Large Electron-dense Granules Research Articles

Morphologic appearance of peripheral blood cells from Zovawk pigs (Sus scrofa domesticus) visualized by transmission electron microscopy.

Ultrastructural information regarding the peripheral blood cells of local (Zovawk) pigs from Mizoram, India, is not available in the scientific literature. The present study was designed to reveal the fine structural details of the blood cells from these local pigs using a transmission electron microscope (TEM). Blood samples were collected from 12 healthy Zovawk pigs of either sex and processed according to a standard protocol. Processed blood samples were then sent to the All India Institute of Medical Sciences, New Delhi, for further processing and imaging under TEM. Different types of blood cells were viewed under TEM, and different characteristics of these cells were assessed. In the present study, erythrocytes are elongated, biconcave, and nucleated without cytoplasmic organelles. Neutrophils are round with 2-5 lobed nuclei surrounded by cytoplasm with an indistinct bilayered nuclear membrane. The cytoplasm is packed with membrane bound round, oval, and elongated cytoplasmic granules. Eosinophils areround to oval with 2-3 lobed nuclei with distinct nuclear membranes. Basophils are spherical and contained small, medium, and large electron-dense granules. Lymphocytes are small, medium, and large and contained all cellular components. Monocytes are irregularly spherical with slight nuclear indentations. The platelets are elongated, oval, or rounded, with a few pseudopods at the cell surface. From the present study, we can conclude that the ultrastructural morphology of blood cells from Zovawk pigs resembles those of other domestic animals. However, a few differences have been observed.

Development and characterization of a canine-specific anti-CD94 (KLRD-1) monoclonal antibody

Natural killer (NK) cells are non-T, non-B lymphocytes are part of the innate immune system and function without prior activation. The human NK cell surface determinant, CD94, plays a critical role in regulation of NK cell activity as a heterodimer with NKG2 subclasses. Canine NK cells are not as well defined as the human and murine equivalents, due in part to the paucity of reagents specific to cell surface markers. Canines possess NK/NKT cells that have similar morphological characteristics to those found in humans, yet little is known about their functional characteristics nor of cell surface expression of CD94. Here, we describe the development and function of a monoclonal antibody (mAb) to canine (ca) CD94. Freshly isolated canine CD94+ cells were CD3+/–, CD8+/–, CD4–, CD21–, CD5low, NKp46+, and were cytotoxic against a canine target cell line. Anti-caCD94 mAb proved useful in enriching NK/NKT cells from PBMC for expansion on CTAC feeder cells in the presence of IL-2 and IL-15. The cultured cells were highly cytolytic with co-expression of NKp46 and reduced expression of CD3. Transmission electron microscopy revealed expanded CD94+ lymphocytes were morphologically large granular lymphocytes with large electron dense granules. Anti-caCD94 (mAb) can serve to enrich NK/NKT cells from dog peripheral blood for ex vivo expansion for HCT and is a potentially valuable reagent for studying NK/NKT regulation in the dog.

Characterization of uncultured giant rod-shaped magnetotactic Gammaproteobacteria from a freshwater pond in Kanazawa, Japan.

Magnetotactic bacteria (MTB) are widespread aquatic bacteria, and are a phylogenetically, physiologically and morphologically heterogeneous group, but they all have the ability to orientate and move along the geomagnetic field using intracellular magnetic organelles called magnetosomes. Isolation and cultivation of novel MTB are necessary for a comprehensive understanding of magnetosome formation and function in divergent MTB. In this study, we enriched a giant rod-shaped magnetotactic bacterium (strain GRS-1) from a freshwater pond in Kanazawa, Japan. Cells of strain GRS-1 were unusually large (~13×~8 µm). They swam in a helical trajectory towards the south pole of a bar magnet by means of a polar bundle of flagella. Another striking feature of GRS-1 was the presence of two distinct intracellular biomineralized structures: large electron-dense granules composed of calcium and long chains of magnetosomes that surround the large calcium granules. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that this strain belongs to the Gammaproteobacteria and represents a new genus of MTB.

