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L Zardi

Publications and source records attributed to L Zardi.

At least 19 recordsLinked to original sources
PubMed2006

High-molecular tenascin-C as an indicator of atypical cells in oral brush biopsies.

Tumour-invasion like wound healing is characterised by the formation of an extracellular matrix with a high tenascin-C content. The tenascin-C molecule undergoes alternative splicing. Analysis using antibody BC2 indicates that especially the high-molecular tenascin-C (hm tn-C) variants are typically tumour-associated, while distribution in normal tissue is restrictive. This study investigated whether hm tn-C is a suitable indicator of atypical cells with invasive potential in oral brush biopsies. One hundred fifty nine consecutive oral brush biopsies with histopathological diagnoses were analysed for the identification of atypical cells. A standardised haematoxylin and eosin staining plus standardised immunocytochemistry using the monoclonal anti-hm tn-C antibody was performed. The bound hm tn-C antibodies were detected with the streptavidine/alkaline phosphatase technique in the autostainer. Conventional cytology produced four false-positives when identifying atypical cells in brush biopsies of inflammatory/benign hyperproliferative mucosa (specificity 96%), while 10 in 52 carcinomas and three of eight recurrences were not identified (sensitivity 78%). Ten of these 13 non-identified tumours could be marked when adding the hm tn-C assay (increasing specificity to 99%). Combining the two assays also reduced the false-positive outcomes from four to one (increasing sensitivity to 95%). The positive and negative predictive values were 92 and 88% for conventional cytology vs 98 and 97% for the dual assay. (1) A 95%-sensitivity proves hm tn-C assisted conventional cytology to be a suitable means of identifying atypical cells in oral brush biopsies. (2) The positive (98%) and negative (97%) predictive values obtained approximate hm tn-C assisted conventional cytology to laminin-5 (100/97%).

Biomarkers, Tumor↗
PubMed2004

Expression of EDA+ and EDB+ fibronectin splice variants in bone.

The extracellular matrix component fibronectin (fn) has fundamental functions in cell attachment, differentiation, proliferation, and migration. Isoforms of cellular fibronectin, named EDA+ fibronectin or embryonal EDB+ fibronectin, are generated by alternative splicing of its mRNA precursors. Little is known about the expression of EDA+ and EDB+ fibronectin splice variants in human bone. The aim of this study was to investigate the expression pattern of fibronectin splice variants in bone cell lines and in different human bone tissue samples (mature bone, early stages of fracture healing, hypotrophic nonunion, osteosarcoma). Analysis was done by immunostaining with recombinant and monoclonal antibodies, qualitative RT-PCR and LightCycler-based real-time quantitative RT-PCR assay. In osteoblast and osteosarcoma cell lines, abundant expression of EDA+ and EDB+ fibronectin was found in immunocytochemistry. High transcription levels of both splice variants mRNA were seen in quantitative RT-PCR in osteosarcoma cell lines. In mature bone, EDA+ and EDB+ were not detectable in immunohistochemistry. Transcription of mRNA in both splice variants was absent in these samples. Early stages of fracture healing and osteosarcoma cell samples exhibited extensive staining for EDA+ and EDB+ fibronectin, and high mRNA levels were found. Both osteosarcoma and bone fracture healing tissue expressed high mRNA levels of the fibronectin splice variants independent of benign or malignant behavior. Low level of EDA+ fibronectin mRNA transcription and focal immunohistochemical staining of EDA+ fibronectin was found in hypotrophic nonunions, whereas EDB+ fibronectin was not detected by immunohistochemistry and qualitative or quantitative PCR. EDA+ fibronectin was found in granulation tissue-forming processes in bone independent from bone-forming activity. EDB+ fibronectin was seen only in high-turnover new osteoid-forming processes like early stages of fracture healing and osteosarcoma and was absent in low-turnover processes like mature bone and hypotrophic nonunion. Both EDA+ and EDB+ fibronectin mark active processes in bone without differentiation between malignant or benign activity. In conclusion, EDA+ and EDB+ fibronectin splice variants are strong markers for active fibrogenetic and osteoid-forming processes in human bones.

Adolescent↗
PubMed2003

Expression of fibronectin splice variants and oncofetal glycosylated fibronectin in the synovial membranes of patients with rheumatoid arthritis and osteoarthritis.

