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We report a case of T-cell chronic lymphocytic leukemia (T-CLL) in which the lymphocytes were small and mature, did not have cytoplasmic granulation, and appeared to be similar to those of B-cell chronic lymphocytic leukemia (B-CLL). Immunophenotyping of the lymphocytes revealed CD2+, CD4+, CD5+, and CD25+ on the cell surface, but expression of the CD3 and TCR-alpha beta molecules was localized only in the cytoplasm. The clinical course was as indolent as that of B-CLL, and the patient was able to undergo open-heart surgery. Thus, we confirmed that true T-CLL is able to exist as a counterpart of B-CLL.

Doxorubicin induces DNA breakage by stabilizing a cleavable topoisomerase II-DNA complex. In contrast, topoisomerase II catalytic inhibitor ICRF-193 and uncoupling inhibitor aclarubicin interfere with the cleavable complex formation. We analysed combination effects of these drugs using two-dimensional flow cytometry of DNA content and the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labelling assay. Both ICRF-193 and aclarubicin attenuated the cytotoxic effect of doxorubicin on HL-60 cells (85% and 46% maximum reduction, respectively), which suggested that doxorubicin exerts its cytotoxic effect at least partially through the topoisomerase II-dependent DNA cleavage. Doxorubicin and ICRF-193 both induced G2 arrest in HL-60 cells, by which they may have reduced the cytotoxic effect of vincristine. Indeed, although ICRF-193 inhibited doxorubicin-induced apoptosis, ICRF-193 and doxorubicin cooperated in arresting HL-60 cells at G2 phase. These results indicated that G2 arrest was caused not only by DNA damage but also through a DNA damage-free, topoisomerase II inactivation-induced pathway. Western blot analysis showed that both types of G2 arrest were mediated by the inhibition of p34cdc2 dephosphorylation.

We examined the effect of a hydroxamic acid-based matrix metalloproteinase inhibitor (KB-R7785), which we previously demonstrated to have a potent ameliorating effect on acute graft-versus-host disease (GVHD), and on the graft-versus-leukaemia (GVL) effect of allogeneic bone marrow transplantation (BMT). KB-R7785 was administered to (C57BL/6 x BALB/c) F1 (CBF1) mice that had been inoculated with IgE-producing B53 hybridoma cells of BALB/c origin as a model tumour, along with or without transplantation of C57BL/6 (B6) bone marrow cells and spleen cells (BMS). Administration of KB-R7785 without BMS significantly prolonged the survival of B53-inoculated CBF1 mice by inhibiting the infiltration of B53 cells into the liver and spleen. Transplantation of B6 BMS without KB-R7785 resulted in the death of most recipients due to acute GVHD while efficiently eliminating B53 cells. Administration of KB-R7785 along with B6 BMS resulted in a 50% survival of B53-inoculated CBF1 mice over 50 d without histological manifestations of acute GVHD or residual B53 cells. These results indicate the beneficial effects of KB-R7785 that inhibit tumour infiltration and prevent acute GVHD while preserving the GVL effect of allogeneic BMT.

A 44-year-old man was admitted because of persistent fever and pancytopenia. Because his bone marrow was hypoplastic and the karyotype of his marrow cells was normal, he was given a diagnosis of aplastic anemia, and treated with glucocorticoids and granulocyte colony-stimulating factor. Splenomegaly was later found and a splenectomy performed: pathological findings on resected tissue specimens disclosed non-Hodgkin's lymphoma, B-cell diffuse large. The patient was transferred to our hospital, where a bone marrow biopsy revealed lymphoma cells infiltrating his hypoplastic marrow. Complex chromosomal abnormalities were detected in marrow cells, but no lymphadenopathy was observed. A diagnosis of primary splenic lymphoma with infiltration of lymphoma cells into bone marrow was made, and chemotherapy was accordingly started. After multiple cycles of chemotherapy, the patient's marrow recovered to a normal state and his karyotype abnormalities disappeared. Six months later, pancytopenia reappeared and lymphoma cells were again detected in the patient's bone marrow. We reasoned that the hypoplastic state of his bone marrow was associated with the lymphoma, and that cytokines, including interferon-gamma, may have been responsible for this association.

We studied clinical effect of a combination therapy with cefozopran (CZOP) and tobramycin (TOB) for infections in 80 patients with hematologic diseases in 15 institutes. Combined doses with CZOP 2 g and TOB 60-90 mg twice a day had been given intravenously. Of the 80 patients, 61 patients (42 with acute leukemia, 10 with malignant lymphoma, 3 with aplastic anemia, 2 with chronic myeloid leukemia, 2 with multiple myeloma, and 2 with myelodysplastic syndrome) were evaluable. Those consisted of 6 patients with septicemia, 49 with suspected septicemia, 3 with pneumonia, and 3 with other infections. Clinical efficacy by the treatment was excellent in 24, good in 17, fair in 9, and poor in 11 patients, and the overall efficacy rate including excellent and good was 67.2%. Microbiologically, 5 of the 6 patients with septicemia (1 coagulase negative Staphylococcus, 2 S. pneumoniae, 1 S. oralis, and 1 E. coli) were responded. The efficacy rate in patients with severe granulocytopenia showing 100/microliter or lesser neutrophil counts during the drug administration was 57.1% (12/21). Side effects and abnormal changes of clinical laboratory findings were observed in 5 patients, and 16 patients, respectively, but most of them were mild. The findings above suggested that the combination therapy with CZOP and TOB is useful as an empiric therapy for severe infections in patients with hematologic diseases.

P-glycoprotein (P-gp), a transmembrane efflux pump encoded by the MDR1 gene, has been found to be expressed in many normal bone marrow and peripheral blood cells. Among normal leukocytes, CD3(-)CD16(+) or CD3(-)CD56(+) lymphocytes, ie, natural killer (NK) cells, express relatively high levels of P-gp, but little is known about P-gp in abnormally expanded NK cells. In this study, we examined the expression and activity of P-gp on NK cells derived from three normal donors, six patients with indolent NK cell-lineage granular lymphocyte-proliferative disorder (NK-GLPD), three patients with aggressive NK cell tumors (one NK cell leukemia and two nasal NK cell lymphoma), and two NK cell lines. By flow cytometric analysis using the monoclonal antibody (MoAb) MRK16 and rhodamine 123 dye (Rh123), P-gp expression and the efflux of Rh123 were found in all NK samples except one NK cell line. The Rh123 efflux of NK cells was inhibited by cyclosporin A (CsA) and its analogue PSC 833, but the aggressive NK tumor cells were less inhibited than were the other NK cells. The percent inhibition of efflux in the normal NK cells, indolent NK-GLPD cells and aggressive NK cell tumors was 81.8% +/- 0. 9%, 93.4% +/- 3.1% and 36.9% +/- 11.7%, respectively, by 1 micromol/L CsA, and 80.2% +/- 3.6%, 91.7% +/- 2.6% and 32.7% +/- 10. 1%, respectively, by 1 micromol/L PSC833. In reverse transcription-polymerase chain reaction (RT-PCR) analysis, the low inhibitory effect of P-gp modulators in aggressive NK cell tumors did not correlate to the expression level of MDR1 gene, multidrug resistance-associated protein gene, or human canalicular multispecific organic anion transporter gene. This phenomenon could be related to the presence of other transporters or to unknown cellular or membrane changes. Some patients with NK cell tumors have been reported to show a highly aggressive clinical course and to be refractory to chemotherapy, and this could be related to the expression of P-gp on NK cells. Our results suggest that, although the inhibitors for P-gp have been used in combination with chemotherapy in some hematologic tumors, these inhibitors may be less effective against aggressive NK cell tumors.

