Beta-cell expression of 65-kDa heat-shock protein mRNA is function- and age-dependent.

This study examined the expression of mRNA coding for the 65-kDa heat-shock protein (HSP) in rat islet cells of different functional states and different ages. In addition, beta cells and non-beta cells purified by fluorescence-activated cell sorting were studied. Total RNA from islet cells and insulin-producing RINm5F cells was isolated and analyzed by Northern blotting using a cDNA probe coding for the human homologue to the mycobacterial 65-kDa HSP, after which blots were quantified by densitometric scanning. Isolated beta cells were found to express 65-kDa HSP mRNA. The expression was increased in Lewis islet cells exposed to heat shock or high glucose concentration, four- and three-fold, respectively (p < 0.01). In isolated beta cells cultured at high glucose concentration a doubling in the content of 65-kDa HSP mRNA was seen compared with islets cultured at low glucose concentration (p < 0.05). In islets from Lewis rats fasted for 24 h, the content of 65-kDa HSP mRNA was 42% lower than in islets isolated from normally fed Lewis rats (p < 0.01). Both in BB rats and Wistar Furth rats the content of 65-kDa HSP mRNA was found to be higher in the 30- and the 60-day-old rats compared with the neonatal animals (p < 0.01). The expression of 65-kDa HSP mRNA was increased in RINm5F cells following heat shock, while no induction was seen after stimulation with glucose, TPA or IBMX. It is concluded that the 65-kDa heat-shock protein belongs to the family of inducible functional antigens in beta cells, which strengthens the interest in 65-kDa HSP as an antigen possibly involved in the initiation of autoimmune beta-cell destruction.     

Adjuvant arthritis and immunity to the mycobacterial 65 kDa heat shock protein.

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)     

Searching for the cartilage-associated mimicry epitope in adjuvant arthritis

Adjuvant arthritis (AA) is a T cell mediated disease which can be induced in genetically susceptible rats by immunization with heat-killed Mycobacterium tuberculosis (Mt) suspended in incomplete Freund's adjuvant. The critical mycobacterial T cell epitope for the induction of AA was previously identified as residues 178-186 of the mycobacterial 65 kDa heat shock protein (Mt. hsp65(178-186)). It was suggested that the development of AA was due to molecular mimicry between a mycobacterial epitope and a cartilage-associated self-antigen. However, until now such cartilage-associated mimicry epitope has not been identified. In this study we designed a computer search profile to predict mimicry self-epitopes, and investigated whether one or more of these self-epitopes could serve as mimicry epitopes in AA. Although several of these self-epitopes were recognized by arthritogenic T cells, no cross-reactivity was found between T cells specific for these self-epitopes and Mt. hsp65(178-186) specific T cells.     

Searching for the Cartilage-associated Mimicry Epitope in Adjuvant Arthritis

Adjuvant arthritis (AA) is a T cell mediated disease which can be induced in genetically susceptible rats by immunization with heat-killed Mycobacterium tuberculosis ( Mt ) suspended in incomplete Freund's adjuvant. The critical mycobacterial T cell epitope for the induction of AA was previously identified as residues 178-186 of the mycobacterial 65 kDa heat shock protein ( Mt. hsp65 178-186 ). It was suggested that the development of AA was due to molecular mimicry between a mycobacterial epitope and a cartilage-associated self-antigen. However, until now such cartilage-associated mimicry epitope has not been identified. In this study we designed a computer search profile to predict mimicry self-epitopes, and investigated whether one or more of these self-epitopes could serve as mimicry epitopes in AA. Although several of these self-epitopes were recognized by arthritogenic T cells, no cross-reactivity was found between T cells specific for these self-epitopes and Mt. hsp65 178-186 specific T cells.     

Modulation of experimental autoimmunity: treatment of adjuvant arthritis by immunization with a recombinant vaccinia virus.

