Hiromitsu Matsuzaki

Coinduction of Nitric-oxide Synthase and Arginase I in Cultured Rat Peritoneal Macrophages and Rat Tissues in Vivo by Lipopolysaccharide

Nitric oxide is synthesized by nitric-oxide synthase from arginine, a common substrate of arginase. Rat peritoneal macrophages were cultured in the presence of bacterial lipopolysaccharide (LPS), and expression of the inducible isoform of nitric-oxide synthase (iNOS) and liver-type arginase (arginase I) was analyzed. mRNAs for iNOS and arginase I were induced by LPS in a dose-dependent manner. iNOS mRNA appeared 2 h after LPS treatment and increased to a near maximum at 8-12 h. On the other hand, arginase I mRNA that was undetectable prior to the treatment began to increase after 4 h with a lag time and reached a maximum at 12 h. Immunoblot analysis showed that iNOS and arginase I proteins were also induced. mRNA for arginase II, an arginase isozyme, was not detected in the LPS-activated peritoneal cells. mRNA for CCAAT/enhancer-binding protein β (C/EBPβ), a transactivator of the arginase I gene, was also induced, and the induction was more rapid than that of arginase I mRNA. Changes in iNOS and arginase I mRNAs were also examined in LPS-injected rats in vivo iNOS mRNA increased rapidly in the lung and spleen, reached a maximum 2-6 h after the LPS treatment, and decreased thereafter. Arginase I mRNA was induced markedly and more slowly in both tissues, reaching a maximum in 12 h. Thus, arginase I appears to have an important role in down-regulating nitric oxide synthesis in murine macrophages by decreasing the availability of arginine, and the induction of arginase I is mediated by C/EBPβ.

An Asian variant of intravascular large B-cell lymphoma: clinical, pathological and cytogenetic approaches to diffuse large B-cell lymphoma associated with haemophagocytic syndrome

Diffuse large B‐cell lymphoma with haemophagocytic syndrome (BCL‐HS) has been reported mainly in Asia and is regarded as a distinct variant of intravascular lymphoma (IVL). However, it is unclear whether all cases of BCL‐HS fall within the framework of IVL and available clinical information is limited. We analysed 25 cases with BCL‐HS, including 11 autopsied cases (median, 66 years; male–female ratio, 1·1:1). The patients presented with fever, anaemia, thrombocytopenia, hepatosplenomegaly, haemophagocytosis, bone marrow invasion, respiratory disturbance and disseminated intravascular coagulopathy, but usually lacked lymphadenopathy, mass formation, neurological abnormalities and skin lesions. The clinical course was aggressive with a median survival of 7 months. The morphological findings were uniform: large lymphoid cells infiltrated vessels and/or sinusoids of the liver, marrow, lung, kidney and other organs. They were positive for CD19, CD20, CD79a and HLA‐DR, but negative for CD10, CD23 and CD30. CD5 was positive in five out of 17 cases. Our critical review indicates that BCL‐HS is the equivalent of the Asian variant of IVL.

Coinduction of Nitric Oxide Synthase, Argininosuccinate Synthetase, and Argininosuccinate Lyase in Lipopolysaccharide-treated Rats RNA BLOT, IMMUNOBLOT, AND IMMUNOHISTOCHEMICAL ANALYSES

Nitric oxide (NO) is synthesized from arginine by nitric oxide synthase (NOS), and citrulline which is generated can be recycled to arginine by argininosuccinate synthetase (AS) and argininosuccinate lyase (AL). Rats were injected with bacterial lipopolysaccharide (LPS), and expression of the inducible isoform of NOS (iNOS), AS, and AL was analyzed. In RNA blot analysis, iNOS mRNA was undetectable before the LPS treatment but was induced by LPS in the lung, heart, liver, and spleen, and less strongly in the skeletal muscle and testis. AS mRNA was induced in the lung and spleen, and AL mRNA was weakly induced in these tissues. AS and AL mRNAs were abundant in the control liver and remained unchanged after the treatment. Kinetic studies showed that iNOS mRNA increased rapidly in both spleen and lung, reached a maximum 2-5 h after the treatment, and decreased thereafter. On the other hand, AS mRNA increased more slowly and reached a maximum in 6-12 h (by about 10-fold in the spleen and 2-fold in the lung). AL mRNA in the spleen and lung increased slowly and remained high up to 24 h. In immunoblot analysis, increase of iNOS protein was evident in the lung, liver, and spleen, and there was an increase of AS protein in the lung and spleen. In immunohistochemical analysis, macrophages in the spleen that were negative for iNOS and AS before LPS treatment were strongly positive for both iNOS and AS after this treatment. As iNOS, AS, and AL were coinduced in rat tissues and cells, citrulline-arginine recycling seems to be important in NO synthesis under the conditions of stimulation.