A forty-eight-year old male diagnosed with acute lymphocytic leukemia was further complicated by Candida krusei fungemia despite introduction of fluconazole prophylaxis during antileukemic therapy. The fungemia subsided after intravenous amphotericin B therapy followed by itraconazole therapy. Multiple ulcerative lesions and a carcinoid lesion were observed in the transverse colon, and it was thought that the C. krusei had invaded from the ulcerative lesions. Azole antifungal agents, especially fluconazole, have frequently been used for therapy and the prophylaxis of fungal infections. Though infections due to fluconazole-resistant Candida species such as C. krusei are a growing problem, in the hematology division of our hospital there is no evidence of an increasing incidence of C. krusei fungemia or an increasing rate of C. krusei in surveillance culture from admitted patients. However, future increases in infections due to fluconazole-resistant Candida species like C. krusei and C. glabrata are entirely possible, making it necessary to identify isolated fungi, perform susceptibility tests on antifungal agents against isolates, measure the serum concentrations of antifungal agents, and then to treat patients with the appropriate therapies.

We report a case in which an aflatoxin-producing strain of Aspergillus flavus (A. flavus) caused systemic aspergillosis in a post-transplant 41-year-old man with acute myeloid leukemia. The leukemia was initially resistant to two courses of induction chemotherapy. The third course of chemotherapy, however, induced complete remission. Thereafter, the patient underwent bone marrow transplantation from his HLA identical brother. Pulmonary aspergillosis was suspected as a complication during induction chemotherapy. Twenty days after the transplant, the patient's absolute neutrophil count had increased to 500/microliter. However, the symptoms of pulmonary aspergillosis were aggravated following neutrophil and monocyte recovery. The patient died of sinus arrest due to complete atrioventricular block 31 days after his transplant. At autopsy, we found that the fungus had invaded the brain, lungs, spleen, kidneys, skin, and myocardium, including the sinoatrial conduction system. There was no sign of acute graft-versus-host disease. A strain of A. flavus was isolated from cultured tissue samples of fungal lesions and shown by thin-layer chromatography to produce aflatoxins. To our knowledge, this is the first case report describing an infection by an aflatoxin-producing A. flavus.

We studied ten cases of Japanese T-cell prolymphocytic leukemia (T-PLL) collected over the last 9 years. Median age was 61 years with a male predominance (M:F, 8:2). The main disease features were splenomegaly, lymphadenopathy, hepatomegaly, skin lesions and serous effusions. The clinical course was progressive with a median survival of 10 months. Immunophenotyping showed that the prolymphocytes had a post-thymic phenotype (TdT-, CD1a-, CD2+, CD3+, CD5+, CD7+) with a predominant CD4+ immunophenotype. Cytogenetic analysis showed no consistent abnormalities. 14q abnormality and trisomy 8q, which are frequently seen in T-PLL of Western countries, were found in only two and zero cases, respectively. We conclude that the clinical and biological characteristics of T-PLL in Japan are almost the same as those in Western countries. However, the cytogenetic findings of T-PLL in Japan might be different.

A 72-year-old man was referred to our hospital because of lymphadenopathy, splenomegaly, and leukocytosis. His WBC count was 54,300/microliter, with 89.6% atypical lymphocytes two to three times the diameter of red blood cells, cleaved nuclei, and one or two nucleoli. A lymph node specimen revealed a vaguely nodular pattern, and the diagnosis of mantle cell lymphoma (MCL) was made. The lymphoma cells appeared smaller and more mature than the leukemic cells. The phenotype of the peripheral blood and the lymph node cells was CD5+ CD10- CD19+ CD20+ and the same rearranged JH bands were detected, suggesting that their lymphocytes were of the same origin. In addition, the phenotype of the leukemic cells was CD23+ CD38+ CD43- CD44+ FMC-7+ micro+ chi+. Cytogenetic analysis revealed complex anomalies but not t(11; 14). Cyclin D1 protein was not detected. Because the lymphocyte morphology of the peripheral blood and lymph nodes was discordant, we speculated that variant large cells had proliferated mainly in the peripheral blood. The patient achieved a partial response after 6 courses of CHOP regimen, and was then placed on a COP regimen. He seemed to have MCL, but the following findings were unusual: marked lymphocytosis at initial presentation, discordant morphology, CD5+ CD10- CD23+ CD43- phenotype with neither t(11; 14) or cyclin D1 over-expression.

In the treatment of severe infections complicated to blood dyscrasia, the efficacy and usefulness of fosfomycin (FOM) in combination with sulbactam (SBT)/cefoperazone (CPZ) were compared between patients receiving FOM in the first followed by SBT/CPZ (Group A) and those receiving both drugs simultaneously (Group B). The following results were obtained. 1. The efficacy rate was 56.3% for Group A and 47.9% for Group B, with no significant difference. 2. The efficacy for patients suspected of the presence of septicemia, the efficacy rate was 57.9% for Group A and 54.3% for Group B, with no significant difference. 3. As for underlying disease, patients with acute myelogenous leukemia were most prevailing. In these patients, the efficacy rate was 57.1% for Group A and 27.3% for Group B, with no statistically significant difference. However, the efficacy rate tended to be higher in Group A. 4. The administration of antibiotics was effective to restore the neutrophil count to 501/microliters or higher in 77.8% and 45.5% of the cases for Groups A and B, respectively, with significantly higher efficacy for Group A. 5. In the safety evaluation a total of 115 cases were included. Side effects and laboratory abnormalities were seen in 3 cases each, but none of them were serious in degree. From these results, it was confirmed that the combination therapy consisting of administration of FOM followed by SBT/CPZ with some interval is effective for severe infections complicated to blood dyscrasia.

