Intracytoplasmic eosinophilic inclusion bodies in benign breast cyst fluids

Intracytoplasmic eosinophilic inclusion bodies (EIBs) observed in cytologic preparations of benign breast cyst fluid aspirates were morphologically similar to the well-known EIBs in urine specimens. EIBs have not been previously described in breast cyst fluids. A chi-square test proved a statistically significant correlation between the presence of intracellular EIBs and cellular degeneration. The possible significance of this finding with regard to the origin of EIBs is discussed.     

Inflammatory Reactions of the Protochordata

Ascidian inflammatory responses are characterized by an influxof blood cells into areas containing foreign material or injuredtissue. The sequestering and removal of foreign substances isa function of their chemical nature and size. Small particulatematerials are removed by phagocytosis; substances too largeto be phagocytosed are encapsulated. Blood cells involved ininflammatory reactions are small undifFerentiated cells (hemoblasts,lymphocytes), hyaline and granular leucocytes and morula cells.     

Ultrastructure,encystment and cyst wall composition of the resting cyst of the peritrich ciliate Opisthonecta henneguyi

The cyst wall of Opisthonecta henneguyi has been studied ultrastructurally and cytochemically by light and electron microscopy, as well as by chemical and electrophoretic analyses, to examine the structure of the cyst wall and its composition. The cyst wall consists of four morphologically distinct layers. The ectocyst is a thin dense layer. The mesocyst is the thickest layer and is composed of a compact material. The endocyst is a thin layer like the ectocyst, but less dense. The granular layer varies in thickness and is composed of a granular material. In the resting cyst, kinetosomes of both oral apparatus and trochal band as well as the myoneme system are maintained, and only cilia are resorbed. The sugars present in the cyst wall are predominantly N-acetylglucosamine (90%) and glucose (10%). The mesocyst is composed of chitin, and the endocyst includes glycoproteins and acid mucopolysaccharides. During secretion of the cyst wall, the endocyst and granular layer are secreted from precursors synthesized "de novo". No cytoplasmic precursors of ectocyst and mesocyst have been detected.     

Histiocytoid change in breast carcinoma: a report of 3 cases with an unusual cytomorphologic pattern of apocrine change

BACKGROUND: Breast carcinomas composed predominantly or exclusively of cells with foamy and/or granular cytoplasm have been termed histiocytoid breast carcinoma. CASES: Three cases of HBC had fine needle aspirates that were moderately cellular and composed of cells with abundant foamy and/or granular cytoplasm, arranged in loosely cohesive groups and dispersed singly. The cells showed subtle cytologic atypia, including nuclear hyperchromasia and slightly irregular nuclear outlines. Definitive cytologic diagnosis was not possible in the 3 cases, and they were reported as "suspicious for malignancy." Core biopsies of 2 cases showed a typical Indian file pattern of invasive lobular carcinoma, while the third case was composed of sheets of discohesive histiocytoid cells admixed with a prominent lymphoid infiltrate. All 3 cases were E-cadherin negative, confirming their lobular nature. CONCLUSION: HBC represents an unusual morphologic pattern of apocrine change that may be seen in lobular and ductal breast carcinomas. Recognition of these lesions is vital in that they may be mistaken for a variety of other entities composed of foamy/granular cells; some of those entities have vastly different implications for treatment and prognosis.     

Tartrate-resistant acid phosphatase facilitates hydroxyl radical formation and colocalizes with phagocytosed Staphylococcus aureus in alveolar macrophages

Tartrate-resistant acid phosphatase (TRAP) is an enzyme expressed specifically in osteoclasts and activated macrophages, two phagocytosing cell types originating from the same hematopoietic stem cells. TRAP contains a binuclear iron centre which has been shown to generate reactive oxygen species (ROS). In this study murine macrophage like cell line RAW-264 overexpressing TRAP was shown to produce elevated levels of hydroxyl radicals compared to parental cells. TRAP transfected cells also had reduced growth rate indicating harmful effects of excessive intracellular ROS levels. Using TRAP specific antibody TRAP protein was shown in alveolar macrophages partially colocalize with late endosomal/lysosomal markers Rab7, Lamp 1 and MHC II molecules that bind antigenic peptides. TRAP also colocalized into compartments where Staphylococcus aureus were phagocytosed. These results suggest that TRAP may have an important biological function in the defence mechanism of macrophages by generating intracellular ROS which would be targeted to destroy phagocytosed foreign material.     

