In silico analyses of heat shock protein 60 and calreticulin to designing a novel vaccine shifting immune response toward T helper 2 in atherosclerosis.

Recent experiments demonstrated that atherosclerosis is a Th1 dominant autoimmune condition, whereas Th2 cells are rarely detected within the atherosclerotic lesions. Several studies have indicated that Th2 type cytokines could be effective in the reduction and stabilization of atherosclerotic plaque. Therefore, the modulation of the adaptive immune response by shifting immune responses toward Th2 cells by a novel vaccine could represent a promising approach to prevent from progression and thromboembolic events in coronary artery disease. In the present study, an in silico approach was applied to design a novel multi-epitope vaccine to elicit a desirable immune response against atherosclerosis. Six novel IL-4 inducing epitopes were selected from HSP60 and calreticulin proteins. To enhance epitope presentation, IL-4 inducing epitopes were linked together by AAY and HEYGAEALERAG linkers. In addition, helper epitopes selected from Tetanus toxin fragment C (TTFrC) were applied to induce CD4+ helper T lymphocytes (HTLs) responses. Moreover, cholera toxin B (CTB) was employed as an adjuvant. A multi-epitope construct was designed based on predicted epitopes which was 320 residues in length. Then, the physico-chemical properties, secondary and tertiary structures, stability, intrinsic protein disorder, solubility and allergenicity of this chimeric protein were analyzed using bioinformatics tools and servers. Based on bioinformatics analysis, a soluble, and non-allergic protein with 35.405kDa molecular weight was designed. Expasy ProtParam classified this chimeric protein as a stable protein. In addition, predicted epitopes in the chimeric vaccine indicated strong potential to induce B-cell mediated immune response and shift immune responses toward protective Th2 immune response. Various in silico analyses indicate that this vaccine is a qualified candidate for improvement of atherosclerosis by inducing immune responses toward T helper 2.

Barberry treatment reduces serum anti-heat shock protein 27 and 60 antibody titres and high-sensitivity c-reactive protein in patients with metabolic syndrome: a double-blind, randomized placebo-controlled trial.

Metabolic syndrome is an important risk factor for cardiovascular disease (CVD). The heat shock proteins (HSPs) are associated with risk factors for CVD. The aim of the present study was to survey the effect of barberry on antibody titres to HSPs and high-sensitivity C-reactive protein (hs-CRP) in patients with metabolic syndrome. In our study, subjects (N=106, 79 women and 27 men, 18-65 years old) with metabolic syndrome were randomized into two groups: a group of patients who received three capsules of barberry and a control group who received three capsules of placebo for 6 weeks. Antibodies against HSPs 27, 60/65 and 70, hs-CRP and lipid profile were determined in patients before (week 0) and after (week 6) intervention. spss software (version 16.0; Inc, Chicago, IL) was used for data analysis. Results showed that barberry had no significant effect on serum level of anti-HSPs 65 and 70. But there was a significant decrease in anti-HSP 27 in both case and control groups (p=0.001 and p<0.001, respectively, in the case and control groups). Barberry decreased significantly anti-HSP 60 in the case group (p=0.03). High-sensitivity CRP was decreased non-significantly (p=0.17) in the case group and increased significantly (p=0.04) in the control group. Barberry decreased significantly low-density lipoprotein and total cholesterol and increased significantly high-density cholesterol (p<0.05). Results of the present study suggested that barberry supplementation in patients with metabolic syndrome decreased significantly anti-HSPs 27 and 60 and hs-CRP levels and improved lipid profile.

[Antibodies to Hsp60 in newborn babies, to whom during the intervention for critical inborn heart failure, autologous cord blood was transfused, in early and late postoperative period].