Both tumor necrosis factor alpha (TNFalpha) and Fas ligand (FasL) have been implicated in the pathogenesis of graft-versus-host disease (GVHD). In this study, we examined the ameliorating effects of neutralizing anti-FasL and/or anti-TNFalpha monoclonal antibody (MoAb) in a lethal acute GVHD model in mice. Whereas the treatment with either anti-FasL or anti-TNFalpha MoAb alone significantly delayed the mortality and improved the body weight, a complete protection was achieved by the administration of both MoAbs. Pathological examination indicated differential effects of anti-FasL or anti-TNFalpha MoAb on GVHD-associated pathologies. Hepatic lesion was improved by anti-FasL but not anti-TNFalpha MoAb. In contrast, intestinal lesion was improved by anti-TNFalpha but not anti-FasL MoAb. Cutaneous and splenic lesions were improved by either MoAb. The combination of both MoAbs improved all these lesions. These results indicate that FasL and TNFalpha differentially contribute to the GVHD pathologies and a complete protection from mortality can be achieved by neutralization of both FasL and TNFalpha.

We report a 45-year-old man with monocytosis and right hemiparesis. The patient suffered from an acute myocardial infarction from which he recovered completely when he was 42 years old. One year prior to his death, he was found to have increase in monocyte count (35.5% of leukocytes) in peripheral blood and splenomegaly; he was admitted to the hematology service of our hospital. He was diagnosed as having chronic myelomonocytic leukemia after bone marrow examination. He was treated with radiation therapy with improvement in splenomegaly. In May of 1995, he had fever, anemia, and thrombocytopenia for which he needed daily blood transfusion. In November of 1995, he had an onset of weakness in his right hand, and neurologic consultation was asked for in November 27, 1995. Neurologic examination revealed a chronically ill japanese man in no acute distress. He was alert and not demented. Higher cerebral functions were intact. Cranial nerve examination revealed right facial paresis of the central type. Motor-wise, he was right hemiparetic. Generalized muscle wasting was noted apparently due to the chronic debilitating disease. Deep tendon reflexes were within normal range in the right upper extremity, but were diminished in other areas. Sensation was intact, and no meningeal signs were noted. Pertinent laboratory findings were as follows: Hb 8 g/dl, RBC 238 x 10(4)/microliter, WBC 2,900/microliter (band 1.0%, seg 18.5%, lym 28.0%, mono 44.0%, Baso 2.5%), Plt 13 x 10(4)/microliter, PT 16.6"/10.9", APTT 44.7"/35.0". CSF contained 87 mg/dl of protein, 155 mg/dl of glucose and 2 mononuclear cells/microliter. Bone marrow was slightly hypercellular with mild increase in blast forms. No chromosome abnormality was found. CT and MRI revealed a large mass in the left fronto-parietal region and the meninges showed marked thickening with enhancement after gadolinium-DTPA in MRI. The patient was treated with glycerol and steroid, but the subsequent course was complicated by a seizure, agitation, and pneumonia. He died from respiratory failure on January 13, 1996. The patient was discussed in a neurologic CPC and the chief discussant arrived at the conclusion that the patient had chronic myelomonocytic leukemia with infiltration of leukemic cells into meninges and the parenchyme of the cerebrum. Thickening of the dura was thought to be in part a reaction to the subdural hematoma as well as to leukemic cells along the meninges. Postmortem examination revealed hypercellular bone marrow with increase in monocytic cells (more than 20%). The lungs showed pneumonia with scattered old tuberculous lesions. The heart showed an old myocardial infarction in the posterior wall of the left ventricle. The brain showed an old chronic subdural hematoma in the left fronto-temporal region and a cystic mass lesion in the left frontoparietal region. The mass was hypercellular and most of them were monocytes. The dura mater showed reactive thickening without leukemic cell infiltration. It was concluded that this patient had chronic myelomonocytic leukemia with a formation of leukemic mass in the brain. Pathologists thought that the mass was a hematogenous spread. It is rare for chronic myelomonocytic leukemia to form a mass lesion in the brain.

The first case of human systemic infection by an Aspergillus flavus isolate demonstrated to produce aflatoxins in vitro and in vivo is described. The patient, a 41-year-old man with acute myelogenous leukaemia, developed a complication of suspected pulmonary Aspergillus infection during remission induction therapy. Antifungal chemotherapy brought about a considerable degree of improvement, but remission of the underlying disease was not attained. Bone marrow transplantation was also not effective. The patient showed recovery from neutropenia but died despite aggressive antifungal chemotherapy. The autopsy revealed lesions in the lungs, myocardium, kidneys, brain, thyroid gland and skin due to a suspected Aspergillus sp. A fungus isolated from the right lung and the skin lesions was identified as A. flavus. Aflatoxins B1, B2 and M1 were detected in culture filtrates of the isolated A. flavus, and in an extract of lung lesions. These aflatoxins are considered to have played an important role in damaging the immune system of the patient through their toxic effects.

T(3;14)(q27;q32) is frequently detected in B-cell non-Hodgkin's lymphomas, especially the diffuse large cell type and the follicular type. The BCL6 gene encoding a putative transcriptional factor which resides on 3q27 rearranges to the immunoglobulin heavy chain (IgH) gene on 14q32 in this chromosomal translocation. The upstream regulatory region of the BCL6 gene is replaced by the IgH gene. Deregulation of the BCL6 gene may contribute to tumourigenesis of these diseases. The rearrangement between the IgH and BCL6 genes generates chimaeric transcripts in which the joining (J) region of the IgH gene fuses to exon 3 of the BCL6 gene. We established a method to detect these chimaeric transcripts by reverse transcriptase polymerase chain reaction (RT-PCR) using the consensus sequence of the J region and the sequence of exon 3 of the BCL6 gene as primers. Using the semi-nested RT-PCR method and a cell line carrying t(3;14)(q27;q32), we detected one lymphoma cell among 10,000 background cells. We detected these chimaeric transcripts in two out of 13 clinical samples by this method. This method can detect t(3;14)(q27;q32) easily, whereas this alteration is frequently overlooked by routine karyotype analysis. Since this technique is sensitive enough to detect a small number of lymphoma cells with this genetic abnormality, it could be employed to detect contaminating lymphoma cells in bone marrow and peripheral blood and minimal residual diseases.

