Biomedical subjects

L Zardi

Publications and source records attributed to L Zardi.

At least 91 records · Page 5Linked to original sources

PubMed1995

Tenascin distribution in human brain tumours.

Using a monoclonal antibody specific for human tenascin (TN), 180 intracranial growths were immunohistochemically studied. In 69 cases of meningioma, neoplastic cells were negative, with some positivity being observed only in the perivascular and the supporting stroma, especially in anaplastic meningiomas. In 57 cases of glioma different degrees of reactivity occurred in both the cellular conglomerates and the stromal components of the tumours. A higher variability in reactivity was observed in anaplastic astrocytomas and glioblastomas. The most constant finding of the study was the staining of the stroma, which was observed in all types of growths, including metastasis, abscess and tuberculoma. The results are consistent with the hypothesis that tenascin is a stromal marker rather than a true marker of malignant tumours. The heterogeneous distribution of TN in anaplastic gliomas may be a factor in the variable response to treatment with radiolabelled anti-TN monoclonal antibodies.

Antibodies, Monoclonal↗

PubMed1995

Extracellular matrix proteins in colorectal carcinomas. Expression of tenascin and fibronectin isoforms.

BACKGROUND: Interactions of tumor cells and extracellular matrix (ECM) components are crucial determinants of tumor cell spreading and metastatic activity. Particularly tenascin (TN) as a member of the adhesion modulating family of ECM and its alternatively spliced isoforms became the matter of interest in ECM changes associated with malignancy. EXPERIMENTAL DESIGN: We analyzed the composition of the stromal- and basement membrane-associated ECM of colorectal adenomas and carcinomas using indirect immunofluorescence. Tenascin was investigated by immunoblot of snap frozen tumor specimens. RESULTS: Fibronectin (FN), TN, and chondroitin sulfate proteoglycan were the major components of the tumor stroma. Normal basement membrane components like laminin (LM), collagen type IV, and heparan sulfate proteoglycan were down-regulated. In the center of the tumor, tumor glands were surrounded by discontinuous basement membranes. At the tumor-host interface and in solid, poorly differentiated tumors, no immunoreactivity with normal basement membrane components was found. However, in cases with pericellular anti-LM staining, LM immunoreactivity was also found at the tumor-host interface. An alternatively spliced isoform of TN with a molecular weight of 330 kDa was found in seven of 15 carcinomas. In four of these cases, an alternatively spliced isoform of FN containing the ED-B segment was present. CONCLUSIONS: The coexpression of alternative splicing of FN and TN suggests that there may be common regulation mechanisms. The matrix composition found in the present study resembles that of healing wounds and probably favors the invasive spread of tumor cells.

Adenoma↗

PubMed1995

TGF beta and bFGF synthesis and localization in Dupuytren's disease (nodular palmar fibromatosis) relative to cellular activity, myofibroblast phenotype and oncofetal variants of fibronectin.

Nodular palmar fibromatosis is a self-limited proliferation of fibro-/myofibroblasts associated with growth factor synthesis and abundant fibronectin extracellular matrix deposition. bFGF and TGF beta are potent modulators of fibro-/myofibroblast proliferation and differentiation. Moreover, in vitro investigations evidenced a TGF beta 1-dependent regulation of alternative splicing of fibronectin mRNA. To investigate a possible implication of these growth factors in the tissue formation process of palmar fibromatosis, TGF beta 1/2 and bFGF synthesis, as well as TGF beta 1/3 and bFGF tissue distribution, is demonstrated by RNA in situ hybridization and/or immunohistochemistry in relation to myofibroblast phenotype development (alpha-smooth muscle actin, desmin immunohistochemistry), expression of different fibronectin isoforms (ED-A+, ED-B+ and oncofetal glycosylated fibronectin immunohistochemistry, fibronectin RNA in situ hybridization) and cellular activity (cyclin RNA in situ hybridization, Ki-67 immunolabelling). The myofibroblast phenotype (alpha-smooth muscle actin, desmin), the growth factor synthesis (TGF beta 1 and 2, bFGF), fibronectin matrix synthesis (RNA in situ hybridization with cDNA) and ED-A+, ED-B+ and oncofetal glycosylated fibronectin immunostaining are exclusively localized in the active proliferative nodules (Ki-67 immunolabelling and cyclin mRNA demonstration). Whereas the growth factor synthesis is restricted to the proliferative areas of the fibromatosis only, TGF beta 1, TGF beta 3 and bFGF proteins can also be detected immunohistochemically with a lower intensity in the surrounding aponeurotic tissue. The spatial correlation of myofibroblast phenotype, TGF beta and bFGF synthesis and the occurrence of the oncofetal molecular fibronectin variants (ED-B+ and oncofetal glycosylated fibronectin) in the active proliferative fibromatosis nodules suggests a pathogentic role of these growth factors and matrix components in the tumorous tissue formation process. The presence of the bFGF and TGF beta 1/3 proteins in fibroblasts neighbouring the proliferative nodules may point to a recruitment of quiescent aponeurotic fibroblasts in the fibromatous tissue formation process.

