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Cytogenetic abnormalities were often observed in primary myelofibrosis patients. The presence of specific cytogenetic abnormalities, such as sole abnormalities of chromosome 13q-, 20q-, or -7/7q-, is reported to have the influence on the prognosis of primary myelofibrosis. We analyzed the data from the prospective survey of Japanese primary myelofibrosis patients which was conducted from 1999 to clarify the impact of cytogenetic abnormalities on the prognosis of primary myelofibrosis. A total of 202 primary myelofibrosis patients had the cytogenetic and the prognostic data. Eighty (40%) out of 202 cases had cytogenetic abnormalities, and an association was evident for platelet counts. Although the presence of an abnormal karyotype did not affect the prognosis, primary myelofibrosis patients with cytogenetic abnormalities other than 13q- and 20q- showed an inferior prognosis compared to patients with a normal karyotype or sole 13q- or 20q- abnormalities. Patients with an unfavorable cytogenetic profile (abnormal cytogenetics other than 13q- or 20q-) also had a greater tendency to transform to leukemia than patients with a favorable cytogenetic profile (normal cytogenetics, sole abnormalities of either chromosome 13q-, or 20q-). Abnormal cytogenetics other than 13q- or 20q- in primary myelofibrosis patients has the poor prognostic effect for both survival and the risk of leukemic transformation.

Our hospital acts as a regional core hospital through inter-hospital collaboration. Geographical information is necessary to guide patients to the other hospitals. Although paper maps, which contain directions, nearest public transportation, etc., are usually provided to guide patients to the hospitals, the geographical information tends to change daily. However, updating the geographical information on the maps is costly. We constructed an electronic geographical information system using the Google Map API ( http://code.google.com/apis/maps/ ) with open source software to improve our ability to collaborate with other clinics.

In order to obtain sequence data for the blood coagulation factor VII and factor X in several mammalian species, we amplified and sequenced the DNA segments of exon VIII from each gene by means of the polymerase chain reaction (PCR) method. The DNA segments from the following species were successfully amplified: factor VII from the rhesus monkey and dog, and factor X from the rhesus monkey, Syrian hamster and rat. In each factor, the nucleotide sequences and predicted primary structures of the protease domain showed a high degree of homology among species; amino acid identities of approximately 68%-92% and 80%-98% were demonstrated among species in factor VII and factor X, respectively. The locations of the active site residues and five Cys residues were evolutionarily conserved in both factors. Interestingly, the amino acids involved in the human genetic variants, both factor VII 304-Arg and factor X 326-Arg, were always conserved across species. The data presented here will be helpful for investigating human genetic variants of factor VII or X, and will provide considerable information for constructing in vitro site-specific mutants of these factors.

Colony-stimulating activity (CSA) was demonstrated in materials taken from a patient suffering from lung cancer associated with excessive leucocytosis. CSA was detected not only in his urine, serum and pleural effusion but also in the supernatant of cell cultures originating from the effusion. The excessive leukocytosis of the patient might be due to a CSA producing tumor. A cell line (KONT) originating from the CSA producing tumor has been maintained for 4 years and shown to produce mouse- and human-CSA.

Human granulocyte colony-stimulating factor (G-CSF) receptors on human acute leukemia cells were investigated using human G-CSF iodolabeled by the lactoperoxidase method. Among various human leukemic cell lines, only cells of myelogenous lineage including HL-60, THP-1 and U937 had one type of high-affinity receptor for G-CSF, as shown by Scatchard analysis. Fresh leukemia cells from 19 patients with acute myelogenous leukemia (AML) were then studied. Specific receptors for G-CSF were demonstrated on blast cells in all 19 cases, the mean number of G-CSF receptors per AML cell ranging from 95 to 1436. G-CSF receptors on AML cells appeared to be a single affinity type, although some variations were observed. The mean number of G-CSF receptors on leukemic cells from patients with either FAB M3 or FAB M2 was greater than that of cells from patients with M1 (p less than 0.01, p less than 0.10, respectively). Moreover, the mean number of receptors for G-CSF on CD13- and CD34-positive AML cells was higher than that on CD13-negative and CD34-positive AML cells (p less than 0.01), and the mean number of G-CSF receptors on CD7-positive AML cells was lower than that for CD7-negative AML cells (p less than 0.10). Since the FAB classification and surface phenotypes reflect maturation stages, our findings indicate that the distribution of G-CSF receptors, even on AML cells, may be related to the maturation process.

We demonstrate the in vivo effects of bacterially synthesized, N-methionine-free recombinant human granulocyte-macrophage colony stimulating factor (rh GM-CSF) using a crab-eating monkey model. Monkeys were treated with cyclophosphamide (60 mg/kg) and administered with rh GM-CSF (30 micrograms/kg/d) subcutaneously (s.c.) for 7 days. Within 12 h, a transient increase of neutrophils (greater than 15.0 x 10(9)/l) was observed, and complete recovery of WBC counts was obtained by d 9 (d 16 in control monkeys). Neutrophils and eosinophils were absolutely increased (greater than 8 x 10(9)/l) on d 10. Readministration of rh GM-CSF (30 micrograms/kg/d, s.c.) for 3 d (including control monkeys) revealed absolute increases of neutrophils, eosinophils, monocytes and platelets. A two-fold increase of granulocyte/macrophage colony-forming units was also seen in the bone marrow, while the number of burst-forming units-erythroid was not affected. These data indicate that rh GM-CSF of this type stimulates granulopoiesis and thrombopoiesis in vivo.

Changes occurring in human haematopoiesis with advancing age were studied using an in vitro haematopoietic colony assay in 22 elderly subjects with unexplained anaemia, and in 15 elderly and 15 young subjects without anaemia. Both elderly groups were found to have significantly lower numbers of bone marrow early erythroid-committed progenitors (BFU-E) than the young controls. The elderly anaemic group also showed significantly lower numbers of granulocyte/macrophage progenitors (CFU-GM) than the young controls. However, the responses of the erythroid-committed progenitors in the elderly groups to erythropoietin and burst-promoting activity were similar to those observed in the young controls. Therefore, it is probable that anaemia tends to occur easily in elderly individuals as a result of reduction of reserves of haematopoietic progenitors with advancing age.

