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.

Structure of 5' region of human tenascin-R gene and characterization of its promoter.

The tenascin-R (TN-R) gene encodes a multidomain extracellular matrix protein belonging to the tenascin family, previously detected only in the central nervous system. In this report, we describe the structure of the 5' region of the human TN-R gene and characterize the activity of its promoter. We cloned two previously unreported nontranslated exons (exons 1 and 2, 539 and 101 bp in length, respectively) separated by a large (> or = 40-kb) intron. The intron between exons 2 and 3 (containing the ATG codon) is 122 kb in length. Tenascin-R transcripts in fetal, adult, and neoplastic human brain contain both exons 1 and 2, as demonstrated by S1 nuclease analysis and reverse transcriptase-polymerase chain reaction. The human TN-R promoter displays relatively unusual features in terms of sequence in that it lacks any TATA box, CAAT box, GC-rich regions, or initiator element. The promoter displays its activity only in cultured cells of neural and glial origin, not in transformed epithelial cells and melanoma cells. All the elements required for the full and cell-specific activity of the promoter are contained in the 57-bp sequence closest to the transcription startpoint.

Rat tenascin-R gene: structure, chromosome location and transcriptional activity of promoter and exon 1.

Tenascin-R is an extracellular matrix protein expressed exclusively in the central nervous system where it is thought to play a relevant role in regulating neurite outgrowth. We have i) cloned the cDNA of the rat tenascin-R 5' region; ii) defined its genomic organization, obtaining the sequence of two novel untranslated exons; iii) mapped the gene to rat chromosome 13q23 and suggested a previously unreported synteny between rat chromosome 13q23, human chromosome 1q24, and mouse chromosome 4E; and iv) sequenced and characterized the elements responsible for its neural cell-restricted transcription. We found that two discrete regions of the rat gene (the first in the proximal promoter, the second in the first exon) are independently able to activate to a high degree the transcription of a reporter gene in either human or rat neuroblastoma cell lines but not in other cell lines. Based on this observation, we re-evaluated the arrangement of transcriptionally active regions in the human tenascin-R gene we recently cloned and found that the human gene also contains an exon sequence able to initiate and sustain transcription independently of promoter sequences.

Targeting by affinity-matured recombinant antibody fragments of an angiogenesis associated fibronectin isoform.

The oncofetal fibronectin (B-FN) isoform is present in vessels of neoplastic tissues during angiogenesis but not in mature vessels. B-FN could therefore provide a target for diagnostic imaging and therapy of cancer. Phage display libraries have been used to isolate human antibody fragments with pan-species recognition of this isoform. We describe the use of these fragments in nude mice to target an aggressive tumor (grafted F9 murine teratocarcinoma). Imaging in real time was done by infrared photodetection of a chemically coupled fluorophore. The targeting was improved by use of affinity-matured fragments with low kinetic dissociation rates (koff = 1.5 x 10(-4) s-1) and also by engineering dimeric fragments via a C-terminal amphipathic helix.

Fibronectin type III5 repeat contains a novel cell adhesion sequence, KLDAPT, which binds activated alpha4beta1 and alpha4beta7 integrins.

The region of fibronectin encompassing type III repeats 4-6 contains a low affinity heparin binding domain, but its physiological significance is not clear. We have studied whether this domain is able to interact with cells as already shown for other heparin binding domains of fibronectin. A computer search based on homologies with known active sites in fibronectin revealed the sequence KLDAPT located in FN-III5. A synthetic peptide containing this sequence induced lymphoid cell adhesion upon treatment with the activating anti-beta1 monoclonal antibody (mAb) TS2/16 or with Mn2+, indicating that KLDAPT was binding to an integrin. A recombinant fragment containing repeat III5 (FN-III5) also mediated adhesion of TS2/16/Mn2+-treated cells while the FN-III6 fragment did not. Soluble KLDAPT peptide inhibited cell adhesion to FN-III5 as well as to a 38-kDa fibronectin fragment and VCAM-1, two previously known ligands for alpha4beta1 integrin. KLDAPT also competed with the binding of soluble alkaline phosphatase-coupled VCAM-Ig to Mn2+-treated alpha4beta1. Furthermore, mAbs anti-alpha4 and anti-alpha4beta7, but not mAbs to other integrins, inhibited cell adhesion to FN-III5 and KLDAPT. These results therefore establish a cell adhesive function for the FN-III5 repeat and show that KLDAPT is a novel fibronectin ligand for activated alpha4 integrins.

