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In the NOD mouse, the onset of beta-cell destruction is associated with spontaneous development of T-lymphocytes reactive to members of the 60 kDa heat shock protein (hsp60) family, including the Mycobacterial (MT) and the human (H) hsp60 molecules. Diabetes in the NOD mouse is a spontaneous tissue-specific autoimmune disease occurring without prior immunization. Therefore, it has been suggested that the anti-hsp60 T cells involved in the autoimmune diabetes of NOD mice might reflect molecular mimicry between MT-hsp60 and a beta-cell tissue specific molecule sharing similar T cell epitopes, the p277 peptide of hsp60 in particular. We cloned and expressed the mouse hsp60 cDNA from a beta-cell tumour. This mouse beta-cell hsp60 cDNA was found to be identical in sequence to the hsp60 of mouse fibroblasts. We further report that NOD spleen cells and an NOD diabetogenic T cell clone C9 responded to the recombinant mouse hsp60 and to its peptide M-p277 to the same extent as to H-hsp60 and H-p277. Splenocytes of mice of other strains did not respond to p277. Moreover, treatment of 3 month old NOD mice with the non-modified self M-p277 peptide was as efficient as H-p277, from which it differs in one amino acid, in halting progression of the disease. Thus, anti-H-p277 T cells modulating diabetes in the NOD mouse are autoreactive, and are targeted at the mouse beta-cell hsp60, which is not tissue specific. These findings raise the question of how a non-tissue specific molecule may be a target of a tissue-specific autoimmune disease.

Background: Behcet's disease (BD) is a multisystem disorder of unknown pathogenesis. The diagnosis is based on a set of international clinical criteria. Previous investigations have suggested that immunological cross-reactivity between peptides within streptococcal heat-shock proteins and human peptides might be involved in the pathogenesis of BD. We tested four peptides from mycobacterial heat-shock proteins to see if they specifically stimulated gamma delta T cells from BD patients. We then investigated this response to see whether it could be used as a laboratory test to diagnose BD., Methods: We used a T-cell proliferative test to assay responses to four mycobacterial 65 kDa heat-shock-protein peptides and to four homologous peptides derived from the sequence of the human 60 kDa heat-shock protein., Findings: We elicited significant gamma delta T-cell responses to the mycobacterial peptides in 25 (76%) of 33 patients with BD, compared with 2 (3.6%) of 55 controls with recurrent oral ulcers, systemic disease, or no disorders. The proportion of BD patients who had false-negative results decreased if the test was done during clinical manifestation of disease activity. There was a correlation between disease activity and T-cell responses. Four homologous peptides from human 60 kDa heat-shock protein also specifically stimulated T cells from patients with BD but with lower stimulation indices., Interpretation: Activation of peripheral-blood mononuclear cells with the four heat-shock-protein peptides elicited significant T-cell proliferative responses by the gamma delta subset of T cells, which may regulate alpha beta T cells. Because these peptides have a high specificity for BD, this assay can be used as a laboratory diagnostic test for BD.

Tolerance for self has appeared incomplete for many self antigens. We have obtained experimental evidence that both for self heat shock proteins and T cell receptor V-gene products, reactive T cells are part of the normal immune repertoire. Furthermore, it has become apparent that stimulation of T cell responsiveness to these antigens, by using peptide immunisation or by transfer of activated T cells, raises resistance to experimentally induced autoimmune arthritis. In addition, available evidence has suggested that these reactivities may be functional during natural processes of disease remission. The observations with regard to heat-shock proteins have indicated that mechanism leading to disease resistance are most efficiently triggered by exposing the immune system to non-self antigens such as bacterial hsp's, which are similar to, but not identical to, self. Experimental evidence has been obtained, that conserved bacterial hsp peptides, may trigger self hsp reactive T cells, with disease suppressive regulatory potential. It is possible that such self hsp reactive T cells, being expanded by recognising bacterial peptides as full agonists, do, in fact, perceive the self epitopes as partial agonists, and therefore have the possibility of displaying downregulatory activity at the site of inflammation. Experiments with peptide analogs of self epitopes, being variants of disease critical T cell epitopes, have indeed suggested that also their activity in modulating disease may comply with the principles of dominant immunological tolerance.

Stress or heat shock proteins (hsp) are a family of approximately two dozen proteins with a high degree of amino acid sequence homology between different species, ranging from prokaryotes to humans, and are representative of a generalized response to environmental and metabolic stressors. Our previous studies showed increased expression of human hsp60 on endothelial cells of arterial intima with atherosclerotic lesions, and elevated levels of serum antibodies (Ab) against hsp65/60 in subjects with carotid atherosclerosis. To investigate the possible involvement of anti-hsp65/60 Ab in endothelial injury, specific hsp-Ab were isolated from human high titer sera by affinity chromatography and probed on heat-shock human umbilical vein endothelial cells. Purified human anti-hsp65/60 Ab reacted specifically with mycobacterial hsp65, human hsp60, and a 60-kD protein band of heat-shocked endothelial cells. High levels of hsp60 mRNA expression in endothelial cells were found between 4 and 12 h after 30 min treatment at 42 degrees C. In immunofluorescence tests, positive staining of heat-stressed endothelial cells was observed not only in the cytoplasm but also on the cell surface. Furthermore, only heat-stressed, but not untreated, Cr-labeled endothelial cells were lysed by anti-hsp65/60 Ab in the presence of complement (complement-mediated cytotoxicity) or peripheral blood mononuclear cells (antibody-dependent cellular cytotoxicity). Control Abs, including human anti-hsp65/60 low titer antiserum, human Ig fraction deprived of hsp65/60 Ab, and mAbs to Factor VIII, alpha-actin, hsp70, and CD3 showed no cytotoxic effect. In conclusion, human serum anti-hsp65 antibodies act as autoantibodies reacting with hsp60 on stressed endothelial cells and are able to mediate endothelial cytotoxicity. Thus, a humoral immune reaction to hsp60 may play an important role in the pathogenesis of atherosclerosis.

Continuous 12-lead electrocardiographic monitoring was performed in 61 patients receiving thrombolytic therapy for an acute myocardial infarction. Coronary angiography within 90 minutes revealed a patent vessel (Thrombolysis in Myocardial Infarction [TIMI] trial 2 or 3) in 44 patients. Early signs of reperfusion were ST-segment normalization (likelihood ratio 16.0), development of terminal T-wave inversion (likelihood ratio 10.6), accelerated idioventricular rhythm (likelihood ratio 6.0), and a twofold increase in ventricular premature complexes (likelihood ratio 2.5). Relief of chest pain after 60 minutes was reported by 96%. During reperfusion of the infarct-related vessel, an increase in ST-segment deviation was recorded in 61% of the patients, whereas 69% had an increase in chest pain preceding the eventual decline.

