RESUMEN
A major concern in the clinical application of cell therapy is the manufacturing cost of cell products, which mainly depends on quality control. The mycoplasma test, an important biological test in cell therapy, takes several weeks to detect a microorganism and is extremely expensive. Furthermore, the manual detection of mycoplasma from images requires high-level expertise. We hypothesized that a mycoplasma identification program using a convolutional neural network could reduce the test time and improve sensitivity. To this end, we developed a program comprising three parts (mycoplasma detection, prediction, and cell counting) that allows users to evaluate the sample and verify infected/non-infected cells identified by the program. In experiments conducted, stained DNA images of positive and negative control using mycoplasma-infected and non-infected Vero cells, respectively, were used as training data, and the program results were compared with those of conventional methods, such as manual counting based on visual observation. The minimum detectable mycoplasma contaminations for manual counting and the proposed program were 10 and 5 CFU (colony-forming unit), respectively, and the test time for manual counting was 20 times that for the proposed program. These results suggest that the proposed system can realize a low-cost and streamlined manufacturing process for cellular products in cell-based research and clinical applications.
Asunto(s)
Aprendizaje Profundo , Mycoplasma , Animales , Chlorocebus aethiops , Mycoplasma/genética , Células VeroRESUMEN
Sessions included an overview of past cell therapy (CT) conferences sponsored by the International Alliance for Biological Standardization (IABS). The sessions highlighted challenges in the field of human pluripotent stem cells (hPSCs) and also addressed specific points on manufacturing, bioanalytics and comparability, tumorigenicity testing, storage, and shipping. Panel discussions complemented the presentations. The conference concluded that a range of new standardization groups is emerging that could help the field, but ways must be found to ensure that these efforts are coordinated. In addition, there are opportunities for regulatory convergence starting with a gap analysis of existing guidelines to determine what might be missing and what issues might be creating divergence. More specific global regulatory guidance, preferably from WHO, would be welcome. IABS and the California Institute for Regenerative Medicine (CIRM) will explore with stakeholders the development of a practical and innovative road map to support early CT product (CTP) developers.
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Tratamiento Basado en Trasplante de Células y Tejidos , Células Madre Pluripotentes , Pruebas de Carcinogenicidad , Guías como Asunto , Humanos , Control de Calidad , Medicina RegenerativaRESUMEN
Human embryonic stem cells (hESCs) and their derivatives are expected to be used in drug discovery, regenerative medicine and the study of human embryogenesis. Because hepatocyte differentiation from hESCs has the potential to recapitulate human liver development in vivo, we employed this differentiation method to investigate the molecular mechanisms underlying human hepatocyte differentiation. A previous study has shown that a gradient of transforming growth factor beta (TGFß) signaling is required to segregate hepatocyte and cholangiocyte lineages from hepatoblasts. Although CCAAT/enhancer binding proteins (c/EBPs) are known to be important transcription factors in liver development, the relationship between TGFß signaling and c/EBP-mediated transcriptional regulation in the hepatoblast fate decision is not well known. To clarify this relationship, we examined whether c/EBPs could determine the hepatoblast fate decision via regulation of TGFß receptor 2 (TGFBR2) expression in the hepatoblast-like cells differentiated from hESCs. We found that TGFBR2 promoter activity was negatively regulated by c/EBPα and positively regulated by c/EBPß. Moreover, c/EBPα overexpression could promote hepatocyte differentiation by suppressing TGFBR2 expression, whereas c/EBPß overexpression could promote cholangiocyte differentiation by enhancing TGFBR2 expression. Our findings demonstrated that c/EBPα and c/EBPß determine the lineage commitment of hepatoblasts by negatively and positively regulating the expression of a common target gene, TGFBR2, respectively.
