Presence Of Crosslinking Research Articles

A novel digitonin/graphene oxide/iron oxide nanocomposite: synthesis, physiochemical characterization and antioxidant activity.

In this work, iron oxide (Fe3O4) magnetic nanoparticles (MNPs) and graphene oxide (GO) nanosheets were prepared via the co-precipitation technique and the Modified Hummer method. Fe3O4 MNPs and GO nanosheets were combined to prepare Fe3O4/GO nanocomposite and subsequently conjugated with Digitonin (DIG) in order to obtain a dual-targeted delivery system based on DIG/Fe3O4/GO nanocomposite. SEM images reveal the presence of Fe3O4 MNPs at a scale of 100nm, exhibiting dispersion between the GO nanosheets. Aggregation of the DIG/Fe3O4/GO nanocomposite was observed at various size scales. The XRD structural analysis confirms the crystal structure of the prepared samples. The Fe3O4 MNPs demonstrated the main XRD-diffracted peaks. Also, GO nanosheets exhibit crystalline characteristics on the (001) and (002) planes. The predominant peaks observed in the DIG/GO/Fe3O4 nanocomposite are attributed to the crystal phases of Fe3O4 MNPs. The FT-IR vibrational modes observed in the GO/DIG/Fe3O4 nanocomposite indicate the presence of crosslinking between GO nanosheet layers and the Fe3O4 MNPs. The antioxidant activity of the prepared samples was measured and the DIG/GO/Fe3O4 nanocomposite demonstrated a significantly high antioxidant activity in both 2-diphenyl-1-picrylhydrazyl (DPPH·) and 2,2-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid (ABTS·+) tests.

Abstract B034: CD27 is a new promising T cell co-stimulatory target for cancer immunotherapy

Abstract Members of the tumor necrosis factor receptor superfamily (TNFRSF) are key co-stimulators of T cells. CD27, a member of the TNFRSF, is expressed only on the surface of lymphocytes, including naive and activated CD4+ and CD8+ T cells as well as NK cells. It enhances T cell activation, proliferation, and differentiation of effector and memory T cells after stimulation with its ligand, CD70. The costimulatory signal of CD27 is mediated via the NFkB pathway but also via the phosphatidylinositol 3 kinase and the protein kinase B pathways. CD27 signaling also influences the innate immune response via direct activation of NK cells and subsequent secretion of interferon-gamma (IFNg). Several published preclinical studies demonstrated that anti-CD27 agonistic monoclonal antibodies can promote T-cell activation and antitumor immunity making CD27 an attractive cancer immunotherapy target. Here we describe the characterization, preclinical development, and selection of our anti-CD27 fully human monoclonal antibody (mAb) lead candidate. We selected this candidate from a library of 147 anti-CD27 mAbs generated after immunization of humanized Trianni® mice with soluble human CD27 extracellular domain (hCD27-ECD). Anti-CD27 mAbs were tested in an accelerated stability study and showed excellent stability parameters for up to 7 days at 4 and 37°C in commonly used formulation buffers. The selected agonist anti-CD27 mAb demonstrated high affinity binding to both human and cynomolgus monkey CD27 and not to mouse CD27. It also demonstrated high specificity against CD27 with no cross-reactivity detected against other members of the TNFRSF. This lead candidate did not block the binding of CD27 natural ligand, CD70 and induced strong NFkB-mediated CD27 signaling in the absence or presence of cross-linking by Fc gamma receptors or secondary cross-linking antibodies. Moreover, this anti-CD27 mAb mediated NFkB activation is significantly potentiated by the addition of a sub-optimal amount of soluble CD70. The anti-CD27 lead mAb induced T cell proliferation and secretion of pro-inflammatory cytokines only in the presence of sub-optimal TCR stimulation in vitro using primary human T cells. It also activated NK cells demonstrated by CD69 expression induction. The anti-CD27 mAb lead candidate showed extended serum half-life in hCD27-KI mice. It also demonstrated a significant antitumor effect as a single agent in human CD27-Knockin mice (hCD27-KI) subcutaneously implanted with MC38 or in NOD-SCID mice subcutaneously implanted with Raji. These preclinical results establish that the selected anti-CD27 mAb is a promising drug candidate and we are actively pursuing its development. Citation Format: Thierry Guillaudeux, Yulia Ovechkina, Kurt Lustig, Shawn Iadonato. CD27 is a new promising T cell co-stimulatory target for cancer immunotherapy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr B034.