A major issue in autologous blood stem cell transplantation (ABSCT) for leukaemia is whether peripheral blood stem cell (PBSC) harvests are less contaminated with leukaemic cells than bone marrow mononuclear cells (BMMNC). We compared leukaemic contamination in PBSC harvests and BMMNC, obtained simultaneously, by using reverse transcriptase polymerase chain reaction (RT-PCR) of leukaemia-specific chimaeric messenger RNA (mRNA), in three patients with Philadelphia chromosome (Ph)-positive acute lymphoblastic leukaemia (ALL), one with Ph-positive acute myelogenous leukaemia (AML), and two with acute promyelocytic leukaemia (APL). Our two-step PCR method employed 'nested primers' in the second step and can detect one leukaemic blast diluted into 10(6) HL-60 cells. In three of four patients with Ph-positive ALL and AML we detected leukaemic contamination in both PBSC harvests and BMMNC. In the remaining patient with ALL, both PBSC harvests and BMMNC were PCR-negative. Both PBSC harvests and BMMNC from one patient with APL were PCR-positive. In contrast, PBSC harvests from another patient with APL, whose BMMNC could not be obtained because of bone marrow necrosis, were PCR-positive after the first course of consolidation chemotherapy, but became PCR-negative after the second course. The present study does not support the hypothesis that PBSC harvests are less contaminated by leukaemic cells than BMMNC, but suggests that PBSC harvests are contaminated when BMMNC are contaminated.

症例1はgenotype 1bのウイルスによるC型慢性肝炎の51歳女性患者．ペグインターフェロン，リバビリン併用療法を施行したところ，投与40週目に血糖値581mg/dlと1型糖尿病を発症し，インスリン療法を施行した．治療前の血糖値，HbA1c値は正常範囲で，抗GAD抗体も陰性であったが，糖尿病発症時には抗GAD抗体は714U/mlと陽転化した．症例2はserotype 1のC型慢性肝炎の58歳男性患者．治療前の血糖値，HbA1c値は正常範囲であったが，抗GAD抗体は陽性を示した．ペグインターフェロン，リバビリン併用療法を施行し，慎重に経過を追ったが糖尿病の発症はみられなかった．当科で抗GAD抗体陰性を確認した後にインターフェロン療法を行った58例のC型慢性肝炎のうち1型糖尿病を発症したのは本報告（症例1）1例のみであった．また抗GAD抗体陽性を確認した後にインターフェロン療法を行ったC型慢性肝炎症例は本報告（症例2）1例のみであった．インターフェロン単独あるいはリバビリン併用療法による1型糖尿病発症は稀ではあるが，大多数が終生のインスリン療法を必要とする重大な合併症であり，その認識は肝要である．しかしながら，C型慢性肝炎に対する抗ウイルス療法により発症する1型糖尿病の予測は極めて困難であり，今後の症例の蓄積と解析が俟たれるところである．

Accumulating evidence suggests that viruses play an important role in the development of diabetes. Although the diabetogenic encephalomyocarditis strain D virus induces diabetes in restricted lines of inbred mice, the susceptibility genes to virus-induced diabetes have not been identified. We report here that novel Tyrosine kinase 2 (Tyk2) gene mutations are present in virus-induced diabetes-sensitive SJL and SWR mice. Mice carrying the mutant Tyk2 gene on the virus-resistant C57BL/6 background are highly sensitive to virus-induced diabetes. Tyk2 gene expression is strongly reduced in Tyk2-mutant mice, associated with low Tyk2 promoter activity, and leads to decreased expression of interferon-inducible genes, resulting in significantly compromised antiviral response. Tyk2-mutant pancreatic β-cells are unresponsive even to high dose of Type I interferon. Reversal of virus-induced diabetes could be achieved by β-cell-specific Tyk2 gene expression. Thus, reduced Tyk2 gene expression in pancreatic β-cells due to natural mutation is responsible for susceptibility to virus-induced diabetes., Diabetes can be caused by viral infections in humans and some inbred mice, suggesting genetic predisposition. Here the authors show that mutations in Tyk2 gene underlie susceptibility to virus-induced diabetes in mice, due to Tyk2 requirement for antiviral response in insulin-producing cells.

Animal models suggest that a deficiency in myeloperoxidase (MPO; EC 1.11.1.7), a lysosomal hemoprotein involved in host defense, may be associated with a decreased level of immunity. A nonsynonymous mutation, resulting in an arginine to cysteine substitution (Arg499Cys or R499C), has been identified in the exon 9 genetic coding region of a Japanese patient with complete MPO deficiency. Genetic analysis revealed that the mRNA of the patient could be correctly transcribed then further translated into a peptide sequence. However, the Western blot analysis confirmed the absence of MPO peptides. An initial screening assay of the patient’s blood exhibited an abnormal hematograph, and no MPO activity was detected. To determine if this mutation might be associated with MPO deficiency, DNA samples for 387 controls were examined. Genetic analysis was performed using standard PCR techniques for amplification and sequencing. None of the control samples possessed the R499C substitution. This mutation is in close proximity to a different mutation (G501S) previously found in another Japanese MPO-deficient patient, and the amino acid, H502, which is strongly involved in heme binding, leading to the speculation that heme binding may play a role in complete MPO deficiency.

In treating elderly non-Hodgkin's lymphoma (NHL) patients, it is particularly important to use drugs that have a low incidence of adverse events and high efficacy. In this multicenter study, THP (pirarubicin)-COP (cyclophosphamide, vincristine, and prednisolone) was compared to two thirds dosage of full CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisolone) regimen with regard to both adverse events and efficacy. For a third group, etoposide (E) was added to the THP-COP regimen (THP-COPE) in order to achieve high dose-intensity. Subjects were 486 previously untreated patients, aged 65 or older (range, 65-92 years; median, 74 years), with NHL. Subjects were randomly assigned to receive THP-COP, two thirds CHOP, or THP-COPE. Four hundred and forty-three patients were assessed for response and followed for 8 years after the last subject registered. The complete remission rates for the THP-COP, CHOP, and THP-COPE groups were 42.5%, 41.4%, and 48.0%, respectively. There was no difference in overall survival or progression-free survival among these 3 groups. In aggressive lymphoma, there was also no difference in complete response (CR) rate (45.3% in THP-COP, 44.9% in CHOP, 48.0% in THP-COPE), overall survival, and progression-free survival among these groups. The 5- and 8-year survival rates for all patients were 29.4% and 18.7%, respectively. The 5- and 8-year survival rates for patients with aggressive lymphoma were 27.4% and 17.4%, respectively. Although long-term survival for patients with aggressive lymphoma on our regimens was not worse compared to previous reports, the CR rate was lower. Because severe adverse events were not observed, higher dose chemotherapy may be directed to achieve better CR rates. In patients with T-cell-type lymphoma, the CR rate was greater after treatment with THP-COP (51.4%) or THP-COPE (57.7%) compared to treatment with CHOP (19.4%). Pirarubicin may be more useful for T-cell lymphoma than doxorubicin. Because adverse cardiac events were reported only in CHOP, adverse cardiac events might be low in the THP group.