Live recombinant vaccinia viruses, expressing antigens from pathogenic microorganisms, are studied for their use as vaccines designed for the protection against infectious diseases. Infections with these vaccinia virus recombinants, expressing proteins or epitopes from viruses, parasites, or bacteria, have resulted in the development of specific neutralizing antibodies or cytotoxic T lymphocytes. Here, we describe the generation of a recombinant vaccinia virus expressing the mycobacterial 65-kDa heat shock protein (HSP65). A vaccinia recombinant virus was constructed by placing the gene for the Mycobacterium bovis BCG HSP65 under control of a vaccinia virus promoter and inserting this mycobacterial gene in the thymidine kinase locus of the vaccinia virus genome. Mycobacterial HSP65 is a critical antigen in the autoimmune model of adjuvant arthritis induced in Lewis rats by the immunization with Mycobacterium tuberculosis. We report the induction of immunity directed to this mycobacterial HSP65 by testing for the presence of specific antibodies and T-cell proliferation. Furthermore, induction of such immunity resulted in a reduction of arthritis severity when given to rats before or, even more interestingly, during development of arthritis. Disease reduction was not found after administration of HSP65 in the absence of vaccinia virus as a vector when given during arthritis development. Therefore, recombinant vaccinia virus may offer new prospectives for specific intervention in autoimmunity.     

Oral administration of type II collagen suppresses pro-inflammatory mediator production by synoviocytes in rats with adjuvant arthritis

The objective of this study was to investigate the effect of the oral administration of type II collagen (CII) on pro-inflammatory mediator production by synoviocytes in rats with adjuvant arthritis (AA). Sprague-Dawley rats were fed with bovine CII either before immunization with Complete Freund's adjuvant (CFA) or after initiation of arthritis. Hind paw secondary swelling was measured and synoviocytes were harvested. Sera from portal vein of oral tolerized rats were collected and in vitro synoviocytes culture or synoviocytes-Peyer's Patches (PP) cells coculture system were developed. Interleukin (IL)-1 activity was measured by a mouse thymocyte activation assayed by MTT dye reduction and tumour necrosis factor (TNF) activity was measured by an L929 cytotoxicity bioassay. Nitric oxide (NO) and malondialdehyde (MDA) levels were measured by biochemical methods. We found that feeding with CII (5, 50 and 500 micro g/kg) for 7 days before immunization significantly suppressed hind paw secondary swelling measured at day 16, 20, 24 and 28 (all P < 0.01) and pro-inflammatory mediator (IL-1, TNF, NO and MDA) production by synoviocytes (all P < 0.01) in rats with AA. Feeding with CII (5, 50 and 500 micro g/kg) for 7 days after initiation of arthritis had a similar effect. CII (1, 10, 100 micro g/ml) had no effect on IL-1 and TNF production by synoviocytes in vitro, but CII 10 micro g/ml suppressed IL-1 and TNF production by synoviocytes-PP cells coculture system (P < 0.01), which was antagonized by anti-TGF-beta antibody (10 micro g/ml) (P < 0.01). Portal serum (1 : 10) from oral tolerized rats suppressed IL-1 and TNF production by synoviocytes (P < 0.01), which was also antagonized by anti-TGF-beta antibody (10 micro g/ml) (P < 0.01). We conclude that oral administration of CII had prophylactic and therapeutic effects on AA and over-production of IL-1, TNF, NO and MDA by synoviocytes was suppressed. Bystander active suppression may be the main mechanism of oral CII in the suppression of synoviocyte function.     

Human T cells recognize mycobacterial heat shock proteins in the context of multiple HLA-DR molecules: studies with healthy subjects vaccinated with Mycobacterium bovis BCG and Mycobacterium leprae.