Synovial fluid and peripheral blood mononuclear cell proliferative responses to the 60-kD human heat shock protein (HSP60) were studied in 23 patients with juvenile chronic arthritis (JCA) and 7 non-JCA control patients. All patients showed active arthritis at the time of study. The patients were divided into two groups according to the presence (group A) or absence (group B) of T lymphocyte reactivity to human HSP60. We show that reactivity to human HSP60 is primarily, though not exclusively, occurring in patients with a remitting course of disease, i.e., the subgroup of HLA-B27 negative JCA patients with an oligoarticular onset. Immunohistochemical analysis of HSP expression in synovial membranes showed a significantly higher intensity of staining in JCA patients than in non-JCA controls. The results suggest that, in accordance with the earlier observation made in experimental models, T lymphocyte reactivity to human HSP60 in this subgroup of JCA patients may be part of T cell regulatory mechanisms that control the development of arthritis.

T cell epitopes of the 65-kD heat shock protein (hsp) were investigated in patients with recurrent oral ulcers (ROU). Peripheral blood mononuclear cells were stimulated with overlapping synthetic peptide (15ers), derived from the sequence of the 65-kD hsp of Mycobacterium tuberculosis. Specific lymphoproliferative responses were stimulated only with peptide 91-105 in ROU, compared with healthy or disease controls (P < 0.01). This was confirmed by studying 760 short term cell lines generated with the 65-kD hsp and then stimulated with the peptides. The frequency of short term cells lines responding to peptide 91-105 in ROU was significantly greater than in healthy (P < 0.0001) or disease controls (P < 0.01). A comparative investigation with the homologous human 60-kD hsp peptide 116-130 also showed significantly greater lymphoproliferative responses in ROU than in healthy (P < 0.01) or disease controls (P < 0.001). The potential involvement of the T cell epitope 91-105 in the pathogenesis of ROU is supported by finding a significant increase in the lymphoproliferative responses stimulated with peptide 91-105 during the stage of ulceration, compared with remission in 9/11 patients studied sequentially (P < 0.05). The results suggest that oral ulceration might be initiated by the microbial hsp peptide 91-105 stimulating the mucosal Langerhans cells, which may generate autoreactive T cell clones primed to the homologous peptide 116-130.

In the epidermis, the major population of T lymphocytes expresses a T-cell receptor (TCR) with V gamma 5 and V delta 1 variable regions, which is unique to this tissue. Roberts et al and Ezquerra et al, also describe a minor population of gamma delta-TCR+ cells in the epidermis that expresses a V gamma 1/V delta 6 TCR. These cells are different from other epidermal T cells in that they "spontaneously" produce cytokines, a result thought to be due to autoreactivity. Over the past 5 years, our laboratory has produced V gamma 1/V delta 6+ T-cell hybridomas from many tissue sources. These spontaneously produce cytokines but also are activated by heat shock protein (HSP-60)-derived peptides. Ezquerra et al report that none of their V gamma 1/V delta 6+ epidermal T-cell lines derived from C3H/HeN mice respond to HSP-60. Of the 99 gamma delta-TCR+ hybridomas we have produced from epidermal T cells of C57BL/6 mice, only one expressed the V gamma 1/V delta 6 TCR. This hybridoma, 70BET-2.12, not only spontaneously produces cytokines, but, unlike the V gamma 1/V delta 6-TCR+ epidermal T cells of Ezquerra et al, it also responds to the whole HSP-60 protein and a 17-mer HSP-60 peptide from M. leprae, producing increased levels of interleukin-2 of up to approximately ten-fold above the spontaneous level. This shows that V gamma 1/V delta 6-TCR+ epidermal T cells can respond to HSP-60. To confirm that 70BET-2.12 expresses TCR genes similar to those of cells that have HSP-60 reactivity, V gamma 1-C gamma 4 and V delta 6-C delta cDNA were produced from RNA isolated from this hybridoma, amplified by the polymerase chain reaction, and sequenced. The gamma and delta TCR gene sequences were similar but not identical to previously published sequences of HSP-60-reactive cells from lymphoid and other organs. No explanation can be found for the discrepancy between our findings and those of others at the level of TCR expression such that other strain-specific factors might be responsible for HSP-60 reactivity.

CD4+ helper T cells recognize antigen in association with MHC class II molecules. To investigate the role of the context of a minimal T cell epitope on presentation and recognition in association with MHC class II molecules, an epitope of the 65 kDa heat shock protein of Mycobacterium tuberculosis was inserted at four different sites in the outer membrane protein PhoE of Escherichia coli. Only some of the constructs could stimulate the epitope-specific T cell clone A2b in vitro. One non-stimulatory construct could be made stimulatory by denaturation, suggesting that the protein conformation prevents correct presentation of the epitope. Other non-stimulatory constructs did not become stimulatory with denaturation. One of these constructs could be made stimulatory by substitution of either one of the two amino acids directly preceding the minimal epitope in the recombinant protein. In synthetic peptides the presence of these upstream residues did not interfere with T cell recognition, suggesting that these residues influence processing of the recombinant protein. Flanking amino acids also influenced induction of a T cell response against the inserted epitope in vivo. These results demonstrate that insertion of minimal T cell epitopes in carrier proteins for the design of vaccines might fail because of the inhibiting effects of flanking residues.

Mycobacterial and homologous human heat shock protein T cell peptide epitopes specific for T lymphocytes in Behçet's disease were investigated for their pathogenicity in Lewis rats. The potential pathogenicity of eight peptides and two controls was assessed by administering the peptides in enriched Freund's adjuvant into the footpads of male Lewis rats. Anterior uveitis which is a major manifestation of Behçet's disease was induced with two out of the four mycobacterial and all four homologous human peptides. The most effective peptides inducing iridocyclitis in 64-75% of rats were peptides with amino acids 336-351 and 136-150, derived from the sequence of the human 60-kD heat shock protein. A few of the rats also showed evidence of focal loss of photoreceptors. These results suggest that selected peptides within heat shock protein 60 kD which function as T cell epitopes in Behçet's disease are capable of inducing uveitis in rats. This supports the view that the peptide T cell determinants may be involved in the pathogenesis of Behçet's disease.

T-T cell interactions have been proposed in postulated network theories of immunoregulation and autoimmunity. Despite previous reports of protection induced by T-cell receptor (TcR)-derived peptides in experimental autoimmunity, no evidence for T-T cell interactions by direct recognition of processed TcRs on native T cells was obtained. Here we report that immunization of rats with overlapping sets of peptides of the TcR alpha or beta chain allowed us to detect immunogenic TcR peptides. Remarkably enough, these TcR peptides appeared to cluster within the hypervariable complementarity-determining regions of the TcR. Immunization of rats with these TcR peptides induced CD4+ TcR peptide-specific T cells, which recognized both rDNA TcR proteins and the original, arthritogenic T cell in a major histocompatibility complex class II-restricted way. These findings indicate that activated T cells can process and present their own TcR in the context of major histocompatibility complex class II molecules and, furthermore, that such peptides can be recognized by TcR variable gene-specific T cells.

