Changes in cell migration-related molecules expressed by thymic microenvironment during experimental Plasmodium berghei infection: consequences on thymocyte development.

We previously showed alterations in the thymus during experimental infection with Plasmodium berghei. Such alterations comprised histological changes, with loss of cortical-medullary limits, and the intrathymic presence of parasites. As the combination of chemokines, adhesion molecules and extracellular matrix (ECM) is critical to appropriate thymocyte development, we analysed the thymic expression of ECM ligands and receptors, as well as chemokines and their respective receptors during the experimental P. berghei infection. Increased expression of ECM components was observed in thymi from infected mice. In contrast, down-regulated surface expression of fibronectin and laminin receptors was observed in thymocytes from these animals. Moreover, in thymi from infected mice there was increased CXCL12 and CXCR4, and a decreased expression of CCL25 and CCR9. An altered thymocyte migration towards ECM elements and chemokines was seen when the thymi from infected mice were analysed. Evaluation of ex vivo migration patterns of CD4/CD8-defined thymocyte subpopulations revealed that double-negative (DN), and CD4(+) and CD8(+) single-positive (SP) cells from P. berghei-infected mice have higher migratory responses compared with controls. Interestingly, increased numbers of DN and SP subpopulations were found in the spleens of infected mice. Overall, we show that the thymic atrophy observed in P. berghei-infected mice is accompanied by thymic microenvironmental changes that comprise altered expression of thymocyte migration-related molecules of the ECM and chemokine protein families, which in turn can alter the thymocyte migration pattern. These thymic disturbances may have consequences for the control of the immune response against this protozoan.

Sarcoglycan immunoreactivity is lacking in infantile hypertrophic pyloric stenosis. A confocal laser scanning microscopic study.

OBJECTIVES: The Dystrophin-Glycoprotein Complex (DGC) is a large multisubunit complex that plays a crucial role in maintaining the structural integrity and physiology of muscle fibers. Dystrophin has been reported to be absent in the pyloric muscle of infantile hypertrophic pyloric stenosis (IHPS) patients. The present study was designed to investigate the other two patterns of DGC (dystroglycan and sarcoglycan complexes) in normal pyloric muscle and their possible modifications in IHPS patients. METHODS: Ten pyloric muscle biopsies were obtained from babies operated for IHPS and five control pylorus biopsy taken at autopsy from cases without gastrointestinal disease. The DGC sub-complexes (beta-dystroglican and beta, delta- sarcoglycans) were localized immunohistochemically using specific monoclonal antibodies. The results were evaluated using a confocal laser scanning microscope. RESULTS: Positive immunolocalization of the two DGC sub complexes was demonstrated in the smooth muscle cells (SMCs) of the pyloric region of control patients. Similarly, a positive immune expression of beta-dystroglican was observed in the pyloric SMCs of IHPS patients. On the other hand a negative immunoreaction for sarcoglycans was recorded within the full thickness of the pyloric SMCs of these patients. CONCLUSIONS: The absence of sarcoglycans within the hypertrophied pyloric muscle may be a predisposing factor in the pathogenesis of IHPS since it could alter the normal physiology of SMCs through the modifications of structural integrity of sarcolemma and signaling between the extracellular and intracellular compartment.

Expression of laminin 5, fibronectin, and epithelium-associated integrins in recurrent aphthous ulcers.

Recurrent aphthous ulceration (RAU) is characterized by an ulcerated lesion that persists longer than traumatic ulcers of similar size. This delayed healing phase of the lesion was investigated for extracellular matrix components and matrix receptors (integrins). The hypothesis tested was that aphthous ulcers may lack key extracellular matrix components, or their receptors, that are necessary for the migration of marginal keratinocytes from the ulcer edge. We immunocytochemically stained biopsy specimens of RAUs and non-involved mucosal specimens from HIV+ and non-infected individuals to investigate the presence and distribution of molecules reported to be associated with reepithelialization of mucosal and cutaneous wounds. Fibronectin, laminin type 5 (kalinin), and integrin subunits beta 1, beta 4, alpha 6, and alpha v were consistently found at the margins of RAU, as they are in traumatic ulcers. The alpha 5 and beta 6 subunits were not always present. We also found alpha v in the intact stratified squamous epithelium adjacent to ulcers. Immunohistochemical stains showed distruption in the deposition of laminin 5 and an apparent lack of fibronectin at the edges of some ulcers. Although these tissue results do not determine which integrin subunits are paired with each other, they do show some alterations in their expression in RAU. Absence of one or more of these molecules at the migrating front may contribute to delayed epithelial regeneration. It is likely that the absence or inappropriate expression of keratinocyte integrins or their extracellular matrix receptors occurs after the causative factors (currently unknown) of the lesion are gone. The reason for the altered expression of these molecules may be related to the secretory products (including lymphokines and proteinases) of the lymphocytic infiltrate.

