Kazuto Tajiri

A rapid and efficient single-cell manipulation method for screening antigen-specific antibody-secreting cells from human peripheral blood.

Antigen-specific human monoclonal antibodies (mAbs) are key candidates for therapeutic agents. However, the availability of a suitable screening system for antigen-specific antibody–secreting cells (ASCs) is limited in humans. Here we present a unique method for detecting individual ASCs using microwell array chips, which enables the analysis of live cells on a single-cell basis and offers a rapid, efficient and high-throughput (up to 234,000 individual cells) system for identifying and recovering objective ASCs. We applied the system to detect and retrieve ASCs for hepatitis B virus and influenza viruses from human peripheral blood lymphocytes and produced human mAbs with virus-neutralizing activities within a week. Furthermore, we show that the system is useful for detecting ASCs for multiple antigens as well as for selection of ASCs secreting high-affinity antibodies on a chip. Our method can open the way for the generation of therapeutic antibodies for individual patients.

Single lymphocyte analysis with a microwell array chip.

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.

Role of liver-infiltrating CD3+CD56+ natural killer T cells in the pathogenesis of nonalcoholic fatty liver disease.

Background/Aims 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. Methods 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. Results 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 V&agr;24+ NKT cells, which produced more IFN-&ggr; and IL-4 as NAS increased. Conclusion 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.

Branched-chain amino acids in liver diseases

Branched chain amino acids (BCAAs) have been shown to affect gene expression, protein metabolism, apoptosis and regeneration of hepatocytes, and insulin resistance. They have also been shown to inhibit the proliferation of liver cancer cells in vitro, and are essential for lymphocyte proliferation and dendritic cell maturation. In patients with advanced chronic liver disease, BCAA concentrations are low, whereas the concentrations of aromatic amino acids such as phenylalanine and tyrosine are high, conditions that may be closely associated with hepatic encephalopathy and the prognosis of these patients. Based on these basic observations, patients with advanced chronic liver disease have been treated clinically with BCAA-rich medicines, with positive effects.

Cell-microarray analysis of antigen-specific B-cells: single cell analysis of antigen receptor expression and specificity.

The authors previously developed a cell‐microarray system that effectively detects antigen‐specific B‐cells by monitoring intracellular Ca2+ 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 single‐cell 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.

Liver physiology and liver diseases in the elderly.

The liver experiences various changes with aging that could affect clinical characteristics and outcomes in patients with liver diseases. Both liver volume and blood flow decrease significantly with age. These changes and decreased cytochrome P450 activity can affect drug metabolism, increasing susceptibility to drug-induced liver injury. Immune responses against pathogens or neoplastic cells are lower in the elderly, although these individuals may be predisposed to autoimmunity through impairment of dendritic cell maturation and reduction of regulatory T cells. These changes in immune functions could alter the pathogenesis of viral hepatitis and autoimmune liver diseases, as well as the development of hepatocellular carcinoma. Moreover, elderly patients have significantly decreased reserve functions of various organs, reducing their tolerability to treatments for liver diseases. Collectively, aged patients show various changes of the liver and other organs that could affect the clinical characteristics and management of liver diseases in these patients.

Rapid isolation of antigen-specific antibody-secreting cells using a chip-based immunospot array

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.

Development of osteomalacia in a post-liver transplant patient receiving adefovir dipivoxil

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 recipients treated with both ADV and tacrolimus.

Role of NKT Cells in the Pathogenesis of NAFLD

Nonalcoholic fatty liver disease (NAFLD) is the most frequent chronic liver disease and shows various inflammatory changes in the liver. Among those inflammatory cells, natural killer T (NKT) cells are found to have a critical role during the disease progression. NKT cells may have a protective role at the early stage with simple steatosis through modification of insulin resistance, whereas they act as a progression factor at the advanced stage with fibrosis. Those processes are thought to depend on interaction between NKT cells and CD1d molecule in the liver.

Analysis of the epitope and neutralizing capacity of human monoclonal antibodies induced by hepatitis B vaccine.

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.