Heat shock proteins (HSP) are considered to be important targets of the immune response to mycobacteria and, as such, relevant to subunit vaccine design. If HSP are major antigens in cell-mediated immunity, they should be recognized in the context of most of the HLA-DR molecules required for presentation of mycobacterial antigens to T cells. We tested peripheral blood mononuclear cells (PBMC) and T-cell lines from Mycobacterium leprae- and M. bovis BCG-vaccinated subjects for proliferation in response to the 18- and 65-kDa HSP of M. leprae, the 65-kDa HSP of M. bovis BCG, and the 70-kDa HSP of M. tuberculosis. Irrespective of HLA types, PBMC showing a strong response to M. leprae proliferated in response to mycobacterial HSP. HLA restriction analysis with T-cell lines showed that the M. leprae 18-kDa HSP was recognized in the context of HLA-DR4, HLA-Dw4, and HLA-DR1 molecules. The T-cell lines recognized the M. leprae 65-kDa HSP in the context of all of the HLA-DR molecules expressed by autologous antigen-presenting cells, i.e., HLA-DR1, HLA-DR2, HLA-DR5, HLA-DR7, and importantly HLA-DR4 (HLA-Dw4 and HLA-Dw14), which is relevant to autoimmunity. The M. tuberculosis 70-kDa antigen was also presented to the T-cell lines by HLA-DR1, HLA-DR2, HLA-DR5, and HLA-DR7 molecules. In addition, this HSP was recognized in the context of the HLA-DRw53 molecule, which is frequently expressed in many regions where leprosy is endemic. The T-cell lines proliferating in response to a given HSP lysed autologous monocytes-macrophages pulsed with that HSP. The results demonstrate that PBMC from individuals immunized with M. leprae respond to mycobacterial HSP and that these HSP are presented to T cells by multiple HLA-DR molecules, a prerequisite for their application in the next generation of subunit vaccines.     

Autoreactive CD4- CD8- alpha beta T cells to vaccinate adjuvant arthritis.

Studies suggested that experimental autoimmune diseases can effectively be prevented and treated by application of normal autoreactive T cells or autoreactive T cells in an attenuated form. In this study, several autoreactive CD4- CD8- T-cell clones (A2, A6, and A13 cells) were isolated for the first time from the draining lymph nodes of Lewis rats with adjuvant arthritis (AA). Surprisingly, intraperitoneal inoculation with A13 cells, but not A2 or A6 cells protected rats from AA both clinically and histologically. It was demonstrated that A13 cells were CD4- CD8- alpha beta T cells, and showed proliferative responses to irradiated syngeneic spleen cells (antigen-presenting cells; APC). Interestingly, A13 cells proliferated against concanavalin A (Con A) and staphylococcal enterotoxin B (SEB), but did not show any proliferation to Mycobacterium tuberculosis (Mt), or its 65 000 MW heat-shock protein (HSP). Rats protected from AA by inoculation with A13 cells showed a specific anti-idiotypic delayed-type hypersensitivity reaction compared with other autoreactive T cells (A2 or A6 cells). These findings demonstrate that AA can be suppressed by autoreactive CD4- CD8- alpha beta T cells, and these cells may be used as therapeutic agents in experimental autoimmunity.     

Antibodies to the mycobacterial 65-kd heat-shock protein are reactive with synovial tissue of adjuvant arthritic rats and patients with rheumatoid arthritis and osteoarthritis.

The expression of 65-kd mycobacterial heat-shock protein (HSP)-related antigens in synovial membrane from rats and humans with arthritis was studied using three monoclonal antibodies and one polyclonal antiserum directed to antigens of mycobacteria. The antibodies labeled synovial tissue sections from both adjuvant arthritis (AA) rats and from patients with either rheumatoid arthritis (RA) or osteoarthritis (OA); especially the synovial lining cells appeared to be positive. The cytoplasmic staining patterns in rats and humans were essentially the same and were not related to the extent of inflammation ie, the size of lymphoid infiltration. In control tissues no cytoplasmic staining was observed. The results suggest a role for a 65-kd HSP or a cross-reactive molecule in the immunopathologic process of arthritic disease.     

