Integrin-Assisted T-Cell Activation on Nanostructured Hydrogels.

Adoptive cell therapy (ACT) has shown very promising results as treatment for cancer in a few clinical trials, such as the complete remissions of otherwise terminal leukemia patients. Nevertheless, the introduction of ACT into clinics requires overcoming not only medical but also technical challenges, such as the ex vivo expansion of large amounts of specific T-cells. Nanostructured surfaces represent a novel T-cell stimulation technique that enables us to fine-tune the density and orientation of activating molecules presented to the cells. In this work, we studied the influence of integrin-mediated cell-adhesion on T-cell activation, proliferation, and differentiation using nanostructured surfaces, which provide a well-defined system at the nanoscale compared with standard cultures. Specifically, we synthesized a polymeric polyethylene glycol (PEG) hydrogel cross-linked with two fibronectin-derived peptides, cyclic Arg-Gly-Asp (cRGD) and cyclic Leu-Asp-Val (cLDV), that are known to activate different integrins. Moreover, the hydrogels were decorated with a quasi-hexagonal array of gold nanoparticles (AuNPs) functionalized with the activating antibody CD3 to initiate T-cell activation. Both cLDV and cRGD hydrogels showed higher T-cell activation (CD69 expression and IL-2 secretion) than nonfunctionalized PEG hydrogels. However, only the cRGD hydrogels clearly supported proliferation giving a higher proportion of cells with memory (CD4+CD45RO+) than naïve (CD4+CD45RA+) phenotypes when interparticle distances smaller than 150 nm were used. Thus, T-cell proliferation can be enhanced by the activation of integrins through the RGD sequence.

Location of a fibronectin domain involved in newt epidermal cell migration.

The interaction of migrating newt epidermal cells with the extracellular matrix protein, fibronectin, was studied. Pieces of nitrocellulose coated with intact human plasma fibronectin or proteolytically derived fragments were implanted into wounded limbs so that the coated nitrocellulose served as wound bed for migrating epidermal cells as they attempted to form a wound epithelium. Epidermal cells migrated very poorly on nitrocellulose pieces coated with (a) a 27-kD amino-terminal heparin-binding fragment, (b) a 46-kD gelatin-binding fragment, (c) a combined 33- and 66-kD carboxy-terminal heparin-binding preparation representing peptide sequences in the A and B chains, respectively, or (d) a 31-kD carboxy-terminal fragment from the A chain, containing a free sulfhydryl group. In contrast, epidermal cells readily migrated onto nitrocellulose coated with a mixture of fragments from the middle of the molecule (80-125kD) that bind neither heparin nor gelatin. Attempts to block migration on fibronectin-coated nitrocellulose using IB10, a monoclonal antibody that blocks Chinese hamster ovary cell attachment to fibronectin, were unsuccessful despite saturation of the epitope against which IB10 is directed. In contrast, a polyclonal anti-fibronectin antibody did inhibit migration. These results show that the ability of fibronectin to support newt epidermal cell migration is not shared equally by all regions of the molecule, but is restricted to a domain in the middle third. They also suggest that the site supporting migration is separate and distinct from the site mediating Chinese hamster ovary cell attachment.

Porphyromonas gingivalis, gamma interferon, and a proapoptotic fibronectin matrix form a synergistic trio that induces c-Jun N-terminal kinase 1-mediated nitric oxide generation and cell death.