Plexiform fibrohistiocytic tumor of bone

Plexiform fibrohistiocytic tumor is an extremely rare soft tissue tumor with a low malignancy potential. The patient is usually a child or a young adolescent and the tumor is usually localized in the upper extremities. We report on a case of a 21-year old male with a plexiform fibrohistiocytic tumor in the left fibula admitted to our hospital due to a swelling and pain in the left lower extremity. Radiologically a lytic lesion in the distal end of left fibula consistent with a non-aggressive lesion with low biological activity was found. Treated with curettage, the specimen revealed plexiform proliferation of mononuclear histiocyte-like cells, multinucleated osteoclast-like cells, and spindle fibroblast-like cells in variable proportions histopathologically. Immunohistochemical stains were positive for CD68 in scattered fashion in histiocytes and giant cells, and spindle like cells showed positivity for smooth muscle actin. Under electron microscopy, rough endoplasmic reticulum and collagen bundles in the spindle cells suggested fibroblastic differentiation. Also multiple large electron-dense lysosomal granules in histiocytoid cells were found. Multinucleated giant cells exhibited osteoclast-like appearance. All these findings suggested plexiform fibrohistiocytic tumor. Interestingly, the tumor was localized in bone. During the follow up for 27 months after the resection, there was no recurrence or metastasis.

Primary structure and cellular localization of callinectin, an antimicrobial peptide from the blue crab

We report the complete amino acid sequence of callinectin, a 32 amino acid, proline-, arginine-rich antimicrobial peptide (AMP) with four cysteines and having the sequence WNSNRRFRVGRPPVVGRPGCVCFRAPCPCSNY-amide. The primary structure of callinectin is highly similar to arasins, AMPs recently identified in the small spider crab ( Hyas araneus). Callinectin exists in three isomers that vary in the functional group on the tryptophan (W) residue. The most prevalent isomer had a hydroxy-N-formylkynurenine group, while the other two isomers had either N-formylkynurenine or hydroxy-tryptophan. Using a sequence highly similar to native callinectin, we chemically synthesized a peptide which we called callinectin-like peptide (CLP). Via immuno-electron microscopy, affinity-purified rabbit antibodies raised to CLP successfully localized the site of callinectin in blue crab hemocytes to the large electron-dense granules that are found primarily in large granule hemocytes.

Concholepas hemocyanin biosynthesis takes place in the hepatopancreas, with hemocytes being involved in its metabolism

Hemocyanins are copper-containing glycoproteins in some molluscs and arthropods, and their best-known function is O(2) transport. We studied the site of their biosynthesis in the gastropod Concholepas concholepas by using immunological and molecular genetic approaches. We performed immunohistochemical staining of various organs, including the mantle, branchia, and hepatopancreas, and detected C. concholepas hemocyanin (CCH) molecules in circulating and tissue-associated hemocytes by electron microscopy. To characterize the hemocytes, we purified them from hemolymph. We identified three types of granular cells. The most abundant type was a phagocyte-like cell with small cytoplasmic granules. The second type contained large electron-dense granules. The third type had vacuoles containing hemocyanin molecules suggesting that synthesis or catabolism occurred inside these cells. Our failure to detect cch-mRNA in hemocytes by reverse transcription with the polymerase chain reaction (RT-PCR) led us to propose that hemocytes instead played a role in CCH metabolism. This hypothesis was supported by colloidal gold staining showing hemocyanin molecules in electron-dense granules inside hemocytes. RT-PCR analysis, complemented by in situ hybridization analyses with single-stranded antisense RNAs as specific probes, demonstrated the presence of cch-mRNA in the hepatopancreas; this was consistent with the specific hybridization signal and confirmed the hepatopancreas as the site of CCH synthesis. Finally, we investigated the possibility that CCH catabolism in hemocytes was involved in the host immune response and in the generation of secondary metabolites such as antimicrobial peptides and phenoloxidase.