OBJECTIVE: The aim of this study was to define and compare the expression of fibronectin (Fn) isoforms in synovial tissue of patients with rheumatoid arthritis (RA) and osteoarthritis (OA). METHODS: Using monoclonal antibodies specific for total Fn, extra domain (ED)-A Fn, ED-B Fn, and oncofetal glycosylated Fn, we studied the expression of the Fn isoforms in synovium. Furthermore, in situ hybridization for the detection of ED-B Fn mRNA including a double labeling technique for the detection of cell type was applied. RESULTS: Strong expression of total Fn, ED-A Fn, oncofetal glycosylated Fn and, to a lesser extent, ED-B Fn could be demonstrated in the synovial lining layer in both RA and OA. Stromal and vessel expression of Fn isoforms was more prominent in RA tissue. Pannus tissue showed strong labeling with ED-B Fn. CONCLUSION: The expression of alternatively spliced isoforms of Fn is associated with tissue remodeling and, as a partial process of this phenomenon, with neovascularization rather than underlying disease, X-ray status, or parameters of acute inflammation. In the lining layer, Fn expression correlates with hyperplasia associated with cell recruitment but not with proliferative status. Most remarkably, the expression of ED-B Fn in pannus tissue seems to be associated with the invasive phenotype described in RA tissue.

Alternative Splicing↗
PubMed2002

Enhancement of the antitumor activity of interleukin-12 by targeted delivery to neovasculature.

Interleukin-12 (IL-12) is a heterodimeric cytokine with potent immunostimulatory activity and anti-angiogenic properties. Its clinical applications are limited, however, by severe side-effects. Here we report that an IL-12 fusion protein, consisting of IL-12 fused to a human antibody fragment specific to the oncofetal ED-B domain of fibronectin, markedly enhances the antitumor activity of this cytokine, as demonstrated in a mouse lung-metastasis model and in two models of mice bearing different aggressive murine tumors. The residual small tumor masses seen in the treated mice were infiltrated with lymphocytes, macrophages, and natural killer cells and had elevated interferon gamma (IFN-gamma). These results are of therapeutic relevance as the ED-B domain of fibronectin, a naturally occurring marker of angiogenesis identical in mouse and man, is expressed in the majority of aggressive solid tumors but is not detectable in normal vessels and tissues.

Animals↗
PubMed2001

Isolation of anti-angiogenesis antibodies from a large combinatorial repertoire by colony filter screening.

We describe here a method, based on iterative colony filter screening, for the rapid isolation of binding specificities from a large synthetic repertoire of human antibody fragments in single-chain Fv configuration. Escherichia coli cells, expressing the library of antibody fragments, are grown on a porous master filter, in contact with a second filter coated with the antigen, onto which antibodies secreted by the bacteria are able to diffuse. Detection of antigen binding on the second filter allows the recovery of a number of E.coli cells, including those expressing the binding specificity of interest, which can be submitted to a second round of screening for the isolation of specific monoclonal antibodies. We tested the methodology using as antigen the ED-B domain of fibronectin, a marker of angiogenesis. From an antibody library of 7 x 10(8) clones, we recovered a number of specifically-binding antibodies of different aminoacid sequence. The antibody clone showing the strongest enzyme-linked immunosorbent assay signal (ME4C) was further characterised. Its epitope on the ED-B domain was mapped using the SPOT synthesis method, which uses a set of decapeptides spanning the antigen sequence synthesised and anchored on cellulose. ME4C binds to the ED-B domain with a dissociation constant K:(d) = 1 x 10(-7) M and specifically stains tumour blood vessels, as shown by immunohistochemical analysis on tumour sections of human and murine origin.

Amino Acid Sequence↗
PubMed2001

Targeted delivery of tissue factor to the ED-B domain of fibronectin, a marker of angiogenesis, mediates the infarction of solid tumors in mice.