A chimeric toxin in which the cell-surface binding domain of Pseudomonas exotoxin A was replaced with mature human granulocyte colony-stimulating factor (G-CSF) was produced in Escherichia coli, purified and tested for its biological activity on the human G-CSF-responsive myeloid leukemia cell line, UT7/GR. This fusion protein, termed G-CSF-PE40, showed potent cytotoxicity in the cell line in a dose-dependent manner. G-CSF-PE40 displaced binding of biotinylated G-CSF to its receptor, and the cytotoxicity of G-CSF-PE40 was neutralized by an excess of wild-type G-CSF, indicating the receptor-mediated effects of this chimeric toxin. When G-CSF-PE40 was injected into normal mice, they showed transient neutropenia but no significant changes in the numbers of red blood cells or platelets. Furthermore, G-CSF-PE40 prolonged the survival of mice transplanted with syngeneic myeloid leukemia cells. These observations suggest that G-CSF-PE40 may be useful in targeted therapy of myeloid leukemia cells expressing G-CSF receptors.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the activation of autoreactive B lymphocytes, which are supposed to carry aberrant signal transduction after the stimulation of B-cell receptor (BCR). To investigate abnormalities in BCR-mediated signaling pathway in lupus B lymphocytes, we analyzed HS1, a molecule downstream of BCR, in 80 Japanese SLE patients. We identified 37 amino acid deletion of HS1 in a 25-year-old female patient, and the aberrant HS1 lacked a part of a functional motif. Analysis of genomic DNA revealed that the aberrant HS1 was caused by exon skipping. Family study showed that the patient as well as her father and sister are heterozygous for the abnormality. WEHI-231 cell, a mouse B cell line, transfected with the aberrant HS1 displayed a significantly increased cell death upon cross-linking of BCR. Additionally, peripheral B lymphocytes from the patient exerted increased apoptosis after BCR stimulation compared to those from control SLE patients. These data suggest that the aberrant HS1 molecule may transmit an accelerated signal after BCR stimulation and may play a role in the activation of autoreactive B lymphocytes.

Tyk2 is activated in response to interleukin-12 (IL-12) and is essential for IL-12-induced T-cell function, including interferon-gamma (IFN-gamma) production and Th1 cell differentiation. Because IL-12 is a stimulatory factor for natural killer (NK) cell-mediated cytotoxicity, we examined whether tyk2 is required for IL-12-induced NK cell activity. IL-12-induced NK cell activity in cells from tyk2-deficient mice was drastically reduced compared to that in cells from wild-type mice. IL-18 shares its biologic functions with IL-12. However, the molecular mechanism of IL-18 signaling, which activates an IL-1 receptor-associated kinase and nuclear translocation of nuclear factor-kappaB, is different from that of IL-12. We next examined whether biologic functions induced by IL-18 are affected by the absence of tyk2. NK cell activity and IFN-gamma production induced by IL-18 were reduced by the absence of tyk2. Moreover, the synergistic effect of IL-12 and IL-18 for the production of IFN-gamma was also abrogated by the absence of tyk2. This was partially due to the absence of any up-regulation of the IL-18 receptor treated with IL-12, and it might suggest the presence of the cross-talk between Jak-Stat and mitogen-activated protein kinase pathways in cytokine signaling.

We report a case of acute myelogenous leukemia (AML) concurrent with untreated chronic lymphocytic leukemia (CLL). An 84-year-old Japanese man was admitted to the Chihaya Hospital with persistent high-grade fever. Morphologic observation of peripheral blood and bone marrow smears revealed a proliferation of blasts and lymphocytosis with small and mature phenotypes. Immunophenotyping of the blast cells revealed CD13+, CD33+, CD34+, and HLA-DR+, and that of the lymphocytes revealed CD5+, CD19+, CD20+, and lambda+ on the cell surface. The peripheral lymphocytes revealed an IgH gene rearrangement. Chromosome analysis of 20 metaphase cells from bone marrow showed numerous abnormalities, containing +8,+11,+21. The patient's disease was diagnosed as AML with trilineage dysplasia concurrent with CLL. The simultaneous occurrence of AML and CLL is extremely rare but should not be overlooked as a possible underlying cause of lymphocyte abnormalities in AML patients.

Summary. To investigate the efficacy and safety of granulocyte colony-stimulating factor (G-CSF) in patients with acute myelogenous leukaemia, a multicentre randomized study was performed. From October 1993 to September 1996, 270 patients with newly diagnosed acute myelogenous leukaemia were randomized to G-CSF or control groups after remission induction therapy. The G-CSF group received G-CSF (Filgrastim) from 48 h after the completing chemotherapy until the absolute neutrophil count exceeded 1·5 × 109/l. The control group did not receive G-CSF unless severe infection occurred. There were 245 evaluable patients (120 and 125 in the G-CSF and control groups respectively). The complete remission rate was similar in the G-CSF and control groups (80·8% versus 76·8%), as was the 5-year probability of disease-free survival (34·5% versus 33·6%) and overall survival (42·7% versus 35·6%). Neutrophil recovery was significantly faster in the G-CSF group than in the control group (12 d versus 18 d, P = 0·0001). The median duration of febrile neutropenia was significantly shorter in the G-CSF group than in the control group (3 d versus 4 d, P = 0·0001). In conclusion, prophylactic administration of G-CSF after remission induction therapy for acute myelogenous leukaemia is safe and useful even in patients without infection on completing chemotherapy.

