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SUMMARY Tumour necrosis factor (TNF) was detected in scrum from mice challenged with Paracoccidioides brasiliensis. The scrum TNF levels of mice challenged with an avirulent strain were significantly higher than those of mice challenged with a virulent strain, and the same was observed for the TNF levels of mice challenged with a cell wall fraction (F1) from the two fungal strains. Fraction F1 consisted of chitin and β-glucan; but although the chitin contents were similar for the two strains, the avirulcnt strain allowed a greater content of β-glucan. The β-glucan, purified from both strains, increased serum TNF levels in an identical dose-dependent manner, whereas purified chitin did not induce scrum TNF levels. P. brasiliensis, the F1 fractions and β-glucan induced macrophages to secrete TNF in vitro. The differences in TNF levels, induced by the different fungal strains, were correlated with the β-glucan concentrations in the cell walls of both the avirulent and virulent strains of P. brasiliensis. These findings support a role for TNF in the pathogenicity of P. brasiliensis.

The antibody response against the antigen sheep red blood cells (SRBC) was investigated in mice pre-treated with formalin-killed Paracoccidioides brasiliensis or with cell wall fractions of the fungus. Pre-treatment with P. brasiliensis, as well as with the Fl fraction and beta-glucan significantly increased the anti-SRBC antibody response in the experimental groups as compared to the control group that received only SRBC. This immunomodulatory effect varied with the different doses employed and with pre-treatment time. We conclude that the cell wall fractions of P. brasiliensis might play an important role in the hypergammaglobulinemia associated with Paracoccidioidomycosis.

Background We have previously explored a therapeutic strategy for specifically targeting the profibrotic activity of IL-13 during experimental pulmonary fibrosis using a fusion protein comprised of human IL-13 and a mutated form of Pseudomonas aeruginosa exotoxin A (IL13-PE) and observed that the intranasal delivery of IL13-PE reduced bleomycin-induced pulmonary fibrosis through its elimination of IL-13-responsive cells in the lung. The aim of the present study was to determine whether the presence of an immune response to P. aeruginosa and/or its exotoxin A (PE) would diminish the anti-fibrotic properties of IL13-PE. Methodology/Principal Findings Fourteen days after P. aeruginosa infection, C57BL/6 mice were injected with bleomycin via the intratracheal route. Other groups of mice received 4 doses of saline or IL13-PE by either intranasal or intraperitoneal application, and were challenged i.t. with bleomycin 28 days later. At day 21 after bleomycin, all mice received either saline vehicle or IL13-PE by the intranasal route and histopatological analyses of whole lung samples were performed at day 28 after bleomycin. Intrapulmonary P. aeruginosa infection promoted a neutralizing IgG2A and IgA antibody response in BALF and serum. Surprisingly, histological analysis showed that a prior P. aeruginosa infection attenuated the development of bleomycin-induced pulmonary fibrosis, which was modestly further attenuated by the intranasal administration of IL13-PE. Although prior intranasal administration of IL13-PE failed to elicit an antibody response, the systemic administration of IL13-PE induced a strong neutralizing antibody response. However, the prior systemic sensitization of mice with IL13-PE did not inhibit the anti-fibrotic effect of IL13-PE in fibrotic mice. Conclusions Thus, IL13-PE therapy in pulmonary fibrosis works regardless of the presence of a humoral immune response to Pseudomonas exotoxin A. Interestingly, a prior infection with P. aeruginosa markedly attenuated the pulmonary fibrotic response suggesting that the immune elicitation by this pathogen exerts anti-fibrotic effects.

Sporothrix schenckii is the etiologic agent of sporotrichosis, a mycosis of world-wide distribution more commonly occurring in tropical regions. The immunological mechanisms involved in the prevention and control of sporotrichosis are not fully understood but apparently include both the humoral and cellular responses. In the present investigation, cellular immunity was evaluated by in vivo and in vitro tests in mice infected with yeast-like forms of S. schenckii. The disease developed systemically and cellular immunity was evaluated for a period of 10 weeks. The soluble antigen utilized in the tests was prepared from yeast form of the fungus through the sonication (20 min: 10 sonications at 50 W at 2-min intervals). Delayed hypersensitivity and lymphocyte transformation tests showed that the cellular immune response was depressed between the 4th and 6th week of infection when the animals were challenged with the soluble fungal antigen. This depression frequently indicates worsening of the disease, with greater involvement of the host. This is a promising field of research for a better understanding of the pathogeny of this mycosis.

Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-27T11:23:56Z No. of bitstreams: 0Bitstream added on 2014-05-27T14:41:02Z : No. of bitstreams: 1 2-s2.0-68449088131.pdf: 934532 bytes, checksum: e15a3c16c445e04e0abca46a1507293f (MD5) Made available in DSpace on 2014-05-27T11:23:56Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-07-16 Background: Protein-calorie malnutrition (PCM) is the most common type of malnutrition. PCM leads to immunodeficiency and consequent increased susceptibility to infectious agents. In addition, responses to prophylactic vaccines depend on nutritional status. This study aims to evaluate the ability of undernourished mice to mount an immune response to a genetic vaccine (pVAXhsp65) against tuberculosis, containing the gene coding for the heat shock protein 65 from mycobacteria. Methods: Young adult female BALB/c mice were fed ad libitum or with 80% of the amount of food consumed by a normal diet group. We initially characterized a mice model of dietary restriction by determining body and spleen weights, hematological parameters and histopathological changes in lymphoid organs. The ability of splenic cells to produce IFN-gamma and IL-4 upon in vitro stimulation with LPS or S. aureus and the serum titer of specific IgG1 and IgG2a anti-hsp65 antibodies after intramuscular immunization with pVAXhsp65 was then tested. Results: Dietary restriction significantly decreased body and spleen weights and also the total lymphocyte count in blood. This restriction also determined a striking atrophy in lymphoid organs as spleen, thymus and lymphoid tissue associated with the small intestine. Specific antibodies were not detected in mice submitted to dietary restriction whereas the well nourished animals produced significant levels of both, IgG1 and IgG2a anti-hsp65. Conclusion: 20% restriction in food intake deeply compromised humoral immunity induced by a genetic vaccine, alerting, therefore, for the relevance of the nutritional condition in vaccination programs based on these kinds of constructs. © 2009 Ishikawa et al; licensee BioMed Central Ltd. Department of Microbiology and Immunology Biosciences Institute São Paulo State University (UNESP), Botucatu, São Paulo 18618-000 Department of Parasitology Biosciences Institute São Paulo State University (UNESP), Botucatu, São Paulo 18618-000 Department of Tropical Diseases Medical School São Paulo State University (UNESP), Botucatu, São Paulo 18618-000 Department of Biochemistry and Immunology University of São Paulo (USP), Ribeirão Preto, São Paulo 14049-900 Department of Microbiology and Immunology Biosciences Institute São Paulo State University (UNESP), Botucatu, São Paulo 18618-000 Department of Parasitology Biosciences Institute São Paulo State University (UNESP), Botucatu, São Paulo 18618-000 Department of Tropical Diseases Medical School São Paulo State University (UNESP), Botucatu, São Paulo 18618-000

The composition of the α-branched-β-hydroxylated fatty acids from Corynebacterium ovis was studied. These acids were fractionated by argentation thin-layer chromatography in three species according to the degree of unsaturation of their O-acetylated methyl ester derivatives; the isolated fractions were analysed by gas chromatography combined with mass spectrometry. Corynomycolic (2-tetradecyl-3-hydroxy octadecanoic), corynomycolenic (2-tetradecyl-3-hydroxy-11-octadecenoic) and corynomycoldienic (2-tetradec-7′-enyl-3-hydroxy-11-octadecenoic) acids were identified; these acids represented the main component of the saturated, mono- and di-unsaturated species, respectively. On the other hand, a short-chain corynomycolic acid (2-tetradecyl-3-hydroxy decanoic acid), C 24 H 48 O 3 was found.

