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Biomedical subjects

A Dolfi

Publications and source records attributed to A Dolfi.

At least 19 recordsLinked to original sources
PubMed2006

Morphological features of ovine embryonic lung fibroblasts cultured on different bioactive scaffolds.

Tissue regeneration with autologous cell transplantation is one of the most important goals in clinical research. In this field, the development of bioactive materials that provide microenvironments for cell-matrix interactions mimicking biological conditions is required. In recent years, many synthetic materials have been developed as scaffolds and many procedures for the surface modification of these materials have been applied using biological molecules. In this study, we analyzed the morphology and the molecule production by ovine embryonic lung fibroblasts cultured on three different sponge-like matrices based on poly(L-lactic acid) (PLLA): agarose/PLLA, crosslinked and uncrosslinked gelatin/PLLA. The matrices were produced by using an emulsion freeze-drying method leading to the formation of sponge-like materials with high porosity and with interconnection between the pores. In vitro MTT test demonstrated that transplanted cells were viable and metabolically active. Morphological analysis revealed that fibroblasts adhered to and penetrated the polymeric structures. Moreover, all the different matrices supported fibroblast production of proteoglycans, glycoproteins, and matrix molecules such as elastin, collagen I, and fibronectin. These data suggest that the tested bioactive scaffolds may support the growth and extracellular matrix molecule production of fibroblasts allowing in vitro connective tissue regeneration.

Animals↗
PubMed2006

Constitutive expression of cyclooxygenase-2 in the neuromuscular compartment of normal human colon.

Prostaglandins regulate various functions throughout the gastrointestinal system. Their biosynthesis depends on cyclooxygenase isoforms, named COX-1 and COX-2. The initial hypothesis that COX-2 is an inducible enzyme has been challenged and its constitutive expression in the stomach has been established. In this study, an immunohistochemical analysis was performed to evaluate the distribution and cellular localization of COX-2 in normal human colon. Colonic surgical specimens were processed for COX-2, protein HuC/HuD, neurofilament, S-100 protein and CD117/c-kit immunodetection. COX-2 protein was found to be constitutively expressed in the colonic wall: detectable amounts were localized in mucosal, submucosal and muscular layers, mainly in the neuromuscular compartment. In particular, COX-2 was expressed in muscularis mucosae, submucosal ganglia, longitudinal muscle layer and myenteric ganglia, the neurons of which displayed different degrees of immunostaining. Intramuscular interstitial cells of Cajal, regarded as important sites for the regulation of enteric neuromuscular activity, were also partly COX-2 immunoreactive. This study provides a detailed mapping of COX-2 expression in human colon, and allows better understanding of the roles played by this isoenzyme in gut physiology.

Blotting, Western↗
PubMed2004

Immunohistochemical demonstration of the small GTPase RhoA on epoxy-resin embedded sections.

The purpose of the present study was to establish a method for light microscopical immunohistochemical localization of the small G protein RhoA on specimens treated and embedded for routine transmission electron microscopy. There are advantages in antigen immunolocalization on resin semi-thin sections compared to cryostat or paraffin sections: the preservation of morphological details, the well-defined immunoprecipitate localization and the possibility to correlate the immunohistochemical results with those obtained by electron microscope on neighbouring sections. These advantages are particularly useful for the subcellular localization of low molecular weight proteins such as RhoA, a small G protein able to cycle from the inactive cytoplasmic form to the plasma membrane-bound active form.

Adult↗
PubMed2002

Monolayers and multilayers of chlorophyll [correction of chlorophyl] a on a mercury electrode.

A novel experimental technique used to investigate chlorophyll films on a hanging mercury drop electrode is described. Two different procedures are employed to prepare self-assembled chlorophyll monolayers and multilayers on the mercury electrode. Upon illuminating the chlorophyll a (Chl)-coated mercury electrode with an appropriate light source, the photocurrents generated by the Chl aggregates are measured under short-circuit conditions in the absence of photoartefacts. The preliminary results obtained by this novel technique are presented.

Chlorophyll↗
PubMed2001

Renal cell cultures for the study of growth factor interactions underlying kidney organogenesis.

