Immunomodulatory activity of hyaluronidase is associated with metabolic adaptations during acute inflammation.

OBJECTIVE AND DESIGN: Investigate survival outcomes, and immunological and metabolomic effects of hyaluronidase (Hz) treatment during mouse models of acute inflammation and sepsis. METHODS: Survival of C57Bl/6 mice was monitored after lethal challenge with lipopolysaccharide (LPS) or cecal and ligation puncture (CLP)-induced sepsis and treated with Hz or saline. Mice were also challenged with LPS and treated with Hz for leukocyte counting, cytokine quantification and determination of metabolomic profiles in the peritoneal fluid. RESULTS: Hz treatment improved survival outcomes after lethal challenge with LPS or CLP-induced sepsis. LPS challenge promoted acute neutrophil accumulation and production of interleukin-1ß (IL-1ß) and IL-6 in the peritoneum, whereas Hz treatment suppressed neutrophil infiltration and cytokine production. We further characterized the metabolomic alterations caused by LPS challenge, which predicted activity of metabolic pathways related to fatty acids and eicosanoids. Hz treatment had a profound effect over the metabolic response, reflected by reductions of the relative levels of fatty acids. CONCLUSION: Collectively, these data demonstrate that Hz treatment is associated with metabolic reprogramming of pathways that sustain the inflammatory response.

Therapeutic effect of DNA vaccine encoding the 60-kDa-heat shock protein from Paracoccidoides brasiliensis on experimental paracoccidioidomycosis in mice.

Paracoccidioidomycosis (PCM) is a systemic mycosis autochthonous to Latin America and endemic to Brazil, which has the majority of the PCM cases. PCM is acquired through the inhalation of propagules of fungi from genus Paracoccidioides spp. and mainly affects the lungs. We have previously shown that P. brasiliensis-infected mice treated with single-dose of recombinant 60-kDa-heat shock protein from P. brasiliensis (rPbHsp60) had a worsening infection in comparison to animals only infected. In this study, we investigate whether the treatment of infected mice with PB_HSP60 gene cloned into a plasmid (pVAX1-PB_HSP60) would result in efficient immune response and better control of the disease. The harmful impact of single-dose therapy with protein was not seen with plasmid preparations. Most importantly, three doses of pVAX1-PB_HSP60 and protein induced a beneficial effect in experimental PCM with a reduction in fungal load and lung injury when compared with infected mice treated with pVAX1 or PBS. The increase of the cytokines IFN-γ, TNF, and IL-17 and the decrease of IL-10 observed after treatment with three doses of pVAX1-PB_HSP60 appears to be responsible for the control of infection. These results open perspectives of the therapeutic use of Hsp60 in PCM.

Recombinant BCG Expressing LTAK63 Adjuvant induces Superior Protection against Mycobacterium tuberculosis.

In order to develop an improved BCG vaccine against tuberculosis we have taken advantage of the adjuvant properties of a non-toxic derivative of Escherichia coli heat labile enterotoxin (LT), LTAK63. We have constructed rBCG strains expressing LTAK63 at different expression levels. Mice immunized with BCG expressing low levels of LTAK63 (rBCG-LTAK63lo) showed higher Th1 cytokines and IL-17 in the lungs, and when challenged intratracheally with Mycobacterium tuberculosis displayed a 2.0-3.0 log reduction in CFU as compared to wild type BCG. Histopathological analysis of lung tissues from protected mice revealed a reduced inflammatory response. Immunization with rBCG-LTAK63lo also protected against a 100-fold higher challenge dose. Mice immunized with rBCG-LTAK63lo produced an increase in TGF-ß as compared with BCG after challenge, with a corresponding reduction in Th1 and Th17 cytokines, as determined by Real Time RT-PCR. Furthermore, rBCG-LTAK63lo also displays protection against challenge with a highly virulent Beijing isolate. Our findings suggest that BCG with low-level expression of the LTAK63 adjuvant induces a stronger immune response in the lungs conferring higher levels of protection, and a novel mechanism subsequently triggers a regulatory immune response, which then limits the pathology. The rBCG-LTAK63lo strain can be the basis of an improved vaccine against tuberculosis.

