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* Clinical Immunology and Allergy Unit, Hadassah University Hospital, Jerusalem, Israel; and
Department of Food Engineering and Biotechnology, Technion, Haifa, Israel
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The role of the 65-kDa heat shock protein (HSP)65 of the MT (7, 8) in the pathogenesis of autoimmune arthritis, both in experimental animals (9, 10) and in humans (11, 12, 13), has been intensively investigated in the past. AA can be passively transferred by a T cell clone reactive to residues 180–188 of the HSP65 (10). An association between T cell responses to HSP65 and early stages of joint inflammation has been also found in patients suffering from rheumatoid arthritis (14, 15, 16). Preimmunization of susceptible rats with the mycobacterial HSP65 leads to resistance to induction of the disease by MT. This protective effect is believed to be mediated by T cells specific for HSP65 (3, 7, 17, 18). Likewise, although arthritic rats develop vigorous T cell responses to self-HSP and to peptide 180–188 of the MT HSP65, neither of these molecules is arthritogenic when administered to arthritis-susceptible rats (19, 20). This suggests that HSP65 may contain different epitopes, some of which are responsible for its pathogenicity, whereas others confer resistance to disease induction (3, 19, 21, 22, 23, 24).
We have previously shown that resistance to AA is due to the presence of Abs against HSP65 and can be transferred to susceptible rats by i.v. infusion of these Igs derived from resistant strains (25). In this study we have analyzed the fine epitope specificity of the anti-HSP Abs of arthritis-susceptible and -resistant rats. We found that resistant BN rats have natural Abs to certain epitopes of the mycobacterial HSP65 that are not found in the young arthritis-susceptible Lewis rats. These Abs are also present in old naive Lewis rats and are acquired by young Lewis rats after recovery from the disease.
Vaccination of Lewis rats with some of these peptide epitopes before disease induction resulted in the production of Abs against the whole molecule as well as resistance to disease induction. These Abs were shown to skew the cytokine profile of mononuclear cells toward an anti-inflammatory response in vitro and to suppress disease upon in vivo treatment.
Analysis of the primary and tertiary structure of the whole HSP65 molecule indicated that the protective peptides are potential B cell epitopes found on the outer surface of the molecule.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female inbred Lewis rats were obtained from Harlan Laboratories (Jerusalem, Israel). Female BN rats were obtained from Harlan Sprague Dawley (Indianapolis, IN).
Ags and Abs
Recombinant HSP65 of MT was a kind gift from Dr. M. Singh (World Health Organization Recombinant Protein Bank, Braunschweig, Germany). Recombinant mammalian HSP60 was purchased from StressGen Biotech (Victoria, British Columbia, Canada). Synthetic 16-mer peptides overlapping by 10 amino acids of HSP65 were a kind gift from Dr. L. Adorini (Roche Milano Ricerche, Milan, Italy) (26). Synthetic 20-mer peptides overlapping by five amino acids of the mammalian HSP60 were a kind gift from Dr. I. Cohen (Weizmann Institute, Rehovot, Israel) (27). Goat anti-rat IgG conjugated to alkaline phosphatase was purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). R83D is an unrelated control rat mAb.
Induction and clinical assessment of AA
Lewis rats were injected s.c. at the base of the tail with 1 mg of MT H37Ra (Difco, Detroit, MI) in CFA (Difco). Severity of arthritis (arthritis index) was assessed every other day by a blinded observer as follows: 0, no arthritis; 1, redness of the joint; 2, redness and swelling of the joint. The ankle and tarsal-metatarsal joints of each paw were scored. A maximum score of 16 could be obtained.
Dot blot assay
Ags were dissolved in PBS and samples of 1 µg were adsorbed on nitrocellulose paper (Sigma-Aldrich, St. Louis, MO). The paper was air-dried and incubated with 1% BSA (Sigma-Aldrich) in PBS for 20 min to block nonspecific binding. The samples were then washed in 0.05% PBS-Tween 20 and incubated with rat sera diluted 1/100 in 1% BSA for 90 min at room temperature (RT). The membranes were washed and incubated with goat anti-rat Ab conjugated to alkaline phosphatase diluted 1/1000 in 1% BSA for 90 min at RT. After rewashing the color reaction was developed by adding a mixture of 5-bromo-4-chloro-3-indolyl phosphate-nitroblue tetrazolium (Sigma-Fast; Sigma-Aldrich). The reaction was stopped by the addition of tap water.
