J. Exp. Med.,
Volume 189, Number 9, May 3, 1999 1507-1512
Humoral and Cell-mediated Autoimmune Reactions to
Human Acidic Ribosomal P2 Protein in Individuals
Sensitized to Aspergillus fumigatus P2 Protein
By
Christina
Mayer,*
Ulrich
Appenzeller,*
Heike
Seelbach,
Gernot
Achatz,§
Hannes
Oberkofler,§
Michael
Breitenbach,§
Kurt
Blaser,*
and
Reto
Crameri*
From the * Swiss Institute of Allergy and Asthma Research (SIAF), CH-7270 Davos, Switzerland; Hochgebirgsklinik, CH-7265 Davos-Wolfgang, Switzerland; and the § Institut für Genetik und
Allgemeine Biologie, Universität Salzburg, A-5020 Salzburg, Austria
 |
Abstract |
A panel of cDNAs encoding allergenic proteins was isolated from an Aspergillus fumigatus
cDNA library displayed on the surface of filamentous phage. Solid phase-immobilized serum
immunoglobulin E (IgE) from A. fumigatus-allergic individuals was used to enrich phage displaying IgE-binding molecules. One of the cDNAs encoded a 11.1-kD protein that was identified as acidic ribosomal phosphoprotein type 2 (P2 protein). The allergen, formally termed
rAsp f 8, shares >62% sequence identity and >84% sequence homology to corresponding eukaryotic P2 proteins, including human P2 protein. The sequences encoding human and fungal
P2 protein were subcloned, expressed in Escherichia coli as His6-tagged fusion proteins, and purified by Ni2+-chelate affinity chromatography. Both recombinant P2 proteins were recognized
by IgE antibodies from allergic individuals sensitized to the A. fumigatus P2 protein and elicited
strong type 1-specific skin reactions in these individuals. Moreover, human and fungal P2 proteins induced proliferative responses in peripheral blood mononuclear cells of A. fumigatus-
allergic subjects sensitized to the fungal P2 protein. These data provide strong evidence for in
vitro and in vivo humoral and cell-mediated autoreactivity to human P2 protein in patients suffering from chronic A. fumigatus allergy.
Key words:
phage display;
cDNA libraries;
IgE;
allergens;
autoimmunity
| |
Introduction |
Aspergillus fumigatus, a ubiquitous mold (1), is considered
an opportunistic pathogen responsible for a vast variety
of pulmonary complications in humans and animals. The
spectrum of diseases associated with the fungus ranges from
mild forms, like saprophytic colonization of the lung and
allergy, to life-threatening diseases, such as invasive systemic aspergillosis or allergic bronchopulmonary aspergillosis (ABPA) (2). These different clinical presentations indicate that not only the virulence of the fungus itself, but also
other underlying conditions, including impaired immune
status, play a role in the development of opportunistic mycoses (3). Cloning and sequencing of allergen-encoding
cDNAs from A. fumigatus (4) have permitted characterization of the biochemical function of some allergens by sequence comparison. The 18-kD protein Asp f 1, a member
of the ribotoxin family (4, 7), represents a major allergen of
the fungus (7, 8) and was found in the urine of patients
with invasive aspergillosis (9). Serologic studies with rAsp f 1 and other A. fumigatus allergens (10) clearly demonstrated
the existence of disease-specific allergens able to elicit IgE
responses exclusively in patients suffering from ABPA (10-
12). Although the pathophysiologic mechanisms leading to
Aspergillus-related pulmonary complications remain largely
unknown, the availability of recombinant allergens from the fungus substantially contributed to improved diagnosis
of the diseases (11). At the molecular level, the cloned
allergens can be subdivided into two categories: secreted
and cytoplasmatic proteins (10). Interestingly, at least one
of the ABPA-specific allergens, manganese-dependent superoxide dismutase (MnSOD), a phylogenetically highly conserved protein (15), also shows cross-reactivity with human
MnSOD (16). Phylogenetically conserved proteins are often involved in fungal allergy (17) and also have the potential to be involved in humoral and cell-mediated autoimmune reactions (15, 18). We have cloned a large panel of
cDNAs encoding allergenic A. fumigatus proteins using
phage surface display technology (5, 6, 19). Here, we describe the sequence and properties of one of these allergens
identified as an A. fumigatus acidic ribosomal phosphoprotein type 2 (P2 protein) by sequence homology. The acidic ribosomal phosphoproteins P0 (38 kD), P1 (13 kD), and P2
(13 kD), contained in the 60 S ribosomal subunit, are
highly conserved among eukaryotes and are required for
the functional activity of the ribosome (20). These proteins
have been reported as antigens capable of inducing IgG antibody responses in systemic lupus erythematosus, the prototypic systemic autoimmune disease (21, 22). Additionally, the P2 proteins from Alternaria alternata and Cladosporium
herbarum have been reported to be minor allergens of these
molds (17). The A. fumigatus P2 protein is recognized by
IgE antibodies of individuals sensitized to the mold and
shows significant humoral cross-reactivity to human P2
protein. Both human and A. fumigatus P2 proteins induce
strong type 1 skin reactions and proliferative responses in
PBMCs of individuals sensitized to the fungal P2 protein.