Blocking of the Ag presentation function of MHC molecules by competitor peptides has been proposed as a potential immunotherapy for MHC-associated autoimmune diseases. Despite the fact that successful inhibition of experimental autoimmune encephalomyelitis (EAE) by coimmunization with competitor peptides had been achieved, it remained questionable whether the in vivo activity of such peptides was solely the result of MHC blockade. In the peptide MBP72-85-induced EAE model in Lewis rats, we designed a single amino acid-substituted analogue of MBP72-85 with a superior MHC binding capacity, and with the capacity to activate encephalitogenic MBP72-85-specific T cells. Subsequently, two well-defined competitor peptides, one EAE related and one non-EAE related, were studied for their respective efficacies to inhibit the in vitro proliferation of an encephalitogenic T cell clone induced by the original MBP72-85 or the superior MHC binding analogue peptide. It appeared that the response to MBP72-85 was inhibited very efficiently by both competitor peptides, whereas the response to the superior MHC binding analogue peptide was not. Co-immunization of either the related or non-related competitor peptide together with MBP72-85 inhibited EAE induction in a concentration-dependent manner. In such protected rats, polyclonal T cell responses against MBP72-85 were dramatically decreased. However, EAE induced by the stronger MHC binding MBP72-85 analogue could not be inhibited by either of the competitor peptides. Moreover, in these rats, T cell priming for both MBP72-85 and the MBP72-85 analogue was not inhibited. These results show that competition for MHC binding in vivo could lead to inhibition of EAE induction.

Immunization of Lewis rats with heat-killed Mycobacterium tuberculosis (Mt) in mineral oil induces adjuvant arthritis (AA), associated with T cell responses to residues 180-188 of the mycobacterial 65-kDa heat shock protein (hsp65). Preimmunization with hsp65 protects rats against AA and other forms of arthritis. Several explanations for these protective effects have been proposed, including enhanced responsiveness to protective epitopes in hsp65, down-regulation of T cell responses to the 180-188 epitope, and activation of self-hsp60-reactive T cells. To assess the potential of these hypotheses, we analyzed hsp65 T cell epitopes recognized after immunization of Lewis rats with Mt or hsp65. Here we identify nine RT1.B1-restricted T cell epitopes in hsp65. Mt immunization induced T cell responses in which the 180-188 epitope was dominant, whereas hsp65 immunization resulted in a co-dominance of this and two further epitopes, 216-225 and 226-235. Two minor epitopes were recognized after hsp65 but not Mt immunization. These results indicate that hsp65 preimmunization does not down-regulate responses to the AA-associated epitope, but does enhance responses to several hsp65 epitopes that are minor or absent after the AA-inducing immunization protocol. Cross-reactive T cell recognition of hsp65 and rat hsp60 was limited to a single epitope (256-265), recognized after hsp65 immunization, but poorly recognized after Mt immunization. This study provides the necessary basis for elucidating the T cell events involved in the protective effects of hsp65 preimmunization.

The outer membrane protein PhoE of Escherichia coli can be used for the expression of foreign antigenic determinants. Previously, a T-cell epitope of the 65 kDa heat shock protein (hsp65) of Mycobacterium tuberculosis, comprising amino acids 180 to 188, was expressed in PhoE. The hybrid protein induced proliferation of epitope-specific T-cell clones in vitro. In this report, the potential of the hybrid protein to induce an in vivo T-cell response against the 180-188 T-cell epitope was assessed. Popliteal lymph node cells, isolated from rats immunized with PhoE containing the hsp65 epitope, showed high proliferative responses to a synthetic peptide consisting of amino acids 180 to 188 of hsp65, indicating that the epitope is immunogenic in the PhoE-associated conformation.

The 60-kD heat shock protein (hsp60) has been implicated in the etiology and pathogenesis of both experimental and naturally occurring autoimmune diseases such as juvenile chronic arthritis (JCA). Human hsp60 is expressed in inflamed synovial tissue, and T lymphocytes both from peripheral blood and synovial fluid show reactivity to human hsp60. Because the anti-hsp60 B lymphocyte response has been less well studied, we have determined the occurrence of IgG anti-human hsp60 antibodies in patients with JCA and various other autoimmune diseases of childhood. Serum IgG anti-human hsp60 antibodies in JCA patients were significantly higher compared with control children (358 and 163 U/mL, respectively). Within the group of JCA patients, the highest antibody titers were found in the subgroup with a polyarticular onset of JCA. IgG anti-human hsp60 antibody levels in synovial fluid were 3- to 4-fold higher compared with paired serum samples. Because this difference was not found for total IgG or for irrelevant antibodies (anti-polyribosylribitol phosphate), this suggests local anti-hsp60 antibody production in the synovial compartment. The occurrence of anti-hsp60 antibodies is not specific for JCA but also is found in children with systemic lupus erythematosus and in cystic fibrosis, whereas mixed connective tissue disease and insulin-dependent diabetes are negative in this respect. Whether the anti-human hsp60 antibodies are directed toward species-specific sequences or to conserved sequences of the hsp60 molecule remains to be determined.

We have previously shown in mice that antibodies can be induced to synthetic malaria peptides conjugated to mycobacterial antigens, such as purified protein derivative (PPD) or heat shock proteins (hsp), and given in the absence of adjuvants after a previous priming with bacille Calmette-Guérin (BCG). In the present study we investigated this model of immunization in the non-human primates, Saimiri sciureus monkeys. Monkeys primed with BCG subcutaneously and then immunized subcutaneously with the Plasmodium falciparum sporozoite (NANP)40 synthetic peptide conjugated to PPD or mycobacterial hsp of 65 or 70 kD, in the absence of adjuvants, produced antipeptide and anti-sporozoite IgG antibodies. Interestingly, the carrier effect of the hsp of 70 kD for the induction of anti-(NANP)40 antibodies was also observed in the absence of a previous priming with BCG. These data suggest that such a vaccination strategy may be applied to humans.