Differential cation regulation of the alpha 5 beta 1 integrin-mediated adhesion of leukemic cells to the central cell-binding domain of fibronectin.

Normal and neoplastic leukocytes interact with the central cell-binding and carboxyl-terminal regions of fibronectin (FN) primarily via the alpha(4) beta(1) and alpha(5) beta(1) integrins. By using a unique centrifugation-based cell adhesion assay and affinity chromatography of the cell surface-labeled integrins, we show in this study that the constitutive alpha(5) beta(1)-dependent attachment of three leukemic cell lines, BV-173, K562, and Nalm-6, to FN or to a 110-kDa central cell-binding fragment of FN was totally inhibited at 37 degrees C by preincubation of the substrate with either antibodies to the arginine-glycine-aspartic acid-containing region (3Fn-9 module) or to the synergistic region (3Fn-9 module) of FN. Similar results were obtained when assays were carried out at 4 degrees C, suggesting that energy-dependent events were not involved. On the other hand, only the antibody against the 3Fn-10 module was able to detach most firmly adherent cells. Constitutive cell attachment to the 110-kDa fragment was cation dependent, with the order of efficacy of the cation being Mn2+ > Mg2+ > Ca2+, and Ca2+ was only effective for BV-173 cells. Antibodies against the alpha(5) beta(1) integrin or the alpha(5) subunit completely impaired cell attachment of all cell lines, whereas several blocking anti-beta(1) subunit antibodies, including 4B4, P4C10, and AIIB2, differentially perturbed cell adhesion of BV-173, depending on which cation was present. These anti-beta(1) blocking antibodies, whose epitopes map to a region distant from the one expected to contain the beta(1) putative cation binding sites, seemed to lock the higher activation state of this integrin attained in the presence of Mg2+ and to preserve it during the subsequent adhesion events irrespectively of the presence of the low avidity state-inducing Ca2+ ion. Because the higher binding avidity displayed by BV-173 could not be explained by a higher degree of preclustering of alpha(5) beta(1) integrin in this cell line compared to K562, these findings suggest that cations might act as allosteric activators of integrin function. Whether this novel cation-dependent parameter of alpha(5) beta(1) integrin-FN interaction might contribute to the growth control and/or tissue dissemination of leukemic cells remains to be determined.

The human tenascin-R gene.

The human tenascin-R gene encodes a multidomain protein belonging to the tenascin family, until now detected only in the central nervous system. During embryo development, tenascin-R is presumed to play a pivotal role in axonal path finding through its adhesive and repulsive properties. Recently, the primary structure of human tenascin-R has been elucidated (Carnemolla, B., Leprini, A., Borsi, L., Querzé, G., Urbini, S., and Zardi, L. (1996) J. Biol. Chem. 271, 8157-8160). As a further step to investigate the role of human tenascin-R, we defined the structure of its gene. The gene, which spans a region of chromosome 1 approximately 85 kilobases in length, consists of 21 exons, ranging in size from 90 to >670 base pairs. The sequence analysis of intron splice donor and acceptor sites revealed that the position of introns in human tenascin-R are precisely conserved in the other two tenascin family members, tenascin-C and tenascin-X. The determination of intronic sequences flanking the exon boundaries will allow investigation of whether mutations may be responsible for altered function of the gene product(s) leading to central nervous system development defects.

Phage antibodies with pan-species recognition of the oncofoetal angiogenesis marker fibronectin ED-B domain.

Fibronectin (FN) exists in several polymorphic forms due to alternative splicing. The B-FN isoform (with ED-B domain inserted by splicing) is present in the stroma of foetal and neoplastic tissues and in adult and neoplastic blood vessels during angiogenesis but is undetectable in mature vessels. This isoform, therefore, represents a promising marker for angiogenesis, as already shown using the mouse monoclonal antibody (MAb) BC-1 directed against an epitope on human B-FN. However, this MAb does not directly recognise the human ED-B domain nor does it recognise B-FN of other species; therefore, it cannot be used as a marker of angiogenesis in animal models. In principle, antibodies directed against the human ED-B domain should provide pan-species markers for angiogenesis as the sequence of this domain is highly conserved in different species (and identical in humans and mice). As it has proved difficult to obtain such antibodies by hybridoma technology, we used phage display technology. Here, we describe the isolation of human antibody fragments against the human ED-B domain that bind to human, mouse and chicken B-FN. As shown by immunohistochemistry, the antibody fragments stain human neoplastic tissues and the human, mouse and chicken neovasculature.

