Antibodies against the human heat shock protein hsp70 in patients with severe coronary artery disease

Heat shock proteins (hsps) play complex role in the function of the immune system, they can activate both humoral and cellular immune response, as well the complement system. Although autoimmunity to hsp70 was implicated in certain autoimmune diseases and other conditions, the exact role of anti-hsp70 antibodies is not known. It was demonstrated by our previous work and other's findings that antibodies against the 60 kDa hsps are strongly associated with coronary atherosclerosis and carotis disease. It is also known that there is increased hsp70 expression at different sites of atherosclerosis. Therefore our aim was to study whether level of anti-hsp70 antibodies correlate with the presence of severe coronary artery disease (CAD). We measured and compared anti-hsp70 IgG antibody levels in CAD patients (n = 99) and healthy subjects (n = 99) with ELISA. The frequency of these antibodies was high in both groups and there was no significant difference in the median level of anti-hsp70 antibodies between patients with severe CAD and controls (653 (400-1141) vs. 630 (326-1152) AU/mL, P = 0.337). Adjustment for age, sex, BMI and lipid parameters did not change this result. Furthermore we did not find a correlation between anti-hsp70 antibody levels and certain risk factors of CAD (age, lipid parameters, body mass index, C-reactive protein, gender, smoking, diabetes and anti-hsp60 antibodies). By contrast, in accordance with our previous findings, anti-hsp60 and anti-hsp65 antibody levels were significantly higher in CAD patients, compared to this control group (p < 0.0001 for both variables). We did not find any correlation between the levels of anti-hsp70 and anti-hsp60 or anti-hsp65 antibodies either in the patients or the controls. The exact role of hsp70 in atherosclerosis is controversial, but we suggest that humoral immunity against human hsp70 does not contribute to coronary atherosclerosis in contrast to antibodies against 60kDa hsps.     

Frequency of anti-hsp60, -65 and -70 antibodies in sera of patients with juvenile idiopathic arthritis

Cross-reactivity between microbial and human heat shock proteins (hsps) led to the concept that hsp might be involved in the etiopathogenesis of autoimmune diseases. We investigated antibodies to recombinant human hsp60, recombinant Mycobacterium bovis hsp65 and to stress-inducible recombinant human hsp70 using enzyme-linked immunosorbent assay (ELISA) in sera of 209 juvenile idiopathic arthritis (JIA) patients and 50 healthy controls. Anti-hsp60 antibodies did not exceed the control level in any JIA patient. The numbers of JIA patients (16/209, 7.6%) who raised anti-hsp65 antibodies was equal to healthy controls (4/50, 8%). Elevated levels of antibodies against hsp70 were found in a cohort of patients with JIA (36.8%) when compared with age-matched healthy individuals (2%). These antibodies were predominantly of IgG isotype in systemic disease and IgM isotype in oligoarthritis. In polyarthritis both IgG and IgM antibodies frequently occurred. Significantly higher anti-hsp70 antibody levels were found in RF-positive JIA patients. The levels of anti-hsp70 antibodies correlated with the severity of disease evaluated on the basis of Steinbrocker's functional classification and rtg staging system. No association between anti-hsp70 antibody levels and ANA, HLA B27 and disease duration (less than 2 years x more than 2 years) was observed except IgM anti-hsp70 antibody where significantly higher levels were also detected in HLA B27-positive patients. The prevalence of anti-hsp70 antibodies is much higher in JIA patients when compared with healthy controls, suggesting their possible role in pathological mechanism of the disease.     

Antibodies against human heat-shock protein (hsp) 60 and mycobacterial hsp65 differ in their antigen specificity and complement-activating ability.

