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In order to clarify the relationship between myelodysplastic morphologic features of marrow cells and prognoses and 1.0 define other prognostic factors, 124 patients with the FAB criteria of myelodysplastic syndrome (MIX) were analysed. These included 57 patients with refractory anemia (RA), 5 RA with ring sideroblasts (RARS), 25 RA with excess of blasts (RAEB), 14 chronic myelomonocytic: leukemia (CMML) and 23 with RAEB in transformation (RAEB in T). Univariate analysis of all MDS patients or those of RA demonstrated that the following factors, which were not reported or fully investigated previously, were significantly associated with prognosis. These included neutrophil alkaline phosphatase (NAP) score (significant only for all MDS), the percentage of marrow erythroblasts and lymphocytes present, the percentage of cells with morphological abnormalities in individual cell lineages and the number of cell lineages showing atypia (significant for all MDS and RA). Multiple regression analysis showed that (%) of marrow erythroblasts, NAP score, hemoglobin levels and number of marrow granulocytes with atypia were significant for predicting the prognosis of all MDS patients while the number of marrow megakaryocytes and granulocytes with atypia were significant for prognosis in the subgroup with RA.

Summary Statins are 3-hydroxy-3-methylglutaryl-co-enzyme A reductase inhibitors of cholesterol biosynthesis, and have been reported to exert pleiotropic effects on cellular signalling and cellular functions involved in inflammation. Recent reports have demonstrated that previous statin therapy reduced the risk of pneumonia or increased survival in patients with community-acquired pneumonia. However, the precise mechanisms responsible for these effects are unclear. In the present study, we examined the effects of statins on cytokine production from lipopolysaccharide (LPS)-stimulated human bronchial epithelial cells (BEAS-2B). Interleukin (IL)-6 and IL-8 mRNA expression and protein secretion in LPS-stimulated cells were inhibited significantly by the lipophilic statin pitavastatin and the hydrophilic statin pravastatin. As these inhibitory effects of statin were negated by adding mevalonate, the anti-inflammatory effects of statins appear to be exerted via the mevalonic cascade. In addition, the activation levels of Ras homologue gene family A (RhoA) in BEAS-2B cells cultured with pitavastatin were significantly lower than those without the statin. These results suggest that statins have anti-inflammatory effects by reducing cytokine production through inhibition of the mevalonic cascade followed by RhoA activation in the lung.

The chemokinetic inhibitory factor (CIF) is a recently described B-cell derived lymphokine that mediates a chemokinetic inhibitory effect on human polymorphonuclear leukocyte (PMN) migration. In the present report the interaction of CIF with the neutrophil plasma membrane was studied. Normal human peripheral blood neutrophils and purified neutrophil plasma membranes selectively removed biologic activity from CIF-containing concentrates obtained during the purification procedure from conditioned medium. Removal was obtained at both 4 degrees C and 37 degrees C. Furthermore, HL-60 cells treated with dimethyl sulfoxide removed CIF activity (granulocyte-like cells) but HL-60 cells treated with 12-O-tetradecanoylphorbol-13-acetate (macrophage-like cells) did not. Purified human blood monocytes, cells from the macrophage-like U-937 cell line and cells from the basophilic leukemia cell line KU-812 did not remove CIF. These studies suggest that neutrophils express specific binding sites for CIF-activity.

Multidrug-resistant Pseudomonas aeruginosa, especially metallo-beta-lactamase (MBL)-producing P. aeruginosa, is an important pathogen in nosocomial infection and emergence of this pathogen has revived interest in polymyxin B (PMB) and colistin (COL). In this study, we evaluated the efficacies of PMB, COL and other antipseudomonal agents against IMP-type MBL-producing P. aeruginosa both in vitro and in vivo. A total of 75 isolates of bla(IMP)-positive P. aeruginosa obtained from clinical specimens (94.6% of isolates demonstrated resistance to beta-lactam, fluoroquinolone and aminoglycoside agents) were evaluated in the in vitro study. More than 90% of the examined isolates were susceptible to PMB (minimum inhibitory concentration for 50/90% of the isolates (MIC(50)/MIC(90)) 4/4 mg/L), although COL was less potent (MIC(50)/MIC(90) 8/16 mg/L). Cyclophosphamide-treated mice were intraperitoneally inoculated with bla(IMP)-positive P. aeruginosa. Treatment with PMB, but not COL, imipenem/cilastatin or aztreonam, significantly improved the survival rate and decreased the number of bacteria in the blood in a dose-dependent manner. Our results indicate that, among the agents studied, PMB is the most effective agent against bla(IMP)-positive P. aeruginosa.

Neutropenic mice with latent trichosporonemia were given various antifungal agents (amphotericin B, fluconazole, itraconazole) or saline to determine which antifungal agent could be useful for prophylaxis. The 3-week-survival rate was 80% in the fluconazole group, 50% in the amphotericin B group, 45% in the itraconazole group, and 30% in the saline group. Compared with the other antifungal agents, fluconazole offered superior prophylaxis against the progression of trichosporonosis fungemia to disseminated disease (P

We examined the protective effect of intratracheal immunization with Pseudomonas aeruginosa pili protein against respiratory infection caused by P. aeruginosa. Mice were immunized intratracheally or subcutaneously with purified pili protein or bovine serum albumin as a control. Intratracheally but not subcutaneously pili protein-immunized mice showed significant improvement of survival after intratracheal challenge with the PAO1 strain. Furthermore, bacterial cell counts in pili protein-immunized murine lungs were significantly decreased compared to controls at 18 h after the challenge. Antipili protein antibody titers in bronchoalveolar lavage fluid of intratracheally pili protein-immunized mice were higher than in bovine serum albumin immunized mice. However, antipili antibody titers were not increased in bronchoalveolar lavage fluid of subcutaneously pili protein-immunized mice, despite the high serum antipili antibody titers. Inoculation of P. aeruginosa induced immediate increases in interleukin-12 and interferon-gamma in bronchoalveolar lavage fluid of pili protein-immunized mice, reflecting an adequate and rapid immune response against P. aeruginosa respiratory tract infection. Our findings suggest that intratracheal pili protein immunization is effective against respiratory tract infection caused by P. aeruginosa in mice.

