Deficiency in WT1-targeting microRNA-125a leads to myeloid malignancies and urogenital abnormalities.

The Wilms' tumor gene WT1 is overexpressed in leukemia and solid tumors and has an oncogenic role in leukemogenesis and tumorigenesis. However, precise regulatory mechanisms of WT1 overexpression remain undetermined. In the present study, microRNA-125a (miR-125a) was identified as a miRNA that suppressed WT1 expression via binding to the WT1-3'UTR. MiR-125a knockout mice overexpressed WT1, developed myeloproliferative disorder (MPD) characterized by expansion of myeloid cells in bone marrow (BM), spleen and peripheral blood, and displayed urogenital abnormalities. Silencing of WT1 expression in hematopoietic stem/progenitor cells of miR-125a knockout MPD mice by short-hairpin RNA inhibited myeloid colony formation in vitro. Furthermore, the incidence and severity of MPD were lower in miR-125a (-/-) mice than in miR-125a (+/-) mice, indicating the operation of compensatory mechanisms for the complete loss of miR-125a. To elucidate the compensatory mechanisms, miRNA array was performed. MiR-486 was occasionally induced in compete loss of miR-125a and inhibited WT1 expression instead of miR-125a, resulting in the cancellation of MPD occurrence. These results showed for the first time the post-transcriptional regulatory mechanisms of WT1 by both miR-125a and miR-486 and should contribute to the elucidation of mechanisms of normal hematopoiesis and kidney development.

In vivo eradication of MLL/ENL leukemia cells by NK cells in the absence of adaptive immunity.

It remains unclear how the immune system affects leukemia development. To clarify the significance of the presence of immune systems in leukemia development, we transferred MLL/ENL leukemia cells into immune-competent or immune-deficient mice without any preconditioning including irradiation. The wild-type mice did not develop leukemia, whereas all the Rag2(-/-)γc(-/-) mice lacking both adaptive immune cells and natural killer (NK) cells developed leukemia, indicating that leukemia cells were immunologically rejected. Interestingly, leukemia cells were also rejected in 60% of the Rag2(-/-) mice that lacked adaptive immune cells but possessed NK cells, suggesting that NK cells play a substantial role in the rejection of leukemia. Moreover, engraftment of leukemia cells was enhanced by NK cell depletion in Rag2(-/-) recipients and inhibited by transfer of NK cells into Rag2(-/-)γc(-/-) recipients. Upregulation of NKG2D (NK group 2, member D) ligands in MLL/ENL leukemia cells caused elimination of leukemia cells by NK cells. Finally, we found that leukemia cells resistant to elimination by NK cells had been selected during leukemia development in Rag2(-/-) recipients. These results demonstrate that NK cells can eradicate MLL/ENL leukemia cells in vivo in the absence of adaptive immunity, thus suggesting that NK cells can play a potent role in immunosurveillance against leukemia.

Inwardly rectifying potassium channel Kir4.1 is localized at the calyx endings of vestibular afferents.

Inwardly rectifying potassium (Kir) channel Kir4.1 (also called Kcnj10) is expressed in various cells such as satellite glial cells. It is suggested that these cells would absorb excess accumulated K(+) from intercellular space which is surrounded by these cell membranes expressing Kir4.1. In the vestibular system, loss of Kir4.1 results in selective degeneration of type I hair cells despite normal development of type II hair cells. The mechanisms underlying this developmental disorder have been unclear, because it was thought that Kir4.1 is only expressed in glial cells throughout the entire nervous system. Here, we show that Kir4.1 is expressed not only in glial cells but also in neurons of the mouse vestibular system. In the vestibular ganglion, Kir4.1 mRNA is transcribed in both satellite cells and neuronal somata, whereas Kir4.1 protein is expressed only in satellite cells. On the other hand, in the vestibular sensory epithelia, Kir4.1 protein is localized at the calyx endings of vestibular afferents, which surround type I hair cells. Kir4.1 protein expression in the vestibular sensory epithelia is detected beginning after birth, and its localization gradually adopts a calyceal shape until type I hair cells are mature. Kir4.1 localized at the calyx endings may play a role in the K(+)-buffering action of vestibular afferents surrounding type I hair cells.

