Mucosubstance histochemistry in control and acid-stressed epidermis of brown bullhead catfish, Ictalurus nebulosus (LeSueur)

Glycoprotein components of epidermal mucous cells in control (pH 6.8) and acid-stressed (pH 5.8, 4.8, 4.0) brown bullhead catfish were studied by histochemical and autoradiographic methods. Following exposure to acid, increased numbers of epidermal mucous cells were noted in all tissue sites studied, particularly dorsal and ventral skin. Mucous cells of control fish and acid-stressed (pH 5.8) fish contained a mixture of neutral and acidic mucosubstances, the latter including both sialomucins and sulfomucins, with sialomucins predominating. An apparent shift toward increased sulfomucin production was observed in skin from fish exposed to pH 4.8 and 4.0. In addition, autoradiographic studies using 35S showed increased labeling of mucous cells in acid-exposed (pH 4.0) epidermis, offering further support for a shift toward sulfomucin production in acid-stressed fish. Absence of appreciable sulfomucin production in younger fish (less than 1 year old) suggested that this shift may be partly age-dependent. The functional significance of increased sulfomucin production in acid-stressed epidermal mucous cells is uncertain but may be related to maintenance of ionic equilibrium.     

Retinoblastoma in a porkfish (Anisotremus virginicus, Linnaeus) and a brown bullhead (Ictalurus nebulosus, Lesueur)

Two cases of retinoblastoma in fish are described. The neoplasms occurred in a porkfish (Anisotremus virginicus, Linnaeus) and in a brown bullhead (Ictalurus nebulosus, Lesueur).     

Characterization of immunoglobulins from the brown bullhead (Ictalurus nebulosus) produced against a naturally occurring bacterial pathogen, Aeromonas hydrophila

The primary immune response in the brown bullhead (Ictalurus nebulosus) to intramuscularly injected, chloroform-killed Aeromonas hydrophila reached an agglutination titer of 128 in 3 wk, and the response to the bacteria plus adjuvant reached a titer of 512 in 5 wk. Anti-Aeromonas hydrophila antibodies from brown bullheads were excluded from Sephadex G-200 and had an immunoelectrophoretic migration pattern toward the cathode. Cleavage of the macromolecule by 2-ME reduction and iodoacetamide alkylation yielded subunits that were resumptively heavy chains of about 50,500 daltons and light chains of about 22,500 daltons. Bullhead antibodies were assumed to be tetrameric in configuration and therefore to have an approximate molecular weight of 600,000 daltons assuming a J chain is present in both early (6 wk) and late (20 wk) immune sera. The agglutinating activity of the antibody was completely eliminated by exposure to 65 degrees C for 30 min. Immunoelectrophoresis showed a single antibody component that moved toward the cathode. The structure and molecular weight of bullhead antibodies appears similar to that observed in channel catfish and other closely related teleost fish.     

Mucosubstance histochemistry in control and acid-stressed epidermis of brown bullhead catfish,lctalurus nebulosus (LeSueur)

Glycoprotein components of epidermal mucous cells in control (pH 6.8) and acid-stressed (pH 5.8, 4.8, 4.0) brown bullhead catfish were studied by histochemical and autoradiographic methods. Following exposure to acid, increased numbers of epidermal mucous cells were noted in all tissue sites studied, particularry dorsal and ventral skin. Mucous cells of control fish and acid-stressed (pH 5.8) fish contained a mixture of neutral and acidic mucosubsf ances, the latter including both sialomucins and sulfomucins, with sialomucins predominating. An apparent shift toward increased sulfomucin production was observed in skin from fish exposed to pH 4.8 and 4.0. In addition, autoradiographic studies using ³⁵S showed increased labeling of mucous cells in acid-exposed (pH 4.0) epidermis, offering further support for a shift toward sulfomucin production in acid-stressed fish. Absence of appreciable sulfomucin production in younger fish (<1 year old) suggested that this shift may be partly age-dependent. The functional significance of increased sulfomucin production in acid-stressed epidermal mucous cells is uncertain but may be related to maintenance of ionic equilibrium.     

