Kojun Setsu

Acute arterial thrombosis due to platelet aggregation in a patient receiving granulocyte colony-stimulating factor

We describe a 44‐year‐old man with non‐Hodgkins lymphoma receiving granulocyte colony‐stimulating factor (G‐CSF) who developed an acute arterial thrombosis. The removed thrombus contained large amounts of platelet aggregation. A rapid increase of platelets and increased adenosine diphosphate (ADP)‐ and collagen‐induced platelet aggregation were observed at the time of the thrombotic event. A challenge test of G‐CSF showed an increase in the platelet count and an augmentation of ADP‐ and collagen‐induced platelet aggregation. In the use of G‐CSF, patients who produce a rapid increase in platelet levels could be at greater risk for thrombotic events and need to be followed‐up carefully.

Purification and characterization of enterotoxin produced by Aeromonas sobria.

We purified the toxin of Aeromonas sobria capable of inducing a positive response in the mouse intestinal loop assay. The purified toxin showed a positive response not only in the loop assay but also in a hemolytic assay. Subsequently, we cloned the toxin gene and demonstrated that the product of this gene possessed both hemolytic and enterotoxic activities. These results showed that the enterotoxin of A. sobria possesses hemolytic activity. Nucleotide sequence determination of the toxin gene and amino acid sequence analysis of the purified toxin revealed that it is synthesized as a precursor composed of 488 amino acid residues, and that the 24 amino‐terminal amino acid residues of the precursor is removed in the mature toxin. As antiserum against the purified toxin neutralized the fluid accumulation induced by living cells not only of A. sobria but also of A. hydrophila, this and antigenically related toxin(s) are thought to play an essential role in the induction of diarrhea by these organisms. The toxin‐injured Chinese hamster ovary (CHO) cells induced the release of intracellular lactose dehydrogenase (LDH). The release of LDH from CHO cells and the lysis of erythrocytes by the toxin were repressed by the addition of dextran to the reaction solution, indicating that the toxin forms pores in the membranes and that the cells were injured by the osmotic gradient developed due to pore formation. However, the histopathological examination of intestinal cells exposed to the toxin showed that it caused fluid accumulation in the mouse intestinal loop without causing cellular damage.

NRAS and BRAF mutation frequency in primary oral mucosal melanoma

Oral mucosal melanoma (OMM) is a fatal sarcoma of unknown etiology. Histological morphology and genetic events are distinct from those of its cutaneous counterpart. Mutation and up-regulation of c-kit has been identified in OMM which may activate downstream molecules such as RAS and RAF. These molecules are involved in the mitogen-activated protein kinase (MAPK) pathway leading to tremendous cell proliferation and survival. NRAS and BRAF mutation and protein expression have been studied in other melanoma subtypes. The purpose of this study was to determine RAS protein expression and NRAS and BRAF mutation in 18 primary OMM cases using immunohistochemistry and mutation analysis. Results showed that RAS is intensely expressed in both in situ and invasive OMMs. However, NRAS mutation was only observed in 2/15 polymerase chain reaction (PCR) amplified cases both of which were silent mutations. On the other hand, BRAF missense mutations were observed only in 1/15 cases with PCR amplification. NRAS and BRAF mutations were independent from previously reported c-kit mutations. The classical V600E BRAF mutation was not found; instead a novel V600L was observed suggesting that the oncogenic event in OMM is different from that in skin melanoma. The low frequency of NRAS and BRAF mutations indicate that these genes are not common, but probable events in OMM pathogenesis, most likely independent of c-kit mutation.

Cadmium reduces adipocyte size and expression levels of adiponectin and Peg1/Mest in adipose tissue

Adipose tissue dysfunction has been associated with diabetogenic effects. The effects of repeated Cd exposure on adipocytes remain largely unknown. We administered Cd at doses of 0, 5, 10, and 20 micromol/kgbw sc for 2 weeks (3.5 times/week) to mice and assessed the possible alteration of epididymal white adipose tissue (WAT), including histological difference, adipocyte differentiation and functional capacity. Whereas hepatic weight did not differ between the control and Cd-exposed groups, WAT weight, as well as adipose cell mass, significantly decreased in a dose-dependent manner in Cd-treated mice. The Cd concentration in WAT significantly increased in Cd-treated groups after 2 weeks of exposure. Next, we examined the effects of Cd on adipocyte differentiation and hypertrophy. Cd exposure significantly decreased the paternally expressed gene 1/Mesoderm-specific transcript mRNA expression levels. Both peroxisome proliferator-activated receptor gamma2 and CCAAT/enhancer-binding protein alpha mRNA expression levels in WAT tended to decrease in the Cd-treated groups. Next, we determined the effects of Cd exposure on the mRNA expression levels of adipose-derived hormones, such as adiponectin and resistin. The adiponectin mRNA expression level in WAT decreased after both 6h and 2 weeks of exposure to a high dose of Cd, and the reduction in resistin mRNA expression levels was observed after 2 weeks of exposure. These results suggest that Cd exposure causes abnormal adipocyte differentiation, expansion, and function, which might lead to development of insulin resistance, hypertension, and cardiovascular disease.