Development and characterization of a novel human CD137 agonistic antibody with anti-tumor activity and a good safety profile in non-human primates.

CD137 (4-1BB, TNFRSF9), an inducible T-cell costimulatory receptor, is expressed on activated T cells, activated NK cells, Treg cells, and several innate immune cells, including DCs, monocytes, neutrophils, mast cells, and eosinophils. In animal models and clinical trials, anti-CD137 agonistic monoclonal antibodies have shown anti-tumor potential, but balancing the efficacy and toxicity of anti-CD137 agonistic monoclonal antibodies is a considerable hindrance for clinical applications. Here, we describe a novel fully human CD137 agonistic antibody (PE0116) generated from immunized harbor H2L2 human transgenic mice. PE0116 is a ligand blocker, which is also the case for Utomilumab (one of the leading CD137 agonistic drugs); PE0116 partially overlaps with Urelumab's recognized epitope. In vitro, PE0116 activates NF-κB signaling, significantly promotes T-cell proliferation, and increases cytokine secretion in the presence of cross-linking. Importantly, PE0116 possesses robust anti-tumor activity in the MC38 tumor model. In vivo, PE0116 exhibits a good safety profile and has typical pharmacokinetic characteristics of an IgG antibody in preclinical studies of non-human primates. In summary, PE0116 is a promising anti-CD137 antibody with a good safety profile in preclinical studies.

The Effect of Aloe vera Extract Variation in Electrospun Polyvinyl Alcohol (PVA)-Aloe vera-Based Nanofiber Membrane

Electrospinning technique is a method to produce nanofiber with high potential difference. Several main parameters in the electrospinning process are solution parameter, such as viscosity, surface tension and conductivity. Those parameters affect the morphology of nanofiber membrane produced by the electrospinning process. This study used 10% PVA in aquadest with several Aloe vera concentration (0, 2.5, 5, 7.5 and 10 % w/v). The viscosity of the solution increased when the concentration of the Aloe vera increased. SEM image of the nanofiber membrane showed that the diameter of the nanofiber increased with the increase of the Aloe vera concentration, with the highest diameter of 379.80 nm on the membrane with Aloe vera concentration of 10% w/v. The result of DSC test showed that there were shifts on the glass transition (Tg) and melting point (Tm) value along with the addition of Aloe vera in the PVA solution. This results was also emphasized with the result of FTIR test which showed the shift on the wavenumber absorbance due to the crosslinking between PVA and Aloe vera. The degradation test indicated that the presence of crosslinking could maintain the nanofiber structure, thus the membrane was not easily degraded.

Comparative study of hydrothermal pretreatment for rice straw and its corresponding mixture of cellulose, xylan, and lignin

We herein investigated the effect of hemicellulose and lignin on the hydrothermal pretreatment of lignocellulosic biomass for glucose production from cellulose in terms of structural cross-linking between cellulose and the above components. A comparison was made between the hydrothermal pretreatment of biomass and a mixture containing the individual model compounds in the same composition ratio. Thus, rice straw biomass and the mixture containing cellulose, xylan, and lignin were treated in an autoclave reactor at temperatures between 150 and 250 °C for 30 min. The obtained products were then subjected to enzymatic hydrolysis. Interestingly, different results were obtained for the two samples, as the presence of cross-linking between cellulose and lignin in the original biomass sample affected the efficiency of hydrothermal pretreatment. A model was therefore proposed to account for the obtained result in accordance with previous knowledge regarding the behavior of these compounds under hydrothermal conditions.

Preparation and relaxation dynamics of ethylene–propylene–diene rubber/clay nanocomposites with crosslinking interfacial design

ABSTRACTEthylene–propylene–diene rubber (EPDM)/clay nanocomposites with crosslinking bonding at the interface were fabricated through the intercalation method involving double‐bond functional groups. For comparison, an organoclay modified with an intercalation agent without double bonds was also prepared. X‐ray diffraction indicated that the EPDM intercalated into the galleries of the nanoclay due to crosslinking with the organic intercalation reagent containing double bonds. According to the dielectric relaxation spectra, the segmental relaxation of EPDM was greatly confined, due to the strong filler/polymer interfacial interaction. And a new relaxation appeared at higher temperature and lower frequency than segmental relaxation when the content of clay with double bonds reached 10 phr; the new relaxation is attributed to interfacial relaxation. Whereas the new relaxation did not appear by adding ordinary organoclay, the dynamic mechanical analysis loss peak of EPDM, corresponding to the glass transition, moved to a higher temperature due to covulcanization. The presence of crosslinking in the EPDM/clay nanocomposites can play a significant role in improving their mechanical properties. © 2017 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2018, 135, 45553.

