# Patent

## Title
Fungus-induced inflammation and eosinophil degranulation

## Abstract

This document relates to methods and materials involved in fungus-induced inflammation and eosinophil degranulation. For example, isolated nucleic acids encoding fungal polypeptides, fungal polypeptides, methods for assessing fungus-induced inflammation, methods for assessing eosinophil degranulation, and methods for identifying inhibitors of fungus-induced inflammation and/or eosinophil degranulation are provided.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a divisional of U.S. application Ser. No. 11/580,454, filed Oct. 13, 2006 now U.S. Pat. No. 7,662,400, which claims the benefit of U.S. Provisional Application Ser. No. 60/726,553, filed Oct. 14, 2005. The disclosure of the prior applications are considered part of (and are incorporated by reference in) the disclosure of this application.


STATEMENT AS TO FEDERALLY SPONSORED RESEARCH
Funding for the work described herein was provided by the federal government under grant number AI049235 awarded by the National Institute of Allergy and Infectious Diseases. The federal government has certain rights in the invention.


BACKGROUND
1. Technical Field
This document relates to methods and materials involved in fungus-induced inflammation and eosinophil degranulation. For example, this document relates to isolated nucleic acids encoding fungal polypeptides, fungal polypeptides, methods for assessing fungus-induced inflammation, methods for assessing eosinophil degranulation, and methods for identifying inhibitors of fungus-induced inflammation and/or eosinophil degranulation.
2. Background Information
The National Center for Health Statistics describes the increasingly expensive health care burden that chronic rhinosinusitis (CRS) inflicts in the United States. With an estimated 18 to 22 million cases and at least 30 million courses of antibiotics per year, CRS is one of the predominant chronic diseases in the U.S. In 1996, there were 26.7 million visits to physicians, hospital offices, and emergency departments for sinusitis—at a total cost of $5.8 billion. Sinusitis significantly impacts quality of life, even when compared to typical chronic debilitating diseases, such as diabetes and congestive heart failure. CRS presents a challenge to various medical specialties, including infectious diseases, ear, nose, and throat (ENT), allergy, asthma, and clinical immunology. The FDA has not approved any medication for effective use in CRS. Many antibiotic treatments are prescribed without objective evidence of infection. Roughly 40,000 patients per year undergo sinus surgery, but controlled evidence about the surgical outcomes is lacking. Even with aggressive medical and surgical therapies, many patients have persistent or recurrent disease, leading to frequent courses of antibiotics and multiple surgical interventions.
SUMMARY
This document relates to methods and materials involved in fungus-induced inflammation and eosinophil degranulation. For example, this document relates to isolated nucleic acids encoding fungal polypeptides, fungal polypeptides, methods for assessing fungus-induced inflammation, methods for assessing eosinophil degranulation, and methods for identifying inhibitors of fungus-induced inflammation and/or eosinophil degranulation.
In general, one aspect of this document features a substantially pure polypeptide comprising, or consisting essentially of, an amino acid sequence at least 95 percent identical to the amino acid sequence set forth in SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34. The polypeptide can comprise the amino acid sequence set forth in SEQ ID NO:10. The polypeptide can comprise an amino acid sequence having 99% identity to the sequence set forth in SEQ ID NO:10. The polypeptide can comprise the amino acid sequence set forth in SEQ ID NO:12 or 22. The polypeptide can comprise an amino acid sequence having 99% identity to the sequence set forth in SEQ ID NO: 12 or 22.
In another aspect, this document features an isolated nucleic acid comprising, or consisting essentially of, a nucleic acid sequence that encodes a polypeptide comprising an amino acid sequence at least 95 percent identical to the amino acid sequence set forth in SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34. The polypeptide can comprise the amino acid sequence set forth in SEQ ID NO:10. The polypeptide can comprise an amino acid sequence having 99% identity to the sequence set forth in SEQ ID NO:10. The polypeptide can comprise the amino acid sequence set forth in SEQ ID NO:12 or 22. The polypeptide can comprise an amino acid sequence having fewer than 5 mismatches as compared to the sequence set forth in SEQ ID NO:10, 12, or 22. The nucleic acid can hybridize under highly stringent hybridization conditions to the nucleic acid sequence set forth in SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, or 33. The nucleic acid can hybridize under highly stringent hybridization conditions to the nucleic acid sequence set forth in SEQ ID NO:9, 11, or 21.
In another aspect, this document features a purified antibody having the ability to bind to a polypeptide comprising, or consisting essentially of, an amino acid sequence at least 95 percent identical to the amino acid sequence set forth in SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34. The antibody can have a dissociation constant that is less than 10−7 for the polypeptide. The polypeptide can be a polypeptide having the sequence set forth in SEQ ID NO:10, 12, or 22.
In another aspect, this document features a method of identifying an inhibitor of fungus-induced eosinophil degranulation. The method comprises, or consists essentially of, determining whether or not a test agent reduces the amount of eosinophil degranulation induced by a preparation comprising a polypeptide having an amino acid sequence at least 95 percent identical to the amino acid sequence set forth in SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34, wherein the reduction indicates that the test agent is the inhibitor. The polypeptide can be a recombinantly produced polypeptide. The amount of eosinophil degranulation can be determined by measuring major basic protein or eosinophil-derived neurotoxin.
In another aspect, this document features a method of identifying an inhibitor of fungus-induced inflammation. The method comprises, or consists essentially of, determining whether or not a test agent reduces the amount of inflammation induced in a mammal by a preparation comprising a polypeptide having an amino acid sequence at least 95 percent identical to the amino acid sequence set forth in SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34, wherein the reduction indicates that the test agent is the inhibitor. The polypeptide can be a recombinantly produced polypeptide.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.