The titre of antibodies to Hsp60 (heat shock protein) enhancement in the blood serum is considered a biological marker of poor state of organism. Comparative investigation was done on the antibodies titre to Hsp60 in the blood serum of a newborn babies, suffering critical inborn heart failure, to whom autologous cord blood or the donor's blood components was transfused, in early and remote postoperative period. In early postoperative period the lowering of the antibodies titre to Hsp60 in the blood serum was observed in comparison with them preoperatively, in a late period (in 2 yrs) in all the blood serum samples investigated antibodies to Hsp60 were not revealed. In 35% of patients, to whom the donor's blood components were transfused, there was registered the enhancement of the antibodies to Hsp60 titre in early postoperative period. High titre of antibodies have associated with enhanced rate of complications. In late postoperative period antibodies to Hsp60 were revealed in 20% of the examined patients.

Comparative antigen-induced gene expression profiles unveil novel aspects of susceptibility/resistance to adjuvant arthritis in rats.

Lewis (LEW) and Wistar Kyoto (WKY) rats of the same major histocompatibility complex (MHC) haplotype (RT.1(l)) display differential susceptibility to adjuvant-induced arthritis (AIA). LEW are susceptible while WKY are resistant to AIA. To gain insights into the mechanistic basis of these disparate outcomes, we compared the gene expression profiles of the draining lymph node cells (LNC) of these two rat strains early (day 7) following a potentially arthritogenic challenge. LNC were tested both ex vivo and after restimulation with the disease-related antigen, mycobacterial heat-shock protein 65. Biotin-labeled fragment cRNA was generated from RNA of LNC and then hybridized with an oligonucleotide-based DNA microarray chip. The differentially expressed genes (DEG) were compared by limiting the false discovery rate to <5% and fold change ≥2.0, and their association with quantitative trait loci (QTL) was analyzed. This analysis revealed overall a more active immune response in WKY than LEW rats. Important differences were observed in the association of DEG with QTL in LEW vs. WKY rats. Both the number of upregulated DEG associated with rat arthritis-QTL and their level of expression were relatively higher in LEW when compared to WKY rat; however, the number of downregulated DEG-associated with rat arthritis-QTL as well as AIA-QTL were found to be higher in WKY than in LEW rats. In conclusion, distinct gene expression profiles define arthritis-susceptible versus resistant phenotype of MHC-compatible inbred rats. These results would advance our understanding of the pathogenesis of autoimmune arthritis and might also offer potential novel targets for therapeutic purposes.

Immunotherapy for TB.

Mycobacterium tuberculosis was one of the first human pathogens to be identified as the cause of a specific disease--TB. TB was also one of the first specific diseases for which immunotherapy was attempted. In more than a century since, multiple different immunotherapies have been attempted, alongside vaccination and antibiotic treatment, with varying degrees of success. Despite this, TB remains a major worldwide health problem that causes nearly 2 million deaths annually and has infected an estimated 2 billion people. A major reason for this is that M. tuberculosis is an ancient human pathogen that has evolved complex strategies for persistence in the human host. It has thus been long understood that, to effectively control TB, we will need to address the ability of the pathogen to establish a persistent, latent infection in most infected individuals. This review discusses what is presently known about the interaction of M. tuberculosis with the immune system, and how this knowledge has been used to design immunotherapeutic strategies.

Microarray-based gene expression profiling reveals the mediators and pathways involved in the anti-arthritic activity of Celastrus-derived Celastrol.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation of the joints. The prolonged use of non-steroidal anti-inflammatory drugs and other newer drugs is associated with severe adverse reactions. Therefore, there is a need for newer anti-arthritic agents. Celastrol, a bioactive component of the Chinese herb Celastrus, possesses anti-arthritic activity as tested in the adjuvant arthritis (AA) model of rheumatoid arthritis (RA). However, the mechanism of action of Celastrol has not been fully defined. We reasoned that microarray analysis of the lymphoid cells of Celastrol-treated arthritic animals might provide vital clues in this regard. We isolated total RNA of the draining lymph node cells (LNCs) of Celastrol-treated (Tc) and vehicle-treated (Tp) arthritic Lewis rats that were restimulated in vitro with the disease-related antigen, mycobacterial heat-shock protein 65 (Bhsp65), and tested it using microarray gene chips. Also tested was RNA from LNCs of control arthritic rats just before any treatment (T0). Seventy six genes involved in various biological functions were differentially regulated by Bhsp65 in LNCs of Tp group, and 19 genes among them were shared by the Tc group. Furthermore, a group of 14 genes was unique to Tc. When Tc and Tp were compared, many of the Bhsp65-induced genes were related to the immune cells, cellular proliferation and inflammatory responses. Our results revealed 10 differentially expressed genes and 14 pathways that constituted the "Celastrol Signature". Our results would help identify novel targets for RA therapy.

