Blood Granulocyte Counts Research Articles

Defining the optimal spacing between repeat radioantibody doses in experimental models

Single doses of radioantibody are effective at treating single cells or small clusters of cancer cells. However, large tumor masses require either multiple doses of radioantibody or a multimodal approach to therapy using two or more therapeutic agents. Timing of the second dose in a multiple cycle scheme or the second treatment in a multimodal protocol will depend on recovery from toxicity associated with the first treatment.BALB/c mice were dosed with a maximal tolerated dose (MTD) of I-131-MN-14 anti-carcinoembryonic antigen immunoglobulin G (IgG) (250 microCi) or F(ab')2 (1.2 mCi). Mice were redosed with the MTD at one of four time points, either Day 28, 35, 42, or 49 after IgG or Day 14, 21, 28, or 35 after F(ab')2. Survival was monitored to determine the earliest time to redose without lethality. Several studies were then performed to identify an accurate measure of true myelorecovery. Mice were bled retroorbitally on the day of the first dose and at weekly intervals thereafter. Total peripheral white blood cell counts, granulocyte counts, and lymphocyte counts were determined for each animal. GR-1hi expression (percentage of positive cells) and mean channel florescence were determined by FACScan analysis of a blood sample incubated with fluorescein isothiocyanate-anti-mouse Ly-6G (GR-1). In other studies, two mice were killed weekly from a group treated with a single MTD of radioantibody. The weights of their spleens and thymus glands were determined. At that time, femoral marrow was collected from these animals and plated in Methocult M3430 methylcellulose medium (Stemcell Technologies, Vancouver, Canada), and total colony-forming cells in culture were determined. Another population of mice was used to assess normal tissue metabolic activity following radioantibody therapy by quantitating the 4-hour utilization of I-125-dUrd.The ability to redose mice with a second MTD of 1-131-IgG or F(ab')2 required 49 days and 35 days, respectively. Granulocyte and lymphocyte counts did not accurately predict myelorecovery from the first dose. Hematopoietic tissue weight, tissue metabolic activity, and marrow colony forming cells all suggested that redosing was possible 1-2 weeks before it could actually be done without lethality. Percent of cells expressing GR-1hi (>60%) and absolute numbers of GR-1hi cells (>1400 cells/mm3) suggested myelorecovery in most animals. A greater degree of accuracy was achieved when trends in GR-1hi expression were noted over 2 or more weeks (i.e., the absolute amount of GR-1hi had to exceed levels in untreated mice, as evidence that the hyperproliferative phase of recovery was complete).The only approach that accurately predicted the ability to retreat with myelosuppressive therapy without risk of lethality was an increase in GR-1hi-positive cells above untreated levels. Other approaches are currently being investigated, including the expression of proliferation antigens (e.g., proliferating cell nuclear antigen and Ki-67) in both murine and human samples and differentiation antigens (CD33 and CD34) in humans.

Defining the optimal spacing between repeat radioantibody doses in experimental models: is there an accurate measurement for hematopoietic recovery?