Identification of phagocytic cells, NK-like cytotoxic cell activity and the production of cellular exudates in the coelomic cavity of adult zebrafish

Coelomic cavity (CC) cells of mature zebrafish harvested by lavage with media or trypsin–EDTA contained 0.80–1.20 × 10 5 and 2.0–3.5 × 10 5 cells, respectively. Media lavage was composed of granulocytes (60–80%), lymphocytes (10–20%), and NCC (4–10%). Granulocytes had large electron dense cytoplasmic paracrystalline granules and a segmented nucleus; they expressed plastin-1, myeloid specific peroxidase and MCSF mRNA; and they were NCAMP-1 +. Lymphocytes had B- and T-cell specific mRNA and were NCAMP-1 − and NCCRP-1 −. NCC were 3 μm, NCAMP-1 + and NCCRP-1 + and did not express B- and T-cell specific mRNA. Additionally, trypsin lavage contained monocytes (marginated chromatin, low nuclear:cytoplasm ratio, sparse cytosolic granules) and macrophages (non-segmented nuclei, no margination of chromatin, abundant electron dense granules). E. coli injected into the CC were phagocytosed in a dose and time dependent fashion by granulocytes, monocytes and macrophages. NCC lysed mammalian target cells and NCAMP-1 expressing hybridoma cells in redirected lysis assays.

An inherited macrothrombocytopenic disorder with abnormal large granules

Inherited giant platelet syndromes are a heterogeneous group of rare bleeding disorders. In the current study, a patient was reported with prolonged bleeding time, thrombocytopenia and giant platelets. Both the patient's red and white cell counts were normal without morphological abnormalities. The electron microscopy of platelets showed abnormal large electron-dense granules in the cytoplasm with exocytosis being easily observed. Her fathers platelets had the same abnormalities. The expression of glycoprotein (GP)Ib, GPIIb and GPIIIa on the surface of the patient's platelets was normal, and her platelet aggregation in response to ADP and ristocetin was also normal. Immunogold probes combined to monoclonal antibodies against GPIIIa, P-selectin or CD63 could not identify any relationship between the aberrant structures and alpha granules or lysosomes of platelet. Serotonin level was normal in the platelets of the patient, indicating the electron opaque granules are not dense bodies. The morphological abnormalities of the platelets from the patient are clearly distinguishable from other hereditary giant platelet disorders. We propose that the abnormal large granules from the patient's platelets probably represent a novel inherited thrombocytopenic disorder.

Role of cellular response in elimination of the monogenean Neoheterobothrium hirame in Japanese flounder Paralichthys olivaceus

Adult worms of the blood-feeding monogenean parasite Neoheterobothrium hirame, which cause anemia in the Japanese flounder Paralichthys olivaceus, attach to the host fish by embedding their posterior part deeply into the host tissue. To investigate the possibility that cellular responses of the host fish can eliminate N. hirame, flounder were experimentally infected with N. hirame larvae and reared in either fed or starved conditions. Mature parasites were identified on the buccal cavity wall of the fish 33 d post-infection (Day 33). Monocytes/macrophages and granulocytes increased rapidly in the blood and infected sites after the appearance of mature parasites. These cells adhered to the tegument of the parasites. In addition, a few cells with large electron-dense granules (DGCs) were observed in the inflammatory foci. On Day 47, the tegument of some parasites collapsed partially and were phagocytosed by the infiltrated host cells. Some infiltrated cells adhered directly to the inner tissues of the parasites. On Day 54, in the fed fish group, the loss of the tegument led to damage of the parasites' inner tissue by a large number of infiltrated cells. In this group, the elimination of the parasites was noted from Day 47 to 54. These observations probably suggest that the cellular response of the host fish destructed the parasite's posterior part embedded in the tissue, thereby eliminating the parasites. On the other hand, a high mortality was observed in the starved group. The starved fish developed much more severe anemia than the fed fish, and the elimination of the parasites was not observed in this group. The results of the present study suggest that flounder can eliminate N. hirame if they are fed sufficiently.

