Mycobacterial Heat Shock Protein Research Articles

Mycobacterial Heat Shock Proteins in Sarcoidosis and Tuberculosis.

Pathological similarities between sarcoidosis (SA) and tuberculosis (TB) suggest the role of mycobacterial antigens in the etiopathogenesis of SA. The Dubaniewicz group revealed that not whole mycobacteria, but Mtb-HSP70, Mtb-HSP 65, and Mtb-HSP16 were detected in the lymph nodes, sera, and precipitated immune complexes in patients with SA and TB. In SA, the Mtb-HSP16 concentration was higher than that of Mtb-HSP70 and that of Mtb-HSP65, whereas in TB, the Mtb-HSP16 level was increased vs. Mtb-HSP70. A high Mtb-HSP16 level, induced by low dose-dependent nitrate/nitrite (NOx), may develop a mycobacterial or propionibacterial genetic dormancy program in SA. In contrast to TB, increased peroxynitrite concentration in supernatants of peripheral blood mononuclear cell cultures treated with Mtb-HSP may explain the low level of NOx detected in SA. In contrast to TB, monocytes in SA were resistant to Mtb-HSP-induced apoptosis, and CD4+T cell apoptosis was increased. Mtb-HSP-induced apoptosis of CD8+T cells was reduced in all tested groups. In Mtb-HSP-stimulated T cells, lower CD8+γδ+IL-4+T cell frequency with increased TNF-α,IL-6,IL-10 and decreased INF-γ,IL-2,IL-4 production were present in SA, as opposed to an increased presence of CD4+γδ+TCR cells with increased TNF-α,IL-6 levels in TB, vs. controls. Mtb-HSP modulating the level of co-stimulatory molecules, regulatory cells, apoptosis, clonal deletion, epitope spread, polyclonal activation and molecular mimicry between human and microbial HSPs may also participate in the induction of autoimmunity, considered in SA. In conclusion, in different genetically predisposed hosts, the same antigens, e.g., Mtb-HSP, may induce the development of TB or SA, including an autoimmune response in sarcoidosis.

Interaction of constituents of MDT regimen for leprosy with Mycobacterium leprae HSP18: impact on its structure and function

Mycobacterium leprae, the causative organism of leprosy, harbors many antigenic proteins, and one such protein is the 18-kDa antigen. This protein belongs to the small heat shock protein family and is commonly known as HSP18. Its chaperone function plays an important role in the growth and survival of M. leprae inside infected hosts. HSP18/18-kDa antigen is often used as a diagnostic marker for determining the efficacy of multidrug therapy (MDT) in leprosy. However, whether MDT drugs (dapsone, clofazimine, and rifampicin) do interact with HSP18 and how these interactions affect its structure and chaperone function is still unclear. Here, we report evidence of HSP18-dapsone/clofazimine/rifampicin interaction and its impact on the structure and chaperone function of HSP18. These three drugs interact efficiently with HSP18 (having submicromolar binding affinity) with 1 : 1 stoichiometry. Binding of these MDT drugs to the 'α-crystallin domain' of HSP18 alters its secondary structure and tryptophan micro-environment. Furthermore, surface hydrophobicity, oligomeric size, and thermostability of the protein are reduced upon interaction with these three drugs. Eventually, all these structural alterations synergistically decrease the chaperone function of HSP18. Interestingly, the effect of rifampicin on the structure, stability, and chaperone function of this mycobacterial small heat shock protein is more pronounced than the other two MDT drugs. This reduction in the chaperone function of HSP18 may additionally abate M. leprae survivability during multidrug treatment. Altogether, this study provides a possible foundation for rational designing and development of suitable HSP18 inhibitors in the context of effective treatment of leprosy.

Oral administration of Hsp65-producing Lactococcus lactis attenuates allergic asthma in a murine model.