Although complement activation appears to have an important role both in the early and late phases of atherosclerosis, the exact mechanism of the initiation of this activation is still unknown. Since injuries of the endothelial cells are known to result in increased stress-protein expression we tested the complement-activating ability of recombinant human 60 kDa heat-shock protein (hsp60). Human hsp60 was found to activate the complement system in normal human serum in a dose-dependent manner. Activation took place through the classical pathway. The lack of complement activation in agammaglobulinemic serum indicates that the classical pathway is triggered by anti-hsp60 antibodies. Hsp60 activated complement in the sera of 74 patients with coronary heart disease as well, and a strong positive correlation (r = 0.459, P < 0.0001) was found between the extent of complement activation and the level of anti-hsp60 IgG antibodies but there was no correlation to the level of anti-hsp65 IgG antibodies. Further distinction between anti-hsp60 and anti-hsp65 antibodies was obtained from competitive ELISA experiments: binding of anti-hsp60 antibodies to hsp60-coated plates was inhibited only by recombinant hsp60 and vice versa. Our present findings indicate that anti-hsp60 and anti-hsp65 antibodies are distinct, showing only partial cross-reactivity. Since complement activation plays an important role in the development of atherosclerosis and the levels of complement-activating anti-hsp60 antibodies are elevated in atherosclerosis-related diseases, our present findings may have important pathological implications.     

Antibody to heat shock protein can be used for early serological monitoring of Helicobacter pylori eradication treatment

Infection with Helicobacter pylori induces humoral immune responses against various antigens of the bacterium. Heat shock proteins (hsps) are immunodominant antigens in various diseases including H. pylori infection. In the present study, we measured the anti-hsp antibody titers in 42 patients with H. pylori-infected peptic ulcers during a bacterial eradication study. The patients were treated with a proton pump inhibitor and antimicrobial agents to eradicate the organism. Their sera were obtained at pretreatment and at 1 month and 6 months after the eradication therapy. The titers of immunoglobulin G antibodies to the H. pylori hsp, whole-cell lysate, and urease (30-kDa subunit) antigens in serum were measured by a capture enzyme-linked immunosorbent assay. The levels of H. pylori hsp60 antibodies in sera collected 1 month after treatment had declined significantly, even when changes in the titers of antibodies to whole-cell and urease antigens were not apparent. These results suggest that measurement of antibodies to H. pylori hsp60 in serum is useful for the early monitoring of the effectiveness of eradication therapy.     

Antibodies to 70 kD and 90 kD heat shock proteins are associated with graft-versus-host disease in peripheral blood stem cell transplant recipients

The development of graft-versus-host disease (GVHD) can be modified by non-MHC factors. Based on our previous studies that showed an involvement of 70kD heat shock protein (hsp70) in the pathology of acute GVHD in a rat model, we determined serum levels of antibodies to hsp70, hsp90 and hsp60 in human recipients after allogeneic peripheral blood stem cell transplantation (PBSCT). Serum levels of these antibodies were correlated with GVHD status in the recipients. Twenty-nine recipients with high-risk haematological malignances, who received G-CSF mobilized allogeneic PBSCT from HLA matched family donors, were evaluated between 30 and 960 days after transplantation. Two recipients had no GVHD, 18 developed acute followed by chronic GVHD and nine developed only chronic GVHD. Patients with acute GVHD had a significant increase in IgM anti-hsp70 and/or anti-hsp90 early (30-90 days) after transplantation. In addition, an increase in IgM anti-hsp70 and/or anti-hsp90 antibodies preceded or accompanied chronic GVHD. Antibody levels returned to normal within the next 400 days in the majority of patients. Anti-hsp60 antibody levels were not different from control levels regardless of GVHD status. This study implies that the development of acute and/or chronic GVHD in humans is accompanied by an increase in anti-hsp70 and anti-hsp90 antibodies. Monitoring levels of anti-hsp70 and anti-hsp90 antibodies in stem cell transplant recipients may serve as a diagnostic tool and help to predict the onset of GVHD.     

Antibody to Heat Shock Protein Can Be Used for Early Serological Monitoring of Helicobacter pylori Eradication Treatment

Infection with Helicobacter pylori induces humoral immune responses against various antigens of the bacterium. Heat shock proteins (hsps) are immunodominant antigens in various diseases including H. pylori infection. In the present study, we measured the anti-hsp antibody titers in 42 patients with H. pylori-infected peptic ulcers during a bacterial eradication study. The patients were treated with a proton pump inhibitor and antimicrobial agents to eradicate the organism. Their sera were obtained at pretreatment and at 1 month and 6 months after the eradication therapy. The titers of immunoglobulin G antibodies to the H. pylori hsp, whole-cell lysate, and urease (30-kDa subunit) antigens in serum were measured by a capture enzyme-linked immunosorbent assay. The levels of H. pylori hsp60 antibodies in sera collected 1 month after treatment had declined significantly, even when changes in the titers of antibodies to whole-cell and urease antigens were not apparent. These results suggest that measurement of antibodies to H. pylori hsp60 in serum is useful for the early monitoring of the effectiveness of eradication therapy.     