DESCRIPTION OF THE DRAWINGS
FIG. 1. Production of IL-5 from PBMC from normal individuals (n=15) and patients with CRS (n=18) cultured with extracts of common environmental fungi.
FIG. 2. Correlation between Alternaria-specific IgE (A) and IgG (B) in sera and Alternaria-induced PBMC production of IL-5 in patients with CRS.
FIG. 3. Serum levels of IgG4 antibodies to Alternaria (left) and Aspergillus (right) in normal individuals and patients with allergic rhinitis (AR) and CRS. Each dot represents one subject Assay sensitivity, 100 μg/L. Statistical analysis by Mann-Whitney U test.
FIG. 4. H&E (A). GMS (B), anti-Alternaria (C), and anti-MSP (D) staining of sinus tissue specimen from a patients with CRS. Arrowheads point to GMS-positive fungi, which are barely detectable by this staining Also note presence of fungal organisms as detected by anti-Alternaria Ab (panel C) and diffuse deposition of MBP (panel D) in sinus mucus, but not in sinus tissue.
FIG. 5. Effects of fungi on eosinophil degranulation. Eosinophils were incubated with culture extracts of various fungi for 3 hours. EDN concentrations in the supernatants were measured by RIA as an indicator of degranulation. *, p<0.05 compared to medium alone, n=5.
FIG. 6. Characterization of activity in Alternaria extract. Panel A, Alternaria extracts were treated at various temperatures before incubation with eosinophils. Panel B, size exclusion chromatography with Superdex 200-10/30 column.
FIG. 7. Mechanism of PAR-2 activation.
FIG. 8. Desensitization of eosinophil calcium response (Panel A) and EDN release (Panel B) by PAR-2 peptides. Cells were preincubated with PAR-2 agonist (SLIGKV; SEQ ID NO:38), PAR-2 antagonist (LSIGKV; SEQ ID NO:35) or control peptide (GLIVKS; SEQ ID NO:36) (all at 100 μM) before stimulation with Alternaria extract (Panel A) or with Alternaria extract, PAF or PMA (Panel B).
FIG. 9. Effects of protease inhibitors on PAR-2 cleavage activity (Panel A) and EDN release activity (Panel B) of Alternaria extract. Alternaria extract, trypsin, or PMA was pretreated with pepstatin A agarose, control agarose, or APMSF, and added to the PAR-2 peptide substrate (Panel A) or eosinophils (Panel B). In Panel B, *, p<0.05 compared to no inhibitors, n=4.
FIG. 10. Effects of fungi on IL-6 production by BEAS-2B cells. BEAS-2B cells were incubated with culture extracts of various fungi for 24 hours. IL-6 concentrations in the supernatants were measured by ELISA. *, p<0.05 compared to medium alone, n=3.
FIG. 11. Effects of an aspartate protease inhibitor, ritonavir, on IL-8 production by BEAS-2B cells. Alternaria extract or TNF-α was pretreated with ritonavir and added to BEAS-2B cells. IL-8 concentrations in the supernatants were measured after 24 hours. Data are normalized to the values without ritonavir as 100%. *, p<0.05 compared to no inhibitor, n=4.
FIG. 12. Panel A. DEAE fractionation of Alternaria extract. Alternaria extract was separated by DEAE anion-exchange chromatography (Buffer A, 20 mM Tris pH 7.5; Buffer B, 20 mM Tris 1M NaCl pH 7.5) and individual fractions were analyzed for their PAR-2 cleavage activity, aspartate protease activity, and eosinophil degranulation activity. Panel B. A silver-stained SDS-PAGE analysis. Lane 1; crude Alternaria extract, Lane 2; DEAE fraction #18 further purified by hydroxyapatite chromatography.
FIG. 13. Morphology of eosinophils incubated with germinating A. alternata (Panel A) and release of EDN by these eosinophils (Panel B). Spores of A. alternata were cultured in RPMI medium with 10% FCS for 12 hours. Freshly isolated eosinophils were added to the wells at indicated eosinophil:spore ratios and incubated for an additional 4 hours. Concentrations of EDN released into the supernatants were measured by ELISA. Data are presented as mean±range from a duplicate experiment. Left panel and right panel in Panel A shows bright field image and anti-MBP immunofluorescence staining (to visualize eosinophils), respectively.
FIG. 14. Morphology of spores from GFP-transformed A. alternata. 
FIG. 15. Growth of A. alternata and production of PAR-2 activating enzyme(s). Spores of GFP-transformed A. alternata (1,000 spore/well of 96-well tissue culture plates) were cultured in HBSS medium supplemented with different concentrations of bovine mucin from submaxillary glands. Fungal growth was quantitated after 48 hours by measuring the intensity of GFP fluorescence in each well (Panel A). Production of PAR-2 activating proteases by fungi into the supernatants was measured at 24 hours or 48 hours by using a fluorescence quenched PAR-2 peptide substrate (Abz-SKGRSLIGK(Dnp)D) (Panel B) (SEQ ID NO:37). Data are presented as mean±SEM from a triplicate experiment.
FIG. 16. Effects of intranasal exposure to fungal antigens or OVA on airway inflammation. Naive mice were exposed intranasally to antigens (250 μg/exposure) without prior sensitization. Alt (cult), Alternaria culture supernatant; Alt (cell), Alternaria cellular extract; Can, Candida extract; Asp, Aspergillus extract.
FIG. 17. Effects of immune cell deficiency on Alternaria-induced airway eosinophilia and early cytokine response. Naive Rag-1 knockout (Rag-1) or wild type (WT) mice were exposed to Alternaria (Alt) intranasally on days 0, 3, and 6. Panel A shows kinetics of airway eosinophilia. Panel B shows early cytokine response 12 hours after the first exposure (i.e. day 0.5), n=4-9.
FIG. 18. Early airway IL-5 production in response to Alternaria exposure. Panel A: BALB/c mice were pretreated by intranasal administration of LPS (1 μg) or PBS on day −3, and then exposed to Alternaria (Alt) on day 0. BAL fluids were collected 12 hours later. Panel B: C3H/HeOuJ or C3H/HeJ mice were exposed to Alternaria or PBS on day 0 without prior treatment. BAL fluids were collected 12 hours later. n=5-6.
FIG. 19. Alternaria extract was pretreated with pepstain A-agarose (Pep A) or control agarose (Cont). Panel A: Mice were intranasally challenged with treated Alternaria extract on day 0, and BAL fluids were analyzed for IL-5 after 12 hours. Panel B: Mice were intranasally challenged with treated Alternaria extract or PBS on days 0, 3, and 6, and BAL fluids were analyzed for eosinophil numbers on day 8. n=4-7.
FIG. 20. Effects of Alternaria DEAE fractions on airway inflammation. Naive mice were exposed intranasally to PBS or DEAE fractions of Alternaria extract without prior sensitization. The fractions used are those described in FIG. 12. n=3.
FIG. 21. Effects of Trichoderma xylanase on eosinophil degranulation (Panel A). Effects of Trichoderma xylanase on IL-5 production in mouse airways (Panel B). Panel A: Eosinophils were incubated with various concentrations of Trichoderma xylanase for 3 hours. EDN concentrations in the supernatants were measured by RIA as an indicator of degranulation. Panel B: Naive BALB/c mice were exposed intranasally to various doses of Trichoderma xylanase. After 12 hours, BAL fluids were collected and the concentrations of IL-5 were measured by ELISA. Mean±range, n=2.
FIG. 22. PBMC proliferation monitored using CFSE labeling. PBMCs from a CRS patient were isolated, labeled with CFSE, and cultured in the presence of 25 μg/ml Alternaria extract (Alt) or medium alone (Med). On days 4 and 7, cells were collected, stained with CD4 PE, and analyzed by FACS. Numbers represent the percentage of CFSElowCD4+ cells among total CD4+ cells.
FIG. 23. Comparison of normal and CRS proliferation using CFSE labeling. PBMCs from a normal individual and a CRS patient were CFSE labeled and cultured with 25 μg/ml Alternaria extract (Alt), 2 μg/ml tetanus toxoid (TT) or medium alone (Med). On day 7, cells were collected, stained with CD4 PE, and analyzed by FACS.
FIG. 24. Temporary deglycosylation and downregulation of PAR-2 by xylanase. Isolated eosinophils were incubated with medium alone (Med) or Aspergillus xylanase (Xyl) for the indicated time. Cells were lysed and analyzed for PAR-2 molecules by anti-PAR-2 antibody (which recognizes the N-terminus of the molecule) and Western blot. The 41 kDa and 70 kDa PAR-2 molecules were deglycosylated by xylanase temporarily. Arrow; PAR-2 core protein, Arrow heads; glycosylated PAR-2 molecules.
FIG. 25. Partial characterization of Alternaria extract. A, Before incubation with eosinophils, aliquots of 100 μg/mL Alternaria and 10 ng/mL IL-5 were heated at 37, 56, or 100° C. for 30 min or were treated at 4° C. for 30 min. Eosinophils were incubated in duplicate with these treated stimuli for 3 hours at 37° C. Results show the mean±SEM from five different eosinophil preparations. B, Size exclusion chromatography used a Superdex 200-10/30 column and produced a broad absorbance peak (smooth line) of the Alternaria culture extract. The dots connected by lines show the levels of EDN release when portions of fractions 21-39 were incubated with eosinophils. The molecular weight calibration of the column is shown above the elution profile.
FIG. 26. A. alternata xylanase was PCR amplified using genomic DNA as template. PCR product was cloned in pQE-30 UA E. coli expression vector. The vector was transformed into the E. coli M15 host strain using electroporation and screened for the 6×-His tag. Strong positive colonies were selected and grown in one-liter culture. After induction with IPTG, proteins were purified by a Ni-NTA column. M; marker, 1; protein from uninduced culture, 2; protein from culture induced with IPTG, 3; following purification with Ni-NTA column.
FIG. 27. Nucleic acid sequence (SEQ ID NO:1) encoding a fungal polypeptide having the amino acid sequence set forth in SEQ ID NO:2.
FIG. 28. Nucleic acid sequence (SEQ ID NO:3) encoding a fungal polypeptide having the amino acid sequence set forth in SEQ ID NO:4.
FIG. 29. Nucleic acid sequence (SEQ ID NO:5) encoding a fungal polypeptide having the amino acid sequence set forth in SEQ ID NO:6.
FIG. 30. Nucleic acid sequence (SEQ ID NO:7) encoding a fungal polypeptide having the amino acid sequence set forth in SEQ ID NO:8.
FIG. 31. Nucleic acid sequence (SEQ ID NO:9) encoding a fungal polypeptide having the amino acid sequence set forth in SEQ ID NO:10.
FIG. 32. Nucleic acid sequence (SEQ ID NO:11) encoding a fungal polypeptide having the amino acid sequence set forth in SEQ ID NO:12.
FIG. 33. Nucleic acid sequence (SEQ ID NO:13) encoding a fungal polypeptide having the amino acid sequence set forth in SEQ ID NO:14.
FIG. 34. Nucleic acid sequence (SEQ ID NO:15) encoding a fungal polypeptide having the amino acid sequence set forth in SEQ ID NO:16.
FIG. 35. Nucleic acid sequence (SEQ ID NO:17) encoding a fungal polypeptide having the amino acid sequence set forth in SEQ ID NO:18.
FIG. 36. Nucleic acid sequence (SEQ ID NO:19) encoding a fungal polypeptide having the amino acid sequence set forth in SEQ ID NO:20.
FIG. 37. Nucleic acid sequence (SEQ ID NO:21) encoding a fungal polypeptide having the amino acid sequence set forth in SEQ ID NO:22.
FIG. 38. Nucleic acid sequence (SEQ ID NO:23) encoding a fungal polypeptide having the amino acid sequence set forth in SEQ ID NO:24.
FIG. 39. Nucleic acid sequence (SEQ ID NO:25) encoding a fungal polypeptide having the amino acid sequence set forth in SEQ ID NO:26.
FIG. 40. Nucleic acid sequence (SEQ ID NO:27) encoding a fungal polypeptide having the amino acid sequence set forth in SEQ ID NO:28.
FIG. 41. Nucleic acid sequence (SEQ ID NO:29) encoding a fungal polypeptide having the amino acid sequence set forth in SEQ ID NO:30.
FIG. 42. Nucleic acid sequence (SEQ ID NO:31) encoding a fungal polypeptide having the amino acid sequence set forth in SEQ ID NO:32.
FIG. 43. Nucleic acid sequence (SEQ ID NO:33) encoding a fungal polypeptide having the amino acid sequence set forth in SEQ ID NO:34.
FIG. 44. PBMC after challenge with isolated Alternaria protein fractions 30.
FIG. 45. PBMC after challenge with isolated Alternaria protein fractions 32.



