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.

The use of MAB 1977 monoclonal antibody for the immunohistochemical localization of beta 1 integrins in paraffin-embedded human kidney.

AIMS AND BACKGROUND: Integrins are widely known cell membrane receptors for extracellular matrix molecules. The beta 1 integrin subgroup is mainly expressed by kidney cells; immunolocalization of these molecules is usually carried out on cryostatic sections. A commercial monoclonal antibody directed against the human beta 1 integrin was tested in order to design a method for the detection of this antigen in formalin-fixed, paraffin-embedded human kidney tissue. METHODS: Specimens obtained from nephrectomies were fixed with 10% formalin and embedded in paraffin. Three different detection protocols were applied after incubation with the anti-human beta 1 integrin monoclonal antibody (MAB 1977): 1) immunoperoxidase with labeled streptavidin biotin (LSAB), using biotinylated secondary antibodies, peroxidase-labeled biotin-streptavidin, and 3,3'-diaminobenzidine tetra-hydrochloride (DAB) as the revealing system; 2) immunoperoxidase with tyramide signal amplification (TSA), using biotinylated secondary antibodies, streptavidin-peroxidase, tyramide, streptavidin-peroxidase again and DAB; 3) indirect immunofluorescence with fluorescein-labeled anti-mouse immunoglobulins. RESULTS: The beat results were obtained with the LSAB detection protocol preceded by a predetection step with proteinase k. Proteinase k pretreatment did not significantly damage the tissue morphology and successfully unmasked beta 1 integrin antigens. Nonspecific background staining was reduced by a blocking step with swine serum. Similar results were obtained with the TSA detection method; however, although lower concentrations of anti-beta 1 integrin immunoglobulins and of secondary biotinylated antibody were employed, there was more undesired background staining than with the LSAB protocol. CONCLUSIONS: The method reported and discussed here may represent a valid tool for research and diagnostic applications based upon detection of beta 1 integrin in paraffin-embedded human tissues.

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.

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)

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.

Effect of low molecular weight heparan sulphate on angiogenesis in the rat cornea after chemical cauterization.

Vascularization of the cornea occurs in many pathological conditions and can result in loss of visual acuity. It is also thought that vascularization predisposes the cornea to reject grafts by facilitating the detection of foreign antigens in donor material. A rat corneal assay for angiogenesis was adopted in the present study to evaluate the possible angiostatic activity of a low molecular weight heparan sulphate (LMW-HS). Corneal lesions were induced by chemical cauterization at 2 mm from the corneoscleral limbus. Rats were randomized to receive two drops/eye four times daily, for 6 days, of a solution of LMW-HS in vehicle (2.5% carboxymethylcellulose), heparin, heparin plus hydrocortisone, or vehicle alone. After a 6 day-treatment period, the eyes were perfused with india ink and the degree of neovascularization was evaluated. In rats treated with vehicle alone a dense vascular network extending from the corneoscleral limbus to the cauterized site was observed; on the contrary, a markedly reduced vascular network was evidenced in animals treated with LMW-HS. The distribution of basic fibroblast growth factor (bFGF) in the cauterized cornea was also evaluated by using an immunohistochemical method. A marked bFGF immunoreactivity was demonstrated in corneal epithelium and stroma of control rats 12-48 hours after the cautery. These results lead to the assumption that LMW-HS could be used in ophthalmology to inhibit corneal neovascularization.

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.

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.

Properties of the esophageal epithelium in relation to organizative patterns of lymphoid infiltrations in the red-eared turtle.

The esophagus of the turtle, like the mucosal surfaces in other species, contains variously sized areas of lymphoid infiltration. The tunica propria and the surface epithelial layer of this area are invaded by the lymphoid cells. The features of the layer of epithelial cells which cover the lymphoid infiltrations are of a special kind: they do not possess vibratile cilia and are able to take up materials flowing into the lumen. The present paper contains further information concerning lymphoid infiltration obtained by histological and histochemical methods. The epithelial layer covering the lymphoid infiltrations is composed of cells with irregularly distributed microvilli, ciliated cells and mucous-secreting cells. After administration of silica and colloidal carbon, the microvillar epithelial cells proved to have these substances inside them, thereby accounting for the pinocytotic activity. The absorbing epithelial cells were not damaged by silica which is a macrophage-toxic agent, while the underlying macrophages are damaged. These results are compared with the features of lymphoid infiltration associated cells in various organs and animals; the hypothesis is proposed that these cells in the esophagus of turtles may originate from the covering epithelial cells.

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.

Changes in the chicken bursa of Fabricius and immune response after treatment with melatonin.

Since melatonin causes considerable reduction in the thymic weight of chickens, the aim of the present study was to find out whether the bursa of Fabricius and the immune response are also affected by this substance. The results show that melatonin also acts on the bursa of Fabricius depending upon the administration route. When injected subcutaneously it does not reduce bursal weight and the histological pattern does not change, while primary and secondary immune responses are significantly lowered. When melatonin is introduced into the bursa, it causes a considerable decrease in bursal weight and the histological pattern is greatly modified. A significant reduction in antibody production is observed involving the primary immune response alone. Transmission electronic microscopy seems to show that melatonin affects the reticulo-epithelial (REp) cells.

Purine metabolism and B-lymphocyte development in the chicken bursa of fabricius.

