Special dyeing, histochemistry, immunohistochemistry and ultrastructure: A study of mast cells/eosinophilic granules cells (MCs/EGC) from Centropomus parallelus intestine.

Intestine mast cells/eosinophilic granule cells (MCs/EGC) of the marine species Centropomus parallelus (fat snook) were first studied using light and electron microscopy techniques. Mast cells are cells from the connective tissue found in almost all organs and tissues of vertebrates. In fish, they appear in greater numbers in parts of their bodies that are exposed to their environment, such as skin, gills and intestine. The granules in fat snook's mast cell contain a variety of substances, such as histamine, heparin, chondroitin sulfate, serotonin, proteases and cytokines. The present study of intestine MCs/EGC was carried out in 20 specimens of fat snook. Samples of tissue were fixed in Bouin solution and in buffered formalin. Ferric hematoxylin - Congo red, pH6 acridine orange, pH2.5 and pH0,5 Alcian Blue (AB), toluidine blue, PAS, AB + PAS and immunohistochemistry protocols were used. In the mucosa and submucosa layers, MCs/EGCs granules with basic contents were evidenced by Congo red staining, and with acid contents granules were identified through pH 2.5 and 0,5 AB, and acridine orange. Basic and acid contents were simultaneously evidenced using ferric hematoxylin - Congo red stain. Metachromasia was observed in both mucosal and submucosal mast cells. Neutral glycoproteins were evidenced by using PAS protocol, glycosaminoglycan through AB and both simultaneously through AB + PAS. In immunohistochemistry assays, MCs/EGC were positive for tryptase, chymase and serotonin. As in mammals, the study of samples fixed in modified Karnovsky for transmission electron microscopy evidenced that most of the MCs granules were spherical and showed varying electron density, as described in previous reports on other teleost fish species. The metachromasia observed and the identification of tryptase, chymase and serotonin suggest a great similarity between fat snook's MCs/EGC and those described in the mucosa of mammals.

Special dyeing, histochemistry, immunohistochemistry and ultrastructure: a study of mast cells/eosinophilic granules cells (MCs/EGC) from centropomus parallelus intestine

Intestine mast cells/eosinophilic granule cells (MCs/EGC) of the marine species Centropomus parallelus (fat snook) were first studied using light and electron microscopy techniques. Mast cells are cells from the connective tissue found in almost all organs and tissues of vertebrates. In fish, they appear in greater numbers in parts of their bodies that are exposed to their environment, such as skin, gills and intestine. The granules in fat snook's mast cell contain a variety of substances, such as histamine, heparin, chondroitin sulfate, serotonin, proteases and cytokines. The present study of intestine MCs/EGC was carried out in 20 specimens of fat snook. Samples of tissue were fixed in Bouin solution and in buffered formalin. Ferric hematoxylin - Congo red, pH6 acridine orange, pH2.5 and pH0,5 Alcian Blue (AB), toluidine blue, PAS, AB + PAS and immunohistochemistry protocols were used. In the mucosa and submucosa layers, MCs/EGCs granules with basic contents were evidenced by Congo red staining, and with acid contents granules were identified through pH 2.5 and 0,5 AB, and acridine orange. Basic and acid contents were simultaneously evidenced using ferric hematoxylin - Congo red stain. Metachromasia was observed in both mucosal and submucosal mast cells. Neutral glycoproteins were evidenced by using PAS protocol, glycosaminoglycan through AB and both simultaneously through AB + PAS. In immunohistochemistry assays, MCs/EGC were positive for tryptase, chymase and serotonin. As in mammals, the study of samples fixed in modified Karnovsky for transmission electron microscopy evidenced that most of the MCs granules were spherical and showed varying electron density, as described in previous reports on other teleost fish species. The metachromasia observed and the identification of tryptase, chymase and serotonin suggest a great similarity between fat snook's MCs/EGC and those described in the mucosa of mammals.

Amifostine Increases FAS and Caspase-3 Expression in Colonic Tissue of Irradiated Mice.