Allergic asthma is a chronic inflammatory lung disease characterized by a Th2-type immune response pattern. The development of nonspecific immunotherapy is one of the primary goals for the control of this disease. In this study, we evaluated the therapeutic effects of Lactococcus lactis-producing mycobacterial heat shock protein 65 (LLHsp65) in an ovalbumin (OVA)-induced allergic asthma model. OVA-challenged BALB/c mice were orally administrated with LLHsp65 for 10 consecutive days. The results demonstrate that LLhsp65 attenuates critical features of allergic inflammation, like airway hyperresponsiveness and mucus production. Likewise, the treatment decreases the pulmonary eosinophilia and the serum level of OVA-specific IgE. In addition to deviating immune responses towards Th1-cytokine profile, increase regulatory T cells, and cytokine levels, such as IL-6 and IL-10. Our results reveal that the mucosal immunotherapy of LLHsp65 significantly reduces the overall burden of airway allergic inflammation, suggesting a promising therapeutic strategy for allergic asthma treatment. This research reveals new perspectives on nonspecific immunotherapy based on the delivery of recombinant proteins by lactic acid bacteria to treat of allergic disorders.

Synthesis, characterization and bio-functionalization of magnetic nanoparticles to improve the diagnosis of tuberculosis

Mycobacterium tuberculosis is the cause of one of the diseases with the highest mortality and morbidity rate in the Americas and in the world. In developing countries, the diagnosis of tuberculosis (TB) is based on baciloscopy and bacteriological cultures. The first method has a low sensitivity, and the second can take several weeks to reach a confirmatory diagnosis. The lack of a rapid diagnosis compromises the efforts to control this disease and favors the transmission of tuberculosis to the susceptible population. In this work, we present the synthesis, amine-silanization, characterization and bio-functionalization of magnetic nanoparticles (MNPs) to develop a sandwich ELISA to detect and concentrate antigens from M. tuberculosis. For this purpose, a recombinant mycobacterial heat shock protein Hsp16.3, which contributes to the persistence of TB, was cloned and expressed in the E. coli system. Polyclonal antibodies anti-Hsp16.3 were produced in a rabbit and in mice. Magnetic nanoparticles were synthesized by co-precipitation, amine-functionalized and characterized by several physical-chemical methods. The XRD, Mossbauer spectroscopy, zeta potential, TEM, and FTIR all proved the successful preparation of the MNPs showing a diffraction crystal diameter of 10.48 ± 2.56 nm, superficial net charge of : +23.57 ± 2.87 mV, characteristic patterns of magnetite and a structure similar to a sphere. Additionally, it showed a magnetization saturation of 37.06 emu.g−1. For the functionalization of nanoparticle surfaces with anti-Hsp16.3, the active ester method was used for bond formation, and parameters such as time of incubation, coupling agents ratio (EDC/NHS) and concentration as well as surface saturation level of amine-silanized MNPs (MNP@Si@NH2) were standardized. Finally, bio-functionalized MNPs were used to detect, fix and concentrate the recombinant antigen Hsp16.3 from M. tuberculosis in a sandwich ELISA-MNP assay.

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.

Serological Testing for Mycobacterial Heat Shock Protein Hsp65 Antibody in Health and Diseases.

Mycobacterial heat shock protein 65 gene (Hsp65) has been widely used for classification of Mycobacterial species, and detection of Mycobacterial genes by molecular methods and has proven useful in identification of Mycobacterial infection in various clinical conditions. Circulating antibody against Mycobacterial hsp65 has been found in many clinical diseases including autoimmune diseases (Crohn’s disease, lupus erythematosus, multiple sclerosis, diabetes, etc.), atherosclerosis and cancers. The prevalence of anti-Hsp65 antibody in the normal healthy population is unknown. We determined the blood levels of antibody against Mycobacterial hsp65 in the normal population represented by 288 blood donors of the American Red Cross and tested the blood of 109 patients with Crohn’s disease and 28 patients with Sjogren’s syndrome for comparison. The seroprevalence of anti-Hsp65 IgG in the normal population of Red Cross donors was 2.8% (8 of 288 positive). The Hsp65 antibody levels were significantly elevated in patients with Crohn’s disease and Sjogren’s syndrome. The prevalence of Hsp65 antibody in Crohn’s disease patients was 67.9% (74 of 109 patients), and 85.7% for Sjogren’s patients (24 of 28 patients). Our data indicate that anti-Hsp65 antibody is rare in the normal population, but frequent in chronic diseases. The presence of circulating Hsp65 antibody reflects an abnormal immune (adaptive) response to Mycobacterial exposure in patients with chronic diseases, thus differentiating the patients with chronic diseases from those clinical mimics.

