Full visible range two-dimensional electronic spectroscopy with high time resolution.

Two-dimensional electronic spectroscopy (2DES) is a powerful method to study coherent and incoherent interactions and dynamics in complex quantum systems by correlating excitation and detection energies in a nonlinear spectroscopy experiment. Such dynamics can be probed with a time resolution limited only by the duration of the employed laser pulses and in a spectral range defined by the pulse spectrum. In the blue spectral range (<500 nm), the generation of sufficiently broadband ultrashort pulses with pulse durations of 10 fs or less has been challenging so far. Here, we present a 2DES setup based on a hollow-core fiber supercontinuum covering the full visible range (400-700 nm). Pulse compression via custom-made chirped mirrors yields a time resolution of <10 fs. The broad spectral coverage, in particular the extension of the pulse spectra into the blue spectral range, unlocks new possibilities for coherent investigations of blue-light absorbing and multichromophoric compounds, as demonstrated by a 2DES measurement of chlorophyll a.

CAR-Ts redirected against the Thomsen-Friedenreich antigen CD176 mediate specific elimination of malignant cells from leukemia and solid tumors.

Introduction: Chimeric antigen receptor-engineered T cells (CAR-Ts) are investigated in various clinical trials for the treatment of cancer entities beyond hematologic malignancies. A major hurdle is the identification of a target antigen with high expression on the tumor but no expression on healthy cells, since "on-target/off-tumor" cytotoxicity is usually intolerable. Approximately 90% of carcinomas and leukemias are positive for the Thomsen-Friedenreich carbohydrate antigen CD176, which is associated with tumor progression, metastasis and therapy resistance. In contrast, CD176 is not accessible for ligand binding on healthy cells due to prolongation by carbohydrate chains or sialylation. Thus, no "on-target/off-tumor" cytotoxicity and low probability of antigen escape is expected for corresponding CD176-CAR-Ts. Methods: Using the anti-CD176 monoclonal antibody (mAb) Nemod-TF2, the presence of CD176 was evaluated on multiple healthy or cancerous tissues and cells. To target CD176, we generated two different 2nd generation CD176-CAR constructs differing in spacer length. Their specificity for CD176 was tested in reporter cells as well as primary CD8+ T cells upon co-cultivation with CD176+ tumor cell lines as models for CD176+ blood and solid cancer entities, as well as after unmasking CD176 on healthy cells by vibrio cholerae neuraminidase (VCN) treatment. Following that, both CD176-CARs were thoroughly examined for their ability to initiate target-specific T-cell signaling and activation, cytokine release, as well as cytotoxicity. Results: Specific expression of CD176 was detected on primary tumor tissues as well as on cell lines from corresponding blood and solid cancer entities. CD176-CARs mediated T-cell signaling (NF-κB activation) and T-cell activation (CD69, CD137 expression) upon recognition of CD176+ cancer cell lines and unmasked CD176, whereby a short spacer enabled superior target recognition. Importantly, they also released effector molecules (e.g. interferon-γ, granzyme B and perforin), mediated cytotoxicity against CD176+ cancer cells, and maintained functionality upon repetitive antigen stimulation. Here, CD176L-CAR-Ts exhibited slightly higher proliferation and mediator-release capacities. Since both CD176-CAR-Ts did not react towards CD176- control cells, their response proved to be target-specific. Discussion: Genetically engineered CD176-CAR-Ts specifically recognize CD176 which is widely expressed on cancer cells. Since CD176 is masked on most healthy cells, this antigen and the corresponding CAR-Ts represent a promising approach for the treatment of various blood and solid cancers while avoiding "on-target/off-tumor" cytotoxicity.

Plasma-Enhanced Atomic Layer Deposition of HfO2 with Substrate Biasing: Thin Films for High-Reflective Mirrors.