DETAILED DESCRIPTION
This document relates to methods and materials involved in fungus-induced inflammation and eosinophil degranulation. For example, this document provides isolated nucleic acids encoding fungal polypeptides, substantially pure fungal polypeptides, methods for assessing fungus-induced inflammation, methods for assessing eosinophil degranulation, and methods for identifying inhibitors of fungus-induced inflammation and/or eosinophil degranulation. This document also provides methods and materials for making and using an antibody that can bind a fungal polypeptide. In addition, this document provides methods and materials for treating a mammal having a fungus-induced inflammatory condition (e.g., CRS).
Fungal Polypeptides and Nucleic Acids Encoding Fungal Polypeptides
This document provides a substantially pure fungal polypeptide. Such fungal polypeptides can have the ability to stimulate eosinophil degranulation and/or inflammation. For example a fungal polypeptide provided herein can have the ability to stimulate eosinophil degranulation in vitro, can have the ability to stimulate inflammation in vivo, or both. The term “substantially pure” with respect to a polypeptide refers to a polypeptide that has been separated from cellular components with which it is naturally accompanied. Typically, a polypeptide provided herein is substantially pure when it is at least 60 percent (e.g., 65, 70, 75, 80, 90, 95, or 99 percent), by weight, free from proteins and naturally-occurring organic molecules with which it is naturally associated. In general, a substantially pure polypeptide will yield a single major band on a non-reducing polyacrylamide gel. In some cases, a substantially pure polypeptide can be a polypeptide preparation that contains one of the polypeptides set forth in FIGS. 27-39 or a polypeptide at least about 80 percent identical to such a polypeptide, while being free of at least one of the other polypeptides set forth in FIGS. 27-39.
The polypeptides provided herein can be at least five amino acids in length (e.g., at least 6, 7, 10, 15, 30, 50, 70, or 100 amino acids in length). A substantially pure polypeptide provided herein can be a polypeptide having a sequence that is at least 80 percent identical to the amino acid sequence set forth in SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34. For example, a polypeptide provided herein can have at least 80, 85, 90, 95, 98, or 99 percent identity to SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, or 26. In some cases, a polypeptide provided herein can have the exact amino acid sequence set forth in SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34.
The percent identity between a particular amino acid sequence and the amino acid sequence set forth in SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34 is determined as follows. First, the amino acid sequences are aligned using the BLAST 2 Sequences (B12seq) program from the stand-alone version of BLASTZ containing BLASTP version 2.0.14. This stand-alone version of BLASTZ can be obtained from Fish & Richardson's web site (e.g., www.fr.com/blast/) or the State University of New York-Old Westbury Library (call number: QH 447.M6714). Instructions explaining how to use the B12seq program can be found in the readme file accompanying BLASTZ. B12seq performs a comparison between two amino acid sequences using the BLASTP algorithm. To compare two amino acid sequences, the options of B12seq are set as follows: -i is set to a file containing the first amino acid sequence to be compared (e.g., C:\seq1.txt); -j is set to a file containing the second amino acid sequence to be compared (e.g., C:\seq2.txt); -p is set to blastp; -o is set to any desired file name (e.g., C:\output.txt); and all other options are left at their default setting. For example, the following command can be used to generate an output file containing a comparison between two amino acid sequences: C:\B12seq-i c:\seq1.txt-j c:\seq2.txt-p blastp-o c:\output.txt. If the two compared sequences share homology, then the designated output file will present those regions of homology as aligned sequences. If the two compared sequences do not share homology, then the designated output file will not present aligned sequences.
Once aligned, the number of matches is determined by counting the number of positions where an identical amino acid residue is presented in both sequences. The percent identity is determined by dividing the number of matches by the length of the full-length amino acid sequence set forth in SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34 followed by multiplying the resulting value by 100. For example, an amino acid sequence that has 144 matches when aligned with the sequence set forth in SEQ ID NO:26 is 96.0 percent identical to the sequence set forth in SEQ ID NO:26 (i.e., 144÷150*100=96.0).
It is noted that the percent identity value is rounded to the nearest tenth. For example, 78.11, 78.12, 78.13, and 78.14 are rounded down to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 are rounded up to 78.2. It also is noted that the length value will always be an integer.
In some cases, a substantially pure polypeptide provided herein can have fewer than 10 (e.g., fewer than 9, 8, 7, 6, 5, 4, 3, or 2) mismatches as compared to the amino acid sequence set forth in SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34. For example, a polypeptide provided herein can have 4, 3, 2, or 1 mismatches as compared to the amino acid sequence set forth in SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34.
A substantially pure polypeptide provided herein can be obtained, for example, by extraction from a natural source (e.g., Alternaria cells), chemical synthesis, or by recombinant production in a host cell. To recombinantly produce a polypeptide provided herein, a nucleic acid sequence encoding the polypeptide can be ligated into an expression vector and used to transform a bacterial or eukaryotic host cell (e.g., insect, yeast, Alternaria, Pichia, or mammalian cells). In general, nucleic acid constructs can include a regulatory sequence operably linked to a nucleic acid sequence encoding a polypeptide provided herein. Regulatory sequences do not typically encode a gene product, but instead affect the expression of the nucleic acid sequence. In bacterial systems, a strain of Escherichia coli such as BL-21 can be used. Suitable E. coli vectors include the pGEX series of vectors (Amersham Biosciences Corp., Piscataway, N.J.) that produce fusion proteins with glutathione S-transferase (GST). Transformed E. coli typically are grown exponentially, and then stimulated with isopropylthio-galactopyranoside (IPTG) prior to harvesting. In general, such fusion proteins can be soluble and can be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. The pGEX vectors can be designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.
In some cases, fungi can be grown in large quantities in vitro, and a polypeptide provided herein that is endogenously produced can be separated and purified using chromatographic methods (e.g., HPL and/or FPLC with a variety of separation matrices). In order to produce recombinant, highly purified forms of a polypeptide provided herein, one method would be to engineer an affinity tag (e.g. 6×Histidine tag) either on the N- or C-terminus of the polypeptide (either via manipulation of the cDNA nucleic acid sequence with PCR mutagenesis, or use of expression vectors containing an affinity tag sequence) to aid in purification. Existing Pichia pastoris expression vectors and purification systems like those from Invitrogen (Carlsbad, Calif.) can be used for production of recombinant fungal polypeptides. Moreover, yeast and fungi are closely related organisms and thus recombinantly produced fungal polypeptides in P. pastoris can have an increased chance of being properly folded and retain post translation (e.g., glycosylation) modifications involved in activity. P. pastoris can be used as described elsewhere (Reichard et al., Appl. Environ. Microbiol., 72(3):1739-48 (2006)). Another method can involve using Alternaria itself as a production system. This can be accomplished by engineering an affinity tag on the desired polypeptide and then employing the LME fungal transformation approaches as described elsewhere (Cho et al., Molecular Plant-Microbe Interact., 19:7-15 (2006)).
In eukaryotic host cells, a number of viral-based expression systems can be utilized to express polypeptides provided herein. A nucleic acid encoding a polypeptide provided herein can be cloned into, for example, a baculoviral vector such as pBlueBac (Invitrogen, Carlsbad, Calif.) and then used to co-transfect insect cells such as Spodoptera frugiperda (Sf9) cells with wild type DNA from Autographa californica multiply enveloped nuclear polyhedrosis virus (AcMNPV). Recombinant viruses producing polypeptides provided herein can be identified by standard methodology. In some cases, a nucleic acid encoding a polypeptide provided herein can be introduced into a SV40, retroviral, or vaccinia based viral vector and used to infect suitable host cells.
Mammalian cell lines that stably express a polypeptide provided herein can be produced using expression vectors with the appropriate control elements and a selectable marker. For example, the eukaryotic expression vectors pCR3.1 (Invitrogen) and p91023(B) (see Wong et al., Science, 228:810-815 (1985)) can be used to express a polypeptide provided herein in, for example, Chinese hamster ovary (CHO) cells, COS-1 cells, human embryonic kidney 293 cells, NIH3T3 cells, BHK21 cells, MDCK cells, and human vascular endothelial cells (HUVEC). Following introduction of the expression vector by electroporation, lipofection, calcium phosphate or calcium chloride co-precipitation, DEAE dextran, or other suitable transfection method, stable cell lines can be selected, e.g., by antibiotic resistance to G418, kanamycin, or hygromycin. In some cases, amplified sequences can be ligated into a mammalian expression vector such as pcDNA3 (Invitrogen) and then transcribed and translated in vitro using wheat germ extract or rabbit reticulocyte lysate.
Polypeptides provided herein can be purified by known chromatographic methods including DEAE ion exchange, gel filtration, and hydroxylapatite chromatography. See, e.g., Van Loon and Weinshilboum, Drug Metab. Dispos., 18:632-638 (1990); and Van Loon et al., Biochem. Pharmacol., 44:775-785 (1992). Polypeptides provided herein can be modified to contain an amino acid sequence that allows the polypeptide to be captured onto an affinity matrix. For example, a tag such as c-myc, hemagglutinin, polyhistidine, or FlagTM (Kodak) can be used to aid polypeptide purification. Such tags can be inserted anywhere within a polypeptide including at either the carboxyl or amino terminus. Other fusions that can be useful include enzymes that aid in the detection of a polypeptide, such as alkaline phosphatase. Immunoaffinity chromatography also can be used to purify polypeptides provided herein.
Any suitable method, such as PCR, can be used to obtain an isolated nucleic acid encoding a polypeptide provided herein. The term “nucleic acid” as used herein encompasses both RNA and DNA, including cDNA, genomic DNA, and synthetic (e.g., chemically synthesized) DNA. The nucleic acid can be double-stranded or single-stranded. Where single-stranded, the nucleic acid can be the sense strand or the antisense strand. In addition, nucleic acid can be circular or linear.
The term “isolated” as used herein with reference to nucleic acid refers to a naturally-occurring nucleic acid that is not immediately contiguous with both of the sequences with which it is immediately contiguous (one on the 5′ end and one on the 3′ end) in the naturally-occurring genome of the organism from which it is derived. For example, an isolated nucleic acid can be, without limitation, a recombinant DNA molecule of any length, provided one of the nucleic acid sequences normally found immediately flanking that recombinant DNA molecule in a naturally-occurring genome is removed or absent. Thus, an isolated nucleic acid includes, without limitation, a recombinant DNA that exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other sequences as well as recombinant DNA that is incorporated into a vector, an autonomously replicating plasmid, a virus (e.g., a retrovirus, adenovirus, or herpes virus), or into the genomic DNA of a prokaryote or eukaryote. In addition, an isolated nucleic acid can include a recombinant DNA molecule that is part of a hybrid or fusion nucleic acid sequence.
The term “isolated” as used herein with reference to nucleic acid also includes any non-naturally-occurring nucleic acid since non-naturally-occurring nucleic acid sequences are not found in nature and do not have immediately contiguous sequences in a naturally-occurring genome. For example, non-naturally-occurring nucleic acid such as an engineered nucleic acid is considered to be isolated nucleic acid. Engineered nucleic acid can be made using common molecular cloning or chemical nucleic acid synthesis techniques. Isolated non-naturally-occurring nucleic acid can be independent of other sequences, or incorporated into a vector, an autonomously replicating plasmid, a virus (e.g., a retrovirus, adenovirus, or herpes virus), or the genomic DNA of a prokaryote or eukaryote. In addition, a non-naturally-occurring nucleic acid can include a nucleic acid molecule that is part of a hybrid or fusion nucleic acid sequence.
It will be apparent to those of skill in the art that a nucleic acid existing among hundreds to millions of other nucleic acid molecules within, for example, cDNA or genomic libraries, or gel slices containing a genomic DNA restriction digest is not to be considered an isolated nucleic acid.
A nucleic acid provided herein can be at least about ten nucleotides in length. For example, the nucleic acid can be about 10, 11, 15-20 (e.g., 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length), 20-50, 50-100 or greater than 100 nucleotides in length (e.g., greater than 150, 200, 250, 300, 350, 400, 450, 500, 750, or 1000 nucleotides in length). Nucleic acids provided herein can be in a sense or antisense orientation, can be identical or complementary to the nucleotide sequence set forth in SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, or 33, and can be DNA, RNA, or nucleic acid analogs. Nucleic acid analogs can be modified at the base moiety, sugar moiety, or phosphate backbone to improve, for example, stability, hybridization, or solubility of the nucleic acid. Modifications at the base moiety include deoxyuridine for deoxythymidine, and 5-methyl-2′-deoxycytidine and 5-bromo-2′-deoxycytidine for deoxycytidine. Modifications of the sugar moiety can include modification of the 2′ hydroxyl of the ribose sugar to form 2′-O-methyl or 2′-O-allyl sugars. The deoxyribose phosphate backbone can be modified to produce morpholino nucleic acids, in which each base moiety is linked to a six membered, morpholino ring, or peptide nucleic acids, in which the deoxyphosphate backbone is replaced by a pseudopeptide backbone and the four bases are retained. See, for example, Summerton and Weller, Antisense Nucleic Acid Drug Dev., 7:187-195 (1997); and Hyrup, et al., Bioorgan. Med. Chem., 4:5-23 (1996). In addition, the deoxyphosphate backbone can be replaced with, for example, a phosphorothioate or phosphorodithioate backbone, a phosphoroamidite, or an alkyl phosphotriester backbone.