During infection and inflammation, bacterial and inflammatory proteases break down extracellular matrices into macromolecular fragments. Fibronectin fragments are associated with disease severity in arthritis and periodontitis. The mechanisms by which these fragments contribute to disease pathogenesis are unclear. One likely mechanism is that fibronectin fragments induce apoptosis of resident cells, which can be further modulated by nitric oxide. Nitric oxide levels are increased at inflammatory sites in periodontitis patients. The aim of this study was to examine whether a proapoptotic fibronectin matrix (AFn) exerts its action by inducing nitric oxide and whether priming by bacterial and inflammatory components exacerbates this mechanism. Our data demonstrate that AFn increased the levels of nitric oxide and inducible nitric oxide synthase (iNOS) dose and time dependently in periodontal ligament (PDL) cells. These effects and apoptosis were inhibited by iNOS suppression and enhanced by iNOS overexpression. Nitric oxide and iNOS induction were paralleled by increased c-Jun N-terminal kinase 1 (JNK-1) phosphorylation. JNK-1 overexpression enhanced the expression of nitric oxide and iNOS, whereas inhibiting JNK-1 by small interfering RNA or a kinase mutant reversed these findings. Priming PDL cells with Porphyromonas gingivalis, its lipopolysaccharide (LPS), or gamma interferon (IFN-gamma) further increased nitric oxide levels and apoptosis. Escherichia coli and Streptococcus mutans induced lesser effects. Gingival fibroblasts and neutrophils responded to a lesser degree to these stimuli, whereas keratinocytes were resistant to apoptosis. Thus, proapoptotic matrices trigger nitric oxide release via JNK-1, promoting further apoptosis in host cells. LPS and IFN-gamma accentuate this mechanism, suggesting that during inflammation, the affected matrices and bacterial and inflammatory components combined exert a greater pathogenic effect on host cells.

Autoantibodies directed to extracellular matrix components in patients with different clinical forms of periodontitis.

BACKGROUND: Periodontal disease occurs in different clinical forms, from mild and easily controllable to more aggressive inflammatory manifestations, with difficult clinical or surgical control. There is evidence that a local autoimmune reaction may participate in the onset and persistence of the aggressive forms of periodontal disease. As the underlying mechanism in this process is not fully understood, we decided to investigate whether patients bearing this form of disease presented higher levels of antibodies directed to extracellular matrix (ECM) components such as type I collagen, fibronectin, and laminin. METHODS: Three groups of patients were selected by clinical criteria: 22 subjects with aggressive periodontitis (AgP) = group A; 18 subjects with chronic periodontitis (CP) = group B; and 10 healthy (H) volunteers without periodontal disease = group C. Autoantibody levels were evaluated in the sera of these patients using the enzyme-linked immunosorbent assay (ELISA) method. RESULTS: The levels of autoantibodies directed to ECM components (type I collagen, fibronectin and laminin) in the sera of patients with AgP and CP were shown to be statistically different (P <0.05). CONCLUSIONS: Although the present findings suggest an involvement of autoantibodies directed to ECM components per se in the pathogeny of certain types of periodontal disease, the available data do not support the classification of the lesions as autoimmune. Nevertheless, the findings open a possibility for the development of an additional method for a differential diagnosis of the aggressive forms of periodontal disease.

Magnetoliposomes: another principle of cell sorting.

Liposomes bearing anti-fibronectin antibodies and associated with ferromagnetic particles bound firmly to the surface of mouse embryo fibroblasts. Upon binding magnetoliposomes, the cells could be sorted in a magnetic field.

Antigen retrieval of basement membrane proteins from archival eye tissues.

Antigen retrieval (AR) methods can unmask tissue antigens that have been altered by fixation, processing, storage, or resin interactions. This is particularly important in the study of archival tissues, because primary fixatives and storage times may vary among specimens. We performed an electron microscopic study of basement membrane components of the aqueous humor drainage pathways from archival eye tissue. AR (heated citrate buffer, pH 6.0, LR White resin) increased the amount of label of collagen IV and fibronectin in tissue fixed in four different fixatives, including those containing glutaraldehyde. Labeling density was approximately doubled after AR for most fixatives, with the largest increase for tissues fixed in 4% paraformaldehyde/2% glutaraldehyde. Duration of storage time for archival tissues did not affect AR results. AR did not change the components of the extracellular matrix labeled; no "new" components were labeled after AR. We conclude that AR in citrate buffer can be used on selected extracellular matrix antigens to enhance label that would otherwise be lost due to fixation and storage.

Immunoelectrophoretic analysis of membrane glycoproteins in cultured human endothelial cells.