BACKGROUND/AIM: The aim of this study was to evaluate the expression of FASL, FAS and FADD and caspase-3 in oesophagus, stomach and colonic tissues of mice irradiated in vivo by immunohistochemistry. MATERIALS AND METHODS: A total of 48 adult male C57BL mice were distributed into four groups: Ami(-)/Rad(-): Mice received 0.5 ml of 0.9% physiological saline solution (PPS) intraperitioneally (i.p.); Ami(+)/Rad(-): mice received amifostine (400mg/kg i.p.) freshly dissolved in double-distilled water; Ami(-)/Rad(+): mice received 0.5 ml of PSS i.p. 30 min before a single whole-body radiation dose of 7 Gy; Ami(+)/Rad(+): mice received 0.5 ml of an aqueous solution of 400 mg/kg amifostine i.p.30 min prior to irradiation. All groups were assigned into subgroups sacrificed at 0.5 h, 1 h, 2 h and 4 h after irradiation. RESULTS: In oesophagus and stomach tissues, we did not observe any difference between Ami(-)/ad(-), Ami(+)/Rad(-), Ami(-)/Rad(+) and Ami(+)/Rad(+) groups in the expression of FASL, FAS and FADD. The colonic tissue was the only to exhibit any difference in the expression of FAS and caspase-3 protein in the Ami(-)/Rad(+)group at 1 and 2 h. Amifostine increased FAS and caspase-3 immunoexpression when compared to the control. Immunoexpression for FASL and FADD was not remarkably different in colonic tissue. CONCLUSION: Taken together, our results demonstrate that amifostine increases FAS and caspase-3 expression in colonic tissue of irradiated mice.

Functional assessment of toad parotoid macroglands: a study based on poison replacement after mechanical compression.

Toads have a pair of parotoid macroglands behind the eyes that secrete poison used in passive defence against predators. These macroglands are composed of juxtaposed alveoli, each one bearing a syncytial gland, all connected to the exterior by ducts. When the parotoids are bitten, the poison is expelled on the predator oral mucosa in the form of jets, causing several pharmacological actions. After poison release, the empty secretory syncytia immediately collapse in the interior of their respective alveoli and gradually start refilling. After parotoid manual compression, simulating a predator's bite, we studied, by means of morphological methods, the replacement of the poison inside the alveoli. The results showed that after compression, a considerable number of alveoli remained intact. In the alveoli that were effectively affected the recovery occurs in different levels, from total to punctual and often restrict to some areas of the syncytia. The severely affected alveoli seem not recover their original functional state. The fact that only a part of the parotoid alveoli is compressed during an attack seems to be crucial for toad survival, since the amphibian, after being bitten by a predator, do not lose all its poison stock, remaining protected in case of new attacks.

Hematological parameters and phagocytic activity in fat snook (Centropomus parallelus) bred in captivity.

The objective of this work was to determine the hematological parameters and the phagocytic capacity of peritoneal macrophages of fat snook related to sex, stage of gonadal maturation and seasonal cycle. Blood was collected from 135 animals (78 females and 57 males) and used for determinations of: erythrocyte number, hematocrit, hemoglobin, erythrocyte indices mean corpuscular volume (MCV) and mean corpuscular hemoglobin concentration (MCHC), total and differential leukocyte counts, and thrombocyte count. The phagocytic capacity and phagocytic index were determined after Saccharomyces cerevisiae inoculation in the peritoneal cavity of the animals. The hematological results according to sex showed that the erythrocyte, total leukocyte and thrombocyte counts were statistically higher in males than females, with the latter showing a higher MCV. Concerning to erythrocyte count, hematocrit and hemoglobin concentration analyzed separately by sex and stage of gonadal maturation, males were found to have significantly elevated values in the mature stage and decreased levels in the resting stage. The results of the erythrocyte and leukocyte series, thrombocytes and phagocytic activity related to seasonal cycle showed significant differences in both sexes, where hematocrit and hemoglobin concentration were lower in winter and higher in the other seasons, mean corpuscular volume was higher in the summer and lower in the winter and fall, total leukocytes and thrombocytes lower in the spring and higher in the fall, lymphocytes low in the winter and summer and high in the spring and phagocytic capacity and phagocytic index high in the summer and low in the winter and fall. The results showed that the hematological values in males are statistically higher than those in females, the erythrocyte values in males increase with the progression of gonadal maturation and that winter is the season of the year least favorable for hematological and phagocytic responses for survival of fat snook kept in captivity. The parameters studied could be utilized in the evaluation of the health status of this species in captivity.

