Application of sedimentation at 1 g on a Ficoll gradient to the separation of bone marrow cells

Sedimentation at unit gravity of human bone marrow, during 15 hours at 4 degrees C on a linear density gradient of Ficoll in culture medium ranging from 1.020 to 1.065 g/ml shows that it exists a differential migration of bone marrow cells subpopulation with a precise mean densities : we find successively : 1.021 +/- 1.10(-3) g/ml for the lymphocytes, 1.024 +/- 2.5.10(-3) g/ml for the non eosinophil granulocytes, 1.025 +/- 2.5.10(-3) g/ml for the metamyelocytes, 1.030 +/- 3.5.10(-3) g/ml for the immature myeloid cells (myeloblasts, promyelocytes, myelocytes), 1.040 +/- 3.10(-3) g/ml for the eosinophil granulocytes, 1.055 +/- 10.10(-3) g/l for the megakaryocytes. The highest percentages of S phase cells, G2 and M phase cells determinated with a cytofluorograph correspond to peaks of immature myeloid cells (myeloblasts, promyelocytes and myelocytes). This method of bone marrow cells separation may be used to study the cell cycle in pathological bone marrows (leukaemia in particular) and to determine the effects and the efficiency of some antimitotics.     

Leucokinetic study. Morphology of the bone marrow and blood after experimental induction of marrow hypoplasia by cyclophosphamide in laboratory rats

Experimental hypoplasia of femoral bone marrow in rats was induced by cyclophosphamide, injected i.p. at various clock hours (08.00, 12.00, 16.00, 20.00, 24.00, and 04.00). Cyclophosphamide-induced neutropenia persisted for six days and was followed by transitory granulocytosis with subsequent decrease in the cont of circulating mature granulocytes. The absolute counts of circulating segmented neutrophils changed in parallel with the absolute counts of segmented neutrophils in the bone marrow. The count of circulating neutrophils was not essentially influenced by the clock hour of cyclophosphamide injection. The toxic action of cyclophosphamide upon the bone marrow exhibited a circadian rhythmicity: the greatest decrease in the count of nucleated marrow cells was found in the morning, and the least, in the evening. The minimal compartment transit times of the development stages of bone-marrow neutrophils, and the daily granulocyte production in the rat were calculated.     

Inhibition of normal granulopoiesis by cytostatic agents.

Rabbits were treated with cyclophosphamide and 5-fluorouracil, myelosuppressive cytostatic agents, applied with a single dose of 1/3 LD50 or daily doses of 1/30 LD50 given for 14 days. Functional tests for evaluation of granulopoiesis were regularly performed at standard intervals and were following: leukocytosis, bone marrow picture with mitotic index, 3H-thymidine incorporation in vitro followed by autoradiography of labeled promyelocytes and myelocytes, serum lysozyme activity, mobilization of granulocyte reserve pool by staphylococcal alpha-toxin, cytochemistry of granulocytes, phagocytosis ability and Nitro-BT reduction. It has been found that 6-10 days after application of cytostatics, a marked depression of proliferation of young granulocyte forms and lowered reserve pool, are regularly observed. This was followed by spontaneous regeneration of granulopoiesis. No changes were noted in functional tests of mature granulocytes in peripheral blood. It is suggested that for investigation of the impairment of granulopoiesis after application of cytostatic agents, most suitable is evaluation of mobilization of the bone marrow reserve pool, lysozyme activity in blood serum and labelling of promyelocytes and myelocytes with 3H-thymidine in vitro.     

