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Autoantibodies against the M2 receptors (M2AChR) have been associated with Dilated Cardiomyopathy (DCM). In the heart, P2×7 receptors influence electrical conduction, coronary circulation and response to ischemia. They can also trigger pro-inflammatory responses and the development of neurological, cardiac and renal disorders. Here, P2×7−/− mice displayed an increased heart rate and ST segment depression, but similar exercise performance when compared to wild type (WT) animals. After immunization with plasmid containing M2AChR cDNA sequence, WT mice produced anti-M2AChR antibodies, while P2×7−/− mice showed an attenuated production. Despite this, WT and P2×7−/− showed left ventricle cavity enlargement and decreased exercise tolerance. Transfer of serum from M2AChR WT immunized mice to näive recipients led to an alteration in heart shape. P2×7−/− mice displayed a significant increase in the frequency of spleen regulatory T cells population, which is mainly composed by the FoxP3+CD25− subset. M2AChR WT immunized mice showed an increase in IL-1β, IFNγ and IL-17 levels in the heart, while P2×7−/− group produced lower amounts of IL-1β and IL-17 and higher amounts of IFNγ. These results pointed to previously unnoticed roles of P2×7 in cardiovascular and immune systems and underscored the participation of IL-17 and IFNγ in the progress of autoimmune DCM.

BACKGROUND 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. OBJECTIVE Mycobacterial protein combined with CpG was used as immunotherapy for airway allergy. METHODS Using an ovalbumin-induced asthma model, mice were sensitized and challenged, and then treated with mycobacterial heat-shock protein (Hsp65) combined with CpG. RESULTS 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. CONCLUSION 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. CLINICAL RELEVANCE Our findings show that the Hsp65/CpG treatment is a promising strategy for consideration in translational studies.

We described a prophylactic and therapeutic effect of a DNA vaccine encoding the Mycobacterium leprae 65-kDa heat shock protein (DNA-hsp65) in experimental murine tuberculosis. However, high homology of the vaccine to the corresponding mammalian hsp60, together with the CpG motifs in the plasmidial vector, could trigger or exacerbate an autoimmune disease. In the present study, we evaluate the potential of DNA-hsp65 vaccination to induce or modulate arthritis in mice genetically selected for acute inflammatory reaction (AIR), either maximal (AIRmax) or minimal (AIRmin). Mice immunized with DNA-hsp65 or injected with the corresponding DNA vector (DNAv) developed no arthritis, whereas pristane injection resulted in arthritis in 62% of AIRmax mice and 7.3% of AIRmin mice. Administered after pristane, DNA-hsp65 downregulated arthritis induction in AIRmax animals. Levels of interleukin (IL)-12 were significantly lower in mice receiving pristane plus DNA-hsp65 or DNAv than in mice receiving pristane alone. However, when mice previously injected with pristane were inoculated with DNA-hsp65 or DNAv, the protective effect was significantly correlated with lower IL-6 and IL-12 levels and higher IL-10 levels. Our results strongly suggest that DNA-hsp65 has no arthritogenic potential and is actually protective against experimentally induced arthritis in mice.

Autoimmune mediated myocardial damage is likely to be a pathogenic mechanism for acquired dilated cardiomyopathies. Evidence confirms that autoantibodies that bind to M(2) muscarinic (M(2)AChR) and beta(1) adrenergic receptors (beta(1)AR) are present in idiopathic dilated cardiomyopathy and Chagasic patients' sera. To elucidate the role of these antibodies in cardiac functional impairment, we used a murine model immunized with plasmids encoding the M(2)AChR or beta(1)AR via gene-gun bombardment. Anti-M(2)AChR and beta(1)AR antibodies were detected over the course of 37 weeks. These antibodies were directed to the second extracellular loop (el2) of both receptors and the third intracellular loop (il3) of the M(2)AChR. Peak antibody titers from weeks 2 to 5 against M(2)AChR-el2 and beta(1)AR-el2 as well as elevated titers against M(2)AChR-il3 were detected. Anti-M(2)AChR-il3 and anti-beta(1)AR-el2 antibodies were predominant in IgG1 subclass immunoglobulins, suggesting a T-helper-2 biased lymphocyte response. Heart morphology and function was assessed by echocardiography over the course of 42 weeks. Data showed progressive decrease in left ventricular (LV) wall thickness and LV mass that was mostly evident for beta(1)AR-immunized mice albeit a small change in LV dimensions. Fractional shortening was altered and values of 41%, 37% and 48% were observed at week 42 for the M(2)AChR, beta(1)AR and control groups respectively. In support of autonomic deregulation, a twofold increase in M(2)AChR and a similar decrease in beta(1)AR density were observed in radioligand saturation assays for both experimental groups. Histological analysis revealed myofibril disarray and fibrosis, pointing towards remodeling as a consequence of the long-term presence of anti-receptor antibodies.

Histoplasma capsulatum is a dimorphic pathogenic fungus that causes a wide spectrum of disease when mycelial fragments are inhaled. Resistance to H. capsulatum is dependent on cellular immunity mediated by T cells and macrophages. Here we standardized the production of extracts containing cell-free antigens (CFAgs) and observed their efficacy in evaluating cellular immunity during murine histoplasmosis. CFAgs induced a more potent delayed-type hypersensitivity (DTH) response in H. capsulatum -infected mice than did histoplasmin—a classical antigen. This DTH response to CFAgs is able to determine the immune status of infected mice and to predict their death. Moreover, CFAgs stimulated spleen cells from immune mice to produce higher amounts of gamma interferon (IFN-γ) in vitro. Finally, immunization with CFAgs protected against a lethal inoculum of H. capsulatum . These results demonstrate that CFAgs may be useful for the evaluation of cellular immune response and as a potential source for the development of a vaccine against histoplasmosis.

Cyclostellettamines A – F (1 – 6) isolated from the sponge Pachychalina sp. and cyclostellettamines G - I, K and L (7 – 11) obtained by synthesis were evaluated in bioassays of antimicrobial activity against susceptible and antibiotic-resistant Staphylococcus aureus, Pseudomonas aeruginosa and antibiotic-susceptible Escherichia coli and Candida albicans, as well as in antimycobacterial activity against Mycobacterium tuberculosis H37Rv bioassays. The results obtained indicated that cyclostellettamines display different antimicrobial activity depending on the alkyl-chain size, suggesting that, if a mechanism-of action is implied, it is dependent on the distance between the two pyridinium moieties of cyclostellettamines.

Inclusion complexes and physical mixtures of isomeric mixture of E/Z (50:50) of 3-(4'-bromo-[1,1'-biphenyl]-4-yl)-3-(4-bromophenyl)-N,N-dimethyl-2-propen-1-amine (BBAP) and b-cyclodextrin (b-CD) in the molar proportion of 1:1 and 1:2 were analyzed by scanning electron microscopy. The dissolution behavior of BBAP and of the inclusion complexes were also evaluated for six hours. By scanning electron microscopy (SEM), it was possible to observe an inclusion complex formed between BBAP and b-CD by co-evaporation, either in the molar proportion of 1:1 or 1:2. In the physical mixtures, no complex was observed as previously detected by physicochemical analysis. The dissolution studies showed that the inclusion complexes BBAP/b-CD 1:1 and 1:2 released respectively 49.07 ± 1.48 and 40.26 ± 3.90% of BBAP during six hours. Free BBAP was less soluble than the inclusion complex and reached 9.00 ± 0.75% of dissolution. Biological assays, such as cytotoxicity to J774 macrophages and to a permanent lung fibroblast cell line (V79), indicated that the BBAP does not exhibit any additional toxic effect with the b-CD complexes. However, the complexes were less cytotoxic to V79 cells than the free form. The BBAP/b-CD inclusion complexes were more effective (MIC) than the free compound on several mycobacteria strains. Similar behavior was observed for BBAP/b-CD complexes and rifampicin, a front-line antitubercular drug, on M. tuberculosis H37Rv growing inside J774 macrophages.

