We report the case of an adult male with progressing focal nodular hyperplasia (FNH). Although im... more We report the case of an adult male with progressing focal nodular hyperplasia (FNH). Although imaging studies suggested that the tumor was a classical FNH, the tumor biopsy showed glutamine synthetase expression and heat shock protein 70 in part of the tumor. As we could not definitely distinguish this case of FNH from early hepatocellular carcinoma (HCC), we performed laparoscopic partial hepatectomy. The surgical resected specimen showed that the tumor had a central scar with vascular and cholangiolar proliferation, which is compatible with FNH. Immunohistochemical analysis showed that the molecular expression pattern was compatible with FNH in the center of the tumor, whereas it partly resembled early HCC in the periphery of the tumor. FNH progression is occasionally found, and the molecular pattern of the progressing area in FNH might resemble that of early HCC due to morphologic and phenotypic changes induced by the regenerative mechanism and the alteration of blood flow. We should carefully observe progressing FNH.
Primary biliary cirrhosis (PBC) is a progressive liver disease for which limited therapies are re... more Primary biliary cirrhosis (PBC) is a progressive liver disease for which limited therapies are recommended. Rituximab, an anti-CD20 monoclonal antibody, is expected to be a useful therapeutic regimen for PBC. Previous studies indicated biochemical and immunological improvement in PBC after rituximab treatment. Although rituximab shows therapeutic potential for PBC, few cases have been reported and histological improvement and long-term outcome remain uncertain. Here, we report a case of PBC in a 66-year-old Japanese female patient who presented with a gastric lymphoma and who had been treated with a regimen containing rituximab for incidental malignant lymphoma. She showed biochemical and immunological improvements, and liver histology before and after rituximab treatment confirmed a decrease in liver inflammation. However, she developed liver cirrhosis a short time after rituximab treatment without biochemical or immunological worsening. Rituximab treatment for PBC might be conside...
We report the case of a patient treated with living donor-related liver transplantation who suffe... more We report the case of a patient treated with living donor-related liver transplantation who suffered from osteomalacia during adefovir dipivoxil (ADV)-containing antiviral therapy for lamivudine-resistant hepatitis B virus infection. The patient had generalized bone pain, with severe hypophosphatemia after 20 mo of ADV therapy. Radiographic studies demonstrated the presence of osteomalacia. The peak plasma ADV level was 38 ng/mL after administration of ADV at 10 mg/d. It was also found that ADV affected the metabolism of tacrolimus, a calcineurin-inhibitor, and caused an increase in the plasma levels of tacrolimus. The disability was reversed with the withdrawal of ADV and with mineral supplementation. ADV can cause an elevation of plasma tacrolimus levels, which may be associated with renal dysfunction. High levels of ADV and tacrolimus can cause nephrotoxicity and osteomalacia. This case highlights the importance of considering a diagnosis of osteomalacia in liver transplantation ...
The International journal of eating disorders, 2006
Liver injury is well known as a complication of anorexia nervosa (AN), and steatosis of the liver... more Liver injury is well known as a complication of anorexia nervosa (AN), and steatosis of the liver has been thought to be the main cause of the injury. However, the precise mechanism for the liver injury is still unclear. We had a chance to examine the histological change of the liver with AN, and found that not only fat deposit but also peroxidated lipid products and irons were recognized in hepatocytes. Our present case demonstrates that oxidative stresses in hepatocytes, which might be associated with iron deposition, could be involved in the pathogenesis of liver injury in patients with AN.
A 47-year-old Japanese man was first admitted to our hospital for 8 days because of an asthma att... more A 47-year-old Japanese man was first admitted to our hospital for 8 days because of an asthma attack. After discharge he changed his diet. On the 12th day after his discharge, he was re-admitted to our hospital because he exhibited transient loss of consciousness with flapping tremor. His plasma ammonia level was extremely high (245 lg/dL; normal, \90 lg/dL), suggesting hepatic encephalopathy. He underwent intravenous administration of branched-chain amino acids (Aminoleban Ò ) and oral administration of lactulose and kanamycin sulfate; however, the hyperammonemia did not improve. Analysis of the amino acids and citrin gene led to the diagnosis of adult-onset type II citrullinemia (CTLN2). Following this diagnosis, the carbohydrate content of his diet was mildly restricted. As a result, his plasma ammonia level markedly improved (ammonia, 40-60 lg/dL) and he became symptom-free without any medication. CTLN2 is a metabolic disorder characterized by increased plasma concentrations of citrulline and ammonia, which occurs by the failure of compensatory mechanisms associated with diet. Here, we report a case of a patient for whom a change in eating habits during his hospitalization disturbed his compensatory mechanism resulting in clinical CTLN2, which was reversed with an appropriate diet.