Nucleic acids provided herein can hybridize, under hybridization conditions, to the sense or antisense strand of a nucleic acid having the nucleotide sequence set forth in SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, or 33. The hybridization conditions can be moderately or highly stringent hybridization conditions.
As used herein, moderately stringent hybridization conditions mean the hybridization is performed at about 42° C. in a hybridization solution containing 25 mM KPO4 (pH 7.4), 5×SSC, 5×Denhart's solution, 50 μg/mL denatured, sonicated salmon sperm DNA, 50% formamide, 10% Dextran sulfate, and 1-15 ng/mL probe (about 5×107 cpm/μg), while the washes are performed at about 50° C. with a wash solution containing 2×SSC and 0.1% sodium dodecyl sulfate.
Highly stringent hybridization conditions mean the hybridization is performed at about 42° C. in a hybridization solution containing 25 mM KPO4 (pH 7.4), 5×SSC, 5×Denhart's solution, 50 μg/mL denatured, sonicated salmon sperm DNA, 50% formamide, 10% Dextran sulfate, and 1-15 ng/mL probe (about 5×107 cpm/μg), while the washes are performed at about 65° C. with a wash solution containing 0.2×SSC and 0.1% sodium dodecyl sulfate.
Hybridization can be done by Southern or Northern analysis to identify a DNA or RNA sequence, respectively, that hybridizes to a probe. The DNA or RNA to be analyzed can be electrophoretically separated on an agarose or polyacrylamide gel, transferred to nitrocellulose, nylon, or other suitable membrane, and hybridized with a probe using standard techniques well known in the art such as those described in sections 7.39-7.52 of Sambrook et al., (1989) Molecular Cloning, second edition, Cold Spring harbor Laboratory, Plainview, N.Y. Typically, a probe is at least about 20 nucleotides in length. For example, a probe corresponding to a 20 nucleotide sequence set forth in SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, or 33 can be used to identify an identical or similar nucleic acid. In addition, probes longer or shorter than 20 nucleotides can be used. A probe can be labeled with a biotin, digoxygenin, an enzyme, or a radioisotope such as 32P.
Isolated nucleic acids provided herein also can be chemically synthesized, either as a single nucleic acid molecule (e.g., using automated DNA synthesis in the 3′ to 5′ direction using phosphoramidite technology) or as a series of oligonucleotides. For example, one or more pairs of long oligonucleotides (e.g., >100 nucleotides) can be synthesized that contain the desired sequence, with each pair containing a short segment of complementarity (e.g., about 15 nucleotides) such that a duplex is formed when the oligonucleotide pair is annealed. DNA polymerase is used to extend the oligonucleotides, resulting in a single, double-stranded nucleic acid molecule per oligonucleotide pair, which then can be ligated into a vector.
Antibodies
An antibody that can bind to a polypeptide provided herein can be made and purified using methods known to those skilled in the art (e.g., the methods described herein). For example, an antibody that can bind to a polypeptide provided herein can be affinity purified from the serum of an animal (e.g., a mouse, rat, rabbit, goat, donkey, horse, duck, or chicken) that received a substantially pure polypeptide provided herein under conditions that illicit an immune response to the polypeptide. In some cases, an antibody that can bind to a polypeptide provided herein can be purified from the supernatant of a B cell hybridoma that produces such an antibody.
An antibody that can bind to a polypeptide provided herein can be monoclonal or polyclonal and can be, for example, a single chain Fv, chimeric antibody, or an Fab fragment.
Fungus-Induced Eosinophil Degranulation
Eosinophils belong to the granulocyte class of white blood cells, and contain cytoplasmic granules that stain with the acidic dye eosin. Eosinophils are the main effectors of antibody-dependent cell-mediated cytotoxicity against multicellular parasites that provoke IgE antibodies. Their role seems to be to engulf and destroy the precipitated antigen-antibody complexes produced in humorally based immune reactions. An elevated eosinophil count usually is seen in allergic reactions, and numerous eosinophils are chemotactically aggregated at sites where antigen-antibody complexes are found.
As used herein, “fungus-induced eosinophil degranulation” refers to eosinophil degranulation in response to one or more antigens from fungal cells (e.g., from fungal cell extracts or fungal culture supernatants). Degranulation is the release of toxic molecules such as eosinophil cationic protein (ECP), eosinophil peroxidase (EPO), and MBP that are contained within eosinophil granules; this release typically causes damage to or death of cells in the vicinity of the degranulating eosinophils.
Eosinophil degranulation can be achieved in vitro as described in the example section herein. In some cases, a fungal preparation (e.g., a fungal cell extract or fungal culture supernatant) can be added to an eosinophil to induce degranulation. As used herein, a “fungal cell extract” is a preparation that contains factors (e.g., polypeptides) found within a fungal cell (e.g., in the cytoplasm, membranes, or organelles of a fungal cell). The term “fungal culture supernatant” refers to media obtained from culturing fungal cells. A fungal culture supernatant can be manipulated to form solid material. For example, a fungal culture supernatant can be obtained by removing fungal organisms from a fungal culture. The resulting supernatant then can be concentrated such that any remaining material (e.g., fungal polypeptides) form concentrated liquid or dry material. This dry material can be a fungal culture extract.
A cell extract or culture supernatant from any suitable type of fungus can be used to induce degranulation, including extracts and supernatants from those fungi listed above (e.g., Alternaria, Candida, Aspergillus, or Cladisporium). Alternaria cell extracts and culture supernatants are particularly useful. These can be obtained by standard laboratory cell culture and extract preparation techniques. Alternatively, fungal cell extracts and culture supernatants are commercially available (e.g., from Greer Laboratories, Lenoir, N.C.). Eosinophils can be obtained by, for example, purification from an individual's blood. Methods for such purification are known in the art.
Eosinophil degranulation can be stimulated in vitro by, for example, incubating a fungal preparation (e.g., a volume of Alternaria culture supernatant or 50 μg/mL of an Alternaria culture supernatant extract) with an eosinophil (e.g., purified eosinophils). Any incubation time (e.g., 1, 2, 3, 4, 5, 6, 7, or more hours) can be used. For example, an incubation time from about 2 to about 6 hours can be used. Any amount of a fungal preparation can be used. For example, the amount of a fungal extract can range from about 10 μg/mL to about 100 mg/mL (e.g., about 50, 100, 200, 300, or more μg/mL). Degranulation can be measured by a number of methods, including those known in the art. Degranulation can be assessed by, for example, measuring the release of markers such as ECP, EPO, MBP, or EDN. Non-limiting examples of methods for measuring marker levels include protein-based methods such as ELISA assays and western blotting. Alternatively, degranulation can be assessed by visual inspection of eosinophils by microscopy (e.g., using an electron microscope) to detect the presence of empty granules.
Identifying an Inhibitor of Fungus-Induced Eosinophil Degranulation and/or Inflammation
This document provides methods and materials that can be used to identify an agent that inhibits fungus-induced eosinophil degranulation and/or inflammation. For example, an inhibitor of fungus-induced eosinophil degranulation can be identified by contacting an eosinophil with a polypeptide provided herein in the presence and absence of a test agent, and measuring levels of degranulation (e.g., by measuring EDN output or MBP output, or by observing empty granules within eosinophils viewed by microscopy). A test agent can be identified as an inhibitor of eosinophil degranulation if the level of degranulation is reduced in the presence of the test agent as compared to the level of degranulation observed in the absence of the test agent. By “reduced” is meant that the level of degranulation in the presence of the test agent is less (e.g., 1% less, 5% less, 10% less, 50% less, 90% less, or 100% less) than the level observed without the test agent.
Molecules belonging to any of a number of classes can be used as test agents. For example, molecules that are polypeptides (i.e., amino acid chains of any length, regardless of modification such as phosphorylation or glycosylation), oligonucleotides, esters, lipids, carbohydrates, and steroids can be used as test agents. Molecules that are protease inhibitors may be particularly useful. Such protease inhibitors can be included within a cocktail of inhibitors (e.g., inhibitor cocktails that are commercially available from Roche Molecular Biochemicals, Indianapolis, Ind.) or can be individual protease inhibitors (e.g., a single serine protease inhibitor such as AEBSF).
In some cases, an inhibitor of fungus-induced inflammation can be identified by contacting an animal model (e.g., a mouse model) with a polypeptide provided herein in the presence and absence of a test agent, and measuring levels of inflammation. A test agent can be identified as an inhibitor of inflammation if the level of inflammation is reduced in the presence of the test agent as compared to the level of inflammation observed in the absence of the test agent.
The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.
EXAMPLES
Example 1
The Abnormal Immunologic Response of CRS Patients to Fungal Antigens
The responses of peripheral blood mononuclear cells (PBMC) from CRS patients to fungal antigens were characterized. The cytokine responses from CRS patients and normal volunteers, when stimulated with extracts from four common environmental fungal species—including Alternaria, Aspergillus, Cladosporium, and Penicillium, were examined. In the Examples section, Alternaria refers to Alternaria alternata unless specified otherwise. In FIG. 1, PBMC from about 90% of the CRS patients, but not those from normal individuals, produced both IL-5 and IL-13 when exposed to Alternaria, Aspergillus, or Cladosporium, but there were no differences in the amounts of these cytokines between allergic and non-allergic CRS patients. In response to Alternaria, PBMC from CRS patients produced about 5-times more IFN-γ than PBMC from normal individuals. Furthermore, levels of serum IgG antibodies to Alternaria and Cladosporium were increased in CRS patients compared to normal individuals (p<0.01), and the increased humoral (serum IgG antibody) response strongly correlated with the increased cellular (IL-5 production) response to Alternaria (r=0.619, p<0.01) (FIG. 2). In contrast, <30% of patients had elevated serum levels of IgE antibody to Alternaria, and there was no correlation between the serum levels IgE antibody and the cellular response to Alternaria. Overall, CRS patients likely exhibit exaggerated humoral and cellular responses, both Th1 and Th2 types, to common airborne fungi, particularly Alternaria. 
The following was performed to determine why <30% of the CRS patients have IgE antibodies to fungi, while about 90% of them exhibit Th2-like PBMC responses. Production of IgE occurs through sequential switching events from μ to γ4 to ε. With chronic antigen exposure, IgG4-switched B memory cells are induced, and these IgG4-switched B memory cells may undergo a secondary switch to IgE. FIG. 3 shows that 60% of the patients with CRS had specific IgG4 antibodies to Alternaria; 20% of patients with seasonal allergic rhinitis (AR) had anti-Alternaria IgG4, and none of the normal individuals did. In contrast, there was no significant difference in the levels of IgG4 antibodies to Aspergillus among the three groups. Thus, patients with CRS may have had an increased exposure to Alternaria, but not to Aspergillus, or they may have had an enhanced “modified Th2 response” to Alternaria, or both.
Epithelial cells are likely participants among the important cellular network of immune and inflammatory responses in the airways. It was found that nasal polyp epithelial cells obtained from CRS patients produce large quantities of IL-8 and GM-CSF. Conditioned media containing GM-CSF markedly enhanced activation of blood eosinophils, suggesting that the products of not only lymphocytes, but also epithelial cells activate airway eosinophils in nasal polyps.
Example 2
Eosinophil Activation and Degranulation in CRS
Asthma and CRS coexist clinically in >50% of patients with CRS. Histologic specimens from refractory CRS patients undergoing endoscopic sinus surgery were examined. Specimens from all CRS patients (22/22) revealed epithelial changes including shedding and basement membrane thickening. Striking eosinophilic inflammation, which did not differ between allergic and non-allergic patients, was also detected in all CRS patients. These findings, coupled with the clinical coexistence of both diseases, suggest that the same pathologic disease process is manifest as CRS in the upper airway and as asthma in the lower airway.
Eosinophilic inflammation in CRS patients was characterized using specific immunological probes. Conventionally, Grocott-methenamine silver (GMS) staining can detect fungi in pathologic specimens; however, this technique can be inconsistent because it lacks sensitivity and specificity. Chitinase is an enzyme, which selectively and specifically binds to chitin in fungal cell walls. Fluorescein-labeled chitinase was used and detected one or more fungal hyphae within the sinus mucus of 54/54 (100%) of consecutive surgical patients with CRS. Fungi were in the airway lumen but not within the airway tissues, suggesting that CRS is not an invasive fungal infection. Because PBMC from CRS patients exhibited vigorous cytokine responses to Alternaria (FIG. 1), a polyclonal antibody to Alternaria was used to investigate the presence of fungi in sinus specimens from CRS patients. Rabbits were immunized with crude Alternaria extract, and as expected, this anti-Alternaria cross-reacted with other fungi, including Aspergillus, Cladosporium, and Penicillium, but not with bacteria. In FIG. 4C, anti-Alternaria antibody clearly visualized fungal hyphae and fungal products in the clusters of inflammatory cells (i.e., eosinophils) within the sinus lumen.