Oral Tolerization with Mycobacterial Heat Shock Protein 65 Reduces Chronic Experimental Atherosclerosis in Aged Mice

Background: Atherosclerosis is a chronic inflammatory disease of the artery wall where both innate and adaptive immunity play important roles. Modulation of the immune response against the stress protein antigen, heat shock protein (HSP) 60, by administration of mycobacterial HSP65 (mbHSP65) orally and/or nasally shows promising therapeutic results in young animals in the sense of less severe experimental atherosclerosis; however, the case of aged animals with already established atherosclerosis has so far never been investigated. Objective: To investigate if mbHSP65 immunization would further accelerate atherosclerotic progression in aged ApoE^-/- mice (18 months old) with already long-established atherosclerosis and if these mice could be orally tolerized against mbHSP65. Methods: Aged wild-type (WT) and ApoE^-/- mice (65 weeks) were immunized and/or orally treated with mbHSP65 and then either kept on normal chow or changed to high-cholesterol diet (HCD). Atherosclerosis was assessed by en face analysis and the number of CD4⁺CD25⁺FoxP3⁺ T regulatory cells (Tregs) was assessed by flow cytometry in lymph node and spleen cells. Total cholesterol and triglyceride levels were determined. Soluble mammalian HSP60 and anti-mouse HSP60 (mHSP60) and anti-mbHSP65 antibodies were detected by enzyme-linked immunosorbent assay. Results: As expected, aged WT mice had only minor lesions in the aorta, which did not change under HCD for 14 weeks. Aged ApoE^-/- mice already had large complicated plaques, which increased in size under HCD. mbHSP65 immunization led to a significant aggravation of atherosclerosis in both WT and ApoE^-/- mice irrespective of the nature of their diet. This increase was accompanied by increased titers of both anti-mHSP60 and anti-mbHSP65 antibodies in the circulation. The increased plaque formation could be significantly diminished with oral mbHSP65 tolerization. An increased number of Tregs and lower or unchanged levels of cholesterol and triglycerides were associated with the reduced size of aortal lesions. Conclusion: Oral tolerization against mbHSP65 could be used both to prevent and to treat chronic atherosclerosis in aged individuals.

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.

Mycobacterial heat shock protein 65 (mbHSP65)-induced atherosclerosis - Preventive oral tolerization and definition of atheroprotective and atherogenic mbHSP65-peptides

Mycobacterial heat shock protein 65 (mbHSP65)-induced atherosclerosis - Preventive oral tolerization and definition of atheroprotective and atherogenic mbHSP65-peptides

Recombinant Bacille Calmette-Guérin coexpressing Ag85B-IFN-γ enhances the cell-mediated immunity in C57BL/6 mice.

The only available vaccine against pulmonary tuberculosis is Bacille Calmette-Guérin (BCG). As the efficacy reported of the vaccine is not up to the mark, there is an urgent need to develop improved anti-tuberculosis vaccines. Antigen 85B (Ag85B) is a very promising vaccine candidate molecule of Mycobacterium tuberculosis and interferon (IFN)-γ and has been considered the most attractive correlate of protective immunity. The aim of this study was to construct a novel recombinant BCG (rBCG) to secrete Ag85B and mouse IFN-γ under control of the Mycobacterial heat shock protein 60 (hsp60) promoter and the antigen signal sequence. Second aim of the present study is to evaluate the immune response in C57BL/6 elicted by the new rBCG. Expression of the fusion protein was readily detectable by western blotting and IFN-γ bioactivity was detected indirectly by enzyme-linked immunosorbent assay (ELISA). Compared with BCG, rBCG::Ag85B-IFN-γ was substantially more active in inducing the production of IFN-γ and tumor necrosis factor (TNF)-α from mouse splenocytes. ELISA analysis for IgG, IgG1 and IgG2c showed that rBCG::Ag85B-IFN-γ induced higher titer of Ag85B and facilitated Th1 type immune response. rBCG::Ag85B-IFN-γ also improved nitric oxide production levels and enhanced antigen-specific splenocyte proliferation. Moreover, rBCG::Ag85B-IFN-γ induced human monocytes such as THP-1 cells to enhance expression of CD80, CD86, CD40 and HLA-DR. Flow cytometry analysis confirmed that rBCG::Ag85B-IFN-γ significantly activated CD4+ T cells. Assessing combinations of IFN-γ, TNF-α and interleukin-2 at the single-cell level by multiparameter flow cytometry, we found that rBCG::Ag85B-IFN-γ improved the multifunctional T cells level in comparison to BCG. In conclusion, the present study indicates that rBCG::Ag85B-IFN-γ increases cell mediated immune response and is a potential candidate vaccine for immunotherapeutic protocols against pulmonary tuberculosis.