We encountered a patient with previously wellcontrolled Wilson disease who experienced fulminant ... more We encountered a patient with previously wellcontrolled Wilson disease who experienced fulminant hepatic failure with hemolytic anemia, possibly caused by the dietary supplement Health Proportion Ò (Jubilant Co., Ltd., Ehime, Japan). A 21-year-old woman was admitted to our hospital with marked liver dysfunction and severe hemolytic anemia. Free serum copper level was elevated at 101 lg/dl, and urinary copper excretion was extremely increased (25,600 lg/day). Plasma exchange and continuous hemodiafiltration were performed to remove serum copper and to treat the hemolytic anemia. However, liver function did not improve, and she underwent liver transplantation on 28th day after admission. Copper and iron contents in the resected liver were high at 851.9 lg and 551.7 lg/dry liver weight (g), respectively, despite the patient having regularly taken D-penicillamine since diagnosis and having a well-controlled copper level 1 year before her admission. Two months before admission, the patient had taken a dietary supplement made from soybeans for 1 month. This supplement was labeled as containing large amounts of copper and iron, and we assume that this caused fulminant hepatic failure with hemolytic crisis in this patient. It is important to be mindful of the micronutrient content of dietary supplements, especially for metabolic disorder patients.
Here we report a new method for isolating antigen-specific antibody-secreting cells (ascs) using ... more Here we report a new method for isolating antigen-specific antibody-secreting cells (ascs) using a microwell array chip, which offers a rapid, efficient and high-throughput (up to 234,000 individual cells) system for the detection and retrieval of cells that secrete antibodies of interest on a single-cell basis. We arrayed a large population of lymphoid cells containing ascs from human peripheral blood on microwell array chips and detected spots with secreted antibodies. this protocol can be completed in less than 7 h, including 3 h of cell culture. the method presented here not only has high sensitivity and specificity comparable with enzyme-linked immunospot (elIspot) but it also overcomes the limitations of elIspot in recovering ascs that can be used to produce antigen-specific human monoclonal antibodies. this method can also be used to detect cells secreting molecules other than antibodies, such as cytokines, and it provides a tool for cell analysis and clinical diagnosis.
European Journal of Gastroenterology & Hepatology, 2009
Natural killer T (NKT) cells have been recently reported to concern with various lipid disorders.... more Natural killer T (NKT) cells have been recently reported to concern with various lipid disorders. The role of NKT cells in the hepatic lipid disorder, nonalcoholic fatty liver disease (NAFLD), has, however, not yet been clarified. To assess the role of NKT cells in the pathogenesis of NAFLD, we analyzed the composition and function of liver-infiltrating cells isolated from liver biopsy specimens of patients with NAFLD. Specimens from 62 patients with NAFLD were studied, and 54 specimens among them reacted immunohistochemically with monoclonal antibodies against various surface markers. Moreover, using flow cytometry, we analyzed surface markers and intracytoplasmic cytokines of intrahepatic CD3+CD56+ cells in 12 patients among them. Among the various populations of liver-infiltrating cells, only the numbers of CD56+ cells were significantly increased as NAFLD disease activity (NAFLD activity score, NAS) increased. Furthermore, expression of CD1d, a ligand for NKT cells, was also increased in NAFLD as NAS increased. Flow cytometric analysis showed that most CD56+ cells were Valpha24+ NKT cells, which produced more IFN-gamma and IL-4 as NAS increased. Intrahepatic CD3+CD56+ NKT cells are increased in NAFLD as NAS increased. These cells may enhance disease activity through cytokine production after the recognition of lipid antigens presented with CD1d in livers of NAFLD.
