Stefania Stefanini

Immunohistochemical localisation of PDE5 in Leydig and myoid cells of prepuberal and adult rat testis

Expression of phosphodiesterase 5 (PDE5) in the rat testis at several pre and postnatal developmental stages was investigated by immunohistochemistry. The enzyme was localised in vascular smooth muscle cells, as well as in Leydig and peritubular cells. The latter were identified as myoid, based on their immunoreactivity to desmin and α-smooth muscle actin. The presence of PDE5 in myoid cells was confirmed by Western blot analysis and immunohistochemistry performed on highly purified cell fractions, obtained from 16-day-old rats. The expression of PDE5 in these somatic cells of rat testis is discussed in view of the roles played by cGMP signal transduction pathways in the mammalian male reproductive function.

Presence and inducibility of peroxisomes in a human glioblastoma cell line.

We investigated the effect of the peroxisomal proliferator (PP) perfluorodecanoic acid (PFDA), alone or in combination with 9-cis-retinoic acid (RX) on the human glioblastoma cell line Lipari (LI). Cell proliferation, apoptotic rate, peroxisome morphology and morphometry, peroxisomal enzyme activities and the presence of peroxisome proliferator-activated receptors (PPARs) were examined. We show that PFDA alone produces pleiotropic effects on LI cells and that RX enhances some of these effects. Peroxisomal number and relative volume, as well as palmitoyl-CoA oxidase activity and protein, are increased by PFDA treatment, with a synergistic effect by RX. The latter, alone or in association with PFDA, induces catalase activity and protein, increases apoptosis and decreases cell proliferation. PPAR isotypes alpha and gamma were detected in LI cells. While the former is apparently unaffected by either treatment, the latter increases in response to PFDA, independent of the presence of RX. The results of this study are discussed in terms of PPARalpha activation and PPARgamma induction by PFDA, by either a direct or an indirect mechanism.

Biogenesis of peroxisomes in fetal liver

Peroxisomes are single membrane‐limited cell organelles that are involved in numerous metabolic functions. Peroxisomes do not contain DNA; the matrix and membrane proteins are encoded by the nuclear genome. It is assumed that new peroxisomes are formed by division of existing organelles. The present article gives an overview of microscopic studies and recent unpublished results dealing with peroxisome biogenesis in mammalian fetal liver and presents data on peroxisomes in oocytes.

DEHP effects on histology and cell proliferation in lung of newborn rats

Di-(2-ethylhexyl)-phthalate (DEHP), the plasticizer employed in the fabrication of polyvinyl chloride, is known to be released by many medical devices, namely endotracheal tubes currently utilised for pulmonary ventilation of pre-term newborns. When experimentally administered, especially to rodents, the phthalate reportedly causes alterations to several tissues, immature animals being even more responsive targets than adult ones. In the present research, female rats were fed with DEHP in the last week of pregnancy and after delivery, and lung of their pups was morphologically and immunohistochemically analysed. We detected significant alveolar simplification (larger but fewer alveoli with decreased septation), with consequent sensible reduction of gas-exchange surface, at several stages of postnatal development, in distal lung parenchyma of DEHP-treated rats. Moreover, the quantification of PCNA-expressing cells demonstrates that in treated pups the proliferation rates of epithelial and mesenchymal cells progressively increased during the first two postnatal weeks, at difference with controls animals, where the highest proliferation levels were reached at postnatal day 7. The obtained results strongly support the hypothesis that DEHP profoundly affects the alveolarization process in mammalian lung.

Immunohistochemical Localization of Peroxisomal Enzymes During Rat Embryonic Development

Peroxisomes are cytoplasmic organelles involved in a variety of metabolic pathways. Thus far, the morphological and biochemical features of peroxisomes have been extensively characterized in adult tissues. However, the existence of congenital peroxisomal disorders, primarily affecting tissue differentiation, emphasizes the importance of these organelles in the early stages of organogenesis. We investigated the occurrence and tissue distribution of three peroxisomal enzymes in rat embryos at various developmental stages. By means of a highly sensitive biotinyl-tyramide protocol, catalase, acyl-CoA oxidase, and ketoacyl-CoA thiolase were detected in embryonic tissues where peroxisomes had not thus far been recognized, i.e., adrenal and pancreatic parenchyma, choroid plexus, neuroblasts of cranial and spinal ganglia and myenteric plexus, and chondroblasts of certain skeletal structures. In other tissues, i.e., gut epithelium and neuroblasts of some CNS areas, they were identified earlier than previously. In select CNS areas, ultrastructural catalase cytochemistry allowed identification of actively proliferating organelles at early developmental stages in several cell types. Our data show that in most organs maturation of peroxisomes parallels the acquirement of specific functions, mainly related to lipid metabolism, thus supporting an involvement of the organelles in tissue differentiation.

Morphometric analysis of liver and kidney peroxisomes in lactating rats and their pups after treatment with the peroxisomal proliferator di‐(2‐ethylexyl)phthalate

Summary— Di‐(2‐ethylexyl)phthalate (DEHP) administered to adult lactating rats from delivery to weaning induces age‐ and organ‐specific modifications of the peroxisomal morphometric parameters (VV, NA and D) in the liver and kidney of both rats and their pups. In both tissues, peroxisomal relative volume and catalase biochemical activity show a similar pattern during the development, as well as under DEHP treatment. Morphometric results suggest that two modalities of peroxisomal proliferation exist, involving: a) increases in both number and mean diameter of the organelles; b) a purely numerical increase of the organelles, accompanied by a remarkable decrement in their mean diameter. A peroxisomal population proliferated through the latter model appears unable to return to normal conditions, following treatment withdrawal. These two proliferation systems, the first implying a swelling and the latter a fragmentation of pre‐existing peroxisomal profiles, are supposed to be tissue‐specific in the adult animal. In particular, in the liver the ‘swelling’ model appears more suitable to explain peroxisome proliferation, while in the kidney this process would follow the ‘fragmentation’ model. Immature animals might instead show in both organs intermediate features of peroxisomal proliferation modalities.