| |
Materials and Methods |
Construction and Screening of an A. fumigatus cDNA Library Displayed on Phage Surface.
Phage displaying IgE-binding proteins
were enriched from an A. fumigatus cDNA library constructed in
phagemid pJuFo (19) and displayed on the surface of filamentous
phage M13 as described (5, 6). Serum IgE from A. fumigatus-sensitized individuals was captured in microtiter plates coated with anti-
human mAb TN 142 and used as ligand for selective enrichment
of allergen-displaying phage (6). Sera used for screening were selected according to case history, skin test reactivity to commercial
A. fumigatus extracts, and specific IgE to A. fumigatus determined
by radioallergosorbent test (RAST) (8). The screening procedure
yielded a wide variety of phage able to bind specifically to human
serum IgE and thus displaying allergenic molecules (5, 6).
Identification of a Clone Encoding A. fumigatus P2 Protein.
Inserts
carried by phage displaying IgE-binding proteins differing in
length were sequenced as described (23) on an ABI prism 373A
sequencer using the D-rhodamine terminator cycle sequencing kit
(Perkin-Elmer) according to the manufacturer's instructions. Both
DNA strands were sequenced using vector-derived primers. Homology searches and sequence comparisons were performed with BLAST and the Genetics Computer Group program FASTA (24).
One clone revealed strong homology with nucleotide sequences
encoding acidic ribosomal phosphoprotein type 2 (see Fig. 1).
View larger version (42K):
[in this window]
[in a new window]
|
Fig. 1.
Alignment of the deduced amino acid sequences A. fumigatus
(A. fum), A. alternata (A. alt), C. herbarum (C. her), and human (H. sap) P2
protein sequences. Identical amino acid residues in at least three of the sequences are noted by shading, and gaps are indicated by dots. Sequence
identity between A. fumigatus, human, C. herbarum, and A. alternata P2
proteins are 62.16, 72.07, and 71.17%, respectively. Numbers after the sequences indicate the residue numbers, without gaps, starting at the NH2-terminal methionine residues.
|
|
Cloning of the Human P2 Protein; Production and Characterization
of Recombinant Proteins.
The cDNA encoding human P2 protein
was amplified by PCR from a commercial human lung cDNA library (Stratagene) using the following primers: 5'-primer, 5'-GCGGATCCATGCGCTACGTCGCCTCCTACC-3'; 3'-primer, 5'-GCTCTAGATTAATCAAAAAGGCCAAATCCC-3'. The
complete cDNA coding for the putative A. fumigatus P2 protein
was amplified from the original clone by PCR using the following primers: 5'-primer, 5'-GCGGATCCATGAAGTACCTCGCAGCTTTCC-3'; 3'-primer, 5'-CCCGGACTTTAAGTCGAAGAGACCGAAGCCC-3'. PCR cycling conditions were 30 cycles of 95°C for 60 s, 57°C for 60 s, and 72°C for 60 s, followed
by a terminal extension cycle at 72°C for 10 min. The amplification products were purified using a commercial kit (QIAquick;
QIAGEN, Inc.), digested with BamHI and XbaI or BamHI and
HindIII, respectively, and ligated to appropriately restricted
pHis6-DHFR (dihydrofolate reductase) vector (4). Ligation mixtures were transformed into Escherichia coli strain M 15; transformants were grown in liquid to verify the nucleotide sequence
(23) and used to produce hexahistidine-tagged recombinant proteins (4, 6). After a single-step purification over Ni2+-chelate affinity columns (6), molecular size and purity of the recombinant
proteins were analyzed by polyacrylamide gradient gels (4.5-20%)
and 1-mg samples lyophilized for long term storage (14).