The analysis of single cells with multiple parameters in flow cytometry or microscopy requires su... more The analysis of single cells with multiple parameters in flow cytometry or microscopy requires suitable combinations of fluorophores and optical filters. The growing demands for the multiplex analysis of cells increase the requirements for developing new fluorophores and techniques. We have developed a novel method of analyzing a large number of cells with multiple parameters on a single-cell basis using a single fluorophore. Cells were arrayed onto a microwell array chip with an array of 45,000 microwells, which could capture single cells, stained with a phycoerythrin (PE)-conjugated antibody to a marker, and analyzed with a cell-scanner. After analysis, we photobleached the PE molecules by irradiating the sample with blue light. Because the fluorescence of PE was not recovered after the photobleaching and the analyzed cells remained in the same microwells on the chip, we could repeatedly stain and analyze the same cells with other markers using PE. We applied a method of analyzing lymphocytes from 100 lL of peripheral blood for cytokine secretion and expression of intracellular proteins as well as for multiple cell surface markers. This novel method enables us to analyze multiple markers with a single fluorophore using a simple apparatus. The method may expand the scope of cytometry. '
B cells are very heterogeneous, consisting of more than 10 9 B-cell clones with distinct specific... more B cells are very heterogeneous, consisting of more than 10 9 B-cell clones with distinct specificities for antigens in each individual. To identify single B cells with antigen specificity, we have been developing cell microarray technology using microwell array chips whose microwells each capture a single B cell. Using microwell array chips, we detected antigen-specific B cells by monitoring antigen-induced intracellular Ca 21 mobilization with a CCD scanner (MAC-CCD system) or the binding of fluorescence-labeled antigen to cells with a confocal laser scanner. We retrieved target cells from the chip, cloned immunoglobulin genes, and produced antigen-specific antibodies. However, these methods present some difficulties: the former technique could not detect cells whose frequency was less than 0.05% and the latter one took a long time to identify the objective cells although it could detect cells at a frequency of 0.01%. Here, we have combined the advantages of these two methods. Monitoring antigen-induced intracellular Ca 21 mobilizations and the binding of fluorescence-labeled antigens simultaneously with a MAC-CCD system enabled us to detect rapidly, antigen-specific B cells whose frequency was less than 0.01% with high efficiency. Our system provides a superior screening system for antigen-specific B cells and extends the horizons of multiparameter singlecell analysis in heterogeneous cell populations. ' 2009 International Society for Advancement of Cytometry
Following genomics and proteomics, cytomics, a novel method of looking at life, has emerged for a... more Following genomics and proteomics, cytomics, a novel method of looking at life, has emerged for analyzing large populations of cells on a single-cell basis with multiple parameters in a quantitative manner. We have developed a highly integrated live-cell microarray system for analyzing the cellular responses of individual cells using a microwell array chip that has 234,000 microwells each of which is just large enough to fit a single cell. Compared with flow cytometry and microscope-based methods, our system can analyze the history of the cellular responses of a large number of cells. We have successfully applied the system to analyze human antigen-specific B-cells and produced human monoclonal antibodies (MoAb) against hepatitis B virus surface antigen. We have also constructed a mouse system to assess hepatitis B virus-neutralization activity and have demonstrated the neutralization activity of our antibodies. Our technology should expand the horizons of cell analysis as well as enable generation of human MoAb for antibody-based therapeutics and diagnosis for infectious diseases such as hepatitis viruses. '
The authors previously developed a cell-microarray system that effectively detects antigen-specif... more The authors previously developed a cell-microarray system that effectively detects antigen-specific B-cells by monitoring intracellular Ca 21 at single cell levels. Here they present a novel method to detect antigen-specific B-cells using cell-microarray system. To detect antigen-specific B-cells, they arrayed live lymphocytes on a chip, stained cells with fluorescence-labeled nonspecific proteins, and analyzed them with a fluorescence scanner to detect nonspecific protein binding to B-cells. They then stained cells with fluorescence-labeled antigen and analyzed them with the scanner. Cells stained with specific antigen, but not with nonspecific proteins, were determined as antigen-specific B-cells and harvested. Antibody cDNA was amplified from retrieved B-cells by singlecell RT-PCR, inserted into expression vectors, and was examined for its specificity by ELISA. They could detect antigen-specific B-cells at a frequency of 0.01% in a model system using transgenic mice that express antibody to hen-egg lysozyme on the surface of B-cells. They applied this system to directly detect hepatitis B virus surface-antigen (HBs-Ag)-specific B-cells from peripheral blood in HBs-Ag-vaccinated volunteers and succeeded in producing HBs-Ag-specific monoclonal antibody. This novel system allows us to identify human antigen-specific B-cells of very low frequency and is a powerful tool to explore the candidates of antibody therapeutics. ' 2007 International
Biochemical and Biophysical Research Communications, 2010
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptors (TRAIL-R1 and T... more Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptors (TRAIL-R1 and TRAIL-R2) are promising targets for tumor therapy. However, their clinical use is limited because some tumors show resistance to TRAIL-treatment. Here, we analyzed epitopes of nine TRAIL-R1-specific human monoclonal antibodies and demonstrated at least five tentative epitopes on human TRAIL-R1. We found that some of the five were post-translationally modified on some tumor cell lines. Interestingly, one of them, an epitope of TR1-272 antibody (TR1-272-epitope) disappeared on the tumor cells that are more susceptible to TRAIL-induced apoptosis compared to TR1-272-epitope positive cells. Treatment of TR1-272-epitope negative cells with TRAIL induced large cluster formation of TRAIL-R1, while treatment of TR1-272-epiope positive cells with TRAIL did not. These results suggest that TR1-272-antibody might distinguish the TRAIL-R1 conformation that could deliver stronger death signals. Further analysis of epitope-appearance and sensitivity to TRAIL should clarify the mechanisms of TRAIL-induced apoptosis of tumor cells and would provide useful information about tumor therapy using TRAIL and TRAIL-R signaling.