To characterize the extent and location of eosinophilic inflammation, antibody to eosinophil major basic protein (MBP) were used. All tissue specimens from CRS patients exhibited intact eosinophils, but diffuse extracellular MBP deposition, as a marker of eosinophil degranulation, was rare. In contrast, all mucus specimens exhibited abundant diffuse extracellular MBP deposition within or around the clusters of eosinophils (FIG. 4D). Thus, release and deposition of the toxic MBP from eosinophils seem to occur mainly within the airway lumen, but not in airway tissues. This observation and the presence of fungal hyphae and fungal products within the airway lumen suggested that the eosinophilic inflammation of CRS may be part of a normal, but clearly exaggerated, immune response to environmental and airborne fungal organisms. The activation mechanisms of eosinophils in vivo in CRS and asthma have been poorly understood.
The following was performed to determine whether human eosinophils have an innate capacity to respond to environmental fungal organisms. Human eosinophils were incubated with extracts from common environmental airborne fungi. As shown in FIG. 5, Alternaria and Penicillium induced remarkable degranulation (e.g., eosinophil-derived neurotoxin (EDN) release) of eosinophils from normal healthy individuals. No opsonization or sensitization with IgE or IgG antibodies was necessary. Alternaria also strongly induced other activation events in eosinophils from healthy individuals, including increases in intracellular calcium concentration ([Ca2+]i), cell surface expression of CD63 and CD11b, and production of IL-8. Alternaria did not induce neutrophil activation, suggesting cellular specificity of the Alternaria response. The Alternaria-induced eosinophil [Ca2+]i response and degranulation was pertussis toxin (PTX)-sensitive. The eosinophil-stimulating activity in Alternaria extract was heat-labile, inactivated by heat treatment at 56° C. for 30 minutes, and had a molecular mass about 30-50 kDa (FIG. 6). Thus, eosinophils, but not neutrophils, likely possess G protein-dependent cellular activation machinery that directly responds to an Alternaria protein or glycoprotein product(s).
The following was performed to examine whether eosinophils can respond to proteases. Protease-activated receptors (PARs) are a unique class of G protein-coupled seven transmembrane receptors, which are activated by proteolytic cleavage of the amino terminus of the receptor itself (FIG. 7). Four members of this family, including PAR-1,-2, -3, and -4, have been described elsewhere. In the case of PAR-2 (FIG. 7), proteolytic cleavage by a certain protease (e.g., trypsin) exposes its new N-terminus (SLIGKV; SEQ ID NO:38), which binds to the ligand-binding site in the second extracellular loop and results in activation of downstream events. Human eosinophils were found to express PAR-2 constitutively and found to be activated by serine and cysteine proteases, such as trypsin and papain, through this receptor. Eosinophils were also activated by a natural mite allergen protease, Der f 1. PAR-2 may serve as an eosinophil receptor to recognize and respond to proteases from allergens, resulting in active release of pro-inflammatory mediators.
Example 3
Test Hypothesis that Fungi Colonized in Paranasal Sinus and Nasal Cavities are Involved in Persistent Eosinophilic Inflammation in CRS
To examine the clinical significance of fungal colonization in CRS, two clinical trials were performed to examine the efficacy of anti-fungal agents. It was hypothesized that anti-fungal agents will reduce the fungal burden in the upper airways, resulting in less antigenic stimulation of immune cells, less airway inflammation, and improved clinical outcomes. The first aim was to establish the safety and demonstrate potential clinical efficacy of intranasal antifungal drug therapy in patients with CRS in a pilot trial. This prospective, open-label trial used amphotericin B as a medical treatment in 51 randomly selected CRS patients. The antifungal was applied intranasally using 20 mL of a 100 μg/mL solution twice daily for a mean of 11 months (minimum of 3 months). Using amphotericin B, improvement of sinusitis symptoms was observed in 38/51 (75%) of patients. Endoscopically, 18/51 (35%) patients became disease free and an additional 20/51 (39%) improved by at least one stage. No effect was seen in 13/51 (25%) patients. The available CT scans pre- and post-treatment (n=12) demonstrated a significant reduction in the inflammatory mucosal thickening. Thus, this open-label pilot trial demonstrated that direct muco-administration of an antifungal drug is both safe and potentially effective to treat patients with CRS.
Second, to address the efficacy of intranasal antifungal agents more objectively, a randomized, placebo-controlled, double-blind, single center trial was performed to treat 30 randomly selected CRS patients. Patients instilled 20 mL amphotericin B (250 μg/mL) or placebo to each nostril twice daily for 6 months. Twenty-four patients completed the 6 months of treatment. Patients receiving amphotericin B showed reduced mucosal thickening on CT scans compared to placebo (p=0.030). Between group comparisons of the changes in the intranasal mucus levels of EDN, as a marker of eosinophilic inflammation, showed a reduction in the amphotericin B group and an increase in the placebo group (p=0.046). The changes in the endoscopic scores improved in the amphotericin B group compared to placebo (p=0.038). While the group comparison showed statistically significant differences, careful examination of individual patient data in the amphotericin B group showed a spectrum of efficacy. Some patients responded well to the treatment, but others not as well. Thus, fungi may be important in the development of CRS in certain patients.
Example 4
Mechanisms and Molecules Involved in Eosinophil Degranulation in Response to Alternaria 
The majority of previous studies in anti-fungal immune responses used the following models: animal infection in in vivo systems (e.g., Candida albicans, Aspergillus fumigatus), or entire fungal hyphae or conidia (e.g., C. albicans, A. fumigatus), a yeast model (e.g., zymosan), and isolated fungal carbohydrate macromolecules (e.g., β-glucan, mannan) in in vitro systems. These studies pointed to roles for TLRs, in particular TLR2 and TLR4, and to other pattern recognition receptors that immune cells, such as macrophages and neutrophils, use to recognize fungi. Because eosinophils express little TLR2 or TLR4 and the active component(s) in Alternaria extract was a heat-labile molecule(s) with an approximate 30-50 kDa molecular mass (FIG. 6), it was speculated that an Alternaria-derived protease(s) (not carbohydrates), interacting with eosinophil PAR-2, may be involved in the eosinophils' responses to Alternaria. Sine no specific small molecule inhibitor for PAR-2 is available, a desensitization approach was used. As shown in FIG. 8, pre-incubation of eosinophils with the PAR-2 agonistic peptide, SLIGKV (SEQ ID NO:38), significantly inhibited the eosinophils' calcium response to Alternaria extract. Similarly, an N-terminal reversed peptide (LSIGKV; SEQ ID NO:35), which is known to inhibit activation of PAR-2, also inhibited the eosinophils' calcium response to Alternaria; a control scramble peptide (GLIVKS; SEQ ID NO:36) showed no effects. Eosinophil degranulation induced by Alternaria extract was also significantly and specifically inhibited by the LSIGKV (SEQ ID NO:35) peptide (FIG. 8, panel B). In contrast, degranulation induced by PAF or PMA was not affected by the LSIGKV (SEQ ID NP:35) peptide. Thus, PAR-2 is likely involved in the eosinophils' calcium and degranulation responses to Alternaria extract.
A search through a current database of known Alternaria allergens did not reveal any relevant proteases. A fluorescent quenched peptide substrate (Abz-SKGRSLIGK(Dnp)D) (SEQ ID NO:37), which spans the trypsin-cleavage site (between R and S) of PAR-2 was synthesized, and used it in an in vitro assay for PAR-2 cleavage and activation. As shown in FIG. 9, trypsin, as positive control, clearly cleaved this peptide, and a serine protease inhibitor, APMSF, inhibited the activity. Alternaria extract also potently cleaved this peptide, but it was insensitive to APMSF. Alternaria' s activity was abolished when aspartate protease(s) was removed from the extract by pepstatin A agarose (FIG. 9); pepstatin A is a highly specific inhibitor for aspartate protease. Furthermore, eosinophil degranulation induced by Alternaria extract was significantly inhibited by pepstatin A agarose, but not by control agarose or APMSF. Thus, an aspartate protease(s) in Alternaria extract, but not a serine protease(s), may be involved in the activation of eosinophils through PAR-2. this observation was confirmed by using other aspartate protease inhibitors, including alkalo-thermophilic bacillus inhibitor (ATBI), nelfinavir, and ritonavir.
Eosinophils may be the only cell that can recognize Alternaria. In FIG. 10, an airway epithelial cell line, BEAS-2B, produced and released IL-6 when incubated with Alternaria extract for 24 hours. Extracts of Aspergillus, Candida, and Penicillium, did not induce IL-6 production; rather, both Aspergillus and Penicillium inhibited the baseline production of IL-6. BEAS-2B stimulated with Alternaria also produced other pro-inflammatory factors such as IL-8 and GM-CSF. This Alternaria-induced IL-6 production was inhibited by ATBI, nelfinavir, ritonavir or pepstatin A-agarose treatment of Alternaria extract by about 60% to 90%; ritonavir results are shown in FIG. 11. In contrast, TNF-α-induced IL-6 production was not affected by these treatments. Furthermore, a peptide antagonist for PAR-2, LSIGKV (SEQ ID NO:35), partially ( ̃40%) but significantly inhibited Alternaria-induced IL-6 production by BEAS-2B cells. Thus, through its aspartate protease activity, Alternaria may activate airway epithelial cells; this activation is partially mediated by PAR-2.
A series of efforts have been initiated to identify and isolate protease(s) from Alternaria. A preliminary biochemical characterization showed that, at pH 7.5, the Alternaria activity towards eosinophils binds to hydroxyapatite, DEAE Sepharose, and phenyl-Sepharose, but not to a variety of cation exchange or lectin columns. In FIG. 12, DEAE fractionation of an Alternaria extract showed a single peak with strong aspartate protease activity, as detected by a malaria aspartate protease substrate. The peak of aspartate protease activity coincided with the peak of the PAR-2 cleavage activity, and the aspartate protease activity paralleled each fraction's ability to induce eosinophil degranulation.
Partial characterization of Alternaria extract. Three strategies were used to begin characterizing the Alternaria products involved in eosinophil degranulation. First, the Alternaria extract was subjected to membrane filtration. After filtration with a YM100 Centricon® membrane, the filtrate stimulated eosinophil degranulation, but the retentate did not. After filtration with a YM10 Centricon® membrane, the retentate stimulated eosinophils, but the filtrate did not. Thus, the eosinophil-stimulatory activity in the Alternaria extract is likely between 10 and 100 kDa. Second, Alternaria extracts, which had been treated at 56° C. or 100° C. for 30 min, did not induce EDN release (FIG. 25A), but extracts treated at 4° C. or 37° C. for 30 min did induce EDN release, suggesting that it is a heat-labile protein(s) or glycoprotein(s). The activity of a cytokine, IL-5, to induce EDN release was abolished by treatment at 100° C., but not by treatment at 56° C. or lower temperatures. Third, size exclusion chromatography was used (FIG. 25B), and the column fractions tested for their abilities to induce eosinophil degranulation. Although the absorbance profile shows a broad peak from fractions 32 though 37, the most potent eosinophil degranulation activity appeared in fraction 32 with a molecular mass about 60 kDa.
PBMCs obtained from a CRS patient were incubated with fractions 30 or 32, and the level of cytokine production was measured (FIGS. 44 and 45).
Polypeptides (e.g., enzymes) implicated in the activation of eosinophils and promotion of eosinophilic inflammation in a murine model were identified. Proteins in HPLC DEAE fraction #18 and the eluate from pepstatin A agarose were trypsin digested, and the resulting peptides were subjected to nLC-microESI-MS/MS analysis using a Finnigan LTQ system (Thermo Electron Corporation, Waltham, Mass.). Peptide mass fingerprinting with SEQUEST software (distributed by Thermo Electron Corporation, Waltham, Mass.) was used to identify peptides existing in these fractions using the resulting peptide mass data and a database of predicted Alternaria brassicicola proteins derived from expressed sequence tags (ESTs) and the A. brassicola whole genome shotgun sequence information. SEQUEST correlates uninterpreted tandem mass spectra of peptides with amino acid sequences from protein and nucleotide databases. SEQUEST will determine the amino acid sequence of the peptide fragments, and thus the full length protein(s) can be identified. Proteins in the database were predicted using ab initio gene finding and protein prediction software FgeneSH (Softberry, Inc., Mount Kisco, N.Y.). SEQUEST is a registered trademark of the University of Washington. SEQUEST uses algorithms described in U.S. Pat. Nos. 6,017,693 and 5,538,897.
The fungal genes encoding these immunostimulatory proteins were identified using the above described approach. The implicated immunostimulatory proteins identified in these fractions were then further annotated by BlastP analysis against the GenbankNR database and the MEROPS peptidase database. The MEROPS database is an information resource for peptidases (also termed proteases, proteinases and proteolytic enzymes) and the proteins that inhibit them and was developed and web accessible at the Sanger Institute, UK. Furthermore, all candidate proteins were subjected to Interpro analysis. InterPro is a database of protein families, domains and functional sites in which identifiable features found in known proteins can be applied to unknown protein sequences. Interpro analysis is web accessible and a public service available at the European Bioinformatics Institute (EMBL-EBI). The annotated proteins include several proteases belonging to S53 and M38families, several predicted glycolytic enzymes, superoxide dismutase, a ribosomal protein, S-adenosyl-homocysteine lyase, and several others (Table 1).