Cytophotometry of granulocytes in chronic granulocytic leukemia patients. Part I. Cell cycle distribution, S-phase transition and quantitative cytochemistry

In the chronic phase of CGL the proportion of granulocytes in S + G2 was lower (18.7 +/- 1.3% in marrow and 16.7 +/- 2.4% in blood) than in normal bone marrow (42.4 +/- 2.9%) as studied by Feulgen-DNA cytophotometry. During the blast crisis the percentage of S + G2 blasts was 39.3 +/- 8.4 in marrow and 38.7 +/- 7.8 in blood which was much higher than in acute myeloblastic leukemia patients (10.8 +/- 1.4 and 5.1 +/- 1.0). Thymidine labelling index values were lower than the percentage of cytophotometrically detected S-phase cells: up to 28% of cells with Feulgen-DNA content corresponding to S-phase did not incorporate 3H-thymidine. The rate of DNA synthesis remained constant during the S-phase but 3H-thymidine uptake increases towards the end of the S-phase. Morphometric parameters and quantitative cytochemical (PAS, Sudan, myeloperoxidase activity) characteristics of polymorphonuclear neutrophils were altered during the chronic phase of the disease but remained in the normal range during the blast crisis. Mature neutrophils in the blast crisis are assumed to originate from normal granulocyte progenitors.     

Suppression and regeneration of rat bone marrow under the influence of methotrexate. An EM study.

Adult male rats were given daily injections of methotrexate for 12 days, during which time specimens of femoral bone marrow were taken for correlative light- and electron-microscopic study. After 12 days methotrexate was discontinued and specimens were taken the marrow recovered from the effect of methotrexate. Normal rat bone marrow was used as a basis of comparison. As methotrexate was administered, cell division ceased and the immature erythroblasts and myelocytes proceeded in the maturational process and a rapid hypocellularity of the marrow developed. Eosinophilic leukocytes, lymphocytes, large fat cells, erythrocytes, and phagocytic reticuloendothelial cells were the only formed elements remaining after 12 days of methotrexate treatment. Two days after methotrexate was discontinued, myeloblasts, myelocytes and proerythroblasts were plentiful, and on the third day the marrow exhibited an erythroid hyperplasia. By the eighth day all cell types were present though the marrow still was hypocellular. 14 days post methotrexate the marrow returned to normal.     

The kinetics of granulopoiesis in long-term mouse bone marrow culture. Part I

The spontaneous stratification in long-term bone marrow cultures was illustrated and quantified. The cultures were separated into three hematopoietic layers: nonadherent cells in the supernatant medium, lightly adherent cells on top of the stromal layer, and remaining cells buried within the stromal layer. The cells of each layer were subcultured for 10 days in plastic tubes that inhibit the formation of a stromal layer. Daily samplings with absolute and differential cell counts were obtained. We identified three families of cell disappearance curves and cell types: CFU-s, hemocytoblasts, myeloblasts, and promyelocytes (G1, 2); myelocytes (G3); and postmitotic granulocytes (G4). Also, the numbers of mitotic and necrotic cells were determined. The longest half-time of CFU-s was 2.5 days. Lacking stromal support, CFU-s disappeared faster than other differentiated cells. Generally, these cells maintained their numbers for the first week of subcultures, which was attributable to a temporarily maintained balance of cell death and fresh cell production. After more than 7 days, there was a rapid decline of all differentiated cell types.     

Estimation of kinetic parameters of neutrophilic, eosinophilic, and basophilic granulocytes in human blood.

Two hematologically normal patients with glioblastoma and six patients with chronic lymphocytic leukemia received continuous 3H-thymidine infusions for 3--10 days. In autoradiographs of blood cell smears taken for 25 days or more after the beginning of 3H-thymidine administration the labeling index and the labeling intensity of granulocytes were determined. A sufficiently high labeling intensity, i.e. a sufficiently long autoradiographic exposure time was found to be critical for obtaining valid and reproducible results. On the basis of certain assumptions discussed in detail, complete labeling of cells with 3H-thymidine followed by autoradiographic evaluation and mathematical analysis of the labeling patterns seems to be a suitable method for estimation of kinetic parameters of postmitotic granulocytes in vivo. The mean intramedullary maturation and storage time was observed to be 115 +/- 7 h or neutrophils, 103 +/- 4 h for eosinophils and 103 +/- 11 h for basophils. The mean relative inflow rate into the blood (or relative turnover rate in the blood) was found to be 4.2 +/- 0.4/h for neutrophils, 4.0 +/- 0.4%/h for eosinophils and 1.2 +/- 0.3%/h for basophils. The mean blood transit time (or blood sojourn time) was estimated to be 25 +/- 2 h or neutrophils, 26 +/- 3 h for eosinophils and 89 +/- 21 h for basophils. Accordingly the half lifes (T 1/2) of granulocytes in the blood were 17.3 +/- 1.4 h for neutrophils, 18.0 +/- 2.1 for eosinophils and 62 +/- 15 h for basophils. Under the quasi steady state conditions of this study the kinetics of granulocytes in the present CLL patients appeared to be normal, despite a marked lymphocytic infiltration of the bone marrow. The apparent discrepancy between these findings and the data obtained with autotransfusion of DFP-labeled granulocytes is discussed.     

