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We have previously observed that in vitro co-incubation of rat pre-pubertal Sertoli cells (SC), or their dialyzed/concentrated secretory products with homologous islets, resulted in significant stimulation of the islet beta-cell mitotic index. Aim of the present work was to assess both the specificity and nature of the mechanisms underlying this phenomenon. For this purpose, first we tested astrocytes (AA), separated and purified from the rat brain cortex, where they are known to release a number of growth factors and neurotrophic cytokines, for co-incubation with the islets. However, under the same experimental conditions used for SC, AA did not induce any changes in the beta-cell life cycle, thereby confirming specificity of SC, with respect to induction of beta-cell mitogenicity. For the second purpose, we examined the products of PD-1, a gene located in the cytoplasm of SC, where it promotes spermatogenesis. By blocking the protein encoded by PD-1, under appropriate culture conditions, we observed that the SC-induced increase in beta-cell mitotic activity lost its statistical significance, which suggested a role of PD-1 with respect to SC-related mitogenic properties on beta-cells. These findings corroborate the idea that SC, by either direct contact, or by means of their secretory products, clearly affect the islet beta-cell mitotic rate. Preliminarily, PD-1 gene, located in the cytoplasm of SC, might be one of the factors involved with the induction of beta-cell mitotic activity. In conclusion, SC-induced beta-cell mitotic activity is specific, seemingly mediated by humoral factors whose acting mechanisms have started being unfolded.

ECM macromolecules create a specific environment that participates in the control of cell proliferation and differentiation during embryogenesis. Quantitative and qualitative alterations in the ECM may depend on several growth factors that modify cell metabolism. Since transforming growth factor beta (TGFbeta) and alpha (TGFalpha) are abundantly expressed during embryonic development in organs in which epithelial-mesenchymal interactions occur, the aim of this study was to determine: a) the effect of TGFbeta on the phenotype of 7 and 14 day chick embryo back skin (CEBS) fibroblasts by evaluating the neosynthesis of GAG, collagen and fibronectin; b) whether TGFalpha and TGFbeta production, in particular TGFbeta3 and TGFbeta4, and the number of TGFbeta receptors change during these two stages of embryonic development. The results show that the neosynthesis of ECM macromolecules, tested using radiolabelled precursors, is increased by TGFbeta. The growth factor generally favours cellular accumulation more than secretion. As far as GAG is concerned, TGFbeta has a greater stimulatory effect on sulphated GAG than on HA. Specific bioassay shows that TGFbeta3 and TGFbeta4 activity is higher in 7 day than 14 day CEBS fibroblasts. Moreover, TGFbeta3 and TGFbeta4 mRNA expression is increased in the first stages of development. Instead, the level of TGFalpha increases in successive developmental stages. Since TGFalpha stimulates the synthesis and secretion of HA, and HA binds and inactivates TGFbeta, the greater quantity of HA in 14 day fibroblasts may contribute to reducing the TGFbeta effect. Overall our data suggest that the production of TGFbeta and TGFalpha are age-dependent and that the balance between the two growth factors may be a mechanism for controlling skin differentiation.

The growth regulatory activity of transforming growth factor beta (TGFbeta) on chick embryo skin fibroblasts was compared in two developmental ages, days 7 and 14. The time course of 3H-thymidine incorporation, an S-phase marker of replication, was determined during 36 hr of TGFbeta treatment. Seven-day-old cells showed a prereplicative phase of 6 hr, and 14-day-old cells showed a prereplicative phase of 12 hr. DNA synthesis peaked at 24 hr in 7-day-old fibroblasts and was 10 times higher than that in 14-day-old fibroblasts. Ornithine decarboxylase (ODC) activity and content of the natural polyamines spermine (Spm), spermidine (Spd), and putrescine (Put) differed during cell cycle. ODC activity peaked at 12 hr in 7-day-old cells and at 6 hr in 14-day-old cells. Its level was two times higher at day 7 and was associated with a greater content of ODC mRNA. The maximum of polyamine (PA) concentration was determined after 12 hr of treatment in 7-day-old cells and after 36 hr in 14-day-old cells. These findings indicate that the TGFbeta proliferative response of embryo fibroblasts changes during development and is associated with activation of the ODC/PA system. Cotreatment with alpha-difluoromethylornithine, an enzyme-activated irreversible inhibitor of ODC, did not reduced growth rate. Inhibition of ODC resulted in levels of Put and Spd comparable to that of quiescent fibroblasts, whereas Spm concentration remained higher. Because an altered ODC metabolism does not convey the effects of TGFbeta on DNA synthesis, the ODC/PA system may not play a role in the pathway of TGFbeta signaling.

All adenoviruses transform rodent cells in vitro, but only cells transformed by serotypes belonging to subgroups A (Ad12) and B (Ad3) are tumorigenic for immunocompetent animals. In these cells, the expression of major histocompatibility complex (MHC) class I antigens is repressed and might allow them to escape from recognition by cytotoxic T lymphocytes and to develop in tumor. Furthermore, these cell lines appear resistant to lysis by natural killer (NK) cells. To determine the E1A domain(s) responsible for these properties several cell lines were created by transforming baby rat kidney cells with a set of plasmids expressing different Ad2/Ad12 hybrid E1A gene products. The class I gene expression was inhibited in cells expressing the Ad12 13S mRNA product and in cells transformed with Ad2/Ad12 hybrid E1A gene product harboring the C-terminal part of the conserved region (CR) 3 of Ad12. Susceptibility of these transformed cell lines to NK cells was determined by cytolytic assays. The results obtained suggest that two of Ad12 E1A domains are required to induce resistance of the cell lines to NK cells.