Human endothelial cells isolated from umbilical cords were solubilized in Triton X-100 and examined by crossed immunoelectrophoresis using rabbit antiserum against endothelial cells. Endogenous labelling of the endothelial cell proteins with 14C-mannose followed by crossed immunoelectrophoresis and autoradiography revealed about 10 immunoprecipitates. Four of these endothelial cell glycoproteins were labelled by lactoperoxidase catalyzed iodination and thus were surface located. Three of the surface located glycoproteins showed reduced electrophoretic mobility after incubation of the endothelial cells with neuraminidase and were therefore sialoglycoproteins. Amphiphilicity of endothelial cell glycoproteins was studied by crossed hydrophobic interaction immunoelectrophoresis with phenyl-Sepharose in the intermediate gel. Amphiphilic proteins also show increasing electrophoretic migration velocity with decreasing concentration of Triton X-100 in the first dimension gels. Five of the endothelial cell glycoproteins were shown to be amphiphilic using these two techniques. Two monoclonal antibodies against the platelet glycoprotein complex IIb-IIIa and glycoprotein IIIa, respectively, reacted with the same precipitate of endothelial cells. When a polyclonal antibody against the platelet glycoprotein complex IIb-IIIa was incorporated into the intermediate gel the position of two endothelial cell precipitates were lowered. One of these was a sialoglycoprotein.

On the possibility of the unification of drug targeting systems. Studies with liposome transport to the mixtures of target antigens.

In order to make the drug targeting system more effective, simple and technological, we suggest creation of drug-bearing conjugates capable of simultaneous binding with different antigenic components of the target via specific antibodies. It is supposed that the targeted therapy should include sequential administration of the mixture of modified antibodies (or other specific vectors) against different components of affected tissue and, upon antibody accumulation in the desired region, administration of modified drugs or drug carrying systems which can recognize and bind with the target via accumulated antibodies due to the interaction between vector modifier and carrier modifier. Using as a model system monolayers consisting of the mixture of extracellular antigens and appropriated antibodies, it was shown that the treatment of the target with the mixture of biotinylated antibodies against all target components and subsequent binding with the target of biotinylated liposomes via avidin permits high liposome accumulation on the monolayer. The binding achieved is always higher than in the case of the utilization of single antibody-bearing liposomes. Besides, the system suggested is very simple and its components can be easily obtained on technological scale in standardized conditions.

Physical and biological effects of a surface coating procedure on polyurethane catheters.

Central venous catheters are widely used in clinical practice; however, complications such as venous thrombosis or infection are frequent. The physical and biological effects of a coating procedure designed to improve the blood-contacting properties of polyurethane central venous catheters (CVCs) were studied. The surface atomic composition of poly(vinyl pyrrolidone) (PVP)-coated or uncoated Pellethane single lumen CVCs was characterized by electron spectroscopy for chemical analysis (ESCA), which confirmed the presence of an oxygen-rich PVP layer on the former material. Topological analysis of both single and triple lumen CVCs by scanning force microscopy (SFM) revealed a very smooth surface in PVP-coated catheters compared to the more frequent surface irregularities found either in uncoated Pellethane or in four additional randomly selected, commercially available triple lumen polyurethane CVCs. The PVP-coated Pellethane showed a strong reduction in either fibrinogen or fibronectin adsorption compared to all other PVP-free polyurethane CVCs. This decreased protein adsorption led to a proportional reduction in protein-mediated adhesion of either Staphylococcus aureus or Staphylococcus epidermidis and in the binding of a monoclonal antibody directed against the cell-binding domain of fibronectin. Increased surface smoothness and hydrophilic properties of polyurethane CVCs might decrease the risk of bacterial colonization and infection.

Vitronectin may mediate staphylococcal adhesion to polymer surfaces in perfusing human cerebrospinal fluid.

Prosthetic devices are frequently used for temporary or permanent drainage of cerebrospinal fluid (CSF), i.e., ventricular catheters with or without external monitoring devices and shunts. Infections constitute a serious complication in the use of biomaterials in contact with CSF; coagulase-negative staphylococci (CNS) are the most common aetiological agents. In the present study, polyvinylchloride (PVC) and PVC with endpoint-attached heparin were exposed to human CSF under perfusion to mimic conditions in vivo. Adhesion of strains of CNS isolated from patients with or without biomaterial-associated infection was determined: (i) after pre-incubation with fibronectin (Fn) or vitronectin (Vn) to block bacterial surface binding structures; and (ii) after preincubation of biomaterials with antibodies to Fn or Vn to block exposure of bacteria-binding domains on these host proteins. Pre-incubation of bacterial cells with Vn significantly reduced subsequent adhesion to polystyrene precoated with Vn 0.5 microg/well. When PVC pre-exposed to CSF was incubated with antibodies to Vn, subsequent bacterial adhesion of a Vn-binding strain, S. epidermidis 5703, was significantly reduced. The study shows that Vn may mediate adhesion of CNS in the presence of CSF. However, strains retrieved from biomaterials did not express binding of Vn or Fn to a higher extent than non-biomaterial-associated strains. Expression of heparin binding under static conditions did not correlate with staphylococcal adhesion to heparinised polymers under perfusion with CSF. The extent of adhesion of staphylococci to heparinised PVC was either reduced or the same as to unheparinised PVC.

