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Biomedical subjects

L Formigli

Publications and source records attributed to L Formigli.

At least 37 records · Page 2Linked to original sources
PubMed1997

Heterogeneity of binding sites and bioeffects of raloxifene on the human leukemic cell line FLG 29.1.

The benzothiophene divarative raloxifene is known to mimic estrogen in human bone remodeling. To investigate the "in vitro" properties of raloxifene on osteoclast precursors, the human leukemic cell line FLG 29.1, which differentiates toward the osteoclastic phenotype, was examined for raloxifene binding and for evidence of its bioeffects. Scatchard and Hill analysis of binding data with the tritiated raloxifene demonstrated the presence of two classes of binding sites in both nuclear and cytosol fractions with Kd values of approximately 1 nM and approximately 5 nM, respectively. In addition, analysis of binding data using tritiated 17 beta E2 as ligand at high concentrations (10-40 nM) and either unlabeled 17 beta E2 or raloxifene as competitors gave similar results demonstrating the presence of type II EBS in these cells. Picomolar concentrations of raloxifene significantly (p < 0.05) inhibited cell proliferation. Moreover, the compound at nanomolar concentrations induced a significant dose- and time-dependent increase of progesterone receptor, and activated apoptotic cell death. These findings clearly demonstrate that raloxifene acts as an estrogen agonist in FLG 29.1 cells, acting through the estrogen receptor and, possibly, via multiple cooperative binding component(s).

Apoptosis↗
PubMed1997

Beneficial effects of the 21-aminosteroid U 74389G on the ischemia-reperfusion damage in pig hearts.

21-Aminosteroids (Lazaroids), acting as free radical scavengers and as membrane stabilizers, proved to have beneficial effects in various pathological conditions. In the present study we explored the effectiveness of one of these compounds, U 74389 G, in protecting pigs myocardium against the ischemia-reperfusion damage induced by transient coronary occlusion achieved by clampling the left anterior descending coronary artery. Animals were divided into three groups: control, untreated and treated. Control animals were operated but not subjected to ischemia-reperfusion; the untreated group underwent to ischemia-reperfusion without pharmacological treatment; while the treated group received the aminosteroid (4 mg/kg) before coronary occlusion and at the time of reperfusion. Specimens of myocardial tissue and blood samples were taken for morphological and biochemical studies. In the ischemic-reperfused myocardium of the untreated animals, the dominant morphological features were neutrophil infiltration, intercellular edema and severe swelling of mitochondria. All these alterations, notably neutrophil infiltration, were attenuated by aminosteroid treatment. As for the biochemical findings, the changes in adenine nucleotides and nucleosides levels, thus the reduction of energy charge, were reversed in the treated, but not in the untreated group. Myocardial concentration of malondialdehyde, which was undetectable in the control group, was raised in all the animals after reperfusion, but this effect was significantly less marked with aminosteroid treatment. In addition, the higher myocardial content of ascorbic acid and the reduced serum potential peroxidation exhibited by the treated animals compared with untreated group indicate an enhanced antioxidant protection induced by aminosteroid administration. On the other hand, the serum levels of myoglobin, cardiac troponin I and creatine kinase MB isoenzyme suggest the ability of the aminosteroid to attenuate the modifications of membrane permeability induced by ischemia-reperfusion injury. All these results lead to the conclusion that aminosteroid treatment, at least in the conditions of the present study, is effective in reducing the morphological and biochemical alterations occurring in ischemic-reperfused myocardium.

Adenosine Diphosphate↗
PubMed1997

Insulin-like growth factor-I stimulates in vitro migration of preosteoclasts across bone endothelial cells.

Little is known about the factors and the mechanisms involved in preosteoclast emigration from the vasculature. In this study, an in vitro model of bone endothelial lining was mimicked by culturing bone endothelial (BBE) cells at confluence on a 3-microm pore polycarbonate membranes. Preosteoclastic (FLG 29.1) cells were then added on top of the BBE cell monolayer and 10 nM insulin-like growth factor-1 (IGF-I) was added below the supporting membrane. Scanning and transmission electron microscopy were used to evaluate the chemotactic responses of preosteoclastic FLG 29.1 cells towards the IGF-I generated gradient. IGF-I potently stimulated chemotaxis in the FLG 29.1 cells, as shown by the migration of the preosteoclastic cells across the underlying BBE and through the intercellular junctions between adjacent endothelial cells. Subsequently, FLG 29.1 cells penetrated the pores of the supporting membrane and reached the lower face of the membrane. Thus, IGF-I, which is abundantly present in the bone tissue microenvironment, may play a paracrine role in the recruitment of the circulating preosteoclasts from the vascular compartment into the bone tissue. This in vitro model, which mimicks the in vivo phenomenon of preosteoclast extravasation, should prove useful in elucidating the molecular mechanisms that underlie this process.