Tuning ion energies in plasma-enhanced atomic layer deposition (PEALD) processes enables fine control over the material properties of functional coatings. The growth, structural, mechanical, and optical properties of HfO2 thin films are presented in detail toward photonic applications. The influence of the film thickness and bias value on the properties of HfO2 thin films deposited at 100 °C using tetrakis(dimethylamino)hafnium (TDMAH) and oxygen plasma using substrate biasing is systematically analyzed. The HfO2 films deposited without a substrate bias show an amorphous microstructure with a low density, low refractive index, high incorporation of residual hydroxyl (OH) content, and high residual tensile stress. The material properties of HfO2 films significantly improved at a low bias voltage due to the interaction with oxygen ions accelerated to the film. Such HfO2 films have a higher density, higher refractive index, and lower residual OH incorporation than films without bias. The mechanical stress becomes compressive depending on the bias values. Further increasing the ion energies by applying a larger substrate bias results in a decrease of the film density, refractive index, and a higher residual OH incorporation as well as crystalline inclusions. The comparable material properties of the HfO2 films have been reported using tris(dimethylamino)cyclopentadienyl hafnium (TDMACpH) in a different apparatus, indicating that this approach can be transferred to various systems and is highly versatile. Finally, the substrate biasing technique has been introduced to deposit stress-compensated, crack- and delamination-free high-reflective (HR) mirrors at 355 and 532 nm wavelengths using HfO2 and SiO2 as high and low refractive index materials, respectively. Such mirrors could not be obtained without the substrate biasing during the deposition because of the high tensile stress of HfO2, leading to cracks in thick multilayer systems. An HR mirror for 532 nm wavelength shows a high reflectance of 99.93%, a residual transmittance of ∼530 ppm, and a low absorption of ∼11 ppm, as well as low scattering losses of ∼4 ppm, high laser-induced damage threshold, low mechanical stress, and high environmental stability.

Variances in Antiviral Memory T-Cell Repertoire of CD45RA- and CD62L-Depleted Lymphocyte Products Reflect the Need of Individual T-Cell Selection Strategies to Reduce the Risk of GvHD while Preserving Antiviral Immunity in Adoptive T-Cell Therapy.

INTRODUCTION: Viral infections and reactivations still remain a cause of morbidity and mortality after hematopoietic stem cell transplantation due to immunodeficiency and immunosuppression. Transfer of unmanipulated donor-derived lymphocytes (DLI) represents a promising strategy for improving cellular immunity but carries the risk of graft versus host disease (GvHD). Depleting alloreactive naïve T cells (TN) from DLIs was implemented to reduce the risk of GvHD induction while preserving antiviral memory T-cell activity. Here, we compared two TN depletion strategies via CD45RA and CD62L expression and investigated the presence of antiviral memory T cells against human adenovirus (AdV) and Epstein-Barr virus (EBV) in the depleted fractions in relation to their functional and immunophenotypic characteristics. METHODS: T-cell responses against ppEBV_EBNA1, ppEBV_Consensus and ppAdV_Hexon within TN-depleted (CD45RA-/CD62L-) and TN-enriched (CD45RA+/CD62L+) fractions were quantified by interferon-gamma (IFN-γ) ELISpot assay after short- and long-term in vitro stimulation. T-cell frequencies and immunophenotypic composition were assessed in all fractions by flow cytometry. Moreover, alloimmune T-cell responses were evaluated by mixed lymphocyte reaction. RESULTS: According to differences in the phenotype composition, antigen-specific T-cell responses in CD45RA- fraction were up to 2 times higher than those in the CD62L- fraction, with the highest increase (up to 4-fold) observed after 7 days for ppEBV_EBNA1-specific T cells. The CD4+ effector memory T cells (TEM) were mainly responsible for EBV_EBNA1- and AdV_Hexon-specific T-cell responses, whereas the main functionally active T cells against ppEBV_Consensus were CD8+ central memory T cells (TCM) and TEM. Moreover, comparison of both depletion strategies indicated that alloreactivity in CD45RA- was lower than that in CD62L- fraction. CONCLUSION: Taken together, our results indicate that CD45RA depletion is a more suitable strategy for generating TN-depleted products consisting of memory T cells against ppEBV_EBNA1 and ppAdV_Hexon than CD62L in terms of depletion effectiveness, T-cell functionality and alloreactivity. To maximally exploit the beneficial effects mediated by antiviral memory T cells in TN-depleted products, depletion methods should be selected individually according to phenotype composition and CD4/CD8 antigen restriction. TN-depleted DLIs may improve the clinical outcome in terms of infections, GvHD, and disease relapse if selection of pathogen-specific donor T cells is not available.

