Yoshimi Kobayashi

Involvement of natural killer cells in patients with myelodysplastic syndrome carrying monosomy 7 revealed by the application of fluorescence in situ hybridization to cells collected by means of fluorescence‐activated cell sorting

Monosomy 7 is the most frequent chromosome abnormality among patients with secondary myelodysplastic syndrome (MDS). We used fluorescence in situ hybridization (FISH) and fluorescence‐activated cell sorting (FACS) in order to clarify the lineage involvement. Four patients, three with de novo MDS and one with secondary MDS, were enrolled in this study. Monosomy 7 was observed in pluripotent stem cells (CD34+Thy‐1+), and in B (CD34+CD19+) and T/natural killer (NK) progenitors (CD34+CD7+). The number of abnormal cells of B (CD19+) and T (CD3+) cells was below the cut‐off value, but approximately 60% of the NK cells (CD3‐CD56+) contained monosomy 7 in three of the patients.

Degranulation of eosinophils mediated by intercellular adhesion molecule-1 and its ligands is involved in adhesion molecule expression on endothelial cells–selective induction of VCAM-1

BACKGROUND Adhesion molecules and eosinophils may play an important role in the pathogenesis of allergic inflammatory reactions. OBJECTIVE We attempted to clarify eosinophil activation, such as degranulation, by signaling through adhesion molecule and to determine whether degranulation is involved in adhesion molecule expression on endothelial cells. METHODS Eosinophils were cultured with or without recombinant soluble intercellular adhesion molecule-1 (ICAM-1), and the levels of eosinophil cationic protein and eosinophil-derived neurotoxin were determined. The influence of these eosinophil granule proteins or supernatant from eosinophil cultured with ICAM-1 on the expression of ICAM-1 or vascular cell adhesion molecule-1 (VCAM-1) on endothelial cells was also examined by flow-cytometric analysis. RESULTS Supernatant levels of eosinophil granule protein were significantly increased by culture for 4 hourss or 16 hours with recombinant soluble ICAM-1, suggesting degranulation by adherence to ICAM-1. Both granule proteins and the supernatants of eosinophils cultured with recombinant soluble ICAM-1 induced expression of ICAM-1 and VCAM-1 on endothelial cells, with the latter showing a more prominant increase. CONCLUSION Degranulation mediated through adherence to endothelial cells by ICAM-1 and its ligands may be involved in the expression of adhesion molecules, such as ICAM-1 or VCAM-1, on these cells. Our finding of the selective induction of VCAM-1 expression suggests that eosinophil adherence to endothelial cells, even if it is because of ICAM-1, may be involved in selective eosinophil recruitment and accumulation at sites of allergic inflammation.

A New Variant Translocation of t(15;17) in a Patient with Acute Promyelocytic Leukemia (M3): t(15;19;17)(q22;p13;q12)

The reciprocal translocation (15;17) is specifically associated with acute promyelocytic leukemia [APL; M3 subtype according to French-American-British (FAB) classification]. A few patients with this disease have complex variant translocations. We describe a patient with M3 carrying t(15;19;17)(q22;p13;q12), which is a new type of variant translocation. The karyotypic interpretation was confirmed by Southern blot analysis with the use of RAR alpha and by fluorescence in situ hybridization (FISH) with the use of painting probes of chromosomes 15, 17, and 19 and a (15;17) translocation DNA probe. The results support the idea that the key event in APL is the formation of fusion gene PML/RAR alpha on the der(15).

A PML/RARA chimeric gene on chromosome 2 in a patient with acute promyelocytic leukemia (M3) associated with a new variant translocation: t(2;15;17)(q21;q22;q21).

We describe a patient with acute promyelocytic leukemia (APL) carrying a new complex variant translocation of t(2;15;17)(q21;q22;q21). The karyotypic interpretation was confirmed by fluorescence in situ hybridization (FISH) with the use of painting probes of chromosomes 2, 15, and 17 and a PML/RARA dual color DNA probe. FISH showed a PML/RARA fusion gene on the der(2) instead of the der(15). These results suggest that the critical event in the development of APL is the formation of a PML/RARA chimeric gene, regardless of its locus in the genome.

Translocation (8;12;21)(q22.1;q24.1;q22.1): A New Masked Type of t(8;21)(q22;q22) in a Patient with Acute Myeloid Leukemia

The translocation t(8;21)(q22;q22) is found in 40% of cases of acute myeloid leukemia (AML) designated as the subtype M2 in the French-American-British (FAB) classification. The 8;21 translocation is clinically of interest because patients with this subtype have a good prognosis. We describe a masked type of the translocation, t(8;12;21)(q22.1;q24.1;q22.1). The translocation was first interpreted as t(8;12)(q22;q24) based on cytogenetics, but was reevaluated as a result of Southern blot and fluorescence in situ hybridization (FISH) analyses.