Analysis of human bone marrow with monoclonal antibodies

In order to study the antigenic phenotype of different hemopoietic cells, we used a series of monoclonal antibodies to investigate normal bone marrow in a standard immunofluorescence assay. The antibodies detected the following antigens: HLA-ABC, beta 2-microglobulin (beta 2m), HLA-DR (Ia), a lymphocyte subset and specific antigen (T and B) HuLy-m2, m3, T lymphocyte antigen (HuLy-m1), lymphocyte T200 antigen (HuLy-m4), a viral-associated antigen (HuLy-m5), and platelet-specific glycoproteins IIb-IIIa (HuPl-m1). The following results were obtained: (a) normoblasts were weakly HLA-ABC+, beta 2m+ and Ia-; all other lymphocyte and platelet antigens were not detected. (b) Myeloid cells at all stages of differentiation (promyelocytes, myelocytes, metamyelocytes, and neutrophils) were HLA-ABC+; beta 2m+; HuLy-m1-, m2-, m3+/- (20%), m4+, m5+/- (20%); HuPl-m1-; in addition, promyelocytes and myelocytes were Ia+ but neutrophils and metamyelocytes were Ia-. (c) Lymphocytes were HLA-ABC+, beta 2m+, Ia+/- (20-30%), HuLy-m1+/- (40-50%), m2+/- (60-70%), m3+, m4+, m5+; Pl-m1-. (d) Platelets and megakaryocytes were HLA-ABC+; beta 2m+; Ia-; HuLy-m1+-, m2-, m3-, m4-, m5-, HuPl-m1+, and the putative "megakaryocyte precursors" were HuPl-m1+, Ia-, HuLy-m1-. The different cell types in bone marrow could readily be distinguished, particularly cells of the myeloid series (Ia and HuLy-m4, m5), lymphocytes (Ia and HuLy-m1, m2, m3), and platelets and their precursor cells (HuPl-m1). This simple method of defining cellular phenotypes in bone marrow has demonstrated the practicality of using monoclonal antibodies to identify marrow cells and should be of diagnostic value.     

Effect of bone marrow suppression on granulocyte opsonin levels.

Levels of serum opsonin for neutrophilic granulocytes were measured in dogs made neutropenic by cyclophosphamide administration. Heat-labile opsonin became elevated within 24 hr following cyclophosphamide (P less than 0.005) and remained elevated over the 4-5 day period of observation (P less than 0.005). In contrast, heat stable opsonin was not significantly effected. Bone marrow suppression by X-ray and busulfan also caused serum opsonin levels to increase. Changes in the levels of IgG, C3, and total hemolytic complement during the course of bone marrow suppression did not correlate with the granulocyte opsonin levels. These findings suggest that serum granulocyte opsonin levels respond to bone marrow suppression and may provide an improved environment for the function of transfused granulocytes.     