Asunto(s)
Proteína alfa Potenciadora de Unión a CCAAT/metabolismo , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Hepatocitos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Animales , Proteína alfa Potenciadora de Unión a CCAAT/biosíntesis , Proteína beta Potenciadora de Unión a CCAAT/biosíntesis , Diferenciación Celular , Linaje de la Célula , Células Cultivadas , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Regulación del Desarrollo de la Expresión Génica , Humanos , Hígado/embriología , Hígado/crecimiento & desarrollo , Hígado/metabolismo , Ratones , Regiones Promotoras Genéticas/genética , Proteínas Serina-Treonina Quinasas/biosíntesis , Proteínas Serina-Treonina Quinasas/genética , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/biosíntesis , Receptores de Factores de Crecimiento Transformadores beta/genética , Transducción de Señal , Transcripción Genética , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Cell therapy involves the administration of a viable somatic cell preparation to a patient for the treatment of a disease or traumatic damage. Because cell therapies are complex and very different from traditional biological products, they present significant challenges for regulatory authorities, manufacturers, developers, health care providers, and patients involved in their application. Like other emerging areas of biomedical research and development, there are many issues where regulatory views and decisions among countries and regions may differ due to minimal scientific evidence to support safety and efficacy, and lack of experience with these novel treatments. A brief overview of the current regulatory landscape for cell-based therapies is presented, and the need for a global effort to develop a set of common principles that may serve to facilitate the regulatory evaluation and market availability of these products is identified. In addition, a number of elements that could form a core consensus package of requirements for evaluating human cell therapy products is presented in the supplemental material which should be read in conjunction with the manuscript.
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Investigación Biomédica/métodos , Técnicas de Cultivo de Célula/métodos , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Ingeniería de Tejidos/métodos , Investigación Biomédica/normas , Investigación Biomédica/tendencias , Tratamiento Basado en Trasplante de Células y Tejidos/normas , Tratamiento Basado en Trasplante de Células y Tejidos/tendencias , Guías como Asunto , Humanos , Control de Calidad , Organización Mundial de la SaludRESUMEN
CC chemokine receptor 3 (CCR3) is expressed selectively in eosinophils, basophils, and some Th2 cells and plays a major role in allergic diseases. A methanol extract from the arils of Myristica fragrans inhibited CC chemokine ligand 11-induced chemotaxis in CCR3-expressing L1.2 cells at 100 µg/mL. From this extract, eight new neolignans, maceneolignans A-H (1-8), were isolated, and their stereostructures were elucidated from their spectroscopic values and chemical properties. Of those constituents, compounds 1, 4, 6, and 8 and (+)-erythro-(7S,8R)-Δ(8')-7-hydroxy-3,4-methylenedioxy-3',5'-dimethoxy-8-O-4'-neolignan (11), (-)-(8R)-Δ(8')-3,4-methylenedioxy-3',5'-dimethoxy-8-O-4'-neolignan (17), (+)-licarin A (20), nectandrin B (25), verrucosin (26), and myristicin (27) inhibited CCR3-mediated chemotaxis at a concentration of 1 µM. Among them, 1 (EC50 1.6 µM), 6 (1.5 µM), and 8 (1.4 µM) showed relatively strong activities, which were comparable to that of a synthetic CCR3 selective antagonist, SB328437 (0.78 µM).