Trials of immunosuppressive therapy have been reported in some case reports of hypoplastic myelodysplastic syndrome (MDS). In this study, we gave immunosuppressive therapies to eight patients with normo- or hyperplastic MDS of refractory anemia subtype without karyotypic abnormalities and analyzed the HLA-DRB1 type or the presence of paroxysmal nocturnal hemoglobinuria (PNH) neutrophils in these patients. Cyclosporin A (CyA) therapy was effective for improving cytopenia in four of the eight MDS patients. While the side effects of CyA were mostly mild and transient, one patient demonstrated karyotypic abnormality following CyA therapy and accelerated to refractory anemia with an excess of blasts. Additional antithymocyte globulin (ATG) therapy was effective in one of three nonresponders to CyA therapy. One patient died due to leukemic transformation after ATG therapy. When we analyzed the correlation between the response to CyA therapy and the HLA-DRB1 type, there were more responders with DRB1*1501 (three of four patients) than without (one of four patients), but a statistically significant difference was not evident between the two groups. In addition, the presence of PNH neutrophils was not correlated with the response to CyA and/or ATG therapy. These results indicate the usefulness of immunosuppressive therapies even for normo- or hyperplastic MDS patients. Further trials using more patients with a long follow-up period would be worthwhile in order to clarify the possibility of disease progression and in order to predict the response of patients.

AML1-MTG8 chimeric oncogene is generated in acute myelogenous leukemia with t(8;21), and seems to be responsible for the pathogenesis of the disease. However, the role of MTG8 is ambiguous. Here we found that MTG8 interacted with the regulatory subunit of type II cyclic AMP-dependent protein kinase (PKA RIIalpha). The binding site of MTG8 was NHR3 domain, and that of RIIalpha was the N-terminus for interacting with PKA anchoring proteins (AKAPs). NHR3 contains a putative alpha-amphipathic helix which is characteristic in binding of AKAPs with RII. Indirect immunofluorescence microscopy showed that MTG8 and RIIalpha were overlapped at the centrosome-Golgi area in lymphocytes. These findings suggest that MTG8 may function as an AKAP at the centrosome-Golgi area in lymphocytes.

The IPSS scoring system is useful to establish the appropriate treatment plan in MDS. Growth factors may alleviate both anemia and neutropenia in some MDS patients. Serum Epo levels and need for transfusion serve as good predictors of the erythroid response to the combination of Epo and G-CSF. Subgroups of MDS patients may respond favorably to immunosuppressive therapies such as CyA and ATG. Low-dose chemotherapy may also improve peripheral blood counts. Platelet counts, bone marrow cellularity, chromosome aberrations, and ringed sideroblasts combine to create a model predicting the response to low-dose ara-C. High-dose chemotherapy may lead to complete remission in about half of MDS patients, but the duration of remission is often short. The only proven curative therapy for MDS is allogeneic stem cell transplantation, resulting in an overall disease-free survival rate of about 40%. Only a minority of patients, however, can undergo allogeneic transplantation, both because of patient age and the availability of suitable donors. Autologous stem cell transplantation may be an option for selected patients who are unable to find allogeneic donors. Because the clinical features of patients with MDS are quite heterogeneous, the development of more accurate predictive models may be necessary to improve the efficacy of treatment.

Aberrant hypermethylation of tumor suppressor genes plays an important role in the development of many tumors. Recently identified new DNA methyltransferase (DNMT) genes, DNMT3A and DNMT3B, code for de novo methyltransferases. To determine the roles of DNMT3A, DNMT3B, as well as DNMT1, in the development of leukemia, competitive polymerase chain reaction (PCR) assays were performed and the expression levels of DNMTs were measured in normal hematopoiesis, 33 cases of acute myelogenous leukemia (AML), and 17 cases of chronic myelogenous leukemia (CML). All genes were constitutively expressed, although at different levels, in T lymphocytes, monocytes, neutrophils, and normal bone marrow cells. Interestingly, DNMT3B was expressed at high levels in CD34(+) bone marrow cells but down-regulated in differentiated cells. In AML, 5.3-, 4.4-, and 11.7-fold mean increases were seen in the levels of DNMT1, 3A, and 3B, respectively, compared with the control bone marrow cells. Although CML cells in the chronic phase did not show significant changes, cells in the acute phase showed 3.2-, 4.5-, and 3.4-fold mean increases in the levels of DNMT1, 3A, and 3B, respectively. Using methylation-specific PCR, it was observed that the p15(INAK4B) gene, a cell cycle regulator, was methylated in 24 of 33 (72%) cases of AML. Furthermore, AML cells with methylated p15(INAK4B) tended to express higher levels of DNMT1 and 3B. In conclusion, DNMTs were substantially overexpressed in leukemia cells in a leukemia type- and stage-specific manner. Up-regulated DNMTs may contribute to the pathogenesis of leukemia by inducing aberrant regional hypermethylation. (Blood. 2001;97:1172-1179)

We studied intracellular cytokines in monocytes by flow cytometry from 28 patients with hematologic malignancies and solid tumors to analyze the role of monokines in the hematologic recovery phase for peripheral blood stem cell harvest. The patients were divided into three groups: the first group, A, had a documented infection; the second group, B, had fever of unknown origin; and the third group, C, was afebrile. We found an increase in intracellular IL-1alpha, IL-6, IL-8 and TNF-alpha positive monocytes as CD14 positive gated cells cultured with lipopolysaccharide in all groups, but no increase was found with medium only when cultured for 4 h. We also found an increase in intracellular IL-1a, IL-6, IL-8 and TNF-alpha positive monocytes cultured with autologous serum for 4 h, but only in group A. The rate of intracellular cytokine positive cells was higher in monocytes cultured with only autologous serum from group A patients compared to those cells from the other groups; the data concerning IL-1a, IL-6 and TNF-alpha reached statistical significance (P0.05). However, increasing intracellular cytokine levels in the control group of patients exhibiting only infectious disease were observed. Thus, it appear that pro-inflammatory intracellular cytokine levels in monocytes are only related to microbial infections.