Massive apoptosis of colonocytes induced by butyrate deprivation overloads resident macrophages and promotes the recruitment of circulating monocytes

Our previous investigations demonstrated a rapid, massive apoptosis of colonocytes after butyrate deprivation. However, while in vitro apoptotic bodies and cells were sludged at the epithelial surface, in vivo they were phagocytosed by the resident macrophages. In the present study the guinea pig colon was perfused in vivo in the presence or absence of butyrate with the aim of identifying the cells involved in the removal of apoptotic material and the method of clearance. Morphological, immunohistochemical and DNA fragmentation analyses were applied. The results demonstrated massive apoptosis of colonocytes in the absence of butyrate. The resident macrophages were tightly clustered below the surface epithelium. Aided by cytoplasmatic projections they phagocytosed and transported apoptotic material from the epithelial intercellular spaces into their bodies. Apparently, the macrophages could not cope with the great amount of apoptotic material they had to eliminate: the recruitment of circulating monocytes occurred. This was revealed by the application of antibodies directed against MAC 387, CD68 (PG-M1), and S-100, which detected distinct monocyte/macrophage populations in the lamina propria. The recruited cells were phenotypically different from resident macrophages, their occurrence being typical in inflamed tissues. In conclusion, butyrate deprivation in vivo led to untimely death of colonocytes and triggered changes in the lamina propria indicative of an inflammatory response.     

Modes of phagocytosis of Gram-positive and Gram-negative bacteria by Spodoptera littoralis granular haemocytes

Haemocytes are the main immunocompetent cells in insect cellular immune reactions. Here, we show that in Spodoptera littoralis, granular haemocytes are the primary phagocyte haemocytes, both in vivo and in vitro. The "trigger" and "zipper" modes of engulfment known in mammal macrophages are active, in vivo, in S. littoralis granular haemocytes, together with macropinocytosis. Lipopolysaccharide as well as lipoteichoic acid inhibit the binding of both Gram-positive (Corynebacterium xerosis) and Gram-negative (Escherichia coli) bacteria on granular haemocytes. In addition, different ligands can inhibit the binding of E. coli. Most of these inhibitors are known as ligands of scavenger receptors in mammal macrophages and we hypothesise that one of the receptors present on S. littoralis granular haemocytes could be a scavenger-like receptor.     

Actin filament nucleation by the bacterial pathogen, Listeria monocytogenes

Shortly after Listeria is phagocytosed by a macrophage, it dissolves the phagosomal membrane and enters the cytoplasm. 1 h later, actin filaments coat the Listeria and then become rearranged to form a tail with which the Listeria moves to the macrophage surface as a prelude to spreading. If infected macrophages are treated with cytochalasin D, all the actin filaments associated with the Listeria break down leaving a fine, fibrillar material that does not decorate with subfragment 1 of myosin. This material is associated with either the surface of the Listeria (the cloud stage) or one end (the tail stage). If the cytochalasin-treated infected macrophages are detergent extracted and then incubated in nuclei-free monomeric actin under polymerizing conditions, actin filaments assemble from the fine, fibrillar material, the result being that each Listeria has actin filaments radiating from its surface like the spokes of a wheel (cloud form) or possesses a long tail of actin filaments formed from the fine, fibrillar material located at one end of the Listeria. Evidence that the fine fibrillar material is involved in nucleating actin assembly comes from a Listeria mutant. Although the mutant replicates at a normal rate in macrophages, actin filaments do not form on its surface (cloud stage) or from one end (tail stage), nor does the bacterium spread. Furthermore it does not form the fine fibrillar material. Evidence that the nucleating material is a secretory product of Listeria and not the macrophage comes from experiments using chloramphenicol, which inhibits protein synthesis in Listeria but not in macrophages. If chloramphenicol is applied 1 h after infection, a time before actin filaments are found attached to the Listeria in untreated macrophages, actin filaments never assemble on the Listeria even when fixed 3 h later. Furthermore the fine fibrillar material is absent, although there is a coat of dense granular material.     

The phagocytic capacity of macrophages in the S phase of the cell cycle

An inflammatory reaction was induced in the peritoneal cavity of mice. Two days later, the peritoneal macrophages, containing a proportion of S-phase (DNA-synthesizing) cells, were harvested and adhered to glass. Then the S-phase macrophages were labeled with [³H]thymidine (radioautography) and the macrophage monolayers were tested with regard to their ability to phagocytose immunoglobulincoated sheep red blood cells (SRBC). The percentages of S-phase macrophages which had phagocytosed SRBC were a little lower than those found for G-phase (G₁ + G₂) cells. Otherwise, the number of phagocytosed SRBC per macrophage was about equal for macrophages in both phases, and they both responded well by increasing the phagocytosis when the SRBC: macrophage ratio was increased. The S-phase macrophages also phagocytosed latex beads and zymosan particles efficiently.     

