Mesenchymal stem cell-mediated immature dendritic cells induce regulatory T cell-based...
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Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576 S427 of immunochromatography test kit for detection of dexamethasone and prednisolone in herbal medicinal products would be fruitful for surveillance in public health consumer protection. The knowledge of this rapid test would possibly be further useful for the develop- ment of various medicinal product testings. doi:10.1016/j.jbiotec.2010.09.593 [P-M.6] Mesenchymal stem cell-mediated immature dendritic cells induce regulatory T cell-based immunosuppressive effect Y.S. Choi ∗ , J.A. Jeong, D.S. Lim CHA University, Republic of Korea Keywords: Dedritic cells; Mesenchymal stem cells; Regulatory T cells; Immunosuppression Mesenchymal stem cells (MSCs) are multipotent cells, possess- ing a direct immunosuppressive effect on T-cell proliferation in vitro. However, application using MSCs in clinical trials has still partially risk, because they have latent ability to be tumors. In this study, we investigated the factors enabling induction of reg- ulatory T (Treg) cells through syngeneic immature dendritic cells (imDCs) using MSCs in vitro and immunosuppressive effect of MSC- mediated imDCs in vivo. We found that CD25 + Foxp3 + Treg cell population remarkably increased after the T-cell priming phase when splenocytes were co-cultured with both imDCs and MSCs, presumably inducing naïve T cells into Treg cells by MSCs and imDCs. In parallel with these, the TGF- secretion was markedly induced from the imDCs+MSCs+splenocytes culture supernatant to a significant level at 72-hr co-culture, compared with MSCs or imDCs+splenocytes co-culture. Moreover, the IFN- secretion, which elevated by imDCs+CD4 T cells co-culture, was dramatically inhibited when CD4 T cells were co-cultured with both imDCs and MSCs, lowering a T helper 1 (Th1) response. Based on these results, using a murine melanoma tumor model, we confirmed that the sub- cutaneous injection of B16 cells induced a perfect tumor incidence in allogeneic recipients when MSC-mediated DCs were coinjected. Additional parameters in vivo were in line with this datum. Con- sequently, these results suggested that immune tolerance with MSCs-mediated DCs could be the immunosuppression induced at least by Foxp3-specific Treg cells. This tool may be further useful in clinical trials in the light of unknown side effects of stem cell therapy. doi:10.1016/j.jbiotec.2010.09.594 [P-M.7] The Role of Fidelity in Engineering Expanded Genetic Codes E.M. Tippmann Cardiff University, United Kingdom Keywords: Expanded Genetic Code; Recoded Amino acids; Protein Glycosylation The role of fidelity in an expanded genetic code is an especially poignant topic given that the production of proteins using recombi- nant technology is limited to a select number of model organisms. E. coli, S. Cerviseae and P. pastoris predominate as the host organ- isms of choice for the expression of genes of interest. The choice between these hosts is guided by balancing factors to optimize proper protein folding, solubility or post-translational processing. Recent reports (Science 2004,303:371;JACS 2004 126:15654) that E. coli is capable of introducing the glycosylated modification trans- lationally represented a significant advance for the production of this important modification for two reasons: 1) human therapeu- tics require specific glycosylated side chains for optimal function and the technology allowed these modifications to be incorporated directly and with site-specificity, and 2) protein glycosylation in E. coli is rare. Recent work (J Biol Chem 284(42):28795-28800.) from our laboratory was followed by the retraction (Science 2009,326:1187;JACS 131:13883) of these reports. Here, an in-depth analysis of the bioorthogonal translation mechanism is undertaken and a paradigm has emerged for successful implementation of the method. doi:10.1016/j.jbiotec.2010.09.595 [P-M.8] Amoxicillin Assay Based on Penicillin Acylase and D- Phenylglycine Aminotransferase T. Rojanarata 1,∗ , P. Opanasopit 1 , T. Ngawhirunpat 1 , C. Saehuan 2 , S. Wiyakrutta 3 , V. Meevootisom 3 1 Faculty of Pharmacy, Silpakorn University, Thailand 2 Faculty of Allied Health Sciences, Naresuan University, Thailand 3 Faculty of Science, Mahidol University, Thailand Keywords: Enzyme; Assay; Amoxicillin This study reports the use of two microbial enzymes for the development of a novel assay of amoxicillin in pharmaceutical preparations. The principle was based the selective cleavage of d-4-hydroxyphenylglycine side chain of amoxicillin by the activ- ity of penicillin acylase. Subsequently, d-4-hydroxyphenylglycine reacted with 2-oxoglutarate, by using d-phenylglycine amino- transferase, to yield the product with high UV absorption namely 4-hydroxybenzoylformate. The amount of amoxicillin was then spectrophotometrically determined as a change in absorbance at 335 nm. In this work, the assay conditions were studied and optimized and the method was validated. The calibration curve presented an excellent linearity with r 2 of 0.9998 (0–100 M amox- icillin). Detection and quantitation limits were 0.77 and 2.55 M, respectively. Good accuracy and precision were obtained when the method was tested with amoxicillin capsules and powder for oral suspension. No interference from common excipients in the for- mulations or degradation products was observed. In addition to simple, fast and inexpensive features of the method, the assay was found to be green since all procedures were performed without the use of any organic solvents or hazardous chemicals which were detrimental to the environment and had a low consumption of reagents. doi:10.1016/j.jbiotec.2010.09.596
Transcript of Mesenchymal stem cell-mediated immature dendritic cells induce regulatory T cell-based...
Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576 S427
of immunochromatography test kit for detection of dexamethasoneand prednisolone in herbal medicinal products would be fruitful forsurveillance in public health consumer protection. The knowledgeof this rapid test would possibly be further useful for the develop-ment of various medicinal product testings.
doi:10.1016/j.jbiotec.2010.09.593
[P-M.6]
Mesenchymal stem cell-mediated immature dendritic cellsinduce regulatory T cell-based immunosuppressive effect
Y.S. Choi ∗, J.A. Jeong, D.S. Lim
CHA University, Republic of KoreaKeywords: Dedritic cells; Mesenchymal stem cells; Regulatory Tcells; Immunosuppression
Mesenchymal stem cells (MSCs) are multipotent cells, possess-ing a direct immunosuppressive effect on T-cell proliferation invitro. However, application using MSCs in clinical trials has stillpartially risk, because they have latent ability to be tumors. Inthis study, we investigated the factors enabling induction of reg-ulatory T (Treg) cells through syngeneic immature dendritic cells(imDCs) using MSCs in vitro and immunosuppressive effect of MSC-mediated imDCs in vivo. We found that CD25+Foxp3+ Treg cellpopulation remarkably increased after the T-cell priming phasewhen splenocytes were co-cultured with both imDCs and MSCs,presumably inducing naïve T cells into Treg cells by MSCs andimDCs. In parallel with these, the TGF-� secretion was markedlyinduced from the imDCs+MSCs+splenocytes culture supernatantto a significant level at 72-hr co-culture, compared with MSCsor imDCs+splenocytes co-culture. Moreover, the IFN-� secretion,which elevated by imDCs+CD4 T cells co-culture, was dramaticallyinhibited when CD4 T cells were co-cultured with both imDCs andMSCs, lowering a T helper 1 (Th1) response. Based on these results,using a murine melanoma tumor model, we confirmed that the sub-cutaneous injection of B16 cells induced a perfect tumor incidencein allogeneic recipients when MSC-mediated DCs were coinjected.Additional parameters in vivo were in line with this datum. Con-sequently, these results suggested that immune tolerance withMSCs-mediated DCs could be the immunosuppression induced atleast by Foxp3-specific Treg cells. This tool may be further usefulin clinical trials in the light of unknown side effects of stem celltherapy.
doi:10.1016/j.jbiotec.2010.09.594
[P-M.7]
The Role of Fidelity in Engineering Expanded Genetic Codes
E.M. Tippmann
Cardiff University, United KingdomKeywords: Expanded Genetic Code; Recoded Amino acids; ProteinGlycosylation
The role of fidelity in an expanded genetic code is an especiallypoignant topic given that the production of proteins using recombi-nant technology is limited to a select number of model organisms.E. coli, S. Cerviseae and P. pastoris predominate as the host organ-isms of choice for the expression of genes of interest. The choicebetween these hosts is guided by balancing factors to optimizeproper protein folding, solubility or post-translational processing.Recent reports (Science 2004,303:371;JACS 2004 126:15654) that
E. coli is capable of introducing the glycosylated modification trans-lationally represented a significant advance for the production ofthis important modification for two reasons: 1) human therapeu-tics require specific glycosylated side chains for optimal functionand the technology allowed these modifications to be incorporateddirectly and with site-specificity, and 2) protein glycosylation inE. coli is rare. Recent work (J Biol Chem 284(42):28795-28800.)from our laboratory was followed by the retraction (Science2009,326:1187;JACS 131:13883) of these reports. Here, an in-depthanalysis of the bioorthogonal translation mechanism is undertakenand a paradigm has emerged for successful implementation of themethod.
doi:10.1016/j.jbiotec.2010.09.595
[P-M.8]
Amoxicillin Assay Based on Penicillin Acylase and D-Phenylglycine Aminotransferase
T. Rojanarata 1,∗, P. Opanasopit 1, T. Ngawhirunpat 1, C. Saehuan 2,S. Wiyakrutta 3, V. Meevootisom 3
1 Faculty of Pharmacy, Silpakorn University, Thailand2 Faculty of Allied Health Sciences, Naresuan University, Thailand3 Faculty of Science, Mahidol University, ThailandKeywords: Enzyme; Assay; Amoxicillin
This study reports the use of two microbial enzymes for thedevelopment of a novel assay of amoxicillin in pharmaceuticalpreparations. The principle was based the selective cleavage ofd-4-hydroxyphenylglycine side chain of amoxicillin by the activ-ity of penicillin acylase. Subsequently, d-4-hydroxyphenylglycinereacted with 2-oxoglutarate, by using d-phenylglycine amino-transferase, to yield the product with high UV absorption namely4-hydroxybenzoylformate. The amount of amoxicillin was thenspectrophotometrically determined as a change in absorbanceat 335 nm. In this work, the assay conditions were studied andoptimized and the method was validated. The calibration curvepresented an excellent linearity with r2 of 0.9998 (0–100 �M amox-icillin). Detection and quantitation limits were 0.77 and 2.55 �M,respectively. Good accuracy and precision were obtained when themethod was tested with amoxicillin capsules and powder for oralsuspension. No interference from common excipients in the for-mulations or degradation products was observed. In addition tosimple, fast and inexpensive features of the method, the assay wasfound to be green since all procedures were performed withoutthe use of any organic solvents or hazardous chemicals which weredetrimental to the environment and had a low consumption ofreagents.
doi:10.1016/j.jbiotec.2010.09.596