SummaryThe factor XII genes of two unrelated factor XII-deficient Japanese families were screened, and two novel mutations were identified. A heterozygous mutation (Q421K) was identified in the gene of a cross-reacting material (CRM)-negative patient with reduced FXII activity (entitled Case 1). No mutations were discovered in the other allele. Case 2 was a CRM-negative patient with severe FXII deficiency. In this case, a homozygous mutation (R123P) was discerned. Expression studies in Chinese Hamster Ovary (CHO) cells demonstrated accumulation of mutant Q421K factor XII in the cell, and insufficient secretion, while the R123P mutant showed lower levels of accumulation than wild-type, and no evidence of secretion in culture supernatant. In the presence of proteasome inhibitor, all types of FXII (wild-type, Q421K, R123P) accumulated in the cells. Protease protection experiments using the microsomal fraction of these cell lines demonstrated that while 20% wild-type FXII (total wild-type:100%) and 10% R123P mutant (total R123P-type: 40%) were resistant to treatment with trypsin, 50% Q421K-type FXII (total Q421K-type:130%) remained resistant to digestion. From these results, we conclude that Q421K is less susceptible to proteasome degradation than wild-type, but is unable to exit the ER efficiently, resulting in insufficient secretion phenotype. In contrast, R123P is susceptible to proteasome degradation and is not secreted.

Using a guide wire before insertion of a sheath is a common procedure with infrequent complications. We report an unusual case of a guide wire having been entrapped by the Chiari network prior to an intended radiofrequency ablation procedure, and which could be observed using intracardiac echocardiography. Using transthoracic echocardiography prior to ablation, this patient had been shown to have a relatively large Chiari network. We caution against using a long guide wire in patients with a large Chiari network.

The membrane TNF-α is known to serve as a precursor of the soluble form of TNF-α. Although it has been reported the biological functions of the membrane TNF-α as a ligand, the outside-to-inside (reverse) signal transmitted through membrane TNF-α is poorly understood. Here we report a novel function mediated by outside-to-inside signal via membrane TNF-α into the cells expressing membrane TNF-α. Activation by anti-TNF-α Ab against membrane TNF-α on human T cell leukemia virus (HTLV) I-infected T cell line, MT-2, or PHA-activated normal human CD4+ T cells resulted in the induction of an adhesion molecule, E-selectin (CD62E), on the cells with the peak of 12–24 h, which completely disappeared by 48 h. When wild-type or mutant membrane TNF-α (R78T/S79T) resistant to proteolytic cleavage was introduced into Jurkat or HeLa cells, E-selectin was induced by the treatment with anti-TNF-α Ab with the similar kinetics. Membrane TNF-α-expressing Jurkat cells also up-regulated E-selectin when brought into cell-to-cell contact with TNF receptor-expressing HeLa cells. Northern blot analysis and RT-PCR analysis showed that the membrane TNF-α-mediated E-selectin expression was up-regulated at the level of transcription. These results not only confirmed our previous findings of reverse signaling through membrane TNF-α, but also presented evidence that E-selectin was inducible in cell types different from endothelial cells. It is strongly suggested that membrane TNF-α is a novel proinflammatory cell surface molecule that transmits bipolar signals in local inflammation.

The effects of subthreshold stimulation (STS) by direct current were investigated in 20 patients with atrioventricular nodal reentrant tachycardia (AVNRT), 27 with atrioventricular reentrant tachycardia (AVRT) and 3 with idiopathic atrial reentrant tachycardia (IART). STS was delivered to each eligible site for ablation prior to radiofrequency application. STS was defined as `positive' if it could terminate the tachycardia or disrupt the conduction of accessory pathways without myocardial capture and defined as `negative' if it could not. Radiofrequency ablation was performed irrespective of a positive or negative result from STS and was successful in all 50 patients. Among the 50 successful ablation sites, STS was positive at 26 sites (11 sites in AVNRT, 12 in AVRT and 3 in IART). STS was positive at 4 sites where ablation failed in 3 patients with AVRT and was negative at 8 sites where ablation was successful in 4 patients with AVNRT and 4 with AVRT. The positive and negative predictive value of STS for the detection of the optimal ablation site were, respectively, 100% and 74% in AVNRT, 73% and 72% in AVRT, and both 100% in IART. STS-guided mapping is a specific method to predict the successful catheter ablation of reentrant supraventricular tachycardia. (Jpn Circ J 2001; 65: 1057 - 1063)

Janus kinases (Jaks) play an important role in signal transduction via cytokine receptors. Tyk2 is a Janus kinase, and we developed tyk2-deficient mice to study the requirement for tyk2 in vivo. Tyk2-deficient mice show no overt developmental abnormalities; however, they display a lack of responsiveness to a small amount of IFNalpha, although a high concentration of IFNalpha can fully transduce its signal even in the absence of tyk2. Furthermore, IL-12-induced T cell function is defective in these mice. In contrast, these mice respond normally to IL-6 and IL-10, both of which activate tyk2 in vitro. These observations demonstrate that tyk2 plays only a restricted role in mediating IFNalpha-dependent signaling while being required in mediating IL-12-dependent biological responses.

Background: The maze operation for atrial fibrillation is effective but highly invasive. We tested, both in vitro and in vivo, a new technique for creating long linear atrial lesions with a custom-made, 25-mm long, stainless-steel, linear probe and a corresponding 500-kHz generator for assistance in the maze operation. Methods: In the in vitro study with the isolated canine atria, the power of the delivered radio-frequency energy and the saline irrigating flow rate were changed independently, and the sizes of the lesions were measured. In the in vivo study radio-frequency energy was delivered to 4 portions (ie, the smooth and trabeculated portions of the right and left atria). The sizes of the lesions were measured, and the histologic features of the lesions were examined. Electrical isolation of the right atrial appendage from the remaining right atrium was attempted by using this linear probe. Results: In the in vitro study the size of the lesion became larger as the delivered power was increased, although the lesion was limited when the flow rate was high. In the in vivo study the size of the lesion was equal at the 4 different sites. Histologic examinations demonstrated linear and transmural lesions, and electrophysiologic examinations revealed conduction block between the right atrial appendage and the remaining right atrium. Conclusions: The new original long linear probe was effective for creating transmural linear atrial lesions with the irrigation method, presenting the possibility of an intraoperative technique that mimics the maze procedure. (J Thorac Cardiovasc Surg 2000;120:164-72)

