Polyclonal antibodies to xenogeneic endothelial cells induce apoptosis and block support of tumor growth in mice.

In this study, we demonstrate that vaccination of rabbits with murine endothelial cells yields polyclonal immunoglobulin (IgG) with potent antiangiogenic activity. The mechanism of this response appears to be through apoptosis of endothelial cells in vitro. Induction of polyclonal IgG in a xenogeneic host may be useful in passive immunotherapy of a variety of cancers. In fact, the antibody showed antitumor activity in three mouse tumor models (murine B16F10 melanoma, murine SVR angiosarcoma, and human DLD-1 colorectal adenocarcinoma). The polyclonal antibody generated here demonstrated utility in radioimaging of tumors in vivo, using positron emission tomography (PET) imaging, and suggested an antitumor effect in vivo. The results suggest that the antitumor effect in vivo may be related to antiangiogenic effects. Furthermore, anti-endothelial cell antibodies such as these could be useful reagents in isolating specific targets that comprise and induce the antiangiogenic effect.

Conjugation of monoclonal antibodies with TETA using activated esters: biological comparison of 64Cu-TETA-1A3 with 64Cu-BAT-2IT-1A3.

A simple method for conjugation of monoclonal antibodies (mAbs) with the chelating agent 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA), has been developed using commercially available reagents. This method involved activation of a single carboxyl group of TETA with N-hydroxysulfosuccinimide and 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide. The resulting activated ester of TETA was reacted with the anti-colorectal carcinoma mAb 1A3 at molar ratios ranging from 10:1 to 100:1 to give immunoconjugates modified with an average of 0.4 to 2.0 functional chelators per antibody molecule. The TETA-1A3 conjugate was labeled with 64Cu at specific activities as high as 15.4 microCi/microgram, and the radiolabeled mAb exhibited high in vitro serum stability and minimal loss of immunoreactivity. The biodistribution of 64Cu-labeled TETA-1A3 in hamsters bearing GW39 human colon carcinoma xenografts was compared to that of 64Cu-BAT-2IT-1A3 (BAT = 6-(p-bromoacetamidobenzyl)-1,4,8,11-tetraazacyclotetradecane-1,4,8,11- tetraacetic acid; 2IT = 2-iminothiolane). Both conjugates showed high tumor uptake (6.60-9.05% injected dose/gram) from 24 to 48 h post-injection and generally similar blood clearance and non-target organ uptakes. Human absorbed dose estimates derived from the hamster biodistribution data showed the critical organs for both conjugates to be the large intestine and the red marrow. Our results suggest that the in vitro and in vivo performance characteristics of 64Cu-TETA-1A3 compare favorably with those of 64Cu-BAT-2IT-1A3 and that further evaluation of the diagnostic and therapeutic efficacy of 64Cu-TETA-1A3 is warranted.

Evaluation of an aqueous drainage glaucoma device constructed of ePTFE.

Aqueous drainage devices for the treatment of glaucoma are subject to the same limitations as most polymeric implants, namely a healing response comprised of chronic inflammation and fibrosis. The most widely used devices are currently made of silicone or polypropylene, materials that exhibit biocompatibility difficulties when they are implanted on the sclera underneath the conjunctiva of the eye. Decreased outflow of aqueous fluid to the conjunctival space caused by the development of a fibrous capsule around the device accounts for at least 20% of aqueous shunts failures. Clearly, the need exists to improve the healing response to aqueous drainage devices, and one approach is to develop new polymers or polymer modifications. Improved devices would elicit a limited fibrotic response while increasing neovascularization around the implant. Previous studies have indicated that denucleation markedly improves the healing characteristics and biocompatibility of expanded polytetrafluoroethylene (ePTFE). We reasoned that altering the design of drainage devices to allow the use of denucleated ePTFE in vivo might minimize fibrosis, thereby improving shunt function. We found that after 8 weeks in vivo, experimental shunt function was equivalent to the Baerveldt shunt, while there was less scarring with increased neovascularizatin. These findings suggest that ePTFE has potential as an improved, long-term alternative material for use in constructing glaucoma shunts.

Characterization of cellular density and determination of neointimal extracellular matrix constituents in human lower extremity vein graft stenoses.

