Heat Shock Protein 65 Research Articles

Clinico-microbiological Profile of Nontuberculous Mycobacterial Keratitis

PurposeTo assess the clinical and microbiological characteristics of nontuberculous mycobacterial (NTM) keratitis and to evaluate their response to medical therapy.MethodsSixteen patients of NTM keratitis were retrospectively reviewed from May 2014 to May 2019. Laboratory diagnosis were made using Ziehl-Nielsen acid-fast staining, routine culture method of isolation of nontuberculous mycobacteria and further identification of species by PCR (polymerase chain reaction)-based DNA sequencing targeting the heat shock protein-65 (hsp-65) gene.ResultsSixteen patients of microbiologically proven NTM keratitis were included. The average age at the time of presentation was 43.56 years (range, 24–73 years). The mean duration of symptoms was 2.23 months. The commonest risk factor was injury with organic material (43.7) followed by ocular surgery (25%). The majority of the nontuberculous mycobacteria were Mycobacterium abscessus (87.6%) followed by M. fortuitum (6.2%) and M. chelonae (6.2%). The in vitro sensitivity showed maximum sensitivity to Amikacin (AMK; 100%) followed by Azithromycin (AZM; 85.7%), and Clarithromycin (CLR; 85.7%). Out of a total of 16 patients, 12 (75%) had total success with medical therapy while 4 (25%) required surgical intervention.ConclusionThis study is focused on rapid and reliable identification of NTM keratitis through PCR-based identification method to enable effective medical management. The antibiotic susceptibility testing of different subspecies of NTM further reduced the need for surgical intervention. The effective role of AMK either alone or in combination with macrolide antibiotics is also highlighted in this study.

HIGHLY SENSITIVE LABEL-FREE ELECTROCHEMICAL DETECTION OF HEAT SHOCK PROTEIN WITH LOW-COST SCREEN-PRINTED ELECTRODES

Heat shock proteins are produced when the organisms are exposed to various environmental stress conditions such as temperature, light, toxins. It is a known fact that in bacteria, which has the HSP gene, antibiotics can trigger the expression of the heat shock protein. However, the response of heat shock protein genes to antibiotics has not been fully clarified in the literature yet, studies are still ongoing. In this study, a novel way for the detection heat shock protein65 was investigated using the electrochemical impedance spectroscopy due to its sensitivity, selectivity, low cost. To do so, heat shock protein65 probe and target were designed and the hybridization behavior of the probe with designed target was studied upon the binding onto screen-printed electrodes. Cyclic voltammetry was performed to analyze surface characterization of secreen printed electrodes and the performance of the screen printed electrodes was tested using electrocehimcal impedance spectroscopy by measuring changes in the charge transfer resistance upon hybridization. Based on obtained results, the designed heat shock protein65 probe was confirmed and an appropriate increase in charge transfer resistance values compliance to the literature proved that the electrochemical impedance spectroscopy can be effectively used to detect heast shock protein65 probe label-free. Results presented here can lead to development of antibiotic susceptibility assay based on the heat shock protein genes in future.