In order to clarify the role of B cells in the development of insulitis and diabetes, B cell-deficient (B(-)) mice treated with streptozocin (STZ) were studied. The extent of insulitis and the cumulative incidence of diabetes were significantly suppressed in B(-) mice (P0.0001), indicating that B cells are crucial for the progression of insulitis and diabetes. Accumulation of both CD4(+) T cells and B cells was observed in islets of B(+) mice, while CD4(+) T cells but not B cells were found in B(-) mice. A few CD8(+) T cells and macrophages were detectable in both types of mice. The immunohistochemical study did not reveal any change in the subpopulations of infiltrating lymphocytes except for the absence of B cells in the B(-) mice. TCR V(beta) gene repertoire usage of islet-infiltrating T cells was restricted to some extent in the B(+) or B(-) mice, but there was no significant difference between the B(+) and B(-) mice, suggesting that the initial islet-reactive T cell response can occur in the absence of B cells. In contrast, TCR clonotype spreading of islet-infiltrating T cells was significantly suppressed in B(-) mice compared with B(+) mice (P0.0001). These data suggest that initial priming of T cells is not impaired and TCR V(beta) repertoire usage is not limited by the lack of B cells, while B cells are important essentially for the spreading of islet-infiltrating clonal T cells in autoimmune diabetic mice induced with STZ.

This pilot study evaluated the efficacy of a new combination chemotherapy with a newly developed nitrosourea derivative ranimustine and evaluated the efficacy of interferon alpha (IFN-alpha) maintenance in previously untreated patients with multiple myeloma (MM). The induction therapy (ROAD-IN) was a 6-week regimen consisting of chemotherapy with ranimustine, vincristine (Oncovin), melphalan (Alkeran) and dexamethasone starting on day 1 and IFN-alpha, which was administered three times weekly for 3 weeks starting on day 22. This was repeated for three cycles. The responders were subsequently randomized into two groups that received or did not receive IFN-alpha as maintenance therapy. Of the 164 patients registered, 161 were evaluated. An objective response to induction therapy was seen in 75% of patients; complete remission (CR) in 38 (24%) and partial remission (PR) in 82 (51%). The median survival for all patients was 3.6 years from registration. The survival of responders (CR + PR) was significantly better than that of non-responders (median survival 4.3 years vs. 1.4 years; 7-year survival rate 32% vs. 9%; P < 0.0001). The IFN-alpha maintenance did not show any advantage for either response duration or survival. This pilot study demonstrated that a comparatively short period of induction therapy with the ROAD-IN regimen produced a rather high response rate and a similar survival rate to those achieved with other longer induction regimens, and that good responders to the initial therapy survived significantly longer than non-responders.

Although Src transformation of NIH3T3 mouse fibroblasts has been shown to be dependent on Ras function, the signaling mechanism whereby Src induces malignant transformation of human epithelial cells still remains unclear. In the present study, we analyzed the functional role of Ras, which acts downstream of Src in intracellular signaling, in the acquisition of fully neoplastic potentials by v-Src–transformed human gallbladder epithelial cells (HAG/src3–1) by infecting these cells with replication-defective adenovirus vector expressing dominant negative H-Ras (AdCARasY57). High efficiency of gene transduction was demonstrated with the adenovirus vector containing β-gal gene insert (AdCALacZ). On infection with AdCARasY57, the activity of mitogen-activated protein (MAP) kinase, a major downstream event triggered by Ras, was markedly inhibited over 7 days, indicating that the inhibition of Ras function by AdCARasY57 remains active during this period. AdCARasY57 did not inhibit the monolayer growth of HAG-1 cells transfected with activated H-ras, but inhibited the HAG/src3–1 cells by 30%, as compared with cells infected with AdCALacZ as a control. This growth inhibition by AdCARasY57 was strengthened nearly twofold on surfaces coated with an antiadhesive polymer (poly 2-hydroxyethylmethacrylate) that can quantitate anchorage-independent growth, and was much more pronounced up to 95% when assayed in soft agar. The HAG/src3–1 cells transfected with β-gal gene produced tumors in nude mice within 4 weeks after implantation, whereas cells infected with AdCARasY57 failed to form tumors during this period. These findings show that Ras function is essential for v-Src–induced anchorage-independent growth in vitro as well as tumorigenesis in vivo, and that mitogenic activity driven by v-Src is not solely dependent on MAP kinase pathway. Because anchorage-independent growth correlates with tumor growth in vivo as well as metastatic potential, targeting Ras would be potentially useful for the treatment of human tumors with elevated Src tyrosine kinase activity. J. Cell. Physiol. 183:221–227, 2000. © 2000 Wiley-Liss, Inc.

Homeobox genes have important roles in haematopoiesis and are regulated in an activated state by the trithorax group (trxG) of genes. In a repressed state, they are regulated by the Polycomb group (PcG) of genes. ENX-1, a putative human PcG gene product, interacts with the proto-oncogene product Vav. We report an investigation of the role of ENX-1 in human haematopoiesis. CD34+ cells mobilized to peripheral blood strongly expressed ENX-1. When stimulated to proliferate, both T and B lymphocytes rapidly up-regulated ENX-1. ENX-1 was expressed in all cell lines of the various lineages examined. When HL-60 cells were differentiated to mature granulocytes with all-trans retinoic acid, ENX-1 was down-regulated. Moreover, ENX-1 antisense oligodeoxynucleotide suppressed DNA synthesis in HL-60 cells. Our data indicate that ENX-1 is involved in the proliferation of both normal and malignant haematopoietic cells.

The role of CD56 in the process of target cell killing by NK cells has been investigated. Addition of NK cells to HuH28 cells, a CD56-expressing cell line, led to inhibition of the growth of the target cells, which exhibited morphological features of apoptosis. These changes were prevented by the addition of a polyclonal anti-NCAM to the cultures. Since neither Fas antigen expression nor apoptotic changes were induced by addition to a mixed culture supernatant of NK and target cells, both the Fas-Fas ligand system and soluble factors do not seem to participate in apoptosis in these circumstances. Increased secretion of interferon-gamma and tumour necrosis factor-alpha by NK cells must therefore have been suppressed by the presence of the polyclonal antibody. These results lead us to conclude that CD56, through homophilic binding, plays an important role in the process of target cell killing by an apoptosis mechanism.