The activity of three enzymes involved in the salvage pathway of purine nucleosides--purine nucleoside phosphorylase (PNP), xanthine dehydrogenase (XDH), and hypoxanthine-guanine phosphoribosyl transferase (HGPRT)--was investigated in cellular fractions of the chicken bursa of Fabricius differentially enriched in epithelial cells or lymphocytes. Markedly increasing levels of PNP and XDH were observed along with the enrichment in epithelial cells together with a slight, though significant, decrease in HGPRT activity. By contrast, a dramatic fall in PNP and XDH activities was detected along with the enrichment in lymphocytes together with a slight, though significant, increase in HGPRT activity. This sharply different distribution of the three enzymes, all sharing hypoxanthine as a substrate, clearly indicates that lymphocytes preferentially channel hypoxanthine into the salvage and interconversion pathways, phosphorylating it to IMP, while epithelial cells rapidly catabolize such a purine base to uric acid. Moreover, epithelial cells, unlike lymphocytes, are able to retain high intracellular levels of both hypoxanthine and inosine. These results support the possibility that epithelial cells contribute to the normal development of bursal lymphocytes by supplying such actively proliferating cells with purine rings and at the same time by preventing them from accumulating potentially toxic high levels of purine nucleotides being able to rapidly eliminate excess hypoxanthine as uric acid from the bursa environment into the bloodstream.

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.

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.

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.

Lympho-epithelial interactions in the turtle Chrysemys scrypta elegans.

The morphological relationships present in the areas of lymphoid infiltration found in the esophagus in Chrysemys scripta elegans were studied by the use of light and transmission electron microscopy. These infiltrations are widespread and can be observed in the lamina propria and the covering epithelium. Electron microscopy reveals the presence of epithelial cells with differing characteristics at the apex of the lymphoid infiltrations. The introduction of horseradish peroxidase (HRP) into the esophageal lumen revealed that these cells possess a micropinocytotic activity. On the basis of the morphological and experimental results obtained, a hypothesis is advanced of the existence, also in the turtle, of a local system of uptake of extraneous material, which induces local and systemic immunological processes.

Reappraisal of histogenesis in the bursal lymphoid follicle of the chicken.

The development of the bursal follicle and the appearance of the follicle-associated epithelial (FAE) cell and the reticuloepithelial (REp) cell were studied. The stages of development of the bursal follicle were observed by light and electron microscopy; an anticytokeratin monoclonal antibody was also used. At the beginning of follicle development, a mesenchymal cell cluster is observed in the tunica propria; the cluster becomes wedged in a niche of the surface epithelium, and gradually it is completely surrounded by the epithelium itself, which closes under the clump of mesenchymal cells. The epithelial cells lying upon the mesenchymal clump become necrotic, and a number of mesenchymal cells bulge out, forming the FAE cells. The epithelial cells that have closed under the mesenchymal nodule become stratified and form the REp cells; they become star-shaped because the medullary-lymphoid cells grow between them. Finally, the cortex is formed, possibly as a result of the migration of medullary cells before they peripheralize. It is concluded that FAE cells are not specialized epithelial cells, as they do not react to an anticytokeratin monoclonal antibody; on the contrary, they are formed by mesenchymal stemcells that bulge into the lumen and change their character after moving into the epithelium. The REp cells appear in the follicular primordium shortly after the bursal follicle begins to develop; the pronounced reactivity of the REp cells to an anticytokeratin monoclonal antibody supports the hypothesis of their epithelial origin.

Ultrastructural and immunocytochemical study on bursal follicle medulla cells in Gallus domesticus.

The use of the monoclonal anti-cytokeratin 6 and 18 antibody Dako CK 1 revealed a marked positivity of reticulo-epithelial cells (REp). Aspecific esterase testing, light microscopy, and electron microscopy were used in order to obtain a comparison between the morphology of the lymphoid follicle medulla and the picture obtained by using the monoclonal antibody CK 1. Results showed that the bursal follicle medulla can be divided into 2 areas: an esterase-positive, cytokeratin-negative centre-medulla, and a more peripheral cytokeratin-positive, esterase-negative area. These 2 regions appear to be separated by a boundary composed of flattened REp cells. Desmosomes were also observed not only among their processes, but also between the latter and the side of the cortico-medullar boundary epithelium which is external with respect to the basal membrane.

Questioning the role of adenosine deaminase in the development of B lymphocytes in chicken bursa.

Adenosine deaminase, a purine metabolic enzyme, was studied in lymphoid tissues of the developing chicken in order to evaluate whether enzyme activity is related to development of the immune system in birds in the same way as for mammals, in which adenosine deaminase is essential for lymphocyte differentiation, especially for the T-cell lineage. Enzyme activity was assayed in thymocytes and bursal lymphocytes at different times during chicken development ranging from the 17th day of embryonic life up to the 50th day after hatching. Adenosine deaminase activity was significantly higher in the bursa than in the thymus, regardless of whether such an activity was expressed per mg protein or per 10(8) cells; moreover, no substantial difference in the relative levels of adenosine deaminase was observed in thymocytes at the various stages of thymus development studied. Significant changes in enzyme activity, however, were found in bursal lymphocytes in which different amounts of adenosine deaminase appeared to be related to definite stages of bursal development and to specific immunological responsiveness of B lymphocytes to intravenously injected antigens. Therefore, if adenosine deaminase does play a role in the functional maturation of the immune system in birds, such a role appears to be related to the differentiation of the B- rather than the T-cell lineage.