Protective efficacy of different strategies employing Mycobacterium leprae heat-shock protein 65 against tuberculosis

Background: Tuberculosis is a major threat to human health. The high disease burden remains unaffected and the appearance of extremely drug-resistant strains in different parts of the world argues in favor of the urgent need for a new effective vaccine. One of the promising candidates is heat-shock protein 65 when used as a genetic vaccine (DNAhsp65). Nonetheless, there are substantial data indicating that BCG, the only available anti-TB vaccine for clinical use, provides other important beneficial effects in immunized infants. Methods: We compared the protective efficacy of BCG and Hsp65 antigens in mice using different strategies: i) BCG, single dose subcutaneously; ii) naked DNAhsp65, four doses, intramuscularly; iii) liposomes containing DNAhsp65, single dose, intranasally; iv) microspheres containing DNAhsp65 or rHsp65, single dose, intramuscularly; and v) prime-boost with subcutaneous BCG and intramuscular DNAhsp65. Results: All the immunization protocols were able to protect mice against infection, with special benefits provided by DNAhsp65 in liposomes and prime-boost strategies. Conclusion: Among the immunization protocols tested, liposomes containing DNAhsp65 represent the most promising strategy for the development of a new anti-TB vaccine.

Chronic low frequency/low volume resistance training reduces pro-inflammatory cytokine protein levels and TLR4 mRNA in rat skeletal muscle

Skeletal muscle is the source of pro- and anti-inflammatory cytokines, and recently, it has been recognized as an important source of interleukin 6 (IL-6), a cytokine that exerts inhibitory effects on several pro-inflammatory cytokines. Although dynamic chronic resistance training has been shown to produce the known ""repeated bout effect"", which abolishes the acute muscle damage, performing of high-intensity resistance training has been regarded highly advisable, at least from the hypertrophy perspective. On the other hand, a more therapeutic, ""non-damaging"" resistance training program, mainly composed of concentric forces, low frequency/low volume of training, and the same exercise, could theoretically benefit the muscle when the main issue is to avoid muscle inflammation (as in the treatment of several ""low-grade"" inflammatory diseases) because the acute effect of each resistance exercise session could be diminished/avoided, at the same time that the muscle is still being overloaded in a concentric manner. However, the benefits of such ""less demanding"" resistance training schedule on the muscle inflammatory profile have never been investigated. Therefore, we assessed the protein expression of IL-6, TNF-alpha, IL-10, IL-10/TNF-alpha ratio, and HSP70 levels and mRNA expression of SCF(beta-TrCP), IL-15, and TLR-4 in the skeletal muscle of rats submitted to resistance training. Briefly, animals were randomly assigned to either a control group (S, n = 8) or a resistance-trained group (T, n = 7). Trained rats were exercised over a duration of 12 weeks (two times per day, two times per week). Detection of IL-6, TNF-alpha, IL-10, and HSP70 protein expression was carried out by western blotting and SCF(beta-TrCP) (SKP Cullin F-Box Protein Ligases), a class of enzymes involved in the ubiquitination of protein substrates to proteasomal degradation, IL-15, and TLR-4 by RT-PCR. Our results show a decreased expression of TNF-alpha and TLR4 mRNA (40 and 60%, respectively; p < 0.05) in the plantar muscle from trained, when compared with control rats. In conclusion, exercise training induced decreased TNF-alpha and TLR-4 expressions, resulting in a modified IL-10/TNF-alpha ratio in the skeletal muscle. These data show that, in healthy rats, 12-week resistance training, predominantly composed of concentric stimuli and low frequency/low volume schedule, down regulates skeletal muscle production of cytokines involved in the onset, maintenance, and regulation of inXammation.