The gene expression profile of preclinical autoimmune arthritis and its modulation by a tolerogenic disease-protective antigenic challenge.

INTRODUCTION: Autoimmune inflammation is a characteristic feature of rheumatoid arthritis (RA) and other autoimmune diseases. In the natural course of human autoimmune diseases, it is rather difficult to pinpoint the precise timing of the initial event that triggers the cascade of pathogenic events that later culminate into clinically overt disease. Therefore, it is a challenge to examine the early preclinical events in these disorders. Animal models are an invaluable resource in this regard. Furthermore, considering the complex nature of the pathogenic immune events in arthritis, microarray analysis offers a versatile tool to define the dynamic patterns of gene expression during the disease course. METHODS: In this study, we defined the profiles of gene expression at different phases of adjuvant arthritis (AA) in Lewis rats and compared them with those of antigen mycobacterial heat shock protein 65 (Bhsp65)-tolerized syngeneic rats. Purified total RNA (100 ng) extracted from the draining lymph node cells was used to generate biotin-labeled fragment cRNA, which was then hybridized with an oligonucleotide-based DNA microarray chip. Significance analysis of microarrays was used to compare gene expression levels between the two different groups by limiting the false discovery rate to < 5%. Some of the data were further analyzed using a fold change ≥2.0 as the cutoff. The gene expression of select genes was validated by quantitative real-time PCR. RESULTS: Intriguingly, the most dramatic changes in gene expression in the draining lymphoid tissue ex vivo were observed at the preclinical (incubation) phase of the disease. The affected genes represented many of the known proteins that participate in the cellular immune response. Interestingly, the preclinical gene expression profile was significantly altered by a disease-modulating, antigen-based tolerogenic regimen. The changes mostly included upregulation of several genes, suggesting that immune tolerance suppressed disease by activating disease-regulating pathways. We identified a molecular signature comprising at least 12 arthritis-related genes altered by Bhsp65-induced tolerance. CONCLUSIONS: This is the first report of microarray analysis in the rat AA model. The results of this study not only advance our understanding of the early phase events in autoimmune arthritis but also help in identifying potential targets for the immunomodulation of RA.

[Preparation oral liposome-encapsulated recombinant Helicobacter pylori heat shock protein 60 vaccine for prevention of Hp infection].

OBJECTIVE: To prepare oral liposome-encapsulated recombinant Helicobacter pylori (Hp) heat shock protein 60 (Hsp60) vaccine and investigate its effect against Hp infection in mice. METHODS: The recombinant vector PET-22(+)/Hsp60 was transformed into BL21(DE3) E.coli. The recombinant protein was purified with Ni-NTA agrose resin and the oral liposome-encapsulated vaccine was prepared with phosphatidyl choline and cholesterols using film method, with the size distribution of the folate liposomes measured by transmission electronic microscopy. BALB/c mice were divided into 5 groups and immunized by intragastric administration of PBS, liposome, rHsp60 plus choleratoxin (CT), liposome-encapsulated rHsp60, and liposome-encapsulated rHsp60 plus CT, respectively, given once a week for 4 weeks. All the mice were challenged by Hp for 3 times within two weeks following the last immunization and sacrificed 3 weeks after the last challenge. Hp detection was performed by fast urease test. Semi-quantitative assessment of the bacterial colonization density observation of the inflammation severity and gastric histopathology were carried out. RESULTS: The soluble expression product accounted for 27% of the total bacterial protein. The purity of recombinant fusion protein was about 95% after purification. The mean size of the folate liposomes was 0.7+/-0.4 mum. PBS or liposome alone showed no immune-enhancing effect, and rHsp60 plus CT, liposome-encapsulated rHsp60 and liposome-encapsulated rHsp60 plus CT had the protective rates against Hp infection of 73.3%, 66.7% and 86.7%, respectively. The latter 3 preparations effected significantly reduced Hp infection and alleviated the inflammation in the gastric mucosa of the mice challenged with Hp. CONCLUSION: The oral liposome may serve as a potential adjuvant for Hp vaccine in preventing Hp infection.

