RGD-integrin Interaction Research Articles

Deletion of the RGD motif from the penton base in oncolytic adenoviruses enhances antitumor efficacy of combined CAR T cell therapy

Oncolytic viruses often face challenges in achieving optimal antitumor immunity as standalone therapies. The penton base RGD-integrin interactions play a significant role in wild-type adenovirus-induced innate immune responses. To modify these responses, we present ISC301, a novel oncolytic adenovirus engineered by deleting the natural RGD motifs in the penton base while incorporating artificial RGD motifs in the fiber knobs. ISC301 demonstrated comparable invitro infectivity, cytotoxic effects, and signaling profiles across various cell types to its parental ICOVIR-5, which retains the penton base RGD motif. In immunodeficient and immunocompetent mouse models, ISC301 exhibited similar invivo antitumor efficacy to ICOVIR-5. However, ISC301 induced higher intratumoral inflammation through NF-κB activation, leading to increased levels of tumor-infiltrating leukocytes and higher proportion of cytotoxic CD8+ Tcells. In addition, ISC301 elicits a heightened pro-inflammatory response in peripheral blood. Importantly, when combined with CAR Tcell therapy, ISC301 exhibited superior antitumor efficacy, surpassing monotherapy outcomes. These findings emphasize the impact of adenoviral modifications on antitumor immune responses. The deletion of penton base RGD motifs enhances ISC301's pro-inflammatory profile and boosts CAR Tcell therapy efficacy. This study enhances understanding of oncolytic virus engineering strategies, positioning ISC301 as a promising candidate for combined immunotherapeutic approaches in cancer treatment.

Adipose-derived stem cell-based anti-inflammatory paracrine factor regulation for the treatment of inflammatory bowel disease

Stem cell-based therapies offer promising avenues for treating inflammatory diseases owing to their immunomodulatory properties. However, challenges persist regarding their survival and efficacy in inflamed tissues. Our study introduces a novel approach by engineering adipose-derived stem cells (ADSCs) to enhance their viability in inflammatory environments and boost the secretion of paracrine factors for treating inflammatory bowel disease (IBD). An arginine-glycine-aspartate peptide-poly (ethylene glycol)-chlorin e6 conjugate (RPC) was synthesized and coupled with ADSCs, resulting in RPC-labeled ADSCs (ARPC). This conjugation strategy employed RGD-integrin interaction to shield stem cells and allowed visualization and tracking using chlorin e6. The engineered ARPC demonstrated enhanced viability and secretion of paracrine factors upon light irradiation, regulating the inflammatory microenvironment. RNA-sequencing analysis unveiled pathways favoring angiogenesis, DNA repair, and exosome secretion in ARPC(+) while downregulating inflammatory pathways. In in vivo models of acute and chronic IBD, ARPC(+) treatment led to reduced inflammation, preserved colon structure, and increased populations of regulatory T cells, highlighting its therapeutic potential. ARPC(+) selectively homed to inflammatory sites, demonstrating its targeted effect. Overall, ARPC(+) exhibits promise as an effective and safe therapeutic strategy for managing inflammatory diseases like IBD by modulating immune responses and creating an anti-inflammatory microenvironment.

Robust bioactive protein-based screws with dual crosslinked network for internal bone fixation

Resorbable fixation systems have gained interest in orthopedics by avoiding secondary surgery and stress shielding, but lack of enough bioactivity. Herein, we fabricated resorbable and bioactive regenerated silk fibroin/gelatin (RSF/G) screws with dual physically and chemically crosslinked networks, which can control the release of Arg-Gly-Asp (RGD) peptide and bear pull-out and shear forces comparable to those of pure RSF and commercial poly (lactic-co-glycolic acid) devices under dry and wet conditions. The controllable swelling properties of the RSF/G screws support implantation although with the high hydrophilic gelatin. Compared to RSF screws, the RSF/G screws have better bioactivity and significantly promote cell proliferation, adhesion, spreading and osteogenic differentiation by RGD-integrin interactions via the focal adhesion kinase/extracellular signal-regulated (FAK/ERK) pathway. Eight weeks after implantation in rats, the RSF/G screws with magnetic resonance imaging(MRI) compatibility remained fixed with tight contact, less inflammatory reaction and more new bone formation compared with pure RSF screws. The results indicate great promise for the application of RSF/G screws in bone fracture fixation.

