Antimycobacterial and cytotoxicity activities of free and liposome-encapsulated 3-(4'-bromo[1,1'-biphenyl-4-yl)-3-(4-bromo-phenyl)-N,N-dimethyl-2-propen-1-amine

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.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Antimycobacterial and cytotoxicity activities of free and liposome-encapsulated 3-(4'-bromo[1,1'-biphenyl-4-yl)-3-(4-bromo-phenyl)-N,N-dimethyl-2-propen-1-amine

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 concentrations334871874CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQSem informaçã

Antimycobacterial and cytotoxicity activities of free and liposome-encapsulated 3-(4'-bromo[1,1'-biphenyl-4-yl)-3-(4-bromo-phenyl)-N,N-dimethyl-2-propen-1-amine

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)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.334871874Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)CNPq_Brasi

A DNA vaccine against tuberculosis based on the 65 kDa heat-shock protein differentially activates human macrophages and dendritic cells

Abstract\ud \ud \ud \ud Background\ud \ud A number of reports have demonstrated that rodents immunized with DNA vaccines can produce antibodies and cellular immune responses presenting a long-lasting protective immunity. These findings have attracted considerable interest in the field of DNA vaccination. We have previously described the prophylactic and therapeutic effects of a DNA vaccine encoding the Mycobacterium leprae 65 kDa heat shock protein (DNA-HSP65) in a murine model of tuberculosis. As DNA vaccines are often less effective in humans, we aimed to find out how the DNA-HSP65 stimulates human immune responses.\ud \ud \ud \ud Methods\ud \ud To address this question, we analysed the activation of both human macrophages and dendritic cells (DCs) cultured with DNA-HSP65. Then, these cells stimulated with the DNA vaccine were evaluated regarding the expression of surface markers, cytokine production and microbicidal activity.\ud \ud \ud \ud Results\ud \ud It was observed that DCs and macrophages presented different ability to uptake DNA vaccine. Under DNA stimulation, macrophages, characterized as CD11b+/CD86+/HLA-DR+, produced high levels of TNF-alpha, IL-6 (pro-inflammatory cytokines), and IL-10 (anti-inflammatory cytokine). Besides, they also presented a microbicidal activity higher than that observed in DCs after infection with M. tuberculosis. On the other hand, DCs, characterized as CD11c+/CD86+/CD123-/BDCA-4+/IFN-alpha-, produced high levels of IL-12 and low levels of TNF-alpha, IL-6 and IL-10. Finally, the DNA-HSP65 vaccine was able to induce proliferation of peripheral blood lymphocytes.\ud \ud \ud \ud Conclusion\ud \ud Our data suggest that the immune response is differently activated by the DNA-HSP65 vaccine in humans. These findings provide important clues to the design of new strategies for using DNA vaccines in human immunotherapy.We thank Dr. Carlos Rodrigo ZárateBladés for helpful suggestions during the course of the studies. We also thank Mrs. Izaíra T. Brandão and Mrs. Ana P. Masson for technical assistance. This study was supported by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Programa Nacional de DST/AIDS do Ministério da Saúde and Conselho Nacional de Pesquisa (CNPq).We thank Dr. Carlos Rodrigo Zárate-Bladés for helpful suggestions during the course of the studies. We also thank Mrs. Izaíra T. Brandão and Mrs. Ana P. Masson for technical assistance. This study was supported by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Programa Nacional de DST/AIDS do Ministério da Saúde and Conselho Nacional de Pesquisa (CNPq)

Improve protective efficacy of a TB DNA-HSP65 vaccine by BCG priming

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

B cells Can Modulate the CD8 Memory T Cell after DNA Vaccination Against Experimental Tuberculosis

Abstract\ud \ud \ud \ud Background\ud \ud Although B cells are important as antigen presenting cells (APC) during the immune response, their role in DNA vaccination models is unknown.\ud \ud \ud \ud Methods\ud \ud 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.\ud \ud \ud \ud Results\ud \ud \ud 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.\ud \ud \ud \ud Conclusions\ud \ud 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.We are thankful to Ana Paula Masson and Izaíra T Brandão for providing the DNA vaccine and recombinant protein. This study was supported by a FAPESP fellowship (05/030873) to LPA.We are thankful to Ana Paula Masson and Izaíra T Brandão for providing the DNA vaccine and recombinant protein. This study was supported by a FAPESP fellowship (05/03087-3) to LPA