The present study was performed in four renal cell lines to evaluate their capability to: (1) produce and express transforming growth factor alpha (TGFalpha), its respective receptor, the epidermal growth factor receptor (EGFr) and the small G protein, RhoA, and (2) exhibit morphogenetic properties when grown on Matri-cell substrates. The cell lines were derived from normal (Madin-Darby canine kidney cells), embryonic (SK-NEP-1 and 293 cells), and cancerous (human renal adenocarcinoma cells) kidneys. TGFalpha messenger ribonucleic acid, evaluated by a nonradioactive in situ hybridization technique, was found to be expressed in all the cell lines. Large amounts of TGFalpha peptide were observed in all four cell lines, while EGFr was highly expressed only in cancerous ACHN and embryonic-tumor SK-NEP-1 cells. RhoA peptide was found in appreciable amounts in SK-NEP-1 and 293 cells (compared to the other two cell lines). The morphogenetic properties of the four cell lines were assessed, by culturing them on Matri-cell dishes: SK-NEP-1 cells alone were found to grow in three-dimensional structures forming clusters and worm-like cellular aggregates. This feature was displayed by SK-NEP-1 cells but not by the other three cell lines, and may be connected with the contemporary presence of RhoA, EGFr, and TGFalpha found in significant amounts only in the SK-NEP-1 cell line.

Animals↗
PubMed2000

A morphological study of the expression of the small G protein RhoA in resting and activated MDCK cells.

The small G protein Rho subfamily controls several cellular events such as growth, movement, proliferation and differentiation by rearranging actin and cytoskeleton proteins. Most of these effects are mediated by the activation of growth factor and extracellular matrix molecule receptors, suggesting a role for Rho molecules in the transduction pathway of these receptors. Despite the importance of Rho peptides in fundamental cellular events, data on their subcellular immunolocalisation are sparse: here we investigated the expression and subcellular localisation of RhoA in resting (cultured on plastic) and activated (Matri-cell or hepatocyte growth factor) MDCK cells by immunoperoxidase and immunogold techniques. Resting MDCK cells contain detectable amounts of RhoA mainly localised in the cytoplasm; RhoA expression is significantly enhanced by Matri-cell substrates that promote translocation of RhoA at the membrane level. This enhancing effect is reduced after exposure to hepatocyte growth factor.

Animals↗
PubMed1999

Laminin and beta1 integrin distribution in the early stages of human kidney development.

Laminin, an extracellular matrix molecule (EMM) widely expressed in the basal laminae, interacts with specific membrane receptors among which the integrin molecules are the best known. During embryo development laminin is the first synthesized EMM and plays a significant role in the morphogenesis of organs in which epithelial-mesenchymal interactions and branching take place. The present study describes the distribution of laminin and of beta1 integrin receptors during the very early stages of human kidney development. The observations were carried out on paraffin sections of human embryos ranging between the 4th and the 7th gestational week. Laminin was detected within the basement membranes of mesonephric duct, vesicles, glomerular vessels and celomic epithelium. The metanephric anlage reacted with anti-laminin immunoglobulins in the basement membrane underlying the ampullae and in few blastemic cap cells. Low levels of beta1 integrin reactivity were found in both the mesonephric and metanephric structures. This study provides for the first time data about the distribution of laminin and beta1 integrin in the early stages of human renal organogenesis suggesting a key role for these molecules in the epithelial-mesenchymal interactions necessary for kidney development.

Adult↗
PubMed1998

The heparan sulfate suleparoide inhibits rat corneal angiogenesis and in vitro neovascularization.