The purification procedure of 6,6′-diesters of trehalose from Corynebacterium diphtheriae was modified and the isolated substance was analysed by mass spectrometry as its permethylated derivative. The fatty acid moiety released from the glycolipid after alkaline hydrolysis was studied by mass spectral analysis of the O-methylated and O-acetylated methyl ester derivatives. By argentation thin-layer chromatography, three species of O-acetylated methyl esters were recognized, corresponding to saturated, mono-unsaturated and di-unsaturated α-branched-β-hydroxylated fatty acids. The double bond was located by ozonolysis of the O-acetylated methyl ester derivatives, by gas chromatography of the reaction product and mass spectrometry of the effluent from the gas chromatograph. The main components of each species of α-branched-β-hydroxylated fatty acids found in the gly colipid fraction of C. diphtheriae were 2-tetradecyl-3-hydroxyoctadecanoic acid (C 32 H 64 O 3 , corynomycolic acid), 2-tetradecyl-3-hydroxy-11-octadecenoic acid (C 32 H 62 O 3 , corynomycolenic acid), 2-tetradec-7′-enyl-3-hydroxy octadecanoic acid (C 32 H 62 O 3 ) and 2-tetradec-7′-enyl-3-hydroxy-11-octadecenoic acid (C 32 H 60 O 3 , corynomycoldienic acid). The glycolipid fraction from C. diphtheriae is obviously a complex mixture of 6,6′-diesters of trehalose.

A component of the acetone-soluble lipids of Nocordia rhodochrous grown on glycerol, was purified by column chromatography on silicic acid and characterized by infrared, nuclear magnetic resonance spectroscopy, optical rotation measurement and product identification after alkaline hydrolysis. Glycerol was the sole water-soluble component and nocardomycolic acids with chain lengths ranging from C 40 to C 44 were the constituent fatty acids identified. On the basis of the evidence obtained, the substance isolated from N. rhodochrous is identified as a mixture of mononocardomycoloylglycerols in which nocardomycolic acids are bound to one of the primary hydroxyl groups of the glycerol molecule.

SUMMARY: The possibility was examined that the toxicity induced in mice by Actinomadura madurae, “Streptomyces pelletieri” and Nocardia brasiliensis was due to lipid and cell-wall constituents. Mice were inoculated intraperitoneally with heat-killed bacteria, lipid extracts and cell-wall preparations emulsified in mineral oil: toxicity was evaluated by recording weight loss and deaths. Killed cells and cell-wall preparations of all three actinomycetes produced a pronounced loss of body weight, tissue necrosis, splenomegaly, a granulomatous inflammation and sometimes death. Mice inoculated with lipid extracts from A. madurae and “S. pelletieri” neither died nor showed toxic effects, but mice injected with lipids isolated from N. brasiliensis did suffer toxic effects. They showed more marked wasting symptoms than observed after inoculation of heat-killed bacteria or of the cell-wall preparation.

A glycolipid fraction was isolated from Corynebacterium ovis (C. pseudotuberculosis) . It had [ α ] D 25 = + 63.2° (C = 0.5, CHCl 3 ) and m.p. 43–46°C; the sugar content was 26%, determined as trehalose. Alkaline hydrolysis of the isolated fraction found trehalose as the sole water-soluble component, while glucose was found only after acid hydrolysis of the aqueous phase. Saturated and unsaturated short-chain mycolic acids with carbon atoms ranging from C 30 to C 36 were the constituents of the fatty acid moiety. The glycolipid fraction of C. ovis is therefore assumed to be a mixture of trehalose esters in which the trehalose molecule is esterified by saturated and unsaturated short-chain (C 30 –C 36 ) mycolic acids.

Trehalose dimycolate (TDM) isolated from Nocardia asteroides induced in mice a severely wasted condition known as cachexia. Intraperitoneal injection of mice with five 10 micrograms doses of TDM in mineral oil at intervals of 2 d killed 90% of the animals within 26 d. Death followed a precipitous weight loss and an inflammatory process in the peritoneal cavity. When mice were injected intraperitoneally with a single 10 micrograms dose of TDM, 48 h later, they had begun to lose weight and exhibited extreme hypertriglyceridaemia and hypoglycaemia. Tumour necrosis factor (or cachectin) was detected in the plasma from animals injected with TDM. This cytokine released by mononuclear phagocytes may be involved in the induction of cachexia by TDM.

Isolation of glycolipids from Nocardia asteroides, N. farcinica, Gordona lentifragmenta and G. bronchialis , by column chromatography of lipid extracts on a 50% (w/w) mixture of silicic acid and silica gel H, is described. The isolated materials were partially characterized by infrared spectroscopy, optical rotation and refractive index measurements, and by identifying the products of alkaline hydrolysis. Analytical studies showed that the glycolipids released only trehalose in the aqueous phase while mycolic acids were the constituent fatty acids identified. The isolated lipids are trehalose esters in which the trehalose molecule is esterified with mycolic acids.