Growth fraction of myelocytes in normal human granulopoiesis

The labelling index (LI) of myelocytes (M) after flash labelling of normal human bone marrow cells with [3H]-thymidine ([3H]TdR) is always lower that the LI obtained for myeloblasts (MB) and for promyelocytes (PM). This fact can be interpreted in two ways: it may mean that the duration of the G1 phase of the cell cycle is longer in M than in MB or PM, or it may mean that the proportion of cells in cycle, i.e., the growth fraction (GF), is lower in the M population than in MB or PM. The evolution of the LI and of the mean number of grains per cell was monitored in [3H]TdR-labelled normal bone marrow during in vitro incubation for 50 hr. The generation time, measured by the halving time of the mean number of grains per cell after flash labelling, was similar for M to that for MB and PM. During continuous labelling, the LI of MB and PM reached 1 and the LI value for M never rose to more than 50% of the values recorded for MB and PM after 30 hr. These findings give support to the second hypothesis, i.e., a lower GF in the M population. Good correlation was found between the LI of M and the proportion of mature polymorphonuclear cells in the bone marrow of normal subjects and of patients with chronic benign neutropenia or hyperleucocytosis. Variations in the M growth fraction could be a medium-term (2-3 days) regulatory factor in granulocyte production.     

Micronuclei in peripheral blood and bone marrow cells of mice exposed to 42 GHz electromagnetic millimeter waves

The genotoxic potential of 42.2 +/- 0.2 GHz electromagnetic millimeter-wave radiation was investigated in adult male BALB/c mice. The radiation was applied to the nasal region of the mice for 30 min/day for 3 consecutive days. The incident power density used was 31.5 +/- 5.0 mW/cm2. The peak specific absorption rate was calculated as 622 +/- 100 W/kg. Groups of mice that were injected with cyclophosphamide (15 mg/kg body weight), a drug used in the treatment of human malignancies, were also included to determine if millimeter-wave radiation exposure had any influence on drug-induced genotoxicity. Concurrent sham-exposed and untreated mice were used as controls. The extent of genotoxicity was assessed from the incidence of micronuclei in polychromatic erythrocytes of peripheral blood and bone marrow cells collected 24 h after treatment. The results indicated that the incidence of micronuclei in 2000 polychromatic erythrocytes was not significantly different among untreated, millimeter wave-exposed, and sham-exposed mice. The group mean incidences were 6.0 +/- 1.6, 5.1 +/- 1.5 and 5.1 +/- 1.3 in peripheral blood and 9.1 +/- 1.1, 9.3 +/- 1.6 and 9.1 +/- 1.6 in bone marrow cells, respectively. Mice that were injected with cyclophosphamide exhibited significantly increased numbers of micronuclei, 14.6 +/- 2.7 in peripheral blood and 21.3 +/- 3.9 in bone marrow cells (P< 0.0001). The drug-induced micronuclei were not significantly different in millimeter wave-exposed and sham-exposed mice; the mean incidences were 14.3 +/- 2.8 and 15.4 +/- 3.0 in peripheral blood and 23.5 +/- 2.3 and 22.1 +/- 2.5 in bone marrow cells, respectively. Thus there was no evidence for the induction of genotoxicity in the peripheral blood and bone marrow cells of mice exposed to electromagnetic millimeter-wave radiation. Also, millimeter-wave radiation exposure did not influence cyclophosphamide-induced micronuclei in either type of cells.     

Protection against gram-negative bacteremia in neutropenic mice with recombinant granulocyte-macrophage colony-stimulating factor

Granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulates production of neutrophils in bone marrow and may decrease the incidence of infection during neutropenia. We evaluated the protective role of recombinant GM-CSF against Pseudomonas aeruginosa challenge in neutropenic mice. CD-1 mice treated with cyclophosphamide on days 1 and 2 of the experiment were given GM-CSF (1, 2, or 4 micrograms/day) starting at day 4 of the experiment according to the following protocol: 1) 1 microgram of GM-CSF 2 hr and 24 hr after challenge; 2) 1 microgram 24 hr before challenge, 2 hr and 24 hr after challenge; 3) 2 micrograms injected 24 hr before and 2 hr after challenge; 4) 2 micrograms given 24 hr before and 2 micrograms given 2 hr and 24 hr after challenge; 5) 4 micrograms administered 2 hr and 24 hr after challenge; and 6) saline and bovine albumin controls. The number of blood neutrophils by days 4 and 5 was similar for GM-CSF-treated and untreated animals. Survival was significantly greater in animals given 2 micrograms of GM-CSF at 24 hr before and at 2 hr and 24 hr after challenge with Pseudomonas. Neutrophils and splenic macrophages obtained from GM-CSF-treated mice (2 micrograms/animal) produced significantly greater amounts of O2- (204 +/- 36 nmoles/10(5) cells) than controls (21 +/- 10 nmoles/10(5) cells). Additionally, neutrophils and macrophages from GM-CSF-treated mice killed significantly more bacteria (P. aeruginosa) in vitro and had a greater number of C3b and Fc receptors (78 +/- 12% and 89 +/- 8%) than did cells obtained from control animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Heterophilic granulocytes of rats during irradiation]

