Cytokinetically based induction chemotherapy and splenectomy for childhood acute nonlymphocytic leukemia

A four-drug regimen, based on cell kinetic principles, induced complete remissions in 68 of 95 children (72%) with acute nonlymphocytic leukemia (ANLL). Patients entered remission after 2-5 weekly cycles of vincristine-daunorubicin (day 1) followed by sequential cytosine arabinoside and 6-azauridine (days 4-7). With continuation therapy of monthly vincristine-doxorubicin-cyclophosphamide, weekly cytosine arabinoside, and daily 6-mercaptopurine, the median duration of complete remission was 10 mo and the median survival time 21 mo. Portal triaditis, evident in 11 of 23 patients with liver biopsies, was associated with long remissions. A larger spleen size (greater than 5 cm) and a higher myeloblast labeling index (greater than 10%) at diagnosis were clearly related to shorter durations of remission. Splenectomy within 1 mo of remission had no statistically significant effect on the frequency of relapse or length of remission. Patients without central nervous system (CNS) leukemia at diagnosis, all treated prophylactically with intrathecal methotrexate, had a low frequency of initial CNS relapse (3/56, 5%). The 2-yr disease-free survival rate is 29% (20 of 68 patients attaining complete remission). fifteen patients have completed 2.5 yr of therapy, and each remains in continuous complete remission, off treatment, for 1+ -36+ mo. This induction chemotherapy was as effective as more intensive regimens, with the advantage of less toxicity and shorter periods of hospitalization.     

Histamine distribution in human basophil secretory granules undergoing FMLP-stimulated secretion and recovery

We examined subcellular histamine localizations in purified human basophils that were stimulated to degranulate with FMLP using an ultrastructural enzyme-affinity technique. Basophils were collected at early (0, 20 seconds, 1 minute) and late (10 minutes to 6 hours) time points poststimulation and were prepared for routine ultrastructural and diamine oxidase-gold (DAO-gold) cytochemical analysis. Histamine was present in unaltered cytoplasmic secretory granules (30.77 gold particles per square micrometer; P < .001 compared with background); specificity controls (histamine absorption, diamine oxidase digestion) abrogated granule labeling for histamine. Altered granules in stimulated cells were not significantly labeled for histamine, as compared with background (P = not significant); unaltered granules in the same cells contained more histamine than altered granules (P < .05). During recovery times, spanning 10 minutes to 6 hours, granules again appeared to be electron-dense and contained histamine (33.49/microns2; P = not significant as compared with unaltered granules in 1-minute FMLP-stimulated cells, and P < .05 as compared with altered granules in 1-minute FMLP-stimulated samples). Other structures devoid of histamine in actively secreting cells included extruded granules and intragranular and extruded Charcot-Leyden crystals. Recovering basophils displayed morphologic evidence of material and membrane conservation, granule content condensation, and biosynthesis. Subcellular histamine-rich sites in actively recovering basophils included condensing granules and collections of cytoplasmic vesicles in three locations: beneath the plasma membrane, adjacent to granules, and in the Golgi region. These studies show that unaltered granules of actively releasing human basophils, as well as similar granules that are reconstituted after FMLP-stimulated degranulation, contain histamine, but that altered granules in stimulated cells undergoing degranulation are devoid of histamine. Reconstitution of histamine-rich granules is associated with DAO-gold-positive cytoplasmic vesicles, suggesting transport of histamine derived from either new synthesis, re-uptake of released histamine, or both, to reconstituted granules.     

Primary thrombocythemia: clonal origin of platelets, erythrocytes, and granulocytes in a GdB/GdMediterranean subject

A patient with primary thrombocythemia, who was heterozygous for glucose-6-phosphate dehydrogenase deficiency (GdB/GdMed), was investigated to test for the clonal origin of this myeloproliferative disorder. In order to assess somatic cell mosaicism in various tissues, we have made use of the different rate of utilization of 2-deoxyglucose- 6-phosphate, an analog of glucose-6-phosphate, by normal glucose-6- phosphate dehydrogenase and by the Mediterranean variant: the results demonstrate that essential thrombocythemia is a clonal disease involving the erythrocytic, granulocytic, and megakaryocytic series, without affecting monocytes, T lymphocytes, and non-T lymphocytes.     