Cytologic findings of atypical adenosis of the breast. A case report

BACKGROUND: Atypical apocrine adenosis, a well-described histopathologic entity, can sometimes be misdiagnosed as carcinoma. Apocrine cells can also appear atypical in cytopathology and be mistaken for carcinoma. Occasional case reports describe false positive cases due to the presence of apocrine cells in a few cases of radial scars and atypical apocrine metaplasia and in a degenerated cyst. CASE: A 37-year-old female underwent ultrasound-guided fine needle aspiration of an ill-defined breast nodule. The aspirate showed clusters and single cells containing abundant granular to focally vacuolated cytoplasm; enlarged, pleomorphic nuclei with irregular nuclear membranes; granular chromatin; and prominent nucleoli. These cells were distinct from and larger than the surrounding ductal and myoepithelial cells. Excision showed a nodular area of atypical apocrine adenosis adjacent to previous biopsy changes, correlating with the cytologic findings. CONCLUSION: Atypical apocrine adenosis can mimic carcinoma in histopathology and cytopathology. One should be cautious when reviewing apocrine cells in cytology, given their atypical features, especially their single, dispersed nature. However, the presence of accompanying benign cellular elements supports a benign diagnosis. Surgical biopsy should be recommended based on the cytologic findings.     

The organization of the wall filaments and characterization of the matrix structures of Toxoplasma gondii cyst form

The encystation process is a key step in Toxoplasma gondii life cycle, allowing the parasite to escape from the host immune system and the transmission among the hosts. A detailed characterization of the formation and structure of the cyst stage is essential for a better knowledge of toxoplasmosis. Here we isolated cysts from mice brains and analysed the cyst wall structure and cyst matrix organization using different electron microscopy techniques. Images obtained showed that the cyst wall presented a filamentous aspect, with circular openings on its surface. The filaments were organized in two layers: a compact one, facing the exterior of the whole cyst and a more loosen one, facing the matrix. Within the cyst wall, we observed tubules and a large number of vesicles. The cyst matrix presented vesicles of different sizes and tubules, which were organized in a network connecting the bradyzoites to each other and to the cyst wall. Large vesicles, with a granular material in their lumen of glycidic nature were observed. Similar vesicles were also found associated with the posterior pole of the bradyzoites and in proximity to the cyst wall.     

Immunocytochemical and ultrastructural diagnosis of a rare mixed apocrine-medullary carcinoma of the breast in a fine needle aspirate

A rare mixed apocrine-medullary mammary carcinoma in a 57-year-old woman was preoperatively diagnosed by fine needle aspiration cytology. The aspirate was characterized by carcinoma cells with an apocrine differentiation and significant cellular atypia admixed with many lymphocytes and plasma cells. These findings were confirmed by histologic examination of the breast tumor and its metastasis in lymph nodes. Electron microscopy (EM) and immunoperoxidase staining for cytokeratin, S-100 protein, epithelial membrane antigen and carcinoembryonic antigen were done on samples of the aspirated material. Although immunostaining was of little help in this case, the EM findings did show many carcinomatous cells of apocrine type in the tumor.     

Fine needle aspiration cytology of mammary carcinoma with osteoclast-like giant cells

Carcinoma with osteoclast-like giant cells (OCGC) is an uncommon neoplasm characterized by giant cells, prominent vascularization, haemorrhage and areas of cribriform epithelial growth with moderate atypia. Multinucleated giant cells (MGC) have been described in several other breast lesions raising an interesting differential diagnosis, mainly with benign disorders. Due to its rarity few cases have been described cytologically. We retrospectively reviewed 13 fine needle aspiration samples from nine patients with this variant of carcinoma. Nine corresponded to breast tumours and four to axillary, liver, subcutaneous and mediastinal metastatic lesions. The expression of CD68 by giant cells was evaluated immunocytochemically in six cases. All patients had a complete pathological study of the breast neoplasm. Smears showed a double component of epithelial and giant cells. Epithelial clusters were predominantly of intermediate size with irregular contours. Most were cohesive but others showed cellular dissociation with scarce to moderate cellular pleomorphism. Giant cells had well defined, deeply stained cytoplasm and round to elongated morphology. Two metastatic cases were devoid of them. Haemosiderin-laden macrophages were common in smears from breast tumours. In the six cases tested CD68 was expressed in MGC. Cytological features of mammary carcinoma with OCGC correlate closely with the histological ones. Most cases are clearly recognizable as malignant but in others cytological atypia may be minimal, mimicking a benign lesion. In difficult cases the presence of haemosiderin-laden macrophages and the histiocytic nature of the MGC are helpful diagnostic features.     