Cell surface proteolysis by serine proteinases enhances RGD-sensitive melanoma cell adhesion on fibrinogen and vitronectin.

Tumor cells avidly secrete various proteinases, and cascades of proteolytic activation occur around the cells. Therefore, cell surface receptors of tumor cells are under the constant influence of proteinases. In this study, the effects of serine proteinases on integrin-medicated cell-matrix interactions were studied in C32TG and Mewo human melanoma cells. These melanoma cells were pretreated with proteinases and their adhesive properties on various substrata were evaluated by cell adhesion assays. Paradoxically, appropriate cell surface proteolysis enhanced the RGD-sensitive cell adhesion on fibrinogen and vitronectin, but not the RGD-insensitive adhesion on type I collagen or laminin. Pretreatment of these cells with 0.1 to 1 microM of trypsin, chymotrypsin, or plasmin for 30 min at 37 degrees C increased the number of spread cells on fibrinogen and vitronectin by 200-300%. The enhancement of cell spreading was not accompanied by up-regulation of the relevant RGD-sensitive integrin expression. Analysis of the cell surface receptor by GRGDSPK-Sepharose affinity chromatography showed that trypsin treatment did not up-regulate alpha v beta 3 integrin, an RGD-sensitive receptor for fibrinogen and vitronectin in the melanoma cells, nor the induced appearance of novel receptors. Treatment of cells with 100 nM proteinases increased cell binding of both monoclonal and polyclonal antibodies against alpha v beta 3 integrin subunits by 70%, but not that of monoclonal antibody against alpha 2, alpha 3, or alpha 6 subunit, indicating that cell surface proteolysis exposed more alpha v beta 3 integrin on the cell surface. These results suggest that exposure of alpha v beta 3 integrin is a part of the mechanisms underlying the serine proteinase-induced enhancement of melanoma cell adhesion on fibrinogen and vitronectin.

Reduced expression of platelet surface glycoprotein receptor IIb/IIIa at hyperthermic temperatures.

BACKGROUND: Hyperthermic temperatures exist from the heat dissipation of the implantable energy source of an artificial heart. This procedure as well as therapies for cancer and thermal injuries pose a new medical problem. Among many reported effects of heat on biologic systems, platelet functions such as maximal aggregation and adhesion are known to be reduced. Using flow cytometry, we have studied platelet dysfunction at elevated temperatures and have gained a mechanistic comprehension of the loss of platelet function. EXPERIMENTAL DESIGN: Platelet rich plasma was incubated at differing temperatures for 1 hour. Immediately after, the platelets were stained using mAb against glycoprotein IIb/IIIa (GPIIb-IIIa) (CD41a) and other platelet surface glycoproteins (GP) involved in aggregation and adhesion. Relative fluorescence intensity was measured using single-labeled, laser flow cytometry to determine changes in GP surface expression. In addition, scanning electron microscopy was used to evaluate morphologic changes. RESULTS: Hyperthermic temperatures between 40 and 44 degrees C significantly lowered the mAb cell surface binding in vitro of GP that participate in aggregation and adhesion. The most dramatic temperature-dependent loss of mAb binding was demonstrated by anti-GPIIb-IIIa, the mAb against the fibrinogen receptor. mAb binding to this receptor at 44 degrees C was decreased to 6.2% of a base-line fluorescence intensity of 654 (arbitrary units). The ADP-induced aggregation of platelets incubated at the same temperature also decreased to 2.1% of maximum aggregation. Other mAb, such as those against the von Willebrand factor receptor (GPIb) (CD42b), the thrombospondin receptor (GPIV) (CD36), and GPIIIa (CD61), also showed statistically significant reduction of mAb binding but to a lesser degree. Finally, scanning electron microscopy as well as side-scatter density plots from flow cytometry revealed that platelets became more spherical after incubation at 44 degrees C. CONCLUSIONS: The significant reduction in mAb binding correlates with functional impairment exhibited during hyperthermic incubation. Our results support the loss of binding ability of surface GP that are involved in aggregation and adhesion as a mechanism of platelet dysfunction upon heating. GPIIb-IIIa appeared the most susceptible to heat and the principal agent in thermal induced loss of platelet function. Significant morphologic changes at 44 degrees C, the critical temperature at which ADP-induced aggregation ceases, may contribute as well.

