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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.

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.

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)

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.

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 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.

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.

IL-4 is a pleiotropic cytokine which exerts its actions on various lineages of hematopoietic and nonhematopoietic cells. This cytokine is one of the central regulators of immunity in health and disease states. An alternative splice variant, in which the second of four exons is omitted, has been recently described and designated as IL-4delta2. The variant has been previously described as a potential naturally occurring antagonist of human IL-4 (hIL-4)-stimulated T cell proliferation. In this study, we investigated the effects of recombinant human (rh) IL-4delta2 on monocytes and B cells. In monocytes, rhIL-4delta2 blocked inhibitory action of hIL-4 on LPS-induced cyclooxygenase-2 expression and subsequent prostaglandin E2 secretion. In B cells, rhIL-4delta2 was an antagonist of the hIL-4-induced synthesis of IgE and expression of CD23. Our results broaden the spectrum of hIL-4-antagonistic activities of rhIL-4delta2, thus creating the background for the potential use of rhIL-4delta2 as a therapeutic anti-hIL-4 agent.

To investigate the relationships of personal hair dye use and environmental factors to myelodysplastic syndromes (MDS), we conducted a case-control study in Japan. A total of 111 MDS cases and 830 controls randomly selected from the residents in the same prefecture of cases using telephone directories responded to a health questionnaire. The odds ratio (OR) for ever having used hair dye was 1.99 (95% confidence interval (CI) 1.17-3.38) and there were statistically significant trends in risk with increasing duration and number of hair dye use. Occupational exposure to organic solvents was marginally associated with the risk of MDS (OR = 1.99; 95% CI 0.97-4.10).

Mitogen-activated protein kinases (MAPKs) are activated by various extracellular stimuli and play an important role in regulating the expression of proinflammatory molecules in monocytes/macrophages. We first questioned whether MAPK activation in involved in cyclooxygenase (COX)-2 expression in lipopolysaccharide (LPS)-stimulated human monocytes. LPS induced the expression of COX-2 protein and COX-2 mRNA as well as the phosphorylation and activation of extracellular signal-regulated protein kinase (ERK)2 and p38 MAPK in monocytes. The induction of COX-2 mRNA, COX-2 protein, and prostaglandin (PG)E2 by LPS was inhibited by the specific inhibitors of ERK and p38 MAPK, suggesting that the activation of ERK2 and p38 MAPK is involved in COX-2 expression in LPS-stimulated monocytes. Since we previously showed that interleukin (IL)-10 and IL-4 similarly inhibited COX-2 expression in LPS-stimulated monocytes, we next questioned whether these cytokines regulate the phosphorylation and activation of ERK2 and p38 MAPK in LPS-stimulated monocytes. Interestingly, LPS-induced phosphorylation and activation of ERK2 was significantly inhibited by IL-4 and IL-10, while that of p38 MAPK was inhibited by IL-10, but not IL-4. These results suggest that the mechanisms of inhibition by IL-10 and IL-4 of the LPS-induced expression of proinflammatory molecules could be ascribed to the regulatory effects of both cytokines on MAPK activation.

The vav proto-oncogene product (Vav), which is specifically expressed in hematopoietic cells, contains multiple structural motifs commonly used by intracellular signaling molecules. Although a variety of stimuli including erythropoietin (Epo) have been shown to tyrosine phosphorylate Vav, little is known about the Vav signal transduction pathway. Here, we have investigated the role of Vav in the Epo signaling pathway by characterizing its interaction with other proteins, using the human Epo-responsive cell line, F-36P. Immunoprecipitation and immunoblot analyses have demonstrated that Vav was associated with the Epo receptor (EpoR) in an Epo-independent manner and was tyrosine-phosphorylated after Epo stimulation. Furthermore, two phosphotyrosine proteins (pp70 and pp100) co-immunoprecipitated with the regulatory subunit of phosphatidylinositol 3-kinase (PI3-kinase) (p85) were identified as EpoR and Vav, respectively. The interaction between Vav and p85 was shown to be mediated through the SH2 domains of p85 by an in vitro binding assay and confirmed by the presence of in vitro PI3-kinase activity associated with Vav. Treatment of the cells with antisense-vav and -p85 abrogated Epo-induced cell proliferation and PI3-kinase activity. Finally, we found that JAK2 was associated with Vav in vivo and that Vav could be tyrosine-phosphorylated by activated JAK2 in vitro. These results suggest the possible role of JAK2 for tyrosine phosphorylation of Vav and involvement of Vav and PI3-kinase in Epo-induced proliferative signals.