DNA, Complementary↗

PubMed1994

The fibronectin isoform containing the ED-B oncofetal domain: a marker of angiogenesis.

Different fibronectin (FN) isoforms are generated by the alternative splicing of 3 regions (ED-A, ED-B and IIICS) of the primary transcript. The FN isoform containing the ED-B sequence, a complete type-III-homology repeat, while having extremely restricted distribution in normal adult tissues, reveals high expression in fetal and tumor tissues. Using the monoclonal antibody (MAb) BC-I, specific for the FN isoform containing the ED-B sequence (B+.FN), we demonstrated here, using immunohistochemical techniques, that while this FN isoform is undetectable in mature vessels, it is highly expressed during angiogenesis both in neoplastic and in normal tissues, as in the case of the functional layer of endometrium during the proliferative phase. B+.FN is thus a marker for the formation of new vessels, and the BC-I MAb may be a useful reagent for evaluating the level of the angiogenetic process in different neoplasms.

Alternative Splicing↗

PubMed1994

Distribution of oncofetal fibronectin isoforms in normal, hyperplastic and neoplastic human breast tissues.

Two different oncofetal fibronectins (FN) have been reported: one, generated by O-glycosylation in the splicing region IIICS that is recognized by monoclonal antibody (MAb) FDC-6, and another, recognized by MAb BC-I, generated by the alternative splicing of the FN pre-mRNA which includes an extra type-III repeat called ED-B. Using these and 2 other MAbs (IST-4 which recognizes all different FN isoforms and IST-6 which recognizes only the FN molecules that do not include the ED-B sequence) we have immunohistochemically studied 171 normal, hyperplastic and neoplastic breast-tissue specimens. Although all normal specimens reacted strongly with MAbs IST-4 and IST-6, they did not show the presence of oncofetal FNs as established by the use of BC-I and FDC-6. In contrast, out of the 97 cases of invasive ductal carcinomas studied, 90 (93%) and 96 (99%) reacted positively with BC-I and FDC-6, respectively, the reaction being observed in the tumoral stroma connective tissue and in tumoral vessels. Furthermore, invasive lobular carcinoma showed less intense and less frequent staining with BC-1 and FDC-6 (10 and 11 out of 14, respectively). We found differences in the distribution of the 2 oncofetal fibronectin isoforms within the same specimens. The most remarkable difference was observed in the tumoral vessels: in invasive ductal carcinoma MAb BC-1 revealed a positive reaction with vessels in 78% of cases while FDC-6 showed such a reaction in only 59% of cases.

Adenocarcinoma, Mucinous↗

PubMed1994

Tenascin distribution in articular cartilage from normal subjects and from patients with osteoarthritis and rheumatoid arthritis.

OBJECTIVE: To determine whether tenascin is present in normal and diseased human cartilage. METHODS: Immunohistochemical and biochemical assays with a monoclonal antibody against all tenascin isoforms (BC-4) were used. RESULTS: Cartilage samples from osteoarthritis and rheumatoid arthritis patients contained increased amounts of tenascin compared with the levels in normal cartilage. Human fetal cartilage was also found to contain tenascin. In normal cartilage explants treated with interleukin-1 beta, tenascin was present in pericellular areas of all layers. Immunolocalization studies revealed that tenascin was most abundant in the superficial layers of osteoarthritic cartilage. Western blot analysis performed from dissociative extracts of diseased cartilage confirmed the presence of subunits of the native molecule. CONCLUSION: Tenascin is increased in arthritic cartilage and is weakly expressed in normal cartilage.