The selective thrombosis of tumor blood vessels, leading to the starvation and subsequent death of tumor cells, is an attractive anticancer strategy. Here we report that a fusion protein, consisting of an antibody fragment specific for the oncofoetal ED-B domain of fibronectin fused to the extracellular domain of tissue factor, selectively targets tumor blood vessels in vivo. Furthermore, this fusion protein mediates the complete and selective infarction of three different types of solid tumors in mice. At the highest doses administered, complete tumor eradication was observed in 30% of the mice treated without apparent side effects. These results are of therapeutic relevance because the ED-B domain of fibronectin, a naturally occurring marker of angiogenesis identical in mouse and man, is expressed in the majority of aggressive solid tumors but is undetectable in normal vessels and tissues.

Animals↗
PubMed2001

Lack of specificity of endoglin expression for tumor blood vessels.

A monoclonal antibody (MAb; A11) has been raised following mouse immunization with cultured human microvascular endothelial cells. The MAb showed a strong positivity within tumor vessels in glioblastoma and breast carcinoma samples, and the distribution was consistent with antigen association with vascular endothelial cells. A purification procedure of the antigen was developed starting from DG-RSV-LT-2, an immortalized human endothelial cell line. Molecular mass, N-terminal sequence of the purified antigen and localization on endothelial cell surface allowed identification with human endoglin (CD105). Flow cytometry analysis of a group of normal and transformed cell lines showed that, besides endothelial cells and myelocytic leukemia cells already shown to be positive, fetal fibroblasts, choriocarcinoma, fibrosarcoma and rhabdomyosarcoma cell lines were also positive for this antigen. Immunohistochemic analysis of several normal adult tissues revealed a more extensive presence of the antigen in normal vessels compared to that described with previously characterized antibodies. In fact, even though the staining was weaker than in tumor tissues, all tissues were found to be positive, at least in microvessels, except for normal breast. Moreover, in some tissues (glands and reproductive tract) a positive reaction was observed in the stroma. Since endoglin has been proposed as a possible target for antiangiogenic therapy in tumor patients and our data demonstrate a sizable amount of endoglin in normal vessels and stroma, its clinical use should be carefully reevaluated.

Animals↗
PubMed2001

Selective targeting of tumour neovasculature by a radiohalogenated human antibody fragment specific for the ED-B domain of fibronectin.

Angiogenesis is a characteristic feature of many aggressive tumours and other disorders. Antibodies capable of binding to new blood vessels, but not to mature vessels, could be used as selective targeting agents for immunoscintigraphic and radioimmunotherapeutic applications. Here we show that scFv(L19), a recombinant human antibody fragment with sub-nanomolar affinity for the ED-B domain of fibronectin, a marker of angiogenesis, can be stably labelled with iodine-125 and astatine-211 with full retention of immunoreactivity, using a trimethyl-stannyl benzoate bifunctional derivative. Biodistribution studies in mice bearing two different types of tumour grafted subcutaneously, followed by ex vivo micro-autoradiographic analysis, revealed that scFv(L19) rapidly localises around tumour blood vessels, but not around normal vessels. Four hours after intravenous injection of the stably radioiodinated scFv(L19), tumour to blood ratios were 6:1 in mice bearing the F9 murine teratocarcinoma and 9:1 in mice bearing an FE8 rat sarcoma. As expected, all other organs (including kidney) contained significantly less radioactivity than the tumour. Since the ED-B domain of fibronectin has an identical sequence in mouse and man, scFv(L19) is a pan-species antibody and the results presented here suggest clinical utility of radiolabelled scFv(L19) for the scintigraphic detection of angiogenesis in vivo. Furthermore, it should now be possible to investigate scFv(L19) for the selective delivery of 211At to the tumour neovasculature, causing the selective death of tumour endothelial cells and tumour collapse.

Animals↗
PubMed2001

Antibody-based targeting of angiogenesis.

The selective targeting of neovasculature opens new avenues for the diagnosis and therapy of angiogenesis-related diseases such as cancer, blinding ocular disorders, and rheumatoid arthritis. Here we review recent advances in the identification of markers of angiogenesis as well as in the isolation and use of antibodies (and their derivatives) for the in vivo targeting of both tumoral and nontumoral neovasculature.

Animals↗
PubMed2000

Expression of emmprin by oral squamous cell carcinoma.

