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.

[Acute myeloblastic leukemia showing pronounced skin infiltration during administration of low-dose cytarabine and etoposide with granulocyte colony-stimulating factor].

A 26-year-old woman was admitted to our hospital for lumbago on November 29, 1995. The white blood cell count was 6,500/microliter with 26.5% myeloblasts and the bone marrow was hyperplastic due to myeloblasts. Myeloblasts were negative for myeloperoxidase and positive for alpha-naphthyl butylate esterase, CD11a (89%), CD11b (38%), CD11c (92%), CD33 (91%) and HLA-DR (58%). Chromosomal abnormalities were recognized: 46, XX, t(9;11) (p22;q23), 45, XX, -7, t(9;11) (p22;q23) and 47, XX, +19, t(9;11) (p22;q23). Acute myeloblastic leukemia (M5a) was diagnosed. Disseminated intravascular coagulation was also present. The patient received induction therapy and achieved remission on January 9, 1996, but myeloblasts increased to 3.6% in bone marrow despite consolidation therapy. Low doses of cytarabine (AraC) and etoposide were instituted on March 7, granulocyte colony-stimulating factor (G-CSF) was started on March 15, and pronounced skin infiltration developed on March 18. The patient received reinduction therapy from April 16 and administration of G-CSF was combined for 2 days, and a marked increment of myeloblasts in the peripheral blood was observed. After discontinuation of G-CSF, myeloblasts decreased and skin infiltration disappeared. However, the patient died of cerebral infiltration on June 30. The response of myeloblasts to G-CSF by in vitro liquid culture was noteworthy. The present case stresses the requirement for great caution to be exercised in the use of G-CSF in patients receiving low dose AraC.

[CD2 and CD8 expression in acute promyelocytic leukemia].

A 34-year-old man was admitted to our hospital for a headache in March, 1995. The patient's hemoglobin was 7.5 g/dl, platelet count was 1.8 x 10(4)/microliter and white blood cell (WBC) count was 12,400/microliters with 99% myeloblasts. Myeloblasts were agranular or hypogranular but electron microscopy revealed microgranules in cytoplasm, and a few faggots were observed. The bone marrow was hyperplastic due to myeloblasts and chromosomal abnormality was recognized: 46, XY, t(15; 17) (q22; q12). PML-RAR alpha with intron 3 breakpoint of the PML locus, and rearrangements of the T-cell receptor beta and gamma genes were detected. These cells were positive for CD2 (63%), CD8 (47%), CD13 (87%) and CD33 (99%). Microgranular variant type of acute promyelocytic leukemia (APL) was diagnosed. Disseminated intravascular coagulation (DIC) was also present. The patient was treated with enocitabine, daunorubicin, 6-mercaptopurine, dalteparin sodium, anti-thrombin III concentrates and gabexate mesilate with prophylactic frozen transfusions of fresh plasma and platelet transfusions for 5 days, but WBC count did not decrease and DIC did not improve. The patient died of cerebral hemorrhage 7 days after diagnosis of APL. APL with CD8 expression has never been reported. We suggest that therapy should be modified in this type of APL and conclusions concerning the most appropriate therapeutic strategy will depend on the results of treatment of similar cases in the future.

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

We describe a 44-year-old man with non-Hodgkin's 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 GCSF. patients who produce a rapid increase in platelet levels could be at greater risk for thrombotic events and need to be followed-up carefully.

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.

[Chylothorax in a patient with 9-year remission of malignant lymphoma].

We describe a 69-year-old man who developed chylothorax after a 9-year remission of malignant lymphoma. The patient was admitted to our hospital and received exploratory laparotomy for ileus in February 1986. Bulky masses in the posterior mediastinum and the retroperitoneum, and also a jejunal tumor were observed. Fibrosis of the liver was also observed. The jejunal tumor was removed and histological findings revealed diffuse large B-cell malignant lymphoma. He was treated by combination chemotherapy and remission was achieved. He was discharged in June and remained in remission, but was readmitted for right pleural effusion in October 1994. Effusion was chylous and the chylomicron level was estimated to be 181 mg/dl. Liver cirrhosis also developed but there was no chylous ascites. Chylorrhea disappeared after continuous aspiration, but recurred in December. Continuous aspiration was ineffective, therefore 10 KE of OK-432 was administered twice into the pleural cavity, and chylorrhea again disappeared. No findings suggestive of malignant lymphoma were not detected by computerized tomography and gallium scintigram. He was discharged in March 1995 and chylothorax has not recurred since. These findings suggest that the fragility of the thoracic duct which had been infiltrated by malignant lymphoma might increase, resulting in rupture, even if in remission.