UNLABELLED: Arterial restenosis has been attributed to a hyperproliferative smooth muscle cell response. Paradoxically, studies of human coronary atherectomy and vein graft stenotic lesions have demonstrated a relatively low nuclear proliferative rate with the majority of the neointimal mass consisting of extracellular matrix. The purpose of the present study was to characterize the cellular density and determine the relative composition of the extracellular matrix protein constituents in stenotic, human lower extremity vein-bypass graft lesions. METHODS: Duplex surveillance of 148 consecutive infrainguinal bypass grafts identified 17 patients with 22 preocclusive autogenous vein graft stenoses (mean graft age 7 months). Morphological analyses of these stenotic lesions were compared with excised samples of 20 greater saphenous vein segments taken at the time of graft implantation from matched control patients. Intimal and medial areas were compared and cell density was determined with fluorescent nuclear (Bisbenzimide) staining. Differential light microscopy with pentachrome staining was performed to determine the relative percent composition of intimal matrix constituents by stereological morphometric (point-count) techniques. RESULTS: The intimal areas for control and stenotic vein segments were 1.64 x 10(6) microm2 and 3.85 x 10(6) microm2, P < 0.0001, whereas the intimal nuclear densities (cells/unit volume) were 1.42 x 10(3) and 1.70 x 10(3) cells/microm2, P = 0.03. respectively. The corresponding medial area and medial nuclear densities were 5.01 x 10(6) microm2, 3.31 x 10(6) microm2; P = 0.08, and 2.27 x 10(3), 3.29 x 10(3); P = 0.001, for control and stenotic specimens, respectively. The intima:media area ratios were much greater, whereas the intimal and medial cell densities were only slightly greater in the stenotic compared with control veins. The relative composition of intimal extracellular matrix proteins of stenotic vein graft segments consisted of 21% cellular (fibrous) material, 33% collagen, and 46% glycosaminoglycan ground substance. CONCLUSION: The intimal lesions responsible for lower extremity vein graft stenosis are more hypertrophic than hyperplastic. Therapies aimed at preventing arterial and vein graft restenosis may thus need to inhibit matrix biosynthetic processes in addition to cellular proliferation.

Denucleation promotes neovascularization of ePTFE in vivo.

Expanded polytetrafluoroethylene (ePTFE) implants are being increasingly used as vascular prostheses and other devices. However the tissue response associated with this and other polymer implants continues to limit any long-term function. One of the major approaches currently being investigated to improve biocompatibility involves surface modification of the base polymer. In this report, we attempted to alter the healing characteristics of ePTFE by denucleation, a process which removes air trapped within the interstices of the material. Additionally, adsorption of extracellular proteins on the denucleated polymer was also tested. After 5 weeks implanted in subcutaneous and epididymal fat sites of rats, the material was explanted and the healing around the implant evaluated histologically. We found that in skin implants, denucleation alone resulted in a substantial reduction in the fibrous capsule which has been previously reported for untreated ePTFE, and an increase in blood vessel development around and within the polymer. Absorption of extracellular matrix proteins prior to implantation resulted in a reduced vascularity of the implants compared with denucleation-only implants. Implants in fat tissue, regardless of treatment, showed very little tissue reaction, either in the number of inflammatory cells, development of a fibrous capsule or neovascularization. These results suggest that the presence of air nuclei within porous material may contribute to the inappropriate healing response associated with these polymers. In addition, they confirm earlier reports that healing around implanted polymers is tissue-specific.

Dynamics of protein-protein docking: cytochrome c and cytochrome c peroxidase revisited.

The dynamics of the docking step in the electron transfer reaction between yeast cytochrome c peroxidase and iso-1-cytochrome c has been studied using the Brownian dynamics method. In particular we have calculated the bimolecular rate constant at which a specific complex, the xray crystalline complex, can form in solution by translational and rotational diffusion in a field of force. Complexation criteria have been assessed based on the simultaneous alignment of three atom-atom contacts, as well as alternative criteria. The proteins are able to align one or two contacts at remarkably high rates, in fact, at rates approaching the diffusion-controlled limit for two spheres reactive over their entire surfaces. Three contacts may align, and hence the specific complex may dock, at rates on the order of 10(8) M(-1) s(-1), which is quite representative of the experimental association rate constant for ET-competent complex(es). The formation of the specific complex is strongly influenced by the favorable electrostatic interaction between these proteins. It is striking that a specific protein-protein complex can form within one order of magnitude as fast as two spherical proteins can touch at any orientation. It remains plausible that the high ET tunneling rate in this system can take place through a single highly favorable specific complex using a single high efficiency pathway. Still the contribution from a nonspecific set of complexes is not ruled out, particularly considering the marginal reproduction of the ionic strength dependence in the formation of the xray complex.