ELISA
Flat-bottom 96-well plates (Corning Glass, Corning, NY) were coated overnight with mammalian HSP60 or mycobacterial HSP65 (10 µg/ml) in carbonate buffer (pH 9.6) at 4°C. After extensive washing with 0.05% PBS-Tween 20 plates were blocked with 1% BSA for 60 min at RT. HSP peptides were attached to plates pretreated with glutaraldehyde according to Kasprzyk et al. (28). Briefly, plates were treated with 100 µl/well of 5% (w/v) glutaraldehyde in PBS for 1 h at RT. Plates were washed thoroughly with PBS and peptides (1 µg/100 µl) were added to each well and incubated overnight at 4°C. Plates were shaken dry and blocked with 1% BSA. Plates coated with either whole HSP or peptides were washed again and incubated with rat sera diluted 1/100 in 0.01% PBS-Tween 20 for 90 min at RT. After rewashing the plates were incubated with goat anti-rat IgG conjugated to alkaline phosphatase for 60 min at RT. The presence of Abs was detected by addition of the substrate p-phenylphosphate (p-NPP; Chemicon, Temecula, CA) to the plates. OD was measured photometrically at 405 nm by an ELISA reader (Microwell System; Organon Teknika, Turnhout, Belgium).
Competitive inhibition was performed by incubation of the Abs with various concentrations (1, 10, and 100 µg/ml) of the inhibitors for 1 h and then tested for the residual binding by ELISA.
Modulation of AA by mycobacterial and mammalian HSP peptides
Mycobacterial and mammalian HSP-derived peptides were tested for their ability to modulate the incidence and severity of AA in Lewis rats. Rats were vaccinated i.p. three times at weekly intervals with 100 µg of each peptide in PBS before induction of AA. Control rats were treated with PBS. Rats were bled for testing Ab presence before injection of MT and 30 days later.
Purification of polyclonal anti-peptide 6 Abs
Naive Lewis rats were vaccinated i.p. three times with 100 µg peptide 6 in PBS at weekly intervals. Rats were bled and their sera (diluted 1/100) were tested for Abs to peptide 6 and to HSP65 by ELISA (as described in Materials and Methods). Polyclonal rat anti-peptide 6 Abs were purified on a 5-ml column of HiTrap NHS-activated Sepharose (Amersham Biosciences, Uppsala, Sweden) to which we coupled peptide 6 covalently, according to the manufacturer’s instructions. Rat serum (1/5 in PBS) was applied on the column and the flow-through was collected. Bound Abs were eluted with 0.1 M glycine-HCl (pH 2.5). Fractions of 1 ml were collected and immediately neutralized with 30 µl 2 M Tris (pH 12) to avoid denaturation. Protein concentration was measured by absorbance at 280 nm, and the protein-rich fractions were dialyzed against PBS and concentrated to 1 mg/ml.
Modulation of AA by anti-peptide 6 polyclonal Abs
Lewis rats were immunized with CFA to induce AA and treated with 10 µg polyclonal anti-peptide 6 Igs, naive Lewis rats polyclonal Igs, or an unrelated rat mAb (R83D). Control rats were treated with PBS. The Abs were injected i.v. on the day of disease induction and i.p. the day after.
Cytokine secretion by murine and human PBMC in vitro
PBMC from naive Lewis rats and human volunteers were
collected with heparin, layered on top of a Ficoll-Paque solution
(Amersham Biosciences), and centrifuged for 25 min at 1800 rpm. PBMC
were collected and washed with PBS. A total of 2 x
106 rat or human cells were incubated in 1 ml
DMEM or RPMI consecutively (Beit-Haemek, Beit-Haemek, Israel) in
a 24-well tissue culture dish (Nunc, Roskilde, Denmark) with 10 ng/ml
LPS (Sigma-Aldrich), 10 µg/ml polyclonal anti-peptide 6 Ab, or 10
µg/ml naive Lewis rat Igs, or without Ag (control), at 37°C and 5%
CO2. After 24 h the supernatants were
collected and kept frozen. Rat and human TNF-
and IL-10 were
measured by ELISA (Quantikine M; R&D Systems, Minneapolis,
MN).