Asunto(s)
Lignanos/aislamiento & purificación , Lignanos/farmacología , Myristica/química , Receptores CCR3/antagonistas & inhibidores , Quimiotaxis/efectos de los fármacos , Eosinófilos/metabolismo , Furanos/farmacología , Humanos , Lignanos/química , Estructura Molecular , EstereoisomerismoRESUMEN
Human malignant melanomas remain associated with dismal prognosis due to their resistance to apoptosis and chemotherapy. There is growing interest in plant oligostilbenoids owing to their pleiotropic biological activities, including anti-inflammatory, antioxidant, and anticancer effects. Recent studies have demonstrated that resveratrol, a well-known stilbenoid from red wine, exhibits cell cycle-disrupting and apoptosis-inducing activities on melanoma cells. The objective of our study was to evaluate the anti-melanoma effect of oligostilbenoids isolated from the bark of Shorea roxburghii. Among the isolates, four resveratrol oligomers, i.e., (-)-hopeaphenol, vaticanol B, hemsleyanol D, and (+)-α-viniferin, possessed more potent antiproliferative action than did resveratrol against SK-MEL-28 melanoma cells. Cell cycle analysis revealed that (-)-hopeaphenol, hemsleyanol D, and (+)-α-viniferin arrested cell division cycle at the G1 phase, whereas vaticanol B had little effect on the cell cycle. In addition, cell proliferation assay also revealed that (+)-α-viniferin induced DNA damage followed by induction of apoptosis in SK-MEL-28 cells, which was confirmed by an increased expression of γ-H2AX and cleaved caspase-3, respectively. The compounds vaticanol B, hemsleyanol D, and resveratrol significantly increased the expression of p21, suggesting that they are able to block cell cycle progression. Moreover, these oligostilbenoids downmodulated cylin D1 expression and extracellular signal-regulated kinase (ERK) activation. Furthermore, hemsleyanol D, (+)-α-viniferin, and resveratrol significantly decreased the expression of cyclin B1, which could also suppress cell cycle progression. The present study thus suggests that these plant oligostilbenoids are effective as therapeutic or chemopreventive agents against melanoma.
Asunto(s)
Antineoplásicos/farmacología , Dipterocarpaceae , Estilbenos/farmacología , Administración Tópica , Apoptosis/efectos de los fármacos , Caspasa 3/metabolismo , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Humanos , Corteza de la PlantaRESUMEN
The development of human cell therapy and gene therapy products has progressed internationally. Efforts have been made to address regulatory challenges in the evaluation of quality, efficacy, and safety of the products. In this forum, updates on the specific challenges in quality, efficacy, and safety of products in the view of international development were shared through the exchange of information and opinions among experts from regulatory authorities, academic institutions, and industry practitioners. Sessions identified specific/critical points to consider for the evaluation of human cell therapy and gene therapy products that are different from conventional biological products; common approaches and practices among regulatory regions were also shared. Certain elements of current international guidelines might not be appropriate to be applied to these products. Further, international discussion on the concept of potency and in vivo tumorigenicity studies, among others, is needed. This forum concluded that the continued collective actions are expected to promote international convergence of regulatory approaches of the products. The Pharmaceuticals and Medical Devices Agency and Japanese Society for Regenerative Medicine jointly convened the forum with support from the National Institutes of Biomedical Innovation, Health and Nutrition. Participants at the forum include 300 experts in and outside of Japan.
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Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Terapia Genética/métodos , Tratamiento Basado en Trasplante de Células y Tejidos/instrumentación , Congresos como Asunto , Terapia Genética/instrumentación , HumanosRESUMEN
A methanol extract of the flowers of Mammea siamensis (Calophyllaceae) was found to inhibit enzymatic activity against aromatase (IC50=16.5 µg/mL). From the extract, two new geranylated coumarins, mammeasins C (1) and D (2), were isolated together with seven coumarins: 8-hydroxy-5-methyl-7-(3,7-dimethyl-octa-2,6-dienyl)-9-(2-methyl-1-oxobutyl)-4,5-dihydropyrano[4,3,2-de]chromen-2-one (9), 8-hydroxy-5-methyl-7-(3,7-dimethyl-octa-2,6-dienyl)-9-(3-methyl-1-oxobutyl)-4,5-dihydropyrano[4,3,2-de]chromen-2-one (10), mammeas A/AA (14), A/AB (15), A/AA cyclo D (18), E/BA (23), and E/BC cyclo D (25). The structures of 1 and 2 were elucidated on the basis of spectroscopic evidence. Among the isolates including 17 previously reported coumarins, 1 (IC50=2.7 µM), 2 (3.6 µM), and mammea B/AB cyclo D (21, 3.1 µM) showed relatively strong inhibitory activities comparable to the activity of the synthetic nonsteroidal aromatase inhibitor aminoglutethimide (2.0 µM).