A simple method of extracting Cap antigen of PCV2b from emulsified vaccines for testing its stability and antigenicity

Oil-based emulsions are commonly used adjuvants for veterinary vaccines. After formulation, it is required to extract protein antigens from emulsified vaccines for testing their stability and antigenicity. To establish a simple method to extract the protein antigens, two emulsified vaccines, designated as Cap-206 and Cap-35, were prepared by formulating a 28-kDa capsid protein (Cap) of PCV2b with ISA 206, a water-in-oil-in-water emulsion, or ISA 35, an oil-in-water emulsion. We found that the freeze-thaw-centrifugation method with steps of freezing at −20 °C for 12 h, thawing at room temperature and centrifuging at 9000×g for 10 min could separate the aqueous phase from Cap-206, and a centrifugation method by centrifuging at 9000×g for 10 min could isolate the aqueous portion from Cap-35. The Cap proteins were recovered from the aqueous phase and could be evaluated for their stability and antigenicity by SDS-PAGE, Western blot and ELISA. The freeze-thaw-centrifugation or the centrifugation method could also be used to recover recombinant mycobacterial heat-shock protein 65, a larger protein with molecular weight of 57-kDa, from ISA 206 or ISA 35 emulsions. The methods could be used to recover protein antigens from oil-based emulsion formulated vaccines for monitoring their stability and antigenicity during vaccine manufacture and storage.

New strategy for testing efficacy of immunotherapeutic compounds for diabetes in vitro.

BackgroundThe valuable role of immunotherapy in treating autoimmune diseases is increasingly recognized by those involved in the research and clinical application of new biopharmaceuticals products. However, many aspects related to the mechanisms of immune-modulated therapies remain to be elucidated in order to explore fully the emerging opportunities. The non-obese diabetic NOD mouse develops insulin-dependent diabetes mellitus spontaneously as a consequence of an autoimmune process in the presence of pathogenic CD4+ T cells that typically exhibit Th17 cell phenotypes. The change of a Th17 phenotype into a pattern of regulatory T cells (Treg) is extremely important in controlling autoimmune diseases. Heat shock proteins (HSPs) are stress-induced proteins with immunoregulatory properties. In the current study, the capacity of Hsp65 and Hsp70 mycobacterial HSPs and a constructed DNA encoded Hsp65 (DNAhsp65) to transform the pattern of the immune response from Th17 into Treg cells has been studied in vitro using co-cultures of antigen presenting cells (APCs) and T cells in NOD mice.ResultsCells harvested from NOD mice and cultured for 48 h (without immunoregulatory compounds) presented with Th1/Th17 patterns and secretions of IL-6, IFN-γ, IL-10 and IL-17 cytokines. The cultured cells from the non-diabetic BALB/C mice exhibited a Th1 pattern and the production of IL 6 and IFN-γ secretions. An up-regulation was observed in the supernatants from the co-cultures of NOD cells that were stimulated with DNAhsp65, Hsp65 or Hsp70 through increased levels of IL-10 secretion and the suppression of IL-6, IFN-γ and IL-17 production. In addition, immunoregulation was demonstrated through IL-17 suppression in the co-culture stimulated by the specific insulin antigen. Moreover, an increase of immunoregulatory compounds were observed in the co-culture through the expression of CD11b+CD86+ activation markers on APCs, as well as the frequency of Treg cells expressing CD4+CD3+ and CD4+CD25hi.ConclusionsThe in vitro observation of Th17 cells differentiating into Tregs in NOD mice could raise the hypothesis that the immune regulatory activity of HSPs could be an efficient strategy for diabetes prevention and treatment.