This paper investigates the ability of macrophages and of non-typically phagocitic cells such as fibroblasts to internalize 51Cr-labelled C. albicans in presence or in absence of lectin concanavalin A (Con A). The results obtained demonstrate that fibroblasts are also able to internalize C. albicans and that this property is potentiated by the presence of Con A. Lectin modifies only the phenotype of the fibroblast, which, poorly attached to the substrate, is globular in shape. Despite reduced cellular spreading, phagocytosis is stimulated by the lectin. In both cell populations, changes in the organization of some cytoskeletal proteins such as tubulin, actin and alpha-actinin are evident during the C. albicans infection; such rearrangements are more evident and longlasting in the fibroblasts treated with Con A.

In the present study, we demonstrate that both interleukin-1 (IL-1) and interleukin-6 (IL-6) induced a significant decrease in glycosaminoglycan (GAG) synthesis and, more strikingly, secretion by 7 and 13 day-old chick embryo skin fibroblasts. We demonstrated that interleukin treatment also inhibited the synthesis of collagenase-digestible proteins (type I collagen). In addition, tissue culture supernatants (conditioned media, CM) were tested for reactivity for IL specific ELISAs and for their ability to stimulate proliferative responses in mouse thymocytes and hybridoma cells. Our findings demonstrate that chick embryo skin fibroblasts spontaneously produce IL-1 and, in even greater amounts, IL-6. Highest levels of interleukin secretion were found in the CM of 13 day-old fibroblasts and the IL-1 beta isoform was predominant over IL-1 alpha. Pretreatment of the fibroblasts with either IL-1 or IL-6 increased the secretion of both cytokines. Increased IL-1 levels were correlated with enhanced IL-1 bioactivity in the CM of IL-6 treated fibroblasts. By contrast, the raised concentrations of IL-1 in the CM of IL-1 treated cells and IL-6 in the CM of IL-1 or IL-6 treated fibroblasts failed to translate into augmented bioactivity. These observations, taken together, indicated that IL-1 and IL-6 are able to regulate the synthesis and secretion of ECM macromolecules of developing connective tissues and the cytokine release by chick embryo skin fibroblasts.(ABSTRACT TRUNCATED AT 250 WORDS)

We have investigated the action of sulphated glycosaminoglycans (GAGs) upon primary 11-day chick embryo skin fibroblasts cultured for various periods of time in the presence or absence of fetal calf serum (FCS). Chondroitin 4- and 6-sulphate (CS) added to serum supplemented medium provoked a decrease in DNA synthesis both in sparse and crowded cultures only at high concentrations (250 micrograms/ml). Synthesis of endocellular and secreted proteins was not affected. In contrast, CS administered to fibroblasts for 24 h in serum-free medium stimulated DNA synthesis. We postulate that the CS stimulating effect seen in serum-free cultures might be antagonized by peptide regulators of cell growth normally present in serum.

Conditioned medium (CM), collected from 7 and 14 days-old chick embryo skin fibroblasts and added to the same cells, increases glycosaminoglycans (GAG) intra- and extracellular accumulation. The factors responsible for GAG enhancement are TGF alpha and TGF beta because they are trypsin and dithiothreitol sensitive, stable or enhanced by heat and transient acidification. Moreover, Sephadex G-75 fractions of CM active on GAG synthesis contain, when analysed on SDS-polyacrylamide gel electrophoresis, two bands that comigrate with TGF alpha and TGF beta and induce NRK cells clone 49F to form large colonies of mean size8.000 microns 2 in soft agar. Since both the factors must be present to induce the formation of large colonies we come to the conclusion that CM contains TGF alpha and TGF beta. The two growth factors have different effects on the accumulation of individual classes of GAG in the ECM. In particular, TGF beta stimulates a marked increase of CS and DS, TGF alpha of HA and DS in the medium. The contemporaneous addition of TGF alpha and TGF beta to 7 days-old fibroblasts produces a pattern of GAG response similar to CM. These embryonic fibroblasts may control their own GAG synthesis and secretion through autocrine TGF alpha and beta activity.

The administration of lectin concanavalin A (Con A) to in vitro cultures of chick embryo fibroblasts caused dose-dependent changes in cell shape, cytoskeleton network and DNA synthesis. After 6 hrs. even a low doses of Con A produced evident effects in serum-free cultures, whereas higher doses were required to cause alterations in cells cultured in serum-added medium. As the sugar competitor mannoside abolishes the effects, it would seem that the lectin acts by binding to transmembrane receptors and that the fibronectin present in the serum engages the receptors so that they are not available to Con A.

The microfilaments and microtubules of cultured chick embryo skin fibroblasts were studied in the presence of exogenous lectins by an indirect immunofluorescence technique. Lectin treatment induced modifications in the arrangement of myosin, actin and tubulin, determined depolymerization of the proteins and caused changes in cell shape and size. The results suggest that the interaction between lectins and their specific membrane receptors triggers a series of changes in the cytoskeletal pattern via transmembrana as yet unknown mechanisms and that these are responsible for the alterations in cell shape and size.

The present research investigated the time required for reconstructing normal microtubular pattern and the phenotype of cells after short-term (30 min.) and long-term (24 hrs.) pretreatment with the lectin concanavalin A (Con A). Short-term pretreatment led to the formation of incomplete tubules within 2 and 24 hrs. in cells cultured in 199 medium alone. The addition of serum to the medium reversed the globular phenotype and allowed the formation of normal microtubules, even after prolonged pretreatment with Con A. Whereas long-term Con A treatment provoked a reduction in DNA synthesis in 199 alone, in serum-added 199 the percentage of 3H-thymidine incorporation of treated cells tended to reach that of controls over time.