Complement-fixing islet cell antibodies in type-1 diabetes can trigger the assembly of the terminal complement complex on human islet cells and are potentially cytotoxic.

Forty-one sera of patients with IDDM (insulin-dependent diabetes mellitus) containing complement-fixing islet cell antibodies were analyzed for their ability to activate TCC (terminal complement complex). Eighteen sera were found to promote deposition of TCC on human islets of pancreatic cryostat sections with a nonhomogeneous pattern of distribution corresponding to that of insulin. Activation of TCC by IDDM serum and binding of this complex to islet cells was confirmed using purified islets. Flow cytometric analysis of islet cell treated with a TCC+ IDDM serum showed IgG binding to the cell surface. The same serum had a cytotoxic effect on islet cells in the presence of human C. These results obtained with a homologous system of C activation by IDDM sera suggest that TCC may contribute, at least in part, to the pancreatic beta cell damage.

Human tenascin-R. Complete primary structure, pre-mRNA alternative splicing and gene localization on chromosome 1q23-q24.

We have established the primary structure of human tenascin-R (TN-R), a component of the extracellular matrix of the central nervous system, by sequencing cDNA clones which cover its complete coding region. The deduced amino acid sequence of human TN-R (1358 amino acids) showed a homology to chicken and rat TN-R of 75 and 93%, respectively. By reverse transcriptase-polymerase chain reaction we have studied the existence of TN-R isoforms generated by pre-mRNA alternative splicing in various human astrocytomas and meningiomas. Our findings demonstrate the existence of a human isoform in which one fibronectin-like repeat is omitted. Northern blot analysis of the poly(A)-rich RNA from different tissues showed two mRNAs having sizes of about 10 and 11 kilobases. Using DNA from a panel of human-hamster and human-mouse somatic cell hybrids and by fluorescence in situ hybridization, we have assigned the gene for human TN-R to the region 1q23-q24. The mouse mutation loop-tail (Lp), which has been proposed as a model for human neural tube defects, maps to region of mouse chromosome 1 syntenic with human 1q23-q24.

Novel self-association fibronectin sites.

In this study, we report a strong interaction between two contiguous proteolytic fragments of fibronectin, each having a mass of about 16 kDa. This interaction was stable in 4 M NaCl and 4 M urea and dissociation of the two fragments required buffers containing 0.5% sodium dodecyl sulphate. After purification, these peptides maintained their ability to interact when mixed. One fragment was made up of type III repeat 4 and part of 5, the other by repeat 6 and part of 5. Such strong interaction between two fibronectin regions may play a role in fibronectin conformation as well as during fibronectin fibril formation.

[Nasal polyps: comparative immunological study of polyps with different histopathologic types]. / I polipi naso-sinusali: studio immunoistologico comparativo di formazioni con differente tipologia istopatologica.

Cellular Infiltrate as well as class I and II HLA molecule expression, on 22 nasal polyps and on 12 samples of corresponding hypsilateral mucous membrane were studied by means of immuno-histological methods. These nasal polyps were classified according to their histopathological structure. Five polyps, with a fibrous connective core infiltrated by cells of the monocyte-macrophage lineage, were classified mixed. The remaining seventeen polyps were characterized by the presence of central oedematous connective tissue infiltrated almost exclusively by eosinophils and either contained (glandular type) or did not contain (oedematous type) glands. A comparative study of different types of nasal polyps and corresponding hypsilateral nasal mucous membranes was carried out on atopic and non-atopic patients. No correlation between atopic status and polyp presence or polyp typology was found. On the other hand, different polyp types appear to have a structural correlation with the corresponding hypsilateral mucous membrane regarding infiltrate cell type, oedematous or fibrous connective tissue presence and expression of on HLA antigen positivity pattern. The characteristic histological structure of hypsilateral mucous membranes in patients with different types of polyps appeared to be brought about by a multifactorial etiology involving mucosal hyperreactivity. Lastly, both polyps and parapolypal nasal mucous membranes were found to be infiltrated mainly in the peripheral subepithelial connective tissue by lymphocytes (55%) as well as by other leukocyte types. The presence of growth factors capable of enhancing an increase of fibroblasts, endothelial cells, together with focal distrupture on the basal membrane, might well be a general mechanism responsible for polyp sprouting.

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.

[Expression of HLA-DR, DP, DQ antigens in fibrous sino-nasal polyps]. / Espressione di antigeni HLA-DR, DP, DQ, in polipi naso-sinusali di tipo fibroso.

