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Mast cells (MC) are phylogenetically old cells which are distributed throughout the human organism and, on the whole, occupy roughly the volume of the spleen. MC have long been recognized as key cells of type I hypersensitivity reactions. Several lines of evidence, however, indicate that they not only express critical effector functions in classic IgE-associated allergic disorders, but also play important roles in host defence against parasites, bacteria and perhaps even viruses. Indeed, it is now clear that MC can contribute to host defence in the context of either acquired or innate immune responses through the release of a myriad of pro-inflammatory and immunoregulatory molecules and the expression of a wide spectrum of surface receptors for cytokines and chemokines. Moreover, there is growing evidence that MC exert distinct non-immunological functions, playing a relevant role in tissue homeostasis, remodeling and fibrosis as well as in the processes of tissue angiogenesis. In this review, we provide a small insight into the biology of human MC and their potential implications in clinical pathology.

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The subcellular distribution of doxorubicin was evaluated in living non-fixed LLC-PK1 cells, which maintain the structural and functional characteristics of the kidney proximal tubule epithelium and also express P-glycoprotein. After 10 min incubation, doxorubicin fluorescence was detectable in the nucleus. The intensity of nuclear fluorescence progressively increased, reaching the maximum at the end of the first hour. Then, the nuclear signal started to decrease and, at 2 h, doxorubicin fluorescence disappeared almost completely from the cell nucleus. Cytoplasmic fluorescent vesicles first appeared in the perinuclear region after 10 min doxorubicin exposure and increased in number and size over a period of 2 h. From 2 to 5 h, fluorescent vesicles moved unidirectionally to the cell periphery. Disappearance of doxorubicin punctate fluorescence in LLC-PK1 cells treated with methylamine or monensin demonstrated that drug accumulation occurred inside acidic compartments. In addition, the cytoplasmic pattern of doxorubicin fluorescence was very similar to that observed upon exposure to the acidotropic tracer LysoSensor Blue. Involvement of P-glycoprotein in doxorubicin handling by LLC-PK1 cells was suggested by modified intracellular doxorubicin distribution after cell incubation with verapamil and vinblastine. Moreover, the fluorescent P-glycoprotein substrate Bodipy FL Verapamil was shown to accumulate in LLC-PK1 cells in a manner that is quite similar to that observed for doxorubicin. P-glycoprotein expression was evaluated by immunoblot using the JSB-1 and C219 monoclonal antibodies. Immunofluorescence analysis was performed using the JSB-1 monoclonal antibody. P-glycoprotein immuno-reactivity was found both on the plasma membrane and intracytoplasmically in a perinuclear position. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed that MDR1 gene was expressed. This study indicates that a rapid intracellular redistribution accompanies the process of doxorubicin uptake by LLC-PK1 cells. Although these cells are non-tumour cells derived from the normal epithelium of the proximal renal tubule, they display a model of doxorubicin redistribution which is characteristic of doxorubicin-resistant tumour cells.

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The process of exocytosis was studied in rat and beige mouse peritoneal mast cells stimulated by adriamycin (ADR) at 37 degrees C and 22 degrees C. ADR induces a non cytotoxic histamine release that is followed by a significant uptake of the drug. Examination was performed by transmission electron microscopy and, at the same time, histamine release and ADR uptake were measured by spectrofluorimetry. ADR accumulation in mast cells was investigated also by fluorescence microscopy. In rat mast cells stimulated at 37 degrees C, the secretory process developed abruptly and was virtually complete after 30 sec. Electron microscopy showed rapid intracytoplasmic channel formation and extrusion of secretory granules; spectrofluorimetry revealed a massive release of histamine and rapid uptake of ADR. In addition, fluorescence microscopy showed mast cells exhibiting an intense orange-yellow fluorescent signal localized at the secretory granules. At 22 degrees C, rat mast cells showed alteration of the granules, cavity formation by fusion of the perigranular membrane and granule discharge due to fusion of the cavity membrane with the cell membrane. Histamine release and ADR uptake proceeded less quickly than at 37 degrees C. Quantitative analysis of rat mast cell ultrastructure demonstrated that histamine release induced by ADR stimulation was achieved by sequential exocytosis. This process presents both morphological and biochemical affinities with the exocytosis induced by basic secretagogues such as compound 48/80. In beige mouse mast cells the process of exocytosis progressed more slowly and was completed after 20 min at 37 degrees C. By electron microscopy, the cytoplasm presented a rigid structure due to abundance of actin-like fibrils. Granule fusion was an uncommon feature and exocytosis was mostly the result of single granule opening to the cell exterior without extrusion of granule matrices.