CD31/PECAM-1 is a ligand for alpha v beta 3 integrin involved in adhesion of leukocytes to endothelium.

To protect the body efficiently from infectious organisms, leukocytes circulate as nonadherent cells in the blood and lymph, and migrate as adherent cells into tissues. Circulating leukocytes in the blood have first to adhere to and then to cross the endothelial lining. CD31/PECAM-1 is an adhesion molecule expressed by vascular endothelial cells, platelets, monocytes, neutrophils, and naive T lymphocytes. It is a transmembrane glycoprotein of the immunoglobulin gene superfamily (IgSF), with six Ig-like homology units mediating leukocyte-endothelial interactions. The adhesive interactions mediated by CD31 are complex and include homophilic (CD31-CD31) or heterophilic (CD31-X) contacts. Soluble, recombinant forms of CD31 allowed us to study the heterophilic interactions in leukocyte adhesion assays. We show that the adhesion molecule alpha v beta 3 integrin is a ligand for CD31. The leukocytes revealed adhesion mediated by the second Ig-like domain of CD31, and this binding was inhibited by alpha v beta 3 integrin-specific antibodies. Moreover alpha v beta 3 was precipitated by recombinant CD31 from cell lysates. These data establish a third IgSF-integrin pair of adhesion molecules, CD31-alpha v beta 3 in addition to VCAM-1, MadCAM-1/alpha 4 integrins, and ICAM/beta 2 integrins, which are major components mediating leukocyte-endothelial adhesion. Identification of a further versatile adhesion pair broadens our current understanding of leukocyte-endothelial interactions and may provide the basis for the treatment of inflammatory disorders and metastasis formation.

Antibodies to the vitronectin receptor (integrin alpha V beta 3) inhibit binding and infection of foot-and-mouth disease virus to cultured cells.

The amino acid sequence Arg-Gly-Asp (RGD) is highly conserved on the VP1 proteins of different serotypes and subtypes of foot-and-mouth disease virus (FMDV) and is essential for cell attachment. This sequence is also found in certain extracellular matrix proteins that bind to a family of cell surface receptors called integrins. Within the Picornaviridae family, enterovirus coxsackievirus A9 also has an RGD motif on its VP1 capsid protein and has recently been shown to utilize the vitronectin receptor integrin alpha V beta 3 as a receptor on monkey kidney cells. Competition binding experiments between type A12 FMDV and coxsackievirus A9 using BHK-21 and LLC-MK2 cells revealed shared receptor specificity between these two viruses. Polyclonal anti-serum to the vitronectin receptor and a monoclonal antibody to the alpha V subunit inhibited both FMDV binding and plaque formation, while a monoclonal antibody to the beta 3 subunit inhibited virus binding. In contrast, antibodies to the fibronectin receptor (alpha 5 beta 1) or to the integrin (alpha V beta 5) had no effect on either binding or plaque formation. These data demonstrate that the alpha V beta 3 vitronectin receptor can function as a receptor for FMDV.

Antigen-independent, integrin-mediated T cell activation.