Generation of the First TCR Transgenic Mouse with CD4(+) T Cells Recognizing an Anti-inflammatory Regulatory T Cell-Inducing Hsp70 Peptide.

Antigen-specific regulatory T cells (Tregs) directed at self-antigens are difficult to study since suitable specific tools to isolate and characterize these cells are lacking. A T cell receptor (TCR)-transgenic mouse would generate possibilities to study such ­antigen-specific T cells. As was shown previously, immunization with the mycobacterial heat shock protein (Hsp) 70-derived peptide B29 and its mouse homologs mB29a and mB29b induced anti-inflammatory responses. Furthermore, B29 induced antigen-­specific Tregs in vivo. To study mB29b-specific Tregs, we isolated the TCR from T cell hybridomas generated against mB29b and produced a TCR transgenic mouse that expresses a MHC-class II restricted mB29b-specific TCR. These TCR transgenic CD4+ T cells were found to cross-react with the B29 epitope as identified with peptide-induced proliferation and IL-2 production. Thus, we have successfully generated a novel mouse model with antigen-specific CD4+ T cells that recognize self and bacterial Hsp 70-derived peptides. With this novel mouse model, it will be possible to study primary antigen-specific T cells with specificity for a regulatory Hsp70 T cell epitope. This will enable the isolation and characterization CD4+CD25+ Tregs with a proven specificity. This will provide useful knowledge of the induction, activation, and mode of action of Hsp70-specific Tregs, for instance, during experimental arthritis.

Immune responses to Mycobacterial heat shock protein 70 accompany self-reactivity to human BiP in rheumatoid arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease, and a member of human heat shock protein (HSP) 70 protein family, Binding Immunoglobulin Protein (BiP), has been identified as an important autoantigen for T and B cells. We herein focused on Mycobacterial (Myc) HSPs and immune responses to MycHSPs in RA patients. Serum titers of antibodies against MycHSP70 were significantly elevated in RA patients and correlated with serum anti-BiP antibody titers. A MycHSP70-derived HLA-DR4 major epitope was identified using the proliferative capacity of RA PBMCs as an indicator. The major epitope, MycHSP70287–306, was located at the corresponding position in the major epitope for human BiP336–355, and a strong correlation was found between the proliferation of PBMCs in response to MycHSP70287–306 and BiP336–355. The immunization of HLA-DR4 transgenic mice with MycHSP70 induced the proliferation of T cells and development of anti-BiP antibodies. In contrast, the oral administration of MycHSP70287–306 resulted in the amelioration of collagen-induced arthritis, serum antibody responses, and T cell proliferation. In conclusion, immune responses to MycHSP70 were associated with adaptive immunity against BiP in RA, and could be an important mechanism underlying the development of autoimmunity.

Dysregulated leukemia inhibitory factor and its receptor regulated signal transducers and activators of transcription 3 pathway: a possible cause for repeated implantation failure in women with dormant genital tuberculosis?

To investigate the influence of dormant Mycobacterium tuberculosis on the expression of various endometrial receptivity markers and leukemia inhibitory factor (LIF)-signal transducers and activators of transcription 3 (STAT3) signaling pathway. Expression of endometrial receptivity markers and LIF-STAT3 signaling in invitro decidualized human endometrial stromal cells (hESC) treated with 65kDa mycobacterial heat shock protein (HSP65) is also explored. A prospective study. Tertiary care hospital and reproductive health research unit. Endometrial tissue samples were collected from 38 women who tested positive for Mycobacterium tuberculosis and 30 normal women with proven fertility undergoing sterilization. Invitro decidualization of hESC was performed. Endometrial biopsies collected from all women during implantation window and treatment of hESC with HSP65. Measurement of various endometrial receptivity markers including αvβ3 integrin, E-cadherin, MECA-79, mucin-1, and pinopodes and LIF/LIFR-STAT3 signaling molecules expressed in the endometrium of women with dormant genital tuberculosis (GTB) during implantation window and measured also in HSP65-treated hESC. Significantly reduced levels of endometrial receptivity markers LIF, LIFR, and pSTAT3 were observed in endometrium of women with dormant GTB as compared with controls. A similar trend was observed under invitro conditions with decreased level of phosphorylated STAT3 in HSP65-treated hESC. However, no change in the expression of endometrial receptivity markers under invitro conditions was observed. Our findings suggest that endometrium of women with dormant GTB is associated with poor receptivity, as evidenced by reduced receptivity markers and aberrant LIF-STAT3 signaling. Invitro treatment of hESC with HSP65 also confirms compromised endometrial decidualization.

