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Expression of TSG-6 during inflammation suppresses pro-inflammatory pathways, i. TSG-6 is the first soluble chemokine-binding protein to be identified in mammals. Anti-resorptive effects: Altered bone turnover is a feature of OA, with pathological changes to the subchondral bone occurring early in the disease process.
Applications The technology is being developed as a biological therapeutic. There is also scope to treat other musculoskeletal indications e. All intellectual property is wholly owned by The University of Manchester. Opportunity The technology will be of interest to biotechnology and pharmaceutical companies. Innovate Quick Links. Connect With Us. This website uses cookies to provide you with the best browsing experience. Powered by GDPR plugin.
Privacy Overview This website uses cookies so that we can provide you with the best user experience possible. Strictly Necessary Cookies Strictly Necessary Cookie should be enabled at all times so that we can save your preferences for cookie settings. Key - umip. Keeping this cookie enabled helps us to improve our website. The column was first washed with 25 column volumes of an endotoxin removal buffer mM NaCl, 0. The recombinant protein was then recovered with about ml of elution buffer mM NaCl, 2 M Urea, and mM imidazole in 20 mM phosphate buffer at pH 7.
Thirty to 35 fractions were pooled around ml. The peak fractions were pooled, and dialyzed against PBS.
To visualize the stained bands, the membrane was washed with ultrapure water. Blood was collected by cardiac puncture at 0, 5 min, 30 min, 1 h, 3 h, 6 h, 12 h, and 24 h after rhTSG-6 injection. Continuous variables were tested for normal distribution by using the Kolmogorov-Smirnov KS test.
For comparison of two groups of non-normally distributed data was performed using Mann-Whitney U test. Comparisons of parameters among the three or more groups were made by one-way ANOVA followed by the Tukey or Newman-Keuls multiple comparison post hoc test for normally distributed data. The plasmid also incorporated Myc-tag and His-tag of sequences at the C-terminus to facilitate detection and purification of the proteins.
An expression plasmid was then used to prepare stable transfectants of CHO cells using a lipofectamine protocol. To identify clones that secreted the recombinant protein, medium from the clones was assayed for expression by Western blotting and the results confirmed by immunocytochemistry of cultures of the clones S1 Fig.
The transfected cells were similar to controls in morphology and the rhTSG-6 was present in the cytoplasm in a punctate distribution consistent with its being processed for secretion Fig 1A. A iii Expression by Western blotting. Monomeric rhTSG-6 was detected after day 3 but not after day 4, apparently because of aggregation.
Abbreviations: IN, input sample; FT, flow through. Surprisingly, however, the yield of the transfected cells sharply decreased between day 3 and day 4.
Also, there was a sharp decrease in the amount of rhTSG-6 recovered from the medium Fig 1C with monomeric protein detected in the medium after 3 days but essentially none after 4 days, apparently because of aggregation Fig 1D. Similar results were obtained in 5 similar experiments not shown. Microscopy of the cultures demonstrated that between days 3 and 4, the CHO formed large clusters of cells Fig 1E. As expected, immunocytochemistry demonstrated that the aggregates contained both rhTSG-6 and hyaluronan binding protein Fig 1F.
The decrease in yield of both cells and rhTSG-6 after day 3 was therefore explained by the well-documented tendency of the protein to bind hyaluronan [ 8 , 43 ] and the fact that the CHO cells, like many cells in culture, are surrounded by a brush border of hyaluronan [ 44 ]. Therefore, the tendency of the rhTSG-6 to aggregate CHO cells in the spinner cultures was enhanced by the decrease in pH Fig 1C that probably reflected decreased gaseous exchange and an accumulation of CO 2 in the medium [ 48 ].
The second measure was to add heparin Table 1 to the medium to compete with the binding of rhTSG-6 to hyaluronan [ 10 , 49 ]. It also increased slightly the amount of protein that was recovered in a monomeric form and correspondingly decreased the aggregated form Fig 2C. But addition of heparin to spinner culture did not prevent the decrease in pH Fig 2D , an observation suggesting that better control of pH would be helpful.
B Effect on yield of rhTSG E Effects on pH of medium.
Tumor necrosis factor-inducible gene 6 protein also known as TNF-stimulated gene 6 protein or TSG-6 is a protein that in humans is encoded by the TNFAIP6. TSG-6 expression is upregulated in many cell types in response to a variety of proinflammatory mediators and growth factors. This protein is detected in several .
We also instituted several additional conditions to improve the yield. We further supplemented the medium with a high level of glucose Table 1 to improve cell yields S2A Fig.