Single doses of radioantibody are effective at treating single cells or small clusters of cancer cells. However, large tumor masses require either multiple doses of radioantibody or a multimodal approach to therapy using two or more therapeutic agents. Timing of the second dose in a multiple cycle scheme or the second treatment in a multimodal protocol will depend on recovery from toxicity associated with the first treatment. BALB/c mice were dosed with a maximal tolerated dose (MTD) of I-131-MN-14 anti-carcinoembryonic antigen immunoglobulin G (IgG) (250 microCi) or F(ab')2 (1.2 mCi). Mice were redosed with the MTD at one of four time points, either Day 28, 35, 42, or 49 after IgG or Day 14, 21, 28, or 35 after F(ab')2. Survival was monitored to determine the earliest time to redose without lethality. Several studies were then performed to identify an accurate measure of true myelorecovery. Mice were bled retroorbitally on the day of the first dose and at weekly intervals thereafter. Total peripheral white blood cell counts, granulocyte counts, and lymphocyte counts were determined for each animal. GR-1hi expression (percentage of positive cells) and mean channel florescence were determined by FACScan analysis of a blood sample incubated with fluorescein isothiocyanate-anti-mouse Ly-6G (GR-1). In other studies, two mice were killed weekly from a group treated with a single MTD of radioantibody. The weights of their spleens and thymus glands were determined. At that time, femoral marrow was collected from these animals and plated in Methocult M3430 methylcellulose medium (Stemcell Technologies, Vancouver, Canada), and total colony-forming cells in culture were determined. Another population of mice was used to assess normal tissue metabolic activity following radioantibody therapy by quantitating the 4-hour utilization of I-125-dUrd. The ability to redose mice with a second MTD of 1-131-IgG or F(ab')2 required 49 days and 35 days, respectively. Granulocyte and lymphocyte counts did not accurately predict myelorecovery from the first dose. Hematopoietic tissue weight, tissue metabolic activity, and marrow colony forming cells all suggested that redosing was possible 1-2 weeks before it could actually be done without lethality. Percent of cells expressing GR-1hi (>60%) and absolute numbers of GR-1hi cells (>1400 cells/mm3) suggested myelorecovery in most animals. A greater degree of accuracy was achieved when trends in GR-1hi expression were noted over 2 or more weeks (i.e., the absolute amount of GR-1hi had to exceed levels in untreated mice, as evidence that the hyperproliferative phase of recovery was complete). The only approach that accurately predicted the ability to retreat with myelosuppressive therapy without risk of lethality was an increase in GR-1hi-positive cells above untreated levels. Other approaches are currently being investigated, including the expression of proliferation antigens (e.g., proliferating cell nuclear antigen and Ki-67) in both murine and human samples and differentiation antigens (CD33 and CD34) in humans.

Erythropoietin, granulocyte-colony stimulating factor, interleukin-1β and interleukin-6 during the normal menstrual cycle

Objective: Menstruation and ovulation are the main events during the menstrual cycle. Menstruation may influence the complete blood count because of its blood loss. Since ovulation is an inflammatory-like phenomenon, white blood cell count may change during ovulation. For these reasons, we have investigated the complete blood count and peripheral serum concentrations of associated cytokines during the normal menstrual cycle. Materials and methods: Normal healthy female volunteers ( N = 9; age, 21–33 years) with menstrual cycles of 25–33 days recorded basal body temperature every day and venous blood samples were collected three times per week. Complete blood count was performed and erythropoietin, granulocyte-colony stimulating factor, interleukin-1β, interleukin-6 and hormones (FSH, LH, estradiol and progesterone) were measured. Results: The red blood cell, total white blood cell, granulocyte and platelet counts showed no statistically significant changes during the menstrual cycle when using analysis of variance with the Scheffe F-test. No changes in erythropoietin, interleukin-1β and interleukin-6 were seen throughout the menstrual cycle. In contrast, serum granulocyte-colony stimulating factor concentrations increased significantly to 28.0 ± 3.1 pg/ml (mean ± S.E.M.) during the ovulatory phase, compared to 10.7 ± 0.6 pg/ml in other phases. Conclusion: These results indicate that menstrual blood loss does not affect the complete blood count and suggest that granulocyte-colony stimulating factor plays an important role in the mechanism of ovulation.

Transgenic expression of PML/RARalpha impairs myelopoiesis.