Apoptosis is involved in both the cellular and humoral immune system destroying tumors. An apoptosis-inducing factor from HL-60 myeloid leukemia cells was obtained, purified, and sequenced. The protein found has been identified as a human complement factor B-derived fragment Bb, although it is known that factor B is able to induce apoptosis in several leukemia cell lines. Monoclonal antibodies against fragment Ba and Bb inhibited the apoptotic activity of factor B. When the purified fragment Bb was used for apoptosis induction, only the anti-Bb antibody inhibited Bb-induced apoptosis, and not the anti-Ba antibody. The apoptosis-inducing activity was found to be enhanced under conditions facilitating the formation of Bb. Blocking TNF/TNFR or FasL/Fas interactions did not interfere with the factor B-induced apoptosis. CD11c (iC3bR) acts as the main subunit of a heterodimer binding to fragment Bb in the apoptosis pathway, and the factor B-derived fragment Bb was found to possess the previously unknown function of inducing apoptosis in leukemic cells through a suicide mechanism of myeloid lineage cells during the differentiation stage.

We vaccinated a refractory essential monoclonal cryoglobulinemia patient with monocyte-derived DCs (Mo-DCs) pulsed with purified cryoglobulin as a tumor antigen. During the vaccinations, his acrocyanosis improved and we were able to reduce the number of hot baths used to treat his symptoms, with no side effects. Furthermore, cryoglobulin-specific proliferative responses were observed after the vaccination. As there was a recurrence of acrocyanosis after the final vaccination, vaccination with Mo-DCs pulsed with purified cryoglobulin would seem to be a useful treatment for refractory essential monoclonal cryoglobulinemia.

Radiofrequency catheter ablation (RFCA) targeting the cavotricuspid isthmus is usually an effective treatment for common atrial flutter (AFL), except in a small subset of patients and the reason for this has yet to be elucidated. The present study investigated the relationship between the outcome of RFCA for common AFL and the anatomy of the right atrium as seen on angiography. Twenty consecutive patients who underwent RFCA for common AFL were divided into 2 groups according to the results of RFCA. Group A comprised 13 patients whose AFL was abolished, fulfilling the criteria of success by the conventional catheter approach, and group B comprised 7 patients whose AFL could not be abolished according to the criteria for success (n=4) or was abolished following an additional superior vena cava approach (n=3). On angiography, the cavotricuspid isthmus was longer (3.5+/-0.5 vs 2.2+/-0.6 cm) and deeper (0.94+/-0.35 vs 0.49+/-0.19 cm) in group B than in group A (both p

Granulocyte colony-stimulating factor (G-CSF) undergoes receptor-mediated internalization into target cells which are normally restricted to neutrophilic granulocytes and their committed progenitor cells, suggesting that it may be applicable as a myeloid cell-targeting vehicle. To test this notion, we constructed a cDNA encoding a human G-CSF/murine stem cell factor (mSCF) chimeric molecule in a mammalian expression vector and transfected NIH3T3 cells with this plasmid. The resulting chimeric cytokine consisted of the entire G-CSF sequences fused to Lys148 of mSCF. It can be released from the surface membrane of NIH3T3 transformants through proteolytic cleavage at Ala164 of mSCF. The culture media conditioned by a number of stable transformants, which were confirmed by an enzyme-linked immunosorbent assay (ELISA) to secrete an hG-CSF derivative, were examined for their ability to stimulate CFU-G-derived colony formation as well as the proliferation of G-CSF-dependent NFS-60 cells. The results indicated that this C-terminus modified version of hG-CSF is as potent as recombinant hG-CSF in both assays.

The maintenance of telomere length is crucial for the survival of cells. Recently, genes for proteins that consist of human telomerase have been cloned and the results have indicated a close relationship between telomerase activity and its gene expression. We studied the mRNA expression of the telomerase-associated genes, hTERT and TEP1, in hematopoietic cells in order to clarify the relation between them and telomerase activity using semiquantitative RT-PCR. In polymorphonuclear cells and monocytes isolated from peripheral blood, which had no detectable telomerase activity, no hTERT mRNA expression was seen. On the other hand, lymphocytes and CD34-positive cells both demonstrated hTERT mRNA expression. TEP1 mRNA was detected in all samples, showing no differential expression. We then assessed hTERT and TEP1 mRNA expression in CD34-positive cells cultured in vitro with growth factors. After 4 weeks of culture, all the cells showed myeloid differentiation and the telomerase activity was downregulated. hTERT mRNA was expressed in CD34-positive cells, but was downregulated in 4-week-cultured cells. TEP1 showed no apparent differential expression. We conclude that hTERT mRNA expression is downregulated in accordance with telomerase downregulation during the course of myeloid differentiation, which suggests that it plays a crucial role in the expression of enzyme activity, while TEP1 has a much smaller role to play, if any.

We report a patient with clonal proliferation of CD3+8+TCRalphabeta+ large granular lymphocytes (LGL) presenting multiple episodes of autoimmune cytopenia, including autoimmune neutropenia, idiopathic thrombocytopenic purpura, autoimmune haemolytic anaemia, and pure red cell aplasia. Each disorder appeared separately or as a combination during an 11-year clinical course. The increase of blood CD3+8+TCRalphabeta+ LGL was detected 6 years after the initial diagnosis of cytopenia, but the absolute number of LGL cells was always < 1.0 x 109/l. LGL cells were of monoclonal origin and had a chromosomal abnormality. LGL cells transiently responded to cyclosporine A therapy, which was also effective on all of these autoimmune cytopenias. Accordingly, an undetectable level of proliferation of a clonal LGL population could cause various autoimmune haemopoietic disorders.

Interferon (IFN)-α therapy may induce, reveal, or exacerbate various autoimmunerelated disorders. We describe a 48-year-old female patient who developed muscle waakness during IFN-α therapy given for hepatitis C virus-associated chronic active hepatitis. This symptom diminished slightly after discontinuation of the therapy, but she then began to complain of Raynaud’s phenomenon, swelling of bilateral dorsal hands and dysphagia. Accompanied by an elevation of serum antinuclear antibody, the subsequent development of polymyositis (PM) and progressive systemic sclerosis was considered to have been induced by the IFN-α.