Molecular characterization of a putative heat shock protein cognate gene in Rhynchosciara americana

An hsc70 homologue gene (Rahsc70) of the diptera Rhynchosciara americana was isolated and characterized. We were able to determine the mRNA sequence from an EST of salivary gland cDNA library, and a Rahsc70 cDNA cassette was used as a probe to isolate the genomic region from a genomic library. The mRNA expression of this gene parallels the 2B puff expansion, suggesting its involvement in protein processing, since this larval period corresponds to a high synthetic activity period. During heat shock stress conditions, hsc70 expression decreased. In situ hybridization of polytene chromosomes showed that the Rahsc70 gene is located near the C3 DNA puff. The cellular localization of Hsc70 protein showed this protein in the cytoplasm and in the nucleus.

L-NAME Treatment Enhances Exercise-induced Content of Myocardial Heat Shock Protein 72 (Hsp72) in Rats

Background/Aim: Nitric oxide (NO) modulates the expression of the chaperone Hsp72 in the heart, and exercise stimulates both NO production and myocardial Hsp72 expression. The main purpose of the study was to investigate whether NO interferes with an exercise-induced myocardial Hsp72 expression. Methods: Male Wistar rats (70-100 days) were divided into control (C, n= 12), L-NAME-treated (L, n= 12), exercise (E, n= 13) and exercise plus L-NAME-treated (EL, n= 20) groups. L-NAME was given in drinking water (700 mg. L(-1)) and the exercise was performed on a treadmill (15-25 m.min(-1), 40-60 min. day(-1)) for seven days. Left ventricle (LV) protein Hsp content, NOS and phosphorylated-NOS (p-NOS) isoforms were measured using Western blotting. The activity of NOS was assayed in LV homogenates by the conversion of [(3)H] L-arginine to [(3)H] L-citrulline. Results: Hsp72 content was increased significantly (223%; p < 0.05) in the E group compared to the C group, but exercise alone did not alter the NOS content, p-NOS isoforms or NOS activity. Contrary to our expectation, L-NAME enhanced (p < 0.05) the exercise-induced Hsp72 content (EL vs. C, L and E groups = 1019%, 548% and 457%, respectively). Although the EL group had increased stimulatory p-eNOS(Ser1177) (over 200%) and decreased inhibitory p-nNOS(Ser852) (similar to 50%) compared to both the E and L groups (p < 0.05), NOS activity was similar in all groups. Conclusions: Our results suggest that exercise-induced cardiac Hsp72 expression does not depend on NO. Conversely, the in vivo L-NAME treatment enhances exercise-induced Hsp72 production. This effect may be due to an increase in cardiac stress. Copyright (C) 2011 S. Karger AG, Basel

Administration of Mycobacterium leprae rHsp65 Aggravates Experimental Autoimmune Uveitis in Mice

The 60kDa heat shock protein family, Hsp60, constitutes an abundant and highly conserved class of molecules that are highly expressed in chronic-inflammatory and autoimmune processes. Experimental autoimmune uveitis [EAU] is a T cell mediated intraocular inflammatory disease that resembles human uveitis. Mycobacterial and homologous Hsp60 peptides induces uveitis in rats, however their participation in aggravating the disease is poorly known. We here evaluate the effects of the Mycobacterium leprae Hsp65 in the development/progression of EAU and the autoimmune response against the eye through the induction of the endogenous disequilibrium by enhancing the entropy of the immunobiological system with the addition of homologous Hsp. B10. RIII mice were immunized subcutaneously with interphotoreceptor retinoid-binding protein [IRBP], followed by intraperitoneally inoculation of M. leprae recombinant Hsp65 [rHsp65]. We evaluated the proliferative response, cytokine production and the percentage of CD4(+)IL-17(+), CD4(+)IFN-gamma(+) and CD4(+)Foxp3(+) cells ex vivo, by flow cytometry. Disease severity was determined by eye histological examination and serum levels of anti-IRBP and anti-Hsp60/65 measured by ELISA. EAU scores increased in the Hsp65 group and were associated with an expansion of CD4(+)IFN-gamma(+) and CD4(+)IL-17(+) T cells, corroborating with higher levels of IFN-gamma. Our data indicate that rHsp65 is one of the managers with a significant impact over the immune response during autoimmunity, skewing it to a pathogenic state, promoting both Th1 and Th17 commitment. It seems comprehensible that the specificity and primary function of Hsp60 molecules can be considered as a potential pathogenic factor acting as a whistleblower announcing chronic-inflammatory diseases progression.