The translocation found in acute promyelocytic leukemia rearranges the promyelocytic leukemia gene (PML) on chromosome 15 with the retinoic acid receptor alpha (RARalpha) on chromosome 17. This yields a fusion transcript, PML/RARalpha, a transcription factor with reported dominant negative functions in the absence of hormone. Clinical remissions induced with all-trans retinoic acid (RA) treatment in acute promyelocytic leukemia are linked to PML/RARalpha expression in leukemic cells. To evaluate the PML/RARalpha role in myelopoiesis, transgenic mice expressing PML/RARalpha were engineered. A full-length PML/RARalpha cDNA driven by the CD11b promoter was expressed in transgenic mice. Expression was confirmed in the bone marrow with a reverse transcription PCR assay. Basal total white blood cell and granulocyte counts did not appreciably differ between PML/RARalpha transgenic and control mice. Cell sorter analysis of CD11b+ bone marrow cells revealed similar CD11b+ populations in transgenic and control mice. However, in vitro clonal growth assays performed on peripheral blood from transgenic versus control mice revealed a marked reduction of myeloid progenitors, especially in those responding to granulocyte/ macrophage colony-stimulating factor. Granulocyte/macrophage colony-stimulating factor and kit ligand cotreatment did not overcome this inhibition. Impaired myelopoiesis in vivo was shown by stressing these mice with sublethal irradiation. Following irradiation, PML/RARalpha transgenic mice, as compared with controls, more rapidly depressed peripheral white blood cell and granulocyte counts. As expected, nearly all control mice (94.4%) survived irradiation, yet this irradiation was lethal to 45.8% of PML/RARalpha transgenic mice. Lethality was associated with more severe leukopenia in transgenic versus control mice. Retinoic acid treatment of irradiated PML/RARalpha mice enhanced granulocyte recovery. These data suggest that abnormal myelopoiesis due to PML/RARalpha expression is an early event in oncogenic transformation.

The effect of inhalation of platelet-activating factor on the pulmonary clearance of 99mTc-DTPA aerosol.

Platelet-activating factor (PAF) is a short-acting, lipid-soluble autocoid, inhalation of which causes an immediate pulmonary vascular sequestration of granulocytes and a peripheral neutropenia. We investigated the effect of PAF inhalation on the pulmonary clearance rate of inhaled 99mTc-DTPA in order to test the hypothesis that the pulmonary sequestration of granulocytes results in acute lung injury. In nine normal nonsmoking adults, the rate of clearance of DTPA, corrected for background activity, was 1.5 (SD 0.7) %/min over the first 10 min after inhalation. Inhalation of 4.8 micrograms PAF abruptly increased the clearance rate to a mean value of 2.3 (1.4) %/min (p < 0.05). No increase in clearance was observed in four nonsmoking subjects who inhaled vehicle only. The mean overall increase after PAF was 87% of the baseline clearance, significantly different (p < 0.05) from the corresponding change in the control group, which was -17%. After PAF, the clearance rate returned to baseline values within 10 min in all subjects. In all subjects who inhaled PAF, but in none who inhaled vehicle, there was an immediate neutropenia of 51 (SD 25) % of the baseline value (p < 0.01). This neutropenia persisted longer than the corresponding accelerated DTPA clearance and was still 74 (36) % of the baseline value at 10 min. Furthermore, there was no correlation between the increase in DTPA clearance induced by PAF inhalation and the decrease in peripheral blood granulocyte count. We conclude that PAF inhalation results in an increase in pulmonary DTPA clearance, probably not mediated by pulmonary vascular granulocyte sequestration.

Tolerability, safety, and acceptability of zidovudine prophylaxis in health care workers.