The "outside-in" signals produced by the interaction of integrin molecules with the extracellular matrix (ECM) trigger a multitude of cellular events. The vitronectin receptor (VNR), an alpha v beta 3 heterodimer, functions as a costimulatory molecule for the activation of a subset of V gamma 1.1/C gamma 4-bearing gamma/delta T cells, which have been postulated to recognize a ubiquitous self-antigen. We addressed the question of whether stimulation of these T cells requires both engagement of the VNR by ECM proteins and engagement of the TCR by its Ag. We introduced into a TCR- but VNR+ mutant T cell hybridoma, TG40 (derived from 2B4), a chimeric molecule that contains the cytoplasmic tail of the TCR zeta-chain fused to the cytoplasmic and transmembrane region of either human CD8 or human CD25. The transfectants expressing the chimeric molecules secreted IL-2 constitutively when the VNR was engaged with a ligand, e.g., provided by ECM proteins present in FCS. This constitutive cytokine secretion could be blocked with mAb directed against the VNR, with or the peptide RGD, or by growth in serum-free medium. VNR-mediated cell activation also induced the phosphorylation of the zeta-chain. Signaling through the zeta-chain was required, as cells transfected with a chimera containing only a 22 amino-acid long, truncated zeta-chain did not secrete IL-2 constitutively. Thus, we demonstrated that the binding of the VNR to ECM protein in the presence of the zeta-chain is sufficient to induce cytokine secretion by T cells and does not require the recognition of an Ag by the TCR. Such integrin-mediated, Ag-independent activation of T cells may play a critical role in the potentiation of inflammatory responses.

Expression of vitronectin receptor on human NK cells and its role in protein phosphorylation, cytokine production, and cell proliferation.

In this paper, we provide evidence that the vitronectin receptor (VNR) alpha v beta 3 is expressed on human NK cells. The presence of this VNR on freshly purified NK cells was demonstrated by flow cytometry analysis, as well as biochemically, after 125I-labeled surface lactoperoxidase labeling and immunoprecipitation. mAbs LM142 and LM609 specific for alpha v and alpha v beta 3, respectively, precipitated a heterodimer of alpha- and beta-chains with approximate molecular masses of 155 and 110 kDa under nonreducing conditions. Under reducing conditions, there was an apparent decrease in the molecular mass of the alpha-chain, which is likely to result from the release of a protein of 20 to 30 kDa linked by internal disulfide bond to the alpha v-chain. Integrin alpha v beta 3 expressed on NK cells became functional, i.e., was able to bind its ligand, vitronectin (VN), only after cellular activation or when costimulation with an additional signal was provided. Thus, NK cells adhered to plastic-immobilized VN only after IL-2 activation, and RGD-containing synthetic peptides or mAbs specific for alpha v beta 3 complex inhibited this binding. To assess the role of the VNR in signal transduction, anti-beta 3 mAb was used to cluster the VNR on NK cells and, thereby, mimic the process that occurs during formation of adhesive contacts. Cross-linking of VNR on fresh NK cells stimulated phosphorylation on tyrosine residues of several intracellular proteins. The major increase in tyrosine phosphorylation was observed in proteins of approximate molecular masses of 75 and 120 kDa. Therefore, signal transduction by the VNR on NK cells induced activation of intracellular protein kinases. Ligand engagement of the VNR on NK cells also costimulated cytokine production and proliferation of NK cells. Binding of NK cells to plastic-immobilized VN served as a costimulus with either anti-Fc gamma RIII or IL-2 to produce IFN-gamma, TNF-alpha, and cell proliferation. Our findings suggest that occupancy and subsequent clustering of VNRs play a role in the activation and function of human NK cells.

Constitutive alpha V beta 3 integrin-mediated adhesion of human lymphoid B cells to vitronectin substrate.

Adherence to cells and matrices participates in lymphocyte migration and tissue localization and contributes to the regulation of growth and differentiation of the lymphoid cells. The adherence is mainly mediated by three families of cell-surface proteins: integrins, immunoglobulin (Ig)-related molecules, and selectins. Integrins recognize Ig-related molecules such as ICAMs as well as fibronectin (FN), vitronectin (VN), and other matrix proteins. In this study, the in vitro adhesive properties of two Epstein-Barr virus-carrying B lymphoblastoid cell lines, IB-4 and NAD-20, were compared. IB-4 cells grow as a monolayer in contrast to NAD-20 cells, which grow as cell clusters. IB-4 cells were found to adhere to the tissue culture vessel through a component of the fetal bovine serum. By using blocking monoclonal antibodies to cell-surface molecules and serum proteins, IB-4 cells were found to use alpha V beta 3 integrin (CD51/CD61) and serum VN as the adhesive molecules. alpha V beta 3 integrin also mediated adhesion of IB-4 cells to human serum VN and to purified VN and FN. This constitutive adherence was not enhanced by phorbol ester treatment and was inhibited by RGD-containing peptides, in contrast to the homotypic adhesion of NAD-20 cells, which was mediated by beta 2 integrin CD11a/CD18 and its ligand ICAM-1 (CD54). Since VN is a component of both lymphoid tissue matrix and plasma, adhesion to this protein may affect functions and activities of B lymphocytes.