Asunto(s)
Inhibidores de la Aromatasa/farmacología , Aromatasa/metabolismo , Cumarinas/farmacología , Flores/química , Mammea/química , Inhibidores de la Aromatasa/química , Inhibidores de la Aromatasa/aislamiento & purificación , Cumarinas/química , Cumarinas/aislamiento & purificación , Relación Dosis-Respuesta a Droga , Humanos , Estructura Molecular , Proteínas Recombinantes/metabolismo , Relación Estructura-ActividadRESUMEN
Essential scientific elements for early product development, evaluation, and control of human cell-based products for cell therapy are addressed in a comprehensive and unifying way. Among them, donor issues (autologous and allogeneic), testing of raw materials, cell banking and cell substrate characterization, testing for adventitious agents (viral safety), and product sterility are very much related to each other. A significant amount of expertise exists in these areas both for traditional biologicals as well as for biotechnology products. Thus, core principles/concepts as well as the testing methodologies are already well defined. Other critical technical elements that are essential but need further discussion in terms of relevant regulatory requirements and testing methods are touched upon very briefly and followed by a detailed discussion (elsewhere).
Asunto(s)
Tratamiento Basado en Trasplante de Células y Tejidos , Humanos , Control de CalidadRESUMEN
The regulation of human cell therapy products is a key factor in their development and use to treat human diseases. In that regard, there is a recognized need for a global effort to develop a set of common principles that may serve to facilitate a convergence of regulatory approaches to ensure the smooth and efficient evaluation of products. This conference, with experts from regulatory agencies, industry, and academia, contributed to the process of developing such a document. Elements that could form a minimum consensus package of requirements for evaluating human cell therapy products were the overall focus of the conference. The important regulatory considerations that are unique to human cell therapy products were highlighted. Sessions addressed specific points that are different from those of traditional biological/biotechnological protein products. Panel discussions complemented the presentations. The conference concluded that most of the current regulatory framework is appropriate for cell therapy, but there are some areas where the application of the requirements for traditional biologicals is inappropriate. In addition, it was agreed that there is a need for international consensus on core regulatory elements, and that one of the major international organizations should take the lead in formulating such a consensus document.
Asunto(s)
Biotecnología/legislación & jurisprudencia , Tratamiento Basado en Trasplante de Células y Tejidos , Productos Biológicos , HumanosRESUMEN
Recently, we reported comparative analysis of glycoproteins which express cancer-specific N-glycans on various cancer cells and identified 24 glycoproteins having polylactosamine (polyLacNAc)-type N-glycans that are abundantly present in malignant cells [ Mitsui et al., J. Pharm. Biomed. Anal. 2012 , 70 , 718 - 726 ]. In the present study, we applied the technique to comparative studies on common glycoproteins present in the matched patient primary and metastatic melanoma cell lines. Metastatic melanoma cells (WM266-4) contained a large amount of polyLacNAc-type N-glycans in comparison with primary melanoma cells (WM115). To identify the glycoproteins expressing these N-glycans, glycopeptides having polyLacNAc-type N-glycans were captured by a Datura stramonium agglutinin (DSA)-immobilized agarose column. The captured glycopeptides were analyzed by LC/MS after removing N-glycans, and some glycoproteins such as basigin, lysosome-associated membrane protein-1 (LAMP-1), and chondroitin sulfate proteoglycan 4 (CSPG4) were identified in both WM115 and WM266-4 cells. The expression level of polyLacNAc of CSPG4 in WM266-4 cells was significantly higher than that in WM115 cells. In addition, sulfation patterns of chondroitin sulfate (CS) chains in CSPG4 showed dramatic changes between these cell lines. These data show that characteristic glycans attached to common proteins observed in different stages of cancer cells will be useful markers for determining degree of malignancies of tumor cells.