ELISA and IgE Immunoblots.
The specific binding properties
of serum IgE from A. fumigatus-sensitized individuals to recombinant fungal and human P2 protein were analyzed by an allergen-specific ELISA (8). Absorbency was measured at 405 nm with a
Molecular Devices reader and optical densities converted into
arbitrary ELISA units (EU/ml) calibrated against an in-house serum
pool arbitrarily defined as 100 EU/ml (8, 11, 12). Values below
1 EU/ml were set as 1 EU/ml for graphic display and nonparametric statistical analysis (Mann-Whitney U test). For IgE immunoblots, proteins were separated on SDS-polyacrylamide gradient
gels (4-20%), transferred to nitrocellulose, incubated with patient
sera diluted 1:10, and processed as described (16, 25).
Proliferative Responses of PBMCs.
PBMCs were isolated from
heparinized peripheral venous blood by Ficoll density gradient
centrifugation, washed three times, and resuspended in RPMI 1640 supplemented with 1 mM sodium pyruvate, 2 mM L-glutamine, 50 µM 2-ME, 1% MEM nonessential amino acids and vitamins, 100 µg/ml streptomycin, 100 U/ml penicillin (all from Life Technologies), and 10% heat-inactivated FCS (Sera-Lab). Samples of 5 × 105 cells/100 µl were stimulated with different concentrations of recombinant A. fumigatus or human P2 protein or with A. fumigatus extract in triplicate for 7 d. Proliferation was measured as incorporation of tritiated thymidine (DuPont-NEN) during the final 16 h
of culture. A stimulation index >3 was considered positive.
Skin Tests with Recombinant P2 Protein.
Recombinant proteins
were dissolved in 0.9% saline at concentrations ranging from 10
5
to 1 µg/ml. Intradermal skin tests were performed on the patient's back by injecting 100 µl test solution containing 1 ng recombinant protein. If no positive reaction could be observed after 15 min, testing was continued by injecting a 10-fold higher amount
of protein. The test was stopped and considered positive when
the wheal surface reached at least half of the histamine wheal size (8, 16) or after injection of 1 µg protein. 0.01% histamine dihydrochloride and 0.9% saline were used as positive and negative controls, respectively (8). An ethical approval for skin testing human subjects with recombinant proteins was obtained from the local ethics committee (Davos, Switzerland). A full explanation of
the procedure was given to all participants, and their written consent was obtained before testing.
| |
Results and Discussion |
Isolation of cDNA Clones and Sequence Analysis.
The cloning technology based on phage surface display of expression
products from cDNA libraries (5, 6, 19) is particularly suitable for selective isolation of cDNAs encoding IgE-binding proteins from complex allergenic systems (26). Starting
from a phage surface-displayed cDNA library generated
from mRNA of A. fumigatus (6), we selectively enriched
phage able to bind human serum IgE from individuals sensitized to the fungus. cDNAs isolated from single phagemids after four rounds of affinity selection, carrying inserts
of different lengths, were sequenced and shown to code for
different allergenic proteins (10). Among these, a clone containing an open reading frame spanning 333 bp (sequence data available from EMBL under accession no. AJ224333)
revealed strong homology with sequences encoding eukaryotic type 2 acidic ribosomal phosphoproteins. The deduced
amino acid sequence of this cDNA clone was homologous
to P2 proteins, showing a high sequence identity to the human (62%), C. herbarum (71%), and A. alternata (72%) P2 proteins (Fig. 1). Acidic ribosomal phosphorylated (P) proteins
have been isolated and characterized from a variety of eukaryotic cells and share significant sequence identity and similarity (20).
Production and Characterization of Recombinant A. fumigatus
and Human P2 Proteins.
Both complete cDNAs encoding
the putative A. fumigatus P2 protein and the human P2 protein were amplified by PCR, subcloned into the high level
expression plasmid pHis6-DHFR, verified by sequencing,
and used to produce hexahistidine-tagged P2 proteins (see
Materials and Methods). The constructs yielded, after single-step purification by Ni2+-chelate affinity chromatography,
33 and 28 mg/liter E. coli culture of virtually pure A. fumigatus and human P2 protein, respectively. Purity was analyzed by reducing, denaturing SDS-PAGE, and Coomassie blue staining. In each preparation, only one band with estimated molecular mass in agreement with the calculated
values of ~12.6 kD for the hexahistidine-tagged A. fumigatus and human P2 protein was visible (data not shown).