Epistatic effects of genes encoding immunoglobulin GM allotypes and interleukin-6 on the production of autoantibodies to 60- and 65-kDa heat-shock proteins

Immunoglobulin GM and KM genes have been associated with antibody responses to a variety of antigens. A promoter-region polymorphism of interleukin-6 (IL-6) gene (-174 G/C) has been shown to be associated with antibody responses to heat-shock proteins (hsp) 60 and hsp65. To examine the possible epistatic effects of these unlinked genetic systems on the autoimmune responses to hsp60 and hsp65, 176 healthy Caucasian subjects from Finland were genotyped for several allelic determinants of GM, KM, and IL-6 genes by PCR-RFLP methods. IgG antibodies to hsp60 and hsp65 were measured by an ELISA. Significant interactive effects of GM f,z and IL-6-174 genotypes were noted for both anti-hsp60 (P=0.002) and anti-hsp65 (P=0.038) antibody levels. Since these autoantibodies have been implicated in susceptibility to coronary heart disease and carotid atherosclerosis, the associations reported here might be relevant to the etiology of these diseases.     

Reduced levels of Hsp70 result in a therapeutic effect of 15-deoxyspergualin on acute graft-versus-host disease in (DA x LEW)F1 rats

We have shown previously that increased levels of hsp70, and antibodies reactive with hsp70 parallel the onset and severity of graft-versus-host disease (GVHD) in a parent --> (DA x LEW)F1 rat model. In this study we have assessed the effect of reducing the levels of the 70 kDa heat shock protein (hsp70), on the morbidity and mortality of acute GVHD in (DA x LEW)F1 rats. The reduction was accomplished by the administration of 15-deoxyspergualin (DSG), an immunosuppressive agent which binds to a constitutively expressed member of the 70 kDa heat shock protein family. DSG administered via three different protocols reduced GVHD-associated morbidity. One of the regimens, which consisted of intermittent DSG administration, also significantly reduced GVHD associated mortality. This DSG treatment reduced hsp70 levels in spleen and lymph nodes, inhibited anti-hsp70 antibody production, and diminished the serum levels of IL-2, IFN-gamma, TNF-alpha, and IL-10. IL-4 levels in the serum did not change during GVHD and were not effected by DSG. These results show that the mechanism of DSG immunosuppressive effect in rat GVHD may involve DSG's capacity to bind to hsp70, which in turn may lead to a decrease in levels of circulating anti-hsp70 antibodies, and reduced production of cytokines.     

Heat shock protein 70 (hsp70) as a major target of the antibody response in patients with pulmonary cryptococcosis

Cryptococcus neoformans causes infection in individuals with defective T cell function, such as AIDS, as well as without underlying disease. It has been suggested that humoral as well as cellular immunity might play an important role in the immune response to C. neoformans infection. We have recently shown, using immunoblotting, that the 70-kD hsp family of C. neoformans was the major target molecule of the humoral response in murine pulmonary cryptococcosis. In this study we also used immunoblotting to define the antibody responses in the sera of 24 patients with pulmonary cryptococcosis: 21 proven and three suspected diagnoses. Anti-C. neoformans hsp70 antibody was detected in 16 of 24 (66.7%) patients with pulmonary cryptococcosis. Fourteen of 17 (82.3%) patients with high antigen titres (> or = 1:8) and two of seven (28.6%) patients with low titres (< or = 1:4) had detectable levels of anti-hsp70 antibody. Sera from patients positive for anti-hsp70 antibody showed high titres in the Eiken latex agglutination test for the detection of serum cryptococcal antigen. Our results indicate that the 70-kD hsp family from C. neoformans appears to be a major target molecule of the humoral response, not only in murine pulmonary cryptococcosis, but also in human patients with pulmonary cryptococcosis.     