Recombinant BCG Expressing LTAK63 Adjuvant induces Superior Protection against Mycobacterium tuberculosis

In order to develop an improved BCG vaccine against tuberculosis we have taken advantage of the adjuvant properties of a non-toxic derivative of Escherichia coli heat labile enterotoxin (LT), LTAK63. We have constructed rBCG strains expressing LTAK63 at different expression levels. Mice immunized with BCG expressing low levels of LTAK63 (rBCG-LTAK63lo) showed higher Th1 cytokines and IL-17 in the lungs, and when challenged intratracheally with Mycobacterium tuberculosis displayed a 2.0–3.0 log reduction in CFU as compared to wild type BCG. Histopathological analysis of lung tissues from protected mice revealed a reduced inflammatory response. Immunization with rBCG-LTAK63lo also protected against a 100-fold higher challenge dose. Mice immunized with rBCG-LTAK63lo produced an increase in TGF-β as compared with BCG after challenge, with a corresponding reduction in Th1 and Th17 cytokines, as determined by Real Time RT-PCR. Furthermore, rBCG-LTAK63lo also displays protection against challenge with a highly virulent Beijing isolate. Our findings suggest that BCG with low-level expression of the LTAK63 adjuvant induces a stronger immune response in the lungs conferring higher levels of protection, and a novel mechanism subsequently triggers a regulatory immune response, which then limits the pathology. The rBCG-LTAK63lo strain can be the basis of an improved vaccine against tuberculosis.

Recombinant BCG expressing LTAK63 adjuvant induces superior protection against mycobacterium tuberculosis

In order to develop an improved BCG vaccine against tuberculosis we have taken advantage of the adjuvant properties of a non-toxic derivative of Escherichia coli heat labile enterotoxin (LT), LTAK63. We have constructed rBCG strains expressing LTAK63 at different expression levels. Mice immunized with BCG expressing low levels of LTAK63 (rBCG-LTAK63(lo)) showed higher Th1 cytokines and IL-17 in the lungs, and when challenged intratracheally with Mycobacterium tuberculosis displayed a 2.0-3.0 log reduction in CFU as compared to wild type BCG. Histopathological analysis of lung tissues from protected mice revealed a reduced inflammatory response. Immunization with rBCG-LTAK63(lo) also protected against a 100-fold higher challenge dose. Mice immunized with rBCG-LTAK63(lo) produced an increase in TGF-beta as compared with BCG after challenge, with a corresponding reduction in Th1 and Th17 cytokines, as determined by Real Time RT-PCR. Furthermore, rBCG-LTAK63(lo) also displays protection against challenge with a highly virulent Beijing isolate. Our findings suggest that BCG with low-level expression of the LTAK63 adjuvant induces a stronger immune response in the lungs conferring higher levels of protection, and a novel mechanism subsequently triggers a regulatory immune response, which then limits the pathology. The rBCG-LTAK63(lo) strain can be the basis of an improved vaccine against tuberculosis.

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

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(+)CD25(hi). 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.

Investigation of local anesthetic and antimycobacterial activity of Ottonia martiana Miq. (Piperaceae).

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.

Synergy of chemotherapy and immunotherapy revealed by a genome-scale analysis of murine tuberculosis.

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.

Mycobacterium tuberculosis expressing phospholipase C subverts PGE2 synthesis and induces necrosis in alveolar macrophages.

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.

Immunotherapy of tuberculosis with Mycobacterium leprae Hsp65 as a DNA vaccine triggers cross-reactive antibodies against mammalian Hsp60 but not pathological autoimmunity.

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.

Modulation of angiogenic factor VEGF by DNA-hsp65 vaccination in a murine CNS tuberculosis model.

Tuberculosis (TB) is a serious public health problem. Development of experimental models and vaccines are essential to elucidate physiopathological mechanisms and to control the disease. Vascular endothelial growth factor (VEGF) is a potent activator of vascular permeability and angiogenesis. VEGF seems to participate in breakdown of the blood brain-barrier (BBB) in tuberculous meningitis (TBM), contributing to worsening of disease. Therefore, the objective here was to extent the characterization of our previously described murine model of central nervous system TB (CNS-TB) by describing the VEGF participation in the CNS disease, and suggesting a vaccination plan in mice. Plasmid encoding DNA protein antigen DNA-hsp65 has been described as a protector against TB infection and was used here to test its effectiveness in the prevention of VEGF production and TB disease. Vaccinated mice and its controls were injected with Mycobacterium bovis bacillus Calmette-Guerin (BCG) in cerebellum. Four weeks after BCG injection, mice were perfused and brains were paraffin-embedded for VEGF expression analysis. We observed VEGF immunohistochemical expression in TBM and granulomas in non-vaccinated mice. The DNA-hsp65 treatment blocked the expression of VEGF in mice TBM. Therefore, our murine model indicated the VEGF participation in the physiopathology of CNS-TB and the potential prevention of the DNA-hsp65 in the disease progression.

Evaluation of the overall IFN-γ and IL-17 pro-inflammatory responses after DNA therapy of tuberculosis.