Allergenic Properties of the P2 Proteins.
As expected from
the selection procedure devoted to isolate allergens from a
phage surface display library, the A. fumigatus P2 protein
was able to bind IgE present in the serum used for screening. However, both A. fumigatus and human P2 proteins
were identified as allergens by ELISA with sera from individuals allergic to A. fumigatus (Fig. 2) and by IgE immunoblots (data not shown). Inhibition experiments showed that
increasing amounts of human recombinant P2 protein
added to the fluid phase were able to inhibit the binding of
serum IgE from patients sensitized to A. fumigatus P2 protein (Fig. 3), demonstrating that the proteins share common IgE-binding epitopes. 14/92 patients studied that
were suffering from ABPA and 6/75 patients that were
allergic to A. fumigatus and suffering from severe atopic
dermatitis studied showed relevant levels of serum IgE antibodies to the A. fumigatus P2 protein, resulting in an incidence of sensitization in the range of 15 and 8%, respectively. Sensitization to P2 protein was not observed in
A. fumigatus-sensitized individuals with mild forms of
atopic dermatitis or in patients without ABPA.
View larger version (18K):
[in this window]
[in a new window]
|
Fig. 2.
Competitive inhibition of IgE binding to solid phase-coated
A. fumigatus recombinant P2 protein. Serum from A. fumigatus-sensitized
patients was preincubated with increasing amounts of recombinant A. fumigatus ( ), human P2 protein ( ), or Asp f 1 as negative control ( ). Preincubated serum samples were transferred to wells coated with A. fumigatus P2
protein, and bound IgE was analyzed by antigen-specific ELISA (8, 16).
|
|
View larger version (14K):
[in this window]
[in a new window]
|
Fig. 3.
20 sera from individuals sensitized to ( ) and 20 sera of individuals lacking IgE to the A. fumigatus P2 protein () were analyzed for
their content of serum IgE able to recognize human P2 protein (A). (B)
IgE binding of the same sera to A. fumigatus P2 protein is shown. Units are
expressed as arbitrary ELISA units (EU/ml) calculated from the absorbency of an in-house reference serum pool, which was set as 100 EU/ml
(8). Bars, mean values.
|
|
Proliferative Responses of PBMC from Individuals Sensitized
to A. fumigatus P2 Protein.
T cell help is required for the
production of allergen-specific IgE (27). Therefore, we
measured the proliferative responses of mononuclear cells
from six individuals sensitized to the A. fumigatus P2 protein
to fungal extract, recombinant A. fumigatus, and human P2
protein. The mean proliferative responses to optimal concentrations of extract, fungal, and human P2 protein were
39,500; 20,092; and 11,790 cpm, respectively (mean SI
11.9, 6.5, and 5.7). The mean proliferative responses of
15,608; 1,601; and 1,273 cpm (mean SI 12.6, 0.8, and 1) to
fungal extract, A. fumigatus, and human P2 protein, respectively, obtained for four individuals sensitized to A. fumigatus lacking IgE antibodies against the fungal P2 protein indicate that the recombinant antigens did not induce
nonspecific effects. Three control individuals did not respond to any of the antigen preparations (mean SI < 1).
Comparison of the values by the rank sum test indicates
highly significant differences (P < 0.01). The proliferative
responses induced by human P2 protein in individuals sensitized to the A. fumigatus P2 protein indicate a pathogenesis
related to autoreactive T cells. Intense local inflammatory
responses to A. fumigatus occurring in the lungs of patients
suffering from ABPA (11, 12) and in the skin of patients
suffering from severe atopic dermatitis might result in release of autoantigens as a consequence of tissue damage due
to the inflammatory process (28). Exposure to autoantigens containing cross-reactive determinants (molecular mimicry)
can recruit the memory T cell repertoire at the site of inflammation where lymphokine expression is upregulated.
These lymphokines can induce expression of MHC II on
naive T cells and upregulate accessory molecules that function as costimulatory signals for T cell activation, creating a
microenvironment in which all requirements for priming a
T cell response are present (29). Molecular mimicry at the
T cell level could be a possible pathogenic mechanism to explain autoaggression remaining confined to the local area
of inflammation (29).