Radicicol improves regeneration of skeletal muscle previously damaged by crotoxin in mice

This work investigates the influence of heat shock proteins (HSPs) on necrosis and subsequent skeletal muscle regeneration induced by crotoxin (CTX), the major component of Crotalus durissus terrificus venom. Mice were treated with radicicol, a HSP inductor, followed by an intramuscular injection of CTX into the gastrocnemius muscle. Treated groups were sacrificed 1, 10 and 21 days after CTX injection. Muscle histological sections were stained with toluidine blue and assayed for acid phosphatase or immunostained with either neuronal cell adhesion molecule (NCAM) or neonatal myosin heavy chain (MHCn). Muscle samples were also submitted to Western blotting analysis. The results show that CTX alone and CTX combined with radicicol induced a similar degree of myofiber necrosis. CTX-injured muscles treated with radicicol had increased cross-sectional areas at 10 and 21 days post-lesion compared with untreated CTX-injured muscles. Additionally, radicicol significantly increased the number of NCAM-positive satellite cells in the gastrocnemius at one day post-CTX injury. CTX-injured Muscles treated with radicicol contained more MHCn-positive regenerating myofibers compared with untreated CTX-injured muscles. These results suggest that HSPs contribute to the regeneration of myofibers damaged by CTX. Additionally, further studies should investigate the potential therapeutic effects of radicicol in skeletal muscles affected by Crotalus venom. (C) 2008 Elsevier Ltd. All rights reserved.

DNAhsp65 vaccination induces protection in mice against Paracoccidioides brasiliensis infection

Heat-shock proteins are Molecules with extensive data showing their potential as immunomodulators of different types of diseases, The gene of HSP65 from Mycobacterium leprae has shown prophylactic and immunotherapeutic effects against a broad arrays of experimental models including tuberculosis, leishmaniasis, arthritis and diabetes. With this in mind, we tested the DNAhsp65 vaccine using an experimental model of Paraccocidiodomycosis, an important endemic mycosis in Latin America. The intramuscular immunization with DNAhsp65 induced, in BALB/c mice, an increase of Th1-levels cytokines and a reduction of fungal burdens resulted in a marked reduction of collagen and lung remodeling. DNAhsp65 may be an attractive candidate for prevention, therapy and as an adjuvant for mycosis treatment. (C) 2008 Elsevier Ltd. All rights reserved.

Immune response to vaccination with DNA-hsp65 in a phase I clinical trial with head and neck cancer patients

DNA-hsp65, a DNA vaccine encoding the 65-kDa heat-shock protein of Mycobacterium leprae (Hsp65) is capable of inducing the reduction of established tumors in mouse models. We conducted a phase I clinical trial of DNA-hsp65 in patients with advanced head and neck carcinoma. In this article, we report on the vaccine`s potential to induce immune responses to Hsp65 and to its human homologue, Hsp60, in these patients. Twenty-one patients with unresectable squamous cell carcinoma of the head and neck received three doses of 150, 400 or 600 mu g naked DNA-hsp65 plasmid by ultrasound-guided intratumoral injection. Vaccination did not increase levels of circulating anti-hsp65 IgG or IgM antibody, or lead to detectable Hsp65-specific cell proliferation or interferon-gamma (IFN-gamma) production by blood mononuclear cells. Frequency of antigen-induced IL-10-producing cells increased after vaccination in 4 of 13 patients analyzed. Five patients showed disease stability or regression following immunization; however, we were unable to detect significant differences between these patients and those with disease progression using these parameters. There was also no increase in antibody or IFN-gamma responses to human Hsp60 in these patients. Our results suggest that although DNA-hsp65 was able to induce some degree of immunostimulation with no evidence of pathological autoimmunity, we were unable to differentiate between patients with different clinical outcomes based on the parameters measured. Future studies should focus on characterizing more reliable correlations between immune response parameters and clinical outcome that may be used as predictors of vaccine success in immunosuppressed individuals. Cancer Gene Therapy (2009) 16, 598-608; doi:10.1038/cgt.2009.9; published online 6 February 2009