Structure analysis
RasMol v. 2.6 program and the known three-dimensional (3D) structure of the GroEL molecule (Escherichia coli HSP60; Brookhaven Protein Data Bank no. 1AON) (29) were used to analyze the position of the relevant epitopes. As the crystal structure of MT HSP65 has not been solved yet, we used a 3D model for the tertiary structure of MT HSP65 based on the crystal structure of the E. coli GroEL (Brookhaven Protein Data Bank no. 1GRL) (30) as template. This model was built by programs for comparative protein modeling (31, 32, 33).
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Results |
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We have previously shown that Igs from naive AA-resistant rats (i.e., BN or Fisher) and from Lewis rats that recovered from AA (post-AA Lewis rats) are able to prevent the induction of AA in naive Lewis rats and that this is due to a subpopulation of anti-HSP65 Abs. To further analyze the fine specificity of this reaction we tested the binding of Igs from these rats with the whole molecule of the mycobacterial HSP65, as well as with a panel of overlapping peptides covering the whole molecule.
We found that Igs from arthritis-resistant rats (i.e., 6- to 8-wk-old
BN rats, 9-mo-old Lewis rats, and post-AA Lewis rats) reacted strongly
with the whole MT HSP65. Igs derived from arthritis-susceptible naive
young (up to 4 mo) Lewis rats did not bind to this molecule in the
native or heat-denatured form (Table I
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Only 10 of the 90 peptides tested were recognized by the Igs tested
(Table I
). Igs from 6-wk-old naive Lewis rats reacted with two peptides
(40 and 63). Four-month-old naive Lewis rats acquire Abs against two
additional peptides (36 and 45), and 9-mo-old naive Lewis rats acquire,
in addition, Abs to peptides 6, 7, and 31, as well as to the whole
HSP65 molecule. The Ab profile of the old Lewis rats is similar to that
found in young naive BN rats, which react in addition with peptide 59.
Lewis rats immunized with CFA (post-AA) reacted in addition with
peptides 21 and 84. Although Igs derived from naive young Lewis rats
interact with some of the peptides of this molecule, they do not
recognize the whole HSP65 molecule, and they do not have any effect on
susceptibility to AA.
As recognition of the whole HSP65 was acquired together with binding to
peptides 6, 7, and 31, we have tested the ability of peptides 6 and 31
(at 1, 10, and 100 µg/ml) to inhibit the binding of Igs from
CFA-immunized Lewis rats to HSP65 by a competitive inhibition ELISA. We
found that 100 µg/ml peptide 6 reduced the binding by 59% and 100
µg/ml peptide 31 reduced it by 40%. The mixture of both peptides
inhibited 75% of the binding to HSP65, indicating that these epitopes
are the dominant epitopes of the anti-HSP Ab response (Fig. 1).
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It has been previously shown that the mycobacterial HSP65 can trigger self-HSP-reactive T cells with disease-suppressive regulatory potential (24). To analyze the anti-mammalian self-HSP60 Ab repertoire of these rats we tested Igs derived from naive and post-AA Lewis rats as well as from naive BN rats for their reactivity with the whole mammalian HSP60 by ELISA.
We found that naive young Lewis rats do not have anti-mammalian
HSP60 Abs, whereas 9-mo-old Lewis rats, young BN rats, and post-AA
Lewis rats had significant binding to the self-HSP molecule (Table II).
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). Vaccination of Lewis rats with HSP-derived peptides
To test whether active vaccination with HSP peptides recognized by protective Igs can prevent AA induction, we vaccinated Lewis rats with the MT HSP65-derived peptides 6, 7, 21, 31, 36, 45, and 84 and with the mammalian HSP60-derived peptide M5 recognized by Abs from resistant Lewis rats. Control vaccination was performed with the following nonreactive mycobacterial HSP65 peptides: peptides 26 (aa 151–166), 28 (aa 163–178), and 70 (aa 415–430).
We found that vaccination of rats with the bacterial peptides 6 and 7
and the mammalian peptide M5 resulted in a significant suppression of
disease severity (Fig. 2). Vaccination
with these protective peptides also resulted in the production of Abs
against peptide 6 as well as against the whole mycobacterial HSP65
molecule (data not shown). Neither protection nor anti-HSP response
was observed after vaccination with mycobacterial peptide 31 as well as
with the rest of the peptides.