Identification of Promiscuous Epitopes from the Mycobacterial 65-Kilodalton Heat Shock Protein Recognized by Human CD4+ T Cells of the Mycobacterium leprae Memory Repertoire

By using a synthetic peptide approach, we mapped epitopes from the mycobacterial 65-kDa heat shock protein (HSP65) recognized by human T cells belonging to the Mycobacterium leprae memory repertoire. A panel of HSP65 reactive CD4(+) T-cell lines and clones were established from healthy donors 8 years after immunization with heat-killed M. leprae and then tested for proliferative reactivity against overlapping peptides comprising both the M. leprae and Mycobacterium tuberculosis HSP65 sequences. The results showed that the antigen-specific T-cell lines and clones established responded to 12 mycobacterial HSP65 peptides, of which 9 peptides represented epitopes crossreactive between the M. tuberculosis and M. leprae HSP65 (amino acids [aa] 61 to 75, 141 to 155, 151 to 165, 331 to 345, 371 to 385, 411 to 425, 431 to 445, 441 to 455, and 501 to 515) and 3 peptides (aa 343 to 355, 417 to 429, and 522 to 534) represented M. leprae HSP65-specific epitopes. Major histocompatibility complex restriction analysis showed that presentation of 9 of the 12 peptides to T cells were restricted by one of the 2 HLA-DR molecules expressed from self HLA-DRB1 genes, whereas 3 peptides with sequences completely identical between the M. leprae and M. tuberculosis HSP65 were presented to T cells by multiple HLA-DR molecules: peptide (aa 61 to 75) was presented by HLA-DR1, -DR2, and -DR7, peptide (aa 141 to 155) was presented by HLA-DR2, -DR7, and -DR53, whereas both HLA-DR2 and -DR4 (Dw4 and Dw14) were able to present peptide (aa 501 to 515) to T cells. In addition, the T-cell lines responding to these peptides in proliferation assays showed cytotoxic activity against autologous monocytes/macrophages pulsed with the same HSP65 peptides. In conclusion, we demonstrated that promiscuous peptide epitopes from the mycobacterial HSP65 antigen can serve as targets for cytotoxic CD4(+) T cells which belong to the human memory T-cell repertoire against M. leprae. The results suggest that such epitopes might be used in the peptide-based design of subunit vaccines against mycobacterial diseases.     

Interleukin-4 inhibits secretion of interleukin-1beta in the response of human cells to mycobacterial heat shock proteins.

Cellular activation induced by Mycobacterium bovis bacillus Calmette-Guérin (BCG) and heat shock proteins (HSP) leads to the production of proinflammatory cytokines such as interleukin-1beta (IL-1beta) and IL-6. In this study, we found that IL-4 significantly suppressed IL-1beta secretion induced by BCG and the 70- and 65-kDa HSP. When exogenous recombinant human IL-4 was added to human mononuclear cells, a dose- and time-related inhibition of the 70-kDa HSP- and BCG-induced IL-1beta secretion was observed. IL-1beta secretion was maximally inhibited at 24 h of culture, and this inhibitory effect was sustained at a later time point of culture (120 h). In addition, IL-2, another T-cell-derived cytokine acting on monocytes, had no effect on IL-1beta secretion induced by either BCG or the 70-kDa HSP, indicating that in these experiments not all cytokines could immunoregulate IL-1beta secretion. This inhibitory effect was not due to a cytotoxic effect of IL-4, since the viabilities of human mononuclear cells were comparable in the presence and absence of IL-4. IL-4 was also able to inhibit the secretion of IL-1beta by mycobacterium-stimulated cells from three rheumatoid arthritis patients. This inhibitory effect of IL-4 was reversed with a blocking anti-IL-4 antibody. Finally, IL-4 inhibited IL-6 secretion by mycobacterium-activated human cells. These results suggest that IL-4 may be important in the regulation of the immune response to mycobacterial antigens.     

Modulation of adjuvant arthritis in Lewis rats by recombinant vaccinia virus expressing the human 60-kilodalton heat shock protein.