Heat-shock proteins induce T-cell regulation of chronic inflammation.

Immune responses to certain heat-shock proteins (HSPs) develop in almost all inflammatory diseases; however, the significance of such responses is only now becoming clear. In experimental disease models, HSPs can prevent or arrest inflammatory damage, and in initial clinical trials in patients with chronic inflammatory disease, HSP-derived peptides have been shown to promote the production of anti-inflammatory cytokines, indicating that HSPs have immunoregulatory potential. In this Review, we discuss the unique characteristics of HSPs that endow them with these immunoregulatory qualities.

Recognition and phagocytosis of multiple periodontopathogenic bacteria by anti-Porphyromonas gingivalis heat-shock protein 60 antisera.

The present study has been performed to evaluate Porphyromonas gingivalis heat shock protein (HSP) 60 as a candidate vaccine to protect against multiple putative periodontopathic bacteria. Mouse anti-P. gingivalis HSP antisera demonstrated the elevated IgG antibody titers against the multiple bacteria tested and cross-reacted with heat-induced bacterial proteins of the target bacteria. The antisera also demonstrated a significantly higher opsonophagocytosis function against all the target bacteria than the control sera (P<0.01). We concluded that P. gingivalis HSP 60 could potentially be developed as a vaccine against multiple periodontopathic bacteria.

[Evidence of antibodies against the chlamydial heat shock protein (cHSP60) in women from "in vitro" fertilization program]. / Prukaz protilátek proti proteinu tepelného soku chlamydií (cHSP60) u zen zarazených do in vitro fertilizacního programu.

OBJECTIVE: A contribution to the role of chlamydial heat shock protein in women from the IVF program. DESIGN: A serological study proving the antibodies against the chlamydial heat shock protein. SETTING: Veterinary Research Institute, Brno, Department of Clinical Microbiology, and Centrum of Reproductive Medicine, Faculty Hospital Brno. METHODS: The IgG antibodies against the chlamydial heat shock protein (cHSP60) and the IgA and IgG antibodies against species specific chlamydial major outer membrane protein (cMOMP) of Chlamydia trachomatis (C. trachomatis) and Chlamydophila pneumoniae (C. pneumoniae) in the blood serum of 70 females being in the fertilization program due to fertility disorders (Group 1) have been estimated and the results compared with those obtained in 50 females suffering from pelvic inflammation disease (PID) (Group 2) and in 51 female blood donors (Group 3) respectively. RESULTS: The anti-cHSP60 antibodies have been ascertained as follows: in 26 women from the first group (37.1%), in 16 of the second group (32.0%) and in 12 (23.5%) of female blood donors. The anti cHSP60-antibody-occurrence and mean index of positivity as well, found in the groups examined, was statistically insignificant. Similarly no difference has been found in the occurrence of the species specific anti-cMOMP antibodies C. trachomatis and C. pneumoniae in the groups examined. The anti-cMOMP antibodies against C. trachomatis and simultaneously against C. pneumoniae have been detected in 21 from 171 women of all groups (12.3%), only against C. trachomatis in 9 (5.3%) and against C. pneumoniae in 73 of them (42.7%). There was considerably higher occurrence of the specific antibodies against C. trachomatis in women with a positive finding of antibodies against the cHSP60, especially in women suffering from gynecological disorders, than in women without such antibodies. CONCLUSION: C. trachomatis has a significant impact on the production of antibodies against the cHSP60. This fact can be documented by considerably higher occurrence of the specific antibodies against C. trachomatis in women with a positive finding of antibodies against the cHSP60, than in women without such antibodies. Preceding infection C. trachomatis and following sensitization with chlamydial heat shock protein indicate an unfavourable prognosis of the reproductive outcome and impairs the perspective of a successful in vitro fertilization. The proof of antibodies against the chlamydial HSP60 can be recommended as a further auxiliary criterion in women suffering from fertility disorders.