TABLE 1







Identified polypeptides.








SEQ ID NO:
Functional Annotation











2
Alternaria alternata endoxylanase -



gi|6179886|gb|AF176570.1|AF176570


4
S-adenosyl-L-homocysteine hydrolase


6
glycosyl hydrolase family 61 (Endo-1,4-beta-glucanase IV/



Cellulase IV)


8
glycosyl hydrolase family 31, alpha-glucosidase


10
peptidase family S53 contains acid-acting endopeptidases


12
peptidase family S53 contains acid-acting endopeptidases


14
contains predicted signal peptide for secretion


16
A. alternata 60S acidic ribosomal protein P1 (Allergen Alt



a12) P49148 GI:1350779


18
Superoxide dismutase


20
contains predicted transmembrane regions


22
Peptidase family M38 (beta-aspartyl dipeptidase family)


24
contains predicted signal peptide and transmembrane



domains


26
Unknown


28
Arginase


30
glyosyl hydrolase family 7 Exoglucanase 1 precursor



(Exoglucanase I) (Exocellobiohydrolase I) (1,4-beta-



cellobiohydrolase I) (Beta-glucancellobiohydrolase I)


32
glycosyl hydrolase family 6 - cellobiohydrolase II


34
cellobiose dehydrogenase









The Alternaria brassicicola nucleic acid sequence for each identified Alternaria alternata candidate along with the predicted Alternaria brassicicola amino acid sequence is set forth in FIGS. 27-39.
Example 5
Production of Immunostimulatory Molecules by Live Alternaria 
Spores of A. alternata were obtained, and the effects of the fungus itself on eosinophil activation were examined. Various numbers of spores were suspended in RPMI medium with 10% FCS and incubated in tissue culture wells for 12 hours to induce germination. A fixed number of isolated human eosinophils were added to the wells and incubated for an additional 4 hours. These eosinophils showed strong conjugate formation with the germinating Alternaria fungal spores (FIG. 13A). Furthermore, these eosinophils became activated and released their granule proteins into the supernatants (FIG. 13B). To characterize the growth pattern and production of immunostimulatory molecules by Alternaria further, GFP-transformed A. alternata were used (FIG. 14). Currently, there is no standardized scientific method to quantitate fungal growth. However, these transformed fungi have a technical advantage; fungal growth can be quantitated by measuring the fluorescence intensity using a plate reader or spectrophotometer. Production of so-called “allergens” by fungi can be significantly increased during and after their germination. FIG. 15B shows that the PAR-2-stimulating enzymatic activity(ies) is clearly produced by A. alternata during their germination and hyphal growth. The growth of fungi (FIG. 15A) and production of PAR-2 activating enzymes (FIG. 15B) dramatically increased when fungi were incubated in the presence of airway mucin. Thus, A. alternata likely produces PAR-activating enzyme(s) during their germination and growth, in particular when they germinate on mucosal surfaces, and eosinophils demonstrate a vigorous inflammatory response against these germinating fungi. The model of a spore/eosinophil mixed culture provides a tool to dissect the role of specific Alternaria molecule(s) in the eosinophil's recognition of and response to this fungus.
The polypeptide having the amino acid sequence set forth in SEQ ID NO:2 was recombinantly produced in E. coli and tested for the ability to stimulate eosinophil degranulation. This polypeptide stimulated eosinophil degranulation, as measured by EDN release, in a concentration-dependent manner.
Example 6
In Vivo Mouse Model of Immune Response to Alternaria 
In FIG. 1, PBMC from CRS patients show increased cellular and humoral immune responses to Alternaria. To dissect the role of immune cells in their responses to fungi, a mouse model was developed. Because CRS patients showed an increased immune response to fungi, BALB/c mice were sensitized to Alternaria by intraperitoneal (i.p) injection of Alternaria extract (Greer Laboratories) and subsequently challenged mice intranasally (i.n.) with the same extract. Mice sensitized and challenged with Alternaria exhibited striking airway eosinophilia. Airway eosinophilia was also observed in mice sensitized with PBS (no antigen) and challenged intranasally with Alternaria. Thus, mice might have an innate ability to produce an airway eosinophilic response to certain fungi, which may be similar to the innate Th2 and eosinophilic responses to helminth parasites in mice.
To test this hypothesis, fungal extracts or OVA (as a control) were administered intranasally to naive mice without prior sensitization on days 0, 3, and 6, and airway inflammation was analyzed on day 8. Mice exposed to culture supernatant or cellular extract of Alternaria exhibited significant airway eosinophilia (FIG. 16). Aspergillus induced mild airway eosinophilia. In contrast, Candida induced neutrophilia, but no eosinophilia. This airway eosinophilia in Alternaria-exposed mice is probably not due to accidental prior sensitization of the animals to Alternaria for the following reasons: 1) mice from different animal vendors showed similar eosinophilic responses; 2) no IgG or IgE antibodies to Alternaria were detected in naive mouse serum; and 3) spleen cells from naive mice cultured with Alternaria antigen did not produce IL-4 or IL-5. In addition, the airway eosinophilic response to Alternaria was reproducible among different strains of mice including BALB/c, C57BL/6, C3H/HeJ, C3H/HeSnJ, and WBB6F 1/J-KitW/KitW-v.
Generally, an intact adaptive immune system, especially the Th2 cells, is needed to develop robust airway eosinophilia in mice sensitized and challenged with OVA as described elsewhere. The contributions of the adaptive immune system in the development of airway eosinophilia in naive Alternaria-exposed mice were investigated. In FIG. 17A, there were no differences in the early eosinophilia (i.e., days 0.5 and 5) between wild-type animals and Rag-1−/−mice, suggesting that an innate immune response mediated the early eosinophilic response to Alternaria. In contrast, an adaptive immune system, presumably T cells, was required for further development of eosinophilia at a later time point (i.e., day 8). When Alternaria was administered only once to the mouse airways, IL-5 and IFN-γ, but not IL-4, were detected in BAL fluids by as early as 3 hours and peaked at 12 hours, suggesting that the early cytokine production does not reflect a typical Th2 pattern. Furthermore, the early IL-5 and IFN-γ responses (12 hours after first exposure) were not reduced in Rag-1−/− mice (FIG. 17B). Rather, IL-5 production was enhanced in Rag-1−/− mice, suggesting that innate immune cells are responsible for this early production of IL-5 and IFN-γ and that adaptive immune cells may show inhibitory effects on this innate response.
Various molecules and their receptors can be involved in this Th2-like airway inflammation in naive mice exposed to Alternaria in vivo (FIG. 16). In mice, a small amount of LPS interacting with TLR4 is a factor in promoting Th2 sensitization to protein antigens as described elsewhere. In addition, the cysteine proteinase gene from Leishmania mexicana has been implicated in the upregulation of Th2 immunity and the downregulation of Th1 immunity to this pathogen in mice. The Alternaria preparation contained a minimal amount of LPS (0.4 ng/mg dry weight); thus, each mouse received 0.1 ng of LPS/challenge. Because this amount of LPS is much smaller than that used previously to promote an airway Th2 response to OVA (i.e., 100 ng/challenge, 74), it is very unlikely that LPS contributes to this model. Also, prior treatment of mouse airways with 1 μg LPS significantly inhibited this early IL-5 production (FIG. 18A). This early IL-5 production was significantly enhanced in mice deficient in TLR-4 (C3H/HeJ) compared to control mice (C3H/HeOuJ) (FIG. 18B). Early IL-5 production was also increased in IL-10 deficient mice compared to wild-type controls (19.1±8.0 vs 7.6±2.8, n=4), suggesting a role for IL-10 to down-regulate the early IL-5 response. Altogether, naïve mice likely show innate IL-5 and eosinophilic responses to airway exposure of Alternaria, and this innate response may be down-regulated by activation of TLR-4 or by production of IL-10.
The in vitro experiments suggested a potential role for Alternaria aspartate protease(s) in the activation of eosinophils (FIG. 9) and airway epithelial cells (FIG. 11). Thus, it is hypothesized that the protease(s) similar to those involved in eosinophil degranulation and airway epithelial cell production of IL-8 in vitro may be involved in the development of airway eosinophilia in vivo in mice. To address this question in vivo, Alternaria extract was treated with pepstatin A-agarose to remove aspartate protease(s) or control agarose (FIG. 9) and was administered to naïve mice. Pepstatin A treatment significantly inhibited both early production of IL-5 at 12 hours and airway eosinophilia on day 8 (FIG. 19). FIG. 20 shows that the same peak fraction from the DEAE fractionation (i.e., Fraction #18 of FIG. 12), which contained strong aspartate protease activity and potently induced eosinophil degranulation, also induced marked airway eosinophilia when administered into nave mice.
Example 7
Effects of Glycolytic Enzyme Homologs on Immune Cell Activation In Vitro and In Vivo
The following was performed to characterize the responses of eosinophils (in vitro) and mouse airways (in vivo) to the homologous enzymes from other fungal species, some of which are commercially available. In Table 1, A. alternata xylanase (a glycolytic enzyme) (AAF05698.1) was identified by pepstatin A-affinity chromatography of an Alternaria extract. Thus, the commercially available xylanase isolated from Trichoderma viride was used (Sigma catalog# X3876), and its biological activity examined. Incubation of isolated human eosinophils with Trichoderma xylanase induced EDN release (FIG. 21A). Instillation of Trichoderma xylanase into the airways of naïve mice induced increases in airway levels of IL-5 in vivo (FIG. 21B); IL-5 production was not inhibited in Rag-1−/− mice. Thus, the fungus-derived immunostimulatory activities are not limited to Alternaria, but are likely shared with certain other fungal species. Furthermore, the eosinophil activation assay in vitro and the mouse airway response in vivo, as well as the airway epithelial cell culture provide models to examine the effects of specific immunostimulatory molecules produced by fungi and to dissect the molecular mechanisms involved in this fungus-immune cell interaction.
Example 8
Characterizing the Airway Immune and Inflammatory Responses to Environmental Fungi in Patients with CRS
PBMC are isolated from CRS patients with or without nasal polyps, AR patients and normal individuals, and their proliferative and cytokine responses to fungal antigens are compared. CD4+ cell proliferation is measured by dilution of the carboxyfluorescein diacetate succinimidyl ester (CFSE). Twenty-five cytokines and chemokines in the supernatants are quantitated simultaneously by a Luminex system.
