Selection of an ASIC1a-blocking combinatorial antibody that protects cells from ischemic death.

Acid-sensing ion channels (ASICs) have emerged as important, albeit challenging therapeutic targets for pain, stroke, etc. One approach to developing therapeutic agents could involve the generation of functional antibodies against these channels. To select such antibodies, we used channels assembled in nanodiscs, such that the target ASIC1a has a configuration as close as possible to its natural state in the plasma membrane. This methodology allowed selection of functional antibodies that inhibit acid-induced opening of the channel in a dose-dependent way. In addition to regulation of pH, these antibodies block the transport of cations, including calcium, thereby preventing acid-induced cell death in vitro and in vivo. As proof of concept for the use of these antibodies to modulate ion channels in vivo, we showed that they potently protect brain cells from death after an ischemic stroke. Thus, the methodology described here should be general, thereby allowing selection of antibodies to other important ASICs, such as those involved in pain, neurodegeneration, and other conditions.

Design and Characterization of a Human Monoclonal Antibody that Modulates Mutant Connexin 26 Hemichannels Implicated in Deafness and Skin Disorders.

Background: Mutations leading to changes in properties, regulation, or expression of connexin-made channels have been implicated in 28 distinct human hereditary diseases. Eight of these result from variants of connexin 26 (Cx26), a protein critically involved in cell-cell signaling in the inner ear and skin. Lack of non-toxic drugs with defined mechanisms of action poses a serious obstacle to therapeutic interventions for diseases caused by mutant connexins. In particular, molecules that specifically modulate connexin hemichannel function without affecting gap junction channels are considered of primary importance for the study of connexin hemichannel role in physiological as well as pathological conditions. Monoclonal antibodies developed in the last three decades have become the most important class of therapeutic biologicals. Recombinant methods permit rapid selection and improvement of monoclonal antibodies from libraries with large diversity. Methods: By screening a combinatorial library of human single-chain fragment variable (scFv) antibodies expressed in phage, we identified a candidate that binds an extracellular epitope of Cx26. We characterized antibody action using a variety of biochemical and biophysical assays in HeLa cells, organotypic cultures of mouse cochlea and human keratinocyte-derived cells. Results: We determined that the antibody is a remarkably efficient, non-toxic, and completely reversible inhibitor of hemichannels formed by connexin 26 and does not affect direct cell-cell communication via gap junction channels. Importantly, we also demonstrate that the antibody efficiently inhibits hyperative mutant Cx26 hemichannels implicated in autosomal dominant non-syndromic hearing impairment accompanied by keratitis and hystrix-like ichthyosis-deafness (KID/HID) syndrome. We solved the crystal structure of the antibody, identified residues that are critical for binding and used molecular dynamics to uncover its mechanism of action. Conclusions: Although further studies will be necessary to validate the effect of the antibody in vivo, the methodology described here can be extended to select antibodies against hemichannels composed by other connexin isoforms and, consequently, to target other pathologies associated with hyperactive hemichannels. Our study highlights the potential of this approach and identifies connexins as therapeutic targets addressable by screening phage display libraries expressing human randomized antibodies.

The role of SATB2 in skeletogenesis and human disease.

Since the discovery of SATB2 (special AT-rich sequence binding protein 2) a decade ago, its pivotal roles in development and tissue regeneration have emerged, particularly in craniofacial patterning and development, palate formation, and osteoblast differentiation and maturation. As a member of the special AT-rich binding proteins family that bind to nuclear matrix-attachment regions (MAR), it also displays functional versatility in central nervous development, especially corpus callosum and pons formation, cancer development and prognosis, as well as in immune regulation. At the molecular level, Satb2 gene expression appears to be tissue and stage-specific, and is regulated by several cytokines and growth factors, such as BMP2/4/7, insulin, CNTF, and LIF via ligand receptor signaling pathways. SATB2 mainly performs a twofold role as a transcription regulator by directly binding to AT-rich sequences in MARs to modulate chromatin remodeling, or through association with other transcription factors to modulate the cis-regulation elements and thus to regulate the expression of down-stream target genes and a wide range of biological processes. This contemporary review provides an exploration of the molecular characteristics and function of SATB2; including its expression and cytokine regulation, its involvement in human disease, and its potential roles in skeletogenesis.

