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Chen-Ta Ho,
Ruei-Zeng Lin,
Rong-Jhe Chen,
Chung-Kuang Chin,
Song-En Gong, Hwan-You Chang,
Hwei-Ling Peng,
Long Hsu,
Tri-Rung Yew,
Shau-Feng Chang,
Cheng-Hsien Liu
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ABSTRACT: A lobule-mimetic cell-patterning technique for on-chip reconstruction of centimetre-scale liver tissue of heterogeneous hepatic and endothelial cells via an enhanced field-induced dielectrophoresis (DEP) trap is demonstrated and reported. By mimicking the basic morphology of liver tissue, the classic hepatic lobule, the lobule-mimetic-stellate-electrodes array was designed for cell patterning. Through DEP manipulation, well-defined and enhanced spatial electric field gradients were created for in-parallel manipulation of massive individual cells. With this liver-cell patterning labchip design, the original randomly distributed hepatic and endothelial cells inside the microfluidic chamber can be manipulated separately and aligned into the desired pattern that mimicks the morphology of liver lobule tissue. Experimental results showed that both hepatic and endothelial cells were orderly guided, snared, and aligned along the field-induced orientation to form the lobule-mimetic pattern. About 95% cell viability of hepatic and endothelial cells was also observed after cell-patterning demonstration via a fluorescent assay technique. The liver function of CYP450-1A1 enzyme activity showed an 80% enhancement for our engineered liver tissue (HepG2+HUVECs) compared to the non-patterned pure HepG2 for two-day culturing.
Lab on a Chip 06/2013; · 5.67 Impact Factor
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04/2013;
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ABSTRACT: Tylophorine, a phenanthroindolizidine alkaloid, is the major medicinal constituent of herb tylophora indica. Tylophorine treatment increased the accumulation of c-Jun protein, a component of activator protein one (AP1), in carcinoma cells. An in vitro kinase assay revealed that the resultant c-Jun phosphorylation was primarily mediated via activated c-Jun NH(2)-terminal protein kinase (JNK). Moreover, flow cytometry indicated that ectopically overexpressed c-Jun in conjunction with tylophorine significantly increased the number of carcinoma cells that were arrested at the G1 phase. The tylophorine-mediated downregulation of cyclin A2 protein levels is known to be involved in the primary G1 arrest. Chromatin immunoprecipitation and reporter assays revealed that tylophorine enhanced the c-Jun downregulation of the cyclin A2 promoter activity upon increased binding of c-Jun to the deregulation AP1 site and decreased binding to the upregulation ATF site in the cyclin A2 promoter, thereby reducing cyclin A2 expression. Further biochemical studies using pharmacological inhibitors and RNA silencing approaches demonstrated that tylophorine-mediated elevation of the c-Jun protein level occurs primarily via two discrete prolonged signaling pathways: 1) the NF-κB/PKCδ_(MKK4)_JNK cascade, which phosphorylates c-Jun and increases its stability by slowing its ubiquitination, and 2) the PI3K_PDK1_PP2A_eEF2 cascade, which sustains eEF2 activity and thus c-Jun protein translation. To the best of our knowledge, this report is the first to demonstrate the involvement of c-Jun in the anti-cancer activity of tylophorine and the release of c-Jun translation from a global translational blockade via the PI3K_PDK1_eEF2 signaling cascade.
Carcinogenesis 02/2013; · 5.70 Impact Factor
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ABSTRACT: Surface IgD and IgM doubly positive cells comprise the major population of B cells in the human immune system. The heavy chain of membrane-bound IgD (mδ) differs from that of IgD (δ) in that mδ contains a C-terminal membrane-anchor peptide. Our group previously proposed that the N-terminal extracellular segment of 27 aa residues of the membrane-anchor peptide of mδ, referred to as the mIg isotype-specific-δ (migis-δ) segment, may provide a unique antigenic site for isotype-specific targeting of mIgD(+) B cells. Here we report the preparation of mouse mAbs specific for human migis-δ. The mAbs bound to human migis-δ-containing recombinant proteins in an ELISA and to mIgD-expressing transfectants of a CHO cell line as analyzed by flow cytometry. MAb 20E6, which binds to an epitope toward the N-terminal of human migis-δ, could stain human B cell line MC116, which expressed mIgD and mIgM. MC116 cells could be induced to undergo apoptosis by treatment with 20E6 in the presence of a second crosslinking antibody. Chimeric 20E6 caused antibody-dependent cellular cytotoxicity of MC116 cells in the presence of human PBMCs as the source of effector cells. In cultures of PBMCs, 20E6 down-regulated the population of mIgD(+) B cells. The production of human IgM by transplanted MC116 cells in NOD-SCID (NOD.CB17-Prkdc(scid)/IcrCrlBltw) mice could be suppressed by 20E6. These results encourage further investigation of the potential of anti-migis-δ mAbs to control mIgD(+) B cells, when such a manipulation may alleviate a disease state.