Cytochemical, immunocytochemical and ultrastructural observations on leukocytes and thrombocytes of fat snook (Centropomus parallelus).

The cytochemical, immunocytochemical and ultrastructural characteristics of leukocytes and thrombocytes in the peripheral blood of the fat snook (Centropomus paralellus) - a fish occurring in Brazil - were investigated. The cytochemical methods were performed to demonstrate four enzymatic reactions - o-toluidine-hydrogen peroxide, naphtol AS-MX phosphate, naphtol AS-BI phosphate and alpha-naphtil acetate to detect myeloperoxidase (MPO), alkaline phosphatase (ALP), acid phosphatase (ACP) and non-specific esterase (α-NAE), respectively - and two non-enzymatic ones - Periodic-Acid Schiff (PAS) and Sudan black B (SBB) to detect the occurrence of glycogen and phospholipids, respectively. Immunocytochemical method utilizing polyclonal rabbit antibody against mammal metalloproteinases (MMPs) 2 and 9 were done. Standard method for Electron Microscopy (EM) was applied for the ultrastructural study. The cytochemical reactions were positive in neutrophils for MPO, ACP, α-NAE, glycogen and phospholipids; in lymphocytes for ACP and α-NAE; in monocytes for ACP and α-NAE and in thrombocytes for ACP, α-NAE and glycogen. Only neutrophils were positive for MMPs 2 and 9, and none of the cells studied were positive for ALP. Ultrastructurally: 1) neutrophil showed a spherical shape with a spherical, indented or lobulated euchromatic nucleus, and cytoplasm containing granules of varied sizes and mitochondria of varied shapes and sizes. The nucleus/cytoplasm relation and the size of granules suggest neutrophil maturation in peripheral blood; 2) lymphocytes showed partially heterochromatic nucleus and minimal cytoplasm; 3) monocytes had long cytoplasmic projections, an indented nucleus, evident nucleolus and cytoplasm with granules of varied sizes and vacuoles; 4) thrombocytes were predominantly elliptical or roughly spherical in shape, had a partially heterochromatic nucleus and cytoplasm containing electron-dense granules, intricate canalicular system and vacuoles occasionally holding phagocytic material.

Cytochemical, immunocytochemical and ultrastructural observations on leukocytes and thrombocytes of fat snook (Centropomus parallelus)

The cytochemical, immunocytochemical and ultrastructural characteristics of leukocytes and thrombocytesin the peripheral blood of the fat snook (Centropomus paralellus) e a fish occurring in Brazil e wereinvestigated. The cytochemical methods were performed to demonstrate four enzymatic reactions e o-toluidine-hydrogen peroxide, naphtol AS-MX phosphate, naphtol AS-BI phosphate and alpha-naphtil acetate to detect myeloperoxidase (MPO), alkaline phosphatase (ALP), acid phosphatase (ACP) and non-specific esterase (a-NAE), respectively e and two non-enzymatic ones e Periodic-Acid Schiff (PAS) and Sudan black B (SBB) to detect the occurrence of glycogen and phospholipids, respectively. Immunocytochemical method utilizing polyclonal rabbit antibody against mammal metalloproteinases (MMPs) 2 and 9 were done. Standard method for Electron Microscopy (EM) was applied for the ultrastructuralstudy.

Phosphorylation and cytoplasmic localization of MAPK p38 during apoptosis signaling in bone marrow granulocytes of mice irradiated in vivo and the role of amifostine in reducing these effects.