MO223MEMBRANOUS NEPHROPATHY ASSOCIATED TO ANKYLOSING SPONDYLITIS: CASE REPORT

Abstract Background and Aims Ankylosing Spondylitis (AS) is a chronic inflammatory rheumatic disease diagnosed by the presence of the HLA-B27 antigen with joint and extra-articular manifestations. Its pathogenesis was initially based on auto-inflammatory phenomena, mainly involving innate immunity. However, many studies carried out in the recent years focus on its adaptive immunity aspect, especially autoimmune. These mechanisms seem to interact with each other, resulting in a complex pathophysiology. The general autoimmune characteristics of AS were investigated by Yuan and al, who reviewed recent reports of autoantibodies levels in AS patients. Their analysis shows that none of the autoantibodies considered in the study meet the criteria to be considered as a biomarker for the disease (including antibodies Anti- :CD74; beta2-Microglobulin; mutated Citrullinated Vimentin (MCV); Heat Shock Protein 65 (HSP65); 14-3-3 eta autoantibodies (14-3-3η); 1A-dependent autoantibody magnesium anti-protein phosphatase (PPM1A); sclerostin (SOST); and Anti-microbial antibodies). Renal involvement in AS is dominated by amyloidosis and IgA nephropathy. In rare cases, this disease has been associated with Membranous Nephropathy (MN). The pathogenetic link between the two disorders remains obscure. However, the recent connection of AS to autoantibodies potentially indicates the involvement of immune complexes formed from these autoantibodies in the development of MN. Method We report the case of a 36-years-old woman with a 7 years history of sacroiliitis, who also developed a pedal edema in May 2020. AS was diagnosed in 2014 by the presence of positive HLA-B27, and clinical and radiological manifestations of bilateral sacroiliitis without extra renal manifestations treated by indometacin during periods of pain . A pure nephrotic syndrome was revealed with a 24h proteinuria of 12g / 24h.The patient’s Albumin levels were at 16g/l, without HTA or hematuria, and with a correct renal function (Creatinine at 60mmol/l). A renal biopsy showed a MN with type I polytypical. Light microscopy showed a thickening of capillary loops, while IF staining revealed granular deposits of IgG along the capillary wall. Investigations of further secondary MNs were negative, and the patient was aPLA2R-negative. Results A treatment by corticosteroids was initiated prior biopsy to her referral , which resulted in pain relief and urinary protein reduction (prot 24h 1.5g/24h). Given her good response to this initial treatment, corticotherapy was maintained. As the levels of inflammation and discomfort were low, the patient did not wish to be treated by biotherapy. Conclusion This case suggests a secondary MN in association with AS.The discovery of new autoantibodies associated with AS opens up promising perspectives, and could potentially lead to the characterization of biomarkers for screening and monitoring this disease. However, more studies are needed in order to improve our understanding of the role played by possible immune complex diseases, (in particular MN), in relation to this Ankylosing spondilitis.

Proposing BCG Vaccination for Mycobacterium avium ss. paratuberculosis (MAP) Associated Autoimmune Diseases.

Bacille Calmette–Guerin (BCG) vaccination is widely practiced around the world to protect against the mycobacterial infection tuberculosis. BCG is also effective against the pathogenic mycobacteria that cause leprosy and Buruli’s ulcer. BCG is part of the standard of care for bladder cancer where, when given as an intravesicular irrigant, BCG acts as an immunomodulating agent and lessens the risk of recurrence. Mycobacterium avium ss. paratuberculosis (MAP) causes a fatal enteritis of ruminant animals and is the putative cause of Crohn’s disease of humans. MAP has been associated with an increasingly long list of inflammatory/autoimmune diseases: Crohn's, sarcoidosis, Blau syndrome, Hashimoto’s thyroiditis, autoimmune diabetes (T1D), multiple sclerosis (MS), rheumatoid arthritis, lupus and Parkinson’s disease. Epidemiologic evidence points to BCG providing a “heterologous” protective effect on assorted autoimmune diseases; studies using BCG vaccination for T1D and MS have shown benefit in these diseases. This article proposes that the positive response to BCG in T1D and MS is due to a mitigating action of BCG upon MAP. Other autoimmune diseases, having a concomitant genetic risk for mycobacterial infection as well as cross-reacting antibodies against mycobacterial heat shock protein 65 (HSP65), could reasonably be considered to respond to BCG vaccination. The rare autoimmune disease, relapsing polychondritis, is one such disease and is offered as an example. Recent studies suggesting a protective role for BCG in Alzheimer’s disease are also explored. BCG-induced energy shift from oxidative phosphorylation to aerobic glycolysis provides the immunomodulating boost to the immune response and also mitigates mycobacterial infection—this cellular mechanism unifies the impact of BCG on the disparate diseases of this article.

Lymphocyte proliferation induced by high-affinity peptides for HLA-B*51:01 in Behçet's uveitis.