T cell recognition of a highly conserved epitope in heat shock protein 60: self-tolerance maintained by TCR distinguishing between asparagine and aspartic acid.

Cross-reactive T cell recognition of self-heat shock proteins (hsp) has been ascribed a regulatory role in inflammatory arthritis in both animal models and human disease. The previous work implies that a repertoire for epitopes in self-hsp60 should exist in normal subjects. Accordingly, we sought to generate self-hsp60-reactive T cell clones from a healthy individual using a highly purified preparation of recombinant human (Hu) hsp60. Epitope mapping using synthetic peptides and truncated constructs indicated that the T cell clones obtained actually recognized hsp60 derived from Escherichia coli. Using a series of alanine-substituted peptides and additional appropriate synthetic peptides, it was demonstrated that the clones maintain self-tolerance because of their sensitivity to an asparagine to aspartic acid sequence difference between E. coli and HuHsp60 in the epitope-containing peptide. In addition, despite substantial conservation of sequence, the homologous peptide from HuHsp60 did not compete with the E. coli-derived peptide for recognition or antagonize responses by acting as an altered peptide ligand. The results suggest that, even when the immune system targets a highly conserved epitope in bacterial hsp60, self-tolerance is maintained. Furthermore, the finding that T cell clones specific for minor contaminant proteins in HuHsp60 preparations can readily be isolated raises the possibility that the HuHsp60 facilitates presentation of antigenic proteins to the immune system.

Folate supplementation reduces serum hsp70 levels in patients with type 2 diabetes.

Type 2 diabetes patients are subject to oxidative stress as a result of hyperglycemia. The aim of this study was to determine whether administration of the antioxidant folic acid, previously shown to reduce homocysteine levels, would reduce circulating levels of Hsp70 while improving the condition of type 2 diabetes patients with microalbuminuria. Plasma homocysteine fell from pretreatment values of 12.9 to 10.3 microM (P < 0.0001). The urine albumin-creatinine ratio fell from 12.4 to 10.4 mg/mM (P = 0.38). Pretreatment Hsp70 levels were higher in patients not taking insulin (5.32 ng/mL) compared with those on insulin (2.44 ng/mL) (P = 0.012). Folic acid supplementation resulted in a significant fall in Hsp70 (5.32 to 2.05 ng/mL) (P = 0.004). There was no change in Hsp70 in those receiving insulin. Folic acid supplementation in non-insulin-treated type 2 diabetes patients, therefore, resulted in a fall in Hsp70, reflecting an improvement in oxidative stress. The data shows that improvement in homocysteine status can lead to a reduction in Hsp70, indicating the possibility of its use as a marker for severity of disease.

Atherosclerosis as a paradigmatic disease of the elderly: role of the immune system.