Allergenicity of the Recombinant P2 Proteins In Vivo.
The
ability of a protein to bind IgE in ELISA and Western blots
provides strong evidence for the allergenicity of the protein. However, the final demonstration that a protein preparation acts as an allergen and therefore possesses biological
activity in vivo is its ability to elicit a type I skin reaction in
sensitized individuals. We have investigated whether the
IgE-mediated cross-reactivity against A. fumigatus and human P2 protein shown in vitro is sufficient to provoke allergic reactions in vivo through skin tests (8, 16). Four individuals with high IgE levels against A. fumigatus P2
protein, four individuals allergic to the fungus lacking IgE responses to the P2 protein, and two nonallergic control individuals were investigated for their ability to respond to
intradermal challenge with recombinant A. fumigatus and
human P2 proteins. As expected, none of the individuals
without detectable IgE antibodies to A. fumigatus P2 protein
reacted against the recombinant protein preparations. A
positive skin reaction to A. fumigatus P2 protein was detected only in individuals who had IgE levels >10 EU/ml to the fungal protein. The amounts of recombinant protein
needed to elicit a classical type I reaction ranged from 1 to
10 ng, depending on the subject. All individuals reacting to
A. fumigatus P2 protein also showed strong skin reaction
to challenges with comparable amounts of human P2 protein
(Fig. 4). These results show that human P2 protein can
cross-link IgE on mast cells in vivo (30) and suggest humoral autoimmune response in some patients suffering
from mold allergy. IgE reactivity to fungal and human P2
protein was, however, only detectable in individuals sensitized to A. fumigatus suffering from ABPA or severe atopic
dermatitis. This was also the case for the IgE autoreactivity
to human MnSOD described earlier (16). Therefore, these
two human proteins may serve as a tool to study the role of
IgE autoreactivity in tissue damage and release of autoantigens at the site of inflammation.
View larger version (96K):
[in this window]
[in a new window]
|
Fig. 4.
Skin test reactivity to recombinant A. fumigatus and human
proteins in a patient sensitized to MnSOD and P2 protein. For intradermal skin tests, 100 µl of the protein solutions (101 µg/ml) was injected
with a syringe. 0.01% histamine dihydrochloride (A) and 0.9% saline (B)
were used as positive and negative controls, respectively. The reactions
show that 10 ng fungal (D) and human (E) P2 protein or fungal (H) and
human (I) MnSOD are able to elicit a wheal that is comparable to the size
of the skin reaction induced by the positive histamine control. C shows
the reaction to a challenge with 1 ng human P2 protein. The patient lacks
IgE to rAsp f 3 (F) and rAsp f 11 (G), two additional A. fumigatus allergens
(10). The absence of reactions to skin challenges with these allergens
demonstrates the specificity of the test.
|
|
| |
Footnotes |
Address correspondence to Reto Crameri, Swiss Institute of Allergy and Asthma Research (SIAF), Obere
Strasse 22, CH-7270 Davos Platz, Switzerland. Phone: 41-81-410-08-48; Fax: 41-81-410-08-40; E-mail:
crameri{at}siaf.unizh.ch
Received for publication 2 February 1999 and in revised form 5 March 1999.
We are grateful to Dr. G. Menz (Hochgebirgsklinik, Davos-Wolfgang, Switzerland) for fruitful discussions
and careful reading of the manuscript. We thank Dr. D. Stüber (Hoffmann LaRoche, Basel, Switzerland) for
providing expression vectors and Dr. C. Heusser (Novartis, Basel, Switzerland) for the TN-142 mAb.
This study was supported in part by Swiss National Science Foundation grants 31-39429.93 and 31-50515.97.
| |
References |
| 1.
|
Mullis, J.,
R. Harvey, and
A. Seaton.
1976.
Sources and incidence of airborne Aspergillus fumigatus.
Clin. Allergy.
6:
209-217
[Medline].
|
| 2.
|
Bardana, E.J..
1980.
The clinical spectrum of aspergillosis. Part
2: classification and description of saprophytic allergic and invasive variants of human diseases.
Crit. Rev. Clin. Lab. Sci.
13:
85-159
.
|
| 3.
|
Romani, L., and
D.H. Howard.
1995.
Mechanisms of resistance to fungal infections.
Curr. Opin. Immunol.