In situ heparin-induced peroxisomal reticulum and biogenesis of peroxisomes in pulmonary intravascular macrophages (PIMs) of caprine lung: an ultrastructural and cytochemical study.

Pulmonary intravascular macrophages (PIMs) contain a unique electron-dense globular surface-coat which is sensitive to heparin treatment, halothane anesthesia, and the digestive effect of lipolytic lipase (LPL), suggesting that the coat is predominantly composed of lipoproteins. In the present study, evidence is presented that heparin, when administered intravenously in goats, potentiated both the translocation of the surface-coat into the vacuolar system and the expansion of the Golgi apparatus. Sequentially, these changes were followed by proliferation of peroxisomes in combination with peroxisomal reticulum (PR), a transient precursor of this organelle. The peroxisomes, as well as PR, reacted positively for catalase after aldehyde fixation and 3,3'-diaminobenzidine (DAB) staining. In addition to their role as phagocytes, the ultrastructural and cytochemical detection of peroxisomes suggests a functional capacity of the PIMs, which may be adaptable to the circulating level of free fatty acids (FAAs).     

In situ heparin‐induced peroxisomal reticulum and biogenesis of peroxisomes in pulmonary intravascular macrophages (PIMs) of caprine lung: an ultrastructural and cytochemical study

Pulmonary intravascular macrophages (PIMs) contain a unique electron‐dense globular surface‐coat which is sensitive to heparin treatment, halothane anesthesia, and the digestive effect of lipolytic lipase (LPL), suggesting that the coat is predominantly composed of lipoproteins. In the present study, evidence is presented that heparin, when administered intravenously in goats, potentiated both the translocation of the surface‐coat into the vacuolar system and the expansion of the Golgi apparatus. Sequentially, these changes were followed by proliferation of peroxisomes in combination with peroxisomal reticulum (PR), a transient precursor of this organelle. The peroxisomes, as well as PR, reacted positively for catalase after aldehyde fixation and 3,3′‐diaminobenzidine (DAB) staining. In addition to their role as phagocytes, the ultrastructural and cytochemical detection of peroxisomes suggests a functional capacity of the PIMs, which may be adaptable to the circulating level of free fatty acids (FAAs). Anat Rec 266:69–80, 2002. © 2002 Wiley‐Liss, Inc.     

Epithelioid hemangioendothelioma of the liver: an ultrastructural study

Epithelioid hemangioendotheliomas are uncommon vascular tumors, mainly observed in lung and soft tissues. Liver involvement is infrequent and, in contrast to the extrahepatic localizations of the tumor, has not been subjected to detailed ultrastructural analysis. This prompted us to report the results of the ultrastructural study of three cases of epithelioid hemangioendothelioma of the liver. Neoplastic proliferation was heterogeneous. Most of neoplastic cells presented features suggestive of endothelial differentiation, including presence of Weibel-Palade bodies and evidence of vasoformative properties demonstrated by formation of both intra- and extracellular vascular channels resembling the normal structures successively observed during embryogenesis and tissue regeneration. A minor cell population, unreported so far, exhibited ultrastructural characters resembling those of pericytes and presented organoid relationship to neoplastic endothelial cells. Tumor spreading along sinusoids induced pseudopeliotic dilatations and led to a progressive disruption of normal liver architecture. In conclusion, the three cases of hepatic epithelioid hemangioendothelioma examined in this work assume many organoid features mimicking the successive steps of normal angioformation and indicative of a high degree of morphological differentiation.     