We evaluated the use of a vaccine formulation based on a mixture of two different PLGA microspheres, composed by faster and slower release profiles, containing DNA encoding hsp65 and the recombinant hsp65 protein, respectively, aiming to DNA priming and protein boost after a single dose vaccination. The combination of PLGA50:50 microspheres containing DNA-hsp65 and trehalose dimycolate (TDM) with PLGA75:25 microspheres containing recombinant hsp65 (prime-boost Me) was able to induce high levels of anti-hsp65 specific antibodies. The serum levels of these specific antibodies remained high during 90 days after vaccination, whereas the DNA Me formulation based only in DNA-hsp65 plus TDM-loaded microspheres was not able to sustain the high antibody levels during the same period. Production of IFN-gamma was significant in animals vaccinated with both formulations, while the prime-boost Me vaccinated mice sustained higher levels of this cytokine during all the evaluation period. Thus, prime-boost strategy by using biodegradable microspheres seems to be a promising strategy to stimulate long-lasting immune response.

A DNA vaccine codifying the mycobacterial hsp65 can prevent infection with Mycobacterium tuberculosis in a prophylactic setting and also therapeutically reduce the number of bacteria in infected mice. The protective mechanism is thought to be related to Th1-mediated events that result in bacterial killing. To determine the best method of hsp65 introduction for vaccination efficacy against tuberculosis (TB), we evaluated the immunogenicity and protection of DNA-hsp65 administered by gene gun bombardment or intramuscular (i.m.) injection of naked DNA. Immunization by gene gun induced immune response with plasmid doses 100-fold lower than those required for intramuscular immunization. However, in contrast to intramuscular immunization, which was protective in these studies, gene gun immunization did not protect BALB/c mice against challenge infection.

Nucleic acid vaccines are an attractive alternative to conventional protein vaccines because of their ability to induce de novo production of antigens in a given tissue after DNA delivery. Although DNA vaccines are highly effective in inducing both cell-mediated and humoral immunity, little is known about the many cell types involved in plasmid DNA uptake in vivo. Here we demonstrate, for the first time, that plasmid DNA can be taken up by both bone marrow and lymph node B cells after intramuscular immunization. Plasmid DNA was also detected in CD11b+ and CD11c+ cells. This phenomenon was not restricted to plasmid DNA encoding mycobacterial 65-kd heat shock protein (pcDNA3-hsp65) because we observed similar results with plasmid-encoding green fluorescent protein (GFP-pEGFP-2C). In addition to plasmid DNA uptake, B cells also express the encoded protein, suggesting that B cells play a role in the immune response after DNA immunization. The biodistribution of plasmid DNA in B cells opens a new perspective in B-cell gene therapy for the in vivo use of plasmid DNA.

Histoplasma capsulatum is a fungus found intracellularly in neutrophils and peripheral blood mononuclear cells (PBMCs), suggesting that it is capable of evading damage and survives inside these cells. In this study, we report that neutrophils from H. capsulatum-infected mice, and human neutrophils and mononuclear cells exposed to H. capsulatum presented less apoptosis than those from noninfected animals or cells exposed to medium only. Moreover, cells harvested from infected animals are resistant to apoptosis induced by dexamethasone - a proapoptotic stimulant. We also show that neutrophils harvested from infected mice and PBMCs from humans exposed to the fungus had a greatly decreased Mac-1 expression. We conclude that H. capsulatum induces an antiapoptotic state on leucocytes, which correlates with decreased cell-surface Mac-1 expression. These facts may represent an escape mechanism for the fungus by delaying cell death and allowing the fungus to survive inside leucocytes.

Control of infection by Mycobacterium tuberculosis is dependent on macrophage activation and efficient migration of effector T-cell populations. Lymphocyte differentiation is associated with changes in cell surface phenotype and alterations in the migratory pattern of these cells. In this study, we investigated the expression of adhesion receptors involved in activation and migration process in experimental tuberculosis. We observed that susceptible BALB/c mice infected with virulent M. tuberculosis by intraperitoneal route presented downmodulation of very late antigen 4 (VLA-4) and unchanged levels of CD18 and CD44hi on peritoneal lymphocytes. On the other hand, lymphocytes from resistant C57BL/6 mice infected by the same route showed unchanged levels of VLA-4 and upregulation of CD18 and CD44hi. However, when BALB/c mice were infected by intratracheal route, lung lymphocytes presented a different pattern of CD18, CD44hi and VLA-4 expression from that observed on peritoneal cells, characterized by unchanged levels of VLA-4 and upregulation of CD18 and CD44hi- coincidentally the same phenotype found on peritoneal cells from C57BL/6. These results suggest that susceptibility and resistance to M. tuberculosis infection, depending on the experimental model, are related to the expression of CD18, CD44hi and VLA-4. Moreover, the microenvironment at the site of infection seems to differentially regulate the expression of these receptors. Thus, the up- or downmodulation of these adhesion receptors is probably associated with differential recruitment of T cells at the site of infection, which may or may not mediate protection in experimental tuberculosis.

The present study reports, for the first time, that the recombinant hsp65 from Mycobacterium leprae (chaperonin 2) displays a proteolytic activity toward oligopeptides. The M. leprae hsp65 proteolytic activity revealed a trypsin-like specificity toward quenched fluorescence peptides derived from dynorphins. When other peptide substrates were used (beta-endorphin, neurotensin, and angiotensin I), the predominant peptide bond cleavages also involved basic amino acids in P(1), although, to a minor extent, the hydrolysis involving hydrophobic and neutral amino acids (G and F) was also observed. The amino acid sequence alignment of the M. leprae hsp65 with Escherichia coli HslVU protease suggested two putative threonine catalytic groups, one in the N-domain (T(136), K(168), and Y(264)) and the other in the C-domain (T(375), K(409), and S(502)). Mutagenesis studies showed that the replacement of K(409) by A caused a complete loss of the proteolytic activity, whereas the mutation of K(168) to A resulted in a 25% loss. These results strongly suggest that the amino acid residues T(375), K(409), and S(502) at the C-domain form the catalytic group that carries out the main proteolytic activity of the M. leprae hsp65. The possible pathophysiological implications of the proteolytic activity of the M. leprae hsp65 are now under investigation in our laboratory.

Objectives Although TB immunotherapy improves the results of conventional drug treatment, the effects of combining chemotherapy and immunotherapy have never been systematically evaluated. We used a comprehensive lung transcriptome analysis to directly compare the activity of combined chemotherapy and immunotherapy with that of single treatments in a mouse model of TB. Methods Mycobacterium tuberculosis-infected mice in the chronic phase of the disease (day 30) received: (i) isoniazid and rifampicin (drugs) daily for 30 days; (ii) DNA immunotherapy (DNA), consisting of four 100 μg injections at 10 day intervals; (iii) both therapies (DNA + drugs); or (iv) saline. The effects were evaluated 10 days after the end of treatment (day 70 post-infection). Results In all groups a systemic reduction in the load of bacilli was observed, bacilli became undetectable in the drugs and DNA + drugs groups, but the whole lung transcriptome analysis showed 867 genes exclusively modulated by the DNA + drugs combination. Gene enrichment analysis indicated that DNA + drugs treatment provided synergistic effects, including the down-regulation of proinflammatory cytokines and mediators of fibrosis, as confirmed by real-time PCR, ELISA, histopathology and hydroxyproline assay. Conclusions Our results provide a molecular basis for the advantages of TB treatment using combined chemotherapy and DNA immunotherapy and demonstrate the synergistic effects obtained with this strategy.

Despite substantial efforts in recent years toward the development of new vaccines and drugs against tuberculosis (TB), success has remained elusive. Immunotherapy of TB with mycobacterial Hsp65 as a DNA vaccine (DNA-hsp65) results in a reduction of systemic bacterial loads and lung tissue damage, but the high homology of Hsp65 with the mammalian protein raises concern that pathological autoimmune responses may also be triggered. We searched for autoimmune responses elicited by DNA-hsp65 immunotherapy in mice chronically infected with TB by evaluating the humoral immune response and comprehensive histopathology using stereology. Cross-reactive antibodies between mycobacterial and mammalian Hsp60/65 were detected; however, no signs of pathological autoimmunity were found up to 60 days after the end of the therapy.