The effect of thermal and thermo-oxidative degradation conditions on rheological, chemical and thermal properties of HDPE

This research evaluates the effect that thermal and thermo-oxidative degradation conditions exert on the rheological, chemical and thermal properties of high-density polyethylene (HDPE). To that end, dynamic oscillatory rheology, solubility tests in xylene, TGA, DSC and FTIR were conducted on HDPE samples subjected to different degradation conditions: atmosphere (air or nitrogen), temperature (150, 175, 200, 225 or 250 °C) and time (10, 30 or 60 min). Thus, under nitrogen atmosphere, chain scission mechanism prevails over the formation of long chain branching/crosslinking phenomena, which is reflected in a decrease in complex viscosity (|η*|) and an increase in crystallinity (χc). Interestingly, under air atmosphere, two rheological responses were observed: a) a well-developed rubbery region, at degradation temperatures of 225 and 250 °C and, b) a so-called second “plateau” in G′ and G″, for lower degradation temperatures. Solubility tests in xylene point out that the rubbery region is consequence of both branching phenomena and the presence of crosslinking; however, the second “plateau” should be solely ascribed to the increase in the branching mechanism. In this case, the decrease in χc is consequence of both increase in branching and formation of degradation products (accordingly supported by FTIR results). Finally, TGA results reveal that thermal and thermo-oxidative degradation shift the characteristic temperatures (T5% and Tmax) to lower values, compared to virgin HDPE.

Development of hydrocolloid Bi-layer dressing with bio-adhesive and non-adhesive properties

Bio-active bi-layer thin film having both bio-adhesive and non-adhesive end composed of polyvinyl alcohol (PVA) and gelatin/chitosan/polyethylene glycol (PEG) blend was developed for biomedical applications especially as an alternative of advanced tissue scaffold. The developed composite film was subjected to mechanical, thermal and physico-chemical characterization such as tensile strength (TS) and elongation at break (Eb), differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FTIR), fluid drainage capacity and biocompatibility. Suitable packaging was also selected and stability study and aging test of the composite film were performed after packing. The incorporation of chitosan and PEG into gelatin showed improved mechanical properties of both TS and Eb, which suggested the occurrence of interaction among gelatin, chitosan and PEG molecules in the composite film. The presence of crosslinking as an interaction of above three polymers was also confirmed by FTIR study. Results from the DSC study suggested increased thermal stability after crosslinking. On the other hand, water uptake studies suggested excellent fluid drainage capability and hydro-stability of the composite film. The proposed dressing also showed excellent biocompatibility. Based on the studies related to the performance with confirmed identity, we concluded that our developed bi-layer film is very potential as an ideal wound dressing material.

Effect of Polymer Membrane Processing Techniques in Chemical and Mechanical Properties of Anion Exchange Membranes for Electrochemical Applications

Anion exchange membranes (AEMs) are promising materials for many electrochemical energy conversion and storage systems such as fuel cells, electrolysers, redox flow batteries, reverse electrolysers, just to name a few. We have observed that membrane-processing techniques affect mechanical and chemical properties of an anion exchange membrane, significantly. Literatures have shown highly conductive AEMs 1 at fuel cell operating conditions, however, a more study is still needed to find a balance between ionic conductivity and mechanical strength of AEMs for practical applications. We used poly(vinyl benzyl chloride)(PPO-b-PVBC) polymer with IEC of 2.6 mmol/g, synthesis published else where1, to understand the effect of polymer processing techniques in ionic conductivity and mechanical properties. Elemental analysis of the membrane after melt pressing for 5, 10 and 20 min of solvent cast membranes showed the decrease in chloride in the membrane by ~55, 44 and 44%; respectively from it’s initial 14.5 %w/w of chlorine in the polymer. The reduced chlorine but oxygen in the membrane indicated the presence of crosslinking in hydrophilic group of the polymer. Ionic conductivity, diffusion of ions and water, and dynamic vapor water sorption experiments were performed to understand ionic transport, tensile stretching of membrane were performed at fuel cell operating conditions to understand membrane strength, stiffness and elongation, and small angle x-ray scattering experiments were performed to understand the membrane morphology. Membranes with higher degree of cross-linking by melt pressed for 40 min, were found to be mechanically robust, but chemically less conductive. The membrane melt pressed for just 10 min, was highly conductive with slightly lower membrane strength. A different degree of cross-linking showed a difference in water uptake (Figure 1). A greater water uptake by membrane had bigger effect of water plasticizing the membrane resulting in poor mechanical performance. A good balance of degree of cross-linking in the membrane to optimize the ionic conductivity, and mechanical properties will be discussed in the presentation. Figure 1. Vapor water uptake by the membrane at 60°C as a function of humidity. SCMP5, SCMP10, and SCMP20 (solvent cast and then melt pressed for 5, 10 and 20 min; respectively in addition to waiting same amount of time for melting the polymer before the application of pressure) Acknowledgement The authors would like to thank the US Army Research Office for the funding under the MURI #W911NF-10-1-0520and Yating Yang and Daniel M. Knauss for providing the polymer for the study.