CD138-negative clonogenic cells are plasma cells but not B cells in some multiple myeloma patients.

Clonogenic multiple myeloma (MM) cells reportedly lacked expression of plasma cell marker CD138. It was also shown that CD19(+) clonotypic B cells can serve as MM progenitor cells in some patients. However, it is unclear whether CD138-negative clonogenic MM plasma cells are identical to clonotypic CD19(+) B cells. We found that in vitro MM colony-forming cells were enriched in CD138(-)CD19(-)CD38(++) plasma cells, while CD19(+) B cells never formed MM colonies in 16 samples examined in this study. We next used the SCID-rab model, which enables engraftment of human MM in vivo. CD138(-)CD19(-)CD38(++) plasma cells engrafted in this model rapidly propagated MM in 3 out of 9 cases, while no engraftment of CD19(+) B cells was detected. In 4 out of 9 cases, CD138(+) plasma cells propagated MM, although more slowly than CD138(-) cells. Finally, we transplanted CD19(+) B cells from 13 MM patients into NOD/SCID IL2Rγc(-/-) mice, but MM did not develop. These results suggest that at least in some MM patients CD138-negative clonogenic cells are plasma cells rather than B cells, and that MM plasma cells including CD138(-) and CD138(+) cells have the potential to propagate MM clones in vivo in the absence of CD19(+) B cells.

Determination of red cells, nucleic acid-containing cells and platelets (RNP Determination) by a crossover analysis of emission DNA/RNA light.

We developed a new flow cytometry analysis method using a crossover analysis of emission light from intracellular DNA/RNA. Both the RNA and the DNA content in each cell were new parameters obtained by fluorescence analysis using acridine orange supravital stain. With this method, two-dimensional diagrams produced by cellular RNA concentration (CRc) and cellular DNA concentration (CDc) enabled clear separation of red cells and platelets, and the diagram (RNP Diagram) also distinguished fluorescently stained blood cells and small particles derived from background dust of the reagent. This study assessed the capability of RNP Determination concerning reticulocyte count and accurate platelet count obtained by the ratio of red cells and platelets. The distance of each event between red cell distribution and platelet distribution was sufficiently large on the diagram, and the two regions did not overlap. Both reticulocyte count and platelet count showed excellent correlation with those obtained by their respective reference methods. In conclusion, this new assay, using RNP Determination, demonstrated great potential for detecting abnormalities of red cells and platelets.

Body fat mass and lean mass as predictors of survival in hemodialysis patients.

A higher body mass index (BMI) is a predictor of better survival in hemodialysis patients, although the relative importance of body fat and lean mass has not been examined in the dialysis population. We performed an observational cohort study in 808 patients with end-stage renal disease on maintenance hemodialysis. At baseline, fat mass was measured by dual-energy X-ray absorptiometry and expressed as fat mass index (FMI; kg/m2). Lean mass index (LMI) was defined as BMI minus FMI. During the mean follow-up period of 53 months, 147 deaths, including 62 cardiovascular (CV) and 85 non-CV fatal events, were recorded. In univariate analysis, LMI was not significantly associated with CV or non-CV death, whereas a higher FMI was predictive of lower risk for non-CV death. Analyses with multivariate Cox models, which took other confounding variables as covariates, indicated the independent associations between a higher LMI and a lower risk of CV death, as well as between a higher FMI and a lower risk of non-CV death. These results indicate that increased fat mass and lean mass were both conditions associated with better outcomes in the dialysis population.

Antiapoptotic function of 17AA(+)WT1 (Wilms' tumor gene) isoforms on the intrinsic apoptosis pathway.