Binding and orientation of fibronectin on polystyrene surfaces using immobilized bacterial adhesin-related peptides.

Fibronectin (FN) is known to bind to bacteria via high affinity receptors on bacterial surfaces known as adhesins. The binding of bacteria to FN is thought to have a key role in foreign device associated infections. For example, previous studies have indicated that Staphylococcus aureus adhesins bind to the 29 kDa NH(3) terminus end of FN, and thereby promote bacteria adherence to surfaces. Recently, the peptide sequences within the S. aureus adhesin molecule that are responsible for FN binding have been identified. Based on these observations, we hypothesize that functional FN can be bound and specifically oriented on polystyrene surfaces using bacterial adhesin-related (BRP-A) peptide. We further hypothesize that monoclonal antibodies that react with specific epitopes on the FN can be used to quantify both FN binding and orientation on these surfaces. Based on this hypothesis, we initiated a systematic investigation of the binding and orientation of FN on polystyrene surfaces using BRP-A peptide. To test this hypothesis, the binding and orientation of the FN to immobilized BRP-A was quantified using (125)I-FN, and monoclonal antibodies. (125)I-FN was used to quantitate FN binding to peptide-coated polystyrene surfaces. The orientation of bound FN was demonstrated by the use of monoclonal antibodies, which are reactive with the amine (N) or carboxyl (C) termini of the FN. The results of our studies demonstrated that when the BRP-A peptide was used to bind FN to surfaces that: 1. functional FN was bound to the peptide; 2. anti-C terminus antibodies bound to the peptide FN; and 3. only limited binding of anti-N terminus antibodies to peptide-bound FN occurred. We believe that the data that indicate an enhanced binding of anti-C antibodies reactive to anti-N antibodies are a result of the FN binding in an oriented manner with the N termini of FN bound tightly to the BRP-A on the polystyrene surface.

Expression of Fc-gamma-RIII and fibronectin in peripheral polymorphonuclear neutrophils with increased response to Fc stimulation in patients with juvenile periodontitis.

The nature of increased luminol-enhanced chemiluminescence (CL) in peripheral polymorphonuclear neutrophils (PMN) in juvenile periodontitis is of pathophysiological interest and may serve as a model for tissue damage caused by granulocytes. Activation of PMNs by opsonized Staphylococcus aureus was compared with that of Actinobacillus actinomycetemcomitans and Bacteroides gingivalis, regarded as being more specific for juvenile periodontitis. The CL was higher in the PMNs from the patients, independently of bacteria and mode of opsonization (autologous serum or gamma-globulin). Fc-gamma-RIII assessed on the washed fragments from peripheral PMNs was significantly (p < or = 0.005) lower in the patients with juvenile periodontitis than in their pair-matched healthy controls, while the content of fibronectin was higher (p < or = 0.032). However, when Fc-gamma-RIII and fibronectin were studied in fresh PMNs by flow cytometry no difference could be found between the two groups. The increased generation of CL of peripheral PMNs found in patients with juvenile periodontitis seems to be independent of humoral factors and of bacterial species and may be related to the properties of the PMN cell membranes.

Fibronectin-like immunoreactivity of the basilar membrane of celloidin-embedded human temporal bone sections.

Dysfunction of the mechanical properties of the basilar membrane is a potential cause of presbycusis. In cases of minimal sensorineural or strial degeneration it is believed to play a major role. The membrane has been shown to be partly composed of fibronectin. Fibronectin immunoreactivity is diminished in aged rats. Mesothelial cell line the perilymphatic surface of the membrane and are reduced in number in the aged rat cochlea. Fibronectin immunoreactivity was examined in human temporal bone sections (6 months to 92 years old). Hematoxylin and eosin stained section (17 to 97 years) were immunoreactivity was demonstrable in the human cochlea, but was not reduced, even in the eldest cases examined The number of mesothelial cells was reduced, however, and was related to the age of the individual, but not to the clinical diagnosis or audiogram shape. These two factors do not, therefore, appear to give rise to hearing losses associated with presbycusis.