Single-Molecule Force Probing of RGD-Binding Integrins on Pancreatic Cancer Cells.

Integrin-targeting arginine-glycine-aspartic acid (RGD)-based nanocarriers have been widely used for tumor imaging, monitoring of tumor development, and delivery of anticancer drugs. However, the thermodynamics of an RGD-integrin formation and dissociation associated with binding dynamics, affinity, and stability remains unclear. Here, we probed the binding strength of the binary complex to live pancreatic cancer cells using single-molecule binding force spectroscopy methods, in which RGD peptides were functionalized on a force probe tip through poly(ethylene glycol) (PEG)-based bifunctional linker molecules. While the density of integrin αV receptors on the cell surface varies more than twofold from cell line to cell line, the individual RGD-integrin complexes exhibited a cell type-independent, monovalent bond strength. The load-dependent bond strength of multivalent RGD-integrin interactions scaled sublinearly with increasing bond number, consistent with the noncooperative, parallel bond model. Furthermore, the multivalent bonds ruptured sequentially either by one or in multiples, and the force strength was comparable to the synchronous rupture force. Comparison of energy landscapes of the bond number revealed a substantial decrease of kinetic off-rates for multivalent bonds, along with the increased width of the potential well and the increased potential barrier height between bound and unbound states, enhancing the stability of the multivalent bonds between them.

Enzymatically functionalized RGD-gelatin scaffolds that recruit host mesenchymal stem cells in vivo and promote bone regeneration

Critical-size bone defects are imposing a substantial biomedical burden. Despite being long regarded as a potential approach to mitigate this burden or an alternative to bone grafts, bone tissue engineering (BTE) has virtually not proceeded to widespread clinical practices. In the BTE field, it is highly required to find a facile method to prepare active scaffolds with tailored biological functions. Here, we immobilized cell adhesive RGD motifs onto gelatin sponge (GS) scaffolds through enzymatic linking. On the basis of the resulting RGD-functionalized GS (RGD/GS) scaffolds, we developed a new and convenient strategy for bone defect repair, in which the scaffolds were first used to recruit mesenchymal stem cells (MSCs) from skeletal muscle, immediately followed by their engraftment into bone defect. We demonstrated significantly enhanced host cells homing into RGD/GS scaffolds as a result of specific RGD-integrin interactions, and the recruited host cells showed a strong osteogenic differentiation potential. After ectopic implantation of cell-laden RGD/GS scaffolds into critical-size mouse bone defects, marked bone tissue regeneration occurred. The presented strategy not only provides an agile route for the preparation of bioactive scaffolds and the construction of osteoinductive bone-graft substitutes, but also avoids or minimizes the complicated and laborious cell isolation, in vitro expansion and cell seeding procedures used in the conventional BTE.

SARS-CoV-2 attachment to host cells is possibly mediated via RGD-integrin interaction in a calcium-dependent manner and suggests pulmonary EDTA chelation therapy as a novel treatment for COVID 19