Sesame oil in injectable gold: two drugs in one?

To investigate the potential anti-inflammatory effects of sesame oil, which is present in the injectable gold preparation Auromyose, the synthesis of tumour necrosis factor alpha (TNF-alpha), prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) by in vitro stimulated blood cells was measured before, during and after 12 weeks of dietary supplementation with 18 g of sesame oil daily in 11 healthy male volunteers. Neither TNF-alpha, PGE2 nor LTB4 production levels showed statistically significant changes during the 12 weeks of dietary supplementation with sesame oil. These results do not suggest an anti-inflammatory effect of sesame oil as present in injectable gold preparations which are used in the treatment of rheumatoid arthritis.      

Rearrangement of immunoglobulin heavy chain genes in T cell acute lymphoblastic leukemia

We studied the arrangement of the immunoglobulin heavy chain genes by Southern blot analysis of DNA freshly obtained from marrow blast cells of 14 children with T cell acute lymphoblastic leukemia (T-ALL) using probes to the C mu and JH gene segments: At least one of the C mu-gene alleles was rearranged in three cases. In two of these, one C mu gene had the germ-line configuration and one was rearranged, whereas both alleles were rearranged in the third case. In one case, a rearranged heavy chain gene hybridized to the C mu-region probe, but not to the JH probe, indicating that the entire JH region had been deleted. These results demonstrate that immunoglobulin heavy chain gene rearrangements are not restricted to B lineage lymphoproliferative diseases in humans.     

Arachidonic acid incorporation by cytoplasmic lipid bodies of human eosinophils

The presence of cytoplasmic lipid bodies in human eosinophils and the participation of these lipid bodies in the metabolism of arachidonic acid by human eosinophils have been studied. Lipid bodies, structures of roughly spherical shape and variable size within the cytoplasm, were identified with transmission electron microscopy by their shape and variable osmiophilia and by their lack of a limiting membrane. While generally absent from eosinophils of normal peripheral blood, lipid bodies were found in tissue eosinophils and in blood eosinophils from patients with eosinophilia. A role for lipid bodies in arachidonic acid metabolism was determined with eosinophils obtained from two eosinophilic patients. After incubation for 30 to 60 minutes with 3H- arachidonic acid, purified eosinophils took up 50% to 98% of the tritium label. By electron microscopic autoradiography, almost all tritium label was localized to lipid bodies. Only 3.6% of the cell- incorporated tritium label was free arachidonic acid, while 5.8% was neutral lipids and 66% was phospholipid. Thus, most of the tritiated arachidonic acid incorporated by human eosinophils was present in esterified form, predominantly as phospholipids, and almost all of the tritiated lipids were localized ultrastructurally to cytoplasmic lipid bodies. These results provide evidence that lipid bodies of human eosinophils have a role in the cellular metabolism of arachidonic acid.     

The origin of IgG production and homogeneous IgG components after allogeneic bone marrow transplantation

Pediatric recipients (n = 25) of an allogeneic bone marrow (BM) graft were selected on the basis of informative IgG allotype (Gm) differences between the BM donor and the recipient. To investigate the kinetics of the appearance of IgG of donor origin and the disappearance of IgG of recipient origin, G1m and G2m allotype levels were quantified in sera obtained at regular intervals between 3 months and 5 years after BM transplantation (BMT). For this quantification, a dot immunobinding assay (DIBA) has been developed. In 19 of 22 informative recipients, the Gm allotype distribution had reached the range of values expected on the basis of the Gm phenotype of the donor within 6 months after BMT. Remarkably, IgG of recipient origin persisted in 15 of 18 informative recipients until last follow up, ie, for several years after BMT. In addition to the origin of total IgG production, the origin of homogeneous IgG components (H-IgG) appearing after BMT was investigated. H-IgG of donor origin could be detected as early as 3 weeks after BMT, but also H-IgG of recipient origin were present in 8 of 13 informative recipients for a period of up to 1 year after BMT. We conclude that host-type IgG-producing cells were not eradicated by the (myeloablative) conditioning regimen and persisted in a high number of graft recipients. It is our hypothesis that lack of graft-versus-host disease (GVHD) in the majority of these recipients results in the persistence of IgG-producing cells of host origin. These observations may be relevant for the evaluation of patients who received allogeneic BMT for the treatment of multiple myeloma.     