The effects of vinblastine on the secretory ameloblasts: an ultrastructural, cytochemical, and immunocytochemical study in the rat incisor

Secretory ameloblasts synthesize the organic matrix of enamel and secrete it at two distinct "putative secretory sites" characterized by membrane infoldings (Nanci and Warshawsky, 1984a). The antimicrotubular agent vinblastine sulphate interferes with secretion. We have examined the effect of this drug on the ameloblast secretory sites and re-evaluated the effect on the intracellular organization of the cell by using conditions that optimize fixation, cytochemistry (ZI0), and immunocytochemistry. Associated with the disappearance of secretory granules and Golgi-related structures from Tomes' process was the loss of membrane infoldings at secretory sites. The Golgi apparatus appeared fragmented and numerous granule clusters were found throughout the cell body. These clusters were often seen in relation to extracellular patches of material in which no crystallites were seen. Immunocytochemistry revealed the presence of enamel proteins in the protein synthetic organelles, including various granule types, in lysosomes and in the extracellular patches. These data suggest that ameloblasts under the effect of vinblastine carry on secretory activities, but the product is not routed to the usual sites. It was confirmed that membrane infoldings characterize the sites where enamel proteins are normally secreted.     

Effects of phorbol ester on Leishmania mexicana amazonensis: an ultrastructural and cytochemical study

Promastigotes and amastigotes of Leishmania mexicana amazonensis, incubated in the presence of 20 ng/ml of 12-O-tetradecanoyl phorbol-13-acetate (TPA), an exogenous protein kinase C activator, developed several membrane and cytoplasmic alterations. Increased exocytic activity was observed especially in the amastigotes which had an enlarged flagellar pocket. Treatment with TPA induced protrusions of the plasma membrane where cytoplasmic elements (ribosomes and sub-pellicular microtubules) were not seen. Freeze-fracture replicas of TPA-treated parasites showed reduction in the density of the intramembranous particles (IMP), which were not seen on either fracture face of the membrane lining the protrusion. Cytochemical observations showed that sterols and anionic sites which bind to filipin and cationized ferritin particles, respectively, can be detected in the membrane lining the protrusions. However, the pattern of distribution of anionic sites, which bind colloidal iron hydroxide particles, and acid phosphatase in the membrane lining the protrusion region differed from the other portions of the plasma membrane.     

Demyelination-induced plasticity in the axon membrane: an ultrastructural cytochemical study of lead neuropathy in the rat

We examined the distribution of ferric ion-ferrocyanide stain (a marker for excitable regions of myelinated fibers) in the lead-induced demyelinating neuropathy of the rat. By electron microscopy, we found that paranodal degeneration resulted in spreading of the reaction product from nodal to internodal axolemma. During repair, nodal-like stained areas formed at the contact zones between preremyelinating Schwann cells. These data suggest that the location and extent of excitable axonal regions are influenced by axoglial relationships. Additionally, some fibers displayed staining at paranodal axolemma adjacent to demyelinated segments, suggesting it might be an alternative site for impulse generation in demyelinated fibers.     

The changing axonal distribution in white muscle of the developing catfish, Ictalurus nebulosus (Lesueur).

The development of the peripheral innervation pattern of the axial white muscle fibres of the brown bullhead, Ictalurus nebulosus (Lesueur), was investigated utilizing silver impregnation. In the younger fish examined, axons were observed to run along the myoseptum and into the body of the myotome. With development, axons were gradually restricted to the myoseptal ends as is the situation in adult bullheads. Possible mechanisms for elimination of the superfluous axons observed in the white muscle of the developing brown bullhead are discussed.     

The spherule cells of Holothuria polii Delle Chiaie, 1823 (Aspidochirota, Holothuroidea) during brown body formation: an ultrastructural study

Spherule cells are specific types of coelomocytes found in both the coelomic fluids and the connective tissues of many echinoderm groups and are characterised by large membrane-bound inclusions which completely fill their cytoplasm. In holothurians they are present in massive number in the coelomic fluids and are employed in brown body formation. Brown bodies are products of encapsulation and mainly consist of phagocytic amoebocytes and spherule cells: they surround foreign particles too large to be ingested by circulating phagocytes. During brown body formation, phagocytic amoebocytes flatten out over the surface of foreign particles to form unpigmented nodules which eventually aggregate into a single brown body in which many spherule cells are entrapped. Morphological modifications of spherule cells were studied in Holothuria polii following the induction of brown body formation by intracoelomic injection of sheep erythrocytes. Our ultrastructural observations provide evidence that the granules undergo typical exocytosis after previous disorganisation of their content and suggest a specific secretory activity for the spherule cells. The possible functional role of the secreted vacuolar material in brown body formation is discussed.     