We related cellular content of DNA topoisomerase (topo) IIalpha and IIbeta with the cell cycle position in proliferating, differentiated, and apoptotic HL-60 cells using two-dimensional flow cytometry. In logarithmically growing HL-60 cells, topo IIalpha increased especially in late S to G2/M phases, although the topo IIbeta level was almost constant throughout the cell cycle. Induction of differentiation by all-trans retinoic acid dramatically reduced the topo IIalpha but not the topo IIbeta level. A new G2/M population containing virtually no topo IIalpha appeared during differentiation and was supposed to be alive and noncycling. Two-dimensional flow cytometry of topo IIalpha or IIbeta staining and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling assay showed that one topo IIbeta epitope situated at the C-terminal end decreased specifically in apoptotic HL-60 cells treated with Ara-C, etoposide, and vincristine. The amounts of a topo IIalpha epitope and another topo IIbeta epitope located at a more central portion were almost equal between apoptotic and nonapoptotic cells. Western blot analysis confirmed that topo IIbeta protein was completely degraded into smaller fragments and lost its C-terminal end during apoptosis. On the contrary, a large portion of topo IIalpha remained of its original size, although both topo IIalpha and IIbeta left from the nuclear fraction in apoptotic cells. Confocal laser microscopy showed nuclear localization of topo IIalpha and IIbeta in growing HL-60 cells. Although topo IIalpha and IIbeta were distributed throughout the cell during mitosis, only topo IIalpha was densely concentrated in the mitotic chromosomes. Both enzymes were dissociated from the genomic DNA even at an early phase of apoptosis and completely separated from the propidium iodide signal of DNA in the advanced stage. Chromatin condensation process in apoptosis is therefore completely topo II-independent and obviously differs from the mitotic one.

For the collection of adequate numbers of peripheral blood stem cells (PBSC) for PBSC transplantation, an accurate quantification of circulating CD34+ stem cells is required for deciding the optimal time of the collection. To enumerate peripheral blood (PB) CD34+ stem cells, the percentage of CD34+ cells in the gated PB mononuclear cells should be multiplied by the percentage of the gated mononuclear cells among white blood cells (WBC) and by the total WBC count. Accordingly, a minor difference in the measured percentage of the CD34+ cells can lead to a major difference in the PB CD34+ cell concentration. In the present study, we measured the concentration of PB CD34+ stem cells with a flow cytometer designed to provide direct absolute counts of cell subsets from a single instrument. Whole blood was stained with a phycoerythrin-conjugated anti-CD34 monoclonal antibody, and, after the lysis of red blood cells, CD34+ cells were counted in a fraction of the lymphocyte and monocyte gate. The accuracy of our method was demonstrated in an experiment in which various dilutions of known numbers of CD34+ leukemic cells were mixed with normal blood; the predicted value of the CD34+ cell count was observed. The concentration of CD34+ cells in leukapheresis products was measured both by our direct assay and an indirect assay that calculates the number from the percentage of CD34+ cells in mononuclear cells, and our assay was shown to produce less variation. Further, our assay showed a significant correlation between the concentration of mobilized CD34+ cells in the PB and the number of harvested CD34+ cells in leukapheresis. These findings indicate that the monitoring of the concentration of PB CD34+ cells by the present method can be used to predict the number of stem cells collected in leukapheresis. This procedure is easy to perform and can be applied to daily monitoring to decide the appropriate timing for harvest of mobilized stem cells.

Proliferating cell nuclear antigen (PCNA) was detected in the peripheral blood mononuclear cells (PBMC) of patients with malignant lymphoma (ML). Twenty-one of 27 patients with ML had PCNA expressing PBMC (5.25+/-4.75% cells), which tended to increase in the advanced clinical stage of ML. PCNA in PBMC extracts was detected in 11 of 16 patients (54.5+/-41.9 ng/ml). The percentage of PCNA-positive cells correlated significantly with the concentration of PCNA in PBMC extracts (P < 0.005). Serum PCNA was detected in 6 of 16 patients (160.1+/-141.1 ng/ml), but did not correlate with the number of PCNA-positive cells. In some cases, the concentration of serum PCNA increased after chemotherapy while the percent PCNA-positive cells decreased. Our finding indicate that detection of PCNA in PBMC appears to help monitoring the extent of disease in ML and the serum PCNA level may be used in therapeutic studies of lymphoma patients.

Tumor necrosis factor (TNF) and Fas ligand (FasL) have been implicated in the pathogenesis of graft-versus-host disease (GVHD), which is a major complication after allogeneic bone marrow transplantation. We examined here the ameliorating effect of a metalloproteinase inhibitor (KB-R7785) that inhibits TNF-alpha and FasL release in a lethal acute GVHD model in mice. Administration of KB-R7785 into (BALB/c x C57BL/6) F1 that received C57BL/6 spleen cells markedly reduced the mortality and weight loss in association with minimal signs of GVHD pathology in the liver, intestine, and hematopoietic tissues. The ameliorating effect of KB-R7785 was superior to that of anti-TNF-alpha antibody. Our results suggest that KB-R7785 could be a potent therapeutic agent for GVHD.

A case of T-cell lymphoma of granular lymphocyte (GL) morphology and CD3+CD4+CD56+ phenotype is reported. The lymphoma originated in the subcutaneous tisse of the right thigh and subsequently infiltrated into various organs including the lymph nodes, peripheral blood, bone marrow, skin, lungs, liver, spleen, and peritoneal cavity, and the patient finally developed a hemophagocytic syndrome. The lymphoma cells were positive for CD2, CD3, CD4, and CD56, and negative for CD8, CD16, CD20, and CD57. An identical clonal rearrangement of the T-cell receptor beta chain genes was detected in cells from a lymph node, peripheral blood, and ascites fluid, indicating the monoclonal proliferation of T cells in such organs. This is a T-cell GL lymphoma, expressing the unusual phenotype of CD3+CD4+CD8-CD16-CD56+, which has not been previously reported. The development of hemophagocytic syndrome has also to our knowledge not previously been reported in T-cell GL lymphoma.

Granular lymphocytes have been characterized as cells with azurophilic granules in the cytoplasm. Patients with increased numbers of granular lymphocytes are designated as granular lymphocyte-proliferative disorders (GLPDs). A variety of haematological abnormalities are associated with T-cell-lineage GLPD. Among these, pure red cell aplasia is frequent, and adequate therapy is required. Seven patients with pure red cell aplasia, or a related condition complicating T-cell-lineage GLPD, were entered into this study. Cyclophosphamide was initiated at a daily oral dose of 100 mg. After 2 weeks the dose was reduced to 50 mg/d, and maintained at that dose. Cyclophosphamide was administered until the lymphocyte count was <1 x 10(9)/l and T-cell receptor-beta gene analysis was used to monitor the response to treatment. All the patients were successfully treated, irrespective of their former treatment. Clinical remission was associated with the disappearance of the abnormal granular lymphocyte clone, as detected by Southern blot hybridization analysis. Therapeutic responses began after 8 weeks, and clinical complete remissions were obtained after 6 months. Oral cyclophosphamide monotherapy can successfully treat the pure red cell aplasia associated with T-cell-lineage GLPD.