CCR3 mRNA Expression in Bronchial Epithelial Cells and Various Cells in Allergic Inflammation

Background: RANTES and eotaxin are important chemokines involved in the activation and migration of eosinophils and are considered to play a major role in allergic inflammation. Methods: In this study, we used RT-PCR to investigate the kinds of cells that express mRNA for CCR3, a common receptor of these chemokines, and eotaxin, a ligand for CCR3. Results: CCR3 mRNA was expressed in eosinophils, peripheral mononuclear cells, an eosinophilic cell line (EoL-1), a bronchial epithelial cell line (NCI-H292), human endothelial cells and nasal washings from patients with allergic rhinitis. Conclusion: These results suggest that the CCR3-eotaxin system plays an important role in generating inflammation, since these substances are expressed not only in cells implicated in activation or migration of eosinophils but also in various other cells involved in allergic inflammation.

Fluorescence In Situ Hybridization Monitoring of BCR-ABL-Positive Neutrophils in Chronic-Phase Chronic Myeloid Leukemia Patients during the Primary Stage of Imatinib Mesylate Therapy

We describe a method for monitoring chronic myeloid leukemia (CML) patients treated with imatinib that uses fluorescence in situ hybridization (FISH) to detect BCR- ABL in peripheral blood (PB) granulocytes. First, we compared this method, termed Neutrophil- FISH, with interphase FISH (i-FISH) analysis of bone marrow (BM), i-FISH analysis of PB mononuclear cells, and conventional cytogenetic analysis (CCA) of BM in 30 consecutive CML patients. We found the percentage of BCR- ABL-positive neutrophils as determined by Neutrophil-FISH to correlate best with the percentage of Philadelphia chromosome-positive metaphases in the BM determined by CCA (y = 0.8818x + 5.7249; r2 = 0.968). We then performed a serial Neutrophil-FISH study of 10 chronic-phase CML patients treated with imatinib and found that the technique could clearly separate imatinib responders from nonresponders within 12 weeks of drug administration. There was a significant difference in the percentages of BCR- ABL-positive neutrophils between responder (mean ± SD, 18.2% ± 11.8%) and nonresponder (82.4% ± 5.1%) groups at 12 weeks (P <.0001, Student t test). Together with real-time quantitative polymerase chain reaction analysis, Neutrophil-FISH represents another useful method for monitoring CML patients during the primary myelosuppressive stage of imatinib therapy because it is a quick, simple, and reliable method for assessing cytogenetic response.

Effect of Roxithromycin on Eotaxin-Primed Reactive Oxygen Species from Eosinophils

Background: The CC chemokine eotaxin not only attracts eosinophils to inflamed sites but also promotes adhesion, degranulation and reactive oxygen species production of eosinophils. Reactive oxygen species released from eosinophils are believed to injure epithelial cells at inflamed sites, resulting in airway hyperresponsiveness. Roxithromycin has been reported to have antiasthmatic effects, although its mechanism of action is not thoroughly understood. Therefore, the effect of roxithromycin on eotaxin-primed reactive oxygen species production from eosinophils was studied. Methods: Reactive oxygen species production by eosinophils cultured with or without roxithromycin was evaluted using luminol-dependnet chemiluminescence. Results: Roxithromycin inhibited the release of reactive oxygen species from eosinophils evoked with the calcium ionophore A23187, regardless of pretreatment with or without eotaxin. Conclusion: Roxithromycin may protect epithelial cells at inflamed sites, at least partly by inhibiting the release of reactive oxygen species from eosinophils.

Multilineage involvement in hypereosinophilic syndrome terminating in granulocytic sarcoma and leukaemic transformation with trisomy 8

Summary. We report a patient with hypereosinophilic syndrome (HES), which, 8 years later, transformed into granulocytic sarcoma in the brain and, subsequently, into acute myelocytic leukaemia. Repeated chromosome analyses showed a normal karyotype, until the time of leukaemic transformation when trisomy 8 was confirmed in cells from the bone marrow and cerebrospinal fluid. The combined techniques of May–Grunwald–Giemsa staining and fluorescence in situ hybridization identified trisomy 8 not only in blasts and eosinophils but also in neutrophils and erythroblasts. Our observation suggests that HES is a multilineage myeloproliferative disorder involving precursors of at least the eosinophil, neutrophil and erythroid lineages.

Expression of VLA-4 on Eosinophils Decreases in Patients with Eosinophilia

Background: It is assumed that the very late antigen-4 (VLA-4) plays a key role in selective migration and accumulation of eosinophils to the allergic inflammatory focus. The regulatory mechanism for VLA-4 expression is poorly understood, as is its relationship between other adhesion molecules. Objective: The aim of the study was to elucidate the relationship between VLA-4 expression and the activation of eosinophils. Methods: The surface expression of VLA-4, Mac-1, ICAM-1, CD4, CD25, CD69, CD89, IL-5 receptor and GM-CSF receptor on eosinophils isolated from the peripheral blood of 15 patients with eosinophilia and 16 healthy volunteers was measured. Results: The surface expression of VLA-4 presented in mean fluorescent intensity by flow-cytometric analysis showed a significant decrease in the patients with eosinophilia (>700 eosinophils/µl) compared to that of the subjects without eosinophilia. On the other hand, the surface expression of Mac-1 was significantly increased in the patients with eosinophilia. There was an inverse correlation between the expression of VLA-4 and that of Mac-1 (r = –0.81) on the eosinophils obtained from the patients with eosinophilia. Conclusion: The changes on the surface expressions of Mac-1 and VLA-4 may be indicating the activation of eosinophils in the patients with eosinophilia and may contribute to their migration to the allergic inflammatory focus.