T cell epitopes of the 65-kDa heat shock protein (HSP) were mapped in patients with Behçet's disease (BD), by stimulating T cells with the overlapping synthetic peptides derived from the sequences of the Mycobacterium tuberculosis 65-kDa HSP. Significant lymphoproliferative responses were stimulated with four HSP peptides in BD, as compared with the related disease (recurrent oral ulcers), unrelated disease, and healthy controls (p < 0.05 to 0.005). In order to assess the relative frequency of sensitized lymphocytes by these peptides, 7353 short term cell lines were generated from the lymphocytes of patients and controls. Peptides 111-125, 154-172, and 311-325 (p < 0.001) and peptide 219-233 (p < 0.02) yielded significantly greater frequency of STCL in BD than in healthy and disease controls. All but peptide 154-172 stimulated only the CD4+ subset of T cells, although there was no evidence that reactivity to the selected peptides is restricted by DR2 to DR7 Ag. HLA-B51 is significantly associated with BD, but there was no evidence that B51 was a restricting element, when B51+ patients were compared with B51- patients with BD, and with B51+ healthy control subjects. A comparative investigation was then carried out between the corresponding mycobacterial and human HSP peptides. Similar or higher lympho-proliferative responses were stimulated by the human peptides compared with the mycobacterial peptides. These results suggest that the four peptide determinants within the 65-kDa HSP might be involved in the pathogenesis of BD. Whereas the high microbial load and associated stress proteins found in oral ulceration of BD may initiate an immune response to these conserved epitopes, expression of autoreactive T cell clones might be stimulated by immunodominant T cell epitopes of endogenous HSP which may induce immunopathologic changes.

The immune system has the potential to utilize a diverse T cell repertoire for the recognition of Ag in the context of MHC molecules. Here we describe the analysis of two rat T cell clones, both of which recognize a synthetic peptide comprised of the arthritis-associated 180-188 amino acid sequence of the mycobacterial 65-kDa heatshock protein (hsp65 180-188), but which differ in the recognition of the naturally processed hsp65. The arthritogenic T cell clone A2b, generated by immunization with whole Mycobacterium tuberculosis, recognized the hsp65 180-188 synthetic peptide as well as the processed hsp65, whereas T cell clone ATL11, generated after immunization with a single amino acid substituted peptide analog of hsp65 180-188, recognized peptide hsp65 180-188 but not the processed hsp65. For both T cell clones the minimal stimulatory sequence was hsp65 180-186. However, within this minimal stimulatory sequence marked differences between the clones were found with regard to peptide residues interacting with the TCR. Furthermore, addition of extra residues at the N terminus of the hsp65 180-186 sequence abrogated the recognition by clone ATL11, but not by A2b. These findings demonstrate that, upon in vivo immunization with a synthetic peptide comprised of a single amino acid variant of a T cell epitope, T cells can be triggered that recognize a peptide comprised of the original epitope sequence, but that do not recognize this epitope in its naturally processed protein fragment. The possibility of triggering such T cells by immunization with synthetic peptides, may well have consequences for the design of peptide vaccines or peptide immunomodulatory agents.

To investigate whether and in which proportion normal individuals experience a priming to microbial heat shock proteins (hsp), the presence of antibodies to two mycobacterial hsp was tested in serum sample from 2- to 4-mo-old children before and at different times after vaccination with the trivalent vaccine against tetanus, diphtheria, and pertussis (DTP). We show that 88.9% of infants vaccinated with DTP developed antibody responses to mycobacterial hsp. Such a response was due to the whole-cell pertussis component of the vaccine, because it was not observed in infants receiving an acellular pertussis vaccine. Antibodies and cells reactive to the mycobacterial 65-kDa hsp were also found in mice immunized with DTP. Interestingly, whole-cell pertussis vaccine-induced anti-hsp antibodies cross-reacted with the Escherichia coli GroEL hsp, and at a some extent with the human 60-kDa hsp, belonging to the same hsp family. These data suggest that priming of the immune system to hsp is a common phenomenon occurring very early in life.

Injection of incomplete Freund's adjuvant (IFA) into the footpads of BALB/c mice induced an acute inflammation. Draining popliteal lymph nodes showed major histocompatibility complex (MHC) class II-restricted proliferation when challenged in vitro with recombinant Mycobacterium bovis 65-kDa heat shock protein (hsp65). alpha beta T cell receptor-positive, CD4+, hsp65-specific T cell lines and clones were generated from these lymph nodes, and 87% of clones responded to a beta galactosidase fusion protein containing residues 238-573 of human hsp60. Seventy percent of these hsp60-responsive clones also responded to a synthetic peptide corresponding to residues 412-423 of the mouse hsp60. This peptide also induced significant responses in IFA-primed lymph node cells but not in lymphoid cells from unimmunized mice. These results demonstrate that T cells specific for epitopes in self hsp60 are activated during inflammatory responses induced in the absence of exogenous bacterial hsp65. The findings of this study may provide a basis for understanding the often reported isolation of mycobacterial hsp65-responsive T cells from inflammatory sites of arthritis patients, and the protective effects of preimmunization with hsp65 in experimental models of arthritis.

Peptide analogues of disease-associated epitopes were studied for inhibition of experimental allergic encephalomyelitis (EAE) and adjuvant arthritis (AA) in Lewis rats. EAE- and AA-associated analogues were selected as competitors because of their in vitro inhibitory activity on proliferation of encephalitogenic and arthritogenic T cells. Although the EAE-associated competitor had a superior major histocompatibility complex (MHC) binding affinity, the AA-associated competitor was a better inhibitor of the in vitro proliferation of arthritogenic T cells. Furthermore, although in vivo EAE was inhibited by both competitors, AA was only inhibited by the AA-associated competitor. Remarkably, in contrast to what was expected of a regular MHC competitor peptide, the AA-associated peptide analogue also prevented AA upon immunization before disease induction and appeared to induce T cell responses that crossreacted with the original disease-associated epitope. Therefore, it is concluded that antigen-specific regulatory mechanisms were involved in synergy with MHC competition. The integration of both qualities into a single "competitor-modulator" analogue peptide may lead to the development of novel, more effective, disease-specific immunomodulatory peptides.

T lymphocyte responses to heterologous or self 65-kD heat shock protein (hsp) have been implicated in the pathogenesis of various forms of arthritis. To delineate the relationship of 65-kD hsp to different synovial fluid (SF) T cell subsets, we stimulated synovial fluid (SFMC) and peripheral blood mononuclear cells (PBMC) from patients with different inflammatory rheumatic diseases and from healthy controls with human or mycobacterial 65-kD hsp, tetanus toxoid (TT), heat-killed or live Yersinia enterocolitica. Phenotyping of the resulting T cell lines revealed an increase of up to 97% TCR-gamma delta+ lymphocytes in the 65-kD hsp-stimulated SF-derived lines. This expansion of TCR-gamma delta+ cells was less pronounced with cultures of PBMC. A preferential expansion of TCR-gamma delta+ cells was also shown after SFMC stimulation with live, but not with heat-killed Yersinia or with TT. We conclude that a common mechanism is involved in the selective expansion of TCR-gamma delta+ lymphocytes upon SFMC infection with live Yersinia or upon contact with 65-kD hsp. Out of a panel of TCR-gamma delta+ T lymphocyte clones (TLC) derived from a human 65-kD hsp-stimulated line, only a minority of TLC proliferated weakly upon restimulation with this antigen in the presence of autologous monocytes, whereas TCR-alpha beta+ TLC responded vigorously to the human 65-kD hsp and in some cases also cross-recognized the mycobacterial hsp homologue and/or heat-killed Yersinia. This implies that additional factors or cells may be present in the milieu of SFMC cultures that propagate the expansion of TCR-gamma delta+ cells in response to 65-kD hsp or live bacteria.