Ultrastructure of the apical glandular region of the scolex of Proteocephalus torulosus (Cestoda: Proteocephalidae)

In the apical glandular region of the adult Proteocephalus torulosus (Batsch, 1786), two types of eccrine gland cells are present. The first type of unicellular gland produces large electron-dense granules of various sizes. The second type contains small electron-dense granules. Most cells form glands with large granules; glands with small granules are infrequent. The secretion of both types of gland cells is concentrated in the apical parts of the cyton and in the ducts opening to the exterior. On the scolex of P. torulosus, there are regional structural differences of the microthrix border. The apical glandular region bears filamentous microtriches only. On the remaining frontal part, surrounding the glandular region, there are blade-like and filamentous microtriches. The lateral parts of the scolex and suckers bear blade-like microtriches. Possible functions of both types of gland cells and different parts of the scolex microthrix border are discussed. The unique structure of the frontal part of the scolex of P. torulosus and its differences from Proteocephalus macrocephalus, P. longicollis and P. percae correlate well with the putative basal phylogenetic position of P. torulosus among European species of Proteocephalus.

Identification of Japanese Flounder Leucocytes Involved in the Host Response to Neoheterobothrium hirame.

Leucocyte types involved in tne cellular response of Japanese flounder Paralichthys olivaceus against the monogenean Neoheterobothrium hirame were identified. With electron microscopy, peripheral blood leucocytes (PBL) of Japanese flounder could be classified into four types : lymphocytes, thrombocytes, monocytes and granulocytes. Granulocytes were positive for acid phosphatase and peroxidase activities, and stained with Sudan black B. In fish infected with N. hirame, the ratios of granulocytes and monocytes significantly increased in the PBL composition. Infiltration of monocytes/macrophages and granulocytes, and cells having large electron-dense granules (DGC) was observed in the infection sites. These cells were often observed adhering to the tegument of N. hirame. Monocytes/macrophages showed particularly higher adherent ability to N. hirame than the other leucocytes under in vitro and in vivo conditions. We conclude that monocytes/macrophages, granulocytes and DGC are major effector cells in the defense of the flounder against N. hirame.

Solid Papillary Carcinoma of the Breast: Report of Two Cases

Two cases of solid papillary carcinoma of the breast were pathologically studied. Subjects included two female patients: a 76-year-old (Case 1) and a 43-year-old (Case 2). Both cases showed solid and papillary proliferation of spindle cells in expanded ducts, perivascular pseudorosettes, eosinophilic fine granules in an abundant cytoplasm, intracellular mucin production, and positivity for chromogranin A. Case 1 showed an aggregation form involving multiple ducts, admixture of signet-ring like cells, mucin pools, and small and large electron-dense granules and flocculent granules in electron microscopy. Case 2 showed a solitary and compact form in a dilated duct and an interlacing bundle pattern.

Solid Papillary Carcinoma of the Breast: Report of Two Cases

Two cases of solid papillary carcinoma of the breast were pathologically studied. Subjects included two female patients: a 76-year-old (Case 1) and a 43-year-old (Case 2). Both cases showed solid and papillary proliferation of spindle cells in expanded ducts, perivascular pseudorosettes, eosinophilic fine granules in an abundant cytoplasm, intracellular mucin production, and positivity for chromogranin A. Case 1 showed an aggregation form involving multiple ducts, admixture of signet-ring like cells, mucin pools, and small and large electron-dense granules and flocculent granules in electron microscopy. Case 2 showed a solitary and compact form in a dilated duct and an interlacing bundle pattern.

Ultrastructural aspects of peptidergic modulation in the peripheral nervous system of Helix pomatia.