We report a case of myelodysplastic syndrome with bone marrow eosinophilia and the chromosomal abnormality, inv(16)(p13q22). Hematologic findings including monocytosis and trilineage myelodysplasia were consistent with chronic myelomonocytic leukemia, and numerous abnormal eosinophils were present in the bone marrow. Chromosomal analysis of all metaphase cells from peripheral blood and bone marrow revealed inv(16)(p13q22), which is well known characteristic of acute myelomonocytic leukemia with eosinophilia (M4Eo). Both monocytes and eosinophils in this case may be derived from common leukemic progenitors affected by inv(16)(p13q22).

Information on the anti-carcinogenic effect of EGCG, the main constituent of the polyphenols present in Japanese green tea leaves, has recently been accumulating. In this report, we evaluate the effect of EGCG on leukemic blast cells from AML patients. The results showed that EGCG inhibited the proliferation of AML cells in all cases examined. Since AML cells might proliferate by autocrine or paracrine growth mechanisms, we also examined the effect of EGCG on the production of GM-CSF from AML cells. Although EGCG did not directly inhibit the production of GM-CSF, it did inhibit the effect of TNF-α or TPA, both of which stimulated AML cells to produce GM-CSF. On the other hand, the modulation of receptors for growth factors might play a role in the proliferation or carcinogenesis of AML cells. We also found that EGCG inhibited the modulation of c- kit , a receptor for stem cell factor, on leukemic cells. These findings suggested that EGCG might be available as a new therapeutic tool for AML patients.

Objective: To compare the sensitivity and specificity of three different assays for diagnosis of invasive candidiasis. Methods: A passive hemagglutination assay (PHA), counter-immunoelectrophoresis assay (CIE), and Cand-tec were used to test sera from 125 patients with hematologic malignancies and 65 other hospitalized patients. The former group included 15 patients with invasive candidiasis, 38 patients with Candida colonization, and 72 patients without candidiasis. Results: Sensitivity/specificity of PHA, CIE, and Cand-tec were 87%/85%, 67%/98%, and 33%/97%, respectively. The measurement of antibody in paired sera, by PHA, was sensitive and specific; however, increased antibody titers usually occurred late in the disease. Conclusions: The combination of PHA and CIE, with a sensitivity of 67% and specificity of 98%, appeared to be the best assays for detection of invasive candidiasis in this cohort. The Cand-tec assay for Candida antigen had poor sensitivity for diagnosis of infection.

Patients with chronic graft-versus-host disease (cGVHD) occasionally suffer from symptoms of xerostomia and xerophthalmia, which are also features of Sjógren's syndrome (SS). To identify differences in the glandular involvement between cGVHD and SS, we measured the proportions of infiltrating lymphocyte subsets and the expression of HLA-DR antigen and cell adhesion molecules in labial salivary glands (LSG). In cGVHD, more than 90% of the infiltrating lymphocytes were T cells with a slight predominance of CD8+ over CD4+ cells. In SS, CD4+ cells were predominant, and B cells accounted for 10-30% of the infiltrating lymphocytes. Ductal epithelial cell associated with lymphocytic infiltration expressed HLA-DR antigen in both cGVHD and SS. In SS alone, HLA-DR antigen expression also occurred without associated lymphocytic infiltration. The expression of adhesion molecules on ductal epithelial cells, especially vascular cell adhesion molecule 1, was more intense in SS than in cGVHD, while that on endothelial cell was similar in cGVHD and SS. These data suggest that the pathogenesis of glandular involvement of cGVHD is different from that of SS.