Long-term administration of macrolide antibiotics reduced the number of lymphocytes in bronchoalveolar lavage fluid in patients with chronic airway inflammatory disease. To evaluate the inflammatory activity of macrolides, their effect on apoptosis of activated lymphocytes isolated from human peripheral blood was compared with that of other antibiotics. Macrolides, including clarithromycin and azithromycin, at a final concentration of 100 μg/ml accelerated apoptosis of activated lymphocytes, while other antibiotics such as fosfomycin sodium, β-lactams — ceftazidime, piperacillin sodium and biapenem, and a quinolone, ofloxacin, did not cause significant induction of apoptosis. Our results suggest that 14- or 15-membered ring macrolides are specifically involved in the augmentation of apoptosis of activated lymphocytes, and this may be of value therapeutically for chronic airway diseases.

Eosinophil granule major basic protein (MBP) is expressed exclusively in eosinophils and basophils in hematopoietic cells. In our previous study, we demonstrated a major positive regulatory role for GATA-1 and a negative regulatory role for GATA-2 in MBP gene transcription. Further analysis of the MBP promoter region identified a C/EBP (CCAAT/enhancer-binding protein) consensus binding site 6 bp upstream of the functional GATA-binding site in the MBP gene. In the cell line HT93A, which is capable of differentiating towards both the eosinophil and neutrophil lineages in response to retinoic acid (RA), C/EBPα mRNA expression decreased significantly concomitant with eosinophilic and neutrophilic differentiation, whereas C/EBPβ expression was markedly increased. Electrophoretic mobility shift assays (EMSAs) showed that recombinant C/EBPβ protein could bind to the potential C/EBP-binding site (bp −90 to −82) in the MBP promoter. Furthermore, we have demonstrated that both C/EBPβ and GATA-1 can bind simultaneously to the C/EBP- and GATA-binding sites in the MBP promoter. To determine the functionality of both the C/EBP- and GATA-binding sites, we analyzed whether C/EBPβ and GATA-1 can stimulate the MBP promoter in the C/EBPβ and GATA-1 negative Jurkat T-cell line. Cotransfection with C/EBPβ and GATA-1 expression vectors produced a 5-fold increase compared with cotransfection with the C/EBPβ or GATA-1 expression vectors individually. In addition, GST pull-down experiments demonstrated a physical interaction between human GATA-1 and C/EBPβ. Expression of FOG ( F riend o f G ATA), which binds to GATA-1 and acts as a cofactor for GATA-binding proteins, decreased transactivation activity of GATA-1 for the MBP promoter in a dose-dependent manner. Our results provide the first evidence that both GATA-1 and C/EBPβ synergistically transactivate the promoter of an eosinophil-specific granule protein gene and that FOG may act as a negative cofactor for the eosinophil lineage, unlike its positively regulatory function for the erythroid and megakaryocyte lineages.

Although peroxidase activity in basophils can be detected by optical and ultrastructural cytochemistry, its characteristics remain to be determined. We have demonstrated the characteristics of peroxidase activity induced in the immature basophil cell line, KU812-F. Ultrastructurally, peroxidase activity was detected in granules as well as in the perinuclear space and endoplasmic reticulum. Immunocytochemistry revealed that KU812-F cells were stained by anti-eosinophil peroxidase antibodies, and eosinophil peroxidase mRNA, not myeloperoxidase, was detected in the cells using Northern hybridization and reverse transcription-polymerase chain reaction. Eosinophil peroxidase can be one of the molecules shared with eosinophils and basophils. The biological function of eosinophil peroxidase detected in basophils remains uncertain.

Acute promyelocytic leukemia (APL) has a specific genetic rearrangement between the retinoic acid receptor (RAR)-α gene and the pml nuclear protein gene. All-trans retinoic acid (ATRA) induces granulocytic differentiation of APL-derived cells and is used to treat APL patients. However, ATRA interacts with normal cells with RAR throughout the entire body, and when used at high doses or over a long duration, it induces several adverse effects. The development of drugs that selectively act on APL cells may contribute to increasing the therapeutic efficacy of APL treatment as well as elucidating the mechanisms of response to ATRA. In this study, 9-cis retinoic acid α-tocopherol ester (9CTT) inhibited the proliferation of APL-derived NB4 and HT93 cells and induced differentiation markers, such as granulocytic maturation, nitroblue tetrazolium reduction, and CD11b expression, in these cells. The effects of 9CTT on non-APL cells, including HL-60 and U937 cells, were much weaker than those on APL cells, and tretinoin tocoferil (TT), which is an α-tocopherol ester of ATRA, did not induce the differentiation of APL cells as effectively as 9CTT. The differentiation-inducing effects of 9CTT were inhibited by RAR antagonists. 9CTT and TT similarly induced the transactivating activity of RARs, but were not effective on RXRs. 9CTT downregulated the expression of PML/RAR-α protein more effectively than TT, which suggests that it may be involved in the selectivity of 9CTT against APL cells. Interestingly, 9CTT enhanced the differentiation of APL cells induced by ATRA, 9-cis retinoic acid, and synthetic retinobenzoic acids. Combined with 1α,25-dihydroxyvitamin D3 (VD3), 9CTT also more than additively induced the differentiation of APL cells. Thus, 9CTT, alone or in combination with other retinoids or VD3, may be useful for the treatment of APL.

Competitive RT-PCR was used to determine the quantitative variation in the expression of deoxycytidine kinase (dCK) gene in childhood leukemic cells. The degree of dCK gene expression varied over a 50-fold range. In two cases in which both primary and relapsed leukemic cells were analysed, decreased expression of dCK gene was found in relapsed leukemic cells. The sequence variation analysis using bisbenzimide/polyethylene glycol electrophoresis demonstrated no sequence alteration of dCK cDNA in all cases. These results indicate that the expression of dCK gene varies in patients and suggests decreased expression of the dCK gene as one of the mechanisms responsible for clinical resistance to ara-C.