The immune response to the mycobacterial 65-kDa heat shock protein (hsp65) is considered an important event in the induction of adjuvant arthritis (AA) in rats; this induction probably occurs through a molecular mimicry mechanism involving cross-reactivity against the rat homolog hsp60. To analyze the role of mammalian molecule hsp60 in arthritis, we generated a recombinant vaccinia virus (hsp60-VV) carrying the human hsp60 gene inserted into the thymidine kinase locus under the control of the 7.5k vaccinia virus promoter. Human hsp60 is almost identical to its rat homolog (97.4% linear amino acid homology) and shares about 50% of amino acid positions with Mycobacterium tuberculosis hsp65. The latter supposedly carries a critical epitope for AA induction that is not present in human hsp60. Infections with hsp60-VV of monkey cell cultures led to the expression of the human hsp60 molecule, as evidenced by immunoblotting analysis with specific monoclonal antibodies. Also, Lewis rats infected with hsp60-VV produced specific antibodies, demonstrating the in vivo expression of human hsp60 in the infected animals. Therefore, we used hsp60-VV to analyze whether the delivery of hsp60 could affect the induction of AA in Lewis rats. hsp60-VV clearly reduced and retarded arthritic symptoms when administered to rats at day 7 after AA induction. In contrast, inoculation of rats with a control recombinant vaccinia virus did not affect the course of the disease. The improvement in AA with hsp60-VV administration was associated with a specific immune response, as determined by the presence of antibodies to hsp60 in the sera and the proliferation induced by hsp60 of T cells from popliteal lymph nodes. These results support a critical role for immunity to heat shock proteins in AA. Since the protective construct is virtually identical to rat homolog hsp60, we conclude that immunity directed to conserved areas of this family of proteins is directly involved in the pathogenesis of AA.     

Antibody responses to the repetitive Plasmodium falciparum antigen Pf332 in humans naturally primed to the parasite

The 65 kD heat shock protein (HSP) has been implicated in the aetiology of recurrent aphthous stomatitis (RAS). We have previously demonstrated that peptide 91-105 derived from the sequence of mycobacterial 65 kD HSP stimulates specifically lymphocytes from patients with RAS. In this investigation, we show that both CD4+ and CD8+ T cells were significantly stimulated with mycobacterial peptide 91-105. In contrast, the human homologous peptide 116-130 stimulated only CD4+ T cells. Inhibition studies showed that CD4+ T cells were class II restricted, whereas CD8+ T cells were class I restricted. We then used truncated or substituted peptides, and demonstrated that residues 95-105 appear to be important, and residue 104(Arg) critical, in stimulating the T cells. Thus, peptide 95- 105 may constitute a T-cell proliferative epitope in RAS. We postulate that the high load of micro-organisms that colonize the oral mucosa may initiate an immune response by the microbial HSP 65-derived peptide 95-105, stimulating the numerous Langerhans cells in the oral mucosa to activate a cross-reacting immune response to the homologous peptide 116-130 within the epithelial HSP 60, initiating the immunopathological changes that lead to RAS.     

Nasal tolerance in experimental autoimmune myasthenia gravis (EAMG): induction of protective tolerance in primed animals

Nasal administration of μg doses of acetylcholine receptor (AChR) is effective in preventing the development of B cell-mediated EAMG in the Lewis rat, a model for human MG. In order to investigate whether nasal administration of AChR modulates ongoing EAMG, Lewis rats were treated nasally with AChR 2 weeks after immunization with AChR and Freund's complete adjuvant. Ten-fold higher amounts of AChR given nasally (600 μg/rat) were required to ameliorate the manifestations of EAMG compared with the amounts necessary for prevention of EAMG. In lymph node cells from rats receiving 600 μg/rat of AChR, AChR-induced proliferation and interferon-gamma (IFN-γ) secretion were reduced compared with control EAMG rats receiving PBS only. The anti-AChR antibodies in rats treated nasally with 600 μg/rat of AChR had lower affinity, reduced proportion of IgG2b and reduced capacity to induce AChR degradation. Numbers of AChR-reactive IFN-γ and tumour necrosis factor-alpha (TNF-α) mRNA-expressing lymph node cells from rats treated nasally with 600 μg/rat of AChR were suppressed, while IL-4, IL-10 and transforming growth factor-beta (TGF-β) mRNA-expressing cells were not affected. Collectively, these data indicate that nasal administration of AChR in ongoing EAMG induced selective suppression of Th1 functions, i.e. IFN-γ and IgG2b production, but no influence on Th2 cell functions. The impaired Th1 functions may result in the production of less myasthenic anti-AChR antibodies and contribute to the amelioration of EAMG severity in rats treated with AChR 600 μg/rat by the nasal route.     