7:
517-523
[Medline].
|
| 4.
|
Moser, M.,
R. Crameri,
G. Menz,
T. Schneider,
T. Dudler,
C. Virchow,
M. Gmachl,
K. Blaser, and
M. Suter.
1992.
Cloning and expression of recombinant Aspergillus fumigatus
allergen I/a (rAsp f I/a) with IgE binding and type I skin test
activity.
J. Immunol
149:
454-460
[Abstract].
|
| 5.
|
Crameri, R.,
R. Jaussi,
G. Menz, and
K. Blaser.
1994.
Display of expression products of cDNA libraries on phage surfaces. A versatile screening system for selective isolation of
genes by specific gene-product/ligand interaction.
Eur. J. Biochem
226:
53-58
[Medline].
|
| 6.
|
Crameri, R., and
K. Blaser.
1996.
Cloning Aspergillus fumigatus allergens by the pJuFo filamentous phage display system.
Int. Arch. Allergy Immunol
110:
41-45
[Medline].
|
| 7.
|
Arruda, L.K.,
T.A. Platts-Mills,
J.W. Fox, and
M.D. Chapman.
1990.
Aspergillus fumigatus allergen I, a major IgE-binding protein, is a member of the mitogillin family of cytotoxins.
J. Exp. Med.
172:
1529-1532
[Abstract/Free Full Text].
|
| 8.
|
Moser, M.,
R. Crameri,
E. Brust,
M. Suter, and
G. Menz.
1994.
Diagnostic value of recombinant Aspergillus fumigatus
allergen I/a for skin testing and serology.
J. Allergy Clin. Immunol.
93:
1-11
[Medline].
|
| 9.
|
Lamy, G.,
M. Moutaouakil,
J.P. Latgé, and
J. Davies.
1991.
Secretion of a potential virulence factor, a fungal ribonucleotoxin, during human aspergillosis infections.
Mol. Microbiol.
5:
1811-1815
[Medline].
|
| 10.
|
Crameri, R..
1998.
Recombinant Aspergillus fumigatus allergens: from the nucleotide sequences to clinical applications.
Int. Arch. Allergy Immunol
115:
99-114
[Medline].
|
| 11.
|
Crameri, R.,
S. Hemmann,
C. Ismail,
G. Menz, and
K. Blaser.
1998.
Disease-specific recombinant allergens for the diagnosis of allergic bronchopulmonary aspergillosis.
Int. Immunol.
10:
1211-1216
[Abstract/Free Full Text].
|
| 12.
|
Hemmann, S.,
W.H. Nikolaizik,
M.H. Schöni,
K. Blaser, and
R. Crameri.
1998.
Differential IgE recognition of
recombinant Aspergillus fumigatus allergens by cystic fibrosis
patients with allergic bronchopulmonary aspergillosis or
Aspergillus allergy.
Eur. J. Immunol
28:
1155-1160
[Medline].
|
| 13.
|
Barnerjee, B.,
V.P. Kurup,
S. Phadnis,
P.A. Greenberger, and
J.N. Fink.
1996.
Molecular cloning and expression of a recombinant Aspergillus fumigatus protein Asp f 2 with significant immunoglobulin E reactivity in allergic bronchopulmonary aspergillosis.
J. Lab. Clin. Med
127:
253-262
[Medline].
|
| 14.
|
Crameri, R.,
J. Lidholm,
H. Grönlund,
D. Stüber,
K. Blaser, and
G. Menz.
1996.
Automated specific IgE assay with recombinant allergens: evaluation of the recombinant Aspergillus fumigatus allergen I in the Pharmacia CAP system.
Clin. Exp. Allergy.
26:
1411-1419
[Medline].
|
| 15.
|
Mayer, C.,
S. Hemmann,
A. Faith,
K. Blaser, and
R. Crameri.
1997.
Cloning, production, characterization and IgE cross-
reactivity of different manganese superoxide dismutases in individuals sensitized to Aspergillus fumigatus.
Int. Arch. Allergy Immunol.
113:
213-215
[Medline].
|
| 16.
|
Crameri, R.,
A. Faith,
S. Hemmann,
R. Jaussi,
C. Ismail,
G. Menz, and
K. Blaser.
1996.
Humoral and cell-mediated autoimmunity in allergy to Aspergillus fumigatus.