We studied p38 phosphorylation and its intracellular localization during p53 and Puma (a p53 upregulated modulator of apoptosis) apoptotic signaling pathway in bone marrow granulocytes in mice irradiated in vivo and the role of the radioprotector amifostine in ameliorating these responses. Sixty-four C57BL mice were randomly assigned in two non-irradiated (Ami-/rad- and Ami+/rad-) and two irradiated (Ami-/rad+ and Ami+/rad+) groups. Animals received 400mg/kg of amifostine i.p. 30 min prior to a single whole body radiation dose of 7Gy. The experiments were performed using immunohistochemistry for caspase-3, cleaved caspase-3, p53, p-p53 (Ser 15), Puma, p38 and p-p38 (Thr 180/Tyr 182) protein expression. In addition transmission electron microscopy was used for ultrastructural characterization of apoptosis. Data showed that: (i) amifostine significantly reduced the number of apoptotic cells, (ii) p-p53 and Puma proteins were strongly immunostained in granulocytes after irradiation (Ami-/rad+), (iii) amifostine decreased the immunostaining of the proteins (Ami+/rad+), (iv) p38 was immunolocalized in physiological conditions in the nucleus and cytoplasm of granulocytes and neither radiation nor amifostine changed the protein immunostaining or its subcellular distribution, but influenced its activation, (v) radiation-induced p38 phosphorylation and its cytoplasmic accumulation during apoptosis signaling in granulocytes after whole body high radiation dose and amifostine markedly reduced these effects.

Cytochemistry and morphology of granulocytes of the caecilian Siphonops annulatus (Amphibia, Gymnophiona)

The caecilians (Amphibia, Gymnophiona) constitute one of the least known groups of terrestrial vertebrates because most species live underground in quite inaccessible environments. Siphonops annulatus is an exclusively fossorial species and is the most extensively distributed caecilian in South America. Little is known of this order concerning circulating granulocytes, including their morphological and cytochemical structure and ultrastructure. This paper is part of a project covering the study of granulocytes in representative species of the order Amphibia. Blood extensions were carried out and submitted to Leishman, Toluidine Blue, Periodic acid Schiff, Sirius Red and hydrogen o-toluidine peroxide methods. Part of the samples was prepared for conventional transmission electron microscopy. Among granular leukocytes, mature and immature neutrophils and eosinophils were identified, plus basophils. The most frequent granulocyte encountered in S. annulatus peripheral blood is the neutrophil. This is a cell with a hyper-segmented nucleus and with a very clear cytoplasm when compared to the eosinophil, which presents large cytoplasmic acidophilic granules. On the other hand, the basophils present basophilic and metachromatic granules. Glycogen was detected in the cytoplasm of the neutrophils and eosinophils, while basic protein rich in amino acids was observed in the eosinophil’s granules. Myeloperoxidase activity was detected in the cytoplasm of the neutrophils and eosinophils. Neutrophils were ultrastructurally detected with three types of small granules: eosinophils with large and small spherical granules and basophils with large spherical granules with lamellate structures.

Amifostine does not prevent activation of TGFbeta1 but induces smad 7 activation in megakaryocytes irradiated in vivo.

Experiments were undertaken to assess the role of amifostine in the activation of latent TGFbeta1 and in the smad proteins cascade (smad 2/3, smad4, smad7), focusing on megakaryocytes, in the bone marrow irradiated in vivo. Non-irradiated megakaryocytes were negative for active TGFbeta1. Immunopositivity to active TGFbeta1 was detected in megakaryocytes 10 days after irradiation in amifostine- treated and untreated marrows. Smad 2/3 and smad 4 were strongly positive in the nucleus of megakaryocytes 10 days after irradiation. At the same time, a predominant hypocellular bone marrow with foci of hematopoiesis was observed with few megakaryocytes. An increase in the number of reticulin fibers was also seen. In amifostine-treated marrows, smad 2/3 and smad4 were not detected in the nucleus but were positive in the cytoplasm of megakaryocytes 10 days after irradiation. Coincidentally, bone marrows were cellular with megakaryocytes. Smad7 immunoexpression was detected in the cytoplasm of megakaryocytes in the non-irradiated, amifostine-treated and in the irradiated, amifostine-treated marrows. Data indicate that amifostine does not prevent latent TGFbeta1 activation in irradiated megakaryocytes. While TGFbeta1 signal transduction occurs in megakaryocytes in untreated bone marrows, it is inhibited in megakaryocytes in amifostine-treated marrows due to the induction of smad 7 activation. This is the first report showing smad 7 activation by amifostine. Our results also suggest a role for TGFbeta1 as an inhibitor of megakaryocytes in vivo.