Despite the enormous efforts displayed globally in the fight against tuberculosis, the disease incidence has modified slightly, which has led to a renewed interest in immunotherapy. In general, successful immunotherapeutic candidates against tuberculosis are agents that can trigger strong, specific pro-inflammatory responses, especially of the T-helper (Th) 1 pattern. However, how these pro-inflammatory agents effectively kill the bacteria without eliciting immunopathology is not well understood. We reasoned that, in addition to the specific immune response elicited by immunotherapy, the evaluation of the overall pro-inflammatory responses should provide additional and valuable information that will be useful in avoiding immunopathology. We evaluated the overall IFN-γ and IL-17 pro-inflammatory responses among CD4(+), CD8(+) and γδ T cells in the lungs of mice that were infected with M. tuberculosis and treated with a DNA vaccine in an immunotherapeutic regimen. Our results demonstrate that mice that effectively combat the pathogen develop a strong, specific Th1 immune response against the therapeutic antigen and have reduced lung inflammation, present in parallel a fine-tuning in the total IFN-γ- and IL-17-mediated immunity in the lungs. This modulation of the total immune response involves reducing the Th17 cell population, augmenting CD8(+) T cells that produce IFN-γ and increasing the total γδ T cell frequency. These results stress the importance of a broad evaluation of not only the specific immune response at the time to evaluate new immune interventional strategies against tuberculosis but also non-conventional T cells, such as γδ T lymphocytes.

Low-dose plasmid DNA treatment increases plasma vasopressin and regulates blood pressure in experimental endotoxemia.

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.

The contribution of a murine CNS-TB model for the understanding of the host-pathogen interactions in the formation of granulomas.

Central nervous system (CNS) tuberculosis (TB) is the most severe form of TB, characterized morphologically by brain granulomas and tuberculous meningitis (TBM). Experimental strategies for the study of the host-pathogen interaction through the analysis of granulomas and its intrinsic molecular mechanisms could provide new insights into the neuropathology of TB. To verify whether cerebellar mycobacterial infection induces the main features of the disease in human CNS and better understand the physiological mechanisms underlying the disease, we injected bacillus Calmette-Guerin (BCG) into the mouse cerebellum. BCG-induced CNS-TB is characterized by the formation of granulomas and TBM, a build up of bacterial loads in these lesions, and microglial recruitment into the lesion sites. In addition, there is an enhanced expression of signaling molecules such as nuclear factor-κB (NF-κB) and there is a presence of inducible nitric oxide synthase (iNOS) in the lesions and surrounding areas. This murine model of cerebellar CNS-TB was characterized by cellular and biochemical immune responses typically found in the human disease. This model could expand our knowledge about granulomas in TB infection of the cerebellum, and help characterize the physiological mechanisms involved with the progression of this serious illness that is responsible for killing millions people every year.

Antimycobacterial activity of 2-phenoxy-1-phenylethanone, a synthetic analogue of neolignan, entrapped in polymeric microparticles.

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.

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

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.

Expression of Mycobacterium leprae HSP65 in tobacco and its effectiveness as an oral treatment in adjuvant-induced arthritis.

Transgenic plants are able to express molecules with antigenic properties. In recent years, this has led the pharmaceutical industry to use plants as alternative systems for the production of recombinant proteins. Plant-produced recombinant proteins can have important applications in therapeutics, such as in the treatment of rheumatoid arthritis (RA). In this study, the mycobacterial HSP65 protein expressed in tobacco plants was found to be effective as a treatment for adjuvant-induced arthritis (AIA). We cloned the hsp65 gene from Mycobacterium leprae into plasmid pCAMBIA 2301 under the control of the double 35S promoter from cauliflower mosaic virus. Agrobacterium tumefaciens bearing the pChsp65 plasmid was used to transform tobacco plants. Incorporation of the hsp65 gene was confirmed by PCR, reverse transcription-PCR, histochemistry, and western blot analyses in several transgenic lines of tobacco plants. Oral treatment of AIA rats with the HSP65 protein allowed them to recover body weight and joint inflammation was reduced. Our results suggest a synergistic effect between the HSP65 expressed protein and metabolites presents in tobacco plants.

A dynamic analysis of tuberculosis dissemination to improve control and surveillance.

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.

Intranasal vaccination with messenger RNA as a new approach in gene therapy: use against tuberculosis.

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.

Helminth coinfection does not affect therapeutic effect of a DNA vaccine in mice harboring tuberculosis.

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-gamma production was correlated with vaccine-induced protection in coinfected mice. Moreover, in S. mansoni-coinfected mice, DNA treatment inhibited in vivo TGF-beta 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.