To evaluate the safety, tolerability, and acceptability of zidovudine used for prevention of human immunodeficiency virus (HIV) transmission to health care workers. Prospective study between January 1989 and December 1992 at a suburban New York City medical center of health care workers who had percutaneous or permucosal exposures to blood or body fluids of HIV-infected patients. The zidovudine regimen offered was 200 mg orally every 4 hours for 42 days. Only 60 (53%) of 113 health care workers chose to take zidovudine, and only 21 (35%) of these health care workers completed the recommended 42-day course of therapy. Men were more likely than women to choose zidovudine therapy (P = .06), and a greater proportion of men completed the full 42 days of zidovudine therapy (P = .09). The most frequent reason for stopping treatment prematurely was clinical adverse reactions (n = 18 [30%]). Overall, clinical adverse events occurred in 44 (73%) of those taking zidovudine, with the most frequent events being nausea (n = 28 [47%]), headache (n = 21 [35%]), and fatigue (n = 18 [30%]). Selected laboratory parameters (hemoglobin level, hematocrit, alanine aminotransferase level, and white blood cell, granulocyte, and platelet counts) were compared at baseline and at week 4. Of interest, platelet counts were significantly more likely to increase by week 4 than to remain the same or decrease (P = .035). This observation may shed light on the mechanism of platelet elevation seen in zidovudine-treated patients infected with HIV. None of the laboratory changes was considered clinically significant. Of the 42 health care workers followed up for 3 months or longer (range, 3 to 32 months), none had undergone HIV antibody seroconversion. Zidovudine therapy is poorly accepted and tolerated by health care workers, at least in the dosage regimen used in this study. Alternative prophylactic regimens are needed.

Decreased β-Endorphin Content in Peripheral Blood Mononuclear Leukocytes from Patients with Crohn′s Disease

Increased activation of lymphocytes in inflammatory bowel disease is reflected by alterations of various immunological functions including enhanced spontaneous secretion of rheumatoid factor by mononuclear cells. Since in rheumatic diseases increased secretion of rheumatoid factor is associated with decreased levels of β-endorphin in circulating blood mononuclear leukocytes, we investigated levels of leukocyte β-endorphin in inflammatory bowel disease and compared them with those in hepatobiliary disorders and in healthy subjects. Levels of β-endorphin were measured in extracts from peripheral blood mononuclear leukocytes by radioimmunoassay. β-Endorphin levels ranged from 0 to 67 pg/106 cells. Mononuclear leukocytes from ulcerative colitis patients contained as much β-endorphin as those from healthy control subjects. In patients with Crohn′s disease, levels of β-endorphin were reduced by as much as roughly 50%. An inverse relationship was found between leukocyte β-endorphin on the one hand and erythrocyte sedimentation rate, blood granulocyte or thrombocyte counts, and C-reactive protein levels in plasma on the other. In patients with various hepatobiliary disorders including fatty liver disease, viral hepatitis, primary biliary cirrhosis, and cryptogenic or alcoholic cirrhosis, β-endorphin levels were not significantly different from the normal range values. Data indicate that leukocyte β-endorphin may be involved in regulation of the systemic inflammatory activity of Crohn′s disease.

Granulocyte function of patients on anti-cancer chemotherapy

Granulocyte function of patients on anti-cancer chemotherapy was investigated. Five bladder cancer patients, who had undergone M-VAC neo-adjuvant, chemotherapy were select for this study. Their tumor stage was T3M0N0 and peripheral blood granulocyte and platelet counts were more than 1500/mm3 and 10 x 10(4)/mm3, respectively, before starting chemotherapy regardless of the course Flow-cytometric analyses were utilized to measure the function of phagocytosis and the ability to produce superoxide (bactericidal function) of granulocytes. During the course peripheral blood count, granulocyte function, urinalysis and blood chemistry were checked every other day and urine and blood culture were done once a week. Although the first course of M-VAC was completed without using rhG-CSF, the second course of M-VAC administration required rhG-CSF when the number of peripheral granulocytes was below 1000/mm3. The function of phagocytosis decreased from the onset of the first course of M-VAC until the nadir of granulocyte number, then recovered close the pre-chemotherapy level by the time when the chemotherapy was completed with the increase in the number of granulocyte but remained relatively low until starting the second course. The function deteriorated more rapidly with the second course than with the first course, but recovered rapidly after administration of rhG-CSF and was significantly higher when the nadir of granulocytes was noted with the first course of M-VAC than with the second course. As for the function of superoxide production, it decreased gradually until the nadir of granulocyte during the first course and then continued to recover toward starting the second course but failed to return to the pre-chemotherapy level.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies on onset patterns in antithyroid drug-induced agranulocytosis