In vivo cell interactions with calcium phosphate bioceramics.

Biointegration, resorption process, and solubility in physiological environments of calcium phosphate materials are scarcely described by ultrastructural studies. In vivo cells interactions with calcium phosphate materials are scarcely described by ultrastructural studies. In vivo cells interactions with calcium phosphate biomaterials are mediated by different proteins from physiological fluid, and in order to observe at the ultrastructural level the cell colonization, the resorption, process and the biointegration, we used in these experiments calcium phosphate materials precoated with fibronectin or not precoated. Two kinds of well determined materials were used for this study, Beta-tricalcium phosphate (B-TCP) and hydroxyapatite (HAP). The implants were soaked in human fibronectin diluted solution and were implanted in the connective tissue of rabbit abdomen. Our results showed that the fibroblasts and macrophagous++ cells interaction with the calcium phosphate crystal (B-TCP and HAP) was more important in the experiments with a fibronectin bilayer. In the presence of fibronectin at the grains surface of the material, cystic cavities' or fibrous encapsulation was suppressed and cells with fibers were in close contact with the material. The presence of fibronectin immediately after implantation seemed to increase the adhesion and the cell colonization. Fibronectin creates an organic interface between crystals and cells, and can promote interactions from cells and biomaterials.

The effect of molecular mobility of supramolecular polymer surfaces on fibroblast adhesion.

The effect of hydrated molecular mobility of polymer surfaces on cell adhesion behavior was investigated. ABA-type block copolymers composed of polyrotaxane (PRX) and hydrophobic anchoring terminal segments were synthesized as a platform of molecularly mobile surfaces. The result of QCM-D measurement in water revealed that the molecularly mobile PRX block copolymer surfaces were higher in hydrated molecular mobility than the corresponding random copolymer surfaces with similar content of hydrophobic methoxy groups. The number of adhering fibroblasts depended on the amount of fibronectin adsorbed from serum but was independent of the molecular mobility. However, the morphology of the adhering fibroblasts was strongly dependent on the extent of molecular mobility in water. These results indicate that molecular mobility on polymer surfaces is one of the significant considerations for regulating cellular responses.

Priming effect of fibronectin fragments on the macrophage inflammatory response: potential contribution to periodontitis.

Fibronectin, an extracellular matrix component, is a substrate for multiple host and bacterial proteinases found in inflamed periodontal sites. In the present study, we investigated the potential contribution of various fibronectin fragments to the inflammatory process of periodontitis. Our results showed that the smaller fragments of fibronectin (30 and 45 kDa) were the most potent inflammatory inducers as they dose-dependently increased the secretion of TNF-α, IL-1ß, and IL-8 by human macrophages. The 120-kDa fragment did not induce the secretion of all the cytokines tested, while intact fibronectin only increased IL-8 secretion and to a lesser extent TNF-α secretion. Cytokine secretion was associated with increased amounts of phosphorylated ERK1/2, JNK2, and p38α MAPK in treated macrophages. The combination of fibronectin or fibronectin fragments with Porphyromonas gingivalis lipopolysaccharide had an additive effect, but no synergism appeared to occur. It was also demonstrated that gingival crevicular fluid samples recovered from patients with moderate to severe periodontitis contained more fibronectin fragments than samples obtained from healthy subjects. Finally, both Arg- and Lys-gingipains purified from P. gingivalis were found to modulate fibronectin fragmentation. In conclusion, we showed that specific fibronectin fragments that may be present in diseased periodontal sites may contribute to maintaining and amplifying the inflammatory state and that P. gingivalis gingipains may be involved in the production of these fragments.

Dynamic control of protein-protein interactions.