Hierarchic micro-patterned porous scaffolds via electrochemical replica-deposition enhance neo-vascularization

Neo-vascularization is a key factor in tissue regeneration within porous scaffolds. Here, we tested the hypothesis that micro-patterned scaffolds, with precisely-designed, open micro-channels, might help endothelial cells to produce intra-scaffold vascular networks. Three series of micro-patterned scaffolds were produced via electrochemical replica-deposition of chitosan and cross-linking. All had regularly-oriented micro-channels (ϕ 500 μm), which differed for the inter-channel spacing, at 600, 700, or 900 μm, respectively. Random-pore scaffolds, using the same technique, were taken as controls. Physical-mechanical characterization revealed high water uptake and favorable elastic mechanical behavior for all scaffolds, slightly reduced in the presence of cross-linking and enhanced with the 700 μm-spaced micro-pattern. At MTT assay, mouse endothelial cell viability was >90% at day 1, 3 and 7, confirmed by visual examination with scanning electron microscopy (SEM). Intra-scaffold cell density, at fluorescence analysis, was higher for the 600 μm-spaced and the 700 μm-spaced micro-patterns over the others. The 700 μm-spaced scaffold was selected for the in vivo testing, to be compared to the random-pore one. Neither type produced an inflammatory reaction; both showed excellent tissue ingrowth. Micro-patterned scaffolds enhanced neo-vascularization, demonstrated by immunofluorescent, semi-quantitative analyses. These findings support the use of micro-patterned porous scaffolds, with adequately spaced micro-channels, to promote neo-vascularization.

Optimizing Collagen Scaffolds for Bone Engineering: Effects of Cross-linking and Mineral Content on Structural Contraction and Osteogenesis.

Osseous defects of the craniofacial skeleton occur frequently in congenital, posttraumatic, and postoncologic deformities. The field of scaffold-based bone engineering emerged to address the limitations of using autologous bone for reconstruction of such circumstances. In this work, the authors evaluate 2 modifications of three-dimensional collagen-glycosaminoglycan scaffolds in an effort to optimize structural integrity and osteogenic induction. Human mesenchymal stem cells (hMSCs) were cultured in osteogenic media on nonmineralized collagen-glycosaminoglycan (C-GAG) and nanoparticulate mineralized collagen-glycosaminoglycan (MC-GAG) type I scaffolds, in the absence and presence of cross-linking. At 1, 7, and 14 days, mRNA expression was analyzed using quantitative real-time -reverse-transcriptase polymerase chain reaction for osteocalcin (OCN) and bone sialoprotein (BSP). Structural contraction was measured by the ability of the scaffolds to maintain their original dimensions. Mineralization was detected by microcomputed tomographic (micro-CT) imaging at 8 weeks. Statistical analyses were performed with Student t-test. Nanoparticulate mineralization of collagen-glycosaminoglycan scaffolds increased expression of both OCN and BSP. Cross-linking of both C-GAG and MC-GAG resulted in decreased osteogenic gene expression; however, structural contraction was significantly decreased after cross-linking. Human mesenchymal stem cells-directed mineralization, detected by micro-CT, was increased in nanoparticulate mineralized scaffolds, although the density of mineralization was decreased in the presence of cross-linking. Optimization of scaffold material is an essential component of moving toward clinically translatable engineered bone. Our current study demonstrates that the combination of nanoparticulate mineralization and chemical cross-linking of C-GAG scaffolds generates a highly osteogenic and structurally stable scaffold.