A therapeutically effective amount of polypeptide or polynucleotide is administered. Interestingly, the expression levels of the M2 macrophage markers were shown to be further increased in the LPS-stimulated Raw As expected, no significant changes of either M1 or M2 ratio were noticed between the SAH and vehicle-treated groups. Wang, M. Conclusions TSG-6 exerted neuroprotection against SAH-induced EBI in rats, mediated in part by skewing the balance of microglial response towards a protective phenotype, thereby preventing excessive tissue damage and improving functional outcomes.
Separate additions of a lipid concentrate or a surfactant polymer Pluronic F68 also improved cell yield but, surprisingly, a combination of the two inhibited the system S2C Fig. Therefore, these supplements were omitted. Supplementation with non-essential amino acids Table 1 protected against the decrease in cell yield between days 3 and 4 compare S2A and S2B Fig. The results of the experiments provided a medium that was defined here as optimized chemically-defined and protein-free medium OCDPF Table 1. In addition to the above measures, we elected to replace the spinner culture system with a bioreactor that allowed control of pH and oxygen.
However, the bioreactor did not provide complete control of the incubation system, since the oxygen concentration fell drastically on day 7 Fig 3A , apparently because of the frequently encountered problem of insufficient gaseous exchange as the cell concentration increased [ 48 ]. The bioreactor did control pH Fig 3B.
Most importantly, the yield of TSG-6 in medium increased with time in culture for 6 or 7 days Fig 3B. A Expansion of cells and oxygen saturation. Western blot of medium with antibodies to hTSG Values in A and B are means of 3 replicates. As a first step in purification, the culture medium was chromatographed on a Ni-chelate column Fig 4A to take advantage of the His-tag engineered into the C-terminus of the protein Fig 1A. After binding of the protein, the column was washed with Triton X, a step shown to remove endotoxin [ 51 ]. The rhTSG-6 was then eluted and the protein chromatographed on an anion exchange column.
The isolated protein was homogeneous as assayed by stained electrophoretic gels and primarily in a monomeric form Fig 4B. The initial levels of endotoxin of about 0. A concentration of 0. A Schematic for the purification steps. D Deglycosylation of the purified rhTSG Arrows indicate deglycosylated forms rhTSG In addition, digestion with N-glycosidase indicated that it was glycosylated Fig 4D. As expected, the apparent molecular weight and the specific staining of the protein decreased with deglycosylation.
The purification of the rhTSG-6 is summarized in Table 2. A 4- to 5-fold purification was obtained with chromatography on the Ni-chelate column with a recovery of The polishing step on the anion exchange column provided a higher yield and the over-all yield from the culture medium was One measure of the biological activity of a secreted glycoprotein like TSG-6 is its metabolic half-life in vivo, since misfolded and unglycosylated proteins tend to be cleared rapidly [ 53 ].
The tendency was confirmed in a further experiment in which a larger number of mice were compared 25 h after infusion of the two proteins Fig 5B. The differences are probably explained by the fact that although both proteins were monomeric as assayed by SDS-PAGE, the rhTSG-6 from the CHO cells is a secreted glycosylated form of the protein whereas the commercial rhTSG-6 was synthesized in mouse myeloma cells and was purified from cell lysates. Values are mean and SEM from 3 mice. TSG-6 was shown to suppress inflammation in a series of different animal models [see 25 ].
Most of the models require some technical expertise. Therefore, we elected to test the rhTSG-6 in a simple model in which inflammation is produced in mice by intravenous injection of the bacterial extract LPS and RT-PCR assays are used to detect the increase in expression of pro-inflammatory cytokines in spleen. As indicated in Fig 6 , there was a large variance in the assay. Also, the maximum amount of rhTSG-6 testable was limited by the solubility limit of the protein about 0.
The similar results were obtained with a control of a commercially available rhTSG-6 in the same assay S6 Fig. A Schematic for the experiment. Symbol: RQ, relative quantity. Synthesis of rhTSG-6 has been an unmet challenge since the protein was first discovered over 20 years ago [ 4 ]. Limited amounts were produced in several systems, but scalable production was not achieved.
The results presented here demonstrate that cellular synthesis of rhTSG-6 is limited by well-known characteristics of the protein. It belongs to a family of hyaluronan binding proteins, termed hyadherins, that extensively interact with proteins in the extracellular matrix [ 46 ]. As demonstrated here, these properties limit production of the recombinant protein by CHO cells because the rhTSG-6 bound to and probably cross-linked the hyaluronan that forms a brush found on most cultured cells [ 44 ].
As a result, the CHO cells formed large aggregates which limited both the propagation of the cells and the amount of rhTSG-6 recovered from the medium. In addition, the tendency of the protein to self-aggregate presented a serious technical challenge. The aggregates may have retained biological activity but they were too difficult to isolate and assay.
Instead, they required more stringent conditions for dissociation see S5 Fig , an observation occasionally made with other protein aggregates see ref. The protocol developed here largely surmounts these problems.