The capability to selectively and reversibly control protein-protein interactions in antibody-doped polypyrrole (PPy) was accomplished by changing the voltage applied to the polymer. Polypyrrole was doped with sulfate polyanions and monoclonal anti-human fibronectin antibodies (alphaFN). The ability to toggle the binding and dissociation of fibronectin (FN) to alphaFN-doped polypyrrole was demonstrated. Staircase potential electrochemical impedance spectroscopy (SPEIS) was performed to characterize the impedance and charge transfer characteristics of the alphaFN-doped PPy as a function of applied voltage, frequency, and FN concentration. Impedance measurements indicated oxidation of alphaFN-doped PPy promoted selective binding of FN to alphaFN antibodies and reduction of the polymer films facilitated FN dissociation. Moreover, SPEIS measurements suggested that the apparent reversibility of antigen binding to antibody-doped PPy is not due to the suppression of hydrophobic binding forces between antibody and antigen. Instead, our data indicate that reversible antigen binding to antibody-doped PPy can be attributed to the minimization of charge in the polymer films during oxidation and reduction. Furthermore, alphaFN-doped PPy was utilized to collect real-time, dynamic measurements of varying FN concentrations in solution by repeatedly binding and releasing FN. Our data demonstrate that antibody-doped PPy represents an electrically controllable sensing platform which can be exploited to collect rapid, repeated measurements of protein concentrations with molecular specificity.

Binding of antibodies in liposomes to extracellular matrix antigens.

We have incorporated antibodies against fibronectin or laminin into liposomes and studied their interaction with insoluble forms of these antigens. The antibodies, after modification by palmitoylchloride, were incorporated into the lipid bilayer by the cholate dialysis method. The antibodies in the liposomes recognized their specific antigen with little reaction to the alternative attachment protein or to albumin (less than 2%). The binding of antibody-containing liposomes to insoluble antigen was inhibited by soluble antibodies to the respective antigens but not by antibodies to other antigens. The affinity constant of the liposome-antibody complex with the antigen was estimated at 1-10 X 10(-9) M liposomes. Thus, antibodies in liposomes retain their reactivity and specificity, and the reaction constant is comparable to that observed for immune complexes.

Fibronectin in polyethylene glycol precipitates: evidence for a role in immune complexes.

Fibronectin is involved in the opsonic clearance of particulate material. It is present in plasma and synovial fluid and thus might be expected to have a role in the clearance of immune complexes. We have investigated this in a study of polyethylene glycol (PEG) precipitable material from the serum of patients with rheumatoid arthritis, systemic lupus erythematosus, and other connective tissue disorders. Fibronectin is a significant component of PEG precipitates but the amount present is influenced by the method of preparation: more precipitates at 4 degrees C than at 20 degrees C. Fibronectin precipitation by PEG was considered to be related to immune complexes because: there was no direct relationship between serum fibronectin levels and the amount present in PEG precipitates; radiolabelled purified isolated fibronectin did not precipitate in 4% PEG; there was a direct relationship between the amount of fibronectin in PEG precipitates and the amounts of immunoglobulin G, A, and M. These results indicate that fibronectin is involved in immune complexes in rheumatic diseases, though they do not show it has an important biological role in these circumstances.

Identification and characterization of a dimeric chicken fibronectin receptor. Subunit-specific monoclonal antibodies to the putative chicken alpha 5 beta 1 integrin.

A fibronectin receptor was isolated from chicken embryo fibroblasts using a cell-binding fragment of chicken fibronectin as an affinity matrix. The synthetic peptide GRGDSP specifically eluted the receptor, whereas the GRGESP control peptide was negative. The purified chicken receptor is a heterodimeric molecule as are all described mammalian integrins. It showed an RGD-dependent binding to fibronectin-coated wells after incorporation into liposomes. Monoclonal antibodies were raised against the two subunits of the receptor. Immunoprecipitations with the beta chain-specific monoclonal antibody U2 beta gave the same pattern of multiple bands as with the "avian integrin complex" specific monoclonal antibody JG22. In contrast, the alpha chain-specific monoclonal antibody U1 alpha only precipitated a dimeric fibronectin receptor. On immunoblots, U2 beta recognized the chicken gizzard integrin beta 1 chain, but U1 alpha did not cross-react with the alpha chain of gizzard integrin. Immunofluorescence labeling of cell cultures with U1 alpha revealed an exclusive colocalization with extracellular fibronectin fibrils, whereas U2 beta showed in addition, diffuse staining of the whole cell surfaces. The alpha chain-specific monoclonal antibody for the first time allowed us to exclusively detect the putative chicken alpha 5 beta 1 integrin among the many beta 1 integrin complexes present on chicken cells.