Ottonia martiana is a plant popularly known in Brazil by the use for toothache. Ethanolic extract (EE), hexane fraction (HF), dichloromethane fraction (DF) and piperovatine obtained from O. martiana were assayed in vitro and in vivo. The acute toxicity of EE was determined, and LD50 values of 164.5 and 65.0 mg/kg by the oral and intraperitoneal routes, respectively, indicated a high toxicity for EE in vivo, explaining its popular use by topical administration only. A local anesthetic-like effect of EE and its fractions was observed in experimental models using pain induction, and such effect involved an analgesic action. The antimycobacterial activity of EE, HF, DF and piperovatine was evaluated against Mycobacterium tuberculosis H37Rv ATCC 27924. EE, HF, DF, and piperovatine showed a potential antimycobacterial effect with MICs of 16.0, 62.0, 62.0 and 8.0 μg/mL, respectively. Piperovatine was more effective than the EE or the other fractions. The selectivity index (SI=IC50/MIC) values calculated for EE, HF, DF and piperovatine based on the MICs and the cytotoxicity against J774 macrophages (IC50 by MTT assay) revealed values of 6.43, 2.34, 1.5 and 9.66, respectively. RESUMO Ottonia martiana é uma planta popularmente conhecida no Brasil pelo uso para dor de dente. Extrato etanólico (EE), fração hexano (HF), fração diclorometano (DF) e piperovatina, obtidos de O. martiana, foram ensaiados in vitro e in vivo. A toxicidade aguda de EE foi determinada e valores de LD50 164,5 e 65,0 mg/kg pelas rotas oral e intraperitoneal, respectivamente, indicaram alta toxicidade para EE in vivo, explicando seu uso popular somente por meio da administração tópica. Um efeito local similar ao anestésico do EE e das frações foi observado em modelos experimentais utilizando indução de dor e tal efeito envolveu uma ação analgésica. A atividade antimicobacteriana de EE, HF, DF e piperovatina foi avaliada contra Mycobacterium tuberculosis H37Rv ATCC 27924. EE, HF, DF e piperovatina mostraram um efeito antimicobacteriano potencial com MICs de 16,0, 62,0, 62,0 e 8,0 μg/mL, respectivamente. Piperovatina foi mais efetiva que EE e as outras frações. Os valores do índice de seletividade (SI=IC50/MIC) calculados para EE, HF, DF e piperovatina, baseados em MICs e na citotoxicidade contra J774 macrófagos (IC50 pelo teste MTT), revelaram valores de 6,43, 2,34, 1,5 e 9,66, respectivamente.

Tuberculosis (TB) remains one of the biggest treats in public health, infecting approximately one-third of the human population and killing almost two million people per year. Prophylaxis and treatment methods present some weakness, and HIV co-infection and resistant strains add complexity to the situation, leading WHO to declare the disease a global emergence. To change this situation, new diagnostics, therapies and prevention strategies are urgently needed, but their development relies on biomarker availably. TB biomarker studies have focused especially on diagnostics, treatment efficacy and prophylaxis success by vaccination. Given the multifactorial complexity of this disease, biosignatures are considered more adequate in TB than isolated markers. Standardizations (including assays, definitions and protocols) could accelerate biomarkers research once it would reduce heterogeneous datasets. Thus, specific databanks and integrated platforms of studies are precious resources to conduct broad research. Consequently, to reach the objective of defining TB biosignatures will demand tools from other areas, such as bioinformatics.

Development and evaluation of new vaccines and immunotherapy against tuberculosis demand a better understanding of the immune mechanisms in this disease. Costimulatory signals and intercellular contact seem to be pivotal in determining whether recognition of antigen by T cells leads to activation or anergy. In this paper, we show that virulent M. tuberculosis H37Rv downmodulates the ex vivo expression of CD18 and CD86 on peritoneal macrophages and VLA-4 on lymphocytes but does not disturb the in vitro production of interleukin (IL)-12 and interferon (IFN)-gamma after intraperitoneal infection. In addition, splenocytes from infected mice produce IL-10, while the expression of cell surface receptors is unchanged. The interplay among IL-12, IFN-gamma and IL-10 in vivo and the downmodulation of cell-surface receptors during the infection at the inflammatory site may contribute to the explanation of the maintenance of infection.

Mice treated with viableMycobacterium tuberculosiswith no glycolipid trehalose dimycolate (TDM) on the outer cell wall (delipidatedM. tuberculosis) by intraperitoneal or intratracheal inoculation presented an intense recruitment of polymorphonuclear cells into the peritoneal cavity and an acute inflammatory reaction in the lungs, respectively. In addition, lung lesions were resolved around the 32nd day after intratracheal inoculation. TDM-loaded biodegradable poly-dl-lactide-coglycolide microspheres as well as TDM-coated charcoal particles induced an intense inflammatory reaction. In addition, high levels of interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), IL-12, IL-10, gamma interferon (IFN-γ), and IL-4 production were detected in lung cells, and nitric oxide (NO) production was high in culture supernatants of bronchoalveolar lavage cells. These in vivo data were confirmed by in vitro experiments using peritoneal macrophages cultured in the presence of TDM adsorbed onto coverslips. High levels of IFN-γ, IL-6, TNF-α, IL-12, IL-10, and NO were detected in the culture supernatants. Our results suggest that TDM contributes to persistence of infection through production of cytokines, which are important for the recruitment of inflammatory cells and maintenance of a granulomatous reaction. In addition, our findings are important for a better understanding of the immunostimulatory activity of TDM and its possible use as an adjuvant in experiments using DNA vaccine or gene therapy against tuberculosis.

The fate of the proteasome-generated peptides depends upon the cytosolic peptidases whose activities ought to be regulated. One of the most important oligopeptide-degrading and -binding proteins in the cytosol is the thimet oligopeptidase (EC 3.4.24.15), ubiquitously found in mammalian tissues. To date, there is no indication whether thimet oligopeptidase activities are physiologically regulated. Here, we present evidences suggesting that the concentration of unbound ATP in the cytosol regulates the thimet oligopeptidase activities both, in vitro and ex vivo. To perform these studies two oligopeptides were used: a quenched fluorescent peptide, which is susceptible to thimet oligopeptidase degradation, and the ovalbumin257−264 (MHC class I ovalbumin epitope), which displays high affinity to the thimet oligopeptidase without being degraded. We also showed that the thimet oligopeptidase undergoes autophosphorylation by ATP, a modification that does not affect the peptidase activity. The autophosphorylation is abolished in the presence of the thimet oligopeptidase substrates, as well as by the effect of a site directed inhibitor of this enzyme, and by the substitution of Glu474 for Asp at the metallo-peptidase motif. Altogether, the results presented here suggest that Zn2+ at the active center of the thimet oligopeptidase is the target for the ATP binding, leading to the inhibition of the enzyme activity, and inducing autophosphorylation. These effects, which depend upon the concentration of the unbound ATP, may help to explain the fate of the proteasomal-generated oligopeptides in the cytosol.

As we seek to develop and evaluate new vaccines against tuberculosis, it is desirable that we understand the mechanisms of protective immunity in our models. Adoptive transfer of protection with hsp65-specific T-cell clones from infected or vaccinated mice into naı̈ve mice had indicated that cytotoxic T cells can make a major contribution to protection. We characterized 28 CD4+CD8−and 28 CD4−CD8+hsp65-specific T-cell clones derived from infected or vaccinated mice. Half of the CD4+CD8−and 64% of the CD4−CD8+clones were cytotoxic. Cytotoxicity was associated with high expression of CD44 and gamma interferon production. Most (86%) of the cytotoxic CD4+CD8−clones lysed target cells via the Fas-FasL pathway, and most (83%) of the cytotoxic CD4−CD8+clones lysed target cells via cytotoxic granules. Only the clones using the granule-mediated pathway caused substantial loss of viability of virulentMycobacterium tuberculosisduring lysis of infected macrophages, and the degree of killing closely correlated with the availability of granule marker enzyme activity. Granule-mediated cytotoxicity thus may have a key role in protection against tuberculosis by delivering mycobactericidal granule contents.