The ultrastructural characteristics of peptidergic peripheral contacts in the snail, Helix pomatia, were investigated, with special attention to the innervation of the heart, buccal mass, and salivary gland by Mytilus inhibitory peptide-immunoreactive neurons. Following the application of correlative light- and electron-microscopic pre-embedding immunocytochemistry, the peripheral tissues reveal a rich innervation by Mytilus inhibitory peptide-immunoreactive elements. These neurons establish three types of neuromuscular contacts in the heart and buccal mass: (1) close (16-20 nm) unspecialized membrane contacts; (2) contacts with a relative wide (40-100 nm) intersynaptic cleft; and (3) labeled varicosties located freely in the extracellular space, far (0. 5-several microm) from the muscle cells. In the salivary gland, the immunoractive profiles contact both the muscular and glandular elements with close (type 1) and wider (type 2) membrane attachments. The great majority of Mytilus inhibitory peptide-immunoreactive profiles contain an ultrastructurally uniform population of large (120-150 nm) electron dense granules. The ultrastructural features of the innervation by Mytilus inhibitory peptide-immunoreactive elements are compared with those established by immunogold labelled FMRFamide-containing profiles in the heart and salivary gland. These latter display similarities in forming the different kinds of intercellular contacts, and differences in the morphological variability of the content of granules in the immunolabeled profiles. The results suggest diverse, non-synaptic modulatory roles of neuropeptides in the peripheral nervous system of Helix pomatia, including localized membrane effects and neurohormonal-like remote global controls, that may also be of significance in orchestrating the effects of neuropeptides released at the same time on different targets.

GH and PRL-Producing Pituitary Adenoma with Neuron-Like Differentiation.

A pituitary adenoma with neuron-like differentiation in the sella turcica is reported. Sections of the tumor showed a mixture of adenoma cells, ganglionic cells, and neuropil-like structures by light microscopy. Both pituitary adenoma cells and large cells recognized as ganglionic cells by H&E were strongly immunoreactive for both growth hormone (GH) and prolactin (PRL), which indicated that these large cells had properties similar to those of pituitary adenoma cells. Furthermore, electron microscopy (EM) revealed characteristic low electron-dense secretory granules as well as GH-type large electron-dense secretory granules in adenoma cells, neuropils, and swollen bulbs of neuronal endings, which indicated that these three populations may be of the same origin. Furthermore, we could not find typical cell bodies of ganglionic cells by EM. These results are consistent with a hypothesis that attempts to explain the origin of the neuronal components by the neuronal differentiation of adenoma cells. Thus, the best designation of our tumor may be "pituitary adenoma with neuron-like differentiation."

MIP-immunoreactive innervation of the snail, Helix pomatia, heart. An ultrastructural study

The ultrastructural characteristics of the innervation established by MIP-(Mytilus inhibitory peptide) immunoreactive neurons was investigated in the heart of the snail, Helix pomatia, applying correlative light- and electron microscopic pre-embedding immunocytochemistry on Vibratome-slices. In both the auricle and ventricle, the muscle fibers receive a rich innervation by MIP-immunoreactive (IR) varicose fibers. However, the innervation is seasonally changing in the two parts of the heart. The varicosities, containing a morphologically uniform population of large (120-150 nm) electron-dense granules, can be found in three different positions in relation to the muscle fibers: (i) close (15-20 nm) but unspecialized membrane connections between MIP-(IR) varicosities and muscle fibers; (ii) MIP-IR varicosities located relatively far (0.5-several microm) from the muscles fibers; (iii) MIP-IR profiles localized freely in the extracellular space among the loosely arranged muscle fibers. A general modulatory role of MIP in regulating the heart activity of Helix is suggested.

Morula-Like Cells in Photo-Symbiotic Clams Harboring Zooxanthellae

Symbiosis is observed between zooxanthellae, symbiotic dinoflagellates, and giant clams and related clams which belong to the families Tridacnidae and Cardiidae. We have previously shown that a photo-symbiotic clam Tridacna crocea has three types of hemocytes, the eosinophilic granular hemocyte with phagocytic activity, the agranular cell with electron lucent granules, and the morula-like cell with large (ca. 2 mum in diameter) colorless granules. The function of the morula-like cell is not clear, but it has not been reported in any other bivalves except photo-symbiotic clams T. crocea and Tridacna maxima. In order to clarify whether it is specific to photo-symbiotic clams or not, we studied hemocytes in the photo-symbiotic clams Tridacna derasa (Tridacnidae), Hippopus hippopus (Tridacnidae) and Corculum cardissa (Cardiidae), and a closely related non-symbiotic clam Fulvia mutica (Cardiidae). The eosinophilic granular hemocytes and the agranular cells were found in all of the clams examined. However, the morula-like cells which were packed with many large electron dense granules (ca. 2 mum in diameter), were observed only in the photo-symbiotic clams. In F. mutica, a closely related non-symbiotic clam, this type of hemocyte was not found. Instead a hemocyte with vacuoles and a few large granules containing peroxidase activity was observed. The large granules of F. mutica varied in size from ca. 1-9 mum in diameter. Present data suggests that the presence of morula-like cells is restricted to photo-symbiotic clams and that the hemocytes associated with the morula-like cells may have some functional relationship to symbiosis with zooxanthellae.