The bone marrow eosinophils of rats subjected to irradiation are capable of secreting a peroxidase system obtained by neutrophils and endotheliocytes. As a result, there appear heterophil granulocytes in the peripheral blood.     

Immunocytochemical localization of lactoferrin in human neutrophils

Summary The subcellular localization of lactoferrin in human neutrophils was studied by an electron-microscopic immunoperoxidase method. This molecule was detected in small granules of blood polymorphonuclear leukocytes. A morphometrical analysis showed that there was no significant difference in the mean size between lactoferrin-positive and myeloperoxidase-negative granules. In contrast, the mean size of myeloperoxidase-positive granules was significantly larger than that of lactoferrin-positive granules. This indicates that lactoferrin is contained in the myeloperoxidase-negative, secondary, granules of human neutrophils. In immature bone marrow mononuclear neutrophils, lactoferrin was present in cytoplasmic granules of somewhat larger size than lactoferrin-positive granules of polymorphonuclear leucocytes. A morphometrical study showed that the mean size of lactoferrin-positive granules was significantly greater in immature bone marrow cells than in polymorphonuclear leucocytes. This indicates that lactoferrin-positive granules decrease in size as the cells mature. Besides cytoplasmic granules, lactoferrin was demonstrated in the Golgi complex and a part of the rough endoplasmic reticulum of immature bone marrow neutrophils, probably myelocytes and early metamyelocytes. These results show that lactoferrin is synthesized and packed into secondary granules in immature bone marrow neutrophils and therefore that the secondary granules are a type of secretory granule.     

A time-course study of chronic paternal cyclophosphamide treatment in rats: effects on pregnancy outcome and the male reproductive and hematologic systems

We have found previously that daily treatment of male rats for 11 wk with low doses of the anticancer drug cyclophosphamide had no apparent effect on male reproductive organ weights, epididymal sperm counts, or serum hormones at the end of the treatment period; yet, upon breeding to untreated females, these males produced a high rate of post-implantation loss and fetal anomalies. The present study was designed to investigate the time course and dose response of the effects of chronic cyclophosphamide treatment on the male reproductive and hematologic systems. Male Sprague-Dawley rats were gavage-fed for 1, 3, 6 and 9 wk with saline (control), or 5.1 (low dose) or 6.8 (high dose) mg/kg/day of cyclophosphamide. After each of the treatment periods, males were mated to determine the effect on pregnancy outcome, then killed, and the effects on the male reproductive and hematologic systems were assessed. After 6 wk of treatment, a sharp increase in mortality was found between the 5.1 and 6.8 mg/kg/day doses of cyclophosphamide. The high dose of cyclophosphamide induced higher levels of pre- and post-implantation loss but fewer fetal anomalies than did the low dose. The low dose of cyclophosphamide did not affect reproductive organ weights; in contrast, the high dose caused decreases in epididymal, ventral prostate, and seminal vesicle weights after 3, 6, and 9 wk. Testicular and epididymal sperm counts were decreased in a dose-dependent manner after 3 wk; in addition, the high dose led to a decrease in epididymal sperm counts after 6 wk of treatment. Another rapidly proliferative tissue, the bone marrow, was dramatically affected by both doses of cyclophosphamide at all time points, with leukocyte counts decreasing to 40% of control by 1 wk. After 9 wk of treatment, effects on the male reproductive system were less marked, compared to earlier time points, whereas those on the hematologic system and pregnancy outcome persisted. Thus chronic low-dose treatment of male rats with cyclophosphamide not only had early and striking effects on the bone marrow and the pregnancy outcome but also affected the male reproductive system in a clear time- and dose-dependent manner.     