The inflammatory cell influx towards the peritoneal cavity in mice inoculated i.p. with live or dead Histoplasma capsulatum or with its subcellular preparations was studied. We also evaluated the effects of dexamethasone (Dexa) or MK886, an inhibitor of leukotriene (LT) biosynthesis, on the recruitment of leukocytes. Live yeast form of fungus (LYH) induced an increase in neutrophils (NE) which was highest 4 to 24 h after inoculation. Mononuclear cell (MN) migration beginning at 24 h with a gradual increase over 48 and 168 h, and an eosinophil (EO) recruitment occurs between 24 and 48 h. NE and EO recruitment induced by dead mycelial form of fungus (DMH) was greater than that observed for dead yeast form of fungus (DYH). A similar leukocyte migration pattern was seen after i.p. injection of the alkali-insoluble fraction (F1) from DYH (F1Y) and F1 from DMH (F1M) this being more active than former. The difference in concentration of β-glucan in DYH and DMH could explain the different inflammatory capacity exhibited by the two forms of H. capsulatum. LT seems to be the principal mediator of leukocyte migration in response to LYH, DYH or DMH or to β-glucan. However, other mediators appear to contribute to NE and EO migration since the treatment with Dexa was more effective in inhibiting cell migration than MK886. Complement dependent leukocyte migration may participate in this recruitment. Treatment with MK886 completely abolished MN cell migration, indicating its dependence on the presence of LT. Keywords: Eosinophils, neutrophils, mononuclear cells, leukotrienes, dexamethasone, MK886, Histoplasma capsulatum, histoplasmosis, β-glucan, inflammation Introduction Histoplasma capsulatum is a dimorphic pathogenic fungus that causes a wide spectrum of disease when condidiospores or mycelial fragments are inhaled (Schwartz, 1981). By far the most common clinical manifestation of acute H. capsulatum infection is pulmonary disease that usually is self-limiting over a period of several weeks and is characterized by chronic granulomatous and suppurative inflammatory reaction. During the last few decades the incidence of histoplasmosis infection has dramatically increased worldwide, mainly as the result of alterations in immune status associated with the acquired immunodeficiency syndrome (AIDS) epidemic, cancer chemotherapy, and organ and bone marrow transplantation (Georgopapakadou & Walsh, 1996). Many factors are associated with the disease, some of them involved within the fungus, such as fungal strain and virulence (Goodwin & Des Prez, 1978) and others related to host competence (Goodwin et al., 1981). Few attempts have been made to determine what cellular factor(s) might be responsible for the inflammatory reaction caused by this fungus and how it could be controlled. The causative organism, H. capsulatum, is a dimorphic fungus with a yeastlike (Y) morphology in tissue or in artificial media at 37°C. However, in soil or artificial media at room temperature it forms a filamentous mycelium (M) that carry the reproductive conidia. In the body, the Y form is found intracellularly in macrophages, where it survives and multiplies. Chemical and ultrastructural studies of the cell walls of the Y and M forms of the fungus revealed that the Y form contained high amounts of α-glucan and low level of β-glucan, galactomannan and chitin, whereas the cell wall of the M form contained high amounts of β-glucan and low level of α-glucan, galactomannan, and chitin (Kanetsuma et al., 1974). We have previously demonstrated such differences in another dimorphic fungus, Paracoccidioides brasiliensis. Only β-glucan from P. brasiliensis was able to trigger attraction of neutrophils (NE) and mononuclear (MN) cells at the inflammatory foci, and the difference in the concentration of this compound in the cell walls of Y and M forms could explain the different inflammatory capacity exhibited by the two forms of the fungus (Carareto-Alves et al., 1987; Silva et al., 1994). Previous studies of the inflammatory response in lungs of humans or experimental animals to pulmonary histoplasmosis have described the cell infiltration in lung tissues (Procknow et al., 1960; Baughman et al., 1986). Although these studies have detailed the serial changes in the inflammatory response to H. capsulatum, no information is available regarding the dynamics of inflammatory cells within lungs and other organs during the course of infection. We believe that the early inflammatory response to live fungus or to its cell wall components is of critical importance to both host and parasite relationship and to the establishment of infection. Inflammatory cells release a wide variety of mediators, including local preformed mediators, newly synthesized metabolites of arachidonic acid, and soluble pro-inflammatory proteins including kinins and cytokines (Stewart, 1995; Lee & Arm, 1991; Xing et al., 1999). Glucocorticoids are potent inhibitors of virtually every type of inflammatory reaction and exert a strong effect on leukocyte recruitment (Flower, 1988; Perretti & Flower, 1993; Barnes, 1998). Dexamethasone (Dexa) is routinely used to modulate negatively cell migration into sites of inflammation and this action is accomplished, in part, by its potent effect on the synthesis of pro-inflammatory cytokines and chemokines coupled to a reduced degree of leukocyte responsiveness (van der Velden, 1998). On the other hand, leukotrienes (LT) are products of arachidonic acid metabolism derived through an oxygenation of the fatty acid and a subsequent dehydrase step by 5-lipoxygenase (Miller et al., 1990). Leukotriene B4 (LTB4) is a potent stimulant of leukocyte functions including the chemotaxis, chemokinesis, and aggregation of polymorphonuclear leukocytes (PMN) (O'Byrne, 1988). Moreover, LTB4, LTD4, LTC4 and LTE4 are chemoatractant for eosinophils (EO) (Faccioli et al., 1991; Woodward et al., 1991; Bureau et al., 1997; O'Byrne, 1997). The compound MK886 (Gillard et al., 1989; Ford-Hutchinson et al., 1991) is a potent inhibitor of LT biosynthesis in NE, both in vivo and in vitro, thus indicating that the compound is suitable for studying the role of LT in a variety of inflammatory situations (Puustinen et al., 1988). The objective of the present investigation was: to characterize the kinetics and type of cells recruited to the peritoneal cavity of mice induced by i.p. injection of live or dead fungus, or by fungal cell wall fractions; to explore the nature of the cell wall components of H. capsulatum involved in inflammatory cell recruitment leading to the development of an intense inflammatory reaction; and to characterize the possible mediators involved in cell migration.

The memory/activated T cells, which mediate the long-lived host response against tuberculosis, in mice immunized with either bacillus Calmette–Guerin (BCG) or mycobacterium heat-shock protein 65 (hsp 65) antigen expressed from plasmid DNA (DNA-hsp 65), were characterized. Protection against Mycobacterium tuberculosis challenge by DNA-hsp 65 vaccination was associated with the presence of lymph node T-cell populations in which CD8+/CD44hi interferon-γ (IFN-γ)-producing/cytotoxic cells were prominent even after 8 or 15 months of plasmid DNA-mediated immunizations, whereas after BCG vaccination the majority were CD4+/CD44lo IFN-γ-producing T cells. When the cells were separated into CD4+CD8− and CD8+CD4− and then into CD44hi and CD44lo types, CD44lo cells were essentially unable to transfer protection in adoptive transfer experiments, the most protective CD44hi cells were CD8+CD4− and those from DNA-vaccinated mice were much more protective than those from BCG-immunized mice. The frequency of protective T cells and the level of protection were increased up to 8 months and decreased after 15 months following DNA or BCG immunizations.

Over the last few years, some of our experiments in which mycobacterial heat-shock protein (HSP) antigens were presented to the immune system as if they were viral antigens have had a significant impact on our understanding of protective immunity against tuberculosis. They have also markedly enhanced the prospects for new vaccines. We now know that the mycobacterial HSP65 antigen can confer protection equal to that from live BCG vaccine in mice.

Immunization by intramuscular injection of plasmid DNA expressing mycobacterial 65-kDa heat shock protein (hsp65) protects mice against challenge with virulent Mycobacterium tuberculosis H37Rv. During infection or after immunization, CD4 + /CD8 − and CD8 + /CD4 − hsp65-reactive T cells increased equally in spleens. During infection, the majority of these cells were weakly CD44 positive (CD44 lo ) and produced interleukin 4 (IL-4) whereas after immunization the majority were highly CD44 positive (CD44 hi ) and produced gamma interferon (IFN-γ). In adoptive transfer of protection to naive mice, the total CD8 + /CD4 − cell population purified from spleens of immunized mice was more protective than that from infected mice. When the cells were separated into CD4 + /CD8 − and CD8 + /CD4 − types and then into CD44 hi and CD44 lo types, CD44 lo cells were essentially unable to transfer protection, the most protective CD44 hi cells were CD8 + /CD4 − , and those from immunized mice were much more protective than those from infected mice. Thus, whereas the CD44 lo IL-4-producing phenotype prevailed during infection, protection was associated with the CD8 + /CD44 hi IFN-γ-producing phenotype that predominated after immunization. This conclusion was confirmed and extended by analysis of 16 hsp65-reactive T-cell clones from infected mice and 16 from immunized mice; the most protective clones, in addition, displayed antigen-specific cytotoxicity.