Haematology, cytochemical and ultrastructural characteristics of blood cells in leopard (Panthera pardus)

The leopard (Panthera pandus) is an endangered Asian big cat found in Thailand and also listed in the CITES, Appendix 1. Blood samples from 17 leopards (six males and 11 females) were collected from the cephalic vein for haematology, cytochemical and ultrastructural studies. Red blood cells (RBCs) were slightly anisocytosis, ranged 5–6.5 μm with 5.6 μm mean diameter. They were easy to form rouleaux, blunt end crenation and some RBCs contained Heinz bodies. The male leopards had a significantly higher packed cell volume (39.3 ± 7.5%) values and absolute eosinophil number (1.658 ± 1.483 × 10⁹/l) than the females (30.7 ± 3.2%; 0.965 ± 0.611 × 10⁹/l). The cytochemical results were: sudanophilia and myeloperoxidase in neutrophils and some monocytes; nonspecific esterase (alpha-naphthyl acetate esterase, ANAE) in the granules of eosinophils, some lymphocytes, some monocytes and platelets; beta-glucuronidase (βG) in granules of basophils, monocytes and some lymphocytes. The ANAE and βG reaction were detected inter-granular of eosinophils. More detailed morphological aspects of all blood cells were observed by means of scanning electron microscope and transmission electron microscope (TEM). The many round granules and homogeneous content under TEM were characteristics of leopard eosinophils. The engulfed RBCs by neutrophils were detected under TEM in one male leopard. This study provides a guide for the haematology, identification of the morphology, cytochemistry and ultrastructure of blood cells in leopards that is useful for zoological veterinarians in leopard conservation.     

Terminal deoxynucleotidyl transferase (TdT) in acute nonlymphocytic leukemia. A clinical, morphologic, cytochemical, immunologic, and ultrastructural study

The classification of acute leukemia is essential for proper therapy and may be based on morphologic, cytochemical, immunologic, or even ultrastructural studies. Terminal deoxynucleotidyl transferase (TdT) is expressed in most patients with acute lymphocytic leukemia (ALL) and a minority of patients with acute nonlymphocytic leukemia (ANLL). Thirteen patients with ANLL and greater than 30% blasts positive for TdT were studied to establish the clinical, light microscopic, cytochemical, immunologic, and ultrastructural correlates of this phenomenon. Most patients demonstrated some morphologic and cytochemical features of monocytic differentiation. On cytochemical stains, nine had greater than 3% Sudan black-positive blasts. Diffuse alpha naphthyl acetate esterase (ANAE) staining of leukemic cells was present in nine cases, though extremely weak in seven. Blasts in ten patients did not express any other markers of lymphoid differentiation except TdT. However, two patient's immature cells bore CD10 common ALL antigen (CALLA) and CD19 (B4). Ultrastructural studies confirmed nonlymphoid differentiation in all ten patients studied, with a prominent monocytic component present in nine. In no case was a second population of lymphoblasts identified to account for TdT positivity. These patients responded poorly to conventional therapy for ANLL, with complete remissions in 3 of 13 (23%). With conventional therapy for ALL, complete remission was achieved in only two of nine (22%) patients. However, four of seven (57%) patients had a complete remission with high-dose cytosine arabinoside regimens. The authors' studies suggest that patients with TdT-positive ANLL represent a distinct subset that usually displays ultrastructural evidence for monocytic differentiation and is clinically significant in that these patients respond poorly to conventional therapy for both ALL and ANLL. Recognition of the monocytic lineage of these cases by light microscopic examination is difficult because they are often poorly differentiated morphologically and express only weak nonspecific esterase positivity.     