The fourth component of complement (C4) in urine was measured in 19 patients with systemic lupus erythematosus (SLE). Urinary C4 was detectable in all SLE patients using an enzyme linked immunosorbent assay. In 11 of 13 patients whose urinary C4 was serially measured, a decrease of urinary C4 was found in parallel with a decrease of disease activity of SLE. In the remaining 2 patients, the amount of C4 increased preceding a flare-up of the disease. The measurement of urinary C4 may be useful in evaluating the disease activity of SLE in individual patients and sometimes in predicting the flare-up of the disease. The specific hemolytic activity of excreted C4 and Western blotting analysis showed that urinary C4 consisted mainly of degraded fragments of C4. In two cases, camostat, a serine protease inhibitor, rapidly decreased the urinary C4 excretion, suggesting that the complement activation occurred in the glomerulus in lupus nephritis.

Forty-eight patients with primary biliary cirrhosis (PBC) and 336 healthy individuals were examined for HLA-DR, DQ and DP alleles, by DNA typing based on the polymerase chain reaction (PCR) sequence-specific oligonucleotide probe (SSOP) method. Frequencies of HLA-DRB1∗1602, DRB1∗0803, DQB1∗0502, DQB1∗0601 and DPB1∗0202 were found to be increased in the patients. These HLA alleles are in linkage disequilibria consisting of DRB1∗1602-DQB1∗0502 and DRB1∗0803-DQB1∗0601-DPB1∗0202 haplotypes. Since the frequencies of DRB1∗1401 and DRB1∗1502, that are in linkage disequilibria with DQB1∗0502 and DQB1∗0601, respectively, were not increased in the patients, the susceptibility to PBC was suggested to be controlled by the HLA-DRB1 locus. In contrast, the frequency of HLA-DQB1∗0302 was decreased in the patients, while the frequency of each DRB1 allele in linkage disequilibria with DQB1∗0302 did not differ significantly between the patient group and control group. This observation suggested that the resistance to PBC may be controlled by the HLA-DQ locus.

The effects of Bestatin (Ubenimex, UBX) on normal hematopoiesis were investigated using long-term bone marrow cultures (LTBMC) to determine whether it might enhance hematopoiesis over a long period, as well as induce the release of cytokines. LTBMC were inoculated with 0.1 or 1.0 microgram/ml of UBX at the onset of culture and was added at each weekly medium change. The cellularity and the content of the progenitors in nonadherent layer were examined each week for 5 weeks; those from the adherent layer were examined at week 5. The number of the nucleated mature cells and that of the myeloid progenitors from the nonadherent layer increased significantly (approximately two-fold) following treatment with UBX vs controls. The total number of colonies and the number of myeloid progenitors, but not that of erythroid progenitors, from the adherent layer increased significantly (about 1.5-fold) following treatment with 1.0 microgram/ml UBX. The level of interleukin-6 (IL-6) in the culture supernatants was significantly increased one day after the addition of 1.0 microgram/ml UBX. Findings indicate that UBX stimulated both the production of mature cells and myeloid progenitors on normal hematopoiesis in LTBMC. This was mediated by an indirect action via IL-6 production.

To investigate the effect of transcatheter arterial embolization (TAE) or lipiodolization (Lip) on additional tumoricidal activities and the immune system, we studied the serum tumor necrosis factor (TNF) activity before and after either TAE or Lip in patients with hepatocellular carcinoma (HCC). No significant differences in the serum basal TNF activities were observed before TAE or Lip among either the normal controls, patients with cirrhosis (LC) or patients with HCC. The serum TNF activities increased in four of 15 patients with HCC after the treatment. On the other hand, the TNF production of peripheral blood mononuclear cells (PBMCs) in five patients with HCC increased from 9.0 ± 3.8 U/ml to 18.8 ± 8.7 U/ml (P