Proliferative response of foetal liver peroxisomes to clofibrate treatment of pregnant rats. A quantitative evaluation

Liver peroxisomes during prenatal development were studied by means of morphological and morphometric analysis in foetuses growing both in untreated and in clofibrate‐treated rats. Pregnant rats were fed a standard diet (25 g/d) containing or not 0.8% clofibrate during the week preceding sacrifice and livers were excised from 13, 15, 17, 19 and 21‐day old foetuses and newborn rats. The morphometric analysis of hepatocyte peroxisomes, performed by a semiautomatic method in specimens incubated with 3,3′ diaminobenzidine, shows that the peroxisome volume density and average diameter in test animals were significantly increased over the control values. While the increase in the average diameter was roughly the same (×1.4) at all examined stages, the volume density increased over the control values particularly in foetuses over 19d‐old and in newborn rats; over the same period the peroxisome numerical density also significantly increased over the control values. Finally, the average diameters of peroxisome profiles in test rats showed a more dispersed distribution (SD 40%) than in control animals (SD 30%). These results demonstrate that clofibrate, given to rats during pregnancy, induces peroxisomal proliferation in the livers of their foetuses. Data are discussed in view of the models proposed for peroxisomal biogenesis.

Liver peroxisomes in newborns from clofibrate-treated rats. II. A biochemical study of the recovery period.

Summary— The fatty‐acyl‐CoAβ‐oxidation (FAO) and catalase activities, as well as membrane fluidity of liver peroxisomes of newborns from normal and clofibrate‐treated rats were studied during the recovery period, ie, throughout the first week of postnatal life. In the test animals the enzyme activities, which are significantly higher than controls at birth return to normal levels showing a somewhat different time course with FAO rapidly decreasing to control values within three days but with catalase still higher than controls at day 6. The half‐life and degradation rate (Kd) of FAO are identical to those calculated by us for the whole organelles and to those reported by others for total catalase in normal or clofibrate‐treated adult animals in the presence of catalase inhibitors. Soluble catalase shows turnover values which are similar though not identical to those of FAO, while total catalase has a very long half‐life and a low Kd. Peroxisomal membrane fluidity, as determined by fluorescence anisotropy of 1‐anilinonaphthalene‐8‐sulfonate (ANS) bound to purified peroxisomal fractions is higher in tests than in controls, recovering normal values within 6 days. Our results demonstrate that liver peroxisomes of rats prenatally exposed to clofibrate return to control conditions within about 1 week. The turnover parameters of enzymes and the membrane fluidity values are discussed in terms of disposal mechanism(s) for the excess of induced peroxisomes.

Maturation of the liver-specific peroxisome versus laminin, collagen IV and integrin expression

The interaction of cells with extracellular matrix components contributes to their specific differentiation. We studied hepatic peroxisomes and their changing features during embryonic development, and we immunolocalized in the same tissue the extracellular matrix components laminin and collagen IV as well as the integrin receptor subunits alpha 1, alpha 2, beta 1, and beta 4. Rat and human embryonic liver peroxisome expression were studied at the light- and electron-microscopic level by means of localizing catalase-, D-amino acid oxidase- and polyamine oxidase activities and by means of the immunocytochemistry of six peroxisomal proteins. The successive import of catalase and the peroxisomal beta-oxidation enzymes, the late appearance of the other enzymes, and the gradual increase of peroxisomal size and number to adult values occurred as asynchronous events. Although still immature, peroxisomes were recognized at every stage examined and coexisted with laminin and collagen IV in both species. The beta 1 integrin subunit was immunodetected as early as at 12.5 days in rat. It was concluded that these extracellular matrix factors may be important for the differentiation of liver parenchyma from the liverbud stage onwards. However, the stepwise maturation of the liver-specific peroxisome suggests the involvement of many other regulating factors.

Interleukin-1-producing plasma cells in close contact with hepatocytes in patients with chronic active hepatitis

BACKGROUND/AIMS In chronic active liver diseases (CALD) with viral aetiology, a population of plasma cells localised in the piecemeal necrosis areas was previously detected by means of autoradiography after in vitro 3H-proline incorporation, a method which proved much more sensitive than conventional immunohistochemical procedures. These plasma cells, characteristically located in niches among hepatocytes, in close contact with collagen fibrils, have been hypothesised to exert a role in fibrogenesis stimulation, and particularly in collagen synthesis, possibly through secretion of lymphokines. Specifically, we investigated the presence of interleukin-1, well known to play a crucial role in inflammation and production of collagen by epithelial cells, and to be present in activated plasma cells of myeloma. METHODS The immunohistochemical localisation of interleukin-1beta in biopsies of patients suffering from chronic active hepatitis was studied, using an affinity-purified rabbit polyclonal antibody. RESULTS The strongest interleukin-1beta immunostaining was observed in the above-described plasma cell population, identified by anti-immunoglobulin antibodies, and 3H-proline incorporation. CONCLUSIONS The ability of plasma cells to produce interleukin-1 during viral CALD suggests that in these pathologies plasma cells play a major role, mainly of paracrine nature. Interleukin-1, possibly together with other mediators, might in turn stimulate the production of collagen. Hepatocytes of the piecemeal necrosis area appear to be possible candidates for this synthesis, as they show a significant labelling after 3H-proline incorporation, which is absent from hepatocytes far from necrotic areas.