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As active vaccination with peptide 6 induced anti-peptide 6 Abs and suppressed the severity of AA we tested the effect of passive treatment with these polyclonal Abs on AA. Lewis rats were immunized with CFA to induce AA and were treated concomitantly i.v. with 10 µg of Lewis rat polyclonal anti-peptide 6 Abs, naive Lewis rat polyclonal Igs, or an unrelated rat mAb (R83D). The same Ab treatment was given i.p. the following day. Control rats received PBS.
We found that treatment with anti-peptide 6 Abs reduced arthritis
severity by 44% on day 22 and by 64% on day 29 (Fig. 3). Treatment with Igs from naive Lewis
rats also reduced the severity of the disease. However, this effect was
not significant statistically. The rat mAb had no effect on the
severity of AA.
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To test whether the protective effect of anti-peptide 6 Abs is
due to their effect on inflammatory cytokines we analyzed the effect of
these Abs on cytokine secretion in vitro. PBMC from naive Lewis rats
and human volunteers were incubated for 24 h at 37°C with
LPS (10 ng/ml), naive Lewis rat polyclonal Igs (10 µg/ml), or
polyclonal anti-peptide 6 Abs (10 µg/ml). Cells in medium without
Ag served as control. Supernatants were collected to measure the
secretion of IL-10 and TNF-.
As can be seen in Table III, the
anti-peptide 6 Abs induced at least a 6-fold higher secretion of
IL-10 and a 3.7-fold higher secretion of TNF- by rat PBMC compared
with the control. The increase in secretion of human IL-10 and TNF-
was 50.3 and 7.4, respectively.
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Analysis of the peptide location on the 3D structure of the HSP65
Fig. 4 shows the location of
bacterial peptides 6 and 31 on a model of the mycobacterial HSP65 based
on the structure of the E. coli GroEL monomer with a space
filling and secondary structure representations (29). As
can be seen in Fig. 4, the linear peptides 6 and 31 are completely
exposed on the outer surface of the HSP molecule.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have found that Abs from arthritis-resistant rats bind to the whole MT HSP65 as well as to several HSP peptides, which are not recognized by Abs from arthritis-susceptible rats.
Vaccination of the susceptible rats with some of these peptides resulted in the production of anti-HSP65 Abs and induced resistance to arthritis. The Abs were found to induce IL-10 secretion in vitro and to suppress arthritis upon in vivo inoculation to naive susceptible rats.
Structural analysis of these protective HSP epitopes revealed that they are potential B cell epitopes, mainly located on the outer surface of the HSP molecule.
It has been previously reported that the T cell response to bacterial HSP shows determinant spreading (22). In the present data we show that there is a clear B cell determinant spreading as well, and this spreading can occur also naturally, namely without intentional vaccination. The B cell epitopes, as we will discuss later, are different from the T cell epitopes.
Abs derived from young naive Lewis rats bound to two bacterial peptides, whereas Abs from 9-mo-old Lewis rats reacted with five additional peptides as well as with the whole molecule. Immunization with CFA resulted in recognition of three additional epitopes. The Ab repertoire of the naive young BN rats was similar to that of naive old Lewis rats.
Although we refer to all the anti-HSP peptide Abs found in naive old Lewis rats and in naive young BN rats as natural Abs, it is possible that they are indeed a response to the exposure of these rats to environmental microorganisms (as "natural" Abs may always be) (34), and that the epitope spreading in response to these pathogens occurs in the BN rat earlier and reaches a higher titer than in the Lewis rat. Lewis rats have to be immunized with CFA to mimic the natural response of the BN rats. The similarity of the Ab repertoire of the naive BN rats to that of the immunized Lewis rats supports this possibility.
The T cell response of the Lewis rat to the bacterial HSP65 has been thoroughly studied. It has been shown that in the early postimmunization stages the Lewis T cells respond to several determinants found in the N terminus as well as in the carboxyl terminus of the molecule, whereas later a shift to recognition of carboxyl-terminal epitopes develops (22). The B cell epitopes, in contrast, were found all along the molecule without any predilection for either the carboxyl or the N terminus of the molecule. This is not surprising, as in the tertiary structure of the molecule the carboxyl- and the N-terminal sites are located in the same domains.