The WT1 gene is overexpressed in human primary leukemia and a wide variety of solid cancers. The WT1 gene is alternatively spliced at two sites, yielding four isoforms: 17AA(+)KTS(+), 17AA(+)KTS(-), 17AA(-)KTS(+), and 17AA(-)KTS(-). Here, we showed that 17AA(+)WT1-specific siRNA induced apoptosis in three WT1-expressing leukemia cell lines (K562, HL-60, and Kasumi-1), but not in WT1-non-expressing lymphoma cell line (Daudi). 17AA(+)WT1-specific siRNA activated caspase-3 and -9 in the intrinsic apoptosis pathway but not caspase-8 in the extrinsic one. On the other hand, 17AA(-)WT1-specific siRNA did not induce apoptosis in the three WT1-expressing cell lines. The apoptosis was associated with activation of proapoptotic Bax, which was activated upstream of the mitochondria. Constitutive expression of 17AA(+)WT1 isoforms inhibited apoptosis of K562 leukemia cells induced by apoptosis-inducing agents, etoposide and doxorubicin, through the protection of mitochondrial membrane damages, and DNA-binding zinc-finger region of 17AA(+)WT1 isoform was essential for the antiapoptotic functions. We further studied the gene(s) whose expression was altered by the expression of 17AA(+)WT1 isoforms and showed that the expression of proapoptotic Bak was decreased by the expression of 17AA(+)KTS(-)WT1 isoform. Taken together, these results indicated that 17AA(+)WT1 isoforms played antiapoptotic roles at some points upstream of the mitochondria in the intrinsic apoptosis pathway.

Japanese collaborative study for fibrinogen assay: variability of the fibrinogen assay between different laboratories does not improve when a common calibrator is used.

Several national and local external quality assurance schemes have been developed to improve the plasma fibrinogen assay in Japan over the past 30 years. Now most commercial calibrant plasma may be calibrated against an International Standard preparation, in order to achieve agreement of results obtained by different laboratories. However, we have never achieved satisfactory results, according to an external quality control survey regarding the fibrinogen assay. Therefore, we distributed two kinds of fibrinogen standards to be used as common calibrators, along with three plasma samples, among 183 general laboratories in Japan. The results of this collaborative study showed that the assigned value for the commercially available calibrators remained problematic. Furthermore, it was concluded that the between-laboratory variability could not be improved beyond a certain degree of standardization, even if a common calibrator was used for the Clauss-derived assay carried out by an automatic coagulometer.

Comparison of haemoglobinometry by WHO Haemoglobin Colour Scale and copper sulphate against haemiglobincyanide reference method.

Although estimation of haemoglobin is essential for diagnosing anaemia and assessing its severity, many health centres in developing countries do not have the facilities for haemoglobinometry. The WHO Haemoglobin Colour Scale (HCS) method is a simple and inexpensive clinical device that was recently developed in order to diagnose anaemia in such centres. In Indonesia, the copper sulphate specific gravity method is used for blood donor screening and also in primary health clinics in the rural and remote areas. In this study, the HCS method is compared with the copper sulphate method and with an earlier paper scale, the Tallquist method, against the standard haemiglobincyanide spectrophotometric method. The HCS method showed an acceptable level of precision and accuracy for use as a reliable screening tool to diagnose anaemia in patients and also for blood donor screening.

Preparation of a purified fibrinogen calibration material for Clauss method and turbidimetric immunoassay possessing biological activity and antigenicity.