Molecular Immunology 08/2012; 53(3):187-97. · 2.90 Impact Factor
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Chia-Han Chan,
Feng-Jung Chen,
Ying-Jung Huang,
Shin-Yu Chen,
Kuo-Liang Liu,
Zhe-Chong Wang,
Hwei-Ling Peng,
Tri-Rung Yew,
Cheng-Hsien Liu,
Gunn-Guang Liou,
Ken Y Hsu, Hwan-You Chang,
Long Hsu
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ABSTRACT: The Klebsiella pneumoniae type 3 fimbriae are mainly composed of MrkA pilins that assemble into a helixlike filament. This study determined the biomechanical properties of the fimbriae and analyzed 11 site-directed MrkA mutants to identify domains that are critical for the properties. Escherichia coli strains expressing type 3 fimbriae with an Ala substitution at either F34, V45, C87, G189, T196, or Y197 resulted in a significant reduction in biofilm formation. The E. coli strain expressing MrkAG189A remained capable of producing a normal number of fimbriae. Although F34A, V45A, T196A, and Y197A substitutions expressed on E. coli strains produced sparse quantities of fimbriae, no fimbriae were observed on the cells expressing MrkAC87A. Further investigations of the mechanical properties of the MrkAG189A fimbriae with optical tweezers revealed that, unlike the wild-type fimbriae, the uncoiling force for MrkAG189A fimbriae was not constant. The MrkAG189A fimbriae also exhibited a lower enthalpy in the differential scanning calorimetry analysis. Together, these findings indicate that the mutant fimbriae are less stable than the wild-type. This study has demonstrated that the C-terminal β strands of MrkA are required for the assembly and structural stability of fimbriae.
Langmuir 04/2012; 28(19):7428-35. · 4.19 Impact Factor
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ABSTRACT: Techniques to separate cells are widely applied in immunology. The technique to separate a specific antigen on a microfluidic platform involves the use of a shear horizontal surface-acoustic-wave (SH-SAW) sensor. With specific antibodies conjugated onto the surface of the SH-SAW sensors, this technique can serve to identify specific cells in bodily fluids. Jurkat cells, used as a target in this work, provide a model of cells in small abundance (1:1000) for isolation and purification with the ultimate goal of targeting even more dilute cells. T cells were separated from a mixed-cell medium on a chip (Jurkat cells/K562 cells, 1/1000). A novel microchamber was developed to capture cells during the purification, which required a large biosample. Cell detection was demonstrated through the performance of genetic identification on the chip.
Journal of the Association for Laboratory Automation 04/2012; · 1.42 Impact Factor
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ABSTRACT: The histidine-containing phosphotransfer protein-B (HptB; PA3345) is an intermediate protein involved in transferring a phosphoryl group from multiple sensor kinases to the response regulator PA3346 in Pseudomonas aeruginosa PAO1. The objective of this study was to elucidate the biological significance of the HptB-PA3346 interaction and the regulatory mechanisms thereafter. The transcription profiling analysis of an hptB knock-out mutant showed that the expression of a number of motility-related genes was altered consistent with the non-swarming phenotype observed for the mutant. Domain analysis indicated that the PA3346 C-terminal region (PA3346C) exhibits ∼30% identity with the anti-σ factor SpoIIAB of Bacillus subtilis. The presence of Ser/Thr protein kinase activity targeting an anti-σ antagonist, PA3347, at Ser-56 was confirmed in PA3346C using an in vitro phosphorelay assay. Furthermore, PA3346C and the anti-σ(28) factor FlgM were found to interact with PA3347 individually both in vivo and in vitro. FlgM displaced PA3346C in binding of PA3347 and was then competitively displaced by σ(28) from the PA3347-FlgM complex, forming a phosphorylation-dependent partner-switching system. The significance of PA3347 phosphorylation in linking the partner-switching system and swarming motility was established by analyzing the swarming phenotype of the PA3347 knock-out mutant and its complement strains.