We experienced a case of Burkitt's lymphoma showing an unusual surface phenotype, CD5 expression, at an early stage of the disease. Initially, this patient showed massive abdominal para-aortic lymph node swelling which rapidly developed into leukemic change. Based on the clinical course and cytogenetic features of lymphoblasts in the bone marrow, which showed t(8;14) and c-myc gene rearrangement, the patient was diagnosed with Burkitt's lymphoma. Combination chemotherapy induced short-term remission, but central nervous system (CNS) involvement developed, followed by a regrowth of lymphoma cells in the bone marrow. The bone marrow at the end stage showed monotonous expansion of large cells with conspicuous vacuolation in the basophilic cytoplasm. The initial lymphoma cells showed pan-B markers and were CD5 positive but weakly CD10 positive; however, the lymphoma cells obtained from the bone marrow at the terminal stage did not express CD5. The chromosomal t(8;14) was seen, and identical rearrangement of immunoglobulin heavy chain joining gene and c-myc gene were detected by Southern blot analysis in the bone marrow lymphoblasts throughout the clinical course. This case is evidence that remarkable transformation of CD5-positive lymphoblasts to CD5-negative lymphoblasts occurred in an identical clone of Burkitt's lymphoma.

Primary familial and congenital polycythaemia (PFCP) is a rare disease characterized by congenital erythrocytosis inherited in an autosomal dominant fashion. Recently, mutations in the erythropoietin receptor (EpoR) have been identified in PFCP families. We describe a Japanese family with an autosomal dominant inheritance of PFCP. An in vitro colony assay demonstrated hypersensitivity of erythroid progenitors to erythropoietin (Epo) in affected family members. Sequence analysis of RT-PCR products amplified from the C-terminal region of EpoR transcripts in affected family members revealed that they were all heterozygous for C and T bases at position 5986, which suggested a genetic mutation (C to T) on one allele of EpoR. This mutation gave rise to a translation termination codon TAG at amino acid 435. Thus, the resulting EpoR is a truncated protein product lacking all 74 amino acids downstream of the mutation. To date, all genetic mutations affecting a family with PFCP, including this one, have been located in the cytoplasmic negative regulatory region of the EpoR. All mutations gave rise to truncated Epo receptors between Tyrosine 427 and Tyrosine 455. The phosphotyrosines in this region of EpoR have been demonstrated to be binding sites for SHP-1 phosphatase. Therefore PFCP is presumably brought about as a result of genetic mutations which cause the loss of the SHP-1 binding site in the cytoplasmic region of EpoR.

Several means of analyzing minimal residual disease (MRD) in leukemia involving the rearranged T cell receptor (TCR) gene have been described. We investigated MRD in leukemia with TCR beta rearrangement by examining TCR beta-chain RNA. A complementary DNA (cDNA) corresponding to the variable region of the TCR beta-chains originating from the peripheral blood or bone marrow from four patients was amplified. Single strand conformation polymorphism (SSCP) analysis of amplified cDNA showed that all four patients had monoclonal leukemia with TCR beta rearrangement; two patients had Vbeta2+ leukemia, another patient had Vbeta14+ leukemia and the other had Vbeta9+ leukemia. Flow cytometry supported this finding. Sequencing of the Vbeta2-complementarity determining region 3 (CDR3), Vbeta9-CDR3 and Vbeta14-CDR3 revealed monoclonality. To investigate MRD using TCR beta-chain RNA, cDNA from each patient was diluted with the cDNA of a healthy person and amplified using a specific CDR3 clonotype primer. A band in the ethidium bromide-stained agarose gel was detected from samples diluted 10,000-fold. SSCP analysis determined which V region gene was utilized in monoclonal leukemic cells. The leukemic cell specific TCR, determined in such a manner, may be a target for immunotherapy. Because the MRD of T cell malignancy can be easily examined once the CDR3 clonotype primer is made, this novel analysis is considered to be a useful method.

The expression of the c-kit gene product has been examined in normal mast cells, mast cell neoplasms, and basophil/mast cell precursors obtained from patients with chronic myelogenous leukaemia (CML). Formalin-fixed, paraffin-embedded sections or smears fixed with formalin vapour were studied by immunohistochemical methods, using a polyclonal antibody against the c-kit gene product. Normal and neoplastic mast cells showed a positive immunoreaction for c-kit gene product, but neoplastic basophil/mast cell precursors from CML patients lacked c-kit gene product by immunohistochemical and flow cytometric methods, even in cells having mast cell granules, together with or without basophil granules. Mast cell tryptase was, however, expressed in normal and neoplastic mast cells and basophil/mast cell precursors containing mast cell granules. In addition, cells of monocyte/macrophage lineage lacked c-kit gene product. These findings indicate that the c-kit gene product may play an important role in the development and function of mast cell but not of cell of basophil and monocyte/macrophage lineage.

New adriamycin (ADR) resistant human leukemic cell lines (KY-ADR1 and KY-ADR2) have been established. KY-ADR1 was selected from a cytosine arabinoside (Ara C) resistant cell line by gradually increasing the concentration of ADR and KY-ADR2 from the parental cell line, KY-821, by the same method. The IC 50 S of both cell lines were 4.3 × 10 −5 and 3.6 × 10 −5 M ADR, respectively. Both lines revealed a similar cross resistance to various anticancer drugs, but KY-ADR1 was resistant to Ara C, whereas KY-ADR2 was sensitive. MDR1 gene was over-expressed and P-glycoprotein was expressed on the cytoplasmic membrane in both lines. Neither verapamil nor cyclosporin A could completely reverse ADR resistance. In addition, no significant changes in topoisomerase II and glutathione-s-transferase levels were detected. These findings indicate that ADR resistance in both cell lines is mainly mediated by P-glycoprotein and some other mechanism may be present. Interestingly, growth of both cell lines was stimulated by natural IL-1 and not affected by TNFα and IFNγ, whereas growth of parental KY-821 was inhibited by these factors. These cell lines will provide new biological aspects on drug resistant leukemic cells.