The vitronectin receptor (alpha v beta 3) is implicated, in cooperation with P-selectin and platelet-activating factor, in the adhesion of monocytes to activated endothelial cells.

In this study we have investigated the presence on endothelial cells of potential glycoprotein receptors, other than P-selectin, which are involved in the adhesion of monocytes at the early stages of activation. We report that the majority of cells binding to thrombin-activated endothelial cells from a peripheral blood mononuclear cell (PBMC) preparation are monocytes. The adhesion of PBMC to thrombin-activated, but not resting, endothelial cells was inhibited (66%) by a monoclonal antibody (mAb) directed against alpha v beta 3. Elutriated monocytes or a monocytic cell line (U937) were also inhibited by this antibody, its F(ab)'2 fragments and three other anti-(alpha v beta 3) mAbs. alpha v beta 3 isolated from endothelial-cell lysates significantly inhibited the adhesion of monocytes and U937 cells to endothelial cells. A peptide motif (RGDF) known to interact with alpha v beta 3 inhibited U937 cell adhesion to activated endothelial cells by 53%. Finally, an anti-(P-selectin) mAb (LYP20) or a platelet-activating factor (PAF)-receptor antagonist (WEB 2086) inhibited monocyte adhesion to activated endothelial cells. This study shows for the first time that alpha v beta 3 is implicated, in addition to P-selectin and PAF, in the adhesion of monocytes to activated endothelial cells.

Integrin alpha v beta 3 antagonists promote tumor regression by inducing apoptosis of angiogenic blood vessels.

A single intravascular injection of a cyclic peptide or monoclonal antibody antagonist of integrin alpha v beta 3 disrupts ongoing angiogenesis on the chick chorioallantoic membrane (CAM). This leads to the rapid regression of histologically distinct human tumors transplanted onto the CAM. Induction of angiogenesis by a tumor or cytokine promotes vascular cell entry into the cell cycle and expression of integrin alpha v beta 3. After angiogenesis is initiated, antagonists of this integrin induce apoptosis of the proliferative angiogenic vascular cells, leaving preexisting quiescent blood vessels unaffected. We demonstrate therefore that ligation of integrin alpha v beta 3 is required for the survival and maturation of newly forming blood vessels, an event essential for the proliferation of tumors.

Epitopes and biological activities of two monoclonal antibodies to platelet integrin alpha IIb beta 3.

We have developed two monoclonal antibodies (MAbs), B6A3 and C4G1. The whole molecules of the two MAbs inhibited in vitro human platelet aggregation induced by either ADP, collagen or thrombin, and their F(ab')2 fragments inhibited ex vivo platelet aggregation induced by ADP in monkey. The concentrations necessary for complete inhibition were 5 and 1 microgram/ml for B6A3 and C4G1, respectively. The Fab fragment of C4G1 but not B6A3 inhibited platelet aggregation. B6A3 and C4G1 bound to activated platelets with dissociation constants of 0.25 and 0.82 nM, respectively. B6A3 recognized an epitope on beta 3, which was sensitive to reduction and alkylation of cystine residues, and C4G1 recognized a conformational epitope on the alpha IIb beta 3 complex, which was sensitive to EDTA. The binding of fibrinogen to activated platelets was inhibited by both MAbs. However, the binding of fibrinogen to isolated alpha IIb beta 3 was inhibited by the whole molecule of C4G1 but not B6A3, although both MAbs bound to the isolated alpha IIb beta 3. The binding of these MAbs to the isolated alpha IIb beta 3 was not inhibited by either Arg Gly Asp Ser (RGDS) or fibrinogen gamma-peptide. In addition, B6A3 but not C4G1 bound to human endothelial cells. These MAbs should contribute to the elucidation of the mechanism of platelet aggregation.