Several proteins have been proposed as candidate auto-antigens in the pathogenesis of Behçet’s disease (BD). In this study, we aimed to confirm the cellular responses to candidate peptide autoantigens with high affinity for the HLA-B*51:01 molecule using computerized binding predictions and molecular dynamics simulations. We identified two new candidate peptides (HSP65PD, derived from heat shock protein-65, and B51PD, derived from HLA-B*51:01) with high-affinity to the HLA-B*51:01 binding pocket using the Immune Epitope Database for Major Histocompatibility Complex-I Binding Prediction and molecular dynamics simulations. The peptide-induced proliferation of lymphocytes from patients with BD, sarcoidosis, Vogt–Koyanagi–Harada disease (VKH) with panuveitis, systemic scleroderma (SSc) without uveitis, and healthy controls (HC) was investigated using the bromodeoxyuridine assay. The proliferative response of leukocytes to HSP65PD was significantly higher in BD (SI 1.92 ± 0.65) than that in sarcoidosis (SI 1.38 ± 0.46), VKH (SI 1.40 ± 0.33), SSc (SI 1.32 ± 0.31), and HC (SI 1.27 ± 0.28) (P = 0.0004, P = 0.0007, P < 0.0001, P < 0.0001, respectively, Mann-Whitney’s U-test). The proliferative response of leukocytes to B51PD was also higher in BD than that in sarcoidosis, VKH, SSc without uveitis, and HC, whereas no significant differences were observed among the five groups in response to a control peptide derived from topoisomerase 1. A significantly higher response to HPS65PD and B51PD was observed in the HLA-B*51:01-positive patients with BD than in the HLA-B*51:01-negative patients. In conclusion, two peptides that had high affinity to HLA-B*51:01 in computerized binding prediction showed significantly higher response in HLA-B*51:01-positive patients with BD, indicating the usefulness of computerized simulations for identifying autoreactive peptides to HLAs.

Inhibition of mouse RM-1 prostate cancer and B16F10 melanoma by the fusion protein of HSP65 & STEAP1 186-193

The research of tumor vaccine plays a crucial role in tumor immunotherapy. This study has constructed and prepared a fusion protein vaccine of heat shock protein 65 (HSP65) and the octapeptide epitope 186-193 of the six transmembrane epithelial antigen of the prostate 1 (STEAP1 186-193), and investigated the inhibitory effect of the fusion protein on mouse RM-1 prostate cancer and B16F10 melanoma xenografts. The fusion protein His-HSP65-STEAP1 186-193 (HHST1), His-HSP65-2×STEAP1 186-193 (HHST2) and His-HSP65-6×STEAP1 186-193 (HHST6) were obtained by setting different copy number of STEAP1 186-193 and adding His purification tag before HSP65. Firstly the inhibitory effect of fusion protein on mouse RM-1 prostate cancer xenografts has been studied, which could be the basis of the study the inhibitory effect of the best fusion protein on mouse B16F10 melanoma xenografts. All studies compared with the fusion protein His-HSP65 (HHSP65), the fusion proteins HHST1, HHST2 and HHST6 all could significantly inhibit the growth of mouse RM-1 prostate cancer xenografts. In addition, the fusion protein HHST2 was proved to be the best compared with the fusion proteins HHST1 and HHST6 (P<0.05). Apart from this, compared with the fusion protein HHSP65, the fusion protein HHST2 also significantly inhibited the growth of mouse beared B16F10 melanoma. The results above indicate that HSP65 and STEAP1 186-193 can significantly inhibit the growth of mouse RM-1 prostate cancer and B16F10 melanoma xenografts, and the appropriate increase of copy number can effectively improve that the fusion protein has an excellent anti-tumor ability.

Genetic Diversity and Prevalence of Nontuberculous Mycobacteria Isolated from Clinical Samples in Tehran, Iran.

The prevalence of nontuberculous mycobacteria (NTM) has increased in tuberculosis (TB)-suspected clinical samples. These bacteria are now recognized as important emerging pathogens, which affect both immunocompetent and immunocompromised individuals. The aim of this study was to evaluate the frequency of NTM in clinical samples and to efficacy of genomic loci as targets for detection of NTM species. This cross-sectional study was performed on 8166 clinical samples to determine the presence of NTM species from April 2013 to December 2015. The phenotypic methods were applied for preliminary NTM identification. The PCR-RFLP assay of heat shock protein-65 (hsp-65) gene and multilocus sequence analysis based on 16S-23S internal transcribes spacer (ITS), rpoB, and 16S rRNA genes were applied for species identification. In a total of 520 isolates from TB-suspected cases, 61 samples (11.7%) were identified as NTM. Overall, Mycobacterium (M.) fortuitum (63.9%) was the most frequently encountered species, followed by M. kansasii (9.8%), M. simiae (9.8%), M. abscessus (6.7%), M. gordonae (4.9%), M. flavescens (3.3%), and M. setense (1.6%). Moreover, sequencing of 16S rRNA and rpoB genes could identify all NTM species. In conclusion, we showed that the samples were infected by six NTM species, and M. fortuitum was the most frequent NTM strain. Based on the findings, 16S rRNA and rpoB genes were superior to ITS gene in identification of NTM species.

