Immunotherapeutic Activities of a DNA Plasmid Carrying the Mycobacterial hsp65 Gene (DNAhsp65).

DNA vaccines have become relevant subject matter, and efforts for their development have been increasing due to their potential as technology platforms applicable for prophylactic and therapeutic approaches for infectious diseases and for cancer treatment, allergies, and autoimmune diseases. This review aimed to summarize current knowledge about the plasmid DNA vaccine carrying the mycobacterial hsp65 gene (DNAhsp65), which demonstrates immunomodulatory and immunoregulatory properties of both the innate and adaptive immune systems. The possible mechanisms associated with the modulation and regulatory role of DNAhsp65 in the control of various conditions is also discussed.

Cattle Tick Rhipicephalus microplus-Host Interface: A Review of Resistant and Susceptible Host Responses.

Ticks are able to transmit tick-borne infectious agents to vertebrate hosts which cause major constraints to public and livestock health. The costs associated with mortality, relapse, treatments, and decreased production yields are economically significant. Ticks adapted to a hematophagous existence after the vertebrate hemostatic system evolved into a multi-layered defense system against foreign invasion (pathogens and ectoparasites), blood loss, and immune responses. Subsequently, ticks evolved by developing an ability to suppress the vertebrate host immune system with a devastating impact particularly for exotic and crossbred cattle. Host genetics defines the immune responsiveness against ticks and tick-borne pathogens. To gain an insight into the naturally acquired resistant and susceptible cattle breed against ticks, studies have been conducted comparing the incidence of tick infestation on bovine hosts from divergent genetic backgrounds. It is well-documented that purebred and crossbred Bos taurus indicus cattle are more resistant to ticks and tick-borne pathogens compared to purebred European Bos taurus taurus cattle. Genetic studies identifying Quantitative Trait Loci markers using microsatellites and SNPs have been inconsistent with very low percentages relating phenotypic variation with tick infestation. Several skin gene expression and immunological studies have been undertaken using different breeds, different samples (peripheral blood, skin with tick feeding), infestation protocols and geographic environments. Susceptible breeds were commonly found to be associated with the increased expression of toll like receptors, MHC Class II, calcium binding proteins, and complement factors with an increased presence of neutrophils in the skin following tick feeding. Resistant breeds had higher levels of T cells present in the skin prior to tick infestation and thus seem to respond to ticks more efficiently. The skin of resistant breeds also contained higher numbers of eosinophils, mast cells and basophils with up-regulated proteases, cathepsins, keratins, collagens and extracellular matrix proteins in response to feeding ticks. Here we review immunological and molecular determinants that explore the cattle tick Rhipicephalus microplus-host resistance phenomenon as well as contemplating new insights and future directions to study tick resistance and susceptibility, in order to facilitate interventions for tick control.

MicroRNA expression signatures in lungs of mice infected with Mycobacterium tuberculosis.

Tuberculosis (TB) is a major public health concern worldwide; however the factors that account for resistance or susceptibility to disease are not completely understood. Although some studies suggest that the differential expression of miRNAs in peripheral blood of TB patients could be useful as biomarkers of active disease, their involvement during the inflammatory process in lungs of infected individuals is unknown. Here, we evaluated the global expression of miRNAs in the lungs of mice experimentally infected with Mycobacterium tuberculosis on 30 and 60 days post-infection. We observed that several miRNAs were differentially expressed compared to uninfected mice. Furthermore, we verified that the expression of miR-135b, miR-21, miR-155, miR-146a, and miR-146b was significantly altered in distinct leukocyte subsets isolated from lungs of infected mice, while genes potentially targeted by those miRNAs were associated with a diversity of immune related molecular pathways. Importantly, we validated the inhibition of Pellino 1 expression by miR-135b in vitro. Overall, this study contributes to the understanding of the dynamics of miRNA expression in lungs during experimental TB and adds further perspectives into the role of miRNAs on the regulation of immune processes such as leukocyte activation.

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.

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.

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.

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.