Robust Identification of Suitable T-Cell Subsets for Personalized CMV-Specific T-Cell Immunotherapy Using CD45RA and CD62L Microbeads.

Viral infections and reactivations remain a serious obstacle to successful hematopoietic stem cell transplantation (HSCT). When antiviral drug treatment fails, adoptive virus-specific T-cell transfer provides an effective alternative. Assuming that naive T cells (TN) are mainly responsible for GvHD, methods were developed to generate naive T-cell-depleted products while preserving immune memory against viral infections. We compared two major strategies to deplete potentially alloreactive T cells: CD45RA and CD62L depletion and analyzed phenotype and functionality of the resulting CD45RA-/CD62L- naive T-cell-depleted as well as CD45RA⁺/CD62L⁺ naive T-cell-enriched fractions in the CMV pp65 and IE1 antigen model. CD45RA depletion resulted in loss of terminally differentiated effector memory T cells re-expressing CD45RA (TEMRA), and CD62L depletion in loss of central memory T cells (TCM). Based on these differences in target cell-dependent and target cell-independent assays, antigen-specific T-cell responses in CD62L-depleted fraction were consistently 3⁻5 fold higher than those in CD45RA-depleted fraction. Interestingly, we also observed high donor variability in the CD45RA-depleted fraction, resulting in a substantial loss of immune memory. Accordingly, we identified donors with expected response (DER) and unexpected response (DUR). Taken together, our results showed that a naive T-cell depletion method should be chosen individually, based on the immunophenotypic composition of the T-cell populations present.

Inhibition of Heme Oxygenase-1 Activity Enhances Wilms Tumor-1-Specific T-Cell Responses in Cancer Immunotherapy.

Wilms tumor protein-1 (WT1) is an attractive target for adoptive T-cell therapy due to its expression in solid tumors and hematologic malignancies. However, T cells recognizing WT1 occur in low frequencies in the peripheral blood of healthy donors, limiting potential therapeutic possibilities. Tin mesoporphyrin (SnMP) is known to inhibit heme oxygenase-1 (HO-1), which has been shown to boost the activation and proliferation of human virus-specific T cells. We analyzed the influence of this effect on the generation of WT1-specific T cells and developed strategies for generating quantities of these cells from healthy donors, sufficient for adoptive T-cell therapies. HO-1 inhibition with SnMP increased WT1-specific T-cell frequencies in 13 (26%) of 50 healthy donors. To assess clinical applicability, we measured the enrichment efficiency of SnMP-treated WT1-specific T cells in response to a WT1-specific peptide pool and a HLA-A*02:01-restricted WT1 peptide by cytokine secretion assay. SnMP treatment resulted in a 28-fold higher enrichment efficacy with equal functionality. In conclusion, pharmacological inhibition of HO-1 activity with SnMP results in more efficient generation of functionally active WT1-specific T cells. This study demonstrates the therapeutic potentials of inhibiting HO-1 with SnMP to enhance antigen-specific T-cell responses in the treatment of cancer patients with WT1-positive disease.

Cord blood-derived T cells allow the generation of a more naïve tumor-reactive cytotoxic T-cell phenotype.

BACKGROUND: Transplantation of hematopoietic stem cells (HSCs) from peripheral blood (PB) or cord blood (CB) is well established. HSCs from CB are associated with a lower risk of graft-versus-host disease (GVHD), but antigen-independent expanded CB- and PB-derived T cells can induce GVHD in allo-HSC recipients. CB-derived cells might be more suitable for adoptive immunotherapy as they have unique T-cell characteristics. Here, we describe functional differences between CB and PB T cells stimulated with different cytokine combinations involved in central T-cell activation. STUDY DESIGN AND METHODS: Isolated CD8+ T cells from CB and PB were stimulated antigen independently with anti-CD3/CD28 stimulator beads or in an antigen-dependent manner with artificial antigen-presenting cells loaded with the HLA-A*02:01-restricted peptide of tumor-associated melanoma antigen recognized by T cells 1 (MART1). CB and PB T cells cultured in the presence of interleukin (IL)-7, IL-15, IL-12, and IL-21 were characterized for T-cell phenotype and specificity, that is, by CD107a, interferon-γ, tumor necrosis factor-α, and IL-2 expression. RESULTS: After antigen-independent stimulation, activated CD8+ CB T cells exhibited stronger proliferation and function than those from PB. After antigenic stimulation, MART1-reactive CB T cells were naïve (CD45RA+CCR7+), cytotoxic, and highly variable in expressing homing marker CD62L. Addition of IL-21 resulted in increased T-cell proliferation, whereas supplementation with IL-12 decreased IL-21-induced expansion, but increased the functionality and cytotoxicity of CB and PB T cells. CONCLUSION: MART1-reactive CB T cells with a more naïve phenotype and improved properties for homing can be generated. The results contribute to better understanding the effects on GVHD and graft versus tumor.