Fibrinogen plays a major role in basic coagulation tests such as prothrombin time (PT), activated partial thromboplastin time (APTT) and thrombin time (TT). These show high interlaboratory variation because of inaccurate instrumental calibration. The same is true of the fibrinogen assay, despite its being a quantitative assay. Most medical laboratories use automated coagulometers and commercially available calibration materials (calibrators) to obtain an accurate fibrinogen value, but, when checked, calibrators have been found to deviate from the assigned value. The Japan Society of Laboratory Medicine (JSLM) has identified the need for a reliable plasma fibrinogen standard. To enhance the reliability of calibrator fibrinogen values and thereby remedy the poor precision and accuracy of plasma fibrinogen testing, we undertook the preparation of a standard calibration material applicable to both the Clauss method and turbidimetric immunoassay (TIA). The calibrator was prepared from fresh human plasma by glycine precipitation and virus inactivation followed by affinity chromatography to remove contaminated plasminogen. In the resulting product, clottable fibrinogen accounted for 95% of total protein and within-run precision showed a CV of less than 1.8%. We believe the preparation will become a candidate material for laboratory and manufacturer use in Japan.

Electrocardiogram is very useful for predicting acute heart failure following myeloablative chemotherapy with hematopoietic stem cell transplantation rescue.

A prospective study was conducted in 71 evaluable patients who received myeloablative hematopoietic stem cell transplantation (HSCT) at our facility from 1995 to 2002, to find a sensitive marker for post-transplant heart failure, including echocardiographic systolic and diastolic markers and QTc interval. QTc was found to be an independent and significant risk factor for acute heart failure (AHF) on multivariate logistic regression analysis (OR 1.5, P=0.01, 95% confidence interval (CI) 1.1-2.0), while no significant differences between patients with AHF and those without AHF were found in age, sex, treatment history, type of conditioning regimen, and echocardiographic systolic and diastolic markers. On further analysis, post-transplant risk of AHF appeared to be increased as QTc was prolonged. The post-transplant risk of AHF in the group with longest QTc on multivariate logistic regression analysis was found to be 9.8 times that in the group with shortest QTc (P=0.04, 95% CI 1.0-100). These results suggest that echocardiographic markers are less valuable predictors of post-transplant AHF, but that prolongation of the QTc, an ECG marker, before HSCT is strongly associated with onset of AHF after HSCT.

International Council for Standardization in Haematology (ICSH) recommendations for "surrogate reference" method for the packed cell volume.

The spun packed cell volume (PCV, hematocrit) is a key measurement on which are based hematology instrument calibration, reference range determination, and assignment of values to calibrators/controls. In 2001, the International Council for Standardization in Haematology (ICSH) recommended a Reference PCV method, which is fully traceable to the ICSH reference hemoglobin method. Because of its complexity, however, this method is impractical for occasional use in routine laboratories and is therefore intended primarily for use by manufacturers of capillary microhematocrit tubes, liquid calibrators, and multichannel analyzers. In response to the need for a simpler method--accessible to all routine laboratories--the ICSH offers this "Surrogate Reference" PCV procedure. It is traceable to the original ICSH Reference PCV method and is based on spun PCVs obtained using borosilicate capillary tubes with an already-known relationship to this reference procedure. This ICSH "Surrogate Reference" PCV method is substantially simpler, thus putting it within the reach of most routine hematology laboratories.

CTAD as a universal anticoagulant.

The feasibility of CTAD (a mixture of citrate, theophylline, adenosine and dipyridamole) as a new anticoagulant for medical laboratory use was studied prospectively. Whole blood anticoagulated with CTAD exhibited results very similar to those of blood anticoagulated with EDTA on complete blood count and automated white cell differential except for a slight decrease in platelet count and mean platelet volume. Chemistry test data for plasma obtained from CTAD whole blood were close to those obtained for matched sera. Among coagulation tests, prothrombin time, activated partial thromboplastin time and fibrinogen concentrations were close to those obtained with citrate plasma. Based on the results, CTAD was judged to be a good candidate as a new anticoagulant.

The HLA-DOB gene displays limited polymorphism with only one amino acid substitution.

