Evaluation of immune responses in mice and sheep inoculated with a live attenuated Brucella melitensis REV1 vaccine produced in bioreactor.

The Brucella melitensis REV1 vaccine is the most widely employed vaccine for prophylaxis against brucellosis in sheep and goats. The objective of vaccination is disease control in herds or preventing infection in farms. In this study, we produced REV1 vaccine with a protocol, based on the use of liquid medium in a bioreactor, that resulted efficient, safe, relatively fast, and cost-effective. The live attenuated vaccine produced was tested in mice and sheep to investigate its immunogenicity and efficacy. Seventy-two female BALB/c mice were obtained and subdivided in 2 groups, one was stimulated with 1 × 106 colony-forming units (CFUs) of B. melitensis while the other with physiological solution alone and acting as control group. Furthermore, 25 sheep were subdivided into 5 groups: four were inoculated with a B. melitensis dose, ranging from 0.6 × 109 and 3.2 × 109 CFUs and the other was the control group. In addition, a serological diagnosis was performed for sheep by rapid serum agglutination and the complement-fixation test. Immunocompetent cells from both experiment were collected at different times post vaccination and immunostained to evaluate innate and adaptive-immune responses. In mice flow cytometry was used to detect macrophages, T lymphocytes, dendritic cells, memory cells, naïve cells, natural killer cells, major histocompatibility complex type II, B lymphocytes, regulatory T lymphocytes, T helper lymphocytes, cytotoxic T lymphocytes and recently activated CD4+ and CD8+ lymphocytes. In sheep, macrophages, T helper cells, cytotoxic T lymphocytes, regulatory T lymphocytes, dendritic cells, memory cells and naïve lymphocytes, by the same method, were analyzed. The results showed, both in mice and sheep, that the live, attenuated REV1 vaccine stimulated all immunocompetent cells tested, with a balanced innate and adaptive response. In the sheep experiment, the administered vaccine dose was very important because, at the lower doses, immunological tolerance tended to disappear, while, at the highest dose, the immunological tolerance remained active for a long period. In our experimental conditions, the optimal vaccine dose for sheep was 3.2 × 109 CFUs, although a good immune response was found using a dose of 1.6 × 109 CFUs. The vaccine produced in this study could be extensively employed in developing countries to control the brucellosis in sheep and goats.

Membrane vesicles mediate pro-angiogenic activity of equine adipose-derived mesenchymal stromal cells.

Multipotent mesenchymal stromal cells (MSCs) have attracted a great deal of interest, due to several distinctive features, including the ability to migrate to damaged tissue and to participate in tissue regeneration. There is increasing evidence that membrane vesicles (MVs), comprising exosomes and shedding vesicles, represent a key component, responsible for many of the paracrine effects of MSCs. The aim of the present study was to establish whether equine adipose-derived MSCs (E-AdMSCs) produce MVs that are capable of influencing angiogenesis, a key step in tissue regeneration. A morphological study was performed using MSC monolayers, prepared for transmission and scanning electron microscopy and on ultracentrifuged MSC supernatants, to identify production of MVs. The ability of MVs to influence angiogenesis was evaluated by means of the rat aortic ring and scratch assays. The results demonstrated that MVs, constitutively produced by E-AdMSCs, are involved in intercellular communication with endothelial cells, stimulating angiogenesis. Although many questions remain regarding their formation, delivery, content and mechanism of action, the present study supports the concept that MVs released by MSCs have the potential to be exploited as a therapeutic tool for regenerative medicine.

Evaluation of storage conditions on equine adipose tissue-derived multipotent mesenchymal stromal cells.

The transplantation of multipotent mesenchymal stromal cells (MSCs) is a potentially promising therapy for the treatment of tendon and ligament injuries and some forms of articular pathology in horses. This study investigated the effects of storage conditions on MSCs. Equine adipose tissue-derived MSCs (eAd-MSCs) were stored at 4 °C and at room temperature (RT) for 24 and 48 h, and viability, doubling time, expression of CD44 and CD90 antigens, clonogenic/differentiation potentials, and karyotype were subsequently evaluated. The eAd-MSC viability was significantly affected by the storage conditions, while doubling time was not significantly altered. The findings indicate (1) that storage at 4 °C is preferable to RT as this results in greater numbers of viable, morphologically normal cells, and (2) that cells should be used within 24 h.