Morphological, cytochemical, and ultrastructural study of thrombocytes and leukocytes in neotropical fish, Brycon orbignyanus Valenciennes, 1850 (Characidae, Bryconinae)

Morphological, cytochemical and ultrastructural studies are important to demonstrate the function of the blood cells, which is very little understood in teleosts. In peripheral blood of 'piracanjuba' Brycon orbignyanus, thrombocytes, lymphocytes, monocytes, neutrophils and heterophils were studied and characterized. Thrombocytes had a fusiform or oval shape with PAS-positive granules. Lymphocytes presented small size with sparse basophilic cytoplasm. Monocytes were large in size, presented basophilic cytoplasm that may be foamy or vacuolated, with non-specific esterase staining. The neutrophils presented lightly neutrophilic granule cytoplasm, with positivity for PAS and peroxidase. The heterophils were large in size, with eosinophilic and basophilic granules cytoplasm and PAS-positive. Transmission electron microscopy study demonstrated that the thrombocytes, lymphocytes and monocytes features were similar to other teleosts. In ultrastructural study only one type of neutrophils was observed. Cytochemical findings indicated that neutrophils and monocytes of B. orbignyanus may be involved in phagocytosis, and neutrophils play an important microbicidal role.     

Morphological, cytochemical, and ultrastructural observations on the blood cells of the reptile Tupinambis merianae (Squamata)

Tupinambis merianae is a lizard of the Brazilian fauna and belongs to the Squamata order. Blood cell data are scarce. Blood samples from six specimens of adult T. merianae were used to evaluate some hematological parameters. For structural analysis, 2 ml of blood was collected in the presence of EDTA. Part of the blood was used for preparing blood smears which were submitted to the methods of Leishman, Laür, and toluidine blue, and the cytochemical reactions of periodic acid-Schiff, sirius red, sudan black B, and ortho-toluidine-H₂O₂. The remainder was centrifuged and the leukocyte buffy coat was fixed in Karnovsky’s fluid for electron microscopy examination. The following blood cells were identified: mature and immature erythrocytes, spherical and elliptical thrombocytes, heterophilic granulocytes, eosinophils and basophils types I and II, lymphocytes, monocytes, and azurophilic cells. The more significant results obtained were: the presence of glycogen in the cytoplasm of the thrombocytes, heterophils, and basophils; the presence of basic polyaminoacid-rich proteins in the granules of heterophils and eosinophils and myeloperoxidase in the granules of the heterophils, eosinophils, and azurophilic cells; and sudanophilic small granules in the heterophils, eosinophils, and azurophilic cells. More detailed morphological aspects of the cells were observed by means of ultrastructural analysis.     

Lactoferrin-deficient neutrophil polymorphonuclear leucocytes in leukaemias: a semiquantitative and ultrastructural cytochemical study

Semiquantitative analysis of lactoferrin deficiency in neutrophil polymorphonuclear leucocytes in various haematological and non-haematological disease was carried out by scoring polymorphonuclear leucocytes stained for lactoferrin by the immunoperoxidase method. The staining patterns for lactoferrin were classified into four types (0-III) based on the intensity of reaction, and the sum of the ratings of 100 polymorphonuclear leucocytes was considered as "lactoferrin score" with a possible range of 0-300. As a result, significantly low lactoferrin-scores were frequently observed in acute leukaemias and the acute phase of chronic leukaemias. Of 35 cases with leukaemias, lactoferrin-negative polymorphonuclear leucocytes (type 0) were observed in the following cases: eight cases of acute myelogenous leukaemia (8/14), a case of chronic myelogenous leukaemia (1/10) in blast crisis, one of acute promyelocytic leukaemia (1/1), one of acute monocytic leukaemia (1/2), and a case of chronic myelomonocytic leukaemia (1/2) in a transitional phase to an acute myelomonocytic leukaemia. In two cases of acute myelogenous leukaemia, in which the majority of polymorphonuclear leucocytes were negative for lactoferrin, ultrastructural cytochemical study revealed total lack of specific granules in these polymorphonuclear leucocytes. This suggests that lactoferrin is localised in the specific granules of neutrophils as has been postulated previously by others.