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.

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We investigated 998 serial Japanese forensic autopsy cases (0-101 years old, mean age 61.7 ± 21.9), with no case selection, using immunohistochemistry to detect cases with progressive supranuclear palsy (PSP). Twenty-nine cases (mean age 82.3 ± 7.2 years, 11 males, 18 females) fulfilled the National Institute of Neuronal Disorders and Stroke (NINDS)-PSP pathological criteria (2.9% of all cases, 4.6% of cases over 60). All had neuronal and glial inclusions in the basal ganglia and brainstem. However, 13 cases had low tau pathology and were categorized as atypical PSP. In addition to PSP pathology, multiple types of astrocytic inclusions and comorbid proteinopathies, particularly a high prevalence of argyrophilic grain disease, were found. All cases had not been diagnosed with PSP and had preserved daily functioning prior to death. However, 14 (48.3%), 11 (37.9%), and 16 (55.2%) cases showed signs of dementia, depressive state, and gait disturbance, respectively. Sixteen accidental death cases (55.2%), including from falls and getting lost, and 11 suicide cases (37.9%) appear to have a relationship with incipient PSP pathology. Cluster analysis using the distribution and amount of 4-repeat-tau pathology classified the cases into three subgroups: Group 1 (10 cases) had typical PSP pathology and seven cases (70.0%) had dementia as the most frequent symptom; Group 2 (7 cases) had significantly higher frequency of gait disorder (6 cases, 85.7%), and less neocortical tau pathology than Group 1; Group 3 (12 cases) had relatively mild PSP pathology and high argyrophilic grain burdens. Granular-shaped astrocytes were the dominant astrocytic inclusion in all cases. We conclude that in forensic cases incipient PSP occurs with a higher prevalence than expected. If these findings can be extrapolated to other population-based cohorts, PSP may be more common than previously thought.

Functional protein-protein interactions between UDPglucuronosyltransferase (UGT)1A isoforms and cytochrome P450 (CYP)3A4 were studied. To this end, UGT1A-catalyzed glucuronidation was assayed in Sf-9 cells that simultaneously expressed UGT and CYP3A4. In the kinetics of UGT1A6-catalyzed glucuronidation of serotonin, both Michaelis constant (K m ) and maximal velocity (V max ) were increased by CYP3A4. When CYP3A4 was coexpressed with either UGT1A1 or 1A7, the V max for the glucuronidation of the irinotecan metabolite (SN-38) was significantly increased. S 50 and K m both which are the substrate concentration giving 0.5 V max were little affected by simultaneous expression of CYP3A4. This study also examined the catalytic properties of the allelic variants of UGT1A1 and 1A7 and their effects on the interaction with CYP3A4. Although the UGT1A1-catalyzing activity of 4-methylumbelliferone glucuronidation was reduced in its variant, UGT1A1*6, the coexpression of CYP3A4 restored the impaired function to a level comparable with the wild type. Similarly, simultaneous expression of CYP3A4 increased the V max of UGT1A7*1 (wild type) and *2 (N129K and R131K), whereas the same was not observed in UGT1A7*3 (N129K, R131K, and W208R). In the kinetics involving different concentrations of UDPglucuronic acid (UDP-GlcUA), the K m for UDP-GlcUA was significantly higher for UGT1A7*2 and *3 than *1. The K m of UGT1A7*1 and *3 was increased by CYP3A4, whereas *2 did not exhibit any such change. These results suggest that (1) CYP3A4 changes the catalytic function of the UGT1A subfamily in a UGT isoformspecific manner and (2) nonsynonymous mutations in UGT1A7*3 reduce not only the ability of UGT to use UDP-GlcUA but also CYP3A4-mediated enhancement of catalytic activity, whereas CYP3A4 is able to restore the UGT1A1*6 function.

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

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Kawasaki Disease (KD) is an acute inflammatory disease that takes the form of systemic vasculitis.Endothelial microparticles (EMPs) have been recognized as an important transcellular delivery system. We hypothesized whether EMPs are involved in vasculitis in acute KD. Fifty patients with acute KD were enrolled, divided into two subgroups: those with coronary artery lesions (CAL) (n = 5) and those without CAL (NCAL) (n = 45). EMPs were measured using flow cytometry, and microRNA (miR) expression profiling was performed by microRNA array. The percentage of EMPs in acute KD was significantly higher than in controls (P < 0.0001). EMPs in patients with CAL rapidly increased after the initial treatment, and was significantly higher than those in NCAL (P < 0.001). In patients with CAL, we identified 2 specific miRs encapsulated in EMPs, hsa-miR-145-5p and hsa-miR-320a, which are predicted to affect monocyte function using in silico analysis, and were demonstrated to upregulate inflammatory cytokine mRNAs in THP-1 monocytes. In situ hybridization confirmed that hsa-miR-145-5p was preferentially expressed in CAL. EMPs may serve as a sensitive marker for the severity of vasculitis in acute KD. Moreover, these 2 specific miRs encapsulated in EMPs might be involved in inflammatory cytokine regulation and the pathogenesis of vasculitis in acute KD.