Stimulation of angiogenesis by canine parathyroid tissue.

BACKGROUND: Autotransplanted parathyroid tissue is capable of inducing neovascularization in vivo, restoring calcium homeostasis with regulatory control. The mechanisms of parathyroid-induced neovascularization remain to be determined. Using an unique three-dimensional in vitro model, we tested the ability of parathyroid tissue to stimulate angiogenesis. METHODS: Healthy 1 mm3 fragments of normal canine parathyroid tissue were cocultured with freshly isolated microvessels that were embedded in a collagen I gel. After 7 days the gels were stained with Gs-1 lectin, a specific marker for rat endothelium. With image analysis the microvessel density (as the percentage of area) was determined. RESULTS: A significant increase in mean microvessel density (20.90% +/- 1.28 versus 16.51% +/- 1.66%) was seen with parathyroid coculture compared with control (n = 17, p < 0.05). There was no difference in microvessel density between controls and microvessels exposed to increasing concentrations of parathyroid hormone or calcium. The density of seeded microvessels influenced the effect of parathyroid stimulation of angiogenesis. The effect was apparent only at low seeding densities. CONCLUSIONS: We conclude that parathyroid tissue intrinsically stimulates angiogenesis in vitro by a secreted product, independent of calcium or parathyroid hormone.

Angiogenic potential of microvessel fragments established in three-dimensional collagen gels.

During angiogenesis, the microvasculature displays both vessel remodeling and expansion under the control of both cellular and extracellular influences. We have evaluated the role of angiogenic and angiostatic molecules on angiogenesis in an in vitro model that more appropriately duplicates the cellular and extracellular components of this process. Freshly isolated microvessel fragments from rat adipose tissue (RFMF) were cultured within three-dimensional collagen I gels. These fragments were characterized at the time of isolation and were composed of vessel segments observed in the microvasculature of fat in situ (i.e., arterioles, venules, and capillaries). Fragments also exhibited characteristic ablumenally associated cells including smooth muscle cells and pericytes. Finally, fragments were encased in an extracellular matrix composed of collagen type IV and collagen type I/III. The elongation of microvascular elements was subsequently evaluated using morphologic and immunocytochemical techniques. The proliferation, migration, and elongation of cellular elements in microvessel fragments from rat adipose tissue was dependent on initial fragment density, matrix density, and required serum. Inclusion of endothelial cell growth factors to microvessel fragments from rat adipose tissue 3-D cultures resulted in the accelerated elongation of tube structures and the expression of von Willebrand factor in cells constituting these tubes. Molecules with reported angiostatic capacity (e.g., transforming growth factor and hydrocortisone) inhibited vessel tube elongation. In vitro methods have been developed to evaluate numerous mechanisms associated with angiogenesis, including endothelial cell proliferation, migration, and phenotypic modulation. Microvascular endothelial cell fragments described in this study represent an in vitro population of cells that accurately duplicate the in vivo microcirculatory elements of fat. The proliferation of cells and elongation of microvascular elements subsequently observed in three-dimensional cultures provides an in vitro model of angiogenesis. Microvascular formation in this system results from pre-existing microvessel fragments unlike tube formation observed when cultured endothelial cells are placed in three-dimensional gels. This form of tube formation from cultured endothelium is more characteristic of vasculogenesis. Thus, the formation of microvascular elements from microvessel fragments provides the opportunity to examine the mechanisms regulating angiogenesis in an in vitro system amenable to precise experimental manipulation.

Reversible endothelial cell relaxation induced by oxygen and glucose deprivation. A model of ischemia in vitro.