We studied the patterns of the onset of antithyroid drug-induced agranulocytosis. From 1975 to 1990, 19,050 patients with Graves' disease receiving treatment with antithyroid drugs were seen at our clinic. For all patients with Graves' disease treated with an antithyroid drug, a routine white blood cell count was done every 2 weeks until euthyroid state was gained, and a count was done once every moth thereafter. Of these, 70 were found to have agranulocytosis. Agranulocytosis was defined as a granulocyte count of 500/mm3 or less. In only 19 of the 70 was agranulocytosis detected after the occurrence of infection (symptomatic: classical agranulocytosis). The remaining 51 patients were asymptomatic when agranulocytosis was detected during routine white blood cell and granulocyte count monitoring. However, 17 of the 51 patients became symptomatic several days after the withdrawal of antithyroid drug treatment (shifted from asymptomatic to symptomatic agranulocytosis). Thirty-four patients had no symptoms of infection throughout the course of the disease (asymptomatic agranulocytosis). In conclusion, 1) We found three patterns in the onset of antithyroid drug-induced agranulocytosis: classical (symptomatic), a shift from asymptomatic to symptomatic, and asymptomatic agranulocytosis, 2) Unexpectedly, classical (symptomatic) agranulocytosis was seen in only 19 of the 70 patients, 3) We were again remained of the importance of routine white blood cell and granulocyte count monitoring.

Lack of Ranitidine Effects on Cyclophosphamide Bon Marrow Toxicity or Metabolism: A Placebo-Controlled Clinical Trial

We previously reported that cimetidine but not ranitidine significantly enhances cyclophosphamide-induced bone marrow toxic effects and the appearance of cyclophosphamide alkylating species in a murine leukemia mouse model, and we advised caution in the use of cimetidine with microsomally metabolized anticancer drugs. Both drugs have been used for the treatment of gastric complications of chemotherapy. Using a randomized, double-blind, crossover study design, we have now evaluated the potential interaction of ranitidine with cyclophosphamide in seven cancer patients, who received two courses of cyclophosphamide, one with ranitidine and one with placebo. Four patients received ranitidine in the first course, and three received placebo. Ranitidine or placebo was started 3 days before a single dose of cyclophosphamide and given for 17 consecutive days. Ranitidine or placebo was given orally (300 mg/d), and cyclophosphamide (600 mg/m2) was given intravenously with [3H]cyclophosphamide (1000 muCi). Cyclophosphamide treatment was repeated at 4 weeks plus or minus 4 days. Blood samples were collected at intervals from 5 minutes to 24 hours after cyclophosphamide treatment and analyzed by thin-layer chromatography and radioassay for the drug and its metabolites. On days 0, 7, 14, and 21 after cyclophosphamide administration, complete blood cell counts, white blood cell differential counts, platelet counts, and SMA-17 were determined. The differences in mean nadir white blood cell counts, granulocyte counts, hemoglobin levels, and hematocrit values during ranitidine versus placebo treatment were not statistically significant. In a statistical but not a clinical sense, mean nadir platelet counts were significantly lower with ranitidine. There was a statistically significant increase in area under the curve for drug concentration in plasma x time (AUC) with ranitidine as well as a statistically significant decrease in the total-body clearance rate of the cyclophosphamide molecule. However, the effect on AUC for the major oncolytic metabolites 4-hydroxycyclophosphamide and phosphoramide mustard was not statistically significant. The lack of toxicologic or metabolic interaction between ranitidine and cyclophosphamide suggests that ranitidine can be used safely with cyclophosphamide.