Lanosterol 14-α-demethylase ( Erg11 protein; Erg11 p), encoded by the ERG11 gene, is the primary target of azoles. Recently, a change in affinity of this enzyme for azoles has been reported as a resistance mechanism in several fungal species. Trichosporon asahii ( T. asahii) is susceptible to fluconazole (FLC). This report identified the ERG11 gene of T. asahii (NCBI accession; [HQ176415][1]). The Erg11 p of T. asahii , presumed from the DNA sequence, was closely related to the Erg11 p of Cryptococcus neoformans . Furthermore, a FLC-susceptible strain was cultured in medium containing FLC at concentrations from 4.0 to 16 μg mL−1 in order to analyze the development of FLC resistance in T. asahii . The degree of resistance was related to the FLC concentration of the growth medium. One highly resistant strain that was cultured in the medium containing 16 μg mL−1 FLC contained 1 point mutation (G1357C) that caused a single amino acid substitution at G453R. This amino acid is highly conserved among major fungal pathogens, and it is in a region very close to the heme-binding domain, which is characteristic of the cytochrome P450 superfamily, the primary target for the azole class of antifungal agents. This amino acid substitution may have caused the high resistance to azoles in T. asahii . [1]: /lookup/external-ref?link_type=GEN&access_num=ddbj-embl-genbank%2FHQ176415&atom=%2Ffemsyr%2F12%2F6%2F662.atom

The perivascular macrophages in the brain are thought to be derived from bone marrow precursor cells. Results presented here demonstrate that immature macrophages obtained from nonadherent spleen cell populations can adhere to cerebrovascular endothelial cell (EC) monolayers and proliferate. The proliferation of these cells can be stimulated by either purified murine granulocyte-macrophage colony-stimulating factor (GM-CSF) or EC conditioned medium but not by IL-3. No proliferation of the GM-CSF- or IL-3-dependent murine cell line IC-2 was observed in the presence of EC conditioned medium. Macrophage-like cells could also be derived from murine bone marrow cells but inhibited by anti-macrophage-CSF (M-CSF or CSF-1). The capacity of EC conditioned medium to induce the proliferation of macrophage-like cells from the spleen is therefore not due to the release of GM-CSF but CSF-1. In concomitant experiments using mature macrophages incubated on EC monolayers, no proliferation was observed. These findings suggest that the growth of perivascular macrophages in the brain may be stimulated by cerebrovascular EC.

In order to apply a simple purging method by heat to autologous bone marrow transplantation (ABMT), we have revaluated the ability to purge clonogenic leukemic cells from the simulated marrow mixture of normal marrow cells and leukemic cell lines (HL-60, Molt-3 and HEL) in vitro by heat, using two different clonogenic assays for normal granulocyte-macrophage progenitors (CFU-GM) and leukemic cell lines. It appeared that in vitro hyperthermia (42°C for 120 min) is able to selectively remove clonogenic leukemic cells from simulated tumor cell-normal marrow mixtures even when leukemic cell concentrations are increased up to 3 × 10 6 cells/ml in vitro , and results in a 4–6 log destruction of clonogenic leukemic cells/ml according to a limiting dilution assay, while leaving half of normal CFU-GM surviving. The hyperthermic purging of clonogenic leukemic cells was not affected in the presence of normal marrow cells in vitro . This high level of clonogenic leukemic cell depletion by heat correlated with that of immunologic and pharmacologic studies. These results suggest that in vitro hyperthermia could be applied effectively and safely for the elimination of residual clonogenic leukemic cells in autologous marrow grafts before ABMT.

Our previous study demonstrated the positive relationship between the gene introduction rate into hematopoietic cell lines by electroporation and the percentage of cells in S-phase. In the present study, granulocyte-macrophage progenitor cells (CFU-C) rich marrow cell fraction were cultured in suspension with IL-3, GM-CSF and G-CSF for 4 days. The number of CFU-C were increased three times after the culture, and 3H-thymidine suicide tests of cultured cells demonstrated that the proportion of CFU-C in S-phase was increased by two to four times. The efficiency of gene transfer into CFU-C with the plasmid pMoZtk (containing the beta-galactosidase gene) by electroporation was nearly doubled by culturing marrow cells with these growth factors. These findings confirm that the introduction rate of the gene into CFU-C by electroporation is more efficient in cell populations with a higher percentage of CFU-C in S-phase.

To test the relationship between DMs and drug resistance in newly established AML cell lines, KY821, and its clone KY821A3, the latter had lost DMs during cloning, were cultured in increasing concentrations of MTX. KY821 became resistant against 2 x 10(-4) M MTX, whereas KY821A3 did against 2 x 10(-5) M MTX in a same period. Enhanced enzyme activities of DHFR were correspondent to the increased DMs numbers and DHFR gene amplification in both resistant clones. The amplified DHFR gene was located on DMs by in situ hybridization. These data indicated that the presence of DMs in KY821 would facilitate the acquisition of drug resistance.

A vincristine-resistant human myelomonocytic leukemic cell line (KY-VCR) was established. KY-VCR exhibited approximately a 2.5 x 10(6)-fold increase in resistance to vincristine compared to the parental cell line. KY-VCR showed a decreased uptake and, an increased efflux of vincristine, and cross-resistance to Adriamycin and Actinomycin D. The M(r) 200,000 membrane glycoprotein was overexpressed in KY-VCR. Furthermore, two antibodies, designated TO73 and TO77, preferentially reacting with KY-VCR were obtained. Enzyme linked immunosorbent study indicated that both antibodies recognized the same epitope and TO77 the wide portion. Immunoprecipitation analysis demonstrated that the antibodies recognized M(r) 65,000 membrane protein, which was distinct from overexpressed glycoprotein in KY-VCR. The induction of membrane protein identified by the antibodies may play a role in drug resistance. KY-VCR cells and two antibodies to them may be very useful for the study of drug resistance and prediction of drug efficacy.