Ultrastructural and autometallographic studies of the nephrocytes of Lithobius forficatus L. (Myriapoda, Chilopoda): role in detoxification of cadmium and lead

Ultrastructural and autometallographic investigations of the nephrocytes of the centipede Lithobius forficatus (Myriapoda : Chilopoda) and their role in detoxification of cadmium and lead are presented. This cell-type exhibits the characteristic features of podocytes, i.e. the typical pedicel-basal lamina complex and the well-developed lysosomal vacuolar system. Ultrastructural study demonstrated that heavy metals, such as cadmium and lead, may cause intracellular injuries. The autometallographic procedure showed that large electron-dense granules, referred to as cytosomes in the ultrastructural study, are the main organelles of the nephrocytes to accumulate heavy metals. Consequently, these cells may play an important role in the detoxification process.

Proteins of morula-like cells in hemolymph of the giant clam, Tridacna derasa.

The morula-like cell, a hemocyte packed with many large (about 3 microns in diameter) electron-dense granules, is found only in the hemolymph of giant clams belonging to the Tridacnidae. To clarify the function of the morula-like cell, we investigated its proteins, especially those found in the large granules. Proteins with molecular weights of 64 kDa, 17 kDa and 7.4 kDa were found to be specific to this type of hemocyte. N-terminal amino acid sequence analysis revealed that the 17-kDa and 7.4-kDa proteins were novel proteins rich in aromatic amino acids. Rabbit polyclonal antibody against a synthetic peptide of the 7.4-kDa protein reacted not only with that protein but also with a larger molecular weight (about 16-kDa) protein in the morula-like cell. Examination of the N-terminal amino acid sequences showed that the 16-kDa protein is distinct from the 17-kDa protein, and Western blot analysis suggested that it is a precursor of the 7.4-kDa protein. The zooxanthellate portion of clam mantle and kidney contained proteins immunoreactive to the antibody, but the azooxanthellate portion of the mantle did not contain any immunoreactive protein. These results suggest that the morula-like cells interact with the zooxanthellae.

Effects of gastrin on the histamine-secretory and proliferative activity of cultured carcinoid cells derived from the stomach of the rodent Mastomys natalensis.

The effects of gastrin on the synthesis and release of histamine and on cellular proliferation were investigated in a homotransplantable carcinoid tumor implanted in the rodent Mastomys natalensis and in cultured cells derived from the tumor. The homotransplanted tumor was immunopositive for histamine, synaptophysin and protein gene product 9.5, and its cells contained numerous secretory granules that were visualized by electron microscopy. When carcinoid cells were cultured in a medium with a high concentration of gastrin-I (10(4) pg/mL) for 7 days, large electron-dense secretory granules were characteristically observed in the cytoplasm. By contrast, only a few such granules and numerous secondary lysosomes were seen in cells that had been cultured in the same medium without gastrin-I. A high concentration of gastrin-I (10(4) pg/mL) significantly increased the release of histamine into the culture medium from the carcinoid cells compared with the control (P < 0.05). Cellular proliferation, as determined by monitoring the incorporation of [methyl-3H]-thymidine into the carcinoid cells increased significantly at lower concentrations of gastrin-I (10(2) and 10(3) pg/mL), (P < 0.05). At higher concentrations (10(4) pg/mL or more), gastrin-I had no effect on proliferation. These findings indicate that gastrin stimulates the synthesis and release of histamine by carcinoid cells, as well as their proliferation.