Granulocyte-macrophage colony-stimulating factor and interleukin-3 in combination: A potent and consistent myelodysplastic syndrome bone marrow stimulant in vitro

In an effort to overcome bone marrow failure in myelodysplastic syndrome (MDS), we have investigated recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3) in phase I-II clinical trials. Although these agents partially increased peripheral blood granulocyte counts, their effect on other hematopoietic lineages was generally sporadic. Since in vitro analysis and in vivo studies in primates indicate that GM-CSF and IL-3 synergistically enhance hematopoietic stem cell proliferation, we evaluated their combined effect on marrow progenitors obtained from ten MDS patients. When used singly, each growth factor stimulated replication of granulocyte-macrophage (CFU-GM) and erythroid (BFU-E) colony-forming cells in a dose-dependent fashion. When colony-stimulating activity was compared at concentrations that maximally amplified individual MDS patients' colony numbers, IL-3 was a more potent stimulant in some patients and GM-CSF in others. When used in combination, IL-3 plus GM-CSF was more effective than each growth factor by itself in five of six patients. Our data indicate that the MDS hematopoietic progenitor stimulatory effect of these growth factors varies from patient to patient. However, the combination of GM-CSF and IL-3 appears to be more potent than the individual molecules in the majority of patients.

Effect of antileukemia chemotherapy on marrow, blood, and oral granulocyte counts

This study was designed to elicit the effects of antileukemia chemotherapy on marrow production, blood carriage, and oral extravasation of granulocytes, and on the phagocytic activity of those harvested from the mouth. Fifteen adult patients with various morphologic forms of acute leukemia were followed through one to four courses of chemotherapy. Oral saline rinse samples were obtained thrice weekly and prepared for enumeration in a hemocytometer. The oral granulocyte counts were compared with concurrent counts in the bone marrow and peripheral blood. Phagocytic activity of the oral granulocytes was measured by the method of Smith and Rommel. The 15 patients were followed through 30 courses of chemotherapy and recovery. During each, there was a drug-induced decrease in marrow, blood, and oral granulocytes that was reversed when therapy was discontinued and bone marrow activity was restored. Phagocytic activity of the oral granulocytes was not perceptibly affected by the antileukemic drugs. Oral granulocyte counts provide a noninvasive method for monitoring the onset and recovery of chemotherapy-induced myelosuppression and granulocytopenia in patients with leukemia.

Cellular-phase fibrinolysis and coronary reperfusion during acute myocardial infarction: A study in patients receiving intravenous streptokinase-methylprednisoline therapy

Activation of plasma plasminogen to plasmin is the objective of current strategies for thrombolytic therapy. Although thrombolytic activity in blood involves a large cellular component as well as that in plasma, the contribution of this cellular phase to clinical thrombolysis has not been examined. Using a 125I-fibrin test tube assay, we determined blood, plasma and calculated cellular phase fibrinolytic activities in 39 patients with acute myocardial infarction before, immediately after, and at 2 hours after therapy with an intravenous streptokinase-methylprednisolone regimen. By coronary angiography and time to peak creatine phosphokinase levels, 32 patients had coronary reperfusion and 7 did not. Before streptokinase therapy, cellular phase activity of patients who reperfused was more than 2-fold greater than that of patients who did not reperfuse (p < 0.001), while plasma activities were identical, suggesting that intrinsic cellular phase activity may be a determinant of the success of subsequent thrombolytic therapy. In both groups, in addition to the expected (and similar) increases in plasma activity, cellular phase activity increased when compared with pre-treatment values (+96% to +248%; p < 0.001), with accompanying increase in blood granulocyte count (+23% to +65%), indicating that blood cells, as well as plasma, are major contributors to streptokinase-mediated fibrinolysis. Cellular phase stimulation was reproduced in a patient receiving streptokinase without methylprednisolone, and by addition of streptokinase to normal blood in vitro , indicating that streptokinase alone could account for these effects. Increased cellular phase activity in patients who reperfused after streptokinase was similar to that in those who did not reperfuse, when pre-treatment values were considered. These findings indicate that initial cellular phase activity in blood may determine subsequent fibrinolytic response, and that there is a significant cellular phase component to the fibrinolytic response to streptokinase, probably mediated by increased numbers of blood neutrophils, with a possible contribution from increased activity of individual neutrophils.