SARS-CoV-2 is a highly contagious virus that has caused serious health crisis world-wide resulting into a pandemic situation. As per the literature, the SARS-CoV-2 is known to exploit humanACE2 receptors (similar toprevious SARS-CoV-1) for gaining entry into the host cell for invasion, infection, multiplication and pathogenesis. However, considering the higher infectivity of SARS-CoV-2 along with the complex etiology and pathophysiological outcomes seen in COVID-19 patients, it seems that there may be an alternate receptor for SARS-CoV-2. I performed comparative protein sequence analysis, database based gene expression profiling, bioinformatics based molecular docking using authentic tools and techniques for unveiling the molecular basis of high infectivity of SARS-CoV-2 as compared to previous known coronaviruses. My study revealed that SARS-CoV-2 (previously known as 2019-nCoV) harbors a RGD motif in its receptor binding domain (RBD) and the motif is absent in all other previously known SARS-CoVs. The RGD motif is well known for its role in cell-attachment and cell-adhesion. My hypothesis is that the SARS-CoV-2 may be (via RGD) exploiting integrins, that have high expression in lungs and all other vital organs, for invading host cells. However, an experimental verification is required. The expression of ACE2, which is a known receptor for SARS-CoV-2, was found to be negligible in lungs. I assume that higher infectivity of SARS-CoV-2 could be due to this RGD-integrin mediated acquired cell-adhesive property. Gene expression profiling revealed that expression of integrins is significantly high in lung cells, in particular αvβ6, α5β1, αvβ8 and an ECM protein, ICAM1. The molecular docking experiment showed the RBD of spike protein binds with integrins precisely at RGD motif in a similar manner as a synthetic RGD peptide binds to integrins as found by other researchers. SARS-CoV-2 spike protein has a number of phosphorylation sites that can induce cAMP, PKC, Tyr signaling pathways. These pathways either activate calcium ion channels or get activated by calcium. In fact, integrins have calcium & metal binding sites that were predicted around and in vicinity of RGD-integrin docking site in our analysis which suggests that RGD-integrins interaction possibly occurs in calcium-dependent manner. The higher expression of integrins in lungs along with their previously known high binding affinity (~KD = 4.0 nM) for virus RGD motif could serve as a possible explanation for high infectivity of SARS-CoV-2. On the contrary, human ACE2 has lower expression in lungs and its high binding affinity (~KD = 15 nM) for spike RBD alone could not manifest significant virus-host attachment. This suggests that besides human ACE2, an additional or alternate receptor for SARS-CoV-2 is likely to exist. A highly relevant evidence never reported earlier which corroborate in favor of RGD-integrins mediated virus-host attachment is an unleashed cytokine storm which causes due to activation of TNF-α and IL-6 activation; and integrins role in their activation is also well established. Altogether, the current study has highlighted possible role of calcium and other divalent ions in RGD-integrins interaction for virus invasion into host cells and suggested that lowering divalent ion in lungs could avert virus-host cells attachment.

Synergism of highly transducible adenovirus encoding heme oxygenase 1 gene and low-dose immunosuppressants for successful outcomes of xenotransplanted pancreatic islet

Pancreatic islet transplantation is a promising strategy for diabetic patients. Unfortunately, host’s immune cells rapidly reject transplanted islets. Cytoprotective gene therapy has been tried to protect them. However, the transduce yield of ex vivo gene delivery into islets is very rare due to the anatomical structure of them. Therefore, we newly designed an Arg-Gly-Asp (RGD) peptide-incorporated adenovirus vector expressing cytoprotective heme oxygenase-1 (HO1) gene (RGD-Adv-HO1) for effective gene therapy to islets via RGD–integrin interaction between adenovirus and islet. After exposure to islets, the transduced RGD-Adv-HO1 did not affect the viability and insulin secretion of islets with higher transduction efficacy. Moreover, HO1 showed cytoprotective effect from the presence of paraquat-induced reactive oxygen species (ROS). When the transduced islets were xenotransplanted into streptozotocin-induced diabetic Balb/c mice, their survival time in vivo was significantly prolonged with curing blood glucose level, which was attributed to the stable expression of HO1. Additionally, we found that RGD-Adv-HO1 showed a good synergistic effect in xenotransplanted islets when accompanied with low dose of immunosuppressants tacrolimus and anti-CD154 monoclonal antibody. Collectively, this new combinatorial remedy of RGD-Adv-HO1 transduction with low dose of immunosuppressive agents could be an effective therapy for successful outcome of islet transplantation.

Supramolecular Self-Assemblies with Nanoscale RGD Clusters Promote Cell Growth and Intracellular Drug Delivery.