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Mast cells stimulated with adriamycin at 4 degrees C underwent a unique exocytotic reaction. Rat peritoneal cells including mast cells were stimulated in vitro with adriamycin (100 micrograms/ml) for 0, 10, 30 or 60 sec and observed by transmission electron microscopy. Early changes could be observed after 10 sec stimulation and consisted in an approximately 5-fold increase (p < 0.001) of 0.05-0.2 micron diameter cytoplasmic vesicles. The Golgi apparatus showed signs of activation and vacuolization. From 10 to 30 sec, cytoplasmic vesicles fused with the perigranular membranes and with the membranes of developing secretory channels. At 60 sec, the number of vesicles and vacuoles diminished to nearly two-fold starting levels. The exocytotic reaction characteristically resulted in the formation of enormously dilated granular cavities. The secretory process appeared incomplete; after 60 sec, in fact, maximal histamine release was 20% and exocytosis could be found in approximately 30% of mast cells. Pre-incubation with vinblastine followed by adriamycin stimulation at 37 degrees C determined a dose-dependent inhibition of histamine release which was accompanied by the ultrastructural appearance of numerous 0.05-0.5 micron cytoplasmic vesicles and by signs of inhibited exocytosis. Our results support the concept that hyperstability of the cortical cytoskeleton coupled with microtubule perturbation would be responsible for the depressed pattern of mast cell exocytosis observed at 4 degrees C. Although stimulation at 4 degrees C induces a paradoxal secretory process, we believe that this approach may represent a useful model for understanding some basic mechanisms of exocytosis in mast cells.

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The sinus endothelial cell architecture has been studied in the mouse lymph node by light and electron microscopy after tissue fixation with an osmium-zinc iodide mixture. This histochemical method allowed visualization of sinus endothelial cells in the medullary and, partly, in the intermediate sinuses. The osmium-zinc iodide reaction also demonstrated fibroblastic reticular cells, which represent the main stromal cell population in the lymph node and form the skeletal framework of the organ as well as the adventitia of sinuses. In the mouse lymph nodes, sinuses were lined by a simple layer of sinus endothelial cells whose integrity and continuity showed interruptions of various extension. In areas devoid of complete sinus endothelial cell lining, fibroblastic reticular cells located in the nearby parenchyma were able to reach the border of the sinus, being thus in direct contact with the lumen content. Dendritic sinus endothelial cells as well as intermediate forms between sinus endothelial cells and fibroblastic reticular cells could be observed. The close structural characteristics that sinus endothelial cells have in common with fibroblastic reticular cells and the finding of transitional cells with intermediate morphology between sinus endothelial cells and fibroblastic reticular cells, suggest a possible origin of sinus endothelial cells by migration and differentiation of fibroblastic reticular cells located in the sinus adventitia.

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The characteristics of doxorubicin handling have been studied in the cultured kidney epithelial cell line LLC-PK1, which has structure and function similar to those of renal tubular cells and expresses P-glycoprotein. The uptake of doxorubicin by LLC-PK1 cells was time dependent, reaching a steady state at about 4 hr, and reduced at low temperature; the initial uptake was saturable. The efflux of doxorubicin from LLC-PK1 cells was also temperature dependent but, even at 37 degrees C, a significant percentage of the drug remained associated with the cells after 180 min, which suggests a strong cellular binding, and the fluorescence microscopy revealed that the drug was concentrated in intracellular organelles. Substances that are substrates for P-glycoprotein, such as verapamil, vinblastine, vincristine and quinidine, significantly increased doxorubicin concentrations in LLC-PK1 cells. Similar results were obtained with the metabolic inhibitors sodium metavanadate and 2,4-dinitrophenol. On the other hand, the uptake was not affected by the classic organic cation transport drugs cimetidine, decynium 22 or decynium 24, nor by the organic anion drug probenecid. These results indicate that, in LLC-PK1 cells, doxorubicin enters by passive diffusion, is trapped in intracellular organelles and then is extruded from cells by a mechanism that probably involves P-glycoprotein. On the contrary, substances that interfere with the renal organic cation or anion secretory system have no effect on doxorubicin net accumulation in these cells.

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Rat and beige mouse peritoneal mast cells, induced to exocytose with the antineoplastic agent adriamycin, extrude their granule remnants in the extracellular medium. These granules are loaded with the fluorescent drug adriamycin and exhibit intense yellow-reddish fluorescent staining. Granules extruded from mast cells were ultimately phagocytosed and could be observed inside the macrophages by fluorescence microscopy. All stages of the internalization process could be followed by electron microscopy. Granules adhering to the cell surface of macrophages were first embraced by short superficial projections, then enveloped by deep surface infoldings, and finally engulfed into the macrophage cytoplasm. Phagocytosis occurred exclusively in macrophages; granules were observed also on the surface of eosinophils and lymphocytes, but never inside these cells. The concentrations of adriamycin in macrophages, measured by spectrofluorimetry, were significantly higher when these cells were incubated with adriamycin and granule remnants in comparison with adriamycin alone. Preincubation with the endocytosis inhibitor cytochalasin B significantly reduced the granule mediated adriamycin uptake. As a consequence of the phagocytosis of adriamycin loaded mast cell granules, macrophages can concentrate the antineoplastic drug. These cells act as reservoirs of adriamycin and could have an important role in both the antitumor and toxic effects of the drug.