J. Exp. Med
184:
265-270
[Abstract/Free Full Text].
|
| 17.
|
Achatz, G.,
H. Oberkofler,
E. Lechnauer,
B. Simon,
A. Unger,
D. Kandler,
C. Ebner,
H. Prillinger,
D. Kraft, and
M. Breitenbach.
1995.
Molecular cloning of major and minor allergens of Alternaria alternata and Cladosporium herbarum.
Mol.
Immunol
32:
213-227
[Medline].
|
| 18.
|
Valenta, R.,
M. Duchêne,
K. Pettenburger,
C. Sillaber,
P. Valent,
P. Bettelheim,
M. Breitenbach,
H. Rumpold,
D. Kraft, and
O. Scheiner.
1991.
Identification of profilin as a
novel pollen allergen; IgE autoreactivity in sensitized individuals.
Science.
253:
557-560
[Abstract/Free Full Text].
|
| 19.
| Crameri, R. 1997. pJuFo: a phage surface display system
for cloning genes based on protein-ligand interaction. In
Gene Cloning and Analysis: Current Innovations. B. Schaefer, editor. Horizon Scientific Press, Wymondham, UK.
29-45.
|
| 20.
|
Rich, B.E., and
J.A. Steitz.
1987.
Human acidic ribosomal
phosphoproteins P0, P1, and P2: analysis of cDNA clones, in
vitro synthesis, and assembly.
Mol. Cell. Biol.
7:
4065-4074
[Abstract/Free Full Text].
|
| 21.
|
Elkon, K.B.,
A.P. Parnassa, and
C.L. Forster.
1985.
Lupus
autoantibodies target ribosomal P proteins.
J. Exp. Med.
162:
459-471
[Abstract/Free Full Text].
|
| 22.
|
Elkon, K.,
S. Skelly,
A. Parnassa,
W. Moller,
W. Danho,
H. Weissbach, and
N. Brot.
1986.
Identification and chemical
synthesis of a ribosomal protein antigenic determinant in systemic lupus erythematosus.
Proc. Natl. Acad. Sci. USA.
83:
7419-7423
[Abstract/Free Full Text].
|
| 23.
|
Sanger, F.,
S. Nicklen, and
A.R. Coulson.
1977.
DNA sequencing with chain-terminating inhibitors.
Proc. Natl. Acad.
Sci. USA.
74:
5463-5467
[Abstract/Free Full Text].
|
| 24.
|
Pearson, W.R., and
D.J. Lipman.
1988.
Improved tools for
biological sequence comparison.
Proc. Natl. Acad. Sci. USA.
85:
2444-2448
[Abstract/Free Full Text].
|
| 25.
|
Hemmann, S.,
K. Blaser, and
R. Crameri.
1997.
Allergens of
Aspergillus fumigatus and Candida boidinii share IgE-binding
epitopes.
Am. J. Respir. Crit. Care Med
156:
1956-1960
[Abstract/Free Full Text].
|
| 26.
|
Crameri, R.,
S. Hemmann, and
K. Blaser.
1996.
pJuFo: a
phagemid for display of cDNA libraries on phage surface
suitable for selective isolation of clones expressing allergens.
Adv. Exp. Med
409:
103-110
.
|
| 27.
|
Zubler, R.H.,
C. Werner-Favre,
L. Wen,
K.I. Sekita, and
C. Straub.
1987.
Theoretical and practical aspects of B-cell activation: murine and human systems.
Immunol. Rev.
99:
281-299
[Medline].
|
| 28.
|
Zhao, Z.S.,
F. Granucci,
L. Yeh,
P.A. Schaffer, and
H. Cantor.
1998.
Molecular mimicry by herpes simplex virus-type 1:
autoimmune disease after viral infection.
Science.
279:
1344-1347
[Abstract/Free Full Text].
|
| 29.
|
Sercarz, E.E.,
P.V. Lehmann,
A. Ametani,
G. Benichou,
A. Miller, and
K. Moudgil.
1993.
Dominance and crypticity
of T cell antigen determinants.
Annu. Rev. Immunol.
11:
729-766
[Medline].
|
| 30.
|
Metzger, H.,
G. Alcaraz,
R. Hohman,
J.P. Kinet,
V. Pribluda, and
R. Quarto.
1986.
The receptor with high affinity
for immunoglobulin E.
Annu. Rev. Immunol
4:
419-470
[Medline].
|
Top
Abstract
Introduction
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