This edited transcript of a presentation at the 'Vaccines Beyond 2000' conference describes a series of investigations by the authors throwing light on the mechanisms of protective immunity against tuberculosis in mice and raising hope for a new kind of vaccine to replace bacille Calmette-Guérin (BCG). DNA encoding only one or a few protein antigens was found capable of conferring persistent protection equal to the effect of BCG. The essential features seem to be an endogenous origin of the antigen within transfected mouse cells which favours the development of CD8+/CD44hi/IFN-gamma-producing T cells with antigen-specific cytotoxicity. Such cells were the most efficient in adoptive transfer of protection from infected or DNA-vaccinated mice to naive mice.

Ergosterol peroxide, a presumed product of the H_2O_2-dependent enzymatic oxidation of ergosterol, has been isolated from yeast forms of the pathogenic fungus Sporothrix schenckii. The substance, which may have a role in fungal virulence, has been characterized mainly using spectroscopic methods (1H and 13C nuclear magnetic resonance and high resolution mass spectra). The purified compound showed a molecular formula of C_28H_44O_3, displaying characteristic features of epidioxy sterols and was reverted to ergosterol when submitted to S. schenckii enzymatic extract.

SummaryType I diabetes is a disease caused by autoimmune destruction of the beta cells in the pancreas that leads to a deficiency in insulin production. The aim of this study was to evaluate the prophylactic potential of a prime-boost strategy involving bacille Calmette–Guérin (BCG) and the pVAXhsp65 vaccine (BCG/DNAhsp65) in diabetes induced by streptozotocin (STZ) in C57BL/6 mice and also in spontaneous type 1 diabetes in non-obese diabetic (NOD) mice. BCG/DNAhsp65 vaccination in NOD mice determined weight gain, protection against hyperglycaemia, decreased islet inflammation, higher levels of cytokine production by the spleen and a reduced number of regulatory T cells in the spleen compared with non-immunized NOD mice. In the STZ model, however, there was no significant difference in the clinical parameters. Although this vaccination strategy did not protect mice in the STZ model, it was very effective in NOD mice. This is the first report demonstrating that a prime-boost strategy could be explored as an immunomodulatory procedure in autoimmune diseases.

Tuberculosis is one of the oldest human diseases, which still killing about 2 million people a year around the world. It is believed that one third of the world population is infected with the bacillus M. tuberculosis, representing a huge and worrying reservoir of the pathogen. Different factors contribute to this disturbing scenario: the only available vaccine, BGC, is inefficient; treatment is too long, causing considerable side effects, generating large non compliance rates and favouring the development of resistant strains; the diagnosis is not always accurate. Although the host-pathogen interaction and immune response against tuberculosis are intensively studied, there are still open questions on this subject. Thus, the development of new diagnostics, vaccines and therapies are extremely affected and harmed.

Made available in DSpace on 2014-12-03T13:10:54Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-01-01Bitstream added on 2014-12-03T13:22:54Z : No. of bitstreams: 1 WOS000326839000001.pdf: 4090704 bytes, checksum: a41f3346235c8b4ace4610f2fd1e6214 (MD5) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) A prime-boost strategy conserving BCG is considered the most promising vaccine to control tuberculosis. A boost with a DNA vaccine containing the mycobacterial gene of a heat shock protein (pVAXhsp65) after BCG priming protected mice against experimental tuberculosis. However, anti-hsp65 immunity could worsen an autoimmune disease due to molecular mimicry. In this investigation, we evaluated the effect of a previous BCG or BCG/pVAXhsp65 immunization on experimental autoimmune encephalomyelitis (EAE) development. Female Lewis rats were immunized with BCG or BCG followed by pVAXhsp65 boosters. The animals underwent EAE induction and were daily evaluated for weight loss and clinical score. They were euthanized during recovery phase to assess immune response and inflammatory infiltration at the central nervous system. Previous immunization did not aggravate or accelerate clinical score or weight loss. In addition, this procedure clearly decreased inflammation in the brain. BCG immunization modulated the host immune response by triggering a significant reduction in IL-10 and IFN-gamma levels induced by myelin basic protein. These data indicated that vaccination protocols with BCG or BCG followed by boosters with pVAXhsp65 did not trigger a deleterious effect on EAE evolution. Univ Estadual Paulista UNESP, Inst Biociencias, Dept Microbiol & Imunol, BR-18618970 Botucatu, SP, Brazil Univ Sao Paulo, Dept Bioquim & Imunol, BR-14049900 Ribeirao Preto, SP, Brazil Univ Estadual Paulista UNESP, Inst Biociencias, Dept Microbiol & Imunol, BR-18618970 Botucatu, SP, Brazil FAPESP: 07/05353-8 CNPq: 473351/2004-8

Mice vaccinated by injection with tumor cells expressing the Mycobacterium leprae gene for hsp65 acquire a remarkably high degree of protection against challenge with Mycobacterium tuberculosis. We used limiting-dilution analysis to assess the frequency of CD4+ CD8- and CD4- CD8+ splenocytes responding to mycobacterial hsp65 in such vaccinated mice. Cells of both phenotypes were present at very high and equal frequencies (approximately 1:100). Vaccination with live Mycobacterium bovis BCG also increased the frequencies of both phenotypes of hsp65-reactive cells equally (to approximately 1:2,500), whereas vaccination procedures that were not protective, with either dead BCG, hsp65 protein in incomplete Freund's adjuvant, or hsp65 mixed with tumor cells, resulted in preferential increase in CD4+ CD8- cells. Twelve CD4+ CD8- and twelve CD4- CD8+ hsp65-responsive T-cell clones were obtained and characterized. All showed conventional antigen recognition via major histocompatibility complex class II and class I pathways but differed in secretion of gamma interferon and interleukin 4 and cytotoxicity. In tests of antimycobacterial activity against M. tuberculosis, both in infected macrophages in vitro and by adoptive transfer of protection with T-cell clones injected into irradiated mice, the most effective clones were the most cytotoxic and secretion of gamma interferon made only a secondary contribution.

Background Although plasmid DNA encoding an antigen from pathogens or tumor cells has been widely studied as vaccine, the use of plasmid vector (without insert) as therapeutic agent requires further investigation. Results Here, we showed that plasmid DNA (pcDNA3) at low doses inhibits the production of IL-6 and TNF-α by lipopolysaccharide (LPS)-stimulated macrophage cell line J774. These findings led us to evaluate whether plasmid DNA could act as an anti-inflammatory agent in a Wistar rat endotoxemia model. Rats injected simultaneously with 1.5 mg/kg of LPS and 10 or 20 μg of plasmid DNA had a remarkable attenuation of mean arterial blood pressure (MAP) drop at 2 hours after treatment when compared with rats injected with LPS only. The beneficial effect of the plasmid DNA on MAP was associated with decreased expression of IL-6 in liver and increased concentration of plasma vasopressin (AVP), a known vasoconstrictor that has been investigated in hemorrhagic shock management. No difference was observed in relation to nitric oxide (NO) production. Conclusion Our results demonstrate for the first time that plasmid DNA vector at low doses presents anti-inflammatory property and constitutes a novel approach with therapeutic potential in inflammatory diseases.