Atopy is an immune disorder in which a Th2 dominant mechanism leads to high IgE levels and the clinical disorder asthma. It has been postulated that the Th1 cytokine IFNgamma, acting through its heterodimeric receptors, IFNgammaR1 and IFNgammaR2, in the induction/proliferation of Th1 cells, might suppress the Th2 responses that may underlie atopic asthma. However, neither murine nor human variants of IFNgamma associate with atopy. Several dysfunctional mutations have been identified in IFNgamma receptor genes (IFNGR1 and IFNGR2) in relation to severe and selective infections with poorly pathogenic organisms. However, little is known about common polymorphisms and their functional role in atopy. To test whether such variants of IFNGR1 and IFNGR2 relate to atopic asthma, we conducted a genetic association study in both British (n = 300) and Japanese (n = 200) populations. An intronic variant of IFNGR1 showed marginal association with total serum IgE levels in the British population compared with those with total IgE levels30 IU/ml and those with120-500 IU/ml [odds ratio = 2.00 (95% CI 1. 00-4.07), P = 0.048]. A coding variant, Gln64Arg of the IFNGR2, also associated with total serum IgE levels in the British population [chi(2) = 5.08, P = 0.024]. Further genetic and functional analyses are needed to clarify the role of variants of IFNgamma receptor genes in atopic immune disorder among different ethnic groups.

A rare case of an inflammatory pseudotumor (IPT) of the liver with reactive arthritis (ReA) is herein described. A 34-year-old woman presented with post-infectious ReA in the bilateral knees and foot joints. As the primary infected site for ReA could not be determined, a systemic survey revealed an IPT in her liver, which was diagnosed histologically. The arthritis and the tumor regressed simultaneously after being treated by indomethacin farnesil 400 mg/day. Although the exact etiology of IPT or ReA still remains unknown, it is hypothesized that the same pathogenetic mechanisms may be involved in the development of IPT and ReA.

Background and Objectives: Gastric carcinoma rarely affects young patients. This study was undertaken in order to clarify the clinicopathological features and prognosis of young patients with gastric carcinoma. Methods: The resected 107 specimens from 105 patients younger than 30 years of age with gastric carcinoma were investigated using hematoxylin and eosin stain. Results: The male:female ratio was 1:1.6. Histologically, poorly differentiated adenocarcinoma was the most common type (94/107, 87.9%) (P < 0.001). Most tumors were located in the middle third of the stomach (P < 0.001). All patients had depressed lesions. The 5-year survival rates of early and advanced gastric carcinoma were 100% (30/30) and 23.5% (8/34), respectively. Conclusions: Characteristic clinicopathological features in young patients, such as gender ratio, tumor location, macroscopic type, and histological type, were different from those in older ones. The prognosis of early gastric carcinoma in young patients was much better than that in older patients, although the prognosis of advanced gastric carcinoma in young patients was worse than that of older patients. These findings seem to indicate that young patients with early gastric carcinoma can tolerate radical treatments well; however, the aggressiveness of lesions are emphasized in patients with advanced gastric carcinoma.

A 63-year-old diabetic man was admitted with general fatigue. Electrocardiogram (ECG) on admission showed complete atrioventricular (AV) blockade associated with prominent bifid T waves. The second component of the bifid T waves was distinguished from U waves by the beat-to-beat varying bifidity and the nadir between the two components located at > or = 1 mm above the isoelectric line. Range of absolute QT interval was 535 to 650 ms. Hypokalemia (3.6 mEq/L) was noted at admission. Partial restoration of the potassium level (3.9 mEq/L) prior to temporary ventricular demand pacing obscured the bifid T waves and attenuated the QT prolongation and dispersion to some extent (absolute QT interval ranging 520 to 620 ms). It was concluded that marked bradycardia caused by complete AV blockade (ie, a junctional escaped rhythm at a rate of 42 beats/min), hypokalemia, and underlying diabetes mellitus contributed in concert to the QT prolongation and dispersion leading to the prominent bifid T waves.

Genetic factors seem to play a significant role in susceptibility to systemic lupus erythematosus (SLE). We previously described the amino acid polymorphism (Val14Met) within the IFN-gamma receptor 1 (IFN-gammaRI), and that the frequency of the Metl4 allele in SLE patients was significantly higher than that of the healthy control population [Tanaka et al. (1999) Immunogenetics 49, 266-271]. We also found an amino acid polymorphism (Gln64Arg) within IFN-gamma receptor 2 (IFN-gammaR2). Since the IFN-gamma receptor is a complex consisting of IFN-gammaR1 and IFN-gammaR2, we searched for the particular combination of two kinds of amino acid polymorphisms found within the IFN-gamma receptor which plays a prominent role in susceptibility to SLE. The greatest risk of the development of SLE was detected in the individuals who had the combination of IFNGR1 Met14/Val14 genotype and IFNGR2 Gln64/Gln64 genotype.

Both the immunological function of lymphocytes and the immunopathogenic mechanisms in autoimmune hepatitis (AIH) are still not well understood. The aim of this study was to clarify the cell types responsible for the hepatocellular damage in AIH. Sera, liver biopsy specimens and peripheral lymphocytes were obtained from AIH patients. Chronic viral hepatitis, type C (C-CH) patients and healthy subjects were also studied as controls. In the liver of untreated AIH patients, the infiltration of CD8-positive cells was predominant. In addition, the serum soluble interleukin 2 receptor (sIL-2R) and soluble CD8 (sCD8) levels in pre-treated AIH patients were high, which thus suggested that the CD8-positive T lymphocytes were in an activated state. The peripheral blood mononuclear cells (PBMC) produced a high level of IFN-γ which reflected a Th1-dominant state of the T cells in the liver. Even after corticosteroid therapy at a sufficient dose, the increased serum sCD8 level did not normalize in AIH patients, while, in contrast, the increased serum sIL-2R level and IFN-γ production of PBMC did normalize. These results therefore suggest that a persistent activated state of CD8-positive lymphocytes and a Th1-dominant state in the pre-treated stage may be closely associated with the immune pathogenesis of AIH.