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Light (LM) and electron microscopy (EM) were used to investigate the structural relationship between enteric nerves and the population of immune cells in the mouse small bowel. By LM, the osmium-zinc iodide procedure was used for visualizing nerve fibers; the incidence of nerve-plasma cell contacts in the mucosa and submucosa was calculated to be approximately 4 times and, respectively, 3 times greater than would be expected by chance alone (P < 0.0001). EM revealed close, synaptic-like contacts between axonal varicosities and plasma cells or B immunoblasts. The results presented here provide systematic quantitative evidence that a structural foundation for communication between nerve fibers and B cells exists in the mouse small bowel.

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The organisation of the stromal cell compartment in the mouse lymph node was studied by light and electron microscopy after tissue impregnation by the zinc iodide-osmium (ZIO) method. Fibroblastic reticular cells (FRCs) represented the main stromal cell population. These cells were located both in the cortical region and in the medulla and exhibited various configurations. In the cortex, FRCs were fusiform in shape and came into close proximity with the floor of the subcapsular sinus. In the medulla, the FRCs were shaped like irregular dendritic cells which formed a complex 3-dimensional network. The FRCs surrounded vascular structures such as capillaries and/or high endothelial venules; in these instances they were organised in a discontinuous sheath-like fashion around the vessel wall. By light and electron microscopy, FRCs have been observed to come in close spatial relationship with a number of cells in the lymph node, including sinus endothelial cells, the endothelium of high endothelial venules and capillaries, various types of lymphocytes, follicular dendritic cells and interdigitating cells. These microanatomical features are consistent with the proposal that FRCs may be involved in the communicative networks between the different lymph node compartments. In particular, the FRCs may be involved in the transport of molecules from the sinus compartment to the high endothelial venules or to the distinct cell populations in the lymphoid parenchyma.

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Intraepidermal free nerve endings were investigated in the mouse snout skin by means of an immunohistochemical procedure using a rabbit antiserum against protein gene product 9.5 (PGP 9.5). Immunoperoxidase reactivity was detected in different subtypes of intraepidermal nerves and cells. The great majority of axons observed in the stratified epithelium were varicose; a small percentage was either smooth (non-varicose) or irregularly shaped. Intraepidermal nerves ended at different levels within the epidermis, often with a terminal knob-like swelling. Various patterns of intraepidermal innervation could be distinguished. Most fibres entering the epidermis originated from large bundles running a horizontal course below the dermo-epidermal junction. Such fibres ascended vertically through the stratified epithelium in a "candelabrum-like" fashion, without emitting collaterals. Other fibres branched profusely and ended in complex intraepidermal neural networks. Less frequently, intraepidermal fibres terminated with large irregularly shaped expansions of different morphologies. Some of these were the intraepidermal continuations of axons within Meissner's corpuscles. Some fibres appeared to come into contact with PGP 9.5-immunoreactive cells (which closely resembled Merkel cells) located in the stratum basale. Rare suprabasal dendritic cells (Langerhans cells?) also became visible.

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BACKGROUND: The mdr-1 gene, which codes for a 170-kd transmembrane glycoprotein (P170), is frequently overexpressed in multidrug resistant (MDR) tumor cell lines and in spontaneous tumors, including leukemia and lymphoma. However, it is also constitutively expressed as a normal gene in normal tissues. METHODS: We used human mdr-1 cDNA and three anti-P170 monoclonal antibodies (MoAbs: MRK-16, C-219 and JSB-1) to investigate the normal peripheral blood leukocyte content of mdr-1 specific mRNA and of P170 through immunocytochemistry and flow cytometry. RESULTS: We did not find any increase in mdr-1-specific mRNA, while small amounts of P170 were easily detectable in about two thirds of the lymphocytes and monocytes and in about one third of the granulocytes. The level of P170 expression in leukocytes was similar to that found in non-MDR tumor cell lines. CONCLUSIONS: mdr-1 is constitutively expressed in human normal leukocytes at levels that cannot significantly affect drug resistance. Accordingly, low-level mdr-1 expression in leukemic cells should not be considered a label of pleiotropic drug resistance.