Plasmodium Sporozoite Interactions with Macrophages In Vitro: a Videomicroscopic Analysis

ABSTRACT. Studies of in vitro interactions between Plasmodium berghei sporozoites and peritoneal macrophages from mice and rats were performed. A videomicroscopic analysis was made of interactions observed by phase-contrast microscopy. Our results showed a diversity of dynamic interactions between sporozoites and macrophages that included no interaction, surface interaction without sporozoite interiorization, active sporozoite penetration, active penetration with subsequent sporozoite escape, macrophage destruction, and the formation of "tethers" or web-like structures by sporozoites that had actively invaded macrophages. Sporozoites are thus clearly capable of actively invading host macrophages and are not restricted to being phagocytosed for interiorization. The formation of "tethers" by the moving sporozoite might function in vivo by anchoring the sporozoite to the cells lining the lumen of the liver sinusoid. Active sporozoite motility appears to be a functional phenomenon involved in sporozoite invasion of host liver cells.     

Plasmodium sporozoite interactions with macrophages in vitro: a videomicroscopic analysis

Studies of in vitro interactions between Plasmodium berghei sporozoites and peritoneal macrophages from mice and rats were performed. A videomicroscopic analysis was made of interactions observed by phase-contrast microscopy. Our results showed a diversity of dynamic interactions between sporozoites and macrophages that included no interaction, surface interaction without sporozoite interiorization, active sporozoite penetration, active penetration with subsequent sporozoite escape, macrophage destruction, and the formation of "tethers" or web-like structures by sporozoites that had actively invaded macrophages. Sporozoites are thus clearly capable of actively invading host macrophages and are not restricted to being phagocytosed for interiorization. The formation of "tethers" by the moving sporozoite might function in vivo by anchoring the sporozoite to the cells lining the lumen of the liver sinusoid. Active sporozoite motility appears to be a functional phenomenon involved in sporozoite invasion of host liver cells.     

Phagocytosis of mast cell granules results in decreased macrophage superoxide production

The mechanism by which phagocytosed mast cell granules (MCGs) inhibit macrophage superoxide production has not been defined. In this study, rat peritoneal macrophages were co-incubated with either isolated intact MCGs or MCG-sonicate, and their respiratory burst capacity and morphology were studied. Co-incubation of macrophages with either intact MCGs or MCG-sonicate resulted in a dose-dependent inhibition of superoxide- mediated cytochrome c reduction. This inhibitory effect was evident within 5 min of incubation and with MCG-sonicate was completely reversed when macrophages were washed prior to activation with PMA. In the case of intact MCGs, the inhibitory effect was only partially reversed by washing after a prolonged co-incubation time. Electron microscopic analyses revealed that MCGs were rapidly phagocytosed by macrophages and were subsequently disintegrated within the phagolysosomes. Assay of MCGs for superoxide dismutase (SOD) revealed the presence of significant activity of this enzyme. A comparison of normal macrophages and those containing phagocytosed MCGs did not reveal a significant difference in total SOD activity. It is speculated that, although there was no significant increase in total SOD activity in macrophages containing phagocytosed MCGs, the phagocytosed MCGs might cause a transient increase in SOD activity within the phagolysosomes. This transient rise in SOD results in scavenging of the newly generated superoxide. Alternatively, MCG inhibition of NADPH oxidase would explain the reported observations.     

Flow cytometric, phase-resolved fluorescence measurement of propidium iodide uptake in macrophages containing phagocytized fluorescent microspheres

BACKGROUND: Spectral interference (overlap) from phagocytosed green-yellow (GY) microspheres in the flow cytometric, red fluorescence emission measurement channel causes errors in quantifying damaged/dead alveolar macrophages by uptake of propidium iodide. METHODS: Particle burdens of uniform GY fluorescent microspheres phagocytosed by rat alveolar macrophages and the discrimination of damaged/dead cells as indexed by propidium iodide uptake were assessed with conventional and phase-sensitive flow cytometry. RESULTS: The fluorescence spectral emission from phagocytosed microspheres partly overlapped the propidium iodide red fluorescence emission and interfered with the measurement of damaged/dead cells when using conventional flow cytometry without subtractive compensation. This caused errors when estimating the percentage of nonviable, propidium iodide-positive, phagocytic macrophages. The interference was eliminated by employing phase-sensitive detection in the red fluorescence measurement channel based on differences in fluorescence lifetimes between the fluorescent microspheres and propidium iodide. Intrinsic cellular autofluorescence, whose fluorescence lifetime is approximately the same as that of the phagocytosed microspheres, also was eliminated in the phase-sensitive detection process. Because there was no detectable spectral interference of propidium iodide in the green fluorescence (phagocytosis) measurement channel, conventional fluorescence detection was employed. CONCLUSIONS: Phase-resolved, red fluorescence emission measurement eliminates spectral overlap errors caused by autofluorescent phagocytes that contain fluorescent microspheres in the analyses of propidium iodide uptake.Cytometry 39:45-55, 2000. Published 2000 Wiley-Liss, Inc.     