We performed a randomized phase II trial comparing low-dose aclarubicin (LC-ACR) with very low-dose cytosine arabinoside (VLD-AC) in 39 consecutive untreated patients with myelodysplastic syndromes (MDS), including refractory anemia (RA), RA with excess of blasts (RAEB) and RAEB in transformation (RAEB-t). Nineteen patients received the VLD-AC therapy; 2 good responses (GR) and 2 partial responses (PR) were obtained in 11 patients with RAEB and RAEB-t, while 2 PR were obtained in 8 RA patients. Eighteen patients received the LD-ACR therapy; 2 GR and 4 PR were obtained in 11 RAEB/RAEB-t patients while 2 PR in 7 RA patients. There was no significant difference in the therapeutic effects and survival between these two groups of patients. These observations suggest that the LD-ACR therapy is effective in some patients with MDS and can be used as an alternative to the low-dose Ara-C therapy.

We report two cases of Philadelphia chromosome (Ph)-positive acute leukemia with definite myeloid markers. Ph was the sole chromosomal abnormality at presentation, and neither eosinophilia, basophilia, thrombocytosis nor hepatosplenomegaly was present. In both cases, Ph+ myeloblasts showed positive stain for myeloperoxidase and naphthol ASD chloroacetate esterase, which fulfilled the FAB criteria of acute myelogenous leukemia (AML). Ph+ myeloblasts co-expressed myeloid and B-lymphoid antigens (CD10, CD13, CD19 and CD33). In case 1, myeloblasts rearranged M-BCR, and the expression of M-BCR/ABL chimeric RNA was demonstrated by using the reverse transcription polymerase chain reaction (RT-PCR). They also clonally rearranged IGH. Ph clone disappeared on cytogenetic analysis in remission, and granulocytes in remission did not have rearranged M-BCR. In case 2, morphocytochemically distinct myeloid and lymphoid blast populations were seen. Myeloblasts and lymphoblasts were enriched >96% as CD19−/CD33+ and CD19+/CD33− populations, respectively. Both of them possessed the identical rearrangement of IGH and M-BCR, indicating a common leukemic progenitor cell origin. Furthermore, m-BCR/ABL was detected in addition to M-BCR/ABL on RT-PCR. Accordingly, both cases were diagnosed as de novo Ph+ acute leukemia rather than as chronic myelogenous leukemia in blastic crisis. Their mixed B-lymphoid/myeloid characteristics strongly suggest that so-called ‘Ph+ AML’ is derived from Ph+ myeloid/B-lymphoid stem cells.

Human Dexter-type culture of bone marrow (BM) cells maintains long-term hematopoiesis in the presence of an adherent stromal layer. These BM adherent cells produce hematopoietic growth factors constitutively or inducively and support developing hematopoietic cells. To elucidate the ability of cytokine production by BM adherent cells, the cytokine levels of the culture supernatant of BM adherent cells were measured by enzyme-linked immunosorbent assays. Constitutive production of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 6 (IL-6) by the unstimulated BM adherent cells was demonstrated. Levels of granulocyte colony-stimulating factor (G-CSF) were below detectable levels (< 10 pg/ml). The IL-6 level was significantly increased in the co-culture supernatant with KG1 cells or U937 cells (P < 0.01: P < 0.05, respectively). Even when the adherent cells and cell line cells were separated by a membrane filter, the IL-6 level was significantly higher than the control culture (P < 0.01). Co-culture with these cell lines was supposed to induce the increased production of IL-6, which was mediated by some soluble cytokines. The GM-CSF level was not increased in the supernatant co-cultured with any of the cell lines, except with K562 cells. However, K562 cells alone secreted a detectable level of GM-CSF and the increased level of GM-CSF was considered to be due to the production of GM-CSF by K562 cells. G-CSF was not detectable in the supernatant co-cultured with any cell line cells. This result indicates that the cytokine production was regulated by the interaction of BM adherent cells and some leukemic cells.