Immune reactivity to the 65-kDa mycobacterial heat shock protein (hsp65) has been associated with arthritis in rats and humans. In this report we evaluated patients with juvenile rheumatoid arthritis for such immunity. A high proportion of affected children showed both antibody and T lymphocyte responses to hsp65 and to two related peptides: the nonapeptide 180-188 sequence of hsp65 and a partially homologous peptide of the cartilage proteoglycan link protein. The titer of circulating antibodies was generally higher in patients with clinically active disease. In contrast to the juvenile rheumatoid arthritis patients, patients with adult rheumatoid arthritis tended to have lower responses of their peripheral blood T lymphocytes to the whole hsp65 molecule. Moreover, the adult rheumatoid arthritis patients did not respond to the peptides. Thus, there appear to be immunological differences between juvenile and adult forms of rheumatoid arthritis related to hsp65 reactivity.

The mycobacterial 65 kDa heat shock protein (HSP65) is of critical significance in the model of adjuvant arthritis (AA). Arthritogenic and protective T cell clones obtained from arthritic rats recognized the 180-188 sequence of HSP65. Previous reports have shown that administration of HSP65 prior to disease induction led to resistance to arthritis in the AA model and in several other models of experimental arthritis. Here, we report the development of immunity to HSP65 and the critical 180-188 epitope during the course of AA. Following Mycobacterium tuberculosis (MT) immunization both antibodies and T cell responses to HSP65 were detected. Proliferative responses to the 180-188 epitope were seen exclusively in the local draining lymph node cells at day 14 after immunization. The anatomical distribution and course of T cell responses to HSP65 and its 180-188 epitope are compatible with T cell regulated control of the disease. Although lower HSP65 antibody levels were observed in the animals with severe arthritis, in individual animals no evidence was obtained for a relationship between development of HSP65 humoral immunity and arthritis severity. Nevertheless, during disease exacerbation, elicited by HSP65 immunization during disease development, elevated T cell responses against HSP65 and its 180-188 epitope were found. In contrast, we obtained evidence that successful transfer of arthritis resistance to naive recipients depends on the transfer of HSP65 specific T cells. On the basis of these results, it seems that HSP65 plays a crucial role in the T cell regulatory events involved in both the induction of, and protection against, AA.(ABSTRACT TRUNCATED AT 250 WORDS)

Heat-shock proteins have been shown to be critical antigens in a number of autoimmune diseases. In human arthritis and in experimentally induced arthritis in animals, disease development was seen to coincide with development of immune reactivity directed against not only bacterial hsp60, but also against its mammalian homologue. We have developed murine monoclonal antibodies after immunization with recombinant human hsp60. Antibodies with unique specificity for mammalian hsp60, not crossreactive with the bacterial counterpart (LK1), and antibodies recognizing both human and bacterial hsp60 (LK2) were selected. Both antibodies recognize epitopes located between amino acid positions 383 and 447 of human hsp60. In immunogold electron microscopy, the mitochondrial localization of hsp60 in HepG2 cells was shown. Furthermore, both LK1 and LK2 showed a raised level of staining in light microscopy immunohistochemistry of synovial membranes in patients with juvenile chronic arthritis. The increased staining for LK1, with a unique specificity for mammalian hsp60, thus unequivocally demonstrates that this is due to a raised level of expression of endogenously produced host hsp60 and not to deposition of bacterial antigens.

In a recent work, we have shown that mycobacterial heat-shock proteins (hsp) of 65-kDa (GroEL-type) and 70-kDa (DnaK-type) acted as carrier molecules in mice, previously primed with Mycobacterium tuberculosis var. bovis (bacillus Calmette-Guérin, BCG), for the induction of high and long-lasting titers of IgG against the repetitive malaria synthetic peptide (NANP)40. Anti-peptide antibodies were induced when the malaria peptide, conjugated to the mycobacterial hsp, was given in the absence of any adjuvants (Lussow et al., Eur. J. Immunol. 1991. 87:2960). In this report, we show that mice immunized with peptides or oligosaccharides conjugated to the 70-kDa hsp produced high titers of IgG antibodies in the absence of any previous priming with BCG. The anti-peptide antibody response persisted for at least 1 year. This adjuvant-free carrier effect of the 70-kDa hsp was T cell dependent, since no anti-peptide nor anti-70-kDa IgG antibodies were induced in athymic nu/nu mice. Previous immunization of mice with the 65-kDa or 70-kDa hsp did not have any negative effect on the induction of anti-peptide IgG antibodies after immunization with hsp-peptide conjugates in the absence of adjuvants. Furthermore, preimmunization with the 65-kDa hsp could substitute for BCG in providing an effective priming for the induction of anti-(NANP) antibodies. Finally, both the 65-kDa and 70-kDa hsp acted as carrier molecules for the induction of IgG antibodies to group C meningococcal oligosaccharides, in the absence of adjuvants. These findings strongly suggest that the use of hsp as carriers in conjugated constructs for the induction of anti-peptide and anti-oligosaccharide antibodies could be of value in the design of new vaccines for eventual use in humans.

By the introduction of single-amino acid substitutions in well-defined T cell epitopes of autoimmunogenic proteins, e.g., mycobacterial heat shock protein (hsp60) in adjuvant arthritis (AA) and myelin basic protein (MBP) in experimental allergic encephalomyelitis (EAE), efficiently blocking MHC binding peptides were selected. Despite the finding that a substituted variant of epitope 180-188 of hsp60 was 'blocking' not only responses of the 180-188 specific arthritogenic T cell A2b, but also responses of the MBP specific encephalitogenic T cell Z1a, in vivo testing of this competitor peptide revealed a very prominent disease inhibitory activity in AA but not in MBP-induced EAE. The selectivity of this peptide in suppressing the disease in which native 180-188 appears to be of critical relevance, offers the possibility of achieving disease specific immunological intervention. Based on the results collected so far, it seems that, in vivo in addition to blocking activity, a variant peptide itself could trigger responses that confer protective activity in AA. Such combined activities may well be required for achieving full in vivo inhibition of a disease in which multiple distinct epitopes may play a role, possibly through presentation by more than one MHC product.