The mode of action of treatment by IgG of haemolytic anaemia induced by an anti-erythrocyte monoclonal antibody

Tolerization of pathogenic antigens is one of the experimental strategies that has been proposed to prevent autoimmune disease. We have investigated here whether neonatal intraperitoneal infection of Lewis rats with Mycobacterium bovis-BCG has any effect on the expression of adjuvant arthritis (AA), an autoimmune disease that is produced by immunization of the rats with dead mycobacteria in mineral oil (i.e. Freund's complete adjuvant (FCA)). We found that neonatal infection with 10⁸ viable BCG bacilli rendered all Lewis rats resistant to the expression of AA after FCA immunization. This BCG-induced protection from reactive arthritis was not seen in Lewis rats infected with smaller inocula (10⁶ BCG bacilli) or if the infection was performed after the neonatal period (e.g. at 3 weeks of age). Neonatal administration of 65-kD mycobacterial heat shock protein (hsp65, a key antigen in the etiopathogenesis of AA) failed to protect Lewis rats from AA; injection of lactoferrin (an autoantigen that may be involved in the physiopathology of autoimmune arthritis) to newborn Lewis rats decreased the severity of AA observed after FCA immunization of the animals. Western blotting revealed that Lewis rats that had acquired resistance to AA also showed changes in their repertoire of antibody specificities; among these alterations was decreased anti-hsp65 reactivity. We conclude that neonatal infection with BCG, but not hsp65 injection, renders Lewis rats resistant to AA and that the phenomenon is associated with change in the repertoire of specificities of circulating antibodies.     

T cell reactivity to an epitope of the mycobacterial 65-kDa heat-shock protein (hsp 65) corresponds with arthritis susceptibility in rats and is regulated by hsp 65-specific cellular responses.

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.     

Immunological characterization of a human homolog of the 65-kilodalton mycobacterial antigen.

A human mitochondrial protein, designated P1 (63 kilodaltons [kDa], shows extensive sequence homology (47% identical residues and an additional approximately 20% conserved changes) to the 65-kDa mycobacterial antigen. To understand the relationship of these proteins, the cross-reactivity of several monoclonal antibodies directed against the 65-kDa Mycobacterium leprae antigen towards human, Chinese hamster, chicken, and bacterial cells has been examined. A number of antibodies (Y1-2, ML 30-A2, and F47-9-1) were found to cross-react with a 63-kDa antigen in vertebrate cell extracts and stained mitochondria in immunofluorescence studies. Some of these antibodies also reacted with a P1-beta-galactosidase fusion protein in recombinant Escherichia coli cells, expressing part of the human P1 protein. These results provide strong evidence that P1 is the mammalian homolog of the 65-kDa antigen. The human P1 protein also shows significant similarity (P less than 0.001) to a number of other bacterial and viral proteins including the pol polyprotein of human immunodeficiency viruses and the penicillin-binding protein of Neisseria gonorrhoeae. The observed similarity between human P1 protein and the major antigenic proteins of pathogenic organisms (e.g., 60- to 65-kDa mycobacterial antigen) suggests its possible involvement in autoimmune diseases (e.g., rheumatoid arthritis) by antigenic mimicry.     