The purpose of this study was to evaluate the inhibitory activity of the heparan sulfate suleparoide on vascular cell growth in vitro and angiogenesis in vivo. Human HUV-EC-C endothelial cell proliferation and microvessel sprouting from cultured rat aortic rings were assayed by the bioreduction of 3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide. The inhibition of the neoforming capillary network in the chorioallantoic membrane of chick embryo (CAM) was evaluated by agarose disks containing suleparoide and applied on the CAM surface. AgNO3/KNO3 injury was used to induce corneal neovascularization and to evaluate the therapeutic effect of topical suleparoide, while the involvement of bFGF in angiogenesis was evidenced by immunohistochemistry of corneal tissue. Quantitation of angiogenesis in the CAM and the cornea was accomplished by image analysis. Suleparoide dose-dependently inhibited HUV-EC-C cell proliferation (50% inhibitory concentration [IC50], 197.5+/-15.2 microg ml-1) and reduced microvessel sprouting in vitro (IC50, 351+/-22 microg ml-1). Likewise, suleparoide 150 microg in agarose disks produced an avascular area of 19.7+/-2.7% of the total area of the CAM (P<0.05 as compared to controls). bFGF levels were significantly enhanced in the cornea after AgNO3/KNO3 injury, and the increase appeared to be time-dependent (25.6+/-1.8 and 43.2+/-7.4%, vs. uninjured controls after 24 hr and 48 hr, respectively, P<0.05). Suleparoide 4.8 mg eye-1 day-1 for six days reduced the length of blood vessels and the area of the cornea infiltrated by them (59.6+/-7.4% decrease vs. controls, P<0.05). These results demonstrate that suleparoide is an active agent against angiogenesis and suggest that the therapeutic effect of the drug could be of value to treat corneal neovascularization.

Allantoin↗
PubMed1996

Immunohistochemical localization of the epidermal growth factor, transforming growth factor alpha, and their receptor in the human mesonephros and metanephros.

The distribution of epidermal growth factor (EGF), transforming growth factor alpha (TGF alpha), and EGF/TGF alpha receptor were studied by means of immunohistochemical methods starting from the very early stages of human embryonic kidney development. Mesonephros and metanephros were examined in order to detect immunoreactive staining in serial sectioned embryos and fetal kidneys. Anti-EGF immunoprecipitates were found in the S-shaped mesonephric vesicles of 6-week old embryos as well as in the mesonephric duct albeit with a lower degree of reactivity. Intense reactivity was observed in the metanephros within the blastemic caps of the same gestational period; the reaction was weaker within the ureteric bud branches. Bowman's capsule, proximal tubules, and collecting ducts were also reactive in the fetal kidney to varying degrees. The distribution of TGF alpha reactivity in the mesonephros was similar to that observed for EGF but with a lower intensity. In contrast, there was no reactivity in the metanephros, at least during the embyronic periods examined. By the 11th week of gestation, an intense reactivity for TGF alpha polipeptide was shown in the fetal kidney at the level of the proximal tubules and Bowman's capsule; distal tubules as well as all urinary structures from the collecting ducts to the pelvis were less reactive. Finally, EGF/TGF alpha receptor reactivity was identified by the 6th week of development, being more intense in the mesonephros at the level of the mesonephric duct cells. In the metanephros, the ureteric bud-derived branches were reactive, whereas most of the blastemic tissue did not stain. By the 11th week, only the collecting ducts and the remaining urinary structures contained reaction products: Reactivity was distributed to the tissues originating from the ureteric bud branching. Taking into account recent advances in knowledge about the biology of growth factors, the hypothesis is proposed that the secretory components (vesicles, glomerulus, and tubules) of renal anlagen might release the growth factors while the cells of the urinary tract (i.e., collecting duct, pelvis, etc.) may be their targets.

Epidermal Growth Factor↗
PubMed1995

The location and the regulation of the type I-iodothyronine 5'-monodeiodinase (type I-MD) in the rat thyroid: studies using a specific anti-type I-MD antibody.

Type I-iodothyronine monodeiodinase (type I-MD) is abundant in thyroid tissue and contributes to the generation of T3 secreted by the gland. The availability of a specific antibody against rat type I-MD (type I-MD Ab) allowed us to directly identify this enzyme in rat thyroid glands, and in a differentiated strain of rat thyroid cells maintained in continuous culture (FRTL-5 cells). FRTL-5 cells maintain many differentiated functions of thyroid cells, including the expression of TSH receptor and thyroid peroxidase. Using an immunohistochemical technique on rat thyroid sections, a clear staining for type I-MD was demonstrated in follicular cells. The degree of immunoreactivity was greater in small follicles containing little amounts of colloid compared to large follicles lined by functionally inactive cells. Using immunofluorescence (IFL), a strong staining for type I-MD was observed in FRTL-5 cells grown in medium containing TSH. Both in vivo and in culture the staining for type I-MD was localised in the cytoplasm of thyroid cells, while nuclei were negative. Interestingly, no surface staining was shown when viable FRTL-5 cells were submitted to the same IFL procedure. TSH deprivation for 7 days was followed by the disappearance of type I-MD. Immunoreactivity for type I-MD was recovered by addition of TSH, forskolin or thyroid stimulating antibody (TSAb) to TSH-deprived FRTL-5 cells. The effect of TSH was prevented by cycloheximide. There was no induction of type I-MD when IGF-I was added to FRTL-5 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Expression of multidrug resistance (mdr) gene(s) in primary lymphoid organs of chicken immune system during embryonic development.