Structural requirements of mono- and multivalent L-selectin blocking aptamers for enhanced receptor inhibition in vitro and in vivo.

L-selectin mediates extravasation of leukocytes from blood into the surrounding tissue during inflammation and is therefore a therapeutical target in certain overwhelming immune reactions. In this study, we characterized an L-selectin specific blocking DNA aptamer with respect to nucleotide composition and target binding. Introduction of deletions and nucleotide exchanges resulted in an optimized DNA sequence but preservation of the IC50 in the low nanomolar range. The inhibitory potential was significantly increased when the aptamer was displayed as a di- and trimer connected via appropriate linker length. Similar to monoclonal antibodies, trimer yielded picomolar IC50 values in a competitive binding assay. In comparison to the monovalent aptamer, the trivalent assembly reduced PBMC interactions to L-selectin ligands 90-fold under shear and exerted superior inhibition of PBMC rolling in vivo. In conclusion, our work demonstrates the feasibility of optimizing aptamer sequences and shows that multivalent ligand presentation enables superior adhesion receptor targeting. FROM THE CLINICAL EDITOR: During inflammation, leukocytes extravasate from blood vessels under chemotaxic signals. The presence of L-selectin on endothelium acts as a mediator for the extravasation process. In this study, the authors investigated an L-selectin specific blocking DNA aptamer in various forms, as inhibitors to leukocyte binding and extravasation. This new approach confirmed the potential use of aptamers in clinical setting.

Shell Cleavable Dendritic Polyglycerol Sulfates Show High Anti-Inflammatory Properties by Inhibiting L-Selectin Binding and Complement Activation.

A new class of fully synthetic shell cleavable multivalent polysulfates is prepared by introducing degradable linkers into a stable biocompatible dendritic polyglycerol scaffold and subsequent sulfation. The sulfated polymers show different degradation profiles, low anticoagulant and high anti-inflammatory properties, are able to efficiently bind to L-selectin and inhibit the complement activation at very low concentrations in vitro.

Iron oxide nanoparticles stabilized with dendritic polyglycerols as selective MRI contrast agents.

Monodisperse small iron oxide nanoparticles functionalized with dendritic polyglycerol (dPG) or dendritic polyglycerol sulfate (dPGS) are prepared. They are highly stable in aqueous solutions as well as physiological media. In particular, oleic acid capped iron oxide particles (core diameter = 11 ± 1 nm) were modified by a ligand exchange process in a one pot synthesis with dPG and dPGS bearing phosphonate as anchor groups. Dynamic light scattering measurements performed in water and different biological media demonstrate that the hydrodynamic diameter of the particles is only slightly increased by the ligand exchange process resulting in a final diameter of less than 30 nm and that the particles are stable in these media. It is also revealed by magnetic resonance studies that their magnetic relaxivity is reduced by the surface modification but it is still sufficient for high contrast magnetic resonance imaging (MRI). Additionally, incubation of dPGS functionalized iron oxide nanoparticles with human umbilical vein endothelial cells showed a 50% survival at 85 nM (concentration of nanoparticles). Surface plasmon resonance (SPR) studies demonstrate that the dPGS functionalized iron oxide nanoparticles inhibit L-selectin ligand binding whereas the particles containing only dPG do not show this effect. Experiments in a flow chamber with human myelogenous leukemia cells confirmed L-selectin inhibition of the dPGS functionalized iron oxide nanoparticles and with that the L-selectin mediated leukocyte adhesion. These results indicate that dPGS functionalized iron oxide nanoparticles are a promising contrast agent for inflamed tissue probed by MRI.