We have studied leukemic cells, derived from acute nonlymphocytic leukemia with basophilic features and basophilic crisis of chronic myelogenous leukemia (CML), by cytochemical and ultrastructural examination and analysis of surface markers. Cytochemical results varied from case to case, while the ultrastructural appearances of the granules were different from normal granules. The granules had more delicate granular matrices with or without myelinoid figures, whorled or scroll matrix, multivesicular bodies structures, theta granules, and crystalloid structures. Leukemic cells in all cases had myeloid surface markers with some degree of variability. In addition, they were occasionally positive for lymphoid markers, but not for CD10 and IgE receptors. The present results show that leukemic cells with basophilic features are heterogeneous in their morphology, cytochemistry and surface markers.

When a human leukemic cell line, HT93 was incubated with all-trans retinoic acid (ATRA), IL-5, or both, this cell line was differentiated into eosinophic lineage, in that an eosinophilic specific granule proteins, major basic protein (MBP) and eosinophil peroxidase (EPO) appeared. Both CD11b and CC chemokine receptor, CCR3 expression were upregulated, while CD71 expression was downregulated by ATRA or ATRA+IL-5. Concomitantly, marked production of eotaxin-2/CCL24 was observed, but no production of eotaxin-1/CCL11 and eotaxin-3/CCL26 was detected. Since only 20 to 30% cells incubated with ATRA became positive for CCR3, CCR3(+) population was enriched by a magnetic activated cell sorter (MACS). Enriched CCR3(+) population produced higher eotaxin-2/CCL24 than the CCR3(-) population, indicating that differentiated eosinophils are capable of producing eotaxin-2/CCL24. During the ATRA-induced differentiation, expression of a transcriptional factor, GATA-1 was significantly increased. Introduction of siRNA against GATA-1 markedly reduced the ATRA-induced differentiation markers including CD11b and CCR3, as well as reduced eotaxin-2/CCL24 production. Finally, ATRA-induced differentiation and eotaxin-2/CCL24 production were greatly enhanced in the GATA-1-overexpressed clones. These results indicate that the ability to produce eotaxin-2/CCL24 is acquired during the differentiation into eosinophilic lineage which is dependent on GATA-1 expression.

A 35-year-old man developed invasive pulmonary aspergillosis (IPA) with severe neutropenia after umbilical cord stem cell transplantation for chronic myelogenous leukemia. Filamentous fungus isolated from his sputum was identified as Aspergillus terreus. Despite systemic amphotericin B (AMPH) administration, IPA progressed. However, intravenous administration of micafungin (MCFG) and oral itraconazole improved clinical data and symptoms, although he later died of massive hemoptysis. Examination of the in vitro susceptibility of this A. terreus isolate to MCFG revealed a good minimum inhibitory concentration and good time-kill assay results compared to AMPH. Thus, MCFG might be useful for IPA caused by A. terreus.

To evaluate the anti-inflammatory action of macrolide antibiotics, we examined whether macrolide antibiotics could induce apoptosis of activated lymphocytes. The proportion of apoptotic cells was augmented by clarithromycin (CLR) and azithromycin (AZM) compared with control. There was no significant difference in Fas and Fas-ligand expression between the control and macrolide-treated groups. CLR and AZM inhibited the expression of Bcl-xL compared with that of control. Our results suggest that CLR and AZM enhance apoptosis of activated lymphocytes by down-regulation of Bcl-xL.

One hundred and seventy-seven strains of Streptococcus pneumoniae derived from respiratory specimens between 1987 and 2001 were evaluated for their antimicrobial susceptibilities and distribution of genes related to penicillin and macrolide resistance. Resistance rates tended to be higher for the 1996–2001 isolates than for the 1987–1995 isolates for all β-lactams tested. For benzylpenicillin the MIC 90 value of the isolates derived between 1996 and 2001 was 1.56 mg/L, while that of strains isolated between 1987 and 1990 was 0.05 mg/L. Furthermore, the number of strains susceptible to macrolides also decreased, but only two strains isolated in 1993 were resistant to levofloxacin of the 177 S. pneumoniae strains tested. When of genes relating to penicillin resistance were analysed using PCR with primers specific to susceptible alleles, although more than 50% of strains from 1987 to 1990 and 1991 to 1995 revealed no mutations in the pbp 1a , 2x and 2b genes, only 30.0% of strains derived between 1996 and 2001 showed no mutations in the pbp gene. Strains having mutations in all three pbp genes ( 1a , 2x and 2b ) by the PCR method increased from only 2.2% in the 1987–1990 derived strains to 27.5% in the 1996–2001 strains. Furthermore, 64.1 and 60.0% of the isolates from 1987 to 1990 and 1991 to 1995, respectively, did not possess either the mefA or ermB by PCR analysis. Conversely, 75.0% of isolates from 1996 to 2001 possessed mefA and/or ermB . These genetic changes may explain the increase in the number of penicillin and macrolide resistant strains. We believe that it is important to evaluate changes in MIC as well as genetic mutations in order to select the most appropriate therapy for S. pneumoniae infections.

The effects of rokitamycin (ROK) and levofloxacin (LEVX) were investigated in a murine model of enterohaemorrhagic Escherichia coli (EHEC) infection. After C3H/HeN mice were inoculated intragastrically with E. coli O157:H7, ROK (20mg/kg) or LEVX (1.2 mg/kg) was administered intragastrically. The death rate of the mice was noted and the faeces were collected to determine viable cell counts of EHEC and for Shiga-like toxins (SLTs) assays. After the mice were sacrificed, the kidneys and colons of some of the mice were removed for histopathological examination. The death rate of mice administered ROK (19%) was significantly lower than that of the control and LEVX-treated groups (80, 93%, respectively). Viable cell counts of EHEC in the faeces of the control and ROK-treated groups were 10 7 and 10 6 CFU/g at day 5 after the infection, respectively. LEVX reduced the bacterial count by less than 100 CFU/g at day 5. The level of SLTs in the faeces from the ROK group were lower than the LEVX-treated and control groups at day 5. The histopathological findings in the kidneys treated with LEVX showed necrotic tubular epithelial cells and those in the colon, inflammatory infiltrates. These were not seen in the ROK-treated group. These results suggested that ROK suppressed release of SLTs from the EHEC and could be useful in the treatment of EHEC infection.