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The distribution of serotonin immunoreactivity in the mouse cerebellar cortex was studied using the indirect antibody peroxidase-antiperoxidase (PAP) technique of Sternberger (1979) on epoxy embedded semithin sections. The great majority of serotonin-positive afferents distribute throughout the Purkinje cell layer and form dense synaptic contacts with the somata of the Purkinje neurons. Only a sparse immunostaining of serotoninergic fibres could be detected at the granular cell and molecular layers. The microanatomical organization of the serotoninergic projections to the mouse cerebellar cortex is quite different from that observed in other animal species. These findings suggest that in the mouse cerebellar cortex, the Purkinje cell population represents the main target for serotoninergic afferents. Our histochemical data provide morphological support for a series of electrophysiological observations which indicate serotonin as a potential modulatory neurotransmitter for Purkinje cell firing activity.

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BACKGROUND: Granulocyte-macrophage colony stimulating factor (GM-CSF) can affect the treatment of acute non-lymphocytic leukaemia (ANLL) by supporting normal haemopoiesis and by enhancing the proliferation and the maturation of leukaemic cells. METHODS: A human recombinant E. Coli synthesized GM-CSF was administered to 7 patients with ANLL at a dose of 5 or 10 micrograms/kg/day for 7 days, prior to antileukaemic treatment. Peripheral blood neutrophil and blast cell count was monitored daily. Changes in marrow blast cell morphologic features, mitotic index, and the reactivity to a panel of monoclonal antibodies were assessed after 3 and 7 days of treatment. RESULTS: Peripheral blood neutrophil and blast cell count increased in 6/7 cases. The mitotic index of marrow cells increased in 6/7 cases. A significant maturation occurred along the granulocytic line (2 cases) and the monocytic line (1 case). Mild to moderate eosinophilia developed in 3/7 cases. Early stem cell markers (CD 34 and HLA-DR) were not lost, or actually increased. Myelomonocytic markers (CD 33, CD 13, and CD 14) rose and fell. Expression of the multidrug resistance-associated 170 kd glycoprotein remained stable. CONCLUSIONS: These data showed that in vivo GM-CSF more consistently enhanced the proliferation than the maturation of leukaemic cells.

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The effect of the monovalent carboxylic ionophore monensin, which mediates a one-for-one exchange of intracellular H+ for extracellular Na+, was investigated in purified rat peritoneal mast cells. Monensin inhibited histamine secretion induced by compound 48/80, adriamycin and the calcium ionophore A23187; the inhibitory effect was maximal when the compound was added at least 10 min before the secretagogues. Washing of cells before addition of the secretagogues did not abolish the inhibitory effect of monensin. On the contrary the carboxylic ionophore was completely ineffective in preventing concanavalin A-induced histamine release. When rat peritoneal mast cells were incubated in the presence of monensin for longer period (up to 5 hours), the substance induced a slow, progressive and dose dependent histamine release, which, at least for lower doses was noncytotoxic. The secretory effect of monensin was still present if the ionophore was washed away after 10 min of incubation, and the incubation continued in drug-free medium. Monensin stimulated histamine secretion was strictly dependent on extracellular Na+ concentrations, and independent on extracellular Ca++.

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A close microanatomical relationship between serotonin-positive mast cells and nerve fibres positive for substance P, calcitonin gene related peptide, vasoactive intestinal polypeptide, and somatostatin has been observed in whole-mount preparations of rat mesentery by an immunofluorescent double-staining procedure. Peptidergic fibres have been shown either to run in close proximity or come in direct contact with mast cells. This supports earlier morphological and immunohistochemical results suggesting an innervation of mast cells and provides a structural foundation for a series of pharmacological studies which outline the influence of various neuropeptides on mast cell secretory activity.

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It has recently been shown that adriamycin is accumulated in mast cells by an active transport system, which closely resembles the outward transport system of multidrug resistant (MDR) cells. The present study was undertaken in order to test the effect of substances which are known to limit or reverse resistance in MDR cells on adriamycin uptake and histamine release in rat peritoneal mast cells. The lysosomotropic amines chloroquine, nicotine, propranolol, atropine, methylamine, ammonium chloride and quinacrine were only slightly effective at very high concentrations; no effect could be observed with the lysosomotropic amine amantadine. The carboxylic ionophores monensin and nigericin were, on the contrary, extremely efficacious; in particular, the effect of monensin was more evident when mast cells were preincubated with the ionophore for 10 or 30 minutes while only a slight inhibition was evident when the two substances were added simultaneously. Removal of monensin from the incubation medium before challenge with adriamycin did not abolish the inhibitory action of the ionophore. Among the tested membrane active agents, Tween 80 and Triton WR-1339 were able to limit adriamycin uptake and histamine release. The microscopical examination showed that in mast cells treated with adriamycin, an intense fluorescence was present in cytoplasmic granules; on the contrary, mast cells preincubated with monensin showed hardly any fluorescence.

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