Auto-antibodies do not influence development of atherosclerotic plaques in rheumatoid arthritis

Background: Many questions remain unanswered about premature atherosclerosis in rheumatoid arthritis (RA). Besides inflammation, some studies have suggested the role of autoantibodies on its pathogenesis. Objective: The aim of this study was to investigate the presence of antibodies against phospholipids, beta2-glycoproteinl (beta2-gpl), lipoprotein lipase, and heat shock proteins (Hsp) in RA patients and to evaluate their possible association with subclinical carotid atherosclerosis. Methods: Seventy-one RA patients and 53 age- and sex-matched controls were selected to perform anticardiolipin antibodies (aCL) (IgG and IgM), anti-beta2-gpl (IgG, IgM, and IgA), anti-lipoprotein lipase (anti-LPL), anti-Hsp 60, and anti-Hsp 65 by ELISA tests. Intima-medial thickness (IMT) of common carotid and presence of plaques were assessed by high-resolution B-mode ultrasonography. Exclusion criteria were smoking, diabetes, and arterial hypertension. Lipoproteins, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), and fibrinogen levels, as well as health assessment questionnaire (HAQ) and disease activity score (DAS) 28 were also evaluated. Results: Age (48.93 +/- 12.31 vs. 45.37 +/- 9.37 years; p = 0.20) and body mass index (BMI) (p = 0.69) were similar in RA and controls, as well as female gender (p = 0.56). The mean IMT was similar between RA and controls (0. 721 +/- 0.16 vs. 0.667 +/- 0.14 turn, p = 0.07) but the frequency of plaques was higher in RA (14.1% vs. 1.9%; p = 0.02). In RA patients, IMT measurements did not differ according to the presence or absence of these antibodies: IgG aCL (0.62 +/- 0.64 vs. 0.72 +/- 0.17 mm, p = 0.24), IgM aCL (0.65 +/- 0.79 vs. 0.73 +/- 0.17 mm, p = 0.33), anti-Hsp 60 (0.78 +/- 0.20 vs. 0.71 +/- 0.16 mm, p = 0.27), anti-Hsp 65 (0.73 +/- 0.16 vs. 0.72 +/- 0.17 mm, p = 0.77), IgG anti-beta2-gpl (0.73 +/- 0.16 vs. 0.71 +/- 0.17 mm, p = 0.72), and anti-CCP (0.71 +/- 0.16 vs. 0.76 +/- 0.20 mm, p = 0.36). In addition, IMT did not correlate with antibodies titers: IgG aCL (r = -0.09, p = 0.47), IgM aCL (r = - 0.15, p = 0.21), anti-Hsp 60 (r = 0.10, p = 0.42), anti-Hsp 65 (r = 0.05, p = 0.69), IgG anti-beta2-gpl (r = - 0.07, p = 0.57), IgM anti-beta2-gpl (r = - 0.05, p = 0.69), IgA anti-beta2-gpl (r = 0.03, p = 0.79), and anti-CCP (r = - 0.07, p = 0.57). RA patients with plaques had a significantly higher age compared to those without plaques (p = 0.001), as well as higher mean IMT (p < 0.001), total cholesterol (p = 0.001), and LDL (p = 0.003). Conclusions: In RA a clear association between all autoantibodies studied herein and increased IMT or presence of plaques was not observed. The great prevalence of carotid atherosclerosis in RA was related to age, total and LDL cholesterol, as identified in normal population. (c) 2008 Elsevier Masson SAS. All rights reserved.

