Purification of immunoglobulins from Serum Using Thiophilic Cellulose Beads

This study evaluates the chromatographic performance of a support obtained by the reaction of mercaptoethanol with cellulose beads activated with divinyl sulfone. Cellulose beads 500-800 microns (pm) in diameter and with a solids content of 3.5% were selected for this study. A two-step sequence of permeation and reaction was used to install thiophilic sites throughout the cross section of the bead. The distribution of thiophilic sites was visualized by immobilizing fluorescent antibodies. Human and porcine serum proteins were separated on the thiophilic support at different linear velocities. Thiophilic cellulose beads were observed to bind human and porcine immunoglobulins (IgG) selectively from serum. Overall total protein recoveries in the range of 85%-99% were obtained with human serum, porcine serum and cell supernatant. Human TgG yields of 75% and 50% were obtained at linear velocities of 1 cmlmin and 3 cmlmin, respectively. Thiophilic cellulose beads were observed to bind monoclonal antibodies from cell culture supematant but yields in the range of 40-508 were obtained. Purity of the products, obtained from a single chro~natographics tep, as judged by electrophoretic analysis was estimated to be greater than 80%

Chromatographic Purification of MAbs with Non-Affinity Supports

The introduction of new protein-based therapeutics such as monoclonal antibodies (MAbs), MAb-based vaccines, growth factors and plasma proteins implies the need to study, characterize, and purify. The separation step is likely to be a bottleneck and cost-effective technology will be needed to rectify it. The currently prevalent matrices for chromatographic separation of immunoglobulins (Igs) are based on Protein A or its recombinant versions (Protein G). They display excellent selectivity and specificity, but are expensive. A Protein A matrix costs $8,000 to 12,000 per L-resin. The typical production column volume is 100 L. The million-dollar matrix is far more expensive than the production hardware

Immunoaffinity Chomatography

Recent devclopments in recombinant DNA technology have enabled the synthesis of valuable therapeutic proteins in bacterial cells as well as in novel eucaryotic expression systems. However, the purification of proteins of interest from either the conventional sources, cell culture, or novel routes in a highly purified form necessitates the development of separation tcchniques capable of recovering proteins from these feed streams in a highly purified form (1,2). Purification of therapeutic proteins from biological sources is usually complicated by the presence of endogenous proteins (2). Purification methodologies based on ion exchange or adsorption serve as excellent prepurification steps, but they fail to resolve complex protein mixtures to yield a homogeneous protein tein product (l).P urification techniques based on affinity interactions between molecules (i.e., immunoaffinity chromatography, IAC) have rapidly evolved using a variety of biological and synthetic ligands (2)

Purification of immunoglobulins from Serum Using Thiophilic Cellulose Beads

This study evaluates the chromatographic performance of a support obtained by the reaction of mercaptoethanol with cellulose beads activated with divinyl sulfone. Cellulose beads 500-800 microns (pm) in diameter and with a solids content of 3.5% were selected for this study. A two-step sequence of permeation and reaction was used to install thiophilic sites throughout the cross section of the bead. The distribution of thiophilic sites was visualized by immobilizing fluorescent antibodies. Human and porcine serum proteins were separated on the thiophilic support at different linear velocities. Thiophilic cellulose beads were observed to bind human and porcine immunoglobulins (IgG) selectively from serum. Overall total protein recoveries in the range of 85%-99% were obtained with human serum, porcine serum and cell supernatant. Human TgG yields of 75% and 50% were obtained at linear velocities of 1 cmlmin and 3 cmlmin, respectively. Thiophilic cellulose beads were observed to bind monoclonal antibodies from cell culture supematant but yields in the range of 40-508 were obtained. Purity of the products, obtained from a single chro~natographics tep, as judged by electrophoretic analysis was estimated to be greater than 80%