Serum heat-shock protein-65 antibody levels are elevated but not associated with disease activity in patients with rheumatoid arthritis and ankylosing spondylitis.

ObjectivesHeat-shock proteins (HSPs) have gained increased interest for their role in autoimmune disorders. These proteins are targeted by the immune system in various autoimmune diseases. The aim of this study was to assess the serum heat-shock protein-65 antibody (anti-HSP65) levels and their clinical significance in patients with rheumatoid arthritis (RA) and ankylosing spondylitis (AS).Patients and methodsA total of 30 patients with RA, 30 patients with AS, and 30 healthy controls were enrolled in this study. All patients were assessed using routine clinical and laboratory evaluations. Serum anti-HSP65 levels were determined by ELISA.ResultsSerum anti-HSP65 levels of both RA and AS patients were significantly higher than those of controls (p=0.014 and p=0.001, respectively). No association was found between serum anti-HSP65 levels and disease activity in either RA or AS patients. There was a significant correlation between anti-HSP65 and anti-cyclic citrullinated peptide levels in patients with RA (p=0.024).ConclusionIn this study, serum anti-HSP65 levels were increased, but not associated with disease activity in both RA and AS patients. These results suggest that HSP antigens may play a role in the pathogenesis. However, further follow-up studies are needed. Identification of target antigens such as HSP65 is vital to developing new immunotherapeutic agents.

Behçet Disease serum is immunoreactive to neurofilament medium which share common epitopes to bacterial HSP-65, a putative trigger

Autoimmune and dysimmune inflammatory mechanisms on a genetically susceptible background are implicated in the etiology of Behçet's Disease (BD). Heat-shock protein-65 (HSP-65) derived from Streptococcus sanguinis was proposed as a triggering factor based on its homology with human HSP-60. However, none of the autoantigens identified so far in sera from BD share common epitopes with bacterial HSP-65 or has a high prevalence. Here, we report that sera from BD patients are immunoreactive against filamentous neuronal processes in the mouse brain, retina and scrotal skin in great majority of patients. By using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) and peptide mass fingerprinting, Western blotting and peptide blocking experiments, we have identified neurofilament medium (NF-M) as the probable antigen for the serologic response observed. Clustal Omega analyses detected significant structural homology between the human NF-M and bacterial HSP-65 corresponding to amino acids 111–126, 213–232 and 304–363 of mycobacterial HSP-65, which were previously identified to induce proliferation of lymphocytes obtained from BD patients. We also found that sera immunoreactive against NF-M cross-reacted with bacterial HSP-65. These findings suggest that NF-M may be involved in autoimmunity in BD due to its molecular mimicry with bacterial HSP-65.

Mycobacterial heat shock protein 65 mediated metabolic shift in decidualization of human endometrial stromal cells

Successful implantation is dependent on the appropriate decidualization of endometrial stromal cells for the establishment of pregnancy in women. Mycobacterial heat shock protein 65 (HSP65) is involved in pathogenesis of the genital tuberculosis (GTB), one of the common causes of infertility in emerging countries. Though implantation failure appears to be the major cause, understanding the status of decidualizaiton process in women diagnosed with GTB has not been thoroughly addressed. We, therefore, explored the effect of HSP65 protein on the endometrial cell metabolism during in vitro decidualization. In order to identify the cellular metabolism of decidual cells with and without HSP65 treatment, proton NMR based characterization of metabolites extracted from cells and culture media were performed. In presence of HSP65, significant reduction in the decidual phenotype of endometrial stromal cells and prolactin expression is suggestive of impairment in decidualization. The intracellular and extracellular metabolic changes in HSP65 treated endometrial stromal cells produced a distinct pattern, reflecting the interaction between the protein and cellular metabolism. HSP65 mediated dysregulation in cellular metabolism is associated with poor decidualization. Besides enriching the present knowledge on metabolic changes underlying stromal cells decidualization, these findings assist in identifying potential molecular causes for decidualization failure in GTB women.

The therapeutic potency of HSP65-GTL in GL261 glioma-bearing mice.