A transmembrane glycoprotein recently identified on some tumor cells, extracellular matrix metalloproteinase inducer (EMMPRIN), has been shown to induce metalloproteinase (MMP) production by peritumor fibroblasts (PTF). We examined biopsy specimens of normal human oral mucosa and oral squamous cell carcinoma (SCC) for expression of EMMPRIN. In normal mucosa, EMMPRIN was expressed at the cell membrane throughout the epithelium with a slight enhancement along the basal cell layer. In oral SCC, EMMPRIN was expressed at the cell membrane throughout the entire lesion. Immunofluorescence microscopy localized EMMPRIN to the cell membrane in a highly invasive oral SCC cell line in agreement with our in vivo observations. Function-blocking antibodies to EMMPRIN significantly inhibited oral SCC cell migration on tenascin-C (TN-C) and fibronectin as well as invasion through a reconstituted basement membrane (RBM). We previously showed that soluble factors from SCC cells and PTF are required for deposition of a TN-C matrix. To determine whether EMMPRIN may modulate the release or expression of these soluble factors, we again used function-blocking antibodies. Antibodies to EMMPRIN completely inhibited the organization of TN-C matrices and partially reduced the deposition of FN matrices by oral SCC cell /PTF co-cultures. In addition, antibodies to EMMPRIN perturbed the expression of MMP-2. Moreover, antibodies to MMP-2 perturbed oral SCC cell invasion of an RBM by approx. 75%. Our results demonstrate that EMMPRIN is highly expressed in oral SCC, facilitates tumor cell motility, and mediates TN-C matrix deposition. Taken together, these results suggest that EMMPRIN may help regulate oral squamous cell carcinoma invasion.

Antigens, CD↗
PubMed2000

The angiogenesis marker ED-B+ fibronectin isoform in intracranial meningiomas.

Fibronectins (FNs), adhesive glycoproteins mainly expressed in the extracellular matrix, are polymorphic molecules whose various isoforms are dependent on alternative splicing patterns. The isoform containing the ED-B sequence and occurring in foetal and neoplastic tissues (oncofoetal or B+FN) has been previously recognized as a marker for angiogenesis. The distribution of this isoform was analyzed in a consecutive series of 134 surgically obtained intracranial meningiomas, using specific monoclonal antibodies. Oncofoetal FN was found to be widely distributed in the vessels of anaplastic meningiomas, with its expression being restricted in the vasculature of the typical subtypes. and absent in the neighbouring cerebral tissue. The ubiquitous vascular expression of B+FN in meningiomatous malignancies might provide a potential target for the in vivo delivery of angiosuppressive agents.

Biomarkers, Tumor↗
PubMed2000

Detection of tenascin-C in surgically excised choroidal neovascular membranes.

BACKGROUND: Increase of tenascin-C (TN-C) expression has been found in pathologic tissues in which angiogenesis occurs. The aim of this study was to investigate TN-C expression in human choroidal neovascularization (CNV). METHODS: Ten choroidal neovascular membranes were surgically removed from 10 patients with age-related macular degeneration (n=6) and multifocal choroiditis (n=4). All membranes underwent immunohistochemical evaluation using monoclonal antibodies against TN-C and factor VIII. RESULTS: All membranes were positive for TN-C, which was abundantly and diffusely expressed in the extracellular matrix. CONCLUSIONS: Our results support the concept that TN-C has a role in cell proliferation and neovascularization in humans. TN-C, as a marker of angiogenesis, may provide novel rationales for the development of pharmacologic therapies for neovascular disorders, particularly CNV.

Biomarkers↗
PubMed1999

A high-affinity human antibody that targets tumoral blood vessels.