Monoacylglycerols containing α-branched-β-hydroxylated fatty acids (mycolic acids) ranging from C 42 to C 50 and from C 60 to C 66 , were isolated from Gordona lentifragmenta and from G. bronchialis , respectively. On the other hand, G. rubropertincta showed only a monoacylglycerol fraction which released non-hydroxylated fatty acids; they were identified as C 16:0 -, C 16:1 ,- C 18:1 - and branched C 19:0 -fatty acids. This last component was identified as 10-methyl octadecanoic acid (tuberculostearic acid).

The possibility that the histological responses produced in paracoccidioidomycosis might be attributed to lipid constituents of cells of Paracoccidioides brasiliensis was investigated. Charcoal particles coated with the lipid extract were prepared and the suspension inoculated intravenously into mice. The lung inflammation was characterized by an intense mononuclear and polymorphonuclear cell infiltrate surrounding the particles from two to eight days after inoculation. Fractionation of lipid extract by preparative thin-layer chromatography allowed the separation of several fractions and it was observed that fractions containing free fatty acids and triglycerides were the most active. The participation of an uncommon fatty acid eliciting an inflammatory reaction is discussed.

Isolation of monoacylglycerol containing only hydroxy acids was achieved after fractionation of the diethyl ether-soluble lipids of Nocardia asteroides by column and thin layer chromatography. The isolated lipid, which was liquid, liberated only glycerol into the aqueous phase, and had (C 50 C 56 ) mycolic acids as the sole constituent fatty acids.

The mechanism by which cord factor (CF), a toxic glycolipid from mycobacteria, induces cachexia was studied in BALB/c mice. Body weight was markedly reduced 48 h after CF administration; the animals became severely wasted and exhibited hypertriglyceridemia, hypoglycemia, and high levels of tumor necrosis factor (TNF) in plasma. After CF administration, a transferable factor which caused cachexia and hypertriglyceridemia in recipient mice was detected in the blood. Dexamethasone partially inhibited the cachexia-inducing action of CF. Conditioned medium from adherent peritoneal cell cultures incubated with CF produced the same wasting symptoms when inoculated intravenously into mice. These studies also demonstrated that adherent peritoneal cells produced a humoral factor in response to CF which was related to CF-induced cachexia. Antiserum to recombinant TNF-alpha prevented the cachectin action in passive-transfer experiments. Our findings indicate that cachectin (TNF) plays a role as a central mediator of the wasting induced by CF.

The influx of inflammatory cells towards the peritoneal cavity in rats inoculated intraperitoneally with subcellular preparations of the fungus Paracoccidioides brasiliensis was studied. In addition to the dead fungus, also fractions F1 of the cell wall, which mainly consisted of polysaccharides and the lipid extract, induced intense cell migration 4 hr after inoculation, with a greatly increased number of polymorphonuclear leucocytes (PMN). Study of the kinetics of cell influx showed that both fraction F1 and the lipid extract initially induced intense PMN migration between the 4th and 24th hr after inoculation of these agents, followed by migration of mononuclear cells (MN) around the 48th hr. We also observed that migration of these cells increased gradually after inoculation of growing doses of fraction F1. The present data suggest that polysaccharides and lipids isolated from P. brasiliensis may participate in the initial phase of the inflammatory response in paracoccidioidomycosis.

A complex mixture of monoacylglycerols, identified as (C30--C36) corynomycoloylglycerols was isolated from the diethyl ether/acetone-soluble lipids of Corynebacterium pseudotuberculosis.

The distribution of an aqueous suspension of cord factor (CF) from Mycobacterium bovis BCG in several mouse organs was examined after intravenous injection, and the correlation between evolution of the inflammatory granulomatous reaction and the presence of CF in these organs was determined. CF was preferentially deposited in the lungs and liver, and the kinetics of the pulmonary and hepatic inflammatory reaction, evaluated by determining the indices for these organs, showed a gradual increase on day 2 after injection, reached a peak around the fifth day, and declined thereafter. Histological analysis showed that on day 5 both the lungs and the liver were diffusely damaged by a mononuclear inflammatory infiltrate arranged in a granulomatous manner and consisting predominantly of histiocytes. CF elimination was more marked in the liver than in the lungs: 2 d after injection 76% of the material deposited in the liver had been eliminated. Little or no CF was detected in the liver and lungs by day 16, when the inflammatory reaction was also substantially decreased. A second CF dose administered 8 d after the first exacerbated the inflammatory process in both the lungs and the liver, indicating that the intensity of this process depends on CF concentration in the lesion site.