Gliomas are the most common type of brain tumor with poor prognosis. Even after combination treatments including surgery, radiation, and chemotherapy, the median survival is around 15 months, calling for novel approaches such as immunotherapy. To develop novel therapeutic approaches, we tried to prepare a candidate vaccine by mixing the recombinant mycobacterial heat-shock protein 65 (HSP65) with GL261 glioma tissue lysate (GTL). Our data showed that HSP65-GTL induced potent cytotoxic T lymphocyte and prolonged the survival of mice bearing GL261 gliomas. Furthermore, HSP65 or HSP65-GTL upregulated mRNA expressions of RORγt and interleukin-17A in spleen cells or draining lymph node cells, respectively, and enhanced the ratios of brain-infiltrating Th17 cells and inflammatory cells, indicating that the antitumor effect of HSP65-GTL was associated with Th17-type immunity.

Tuberculosis and Cardiovascular Disease: Linking the Epidemics.

The burden of tuberculosis and cardiovascular disease (CVD) is enormous worldwide. CVD rates are rapidly increasing in low- and middle-income countries. Public health programs have been challenged with the overlapping tuberculosis and CVD epidemics. Monocyte/macrophages, lymphocytes and cytokines involved in cellular mediated immune responses against Mycobacterium tuberculosis are also main drivers of atherogenesis, suggesting a potential pathogenic role of tuberculosis in CVD via mechanisms that have been described for other pathogens that establish chronic infection and latency. Studies have shown a pro-atherogenic effect of antibody-mediated responses against mycobacterial heat shock protein-65 through cross reaction with self-antigens in human vessels. Furthermore, subsets of mycobacteria actively replicate during latent tuberculosis infection (LTBI), and recent studies suggest that LTBI is associated with persistent chronic inflammation that may lead to CVD. Recent epidemiologic work has shown that the risk of CVD in persons who develop tuberculosis is higher than in persons without a history of tuberculosis, even several years after recovery from tuberculosis. Together, these data suggest that tuberculosis may play a role in the pathogenesis of CVD. Further research to investigate a potential link between tuberculosis and CVD is warranted.

Heat shock protein 65 promotes atherosclerosis through impairing the properties of high density lipoprotein

Aim: To explicit whether the functions of high density lipoprotein (HDL) are impaired in murine atherosclerosis by subcutaneous immunization with recombinant mycobacterial heat shock protein 65 (HSP65). Methods: C57BL/6 mice were fed a normal chow-diet with non immunization as normal group. ApoE knockout (ApoE−/−) mice on high-fat diet were randomly divided into three groups (n = 8) and immunized subcutaneously with different concentrations of HSP65 or phosphate-buffered saline (PBS). All animals were treated for 16 weeks. Reverse cholesterol efflux, the anti-oxidant and anti-inflammatory functions of HDL were assayed. Hepatocytes and peritoneal macrophages were isolated to examine the expression of cholesterol transport regulating proteins, including SR-B1, ABCA1, ABCG1, PPAR-γ and LXR-α. Results: In HSP65-immunized mice, paraoxonase1 (PON1) activity and the expression of IL-10 were reduced, while High-density lipoprotein inflammatory index (HII), myeloperoxidase (MPO) activity, and the expression of IFN-γ were elevated gradually. The MPO/PON1 ratio amount was significantly higher in HSP65-immunized group than in normal or PBS-immunized group. In addition, compared with normal or PBS-immunized group, cholesterol efflux rate and the expression of regulating proteins were markedly decreased in HSP65-immunized group. The mice immunized with HSP65 developed significantly larger aorta atherosclerotic plaques when compared with PBS-treated littermates. The high MPO/PON1 ratio was correlated with HII, cholesterol efflux rate and atherosclerotic plaques. Conclusions: This study demonstrates that subcutaneous immunization with HSP65 impairs the properties of HDL, which may contribute to its important pathogenic role of HSP65 in atherogenesis. Also, MPO/PON1 ratio may be a predictor of AS.

SUBCUTANEOUS IMMUNIZATION WITH RECOMBINANT HEAT SHOCK PROTEIN 65 IMPAIRS THE PROPERTIES OF HIGH DENSITY LIPOPROTEIN IN APOLIPOPROTEIN E KNOCKOUT MICE

Heat shock protein 65 (HSP65), as a major autoantigen, contributes to the development of atherosclerosis through immuno-inflammatory reactions. Whether the functions of high density lipoprotein (HDL) are impaired in atherosclerosis mice by subcutaneous immunization with HSP65 remains unclear.

