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.

Single-cell microarray for analyzing cellular response.

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.

Reduction of blood glucose level by orexins in fasting normal and streptozotocin-diabetic mice.

Orexin-A and orexin-B are neuropeptides implicated in the maintenance of energy homeostasis. In the present study, we examined the effects of orexins on blood glucose levels in response to fasting in normal and streptozotocin-induced diabetic mice. After the injection of orexin-A and orexin-B (0.01-1 nmol/kg, i.v.), the blood glucose levels in both normal mice and diabetic mice in the fasting state decreased. In contrast, neither orexin-A nor orexin-B affected the glucose levels in the animals allowed free access to food. Intracerebroventricular administration of orexin-A and orexin-B was associated with glucose-lowering effects in fasting diabetic mice. The serum insulin level did not significantly change following the administration of orexin-A or orexin-B, in either the normal or the diabetic mice in the fasting state. These results demonstrate that orexins lower the blood glucose levels exclusively in the fasting state. The orexins may stimulate some neural and hormonal network and thereby promote blood glucose utilization.