Profiling the Proteome of the Venom from the Social Wasp Polybia paulista: A Clue to Understand the Envenoming Mechanism

The study reported here is a classical bottom-up proteomic approach where proteins from wasp venom were extracted and separated by 2-DE; the individual protein spots were proteolytically digested and subsequently identified by using tandem mass spectrometry and database query with the protein search engine MASCOT. Eighty-four venom proteins belonging to 12 different molecular functions were identified. These proteins were classified into three groups; the first is constituted of typical venom proteins: antigens-5, hyaluronidases, phospholipases, heat shock proteins, metalloproteinases, metalloproteinase-desintegrin like proteins, serine proteinases, proteinase inhibitors, vascular endothelial growth factor-related protein, arginine kinases, Sol i-II and -II like proteins, alpha-glucosidase, and superoxide dismutases. The second contained proteins structurally related to the muscles that involves the venom reservoir. The third group, associated with the housekeeping of cells from venom glands, was composed of enzymes, membrane proteins of different types, and transcriptional factors. The composition of P. paulista venom permits us to hypothesize about a general envenoming mechanism based on five actions: (i) diffusion of venom through the tissues and to the blood, (ii) tissue, (iii) hemolysis, (iv) inflammation, and (v) allergy-played by antigen-5, PLA1, hyaluronidase, HSP 60, HSP 90, and arginine kinases.

Hypertonic saline reduces metalloproteinase expression in liver during pancreatitis

P>We recently demonstrated that hypertonic saline reduces inflammation and mortality in acute pancreatitis. The present study investigated the effects of hypertonic saline in metalloproteinase (MMP) regulation and pancreatitis-associated hepatic injury. Wistar rats were divided into four groups: (i) control, not subjected to insult or treatment; (ii) no treatment (NT), induction of pancreatitis (retrograde infusion of 2.5% sodium taurocholate (1.0 mL/kg)), but no further treatment; (iii) normal saline (NS), induction of pancreatitis and treatment with normal saline (0.9% NaCl, 34 mL/kg, i.v. bolus, 1 h after the induction of pancreatitis); and (iv) hypertonic saline (HS), induction of pancreatitis and treatment with hypertonic saline (7.5% NaCl, 4 mL/kg administered over a period of 5 min, 1 h after the induction of pancreatitis). In all four groups, 4, 12 and 24 h after the induction of pancreatitis, liver tissue samples were assayed to determine levels of MMP-2, MMP-9, 47 kDa heat shock protein (HSP47) and collagen (Type I and III). Compared with the control group, MMP-9 expression and activity was increased twofold in the NS and NT groups 4 and 12 h after the induction of pancreatitis, but remained at basal levels in the HS group. In contrast, MMP-2 expression was increased twofold 12 h after the induction of pancreatitis only in the NS group, whereas the expression of HSP47 was increased 4 h after the induction of pancreatitis in the NS and NT groups. Greater extracellular matrix remodelling occurred in the NS and NT groups compared with the HS group, probably as a result of the hepatic wound-healing response to repeated injury. However, the collagen content in hepatic tissue remained at basal levels in the HS group. In conclusion, the results of the present study indicate that hypertonic saline is hepatoprotective and reduces hepatic remodelling, maintaining the integrity of the hepatic extracellular matrix during pancreatitis. Hypertonic saline-mediated regulation of MMP expression may have clinical relevance in pancreatitis-associated liver injury.

Vaccination, atherosclerosis and systemic lupus erythematosus

Atherosclerosis is an inflammatory disease, leading to the formation of pro-inflammatory and pro-oxidative lipids that generate an immune response. Several antigens have been shown to activate the immune response and affect the development of atherogenesis. Systemic lupus erythematosus is an autoimmune and inflammatory disease strongly associated with premature development of atherosclerotic plaques. Modulation of the immune system could represent a useful approach to prevent and/or treat atherosclerosis. A vaccination-based approach might be a useful, effective tool in the modern arsenal of cardiovascular therapies and could be used on a large scale at a low cost. In non-systemic lupus erythematosus populations, vaccines against oxidized low-density lipoprotein, beta-2-glycoprotein I, heat shock proteins, lipoproteins, cholesterol, molecules involved in cholesterol metabolism, and other molecules (CD99, vascular endothelial growth factor-receptor, and interleukin-2) have been tested, with promising results. However, there are no studies of vaccination against atherosclerosis in systemic lupus erythematosus. Lupus (2009) 18, 1209-1212.