Angiogenesis is a characteristic feature of many aggressive tumors and of other relevant disorders. Molecules capable of specifically binding to new-forming blood vessels, but not to mature vessels, could be used as selective vehicles and would, therefore, open diagnostic and therapeutic opportunities. We have studied the distribution of the ED-B oncofetal domain of fibronectin, a marker of angiogenesis, in four different tumor animal models: the F9 murine teratocarcinoma, SKMEL-28 human melanoma, N592 human small cell lung carcinoma, and C51 human colon carcinoma. In all of these experimental models we observed accumulation of the fibronectin isoform containing the ED-B domain around neovascular structures when the tumors were in the exponentially growing phase, but not in the slow-growing phase. Then we performed biodistribution studies in mice bearing a subcutaneously implanted F9 murine teratocarcinoma, using a high-affinity human antibody fragment (L19) directed against the ED-B domain of fibronectin. Radiolabeled L19, but not an irrelevant anti-lysozyme antibody fragment (D1.3), efficiently localizes in the tumoral vessels. The maximal dose of L19 accumulated in the tumor was observed 3 hours after injection (8.2% injected dose per gram). By virtue of the rapid clearance of the antibody fragment from the circulation, tumor-to-blood ratios of 1.9, 3.7, and 11.8 were obtained at 3, 5, and 24 hours, respectively. The tumor-targeting performance of L19 was not dose-dependent in the 0.7 to 10 microg range of injected antibody. The integral of the radioactivity localized in tumoral vessels over 24 hours was greater than 70-fold higher than the integral of the radioactivity in blood over the same time period, normalized per gram of tissue or fluid. These findings quantitatively show that new-forming blood vessels can selectively be targeted in vivo using specific antibodies, and suggest that L19 may be of clinical utility for the immunoscintigraphic detection of angiogenesis in patients.

Animals↗
PubMed1999

Cooperative activity of alpha4beta1 and alpha4beta7 integrins in mediating human B-cell lymphoma adhesion and chemotaxis on fibronectin through recognition of multiple synergizing binding sites within the central cell-binding domain.

We have quantitated the relative contributions of the constitutively active alpha4beta1 and alpha4beta7 integrins and the domains embodying their cognate binding sites in mediating human B-cell lymphoma adhesion and chemotaxis on fibronectin. By cooperating, the central cell-binding and IIICS carboxy-terminal domains were entirely responsible for the adhesion activity displayed by fibronectin, and their relative contribution to this process was estimated to be 30% versus 70%. Assessment of the leukocyte-substrate binding strength (ie, dynes/cell) indicated a 10-fold higher avidity of the cell-IIICS domain interaction. The two integrins interchangeably recognized both domains, but differed quantitatively in their participation in the adhesive event, as well as in domain preference. The use of 3Fn (according to the nomenclature proposed by Bork and Koonin [Curr Opin Struct Biol 6:366, 1996] for the type III fibronectin modules) module-specific antibodies and recombinant polypeptides showed that alpha4 integrins recognized both the RGD sequence (3Fn10) and an apparently novel synergistic site located within the 3Fn8 module; even in this case, the integrins displayed a distinct binding site preference. Interleukin-1beta (IL-1beta)/IL-2-induced chemotaxis also involved cooperative function of the central cell-binding and IIICS domains, but the mechanisms regulating this phenomenon differed markedly from those controlling cell adhesion. First, the relative contribution of the individual domains was comparable, but neither of the individual domains promoted migration to the extent observed on intact fibronectin. Secondly, alpha4beta1 and alpha4beta7 integrins were both involved in the domain-binding necessary for initiation of migration, but the relative contribution of each receptor in the chemotactic process was less disparate than for initial cell adhesion. Thirdly, the mode by which chemotactic B-lymphoma movement was supported by the central cell-binding domain differed from that sustaining cell adhesion in that it involved independent recognition of either the 3Fn8 or the 3Fn9 module, which acted in synergy with the 3Fn10 module. Our data provide novel evidence concerning the relative importance of the constitutively active alpha4beta1 and alpha4beta7 integrins for the interaction of B-cell lymphoma cells with fibronectin, and they emphasize a multiple and diverse recognition of sites responsible for either anchorage or locomotion of tumor leukocytes on this matrix molecule.

Binding Sites↗
PubMed1999

Increased binding affinity and valence of recombinant antibody fragments lead to improved targeting of tumoral angiogenesis.