In this work, we reported the generation of a novel supramolecular hydrogelator from a peptide derivative which consisted of a structural motif (e.g., Fc-FF) for supramolecular self-assembly and a functional moiety (e.g., RGD) for integrin binding. Following self-assembly in water at neutral pH, this molecule first tended to form metastable spherical aggregates, which subsequently underwent a morphological transformation to form high-aspect-ratio nanostructures over 2 h when aged at room temperature. More importantly, because of the presence of nanoscale RGD clusters on the surface of nanostructures, the self-assembled nanomaterials (e.g., nanoparticles and nanofibers) can be potentially used as a biomimetic matrix for cell culture and as a vector for cell-targeting drug delivery via multivalent RGD-integrin interactions.

Insights into the posttranslational assembly of the Mo-, S- and Cu-containing cluster in the active site of CO dehydrogenase of Oligotropha carboxidovorans.

Oligotropha carboxidovorans is characterized by the aerobic chemolithoautotrophic utilization of CO. CO oxidation by CO dehydrogenase proceeds at a unique bimetallic [CuSMoO2] cluster which matures posttranslationally while integrated into the completely folded apoenzyme. Kanamycin insertional mutants in coxE, coxF and coxG were characterized with respect to growth, expression of CO dehydrogenase, and the type of metal center present. These data along with sequence information were taken to delineate a model of metal cluster assembly. Biosynthesis starts with the MgATP-dependent, reductive sulfuration of [MoVIO3] to [MoVO2SH] which entails the AAA+-ATPase chaperone CoxD. Then MoV is reoxidized and Cu1+-ion is integrated. Copper is supplied by the soluble CoxF protein which forms a complex with the membrane-bound von Willebrand protein CoxE through RGD–integrin interactions and enables the reduction of CoxF-bound Cu2+, employing electrons from respiration. Copper appears as Cu2+-phytate, is mobilized through the phytase activity of CoxF and then transferred to the CoxF putative copper-binding site. The coxG gene does not participate in the maturation of the bimetallic cluster. Mutants in coxG retained the ability to utilize CO, although at a lower growth rate. They contained a regular CO dehydrogenase with a functional catalytic site. The presence of a pleckstrin homology (PH) domain on CoxG and the observed growth rates suggest a role of the PH domain in recruiting CO dehydrogenase to the cytoplasmic membrane enabling electron transfer from the enzyme to the respiratory chain. CoxD, CoxE and CoxF combine motifs of a DEAD-box RNA helicase which would explain their mutual translation.

Improving detection specificity of iron oxide nanoparticles (IONPs) using the SWIFT sequence with long T2 suppression

In order to improve the detection specificity of iron oxide nanoparticles (IONPs) delivered to tumors, we embedded saturation pulses into the sweep imaging using Fourier transformation (SWIFT) sequence to suppress long T2 tissues and fat. Simulation of the Bloch equation was first conducted to study behavior of the saturation pulses of various lengths under different T2 and off-resonance conditions. MR experiments were then conducted using in vivo mouse xenografts and a phantom consisting of IONPs, vegetable oil, and explanted tumor specimen, without and with long T2 suppression under a 7T magnetic field. For the in vivo study, arginine-glycine-aspartate (RGD) coated 10nm IONPs (RGD-IONPs) were delivered to tumors implanted in nude mice through both intra-tumor and intravenous injections. Histological studies confirmed that RGD-IONPs efficiently homed to tumors through RGD-integrin interaction. Compared to conventional SWIFT, the proposed method resulted in sufficient suppression on long T2 species but less influence on short T2 species. For both the in vivo and ex vivo studies, significantly improved contrast-to-noise ratio (CNR) was achieved between the IONPs and the long T2 species.