Acute myeloblastic 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.

Richter's syndrome showing pronounced lymphadenopathy in response to administration of granulocyte colony-stimulating factor.

A patient with Richter's syndrome developed rapid generalized lymph node enlargement with a decrease of peripheral blood lymphocytes after recombinant human granulocyte colony-stimulating factor (rhG-CSF) therapy for neutropenia induced by chemotherapy. The lymphadenopathy subsided spontaneously following discontinuation of rhG-CSF medication. Reinstitution of rhG-CSF therapy was followed by the same response as during initial therapy. Histopathologically, the lesions were characteristic of diffuse large cell lymphoma (DLL) with no evidence of myeloid cell involvement. No spontaneous contraction of enlarged lymph nodes followed withdrawal of the second course, but the enlargement subsided with chemotherapy. The patient died of myocardial infarction. All residual tumors examined post mortem presented microscopic features of small lymphocytic lymphoma (SLL), and G-CSF receptor was demonstrated on these neoplastic cells by Northern blot hybridization analysis. This observation indicates that some B cell malignancies may retain G-CSF receptor and respond to G-CSF.

[Extramedullary pleural myelo-megakaryoblastic crisis during hematological chronic phase in chronic myeloid leukemia].

A 77-year-old man, in whom chronic myeloid leukemia (CML) had been diagnosed in October 1990, was admitted to hospital with right chest pain in November 1992. Bone marrow examination revealed the chronic phase of CML. Chest X-ray showed right pleural effusion. The cells from pleural effusion were positive for CD7, CD13, CD33, CD41a, including CD33, CD41a-double positive cells in 57.5%. Southern blot analysis revealed 3'bcr rearrangement. Electron microscopic examination showed the presence of platelet peroxidase. An abnormal karyotype with various additional chromosomes was observed. This is a rare case of extramedullary pleural myelo-megakaryoblastic biphenotypic crisis during the chronic phase of CML.

Adult T-cell leukaemia/lymphoma featuring a large granular lymphocyte leukaemia morphologically.

A 70-year-old man from an endemic area of human T-cell lymphotropic virus type I (HTLV-I) developed rapid generalized lymphadenopathy and abdominal tumours. The white blood cell count was 198.3 x 10(9)/l with 93% lymphocytes, 66.3% of which expressed large granular lymphocytes (LGLs). Bone marrow and lymph nodes were also infiltrated by LGLs. Surface markers were positive for CD4, CD25 and HLA-DR, and negative for CD3, CD8, CD16, CD56 and CD57. A monoclonal integration of HTLV-I proviral DNA was demonstrated on these LGLs by Southern blot hybridization analysis. This fact indicates that some adult T-cell leukaemia/lymphoma may morphologically present LGL leukaemia.

Histochemical and immunohistochemical analyses of primary carcinoma of the liver.

Hematoxylin and eosin (H-E) stained liver sections of 47 autopsy cases of hepatic malignancies were examined. There were 43 cases of hepatocellular carcinoma (subtypes of 30 trabecular, 7 solid, 5 pseudoglandular, and one scirrhous carcinoma), 3 of cholangiocellular carcinoma and one of mixed carcinoma. After immunohistochemical staining, benign hepatocytes reacted positively with anti-epithelial membrane antigen (EMA). Hepatocellular carcinoma cells reacted more weakly than benign hepatocytes. It was noted that the microtubular structure, which could not be demonstrated even by alcian blue or cationic ferric hydroxide colloid stabilized with cacodylate (Fe-CaC), was clearly detected with anti-EMA. The EMA-positive microtubular structures may indicate terminal cholangiolar differentiation. Based on EMA, seven more cases formerly classified as hepatocellular carcinoma by H-E were reclassified as mixed carcinoma, totaling eight (17.0%). The histologic classification of "mixed carcinoma" has been 1.5 to 2.0% of primary liver cancers in Japan, but we suggest there may be more cases of "mixed carcinoma" identified in the future. In conclusion, we emphasize that EMA staining is useful for more accurate classification of hepatic tumors.