Adult↗
PubMed1997

Vitamin E protects human skeletal muscle from damage during surgical ischemia-reperfusion.

PURPOSE: The biochemical and morphological alterations induced in lower limb skeletal muscle by ischemia-reperfusion (I-R) during aortic surgery and the effect of vitamin E pretreatment were investigated. METHODS: Two groups of patients undergoing aortic aneurysm resection, one untreated and one treated with vitamin E, were examined. Quadricep muscle biopsies were taken after induction of anesthesia, at the end of ischemia, and after reperfusion. The malondialdehyde (MDA) content and morphology of biopsies were examined to assess peroxidative processes. RESULTS: Ischemia did not induce an increase in MDA content but did increase neutrophil infiltration in muscle fibers of untreated patients. Reperfusion led to a significant increase in MDA content and to intermyofibrillar edema and mitochondrial swelling. The MDA content was not increased during ischemia and neutrophil infiltration was minimal in vitamin E treated patients. At reperfusion, the MDA content, the ultrastructural injuries and neutrophil infiltration were significantly reduced by the treatment. CONCLUSIONS: Vitamin E is effective in reducing the oxidative muscle damage occurring after a period of I-R.

Aged↗
PubMed1997

HPE cells: a clonal endothelial cell line established from human parathyroid tissue (human parathyroid cell line).

We report the culture and cloning of human endothelial cells derived from parathyroid tissue surgically removed from a patient affected by Multiple Endocrine Neoplasia Type 1 syndrome. These cells, known as HPE, have been isolated and maintained in culture by serial passages for more than 15 months. The clonal cell line grows in a medium containing serum substitutes which favour endothelial cell growth. HPE cells replicate with a mean doubling time of 120 h, showing typical functional and morphological features of endothelial cells, such as uptake of acetylated low density lipoprotein and positive reaction for Factor VIII-Related Antigen. Basic fibroblast growth factor, vascular endothelial growth factor, insulin-like growth factor type I and ascorbic acid stimulate cell proliferation, whereas transforming growth factor beta and heparin act as inhibitory factors. Prostaglandin E2, secretin and epinephrine increased cAMP production, while human parathyroid hormone, histamine and glucagon were inert. Cells were found to express pro-collagen alpha 1 (type I) mRNA. In HPE cells Restriction Fragments Length Polymorphism and PCR analysis did not show allelic loss at chromosome 11q12-13, known to be a typical feature of MEN 1 parathyroid tumors. These cells are the first example of an established normal human clonal cell line with an endothelial phenotype.

Cell Division↗
PubMed1997

Vitamin E prevents neutrophil accumulation and attenuates tissue damage in ischemic-reperfused human skeletal muscle.

Neutrophil accumulation and the consequent production of oxygen-derived free radicals are involved in the pathogenesis of Ischemia-Reperfusion syndrome. In this study we investigated whether a treatment with Vitamin E, which has antioxidant properties, could attenuate the tissue damage by interfering with the influx of neutrophils within the ischemic and reperfused human skeletal muscle. To this purpose, patients undergoing aortic cross-clamping during the surgical repair of aortic abdominal aneurysm were studied as a model of ischemia-reperfusion of the lower limb muscles. Muscle biopsies from the right femoral quadriceps of patients not receiving and receiving Vitamin E pretreatment before surgery were taken: a) after the induction of anaesthesia, as control samples, and b) after a period of ischemia followed by 30 min of reperfusion. The tissue samples were either routinely processed for morphological study and immunohistochemical analysis to detect an altered expression of specific endothelial adhesion proteins, such as E-selectin and ICAM-1. The results obtained showed that Vitamin E administration was able to prevent the accumulation of neutrophils within the ischemic and reperfused muscle. This beneficial effect of Vitamin E was due to its ability to hinder the expression of E-selectin and ICAM-1, molecules known to increase the adhesiveness of endothelium to circulating neutrophils. After treatment with Vitamin E a marked attenuation of the reperfusion injury was also evident. In conclusion, Vitamin E treatment may be considered a valuable tool for protection against the ischemia-reperfusion damage of human skeletal muscle.

Aged↗
PubMed1996

Characterization and function of the receptor for IGF-I in human preosteoclastic cells.