We performed immunocytochemistry to detect mdr1 and mdr3 P-glycoproteins (P-gps) in 81 patients with acute and chronic leukemia, using the mdr1 P-gp-specific monoclonal antibody (MoAb) MRK16, and the mdr3 P-gp-specific MDR3M. Immunoreactivity for the mdr1 gene product was positive in 27 out of 81 (33%) patients. Immunoreactivity with the anti-mdr3 P-gp MoAb was positive in 20 out of 81 (25%) patients. Of 54 patients with acute leukemia, 17 (31%) were positive for mdr1 P-gp and 8 (15%) for mdr3 P-gp. A high proportion (60%) of patients with chronic lymphocytic leukemia (CLL) were mdr3 P-gp positive. Of the patients with granular-lymphocyte proliferative disorder (GLPD), a chronic T-cell or natural killer cell leukemia, 8/17 (47%) were positive for mdr1 P-gp and 6/17 (35%) for mdr3 P-gp. Of 23 patients with chronic leukemia (CLL and GLPD), 10 (37%) were positive for mdr1 P-gp and 12 (44%) for mdr3 P-gp. To clarify the function of the mdr3 P-gp, we examined the intracellular rhodamine123 (Rh123) levels of mdr1 P-gp-negative and mdr3 P-gp-positive leukemic cells from patients with acute lymphocytic leukaemia, on the addition of 10 microM cyclosporin A (CyA). The addition of CyA led to significant increases in intracellular Rh123 levels in mdr1 P-gp-negative and mdr3 P-gp-positive leukemic cells. Results of the assay for dye efflux suggested that the mdr3 P-gp has a role in drug resistance, and functional drug-efflux capacity. In 31 acute leukemia patients at initial diagnosis, mdr1 or mdr3 P-gp expression correlated significantly to an outcome of complete remission (CR). In 54 acute leukemia patients, exposure to precytotoxic agents correlated significantly to expression, with a significant higher number of patients mdr1 or mdr3 P-gp positive than negative. In the 54 patients with acute leukemia, mdr1 P-gp expression correlated to mdr3 P-gp expression significantly (p=0.0007). In the 27 patients with chronic leukemia (CLL and GLPD), mdr1 and mdr3 P-gp expression did not correlate to exposure to precytotoxic agents, nor did mdr1 P-gp expression correlate to mdr3 P-gp expression. It may be speculated that precytotoxic agents induced mdr1 and mdr3 P-gp expression in acute leukemia; however, in chronic leukemia, both P-gps were expressed independently of exposure to precytotoxic agents.

The FHIT (fragile histidine triad) gene on chromosome 3p14 is a candidate tumor suppressor gene, and its transcripts are shown to be abnormal in several human cancers. We examined 40 leukemia samples for the alterations of FHIT transcripts by reverse transcriptase-polymerase chain reaction (RT-PCR) and direct sequencing. Intact FHIT mRNA was not detected in two patients with acute myeloid leukemia (AML) and in one patient with chronic lymphocytic leukemia (CLL). The three cases expressed only an aberrant FHIT mRNA lacking exons 3 to 6 (FHIT delta 3-6 mRNA), which could encode a polypeptide of 13 amino acids. Southern blot analysis on two samples from these cases showed no rearrangements of the FHIT gene. Although intact FHIT mRNA was detected as the main band in the remaining 37 samples, 33 of them (14 of 14 AML, 11 of 13 chronic myeloid leukemia, five of five acute lymphocytic leukemia, and three of five CLL) expressed aberrant FHIT delta 3-6 mRNA. We barely detected the FHIT delta 3-6 mRNA in only one of 25 normal control samples. Our results suggest that loss of the normal FHIT function may be involved in the genesis of at least some human leukemias and that expression of aberrant FHIT transcripts is rather specific and frequent in leukemia samples.

The lymphoproliferative disease of granular lymphocytes (LDGL), also referred to as LGL leukemia, is a heterogeneous disorder, but is clinically, morphologically, and immunologically distinct. Although LDGL has recently been included in the revised classification of lymphomas as an independent clinical entity, no consensus exists on the criteria to establish the diagnosis. The aim of this report was to refine the parameters needed to make the diagnosis of LDGL. We studied 11 patients with chronic granular lymphocytosis selected from among 195 cases observed by our institutions from three different geographic areas (North America, Europe, and Asia). These cases did not meet the current criteria for inclusion in LDGL, since all patients had less than 2,000 GL/microL. However, in each of these patients, we found evidence for expansion of a discrete GL population. Clonal rearrangement of the T-cell receptor (TCR) beta gene was found in peripheral blood mononuclear cells (PBMC) of all nine patients with CD3+ LDGL. Using recently generated monoclonal antibodies (MoAbs) against the TCR V beta gene regions, we identified a unique TCR V beta on GL from each of three patients studied. In two patients with CD3- LDGL, we also identified a restricted pattern of reactivity, by staining with MoAbs against p58 antigen found on normal natural killer (NK) cells. The clinical features of these 11 patients with relatively low absolute number of GL were similar to those reported previously for patients with greater than 2,000 GL/microL. These data demonstrate that newer techniques such as MoAbs against V beta gene regions and p58 molecules and molecular analyses are useful to identify expansions of discrete GL proliferations. Demonstration of an expansion of a restricted GL subset is evidence for the diagnosis of LDGL, even in patients with a relatively low GL count. Our results also contribute to distinguish between the end of normality and the beginning of pathology in the broad spectrum of GL lymphocytoses.

1. During the process of cell death, rises in cytosolic Ca2+ concentration ([Ca2+]i) together with structural changes were investigated in target cells attacked by purified CD3-,CD16+ human natural killer (NK) cells. 2. In the target cell line K562, a rapid [Ca2+]i rise to 1-2 microM occurred a few minutes after NK cell-target cell contact, immediately followed by leakage of the Ca2+ indicator dye fura-2 from the cell. Cells were permeabilized, but their chromatin was not fragmented. The changes were basically consistent with those seen in necrosis induced by activated complement. 3. In the target cell line MOLT-4, which expressed the apoptosis-inducing surface antigen Fas much more strongly than K562, the majority of attacked cells displayed a [Ca2+]i rise to 0.7-1 microM followed by a slow decline, often associated with diminishing [Ca2+]i oscillations. As a whole, [Ca2+]i remained higher than 150 nM for at least 1.5-3 h (approximately 100 nM in control cells). 4. MOLT-4 cells attacked by NK cells became bubble shaped within 20 min of the main [Ca2+]i rise reaching its peak, and then both the cell and chromatin were fragmented into small pieces. These findings were basically consistent with those in apoptosis induced by a monoclonal antibody against the surface antigen Fas. 5. NK cells induced both necrosis and apoptosis in cell lines insensitive to NK cells in the presence of an antibody against the major histocompatibility complex class I (antibody-dependent cell-mediated cytotoxicity, ADCC). The distinct Ca2+ responses patterns described above corresponded to necrosis or apoptosis in different cells stimulated by the common ADCC pathway. 6. Human NK cells were found to be capable of inducing necrosis (membrane damage) or apoptosis (nuclear damage) depending on the target cell types. The characteristic Ca2+ response profile was a good indicator for distinguishing between the modes of cell death induced by the cytotoxicity of the killer cells.