Biochemical and Biophysical Research Communications, 2008
A Vb TCR repertoire is analyzed for understanding the T-cell population in the immune response. H... more A Vb TCR repertoire is analyzed for understanding the T-cell population in the immune response. However, the TCR repertoire of the Va-Vb pair is difficult to analyze because no suitable analytical method is available. Here, we have applied the single-cell 5 0 -RACE method for amplifying TCR cDNAs from single T-cells and analyzed the repertoire of Va-Vb pairs in human T-cells that responded to a superantigen, SEB. We found that the TCR Vb profile of the SEB-stimulated CD4 + T-cells was in accordance with the previous reports, that the TCR Va profile also exhibited a prominent difference, and that the TCR Va-Vb pairs of the SEB-responding T-cells were promiscuous. We have also found a significant dual TCRa expression in single T-cells. This is the first report of a comprehensive analysis of the functional repertoire of Va-Vb pairs at the single T-cell level. This novel method may contribute to TCR-based immunotherapeutics.
Hepatitis B virus (HBV) is an infectious agent that is a significant worldwide public health issu... more Hepatitis B virus (HBV) is an infectious agent that is a significant worldwide public health issue. However, the mechanism by which vaccination-induced antibodies prevent HBV infection remains unclear. To investigate the mechanism by which antibodies induced by hepatitis B surface Ag (HBsAg)-vaccination prevent HBV infection in humans, we prepared human monoclonal antibodies (mAbs) against HBsAg using a novel cell-microarray system from peripheral blood B-lymphocytes from vaccinated individuals. We then characterized the IgG subclass, L-chain subtype, and V-gene repertoire of the H/L-chain, as well as affinities of each of these mAbs. We also determined the epitopes of the individual mAbs using synthesized peptides, and the HBV-neutralizing activities of mAbs using the hepatocyte cell line HepaRG. Consequently, IgG1 and kappa chain was mainly used as the mAbs for HBsAg. Seventy percent of the mAbs bound to the loop domain of the small-HBsAg and showed greater neutralizing activities. There were no relationships between their affinities and neutralization activities. A combination of mAbs recognizing the first loop domain showed a synergistic effect on HBV-neutralizing activity that surpassed conventional hepatitis B-Ig (HBIG) in the HepaRG cell line assay. These results may contribute to the development of effective mAb treatment against HBV infection replacing conventional HBIG administration.
The extra-cellular domain of the influenza virus matrix protein 2 (M2e) is highly conserved betwe... more The extra-cellular domain of the influenza virus matrix protein 2 (M2e) is highly conserved between influenza A virus strains compared to hemagglutinin and neuraminidase, and has long been viewed as a potential and universal vaccine target. M2e induces no or only weak and transient immune responses following infection, making it difficult to detect M2e-specific antibodies producing B-cells in human peripheral blood lymphocytes. Recently, using a single-cell manipulation method, immunospot array assay on a chip (ISAAC), we obtained an M2e-specific human antibody (Ab1-10) from the peripheral blood of a healthy volunteer. In this report, we have demonstrate that Ab1-10 reacted not only to seasonal influenza A viruses, but also to pandemic (H1N1) 2009 virus (2009 H1N1) and highly pathogenic avian influenza A virus, and that the antibody-bound M2e of 2009 H1N1 inactivated the virus with high affinity (∼10(-10)M). More importantly, it inhibited 2009 H1N1 viral propagation in vitro. These results suggest that Ab1-10 might be a potential candidate for antibody therapeutics for a wide range of influenza A viruses.