[Construction and identification of recombinant retroviral vector containing human interleukin 1 receptor antagonist and its expression in osteoarthritic human articular chondrocytes].

OBJECTIVE: To construct the retroviral vector containing human interleukin 1 receptor antagonist (IL-1Ra) and to investigate the property of the transfected articular chondrocytes from osteoarthritic patients in vitro. METHODS: Retroviral vector PLXRN carrying IL-1Ra (PLXRN-IL-1Ra) gene was constructed by inserting IL-1Ra gene at the sites of Sal I and BamH I. The recombinant retroviral plasmid was homologously recombinated in bacterial cells. After screening and amplification, the recombinant retroviral plasmid was obtained and transfected into PT67 cells. The replication-defective retrovirus PLXRN-IL-1Ra was packed and amplified in the PT67 cells. Viral titer was determined by infecting NIH/3T3 cells with serially diluted viral supernatants produced with a control vector. Experiments were divided into 3 groups: non-transduced group (group A), PLXRN transduction group (group B), PLXRN-IL-1Ra transduction group (group C). Primary articular chondrocytes from osteoarthritic patients were transduced with PLXRN and PLXRN-IL-1Ra. The positive chondrocytes clones, which were G418-resistant, were cultured for 3-4 weeks after being selected by G418. The expression of IL-1Ra mRNA in the chondrocytes was determined by RT-PCR. Levels of IL-1Ra protein synthesis in the supernatants were measured by ELISA. RESULTS: Restrictive endonuclease identification and gene sequencing confirmed that the recombinant contained IL-1Ra cDNA. Virus titer could reach 3 x 10(4) CFU/mL. Primary chondrocytes cultured in vitro were polygonal or spindle and were stained with purple particles by toluidine blue staining. After stable transduction into the chondrocytes the 311 bp fragment of IL-1Ra was detected in group C by semi-quantitative RT-PCR. ELISA showed that IL-1Ra in supernatants of the group A and group B were below the level of detection. The concentrations were (60.47 +/- 15.13) ng/L in group C. There were significant differences between gene transduction group and control groups (P < 0.05). CONCLUSION: The construction of recombinant retrovirus vector by homologous recombination in bacterial cells can be quickly and easily performed. Stable and effective expression of IL-1Ra can be achieved by transduction with retroviral vectors in osteoarthritic articular chondrocytes, indicating potential utility in gene therapy for osteoarthritis.

Stimulation of osteogenic differentiation and inhibition of adipogenic differentiation in bone marrow stromal cells by alendronate via ERK and JNK activation.

To elucidate the mechanism of the effect of bisphosphonates on bone metabolism, we investigated the effect of alendronate, a widely used bisphosphonate, on osteogenic and adipogenic differentiation in bone marrow stromal cells (BMSCs) derived from ovariectomized SD rats. Alendronate treatment not only increased the mRNA level of bone morphogenetic protein-2, runt-related transcription factor 2, osteopontin, bone sialoprotein, and alkaline phosphatase activity after osteogenic induction, but also decreased the mRNA level of peroxisome proliferator activated receptor gamma 2 and total droplet number indicated by Oil Red O staining after adipogenic induction. The effect of alendronate treatment was dose-dependent, and the difference of the osteogenic or the adipogenic potential between the treated group and the non-treated group was statistically significant (p<0.001). The MAPK-specific inhibitors, PD98059 and SP600125, but not the p38-specific inhibitor, blocked the alendronate-induced regulation of BMSC differentiation. Analysis of BMSCs induced in the presence of alendronate revealed an immediate increase in ERK and JNK phosphorylation. Taken together, these data suggest that alendronate acts on BMSCs to stimulate osteogenic differentiation and inhibit adipogenic differentiation in a dose-dependent manner; this effect is mediated via activating ERK and JNK.