Thymus and T cells are not essential for rat leucopoiesis

It is generally held that T lymphocytes take part in the regulation of haemopoiesis. We have examined whether, in vivo, the influence of thymus of functional T cells is dispensable for the steady-state or accelerated formation of granulocytes (and in some experiments macrophages), utilizing a rat model. Untreated normal and athymic 'nude' rats had similar blood and marrow granulocyte counts and marrow proliferative activity. Bone marrow regeneration after two cytotoxic insults to the two kinds of rats gave no clues to an important regulatory role for thymic factors or T cells. Nor were such clues obtained in experiments where bone marrow cells from normal rats were cultured in vivo in diffusion chambers (DC), in either normal rats or rats undergoing a graft-versus-host (GvH) reaction (with supranormal serum levels of cytokines). On the other hand, when marrow cells from athymic rats were similarly cultured in DC, small but significant differences in leucopoiesis occurred between the three kinds of DC hosts: Marrow cells lacking functional T cells generated fewer proliferative granulocytes and had a lower proliferative activity when cultured in athymic than in normal hosts, whereas the proliferative granulocytes were most numerous in chambers carried by GvH rats. No differences were found for macrophages and eosinophilic granulocytes. The results indicate that thymic hormones or T cells or both can stimulate granulopoiesis. They apparently play no indispensable role in short-term leucopoiesis, however, since their influence was weak in our experimental models. Consequently, marked effects seen in vitro in simplified cellular systems may lose some importance in the more complex in vivo setting.

Maternal alloimmunization to HLA, platelet and granulocyte‐specific antigens during pregnancy: its influence on cord blood granulocyte and platelet counts

In order to obtain an estimate of the frequency of platelet-specific and granulocyte-specific antibodies and of the effect of such antibodies on the platelet count and granulocyte count of the newborn infant, serum from 147 women in their second or subsequent pregnancies was tested. No platelet-specific antibodies were found but 29 of the women had granulocyte-specific antibodies and the corresponding infants had granulocyte counts which were significantly lower than those of infants without antibodies. HLA antibodies were found in the sera of 57 women but were not associated with diminished platelet or granulocyte counts in the corresponding infants. Maternal granulocyte antibodies may be an underestimated contributory factor in the pathogenesis of neonatal neutropenia.

遺伝子組換えヒトか粒球増殖因子（Ｇ‐ＣＳＦ）の癌治療への応用

Recombinant human granulocyte colony-stimulating factor (Re Hu G-CSF) was prepared and its stimulating effect on granulocytopoiesis was examined in mice. Human G-CSF was purified to homogeneity from conditioned media of a G-CSF producing cell line. The amino-terminal sequence was determined. By using oligonucleotides as probes, which were proposed by the amino acid sequence, a cDNA library preparcd from human macrophages was screened. The cloned G-CSF cDNA was expresscd in E. coli K12MM294, and the mature protein was purified to homogeneity. Mice were given intraperitoneal injections of Re Hu G-CSF everyday for 14 days. Peripheral blood granulocyte counts were examined after 4, 8, 12, and 14 days of injection. Mice were sacrificed on the 14th day for histologic examinations of bone marrow, and spleen. Granulocyte counts began to increase on the 4th day and reached about 80, 000/mm on the 14th day. Cells of granulocyte lineage were markedly increased in the bone marrow and spleen. Granulocyte precursors (CFU-C) were remarkably increased in the spleen. When mice were treated with 5-fluorouracil, cyclophosphamide, or irradiation, the period of granulocytopenia was significantly shortened by subcutaneous injections of Re Hu G-CSF.These results suggest that human G-CSF play a central role in granulocyte production in vivo. The ability of Re Hu G-CSF to stimulate granulocyte production implies that this factor will be clinically useful in neutropenic patients treated with anti-cancer agents or irradiation.