Endothelial cells (EC) cultured on polymerized silicone deform the underlying substrate, producing microscopically visible wrinkles. This has been interpreted as cellular contraction, and we have previously concluded that EC normally maintain an active contractile tone. Since in ischemic tissues capillaries become "paralyzed" and lose their tone, we decided to examine the effects of glucose and/or oxygen deprivation on EC contractility. Contracting cultures with wrinkled silicone substrates were exposed to complete anoxia with or without exogenous glucose and followed by time-lapse photography. Under either glucose-or oxygen-free conditions, contraction was maintained for up to 4 days. If, however, both oxygen and glucose were removed, cellular contraction was reversed. After a period of 2 to 4 hours substrate wrinkles gradually disappeared, until by 3 to 7 hours, few to no wrinkles remained. Furthermore, within 10 minutes of restoration to normal oxygen (but not glucose) levels, substrate wrinkling reappeared. F-actin microfilament patterns and cell number per unit area were also altered by glucose and oxygen deprivation. Similar results were obtained using large or small vessel EC. We conclude that in the absence of glucose and oxygen EC lose their contractile tone, and that tone can be re-established upon re-exposure to oxygen. These findings should have implications for the pathogenesis of capillary paralysis in ischemia.

A new strategic planning model for a community-based nursing services program.

Models provide a way of envisioning effective action within a process. This paper discusses the application of the Boswell-Hensley Strategic Planning Model in the development of a community-based nursing service program. While the concepts, processes, and examples represented in this model are aimed specifically at community-based nursing service, the model can be applied to other health care services. The Boswell-Hensley model works on the assumption that humans with kindred motivations can agree on mutual goals and form rewarding partnerships that in the long term will advance the collective interest.

Acute endothelial cell contraction in vitro: a comparison with vascular smooth muscle cells and fibroblasts.

The contractile responses of cultured rat and calf endothelial cells (EC), vascular smooth muscle cells (VSMC), and fibroblasts (FB) to vasoactive mediators (thrombin, serotonin, bradykinin, and histamine), forskolin, and cytochalasin B were compared. Cells were grown on a pliable silicone membrane, and contraction was assessed, using time-lapse video microscopy, by recording changes in the wrinkling of the silicone as the cells exerted tension on the surface. We found that all cells contracted in the presence of serum or thrombin and that VSMC and FB also contracted with serotonin stimulation. Bradykinin and histamine were not contractants in this system. Discrepancies between these results and reports of changes in permeability of endothelial layers in vitro and in vivo may be due to (1) the vascular segment from which EC were studied or (2) the possibility that certain mediators may provoke a noncontractile response that results in gap formation. Thus changes in vascular permeability, which occur during inflammation, may have both contractile and noncontractile components. Forskolin, known to indirectly inhibit myosin light-chain kinase activity, and cytochalasin B were potent relaxants, suggesting a similar smooth muscle-like contractile mechanism for all three cell types.

Analysis of tegumental surface proteins of Schistosoma mansoni primary sporocysts.

Axenically transformed primary sporocysts of Schistosoma mansoni (NMRI strain) were labeled with 125I in an effort to identify sporocyst proteins exposed at the tegumental surface. Using the 125I activating reagent, 1,3,4,6-tetrachloro-3 alpha,6 alpha-diphenylglycoluril, in conjunction with SDS-PAGE and autoradiography, up to 12 bands were radiolabeled out of 60 components visualized by silver staining. Labeled proteins ranged in apparent Mr from greater than 200 to less than 12 kDa. Pronase treatment of living sporocysts after radioiodination removed all labeled material, suggesting that only surface proteins were being iodinated. Western blot analysis employing 5 monoclonal antibodies (MAB's) to sporocyst surface antigens revealed a wide range of reactivities which produced banding patterns closely reflecting autoradiograms of identical samples. The concomitant removal by Pronase of immunoreactive and radiolabeled surface proteins with identical Mr in the range of 90-130 kDa suggests that epitopes recognized by these antibodies are associated with these higher molecular weight surface proteins. However, although Pronase removes all labeled surface proteins, substantial nonradiolabeled, immunoreactive material with Mr less than 90 kDa still remains on enzyme-treated parasites. This indicates that MAB-reactive epitopes, in addition to their occurrence with surface proteins, are also associated with either unlabeled, protease-resistant surface components or internal antigens. The immunohistochemical localization of antibody-reactive material in gland-like structures within sporocysts supports an internal source for nonradiolabeled, immunoreactive components. Finally, the periodate sensitivity of the epitopes recognized by all tested MAB's suggests that carbohydrate moieties may represent a common and extremely immunogenic constituent of the sporocyst surface.(ABSTRACT TRUNCATED AT 250 WORDS)

Widespread antigenic cross-reactivity between plasma proteins of a gastropod, and its trematode parasite.