We report the case of an adult male with progressing focal nodular hyperplasia (FNH). Although im... more We report the case of an adult male with progressing focal nodular hyperplasia (FNH). Although imaging studies suggested that the tumor was a classical FNH, the tumor biopsy showed glutamine synthetase expression and heat shock protein 70 in part of the tumor. As we could not definitely distinguish this case of FNH from early hepatocellular carcinoma (HCC), we performed laparoscopic partial hepatectomy. The surgical resected specimen showed that the tumor had a central scar with vascular and cholangiolar proliferation, which is compatible with FNH. Immunohistochemical analysis showed that the molecular expression pattern was compatible with FNH in the center of the tumor, whereas it partly resembled early HCC in the periphery of the tumor. FNH progression is occasionally found, and the molecular pattern of the progressing area in FNH might resemble that of early HCC due to morphologic and phenotypic changes induced by the regenerative mechanism and the alteration of blood flow. We should carefully observe progressing FNH.
Primary biliary cirrhosis (PBC) is a progressive liver disease for which limited therapies are re... more Primary biliary cirrhosis (PBC) is a progressive liver disease for which limited therapies are recommended. Rituximab, an anti-CD20 monoclonal antibody, is expected to be a useful therapeutic regimen for PBC. Previous studies indicated biochemical and immunological improvement in PBC after rituximab treatment. Although rituximab shows therapeutic potential for PBC, few cases have been reported and histological improvement and long-term outcome remain uncertain. Here, we report a case of PBC in a 66-year-old Japanese female patient who presented with a gastric lymphoma and who had been treated with a regimen containing rituximab for incidental malignant lymphoma. She showed biochemical and immunological improvements, and liver histology before and after rituximab treatment confirmed a decrease in liver inflammation. However, she developed liver cirrhosis a short time after rituximab treatment without biochemical or immunological worsening. Rituximab treatment for PBC might be conside...
We report the case of a patient treated with living donor-related liver transplantation who suffe... more We report the case of a patient treated with living donor-related liver transplantation who suffered from osteomalacia during adefovir dipivoxil (ADV)-containing antiviral therapy for lamivudine-resistant hepatitis B virus infection. The patient had generalized bone pain, with severe hypophosphatemia after 20 mo of ADV therapy. Radiographic studies demonstrated the presence of osteomalacia. The peak plasma ADV level was 38 ng/mL after administration of ADV at 10 mg/d. It was also found that ADV affected the metabolism of tacrolimus, a calcineurin-inhibitor, and caused an increase in the plasma levels of tacrolimus. The disability was reversed with the withdrawal of ADV and with mineral supplementation. ADV can cause an elevation of plasma tacrolimus levels, which may be associated with renal dysfunction. High levels of ADV and tacrolimus can cause nephrotoxicity and osteomalacia. This case highlights the importance of considering a diagnosis of osteomalacia in liver transplantation ...
The International journal of eating disorders, 2006
Liver injury is well known as a complication of anorexia nervosa (AN), and steatosis of the liver... more Liver injury is well known as a complication of anorexia nervosa (AN), and steatosis of the liver has been thought to be the main cause of the injury. However, the precise mechanism for the liver injury is still unclear. We had a chance to examine the histological change of the liver with AN, and found that not only fat deposit but also peroxidated lipid products and irons were recognized in hepatocytes. Our present case demonstrates that oxidative stresses in hepatocytes, which might be associated with iron deposition, could be involved in the pathogenesis of liver injury in patients with AN.
A 47-year-old Japanese man was first admitted to our hospital for 8 days because of an asthma att... more A 47-year-old Japanese man was first admitted to our hospital for 8 days because of an asthma attack. After discharge he changed his diet. On the 12th day after his discharge, he was re-admitted to our hospital because he exhibited transient loss of consciousness with flapping tremor. His plasma ammonia level was extremely high (245 lg/dL; normal, \90 lg/dL), suggesting hepatic encephalopathy. He underwent intravenous administration of branched-chain amino acids (Aminoleban Ò ) and oral administration of lactulose and kanamycin sulfate; however, the hyperammonemia did not improve. Analysis of the amino acids and citrin gene led to the diagnosis of adult-onset type II citrullinemia (CTLN2). Following this diagnosis, the carbohydrate content of his diet was mildly restricted. As a result, his plasma ammonia level markedly improved (ammonia, 40-60 lg/dL) and he became symptom-free without any medication. CTLN2 is a metabolic disorder characterized by increased plasma concentrations of citrulline and ammonia, which occurs by the failure of compensatory mechanisms associated with diet. Here, we report a case of a patient for whom a change in eating habits during his hospitalization disturbed his compensatory mechanism resulting in clinical CTLN2, which was reversed with an appropriate diet.