Both protective immunity and immunopathology induced by mycobacteria are dependent on Ag-specific, CD4+ MHC class II-restricted T lymphocytes. The identification of Ag recognized by T cells is fundamental to the understanding of protective and pathologic immunity as well as to the design of effective immunoprophylaxis and immunotherapy strategies. Although some T cell clones are known to respond to recombinant mycobacterial heat shock proteins (hsp) like hsp3 65, the specificity of most T cells has remained unknown. We therefore have undertaken a specificity analysis of 48 well defined Mycobacterium leprae- and/or Mycobacterium tuberculosis-reactive (Th-1-like) T cell clones. Most clones (n = 44) were derived from different leprosy patients, and the remainder from one healthy control. Their HLA restriction molecules were DR2, DR3, DR4, DR5, DR7, DQ, or DP. T cell clones were stimulated with large numbers (n = 20 to 40) of mycobacterial SDS-PAGE-separated fractions bound to nitrocellulose. Each clone recognized a single fraction or peak with a particular Mr range. Some of the clones (n = 7) recognized the fraction that contained the hsp 65 as confirmed with the recombinant Ag. Most clones (n = 41), however, responded to Ag other than the hsp 65. Nine clones responded to a 67- to 80-kDa fraction. Five of them responded also to an ATP-purified, 70-kDa M. leprae protein, but only one of these five (that was HLA-DR2 restricted and cross-reactive with M. tuberculosis) recognized the recombinant C-terminal half (amino acids 278-621) of the M. leprae hsp 70 molecule and also recognized the recombinant M. tuberculosis hsp 70. We therefore have used the 5' part of the M. leprae hsp 70 gene that we have cloned recently. This fragment (that encodes amino acids 6-279) was indeed recognized by the other four M. leprae-specific T cells that were all HLA-DR3 restricted and did not cross-react with the highly homologous (95%) M. tuberculosis hsp 70. These results suggest that this novel fragment is a relevant T cell-stimulating Ag for leprosy patients. A panel of other recombinant Ag, including hsp 18 was tested. The majority of T cell clones appeared to recognize antigenic fractions distinct from hsp. In conclusion, T cells of leprosy patients see a large variety of different Ag including non-hsp, and one newly recognized moiety is the N-terminal M. leprae hsp 70 fragment.(ABSTRACT TRUNCATED AT 400 WORDS)

We have previously shown that the priming of mice with live Mycobacterium tuberculosis var. bovis (Bacillus Calmette-Guérin, BCG) and immunization with the repetitive malaria synthetic peptide (NANP)40 conjugated to purified protein derivative (PPD), led to the induction of high and long-lasting titers of anti-peptide IgG antibodies, overcoming the requirement of adjuvants and the genetic restriction of the antibody response to the peptide (Lussow et al., Proc. Natl. Acad. Sci. USA 1990. 87:2960). This initial work led us to the following observations. BCG had to be live for priming to lead to the induction of anti-peptide antibodies. Surprisingly, priming with other living microorganisms which chronically infect the macrophage (e.g. Salmonella typhimurium and Leishmania major) also induced anti-peptide antibodies in mice immunized with PPD-(NANP)40 conjugate. It was, thus, hypothesized that molecules expressed during active infection and also known to be highly conserved between species, namely the heat-shock proteins (hsp), could mediate the T cell sensitization required for the production of anti-peptide antibodies. In fact, when the PPD protion of the conjugate was replaced by a highly purified recombinant protein corresponding to the 65-kDa (GroEL-type) hsp of M. bovis, this resulted in the production of anti-(NANP) IgG antibodies in BCG-primed mice, irrespective of the major histocompatibility complex-controlled responsiveness to the (NANP) sequence itself. Further, similar induction of anti-peptide antibody response was also obtained with a recombinant 70-kDa (DnaK-type) hsp of M. tuberculosis, but not with a small molecular mass (18 kDa) of M. leprae. Finally, an adjuvant-free carrier effect for anti-peptide IgG antibody production in BCG-primed mice, was also exerted by the GroEL hsp of Escherichia coli. This finding that hsp can act as carrier molecules without requiring conventional adjuvants is of potential importance in the development of vaccine strategies.

CD4+ T cell clones specific for the mycobacterial hsp 65 were obtained from synovial fluid of a DR4 homozygous rheumatoid arthritis (RA) patient. A stimulatory epitope was defined using both deletion mutants of the mycobacterial hsp 65 and synthetic peptides and proved to be in a highly conserved region of the molecule. Despite this, however, there was no recognition by these clones of either the recombinant human homologue of mycobacterial hsp 65, P60, nor of a synthetic peptide containing an amino acid sequence from P60 corresponding to the epitope defined in the mycobacterial hsp 65. When the pattern of HLA restriction shown by the hsp-65-specific T cell clones was investigated, all clones tested proved to be restricted by HLA-DP rather than the more usual HLA-DR. Inhibition experiments suggested that this restriction also applied to the polyclonal synovial T cell response to hsp 65, but not to other antigens. Exclusive restriction of T cell recognition of an antigen by HLA-DP has not been reported previously, and strongly suggests that in this case the T cell repertoire for recognizing hsp 65 in the context of DR4 is deficient. Such an association between DR4 and the inability to respond to an immunodominant bacterial antigen may have implications for the pathogenesis of RA.

Humoral and cellular immune reactions to heat-shock proteins have been implicated in the pathogenesis of arthritis. Heat-shock proteins occur in bacteria as well as all eukaryotes and have been highly conserved during evolution. Cross-reactivity between bacterial and human heat-shock proteins induced at the site of inflammation may underlie the pathogenesis of some forms of arthritis. In order to test this hypothesis, we raised and cloned a Yersinia-specific T cell line from the synovial fluid lymphocytes of a patient with Yersinia-induced reactive arthritis. From this line we obtained a CD4+ T cell clone that proliferated in response to Yersinia antigens and both to the mycobacterial and the human 65-kDa heat-shock protein. This T cell clone also proliferated in response to autologous heat-stressed antigen-presenting cells as well as to synovial fluid mononuclear cells from the inflamed joint, thus showing true autoreactivity against endogenously synthetized self-antigen. These results demonstrate the induction of an autoimmune T cell response by a natural bacterial infection and support the important role of heat-shock proteins in the pathogenesis of immune-mediated arthritis.

A postulated mechanism for autoimmune disorders is that the immunoreactivity develops against bacterial antigens which show a high degree of sequence homology with mammalian proteins. The mycobacterial 65 kD heat shock protein (hsp) has been implicated in several forms of arthritis. Substantial amounts of the human 60 kD homologue (hsp60) were produced by insertion of the gene into Escherichia coli. To investigate the hypothesis that T-cell reactivity is directed against the endogenous hsp, T-cell proliferation of synovial-fluid and peripheral-blood mononuclear cells in response to hsp60 was studied in samples from six patients with juvenile chronic arthritis (JCA) and nine adult patients with rheumatoid arthritis (RA). There was no T-lymphocyte proliferative response to purified fractions of hsp60 in mononuclear cells from RA patients or healthy children and young adults. However, both synovial-fluid and peripheral-blood mononuclear cells from JCA patients showed substantial proliferative responses. There was a significant correlation between the stimulation indices for human hsp60 and for mycobacterial hsp65 (r = 0.948, p less than 0.02). A similar correlation for hsp60 and mycobacterial hsp70 did not achieve significance. Immunohistochemistry showed that hsp65 and hsp70 homologues were expressed in the synovial membrane in these patients but not in controls. These findings suggest a sequence of events in which hsps become expressed during synovial inflammation and function as autoantigens. In JCA this may be manifested by specific T-cell reactivity which apparently is lost in the more bone-eroding and non-remitting adult disease.