Evidence obtained earlier with immunofluorescence and immunoelectron microscopy was interpreted to imply molecular mimicry of the host Biomphalaria glabrata by the parasite Schistosoma mansoni. Using Western Blotting, we find that "mimicry" is due to widespread shared epitopes. Furthermore, at least one individual plasma protein of B. glabrata shares epitopes with the majority of plasma proteins in the mollusc. The widespread antigenic determinants may be carbohydrates. Caution is warranted when antisera, raised in mammals against heterogeneous invertebrate extracts, are used to screen for related antigenic determinants.

Lectin-dependent cell-mediated cytotoxicity in an invertebrate model: Con A does not act as a bridge.

The plant lectin concanavalin A (Con A) has been used in an invertebrate model of lectin-dependent cell-mediated cytotoxicity (LDCC). Macrophage-like cells from the susceptible host snail Biomphalaria glabrata become cytotoxic effectors when they encounter sporocysts of the parasitic trematode Schistosoma mansoni that have been treated with Con A. The sugar alpha-methyl mannoside and rabbit anti-Con A antibodies fail to block this LDCC. Con A is effective only when the target, not the effector cell, has been exposed to it. These results constitute evidence against the molecular bridging hypothesis and support the notion that surface modulation of the target may be the stimulus that provokes cytotoxicity. Results from this invertebrate model are discussed in the context of murine T lymphocyte LDCC.

Schistosoma mansoni: lectin-dependent cytotoxicity of hemocytes from susceptible host snails, Biomphalaria glabrata.

Normally benign hemocytes from a strain (M-line) of the snail, Biomphalaria glabrata, susceptible to Schistosoma mansoni, became cytotoxic toward the sporocyst stage if the parasite was first treated with the lectin, concanavalin A. Concanavalin A binding was inhibitable with alpha-methyl mannoside and killing was dose-dependent. Maximal levels of concanavalin A-induced cytotoxicity were comparable with levels observed when hemocytes from a resistant snail strain (13-16-R1) encountered untreated sporocysts. Induction of the cytotoxic response did not occur if hemocytes alone were pretreated with the lectin. A unique method incorporating ultraviolet microscopy and the vital fluorescent dye, eosin Y, was used for discriminating between live and dead sporocysts. This model may prove useful in understanding mechanisms used by invertebrate effector cells in recognition and killing of invading organisms.

Plasma components which mediate cellular defences in the gastropod mollusc Biomphalaria glabrata.

Blood plasmas from certain strains of Biomphalaria glabrata are known to have components which facilitate a hemocyte-effected cytotoxic response against encapsulated Schistosoma mansoni sporocysts. The possible identity of the factor(s) has been investigated. Sporocysts placed in snail plasma rapidly acquire a wide variety of host plasma antigens, at least some of which are displayed on the parasite surface. Plasmas from strains of snail resistant to the parasite agglutinate fixed sporocysts, while plasmas from susceptible strains fail to do so. Fixed sporocysts incubated in plasma bind selectively a subpopulation of plasma antigens; some are bound uniquely in resistant plasma. Another resembles a hemagglutinin from snail plasma. These and other recently acquired data are discussed in light of increasing evidence for defensive roles of multivalent lectins.

Isolation, characterization and functional assessment of a hemagglutinin from the plasma of Biomphalaria glabrata, intermediate host of Schistosoma mansoni.

An agglutinin from the plasma of Biomphalaria glabrata, vector of Schistosoma mansoni, has been isolated and partially characterized. Its isolation is a simple two-step process, yielding an essentially pure preparation that can then be manipulated free from other plasma components. It is a large glycoprotein (10(6) daltons), found in four of the five snail strains tested, and possesses affinity for galactose-type sugar residues. Based upon criteria such as molecular weight, carbohydrate and erythrocyte specificity, physical stability and anatomical source, this agglutinin is considered to be distinct from others reported from this mollusc. Involvement of the agglutinin in the host-parasite interaction is discussed.