Luciana P. Almeida

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

BackgroundmRNAs 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.ResultsWe 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).ConclusionsTaken 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.

Endonucleases: tools to correct the dystrophin gene

Various endonucleases can be engineered to induce double‐strand breaks (DSBs) in chosen DNA sequences. These DSBs are spontaneously repaired by nonhomologous‐end‐joining, resulting in micro‐insertions or micro‐deletions (INDELs). We detected, characterized and quantified the frequency of INDELs produced by one meganuclease (MGN) targeting the RAG1 gene, six MGNs targeting three introns of the human dystrophin gene and one pair of zinc finger nucleases (ZFNs) targeting exon 50 of the human dystrophin gene. The experiments were performed in human cells (i.e. 293 T cells, myoblasts and myotubes).

Influence of storage time and temperature on pleural fluid adenosine deaminase determination

Objectives and background:  The determination of adenosine deaminase (ADA) activity in pleural fluid is important for differentiation of pleural effusions and diagnosing pleural tuberculosis. Although measurement of ADA is simple and inexpensive, controversies exist regarding potential errors caused by time elapsed between sample collection and analysis, storage temperature and the use of anticoagulants. The objective of this study was to evaluate the influence of storage time (1, 3, 7, 10 and 28 days) and temperature (4°C and −20°C) on the determination of ADA in pleural fluid samples collected in EDTA and sent at ambient temperature to the laboratory for initial processing within 1 h of collection.

Activity-Regulated Cytoskeleton-Associated Protein Controls AMPAR Endocytosis through a Direct Interaction with Clathrin-Adaptor Protein 2

Abstract The activity-regulated cytoskeleton-associated (Arc) protein controls synaptic strength by facilitating AMPA receptor (AMPAR) endocytosis. Here we demonstrate that Arc targets AMPAR to be internalized through a direct interaction with the clathrin-adaptor protein 2 (AP-2). We show that Arc overexpression in dissociated hippocampal neurons obtained from C57BL/6 mouse reduces the density of AMPAR GluA1 subunits at the cell surface and reduces the amplitude and rectification of AMPAR-mediated miniature-EPSCs (mEPSCs). Mutations of Arc, that prevent the AP-2 interaction reduce Arc-mediated endocytosis of GluA1 and abolish the reduction in AMPAR-mediated mEPSC amplitude and rectification. Depletion of the AP-2 subunit µ2 blocks the Arc-mediated reduction in mEPSC amplitude, an effect that is restored by reintroducing µ2. The Arc–AP-2 interaction plays an important role in homeostatic synaptic scaling as the Arc-dependent decrease in mEPSC amplitude, induced by a chronic increase in neuronal activity, is inhibited by AP-2 depletion. These data provide a mechanism to explain how activity-dependent expression of Arc decisively controls the fate of AMPAR at the cell surface and modulates synaptic strength, via the direct interaction with the endocytic clathrin adaptor AP-2.

Tissue distribution of DNA-Hsp65/TDM-loaded PLGA microspheres and uptake by phagocytic cells

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.

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

BackgroundAlthough B cells are important as antigen presenting cells (APC) during the immune response, their role in DNA vaccination models is unknown.MethodsIn this study in vitro and in vivo experiments were performed to evaluate the ability of B cells to protect mice against Mycobacterium tuberculosis challenge.ResultsIn 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.ConclusionsThese 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.

B cells expressing IL-10 mRNA modulate memory T cells after DNA-Hsp65 immunization.

In DNA vaccines, the gene of interest is cloned into a bacterial plasmid that is engineered to induce protein production for long periods in eukaryotic cells. Previous research has shown that the intramuscular immunization of BALB/c mice with a naked plasmid DNA fragment encoding the Mycobacterium leprae 65-kDa heat-shock protein (pcDNA3-Hsp65) induces protection against M. tuberculosis challenge. A key stage in the protective immune response after immunization is the generation of memory T cells. Previously, we have shown that B cells capture plasmid DNA-Hsp65 and thereby modulate the formation of CD8+ memory T cells after M. tuberculosis challenge in mice. Therefore, clarifying how B cells act as part of the protective immune response after DNA immunization is important for the development of more-effective vaccines. The aim of this study was to investigate the mechanisms by which B cells modulate memory T cells after DNA-Hsp65 immunization. C57BL/6 and BKO mice were injected three times, at 15-day intervals, with 100 µg naked pcDNA-Hsp65 per mouse. Thirty days after immunization, the percentages of effector memory T (TEM) cells (CD4+ and CD8+/CD44high/CD62Llow) and memory CD8+ T cells (CD8+/CD44high/CD62Llow/CD127+) were measured with flow cytometry. Interferon γ, interleukin 12 (IL-12), and IL-10 mRNAs were also quantified in whole spleen cells and purified B cells (CD43−) with real-time qPCR. Our data suggest that a B-cell subpopulation expressing IL-10 downregulated proinflammatory cytokine expression in the spleen, increasing the survival of CD4+ TEM cells and CD8+ TEM/CD127+ cells.

