTS: Culture supernatants of activated MCs induced HLA-DR, CD80, CD83, and CD86 molecules on immature DCs. Concomitantly, MCs supernatants up-regulated CCR7 expression on DCs. Moreover, MCs culture supernatants modulated the cytokine production profile of LPS-treated DCs by increasing the production of IL-10 and decreasing that of TNF-alpha and IL-12. DCs primed with MCs supernatant promoted the development of allogeneic naïve CD4+ T cells into effectors producing significantly higher levels of IL-10. CONCLUSION: Taken together, the data suggest that MC activation regulates the nature of the primary T cell response to the Ag. We are currently investigating in which conditions MCs might promote the development of Th1, Th2 or T regulatory cells.

TUESDAY

Immunological and Biochemical Characterization of Recombinant Group 1 Dust Mite Allergens N. K. Soni1, S. T. Lyngstrand1, S. Patkar2, L. L. H. Christensen2, M. Schlein3, E. P. Friis4, V. Batori1, P. S. Skov5, L. K. Poulsen6, E. L. Roggen1, U. Bødtger6, H. Draborg1; 1Protein Screening, Novozymes A/S, Bagsvaerd, DENMARK, 2Protein Chemistry, Novozymes A/S, Bagsvaerd, DENMARK, 3Structure and Biophysical Chemistry, Novo Nordic A/S, Bagsvaerd, DENMARK, 4Protein Design, Novozymes A/S, Bagsvaerd, DENMARK, 5Reference laboratory, Copenhagen, DENMARK, 6Allergy Clinic, National University Hospital, Copenhagen, DENMARK. RATIONALE: Recombinant allergens can be safer alternatives to extract-based immunotherapies. Novozymes has recombinantly expressed the cysteine-protease Der p 1 from the house dust mite (HDM) D. pteronyssinus, and hypoallergenic variants thereof. METHODS: pro-Der p 1 was expressed in S. cerevisiae by fusion to a fungal signal-peptide. Mutation of the N-glycosylation site, resulted in production of un-glycosylated pro-Der p 1, which was purified by ultra-filtration followed by two chromatographic steps. The pro-peptide was removed and mature recombinant (r-) Der p 1 was obtained. r-Der p 1 was compared to natural (n-) Der p 1, from mites, in biochemical and immunological assays, using blood samples from 23 HDM allergic donors. Putative B-cell epitopes were predicted by in silico epitope mapping and in vitro by phage display. Predicted epitopes were changed by protein engineering and hypoallergenic variants were expressed, purified and screened. RESULTS: r-Der p1 has cysteine-protease activity, correct N-terminal sequence and displays the same CD-specter as n-Der p 1. In addition, r-Der p 1 had similar IgE binding, T cell activation and histamine release profile as nDer p 1. Several hypoallergenic variants were identified, with reduced ability to bind patient IgE, and release histamine (up to 6-fold) from blood basophils. The hypoallergenic variants maintained the ability to activate T cells. CONCLUSIONS: r-Der p 1 expressed in S. cerevisiae is biochemically and immunologically equivalent to n-Der p 1, and may provide a safer alternative to HDM-extract based immunotherapy. Additionally, several hypoallergenic Der p 1 variants were identified, with the potential for producing even safer therapies. Funding: Novozymes A/S

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