Stimulated PBMC are stained with antibodies for cell surface markers and intracellular cytokines, and are analyzed by FACS to identify cells producing IL-5, IL-13, and IFN-γ. Special attention is focused on whether CD4+ T cells and CD56+ NK cells produce these cytokines.
Subjects. Patients with CRS are studied, using patients with AR and normal individuals as controls. Patients who received systemic glucocorticoids during the past 4 weeks, who are smokers, or who were diagnosed with an immunodeficiency or cystic fibrosis are excluded. The diagnosis of CRS is made based on the fulfillment of all three criteria: i) 2 or more of the following symptoms for more than 12 weeks—anterior or posterior mucopurulent drainage, nasal obstruction, facial pain-pressure-fullness, and decreased sense of smell; ii) anterior rhinoscopy or nasal endoscopy to document signs of inflammation; and iii) sinus CT scan demonstrating isolated or diffuse mucosal thickening. CRS with nasal polyps (CRSwNP) is defined as those CRS patients who now have or who had nasal polyps in the middle meatus, as determined by anterior rhinoscopy or nasal endoscopy. CRS without nasal polyps (CRSsNP) is defined as CRS patients who fulfill all three criteria for CRS as described above, but who do not have demonstrable nasal polyps in the middle meatus both in the past and at present.
Seasonal allergic rhinitis (AR) to ragweed. The clinical diagnosis of AR is established by history, where patients describe the typical seasonal signs of nose itching, sneezing and clear rhinorrhea, and is confirmed with a positive skin test and/or elevated specific serum IgE level for short ragweed antigen. Patients with AR are to have no history or symptoms of CRS or asthma and are to have normal lung function.
Normal Controls. The normal controls are healthy individuals with no history of allergy or asthma and negative skin prick test results to fungi and common aeroallergens.
Demographic Characterization of Patients and Normal Individuals.
Questionnaire: Each patient is asked to complete the questionnaire regarding the history of his or her sinus symptoms, aspirin sensitivity, sinus operations, and recently used and current medications. Patients are also asked regarding their history of asthma and AR, smoking habits, and use of allergen immunotherapy.
Skin tests: Skin prick tests are performed with a battery of 18 commercially available fungal extracts and 8 common aeroallergen extracts, including Dermatophagoides pteronyssinus, D. farinae, cockroach, short ragweed pollen, mixed grass pollen, mixed tree pollen, cat epithelium, and dog dander.
Total and specific IgE: Total serum IgE is measured by two-site ELISA. Allergen-specific IgE antibody levels are determined by RAST using 8 fungal allergens and 8 common aeroallergens.
Assessment of CRS: To assess the extent of the CRS, symptoms and quality of life (QOL) are scored according to the Symptom Score (0-10 visual analogue scale of 6 sinusitis-related symptoms and Gliklich and Metson QOL Score. Sinus CT scans are scored according to CT scoring systems described elsewhere (e.g., the Lund-Mackay staging system and the digital analysis of scanned images).
Sample Size
Given the conservative assumption that IL-5 is produced by PBMC from >83% of the patients with CRS and is produced in 36% of the normal controls, we are to have 80% power with a probability of a type 1 error rate of 0.05 with 20 patients in each group. Therefore, 20 CRSwNP, 20 CRSsNP, 20 AR, and 20 normal controls are recruited.
Cell Proliferation and Cytokine Production by PBMC
PBMC are cultured for 24 hours or 96 hours (for cytokine assay) or for 168 hours (for proliferation assay) with or without 25 μg/mL extracts of Alternaria, Aspergillus, Cladosporium, and short ragweed (Greer Laboratories), 2 μg/mL tetanus toxoid, or 5 μg/mL Con-A. The optimal concentrations of antigens and duration of culture have been determined elsewhere. The concentrations of a panel of 25 cytokines and chemokines (IL-10, IL-Ra, IL-2, IL-2R, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12p40/p70, IL-13, IL-15, IL-17, TNF-α, IFN-α, IFN-γ, GM-CSF, MIP-1α, MIP-β, IP-10, MIG, eotaxin, RANTES, MCP-1) are measured by a Luminex 100 IS system (Upstate) and 25-plex antibody bead kit (BioSource International). The differences in the amounts of individual cytokine/chemokines among the groups are analyzed by Mann-Whitney U test. The pattern and cluster of cytokine production in each subject group are analyzed by Spotfire DecisionSite software (Somerville). For the CD4+ T cell proliferation assay, PBMC are labeled with 5 mM CFSE for 10 min before addition of antigens. After culture, PBMC are stained with PE-conjugated anti-CD4 and analyzed by FACS; CFSE dye is diluted in the proliferating population of the CD4+ T cells, and the numbers of cells that have proliferated per 1,000 CD4+ T cells are determined.
A pilot study showed that when PBMCs from a CRS patient were stimulated with Alternaria extract, a population of CFSElow CD4+ T cells emerged by day 4, and represented 66.9% of total CD4+ T cells on day 7 (FIG. 22); no changes were observed in PBMCs cultured in medium alone. A side-by-side comparison of a normal individual and a CRS patient in a separate experiment (compared on day 7) showed that a higher proportion of CD4+ cells were CFSElow in the CRS patient than those in the normal individual (43.2% vs. 4.8%) (FIG. 23). In contrast, many CD4+ cells were CFSElow in both the CRS patient and normal individual when they were stimulated with tetanus toxoid (43.2% vs. 47.9%).
FACS Analyses of Cytokine Producing Cells
The PBMCs producing IL-5, IL-13 and IFN-γ are analyzed by FACS. IL-5 is likely produced by CD4+ T cells, CD8+ T cells, and CD56+ NK cells. Thus, FITC-conjugated antibodies are used for these cell surface markers and PE-conjugated antibodies to IL-4, IL-5, IL-13, and IFN-γ to identify cytokine-producing cells. After stimulation with antigens, PBMC are re-stimulated with ionomycin plus PMA in the presence of brefeldin A. Cell surface antigens are stained with FITC-conjugated anti-CD3, CD4, CD8 or CD56 (Becton Dickinson). After washing, cells are fixed and permeabilized simultaneously by Cytofix/Cytoperm solution (Pharmingen), and stained with PE-conjugated anti-cytokine or control mouse Ig.
In Vitro Organ Culture of Sinus Tissue Specimens from CRS Patients Produce Distinctive Pro-Inflammatory Cytokines
Large quantities of sinus tissue specimens are obtained from CRS patients during endoscopic sinus surgery. Specimens from the ethmoid sinuses of normal individuals (non-allergic, no asthma, no CRS) undergoing septoplasty procedures are used as a negative control. Other disease control specimens are obtained from patients with AR, who undergo septoplasty.
To examine the immunological responses by sinus mucosa to fungi, an organ culture system is used, rather than isolated mononuclear cells. Organ culture can allow for the study the mucosal immune responses and tolerance that are likely be mediated by a complex network of epithelial cells, antigen presenting cells, lymphocytes and potentially other mucosal resident cells, and each cellular component may play an role. Tissues are minced into 5-mm pieces, and then cultured with fungal extracts (e.g., Alternaria, Cladosporium, Aspergillus), Con-A or tetanus toxoid for 24 hours or 96 hours. First, the concentrations of 25 cytokines and chemokines, including IL-10, in the supernatants are analyzed by a Luminex system. The concentration of TGF-β is measured by ELISA. Second, once several cytokines (e.g. IL-5) are verified to be produced at elevated levels during the CRS organ culture, the cell types that produce these cytokines are identified. After antigenic stimulation for 96 hours, the tissue specimens are treated with a cocktail of highly pure collagenases (Blendzyme 3, Roche). In preliminary studies, the yield was 12 to 70×106 cells/specimen, and the viability was 65 ̃95%. The single cell suspension are recovered after passing through a nylon mesh with 100 μm pore size. The cell types (CD4+, CD8+, CD56+) producing cytokines (IL-5, IL-13, IFN-γ) are analyzed by intracellular cytokine staining and FACS analysis.
Subjects. Patients with CRS, who are undergoing endoscopic sinus surgery, are studied, using normal individuals as controls. The criteria for CRS patients and normal individuals are the same as described above. The patients with CRSwNP are enrolled because the patients with CRSwNP tend to have more expensive disease than those with CRSsNP. For this study, patients who are not using nasal or inhaled steroids for 4 weeks before the surgery are specifically selected. The goal is to detect at least 1.5 SD differences in means between two groups as significant with 80% power with a probability of a type I error rate of 0.05. Therefore, tissues from 7 CRS patients and 7 normal controls for each of the 3 experiments are obtained. Because the sample size is not based on preliminary data, a second power calculation is performed once 7 subjects in each group have completed the study. If there is a risk for type II error, the sample size is increased.
Analyses of the functions of CD4+CD25+regulatory T cells. CD4+ T cells are isolated from single cell suspensions of sinus tissue fragments by negative immunomagnetic selection, followed by positive selection for CD25+ cells by magnetic cell sorting (StemCell Technologies). Isolated CD4+CD25− cells are incubated with serial dilutions of isolated CD4+CD25+ cells in the presence of autologous irradiated mononuclear cells for 96 hours and in the presence or absence of fungal extract (e.g. Alternaria). The production of cytokines (IL-5, IL-13, IFN-γ) in the supernatant is measured by ELISA, and the proliferation of CFSE-labeled CD4+CD25− cells is examined. In some experiments, antibodies to IL-10 and IL-10Rα-chain and a soluble TGF-βR11-Fc chimeric protein (all from R&D systems) are included in the culture to examine the role of IL-10 and TGF-β to dampen the cytokine and proliferative responses.
In Vivo Intranasal Challenge with Alternaria in CRS Patients
Subjects. CRS patients without demonstrable IgE antibodies to Alternaria are studied using CRS patients with IgE antibodies to Alternaria and normal individuals as controls. The criteria for CRS patients and normal individuals are the same as described above, and patients who are not on nasal or inhaled steroids for 4 weeks before the study are selected. The presence or absence of IgE antibodies to Alternaria is examined by both skin tests and IgE RAST. About 30% of patients with CRS have demonstrable IgE antibodies to Alternaria. Asthma is not required for inclusion; if CRS patients do have a history of asthma, they may be included in the study if their asthma is mild as defined by all of the following parameters; (1) a baseline FEV1 of more than or equal to 80% of predicted, (2) no need for any maintenance therapy for asthma with inhaled steroids, long-acting bronchodilators, or systemic steroids, (3) no need for treatment with theophylline or leukotriene inhibitors on daily basis, and (4) no history of emergency room visits or hospitalization because of asthma in the last ten years. Based on preliminary data, for a dichotomous endpoint (e.g., detectable level of IL-5), a sample-size of n=10 per group provides statistical power of 84% to detect a difference between groups. Statistical power is increased when data are analyzed as continuous variables. 10 subjects are recruited for each of the 3 groups.