Asunto(s)
Amino Azúcares/química , Glicoproteínas/metabolismo , Melanoma/metabolismo , Polisacáridos/metabolismo , Secuencia de Aminoácidos , Western Blotting , Secuencia de Carbohidratos , Línea Celular Tumoral , Cromatografía de Afinidad/métodos , Cromatografía Líquida de Alta Presión , Electroforesis en Gel de Poliacrilamida , Humanos , Espectrometría de Masas/métodos , Melanoma/patología , Datos de Secuencia Molecular , Metástasis de la Neoplasia , Polisacáridos/químicaRESUMEN
We have identified novel CE conditions for the separation of 7-amino-4-methylcoumarin-labeled monosaccharides and oligosaccharides from glycoproteins. Using a neutrally coated capillary and alkaline borate buffer containing hydroxypropylcellulose and ACN, saccharide derivatives form anionic borate complexes, which move from the cathode to the anode in an electric field and are detected near the anodic end. Excess labeling reagents and other fluorescent products remain at the cathodic end. Fluorimetric detection using an LED as a light source enables determination of monosaccharide derivatives with good linearity between at least 0.4 and 400 µM, may correspond to 140 amol to 140 fmol. The lower LOD (S/N = 5) is only 80 nM in the sample solution (ca. 28 amol). The results were comparable to reported values using fluorometric detection LC. The method was also applied to the analysis of oligosaccharides that were enzymatically released from glycoproteins. Fine resolution enables profiling of glycans in glycoproteins. The applicability of the method was examined by applying it to other derivatives labeled with nonacidic tags such as ethyl p-aminobenzoate- and 2-aminoacridone-labeled saccharides.
Asunto(s)
Electroforesis Capilar/métodos , Glicoproteínas/química , Monosacáridos/análisis , Oligosacáridos/análisis , Animales , Bovinos , Pollos , Cumarinas/química , Humanos , Límite de Detección , Monosacáridos/química , Oligosacáridos/químicaRESUMEN
Hepatocyte-like cells from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are expected to be a useful source of cells drug discovery. Although we recently reported that hepatic commitment is promoted by transduction of SOX17 and HEX into human ESC- and iPSC-derived cells, these hepatocyte-like cells were not sufficiently mature for drug screening. To promote hepatic maturation, we utilized transduction of the hepatocyte nuclear factor 4α (HNF4α) gene, which is known as a master regulator of liver-specific gene expression. Adenovirus vector-mediated overexpression of HNF4α in hepatoblasts induced by SOX17 and HEX transduction led to upregulation of epithelial and mature hepatic markers such as cytochrome P450 (CYP) enzymes, and promoted hepatic maturation by activating the mesenchymal-to-epithelial transition (MET). Thus HNF4α might play an important role in the hepatic differentiation from human ESC-derived hepatoblasts by activating the MET. Furthermore, the hepatocyte like-cells could catalyze the toxication of several compounds. Our method would be a valuable tool for the efficient generation of functional hepatocytes derived from human ESCs and iPSCs, and the hepatocyte-like cells could be used for predicting drug toxicity.