Journal of Biological Chemistry 11/2011; 287(3):1903-14. · 4.77 Impact Factor
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ABSTRACT: In this study, a CdSe/ZnS quantum dot (QD)-based immunosensor using a simple optical system for human serum albumin (HSA) detection is developed. Monoclonal anti-HSA (AHSA) immobilized on 3-aminopropyltriethoxysilane (APTES)-modified glass was used to capture HSA specifically. Bovine serum albumin (BSA) was used to block non-specific sites. The solution, containing AHSA-QD complex prepared by mixing biotinylated polyclonal anti-HSA and streptavidin coated QD, was used to conjugate with the HSA molecules captured on AHSA/BSA/APTES-modified glass for the modification of HSA with QD. A simple optical system, comprising a diode laser (405 nm), an optical lens, a 515-nm-long pass filter, and an Si-photodiode, was used to detect fluorescence and convert it to photocurrent. The current intensity was determined by the amount of QD specifically conjugated with HSA, and was therefore HSA-concentration-dependent and could be used to quantify HSA concentration. The detection limit of the pure QD solution was ~3.5×10(-12) M, and the detection limit for the CdSe/ZnS QD-based immunosensor developed in this study was approximately 3.2×10(-5) mg/ml. This small optical biosensing system shows considerable potential for future applications of on-chip liver-function detection.
Biosensors & bioelectronics 11/2011; 34(1):286-90. · 5.43 Impact Factor
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ABSTRACT: In this study, electrical impedimetric biosensors composed of Au-electrodes were fabricated for the quantitative detection of human serum albumin (HSA), an essential biomarker of liver function. The Au-electrodes were fabricated via a single-step photolithography process, and can be easily integrated in biochips for assessing liver function in the future. The glass sensing surface between two adjacent Au-electrodes was modified with 3-aminopropyltriethoxysilane (APTES) to improve the biocompatibility for its subsequent binding to anti-human serum albumin (AHSA). The sensing surface without AHSA binding was blocked using skim milk powders, preventing possible non-specific bonding HSA conjugation. Biosensors were used to measure HSA concentration for liver function detection. The impedance between two adjacent Au-electrodes of the biosensors applied with various HSA concentrations was directly measured, and quantified using an electrochemical impedance spectroscopy system under AC conditions. The results of plotting both values in log scales indicated the impedance increased linearly with HSA conjugation increase. The limit of HSA detection was about 2'10(-4)mg/ml using the electrochemical impedimetric biosensor proposed in this work. This study demonstrates the feasibility of using electrochemical impedimetry as a bio-sensing mechanism to quantify human serum albumin concentration. The sensor proposed in this work also displays great potential for assessing liver function because of its simple detection mechanism, ease of biochip integration, and low cost.
Biosensors & bioelectronics 07/2011; 28(1):368-72. · 5.43 Impact Factor
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ABSTRACT: Bacteria monitoring is essential for many industrial manufacturing processes, particularly those involving in food, biopharmaceuticals, and semiconductor production. Firefly luciferase ATP luminescence assay is a rapid and simple bacteria detection method. However, the detection limit of this assay for Escherichia coli is approximately 10(4) colony-forming units (CFU), which is insufficient for many applications. This study aims to improve the assay sensitivity by simultaneous conversion of PP(i) and AMP, two products of the luciferase reaction, back to ATP to form two chain-reaction loops. Because each consumed ATP continuously produces two new ATP molecules, this approach can achieve exponential amplification of ATP. Two consecutive enzyme reactions were employed to regenerate AMP into ATP: adenylate kinase converting AMP into ADP using UTP as the energy source, and acetate kinase catalyzing acetyl phosphate and ADP into ATP. The PP(i)-recycling loop was completed using ATP sulfurylase and adenosine 5' phosphosulfate. The modification maintains good quantification linearity in the ATP luminescence assay and greatly increases its bacteria detection sensitivity. This improved method can detect bacteria concentrations of fewer than 10 CFU. This exponential ATP amplification assay will benefit bacteria monitoring in public health and manufacturing processes that require high-quality water.