Expression of lymphokine genes including interleukin 2(IL-2), interleukin 3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon γ (IFN-g), tumor necrosis factor (TNF) and lymphotoxin (LT) were sequentially monitored in peripheral blood mononuclear cells by Northern blot analysis after stimulation with phytohemagglutinin (PHA). The pattern of the expression of lymphokine genes following PHA stimulation as categorized into two types. Type 1 was characterized by rapid appearance of mRNA and by early maximum accumulation. Type 2 was characterized by the prolonged expression and late peak time. Lymphokines including IL-2, IL-3 and GM-CSF belong to type 1 and IFN-g, TNF and LT belong to type 2. Since lymphokines in type 1 are known to act on hematopoiesis in a stimulatory manner, whereas type 2 show an inhibitory action, this sequential expression of lymphokines following mitogen stimulation may reflect some biological feedback mechanism.

Okadaic acid is a potent tumor promoter and an inhibitor of serine/threonine-specific protein phosphatases. We studied the effect of okadaic acid in human T cell activation and phosphorylation of internal substrates. Okadaic acid at up to 4 nM enhanced phorbol myristate acetate (PMA)-induced proliferation and CD25 (IL-2 receptor, p55) expression, although it showed no activation by itself. Okadaic acid induced hyperphosphorylation of a 60 kDa protein in T cells as well as non-T cells, as reported in fibroblasts and keratinocytes. Preincubation with 4 nM okadaic acid enhanced PMA induced phosphorylation of the 80 kDa protein, an internal substrate of protein kinase C in T cells. These results suggest that okadaic acid inhibited dephosphorylation of protein kinase C specific substrates, and as a result, enhanced T cell activation mediated by protein kinase C pathway.

We quantitated the frequency of B lymphocytes capable of producing antibodies to HTLV-1 in the peripheral blood from patients with HAM/TSP, non-HAM/TSP HTLV-1 carriers and seronegative healthy subjects. Epstein-Barr virus (EBV) was used as a polyclonal activator of B lymphocytes in a limiting dilution condition. We found that B lymphocytes committed to the production of monoreactive-IgG and -IgA antibodies to recombinant HTLV-1 (gag + env) hybrid protein were significantly increased in a number in patients with HAM/TSP as compared to non-HAM/TSP HTLV-1 carriers and seronegative healthy subjects. By transforming these B lymphocytes with EBV and fusing them with human-mouse heteromyeloma (F3B6), a stable hybridoma producing IgG monoclonal antibody (mAb) to HTLV-1 (gag + env) protein was generated from a patient with HAM/TSP. This mAb (IgG1, κ ), designated F31.1, specifically bound to the amino acid residues from 235 to 254 of HTLV-1 envelope glycoproteins (gp46) with high affinity ( K d = 4.0 × 10 −9 mol/1). These data indicate that the antigen-driven process of B lymphocytes maturation by HTLV-1 antigens is markedly increased in patients with HAM/TSP.

We measured granulocyte colony-stimulating factor (G-CSF) levels in cord blood of 59 normal full-term neonates immediately after birth and the subsequent changes in G-CSF levels of 16 cases by our modified enzyme-linked immunosorbent assay (ELISA) for G CSF. Ten out of 59 cases examined (17%) showed G-CSF levels in cord blood after delivery between 20 and 57 pg/ml, although in the remaining cases, the G-CSF levels were below 20 pg/ml, which was our minimal detection level. A direct relationship between G-CSF levels and white blood cell count, absolute granulocyte numbers in cord blood, gestation age or weight was not observed. Although G-CSF levels in cord blood after delivery remained below 20 pg/ml in 5 cases out of 16 tested, those in the remaining 11 cases (69%) subsequently became elevated after delivery. The peak G-CSF level in cord blood after delivery ranged from 26–364 pg/ml, and the time of it was between 4.5 and 18 h. As G-CSF level per wet weight of placenta was high (124 × 29 pg/ml), these subsequent elevations of G-CSF in cord blood after delivery may result from a gradual influx from the placenta. G-CSF / cord blood / placenta / ELISA