When a new hypothesis about the etiology and pathogenesis of a disease is developed, there is always the danger that it will be presented as the only acceptable explanation for the occurrence of a given pathologic condition. In view of the well-proven multifactoral pathogenesis of atherosclerosis, we would like to emphasize that we are not postulating that immunity to HSP60 is the only cause of atherogenesis, especially in the later stages where there are clinically-apparent sequelae, such as myocardial infarction, stroke, and other atherosclerosis-dependent symptoms. In this article, we summarized some of the experimental and clinical data that we and others have collected in support of the concept that atherosclerosis is a good example of pleotropic antagonism, and postulated that age-dependent diseases are the price we pay for genetic traits established by natural selection to assure maximum survival until the age of reproduction, the effects of which may, however, become deleterious later in life. In the present case, the cost we pay for protective immunity to microbial and altered autologous HSP60 is the risk of cross-reactivity with HSP60 expressed by arterial endothelial cells that are subjected to stress factors already known as classical atherosclerosis risk factors. We showed that the first inflammatory stage of atherosclerosis starts early in life, long before it becomes clinically apparent. More severe lesions that lead to atherosclerosis-dependent organ-specific or systemic symptoms will only occur if classical atherosclerosis risk factors, especially those involving the cholesterol metabolism, remain present.

Induction of heat shock proteins fails to produce protection against trypsin-induced acute pancreatitis in rats.

Heat shock proteins (HSPs) are necessary in the synthesis, degradation, folding, transport, and translocation of different proteins. It is well known that the increased expression of HSPs may have a protective effect against cerulein-induced pancreatitis in rats or against choline-deficient ethionine-supplemented diet model pancreatitis in mice. The aim of this study was to investigate the potential effects of HSP preinduction by cold or hot water immersion on trypsin-induced acute pancreatitis in rats. Trypsin was injected into the interlobular tissue of the duodenal part of the pancreas at the peak level of HSP synthesis, as determined by Western blot analysis. The rats were sacrificed by exsanguination through the abdominal aorta 6 h after the trypsin injection. The serum amylase activity, the tumor necrosis factor-alpha, interleukin-1, and interleukin-6 levels, the pancreatic weight/body weight ratio, and the pancreatic contents of DNA, protein, amylase, lipase, and trypsinogen were measured. A biopsy for histology was taken. Hot water immersion significantly elevated the HSP72 expression, while cold water immersion significantly increased the HSP60 expression. Cold water immersion pretreatment ameliorated the pancreatic edema in trypsin-induced pancreatitis, however this was not due to the HSP60. Hot water immersion pretreatment did not have any effect on the measured parameters in trypsin-induced pancreatitis. The findings suggest that the induction of HSP60 or HSP72 are not enough to protect rats against the early phase of this localized necrohemorrhagic pancreatitis model.

[Association of heat-shock protein and uveitis].

Heat-shock protein 60 derived from Yersinia enterocolitica (Yersinia HSP 60) and bovine retinal HSP (Retina HSP 60) were previously identified by immunological cross-reaction and a high degree of common antigenicity, and a specific antibody against both proteins was detected in the sera of uveitis patients. We report here an attempt to isolate and purify Retina HSP 60 and Yersinia HSP 60. Both Retina HSP 60 and Yersinia HSP 60 showed an enriched content of glycine of approximately 60 to 80%. Lewis rats were inculated with 50 or 100 micrograms of purified Retina or Yersinia HSP 60 emulsified in complete Freund's adjuvant. In 50 to 60% of those inoculated with Retina HSP 60, uveoretinitis was observed about 13 days after inoculation, with massive infiltration of lymphocytes and polymorphonuclear neutrophils in the iris, ciliary body, and retinal tissue. Rats inoculated with Yersinia HSP 60 did not develop ocular inflammation. Lymphocyte proliferation assay was performed to investigate cellular immunoresponses in the rats that developed ocular inflammation after immunization with Retina HSP 60. The results showed significantly higher response to the Retina and Yersinia HSP 60 than to either S-antigen or interphotoreceptor retinoid-binding protein (IRBP), which are known to induce ocular inflammation. Cross-reaction between Retina and Yersinia HSP 60 is suggested. This study suggests that the HSP 60 molecules may be involved in the pathogenesis of intraocular inflammation.