Analytical Biochemistry 07/2011; 418(1):19-23. · 3.00 Impact Factor
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ABSTRACT: An investigation of alkaloids present in the leaves and stems of Tylophora ovata led to the isolation of two new septicine alkaloids and one new phenanthroindolizidine alkaloid, tylophovatines A, B, C (1, 2, and 5), respectively, together with two known septicine and six known phenanthroindolizidine alkaloids. The structures of the new alkaloids 1, 2, and 5 were established by means of spectroscopic analyses. These eleven alkaloids show in vitro anti-inflammatory activities with IC₅₀ values ranging from 84 nM to 20.6 μM through their suppression of nitric oxide production in RAW264.7 cells stimulated by lipopolysaccharide and interferon-γ. Moreover, these substances display growth inhibition in HONE-1, NUGC-3, HepG2, SF-268, MCF-7, and NCI-H460 cancer cell lines, with GI₅₀ values ranging from 4 nM to 24.2 μM. In addition, tylophovatine C (5) and 13a(S)-(+)-tylophorine (7) were found to exhibit potent in vivo anti-inflammation activities in a rat paw edema model. Finally, structure–activity relationships were probed by using the isolated phenanthroindolizidines and septicines. Phenanthroindolizidines are suggested to be divided into cytotoxic agents (e.g., 10 and 11) and anti-inflammation based anticancer agents (e.g., 5–9).
Planta Medica 07/2011; 77(17):1932-8. · 2.15 Impact Factor
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ABSTRACT: Many tumor cells are capable of migrating through endothelial cell (EC) junctions and disintegrating sub-endothelial extracellular matrix to achieve extravasation. We demonstrate in this study that certain solid tumor cells can induce EC apoptosis to facilitate their escape from the circulation. The EC apoptosis is triggered by elevated intracellular reactive oxygen species (ROS) levels and direct contacts with tumor cells are required. Treating ECs with antioxidants, such as ascorbate and N-acetyl-L-cysteine (NAC), and a glutathione precursor can rescue the ECs from tumor-induced apoptosis and reduce the number of tumor cells migrating across endothelial barriers. NAD(P)H oxidase was identified as the major ROS producer in the event since inhibitors and small interference RNA specific to the enzyme could abrogate the tumor-induced ROS production and hence EC death. This study also provides evidence showing that the interaction between tumor and EC increases intracellular Ca(2+) concentration and activates protein kinase C (PKC) activity, which leads to NAD(P)H oxidase activation through the serine-phosphorylation of p47(phox) subunit. These findings suggest that blocking the tumor-induced EC apoptosis is a potential way to prevent tumor metastasis.
Journal of Cellular Physiology 07/2011; 226(7):1750-62. · 3.87 Impact Factor
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ABSTRACT: All human organs consist of multiple types of cells organized in a complex pattern to meet specific functional needs. One possible approach for reconstructing human organs in vitro is to generate cell sheets of a specific pattern and later stack them systematically by layer into a three-dimensional organoid. However, many commonly used cell patterning techniques suffer drawbacks such as dependence on sophisticated instruments and manipulation of cells under suboptimal growth conditions. Here, we describe a simple cell patterning method that may overcome these problems. This method is based on magnetic force and photoresponsive poly (ethylene glycol) diacrylate (PEG-DA) hydrogels. The PEG-DA hydrogel was magnetized by mixing with iron ferrous microparticles and then fabricated into blocks with a specific pattern by photolithography. The resolution of the hydrogel empty space pattern was approximately 150 μm and the generated hydrogel blocks can be remotely manipulated with a magnet. The magnetic PEG-DA blocks were used as a stencil to define the area for cell adhesion in the cell culture dish, and the second types of cells could be seeded after the magnetic block was removed to create heterotypic cell patterns. Cell viability assay has demonstrated that magnetic PEG-DA and the patterning process produced negligible effects on cell growth. Together, our results indicate that this magnetic hydrogel-based cell patterning method is simple to perform and is a useful tool for tissue surrogate assembly for disease mechanism study and drug screening.
Tissue Engineering Part C Methods 05/2011; 17(8):871-7. · 4.64 Impact Factor
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Feng-Jung Chen,
Chia-Han Chan,
Ying-Jung Huang,
Kuo-Liang Liu,
Hwei-Ling Peng, Hwan-You Chang,
Gunn-Guang Liou,
Tri-Rung Yew,
Cheng-Hsien Liu,
Ken Y Hsu,
Long Hsu
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ABSTRACT: This study investigated the structural and mechanical properties of Klebsiella pneumoniae type 3 fimbriae, which constitute a known virulence factor for the bacterium. Transmission electron microscopy and optical tweezers were used to understand the ability of the bacterium to survive flushes. An individual K. pneumoniae type 3 fimbria exhibited a helix-like structure with a pitch of 4.1 nm and a three-phase force-extension curve. The fimbria was first nonlinearly stretched with increasing force. Then, it started to uncoil and extended several micrometers at a fixed force of 66 ± 4 pN (n = 22). Finally, the extension of the fimbria shifted to the third phase, with a characteristic force of 102 ± 9 pN (n = 14) at the inflection point. Compared with the P fimbriae and type 1 fimbriae of uropathogenic Escherichia coli, K. pneumoniae type 3 fimbriae have a larger pitch in the helix-like structure and stronger uncoiling and characteristic forces.
