Franco Mallardi

Suggestive evidence for a microanatomical relationship between mast cells and nerve fibres containing substance P, calcitonin gene related peptide, vasoactive intestinal polypeptide, and somatostatin in the rat mesentery.

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.

The Fluorescent Probe Bodipy-FL-Verapamil Is a Substrate for Both P-glycoprotein and Multidrug Resistance-related Protein (MRP)-1

Several fluorescent probes have been used in functional studies to analyze drug transport in multidrug-resistant cells by fluorescent microscopy. Because many of these molecules have some drawbacks, such as toxicity, nonspecific background, or accumulation in mitochondria, new fluorescent compounds have been proposed as more useful tools. Among these substances, Bodipy-FL-Verapamil, a fluorescent conjugate of the drug efflux blocker verapamil, has been used to study P-glycoprotein activity in different cell types. In this study we tested by fluorescent microscopy the accumulation of Bodipy-FL- Verapamil in cell lines that overexpress either P-glycoprotein (P-gp) or multidrug resistance-related protein 1 (MRP1). Expression of P-gp and MRP1 was evaluated at the mRNA level by RT-PCR technique and at the protein level by flow cytometric analysis using C219 and MRP-m6 monoclonal antibodies. Results indicate that Bodipy-FL-Verapamil is actually a substrate for both proteins. As a consequence, any conclusion about P-gp activity obtained by the use of Bodipy-FL-Verapamil as fluorescent tracer should be interpreted with caution.

Stromal cell organisation in the mouse lymph node. A light and electron microscopic investigation using the zinc iodide‐osmium technique

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.

Kinetics of doxorubicin handling in the LLC-PK1 kidney epithelial cell line is mediated by both vesicle formation and P-glycoprotein drug transport

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.

Recombinant human alpha‐2a interferon promotes an atypical process of mast cell secretion with ultrastructural features suggestive for piecemeal degranulation

Purified mast cells (MC), isolated from the rat peritoneum, were stimulated in vitro with recombinant human IFN‐α 2a (rhIFN‐α 2a) and studied ultrastructurally. Quantitative determination of histamine release was also performed. The following ultrastructural features were observed: (1) dilation of single granules, leading to the formation of cytoplasmic cavities filled with dissolved or eroded matrices; (2) induction of partially empty, non‐fused granule containers close to unaltered resting granules, a process very suggestive of piecemeal degranulation; (3) focal exocytosis, characterized by opening of single granules to the cell exterior and/or fusion of a few granules into small secretory channels. Histamine release was slightly increased in rhIFN‐α 2a‐treated MC, although not to significant levels. These results indicate that rhIFN‐α 2a induces a characteristic pattern of degranulation in rat peritoneal MC and that a small proportion of rhIFN‐α 2a‐stimulated MC shows ultrastructural features suggestive for piecemeal degranulation.

Granule changes of human and murine endocrine cells in the gastrointestinal epithelia are characteristic of piecemeal degranulation

Piecemeal degranulation is a unique pattern of cell secretion that consists of a slow release of granule contents from cytoplasmic secretory granules, which leaves empty chambers that do not fuse with each other or with the plasma membrane. To our knowledge, no cell types other than mast cells, basophils, and eosinophils have been reported in the literature to show morphological features of piecemeal degranulation. In the present study we provide evidence for ultrastructural morphologies characteristic of piecemeal degranulation in entero‐endocrine cells of the human and murine gastrointestinal epithelia. Human biopsy samples were taken from the mucosa of the distal duodenum, proximal jejunum, and colon in 10 patients undergoing endoscopic examination for malabsorption, diarrhea, and/or abdominal pain. Murine gastrointestinal samples were obtained from 10 adult C57 mice. All specimens were prepared for transmission electron microscopy (TEM) according to standard protocols. Results showed that different types of gastrointestinal entero‐endocrine cells, in both humans and mice, were recognizable with ultrastructural features diagnostic for piecemeal degranulation, including specific granule and cytoplasmic changes. In the granules, the content was found to be loosely packed or diminished. Notably, altered granules did not fuse with each other or with the plasma membrane, and were characteristically intermingled with normal, resting granules. At times, the release events transformed the granules into enlarged, empty containers. Numerous entero‐endocrine cells presented a rich supply of membrane‐bound vesicles (50–200 nm in diameter) that were free in the cytoplasm or attached to granules. This finding of piecemeal degranulation in gastrointestinal entero‐endocrine cells suggests that such a secretory model might be a general degranulation pattern in cells involved in paracrine‐endocrine secretion. Anat Rec 268:353–359, 2002.

Apposition of enteric nerve fibers to plasma cells and immunoblasts in the mouse small bowel

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.

Amelioration of 4'-Epidoxorubicin-induced cardiotoxicity by sodium cromoglycate.

Epirubicin induces an important noncytotoxic release of histamine from rat peritoneal cells in vitro. This exocytotic response is inhibited by sodium cromoglycate, similarly to that elicited by the classic mast cell secretagogue, compound 48/80. Mast cells obtained from the peritoneal cavities of rats treated with epirubicin in vivo were extensively degranulated; in contrast, samples obtained from rats pretreated with sodium cromoglycate showed normal appearing mast cells. When injected i.p., immediately before the antineoplastic agent, cromolyn significantly improved the survival time and the microscopic appearance of myocardial tissues of epirubicin-treated mice. The results indicate that histamine release could play an important role in the pathogenesis of anthracycline-induced cardiotoxicity.

Serotoninergic fibres form dense synaptic contacts with Purkinje cells in the mouse cerebellar cortex — An immunohistochemical study

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.

Effect of the carboxylic ionophore monensin on histamine release from rat peritoneal mast cells

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++.