Granulomatous reactions induced by lipid extracts from the dermatophyte fungi Fonsecaea pedrosoi, Fonsecaea compactum, Cladosporium carrionii and Phialophora verrucosum, the causal organisms of chromoblastomycosis, were studied. Charcoal particles coated with the lipid extracts were prepared and injected intravenously into mice. Inflammation was characterized by an intense mononuclear cell infiltrate that lodged in the lung from 4 to 8 d after inoculation.

C57B1/6 isogenic mice infected with Paracoccidioides brasiliensis strains showed a disruption in the expression of Ia antigen. Expression slowly decreased during the course of the infection with a slight variation dependent on the route of inoculation and the fungal strain used, but production of interferon-gamma and tumor necrosis factor-alpha were observed. Suppression of Ia antigen expression and depression of the immunoproliferative responses of spleen cells were strongly correlated with nitric oxide levels. These parameters were inhibited when the animals were treated with nitro-L-arginine, which resulted in inhibition the activation of nitric oxide (NO) production. Analysis of the data showed that changes in the expression of the Ia antigen occur in P. brasiliensis infection and are strongly correlated with NO levels. These phenomena may be interrelated and reflect macrophage activation that contributes to the control of the disease and to the immunosuppression observed during the course of the infection.

Made available in DSpace on 2022-04-28T20:55:48Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-01-01 Heat-shock proteins (HSPs) are currently one of the most promising targets for the development of immunotherapy against tumours and autoimmune disorders. This protein family has the capacity to activate or modulate the function of different immune system cells. They induce the activation of monocytes, macrophages and dendritic cells, and contribute to cross-priming, an important mechanism of presentation of exogenous antigen in the context of MHC class I molecules. These various immunological properties of HSP have encouraged their use in several clinical trials. Nevertheless, an important issue regarding these proteins is whether the high homology among HSPs across different species may trigger the breakdown of immune tolerance and induce autoimmune diseases. We have developed a DNA vaccine codifying the Mycobacterium leprae Hsp65 (DNAhsp65), which showed to be highly immunogenic and protective against experimental tuberculosis. Here, we address the question of whether DNAhsp65 immunization could induce pathological autoimmunity in mice. Our results show that DNAhsp65 vaccination induced antibodies that can recognize the human Hsp60 but did not induce harmful effects in 16 different organs analysed by histopathology up to 210 days after vaccination. We also showed that anti-DNA antibodies were not elicited after DNA vaccination. The results are important for the development of both HSP and DNA-based immunomodulatory agents. Copyright © by BIOLIFE, s.a.s. Department of Biochemistry and Immunology Medicine School of Ribeirão Preto University of São Paulo, São Paulo Department of Clinical Analyses Faculty of Pharmaceutical Sciences São Paulo State University, Araraquara, São Paulo Department of Microbiology and Immunology Biosciences Institute São Paulo State University, Botucatu, São Paulo Department of Pathology University of São Paulo Medical School, São Paulo Department of Pathology School of Medicine of Ribeirão Preto University of São Paulo, Ribeirão Preto, São Paulo Heart Institute (InCor) University of São Paulo Medical School, São Paulo Institute for Investigation in Immunology National Institute of Science and Technology - INCT, São Paulo Instituto do Coração Laboratório de Imunologia Universidade de São Paulo, Av Dr Eneas de Carvalho Aguiar, 44 bloco II, 9 andar, Cerqueira César, São Paulo - SP 05403-001 Department of Clinical Analyses Faculty of Pharmaceutical Sciences São Paulo State University, Araraquara, São Paulo Department of Microbiology and Immunology Biosciences Institute São Paulo State University, Botucatu, São Paulo

Recent findings in mice have changed our perception of how protective immunity works in tuberculosis and hold promise for the rapid development of new vaccines. For example, we now know: (1) that a single mycobacterial protein antigen can be sufficient to generate powerful protective immunity, provided that it is presented to the immune system in the right way; (2) that the expression of protection depends on cytotoxic antigen-specific T cells; (3) that the identity of the antigen may be less important than the mode of presentation, and (4) that injection of DNA encoding the antigen (DNA vaccination) is a superior way of raising protective immunity compared to injection of the antigen itself. These advances are timely because there is an urgent need for a new vaccine against tuberculosis. There continue to be about 3 million deaths from tuberculosis every year worldwide and increasingly the causative bacteria are multidrug resistant.

