Laurelúcia Orive Lunardi

Cytochemical characterization of eosinophilic leukocytes circulating in the blood of the turtle (Chrysemys dorbignih).

Eosinophils and neutrophils are granulocytic leukocytes that are present in the blood of most vertebrates. Studies have been performed on lower vertebrates to understand the biological roles of the cells in defense mechanisms and to establish phylogenetic studies and new experimental models. Whether these 2 cell types exist in reptiles is a matter of controversy. In the blood of turtles there are 2 types of granulocytes that exhibit eosinophilia, one of them with round cytoplasmic granules and the other with elongated cytoplasmic granules. It has been suggested that these cells may be eosinophils in different stages of maturation but they also may be distinct cell types, i.e. eosinophils and neutrophils. In the present study, we characterized the 2 types of granulocytes that are present in the blood of Chrysemys dorbignih, using cytochemical techniques. Type I eosinophils showed activity of nonspecific esterase, peroxidase activity that is resistant to KCN, and basic proteins. Type II eosinophils exhibited activity of trimetaphosphatase, alkaline phosphatase, nonspecific esterase, peroxidase that is sensitive to KCN, and basic proteins. These observations indicate the existence of 2 distinct cell types in the blood of Chrysemys dorbignih, type I and type II eosinophils, that correspond to eosinophils and heterophils (neutrophils) of mammals and other vertebrates.

Tumor Necrosis Factor Alpha Immunoreactivity of Rat Peritoneal Mast Cell Granules Decreases during Early Secretion Induced by Compound 48/80: An Ultrastructural Immunogold Morphometric Analysis

We used fast (seconds) and ultrafast (milliseconds) microwave energy-assisted chemical fixation protocols, postembedding immunogold staining, and a morphometric analysis to investigate the early morphological changes and the TNF-alpha immunoreactivity in the cytoplasmic granules of rat peritoneal mast cells that had been stimulated to secrete by exposure to compound 48/80. Exposure to compound 48/80 induced the development of increased numbers of cytoplasmic granules that exhibited decreased electron density; these granules often also appeared swollen. These granule alterations were accompanied by a significantly decreased proportion of granules that were positive for TNF-alpha immunoreactivity. We also calculated the density of TNF-alpha labeling/mu 2 in both dense (unaltered) and altered granules in specimens. TNF-alpha immunoreactivity was present in dense granules (regardless of whether or not the specimens had been stimulated with compound 48/80) and in cells that were fixed with either fast or ultrafast microwave energy. However, altered granules exhibited a decreased density of TNF-alpha label. These findings show that changes in the immunolocalization and/or density of TNF-alpha immunoreactivity occur very rapidly upon stimulation of rat peritoneal mast cells with compound 48/80.

Phagocytosis of PLGA Microparticles in Rat Peritoneal Exudate Cells: A Time-Dependent Study

With the purpose of enhancing the efficacy of microparticle-encapsulated therapeutic agents, in this study we evaluated the phagocytic ability of rat peritoneal exudate cells and the preferential location of poly(d,l-lactide-co-glycolic acid) (PLGA) microparticles inside these cells. The microparticles used were produced by a solvent evaporation method and were characterized by dynamic light scattering (DLS), transmission electron microscopy (TEM), and scanning electron microscopy (SEM). Size distribution analysis using DLS and SEM showed that the particles were spherical, with diameters falling between 0.5 and 1.5 mum. Results from cell adhesion by SEM assay, indicated that the PLGA microparticles are not toxic to cells and do not cause any distinct damage to them as confirmed by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. Among the large variety of cell populations found in the peritoneal exudates (neutrophils, eosinophils, monocytes, and macrophages), TEM showed that only the latter phagocytosed PLGA microparticles, in a time-dependent manner. The results obtained indicate that the microparticles studied show merits as possible carriers of drugs for intracellular delivery.

Morphological Characterization of the Leukocytes in Circulating Blood of the Turtle (Phrynops hilarii)

La especie de tortuga Phrynops hilarii no ha sido aun bien descrita en la literatura. Fue propuesto en este estudio la caracterizacion de leucocitos de sangre de este animal coloreados con el metodo de Leishman y analizados con microscopias de luz y electronica. No fue observado ningun tipo celular con caracteristicas similares a los neutrofilos de mamiferos. Los resultados indican que los heterofilos tienen analogia morfofuncional con otros neutrofilos presentes en el grupo de los mamiferos. Esta conclusion es sustentada por varios estudios recientes encontrados en la literatura

IMMUNOCYTOCHEMICAL LOCALIZATION OF CHYMASE TO CYTOPLASMIC VESICLES AFTER RAT PERITONEAL MAST CELL STIMULATION BY COMPOUND 48/80

The subcellular events responsible for release of mediators by mast cells may help to clarify roles for mast cells in health and disease. In this study we show that the granule-associated protease chymase is also within cytoplasmic vesicles in appropriately stimulated rat peritoneal mast cells. Rat peritoneal mast cells were recovered before or 1–10 sec after exposure to the secretogogue compound 48/80 (10 μg/ml) and then were examined by radioimmunoassay to quantify histamine release or were processed, using routine methods for postembedding immunoelectron microscopy, to identify the subcellular localization of chymase. In comparison to unstimulated cells, compound 48/80 stimulated cells in two independent experiments showed an increase (15%, 28%) in the surface area of the cell and a decrease (12%, 6%) in the surface area of the total granule compartment before degranulation channel formation. These global cellular changes occurred in a background of transient but significant (p<0.01) increases in the area and number of chymase-immunoreactive vesicles per μ2 cytoplasm. These changes were detectable at 5 or 7 sec after stimulation with compound 48/80 but returned to near prestimulation levels by 9 or 10 sec after addition of compound 48/80 (total cumulative histamine release was 28% by 8 sec and 47% by 14 sec). These observations suggest that vesicles participate in the early stages of regulated secretion of chymase from rat peritoneal mast cells.