A comparison between the published dominant T cell epitopes upon immunization with MT (22) and the B cell epitopes described here in naive Lewis rats did not reveal common epitope recognition. On the contrary, the lack of natural Abs to certain peptide epitopes like 6, 7, or 31 in the naive Lewis rat is associated with an early T cell response to these epitopes upon immunization, whereas the presence of Abs to peptides like 40 and 63 is associated with lack of an early T cell response. Based on these correlations we suggest that the presence of natural Abs to certain epitopes may actually inhibit T cell response to them, whereas the lack of Abs enables the T cells to respond to these epitopes. For example, AA-susceptible Lewis rats lack natural Abs to the bacterial peptide 31 and can therefore develop a T cell response to this peptide; these pathogenic T cells may induce arthritis.
The nature of the B cell epitopes and the correlation between recognition of certain epitopes and the whole molecule can be better understood from the primary and tertiary structure analysis of the molecule. Based on the primary structure of MT HSP65, we looked for potential B cell epitopes composed of nine amino acids or more that include neither proline (P), which can cause secondary structure molecular bending, nor any short-chain residue (glycine (G), alanine (A), and serine (S), molecular spacers). This model was used previously by Warren et al. (35) to locate potential B cell epitopes in the myelin basic protein.
A similar analysis of the HSP65 molecule identified six peptides of
consecutive long-chain residues (side chains of two carbons or more)
that satisfied the same structural rules (Table IV).
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experimental peptides). Interestingly, two more epitopes that were
recognized by these Abs (peptides 31 and 45) can be included if we
allow the presence of only one glycine residue in the epitope sequence
of at least 12 amino acids. We did not notice any particularity concerning the secondary structure and the repartition of hydrophobic/polar residues in these epitopes (both experimentally and computer recognized). Generally, the experimentally recognized epitopes tend to be hydrophobic (9–12 hydrophobic residues of 16) except for peptide 59, which is highly polar (13 residues of 16).
Tertiary structure plays an important role for B cell epitope
recognition. To better understand the implications of the tertiary
structure of MT HSP65 and to locate these different amino acid
sequences on the whole molecule, we used a model for the tertiary
structure of MT HSP65 based on the crystal structure of E.
coli GroEL (Fig. 4).
Structure analysis confirms that the experimentally recognized linear epitopes are mainly located on the surface of the protein and can provide a potential site for Ab binding. Peptides 6, 7, 21, 31, and 59 were those that were found to be the most exposed, whereas peptides 36, 40, 45, 63, and 84 are only partially exposed in the MT HSP65 molecule.
Recognition of the exposed peptides 6, 7, and 31 by Abs from 9-mo-old Lewis rats, as well as young BN and post-AA Lewis rats, was associated with binding to the whole molecule, whereas recognition of the partially exposed peptides by Abs from young (6-wk-old and 4-mo-old) Lewis rats was not associated with recognition of the whole HSP molecule.
As we have tested only linear epitopes from the HSP molecule, there are probably many additional conformational nonlinear epitopes recognized by the anti-HSP Abs that we have not depicted. The relevance of the epitopes recognized by Abs that do not bind to the whole HSP molecule is less clear. As T cells respond to linear nonconformational epitopes processed by APCs, it is possible that such T cell help will lead to the production of Abs to epitopes that are not completely exposed without inducing an anti-HSP response; alternatively, these Abs may be cross-reactive Abs, originally directed at other peptides.
HSPs are a family of highly conserved proteins. An amino acid
comparison of HSP65 molecules among >70 microbial species shows that
most of the mycobacterial epitopes recognized by the murine Abs are
relatively conserved (data not shown). There is also 
50% amino acid
identity between the mycobacterial HSP65 and the mammalian HSP60
(36). Despite this homology, most of the bacterial
peptides that were found to be recognized by the anti-MT HSP65 Abs
did not share high homology with the mammalian HSP. This may be due to
the tolerance to self that protects the rats from developing an
autoimmune autoantibody response to their own HSP60.
Analysis of the anti-self (rat) HSP Ab repertoire indeed showed that there is a limited number of self-HSP molecule epitopes recognized by the rat Igs. Naive young Lewis rats did not respond to any self-HSP peptide or to the whole molecule. BN and post-AA Lewis rats that reacted with 8–10 bacterial HSP epitopes responded to only two epitopes in the self-HSP, the mammalian peptides M5 and M30, as well as to the whole self-HSP molecule.