Helminths are known to elicit a wide range of immunomodulation characterized by dominant Th2-type immune responses. Our group previously showed that a DNA vaccine encoding the mycobacterial 65-kDa heat shock protein (DNA-hsp65) showed immunomodulatory properties. We also showed, using a helminth-tuberculosis (TB) co-infection model, that the DNA-hsp65 vaccine protected mice against TB. We next investigated the mechanistic role of the vaccine during helminth-TB co-infection. Clinically, helminth infection causes type 2 granulomas in the lung. Mice were immunized with DNA-hsp65 while they were submitted to the type 2 granuloma induction protocol by Schistosoma mansoni eggs infusion. In this work we investigated the effects of DNA-hsp65 on the pathology and immune response during the development of type 2 granuloma induced by S. mansoni eggs. Histologic analyses of lung parenchyma showed that the DNA-hsp65 vaccine protected mice against exacerbated fibrosis induced by Schistosoma eggs, and decreased the size of the granulomas. These changes were correlated with a reduction in the number of T cells specific for the egg antigens in the lung and also with modulation of Th2 cytokine expression. Taken together, our results showed that the adjuvant properties of the DNA-hsp65 vaccine regulated the immune response in this Th2 model, and resulted in a preserved lung parenchyma.

Antimycobacterial and cytotoxicity activity of synthetic and natural compounds. Secondary metabolites from Curvularia eragrostidis and Drechslera dematioidea, Clusia sp. floral resin, alkaloids from Pilocarpus alatus, salicylideneanilines, piperidine amides, the amine 1-cinnamylpiperazine and chiral pyridinium salts were assayed on Mycobacterium tuberculosis H37Rv. N-(salicylidene)-2-hydroxyaniline was the most effective compound with a minimal inhibitory concentration (MIC) of 8 µmol/L. Dihydrocurvularin was moderately effective with a MIC of 40 µmol/L. Clusia sp. floral resin and a gallocatechin-epigallocatechin mixture showed MIC of 0.02 g/L and 38 µmol/L, respectively. The cytotoxicity was evaluated for N-(salicylidene)-2-hydroxyaniline, curvularin, dihydrocurvularin and Clusia sp. floral resin, and the selectivity indexes were > 125, 0.47, 0.75 and 5, respectively.

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

BackgroundThe greatest challenges in vaccine development include optimization of DNA vaccines for use in humans, creation of effective single-dose vaccines, development of delivery systems that do not involve live viruses, and the identification of effective new adjuvants. Herein, we describe a novel, simple technique for efficiently vaccinating mice against tuberculosis (TB). Our technique consists of a single-dose, genetic vaccine formulation of DNA-hsp65 complexed with cationic liposomes and administered intranasally.ResultsWe developed a novel and non-toxic formulation of cationic liposomes, in which the DNA-hsp65 vaccine was entrapped (ENTR-hsp65) or complexed (COMP-hsp65), and used to immunize mice by intramuscular or intranasal routes. Although both liposome formulations induced a typical Th1 pattern of immune response, the intramuscular route of delivery did not reduce the number of bacilli. However, a single intranasal immunization with COMP-hsp65, carrying as few as 25 μg of plasmid DNA, leads to a remarkable reduction of the amount of bacilli in lungs. These effects were accompanied by increasing levels of IFN-γ and lung parenchyma preservation, results similar to those found in mice vaccinated intramuscularly four times with naked DNA-hsp65 (total of 400 μg).ConclusionOur objective was to overcome the significant obstacles currently facing DNA vaccine development. Our results in the mouse TB model showed that a single intranasal dose of COMP-hsp65 elicited a cellular immune response that was as strong as that induced by four intramuscular doses of naked-DNA. This formulation allowed a 16-fold reduction in the amount of DNA administered. Moreover, we demonstrated that this vaccine is safe, biocompatible, stable, and easily manufactured at a low cost. We believe that this strategy can be applied to human vaccines to TB in a single dose or in prime-boost protocols, leading to a tremendous impact on the control of this infectious disease.