Adjuvant arthritis (AA) can be induced in genetically susceptible rats by immunization with heat-killed mycobacteria suspended in mineral oil. From our analysis of arthritogenic T cell clone A2b, obtained from an arthritic Lewis rat and specific for the 180-188 epitope of mycobacterial 65-kDa heat-shock protein (hsp 65), the possible origin of AA was explained by the existence of a molecular mimicry of the 180-188 epitope with a cartilage-associated self antigen. We now have shown that Lewis rats respond to the 180-188 epitope after Mycobacterium tuberculosis immunization and that arthritis-resistant Fisher and (Lewis x Fisher)F1 rats, although major histocompatibility complex class II identical with Lewis, do not respond to this epitope. However, in rare cases of arthritis in Fisher rats, responses to the epitope were seen. We obtained no evidence for a defect at the level of antigen processing and presentation or for suppression in Fisher rats. Thus, non-responsiveness in Fisher rats was likely due to a difference at the level of the T cell repertoire. Previously, we have reported that pretreatment with hsp 65 in experimental arthritis, and not only in AA, caused resistance to arthritis induction. We now present evidence that immunization with hsp 65 or in vitro stimulation with hsp 65 may lead to inhibition of responses specific for epitope 180-188. Thus the hsp 65-induced resistance to arthritis is probably caused by the induction of regulatory control specifically targeted at the 180-188 epitope. Especially in rats that tend to focus their responses on the critical 180-188 sequence, such as Lewis, regulation seems to develop following immunization with hsp 65. Since recent evidence suggests that hsp 65 and also the 180-188 epitope have a role in human arthritic conditions, the present findings are expected to contribute to further experimentation directed at exploiting hsp 65 or its epitopes for the development of new therapeutical approaches in humans.

The etiology of Behcet's syndrome (BS) is unknown, but a number of streptococcal species have been implicated. A hypothesis was postulated that a shared antigen, such as a stress protein, might account for some of these findings. Indeed, a rabbit antiserum against a 65-kDa heat shock protein of Mycobacterium tuberculosis revealed a corresponding 65-kDa band with all six Streptococcus sanguis strains examined and S. pyogenes but not with S. salivarius. By applying a panel of nine monoclonal antibodies to the mycobacterial 65-kDa heat shock protein, an approximately 65-kDa antigen was identified in the uncommon serotypes of S. sanguis ST3 and H.83 and one with a different Mr was identified in KTH-1 and S. pyogenes. Monoclonal antibodies Y1.2, C1.1, II H9, and ML30, which reacted with these streptococci, recognize residues 11 to 27, 88 to 123, 107 to 122, and 276 to 297 of the 65-kDa heat shock protein, respectively, suggesting that these residues are conserved among some uncommon serotypes of S. sanguis and S. pyogenes. Immunoblot analyses of sera from patients with BS for immunoglobulin A (IgA) and IgG antibodies revealed bands of 65 to 70 kDa with the mycobacterial heat shock protein, S. sanguis strains, and S. pyogenes, although these reactivities were also found to a lesser extent in controls. A 65- to 70-kDa band was found more frequently with S. sanguis KTH-2 or KTH-3 and IgA in serum from patients with BS than with serum from controls (P less than 0.02). Antibodies in serum were then studied by a radioimmunoassay, and in patients with BS this revealed significantly raised IgA antibodies to the recombinant 65-kDa mycobacterial heat shock protein and to soluble protein extracts of S. sanguis ST3, KTH-1, KTH-2, and KTH-3. Whereas significant anti-65-kDa heat shock protein and anti-S. sanguis ST3 antibodies were also found in sera from patients with rheumatoid arthritis and recurrent oral ulcers, the anti-S. sanguis KTH-1, KTH-2, and KTH-3 antibodies were confined to BS. The results are consistent with the hypothesis that some of the streptococcal antigens are associated with heat shock or stress proteins, which will need to be formally established by isolating heat shock proteins from streptococci.

Natural antibodies to 65 kD heat shock protein (hsp65) of Mycobacterium bovis were found in the sera of Lewis rats. The levels of these natural hsp65 antibodies differed substantially between the individual rats. Each rat was subsequently tested for its susceptibility to develop arthritis following injection of M. tuberculosis in incomplete Freund adjuvant. It was found that the incidence and severity of the induced arthritis did not differ between groups of Lewis rats with relatively high and relatively low natural antibody levels to hsp65. Inoculation of rats without natural antibodies to hsp65 with intestinal contents did not induce hsp65 antibodies, although the rats were able to respond to the antigen.

Several prominent mycobacterial protein antigens involved in antibody and T cell responses have been identified as members of highly conserved heat shock protein families. In particular, immune responses to the mycobacterial 65 kD heat shock protein (hsp65) have been implicated in the pathogenesis of autoimmune diseases both in experimental animal models and in man. Additionally, hsp65 has been shown to modulate the course of autoimmune disease in such experimental animal systems. In this report, we have examined the synthesis of heat shock proteins by a fast growing mycobacterial strain, M. vaccae, in heat stressed cultures and used the pristane induced arthritis model to investigate the immunoprophylactic and immunotherapeutic potential of heat killed M. vaccae. Heat shock of M. vaccae cultures at 48 degrees C demonstrated a 43-fold increase in hsp65 over that expressed at 37 degrees C. It is therefore suggested that heat killed M. vaccae contains sufficient hsp that can be presented in the context of appropriate adjuvant properties for use as an effective immunomodulatory agent. Immunisation experiments with M. vaccae revealed that protection or exacerbation of pristane induced arthritis was dependent on the dose (given in an oil or aqueous suspension), route and time of immunisation. In addition, it was demonstrated that the development of arthritis correlated with high levels of agalactosyl IgG and that "protected" animals had significantly depressed levels.

We have analyzed the cellular and humoral immunity to the mycobacterial 65 KDa heat shock protein (hsp65) in a group of Freund's Adjuvant-immunized rats with a limited susceptibility to Adjuvant arthritis. According to the arthritis indices during the period of study (35 days), two different groups of rats could be distinguished; a) autoimmune Adjuvant arthritic rats (AA), and b) Non-arthritic animals (NA), including both rats which did not display any disease symptoms and rats suffering mild transient inflammation. The cellular response to the immunizing agent (Mycobacterium tuberculosis) or the mitogen Concanavalin A was comparable between both groups of rats. However, we detected an impaired cellular response to the individual hsp65 antigen in the animals that did not develop the disease. On the contrary, the level of hsp65-specific antibodies was much higher in NA animals than in AA rats suggesting a protective role for the hsp65 specific antibodies.

