Tomio Kamitani

Purification of calcium-sensitive regulatory protein of platelets which inhibits the gelation of actin

Abstract Ca2+-sensitive regulatory protein of human platelets which inhibits the gelation of actin was purified by DEAE-Sepharose and an affinity column using actin as a ligand. The protein was a single polypeptide chain with an average molecular weight of 90,000 and it bound to actin and inhibited its gelation at concentration from 10−6–10−7M of free calcium. Since the protein existed in the form of a complex with actin even though at concentration lower than 10−7M of free calcium, binding and dissociation of actin and the protein appeared to be dependent on the concentration of free calcium, and complete dissociation was not seen.

Serum IL-18 levels in patients with various hematological disorders.

Interleukin-18 (IL-18), originary called interferon (IFN γ )-inducing factor, is a recently cloned cytokine of approximately 18 kDa. Its major activity is the induction of IFNγ production from anti-CD3-activated Thl cells, especially in the presence of IL-12. Recently, the human IL-18 cDNA was cloned and expressed in Escherichia coli [1]. It has been revealed that human IL-18 has the capacity to stimulate cytotoxic natural killer (NK) cell activity and stimulates T-cells to produce IFN γ , IL-2, and granulocyte/ macrophage colony-stimulating factor [1]. It is reported that IL-18 is produced by Kupfer cells, activated macrophages, keratinocytes, intestinal epithelial cells, osteoblasts, adrenal cortex cells and murine dincephalon [2, 3]. Taniguchiet al. reported that the IL-18 levels from two groups of acute lymphocytic leukemia (ALL) and chronic myelocytic leukemia (CML) patients is significantly increased compared to the control group [4]. We evaluated the serum IL-18 levels in patients with multiple myeloma (MM), myelodysplastic syndrome (MDS), acute non-lymphocytic leukemia (ANLL), ALL, aplastic anemia (AA), idiopathic thrombocytopenic purpura (ITP), Hodgkin’s disease (HD), non-Hodgkin’s lymphoma (NHL), adult T-cell leukemia (ATL), CML and hemophagocytic syndrome (HPS) in this study. On entry all patients had evidence of active disease and had not received previous therapy. The human serum concentration of IL-18 was measured using an enzyme-linked immunoabsorbent assay kit for IL-18 (MBL,

Automated Analyzer Evaluation of Reticulocytes in Bone Marrow and Peripheral Blood of Hematologic Disorders

The R-3000 reticulocyte analyzer uses flow cytometry with an argon laser as its light source. This analyzer stains residual RNA with auramine O to provide a reticulocyte maturation differential. Using the R-3000, we analyzed 119 samples of bone marrow (BM) and peripheral blood (PB) from 111 patients with hematologic disorders. Parameters were reticulocytes, immature reticulocyte fraction (IRF) percentage in BM and PB, BM/PB reticulocyte ratio, and BM/PB IRF ratio. Reticulocytes and IRF percentage in BM were significantly higher than in PB (p < 0.01). There was also a good correlation between reticulocyte percentages in BM and in PB (r = 0.81). Patients were classified into a normal group (without anemia) and an anemia group. Furthermore, the anemia group was classified into three groups: group 1: cases with hematopoietic dysfunction; group 2: cases in bone marrow recovery phase after chemotherapy and hematopoietic stem cell transplantation, and hematologic disorders with bone marrow accelerative phase, and group 3: cases with ineffective hematopoiesis (myelodysplastic syndrome). The mean reticulocyte percentage of the normal group was 2.3 ± 1.1%, which was close to the normal value in BM. The BM/PB reticulocyte ratio of group 3 was statistically higher than that of groups 1 and 2. This indicates that group 3 had ineffective erythropoiesis and that the BM/PB ratio is a useful indicator for the diagnosis of myelodysplastic syndrome.

Lymphoma in Castleman's disease, acute lymphocytic leukemia, adult T-cell leukemia and cutaneous T-cell lymphoma accompanied with high serum soluble Fas ligand levels.

The Fas ligand (FasL) is a member of the tumor necrosis factor family [1]. Cleavage of membrane-bound FasL by a metalloproteinase-like enzyme results in the formation of soluble FasL (sFasL) [2]. sFasL binds to Fas antigen and induces apoptosis which plays a major role during development and in homeostasis against Fas-expressing cells, and is considered to work as a pathological agent in systemic tissue injury [3]. FasL is a 40 kDa glycoprotein that is expressed on the cell membrane of activated cytotoxic cells, including CD4 + and CD8+ T cells and natural killer (NK) cells [4, 5]. A recent report indicated that sFasL is present in the supernatant of activated human peripheral blood T cells [6]. Tanakaet al. reported that serum FasL concentrations were highly elevated in patients with large granular lymphocytic leukemia and NK lymphoma, but were undetectable in patients with adult T-cell leukemia, acute myelogenous leukemia, acute promyelocytic leukemia, acute lymphocytic leukemia, or Tand B-cell lymphoma [2]. Furthermore, it was reported that the serum sFasL level was high on admission, but rapidly decreased to the reference range following chemotherapy in

Urinary trypsin inhibitor levels in the urine of patients with haematological malignancies.