Immunolocalization of myosin Va in the developing nervous system of embryonic chicks

Myosins are molecular motors associated with the actin cytoskeleton that participate in the mechanisms of cellular motility. During the development of the nervous system, migration of nerve cells to specific sites, extension of growth cones, and axonal transport are dramatic manifestations of cellular motility. We demonstrate, via immunoblots, the expression of myosin Va during early stages of embryonic development in chicks, extending from the blastocyst period to the beginning of the fetal period. The expression of myosin Va in specific regions and cellular structures of the nervous system during these early stages was determined by immunocytochemistry using a polyclonal antibody. Whole mounts of chick embryos at 24–30-h stages showed intense immunoreactivity of the neural tube in formation along its full extent. Cross-sections at these stages of development showed strong labeling in neuroepithelial cells at the basal and apical regions of the neural tube wall. Embryos at more advanced periods of development (48h and 72 h) showed distinctive immunolabeling of neuroepithelial cells, neuroblasts and their cytoplasmic extensions in the mantle layer of the stratified neural tube wall, and neuroblasts and their cytoplasmic extensions in the internal wall of the optic cup, as well as a striking labeling of cells in the apparent nuclei of cranial nerves and budding fibers. These immunolocalization studies indicate temporal and site-specific expression of myosin Va during chick embryo development, suggesting that myosin Va expression is related to recruitment for specific cellular tasks.

Decalcification Dynamic of Dog Mineralized Tissue by Microwaves

El objetivo de este articulo fue presentar, un metodo de descalcificacion dinamico de tejido mineralizado de perros, dientes y mandibula, comparando el metodo de descalcificacion tradicional e irradiacion en horno de microondas, analizando la liberacion de calcio en espectro fotometro de absorcion atomica. Usamos como agente descalcificante, solucion de EDTA y acido nitrico. Los resultados mostraron que los fragmentos descalcificados con acido nitrico despues de 15 dias, ya podian ser preparados para analisis histologico, el diente al ser irradiado tardo 65 dias para ser descalcificado. El EDTA descalcifico lentamente, en relacion al acido nitrico. Las observaciones histologicas de las muestras irradiadas mostraron una excelente preservacion de las caracteristicas morfologicas independiente del agente descacificador usado

Immune Cells Depletion During Wound Healing as a Long-Term Effect of Undernutrition

La desnutricion postnatal esta asociada a un conjunto de secuelas agudas y cronicas, y su recuperacion es aun asunto controvertido. En Brasil, a pesar de estar disminuyendo la desnutricion, algunos estudios han demostrado que alrededor del 31% de los ninos presentan malnutricion moderada o severa. El objetivo del presente estudio fue inducir una desnutricion precoz en ratas y observar los efectos inmediatos (desnutricion) y permanentes (depues de la recuperacion) sobre las celulas de defensa involucradas en la cicatrizacion. La desnutricion fue provocada separando las crias de sus madres, por 10 horas diarias, durante el periodo de lactancia (21 dias de nacidos). Como contoles fueron usadas ratas de la misma edad no sometidas a restricion lactea. Los estados de desnutricion y de recuperacion fueron evaluados por el peso corporal. Las heridas fueron realizadas en la piel rasurada del dorso de los animales desnutridos, recuperados y controles, bajo anestesia con tribromoetanol. Los animales fueron sacrificados 1, 3, 7 y 14 dias despues de la cirugia y los tejidos de la region cicatricial fueron procesados histologicamente y examinados al microscopio optico. Los resultados mostraron que: 1) el numero de neutrofilos, linfocitos y macrofagos en la area de cicatrizacion, fue menor en los animales desnutridos y en recuperacion que en los controles; 2) enlos animales recuperados hubo una deficiencia basal en el numero de linfocitos y macrofagos, lo que no ocurrio en los animales con desnutricion aguda. Estos resultados permitieron concluir que los animales sometidos a desnutricion postnatal mostraron una recuperacion completa en el peso fisico despues del periodo de recuperacion nutricional, pero que la region de cicatrizacion mostro menor densidad de celulas de defensa, sugiriendo secuelas permanentes de la desnutricion postnatal

Characterization of Blood Mononuclear Phagocytes in Phrynops hilarii (Chelonia Chelidae)

La localization de la actividad de la peroxidasa en diversas regiones de la celula se utiliza como criterio para la clasificacion de la etapa de maduracion de fagocitos mononucleares. Una reaccion positiva de peroxidasa indica la presencia de monoblastos, promonocitos, monocitos y macrofagos. En este estudio fue evaluada la actividad de la peroxidasa de los fagocitos mononucleares de la sangre de la tortuga Phrynops Hilarii detectada en diversas etapas de la diferenciacion. Las actuales observaciones sugieren que, en tortugas, la diferenciacion de fagocitos mononucleares ocurre en la circulacion de la sangre, en contraste a los mamiferos, donde estan solamente los monocitos en la sangre circulante y la diferenciacion de los macrofagos ocurre en otras partes del cuerpo

Microwave Fixation in Rat Fetuses Tissues: Histological and Immunohistochemical Analysis

El objetivo de este estudio fue evaluar la accion de las microondas en la fijacion de los tejidos dermico y cartilaginoso de fetos de ratas, usando para el analisis metodos histologico e inmunohistoquimico. Fue posible concluir en este estudio usando la rata como modelo experimental, que los dos metodos empleados para la recuperacion antigenica representan excelentes medios para el uso del anticuerpo Ki67, en el analisis inmunohistoquimico