Using a coculture system, we have recently demonstrated that insulin-like growth factor I (IGF-I) is a mediator of preosteoclastic cell migration toward bone-derived endothelial cells. To better characterize the mechanisms of IGF-I action on preosteoclastic cells we evaluated the expression of type I IGFs receptor in the human leukemic cell line, FLG 29.1, which differentiates toward the osteoclastic phenotype following phorbol ester (TPA) treatment. Scatchard analysis of 125I-labeled IGF-I to FLG 29.1 cells revealed the presence of a single high affinity binding site in both untreated and TPA-treated cells with a similar Kd value (0.3 +/- 0.2 nmol/L and 0.4 +/- 0.1 nmol/L, respectively). In untreated cells, IGF-I binding capacity (1.43 +/- 0.41 fmol/10(6) cells) was significantly (p < 0.05) lower than in TPA-treated cells (2.62 +/- 0.87 fmol/10(6) cells). Competition analyses and crosslinking studies revealed the presence of type I IGF receptor both in untreated and TPA-treated cells. Northern analysis demonstrated that mRNA for IGF-I receptor was expressed by both untreated and TPA-treated FLG 29.1 cells. In addition, FLG 29.1 cells released in the conditioned medium IGFBP-2 and IGFBP-4, whose expression was increased by TPA treatment as demonstrated by ligand and immunoblot analyses. The previous observations of chemotactic action of IGF-I on FLG 29.1 cells was confirmed by ultrastructural observations. Indeed, these cells revealed a marked migratory activity in response to nanomolar concentrations of IGF-I. In addition, the IGF-I receptor alpha IR-3 antiserum inhibited the IGF-I-induced FLG 29.1 cell's migratory activity. These findings clearly show that type IIGF receptor is expressed by osteoclast precursors and that IGF-I induces migration of these through the binding to type I IGF receptors. Binding proteins expressed by osteoclast precursors may play an autocrine role in modulating the IGF-I bioeffects.

Adult↗
PubMed1996

Radicular cysts are involved in the recruitment of osteoclast precursors.

In consideration of the close relationship between radicular cysts and alveolar bone, it is important to evaluate the potential involvement of the neighbouring bone tissue in such lesions. In the present study, using cytochemical, immunocytochemical and morphological analyses, presumptive osteoclast precursors were revealed in the connective tissue of radicular cyst capsules. The osteoclastic nature of these cells was postulated by their positive staining for the enzyme tartrate-resistant acid phosphatase (TRAP) and by the expression of vitronectin receptor (VnR) on their cell surface. However, these cells did not express the vacuolar-type proton pump, suggesting that they may represent early osteoclast precursors infiltrating the cyst capsule. Cysts also contained activated small blood vessels whose endothelial cells expressed the VnR. This integrin receptor is important in the adhesion of preosteoclasts to the endothelial lining, a necessary step for their emigration out of the vasculature. Therefore, the intracystic vessels could represent a substrate for preosteoclast recruitment. These precursor cells may then reach the perialveolar bone surface and contribute to bone demolition together with those recruited by the resorbing surfaces. The bone-destroying potential of radicular cysts was confirmed by the presence of numerous osteoclasts with large resorption areas on the perialveolar bone surfaces exposed to the cyst capsules. The resorbed surfaces were usually located around the vascular canals of the Haversian systems.

Acid Phosphatase↗
PubMed1996

Ultrastructural evidence of myocardial alterations in the course of heterotopic heart transplantation.

Using a novel model of heterotopic rat heart transplantation, the present study was undertaken to evaluate whether parenchymal and microvascular alterations of the ischemic and reperfused myocardium occurred and could be related to local neutrophil infiltration. In such a model, hearts were rapidly excised from donor rats, maintained in a cold saline solution at 4 degrees C and then reimplanted in recipient animals. Muscle biopsies of the ischemic and reperfused myocardium were analysed by ultrastructural and immunohistochemical techniques. Although the cold storage of the hearts provided a good protection against the ischemic insults, reperfusion with the recipient blood caused severe myocardial cell injury and microvascular damage. In particular, the microvascular endothelium showed numerous discontinuities due to the partial destruction of endothelial cell. The altered endothelial integrity was associated with aggregation and adhesion of platelets to the luminal surface. Contrary to other models of ischemia and reperfusion, where neutrophils are considered the major source of oxygen radicals and cellular dysfunctions at reperfusion, in our samples the burst of these toxic metabolites did not originate from such cells. In fact, no neutrophils were seen to accumulate within the ischemic as well as reperfused myocardium. Accordingly, the microvascular endothelium did not express E-selectin, an adhesive molecule which is responsible for the increased adherence and emigration of neutrophils in the microvasculature.