An intensive chemotherapy of adult T-cell leukemia/lymphoma: CHOP followed by etoposide, vindesine, ranimustine, and mitoxantrone with granulocyte colony-stimulating factor support.

SUMMARY An intensive combination chemotherapy regimen supported by granulocyte colony-stimulating factor (G-CSF) was evaluated in adult T-cell leukemia/lymphoma (ATLL) patients in a multiinstitutional, cooperative study. Vincristine 1 mg/m2 i.v. day 1, Adriamycin 40 mg/m2 i.v. day 1, cyclophosphamide 400 mg/m2 i.v. day 1, prednisolone 40 mg/m2 i.v. days 1 to 3 and 8 to 10, etoposide 35 mg/m2 i.v. days 1 to 8, vindesine 2 mg/m2 i.v. day 8, ranimustine 50 mg/m2 i.v. day 8, mitoxantrone 7 mg/m2 i.v. day 8, and G-CSF 50 mg/m2 s.c. days 9 to 21 were given for 2 to 4 courses every 3 weeks to 83 patients with ATLL. Complete remission (CR) and partial remission (PR) were achieved in 35.8 and 38.3 percent, respectively, of 81 evaluable patients. The median survival of all patients was 8.5 months, with a predicted 3-year survival of 13.5 percent by the Kaplan-Meier method. The median duration of response was 7.6 months (range 0.2-42.7), and 13 patients were alive. Their median survival time was 29.1 months (range 19.2-44.7). In 67.6 percent of courses, white blood cell (WBC) nadirs were < 1.0 x 10(9)/L. Days required for the recovery of WBC from the nadir to > 1.0 x 10(9)/L were <5 days in 71.4 percent of the treatment courses. The G-CSF supported an intensified chemotherapy regimen for ATLL and yielded better response rate and longer survival compared to previous reports in Japan. Because duration of remission is still short, further studies of postremission therapy or other strategies are warranted.

Expression of Bcl-2 family of proteins in fresh myeloma cells

Members of the Bcl-2 family of proteins Second Department of Internal Medicine, Kumamoto University School of Medicine, Honjo 1-1-1, Kumamoto 860-8556, Japan Bcl-2, Bcl-XL, Bcl-Xs and Bax, are considered to play important roles in the regulation of apoptosis and drug resistance. To understand the significance of these proteins in fresh human myeloma cells, expression of Bcl-2 family of proteins was analyzed by Western blotting in 17 cases with multiple myeloma (MM) and three cases with plasma cell leukemia (PCL). Bcl-2 and Bcl-XL were found in 12 and nine samples, respectively. All PCL cases showed co-expression of Bcl-2 and Bcl-XL. Analysis of MM cases showed that Bcl-2 was preferentially expressed in samples from cases with early clinical stage while Bcl-XL tended to be expressed in samples from cases at advanced clinical stage. Bcl-XL was significantly expressed in tumor cells from cases with extramedullar lesions. There was no correlation between the expression levels of Bcl-2 or Bcl-XL and preceding chemotherapy. Expression of Bax was found in only one patient who had pleural effusion caused by invasion of myeloma cells and a high serum LDH level. Survival analysis revealed that there was no statistical significance in expression of Bcl-2 or Bcl-XL although Bcl-XL tended to be expressed in cases with poor prognosis. These findings indicate that expression of Bcl-2 family of proteins is heterogeneously regulated in fresh myeloma cells. Expression of Bcl-XL and Bcl-2 may correlate with extramedullar invasion and early stage of the disease, respectively. Absence of Bax in myeloma cells may contribute to low sensitivity of myeloma cells to anti-cancer agents since Bax is reported to mediate cytotoxicity of some anti-cancer drugs.

Monoclonal gammopathies in adult T-cell leukemia.

Age and sex distributions of monoclonal gammopathy were studied for 12,196 healthy controls and 2056 patients with various malignancies. The frequency of monoclonal component in adult T‐cell leukemia (ATL) patients was found to be much higher than that in patients with other malignancies and in healthy controls. This suggested that the occurrence of ATL and monoclonal gammopathy is not coincidental. Three cases of ATL with monoclonal gammopathy are also reported.