We investigated the mechanism by which clindamycin (CLI) modulates cytokine induction after lipopolysaccharide (LPS) stimulation. Although CLI decreased the intracellular expression levels of tumor necrosis factor alpha and interleukin 1β (IL-1β) and increased IL-6 expression in macrophages, cytokine mRNA expression levels were similar in CLI-treated and untreated groups. Our findings suggest that CLI modulates cytokine production in LPS-stimulated macrophages.

Previously we isolated and characterized a membrane-bound, arginine-specific serine protease from pig intestinal mucosa [J. Biol. Chem. 269, 32985-32991 (1994)]. For further characterization of this type of enzyme, we cloned a cDNA from rat intestinal mucosa encoding the precursor of a similar protease. The partial amino acid sequences determined for the pig enzyme were found to be shared almost completely by the rat enzyme. The serine protease domain of the rat enzyme, heterologously expressed in Escherichia coli, specifically cleaved Arg (or Lys)-X bonds with a marked preference for Arg-Arg or Arg-Lys, similar to the pig enzyme. The mRNA for the rat enzyme was shown to be distributed mainly in intestine, and the enzyme was detected in the duodenal mucosa as a 70 kDa protein. Immunohistochemical analysis of the small intestinal tissue showed that the enzyme is localized mainly on brushborder membranes.

We investigated the modulation of the expression and phosphatase activity of SHP-1 during granulocytic differentiation of human myeloid leukemia cell line, HL60 and t(15;17) positive APL cell line, HT93, in response to all-trans retinoic acid (ATRA) or dimethylsulfoxide (DMSO). ATRA induced differentiation in both cell lines which was associated with marked growth inhibition and S-phase reduction. On the other hand, DMSO induced it only in HL60 without obvious growth inhibition and S-phase reduction. The expression and phosphatase activity of SHP-1 were upregulated only when the 2 cell lines were differentiated to granulocytes. Furthermore, the changes were not dependent on the inducers or the growth inhibition. These findings suggest that SHP-1 is involved in common myeloid differentiation, and that upregulation of SHP-1 is not always related to myeloid cell growth.

Treatment of septicemia caused by Escherichia coli with ceftazidime (CAZ) may be associated with the development of septic shock due to the release of bacterial lipopolysaccharide. We examined the suppressive effect of clindamycin (CLDM) on CAZ-induced release of endotoxin by cultured E. coli and the subsequent production of inflammatory cytokines (tumor necrosis factor alpha [TNF-α] and interleukin-1β [IL-1β]). E. coli ATCC 12014 was incubated in inactivated horse serum with or without CLDM for 1, 4, or 18 h, followed by the addition of CAZ and collection of the culture supernatant at 0, 1, and 2 h. The concentration of endotoxin in each sample was measured by a chromogenic Limulus test. Another portion of the culture supernatant was added to THP-1 cell culture and incubated for 4 h, and the concentrations of TNF-α and IL-1β in the supernatant were measured by an enzyme-linked immunosorbent assay. In the control group (no CLDM), CAZ administration resulted in significant increases in endotoxin, TNF-α, and IL-1β concentrations. Pretreatment of E. coli with CLDM for 4 or 18 h before the addition of CAZ significantly suppressed the concentrations of endotoxin, TNF-α, and IL-1β in a time-dependent manner. In addition, CAZ treatment transformed E. coli from rod-shaped bacteria to filament-like structures, as determined by electron microscopy, while pretreatment with CLDM prevented these morphological changes. Our in vitro studies showed that CAZ-induced release of large quantities of endotoxin by E. coli could be suppressed by prior administration of CLDM.

The viability of the coccoid forms of Helicobacter pylori was evaluated by assessing protein synthesis. Metabolic labeling studies showed the synthesis of proteins and the specific protein profiles of H. pylori coccoids produced under various conditions. Harsh conditions such as aerobiosis and starvation (lack of horse serum) in the culture did not affect the synthesis of proteins in the coccoids. Lowering of the pH to that of gastric secretions induced expression of several proteins in the coccoids. However, the coccoids produced under prolonged microaerobic conditions exhibited a profile of acid stress-induced protein expression different from that induced by aerobiosis or starvation. Our data suggest that coccoid H. pylori exhibits diversity in viability following exposure to different stresses and that the response to acid stress of coccoid H. pylori could be involved in infection of the host stomach.

YU-311 is a monoclonal antibody reacting with cytosine arabinoside (AraC)-resistant human leukemic cell line and identifies a 92 kDa membrane protein. We have examined YU-311 reactivity with various hematopoietic disorders by an immunohistochemical method and evaluated a correlation between YU-311 expression and refractoriness to chemotherapy, retrospectively. YU-311 reacted with AraC-resistant human leukemia cell lines, in which a 92 kDa membrane protein was identified by Western blotting, whereas drug-resistant cell lines to other than AraC failed to express YU-311 antigen. The frequency of YU-311 positivity was significantly increased in relapsed cases. Only five cases were positive for YU-311 at diagnosis and 24 cases at relapse. Unexpectedly, only eight cases of relapsed leukemia/lymphoma expressed YU-311 and P-glycoprotein simultaneously. Most of the YU-311-positive relapsed cases showed clinical refractoriness for chemotherapy and then failed to induct complete remission or relapsed at short periods with short disease-free duration. These findings indicate that YU-311 expression is closely associated with some aspects of drug resistance, especially with AraC resistance.