Secondary metabolites from the sponges Aplysina fistularis and Dysidea sp. and the antituberculosis activity of 11-ketofistularin-3

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.American Society of Pharmacognosy FoundationFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)BIOTA/FAPESP - BIOprospecTAUSP - PIBIC/CNPqFAPES

Free 2-propen-1-amine derivative and inclusion complexes with beta-cyclodextrin: scanning electron microscopy, dissolution, cytotoxicity and antimycobacterial activity

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 beta-cyclodextrin (beta-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 beta-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/beta-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 beta-CD complexes. However, the complexes were less cytotoxic to V79 cells than the free form. The BBAP/beta-CD inclusion complexes were more effective (MIC) than the free compound on several mycobacteria strains. Similar behavior was observed for BBAP/beta-CD complexes and rifampicin, a front-line antitubercular drug, on M. tuberculosis H37Rv growing inside J774 macrophages.Complexos de inclusões e misturas físicas contendo mistura isomérica E/Z (50:50) de 3-(4'-bromo-[1,1'-bifenil]-4-il)-3-(4-bromofenil)-N,N-dimetil-2-propen-1-amina (BBAP) e beta-ciclodextrina (b-CD) nas proporções molares de 1:1 e 1:2 foram analisados por microscopia eletrônica de varredura (SEM). O perfil de dissolução do BBAP e dos complexos de inclusões foram também avaliados durante 6 horas. Por microscopia eletrônica de varredura foi possível observar os complexos de inclusões formados entre BBAP e beta-CD por co-evaporação nas proporções molares de 1:1 e 1:2. Como previamente detectado pela caracterização físico-química, na mistura física não se observou a presença de complexo de inclusão. Os estudos de dissolução mostraram que os complexos de inclusões 1:1 e 1:2 liberaram, respectivamente 49.07 ± 1.48 e 40.26 ± 3.90% de BBAP durante 6 horas. BBAP na forma livre foi menos solúvel que os complexos de inclusões e atingiu 9.00 ± 0.75% de dissolução. Os ensaios de citotoxicidade em macrófagos J774 e em uma linhagem de células fibroblásticas de pulmão (V79) indicaram que o BBAP não exibiu efeito tóxico adicional quando complexado com beta-CD. Entretanto, os complexos de inclusões foram menos tóxicos para células V79 que BBAP na forma livre. Os complexos de inclusões BBAP/beta-CD foram mais efetivos (CIM) que o composto livre em várias cepas de micobactérias. Resultados semelhantes foram observados sobre M. tuberculosis H37Rv intracelular para os complexos de inclusões BBAP/b-CD e rifampicina, uma droga anti-tuberculose de primeira linha.682689Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Therapeutic Efficacy of Cintredekin Besudotox (IL13-PE38QQR) in Murine Lung Fibrosis Is Unaffected by Immunity to Pseudomonas aeruginosa Exotoxin A

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.National Institutes of Health (NIH)Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)NeoPharm, In

Comparison of different delivery systems of DNA vaccination for the induction of protection against tuberculosis in mice and guinea pigs

The great challenges for researchers working in the field of vaccinology are optimizing DNA vaccines for use in humans or large animals and creating effective single-dose vaccines using appropriated controlled delivery systems. Plasmid DNA encoding the heat-shock protein 65 (hsp65) (DNAhsp65) has been shown to induce protective and therapeutic immune responses in a murine model of tuberculosis (TB). Despite the success of naked DNAhsp65-based vaccine to protect mice against TB, it requires multiple doses of high amounts of DNA for effective immunization. In order to optimize this DNA vaccine and simplify the vaccination schedule, we coencapsulated DNAhsp65 and the adjuvant trehalose dimycolate (TDM) into biodegradable poly (DL-lactide-co-glycolide) (PLGA) microspheres for a single dose administration. Moreover, a single-shot prime-boost vaccine formulation based on a mixture of two different PLGA microspheres, presenting faster and slower release of, respectively, DNAhsp65 and the recombinant hsp65 protein was also developed. These formulations were tested in mice as well as in guinea pigs by comparison with the efficacy and toxicity induced by the naked DNA preparation or BCG. The single-shot prime-boost formulation clearly presented good efficacy and diminished lung pathology in both mice and guinea pigs