The HLA-DO molecule is a non-classical class II heterodimer composed of alpha and beta chains. We have previously recognized that all eight of the allelic variations of the HLA-DOA gene represent non-synonymous amino acid substitution. In the present study, to analyze genetic polymorphism and allelic variation of the HLA-DOB gene which may affect the efficiency of class II restricted antigen presentation thereby being involved in the susceptibility of HLA associated diseases, we conducted direct DNA sequencing of HLA-DOB in 36 HLA class II homozygous typing cells and identified six new allelic variations (DOB*0101101, *0101102, *01012, *01022, *0104101 and *0104102) including five single nucleotide polymorphisms with only one amino acid substitution. Furthermore, strong linkage disequilibrium was detected between DOB*01022 and DRB1*1502 only, with no linkage disequilibrium between the DOA and the DOB genes. The HLA-DOB gene has been identified in other mammals, and their nucleotide sequences are well conserved. These facts suggest that limited polymorphism in the DOB gene is profitable to execute their unique function as a co chaperone and so strong selective pressure is operating to prevent generic variation against the DOB molecule interacting with the DM molecule and thus maintaining the specified immunological function of regulating antigen presentation.

Restructuring of international council for standardization in haematology (ICSH) in Asia.

Standardization and harmonization in Laboratory testing are a key issue in the midst of globalization era, because most of laboratory testing has been currently achieved with various kinds of automated systems. In the developed countries, automated systems with highly-regulated principles are commonly used in the routine laboratory. However, there are so many undeveloped and developing countries in Asia that diversity of testing levels can be observed in the area. Some laboratories use glass chamber method for blood cell counting, while other laboratory use semi-automated or fully automated analyzers for complete blood count. International standardization on Hematology is focused on the developed system but not for the developing system. Established standardized documents therefore whould not be unsuitable for Asian societies. In the context, International Council for Standardization in Hematology (ICSH) changed its rules to adjust our Asian Societies and ICSH started to restructure the body. International ICSH society is divided into 5 region sub-groups. Asian area is able to possess one new sub-society, ICSH-Asia. Its reconstruction work has been just started with Asain colleagues, and we are now extending the new societies to discuss Asian problems on the quality of hematology testing.

E-cadherin expression in lymph nodes of three patients with non-Hodgkin's lymphoma.

The E-cadherins are a family of cell-cell adhesion molecules. These molecules exhibit Ca2+ dependent cell adhesion and are expressed on epithelial cells. E-cadherin levels in serum are known to be significantly elevated in patients with epithelial carcinomas. We determined serum E-cadherin levels in 30 patients with non-Hodgkin's lymphoma (NHL) using an enzyme immunoassay and then investigated whether E-cadherin is expressed on lymphoma cells in lymph nodes of three cases selected to analyze from 15 cases of serum E-cadherin levels over mean + 2SD with monoclonal antibody immunohistochemistry. Results indicated that E-cadherin antigen is expressed on the lymphoma cells in these three patients with NHL, and that soluble E-cadherin might be released into blood from lymphoma cells. Expression of E-cadherin may contribute to the morphological appearance of some malignant lymphoma, although no conclusion can be drawn based on such small number of patients analyzed.

Clinical significance of serum E-cadherin levels in patients with haematological malignancies.

E-cadherin is a transmembrane glycoprotein that mediates Ca2+-dependent intracellular adhesion in normal epithelial cells. E-cadherin levels in serum are known to be significantly elevated in patients with epithelial carcinomas. However, the role of E-cadherin in haematopoietic cells is less clear. In this study, serum E-cadherin levels were therefore determined in patients with acute or chronic leukaemia, malignant lymphoma or myelodysplastic syndromes. Significant elevation of serum E-cadherin levels was detected in patients with haematological malignancies, and between types of acute leukaemias or subtypes of myelodysplastic syndromes, stages of malignant lymphoma, and phases of chronic leukaemia, respectively, compared with those in healthy adult volunteers. These findings suggest that E-cadherin might be expressed in malignant haematopoietic cells and might be useful as a diagnostic indicator in haematological malignancies.