Recently, Pharmacogenomics and Pharmacodynamics are performed in clinical trials. However, the quality and quantity of those pharmacodynamic evaluations is not sufficient. Especially, the cytokine evaluation methods must be adequately constructed as soon as possible in order to avoid the adverse-reaction accident,which was unfortunately happened in the phase I trial performed in Britain in March, 2006. Therefore, we examined the new cytokine evaluation method which uses the flow beads array assay for the pharmacodynamic evaluation. We stimulated peripheral blood mononuclear cells(PBMC) collected from 4 healthy volunteers and 4 allergic patients with Japanese cedar pollen, and then measured those cytokines production with Enzyme Linked Immunosorbent Assay (ELISA) and the flow beads array assay. Th2 cytokines production was detected from PBMC from allergic patients with Japanese cedar pollen after antigen-specific stimulation for 7 days. Th2 cytokines could not be detected from PBMC from healthy volunteers. The results of ELISA and the flow beads array assay on cytokines production were closely correlated. We confirmed that the flow beads array analysis is beneficial for the evaluation of clinical trials in its cost performance and operation efficiency.
The transplantation medicine is divided two categories, one is the organ transplantation, and the other is hematopoietic stem cell transplantation (HSCT). The discovery of HLA in the 1950s was one of the most important new findings in the area of transplantation. In organ transplantation, HLA alloantibodies (Abs) are associated with chronic rejection and poorer graft survival. The most important research is to find out the immunodominant HLA antibodies and the strength of it. On the other hand, in HSCT, the key factor is the matching of histocompatibility antigens. The matching high resolution level of HLA between donor and recipient lead an improvement of recipient’s survival. Luminex technology is a new flow cytometry dual-laser system enabling us to analyze numerous reactions in a tube or well. It is a multiplexed data acquisition and analysis platform of microbeads-based assays that performs simultaneous measurements of up to 100 different analyses. In the histocompatibility field, individual sets of beads are modified with reactive components such as antigens in order to perform HLA antibodies identification or with oligonucleotides in order to perform HLA typing after reverse PCR-SSO. Thus beads are the equivalent of a panel of HLA typed lymphocytes (for panel reactive antibodies determination and antibody identification) or equivalent to a large set of probes selected to assign HLA typing. This genotyping method allows to define the HLA alleles at the four-digital level with a frequency of more than 0.1% in the Japanese population. The HLA antibodies detection system by using the luminex technique has recently been noted to be more sensitive than other procedures.
It has become clear that HLA antibodies cause hyper acute and acute rejection of transplanted kidneys. Moreover, there are some reports that in patients whose transplant kidneys were rejected, HLA antibodies became positive before the transplant kidneys dysfunction, and therefore HLA antibodies participated in the chronic rejection. Using the conventional LCT method to identify the HLA antibody, a satisfactory result was not always provided in spite of complicated operation. However, because the FlowPRATM method using microbead was developed in recent years, we examined the presence and specificity of HLA antibodies using this newer method in a kidney transplant case in our hospital. The objects were 34 cases. We used the FlowPRA Screening Test and Single Antigen Test (One Lambda) for identification of HLA antibodies. 14 out of 34 cases were antibody positive, including 7 before transplant positive and 7 after transplant positive. 2 out of before transplant positive cases became graft loss at the early phase after transplant and donor specific antibody positive. The other 5 cases are functioning well, because the HLA antibodies are donor nonspecific. On the other hand, the kidney function of all cases that became positive after transplant was worse, and in 5 cases, have received the hemo-dialysis therapy again and the donor specific HLA antibodies were detected. The donor specific HLA antibody was suggested on the reason for the dysfunction of a transplant kidney, and it was thought that periodical HLA antibody examinations using the FlowPRA method before and after transplantation were important. The FlowPRA method can be performed more easily, precisely and specifically than the LCT method. Moreover, it is more reliable on a test method. The establishment of the rescue therapy after HLA antibody positive is expected, and MMF therapy is suggested to be effective.