Adult↗

PubMed1994

Distribution of ED-A and ED-B containing fibronectin isoforms in Dupuytren's disease.

Different fibronectin (FN) isoforms arise via alternate splicing of a single gene transcript in a cell- and tissue-specific manner. Antibodies were used to evaluate the presence and distribution of FN and its isoforms in Dupuytren's diseased and normal palmar fascia. Immunolocalization studies show extracellular FN fibrils, including FN isoforms containing extra domains A (A-FN) and B (B-FN), in proliferative and involutional stage Dupuytren's diseased tissue. However, B-FN appears less abundant and more restricted in its distribution as compared to A-FN or total FN. Total FN and A-FN are significantly reduced in residual tissue, while B-FN is not present. A-FN and B-FN are not present in normal palmar fascia, while total FN staining is slight and restricted to the loose connective tissue surrounding the large, parallel bundles of collagen fibers. The presence of A-FN and B-FN in Dupuytren's diseased palmar fascia represents a disease-induced appearance of these FN isoforms and further evidence of an association between Dupuytren's disease and wound healing.

Antibodies, Monoclonal↗

PubMed1994

Cell-cycle dependent alternative splicing of the tenascin primary transcript.

Functionally different tenascin (TN) isoforms may be generated by alternative splicing of the TN primary transcript. In fact, it has been demonstrated that only the larger TN isoform containing the alternatively spliced region induces loss of focal adhesion in cultured cells and seems able to facilitate cell migration. Recent studies have shown that the higher molecular mass TN isoform is a marker of stromal cell proliferation in hyperplastic and neoplastic breast tissues. This finding prompted us to study the pattern of TN alternative splicing in proliferating and non-proliferating cultured fibroblasts. Here, we show that the mitogenic stimulation of fibroblasts with serum or cytokines leads to an early and striking modification in the steady-state levels of the two major TN mRNAs. We also show that de novo protein synthesis is not necessary for this modification, indicating that it is a "primary response" event. Similarly, mitogenic stimulation induces changes both in synthesis and accumulation of the different TN isoforms.

Alternative Splicing↗

PubMed1994

Tenascin isoforms: possible targets for diagnosis and therapy of cancer and mechanisms regulating their expression.

Functionally different tenascin (TN) isoforms containing varying numbers of III homology repeats are generated by alternative splicing of a single TN primary transcript. It has recently been reported that the larger TN isoform is, in general, more expressed in neoplastic tissues than in the normal tissues from which the tumor originates. This is due, at least in breast lesions, to the high proliferative activity of stromal elements. In fact, TN splicing is cell-cycle dependent, thus offering a viable system to study the molecular mechanisms that regulate alternative splicing and suggesting that cell-cycle dependent modifications in the splicing pattern of primary transcripts (which very likely are not limited to the TN pre-mRNA) may also be a cell-cycle regulatory mechanism. Furthermore, the very high accumulation of the larger TN isoform in neoplasia allows wider diagnostic and therapeutic monoclonal antibodies specific for the larger TN isoforms be considered for a number of tumors.

Antibodies, Monoclonal↗

PubMed1993

Production and characterization of monoclonal antibodies specific for different epitopes of human tenascin.

We have obtained and characterized 11 monoclonal antibodies (mAbs) specific for different domains of human tenascin (TN). Five of these mAbs reacted with epitopes contained in the TN area that undergoes alternative splicing and are thus able to recognize specific TN isoforms. These mAbs are a useful tool to study the expression and distribution of TN and its different isoforms in normal and pathological tissues.

Antibodies, Monoclonal↗

PubMed1993

Human amnion epithelial cells assemble tenascins and three fibronectin isoforms in the extracellular matrix.

Monoclonal antibodies (MAb) were used to show that cultured human amnion epithelial (HuA) cells produce tenascins (Tn) and isoforms of cellular fibronectin (cFn). Tn polypeptides of M(r) 280,000 and 190,000, assembled into extracellular matrix (ECM) but not secreted into the culture medium by HuA cells, were electrophoretically similar to those produced by human fibroblasts as revealed with domain-specific MAbs. The results suggested that most Fn produced by HuA cells contained the extradomain (ED) A and an oncofetal domain but only a minor fraction EDB. In immunofluorescence Tn and Fn were seen in different cytoplasmic granules upon monensin-induced intracellular accumulation. Tn appeared to be deposited in the ECM in colocalization with Fn but distinctly slower. The present results show that cultured normal human epithelial cells synthesize Tn and three isoforms of cFn and secrete them by using different cytoplasmic pathways.