The presence of a multidrug resistance (MDR) related protein, P-170, in normal and pathological lymphoid cells has been described. The present report evaluates the expression of the mdr 1 gene by using the reverse Polymerase Chain Reaction (PCR) on cells obtained from the thymus and bursa of chicken embryos starting from day 12 until hatching. Results show that the thymic cells are positive from day 12 to the end of the observation period. In contrast, mdr mRNA was detected in the bursa from day 14 to day 17 of embryonic life. Possible relationships between the expression of mdr and the development of T and B lymphocytes are discussed.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1993

In vitro morpho-functional analysis of pancreatic islets isolated from the domestic chicken.

The technique whereby islets were isolated from the pancreas of chicken and their in vitro histological and functional characterization are described in this paper. Our isolation procedure consisted of two steps: initially the pancreas removed from the chicken was perfused with a 2% solution of collagenase and enzymatic digestion was then carried out using the same solution. After this, density gradient separation was performed on the digested tissue, by means of differing Histopaque solutions: at the end of the separation, the islets were studied by light microscopy after treatment with diphenylthiocarbazone, which selectively stains beta-cells, and by scanning and transmission electron microscopy. The results reported here show the pattern closely resembled that encountered when islets were studied in situ. The beta-cells of the islets proved in vitro to preserve their functional capability of producing insulin even after stimulation with glucose or arginine.

Animals↗
PubMed1993

Involvement of basic fibroblast growth factor in suramin-induced inhibition of V79/AP4 fibroblast cell proliferation.

The V79/AP4 Chinese hamster fibroblasts were densely stained with the anti-basic fibroblast growth factor (bFGF) antibody demonstrating an endogenous production of the peptide. The in vitro proliferation of these cells was stimulated by exogenous bFGF and the maximum growth (259% increase in 3H-thymidine incorporation into DNA) was reached with bFGF 10 ng ml-1. Inhibition of bFGF-mediated mitogenic pathway was obtained with a 15-mer antisense oligodeoxynucleotide targeted against bFGF mRNA and with suramin, a drug which blocks the biological activity of heparin-binding growth factors. bFGF antisense oligomer reduced the synthesis of DNA by 79.5 and 89.5% at 20 and 60 microM, respectively; this effect was reversed by the addition of exogenous bFGF to the culture medium. A short-term exposure to suramin 300 micrograms ml-1 produced a modest reduction in 3H-thymidine incorporation but suppressed the mitogenic effect of bFGF on V79/AP4 cells. In cells treated with suramin 300 micrograms ml-1 the drug concentration increased linearly over 3 days, reaching 13.15 micrograms mg-1 of protein; cell proliferation was inhibited in a dose-related manner as evaluated by the colony formation assay (IC50: 344.22 micrograms ml-1) and by the number of mitoses observed in culture. Furthermore, the drug induced ultrastructural alterations, consisting of perinuclear cisternae swelling, chromatin condensation, nucleolar segregation and cytoplasmic vacuolations. These findings demonstrated that the endogenous production of bFGF plays an important role in V79/AP4 fibroblasts proliferation, and the inhibition of bFGF-mediated mitogenic signalling with bFGF antisense oligomer or suramin is an effective mean of reducing cell growth.

Animals↗
PubMed1993

Scanning electron microscopy of multidrug resistant cells in haematological and mammary malignancies.

