Assessment of the acid phosphatase CP01850 from Corynebacterium pseudotuberculosis in DNA and subunit vaccine formulations against caseous lymphadenitis / Avaliação da fosfatase ácida recombinante CP01850 de Corynebacterium pseudotuberculosis em formulações vacinais de subunidade e de DNA contra a linfadenite caseosa

The target cp1002_RS01850 from Corynebacterium pseudotuberculosis was used to construct a DNA and recombinant subunit vaccine against caseous lymphadenitis. Recombinant protein rCP01850 was expressed in Escherichia coli using pAE vector, and DNA vaccine was engineered with pTARGET vector. BALB/c mice were divided in five groups containing eight animals each, inoculated with: pTARGET/cp01850 as DNA vaccine (G1); rCP01850 plus Al (OH)3 as recombinant subunit vaccine (G2); pTARGET/cp01850 and a boost with rCP01850 plus Al (OH)3 (G3); pTARGET (G4); or Al (OH)3 (G5). Mice were inoculated and blood samples were collected on days 0, 21, and 42 for the analysis of total IgG, IgG1 and IgG2a by ELISA. In each group, five animals were challenged with Mic-6 C. pseudotuberculosis strain, and three were used for cytokine quantification by qPCR. Although no group has been protected by vaccines against lethal challenge, G2 showed an increase in the survival rate after challenge. Significantly higher levels of IL-4, IL-12, IFN-γ, total IgG, IgG1 and IgG2a were also detected for G2, evidencing a mixed Th1/Th2 immunological profile. In conclusion, despite no protection level provided by different vaccinal strategies using cp1002_RS01850 from C. pseudotuberculosis, G2 developed a Th1/Th2 immune response with an increase in survival rate.(AU)

O alvo cp1002_RS01850 de Corynebacterium pseudotuberculosis foi utilizado para construir uma vacina recombinante de subunidade e de DNA contra a linfadenite caseosa. A proteína recombinante rCP01850 foi expressa em Escherichia coli usando o vetor pAE, e a vacina de DNA foi construída com o vetor pTARGET. Camundongos BALB/c foram divididos em grupos de oito animais, inoculados com: pTARGET/cp01850 como vacina de DNA (G1); rCP01850 e Al (OH)3 como vacina recombinante de subunidade (G2); pTARGET/cp01850 e um boost com rCP01850 e Al (OH)3 (G3); pTARGET (G4); ou Al (OH)3 (G5). Os animais foram inoculados e amostras de sangue foram coletadas nos dias 0, 21, e 42 do experimento para a análise de IgG total, IgG1 e IgG2a por ELISA. De cada grupo, cinco animais foram desafiados com a cepa Mic-6 de C. pseudotuberculosis, e três foram usados para a quantificação de citocinas por qPCR. Apesar de nenhum grupo ter sido protegido pelas vacinas testadas contra o desafio letal, G2 apresentou taxa de sobrevida e níveis de IL-4, IL-12, IFN-γ, IgG total, IgG1 e IgG2a significativamente mais altos, evidenciando um perfil imunológico misto Th1/Th2. Conclui-se que apesar das diferentes estratégias vacinais utilizando cp1002_RS01850 de C. pseudotuberculosis não terem sido capazes de gerar proteção, G2 desenvolveu uma resposta Th1/Th2 e elevou a taxa de sobrevida.(AU)

KGF Enhances Oral Epithelial Adhesion and Rete Peg Elongation via Integrins.

Oral epithelial adhesion to the lamina propria underlies the physiologic function of the oral mucosa and contributes to resisting bacterial invasion, preventing body fluid loss, and maintaining routine chewing; thus, understanding the factors that positively influence oral epithelial adhesion is a research topic of great interest. Rete pegs contribute to oral epithelial adhesion by enlarging the contact areas, whereas integrins are the major molecules that mediate epithelial cell adhesion to the basement membrane. Keratinocyte growth factor (KGF) can promote both rete peg elongation in the skin and the expression of integrins in various cell types. Herein, we tested the effects of submucosal injection of KGF in the ventral surfaces of rat tongues on oral epithelial adhesion. The data confirmed that topical injection of KGF elevated the adhesive forces, elongated the rete pegs, and increased the abundance of integrins, KGF, and KGF receptor on the rat tongue ventral surface. However, HYD-1 (Lys-Ile-Lys-Met-Val-Ile-Ser-Trp-Lys-Gly), an integrin antagonist, inhibited the KGF-enhanced epithelial adhesion and rete peg elongation. Moreover, KGF promoted the expression of integrin subunits α6, ß4, α3, and ß1 in human immortalized oral epithelial cells in 2- and 3-dimensional culture systems. In vitro cell attachment assays demonstrated that HYD-1 inhibited the adhesion of human immortalized oral epithelial cells on Matrigel. Strikingly, the expression of integrins, KGF, and KGFR in human masticatory mucosae with longer rete pegs was more abundant than that in the lining mucosae with shorter rete pegs. In addition, rete peg lengths were positively correlated with the expression levels of integrins, KGF, and KGF receptor. These findings indicate that KGF strengthens oral epithelial adhesion and rete peg elongation via integrins.