Vitronectin-driven human keratinocyte locomotion is mediated by the alpha v beta 5 integrin receptor.

Vitronectin is a soluble serum factor that is known to promote epiboly of keratinocytes in explant cultures and enhance cell spreading and attachment to matrix. Recently, vitronectin was demonstrated to promote human keratinocyte locomotion. The mechanism(s) by which vitronectin enhances keratinocyte migration is unknown. In this study, we quantitated the vitronectin-driven migration of human keratinocytes in the presence of antibodies to vitronectin receptors. We found that vitronectin's effect of promoting human keratinocyte migration was inhibited by antibody-directed against the alpha v beta 5 receptor. In addition, we surface-labeled human keratinocytes, chromatographed extracts of the cell membranes on a vitronectin column, and then immunoprecipitated the bound and eluted proteins with antibodies to specific vitronectin receptors. We identified the vitronectin receptors on human keratinocytes as bands of 150,000 and 100,000 daltons without reduction and as 125,000 and 110,000 daltons under reducing conditions. Immunoprecipitation with specific antibodies identified the major receptor to be the alpha v beta 5 integrin. In addition, we quantitated vitronectin-driven migration of human keratinocytes in the presence of Arg-Gly-Asp (RGD) and control peptides. We found that the presence of RGD, but not control peptide, inhibited vitronectin-driven migration of human keratinocytes. These studies demonstrate that human keratinocytes express vitronectin receptors and use the alpha v beta 5 receptor for cellular locomotion.

A monoclonal antibody inhibits adhesion to fibronectin and vitronectin of a colon carcinoma cell line and recognizes the integrins alpha v beta 3, alpha v beta 5, and alpha v beta 6.

Using whole viable human colon carcinoma HT29 cells as immunogen, we produced a monoclonal antibody (mAb) termed 69-6-5. The antibody was functionally selected on its anti-cell-spreading activity. By immunoprecipitation of surface radiolabeled cell lysates from HT29-D4 cells (an HT29 cell clone), mAb 69-6-5 recognized a molecular complex resembling integrin heterodimers. Sequential immunodepletions with mAb to the integrin alpha v subunit demonstrated that this complex was composed of alpha v-containing integrins. Accordingly, mAb 69-6-5 reacted with integrin alpha v beta 3 immunopurified from melanoma cells and integrins alpha v beta 5 and alpha v beta 6 immunopurified from pancreatic carcinoma cells. In cell adhesion assays, the 69-6-5 mAb was able to inhibit strongly in a dose-dependent manner arginine-glycine-aspartic acid-mediated adhesion of HT29-D4 cells to vitronectin, fibronectin, or ProNectin F but not to laminin or collagen. Immunoprecipitations with beta chain-specific antisera indicated that these cells express integrins alpha v beta 5 (receptor for vitronectin) and alpha v beta 6 (receptor for fibronectin) but neither alpha v beta 1 nor alpha v beta 3. In summary, these results indicated that mAb 69-6-5 reacts with several alpha v integrins and that it can effectively interfere with the adhesive functions of at least alpha v beta 5 and alpha v beta 6, which represent the major receptors on HT29-D4 cells responsible for their adhesion on vitronectin and fibronectin.

Requirement of vascular integrin alpha v beta 3 for angiogenesis.

Angiogenesis depends on the adhesive interactions of vascular cells. The adhesion receptor integrin alpha v beta 3 was identified as a marker of angiogenic vascular tissue. Integrin alpha v beta 3 was expressed on blood vessels in human wound granulation tissue but not in normal skin, and it showed a fourfold increase in expression during angiogenesis on the chick chorioallantoic membrane. In the latter assay, a monoclonal antibody to alpha v beta 3 blocked angiogenesis induced by basic fibroblast growth factor, tumor necrosis factor-alpha, and human melanoma fragments but had no effect on preexisting vessels. These findings suggest that alpha v beta 3 may be a useful therapeutic target for diseases characterized by neovascularization.

Localization of thrombospondin and its cysteine-serine-valine-threonine-cysteine-glycine-specific receptor in human breast carcinoma.

