Detection of cellular response by measuring intracellular calcium, (Ca2+)i with Ca2+-dependent fluorescent dye are standard approaches to detect ligand-stimulated cells and to study signaling through ligand/receptor interaction. We describe a single-cell microarray system to analyze cellular response of individual cells such as lymphocytes using microchamber array chips. The single-cell microarray chip is made from polystyrene with over 30,000 microchambers, which can accommodate only single cells. Lymphocytes derived from mouse spleen or human blood were spread on the microarray, and over 80% of the microchambers achieved single-cell status. Stimulation of B-cells through antigen receptors on the microarray allowed us to detect activated B-cells by comparing the states of single B-cells before and after stimulation with antigen, which is disabled for flow cytometry. In addition, this novel method demonstrated retrieval of positive single B-cells from microchambers by a micromanipulator and achieved antibody DNA analysis. The system is suitable for high-throughput analysis of intracellular Ca2+ response at the single-cell level and is applicable to screen antigen-specific lymphocytes for making specific monoclonal antibody.

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. '

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'Immunoglobulin G4 (IgG4)-related disease' is a new clinical concept of multi-organ diseases, with Mikulicz's disease (MD) being a clinical phenotype of IgG4-related disease. To clarify the clinical characteristics of respiratory involvement associated with IgG4-related MD, we retrospectively assessed 25 patients with MD, 11 (44%) of whom had allergic symptoms, and 7 (28%) of whom complained of respiratory problems. Thirteen patients (52%) presented with pulmonary and/or mediastinal lesions (P-MD) on chest computed tomography (CT), and 11 (44%) had lesions limited to the lacrimal and/or salivary glands (L-MD). Mean serum total protein, IgG, and IgG4 concentrations were significantly higher and CH50 was significantly lower in the P-MD than in the L-MD group. Immune complex was present only in the P-MD group. Chest CT images showed bronchial wall thickening, consolidation, nodule(s), interlobular thickening, ground glass opacity, pleural thickening/effusion, and mediastinal lymphadenopathy. Five of seven patients who underwent histological examination of the lungs had abundant IgG4-positive plasma cell infiltrates (IgG4/IgG-positive plasma cells >40%), but the other two did not. These findings suggest that respiratory lesions are not rare in patients with IgG4-related MD, and that they present with various manifestations. IgG4-related MD should be differentiated from similar diseases, such as sarcoidosis, bronchial asthma, Sjögren's syndrome, and malignant lymphoma.

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. To date, many investigators have developed various methods to detect antigenspecific B-cells using fluorescence-labeled antigen. However, nonspecific binding of fluorescence-labeled antigen (0.1%-1% in B-cells) makes it difficult to detect antigen-specific cells by hiding specific signals in background noise. In this study, we have developed a cell-microarray system that consists of microwell-array chips and a cell scanner. Microwell-array chips with a regular array of 45,000 or 234,000 wells that can accommodate only single lymphocytes enable us to analyze cells on a single cell basis (13). Since the position of each cell is fixed on the chip, it allows us to analyze the same single cell repeatedly. Here, we show that we can identify antigen-specific B-cells using this cell-microarray system by distinguishing signals from background