RESUMO O presente trabalho avaliou o papel do baço no armazenamento e na reativação das linhagens de células B, representadas por células IgM positivas imunomarcadas no tecido esplênico, bem como a funcionalidade dessas células, sobre a cinética dos linfócitos e na produção sistêmica de anticorpos em tilápias-do-nilo (Oreochromis niloticus). Foram separados dois grupos: grupo memória, constituído por peixes previamente imunizados com hemácia de carneiro a 2,5%, para a geração da memória imune, e o grupo naive, que recebeu o mesmo volume de solução salina a 0,65%. Após 32 dias, os dois grupos foram submetidos a uma nova dose do antígeno na mesma concentração, volume e via de inoculação. A reativação dos clones de memória foi evidenciada pelo aumento do número de células IgM positivas no baço do grupo memória no dia zero/pré-imune. Além disso, o mesmo grupo apresentou aumento dos títulos de anticorpos séricos no 14º dia e no número absoluto de linfócitos no 21º dia em relação ao grupo naive. Esses resultados sugerem que o baço não seja apenas um local de armazenamento, mas também de reativação de células B de memória em tilápia-do-nilo.

ABSTRACT This work aimed to evaluate the role of the spleen in storage and reactivation of the memory B cells, represented by IgM positive cells and the systemic production of sheep antibodies anti-red cell in Nile tilapia (Oreochromis niloticus). Two groups were established: the memory group, containing fish previously immunized with a 2,5% sheep anti-red cell, to generate the immune memory; and the naive group, containing fish that received a 0,65% saline solution. After 32 days, both groups were subjected to a new dose of the same antigen at the same concentration, volume, and inoculation via. The memory clones reactivation was correlated to the increase of the IgM positive cells in the spleen in the memory group at 0 day. The memory group showed an increase in the absolute number of lymphocytes at 21 days and an increase in the antibodies at 14 days after inoculation when compared to the naive group. The results suggest that the spleen may be a storage and reactivation place of memory B cells in Nile tilapia.

O efeito da idade da matriz sobre o desenvolvimento dos ossos longos foi estudado em embriões de frango de 12 dias de incubação até o nascimento. Ovos férteis foram incubados e distribuídos em delineamento inteiramente casualizado em arranjo fatorial 2 x 6 (duas idades de matriz - 38 e 60 semanas e seis dias de incubação - 12, 14, 16, 18, 20 e 21 dias). Determinou-se a atividade enzimática das fosfatase alcalina e ácida-resistente ao tartrato no peso e nas epífises da tíbia, no comprimento e na largura da tíbia e do fêmur. A atividade das fosfatases não foi afetada pela idade da matriz. O peso absoluto e a largura de fêmur e tíbia foram maiores nos embriões das matrizes com 60 semanas de idade. Atividade enzimática e medidas morfométricas aumentaram com o dia de incubação independentemente da idade da matriz. Concluiu-se que o processo de ossificação endocondral durante os dois últimos terços de desenvolvimento embrionário não foi influenciado pela idade das matrizes. No entanto, em termos de peso absoluto, os ossos longos de embriões provenientes de matrizes velhas foram mais pesados o que foi associado à maior largura e não ao maior comprimento dos ossos.

The effect of breeder age on long bone development was studied in chicken embryos from 12 days of incubation until hatching. Fertile eggs were incubated and randomly assigned in a 2 x 6 factorial arrangement (two breeder ages - 38 and 60 weeks and six incubation days - 12, 14, 16, 18, 20, and 21). Enzymatic activity of acid and alkaline phosphatases in tibial epiphyses and weights as well as length and width in tibias and femurs of the embryos were determined. Tartrate-resistant acid and alkaline phosphatases activity in epiphyses was not affected by breeder age. Absolute weight and width of femur and tibia were larger in 60-week-old embryos compared to 38-week-old. Enzymatic activity and morphometric measurements increased with incubation day, independently of breeder age. The results showed that the process of endochondral ossification during the last two thirds of embryo development was not influenced by the age of the breeders. Although, in terms of absolute weight, the long bones of embryos from older breeders were heavier, which was associated with the larger width of the bones, but and not with their length.

Endochondral calcification involves the participation of matrix vesicles (MVs), but it remains unclear whether calcification ectopically induced by implants of demineralized bone matrix also proceeds via MVs. Ectopic bone formation was induced by implanting rat demineralized diaphyseal bone matrix into the dorsal subcutaneous tissue of Wistar rats and was examined histologically and biochemically. Budding of MVs from chondrocytes was observed to serve as nucleation sites for mineralization during induced ectopic osteogenesis, presenting a diameter with Gaussian distribution with a median of 306 ± 103 nm. While the role of tissue-nonspecific alkaline phosphatase (TNAP) during mineralization involves hydrolysis of inorganic pyrophosphate (PPi), it is unclear how the microenvironment of MV may affect the ability of TNAP to hydrolyze the variety of substrates present at sites of mineralization. We show that the implants contain high levels of TNAP capable of hydrolyzing p-nitrophenylphosphate (pNPP), ATP and PPi. The catalytic properties of glycosyl phosphatidylinositol-anchored, polidocanol-solubilized and phosphatidylinositol-specific phospholipase C-released TNAP were compared using pNPP, ATP and PPi as substrates. While the enzymatic efficiency (k cat/Km) remained comparable between polidocanol-solubilized and membrane-bound TNAP for all three substrates, the k cat/Km for the phosphatidylinositol-specific phospholipase C-solubilized enzyme increased approximately 108-, 56-, and 556-fold for pNPP, ATP and PPi, respectively, compared to the membrane-bound enzyme. Our data are consistent with the involvement of MVs during ectopic calcification and also suggest that the location of TNAP on the membrane of MVs may play a role in determining substrate selectivity in this micro-compartment.