Artificial RGD receptor molecules

Integrins play a pivotal role in cell–cell adhesion, signalling and apoptosis. Many extracellular proteins use the RGD sequence (arginine–glycine–aspartate) as a key to dock onto and unlock their respective binding partners at the cell membrane (αVβ3-, aIIbβ3- and a5β1-integrin). Here, the RGD signal is transduced into the cytoplasm and triggers a variety of biological events such as blood coagulation, cell–matrix binding, cell differentiation and angiogenesis. A misfunction of this recognition system causes severe diseases, rendering the RGD recognition system an attractive drug target. Inhibition of RGD–integrin interactions can be reached in two different ways, by blocking integrins with RGD mimetics or by capping RGD-containing proteins by artificial RGD receptors. This review provides an overview over the very young history of artificial RGD receptor development, beginning with early research in arginine recognition, over the discovery of the first primitive RGD receptor until the present state of research and future prospects.

Improved Adenovirus Type 5 Vector-Mediated Transduction of Resistant Cells by Piggybacking on Coxsackie B-Adenovirus Receptor-Pseudotyped Baculovirus

Taking advantage of the wide tropism of baculoviruses (BVs), we constructed a recombinant BV (BV(CAR)) pseudotyped with human coxsackie B-adenovirus receptor (CAR), the high-affinity attachment receptor for adenovirus type 5 (Ad5), and used the strategy of piggybacking Ad5-green fluorescent protein (Ad5GFP) vector on BV(CAR) to transduce various cells refractory to Ad5 infection. We found that transduction of all cells tested, including human primary cells and cancer cell lines, was significantly improved using the BV(CAR)-Ad5GFP biviral complex compared to that obtained with Ad5GFP or BV(CAR)GFP alone. We determined the optimal conditions for the formation of the complex and found that a high level of BV(CAR)-Ad5GFP-mediated transduction occurred at relatively low adenovirus vector doses, compared with transduction by Ad5GFP alone. The increase in transduction was dependent on the direct coupling of BV(CAR) to Ad5GFP via CAR-fiber knob interaction, and the cell attachment of the BV(CAR)-Ad5GFP complex was mediated by the baculoviral envelope glycoprotein gp64. Analysis of the virus-cell binding reaction indicated that the presence of BV(CAR) in the complex provided kinetic benefits to Ad5GFP compared to the effects with Ad5GFP alone. The endocytic pathway of BV(CAR)-Ad5GFP did not require Ad5 penton base RGD-integrin interaction. Biodistribution of BV(CAR)-Ad5Luc complex in vivo was studied by intravenous administration to nude BALB/c mice and compared to Ad5Luc injected alone. No significant difference in viscerotropism was found between the two inocula, and the liver remained the preferred localization. In vitro, coagulation factor X drastically increased the Ad5GFP-mediated transduction of CAR-negative cells but had no effect on the efficiency of transduction by the BV(CAR)-Ad5GFP complex. Various situations in vitro or ex vivo in which our BV(CAR)-Ad5 duo could be advantageously used as gene transfer biviral vector are discussed.

Measuring Cell Adhesion on RGD‐Modified, Self‐Assembled PEG Monolayers Using the Quartz Crystal Microbalance Technique

In this study, the suitability of a flow-through quartz crystal microbalance system for the detection of the adhesion of rMSCs and 3T3-L1 fibroblasts on different surfaces is demonstrated. Frequency shifts for rMSCs of -6.7 mHz x cell(-1) and -2.0 mHz x cell(-1) for 3T3-L1 cells could be detected on non-modified gold sensors, revealing that the frequency shift per cell is comparable to that of a static setup. Modifying the sensor surface with SAMs of thioalkylated omega-amine-terminated PEG derivatives led to cell-adhesion-resistant surfaces. Total frequency shifts of only -20 +/- 7 Hz showed that protein adsorption was also significantly reduced. Attaching 35 pmol x mm(-2) of the GRGDS cell adhesion motif to the SAMs induced specific cell adhesion due to RGD-integrin interactions; the resonance frequency dropped by 3.4 mHz x cell(-1). Furthermore, the kinetics of cell detachment could be determined. The corresponding processes were completed after 10 min for trypsin, and not before 90 min with GRGDS. Moreover, the detectability of cell adhesion was shown to increase after the addition of manganese cations. The total decrease in the resonance frequency was almost 80 Hz in the presence of Mn(2+) (6.4 mHz x cell(-1)). [image: see text] Staining the cytoskeleton of the rMSCs shows that the GRGDS-modified surfaces are almost completely covered with well-spread cells.