High-resolution electron microscopical study of cyst walls of Entamoeba spp

Knowledge of the fine structural organization, molecular composition and permeability properties of the cell surface of intestinal protozoan cysts is important to understand the biologic basis of their resistance. Recent studies on the biology of the cyst walls of Entamoeba histolytica and Entamoeba invadens have considerably advanced knowledge on the cellular processes involved in the transport and surface deposition of the main cyst wall components. Using transmission electron microscopy, cytochemistry, scanning electron microscopy and freeze-fracture techniques, we have obtained new information. In mature cysts the permeability of Entamoeba cysts is limited to small molecules not by the cyst wall, but by the plasma membrane, as demonstrated with the use of ruthenium red as an electron-dense tracer. Cell walls of E. histolytica cysts are made up of five to seven layers of unordered fibrils 7-8 nm thick. Alcian blue stains a regular mesh of fibrils approximately 4 nm thick, running perpendicularly to the cyst wall. In addition, abundant ionogenic groups are seen in cyst walls treated with cationized ferritin. In the mature cysts of E. histolytica and E. invadens small cytoplasmic vesicles with granular material were in close contact with the plasma membrane, suggesting a process of fusion and deposition of granular material to the cell wall. The plasma membrane of mature cysts is devoid of intramembrane particles when analyzed with the freeze-fracture technique. When viewed with scanning electron microscopy the surface of E. histolytica cysts clearly differs from that of Entamoeba coli and E. invadens.     

Neutrophil Response to Pseudomonas aeruginosa in Respiratory Infection

Sputum from patients with acute exacerbation of respiratory infection by Pseudomonas aeruginosa was observed under the electron microscope. External to the cell wall of P. aeruginosa a granular, electron-dense material was observed which is suggestive of capsule. It is supposed that stabilization of capsule occurred by the host antibody, which was produced due to chronic infection by P. aeruginosa. Mucoid type of microcolonies were observed with a fibrous matrix of exopolysaccharide. Other types of microcolonies were surrounded by granular substances or fine fibers. Neutrophil was found to be partially surrounding the microcolony in an attempt to defense. Debris was formed mainly by the destruction of the neutrophil. Most neutrophils were found full of phagocytosed debris; in contrast only a few neutrophils were found to have phagocytosed P. aeruginosa. This study concludes that instead of phagocytosing bacteria, neutrophil phagocytosed debris and bacteria were not completely eradicated. Therefore, this might be one of the factors in the pathogenesis of respiratory infection and persistent colonization by P. aeruginosa.     

Tubular lysosomes accompany stimulated pinocytosis in macrophages

A network of tubular lysosomes extends through the cytoplasm of J774.2 macrophages and phorbol ester-treated mouse peritoneal macrophages. The presence of this network is dependent upon the integrity of cytoplasmic microtubules and correlates with high cellular rates of accumulation of Lucifer Yellow (LY), a marker of fluid phase pinocytosis. We tested the hypothesis that the efficiency of LY transfer between the pinosomal and lysosomal compartments is increased in the presence of tubular lysosomes by asking how conditions that deplete the tubular lysosome network affect pinocytic accumulation of LY. Tubular lysosomes were disassembled in cells treated with microtubule-depolymerizing drugs or in cells that had phagocytosed latex beads. In unstimulated peritoneal macrophages, which normally contain few tubular lysosomes and which exhibit relatively inefficient transfer of pinocytosed LY to lysosomes, such treatments had little effect on pinocytosis. However, in J774 macrophages and phorbol ester-stimulated peritoneal macrophages, these treatments markedly reduced the efficiency of pinocytic accumulation of LY. We conclude that a basal level of solute accumulation via pinocytosis proceeds independently of the tubular lysosomes, and that an extended tubular lysosomal network contributes to the elevated rates of solute accumulation that accompany macrophage stimulation. Moreover, we suggest that the transformed mouse macrophage cell line J774 exhibits this stimulated pinocytosis constitutively.