Natural killer (NK) cells are a distinct non-T, non-B lineage of lymphocytes that mediate major histocompatibility complex-unrestricted cytotoxicity. Morphologically they are large granular lymphocytes, and phenotypically they commonly express CD16 and CD56 antigens, without expressing cell surface CD3. Although the developmental pathway of NK cells is not fully understood, they arise from CD34+ hematopoietic stem cells and, at least in part, differentiate in the bone marrow. They gain byctoplasmic CD3 gamma delta epsilon zeta antigens during maturation, and lose cytoplasmic CD3 gamma delta epsilon thereafter until the terminal maturation. Lymphoproliferative disorders of NK cells include NK cell-lineage granular lymphocyte-proliferative disorders (NK-GLPD), NK-cell lymphoma, and acute leukemia of NK-cell lineage. NK-GLPD are relatively rare. Most patients exhibit a chronic indolent clinical course, and do not require specific treatment. However, some patients exhibit an aggressive clinical course, and die of the disease despite extensive chemotherapy. This aggressive type NK-GLPD is caused by Epstein-Barr virus (EBV). Patients with NK-cell lymphoma are rare, and often exhibit necrotic lesion and angiocentric morphology. This tumor is mainly found in the nasal tract, but the true incidence of NK-cell lymphoma in nasal lymphomas is not known. Probably many lymphomas arising from the nasal cavity, but not from paranasal sinuses, are of NK-cell lineage. NK-cell lymphoma is also caused by EBV, and is resistant to combination chemotherapy. Acute leukemia of NK-cell lineage is very rare. Several cases of acute lymphoblastic leukemia and a single case of blast crisis of chronic myelogenous leukemia have been documented to have leukemic blasts characteristic of NK cells. However, the precise lineage and differentiation stage of the leukemic blasts have not been delineated.

We analysed p53 mutations in 24 patients with myelodysplastic syndrome (MDS) and overt acute myeloid leukaemia after a period of MDS, using polymerase chain reaction-single strand conformation polymorphism analysis. In exons 5 to 8, mobility shifts were detected in five of the 24 patients. Sequence analysis was subsequently performed, and four missense mutations (16.7%) and one silent nucleotide substitution were identified. Patients harbouring mutations were characterized as having advanced disease. Loss of the wild type allele was observed in three of the four patients with missense mutations. No mobility shifts of the N-ras or FMS gene were detected in these four patients. We next analysed the correlation of the p53 mutations with the progression of MDS in three patients. The mutation was accompanied by the progression in two of the three patients. These findings suggest that mutations of the p53 gene are associated with progression in some cases of MDS, while being compatible with stable disease or clonal evolution in others.

Malignant lymphomas arising from the nasal cavity have been considered to be derived from T cells, but recent surface marker studies suggest that more than half of the lymphomas are derived from natural killer (NK) cells. Here we describe a case of nasal lymphoma whose lymphoma cells were identified as NK cells by morphological, phenotypic, immunogenotypic, and functional studies. We believe this is the first study with functional evidence of NK activity. When compared with normal freshly isolated NK cells or activated NK cells, the surface phenotypes and NK activity of the patient tumour cells were those of the activated, but not resting, NK cells. Also, Epstein-Barr virus (EBV) was detected in the tumour cells and the lymphoma cells were found to be monoclonally expanded. The patient's lymphoma cells also expressed EBV receptor CD21 (CR2) and CD30 (Ki-1) that have not been described on normal NK cells. We therefore examined highly enriched NK cells of normal donors, and found that some resting and/or activated NK cells express these antigens.

The effects of recombinant interleukin-5 (rIL-5) on leukemic blasts obtained from 22 acute myeloid leukemia (AML) patients were investigated. Recombinant IL-5 stimulated leukemic colony formation in the leukemic blasts of 50% of the patients (11/22), and in 7 of these 11 cell cultures leukemic eosinophilic colonies were induced. Eosinophilic cell induction was associated with cellular proliferation, that is, colony or cluster formation. Leukemic blasts cultured with rIL-5 and forming eosinophilic colonies no longer suppressed normal granulocyte-macrophage colony formation, suggesting that functional differentiation of these leukemic blasts took place. Induction of this type of functional differentiation of leukemic blasts may be of clinical value in promoting normal hematopoiesis.

We studied telomeric DNA in leukemic cells as well as in normal T cells, B cells, monocytes, polymorphonuclear leukocytes, and bone marrow hematopoietic progenitor cells. No marked differences were observed in the sizes of the telomeric repeats in the various populations of normal blood cells obtained from donors in their twenties to sixties, and the telomere length ranged between 8.5 and 9.0 kb. The leukemic cells of 12 patients with acute leukemia (seven with myeloid and five with lymphoid leukemia) showed a variable reduction in the length of telomeric DNA, ranging from 2.7 to 6.4 kb. The average telomere length was 4.8 and 4.7 kb in myeloid and lymphoid leukemia, respectively, while the telomere length in peripheral blood mononuclear cells obtained from the same patients during complete remission was 8.5 and 7.9 kb, respectively. When the same Southern blots were hybridized with Alu or alphoid sequences, no marked changes in the sizes of the repetitive DNA sequences were observed, indicating that the DNA abnormality in the leukemic cells was specific to the telomere region. Investigation of telomeric DNA changes may be helpful in determining the biological properties of leukemic cells.

In normal peripheral blood natural killer (NK) cells, the subset of CD16+ CD56+ cells is predominant, and that of CD16+ CD56- cells is rarely present. Because we have found the expansion of CD16+ CD56- NK cells in the peripheral blood of two patients with NK cell-lineage granular lymphocyte-proliferative disorders (NK-GLPD), the clinical findings and cellular characteristics of these patients were compared with those of CD16+ CD56+ NK-GLPD patients. Although CD16+ CD56- and CD16+ CD56+ NK-GLPD cells were morphologically different, clinical findings and courses, and NK activity did not differ significantly. Because strong NK activity was demonstrated in CD16+ CD56- NK-GLPD cells, the CD56 antigen, one of the adhesion molecules, did not seem to play a major role in NK cell-mediated cytotoxicity. The CD56 antigen is known to be more strongly expressed by immature NK cells than by mature NK cells. However, because interleukin 2-activated CD16+ CD56- NK-GLPD cells rapidly expressed the CD56 antigen, the degree of CD56 antigen expression did not always correlate with the maturity of NK cells.