The formation of new blood vessels (angiogenesis) is an important step in tumor progression. Molecules capable of selectively targeting markers of angiogenesis may offer opportunities for the in vivo imaging of aggressive tumors and for the delivery of toxic agents to the tumoral vasculature. Using antibody phage display libraries and combinatorial mutagenesis, we isolated single-chain Fv antibody fragments, which recognize with different affinities the same epitope of the ED-B domain of fibronectin, a marker of angiogenesis. Two single-chain Fv fragments, E1 and L19, with dissociation constants of 41 nM and 0.054 nM, respectively, were investigated for their ability to target F9 murine teratocarcinoma grafted s.c. in nude mice when injected i.v. in either monomeric or homodimeric form (Mr 27,000 and 54,000, respectively). Biodistribution studies, performed at two time points (4 h and 24 h) with radiolabeled samples, showed that the higher affinity antibody targets the tumor significantly better than the lower affinity one, in terms both of tumor:organ ratios and of the amounts of antibody delivered to the tumor. In particular, more than 20% of the injected dose of dimeric L19 accumulated per gram of tumor at 4 h; the tumor:organ ratios at 4 h and 24 h were in the (2.1-8.6):1 and (10.3-29.4):1 range, respectively. This study demonstrates that, although vasculature represents only a small fraction of the total tumor mass, anti-ED-B antibodies can selectively target tumors in vivo and that this process is particularly efficient if very high-affinity binders are used.

Animals↗
PubMed1999

Cooperative role for activated alpha4 beta1 integrin and chondroitin sulfate proteoglycans in cell adhesion to the heparin III domain of fibronectin. Identification of a novel heparin and cell binding sequence in repeat III5.

We recently reported that the heparin (Hep) III domain of fibronectin contains the H2 cell adhesion site in repeat III5 which binds activated alpha4 integrins. We have now further characterized the heparin and cell binding activities of this domain. A recombinant fragment containing repeats III4-III5 (FN-III4-5) induced Jurkat cell adhesion upon integrin activation with Mn2+ or TS2/16 monoclonal antibody (anti-beta1). Adhesion of Mn2+-treated cells to FN-III4-5 or FN-III5 fragments was inhibited by chondroitinase ABC and ACII but not by the anti-alpha4 monoclonal antibody HP2/1. In contrast, HP2/1 completely blocked adhesion of TS2/16-treated cells while chondroitinase had a partial (FN-III4-5) or minor (FN-III5) effect. Thus, the role of each receptor depended on the stimulus used to activate alpha4 beta1. The combination of HP2/1 and chondroitinase at dilutions which did not inhibit when used individually abolished adhesion of Mn2+ or TS2/16-treated cells to both fragments, indicating a cooperative effect between alpha4beta1 and chondroitin sulfate proteoglycans (CSPG). Furthermore, we have identified a 20-amino acid sequence in III5 (HBP/III5) which binds heparin and induces cell adhesion via CSPG exclusively. Although soluble HBP/III5 was a poor inhibitor, when combined with H2, it abolished adhesion to FN-III4-5 and FN-III5 fragments. These results establish that adhesion to the Hep III domain involves the cooperation of activated alpha4 beta1 and CSPG and show that HBP/III5 is a novel heparin and CSPG-binding site contributing to cell adhesion to this domain.

Amino Acid Sequence↗
PubMed1999

Expression of EDA/EDB isoforms of fibronectin in papillary carcinoma of the thyroid.

Cellular fibronectins containing the extracellular domain A or B (EDA and EDB) are particularly abundant in fetal and neoplastic tissues. The presence of EDA and EDB was investigated in 28 cases of papillary carcinoma of the thyroid using IST-9 and BC-1 monoclonal antibodies. Immunostaining for EDA and EDB was detected in tumour stroma, in tumour basement membranes, and in tumour blood vessels. EDA was present in 27 of the 28 cases, in 20 of which more than 75 per cent of the tumour stroma was stained. Immunostaining for EDB was detected in 23 of the 28 cases and was less pronounced than that for EDA, being present in less than 25 per cent of the tumour stroma in most cases. Reactivity for EDA/EDB was not observed in the adjacent normal thyroid in any of the cases investigated. In a group of 20 non-papillary tumours, immunostaining for EDA was present in the stroma of three follicular carcinomas (one minimally and two widely invasive), one medullary carcinoma, and 5 of 16 follicular adenomas; expression of EDB was more restricted, being present in only the two cases of widely invasive follicular carcinoma. The presence of EDA and EDB was not correlated with the extent of fibrosis or the degree of tumour cell differentiation. Immunoreactivity was already present in microcarcinomas. These observations raise the possibility that the production of oncofetal fibronectins is an important step in papillary carcinoma tumourigenesis, perhaps facilitating adhesion and spreading of tumour cells.

Carcinoma, Papillary↗