In order to clarify the function of P210 bcr/abl oncogene in leukemogenesis, IL-3 dependent murine hematopoietic cell line, FDC-P2, was transfected with the plasmid containing cDNA of P210 bcr/abl oncogene (pGD'210) or murine IL-3 (pcDmIL3) by electroporation. Four out of five pGD'210 transfected clones as well as FDC-P2 transfected with pcDmIL3, acquired autonomous proliferation (i.e. lost the requirement for IL-3 supplementation). The expression of bcr/abl oncogene was weak in one clone, which remained dependent on IL-3. Unlike pcDmIL3 transfectants, which secrete IL-3 into the supernatant, IL-3 was not demonstrated in the culture supernatant of pGD'210 transfected FDC-P2. These findings suggest that P210 bcr/abl oncogene is directly associated with autonomous proliferation, which is the first process of leukemogenesis.

Immunoglobulin E (IgE) is central to the induction of allergic diseases through its binding to the high-affinity receptor (Fc epsilon R1) on mast cells and basophils. Crosslinking by allergens of the bound IgE leads to the release of various inflammatory mediators. IgE production by B cells requires a physical interaction with T cells, involving a number of surface adhesion molecules, as well as the soluble factors interleukin-4 (IL-4) and IL-13 (ref. 5) produced by T cells, basophils and mast cells. Here we report that, in the presence of IL-4, mast and basophilic cell lines can provide the cell contact signals that are required for IgE synthesis. The human cell lines HMC-1 (mast) and KU812 (basophilic) both express the ligand for CD40 (CD40L) which is shown to be responsible for the IgE production. Moreover, freshly isolated purified human lung mast cells and blood basophils are also shown to express CD40L and to induce IgE production. This evidence suggests that mast cells and basophils may therefore play a key role in allergy not only by producing inflammatory mediators, but also by directly regulating IgE production independently of T cells.

Human myelomonocytic leukemic cell line, designated as KY-821, and its sublines KY-Ra, KY-VCR, and KY-MTX, which were resistant to cytosine arabinoside, vincristine, and methotrexate, respectively, were compared for response to various hematopoietic growth factors. Cells of KY-Ra and KY-VCR proliferated in response to natural interleukin-1 (nIL-1), whereas the proliferation of KY-821 and KY-MTX was inhibited. Unexpectedly, recombinant IL-1 alpha and IL-1 beta had no effect on the proliferation of each cell line. The effect of nIL-1 was partially deleted by an addition of optimal anti-IL-1. Supernatants of each cell line had no IL-1 activity. Interferon gamma (IFN gamma) and tumor necrosis factor alpha (TNF alpha) also had an inhibitory effect for KY-821 and KY-MTX, but lacked such effect in KY-RA and KY-VCR. nIL-1, IFN gamma and TNF alpha could not differentiate between any of the cell lines but IFN gamma and TNF alpha induced monocytic surface antigens. In addition, there was no difference in the number of IL-1 and TNF alpha receptors in each cell line. These results indicate that there is a difference in biological effects between nIL-1 and recombinant IL-1 species and acquirement of resistance for some types of drugs may associate closely with different responses to hematopoietic growth factors, probably through altered postmembranous transduction.

We have carried out cytochemical and ultrastructural examination of human leukemic cells showing basophil/mast cell features derived from patients with acute myelogenous leukemia or basophilic crisis in chronic myelogenous leukemia. Leukemic cells in each case initially showed meta-chromasia with toluidine blue and various degrees of positivity for astra blue. Other cytochemical results showed considerable variety among cases. The number of granules increased in short-term culture in every case. Ultrastructurally, small membrane-bound granules with or without myelinoid bodies or glycogen particles were present in immature blasts, followed by production of other granule types. In some cases, leukemic cells before and after liquid culture contained the typical basophil granules with or without myelinoid bodies, but the matrix was more loose than normal. Granules showing whorl or scroll matrix profiles, which were typical for mast cells, were present in two cases. In one case, immatue leukemic cells contained theta granules, and some mature forms after short term culture contained typical basophil/mast cell granules as well as theta granules. Leukemic cells occasionally contained multivesicular granules predominantly. These results indicate that leukemic cells with basophil/ mast cell features show a heterogeneous configuration and contain abnormal granules differing from normal ones. This abnormal granulopoiesis may be attributable to the results of leukemic events and may be a hallmark for recognition of leukemic basophils/mast cells. Acta Pathol Jpn 41: 531–539, 1991.

Blast cell colonies that we have developed in our laboratory have proved to be effective in studies of the mechanisms of stem cell proliferation. The progenitors of blast cell colonies appear to be the earliest to be identified in culture and reside in the G0 phase of the cell cycle in the steady-state marrow. Using a “mapping” technique, we have characterized the biological properties of the progenitors for the blast cell colony-forming cells and the mechanisms of growth factor interactions supporting stem cell proliferation. When the blast cell colony-forming cells remain dormant in the steady-state marrow, they do not appear to require the presence of early-acting factors such as interleukin (IL)-1, IL-3, IL-6, or granulocyte colony-stimulating factor (G-CSF) for their survival. Our evidence indicates that IL-6, G-CSF, and leukemia-inhibitory factor (LIF)/differentiation-inhibiting activity (DIA) are involved in the initiation of cell divisions of the dormant stem cells. Once stem cells exit from G0, their proliferation is supported by IL-3 and/or IL-4. Transforming TGFβ has been shown to preferentially inhibit colony formation from early progenitors. We have observed that the inhibitory effect of TGFβ on colony formation from dormant stem cells may be abrogated by IL-6 or G-CSF. Stem cell proliferation may be regulated by interactions of stimulatory and inhibitory factors.