Administration of IL-7 to normal mice stimulates B-lymphopoiesis and peripheral lymphadenopathy.

Normal mice were injected with IL-7 (500 ng, twice daily) for various periods of time up to 6 days and the cellularity and phenotypic composition of the thymus, spleen, lymph node, and bone marrow was assessed. After 6 days of treatment, significant increases in the cellularity of the spleen, lymph node, and bone marrow were observed which returned to the normal range within 6 days after cessation of treatment. After 3 days of IL-7 treatment, increased numbers of B220+/surface(s) IgM- bone marrow cells were observed. After 6 days of treatment, these numbers were still further increased and a significant population of B220+/sIgM- cells were observed in the spleen. The numbers of c mu+/sIgM- cells were also increased in the IL-7-treated mice. Analysis of the expression of B220 and BP-1 on the sIgM- bone marrow cells revealed that the B220+/BP-1+ population was dramatically increased after IL-7 treatment and the size of the B220+/BP-1- population did not differ from control mice. The pre-B cell numbers declined rapidly after the cessation of IL-7 treatment. After 6 days of IL-7 treatment, a twofold increase in the number of B cells in the spleen and lymph node was observed. The B cell numbers declined to normal values within 6 days after the cessation of IL-7 administration. In the spleens of the IL-7-treated mice, there was a significant increase in the number of B cells with an immature phenotype (e.g., sIgMhi/sIgDlo, decreased levels of Ia and FcR expression). The numbers of CD8+ and CD4+ T cells were also increased in the lymph node and spleen of the IL-7-treated mice. These numbers declined to normal levels after the cessation of IL-7 treatment.     

Effect of combined therapy with hyperbaric oxygen and antioxidant on infarct volume after permanent focal cerebral ischemia

The aim of the present study was to evaluate the efficiency of combination of hyperbaric oxygen (HBO) and an antioxidant on permanent focal cerebral ischemia. Male Wistar rats underwent permanent middle cerebral artery occlusion (MCAO). Then, animals were randomly assigned to one of four groups: the control group (n=9) received no treatment, HBO group (n=9) was treated for 90 min at 2.5 absolute atmosphere for 3 days, the U-74389G group (n=8) received single U-74389G injection (3 mg/kg), the HBO + U-74389G group (n=8) received both HBO and U-74389G treatments. Treatments were initiated within the first 10 min after MCAO. After 3 days, the infarct volumes in rat brains were measured. The infarct ratios were 25.6+/-6.5 % for the control group, 21.9+/-6.4 % for the HBO group, 15.7+/-5.7 % for U-74389G group and 12.5+/-3.8 % for HBO + U74389G group. The infarct volumes were significantly reduced in rats treated with U-74389G (p<0.05) and combination therapy (p<0.05). HBO failed to reduce infarct volume significantly. We concluded that 1) U-74389G is more beneficial than HBO on permanent MCAO in rats, and 2) a combined therapy failed to significantly improve infarct volume more than either single treatment.     

gamma-Glutamyltranspeptidase activity in intact leukocytes: flow cytometric analysis and sorting

A fluorescent method developed for visualizing gamma-glutamyltranspeptidase (GGT) in intact liver cells was adapted to leukocytes and used in a multiparameter flow cytometric study of blood and bone marrow cells from rats with subcutaneous implants of mammary carcinoma 5A. The severe granulocytosis caused by this non-metastatic tumor was preceded by a progressive rise in the percentage of leukocytes with high GGT fluorescence. Both granulocytes and small, immature cells of bone marrow showed increased GGT expression, whereas in blood this increase was attributable entirely to mature granulocytes. At 28 days (but not yet at 14 days) after carcinoma implantation, 20-30% of blood or bone marrow granulocytes constituted a distinct subpopulation in that their GGT fluorescence intensity range was much higher and did not overlap with the range for the rest of the population. The results indicate that fluorescent GGT assay of intact leukocytes provides a useful probe for flow cytometric analysis of population heterogeneity in leukoproliferative disorders.