Processability and mechanical properties of extensively recycled high density polyethylene

In plastics industry it is a common practice to mechanically recycle waste material arising from production. However, while plastics are mechanically recycled, their mechanical properties change. These changes may affect material processing conditions and quality of the end products; therefore they need to be quantified. In this study, mechanical recycling of high density polyethylene (HDPE) was simulated by one-hundred (100) consecutive extrusions cycles. During extrusion, processability of virgin HDPE and its recyclates was studied by recording the processing conditions, i.e. melt pressure and extrusion torque, while after preparation of the recyclates, melt flow index measurements (MFI), small amplitude oscillatory shear tests (rheological properties), and differential scanning calorimetry measurements (DSC) of thermal properties were performed. Also, mechanical properties in solid state were characterized in terms of hardness and modulus measured by nanoindentation, and finally, shear creep compliance was measured to characterize the materials' time-dependent mechanical properties and its durability in solid state. In addition, gel permeation chromatography (GPC) and solubility tests were implemented to study changes in the material structure.The results on rheological and MFI measurements indicate significant structural changes in the material that occurred during the first 30 extrusion cycles. Those changes affect material processability which is as well supported by the recorded processing pressure and torque. On the other hand, processing did not significantly affect material thermal properties. Results on hardness and modulus show deterioration of the material mechanical properties after 10th reprocessing cycle. Similarly, shear creep compliance measurements showed an unfavourable effect of mechanical recycling on the time-dependent mechanical properties, particularly after the 30th extrusion cycle. In addition, results suggested chain branching as a dominating mechanism through first 30 extrusion cycles, domination of chain scission afterwards and also presence of cross-linking after 60th extrusion cycle.

Effect of Monomer Dosing Rate in the Preparation of Mesoporous Polystyrene Nanoparticles by Semicontinuous Heterophase Polymerization

The semicontinuous heterophase polymerization of styrene in the presence of cross-linking and porogen agents was carried out. Latexes with close to 20% solid content, which contained mesoporous nanoparticles with 28 nm in average diameters, up to 0.5 cm3/g in porosity and 6–8 nm in pore diameters were obtained. By varying the monomer dosing rate over the micellar solution, an unexpected direct dependence of instantaneous conversion on the monomer dosing rate was found. This was ascribed to the higher average number of radicals per particle attained in the polymerization at the higher dosing rate, which in turn would arise from the higher gel percentage in the polymer. It is believed that the cross-linked chains prevent encounters between radicals, delaying the bimolecular termination reactions and allowing the existence of more than one radical inside the particles, which in turn increases the propagation rate.

Adsorption of Waste Metal Cr(VI) with Composite Membranes (Chitosan-Silica Rice Husks)

Chromium compounds are widely used in modern industry. Many of these compounds are dumped into the surrounding environment. Membrane technology is more efficient and effective than conventional methods for waste treatment. The research objective was to make a membrane separation process that can be applied to Cr(VI). Membranes were made from chitosan and silica rice husks. Variations of chitosan and silica rice husk used (g) are 2:1 (A1), 2:2 (A2), 3:1 (B1), and 3:2 (B2). The membrane was made by using an inverted phase technique. Results of SEM characterization of membranes showed that B2 has the largest pores at 2.58 μm. The FTIR characterization results indicate the presence of crosslinking between chitosan with silica rice husk with the appearance of Si-O adsorption band at wavelength 1122980/cm. A1 membrane, with the smallest pore size has the greatest rejection value towards Cr(VI) which is 70%. The result of this research showed that the composite membrane of silica rice husk was effective enough to adsorb metal Cr(VI) with an average adsorption capacity of 1665.85 mg/g.

Low flammability, foam-like materials based on ammonium alginate and sodium montmorillonite clay

Low flammability, foam-like materials based on bio-based, renewable ammonium alginate and sodium montmorillonite clay were fabricated through a simple, environmentally-friendly freeze-drying process in which water is used as solvent. These materials exhibit mechanical properties similar to those of rigid PU foams or balsa; the compressive modulus (1–97 MPa) and density (0.047–0.174 g/cm3) increase with increasing solids content, with an associated change from a layered to network microstructure structure. Calcium ions, either added directly or indirectly via CaCO3/gluconolactone (GDL) are an effective crosslinking agent for alginate, with the GDL route providing the greatest enhancement of mechanical properties. The thermal stability of the aerogels is also enhanced by the presence of crosslinking. The alginate/clay aerogels possess inherently low flammability, as measured by cone calorimetry, with heat release values decreasing as the proportion of clay in the composites is increased.