Hepatocyte growth factor (HGF) is a potent growth factor for various epithelial cells including mature hepatocytes and renal tubular cells. When 70% of the rat liver was excised, HGF mRNA in the intact lung markedly increased at 6 h later, then decrease to normal levels at 24 h. A similar marked increase of HGF mRNA was found in the lung of rats with hepatitis induced by CCl4. Moreover HGF mRNA in the intact lung also increased to about a 5 times higher level than the normal, within 12 h after unilateral nephrectomy. Isolated alveolar macrophages significantly expressed HGF mRNA, yet the amount remained unchanged after injury of the liver. The marked increase of HGF mRNA in lungs of partially hepatectomized rats remained even after removal of alveolar macrophages. In situ hybridization showed a marked increase of HGF mRNA signal found in endothelial cells in the lung after partial hepatectomy. We postulate that endothelial cells in the lung recognize damage of distal organs through a mediator and that lung-derived HGF may contribute to tissue repair or regeneration of injured organs, through endocrine-related mechanisms.

Two distinct low-molecular-weight growth inhibitory activities were isolated from supernatants of a density-inhibited, tumorigenic V79 Chinese hamster cell line. By chromatographic analyses, one of these was purified to homogeneity and eventually proved to be thymidine (dThd). In order to investigate the biological role of dThd in a density-inhibited culture of these cells, a dThd-kinase deficient (TK−) clone resistant to the excess of dThd was isolated from V79 cells and the effect of the supernatants on growth of these TK− or TK-proficient (TK+) cells was examined. As a result, the growth of TK− cells was not inhibited but enhanced by the supernatant at the concentrations which significantly inhibited the growth of TK+ cells. Such TK-dependent differential responses to supernatants suggest the presence of deoxyribonucleosides including a high level of dThd in the supernatants. Since it is unlikely that dThd might derive from denatured DNA of dead cells, an accumulation of endogenous dThd in confluent culture appears to be responsible for dThd triphosphates which are synthesized de novo, degraded and excreted into the medium rather than incorporated into DNA as a consequence of aberrant growth in the presence of certain growth inhibitors produced by density-inhibited V79 cells.

To determine whether there is an intrinsic defect in T cells from patients with systemic lupus erythematosus (SLE), we studied signal transduction systems, assaying the total protein kinase C (PKC) levels and the phorbol myristate acetate (PMA)-induced activation of PKC in PHA-treated T cells. T cells from SLE patients showed a decrease in proliferation in response to PMA, but not to PHA, thereby suggesting the existence of an intrinsic abnormality in the PKC-mediated activation pathway. Total PKC activity in the T cells from SLE patients was significantly decreased. Although stimulation with PMA induced a translocation of PKC from the cytosol to the particulate fraction, translocated PKC activity after 2 nM PMA treatment was decreased in the SLE T cells. Furthermore, PMA-induced phosphorylation of 80-kDa substrates was also decreased in SLE T cells. These results suggest that there is a reduced PKC activity and an impaired PKC activation in response to PMA in the SLE T cells, a finding which may explain, if partially, the defect in T cell activation in patients with SLE.

C4b-binding protein (C4bp) is a serum glycoprotein that is one of the regulators of the complement activation (RCA) family. This protein is composed of structurally related 70-kDa (alpha chain) and 45-kDa (beta chain) polypeptides. The alpha chain of C4bp (C4bp alpha) consists of eight short consensus repeats (SCR), which constitute the amino-terminal 491 residues. Human C4bp is also one of the acute-phase reactants. In order to clarify the genetic basis of the SCR and to understand the regulatory mechanisms of C4bp synthesis, we isolated 6 genomic DNA clones covering all of the human C4bp alpha gene. This gene consists of 12 exons and spans about 40 kb. Each of the SCRs is encoded by a single exon, except for the second SCR (SCR II), which is encoded by two separate exons, demonstrating that human C4bp alpha has a split SCR at the genomic level. The 5' flanking region was sequenced up to 380 bases upstream from the putative transcription initiation site. Several possible binding sites for transcription factors were identified.