Intranasal challenge and sample collection. Intranasal challenge with Alternaria is performed as described elsewhere. Briefly, before nasal challenge, CRS patients with IgE antibodies to Alternaria undergo endpoint titration to establish the optimal dose for starting their intranasal challenge. Endpoint titration is performed by a skin prick test with escalating or decreasing dosages of Alternaria extract (ALK Abello, product#ALTE21P41L) starting at 18 PNU/mL. If there is no reaction (wheal and flare) at 18 PNU/mL, the next higher concentration is tested until a wheal and flare response occurs. If there is a reaction at 18 PNU/mL, the next lowest concentration is tested until no wheal and flare develops. The starting dosage for the nasal challenge for CRS patients with anti-Alternaria IgE antibody is the highest concentration that causes no wheal and flare response. CRS patients who do not have IgE antibody to Alternaria (i.e., both skin test negative and RAST negative) or normal individuals are started at 18 PNU/mL. For nasal challenge, the Alternaria extract (ALK Abello, product# ALTE21P41L) is administered by a metered nasal spray pump (Callipot) that delivers 0.1 mL of extract per nostril. If no reaction occurs, it is proceed with a 3-fold higher concentration (e.g. 54 PNU/mL) up to 40,000 PNU/mL. The interval between each challenge is 15 minutes. The cumulative dose of Alternaria received by each subject is <12,000 PNU. The nasal lavage specimens are collected before and 24 hours after the challenge. Three milliliters of saline are introduced into each nostril, and secretions are collected into a container. The specimens are processed immediately for cell count and differentials, and supernatants are stored for cytokine and eosinophil granule protein assays. The peak expiratory flow rate (PEFR) is measured at baseline and after each dose. A pulmonary function test (flow volume loop) is performed with measurement of forced expiratory volume 1 (FEV 1) before, immediately after, and 24 hours after the escalating intranasal challenge protocol. There is a stopping rule in place. At baseline and after each challenge, all subjects are asked for their symptoms. These symptoms (nasal blockage, nasal discharge, number of sneezes, nasal itching, difficulty breathing, cough or wheezing) are recorded on a four-point scale (0 to 3). The total symptom score is calculated as the sum of the individual symptom scores. The nasal challenge is stopped at the dosage of Alternaria extract that produces either: i) 1 mL of nasal secretions or more than 5 sneezes within 15 minutes, ii) a symptom score of 3 for two or more of the symptoms mentioned above, or iii) difficulty breathing with a decrease of the PEFR or FEV1 by 15% or more.
Samples and data obtained. Nasal lavage fluids are collected from study subjects before and 24 hours after intranasal challenge, and the total leukocyte counts and differentials are determined. The concentrations of cytokines/chemokines, including IL-4, IL-5, IL-13, IFN-γ, TNF-α, IL-10, and eotaxin, in nasal lavage fluids are quantitated by specific ELISA (Endogen). The sensitivity of these ELISA is generally <0.7 pg/mL. Eosinophil granule MBP and EDN are analyzed by RIA to monitor eosinophilic inflammation.
Example 9
Identifying Alternaria Products that Trigger Profound Th2-Like Inflammation In Vitro in Human Airway Cells and In Vivo in Mouse Airways
The following describes methods and materials for producing recombinant candidate A. alternata immunomodulatory proteins and characterizing their immune responses in vitro and in vivo. Purified recombinant forms of the Alternaria protein candidates are produced. These proteins are used to perform various in vitro and in vivo immunological assays and to elucidate the role of these proteins individually and in concert in CRS pathogenesis.
Candidate proteins identified is Table 1 are expressed recombinantly. Constructs are made to consist of the following: 1) the trpC and ToxA promoter, 2) a PCR amplified cDNA or genomic region from A. alternata corresponding to the full-length candidate genes of the enzymes, and 3) a PCR generated histidine tag (e.g., 6×-His) engineered just prior to the stop codon (C-terminus) to aid in purification. These constructs are then introduced into A. alternata protoplasts using standard polyethylene glycol (PEG)-mediated fungal transformation approaches. Individual mutants are grown in potato dextrose broth with hygromycin, and expression levels of the introduced genes are verified using RT-PCR or northern blotting, and SDS-PAGE. Individual mutants exhibiting high-level expression of the protein of interest are grown in larger amounts, culture filtrates are purified, and Immobilized Metal Affinity Chromatography (IMAC) for the histidine-tagged protein purification involves using a HPLC system and Ni Sepharose chromatography.
Alternatively, routine recombinant protein expression systems with organisms like E. coli and Pichia pastoris are used. For example, E. coli was used to produce one of eight candidates, A. alternata xylanase (AAF05698.1) (FIG. 26).
In Vitro and In Vivo Assays for Activity of Recombinant Alternaria Proteins.
Eosinophil [Ca2+]i response and degranulation. For degranulation, isolated eosinophils are incubated with different concentrations of recombinant proteins (10 ng/mL−1 mg/mL) for 3 hours, and EDN released into supernatants is measured by RIA to indicate degranulation. Changes in [Ca2+]i are measured using FACS analysis and eosinophils loaded with a calcium indicator, indo-1. The involvement of PAR-2 and proteolytic/glycolytic enzymes is verified by a PAR-2 peptide antagonist, LSIGKV (SEQ ID NO:35), and enzyme inhibitors, such as pepstatin A-agarose, ATBI, ritonavir, and allosamidine. The active cleavage of PAR-2 is verified by fluorescent quenched peptide substrate [Abz-SKGRSLIGK(Dnp)D] (SEQ ID NO:37) and by analysis of stimulated eosinophils by FACS and immunoblot using anti-PAR-2 antibody (which recognizes the N-terminus of PAR-2). Although unlikely, the involvement of TLR2 or TLR4/CD14 is examined using blocking antibodies to these molecules (eBioscience).
Epithelial cell production of cytokines. The airway epithelial cell line, BEAS-2B, is stimulated with different concentrations of recombinant proteins for 24 hours, similarly to Alternaria crude extract experiments in FIGS. 10 and 11. Cytokines, including IL-8 and IL-6, released into supernatant are measured by ELISA. The epithelial cells' PAR-2 is analyzed similarly to the analysis for eosinophils.
Cytokine responses and airway eosinophilia in mouse airways in vivo. Naïve mice are exposed intranasally to recombinant proteins (1 μg-100 μg/challenge) on days 0, 3, and 6 (see FIGS. 16 and 20). At 12 hours after the first challenge, on day 5, or day 8, the trachea is cannulated, and the lung is lavaged with 0.5 mL of HBSS. Total numbers of cells and differentials in BAL fluids are determined. Supernatants are collected, and the concentrations of cytokines (IL-5, IL-4, IL-13, IFN-γ) are measured by ELISA. Tissue samples of the lungs are examined histologically. Blood is collected by cardiac puncture on day 8 to quantitate IgE and IgG antibodies to recombinant proteins.
Cellular and humoral immune responses by CRS patients. PBMC are isolated from normal individuals and CRS patients by using the same criteria as described above. PBMC are incubated with serial dilutions of recombinant proteins for 24 hours (for IL-4), for 96 hours (for IL-5, IL-13, and IFN-γ), or for 168 hours for CFSE-based CD4+ T cell proliferation assay as described above. Serum concentrations of IgE, IgG, and IgG4 antibody to recombinant proteins are measured by immunoassay and western blot.
Development of A. alternata Knockout (KO) Mutants for Specific Immunostimulatory Proteins and Analyses of Immune Responses In Vitro and In Vivo with Whole Fungi and Fungal Products.
KO mutants are generated for each candidate immunostimulatory protein. First, the secreted products from KO A. alternata are used to deduce whether the absence of a specific protein significantly affects the activation of immune cells in vitro and in vivo. Second, similar experiments with whole fungus (i.e., fungal spores and fungal hyphae) are compare the immune responses triggered by KO to the wild type.
Fungal mutant generation. The LME approach is used as described above to disrupt the target genes. The LME constructs consistently produce stable transformants for diverse categories of genes. Typically, when using the LME constructs, 100% of the transformants are targeted gene disruption mutants compared to inconsistent transformation and usually less than 10% targeted gene disruption with circular plasmid disruption constructs. All mutants are subjected to molecular characterization to confirm that gene(s) are disrupted.
In vitro and in vivo assays. Wild-type and KO Alternaria are cultured in liquid medium. Proteins released from these fungi into supernatants are analyzed for their immunostimulatory activities in vitro with eosinophils and BEAS-2B cells and in vivo mouse airways as described above. Spores are collected from wild-type and KO Alternaria. These spores are cultured in vitro in HBSS medium with airway mucin and allowed to germinate. Eosinophils are added, and their responses to wild-type and KO Alternaria are examined as in FIG. 13.
Example 10
Inhibiting Alternaria-Induced Eosinophilic Degranulation
To monitor eosinophil function in response to extracts from Alternaria, degranulation of human eosinophils was measured by quantitating released eosinophil-derived neurotoxin (EDN) and/or MBP. In brief, freshly isolated eosinophils were suspended in HBSS with 25 mM HEPES and 0.01% gelatin at 5×105 cells/mL. Eosinophils and stimuli were incubated in 96-well tissue culture plates for 3 hours at 37° C. and 5% CO2. Cell-free supernatants were stored at −20° C. A specific RIA quantitated eosinophil degranulation by measuring the concentration of EDN in the supernatants. The following inhibited Alternaria-induced eosinophilic degranulation: CV6209 (PAF receptor antagonist), heparin, EDTA, EGTA, pepstatin agarose, PAR2-inhibitory peptide, Jasplakinlide (actin inhibitor), and Lanthunum (Ca channel inhibitor). The following did not inhibit eosinophilic degranulation: Chymostatin, Chloroquine, Phosphoramidon, APSF, Calpastatin, Antipain, Bestatin, Leupeptin, Pefabloc SC, Aprotinin, Cytochalasin B, Colchitin, E64, Calpain inhibitor, SB203580 (p38 MAPK inhibitor), Genistein, Wortmannin, Ro-31-8220, Rottelrin, GF109203X, PD98059 (ERK inhibitor), Cyclosporin A, FK 506, W-7, and TLCK.
OTHER EMBODIMENTS
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

## Claims

1. A substantially purified polypeptide comprising the amino acid sequence set forth in SEQ ID NO:8.