High positive frequency of antibodies to metallothionein and heat shock protein 70 in sera of patients with metal allergy

Two principal types of stress protein, heat shock proteins (hsps) and metallothionein (MT), are induced in cells responding to a variety of stresses. They play an important role in protecting cells from these stresses. However, many reports indicate that antibodies to hsps are present in human serum and are associated with several autoimmunity diseases. Metals, which are commonly allergenic to humans, induce both MT and hsp70 (one of the hsps family). Until now, there has been no report of any antibody to MT in human serum. In the present study, serum samples from healthy controls (Group I), and patients suffering from atopic dermatitis without (Group II) or with (Group III) metal allergy, were measured for antibodies to MT and hsp70, using an enzyme-linked immunosorbent assay (ELISA). Metal allergy was confirmed by patch testing. We first found that antibody to MT exists in human serum. We also found a high positive frequency of antibody to MT (51.3%) and to hsp70 (43.6%) in the sera of Group III, compared to those of Group I (3.8% and 5.1%) or Group II (6.4% and 5.1%). Furthermore, there was a strong positive correlation between antibody to MT and antibody to hsp70 in Group III (P = 0.0013), but not in Group I and Group II. Our results indicate that antibody to MT exists in human serum, as do antibodies to hsps, and suggest that elevated levels of MT and hsp70 antibodies are associated with metal allergy in atopic patients.     

Serum antibodies against the heat shock protein 60 are elevated in patients with osteosarcoma

Osteosarcoma is the most frequent malignant bone tumor, mainly occurring in the second and third decade of life. Diagnosis is limited to clinical symptoms, radiology and histology, but so far no diagnostic laboratory tests are available. Heat shock proteins (hsp), highly conserved proteins performing vital intracellular chaperoning functions and preventing cells from death, have been shown to be involved in tumor immunity. We analyzed 75 sera from 23 patients with high-grade osteosarcoma, 8 patients with chondrosarcoma, 10 patients with Ewing's sarcoma, 5 patients with soft tissue sarcoma, 11 patients with benign bone tumors at the time of diagnosis and from 18 healthy controls with an indirect one-site enzyme linked immunosorbent assay (ELISA) for the presence of anti-hsp60 and 70 antibodies. In these assays 10/23 osteosarcoma patients (43%) had anti-hsp60 antibodies with a mean +/- S.D. titer of 0.382 +/- 0.243 U/ml. Only one of the 18 healthy controls (1/18, 5.6%; titer 0.22 U/ml), two of the Ewing's sarcoma patients (2/10, 20%; titer 0.2 +/- 0.09 U/ml), two of the patients with a benign bone tumor (2/11, 18%; titer 0.22 +/- 0.16 U/ml) and one of the chondrosarcoma patients (1/8, 12.5%; titer 0.14 U/ml) were positive, whereas all others, including all soft tissue sarcomas were negative throughout. Anti-hsp60 antibodies in patients with osteosarcoma are therefore significantly increased (p < 0.05). 19/23 (83%) of osteosarcoma biopsy specimens expressed hsp60 immunohistochemically and all specimens from patients with a positive anti-hsp60 serum titer expressed hsp60. The level of the anti-hsp60 antibodies did not correlate with clinical parameters such as response to preoperative chemotherapy, duration of symptoms, age, gender, tumor size, serum alkaline-phosphatase levels and metastases. Although no difference in anti-hsp70 antibodies could be observed between sera from patients and healthy controls, a positive correlation was found for the presence of anti-hsp70 serum antibodies and lung metastases at the time of diagnosis in osteosarcoma patients. These data suggest an increase of anti-hsp60 antibodies at the time of first diagnosis of osteosarcoma. These findings should therefore give rise to further investigations on a group of new markers for the diagnosis of osteosarcoma.     

Levels of antibodies against C1q and 60 kDa family of heat shock proteins in the sera of patients with various autoimmune diseases

Previously a strong positive correlation was found between antibodies to C1q (C1qAb) and antibodies against human heat shock protein (hsp60) and mycobacterial hsp65 in HIV infected patients. Here the levels of these antibodies were measured in the sera of patients with different autoimmune diseases (122 systemic lupus erythematosus (SLE), 55 systemic sclerosis, 33 undifferentiated connective tissue disease (UCTD), 27 primary Raynaud syndrome, 21 rheumatoid arthritis (RA), 14 polymyositis/dermatomyositis (PM/DM), and 192 healthy blood donors. The prevalence of IgG C1qAb was found to be high (P<0.0001 as compared to the healthy controls) only in the SLE group. The levels of the anti-hsp60 (P=0.0094) and anti-hsp65 (P=0.0108) antibodies were high only in the UCTD patients. No correlation was found between the C1qAb and anti-hsp antibodies in any group except a significant (P=0.011) positive correlation between C1qAb and hsp65 antibodies in the patients with UCTD. These findings indicate that the autoantibodies against C1q are heterogeneous: in different diseases different types of C1qAb may dominate.     