Vaccination with the repeat β-hCG C-terminal peptide carried by heat shock protein-65 (HSP65) for inducing antitumor effects

The β-subunit of human chorionic gonadotropin (β-hCG) is ectopically expressed in various types of cancer and has been utilized as an antigenic target in anti-cancer vaccines. In view of the low immunogenicity of this self-peptide, we designed a method based on the isocaudamer technique to generate 14 tandem repeats of the 10-residue sequence X of β-hCG (109-118). These tandemly repeated copies were then combined with β-hCG C-terminal 37 peptides (CTP37) and finally fused to mycobacterial heat-shock protein 65 (HSP65) to construct a fusion protein HSP65-X14-βhCGCTP37 as an immunogen. In this study, BALB/c female mice were immunized via subcutaneous injection of the designed protein. Humoral immune and cellular immune responses were effectively elicited. A high titer of anti-β-hCG antibody was detected in immunized mice sera by enzyme-linked immunosorbent assay and verified by Western blot analysis. The fusion protein, HSP65-X14-β-hCGCTP37, effectively inhibited the growth of Ehrlich ascites carcinoma in mice. These results suggest that HSP65-X14-βhCGCTP37 may be an effective tumor vaccine, and the use of multiple tandem repeats of a certain epitope is an effective method to overcome the low immunogenicity of self-peptide antigens.

Celastrus Treatment Modulates Antigen-Induced Gene Expression in Lymphoid Cells of Arthritic Rats

Rheumatoid arthritis (RA) is a debilitating autoimmune disease of global prevalence and the disease process primarily targets the synovial joints. Despite improvements in the treatment of RA over the past decade, there still is a need for new therapeutic agents that are efficacious, less expensive, and free of severe adverse reactions. Celastrus has been used in China for centuries for the treatment of rheumatic diseases. Furthermore, we previously reported that ethanol extract of Celastrus aculeatus Merr. (Celastrus) attenuates adjuvant-induced arthritis (AA) in rats. However, the mechanisms underlying the anti-arthritic activity of Celastrus have not yet been fully defined. We reasoned that microarray analysis might offer useful insights into the pathways and molecules targeted by Celastrus. We compared the gene expression profiles of the draining lymph node cells (LNC) of Celastrus-treated (Tc) versus water-treated (Tw) rats, and each group with untreated arthritic rats (T(0)). LNC were restimulated with mycobacterial heat shock protein-65 (Bhsp65). We identified 104 differentially expressed genes (DEG) (8 upregulated, 96 downregulated) when comparing Tc with T(0) rats, in contrast to 28 (12 upregulated, 16 downregulated) when comparing Tw and T(0) rats. Further, 20 genes (6 upregulated, 14 downregulated) were shared by both Tw and Tc groups. Thus, Celastrus treatment (Tc) significantly downregulated a large proportion of genes compared to controls (Tw). The DEG were mainly associated with the processes of immune response, cell proliferation and apoptosis, and cell signaling. These results provide novel insights into the mechanism of Celastrus anti-arthritic activity, and unravel potential therapeutic targets for arthritis.

Nasal immunization with different forms of heat shock protein-65 reduced high-cholesterol-diet-driven rabbit atherosclerosis

Heat shock protein-65 (Hsp65) is an important pro-atherogenic factor, but nasal immunization of Hsp65 can induce immune tolerance and reduce atherosclerotic inflammation. Here, we describe the effects of different forms of Hsp65 antigen inoculated, i.e. Hsp65 DNA, Hsp65 protein, Hsp65 DNA prime combined with Hsp65 protein boost, on high-cholesterol-diet-driven rabbit atherosclerosis lesions. The results showed that sera anti-Hsp65 IgG antibodies were lower induced and protective anti-Hsp65 IgA in nasal cavity and lung were induced after rabbits were immunized with different forms of Hsp65. However, the avidity between Hsp65 IgG antibody and antigen is different. Hsp65 DNA prime–protein boost could further improve the avidity compared with Hsp65 DNA immunization alone, but only immunization with Hsp65 protein alone obtain the best avidity. In addition, more protective IL-10 and less adverse IFN-γ were produced and sera TC and LDL-C were decreased obviously at different extents in immunized groups. As a result, atherosclerosis lesions were significantly attenuated, but only nasal immunization with Hsp65 protein alone may be the best effective form of Hsp65 antigen to control atherosclerotic lesions. The results demonstrated that different forms of Hsp65 antigen could produce different effects in the treatment of atherosclerosis. Immunization mucosally with Hsp65 protein could be a promising therapeutic method for atherosclerosis.