Alternative Splicing↗

PubMed1993

Steady-state levels of different tenascin mRNAs in various normal human tissues.

Northern blot analysis of TN mRNA from different human tissues shows two major bands of about 6 and 8 kb which correspond to two different mRNAs generated by alternative splicing of the primary transcript. In liver, pancreas and kidney only the 6 kb TN mRNA was detectable. The highest levels of 8 kb TN mRNA were observed in placenta and skin representing 30% and 52% of total TN mRNA, respectively. In all other tissues tested the 8 kb TN mRNA represented less than 20% of total TN mRNA.

Blotting, Northern↗

PubMed1992

The inclusion of the type III repeat ED-B in the fibronectin molecule generates conformational modifications that unmask a cryptic sequence.

We have previously reported an anti-fibronectin monoclonal antibody (mAb) (BC-1) which reacts with an ED-B-containing beta-galactosidase-fibronectin fusion protein but not with an identical beta-galactosidase-fibronectin fusion protein in which the ED-B sequence is omitted. In further experiments aimed at localizing more precisely the epitope recognized by this mAb, we demonstrate that 1) the mAb BC-1 is indeed specific for ED-B-containing fibronectin (FN) molecules even though the epitope recognized by this mAb is localized on the type III homology repeat 7 (the one which precedes the ED-B sequence) and 2) in fibronectin molecules lacking the ED-B sequence, this epitope is masked. We further demonstrate that, to mask the epitope recognized by the mAb BC-1, the presence of at least half of the FN type III homology repeat 9 is necessary. We also report the production of the mAb IST-6 which recognizes only FN molecules in which the ED-B sequence is lacking. These data clearly demonstrate that the presence of the ED-B sequence within FN molecules generates conformational modification in the central part of the molecules that unmasks previously cryptic sequences and masks others.

Amino Acid Sequence↗

PubMed1992

Expression of different tenascin isoforms in normal, hyperplastic and neoplastic human breast tissues.

Functionally different tenascin (TN) isoforms, containing varying numbers of a 91 amino-acid motif resembling the fibronectin type-III homology repeat, may be generated by alternative splicing of the TN primary transcript. In fact, only the TN isoform containing the alternatively spliced region can induce loss of focal adhesion in cultured cells and seems to be able to facilitate cell migration. We examined the patterns of alternative splicing of the TN primary transcript in normal, hyperplastic and neoplastic breast tissues, and found that, in all the invasive breast carcinomas analyzed, the relative amount of TN mRNA in which the alternatively spliced region was included was about 10 times higher than in RNA from normal breast tissues. A similar result was observed in phyllodes tumors and in those fibroadenomas which showed very high stromal cellularity. Western-blot analysis using different monoclonal antibodies showed the same pattern as that seen in Northern blotting. The data reported here suggest that, in the breast, expression of the high-molecular-mass TN isoform is a marker of stromal element proliferation and that, in invasive breast carcinomas, this TN isoform could play a role in generating a permissive environment for proliferation, invasion and metastasis of neoplastic epithelial cells.

Blotting, Northern↗

PubMed1992

A simple procedure for tenascin purification.

Here we describe a two-step procedure for purification of human tenascin from conditioned medium of the SK-MEL-28 human melanoma cell line. The first step consists in passing the conditioned media through two chromatography columns connected in sequence. The first is a large capacity gelatin--Sepharose affinity chromatography column (to remove fibronectin), the second, over which the unbound material from the first column flows directly, is a hydroxyapatite chromatography column. Under these conditions, all tenascin present in the conditioned medium binds to the hydroxyapatite chromatography column from which it is then eluted by a 5-300 mM sodium phosphate gradient. With this step, we obtain a crude tenascin preparation, concentrated about 20 times with respect to the starting conditioned medium, and in which tenascin represents more than 50% of the total protein. The second step consists of two sequential precipitations with 6% and 12.8% poly(ethylene glycol). After this step, tenascin is more than 95% pure and does not show any contamination of chondroitin-sulfate-containing proteoglycans that are known to bind to it. From 21 medium we obtain about 3-4 mg tenascin which corresponds to a yield of about 40-50%. This procedure gives a higher yield, is simpler with respect to procedures previously described, avoids the exposure of the protein to denaturing agents or harsh conditions and could be used for purification of tenascin from the conditioned media of other cell lines. Thus, this procedure may represent a simple and useful tool for the preparation of tenascin to study its biological functions.