Role of the Bowel Flora for Development of Immunity to hsp 65 and Arthritis in Three Experimental Models

An infectious aetiology in rheumatoid arthritis (RA) has for long been suggested, although no conclusive evidence for this is at present available. Lately a large interest has been devoted to the involvement of heat shock proteins (hsps) in autoimmune disorders due to their conserved structure and immunogenic properties. Immunity to hsps has been observed both in human autoimmune conditions and in experimental models of autoimmune disease. We have studied the role of the bacterial flora and hsp immunity in the arthritic response in three experimental models of arthritis; type II collagen arthritis (CIA), adjuvant arthritis (AA) and oil induced arthritis (OIA); by using germ free and conventional DA rats.
A high incidence of severe arthritis developed in all the models evaluated irrespectively of whether the animals were in the conventional or germ free state. This confirms earlier results which show a minor effect of the bacterial flora in CIA and AA in high responder strains. These results also show that a severe OIA can develop in germ free animals. Despite the severe arthritic response induced, no serum antibody levels to hsp 65 could be detected in the germ free animals, which was in contrast to the conventional animals where a positive anti-hsp 65 serum response was seen in 35–80% of the animals with CIA, AA or OIA.
These results show that development of a humoral response to hsp 65 in these models of arthritis is dependent on the presence of a bacterial flora. Further, the lack of humoral immunity in germ free animals despite a severe arthritic response indicates that humoral immunity to hsp 65 is not involved in development of disease in these three models of experimental arthritis.     

Immune response toChlamydia trachomatis heat-shock protein in infertile female patients and influence ofChlamydia pneumoniae antibodies

A total of 446 sera from 245 patients with primary or secondary infertility, all of whom were examined laparoscopically, 117 patients withChlamydia trachomatis-positive cervical swabs, and 84 control persons (50 obstetric patients and 34 female blood donors) were tested for antibodies toChlamydia trachomatis and toChlamydia pneumoniae with the microimmunofluorescence (MIF) test. MIF test antibody rates were highest in patients with complete tubal occlusion (73%) and in patients with provenChlamydia trachomatis infection (74%), whereas only 9 to 10% of the control group showedChlamydia trachomatis antibodies. Reaction to the 60 kDa antigen ofChlamydia trachomatis, a heat-shock protein (hsp) analogue, has been suggested as a possible marker for the development of chronic sequelae afterChlamydia trachomatis infection. Immunoblot analysis of 222 sera (169 infertility patients, 20 antigen-positive patients, and 33 mothers) showed a significantly higher anti-hsp antibody rate in patients with complete tubal occlusion than in infertility patients with normal fallopian tubes (76% vs. 19%, p<0.001). The presence of antibodies not only toChlamydia trachomatis but also toChlamydia pneumoniae in the MIF test was associated with a significantly higher rate of anti-hsp antibodies and with complete tubal occlusion. This association did not appear to be due to cross-reactivity betweenChlamydia pneumoniae and Chlamydia trachomatis antibodies in the MIF test.     

Preferential Binding withEscherichia colihsp60 of Antibodies Prevalent in Sera from Patients with Rheumatoid Arthritis

One hundred thirty-two patients with various connective tissue disorders, including 60 with rheumatoid arthritis (RA), had antibodies against human as well asEscherichia colihsp60 in titers significantly higher than those of normal controls. There was a correlation between titers of antibody to human hsp60 and those toE. colihsp60. Levels of antibodies against human andE. colihsp60 were lower in joint fluids than in sera, indicating little production of antibodies in the joint. Antibodies affinity-purified withE. colihsp60 bound strongly with the homologous hsp60, but weakly with human hsp60. However, antibodies affinity-purified with human hsp60 bound comparably with bothE. colihsp60 and human hsp60. Antibodies affinity-purified withMycobacterium tuberculosishsp65 bound to human hsp60 with a reactivity similar to the reactivity of those affinity-purified with human hsp60. The reactivity to the three hsp60 species was lost when sera were absorbed withE. colihsp60, while the reactivity toE. colihsp60 remained after extensive absorption withM. tuberculosishsp65 or human hsp60. These results indicate that anti-hsp60 antibodies in patients with RA and other connective tissue disorders are raised by infection with intestinal microorganisms such asE. coli.They may represent another example of autoimmune responses triggered by antigenic mimicry of host proteins to microbes and suggest that the reactivity of antibodies from RA patients withM. tuberculosishsp65 might have been a cross-reaction with theE. colihomologue.     