M. paratuberculosisHeat Shock Protein 65 and Human Diseases: Bridging Infection and Autoimmunity

Mycobacterium avium subspecies paratuberculosis (MAP) is the known infectious cause of Johne's disease, an enteric inflammatory disease mostly studied in ruminant animals. MAP has also been implicated in the very similar Crohn's disease of humans as well as sarcoidosis. Recently, MAP has been associated with juvenile sarcoidosis (Blau syndrome), autoimmune diabetes, autoimmune thyroiditis, and multiple sclerosis. While it is intuitive to implicate MAP in granulomatous diseases where the microbe participates in the granuloma, it is more difficult to assign a role for MAP in diseases where autoantibodies are a primary feature. MAP may trigger autoimmune antibodies via its heat shock proteins. Mycobacterial heat shock protein 65 (HSP65) is an immunodominant protein that shares sequential and conformational elements with several human host proteins. This molecular mimicry is the proposed etiopathology by which MAP stimulates autoantibodies associated with autoimmune (type 1) diabetes, autoimmune (Hashimoto's) thyroiditis, and multiple sclerosis. This paper proposes that MAP is a source of mycobacterial HSP65 and acts as a trigger of autoimmune disease.

Role of Heat Shock Protein Derived from Streptococcus sanguinis in Behcet?s Disease

Patients with Behcet’s disease (BD) have hypersensitivity against oral streptococci increased in their oral cavity. Heat shock protein-65/60 (Hsp-65/60) derived from S. sanguinis and the damaged mammalian tissues, respectively, are supposed to play an important role in BD pathogenesis. The antigen presenting cells taken Hsp-65 via Toll-like receptors in the oral lesion are carried to the local lesions by blood flow and they are known to lead T cells undergo apoptosis in the regional delayed type hypersensitive reaction. The human Hsp-60 peptide (336-351 aa) combined with recombinant cholera toxin B subunit was reported to be a therapeutic agent for BD patients with advanced uveitis. Here, we recognized that the peptide (249-264 aa; designated LO1 and 311-326aa: UK) of Hsp-65/60 exhibiting highly homologous to the T cell-epitope lead CD4+ and CD8+ T cell apoptosis in peripheral blood mononuclear cells from BD patients. In BD patients IL-8 and IL-12 production from activated T cells was significantly inhibited by LO1 and UK peptides. The results suggest that the peptides of Hsp-65/60 are able to be therapeutic agents for active BD patients.

Effects of nasal immunization of multi-target preventive vaccines on atherosclerosis

Previous investigations have demonstrated that anti-inflammatory or lipid-lowering treatments could be useful for alleviating morbidity and mortality of atherosclerotic cardiovascular diseases. However, whether a vaccine designed to target inflammation and lipid simultaneously is more powerful to control the process of atherosclerosis remain to be unknown. Here, a vaccine was designed to target heat shock protein-65(Hsp65) and cholesteryl ester transfer protein (CETP) simultaneously and the effects of nasal immunization of multi-target vaccine on high-cholesterol-diet-driven rabbit atherosclerosis lesions were evaluated. Sera, nasal lavages and lung washes were used to ELISA assay for the analysis of IgG and IgA against Hsp65 and CETP. Sera were also used to the analysis of the avidity of combination of anti-Hsp65 and anti-CETP IgG antibodies with corresponding antigen, cytokines IL-10 and IFN-γ, and lipoproteins. In addition, aortas were harvested for analysis of atherosclerotic lesions. The results showed that lower and lasting specific anti-Hsp65 IgG and high anti-CETP IgG in sera and protective anti-Hsp65 and anti-CETP IgA in nasal cavity and lung were induced, the avidity of combination of anti-Hsp65 and anti-CETP IgG with antigen were higher, and more protective IL-10 and less adverse IFN-γ were produced. In addition, sera TC, and LDL-C were decreased. As a result, the size of aorta atherosclerotic plaques was significantly reduced. We conclude that multifaceted vaccine combining lipid-regulating with anti-inflammation was a potential remedy, especially for atherosclerosis with complicated etiology.