Asunto(s)
Células Madre Embrionarias/citología , Factor Nuclear 4 del Hepatocito/genética , Hepatocitos/citología , Células Madre Pluripotentes Inducidas/citología , Transducción Genética , Diferenciación Celular , Línea Celular , Células Madre Embrionarias/metabolismo , Transición Epitelial-Mesenquimal/genética , Técnicas de Transferencia de Gen , Factor Nuclear 4 del Hepatocito/metabolismo , Hepatocitos/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Proteínas Proto-Oncogénicas c-met/metabolismo , Factores de TiempoRESUMEN
BACKGROUND: Adipose tissues contain populations of pluripotent mesenchymal stem cells that also secrete various cytokines and growth factors to support repair of damaged tissues. In this study, we examined the role of oxidative stress on human adipose-derived multilineage progenitor cells (hADMPCs) in neurite outgrowth in cells of the rat pheochromocytoma cell line (PC12). RESULTS: We found that glutathione depletion in hADMPCs, caused by treatment with buthionine sulfoximine (BSO), resulted in the promotion of neurite outgrowth in PC12 cells through upregulation of bone morphogenetic protein 2 (BMP2) and fibroblast growth factor 2 (FGF2) transcription in, and secretion from, hADMPCs. Addition of N-acetylcysteine, a precursor of the intracellular antioxidant glutathione, suppressed the BSO-mediated upregulation of BMP2 and FGF2. Moreover, BSO treatment caused phosphorylation of p38 MAPK in hADMPCs. Inhibition of p38 MAPK was sufficient to suppress BMP2 and FGF2 expression, while this expression was significantly upregulated by overexpression of a constitutively active form of MKK6, which is an upstream molecule from p38 MAPK. CONCLUSIONS: Our results clearly suggest that glutathione depletion, followed by accumulation of reactive oxygen species, stimulates the activation of p38 MAPK and subsequent expression of BMP2 and FGF2 in hADMPCs. Thus, transplantation of hADMPCs into neurodegenerative lesions such as stroke and Parkinson's disease, in which the transplanted hADMPCs are exposed to oxidative stress, can be the basis for simple and safe therapies.
Asunto(s)
Butionina Sulfoximina/farmacología , Estrés Oxidativo/efectos de los fármacos , Células Madre/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Acetilcisteína/farmacología , Animales , Proteína Morfogenética Ósea 2/genética , Proteína Morfogenética Ósea 2/metabolismo , Diferenciación Celular/efectos de los fármacos , Línea Celular , Factor 2 de Crecimiento de Fibroblastos/genética , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Glutatión/metabolismo , Humanos , MAP Quinasa Quinasa 6/metabolismo , Neuritas/efectos de los fármacos , Neuritas/metabolismo , Células PC12 , Fosforilación , Ratas , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Proteínas Smad/metabolismo , Células Madre/citología , Células Madre/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidoresRESUMEN
BACKGROUND & AIMS: Hepatocyte-like cells differentiated from human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) can be utilized as a tool for screening for hepatotoxicity in the early phase of pharmaceutical development. We have recently reported that hepatic differentiation is promoted by sequential transduction of SOX17, HEX, and HNF4α into hESC- or hiPSC-derived cells, but further maturation of hepatocyte-like cells is required for widespread use of drug screening. METHODS: To screen for hepatic differentiation-promoting factors, we tested the seven candidate genes related to liver development. RESULTS: The combination of two transcription factors, FOXA2 and HNF1α, promoted efficient hepatic differentiation from hESCs and hiPSCs. The expression profile of hepatocyte-related genes (such as genes encoding cytochrome P450 enzymes, conjugating enzymes, hepatic transporters, and hepatic nuclear receptors) achieved with FOXA2 and HNF1α transduction was comparable to that obtained in primary human hepatocytes. The hepatocyte-like cells generated by FOXA2 and HNF1α transduction exerted various hepatocyte functions including albumin and urea secretion, and the uptake of indocyanine green and low density lipoprotein. Moreover, these cells had the capacity to metabolize all nine tested drugs and were successfully employed to evaluate drug-induced cytotoxicity. CONCLUSIONS: Our method employing the transduction of FOXA2 and HNF1α represents a useful tool for the efficient generation of metabolically functional hepatocytes from hESCs and hiPSCs, and the screening of drug-induced cytotoxicity.