Remodeling of chromatin under low intensity diffuse ultrasound

A variety of mechanotransduction pathways mediate the response of fibroblasts or chondrocytes to ultrasound stimulation. In addition, regulatory pathways that co-ordinate stimulus-specific cellular responses are likely to exist. In this study, analysis was confined to the hypothesis that ultrasound stimulation (US) influences the chromatin structure, and that these changes may reflect a regulatory pathway that connects nuclear architecture, chromatin structure and gene expression. Murine fibroblasts seeded on tissue culture plates were stimulated with US (5.0 MHz (14 kPa), 51- s per application) and the thermal denaturation profiles of nuclei isolated from fibroblasts were assessed by dynamic scanning calorimetry (DSC). When compared to the thermal profiles obtained from the nuclei of non-stimulated cells, the nuclei obtained from stimulated cells showed a change in peak profiles and peak areas, which is indicative of chromatin remodeling. Independently, US was also observed to impact the histone (H1):chromatin association as measured indirectly by DAPI staining. Based on our work, it appears plausible that US can produce a remodeling of chromatin, thus triggering signal cascade and other intracellular mechanisms

Preconditioning of mesenchymal stromal cells with low-intensity ultrasound: influence on chondrogenesis and directed SOX9 signaling pathways

Background: Continuous low-intensity ultrasound (cLIUS) facilitates the chondrogenic differentiation of human mesenchymal stromal cells (MSCs) in the absence of exogenously added transforming growth factor-beta (TGFβ) by upregulating the expression of transcription factor SOX9, a master regulator of chondrogenesis. The present study evaluated the molecular events associated with the signaling pathways impacting SOX9 gene and protein expression under cLIUS. Methods: Human bone marrow-derived MSCs were exposed to cLIUS stimulation at 14 kPa (5 MHz, 2.5 Vpp) for 5 min. The gene and protein expression of SOX9 was evaluated. The specificity of SOX9 upregulation under cLIUS was determined by treating the MSCs with small molecule inhibitors of select signaling molecules, followed by cLIUS treatment. Signaling events regulating SOX9 expression under cLIUS were analyzed by gene expression, immunofluorescence staining, and western blotting. Results: cLIUS upregulated the gene expression of SOX9 and enhanced the nuclear localization of SOX9 protein when compared to non-cLIUS-stimulated control. cLIUS was noted to enhance the phosphorylation of the signaling molecule ERK1/2. Inhibition of MEK/ERK1/2 by PD98059 resulted in the effective abrogation of cLIUS-induced SOX9 expression, indicating that cLIUS-induced SOX9 upregulation was dependent on the phosphorylation of ERK1/2. Inhibition of integrin and TRPV4, the upstream cell-surface effectors of ERK1/2, did not inhibit the phosphorylation of ERK1/2 and therefore did not abrogate cLIUS-induced SOX9 expression, thereby suggesting the involvement of other mechanoreceptors. Consequently, the effect of cLIUS on the actin cytoskeleton, a mechanosensitive receptor regulating SOX9, was evaluated. Diffused and disrupted actin fibers observed in MSCs under cLIUS closely resembled actin disruption by treatment with cytoskeletal drug Y27632, which is known to increase the gene expression of SOX9. The upregulation of SOX9 under cLIUS was, therefore, related to cLIUS-induced actin reorganization. SOX9 upregulation induced by actin reorganization was also found to be dependent on the phosphorylation of ERK1/2. Conclusions: Collectively, preconditioning of MSCs by cLIUS resulted in the nuclear localization of SOX9, phosphorylation of ERK1/2 and disruption of actin filaments, and the expression of SOX9 was dependent on the phosphorylation of ERK1/2 under cLIUS

The Effect of Ultrasound Stimulation on the Cytoskeletal Organization of Chondrocytes Seeded In 3D Matrices

The impact of low intensity diffuse ultrasound (LIDUS) stimulation on the cytoskeletal organization of chondrocytes seeded in 3D scaffolds was evaluated. Chondrocytes seeded on 3D chitosan matrices were exposed to LIDUS at 5.0 MHz (~15kPa, 51-secs, 4-applications/day) in order to study the organization of actin, tubulin and vimentin. The results showed that actin presented a cytosolic punctuated distribution, tubulin presented a quasi parallel organization of microtubules whereas vimentin distribution was unaffected. Chondrocytes seeded on 3D scaffolds responded to US stimulation by the disruption of actin stress fibers and were sensitive to the presence of ROCK inhibitor (Y27632). The gene expression of ROCK-I, a key element in the formation of stress fibers and mDia1, was significantly up-regulated under the application of US. We conclude that the results of both the cytoskeletal analyses and gene expression support the argument that the presence of punctuated actin upon US stimulation was accompanied by the up-regulation of the RhoA/ROCK pathway