KU-812-F is a subclone of KU-812, which has cytological features similar to the parent clone, and which carries the Philadelphia chromosome. We studied the effects of various chemical agents and serum- free culture (SF-C) condition on its differentiation. The KU-812-F subclone differentiated into macrophage-like cells with phorbol myristate acetate (PMA), but not with other agents. Unexpectedly, under SF-C, KU-812-F cells differentiated into mature basophil-like cells, and the histamine content of cell lysates increased in proportion to maturation. The addition of the condensed supernatant of SF-C promoted the cloning efficiency of KU-812-F cells in semisolid SF-C, whereas the cloning efficiency was reduced in SF-C alone. KU-812-F cells expressed myelomonocytic antigens. Additionally, My4 was induced with PMA and SF- C, but less often. HLA-DR was not expressed in any culture conditions, despite blastic morphology of KU-812-F cells or macrophage-like cells induced with PMA. The IgE receptor could not be demonstrated under any conditions. Correspondingly, immature and mature KU-812-F cells did not respond to anti-IgE or concanavalin A (con A), while histamine release was induced with PMA. In conclusion, KU-812-F belongs to the myeloid cell lineage and is at least a bipotential cell that can differentiate into basophils and macrophage-like cells. Although the functions appear to be dissimilar to those of normal basophils, the KU-812-F cell line may be a good model for basophil differentiation. Moreover, KU-812-F cells may provide new insights because they grow in semisolid culture by an autostimulating mechanism.

Due to an increase in mature neutrophils, a 72-year-old female with gall bladder cancer showed leukocytosis of up to 1.32 X 10(11)/l; hypercalcemia was up to 7.7 mEq/l in her terminal stage. Leukocyte counts and calcium values increased as the tumor progressed. There was no sign of infection or bone marrow metastasis. Cultured cells from the tumor tissue produced high colony-stimulating factor (CSF) activity into the supernatant. The tumor cell-conditioned medium stimulated exclusively granulocytic colonies. The study of this patient shows that leukocytosis was caused by the CSF produced by tumor cells. Approximately 90% of CSF activity was lost by heat treatment at 60 degrees C for 30 min. The CSF was stable over a pH range of 3-11 and was inactivated by treatment with proteolytic enzymes, but was not affected by treatment with DNase or neuraminidase. Molecular weight of the CSF, demonstrated by fractionation using Sephacryl S-200, was approximately 27,000 to 30,000.

Interleukin-4 (IL-4), which was originally identified as a B-cell growth factor, has been shown to produce diverse effects on hemopoietic progenitors. The present study investigated the effects of purified recombinant murine IL-4 on early hemopoetic progenitors in methylcellulose culture. IL-4 supported the formation of blast cell colonies and small granulocyte/macrophage (GM) colonies in cultures of marrow and spleen cells of normal mice as well as spleen cells of mice treated with 150 mg/kg 5-fluorouracil (5-FU) 4 days earlier. When the blast cell colonies were individually picked and replated in cultures containing WEHI-3 conditioned medium and erythropoietin (Ep), a variety of colonies were seen, including mixed erythroid colonies, indicating the multipotent nature of the blast cell colonies supported by IL-4. To test whether or not IL-4 affects multipotent progenitors directly, we replated pooled blast cells in cultures under varying conditions. In the presence of Ep, both IL-3 and IL-4 supported a similar number of granulocyte/erythrocyte/macrophage/megakaryocyte (GEMM) colonies. However, the number of GM colonies supported by IL-4 was significantly smaller than that supported by IL-3. When colony-supporting abilities of IL-4 and IL-3 were compared using day-4 post-5-FU spleen and day-2 post-5-FU marrow cells, IL-4 supported the formation of fewer blast cell colonies than did IL-3. IL-4 and IL-6 revealed synergy in support of colony formation from day 2 post-5-FU marrow cells. These results indicate that murine IL-4 is another direct-acting multilineage colony-stimulating factor (multi-CSF), similar to IL-3, that acts on primitive hemopoietic progenitors.

We treated KU812 cells from different passage levels with compounds which induce differentiation of other leukemic cell lines. Early passages contained small subpopulations of basophil and erythrocyte-like cells, but later passages consisted of more uniform cells with a myelomonocytic phenotype. When cultured with 1 nM actinomycin D, the cells became nonproliferative, developed pale cytoplasm and segmented nuclei, and lost nonspecific esterase activity. Thus, KU812 cells can be induced to mature to cells with a myelomonocytic phenotype. However, their expression of previously reported basophil characteristics diminishes with continued passage, and may be a capability of only a subpopulation of the cells.

The application of hyperthermia to the treatment of neoplastic disease has focused on solid tumors. Since the hyperthermic sensitivity of human acute leukemia cells is not known, we have studied the in vitro response of human leukemic progenitor cells (L-CFU) to hyperthermia using a quantitative assay system for L-CFU. Human L-CFU were found to be more sensitive than committed normal myeloid progenitor cells to hyperthermic killing (41 to 42 degrees C). In addition, in the five acute myelogenous leukemic patients studied, it was shown that their leukemic progenitor cells--all types were studied according to the French-American-British diagnosis--were unable to form colonies when exposed to a temperature of 42 degrees C for 60 minutes, whereas the residual normal clones suppressed by the leukemic cell population were found to recover and to form more colonies in vitro as compared with untreated leukemic marrows. This strongly suggests that in vitro hyperthermia may selectively purge residual leukemic cells, especially L-CFU in stored remission bone marrow before autologous bone marrow transplantation.

A new myeloid cell line (KU812) was established from a patient with blastic crisis of chronic myelogenous leukemia. His blasts were morphologically characteristic of immature basophils and basophil colonies were grown in agar culture of the blood mononuclear cells. Suspension culture of his blood cells was continued for more than 2.5 years. The KU812 cells morphologically showed a fine reticular nuclei with nucleoli, and some of them contained metachromatic granules with toluidine blue (TB) staining. These granules were positive for astra blue (AB) staining. Immunological marker studies revealed that there were no lymphoid characters except Fc receptors. The KU812 cells grew colonies in in vitro agar cultures, which were proved to be composed of basophils by TB staining and AB staining. Cytogenetical analysis showed marked aneuploidy and was positive for the Philadelphia chromosome (Ph1). The cell lysate was proved to contain histamine. These data suggest that KU812 is a cell line from leukemic basophil precursors. This is the first human basophil cell line, and KU812 will be useful in clarifying the mechanism of basophilic differentiation of the stem cells.