Animals↗
PubMed1995

In vitro structural and functional relationships between preosteoclastic and bone endothelial cells: a juxtacrine model for migration and adhesion of osteoclast precursors.

The role of vascularization in the process of bone resorption has not been clarified. The interactions between vascular endothelium and osteoclast progenitors were analyzed using clonal cell lines of bone-derived endothelial and preosteoclastic cells. Insulin-like growth factor I is a major chemotactic stimulator of preosteoclastic cell migration mediated by bone endothelial cells. Osteoclast precursors rapidly adhered to bone endothelial monolayers. This phenomenon appeared to be cell-specific and mediated through the binding of vitronectin and fibronectin receptors to fibronectin. In addition, direct contact with bone endothelial cells induced osteoclast progenitors to differentiate into more mature elements, with the tendency to cluster together to form large multinucleated cells. These findings demonstrated specific in vitro interactions between bone endothelial cells and osteoclast progenitors, offering a new model for understanding the molecular mechanisms which direct the processes of osteoclast recruitment and ontogeny.

Animals↗
PubMed1995

Functional and structural interactions between osteoblastic and preosteoclastic cells in vitro.

Osteoblasts are involved in the bone resorption process by regulating osteoclast maturation and activity. In order to elucidate the mechanisms underlying osteoblast/preosteoclast cell interactions, we developed an in vitro model of co-cultured human clonal cell lines of osteoclast precursors (FLG 29.1) and osteoblastic cells (Saos-2), and evaluated the migratory, adhesive, cytochemical, morphological, and biochemical properties of the co-cultured cells. In Boyden chemotactic chambers, FLG 29.1 cells exhibited a marked migratory response toward the Saos-2 cells. Moreover, they preferentially adhered to the osteoblastic monolayer. Direct co-culture of the two cell types induced: (1) positive staining for tartrate-resistant acid phosphatase in FLG 29.1 cells; (2) a decrease of the alkaline phosphatase activity expressed by Saos-2 cells; (3) the appearance of typical ultrastructural features of mature osteoclasts in FLG 29.1 cells; (4) the release into the culture medium of granulocyte-macrophage colony stimulating factor. The addition of parathyroid hormone to the co-culture further potentiated the differentiation of the preosteoclasts, the cells tending to fuse into large multinucleated elements. These in vitro interactions between osteoblasts and osteoclast precursors offer a new model for studying the mechanisms that control osteoclastogenesis in bone tissue.

Adult↗
PubMed1995

Osteolytic processes in human radicular cysts: morphological and biochemical results.

In an attempt to verify the nature of bone resorption processes on the bone surrounding radicular cysts, fragments of cysts with the adjacent bone tissue were studied by morphological, cytochemical and ultrastructural methods. Cyst fluid was analysed for its content of cytokines with osteolytic activity. The cyst wall exhibited several connective tissue extensions which penetrated the adjacent bone. Numerous multinucleated tartrate-resistant acid phosphatase (TRAP)-positive cells were seen at the tip of the intraosseous extensions of the cyst capsule and in direct contact with the bone tissue. Typical resorption lacunae were identified on the bone surface by scanning electron microscopy. Moreover, mononuclear TRAP-positive cells were seen within the cyst capsule. High levels of prostaglandin E2 (PGE2) and interleukin-6 (IL-6) were detected in the cyst fluids. In conclusion, active bone resorption may contribute significantly to the growth of these lesions within the jaws.

Acid Phosphatase↗
PubMed1994

Multiple pregnancy induction and selective fetal reduction in high genetic risk couples.

Couples at risk for an inherited disorder often have several pregnancy interruptions because of affected fetuses and difficulty in achieving their desired family. We evaluated the efficiency and acceptability of selective fetal reduction after chorionic villus sampling (CVS) of multiple pregnancy induced by ovarian stimulation and gamete intra-Fallopian transfer (GIFT). This approach has been offered to nine patients at risk of Mendelian diseases and one patient carrier of reciprocal translocation. The acceptance has been very high (90%). One patient at risk of an autosomic recessive disease opted for artificial donor insemination, one conceived spontaneously, and one was a poor responder to ovarian stimulation. Seven patients actually underwent a single GIFT procedure with six achieving pregnancy (86%), all but two being a multiple pregnancy (67%). All pregnancies concluded uneventfully at term and newborns were alive and healthy. Prenatal diagnosis, including fetal karyotyping in all cases, was performed at 8.5-11.5 weeks of gestation and confirmed either on amniotic fluid aspirated at reduction or at birth. The number of fetuses was reduced to one or two because the genetic disease was present and/or to reduce the risk of premature delivery and improve the likelihood of successful pregnancy. The new approach seems to be highly effective and might be considered a practical and useful alternative to preimplantation genetic diagnosis.