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.

Attenuation of experimental asthma by mycobacterial protein combined with CpG requires a TLR9-dependent IFN-γ-CCR2 signalling circuit.

Allergic asthma is a chronic pulmonary disease characterized by a Th2 inflammatory response. The modulation of a Th2 immune response based on immune deviation to a Th1 pattern or induction and migration of regulatory T cells to the lungs constitutes one of the major therapeutic approaches that is being investigated for the treatment of allergic asthma. The potentials of Mycobacterium leprae 65-kD heat-shock protein or Toll-like receptor 9 ligand (CpG oligodeoxynucleotides) as immune modulators for the treatment of airway allergic disease have been studied individually. Mycobacterial protein combined with CpG was used as immunotherapy for airway allergy. Using an ovalbumin-induced asthma model, mice were sensitized and challenged, and then treated with mycobacterial heat-shock protein (Hsp65) combined with CpG. The treatment of mice with established allergy led to the attenuation of eosinophilia, Th2 cytokines and airway hyperresponsiveness. Hsp65 plus CpG treatment also induced an increase in OVA-specific IFN-γ levels and in the frequency of lung inflammatory monocytes. Moreover, we show that the reduction of eosinophilia and the recruitment of inflammatory monocytes to the lungs required early triggering of TLR9, IFN-γ and CCR2 by immunotherapy components. In addition to immune deviation to a Th1 response in the modulation of Th2 allergic inflammation, our findings also attribute an important role to the innate response mediated by TLR9, associated with the recruitment of CCR2-dependent monocytes. Our findings show that the Hsp65/CpG treatment is a promising strategy for consideration in translational studies.

Sulfate Reducing Bacteria and Mycobacteria Dominate the Biofilm Communities in a Chloraminated Drinking Water Distribution System.

The quantity and composition of bacterial biofilms growing on 10 water mains from a full-scale chloraminated water distribution system were analyzed using real-time PCR targeting the 16S rRNA gene and next-generation, high-throughput Illumina sequencing. Water mains with corrosion tubercles supported the greatest amount of bacterial biomass (n = 25; geometric mean = 2.5 × 10(7) copies cm(-2)), which was significantly higher (P = 0.04) than cement-lined cast-iron mains (n = 6; geometric mean = 2.0 × 10(6) copies cm(-2)). Despite spatial variation of community composition and bacterial abundance in water main biofilms, the communities on the interior main surfaces were surprisingly similar, containing a core group of operational taxonomic units (OTUs) assigned to only 17 different genera. Bacteria from the genus Mycobacterium dominated all communities at the main wall-bulk water interface (25-78% of the community), regardless of main age, estimated water age, main material, and the presence of corrosion products. Further sequencing of the mycobacterial heat shock protein gene (hsp65) provided species-level taxonomic resolution of mycobacteria. The two dominant Mycobacteria present, M. frederiksbergense (arithmetic mean = 85.7% of hsp65 sequences) and M. aurum (arithmetic mean = 6.5% of hsp65 sequences), are generally considered to be nonpathogenic. Two opportunistic pathogens, however, were detected at low numbers: M. hemophilum (arithmetic mean = 1.5% of hsp65 sequences) and M. abscessus (arithmetic mean = 0.006% of hsp65 sequences). Sulfate-reducing bacteria from the genus Desulfovibrio, which have been implicated in microbially influenced corrosion, dominated all communities located underneath corrosion tubercules (arithmetic mean = 67.5% of the community). This research provides novel insights into the quantity and composition of biofilms in full-scale drinking water distribution systems, which is critical for assessing the risks to public health and to the water supply infrastructure.