To see whether the anti-HSP protective Abs can be induced by vaccination with the peptides recognized by these Abs, we have vaccinated Lewis rats with these peptides without Freund’s adjuvant. Vaccination with three peptides (bacterial peptides 6 and 7 and mammalian peptide 5) led to production of Abs against bacterial peptide 6 as well as to an anti-HSP response, confirming the hypothesis that Abs against a peptide located on the surface of the molecule will lead to recognition of the whole molecule. Induction of these Abs was also associated with disease resistance. Preliminary results have shown that vaccination with peptide 6 also suppresses collagen arthritis in the Lewis rat and that treatment with anti-peptide 6 Ab suppresses collagen arthritis in the DBA/1j mouse (data not shown).
As we have shown in this work, there was a clear correlation between disease resistance and the presence of anti-HSP Abs. Young naive Lewis rats did not have detectable Abs against the HSP molecule, whereas 9-mo-old Lewis rats developed these Abs in a significant titer. Parallel to the development of the anti-HSP response the old rats also became resistant to induction of arthritis. Young Lewis rats acquired both the Abs and disease resistance after immunization with CFA or with certain HSP epitopes, and the naturally resistant BN rats had anti-HSP Abs spontaneously, without the need for immunization.
The mechanism of disease resistance induced by the natural as well as the induced anti-HSP Abs has not been yet clarified. It is possible that the Abs against the MT HSP inhibit the early steps of induction of pathogenic T cells to the peptide by intervening in the Ag processing or the T cell recognition of the pathogenic epitopes. Alternatively, they may prevent the effector steps of the pathogenic response by binding to the self-HSP cross-reacting target Ag.
Expression of the mammalian (or self) HSP is up-regulated in inflamed synovia of rats with AA (37), and cross-reactive immune recognition has been found between the mycobacterial HSP65 and endogenous self-HSP60 at the T cell level (13, 38). As the anti-self-HSP Abs were found only in the resistant rats, it is possible that Abs that cross-react with the self-HSP may hide it from the pathogenic T cells and thus act as protective Abs. However, the preliminary results showing that the vaccination suppresses murine collagen arthritis as well suggests that the mechanism is not directly related to the induction of the disease by the HSP Ag.
Another possibility is that Abs against the HSP molecule suppress
inflammation by inhibiting the proinflammatory effect of the HSP on the
innate immune system. Mycobacterial HSP65 has been shown to
induce release of proinflammatory cytokines from human monocytic cells
(39), and the mammalian HSP60 has been shown to synergize
with IFN-
and to promote proinflammatory cytokines like IL-12 and
IL-15 (40). Analysis of the effect of the protective Abs
on cytokine secretion by murine and human mononuclear cells showed that
the Abs induce secretion of both IL-10 and TNF-, and that the
increase in IL-10 secretion by murine and human cells was 1.6- to
6.8-fold greater than that of TNF-. The increase in IL-10
secretion in the inflammatory site can skew the local cytokine profile
from an inflammatory to an anti-inflammatory response and thus
explain the mechanism of protection against inflammation by these Abs.
The mechanism of induction of the various cytokines by anti-peptide
6 Abs is now under study and the preliminary results show that the
effect of the Abs is in the level of mRNA of these cytokines.
It is interesting to notice that one of the two self-HSP60 epitopes is
the self-peptide 5, which is the rat homologous epitope of the
bacterial protective peptide 6. Moreover, vaccination with the
bacterial peptides 6 and 7 and with the mammalian peptide 5 led to the
production of antibacterial peptide 6 and antibacterial HSP65 Abs as
well as protection against disease induction. Looking at the primary
structure of these three peptides leads to the conclusion that they
express a common motif (VEWGP), which might be the protective motif of
these peptides (Table V).
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Yaakov Naparstek, Clinical Immunology and Allergy Unit, Hadassah University Hospital, P.O. Box 1200, Jerusalem 91120, Israel. E-mail address: yakovn{at}cc.huji.ac.i
3 Abbreviations used in this paper: AA, adjuvant arthritis; HSP, heat shock protein; RT, room temperature; 3D, three-dimensional; MT, Mycobacterium tuberculosis.
Received for publication December 13, 2001. Accepted for publication April 10, 2002.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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:E
heterodimers in DRA transgenic mice hinder expression of E:E molecules and are more efficient in antigen presentation. Int. Immunol. 7:1927.This article has been cited by other articles:
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