We encountered a patient with previously wellcontrolled Wilson disease who experienced fulminant ... more We encountered a patient with previously wellcontrolled Wilson disease who experienced fulminant hepatic failure with hemolytic anemia, possibly caused by the dietary supplement Health Proportion Ò (Jubilant Co., Ltd., Ehime, Japan). A 21-year-old woman was admitted to our hospital with marked liver dysfunction and severe hemolytic anemia. Free serum copper level was elevated at 101 lg/dl, and urinary copper excretion was extremely increased (25,600 lg/day). Plasma exchange and continuous hemodiafiltration were performed to remove serum copper and to treat the hemolytic anemia. However, liver function did not improve, and she underwent liver transplantation on 28th day after admission. Copper and iron contents in the resected liver were high at 851.9 lg and 551.7 lg/dry liver weight (g), respectively, despite the patient having regularly taken D-penicillamine since diagnosis and having a well-controlled copper level 1 year before her admission. Two months before admission, the patient had taken a dietary supplement made from soybeans for 1 month. This supplement was labeled as containing large amounts of copper and iron, and we assume that this caused fulminant hepatic failure with hemolytic crisis in this patient. It is important to be mindful of the micronutrient content of dietary supplements, especially for metabolic disorder patients.
Here we report a new method for isolating antigen-specific antibody-secreting cells (ascs) using ... more Here we report a new method for isolating antigen-specific antibody-secreting cells (ascs) using a microwell array chip, which offers a rapid, efficient and high-throughput (up to 234,000 individual cells) system for the detection and retrieval of cells that secrete antibodies of interest on a single-cell basis. We arrayed a large population of lymphoid cells containing ascs from human peripheral blood on microwell array chips and detected spots with secreted antibodies. this protocol can be completed in less than 7 h, including 3 h of cell culture. the method presented here not only has high sensitivity and specificity comparable with enzyme-linked immunospot (elIspot) but it also overcomes the limitations of elIspot in recovering ascs that can be used to produce antigen-specific human monoclonal antibodies. this method can also be used to detect cells secreting molecules other than antibodies, such as cytokines, and it provides a tool for cell analysis and clinical diagnosis.
European Journal of Gastroenterology & Hepatology, 2009
Natural killer T (NKT) cells have been recently reported to concern with various lipid disorders.... more Natural killer T (NKT) cells have been recently reported to concern with various lipid disorders. The role of NKT cells in the hepatic lipid disorder, nonalcoholic fatty liver disease (NAFLD), has, however, not yet been clarified. To assess the role of NKT cells in the pathogenesis of NAFLD, we analyzed the composition and function of liver-infiltrating cells isolated from liver biopsy specimens of patients with NAFLD. Specimens from 62 patients with NAFLD were studied, and 54 specimens among them reacted immunohistochemically with monoclonal antibodies against various surface markers. Moreover, using flow cytometry, we analyzed surface markers and intracytoplasmic cytokines of intrahepatic CD3+CD56+ cells in 12 patients among them. Among the various populations of liver-infiltrating cells, only the numbers of CD56+ cells were significantly increased as NAFLD disease activity (NAFLD activity score, NAS) increased. Furthermore, expression of CD1d, a ligand for NKT cells, was also increased in NAFLD as NAS increased. Flow cytometric analysis showed that most CD56+ cells were Valpha24+ NKT cells, which produced more IFN-gamma and IL-4 as NAS increased. Intrahepatic CD3+CD56+ NKT cells are increased in NAFLD as NAS increased. These cells may enhance disease activity through cytokine production after the recognition of lipid antigens presented with CD1d in livers of NAFLD.
