Immunogenicity and Protection by DnaK and SpaA Recombinant Proteins Against Erysipelothrix Rhusiopathiae in a Murine Model.

BACKGROUND/AIMS: Swine erysipelas is a disease caused by Erysipelothrix rhusiopathiae, a Gram-positive bacillus, which has great economic importance because it leads to the loss of the swine herd. To control this disease, animals are immunized with a cellular vaccine of killed or attenuated E. rhusiopathiae, but even with herd vaccination, cases of swine erysipelas outbreaks have been reported in the United States, China and Japan, leading to the search for other antigenic components of the bacteria that may promote greater protection against E. rhusiopathiae. The surface protein SpaA from E. rhusiopathiae has been shown to be a candidate to constitute a subunit vaccine, since it has already been reported to induce a host immune response against the bacterium. DnaK, a hsp70 molecular chaperone, also seems to be a good candidate in the composition of a vaccine, as it has been demonstrated to be an antigenic protein of the bacteria. METHODS: This work evaluated the immunogenicity and protection induced by the E. rhusiopathiaee SpaA and DnaK recombinant proteins in a murine model, by intramuscular administration to mice with two doses of 100 µg at 21-day interval between them. The candidate proteins were tested either separately and together, compared with the commercial vaccine and the non-vaccination condition, and mice were challenged with a virulent strain of E. rhusiopathiae. Serum was collected to assess the produced antibodies and peripheral blood cells, whereas spleen and kidney tissues were assayed for E. rhusiopathiae presence by colony counting. RESULTS: A survival curve of the animals was performed, which confirmed the protection induced by the proteins. IgG antibodies increased in the animal serum inoculated with the proteins when compared to the control, and a significant delay in disease symptoms was observed. CONCLUSION: These results suggest that E. rhusiopathiae DnaK and SpaA are immunogenic in mice and interfere with the disease development.

Efficacy of instillation of MB49 cells and thermoreversible polymeric gel in urothelial bladder carcinoma immunization.

BACKGROUND: Activating the immune system for therapeutic benefit has long been a goal in immunology, especially in cancer treatment, but the low immunogenicity of antitumor vaccines remains a limiting factor in the fight against malignant neoplasms. The increase in the immunogenicity of weak antigens using biodegradable polymers, such as chitosan, has been observed in the field of cancer immunotherapy. However, the effects of the vaccine using a combination of tumor cells and a thermoreversible delivery system based on chitosan in bladder cancer models, mainly using the intravesical route to stimulate the antitumor immune response, are unknown. We propose to evaluate the efficacy of a polymeric gel matrix (TPG) formed by poloxamer 407 and chitosan, associated with MB49 cells, as an intravesical antitumor vaccine using a C57BL/6 murine model of bladder urothelial carcinoma. The effectiveness of immunization was analyzed with the formation of three experimental groups: Control, TPG and TPG + MB49. In the vaccination phase, the TPG + MB49 group underwent a traumatic injury to the bladder wall with immediate intravesical instillation of the vaccine compound containing MB49 cells embedded in TPG. The TPG group was subjected to the same procedures using the compound containing the gel diluted in medium, and the control group using only the medium. After 21 days, the animals were challenged with tumor induction. RESULTS: In vitro tests showed loss of viability and inability to proliferate after exposure to TPG. In vivo tests showed that animals previously immunized with TPG + MB49 had higher cumulative survival, as well as significantly lower bladder weight and size in contrast to the other two groups that did not show a statistically different tumor evolution. In addition, the splenocytes of these animals also showed a higher rate of antitumor cytotoxicity in relation to the TPG and control groups. CONCLUSIONS: We can conclude that MB49 cells embedded in a polymeric thermoreversible gel matrix with chitosan used in the form of an intravesical vaccine are able to stimulate the immune response and affect the development of the bladder tumor in an orthotopic and syngeneic C57BL/6 murine model.

Mitochondrial heat shock protein mortalin as potential target for therapies based on oxidative stress.

BACKGROUND: Treatments based on production of reactive oxygen species for bladder cancer such as photodynamic therapy (PDT) have been marginalized due to low specificity and the existence of resistance mainly associated with the up-regulation of Heat Shock Proteins (HSPs). To overcome these barriers, the establishment of strategies combining PDTs with HSP inhibitors may be promising and the identification of HSPs involved with oxidative stress from bladder tumors in animal models represents a key step in this direction. MATERIALS: Thus, the present study aims to identify cytosolic and mitochondrial HSPs up expressed in murine bladder tumors and in the urothelial carcinoma cell line MB49 by qRT-PCR screening, and to analyze the importance of the activity of the HSPs associated with oxidative stress protection in the survival of the MB49 using strategy of inhibition in vitro. RESULTS: Results showed that both tumor tissues and MB49 cells in culture had significant overexpression of the mitochondrial HSPA9 (mortalin) and HSP60 mRNAs, while the cytosolic HSP90 was overexpressed only in the tumor. The effect of mortalin in the MB49 cells survival under oxidative stress was evaluated in vitro in presence of the specific inhibitor MKT-077 and H2O2. The findings showed that MB49 viability was permanently reduced by the MKT-077 in a dose-dependent manner by inducing apoptosis or necrosis, mainly under oxidative stress conditions. CONCLUSION: Results suggest that mortalin is preferentially expressed in the MB49 cancer model and plays a key role in tumoral survival, especially under oxidative stress, making this HSP a potential target for an alternative treatment combining PDT with HSP inhibitors.