Remodeling of chromatin under low intensity diffuse ultrasound

A variety of mechanotransduction pathways mediate the response of fibroblasts or chondrocytes to ultrasound stimulation. In addition, regulatory pathways that co-ordinate stimulus-specific cellular responses are likely to exist. In this study, analysis was confined to the hypothesis that ultrasound stimulation (US) influences the chromatin structure, and that these changes may reflect a regulatory pathway that connects nuclear architecture, chromatin structure and gene expression. Murine fibroblasts seeded on tissue culture plates were stimulated with US (5.0 MHz (14 kPa), 51- s per application) and the thermal denaturation profiles of nuclei isolated from fibroblasts were assessed by dynamic scanning calorimetry (DSC). When compared to the thermal profiles obtained from the nuclei of non-stimulated cells, the nuclei obtained from stimulated cells showed a change in peak profiles and peak areas, which is indicative of chromatin remodeling. Independently, US was also observed to impact the histone (H1):chromatin association as measured indirectly by DAPI staining. Based on our work, it appears plausible that US can produce a remodeling of chromatin, thus triggering signal cascade and other intracellular mechanisms

Development of biomaterial scaffold for nerve tissue engineering: Biomaterial mediated neural regeneration

Neural tissue repair and regeneration strategies have received a great deal of attention because it directly affects the quality of the patient's life. There are many scientific challenges to regenerate nerve while using conventional autologous nerve grafts and from the newly developed therapeutic strategies for the reconstruction of damaged nerves. Recent advancements in nerve regeneration have involved the application of tissue engineering principles and this has evolved a new perspective to neural therapy. The success of neural tissue engineering is mainly based on the regulation of cell behavior and tissue progression through the development of a synthetic scaffold that is analogous to the natural extracellular matrix and can support three-dimensional cell cultures. As the natural extracellular matrix provides an ideal environment for topographical, electrical and chemical cues to the adhesion and proliferation of neural cells, there exists a need to develop a synthetic scaffold that would be biocompatible, immunologically inert, conducting, biodegradable, and infection-resistant biomaterial to support neurite outgrowth. This review outlines the rationale for effective neural tissue engineering through the use of suitable biomaterials and scaffolding techniques for fabrication of a construct that would allow the neurons to adhere, proliferate and eventually form nerves

Continuous low-intensity ultrasound attenuates IL-6 and TNFα-induced catabolic effects and repairs chondral fissures in bovine osteochondral explants

Background: Cartilage repair outcomes are compromised in a pro-inflammatory environment; therefore, the mitigation of pro-inflammatory responses is beneficial. Treatment with continuous low-intensity ultrasound (cLIUS) at the resonant frequency of 5 MHz is proposed for the repair of chondral fissures under pro-inflammatory conditions. Methods: Bovine osteochondral explants, concentrically incised to create chondral fissures, were maintained under cLIUS (14 kPa (5 MHz, 2.5 Vpp), 20 min, 4 times/day) for a period of 28 days in the presence or absence of cytokines, interleukin-6 (IL-6) or tumor necrosis factor (TNF)α. Outcome assessments included histological and immunohistochemical staining of the explants; and the expression of catabolic and anabolic genes by qRT-PCR in bovine chondrocytes. Cell migration was assessed by scratch assays, and by visualizing migrating cells into the hydrogel core of cartilage-hydrogel constructs. Results: Both in the presence and absence of cytokines, higher percent apposition along with closure of fissures were noted in cLIUS-stimulated explants as compared to non-cLIUS-stimulated explants on day 14. On day 28, the percent apposition was not significantly different between unstimulated and cLIUS-stimulated explants exposed to cytokines. As compared to non-cLIUS-stimulated controls, on day 28, cLIUS preserved the distribution of proteoglycans and collagen II in explants despite exposure to cytokines. cLIUS enhanced the cell migration irrespective of cytokine treatment. IL-6 or TNFα-induced increases in MMP13 and ADAMTS4 gene expression was rescued by cLIUS stimulation in chondrocytes. Under cLIUS, TNFα-induced increase in NF-κB expression was suppressed, and the expression of collagen II and TIMP1 genes were upregulated. Conclusion: cLIUS repaired chondral fissures, and elicited pro-anabolic and anti-catabolic effects, thus demonstrating the potential of cLIUS in improving cartilage repair outcomes