CD4(+) Foxp3(+) regulatory T cells inhibit the production of interferon-γ, which is the major mediator of protection against Mycobacterium tuberculosis infection. In this study, we evaluated whether the protection conferred by three different vaccines against tuberculosis was associated with the number of spleen and lung regulatory T cells. We observed that after homologous immunization with the 65 000 molecular weight heat-shock protein (hsp 65) DNA vaccine, there was a significantly higher number of spleen CD4(+) Foxp3(+) cells compared with non-immunized mice. Heterologous immunization using bacillus Calmette-Guérin (BCG) to prime and DNA-hsp 65 to boost (BCG/DNA-hsp 65) or BCG to prime and culture filtrate proteins (CFP)-CpG to boost (BCG/CFP-CpG) induced a significantly higher ratio of spleen CD4(+) /CD4(+) Foxp3(+) cells compared with non-immunized mice. In addition, the protection conferred by either the BCG/DNA-hsp 65 or the BCG/CFP-CpG vaccines was significant compared with the DNA-hsp 65 vaccine. Despite the higher ratio of spleen CD4(+) /CD4(+) Foxp3(+) cells found in BCG/DNA-hsp 65-immunized or BCG/CFP-CpG-immunized mice, the lungs of both groups of mice were better preserved than those of DNA-hsp 65-immunized mice. These results confirm the protective efficacy of BCG/DNA-hsp 65 and BCG/CFP-CpG heterologous prime-boost vaccines and the DNA-hsp 65 homologous vaccine. Additionally, the prime-boost regimens assayed here represent a promising strategy for the development of new vaccines to protect against tuberculosis because they probably induce a proper ratio of CD4(+) and regulatory (CD4(+) Foxp3(+) ) cells during the immunization regimen. In this study, this ratio was associated with a reduced number of regulatory cells and no injury to the lungs.

Conventional treatment of tuberculosis (TB) demands a long course therapy (6 months), known to originate multiple drug resistant strains (MDR-TB), which emphasizes the urgent need for new antituberculous drugs. The purpose of this study was to investigate a novel treatment for TB meant to improve patient compliance by reducing drug dosage frequency. Polymeric microparticles containing the synthetic analogue of neolignan, 1-phenyl-2-phenoxiethanone (LS-2), were obtained by a method of emulsification and solvent evaporation and chemically characterized. Only representative LS-2-loaded microparticles were considered for further studies involving experimental murine TB induced by Mycobacterium tuberculosis H37Rv ATCC 27294. The LS-2-loaded microparticles were spherical in shape, had a smooth wall and showed an encapsulation efficiency of 93% in addition to displaying sustained release. Chemotherapeutic potential of LS-2 entrapped in microparticles was comparable to control groups. These findings are encouraging and indicate that LS-2-loaded microparticles are a potential alternative to conventional chemotherapy of TB.

Objectives The study's aims were to evaluate the antimycobacterial activity of 13 synthetic neolignan analogues and to perform structure activity relationship analysis (SAR). The cytotoxicity of the compound 2-phenoxy-1-phenylethanone (LS-2, 1) in mammalian cells, such as the acute toxicity in mice, was also evaluated. Methods The extra and intracellular antimycobacterial activity was evaluated on Mycobacterium tuberculosis H37Rv. Cytotoxicity studies were performed using V79 cells, J774 macrophages and rat hepatocytes. Additionally, the in-vivo acute toxicity was tested in mice. The SAR analysis was performed by Principal Component Analysis (PCA). Key findings Among the 13 analogues tested, LS-2 (1) was the most effective, showing promising antimycobacterial activity and very low cytotoxicity in V79 cells and in J774 macrophages, while no toxicity was observed in rat hepatocytes. The selectivity index (SI) of LS-2 (1) was 91 and the calculated LD50 was 1870 mg/kg, highlighting the very low toxicity in mice. SAR analysis showed that the highest electrophilicity and the lowest molar volume are physical-chemical characteristics important for the antimycobacterial activity of the LS-2 (1). Conclusions LS-2 (1) showed promising antimycobacterial activity and very weak cytotoxicity in cell culture, as well as an absence of toxicity in primary culture of hepatocytes. In the acute toxicity study there was an indication of absence of toxicity on murine models, in vivo.

In 1882 Robert Koch identifiedMycobacterium tuberculosisas the causative agent of tuberculosis (TB), a disease as ancient as humanity. Although there has been more than 125 years of scientific effort aimed at understanding the disease, serious problems in TB persist that contribute to the estimated 1/3 of the world population infected with this pathogen. Nonetheless, during the first decade of the 21st century, there were new advances in the fight against TB. The development of high-throughput technologies is one of the major contributors to this advance, because it allows for a global vision of the biological phenomenon. This paper analyzes how transcriptomics are supporting the translation of basic research into therapies by resolving three key issues in the fight against TB: (a) the discovery of biomarkers, (b) the explanation of the variability of protection conferred by BCG vaccination, and (c) the development of new immunotherapeutic strategies to treat TB.

We investigated the capacity of an alkali-insoluble cell wall polysaccharide fraction (F1) of Paracoccidioides brasiliensis to induce rat polymorphonuclear neutrophil (PMN) migratory and chemiluminescence (CL) responses. Normal rat serum pre-incubated with F1 induced a chemotactic neutrophil response which was fully abolished by heat-inactivation. The participation of the alternative complement pathway was more effective than that of the classical pathway since depletion of factor B by heating at 50°C reduced PMN migration, whereas blockade of the classical pathway with EGTA left the migratory response practically unchanged. Opsonized serum F1 induced a significant release of oxygen radicals from PMN as measured by CL. The complement system was also found to be involved in this activity since serum inactivation at 56°C altered the CL response. In addition to complement-derived fragments, other serum opsonins, probably cross-reacting antibodies, were required for optimal interaction between PMN and opsoniz...

Of the hundreds of new tuberculosis (TB) vaccine candidates, some have therapeutic value in addition to their prophylactic properties. This is the case for the DNA vaccine encoding heat-shock protein 65 (DNAhsp65) from Mycobacterium leprae. However, there are concerns about the use of DNA vaccines in certain populations such as newborns and pregnant women. Thus, the optimization of vaccination strategies that circumvent this limitation is a priority. This study evaluated the efficacy of a single dose subunit vaccine based on recombinant Hsp65 protein against infection with M. tuberculosis H37Rv. The Hsp65 protein in this study was either associated or not with immunostimulants, and was encapsulated in biodegradable PLGA microspheres. Our results demonstrate that the protein was entrapped in microspheres of adequate diameter to be engulfed by phagocytes. Mice vaccinated with a single dose of Hsp65-microspheres or Hsp65+CpG-microspheres developed both humoral and cellular-specific immune responses. However, they did not protect mice against challenge with M. tuberculosis. By contrast, Hsp65+KLK-microspheres induced specific immune responses that reduced bacilli loads and minimized lung parenchyma damage. These data suggest that a subunit vaccine based on recombinant protein Hsp65 is feasible.

Background: Detailed analysis of the dynamic interactions among biological, environmental, social, and economic factors that favour the spread of certain diseases is extremely useful for designing effective control strategies. Diseases like tuberculosis that kills somebody every 15 seconds in the world, require methods that take into account the disease dynamics to design truly efficient control and surveillance strategies. The usual and well established statistical approaches provide insights into the cause-effect relationships that favour disease transmission but they only estimate risk areas, spatial or temporal trends. Here we introduce a novel approach that allows figuring out the dynamical behaviour of the disease spreading. This information can subsequently be used to validate mathematical models of the dissemination process from which the underlying mechanisms that are responsible for this spreading could be inferred. Methodology/Principal Findings: The method presented here is based on the analysis of the spread of tuberculosis in a Brazilian endemic city during five consecutive years. The detailed analysis of the spatio-temporal correlation of the yearly geo-referenced data, using different characteristic times of the disease evolution, allowed us to trace the temporal path of the aetiological agent, to locate the sources of infection, and to characterize the dynamics of disease spreading. Consequently, the method also allowed for the identification of socio-economic factors that influence the process. Conclusions/Significance: The information obtained can contribute to more effective budget allocation, drug distribution and recruitment of human skilled resources, as well as guiding the design of vaccination programs. We propose that this novel strategy can also be applied to the evaluation of other diseases as well as other social processes. © 2010 Zorzenon dos Santos et al. Fil: Zorzenon dos Santos, Rita M.. Universidade Federal de Pernambuco; Brasil Fil: Amador, Ana. Universidade Federal de Pernambuco; Brasil. Universidad de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; Argentina Fil: de Souza, Wayner V.. Fundación Oswaldo Cruz; Brasil Fil: de Albuquerque, Maria Fatima P. M.. Universidade Federal de Pernambuco; Brasil. Fundación Oswaldo Cruz; Brasil Fil: Ponce Dawson, Silvina Martha. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; Argentina Fil: Ruffino-Netto, Antonio. Universidade de Sao Paulo; Brasil Fil: Zárate-Bladés, Carlos R.. Universidade de Sao Paulo; Brasil Fil: Silva, Celio L.. Universidade de Sao Paulo; Brasil