A 57-year-old man was admitted because of hypergammaglobulinemia which was initially pointed out by annual complete physical examination. No significant abnormal findings were observed except polyclonal hypergammaglobulinemia at that time (IgG; 2,662 mg/dl, IgA; 422 mg/dl). Seven months later, he has progressed to show thrombocytopenia. The laboratory data showing the reduction of peripheral CD 4-positive T cells (CD 4; 0.2 x 10(9)/l, CD 4/8 ratio; 0.19) and positive serum HIV antibody revealed that his hypergammaglobulinemia and thrombocytopenia were resulted from HIV infection. The peripheral platelet count decreased to 28 x 10(9)/l at minimal point, however, it recovered to 160 x 10(9)/l within 7 months without treatment. His good performance status has been still maintained over 4 years. His whole clinical course suggests that there is no significant correlation between peripheral platelet counts, serum gammaglobulin levels or the amounts of PAIgG in this case. This case proposes that we should also consider a possibility of HIV infection for the patients showing hypergammaglobulinemia.

Interleukin-11 (IL-11), a stromal cell-derived cytokine, has been known to act widely in hematopoietic and non-hematopoietic systems. IL-11 supports the growth of certain types of plasmacytoma and hybridoma cells, acts with interleukin-3 (IL-3) in shortening the Go period of early progenitors. IL-11 supports megakaryocyte colony formation and maturation, and acts as an autocrine growth factor in megakaryoblastic cell lines. In addition, IL-11 stimulates erythrocytopoiesis, enhances antigen-specific antibody responses, induces the synthesis of acute phase proteins, inhibits lipoprotein lipase activity and adipocyte differentiation, and promotes neuronal development. Administration of rhIL-11 to mice resulted in an increase of neutrophils and platelets. The human IL-11 gene is localized at 19q13.3-13.4, and codes 199 amino acids and 23 kDa with no N glycosylation. Its receptor and signal transduction share partially those of interleukin-6 (IL-6). Further analysis of its role in normal and pathological state is necessary to determine the exact function and its application for clinical uses.

We have recently reported that a combination of lymphokine-activated killer (LAK) cells and bispecific antibodies (BsAb) efficiently lysed autologous and allogeneic leukemic blasts that had surface antigens reactive with the BsAb. The effector cells used in that experiment were peripheral blood mononuclear cells stimulated with interleukin-2 (IL-2) for 2 weeks, with the initial addition of anti-CD3 moAb; these were termed T3-LAK effector cells. In this study, we examined the effects of T3-LAK cells and BsAb on autologous normal CD34+ BM cells in both cytotoxicity and colony formation assays. When T3-LAK cells were incubated with CD34+ BM cells, low levels of cytotoxicity were induced against the CD34+ BM cells and the cytotoxicity was enhanced by the addition of anti-CD3 Fab' x anti-CD 13 Fab' BsAb but not by the addition of anti-CD3 Fab' x anti-CD10 Fab' BsAb. This enhancement appeared to be due to the lysis of CD34+CD13+ BM cells. When T3-LAK cells were preincubated with CD34+ BM cells in the presence or absence of the BsAb and plated for colony assay, neither the T3-LAK cells nor the BsAb affected granulocyte-macrophage or mixed-cell colony formation by CD34+ BM cells. Taken together with our previous finding that T3-LAK cells used in combination with the BsAb markedly inhibited colony formation by leukemic progenitor cells, these results indicate that this combination provides a potential new strategy for CD34+ BM cell purging in autologous BMT.

Various types of cytokines have been used in in vitro experiments to generate cytokine-induced killer (CIK) cells that are reactive to patient acute myeloid leukemia (AML) cells. Of these CIK cells, interleukin-2 (IL-2)-activated peripheral blood mononuclear cells, i.e., lymphokine-activated killer (LAK) cells, with the initial addition of the anti-CD3 monoclonal antibody (T3 LAK cells), are the most potent cytotoxic lymphocytes, and have marked proliferative capacity. The cytotoxicity of such T3 LAK cells against CD13+ AML cells is further enhanced by the addition of anti-CD3 x anti-CD13 bispecific antibody (BsAb) during the cytotoxicity assay. The combined use of T3 LAK cells and the BsAb can be used for ex vivo purging of CD13+ AML cells in autologous bone marrow transplantation. Other cytokines, such as IL-7 or IL-7 in combination with IL-2, or newly identified cytokines, will also be tested in attempts to obtain more specific and more potent effector cells. Studies of methods to increase the susceptibility of AML cells to CIK are also required.

The immunological findings and clinical course of 33 patients with granular lymphocyte-proliferative disorders (GLPD) are presented. Based on the surface phenotypes of peripheral blood granular lymphocytes (GL), the GLPD were divided into two groups, namely CD3+ T cell-lineage GLPD (T-GLPD) and CD3- CD16+ NK cell-lineage GLPD (NK-GLPD). Twenty-one patients had T-GLPD, and 12 had NK-GLPD. One patient with T-GLPD and two patients with NK-GLPD had progressive clinical courses and died of the disease despite receiving combination chemotherapy. Among eleven patients analysed for the expression of multidrug resistance P-glycoprotein, six of eight patients with T-GLPD and all three patients with NK-GLPD clearly expressed P-glycoprotein. Since patients with immature NK-GLPD appear to undergo a progressive clinical course, we suggest that therapeutic trials using P-glycoprotein blockers in addition to chemotherapy might be beneficial for such patients.

A 75-year-old man was admitted to our hospital on June 1st, 1993, because of nasal obstruction, epistaxis, fever, night sweats and weight loss. Examination disclosed a 2-cm white necrotic mass in the nasal septum, and a biopsy disclosed non-Hodgkin's lymphoma, diffuse, mixed-type. Imprint smears showed cytoplasmic azurophilic granules in the tumor cells. Dense granules were demonstrated by electron microscopy. The tumor cells were CD1-2+3-4-7+8-16+56+57-, and T cell receptor genes were in germline configuration. NK activity against K562 was strongly positive. Based on morphologic, phenotypic, immunogenotypic, and cytotoxic findings, the tumor cells seemed to be derived from activated NK cells. Because the tumor cells were positive for the EB virus and CD21 antigen, EB virus seemed to have infected CD21-positive NK cells and transformed them. MDR P-glycoprotein was also positive. This finding may explain why nasal lymphomas are resistant to chemotherapy and have a poor prognosis.