Abortion, Induced↗
PubMed1994

Ischemia-reperfusion of human skeletal muscle during aortoiliac surgery: effects of acetylcarnitine.

Our previous study on human skeletal muscle undergoing ischemia and reperfusion has revealed that granulocytes, which infiltrate the muscle tissue in large numbers, play an important role in mediating fibre injuries by producing superoxide anion (O2-) which is responsible for membrane lipid peroxidation. In the current study, five patients undergoing aortic reconstructive surgery were given acetyl-carnitine (2 mg/kg i.v. plus 1 mg/kg/min for 30 min) prior to the induction of ischemia. Muscle biopsies and blood samples were examined: a) after anaesthesia; b) at the end of ischemia; and c) 30 min after reperfusion, with the aim of elucidating whether acetylcarnitine could prevent the infiltration and/or the activation of granulocytes and eventually skeletal muscle injuries. During ischemia and reperfusion complement activation recruited numerous granulocytes into the muscle tissue, but, contrary to the untreated samples, the ability for O2(-)-generation of these cells remained at low levels and was comparable to that of ischemia even when molecular O2 was reintroduced to the tissue. Accordingly, the morphological changes of the postischemic muscle fibers were substantially reduced when compared to the untreated samples; in fact, the mitochondrial swelling was only moderate and the intramitochondrial dense bodies were small and scarce. The current findings support a positive role of acetyl-carnitine in ameliorating the ischemia-reperfusion (I-R)-induced damage of human skeletal muscle.

Acetylcarnitine↗
PubMed1992

Adhesion, growth, and matrix production by osteoblasts on collagen substrata.

A number of studies have demonstrated the pivotal role of collagen molecules in modulating cell growth and differentiation. In order to analyze the direct effects of collagen type I on the osteoblastic phenotype, we have devised an in vitro culture system for studying the interactions between bovine collagen type I and Saos-2 cells, a human osteoblastic cell line. Saos-2 cells were cultured both on top of collagen-coated culture dishes as well as inside a three-dimensional collagen network. Plating on dishes treated with collagen induced maximal adhesion of Saos-2 cells after 24-hour incubation. Cells cultured on collagen gel matrix expressed about 2.5-fold more alkaline phosphatase when compared with untreated plastic dishes. On collagen-coated dishes the responsiveness of Saos-2 cells to parathyroid hormone was decreased, whereas no modifications were observed in the effect of vasoactive intestinal peptide on these cells. Using a microfluorimetric measurement of DNA, an increase of proliferation was observed in Saos-2 cells cultured on collagen gel. Saos-2 cells were also able to colonize collagen sponges and in this three-dimensional network they were able to synthesize osteocalcin, as assessed both by immunocytochemistry and radioimmunoassay. In this study we have demonstrated that bovine collagen type I exhibits favorable effects on attachment and functional and growth activities of a human osteoblastic cell line, encouraging its use as a bone graft material.

Alkaline Phosphatase↗
PubMed1992

Neutrophils as mediators of human skeletal muscle ischemia-reperfusion syndrome.

Nine patients with aortic aneurysm undergoing arterial reconstruction with temporary aortic occlusion were studied. Since a typical condition of ischemia-reperfusion of the muscles of the lower limbs was created during this surgery, muscle biopsies from the right femoral quadriceps as well as blood samples from the homolateral saphenous vein were taken: (1) before clamping of the aorta, (2) just before declamping, and (3) 30 minutes after reperfusion. Light microscopy revealed a consistent granulocyte infiltration in the ischemic and reperfused skeletal muscle. Ultrastructural damage to the muscle fibers was seen during ischemia and became more severe upon reperfusion. The recruitment of granulocytes into the muscle tissue paralleled the activation of the blood complement system and an increase in circulating neutrophils. Although a spontaneous superoxide anion (O2-) generation from such granulocytes cannot be proved, upon stimulation with formyl-methionyl-leucyl-phenylalanine neutrophils showed a reduced ability in O2 free radical production at the end of ischemia and enhanced O2- generation at reperfusion as compared with the controls. All these findings indicate an active role of granulocytes in the genesis of reperfusion-induced tissue injuries.

Aged↗