The Common marmoset becomes increasingly important in the development of recombinant monoclonal antibody drugs, which react with only primates, because of both their relatively close immunological relationship with humans and their relative small size. However, there is little information about cross-reactivity of commercial monoclonal antibodies with Common marmosets for lymphocyte subset analysis. In this study,we compared cross-reactivity with Common marmosets, Cynomolgus monkeys and humans using 19 commercial monoclonal antibodies. Furthermore, we optimized the method of preparation of Common marmoset peripheral blood in flow cytometric analysis. Eleven of 19 monoclonal antibodies that react with human or monkey antigens of CD-defined molecules were cross-reactive with Common marmoset antigens. Those antibodies that cross-reacted with Common marmosets were sufficient for lymphocyte subset analysis. Five lysing solutions were used for preparation of Common marmoset peripheral blood. By using Red Blood Cell Lysing Buffer, Common marmoset peripheral blood cells can be separated to each fraction of cells while maintaining cell viability. The minimum quantity of Common marmoset peripheral blood was only 25 to 50 μl for flow cytometric analysis of lymphocyte subsets. This flow cytometric method using Common marmoset peripheral blood can be used to speculate on the potential immunotoxic effect of monoclonal antibody drugs on humans.
Presence of cancer stem cell (CSC) was demonstrated in some kinds of cancer tissues and cell lines. A cancer recurrence is considered with intervention of CSC resistant to anti-cancer drug and understanding of CSC characteristics leads to development of an effective therapy. We reviewed the results of genome-wide gene expression analysis of side population (SP) cells of solid cancers, especially from a head and neck origin,for the purpose of a search of candidate gene marker(s) of cancer stem cells. SP cells, enriched with stem cells, were observed in head and neck cancer cell lines. The genes which most strongly varied in expression between SP cell /Non-SP cells were neurogenetic, cutaneous disease relations and ATP-binding cassette,sub-family G, member 2 (ABCG2), was overexpressed at the highest level. The genes showed expression profiles similar to ABCG2 were cancerous testiculus antigen and several transcription factors. Annexin family genes showed reverse to ABCG2 expression patterns. The genes, those whom included in cancer SP cells,not in normal cells in particular, were categories of inhibit cell growth, metabolism, and biosynthesis, and are candidate gene markers of an effective treatment with anti-cancer drug. Microarray screened genes are analyzed in detail, and it is necessary to clarify characteristics of cancer stem cells in future.
Chinese hamster ovary (CHO) cell is widely adopted in industry for production of desired proteins such as monoclonal antibodies, hormones and interferon. However, stable expression of a desired protein in CHO cells requires a time-consuming step of antibiotic resistance-based cell cloning. Recently, we have developed a retrovirus-based system for stable expression of a desired protein in the CHO-S cell, a derivative of CHO cell. Although CHO cells are resistant to retrovirus infection, stable expression of ecotropic retrovirus receptor (EcoR) in those cells enabled infection. We constructed the retroviral vector pGX GFP, which expresses the desired protein as a GST fusion construct and the GFP as a marker protein. Multiple infection of retrovirus helped to increase the amount of the desired protein in single cells, and collection of GFP-high cell population by flow cytometry facilitated to obtain cell population expressing the desired protein at high levels. This system made it possible to obtain the reasonable amount of the desired protein (100µg) in about two weeks. In this review, we will discuss the advantage and the point of issue of this system.
Rheumatoid Arthritis (RA) is one of the common autoimmune disease and remarkably decline patient's quality of life (QOL) by destruction of the joint when the disease progress and became untreatable. To arrest progression of the disease, rapid and precise diagnosis becomes an important matter. We use suspension-array analysis to find out candidate factors, those foresight the fate of the disease. Interestingly, peripheral CD4+CD25bright regulatory T cell subset, which plays an important role to suppress inflammatory reactions,were identical between normal control and severe RA patients. However suspension-array analysis on 17 cytokines revealed decreased production of IL-10 and elevated production of IFN-γ. Our findings suggest the possibility that the abrogation of regulatory T cell function deteriorate pathogenesis of rheumatoid arthritis.
We have developed a series of single-cell-based analysis called ‘on-chip cellomics', consisting of singlecell-based sorting for purifying target cells, single-cell-based cultivation on chip for fully geometric/temporal control of target cells, and single-cell-based genome/proteome analysis for identifying target biomarkers of epigenetic phenomenon. For ‘on-chip cellomics' study, non-destructive cell sorting is the most important for the following re-cultivation of target cells on the cultivation chips keeping their intact condition and information. We thus have proposed and examined four approaches of non-destructive cell purification; aptamermagnet microbeads method exploiting digestable DNA/RNA-based aptamer probes, apoptosis method using spot irradiation of focused laser beam to promote apoptosis on cells except for target cells, removable fluorescent probe detection method with disposable microchip, and ultra high-speed camera image detection method with disposable microchip. The results indicated those four methods are non-destructive and can be used for the sufficient selective cell purification for the following on-chip cell cultivation. In this paper, we introduce briefly about our results and potential of those cell purification methods and compare the advantages and defects of those methods.