The urinary trypsin inhibitor (UTI) levels in the urine of patients with various haematological malignancies were determined, using automated latex agglutination immunoturbidimetry. The mean UTI levels in urine in acute non-lymphocytic leukaemia, myelodysplastic syndrome, non-Hodgkins lymphoma, and multiple myeloma groups were significantly elevated, compared with the normal control group. It was found that the UTI level in urine changed from an elevated value to a normal value with haematological improvement by chemotherapy in a patient with myelodysplastic syndrome included in a previous study. These results suggest tha

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-Hodgkins 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.

Analysis of IL-18 bioactivity and IL-18 mRNA in three patients with adult T-cell leukaemia, acute mixed lineage leukaemia, and acute lymphocytic leukaemia accompanied with high serum IL-18 levels.

Interleukin-18 (IL-18) bioactivity in sera and IL-18 mRNA expression in leukaemia cells of three patients with adult T-cell leukaemia (ATL), acute mixed lineage leukaemia (AMLL) and acute lymphocytic leukaemia (ALL) accompanied with high serum IL-18 levels have been analysed. There was little serum IL-18 bioactivity in the three patients with ATL, AMLL and ALL, while IL-18 mRNA expression was detected in leukaemia cells of all three patients.

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.

Analysis of Serum Sialyl-Lewis x Antigen Levels in Acute Leukemias, Myelodysplastic Syndromes, Myeloproliferative Disorders and Malignant Lymphomas

Sialyl-Lewis is a sialylated and fucosylated lactosamine belonging to a group of oligosaccharides recognized as ligands by human selectins. Its antigen, which has long and complicated carbohydrate structures on the cell surface membrane, has been shown to be one of the most important cancer-associated antigens. Sialyl-Lewis is synthesized by a series of glycosyltransferases and has a polysaccharide determinant carried by so-called type 2 chain carbohydrates, the characteristic feature of which is a backbone structure composed of the Galb1 ! 4GlcNAcb repeating unit. Sialyl-Lewis plays a useful role in the diagnosis of a variety of human cancers originating in the lung, ovary, breast and gastrointestinal tract [1,2]. Levels of expression of sialyl-Lewis are increased in various cancers compared to those in corresponding normal tissues [3], and the sialyl-Lewis moiety on circulating tumor cells tend to decrease after treatment including curative surgery and to increase again in cases of relapse [4]. Sialyl-Lewis level in the serum of cancer patients has been known to be a tumor marker for over a decade [4,5]. These findings suggest that change in sialyl-Lewis expression in tumor cells might be important in the disease activity of metastasis of cancer or for predicting its outcome. SialylLewis antigen is assumed to be the binding epitope of E-selectin and P-selectin in normal human neutrophils and myelocytic leukemia HL60 cells [6]. It has been demonstrated that sialyl-Lewis antigen is expressed on monocytes, granulocyte-macrophage-committed cells [7], some lymphocyte subsets [8–10], CD34-positive leukemic blasts [11] and bone marrow hemopoietic progenitor cells [12], and CD10-positive normal bone marrow cells [13]. Recently a variant type of sialyl-Lewis was found to be expressed in adult T-cell leukemia cells [1]. However, the presence of serum sialyl-Lewis levels in patients with various hematologic malignancies has rarely been described, although Cavenagh et al. [14] found expression of sialyl-Lewis antigen in 10 patients with acute myeloid leukemia. We therefore investigated whether serum sialylLewis antigen levels are elevated in patients with various hematologic malignancies compared to those in normal controls, and whether differences exist in serum sialylLewis antigen levels among groups of different malignancies. We determined serum concentrations of sialyl-Lewis using an enzyme immunoassay with a twostep sandwich assay kit for sialyl-Lewis (CSLEX, Nittobo, Fukushima, Japan) [2,15] in 129 patients with acute leukemias, myelodysplastic syndromes, myeloproliferative disorders or malignant lymphomas. Blood samples from 19 with acute nonlymphocytic leukemia (ANLL), 7 with acute lymphocytic leukemia (ALL), 36 with myelodysplastic syndromes (MDS), 19 with myeloproliferative disorders (MPD), 11 with adult T-cell leukemia/lymphoma (ATL/L), 36 with non-Hodgkin’s lymphoma (NHL), and 4 with Hodgkin’s lymphoma (HL)

Evaluation of use of the optional unit QA-810V for the determination of five-part leukocyte differentials.

The newly developed QA-810V is an optional unit for the determination of five-part white blood cell differentials. It can be used together with the same manufacturers haematology analyser which has been used in relatively small-sized laboratories. The present study evaluates the basic performance of the QA-810V and the MEK-8118 haematology analyser using routinely obtained blood specimens treated with ethylenedioaminetetraacetic acid-2K. In this evaluation, reproducibility was good and little carryover was found. Accurate measurements were possible for up to 24h of storage. Storage at 4 °C yielded more stable measurements of complete blood counts and five-part differentials than storage at room temperature. A good correlation between findings with the MEK-8118 haematology analyser and those with the SE-9000 haematology analyser was found for complete blood counts. The leukocyte differential obtained with the QA-810V correlated well with eye counts, with r > 0.9 for percentages of neutrophils, lymphocytes and eosinophils. Scattergrams obtained with the QA-810V reflected the presence of abnormal cells. The performance of the QA-810V was excellent and it can improve the quality of testing in clinical laboratories.