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

BackgroundAlthough 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.ResultsHere, 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.ConclusionOur 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.

Abstract 4244: Epigenetic regulation by SET protein in HNSCC: histone acetylation versus DNA methylation.

Background: Epigenetic regulation is essential in the control of both normal cellular processes and cancer development. DNA methylation and histone acetylation are involved in chromatin remodeling and gene transcription control. Recently, we reported that SET protein, a member of the acetyltransferase inhibitor complex, is strongly accumulated in HNSCC. However, SET action in gene expression is unknown, so we assessed SET role in the control of epigenetic modifications. Materials and Methods: HNSCC (HN6, HN12 and HN13), HEK293 and NOK-SI cell lines were used. SET overexpression was obtained in HEK293 and NOK-SI cells using pcDNA3.1 with SET cDNA, and SET knockdown in HNSCC cells using siRNA or shRNA. Human transcription factors RT² Profiler™ PCR Array profile (TF) and human tumor suppressor genes (TSG) EpiTect® DNA Methyl qPCR Array System (SABioscience-Qiagen) were used to determine the SET action in gene transcription and DNA methylation. Quantitative real time PCR (qPCR), chromatin immunoprecipitation (ChIP), immunofluorescence, Western blotting and DNA methyltransferases (DNMT) activities (EpiSeeker DNMT Activity Quantification Kit; Abcam) assays were used. Trichostatin A (TSA, 100 ng/mL), which increases histone acetylation, or 5-aza-2-deoxycytidine (5-aza, 1ug/mL), which decreases DNA methylation, were used for epigenetic modifications. Results: TFs mRNA analysis showed only 15 genes up-regulated and 65 down-regulated in HEK293/SET cells. Eight down-regulated (ATF2, CTNNB1, HIF-1A, NFATC3, RELA and STAT1) and two up-regulated (ATF3 and MYB) genes were also assessed by qPCR and similar mRNA profile was obtained. HNSCC SET knockdown cells presented opposite mRNA profile. ChIP analysis was performed for nine genes and HIF-1A, NFATC3 and STAT1 promoters showed interaction with SET. However, NFATC3 and STAT1 protein levels were not altered. DNA methylation profile of TSG showed that SET overexpression in HEK293 and NOK-SI cells promoted hypomethylation whereas SET knockdown in HN12 and HN13 cells promoted hypermethylation. HN6, HN12 and HN13 control cells, which present constitutive SET accumulation, presented a methylation loss pattern similar to the observed in HEK293 and NOK-SI cells with SET overexpression. GSTP1, PTEN and TP16 mRNAs levels decreased in SET-overexpressing cells despite the DNA demethylation status. To assess which modification is the main SET mechanism in the gene expression regulation, we performed qPCR in cell lines treated with TSA or 5-aza. TSA treatment reversed the SET overexpression effect in GSTP1, HIF-1A, NFATC3, PTEN, TP16 and STAT1 mRNA whereas 5-aza did not reverse. SET overexpression promoted increase in DNMT activities accompanied by increase in DNMT-1 protein. Conclusions: SET accumulation promotes transcriptional repression through histone hypoacetylation regulation and DNA demethylation is not associated with loss of DNMTs activities. Citation Format: Luciana Almeida, Maryna Tannous, Camila Matsumoto, Lais Sobral, Carlos Curti, Andreia Leopoldino. Epigenetic regulation by SET protein in HNSCC: histone acetylation versus DNA methylation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4244. doi:10.1158/1538-7445.AM2013-4244

Antigen-presenting cells transfected with Hsp65 messenger RNA fail to treat experimental tuberculosis

In the last several years, the use of dendritic cells has been studied as a therapeutic strategy against tumors. Dendritic cells can be pulsed with peptides or full-length protein, or they can be transfected with DNA or RNA. However, comparative studies suggest that transfecting dendritic cells with messenger RNA (mRNA) is superior to other antigen-loading techniques in generating immunocompetent dendritic cells. In the present study, we evaluated a new therapeutic strategy to fight tuberculosis using dendritic cells and macrophages transfected with Hsp65 mRNA. First, we demonstrated that antigen-presenting cells transfected with Hsp65 mRNA exhibit a higher level of expression of co-stimulatory molecules, suggesting that Hsp65 mRNA has immunostimulatory properties. We also demonstrated that spleen cells obtained from animals immunized with mock and Hsp65 mRNA-transfected dendritic cells were able to generate a mixed Th1/Th2 response with production not only of IFN-γ but also of IL-5 and IL-10. In contrast, cells recovered from mice immunized with Hsp65 mRNA-transfected macrophages were able to produce only IL-5. When mice were infected with Mycobacterium tuberculosis and treated with antigen-presenting cells transfected with Hsp65 mRNA (therapeutic immunization), we did not detect any decrease in the lung bacterial load or any preservation of the lung parenchyma, indicating the inability of transfected cells to confer curative effects against tuberculosis. In spite of the lack of therapeutic efficacy, this study reports for the first time the use of antigen-presenting cells transfected with mRNA in experimental tuberculosis.