Poly(acrylamide) films at the solvent-induced glass transition: adhesion, tribology, and the influence of crosslinking

Adhesive and nanotribological properties of end-grafted poly(acrylamide) (PAAm) films with various degrees of crosslinking, and in the presence of solvents over a broad spectrum of quality, were investigated by means of colloidal-probe atomic force microscopy. The solvent consisted of a mixture of water (good solvent for PAAm) and methanol (bad solvent for PAAm). Adhesion measurements carried out on brush (uncrosslinked) structures revealed significant pull-off forces in solvent mixtures that placed the polymer at its glass transition. These pull-off forces, which were orders of magnitude higher than those measured in either pure solvent, were significantly reduced in the presence of crosslinking. The nanostructures of PAAm films with different crosslinking degrees were elucidated in their maximum collapsed state by means of atomic force microscopy, and the parameters influencing adhesive properties of films with brush structures at their glass transition were investigated. Complex nanotribological behavior of PAAm films was observed, and found to result from the interplay of film structure and adhesion forces, which were influenced by both crosslinking degree and solvent quality.

Structural and mechanical properties of Laponite–PEG hybrid films

Inorganic/organic hybrids were obtained by the sol–gel type organic modification reaction of Laponite sidewalls with poly(ethylene glycol) (PEG) bearing alkoxysiloxy terminal functionality. By casting an aqueous dispersion of the hybrid, the flexible and transparent hybrid films were obtained. Regardless of the inorganic/organic component ratio, the hybrid film had the ordered structure of Laponite in-plane flat arrays. The mechanical strength of hybrid films was drastically improved by the presence of cross-linking among alkoxysilyl functionalities of PEG terminals and the absence of PEG crystallines. Hybrid films, especially those that consisted of PEG with short chain, showed good mechanical properties that originate from quasi-homogeneous dispersion of components due to anchoring of PEG terminal to Laponite sidewall and interaction of PEG to Laponite surface.

Controlled synthesis of water-compatible molecularly imprinted polymer microspheres with ultrathin hydrophilic polymer shells via surface-initiated reversible addition-fragmentation chain transfer polymerization

The first controlled synthesis of pure water-compatible molecularly imprinted polymer (MIP) microspheres with ultrathin hydrophilic poly(2-hydroxyethyl methacrylate) (PHEMA) shells (including both PHEMA brushes and lightly crosslinked PHEMA hydrogel layer) via surface-initiated reversible addition-fragmentation chain transfer (RAFT) polymerization is described. The presence of ultrathin hydrophilic polymer shells on the MIP microspheres was confirmed by SEM, FT-IR, fluorescent labeling treatment, contact angle studies, and water dispersion stability test, and some quantitative information including the thickness of the grafted hydrophilic polymer layers as well as the molecular weights and polydispersities of the grafted polymer brushes and their grafting densities was provided. The facile surface-grafting of both PHEMA brushes and PHEMA hydrogel layer on the MIP microspheres proved to be highly efficient for improving their surface hydrophilicity and suppressing the hydrophobically driven nonspecific interactions between the MIPs and template molecules, leading to MIPs with pure water-compatible binding properties. The findings presented here not only prove the general applicability of the controlled hydrophilic polymer brushes-grafting approach in obtaining pure water-compatible MIPs, but also largely extend the scope of this versatile surface-grafting approach through the controlled surface-grafting of hydrophilic polymer hydrogel layer onto the MIPs. Moreover, the significant effect of the chain length of the grafted polymer brushes and the presence of crosslinking in the grafted polymer shells on the surface hydrophilicity and water-compatibility of the MIP microspheres was also demonstrated for the first time, which is of great importance for the rational design of water-compatible MIPs by using this controlled surface-grafting approach.

The Inhibitory Receptor FcgRIIB Is Overexpressed, and Its Ligation by Anti- FcgRIIB Antibodies Suppresses IgM Production and Induces Apoptosis in Waldenstrom's Macroglobulinemia.