Promotion of osteogenesis through beta-catenin signaling by desferrioxamine.

Desferrioxamine, an iron chelator with "hypoxia-mimetic" activity, promotes bone mineralization when used in aluminum-overloaded dialysis patients. However, the effect of desferrioxamine on osteoblastic differentiation from pluripotent mesenchymal stem cells (MSCs) has not been reported. In this study, pluripotent human MSCs and murine mesenchymal C3H10T1/2 cells were simultaneously treated with desferrioxamine and bone morphogenetic protein-2 (BMP2). In BMP2-treated MSCs, desferrioxamine levels of 15 microMu were found to increase alkaline phosphatase (ALP) activity and calcium deposition, which were the markers of osteoblastic differentiation. These effects of desferrioxamine were accompanied by promoted phosphorylation of glycogen synthase kinase 3beta (GSK-3beta) and increased beta-catenin protein content, a direct GSK-3beta substrate. Knockdown of beta-catenin by RNA interference eliminates this positive effect of desferrioxamine on ALP activity. Taken together, these data demonstrate that desferrioxamine plays a direct role in the differentiation of mesenchymal stem cells by activating beta-catenin signaling cascades.

Evaluation of the zein/inorganics composite on biocompatibility and osteoblastic differentiation.

We have previously studied the biocompatibility and mechanical properties of porous zein scaffolds. We based the study on the concept that composite scaffold materials, especially when combined with inorganic materials, are more suited to the mechanical and physiological demands of the host tissue than individual scaffold materials. We investigated the effect of zein/inorganic composite on the physical and biological properties of porous zein scaffolds, which were fabricated by salt-leaching. The composite was prepared by immersion in simulated body fluid. The hydroxyapatite (HA)-coated zein scaffold had the same porosity as the zein scaffold (over 75%). Using scanning electron microscopy, it was established that the morphology of pores located on the surface and within the porous scaffolds showed equally good pore interconnectivity with zein. However, the compressive Young's modulus decreased from 240.1+/-96.8 to 34.4+/-12.6MPa, and compressive strength decreased from 7.8+/-1.2 to 4.2+/-0.8MPa. From the in vitro test with human bone marrow stroma cells, the osteoblastic differentiation on the surface of the HA-coated zein scaffold was increased, as expressed by the alkaline phosphatase activity and reverse transcription-polymerase chain reaction analysis for marker genes. From both the mechanical and biological evaluations, the HA-coated zein scaffold was found to be the optimal biomaterial for bone tissue engineering.

Immunomodulatory and osteogenic differentiation effects of mesenchymal stem cells by adenovirus-mediated coexpression of CTLA4Ig and BMP2.

Many reports have previously utilized a human bone morphogenetic protein 2 (BMP2)-expressing recombinant adenoviral vector (AdBMP2) and mesenchymal stem cells (MSCs) for osteoinductive gene therapy. However, immunosuppression is essential for osteoinduction by AdBMP2, and this is one of the major impediments to its clinical use. In the present study, in vitro propagated MSCs were transduced using an adenoviral (Ad) vector to express the gene encoding cytotoxic T lymphocyte antigen 4-immunoglobulin (CTLA4Ig). Lymphocyte response was induced by allogeneic-irradiated MSCs as stimulators. We also examined the effects of cotransfection with a combination of the CTLA4Ig and the BMP2 gene on osteoblastic cell differentiation. The results showed that BMP2 gene transfected MSC elicited significant stimulatory responses, and one-way MLR reactions were significantly blunted by CTLA4Ig. Further study demonstrates that cotransfection of MSCs with the combination of the CTLA4Ig and the BMP2 gene stimulates osteoblastic cell differentiation in vitro. The findings suggest that genetic engineering of MSCs to express an immunosuppressive molecule in combination with an osteogenic protein gene may have potential application in the treatment of several genetic diseases and in bone reconstruction.