Background mRNAs are highly versatile, non-toxic molecules that are easy to produce and store, which can allow transient protein expression in all cell types. The safety aspects of mRNA-based treatments in gene therapy make this molecule one of the most promising active components of therapeutic or prophylactic methods. The use of mRNA as strategy for the stimulation of the immune system has been used mainly in current strategies for the cancer treatment but until now no one tested this molecule as vaccine for infectious disease. Results We produce messenger RNA of Hsp65 protein from Mycobacterium leprae and show that vaccination of mice with a single dose of 10 μg of naked mRNA-Hsp65 through intranasal route was able to induce protection against subsequent challenge with virulent strain of Mycobacterium tuberculosis. Moreover it was shown that this immunization was associated with specific production of IL-10 and TNF-alpha in spleen. In order to determine if antigen presenting cells (APCs) present in the lung are capable of capture the mRNA, labeled mRNA-Hsp65 was administered by intranasal route and lung APCs were analyzed by flow cytometry. These experiments showed that after 30 minutes until 8 hours the populations of CD11c+, CD11b+ and CD19+ cells were able to capture the mRNA. We also demonstrated in vitro that mRNA-Hsp65 leads nitric oxide (NO) production through Toll-like receptor 7 (TLR7). Conclusions Taken together, our results showed a novel and efficient strategy to control experimental tuberculosis, besides opening novel perspectives for the use of mRNA in vaccines against infectious diseases and clarifying the mechanisms involved in the disease protection we noticed as well.

Made available in DSpace on 2013-09-27T14:54:31Z (GMT). No. of bitstreams: 1 WOS000273688300001.pdf: 288439 bytes, checksum: 6044356f046cdd0c18b0c237b16a5877 (MD5) Previous issue date: 2010-02-01 Made available in DSpace on 2013-09-30T18:38:39Z (GMT). No. of bitstreams: 1 WOS000273688300001.pdf: 288439 bytes, checksum: 6044356f046cdd0c18b0c237b16a5877 (MD5) Previous issue date: 2010-02-01 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T13:51:14Z No. of bitstreams: 1 WOS000273688300001.pdf: 288439 bytes, checksum: 6044356f046cdd0c18b0c237b16a5877 (MD5) Made available in DSpace on 2014-05-20T13:51:14Z (GMT). No. of bitstreams: 1 WOS000273688300001.pdf: 288439 bytes, checksum: 6044356f046cdd0c18b0c237b16a5877 (MD5) Previous issue date: 2010-02-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) 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. São Paulo State Univ UNESP, Dept Microbiol Immunol, Biosci Inst, BR-18618000 São Paulo, Brazil São Paulo State Univ UNESP, Dept Morphol, Biosci Inst, BR-18618000 São Paulo, Brazil Univ São Paulo, Dept Biochem & Immunol, São Paulo, Brazil São Paulo State Univ UNESP, Dept Microbiol Immunol, Biosci Inst, BR-18618000 São Paulo, Brazil São Paulo State Univ UNESP, Dept Morphol, Biosci Inst, BR-18618000 São Paulo, Brazil FAPESP: 03/06348-7

Bordetella pertussis is a gram-negative bacillus that causes the highly contagious disease known as pertussis or whooping cough. Antibody response in children may vary depending on the vaccination schedule and the product used. In this study, we have analyzed the antibody response of cellular pertussis vaccinated children against B. pertussis strains and their virulence factors, such as pertussis toxin, pertactin, and filamentous hemagglutinin. After the completion of the immunization process, according to the Brazilian vaccination program, children serum samples were collected at different periods of time, and tested for the presence of specific antibodies and antigenic cross-reactivity. Results obtained show that children immunized with three doses of the Brazilian whole-cell pertussis vaccine present high levels of serum antibodies capable of recognizing the majority of the components present in vaccinal and non-vaccinal B. pertussis strains and their virulence factors for at least 2 years after the completion of the immunization procedure.

Prostaglandins (PGs) and leukotrienes (LTs) are produced in Mycobacterium tuberculosis (Mtb)-infected lungs and have immune suppressive and protective effects, respectively. Considering that both of these mediators are produced during mycobacterial infection, we investigated the specific and relative biological importance of each in regulating host response in experimental tuberculosis. Administration of celecoxib, which was found to reduce lung levels of PGE 2 and increase LTB 4 , enhanced the 60-day survival of Mtb-infected mice in 14%. However administration of MK-886, which reduced levels of LTB 4 but did not enhance PGE 2 , reduced 60-day survival from 86% to 43% in Mtb-infected mice, and increased lung bacterial burden. MK-886 plus celecoxib reduced survival to a lesser extent than MK-886 alone. MK-886- and MK-886 plus celecoxib-treated animals exhibited reduced levels of the protective interleukin-12 and gamma-interferon. Our findings indicate that in this model, the protective effect of LTs dominates over the suppressive effect of PGs.

Submitted by Guilherme Lemeszenski (guilherme@nead.unesp.br) on 2013-08-22T18:52:50Z No. of bitstreams: 1 S0100-879X2010000700006.pdf: 1207920 bytes, checksum: bdfd880a5c652f478c772b02369b88e6 (MD5) Made available in DSpace on 2013-08-22T18:52:50Z (GMT). No. of bitstreams: 1 S0100-879X2010000700006.pdf: 1207920 bytes, checksum: bdfd880a5c652f478c772b02369b88e6 (MD5) Previous issue date: 2010-07-01 Made available in DSpace on 2013-09-30T18:07:10Z (GMT). No. of bitstreams: 2 S0100-879X2010000700006.pdf: 1207920 bytes, checksum: bdfd880a5c652f478c772b02369b88e6 (MD5) S0100-879X2010000700006.pdf.txt: 26142 bytes, checksum: c72a8913ddc1306036955a9f6666f63c (MD5) Previous issue date: 2010-07-01 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T13:24:05Z No. of bitstreams: 2 S0100-879X2010000700006.pdf: 1207920 bytes, checksum: bdfd880a5c652f478c772b02369b88e6 (MD5) S0100-879X2010000700006.pdf.txt: 26142 bytes, checksum: c72a8913ddc1306036955a9f6666f63c (MD5) Made available in DSpace on 2014-05-20T13:24:05Z (GMT). No. of bitstreams: 2 S0100-879X2010000700006.pdf: 1207920 bytes, checksum: bdfd880a5c652f478c772b02369b88e6 (MD5) S0100-879X2010000700006.pdf.txt: 26142 bytes, checksum: c72a8913ddc1306036955a9f6666f63c (MD5) Previous issue date: 2010-07-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Leukotrienes are reported to be potent proinflammatory mediators that play a role in the development of several inflammatory diseases such as asthma, rheumatoid arthritis and periodontal disease. Leukotrienes have also been associated with protection against infectious diseases. However, the role of leukotrienes in Mycobacterium tuberculosis infection is not understood. To answer this question, we studied the role of leukotrienes in the protective immune response conferred by prime-boost heterologous immunization against tuberculosis. We immunized BALB/c mice (4-11/group) with subcutaneous BCG vaccine (1 x 10(5) M. bovis BCG) (prime) followed by intramuscular DNA-HSP65 vaccine (100 µg) (boost). During the 30 days following the challenge, the animals were treated by gavage daily with MK-886 (5 mg·kg-1·day-1) to inhibit leukotriene synthesis. We showed that MK-886-treated mice were more susceptible to M. tuberculosis infection by counting the number of M. tuberculosis colony-forming units in lungs. The histopathological analysis showed an impaired influx of leukocytes to the lungs of MK-886-treated mice after infection, confirming the involvement of leukotrienes in the protective immune response against experimental tuberculosis. However, prime-boost-immunized mice treated with MK-886 remained protected after challenge with M. tuberculosis, suggesting that leukotrienes are not required for the protective effect elicited by immunization. Protection against M. tuberculosis challenge achieved by prime-boost immunization in the absence of leukotrienes was accompanied by an increase in IL-17 production in the lungs of these animals, as measured by ELISA. Therefore, these data suggest that the production of IL-17 in MK-886-treated, immunized mice could contribute to the generation of a protective immune response after infection with M. tuberculosis. Universidade de São Paulo Departamento de Bioquímica e Imunologia Núcleo de Pesquisas em Tuberculose Universidade de São Paulo Faculdade de Medicina de Ribeirão Preto Departamento de Patologia Universidade Estadual Paulista Faculdade de Ciências Farmacêuticas Departamento de Ciências Biológicas Universidade de São Paulo Faculdade de Ciências Farmacêuticas de Ribeirão Preto Departamento de Análises Clínicas, Toxicológicas e Bromatológicas Universidade Estadual Paulista Faculdade de Ciências Farmacêuticas Departamento de Ciências Biológicas FAPESP: 07/02407-0