Abstract 3736Poster Board III-672 IntroductionWaldenstrom's macroglobulinemia (WM) is a lymphoplasmacytic lymphoma characterized by overproduction of a monoclonal IgM paraprotein which can produce morbidity including hyperviscosity, as well as autoimmune related neuropathy, hemolysis, and thrombocytopenia. Therefore approaches aimed at both suppressing IgM production, as well as selectively inducing apoptosis of WM cells represent an ideal treatment strategy for WM. FcgRIIB is an inhibitory receptor that is expressed on B-cells, and whose expression we recently identified as highly over-expressed in WM. Importantly, FcgRIIB possesses an immunoreceptor tyrosine-based inhibitory motif (ITIM), and which becomes phosphorylated at Tyr 292 upon activation, and is then followed by inhibition of BCR signaling and induction of apoptosis. We therefore validated the expression of this receptor in WM, and examined the impact of its ligation on tumor cell killing, IgM secretion and downstream signaling events in WM cells. Patients and MethodsBone marrow lymphoplasmacytic cells (LPC) from 12 WM patients which were sorted for CD19+ and CD138+, and BCWM.1 WM cells were subjected to real-time PCR and flow cytometric analysis. Cells were then subjected to co-culture with anti- FcgRIIB (AT10, 7.3) or control antibodies for 24-48 hours, and their effects on survival, IgM production and downstream signaling were assessed. ResultsReal-time PCR and flow-cytometric analysis demonstrated strong expression of FcgRIIB in WM patient bone marrow CD19+ and CD138+ cells, thus confirming our recent microarray results. Importantly, the expression of FcgRIIB in WM LPC correlated with the memory B-cell marker CD27. Anti-FcgRIIB antibody treatment dramatically reduced constitutive, and/or IL-6 induced IgM production in CD19+ and CD138+ sorted primary WM LPC, as well as CD32hiCD138hi BCWM.1 cells. This effect was observed in some experiments at an early time point that had not effected survival. Among primary CD138+ WM LPC and CD32hiCD138hi expressing BCWM.1 cells, treatment with anti-FcgRIIB antibodies for 48 hours led to increased apoptosis in 10 of 12 patients, as assessed by Annexin V and PI staining which occurred despite blockade with a pan-caspase inhibitor, and was even more pronounced when anti-FcgRIIB antibodies were cross-linked. Western blot analysis revealed that treatment of CD32hiCD138hi expressing BCWM.1 cells with cross-linked anti- FcgRIIB antibodies led to phosphorylation of Tyr 292 of the FcgRIIB ITIM which was not observed in the absence of cross-linking. Binding of FcgRIIB by both the AT10 and 7.3 antibodies resulted in dephosphorylation of Akt, which was further reduced in the presence of cross-linking. Coincident with the above, the pro-apoptotic molecule JNK also underwent phosphorylation in the presence of anti-FcgRIIB binding. ConclusionTaken together, these studies validate our previous microarray data by showing that the inhibitory receptor FcgRIIB is strongly expressed on LPC from WM patients, and ligation thereof leads to suppression of IgM production and induction of apoptosis thereby identifying FcgRIIB as a novel therapeutic target in WM. Disclosures:No relevant conflicts of interest to declare.

In Vitro Characterization of a Collagen Scaffold Enzymatically Cross-Linked with a Tailored Elastin-like Polymer

Collagen, the main structural component of the extracellular matrix (ECM), provides tensile stiffness to different structures and organs against rupture. However, collagen tissue-engineered implants are hereto still lacking in mechanical strength. Attempts to create stiffer scaffolds have resulted in increased brittleness of the material, reducing the versatility of the original component. The hypothesis behind this research is that the introduction of an elastic element in the scaffold will enhance the mechanical properties of the collagen-based scaffolds, as elastin does in the ECM to prevent irreversible deformation. In this study, an elastin-like polymer (ELP) designed and synthesized using recombinant DNA methodology is used with the view to providing increased proteolytic resistance and increased functionality to the scaffolds by carrying specific sequences for microbial transglutaminase cross-linking, endothelial cell adhesion, and drug delivery. Evaluation of the effects that cross-linking ELP-collagen has on the physicochemical properties of the scaffold such as porosity, presence of cross-linking, thermal behavior, and mechanical strength demonstrated that the introduction of enzymatically resistant covalent bonds between collagen and ELP increases the mechanical strength of the scaffolds in a dose-dependent manner without significantly affecting the porosity or thermal properties of the original scaffold. Importantly, the scaffolds also showed selective behavior, in a dose (ELP)-dependent manner toward human umbilical vein endothelial cells and smooth muscle cells when compared to fibroblasts.