Anti-Hsp65 antibodies recognize M proteins of group A streptococci.

Group A streptococcal M protein and the mycobacterial heat shock protein, hsp65, are strong bacterial immunogens that have been linked to arthritis and autoimmunity. Recent evidence has shown that streptococcal arthritis and adjuvant arthritis may be related to epitopes shared between group A streptococci and hsp65. We investigated the possibility that immunological similarities were shared between streptococcal M protein and hsp65. Antibodies against the 65-kDa heat shock protein of Mycobacterium tuberculosis were tested for reactivity with group A streptococci and purified recombinant M proteins (rM5 and rM6). Rabbit polyclonal anti-hsp65 serum was highly reactive with M type 5 Streptococcus pyogenes and rM5 and rM6 proteins in an enzyme-linked immunosorbent assay (ELISA). A mouse anti-hsp65 monoclonal antibody (MAb), IIC8, reacted with streptococcal M types 5, 6, 19, 24, and 49 in an ELISA but showed no reactivity with an isogenic streptococcal mutant which did not express M protein. Anti-hsp65 MAb IIC8 recognized rM5 and rM6 proteins in the ELISA, and MAbs IIC8 and IIH9 reacted strongly with rM6 protein in Western immunoblots. The binding of M protein by anti-hsp65 MAbs was shown to be inhibited by both hsp65 and M protein. These data show that anti-hsp65 antibodies recognize streptococcal M proteins.     

Secretory granule autoantigen in insulin-dependent diabetes mellitus is related to 62 kDa heat-shock protein (hsp60).

Recent studies in NOD mice suggest that cellular and humoral responses against beta cell protein(s) cross-reactive with mycobacterial heat-shock protein, hsp60, are implicated in the development of autoimmune diabetes. However, this putative, hsp60-related autoantigen has not yet been identified nor have the preceding events triggering the autoimmunity against it. Our recent studies show that antibodies to the mammalian hsp60 bind specifically to the 62 kDa protein located to insulin secretory granules and mitochondria of pancreatic beta cells of healthy mice [1]. In islets of prediabetic NOD mice affected by insulitis, the cellular distribution of this hsp60-related antigen was found to be altered. In the present report, we have examined whether this endogenous hsp60-related protein of secretory granules serves as an autoantigen in type I diabetes. The results of Western blot analysis indicate that diabetic mice sera show reactivity to a 62 kDa islet cell antigen. The NOD mice sera that were positive in detection of the 62 kDa islet cell antigen were also able to recognize the recombinant human hsp60. Immunogold electron microscopy revealed that diabetic NOD mouse sera, cross-reactive to human recombinant hsp60, recognize the antigen located in secretory granules of beta cells. Double-immunogold labelling demonstrated that antigens recognized by both diabetic NOD mice sera and monoclonal hsp60 antibodies co-localized in the same secretory granules of beta cells. Preincubation of islet cell sections with one type of antibody blocks subsequent binding of the other, indicating that epitopes recognized by both antisera on these proteins are shared. Moreover, preadsorption of diabetic sera with the recombinant human hsp60 abolished labelling of secretory granules. These results indicate that the hsp60-related protein of beta cell secretory granules is an autoantigen in type I diabetes in NOD mice.     

