M. Lupetti

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

The distribution of epidermal growth factor (EGF), transforming growth factor α (TGFα), and EGF/TGFα 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. Bowmans capsule, proximal tubules, and collecting ducts were also reactive in the fetal kidney to varying degrees. The distribution of TGFα 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 embryonic periods examined. By the 11th week of gestation, an intense reactivity for TGFα polipeptide was shown in the fetal kidney at the level of the proximal tubules and Bowmans capsule; distal tubules as well as all urinary structures from the collecting ducts to the pelvis were less reactive. Finally, EGF/TGFα 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)

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.

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.

A contribution to the study of lymphopoiesis in the bursa of Fabricius in Gallus domesticus.

SUMMARY It has been proposed that the anatomical connection between the bursa of Fabricius and the cloaca is the pathway for unknown intestinal factors which are necessary for the induction of normal bursal lymphopoiesis. It has also been suggested that normal lymphopoiesis occurs only if nerve and vascular connections are intact. Experiments were performed to test these hypotheses. To test the influence of the intestinal contents, the bursal stalk was cut on the 16th day of incubation or at hatching. In this way, contact between the bursa and the intestinal flow in embryos was avoided and bacterial contamination of the bursa at hatching was also avoided. No change in the bursal follicles was observed. To study the influence of the nervous system on bursal lymphopoiesis, the bursa was isolated from the cloaca at hatching, and by maintaining vascularization the bursa was sutured to the peritoneum of the abdominal wall after scratching the contact surfaces. Once a new vascular network was established, me fragment of bursa was completely isolated from its normal anatomical site, causing interruption of the blood vessels and nerves of the bursa. The histological appearance of the bursa was not changed. It would appear that the integrity of the anatomical relation between bursa and cloaca and an intact nerve supply is not necessary for normal lymphopoiesis to occur in the bursa of Fabricius. In contrast, sufficient vascularization appears to be essential.

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.

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.

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.

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 desmonsomes 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.

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 β1 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 β1 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 β1 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 best 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 31 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-β1 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 β1 integrin in paraffin-embedded human tissues.