Asunto(s)
Células Madre Embrionarias/citología , Factor Nuclear 1-alfa del Hepatocito/genética , Factor Nuclear 3-beta del Hepatocito/genética , Hepatocitos/citología , Hepatocitos/metabolismo , Células Madre Pluripotentes Inducidas/citología , Bupropión/metabolismo , Diferenciación Celular , Línea Celular , Células Madre Embrionarias/metabolismo , Etanolaminas/metabolismo , Técnicas de Transferencia de Gen , Factor Nuclear 1-alfa del Hepatocito/metabolismo , Factor Nuclear 3-beta del Hepatocito/metabolismo , Hepatocitos/enzimología , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Midazolam/metabolismo , Paclitaxel/metabolismo , Fenacetina/metabolismo , Testosterona/metabolismo , Tolbutamida/metabolismo , Transducción GenéticaRESUMEN
Minor N-linked glycans containing N-glycolylneuraminic acid residues and/or α-Gal epitopes (i.e., galactose-α1,3-galactose residues) have been reported to be present in recombinant monoclonal antibody (mAb) therapeutics. These contaminations are due to their production processes using nonhuman mammalian cell lines in culture media containing animal-derived materials. In case of the treatment of tumors, we inevitably use such mAbs by careful risk-benefit considerations to prolong patients' lives. However, expanding their clinical applications such as for rheumatism, asthma, and analgesia demands more careful evaluation of the product characteristics. The present work for detailed evaluations of N-glycans demonstrates the methods using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) and a combination of high-performance liquid chromatography and electrospray ionization time-of-flight mass spectrometry. The CE-LIF method provides excellent separation of both major and minor N-glycans from six commercial mAb pharmaceuticals within 30 min and clearly indicates that a possible trigger of immunogenicity in humans due to the presence of nonhuman N-glycans is present. We strongly believe that the proposed method will be a powerful tool for the analysis of N-glycans of recombinant mAb products in various development stages, such as clone selection, process control, and routine release testing to ensure safety and efficacy of the products.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Electroforesis Capilar , Polisacáridos/análisis , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/genética , Secuencia de Carbohidratos , Cromatografía Líquida de Alta Presión , Galactosa/análisis , Glicosilación , Rayos Láser , Datos de Secuencia Molecular , Ácidos Neuramínicos/análisis , Polisacáridos/aislamiento & purificación , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
The oligosaccharide structure is very important in biopharmaceuticals because of its effects on protein function, including efficacy and half-life. N-glycolylneuraminic acid (Neu5Gc) and Galα1-3Gal (α-Gal) residues are known to show immunogenicity in humans. It is now understood that murine cell lines, such as NS0 or SP2, which are typically used for biopharmaceutical manufacture, produce proteins containing Neu5Gc and α-Gal residues. The expression of these specific residues is affected by the cell line and culture conditions. Therefore, monitoring and controlling the levels of these epitopes are important for the quality control of biopharmaceuticals. To detect the two epitopes on a therapeutic antibody produced by NS0 cells, we applied partial-filling capillary electrophoresis using anti-Neu5Gc antibody and α-galactosidase. In the anti-Neu5Gc antibody filling method, one minor glycan peak with Neu5Gc residues at the nonreducing end disappeared specifically from the electropherogram. In the α-galactosidase filling method, some minor peaks with α1,3-linked Gal residues disappeared. However, in a therapeutic antibody from Chinese hamster ovary cells, no peaks disappeared with the two methods. These results show this method can be used to specifically detect and quantify the two epitopes on biopharmaceuticals with high sensitivity.
Asunto(s)
Anticuerpos Monoclonales , Disacáridos , Epítopos , Ácidos Neuramínicos , Anticuerpos Monoclonales/análisis , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/uso terapéutico , Línea Celular Tumoral , Disacáridos/inmunología , Disacáridos/aislamiento & purificación , Electroforesis Capilar , Epítopos/química , Epítopos/inmunología , Epítopos/aislamiento & purificación , Humanos , Ácidos Neuramínicos/inmunología , Ácidos Neuramínicos/aislamiento & purificaciónRESUMEN
We developed an automated apparatus for rapid releasing of O-glycans from mucin-type glycoproteins [Anal. Biochem. 371 (2007) 52-61; Anal. Chem. 82 (2010) 7436-7443] and applied the device to analyze them in some cancer cell lines [J. Proteome Res. 8 (2009) 521-537]. We also found that the device is useful to release glycosaminoglycans from proteoglycans [Anal. Biochem. 362 (2007) 245-251]. Based on these studies, we developed a method for one-pot analysis of mucin-type glycans and glycosaminoglycans after releasing them from total protein pool obtained from some cancer cell lines. Mucin-type glycans were analyzed by a combination of high-performance liquid chromatography and mass spectrometry techniques, and glycosaminoglycans were analyzed by capillary electrophoresis as fluorescent-labeled unsaturated disaccharides after digestion with specific eliminases followed by fluorescent labeling. Ten cancer cell lines, including blood cancer cells as well as epithelial cancer cells, were used to assess the method. The results clearly revealed that both mucin-type glycans and glycosaminoglycans showed quite interesting profiles. Thus, the current technique will be a powerful tool for discovery of glycan markers of diseases.