Polyglycerolsulfate functionalized gold nanorods as optoacoustic signal nanoamplifiers for in vivo bioimaging of rheumatoid arthritis.

We have synthesized a targeted imaging agent for rheumatoid arthritis based on polysulfated gold nanorods. The CTAB layer on gold nanorods was first replaced with PEG-thiol and then with dendritic polyglycerolsulfate at elevated temperature, which resulted in significantly reduced cytotoxicity compared to polyanionic gold nanorods functionalized by non-covalent approaches. In addition to classical characterization methods, we have established a facile UV-VIS based BaCl2 agglomeration assay to confirm a quantitative removal of unbound ligand. With the help of a competitive surface plasmon resonance-based L-selectin binding assay and a leukocyte adhesion-based flow cell assay, we have demonstrated the high inflammation targeting potential of the synthesized gold nanorods in vitro. In combination with the surface plasmon resonance band of AuNRs at 780 nm, these findings permitted the imaging of inflammation in an in vivo mouse model for rheumatoid arthritis with high contrast using multispectral optoacoustic tomography. The study offers a robust method for otherwise difficult to obtain covalently functionalized polyanionic gold nanorods, which are suitable for biological applications as well as a low-cost, actively targeted, and high contrast imaging agent for the diagnosis of rheumatoid arthritis. This paves the way for further research in other inflammation associated pathologies, in particular, when photothermal therapy can be applied.

Heterotropic modulation of selectin affinity by allosteric antibodies affects leukocyte rolling.

Selectins are a family of adhesion receptors designed for efficient leukocyte tethering to the endothelium under shear. As a key property to resist premature bond disruption, selectin adhesiveness is enhanced by tensile forces that promote the conversion of a bent into an extended conformation of the N-terminal lectin and epidermal growth factor-like domains. Conformation-specific Abs have been invaluable in deciphering the activation mechanism of integrins, but similar reagents are not available for selectins. In this study, we show that the anti-human L-selectin mAbs DREG-55 and LAM1-5 but not DREG-56, DREG-200, or LAM1-1 heterotropically modulate adhesion presumably by stabilizing the extended receptor conformation. Force-free affinity assays, flow chamber, and microkinetic studies reveal a ligand-specific modulation of L-selectin affinity by DREG-55 mAb, resulting in a dramatic decrease of rolling velocity under flow. Furthermore, secondary tethering of polymorphonuclear cells was blocked by DREG-200 but significantly boosted by DREG-55 mAb. The results emphasize the need for a new classification for selectin Abs and introduce the new concept of heterotropic modulation of receptor function.

Linear center-of-mass dynamics emerge from non-linear leg-spring properties in human hopping.

Given the almost linear relationship between ground-reaction force and leg length, bouncy gaits are commonly described using spring-mass models with constant leg-spring parameters. In biological systems, however, spring-like properties of limbs may change over time. Therefore, it was investigated how much variation of leg-spring parameters is present during vertical human hopping. In order to do so, rest-length and stiffness profiles were estimated from ground-reaction forces and center-of-mass dynamics measured in human hopping. Trials included five hopping frequencies ranging from 1.2 to 3.6 Hz. Results show that, even though stiffness and rest length vary during stance, for most frequencies the center-of-mass dynamics still resemble those of a linear spring-mass hopper. Rest-length and stiffness profiles differ for slow and fast hopping. Furthermore, at 1.2 Hz two distinct control schemes were observed.

Carbohydrate-PNA and aptamer-PNA conjugates for the spatial screening of lectins and lectin assemblies.