Down-Regulation of CD98 in Melphalan-Resistant Myeloma Cells with Reduced Drug Uptake

Although melphalan has been used as a therapeutic reagent for multiple myeloma, many patients become refractory. To elucidate the mechanism of resistance to melphalan, we generated a melphalan-resistant myeloma cell line, KHM-11EMS, by treating a parental line, KHM-11, with a mutagen, ethylmethanesulfonate. KHM-11EMS is 55 times more resistant to melphalan. γ-Glutamylcysteine synthetase, P-glycoprotein, multidrug-resistance-associated protein, lung-resistance-related protein and the Bcl-2 family of proteins were not responsible for the drug resistance in KHM-11EMS. Intracellular incorporation of melphalan to myeloma cells was determined by using [14C]-labeled melphalan. Accumulation of melphalan in KHM-11EMS was 43% of KHM-11, while the efflux rates were comparable in both cell lines. The uptake of melphalan was inhibited by the addition of L-phenylalanine, indicating that melphalan is incorporated through the L-phenylalanine transporter as reported previously. Expression of CD98, which was recently cloned as an L-phenylalanine transporter, was 6-fold decreased in KHM-11EMS, suggesting that CD98 may be correlated with the incorporation of melphalan. CD98 expression and incorporation of melphalan were analyzed in fresh purified myeloma cells from 5 patients. All myeloma cells from 4 cases expressed CD98 at a high level and incorporated melphalan. However, tumor cells from 1 case expressed CD98 at low levels and did not incorporate melphalan. Taken together, reduced melphalan uptake could be responsible for the drug resistance in KHM-11EMS, and down-regulation of CD98 may be related to this phenomenon. Further investigation of the correlation between impaired drug uptake and down-regulation of CD98 in myeloma cells should be important to understand the mechanism of resistance to melphalan.

Hyperammonemia in multiple myeloma.

Two patients with multiple myeloma who appeared to be producing ammonia are reported. Both patients showed hyperammonemia and amino acid disturbances, such as a low Fischer ratio. One patient had Bence Jones protein (lambda) type myeloma and became comatose, but the hyperammonemia and disturbance of consciousness were improved by chemotherapy for the myeloma. The other patient had IgA kappa type myeloma and somnolence and died of malignant pleurisy despite intensive chemotherapy. Autopsy showed widespread multiple myeloma and an almost normal liver. Ammonia levels in the supernatant of cultured myeloma cells from the patients pleural effusion increased almost linearly from the time of cell seeding. These observations showed that ammonia was produced at a high level by these human myeloma cells. We also found that one of the common myeloma cell lines, RPMI 8226, could produce ammonia as well.

Ectopic production of salivary‐type amylase by a lga‐λ‐type multiple myeloma

A patient with IgA‐λ‐type multiple myeloma who appeared to be secreting salivary‐type amylase ectopically is reported. Both the patients serum and urine contained high levels of amylase. Amylase activity in the supernatant of cultured myeloma cells, obtained from the patients pleural effusion while he had malignant pleuritis, increased almost linearly from the time of cell seeding. The presence of IgA and S‐type amylase in the myeloma cells was demonstrated cytochemically and by immunoelectron microscopy. These observations showed that amylase was produced by these human myeloma cells.

CCAAT/enhancer-binding protein β is required for activation of genes for ornithine cycle enzymes by glucocorticoids and glucagon in primary-cultured hepatocytes

Transcription of genes for enzymes of the ornithine cycle is activated by hormones such as glucocorticoids and glucagon. Promoters and enhancers of several genes for the enzymes interact with the CCAAT/enhancer‐binding protein (C/EBP) family of transcription factors, and C/EBPβ has been suggested to mediate glucocorticoid response of the gene for arginase, the last enzyme of the cycle. To determine the contribution of C/EBPβ to hormonal regulation of genes for ornithine cycle enzymes, we examined mice with targeted disruption of the C/EBPβ gene. Induction of genes for the enzymes by intraperitoneal injection of dexamethasone and glucagon was almost intact in the liver of C/EBPβ‐deficient mice. On the other hand, in primary‐cultured hepatocytes derived from C/EBPβ‐deficient mice, induction of genes for the first enzyme carbamylphosphate synthetase, as well as for arginase, in response to dexamethasone and/or glucagon was severely impaired. Therefore, C/EBPβ is required for hormonal induction of the genes for ornithine cycle enzymes in primary‐cultured hepatocytes, while the deficiency of C/EBPβ is compensated for in vivo.