Effects of vaccination with heat shock proteins on streptozotocin induced diabetes in histidine decarboxylase knockout mice

Rationale: Type 1 diabetes mellitus (T1D) is an immune mediated disease in which heat shock proteins (hsps) may be involved in the development of the disease. Furthermore, vaccination with different hsps prevented the development of multiple low-dose streptozotocin (STZ) induced autoimmune diabetes in C57BL/KSJ mice. Histamine influences many aspects of the immune response, including Th1/Th2 balance and antibody production. Therefore, a study of diabetes-related immune processes was considered of interest in histidine decarboxylase knockout (HDC-KO) mice. Aim of the study: The aim of our study was i) to characterize antibody production in response to vaccination with p277 or hsp65 in wild type (WT) BALB/c and HDC-KO mice, and ii) to establish a possible correlation between vaccination and the changes in the pattern of STZ diabetes. Materials and methods: An ELISA was employed to measure the hsp65- and p277-specific antibody levels. To induce diabetes multiple low-dose of STZ was used. Results: Vaccination with p277 and hsp65 altered the pattern of STZ diabetes both in HDC-KO and WT animals, characterized by a transient increase followed by sustained reduction of blood sugar levels as compared to controls. However, vaccination with hsp65 and p277 caused a significant anti-p277 and anti-hsp65 antibody level increase only in WT animals. Conclusion: Multiple low-doses of STZ were able to induce diabetes in HDC-KO mice and the development of diabetes was prevented by vaccination with hsps. This protection developed in spite of the fact that vaccination caused a significant antibody level increase only in WT animals. To explain the therapeutic effect of vaccination we need further examination of the HDC KO strain. Received 23 July 2007; returned for revision 21 October 2007; received from final revision 25 November 2007; accepted by I. Ahnfelt-R?nne 7 December 2007     

Immune Response to Chlamydial 60-Kilodalton Heat Shock Protein in Tears from Nepali Trachoma Patients

Although the host immune response to the 60-kDa chlamydial heat shock protein (hsp60) has been implicated in trachoma pathogenesis, no studies have examined mucosal immune responses to hsp60 in populations for which chlamydia is endemic. Tears and sera from Nepali villagers were reacted against hsp60 fusion proteins, whole hsp60, and the major outer membrane protein (MOMP). Tears from villagers without disease were anti-hsp60 immunoglobulin G (IgG) reactive in 6 (38%) of 16 villagers compared with 36 (90%) of 40 with follicular trachoma (TF) (P < 0.001); 47 (89%) of 53 with inflammatory trachoma (TI) (P < 0.001); and 31 (84%) of 37 with conjunctival scarring (TS) (P = 0.002). By multivariate analysis, odds ratios for tear hsp60 IgG immunoreactivity in villagers with TF, TI, and TS were 49.2 (confidence interval [CI], 2.7 to 898), 22.6 (CI, 3 to 170), and 13.6 (CI, 1.4 to 133), respectively. There were no significant differences for tear IgA or secretory IgA (sIgA) reactivity to hsp60 or for tear sIgA and IgG reactivity to MOMP. Serum anti-hsp60 IgG immunoreactivity was associated with TI only. These data suggest that anti-hsp60 IgG immunoreactivity represents largely locally derived antibodies, which may promote disease pathology. In contrast, nonspecific high rates of anti-hsp60 sIgA antibodies suggest chronic or repeat stimulation from an endemic source of organisms.     

Natural autoantibodies against heat-shock proteins hsp70 and gp96: implications for immunotherapy using heat-shock proteins

Immunization of mice with cognate cancer-derived heat-shock protein (hsp) preparations leads to protection from cancer growth. As hsp used for vaccination or therapy are derived from autologous cancers, questions of pathological autoimmunity are of immense significance for the ongoing translation of this approach to therapy of human cancer. Employing the sera of normal adult mice as the first antibody, highly sensitive immunoblotting revealed the presence of anti-hsp natural autoantibodies in healthy animals. Natural autoantibodies of the immunoglobulin D (IgD) isotype bind to gp96, whereas hsp70 was recognized by IgD and IgM autoantibodies. Neither hsp was recognized by the IgA, IgE or IgG immunoglobulins contained in the serum. The antigen-antibody recognition was titratable and dependent on the integrity of the IgD molecule. Sera from only a subset of the animals tested were found to be positive for autoantibodies against gp96 and hsp70, and individual and strain-specific variations were detected. Injection of gp96 into healthy mice did not show sustained or consistent anti-gp96 IgD antibody response, class switching, toxicity or pathological autoimmunity. IgD autoantibodies against gp96 and hsp70 were also not detected in the autoimmune lpr mice. These observations show the existence of a measured and tightly regulated natural immune response to hsp.