Nucleic acid architectures offer intriguing opportunities for the interrogation of structural properties of protein receptors. In this study, we performed a DNA-programmed spatial screening to characterize two functionally distinct receptor systems: 1) structurally well-defined Ricinus communis agglutinin (RCA(120)), and 2) rather ill-defined assemblies of L-selectin on nanoparticles and leukocytes. A robust synthesis route that allowed the attachment both of carbohydrate ligands-such as N-acetyllactosamine (LacNAc), sialyl-Lewis-X (sLe(X)), and mannose-and of a DNA aptamer to PNAs was developed. A systematically assembled series of different PNA-DNA complexes served as multivalent scaffolds to control the spatial alignments of appended lectin ligands. The spatial screening of the binding sites of RCA(120) was in agreement with the crystal structure analysis. The study revealed that two appropriately presented LacNAc ligands suffice to provide unprecedented RCA(120) affinity (K(D) = 4 µM). In addition, a potential secondary binding site was identified. Less dramatic binding enhancements were obtained when the more flexible L-selectin assemblies were probed. This study involved the bivalent display both of the weak-affinity sLe(X) ligand and of a high-affinity DNA aptamer. Bivalent presentation led to rather modest (sixfold or less) enhancements of binding when the self-assemblies were targeted against L-selectin on gold nanoparticles. Spatial screening of L-selectin on the surfaces of leukocytes showed higher affinity enhancements (25-fold). This and the distance-activity relationships indicated that leukocytes permit dense clustering of L-selectin.

The toxin component of targeted anti-tumor toxins determines their efficacy increase by saponins.

Tumor-targeting protein toxins are composed of a toxic enzyme coupled to a specific cell binding domain that targets cancer-associated antigens. The anti-tumor treatment by targeted toxins is accompanied by dose-limiting side effects. The future prospects of targeted toxins for therapeutic use in humans will be determined by reduce side effects. Certain plant secondary metabolites (saponins) were shown to increase the efficacy of a particular epidermal growth factor receptor (EGFR)-targeted toxin, paralleled by a tremendous decrease of side effects. This study was conducted in order to investigate the effects of substituting different toxin moieties fused to an EGF ligand binding domain on the augmentative ability of saponins for each against therapeutic potential of the saponin-mediated efficacy increase for different anti-tumor toxins targeting the EGFR. We designed several EGFR-targeted toxins varying in the toxic moiety. Each targeted toxin was used in combination with a purified saponin (SA1641), isolated from the ornamental plant Gypsophila paniculata L. SA1641 was characterized and the SA1641-mediated efficacy increase was investigated on EGFR-transfected NIH-3T3 cells. We observed a high dependency of the SA1641-mediated efficacy increase on the nature of toxin used for the construction of the targeted toxin, indicating high specificity. Structural alignments revealed a high homology between saporin and dianthin-30, the two toxic moieties that benefit most from the combination with SA1641. We further demonstrate that SA1641 did not influence the plasma membrane permeability, indicating an intracellular interaction of SA1641 and the toxin components of targeted toxins. Surface plasmon resonance measurements point to a transient binding of SA1641 to the toxin components of targeted toxins.

L-selectin--a dynamic regulator of leukocyte migration.

The leukocytic cell adhesion receptor L-selectin mediates the initial step of the adhesion cascade, the capture and rolling of leukocytes on endothelial cells. This event enables leukocytes to migrate out of the vasculature into surrounding tissues during inflammation and immune surveillance. Distinct domains of L-selectin contribute to proper leukocyte migration. In this review, we discuss the contributions of these domains with respect to L-selectin function: the regulation by serine phosphorylation of the cytoplasmic tail, the role of the transmembrane domain in receptor positioning on the cell surface as well as the N-glycosylation of the extracellular part and the identification of novel binding partners.

Stance leg control: variation of leg parameters supports stable hopping.

The spring-loaded inverted pendulum describes the planar center-of-mass dynamics of legged locomotion. This model features linear springs with constant parameters as legs. In biological systems, however, spring-like properties of limbs can change over time. Therefore, in this study, it is asked how variation of spring parameters during ground contact would affect the dynamics of the spring-mass model. Neglecting damping initially, it is found that decreasing stiffness and increasing rest length of the leg during a stance phase are required for orbitally stable hopping. With damping, stable hopping is found for a larger region of rest-length rates and stiffness rates. Here, also increasing stiffness and decreasing rest length can result in stable hopping. Within the predicted range of leg parameter variations for stable hopping, there is no need for precise parameter tuning. Since hopping gaits form a subset of the running gaits (with vanishing horizontal velocity), these results may help to improve leg design in robots and prostheses.