Journal of bacteriology 01/2011; 193(7):1718-25. · 3.94 Impact Factor
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ABSTRACT: Vibrio vulnificus secretes a multifunctional cytotoxin RtxA (VvRtxA), which plays a major role in the bacterial pathogenesis. The lack of an efficient VvRtxA detection tool has hampered the progress of V. vulnificus pathogenesis research. This study aims to isolate VvRtxA specific single-chain variable fragments (scFv) to serve as a detection agent. The VvRtxA C-terminal Gly-Asp (GD) repeat-containing region, which has been implicated for calcium binding and target cell recognition, was chosen as an antigen to screen a scFv phage display library. A scFv clone that reacted positively to VvRtxA was successfully obtained. Using the isolated scFv, a cell-based enzyme-linked immunosorbent assay was established for detecting cell-associated VvRtxA toxin in V. vulnificus infected HEp-2 cells. The result is consistent with previous observations that secretion of VvRtxA toxin is time-dependent on bacteria contacting with host cells. Utilization of scFv for VvRtxA toxin detection provides an applicable strategy devoid of conventional immunization.
Journal of microbiological methods 11/2010; 84(1):94-100. · 2.43 Impact Factor
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ABSTRACT: UDP-glucose dehydrogenase (UGDH) catalyzes oxidation of UDP-glucose to yield UDP-glucuronic acid, a precursor of hyaluronic acid (HA) and other glycosaminoglycans (GAGs) in extracellular matrix. Although association of extracellular matrix with cell proliferation and migration has been well documented, the importance of UGDH in these behaviors is not clear. Using UGDH-specific small interference RNA to treat HCT-8 colorectal carcinoma cells, a decrease in both mRNA and protein levels of UGDH, as well as the cellular UDP-glucuronic acid and GAG production was observed. Treatment of HCT-8 cells with either UGDH-specific siRNA or HA synthesis inhibitor 4-methylumbelliferone effectively delayed cell aggregation into multicellular spheroids and impaired cell motility in both three-dimensional collagen gel and transwell migration assays. The reduction in cell aggregation and migration rates could be restored by addition of exogenous HA. These results indicate that UGDH can regulate cell motility through the production of GAG. The enzyme may be a potential target for therapeutic intervention of colorectal cancers.
Experimental Cell Research 10/2010; 316(17):2893-902. · 3.58 Impact Factor
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ABSTRACT: Negative dielectrophoretic (n-DEP) cell manipulation is an efficient way to pattern human liver cells on micro-electrode arrays. Maintaining cell viability is an important objective for this approach. This study investigates the effect of low conductivity medium and the optimally designed microchip on cell viability and cell adhesion. To explore the influence of conductivity on cell viability and cell adhesion, we have used earlier reported dielectrophoresis (DEP) buffer with a conductivity of 10.2 mS/m and three formulated media with conductivity of 9.02 (M1), 8.14 (M2), 9.55 (M3) mS/m. The earlier reported isotonic sucrose/dextrose buffer (DEP buffer) used for DEP manipulation has the drawback of poor cell adhesion and cell viability. A microchip prototype with well-defined positioning of titanium electrode arrays was designed and fabricated on a glass substrate. The gap between the radial electrodes was accurately determined to achieve good cell patterning performance. Parameters such as dimension of positioning electrode, amplitude, and frequency of voltage signal were investigated to optimize the performance of the microchip.
Biotechnology Journal 10/2010; 5(10):1005-15.