HSP65 DNA as therapeutic strategy to treat experimental paracoccidioidomycosis

The conventional treatment for paracoccidioidomycosis, the most prevalent mycosis in Latin America, involves long periods of therapy resulting in sequels and high frequency of relapses. The search for new alternatives of treatment is necessary. Previously, we have demonstrated that the hsp65 gene from Mycobacterium leprae shows prophylactic effects against murine paracoccidioidomycosis. Here, we tested the DNAhsp65 immunotherapy in BALB/c mice infected with Paracoccidioides brasiliensis, the agent of paracoccidioidomycosis. We observed an increase of Th1 cytokines accompanied by a reduction in fungal burden and pulmonary injury. These results provide new prospects for immunotherapy of paracoccidioidomycosis and other mycoses. (C) 2009 Elsevier Ltd. All rights reserved.

Neonatal BCG Immunization Followed by DNAhsp65 Boosters: Highly Immunogenic but not Protective Against Tuberculosis - a Paradoxical Effect of the Vector?

A new tuberculosis vaccine is urgently needed. Prime-boost strategies are considered very promising and the inclusion of BCG is highly desirable. In this investigation, we tested the protective efficacy of BCG delivered in the neonatal period followed by boosters in the adult phase with a DNA vaccine containing the hsp65 gene from Mycobacterium leprae (pVAXhsp65). Immune responses were characterized by serum anti-hsp65 antibody levels and IFN-gamma and IL-5 production by the spleen. Amounts of these cytokines were also determined in lung homogenates. Protective efficacy was established by the number of colony-forming units (CFU) and histopathological analysis of the lungs after challenge with Mycobacterium tuberculosis. Immunization with BCG alone triggered a significant reduction of CFU in the lungs and also clearly preserved the pulmonary parenchyma. BCG priming also increased the immunogenicity of pVAXhsp65. However, boosters with pVAXhsp65 or the empty vector abolished the protective efficacy of BCG. Also, higher IL-5 levels were produced by spleen and lungs after DNA boosters. These results demonstrated that neonatal BCG immunization followed by DNAhsp65 boosters is highly immunogenic but is not protective against tuberculosis.

Immunization with pVAXhsp65 Decreases Inflammation and Modulates Immune Response in Experimental Encephalomyelitis

Background: A DNA vaccine (pVAXhsp65) containing the gene of a heat-shock protein (hsp65) from Mycobacterium leprae showed high immunogenicity and protective efficacy against tuberculosis in BALB/c mice. A possible deleterious effect related to autoimmunity needed to be tested because hsp65 is highly homologous to the correspondent mammalian protein. In this investigation we tested the effect of a previous immunization with DNAhsp65 in the development of experimental autoimmune encephalomyelitis (EAE), a rat model of multiple sclerosis. Methods: Female Lewis rats were immunized with 3 pVAXhsp65 doses by intramuscular route. Fifteen days after the last DNA dose the animals were evaluated for specific immunity or submitted to induction of EAE. Animals were evaluated daily for weight loss and clinical score, and euthanized during the recovery phase to assess the immune response and inflammatory infiltration at the central nervous system. Results: Immunization with pVAXhsp65 induced a specific immune response characterized by production of IgG(2b) anti-hsp65 antibodies and IFN-gamma secretion. Previous immunization with pVAXhsp65 did not change EAE clinical manifestations (weight and clinical score). However, the vaccine clearly decreased brain and lumbar spinal cord inflammation. In addition, it downmodulated IFN-gamma and IL-10 production by peripheral lymphoid organs. Conclusion: Our data demonstrated that this vaccine does not trigger a deleterious effect on EAE development and also points to a potential protective effect. Copyright (C) 2010 S. Karger AG, Basel