Asunto(s)
Biomarcadores de Tumor/análisis , Glicosaminoglicanos/análisis , Neoplasias/química , Polisacáridos/análisis , Línea Celular Tumoral , Cromatografía de Afinidad , Cromatografía Líquida de Alta Presión , Electroforesis Capilar , Humanos , Neoplasias/patología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Espectrometría de Masas en TándemRESUMEN
A methanol extract of the bark of Shorea roxburghii (Dipterocarpaceae) was found to inhibit plasma glucose elevation in sucrose-loaded mice. From the extract, three new 3-ethyl-4-phenyl-3,4-dihydroisocoumarins, 1'S-dihydrophayomphenol A(2) (1) and phayomphenols B(1) (2) and B(2) (3), were isolated together with 24 known compounds including 20 stilbenoids and oligostilbenoids. The structures of 1-3 were determined on the basis of their spectroscopic properties as well as of chemical evidences. Among the isolates, (-)-hopeaphenol (6), hemsleyanol D (8), (+)-α-viniferin (15), and (-)-balanocarpol (18) showed inhibitory activity against plasma glucose elevation in sucrose-loaded rats at doses of 100-200mg/kg, p.o. To clarify the mode of action of the antihyperglycemic property, effects of these oligostilbenoids on gastric emptying in mice, those on glucose uptake in isolated intestinal tissues as well as inhibitory activities against rat intestinal α-glucosidase and rat lens aldose reductase were examined.
Asunto(s)
Dipterocarpaceae/química , Hipoglucemiantes/química , Isocumarinas/química , Estilbenos/química , Aldehído Reductasa/antagonistas & inhibidores , Aldehído Reductasa/metabolismo , Animales , Metabolismo de los Hidratos de Carbono/efectos de los fármacos , Inhibidores de Glicósido Hidrolasas , Hipoglucemiantes/aislamiento & purificación , Hipoglucemiantes/farmacología , Isocumarinas/aislamiento & purificación , Isocumarinas/farmacología , Espectroscopía de Resonancia Magnética , Metanol/química , Ratones , Conformación Molecular , Corteza de la Planta/química , Ratas , Estilbenos/aislamiento & purificación , Estilbenos/farmacología , alfa-Glucosidasas/metabolismoRESUMEN
A methanol extract of the flowers of Mammea siamensis (Calophyllaceae) was found to inhibit nitric oxide (NO) production in lipopolysaccharide-activated RAW264.7 cells. From the extract, two new geranylated coumarins, mammeasins A (1) and B (2), were isolated together with 17 known compounds including 15 coumarins. The structures of 1 and 2 were determined on the basis of their spectroscopic properties as well as of their chemical evidence. Among the isolates, 1 (IC(50) = 1.8 µM), 2 (6.4 µM), surangins B (3, 5.0 µM), C (4, 6.8 µM), and D (5, 6.2 µM), kayeassamins E (7, 6.1 µM), F (8, 6.0 µM), and G (9, 0.8 µM), mammea A/AD (11, 1.3 µM), and mammea E/BB (16, 7.9 µM) showed NO production inhibitory activity. Compounds 1, 9, and 11 were found to inhibit induction of inducible nitric oxide synthase (iNOS). With regard to mechanism of action of these active constituents (1, 9, and 11), suppression of STAT1 activation is suggested to be mainly involved in their suppression of iNOS induction.