The development of arthritis induced in mice by intraperitoneal injection of the non-antigenic mineral oil, 2,6,10,14-tetramethylpentadecane (pristane), was shown to depend on an intact immune response possibly to a heat-shock protein (hsp) in the synovium. Initial experiments suggested that some crucial event in the development of arthritis takes place early after pristane injection. First, irradiated pristane-treated mice failed to develop arthritis unless they were reconstituted with spleen cells from normal donors within 25 days of irradiation. Second, mice irradiated up to 50 days after pristane injection, but not later, did not develop arthritis. Evidence for the involvement of an immune response to heat-shock protein (hsp) comes from the finding that mice injected with mycobacterial 65-kDa hsp prior to pristane challenge had a reduced incidence of arthritis in contrast to animals pre-immunized with the E. coli hsp equivalent GroEL or with bovine serum albumin. Other experiments revealed that T cells from mice with gross morphologically defined arthritis proliferated strongly to hsp65 and to normal joint antigens, whereas T cells from animals treated with pristane which did not develop arthritis gave much smaller responses. Mice which developed arthritis also had elevated levels of anti-hsp65 IgG in comparison with non-arthritic animals. These findings strongly suggest that autoimmune reactions to an antigen which cross-reacts with hsp65 are generated in pristane-induced arthritis. It is considered that the autoimmune response is directed to a synovial antigen and that pre-immunization with hsp65 protects the animals from the development of pristane-induced arthritis by altering the specificity or quality of the immune response to this antigen.

Reversed-phase high-performance liquid chromatography with microbore columns (50 X 1.0 mm) was used effectively for the separation and analysis of proteins down to 1 ng at flow-rates of 0.1-0.2 ml/min. With the use of standard low-pressure gradient HPLC equipment, the peak volumes were five times smaller when compared with a conventional column at equal chromatographic efficiencies and analysis time. The sensitivity of detection was further increased by a reduction in solvent peaks, resulting in a 20-fold overall increase.

Biological membranes have as a major function the compartmentation of biological processes in cells and organelles. They consist of a bilayer of phospholipid molecules in which proteins are embedded. These integral membrane proteins, which cross the bilayer once or several times, generally have a higher than average hydrophobicity and tend to aggregate. Detergents are needed to remove integral membrane proteins from the lipid bilayer and they have to be present during further chromatographic purification. Predominantly, four modes of HPLC have been used alone or in combination for the purification of integral membrane proteins. These are based on differences of proteins in size (size-exclusion chromatography, SEC), electrostatic interaction (ion-exchange chromatography, IEC), bioaffinity (bioaffinity chromatography, BAC) and hydrophobic interaction (reversed-phase chromatography, RPC, and hydrophobic-interaction chromatography, HIC). SEC, IEC, BAC and HIC are used under relatively mild conditions, and buffer systems generally contain a non-ionic detergent. RPC generally has a denaturing effect on the protein and should preferably be used for the purification of integral membrane proteins smaller than 50 kD.

We report that streptococcal cell wall (SCW)-induced arthritis in rats, a T cell-dependent chronic, erosive polyarthritis, can be prevented by pretreatment of the rats with the mycobacterial 65-kD heat shock protein. This 65-kD protein shows extensive amino acid homology with prokaryotic and eukaryotic 65-kD heat shock proteins and is a ubiquitous bacterial common antigen. Both the clinical and histopathologic manifestations of the arthritis were prevented completely when rats were pretreated with 50 micrograms of 65-kD protein intraperitoneally at 35, 25, 15, or 5 d before administration of SCW. In such protected rats, SCW-specific T cell responses were suppressed, as compared with responses in arthritic rats. Pretreatment with 65-kD protein had no effect on the production of antibodies against SCW, on a nonspecific inflammatory reaction (zymosan-induced arthritis), or on general cellular immunity in vivo (delayed type hypersensitivity reaction to a nonrelated protein antigen). Furthermore, the protection against SCW arthritis was transferable by splenic T cells to naive recipients. Our data show that pretreatment with the 65-kD mycobacterial heat shock protein protects rats against a subsequent bacterium-induced arthritis. This protection is immunologically specific and resides in the lymphoid cell population.

Arthritis - induced experimentally in rats by immunization with mycobacteria has been shown to depend on specific T cell recognition of an epitope present on the mycobacterial 65-kD heat-shock protein. This particular epitope has been observed to have a structural mimicry with a cartilage-associated molecule present in the joints. Since the bacterial heat-shock proteins and the cartilage-associated molecules are of a conserved nature, one might infer from the experimental model that in humans similar mimicry could play a role in the initiation of autoimmune arthritis. Recent findings from the analysis of immunological reactivity to the 65-kD in rheumatoid arthritis patients seem to support such a role for the mycobacterial 65-kD heat-shock protein in human disease.

Nine reversed-phase materials with various bonded phases from different suppliers were studied for the separation of hydrophilic proteins with two solvent systems. Protein retention, resolution and recovery were not correlated with the nature of the hydrocarbonaceous ligand. Peak volumes increased with molecular weight, which led to broad, irregular peaks for the larger proteins on some columns. Four columns that performed equally well were selected for the purification of hydrophobic Sendai virus membrane proteins. In this case, more distinct differences were found between columns. Recovery of the membrane proteins strongly depended on the combination of column and solvent systems.

Virus envelope proteins were isolated from Triton X-100 extracts of purified Sendai virions by gel-filtration, ion-exchange and reversed-phase high-performance liquid chromatography (HPLC). The fusion protein F, the matrix protein M and the tetrameric and dimeric form of the HN protein were isolated by gel-filtration HPLC with a solvent containing 0.1% sodium dodecyl sulphate. HN and F were also isolated by ion-exchange HPLC with 0.1% Triton X-100 in the eluent. Reversed-phase HPLC was performed on a C1 column with acetonitrile as the organic solvent. Especially the F1 and F2 component of the fusion protein F were obtained in pure form. The immunological activity of the proteins after HPLC was determined with an enzyme-linked immunosorbent assay (ELISA). After gel-filtration and ion-exchange HPLC, proteins still reacted with antiserum to the intact virus while proteins purified by reversed-phase HPLC did not react.

Sendai virus envelope proteins were isolated by reversed-phase high-performance liquid chromatography. The F (F1 and F2, connected by disulphide bonds), M and HN proteins were extracted from purified Sendai virions with Triton X-100. After removal of the detergent from the extract with Amberlite XAD-2 and reduction of the proteins, separation was performed on a small (10-nm) and on a larger (30-nm) pore size C18 column. Proteins were eluted with a gradient of an ethanol 1-butanol mixture in 12 mM hydrochloric acid. On the 10-nm pore size column, F2 was completely recovered in pure form, whereas the recoveries of the other proteins were low (5-25%). Similar results were obtained with the 30-nm pore size column, except for protein F1 of which the yield was higher (50%).