The in vitro production of macrophage colony-stimulating factor (M-CSF) was studied in 14 human lymphoblastoid cell lines and their lineages were ascertained by surface phenotype analysis. M-CSF gene transcripts were detected in a T-lymphocyte-derived cell line (CCRF-CEM) and 3 B-lymphoblastoid cell lines (IM-9, BALL-1, and CCRF-SB) by Northern-blot analysis. The secretion of M-CSF protein into the culture supernatant by each cell line was also studied using an enzyme-linked immunosorbent assay for M-CSF. The 4 cell lines which expressed the M-CSF gene secreted considerable amounts of M-CSF into their culture supernatants, while the 10 cell lines without M-CSF gene expression did not do so. The cell lines which constitutively produced M-CSF were then subjected to Southern-blot analysis of the M-CSF gene structure, and all 14 cell lines were examined for infection by the Epstein-Barr virus. Neither structural changes nor amplification of the M-CSF gene were detected, and Epstein-Barr virus infection was found to be not directly related to M-CSF production.

Factor IX Nagoya 3 (IX Nagoya 3) is a natural mutant of factor IX recognized in a patient with moderately severe hemophilia B. The patient had 0.60 units/ml of factor IX antigen and 2–5 % of clotting activity. IX Nagoya 3 was purified from the patient's plasma by immunoaffinity chromatography with an anti-factor IX monoclonal antibody column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that the treatment of IX Nagoya 3 with factor XIa/calcium ions resulted in cleavage only at the Arg180-Val181 bond. The amino acid sequence analysis of one of the lysyl endopeptidase peptides derived from IX Nagoya 3 revealed that Arg-145 is replaced by His. This substitution impairs the cleavage between the light chain and the activation peptide by factor XIa/calcium ions.

To study the therapeutic effect of low-dose aclarubicin (ACR), we carried out comparative treatment of 15 patients with myelodysplastic syndrome (MDS) and atypical leukemia using this drug. Complete remission (CR) was achieved in three patients with RAEB-t and one patient with AML, partial remission was obtained in one patient with RAEB and hematological improvement in one patient with refractory anemia (RA). Interestingly, prolonged CR for more than 26 months with persistent chromosomal abnormalities was observed in a case of AML, which progressed from RA. Myelosuppression caused by low-dose ACR was milder than that caused by low-dose Ara-C. Furthermore, in vitro studies indicated that ACR induced differentiation of bone marrow cells from one patient with MDS. From these observations, it is suggested that low-dose ACR may be an alternative to low-dose Ara-C for treatment of MDS, and that the in vivo effect of ACR may be mediated by the differentiation of abnormal hemopoietic clones.

We studied valvular regurgitation in patients with systemic lupus erythematosus (SLE) by color Doppler echocardiography. Because valvular regurgitation occurs frequently in normal subjects, 1,2 we matched the patients by age with a control group. Furthermore, the same person examined both patients and normal subjects using the same Doppler echocardiographic apparatus.

Factor IX BM Nagoya (IX Nagoya) is a natural mutant of factor IX responsible for severe hemophilia B. A patient with this mutant is characterized by a markedly prolonged ox brain prothrombin time. IX Nagoya was purified from the patient's plasma by immunoaffinity chromatography with an anti-factor IX monoclonal antibody column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that treatment of IX Nagoya with factor XIa/Ca2+ resulted in cleavage only at the Arg145-Ala146 bond. Reversed-phase high performance liquid chromatography of a trypsin digest of IX Nagoya showed an aberrant peptide, which was further digested with proteinase Asp-N. Primary structure analysis of one of the Asp-N peptides revealed that Arg180 is replaced by Trp. An essentially complete (99%) amino acid sequence of IX Nagoya was obtained by sequencing fragments derived from a lysyl endopeptidase digest in which no other substitutions in the catalytic triad or substrate binding site were found. We also found that IX Nagoya is activated by α-chymotrypsin or rat mast cell chymase by monitoring the rate of factor X activation using a fluorogenic peptide substrate in the presence of factor VIII, phospholipids, and Ca2+. These results indicate that the substitution of Arg180 by Trp impairs the cleavage by factor XIa required for activation of this zymogen and that the substitution causes hemophilia BM.