Physicochemical and biological properties of an extracellular serine protease of Aeromonas sobria.

Previously, we cloned a protease gene of Aeromonas sobria, determined its nucleotide sequence and established a method of purifying its product. In this study, we examined the properties of the purified protease. The protease was temperature‐labile and had an optimal pH of 7.5. Metallo‐protease inhibitors and a cysteine protease inhibitor did not block the proteolytic activity of the enzyme. The treatment with reagents to modify sulfhydryl group did not reduce the activity. But, serine protease inhibitors did, showing that it was a serine protease. Subsequently, we examined the ability of the protease to enhance vascular permeability in dorsal skin. The protease showed activity and the reaction was inhibited by a simultaneously injected antihistaminic agent. Histopathological examination showed that mast cells appeared around the site where the protease was injected. These findings show that the vascular permeability‐enhancing effect of the protease is due to histamine released at the site. Furthermore, we found that a soybean trypsin inhibitor (Kunitz) did not block the proteolytic action of the protease in vitro, but inhibited its vascular permeability‐enhancing activity in skin. This suggests that a trypsin‐like protease from skin mediates the activity of the protease to enhance its vascular permeability.

Extra Y chromosome in T-cell acute lymphoblastic leukemia

We describe a 66-year-old man who developed T-cell acute lymphoblastic leukemia (ALL) with a sole clonal chromosomal abnormality of 47,XY,+Y. Leukemic cells were positive for CD2, CD7, terminal deoxynucleotidyl transferase and cytoplasmic CD3. T-cell receptor beta, gamma, and delta genes remained germline configurations. The bone marrow aspirate was 47,XY,+Y in all metaphase cells observed. The patient achieved complete remission by chemotherapy, and the bone marrow cells and the phytohemagglutinin stimulated peripheral blood lymphocytes showed a normal karyotype of 46,XY at that time. This fact suggests that an extra Y chromosome may be a kind of new chromosomal abnormality of T-cell ALL.

Acute Myeloblasts Leukemia Associated with an Intermediate State Between the Healthy Carrier State and Adult T-cell Leukemia

This report describes an intermediate state between the human T-cell lymphotropic virus type I (HTLV-I) healthy carrier and adult T-cell Leukemia (ATL) who developed acute myeloblastic leukemia (AML, FAB subtype M2). The polyclonal integration of HTLV-I proviral DNA was demonstrated in the peripheral blood lymphoid cells, whereas AML cells had no HTLV-I proviral DNA. The patient achieved remission after combination chemotherapy but cells with lobulated nuclei persist at a low level and the polyclonal integration of HTLV-I proviral DNA is still demonstrated. We suggest that the patients with the integration of HTLV-I proviral DNA might develop secondary neoplasms more frequently than healthy carriers and this case stresses the need to exercise caution with these patients. The relationship between HTLV-I and AML is briefly discussed.

Successful Treatment of a 93-Year-Old Patient with Hypoplastic Acute Monocytic Leukemia Using Macrophage Colony-Stimulating Factor

A 93-year-old patient with hypoplastic acute monocytic leukemia (AMoL) achieved a complete remission after macrophage colony-stimulating factor (M-CSF) therapy. Initially, the patient was treated with a low dose of cytarabine, but this treatment proved ineffective. M-CSF was administered for 14 days by drip intravenous infusion, 800 x 10(4) units per day. After a gradual decrease in the number of leukemic cells, a rapid increase in neutrophils was observed in the peripheral blood, and a bone marrow examination 22 days after discontinuation of M-CSF medication revealed a complete remission. These findings suggest that M-CSF may be useful in treating some elderly patients who have hypoplastic AMoL.