Recombinant osteopontin fragment coating on hydroxyapatite for enhanced osteoblast-like cell responses

Surface engineering approaches using biomimetic peptides provide promising strategies to improve the bioactivity of materials. In this study, to improve the bioactivity of hydroxyapatite (HA), a recombinant osteopontin fragment (rhOPN) was devised so as to retain its specific binding domains to HA, as well as to cells, in order that it function as a bridge between the two. A protein adsorption assay was performed to determine the optimal coating conditions, and the bioactivity of rhOPN coated HA was evaluated by conducting various in vitro cellular assays, such as the evaluation of cellular attachment, proliferation and differentiation. The rhOPN was adsorbed to the HA by an electrostatic attraction which was facilitated by the former’s HA binding domain. The osteoblast-like cell attachment was significantly simulated through the RGD-integrin interaction. Moreover, HOS cells attached to the adsorbed rhOPN ligands on the HA surfaces showed significantly enhanced proliferation and differentiation behaviors (P < 0.05). These enhanced cellular responses can be attributed to changes in gene expression via the signal transduction cascade initiated by the RGD-integrin interaction.

Cα-H···O=C hydrogen bonds contribute to the specificity of RGD cell-adhesion interactions

BackgroundThe Arg-Gly-Asp (RGD) cell adhesion sequence occurs in several extracellular matrix molecules known to interact with integrin cell-surface receptors. Recently published crystal structures of the extracellular regions of two integrins in complex with peptides containing or mimicking the RGD sequence have identified the Arg and Asp residues as key specificity determinants for integrin recognition, through hydrogen bonding and metal coordination interactions. The central Gly residue also appears to be in close contact with the integrin surface in these structures.ResultsWhen hydrogen atoms are modelled on the central Gly residue with standard stereochemistry, the interaction between this residue and a carbonyl group in the integrin surface shows all the hallmarks of Cα-H···O=C hydrogen bonding, as seen in the collagen triple helix and in many crystal structures of small organic molecules. Moreover, molecular dynamic simulations of the docking of RGD-containing fragments on integrin surfaces support the occurrence of these interactions. There appears to be an array of four weak and conventional hydrogen bonds lining up the RGD residues with main chain carbonyl groups in the integrin surface.ConclusionsThe occurrence of weak Cα-H···O=C hydrogen bonds in the RGD-integrin interaction highlights the importance of the conserved Gly residue in the RGD motif and its contribution to integrin-ligand binding specificity. Our analysis shows how weak hydrogen bonds may also play important biological roles by contributing to the specificity of macromolecular recognition.

Deletion of Penton RGD Motifs Affects the Efficiency of both the Internalization and the Endosome Escape of Viral Particles Containing Adenovirus Serotype 5 or 35 Fiber Knobs

Adenovirus (Ad) vectors are widely used for gene delivery in vitro and in vivo. A solid understanding of the biology of this virus is imperative for the development of novel, effective, and safe vectors. For the group C adenovirus serotypes 2 and 5 that use CAR as a primary attachment receptor, it is known that the penton base RGD motifs interact with cellular integrins and that this interaction promotes virus internalization. However, the RGD motif's impact on the efficiency of postinternalization steps, such as the escape of the virus particle from the endosome, is less defined. Furthermore, the role of penton-integrin interactions remains unknown for new vectors possessing group B Ad fiber knobs that use CD46 as a primary virus attachment receptor. In this study, we used vectors with the RGD motif deleted that contained Ad5 and B-group Ad35 fiber knobs and long fiber shafts and studied the role of RGD-integrin interactions in virus internalization and endosome escape. The deletion of the RGD motif in the penton base did not affect virus attachment, regardless of the type of cellular receptor used for attachment. RGD motif deletion, however, significantly reduced the rate of virus internalization for both the Ad5 and Ad35 fiber knob-containing vectors. This study also demonstrates the role of penton RGD motifs in facilitating the endosome escape step of virus infection and indicates that penton-integrin interactions are involved in internalization of capsid-chimeric CD46-interacting Ads with long fiber shafts.