Multidrug resistance (MDR) is frequently found in haematologic malignancies. It has been shown that MDR is often related to the expression of a membrane glycoprotein (P-170) which actively pumps many hydrophobic agents out of the cells. Previous electron microscopic investigations revealed morphological differences between P-388 resistant and P-388 sensible cell membranes, but the modulation of the membrane morphology seems to be related to the tumor-cell environment. In order to establish if morphological differences exist between sensitive and resistant cells, both sensitive and resistant strains from three different cell lines were studied by scanning electron microscopy: human leukaemia CEM and vinblastine resistant cells (CEM/VBL100), human breast cancer MCF-7 and mice leukaemia P-388 with the doxorubicin resistant strains (MCF-7/DX and P-388/DX, respectively). The surface of the membranes of the sensitive cells was regular, unlike the resistant ones which proved to be irregular, endowed with long villus-like processes or numerous folds and ruffles. The addition of albumin to the culture medium induced a shift from the resistant to the sensitive phenotype, thus suggesting that the P-388/DX morphology may be linked to the concentration of protein in the culture medium. Exposure to DX, verapamil (VRP) or monoclonal antibody against P-170 (mAb-57) did not modify the surface of the resistant strains, demonstrating that surface irregularities are probably not linked to P-glycoprotein function. Blasts from four P-170 positive leukaemic patients were also analyzed: an irregular shape was always found.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1991

Distribution of the 1,25 dihydroxy-vitamin D3 receptor in the bursa of Fabricius of chicken.

The vitamin D3 metabolite 1,25(OH)2D3 is probably involved in B lymphocyte ontogeny. We therefore determined the distribution of the 1,25(OH)2D3 receptor in the bursa of Fabricius and spleen cells of 7-day-old chicks, by immunohistochemistry using a monoclonal antibody against the chick intestinal cell 1,25(OH)2D3 receptor. The bursa cells of young (7-day-old) chicks contained large amounts of receptor while the spleen cells did not. The bursa cells of older (35-day-old) chicks contained fewer receptors, but the number of receptors in the spleen increased.

Aging↗
PubMed1991

Comparative activity of doxorubicin and its major metabolite, doxorubicinol, on V79/AP4 fibroblasts: a morphofunctional study.

Doxorubicin (DXR), an anthracycline antineoplastic drug, is mainly metabolized to the C-13 dihydroderivative doxorubicinol (DXR-ol), which displays cytotoxic activity on various cell lines. To better characterize the cytotoxic activity of this metabolite, we have studied the effect of DXR (0.1-10 micrograms/ml) or DXR-ol (1-100 micrograms/ml) on the transformed fibroblast cell line V79/AP4 by means of the clonogenic assay, cytofluorescence, and light and electron microscopy. Both DXR and DXR-ol displayed a dose-dependent inhibition of colony formation with an IC50 factor DXR-ol/DXR of 19.5. A striking nuclear fluorescence was observed after DXR but not after DXR-ol. A low number of mitoses and a decrease in nucleoli staining affinity were the most evident alterations induced by DXR. Electron microscopy showed both nuclear and cytoplasmic changes in DXR treated cells: nucleolar segregation, cytoplasmic vacuoles, and mitochondrial swelling with dense needle-shaped material were observed. Exposure to formic acid confirmed the calcific nature of the mitochondrial bodies. Only the highest dose of DXR-ol brought about nuclear and cytoplasmic ultrastructural changes similar to those induced by DXR. Our data describe new in vitro findings on the cytotoxicity and morphological alterations induced by both DXR and DXR-ol, with a lower activity of DXR-ol against V79/AP4 fibroblasts.

Animals↗
PubMed1990

In vitro morphological characterization of the bursal reticuloepithelial (REp) cells of chicken.

The REp cells of the bursa follicle medulla of chicken were isolated in vitro. Culture of the REp cells was maintained over a period of 10 days and the cells were observed at 3 and 10 days by means of transmission electron microscopy (TEM) and immunofluorescence. The use of an anticytokeratin monoclonal antibody confirmed their epithelial nature. TEM observations showed the presence of desmosomes and tonofilaments, which are characteristic of epithelial cells. Furthermore, to some extent the cells regenerated in vitro the network they form in vivo. Though the growth rate becomes slower with time, the features of the REp cells do not significantly change.

Animals↗