Background Helminthiasis and tuberculosis (TB) coincide geographically and there is much interest in exploring how concurrent worm infections might alter immune responses against bacilli and might necessitate altered therapeutic approaches. A DNA vaccine that codifies heat shock protein Hsp65 from M. leprae (DNAhsp65) has been used in therapy during experimental tuberculosis. This study focused on the impact of the co-existence of worms and TB on the therapeutic effects of DNAhsp65. Methodology/Principal Findings Mice were infected with Toxocara canis or with Schistosoma mansoni, followed by coinfection with M. tuberculosis and treatment with DNAhsp65. While T. canis infection did not increase vulnerability to pulmonary TB, S. mansoni enhanced susceptibility to TB as shown by higher numbers of bacteria in the lungs and spleen, which was associated with an increase in Th2 and regulatory cytokines. However, in coinfected mice, the therapeutic effect of DNAhsp65 was not abrogated, as indicated by colony forming units and analysis of histopathological changes. In vitro studies indicated that Hsp65-specific IFN-γ production was correlated with vaccine-induced protection in coinfected mice. Moreover, in S. mansoni-coinfected mice, DNA treatment inhibited in vivo TGF-β and IL-10 production, which could be associated with long-term protection. Conclusions/Significance We have demonstrated that the therapeutic effects of DNAhsp65 in experimental TB infection are persistent in the presence of an unrelated Th2 immune response induced by helminth infections., Author Summary From 14 diseases considered by WHO as Neglected Tropical Diseases, four involve helminth infections, such as schistosomiasis and soil-transmitted helminthiasis. Toxocariasis is a soil-transmitted worm highly prevalent in many developing countries, while schistosomiasis causes an annual mortality of 14,000 deaths per year, with 200–300 million infected people and 10% at risk of infection worldwide. Additionally, tuberculosis (TB) remains one of the leading causes of morbidity and mortality in many settings, particularly in the world's poorest countries. Mycobacteria and helminths are co-endemic and induce opposing patterns of immune responses in the host, recognized as Th1 and Th2 respectively. These co-existing patterns could be associated with the failure of TB vaccines. In this sense, we investigated the inflammatory and immune response in a coinfection model with T. canis or S. mansoni and M. tuberculosis analyzing the effects of an immunotherapy that has previously shown efficacy in experimental TB. This immunotherapy is based on a DNA vaccine that codifies a mycobacterial heat shock protein (hsp65), which can prevent TB in a prophylactic and also therapeutic setting. In this work, we show that helminth coinfection does not abrogate the therapeutic effects of DNAhsp65 vaccine against TB.

The antimycobacterial activity of 3-(4'-bromo[1,1'-biphenyl-4-yl)-3-(4-bromo-phenyl)-N,N-dimethyl-2-propen-1-amine (BBAP), free or incorporated in preformed liposomes, on extracellular M. tuberculosis H37Rv was 8 and 25 μM (MIC), respectively. Extracellular antimycobacterial activity was not significantly improved by entrapment of BBAP in liposomes, but there was a 6.1-fold reduction of BBAP cytotoxicity on J774 macrophages. Liposomal BBAP or its free form showed IC50 values of 165 and 27 μM, resulting in a selectivity index (SI=IC50/MIC) of 3.4 and 6.6, respectively. Free BBAP in concentrations from 10 to 80 μM were quite effective in eliminating intracellular M. tuberculosis while liposomal formulation was less effective at these concentrations.

The present investigation reports the isolation of aeroplysinin-2, 2-(3,5-dibromo-4-methoxyphenyl)-N,N,N-trimethyletanamonium, 7,9-dibromo-10-hydroxy-8-methoxy-1-oxa-2-azaspiro[4.5]deca-2,6,8-trien-3-carboxylic acid and its methyl ester, 11-oxoaerothionin, aerothionin, 11-keto-12-hydroxyaerothionin, 11-ketofistularin-3 and fistularin-3 from Aplysina fistularis, as well as of furodysinin lactone and 9α,11α-epoxicholest-7-en-3β,5α,6α,10-tetrol-6-acetate from Dysidea sp. Although the extracts of both sponges displayed antituberculosis activity, only 11-ketofistularin-3 isolated from A. fistularis displayed antimycobacterial activity against Mycobacterium tuberculosis H34Rv, with MIC at 16 μg/mL and SI of 40, a result that reinforce that fistularin-3 derivatives are interesting leads for the development of antituberculosis drugs.

The roles of tumor necrosis factor (TNF) and macrophage activation in clearance of Nocardia brasiliensis from BALB/c mouse livers and spleens were evaluated. TNF activity was detectable in sera from animals at all stages of infection. Treatment of infected mice with an antiserum against TNF significantly enhanced the experimental infection as judged by enumeration of CFU in the spleens and livers of infected mice. In another set of experiments, a population of activated macrophages from the peritoneal cavities of N. brasiliensis-infected mice was studied by using a cytostatic assay. The observed cytotoxic activity of these activated macrophages against L929 cells was mediated by TNF, since this activity was inhibited by anti-TNF antiserum treatment. The level of TNF activity generated in vitro in the presence of lipopolysaccharide (LPS) by peritoneal macrophages from infected mice was higher than that of adherent peritoneal cells obtained from normal mice after challenge with LPS. When the nocardiacidal activity of peritoneal cells from N. brasiliensis-infected mice was estimated in vitro, a significant decrease in the number of CFU recovered was observed. Moreover, nocardiacidal activity of peritoneal cells obtained from N. brasiliensis-infected mice previously treated with anti-TNF antiserum was significantly reduced compared with the activity of cells obtained from infected mice previously treated with normal rabbit serum and that of cells from uninfected mice. These data suggest a role for TNF in resistance to N. brasiliensis infection.

Mycobacterium leprae lives free in the cytoplasm in infected macrophages. To test if an M. leprae antigen released into the cytoplasm would associate with major histocompatibility complex (MHC) class II we introduced the gene encoding the 65 kDa heat-shock protein (ML65hsp) into a retroviral shuttle vector (pZIPNeoSV(X)) and transfected the murine macrophage cell line J774G8. S1 nuclease mapping and Western blot analysis of the transfected cell line (CJ11) showed that specific messenger RNA and ML65hsp antigen were stably expressed. Presence of antigen at the cell surface was demonstrated by flow cytometric analysis with specific monoclonal antibodies (mAb). Antigen-specific T lymphocytes were stimulated by CJ11 cells to proliferate and release interleukins (IL-2 and IL-3). These responses were blocked by mAbs specific for either MHC class II or for the mycobacterial antigen. The endogenous antigen was also recognised by MHC class I-dependent cytotoxic T cells; cytotoxicity was inhibited by mAbs against either MHC class I molecules or ML65hsp. Thus, production of ML65hsp within the host cytoplasm resulted in association of the antigen with both MHC class I and MHC class II antigen-presenting structures and evoked both lymphocyte proliferation and cytotoxicity towards the antigen-presenting cell. These findings may be relevant to the development of recombinant subunit vaccines against intracellular pathogens.