Mediating specific cell adhesion to low-adhesive diblock copolymers by instant modification with cyclic RGD peptides

One promising strategy to control the interactions between biomaterial surfaces and attaching cells involves the covalent grafting of adhesion peptides to polymers on which protein adsorption, which mediates unspecific cell adhesion, is essentially suppressed. This study demonstrates a surface modification concept for the covalent anchoring of RGD peptides to reactive diblock copolymers based on monoamine poly(ethylene glycol)-block-poly(D,L-lactic acid) (H2N-PEG-PLA). Films of both the amine-reactive (ST-NH-PEG2PLA20) and the thiol-reactive derivative (MP-NH-PEG2PLA40) were modified with cyclic αvβ3/αvβ5 integrin subtype specific RGD peptides simply by incubation of the films with buffered solutions of the peptides. Human osteoblasts known to express these integrins were used to determine cell–polymer interactions. The adhesion experiments revealed significantly increased cell numbers and cell spreading on the RGD-modified surfaces mediated by RGD–integrin-interactions.

Mechanical regulation of HB-GAM expression in bone cells

Bone adaption upon mechanical stimulation is accompanied by changes in gene expression. In this context we investigated the influence of mechanical loading on heparin binding growth associated molecule (HB-GAM) expression, an extracellular matrix molecule which in cell culture has been shown to stimulate the differentiation of osteoblasts. We obtained information on the participating signal transduction pathways using a mitogenic loading regimen. Specific inhibitors of various signal transduction pathways were added to loaded cells and to unloaded controls. By semi-quantitative PCR studies we demonstrated a rapid decrease of HB-GAM expression in primary osteoblasts and SaOs-2 cells by 20–30% upon mechanical loading within 30min. We showed that the RGD–integrin interaction is involved in the regulation of HB-GAM expression. Furthermore, integrity of the cytoskeleton, stretch-activated, and voltage-sensitive Ca2+ channels as well as gap junctional communication are necessary for the downregulation of HB-GAM expression by mechanical loading.

Efficient transduction of murine B lymphocytes and B lymphoma lines by modified adenoviral vectors: enhancement via targeting to FcR and heparan-containing proteins.

Murine lymphocytes are relatively refractory to efficient transfection or retroviral gene transduction. Adenovirus has been used as a vector to transduce a wide variety of cell types. Several advantages of adenoviruses are their ability to transduce non-cycling cells and to transduce the majority of cells in a population. Unfortunately, lymphocytes are not susceptible to infection with conventional adenovirus. Therefore, to express genes efficiently in murine B cells, we tested the ability of genetically modified adenovirus to transduce the beta-galactosidase gene. We found that adenovirus containing polylysine in the fiber knob was able to efficiently transduce lipopolysaccharide (LPS)-activated splenic B cells and the B lymphoma line M12.4.1; greater than 80% of the cells expressed beta-galactosidase activity. However, small resting B cells did not express activity unless treated with LPS after infection. This transduction was mediated by interaction with charged molecules since heparan-sulfate, and to a lesser degree chondroitan sulfate, inhibited the transduction. In addition, adenovirus containing a FLAG epitope in the fiber protein was used to target the FcR expressed on B cells using an anti-FLAG antibody. In the presence of anti-FLAG, the modified adenovirus was able to efficiently transduce LPS-activated B cells and several B cell lymphoma lines. Interestingly, in the absence of anti-FLAG, there was low level transduction in the LPS-blasts and in M12.4.1 that was not inhibited by soluble adenovirus fiber protein or agents that block RGD-integrin interactions. These results demonstrate that modified adenovirus efficiently transduce B lymphocytes which will be critical for targeting genes to normal or malignant B cells.