The analysis of single cells with multiple parameters in flow cytometry or microscopy requires su... more The analysis of single cells with multiple parameters in flow cytometry or microscopy requires suitable combinations of fluorophores and optical filters. The growing demands for the multiplex analysis of cells increase the requirements for developing new fluorophores and techniques. We have developed a novel method of analyzing a large number of cells with multiple parameters on a single-cell basis using a single fluorophore. Cells were arrayed onto a microwell array chip with an array of 45,000 microwells, which could capture single cells, stained with a phycoerythrin (PE)-conjugated antibody to a marker, and analyzed with a cell-scanner. After analysis, we photobleached the PE molecules by irradiating the sample with blue light. Because the fluorescence of PE was not recovered after the photobleaching and the analyzed cells remained in the same microwells on the chip, we could repeatedly stain and analyze the same cells with other markers using PE. We applied a method of analyzing lymphocytes from 100 lL of peripheral blood for cytokine secretion and expression of intracellular proteins as well as for multiple cell surface markers. This novel method enables us to analyze multiple markers with a single fluorophore using a simple apparatus. The method may expand the scope of cytometry. '
B cells are very heterogeneous, consisting of more than 10 9 B-cell clones with distinct specific... more B cells are very heterogeneous, consisting of more than 10 9 B-cell clones with distinct specificities for antigens in each individual. To identify single B cells with antigen specificity, we have been developing cell microarray technology using microwell array chips whose microwells each capture a single B cell. Using microwell array chips, we detected antigen-specific B cells by monitoring antigen-induced intracellular Ca 21 mobilization with a CCD scanner (MAC-CCD system) or the binding of fluorescence-labeled antigen to cells with a confocal laser scanner. We retrieved target cells from the chip, cloned immunoglobulin genes, and produced antigen-specific antibodies. However, these methods present some difficulties: the former technique could not detect cells whose frequency was less than 0.05% and the latter one took a long time to identify the objective cells although it could detect cells at a frequency of 0.01%. Here, we have combined the advantages of these two methods. Monitoring antigen-induced intracellular Ca 21 mobilizations and the binding of fluorescence-labeled antigens simultaneously with a MAC-CCD system enabled us to detect rapidly, antigen-specific B cells whose frequency was less than 0.01% with high efficiency. Our system provides a superior screening system for antigen-specific B cells and extends the horizons of multiparameter singlecell analysis in heterogeneous cell populations. ' 2009 International Society for Advancement of Cytometry
Following genomics and proteomics, cytomics, a novel method of looking at life, has emerged for a... more Following genomics and proteomics, cytomics, a novel method of looking at life, has emerged for analyzing large populations of cells on a single-cell basis with multiple parameters in a quantitative manner. We have developed a highly integrated live-cell microarray system for analyzing the cellular responses of individual cells using a microwell array chip that has 234,000 microwells each of which is just large enough to fit a single cell. Compared with flow cytometry and microscope-based methods, our system can analyze the history of the cellular responses of a large number of cells. We have successfully applied the system to analyze human antigen-specific B-cells and produced human monoclonal antibodies (MoAb) against hepatitis B virus surface antigen. We have also constructed a mouse system to assess hepatitis B virus-neutralization activity and have demonstrated the neutralization activity of our antibodies. Our technology should expand the horizons of cell analysis as well as enable generation of human MoAb for antibody-based therapeutics and diagnosis for infectious diseases such as hepatitis viruses. '
The authors previously developed a cell-microarray system that effectively detects antigen-specif... more The authors previously developed a cell-microarray system that effectively detects antigen-specific B-cells by monitoring intracellular Ca 21 at single cell levels. Here they present a novel method to detect antigen-specific B-cells using cell-microarray system. To detect antigen-specific B-cells, they arrayed live lymphocytes on a chip, stained cells with fluorescence-labeled nonspecific proteins, and analyzed them with a fluorescence scanner to detect nonspecific protein binding to B-cells. They then stained cells with fluorescence-labeled antigen and analyzed them with the scanner. Cells stained with specific antigen, but not with nonspecific proteins, were determined as antigen-specific B-cells and harvested. Antibody cDNA was amplified from retrieved B-cells by singlecell RT-PCR, inserted into expression vectors, and was examined for its specificity by ELISA. They could detect antigen-specific B-cells at a frequency of 0.01% in a model system using transgenic mice that express antibody to hen-egg lysozyme on the surface of B-cells. They applied this system to directly detect hepatitis B virus surface-antigen (HBs-Ag)-specific B-cells from peripheral blood in HBs-Ag-vaccinated volunteers and succeeded in producing HBs-Ag-specific monoclonal antibody. This novel system allows us to identify human antigen-specific B-cells of very low frequency and is a powerful tool to explore the candidates of antibody therapeutics. ' 2007 International
Biochemical and Biophysical Research Communications, 2010
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptors (TRAIL-R1 and T... more Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptors (TRAIL-R1 and TRAIL-R2) are promising targets for tumor therapy. However, their clinical use is limited because some tumors show resistance to TRAIL-treatment. Here, we analyzed epitopes of nine TRAIL-R1-specific human monoclonal antibodies and demonstrated at least five tentative epitopes on human TRAIL-R1. We found that some of the five were post-translationally modified on some tumor cell lines. Interestingly, one of them, an epitope of TR1-272 antibody (TR1-272-epitope) disappeared on the tumor cells that are more susceptible to TRAIL-induced apoptosis compared to TR1-272-epitope positive cells. Treatment of TR1-272-epitope negative cells with TRAIL induced large cluster formation of TRAIL-R1, while treatment of TR1-272-epiope positive cells with TRAIL did not. These results suggest that TR1-272-antibody might distinguish the TRAIL-R1 conformation that could deliver stronger death signals. Further analysis of epitope-appearance and sensitivity to TRAIL should clarify the mechanisms of TRAIL-induced apoptosis of tumor cells and would provide useful information about tumor therapy using TRAIL and TRAIL-R signaling.