BACKGROUND: Thrombospondin (TSP), a cell-matrix adhesion protein, and cysteine-serine-valine-threonine-cysteine-glycine (CSVTCG), a major TSP cell adhesive domain, have recently been shown to play a role in tumor cell metastasis. In this study we immunohistochemically localized TSP and its newly discovered CSVTCG-specific receptor in normal, benign, and neoplastic breast tissues. EXPERIMENTAL DESIGN: Paraffin sections of normal, benign, and neoplastic breast tissue were examined immunohistochemically for the presence of TSP and its CSVTCG-specific receptor using the avidin-biotin complex immunoperoxidase staining procedure. RESULTS: Positive staining using polyclonal antibodies for TSP and its tumor cell adhesion receptor, isolated from a human adenocarcinoma of the lung, was observed in all primary breast ductal carcinomas examined (N = 11). In contrast, all benign lesions and normal breast tissue stained negative for TSP and its receptor with the exception of two fibrocystic breast samples with hyperplasia. One of the samples showed strong TSP staining of ductal apocrine cells, whereas the other showed apical receptor staining of hyperplastic ductal cells. The negatively staining normal and benign tissues consisted of 1 normal breast, 1 gynecomastia, 5 fibroadenomas, and 6 fibrocystic samples. Positive staining for TSP in ductal carcinoma was only localized in the dense stromal collagen adjacent to tumor, whereas the TSP receptor localized to the tumor cells. Consistent with these immunohistochemical staining results was the observation that protein extracts of breast carcinoma cells contained receptor with no detectable TSP as revealed by Western blotting. Capillary endothelium was focally positive for receptor in regions proximal to ductal epithelium in 8 of 11 neoplastic tissues and in 6 of 14 benign samples. CONCLUSIONS: Our results indicate that increasing expression of stromal TSP and the CSVTCG-specific TSP receptor in ductal epithelium correlates with neoplastic transformation. In addition, our results indicate that both malignant and benign breast tissue can stimulate surrounding capillaries to express the TSP receptor, whereas only carcinoma has the capacity to stimulate surrounding nonendothelial stromal cells, such as myofibroblasts, to secrete a TSP-rich matrix that may contribute to the desmoplastic stromal reaction characteristic of ductal carcinoma tumor. The TSP-rich matrix may then promote tumor cell attachment, migration, and angiogenesis, factors important in tumor growth. The receptor-rich capillary endothelium may promote the cell adhesive interactions important in tumor intravasation. Taken together the results of this study provide a rational basis for a role of TSP in tumor angiogenesis and metastasis.

Bioactive Arg-Gly-Asp conformations in anti-integrin GPIIb-IIIa antibodies.

Antibodies can mimic the biological function of physiological ligands, yet few examples indicate the structural similarity between antibodies and the ligands that they mimic. Originally, the competition of antibodies for ligand binding sites was conjectured to be through similar three-dimensional conformations, which represent the "internal image" of the given ligand. Here we show that residues in a complementary determining region (CDR) can adopt the same bioactive structures observed in ligands. Structure-function studies of three anti-GPIIb-IIIa murine monoclonal antibodies, PAC-1, LJ-CP3, and OP-G2, indicate that the RYD sequence in their H-CDR3 domain occupies the same conformational space as RGD in conformationally constrained, bioactive, GPIIb-IIIa cell-surface adhesion ligands. The relative location of the guanidinium and carboxylate groups in the RXD regions is identified as an important recognition feature, and the conformational space occupied by this region in the antibodies is only slightly larger than that in the most bioactive peptides. Additionally, we show that antibodies can unveil other potential bioactive sequences, which may impart specificity. Thus antibodies are an exquisite probe for identifying motifs of short adhesion stretches, thereby revealing amino acid sequences and restricted geometries that might be used as lead compounds in drug design.

Use of enzyme-linked immunosorbent assay (ELISA) for detection and quantification of monoclonal antibodies.

The thrust of the present work is the demonstration of the feasibility of using an enzyme-linked immunosorbent assay (ELISA) for the detection and quantification of a monoclonal antibody human serum. The antibodies used, 7E3 and D3GP3, have been proposed as platelet receptor blockers, being directed specifically against the platelet membrane receptors (glycoprotein IIb/IIIa) and thus of significance in the management of patients at a very hgih risk of thrombotic occlusion. It is shown that 7E3 is more easily adsorbed than D3GP3 and hence has a higher potential for the stated application.