A Ca2+ imaging method has been used to demonstrate simultaneously the magnitude and time course of the increase in the intracellular Ca2+ concentration ([Ca2+]i) in 10-30 individual human peripheral T cells following stimulation by anti-CD4 or anti-CD8 monoclonal antibody (MAb) as well as anti-CD3 MAb. The rise in [Ca2+]i began within 10 s of the introduction of the MAb and reached a peak of 240 nM (mean of 73 cells) in 20-40 s. The peak was followed by a slow decrease persisting for 6-8 min. Comparing Ca2+ responses in the presence and absence of external Ca2+, the rise in [Ca2+]i was found to be caused by both transient intracellular Ca2+ release and a long-lasting Ca2+ influx from outside the cell. Cross-linking of CD4 or CD8 using anti-IgG antibody augmented the response in individual cells, as seen in the higher peak (365-390 nM) and the longer duration (over 10 min). Simultaneous stimulation of CD3 and CD4 did not cause a summation of Ca2+ responses but caused a suppression in the CD3-mediated Ca2+ response. The results support the view that CD4 and CD8 play a role in signal transduction for T cell activation and that the CD4-derived signal interferes with the CD3-derived signal at some stage in the signalling pathway causing the Ca2+ response.

We identified CD3 and myeloperoxidase (MPO) antigens in the cytoplasm of leukemic cells from 15 patients with CD7+ 4- 8- 1- leukemia. Seven patients, five of whom had a mediastinal mass, had only cytoplasmic CD3 (cCD3) antigen; these seven were regarded as possibly being of T-lineage type. Six patients, one of whom had a mediastinal mass, showed both cCD3 and MPO; they were considered to have mixed lineage type. Two patients had neither cCD3 nor cytoplasmic myeloid antigens; they were therefore considered to have stem cell type. These two latter types were considered as the subgroups: mixed lineage or stem cell type. Patients with T-lineage type were good responders to L-17M therapy; four out of five who received L-17M therapy achieved complete remission. There were significant differences between the two subgroups in relapse-free survival, patients with T-lineage type exhibiting better prognosis than other types (p < 0.05).

The hematological and immunological findings and clinical courses of 33 patients (13 male, 20 female; median age at presentation, 60 years) with granular lymphocyte-proliferative disorders (GLPD) are presented. Based on the surface phenotypes of peripheral blood granular lymphocytes (GL), the GLPD were divided into CD3+ T cell-lineage GLPD (T-GLPD) and CD3- CD16+ natural killer (NK) cell-lineage GLPD (NK-GLPD). Twenty-one patients had T-GLPD, and 12 had NK-GLPD. One patient with T-GLPD and two patients with NK-GLPD had progressive clinical courses and died of the disease despite receiving combination chemotherapy. Twelve patients with T-GLPD were found to have severe anemia at presentation or during the course of the disease; four of them fulfilled the diagnostic criteria of pure red cell aplasia, and the others had closely related conditions. Six of these 12 patients were treated with cyclophosphamide, and all responded to the treatment. In 16 patients, the clinical course was stable, and spontaneous regression was observed in two patients. Since some of the patients with NK-GLPD had stable clinical courses while some had progressive clinical courses, clinical findings in these two groups were compared. We found, taking into consideration our cases and those reviewed in the literature, that age less than 40 years, fever, lymph node swelling, hepatosplenomegaly, and GL with CD16(Leu-11)-CD56+CD57- phenotype and low or absent antibody-dependent cellular cytotoxicity seemed to be predictors of a progressive clinical course.

Previous studies have reported that megakaryocyte progenitors in myeloproliferative disorders (MPD) formed spontaneous megakaryocyte colonies without the addition of megakaryocyte colony-stimulating factor (Meg-CSF). To determine whether this spontaneous colony formation is due to autocrine proliferation of MPD megakaryocyte progenitors or to hypersensitivity to Meg-CSF that might exist in the culture system, we investigated colony-forming unit-megakaryocytes (CFU-Meg) in the peripheral blood of 11 MPD patients, using serum-free cultures. Spontaneous megakaryocyte colonies were observed in serum-free cultures of nonadherent mononuclear cells (NAdMNC) obtained from MPD patients with thrombocytosis, whereas the NAdMNC of MPD patients without thrombocytosis, that of patients with reactive thrombocytosis and normal subjects never formed spontaneous colonies. However, the spontaneous colonies from MPD patients with thrombocytosis disappeared in cultures using highly purified CD34-positive cells as target cells. To study the hypersensitivity of megakaryocyte progenitors to Meg-CSF, dose-response experiments were performed with interleukin-3 (IL-3). CFU-Meg from MPD patients with thrombocytosis showed maximal growth at the concentrations of IL-3 lower than those for normal subjects. CFU-Meg of MPD patients without thrombocytosis and that of patients with reactive thrombocytosis showed the same colony growth response to IL-3 as that of normal subjects. This result indicates that the CFU-Meg of MPD patients with thrombocytosis are hypersensitive to IL-3. It also suggests that spontaneous colony formation by NAdMNC is not due to the autocrine growth of megakaryocyte progenitors but is due to the hypersensitivity of megakaryocyte progenitors to Meg-CSF, such as IL-3, released by accessory cells. Furthermore, it is possible that such hypersensitivity of CFU-Meg to IL-3 might be a pathogenic factor in MPD with accompanying thrombocytosis.

The broken ends of chromosomes are unstable, and tandem fusion of telomeres has been observed in some tumors. Using Southern blot analysis, we report here telomeric DNA changes in hematologic cells. There was some variation in the length of the telomeric DNA in normal peripheral blood mononuclear cells obtained from four different individuals, ranging from 10 to 12 kilobases (kb), but there was little difference in signal strength. In two cell lines tested, HL-60 and K562, there was a telomeric sequence reduction of 2-4 kb and there was also a diminution of signal intensity. Reduction of the telomeric DNA array was also observed in two leukemic cases tested. The peak telomere length of the leukemic cells was 5 and 4 kb before and 10 and 7 kb after treatment, respectively, and in one case there was also a reduction in copy numbers of about 50%. Since no remarkable changes were detected in the Alu and alphoid sequences in either normal or leukemic cells, it appeared that the DNA change was specific to telomeric regions. Assessment of telomeric DNA changes may aid in determining the biological significance of leukemic cells.

The mechanism of therapeutic effect of anti-lymphocyte globulin (ALG) and cyclosporin A (CyA) on patients with aplastic anemia was studied. When peripheral CD8 positive cells obtained before therapy were cocultured with bone marrow CD34 positive cells obtained after hematological recovery, the number of colony forming unit granulocyte-macrophage (CFU-GM) and burst forming unit erythroid (BFU-E) were decreased. This result indicates that ALG and CyA inhibits CD8 positive cells which suppress the growth of progenitor cells, resulting in the recovery of hematopoiesis. Next we investigated the plasma concentrations of cytokines including G-CSF, GM-CSF, IL-6, IL-1 alpha and IL-1 beta after ALG treatment. Although the elevation of plasma concentrations of G-CSF and GM-CSF after ALG treatment were found in 11 of 13 patients and in 2 of 13 patients respectively, cytokine production by ALG appeared to be unrelated to the therapeutic effect of ALG for aplastic anemia.