Biochemical and Biophysical Research Communications, 2008
A Vb TCR repertoire is analyzed for understanding the T-cell population in the immune response. H... more A Vb TCR repertoire is analyzed for understanding the T-cell population in the immune response. However, the TCR repertoire of the Va-Vb pair is difficult to analyze because no suitable analytical method is available. Here, we have applied the single-cell 5 0 -RACE method for amplifying TCR cDNAs from single T-cells and analyzed the repertoire of Va-Vb pairs in human T-cells that responded to a superantigen, SEB. We found that the TCR Vb profile of the SEB-stimulated CD4 + T-cells was in accordance with the previous reports, that the TCR Va profile also exhibited a prominent difference, and that the TCR Va-Vb pairs of the SEB-responding T-cells were promiscuous. We have also found a significant dual TCRa expression in single T-cells. This is the first report of a comprehensive analysis of the functional repertoire of Va-Vb pairs at the single T-cell level. This novel method may contribute to TCR-based immunotherapeutics.
Hepatitis B virus (HBV) is an infectious agent that is a significant worldwide public health issu... more Hepatitis B virus (HBV) is an infectious agent that is a significant worldwide public health issue. However, the mechanism by which vaccination-induced antibodies prevent HBV infection remains unclear. To investigate the mechanism by which antibodies induced by hepatitis B surface Ag (HBsAg)-vaccination prevent HBV infection in humans, we prepared human monoclonal antibodies (mAbs) against HBsAg using a novel cell-microarray system from peripheral blood B-lymphocytes from vaccinated individuals. We then characterized the IgG subclass, L-chain subtype, and V-gene repertoire of the H/L-chain, as well as affinities of each of these mAbs. We also determined the epitopes of the individual mAbs using synthesized peptides, and the HBV-neutralizing activities of mAbs using the hepatocyte cell line HepaRG. Consequently, IgG1 and kappa chain was mainly used as the mAbs for HBsAg. Seventy percent of the mAbs bound to the loop domain of the small-HBsAg and showed greater neutralizing activities. There were no relationships between their affinities and neutralization activities. A combination of mAbs recognizing the first loop domain showed a synergistic effect on HBV-neutralizing activity that surpassed conventional hepatitis B-Ig (HBIG) in the HepaRG cell line assay. These results may contribute to the development of effective mAb treatment against HBV infection replacing conventional HBIG administration.
The extra-cellular domain of the influenza virus matrix protein 2 (M2e) is highly conserved betwe... more The extra-cellular domain of the influenza virus matrix protein 2 (M2e) is highly conserved between influenza A virus strains compared to hemagglutinin and neuraminidase, and has long been viewed as a potential and universal vaccine target. M2e induces no or only weak and transient immune responses following infection, making it difficult to detect M2e-specific antibodies producing B-cells in human peripheral blood lymphocytes. Recently, using a single-cell manipulation method, immunospot array assay on a chip (ISAAC), we obtained an M2e-specific human antibody (Ab1-10) from the peripheral blood of a healthy volunteer. In this report, we have demonstrate that Ab1-10 reacted not only to seasonal influenza A viruses, but also to pandemic (H1N1) 2009 virus (2009 H1N1) and highly pathogenic avian influenza A virus, and that the antibody-bound M2e of 2009 H1N1 inactivated the virus with high affinity (∼10(-10)M). More importantly, it inhibited 2009 H1N1 viral propagation in vitro. These results suggest that Ab1-10 might be a potential candidate for antibody therapeutics for a wide range of influenza A viruses.
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Papers by Kazuto Tajiri