Macrophage colony-stimulating factor induces substantial osteoclast generation and bone resorption in human bone marrow cultures

Macrophage colony-stimulating factor (M-CSF) is essential for murine osteoclast formation and its role in human hematopoiesis in vitro is not fully defined. Therefore, we have investigated the effect of M-CSF on the formation of human osteoclasts in vitro. M-CSF was found to induce substantial bone resorption and osteoclast formation in a dose- responsive and time-dependent manner above that induced by 1,25 dihydroxyvitamin D3 (1,25 vitamin D3) in cultures of human bone marrow (BM) stromal cells sedimented onto devitalized bone. By day 14 there was a mean of approximately 50% of the surfaces of the bone slices resorbed compared with only 6% in cultures treated with 1,25 vitamin D3 alone. Osteoclasts were identified as 23c6+ cells (an antibody that recognizes the vitronectin receptor), 87.5% of which coexpressed the calcitonin receptor. The number of 23c6+ cells correlated strongly with bone resorption spatially, and in a dose-responsive and time-dependent manner; the correlation coefficient in cultures treated with 1,25 vitamin D3 alone was 0.856 and those treated with both M-CSF and 1,25 vitamin D3 was 0.880. Granulocyte-macrophage colony-stimulating factor, IL-1 beta, IL-3, IL-6, tumor necrosis factor-alpha, transforming growth factor-beta, leukemia inhibitory factor, and IL-11 did not increase bone resorption above that in 1,25 vitamin D3-treated cultures. We also found that 1,25 vitamin D3 increased, to a minor but significant degree, both bone resorption and the concentration of M-CSF in the culture supernatants above that in vehicle-treated cultures, indicating that M-CSF is present in our BM cultures, but that there is insufficient to induce substantial osteoclast formation. These results define a critical role for M-CSF in the formation of human osteoclasts.     

Inhibition of CFU-NM and CFU-EOS by mature granulocytes

Approximately half of the colony-forming units-culture (CFU-C) from normal peripheral blood are eosinophilic. The purpose of our study was to determine: (1) whether progenitor cells committed to eosinophil or neutrophil maturation would be differentially affected by feedback inhibition, and (2) whether mature eosinophils added to the feeder layers of the culture would inhibit the proliferation of CFU-C in a manner similar to that described for neutrophils. Concentrated eosinophils and neutrophils, obtained by separation on a metrizamide gradient, were added to feeder layers containing either 10(6) autologous whole mononuclear cells (WMNC) or 0.1 ml of leukocyte conditioned media (LCM). The average number of colonies was 123/10(6) nonadherent cells (NAC) cultured. When neutrophils or eosinophils were added to the WMNC feeder layer, the percent inhibition of growth was 40.2% +/- 1.6% (mean +/- SEM) and 42.3% +/- 5.4%, respectively, but the ratio of neutrophil to eosinophil colonies remained constant. No effect was seen when neutrophils or eosinophils were added to an LCM feeder layer. Thus, it appears that the differential control of neutrophil versus eosinophil production in vitro is not regulated through feedback inhibition by mature granulocytes. In addition, these studies suggest that eosinophils, as well as neutrophils, cause inhibition of CFU-C growth when intact cells are the source of colony-stimulating factor (CSF).