In the last years nasal polyps have been studied by several authors with different methodologies; however, their etiology is still unclear. In this paper we have analyzed in four nasal polyps of fibrous type, the HLA class II (HLA-DR, DP, DQ) molecule expression by means of immunohistochemical techniques (immunoperoxidase and immunophosphatase). A strong inflammatory cell infiltration, a percentage increase of both HLA-DR+ and HLA-DQ+ cells (normal nasal mucous membrane stroma infiltrating cells: DR+ < 40%, DP+ < 2%, DQ+ < 3%; fibrous polyps infiltrating cells: DR+ = 68%, DP+ < 2, DQ+ = 7%) as well as a clear positivity for DR expression of both surface and glandular epithelia were observed in all polyps. Furthermore, in the stalk area of one of the studied polyps DR+DQ+ cells with macrophagic features and having tight. connections with the vessels were observed. The scanty vascularization with the presence of activated mononuclear and mast cells might be responsible for polyp growth by locally producing an anomalous concentration of growth factors.

In situ lung perfusion with cisplatin. An experimental study.

BACKGROUND: This research work was planned to evaluate the soundness of in situ lung perfusion as a regional administration modality of chemotherapeutic agents. METHODS: Sixteen pigs were randomly divided into four groups and received cisplatin (2.5 mg/kg) through the pulmonary artery using one of the following techniques: stop-flow (Group 1); stop-flow/out-flow occlusion (Group 2); lung perfusion (Group 3); or lung perfusion with 5 mg/kg of infused drug (Group 4). Serial blood (carotid, pulmonary artery and vein) and tissue samples (lung and mediastinal node), were taken before, at completion of, and 5, 10, 15, 30, 60, 120, 180 and 240 minutes after cisplatin infusion for blood gas and platinum content measurement. Blood circulation was restored to the treated organ (for 60 minutes). The animals were killed, and specimens from lung, thyroid, esophagus, heart, liver, spleen, adrenal glands, kidney, bone marrow, stomach, ileum, colon, psoas muscle, and skin were obtained. Platinum concentrations in plasma, plasma ultrafiltrate (free platinum) urine, and tissues were determined by flameless atomic absorption spectroscopy. Lung damage was evaluated by light and electron microscopic examination. RESULTS: Greater systemic plasma, lower pulmonary plasma, and tissue platinum levels were detected when cisplatin was given using the stop-flow technique with respect to the other administration modalities. No significant difference in regional and systemic platinum exposure was found between Groups 2 and 3. However, lung perfusion resulted in higher mediastinal node and lower bone marrow platinum values. Morphologic alterations and impairment of gas exchanges in the treated lung were not dependent on the applied infusion technique. CONCLUSION: This study provides the pharmacokinetic rationale for the application of lung perfusion to patients with pulmonary metastases.

[Immunohistochemical study of the nasal mucosa of patients with fibrous polyps]. / Studio immunoistochimico della mucosa nasale in pazienti con poliposi di tipo fibroso.

Nasal polyps constitute a common pathology, but their etiology is not yet clear. In the present report, by means of immunohistochemical methods, both cell infiltrate and HLA-DR molecule expression were analysed in nasal mucous membrane fragments biopsied in the vicinity of fibrous polyps. Both surface and gland epithelia were positive for HLA-DR antigens in all sample whereas the epithelium of control nasal mucous membranes appeared to be negative. In addition only little delimited portions of the basement lamina seemed disrupted by an intense traffic of infiltrating cells. The poor venous drainage, the presence of activated mononuclear and mast cells capable of releasing soluble growth factors, together with basal lamina interruptions, might be factors for polyp sprouting.

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.

An experimental study to examine the patency and tissue response of two types of biosynthetic graft used as a replacement for porcine inferior vena cava.

This experimental study has been carried out to evaluate biosynthetic grafts as vascular substitutes. Tubular segments of 35 x 8 mm made of (1) tanned ovine collagen and integral polyester mesh, either of the first (Omniflow I) or second generation (Omniflow II), or (2) polytetrafluoroethylene (e-PTFE), have been sutured in the infrarenal inferior vena cava of pigs, and removed 1 hour, 7, 14, 28, 56 and 112 days after implantation. The patency rate of biosynthetic grafts was higher than that of e-PTFE grafts (p < 0.01). There was no significant difference between the patency of the first generation and second generation collagen grafts. These results indicate that biosynthetic prostheses may be suitable vascular substitutes in low flow and low pressure systems. Improvements in the collagen inner cover structure (Omniflow II vs. Omniflow I), producing greater mechanical endurance, did not enhance long-term patency or the healing patterns of biosynthetic grafts.