Electrophoretic isolation of saponin fractions from Saponinum album and their evaluation in synergistically enhancing the receptor-specific cytotoxicity of targeted toxins.

Saponinum album (SA) is a commercial mixture of saponins isolated from Gypsophila species. In the previously published work, we reported that SA dramatically improves the inhibition of tumor growth by targeted toxins in mice in a synergistic way. Here we report a simplified electrophoretic method for the isolation of a highly effective fraction of SA with a relative electrophoretic mobility to the dye front (R(f) ) of 0.63 from the mixture. In total, four different fractions were separated at a preparative scale, and evaluated by ESI-MS, HPLC and TLC analysis. Electrophoretic mobility and electrochemical properties of the different fractions of saponins from SA were set into relation to their ability to enhance the cytotoxicity of epidermal growth factor (EGF)-based targeted toxins. We here treated HER-14 cells, which are NIH-3T3 Swiss mouse embryo cells transfected with the human EGF receptor. Untransfected NIH-3T3 cells served as control. The major bulk of SA (72.3%) (R(f) =0.78) migrated the farthest and was found to be significantly ineffective (p<0.05) in enhancing the cytotoxicity of the targeted toxin, while the second fraction (R(f) =0.63) showed an enhancement of 9800-fold. The third (R(f) =0.56) had an enhancement factor of 3200, the fourth (R(f) =0.08) was again significantly ineffective (p<0.05) in exhibiting any enhancement of cytotoxicity.

The role of dimension in multivalent binding events: structure-activity relationship of dendritic polyglycerol sulfate binding to L-selectin in correlation with size and surface charge density.

L-, P-, and E-Selectin are cell adhesion molecules that play a crucial role in leukocyte recruitment from the blood stream to the afflicted tissue in an acute and chronic inflammatory setting. Since selectins mediate the initial contact of leukocytes to the vascular endothelium, they have evolved as a valuable therapeutic target in diseases related to inflammation by inhibition of the physiological selectin-ligand interactions. In a previous study, it was demonstrated that dPGS, a fully synthetic heparin analogue, works as an efficient inhibitor towards L- and P-selectin in vitro as well as in vivo. Herein, the focus is directed towards the effect of size and charge density of the polyanion. The efficiency of L-selectin inhibition via an SPR-based in vitro assay and a cell-based flow chamber assay is investigated with dPGS ranging from approximately 4 to 2000 kDa. SPR measurements show that the inhibitory potential of highly sulfated dPGS increases with size and charge density. Thereby, IC(50) values from the micromolar to the low picomolar range are determined. The same tendency could be observed in a cell-based flow chamber assay with three representative dPGS samples. This structure-affinity relationship of dPGS suggests that the strong inhibitory potential of dPGS is not only based on the strong electrostatic interaction with areas of cationic surface potential on L-selectin but is also due to a steric shielding of the carbohydrate binding site by large, flexible dPGS particles.

Multivalent presentation of mannose on hyperbranched polyglycerol and their interaction with concanavalin A lectin.

We describe the synthesis of multivalent mannose derivatives by using hyperbranched polyglycerols (hPG) as a scaffold with different linker structures. Grafting of protected mannose (Man) units is achieved by using Cu(I) -catalyzed Huisgen click chemistry with either an anomeric azide or propargyl ether onto complementarily functionalized alkyne or azido polymer surfaces. NMR spectroscopy, dynamic light scattering (DLS), IR spectroscopy, size-exclusion chromatography (SEC), and elemental analysis have been used to characterize the hPG-Man compounds. The surface availability and bioactivity of Man-modified polymers were evaluated by using a competitive surface plasmon resonance (SPR)-based binding assay by interactions of the glycopolymers with concanavalin A (Con A), a lectin that binds mannose containing molecules. The results indicated that the novel glycoarchitectures presented in this work are efficient inhibitors of Con A-mannose recognition and resulted in inhibitor concentrations (mean IC(50)) from the micro- to the nanomolar range, whereas the corresponding monovalent mannoside (methyl-Man) requires millimolar concentrations. The results provide an interesting structure-activity relationship for libraries of materials that differ in the linkage of the sugar moiety presented on a biocompatible polyglycerol scaffold.