Vaccines are considered by many to be one of the most successful medical interventions against infectious diseases. But many significant obstacles remain, such as optimizing DNA vaccines for use in humans or large animals. The amount of doses, route and easiness of administration are also important points to consider in the design of new DNA vaccines. Heterologous prime-boost regimens probably represent the best hope for an improved DNA vaccine strategy. In this study, we have shown that heterologous prime-boost vaccination against tuberculosis (TB) using intranasal BCG priming/DNA-HSP65 boosting (BCGin/DNA) provided significantly greater protection than that afforded by a single subcutaneous or intranasal dose of BCG. In addition, BCGin/DNA immunization was also more efficient in controlling bacterial loads than were the other prime-boost schedules evaluated or three doses of DNA-HSP65 as a naked DNA. The single dose of DNA-HSP65 booster enhanced the immunogenicity of a single subcutaneous BCG vaccination, as evidenced by the significantly higher serum levels of anti-Hsp65 IgG2a Th1-induced antibodies, as well as by the significantly greater production of IFN-γ by antigen-specific spleen cells. The BCG prime/DNA-HSP65 booster was also associated with better preservation of lung parenchyma.The improvement of the protective effect of BCG vaccine mediated by a DNA-HSP65 booster suggests that our strategy may hold promise as a safe and effective vaccine against TB.

Background Although B cells are important as antigen presenting cells (APC) during the immune response, their role in DNA vaccination models is unknown. Methods In this study in vitro and in vivo experiments were performed to evaluate the ability of B cells to protect mice against Mycobacterium tuberculosis challenge. Results In vitro and in vivo studies showed that B cells efficiently present antigens after naked plasmid pcDNA3 encoding M. leprae 65-kDa heat shock protein (pcDNA3-Hsp65) internalization and protect B knock-out (BKO) mice against Mycobacterium tuberculosis infection. pcDNA3-Hsp65-transfected B cells adoptively transferred into BKO mice rescued the memory phenotypes and reduced the number of CFU compared to wild-type mice. Conclusions These data not only suggest that B cells play an important role in the induction of CD8 T cells but also that they improve bacterial clearance in DNA vaccine model.

This study aimed to demonstrate that microspheres, used as delivery vehicle of DNA-Hsp65/TDM [plasmid DNA encoding heat shock protein 65 (Hsp65) coencapsulated with trehalose dimycolate (TDM) into PLGA microspheres], are widely spread among several organs after intramuscular administration in BALB/c mice. In general, we showed that these particles were phagocytosed by antigen presenting cells, such as macrophages and dendritic cells. Besides, it was demonstrated herein that draining lymph node cells presented a significant increase in the number of cells expressing costimulatory molecules (CD80 and CD86) and MHC class II, and also that the administration of the DNA-Hsp65/TDM and vector/TDM formulations resulted in the up-regulation of CD80, CD86 and MHC class II expression when compared to control formulations (vector/TDM and empty). Regarding the intracellular trafficking we observed that following phagocytosis, the microspheres were not found in the late endosomes and/or lysosomes, until 15 days after internalization, and we suggest that these constructions were hydrolysed in early compartments. Overall, these data expand our knowledge on PLGA [poly (lactic-co- glycolic acid)] microspheres as gene carriers in vaccination strategies, as well as open perspectives for their potential use in clinical practice.

Background: Phospholipases C (PLCs) are virulence factors found in several bacteria. In Mycobacterium tuberculosis (Mtb) they exhibit cytotoxic effects on macrophages, but the mechanisms involved in PLC-induced cell death are not fully understood. It has been reported that induction of cell necrosis by virulent Mtb is coordinated by subversion of PGE2, an essential factor in cell membrane protection. Results: Using two Mtb clinical isolates carrying genetic variations in PLC genes, we show that the isolate 97-1505, which bears plcA and plcB genes, is more resistant to alveolar macrophage microbicidal activity than the isolate 97-1200, which has all PLC genes deleted. The isolate 97-1505 also induced higher rates of alveolar macrophage necrosis, and likewise inhibited COX-2 expression and PGE2 production. To address the direct effect of mycobacterial PLC on cell necrosis and PGE2 inhibition, both isolates were treated with PLC inhibitors prior to macrophage infection. Interestingly, inhibition of PLCs affected the ability of the isolate 97-1505 to induce necrosis, leading to cell death rates similar to those induced by the isolate 97-1200. Finally, PGE2 production by Mtb 97-1505-infected macrophages was restored to levels similar to those produced by 97-1200-infected cells. Conclusions: Mycobacterium tuberculosis bearing PLCs genes induces alveolar macrophage necrosis, which is associated to subversion of PGE2 production.