DNA aneuploidy (DA) has been considered as a potential cancer biomarker for a long time. Recently, quick, easy and automated sample preparation procedure for flow cytometric (FCM) DNA ploidy analysis has been developed, properly complying with the guidelines for FCM analysis of DA established by Japan Cytometry Society. Therefore, we have investigated the capability of the new procedure for use in clinical practice, and compared it with a conventional manual procedure using lung tissues. There was no major difference between the new and conventional procedures in DA detection with lung tumor. On the other hand, the new procedure had a higher incidence of “shoulder” formation or large CV of 2C peak in general and false DA peak (pseudo-DA) for non-tumor tissues than the conventional procedure. Overall, these results demonstrate that the new procedure is useful to detect DA and confirm the validity of DA as a cancer marker in clinical settings although improvement is required for suppressing pseudo-DA.
DNA ploidy analysis is one of the two principal applications of flow cytometry, along with cell surface antigen analysis. DNA ploidy analysis is positive in numerous tumors, but no clear conclusion can be obtained in some tumors. This may be attributed in part to measuring inconsistency and interpretation bias in the analysis. As the measurement method is complex for DNA ploidy analysis of solid tumors, it is difficult to conduct the analysis as a routine clinical procedure. Hence, we have developed the system named “High DNA Content Measuring Flow Cytometer” with a tissue pretreatment function, and a freeze-dried reagent kit for cell staining, in order to perform DNA ploidy analysis easily and reproducibly in non-specialized laboratories. Biopsy specimens from brain tumors of 87 glioma patients who underwent open surgery were analyzed to evaluate the utility of our system. We were able to automatically analyze the biopsy specimens with the system within ten minutes. We compared the results of the same pretreated samples between our system and the conventional method using a common flow cytometer, confirming that the malignancy index was well correlated between them (R=0.975). These results demonstrated that our fully automated DNA ploidy analyzer is useful for rapid determination of glioma presence in surgical biopsy specimens.
Interleukin (IL-) 27 is a member of the IL-12 cytokine family and is unique in that the cytokine has both immunostimulatory and immunosuppressive effects. While IL-27 is able to stimulate IFN-γ-production in T cells and NK cells, it suppresses activation of macrophages and dendritic cells. IL-27 also induces differentiation of IL-10-producing CD4+ T cell population, Tr1. Given the immunosuppressive effects of IL-27, IL-27-defficient or IL-27 receptor-deficient mice exhibited excessive, and often lethal, inflammation during infection with protozoa or mycobacterium. IL-27 was able to enhance anti-tumor activities of CD8+ T cells. IL-27 receptor-deficient dendritic cells exhibited enhanced antigen-presenting activities with augmented expression of co-stimulatory molecules and were able to induce higher tumor-killing activities by CD8+ T cells. Combination of IL-27 receptor-sufficient CD8+ T cells with IL-27 receptor-deficient activated dendritic cells efficiently led to high tumor-killing activity by CD8+ T cells. IL-27 thus is regarded as an immune checkpoint molecule to suppress excessive immune responses and is a potent target for development of novel immunosuppressive drug and/or for adjuvant discovery.
Hemagglutination, the classical laboratory technique for blood group identification, is simple and sufficient for routine ABO blood grouping. As a qualitative assay, however, hemagglutination has certain limitations: it cannot be relied upon to quantify ABO antigens on red blood cells or indicate the ABO blood type of various subgroups. On the other hand, flow cytometry (FCM) can quantify A and B antigens on red blood cells. Here, we examined the expression patterns and levels of A and B antigens using FCM in patients with the mosaic A blood subgroup. The histogram pattern of A antigen revealed a bimodal distribution of fluorescence intensity with two peaks corresponding to group A and O blood cells. Furthermore, we examined the expression patterns and levels of A and B antigens using FCM in patients with the cisA2B3 blood type. The histogram pattern of A antigen by FCM analysis showed a relative sharp spike with a mean fluorescence intensity (MFI) of 1,000 versus 2,000 in controls. In contrast, the histogram pattern of B antigen by FCM analysis was relatively wide, with a low MFI compared with controls. These findings suggest that FCM analysis of A and B antigens is a useful tool in the identifying ABO subgroups.
Thrombopoietin (TPO) was first identified as a cytokine that promotes megakaryocyte proliferation, maturation and platelet production. In the murine hematopoietic system, the TPO receptor MPL is expressed not only in megakaryocyte progenitors and their progeny but also in hematopoietic stem/progenitor cells (HSPCs). In addition, in the murine hematopoietic stem cell (HSC) compartment, TPO/MPL signaling plays an important role in the maintenance of adult quiescent HSCs. However, the role of TPO/MPL signaling in the human primitive HSC compartment has not yet been elucidated. Previously, we identified CD34-negative (CD34-) SCID-repopulating cells (SRCs) in human cord blood using an intra-bone marrow injection technique. In addition, we clearly demonstrated that human CD34+/- HSPCs expressed MPL via flow cytometry. Therefore, in order to investigate the role of the MPL expression in the human primitive CD34+/- HSC compartment, we performed a serial (primary, secondary and tertiary) transplantation analysis of CD34+/-MPL+/- cells using NOG mice for over a year. Our results revealed that all four fractions of cells had primary human cell repopulation abilities. However, only the CD34+/-MPL- cells showed tertiary human cell repopulation abilities. These results clearly indicate that the CD34+/- SRCs not expressing MPL sustain long-term (> 1 year) human cell repopulation in NOG mice. Collectively, these findings highlight a new concept that CD34-MPL- SRCs (HSCs) reside at the apex of the human HSC hierarchy.
Kidney develops from the three types of precursors, including nephron progenitors, ureteric bud and stromal progenitors. Mutual interaction of these progenitors enables concomitant differentiation and maintenance of nephron progenitors, thereby enabling the development of well-organized kidney structure with sufficient number of nephrons. Recently we established the way to selectively induce nephron progenitors from pluripotent stem cells, which showed the first evidence of reconstructing 3-dimensional nephron structure, including both glomerular podocytes and nephric tubules. However, it remains to be elucidated how the ureteric bud and stromal progenitor lineages are induced and reconstructed into the kidney organoid that equips organotypic architecture. Here we review the current situation of kidney reconstruction from pluripotent stem cells and discuss the limitations to overcome to achieve the physiological functions.
Patient-derived xenograft (PDX) models can be created with transplantation of cancerous cells or tissues from patient’s primary tumors into immunodeficient mice. PDX technology leads to the breakthrough with the introduction of novel highly immunodeficient mice such as NOG (NOD/Scid/IL2Rγnull), NSG (NOD/Scid/IL2Rγnull), and NOJ (NOD/Scid/Jak3null) mice. As PDX has been shown to conserve human original tumor characteristics, it is now getting standard methods to evaluate the drug sensitivity, and PDX library is now established in many organization with integrated genomic signature. These PDX data base are getting powerful tool for advancing Precision Cancer Medicine.