口服鸡II型胶原治疗大鼠实验性类风湿性关节炎的研究

本文研究口服鸡II型胶原 (CCII)治疗大鼠类风湿性关节炎的作用。以CCII和完全弗氏佐剂免疫Wistar大鼠 ,建立大鼠的类风湿性关节炎 (RA )模型。用CCII进行口服 ,观察实验组和对照组动物关节炎的发病程度。并以ELISA法对发病大鼠血清中的抗CCII抗体的水平进行检测。结果表明 ,口服CCII可以减轻大鼠关节炎的发病程度。其中口服 6μg、 60 μgCCII的实验组大鼠关节炎指数均较对照组明显减低 ,且差异有显著性意义 (P <0 0 1;P <0 0 5 )。而口服 60 0 μgCCII的实验组大鼠关节炎指数虽较对照组大鼠降低 ,但二者相比 ,无显著性差异 (P >0 0 5 )。ELISA检测结果显示 :口服低剂量 (6μg和 60μg )可溶性CCII对大鼠体内抗CCII抗体的产生有一定的抑制作用。口服CCII对关节炎大鼠的发病程度有较明显的抑制作用 ,为应用口服耐受治疗RA等自身免疫性疾病提供了实验基础     

Reactive oxygen species (ROS), but not nitric oxide (NO), contribute to strain differences in the susceptibility to experimental arthritis in rats

There is extensive evidence for the critical role of reactive oxygen species (ROS) and nitric oxide (NO) produced by phagocytes in development of inflammatory processes and pathogenesis of numerous diseases, including rheumatoid arthritis (RA). Apart from their function as mediators of inflammation and tissue damage, recent research supports their role as signaling and regulatory molecules. In the present study we have investigated the production of ROS and NO over the course of adjuvant arthritis (AA) and oil-induced arthritis (OIA), by resident peritoneal macrophages of two rat strains: Dark Agouti (DA), susceptible, and Albino Oxford (AO), resistant to induction of AA and OIA. We have compared levels of ROS and NO produced by susceptible vs. resistant rat strain, and investigated their relevancy for arthritis development and severity. In addition, we have stimulated macrophages in vitro with Mycobacterium bovis BCG, and two heat shock proteins (HSP): endogenous HSP47 and mycobacterial HSP71 (mHSP71). Our results suggest a possible contribution of increased ROS production to arthritis resistance of AO rats. The ROS production in AO rats is potentiated by endogenous HSP47, but not with mycobacterial cell and mHSP71, suggesting HSP47 participates in AA control. We have found no fundamental relationship between the magnitude of NO production and AA and OIA susceptibility and severity, suggesting that NO has no effector role in AA and OIA. Our results advocate a regulatory type action of NO molecule might be more significant in arthritis development.     

A Monoclonal Antibody to the Mycobacterial 65kDa Heat Shock Protein (ML 30) Binds to Cells in Normal and Arthritic Joints of Rats

A monoclonal antibody reactive with the mycobacterial 65 kDa heat shock protein (ML 30) was investigated for reactivity with biopsies from normal rat joints and with inflamed joints due to adjuvant arthritis (AA) or collagen induced arthritis (CIA). Immunohistochemical stainings with the anti-hsp 65 antibody on paraffin sections from normal rat joints revealed a weak but exclusive staining of cells within the synovial lining. Also normal chondrocytes and bone marrow cells showed occasional staining. In biopsies from inflamed joints obtained from rats suffering from AA or CIA, an intense staining with ML 30 was seen within the cartilage-pannus junction as well as sites of bone erosion. An increased staining, compared with the normal, was also seen in chondrocytes of the eroded cartilage and in some bone marrow cells. No staining with ML 30 was seen in biopsies from inflammatory lesions due to delayed type hypersensitivity reactions in the skin of rats. Reactivity of ML 30 was also seen in a Western blot assay performed on lysates from inflamed synovia from rats with CIA, preferentially with a component slightly below 60 kDa in molecular weight. The demonstration of epitopes cross-reactive with hsp 65 of mycobacteria in normal and, in higher quantity, in arthritic rat joints, suggests, together with our preliminary biochemical findings, that a recently identified mammalian counterpart to bacterial hsp 65 is both preferentially expressed in normal joints and subject to increased expression in arthritis of different aetiologies.