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ABSTRACT: The preserved fungal species Antrodia camphorata has diverse health-promoting effects and has been popularly used in East Asia as a traditional herb. We isolated a volatile compound from the culture medium of A. camphorata and identified it as gamma-dodecalactone (gamma-DDL). Cytomic screening for immune-modulating activity revealed that gamma-DDL can activate human NK cells to express the early activation marker CD69. Further experiments showed that gamma-DDL not only can induce NK cells to express CD69 but also stimulate NK cells to secrete cytotoxic molecules (FasL and granzyme B) and Th1 cytokines (TNF-alpha and INF-gamma). Measuring the distribution of gamma-DDL in the subcellular compartments of NK cells revealed that gamma-DDL has been converted to 4-hydroxydodecanoic acid (an acyclic isomer of gamma-DDL) in a time-dependent manner in the cytoplasm. Synthetic (R,S)-4-hydroxydodecanoic acid activated NK cells to express CD69 mRNA within 10min, in contrast to gamma-DDL, which activated NK cells to express CD69 within 50min. This faster activation suggests that gamma-DDL has converted to 4-hydroxydodecanoic acid and to stimulate the NK cells to express CD69. Optically pure (R)-(+)-4-hydroxydodecanoic acid and (S)-(-)-4-hydroxydodecanoic acid were obtained via: (1) synthesis of its diastereomeric esters of (R,S)-4-hydroxydodecanoic (R)-(-)-2-phenylpropionate; (2) separation of diastereomers via preparative HPLC, and (3) subsequent hydrolysis of the obtained optical pure ester of (R)-(+)-4-hydroxydodecanoic acid (R)-(-)-2-phenylpropionate and (R)-(-)-4-hydroxydodecanoic acid (R)-(-)-2-phenylpropionate, respectively. Further assays of NK cells activation using each enantiomer showed that only the (R)-(+)-4-hydroxydodecanoic acid can activate NK cells.
Bioorganic & medicinal chemistry 09/2010; 18(18):6896-904. · 2.82 Impact Factor
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ABSTRACT: The manufacturing processes of many electronic and medical products demand the use of high-quality water. Hence the water supply systems for these processes are required to be examined regularly for the presence of microorganisms and microbial biofilms. Among commonly used bacteria detection approaches, the ATP luminescence assay is a rapid, sensitive, and easy to perform method. The aim of this study is to investigate whether ATP regeneration from inorganic pyrophosphate, a product of the ATP luminescence assay, can stabilize the bioluminescence signals in ATP detection. ADPglc pyrophosphorylase (AGPPase), which catalyzes the synthesis of ATP from PP(i) in the presence of ADPglc, was selected because the system yields much lower luminescence background than the commercially available ATP sulfurylase/adenosine 5'-phosphosulfate (APS) system which was broadly used in pyrosequencing technology. The AGPPase-based assay could be used to measure both PP(i) and ATP quantitatively and shows 1.5- to 4.0-fold slight increases in a 10-min assay. The method could also be used to stabilize the luminescence signals in detection of Escherichia coli, Pseudomonas aeruginosa, and Bacillus cereus in either broth or biofilm. These findings suggest that the AGPPase-based ATP regeneration system will find many practical applications such as detection of bacterial biofilm in water pipelines.
Analytical Biochemistry 04/2010; 399(2):168-73. · 3.00 Impact Factor
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ABSTRACT: Membrane-bound IgE (mIgE) is part of the IgE-BCR and is essential for generating isotype-specific IgE responses. On mIgE(+) B cells, the membrane-bound epsilon-chain (mepsilon) exists predominantly in the long isoform, mepsilon(L), containing an extra 52 aa CepsilonmX domain between CH4 and the C-terminal membrane-anchoring segment; the short isoform of mepsilon, mepsilon(S), exists in minor proportions. CepsilonmX thus provides an attractive site for immunologic targeting of mIgE(+) B cells. In this study, we show that nine newly prepared CepsilonmX-specific mAbs, as well as the previously reported a20, bound to mIgE.Fc(L)-expressing CHO cells, while only 4B12 and 26H2 bound to mIgE.Fc(L)-expressing B cell line Ramos cells. The mAb 4B12 bound to the N-terminal part, 26H2 the middle part, and all others the C-terminal part of CepsilonmX. Expression of Igalpha and Igbeta on the mIgE.Fc(L)-CHO cells reduces the binding of a20 to CepsilonmX as compared with that of 4B12 and 26H2. The chimeric mAbs c4B12 and c26H2, when cross-linked by secondary antibodies, lysed mIgE.Fc(L)-Ramos cells by apoptosis through a BCR-dependent caspase pathway. Using PBMCs as the source of effector cells, c4B12 and c26H2 demonstrated Ab-dependent cellular cytotoxicity toward mIgE.Fc(L)-Ramos cells in a dose-dependent fashion. In cultures of PBMCs from atopic dermatitis patients, c4B12 and c26H2 inhibited the synthesis of IgE driven by anti-CD40 and IL-4. These results suggest that 4B12 and 26H2 and an immunogen using the peptide segments recognized by these mAbs are potentially useful for targeting mIgE(+) B cells to control IgE production.
The Journal of Immunology 02/2010; 184(4):1748-56. · 5.79 Impact Factor