Cell Adhesion Molecules, Neuronal↗

PubMed1992

Comparison of human tenascin expression in normal, simian-virus-40-transformed and tumor-derived cell lines.

Tenascin is a polymorphic high-molecular-mass extracellular-matrix glycoprotein composed of six similar subunits. Using two-domain-specific anti-tenascin monoclonal antibodies, we have studied the expression and distribution of tenascin in four cultured normal human fibroblasts, two simian-virus-40-(SV40)-transformed and three tumor-derived (melanoma, rhabdomyosarcoma and fibrosarcoma) cell lines. We found that (a) cultured normal human fibroblasts accumulate considerable amounts of tenascin and retain 60-90% in the extracellular matrix, while they release the remainder into the tissue-culture medium; (b) of the two SV40-transformed counterparts we have tested, the AG-280 cell line accumulates no detectable amounts of tenascin and the WI-38-VA cell line accumulates about 10-times less tenascin than its normal counterpart and releases about 90% of it into the culture medium; (c) some tumor-derived cell lines accumulate considerable amounts of tenascin, but in these cases, more than 90% is released into the culture media; (d) in normal human fibroblasts, two major tenascin isoforms, generated by alternative splicing of the mRNA precursor, are detectable (280 kDa and 190 kDa, respectively) and the lower-molecular-mass tenascin isoform is accumulated preferentially in the extracellular matrix; (e) in SV40-transformed or tumor-derived cell lines, only the higher-molecular-mass isoform is detectable and it is more sialylated than the tenascin produced by the normal human fibroblast cell lines.

Blotting, Northern↗

PubMed1992

Differential expression of the fibronectin isoform containing the ED-B oncofetal domain in normal human fibroblast cell lines originating from different tissues.

Fibronectin (FN) polymorphism is due both to alternative splicing of three sequences (ED-A, ED-B, and IIICS) of the primary transcript and to post-translational modifications. The FN isoform containing the ED-B sequence (B-FN), while having an extremely restricted distribution in normal adult tissues, has a high expression in fetal and tumor tissues. On a panel of non-fetal skin, fetal skin, and fetal lung fibroblast cell lines we have studied, through S1-nuclease protection analysis, the expression of the ED-B containing FN mRNA as well as the expression of the ED-B containing FN isoform through immunoblotting and immunofluorescence techniques, using domain specific monoclonal antibodies. The results show that the expression of B-FN in the different fibroblast cell lines has an extremely great variability depending on the developmental stage of the donor and on the tissue of origin. Moreover, we found that SV-40-transformed fibroblasts present a higher expression of B-FN mRNA with respect to their normal counterparts. An increase in the relative amount of the B-FN isoform in normal human fibroblasts was also obtained by treatment with transforming growth factor-beta.

Antigens, Neoplasm↗

PubMed1991

Comparative analysis of the expression of the extracellular matrix protein tenascin in normal human fetal, adult and tumor tissues.

Tenascin (TN) is a high-molecular-mass oligomeric glycoprotein of the extracellular matrix (ECM) endowed with a programmed expression in embryonic life and a neo-expression in the interstitium of some malignancies. Using monoclonal antibodies (MAbs) which identify human tenascin, we have conducted an extensive immunohistochemical analysis of TN expression in normal fetal and adult human tissues as well as in a wide variety of human tumors. Results of this study demonstrate that TN (1) is detectable in embryonic and fetal tissues at least from the 10th week of gestation; (2) is present in the interstitium of a variety of adult tissues of different embryonic origin; (3) may be neo-expressed in the stroma of benign and malignant tumors; (4) has the ability to accumulate in a highly variable manner in the ECM of tumors of the same and of different histotypes.

Adult↗