Myeloid surface antigen abnormalities in myelodysplasia: relation to prognosis and modification by 13-cis retinoic acid.

The relation between prognosis and lineage specific surface antigen expression on peripheral blood granulocytes and monocytes was studied using monoclonal antibodies and flow cytometry in 37 patients with myelodysplastic syndromes (MDS). Abnormalities in antigen expression were summarised as a score, and cases were divided into low (few abnormalities) and high (many abnormalities) groups. Survival was significantly worse in the "high" group (logrank chi 2 = 5.793, p = 0.016), this group having a median survival of 31 weeks, compared with more than 67 weeks in the "low" group. No correlations were found between the score and any of the following: peripheral blood platelet and granulocyte count; FAB subtype; bone marrow blast cells and sideroblast count, or erythroid and myeloid progenitor growth. Antigen expression was also studied in six further cases of MDS before and after six weeks of treatment with 13-cis retinoic acid (CRA), 20 mg given orally, and a comparison was made with six untreated patients studied before and after a similar time interval. In the treated group 58% of initially abnormal measurements reverted to normal, compared with 24% in the untreated group. Five of the six treated patients showed a decrease in the score, whereas only two of the six improved in the untreated group. The data indicate that myeloid antigen expression is a useful indicator of prognosis in MDS, and that antigen expression may be affected by treatment.

The effect of pretreatment with thio-TEPA and cytosine arabinoside on megakaryocytopoiesis in rats given a sublethal dose of thio-TEPA

The present study was performed on rats, mainly to examine the so-called priming effect on megakaryocytopoiesis. One group of animals received 2 or 4 mg thio-TEPA or 200 mg cytosine arabinoside/kg body weight (the pretreatment) 2.5 days or 8 days prior to a dose of 10 mg thio-TEPA/kg body weight (the challenge dose). Another group received a pretreatment dose of 1 mg melphalan/kg body weight 2.5 days prior to a challenge dose of 3 mg melphalan/kg body weight. The number of bone marrow megakaryocytes, blood platelet production, mean platelet volume, blood platelet counts, leucocyte and granulocyte counts were examined on days 2, 4, 7, 10, 13, 16 and 20 after the challenge dose. The gut mucosa (number of mucosal crypts in terminal jejunum) and survival were studied in animals receiving pretreatment 2.5 days prior to a challenge dose of about LD100 for thio-TEPA and melphalan. No systematic differences were observed whether the animals received pretreatment prior to the challenge dose or not. Thus, no priming effect was observed.

In vitro production of granulocyte-macrophage colony-stimulating activity by murine bone marrow and spleen following vinblastine administration in vivo.

The production of granulocyte-macrophage colony-stimulating activity (CSA) by isolated murine femur shafts, non-dispersed bone marrow and spleens was assessed following administration of Vinblastine (VLB). These organs were removed from 2 h to 10 days post-VLB and allowed to condition endotoxin-free medium for 48 h. CSA activity was assessed using a soft agar cloning system. The data demonstrate that CSA elaboration was maximal 24 h post-VLB, corresponding to the nadir of bone marrow GM-CFC, and 6 h after splenic reached a minimum. No relationship between peripheral blood granulocyte count or serum endotoxin levels were observed.

Efficacy and clinical cross-resistance of a new combination therapy (AMSA/VP16) in previously treated patients with acute nonlymphocytic leukemia.

We investigated the tolerance, efficacy, and clinical cross-resistance of a new combination chemotherapy in 38 patients with previously treated acute myeloblastic leukemia (AML). It consisted of 120 mg2/d 4'(9-acridinylamino) methanesulfon-m-Anisidide (m-AMSA) in a one-hour infusion and 80 mg/m2/d etoposide (VP-16) in a 24-hour infusion, both administered for 5 days. The first 27 patients also received vinblastine, 6 mg/m2 on day 8, but this therapy was discontinued because of intestinal complications. Thirteen of 23 patients (56%) at first or subsequent relapse and five of 15 patients (33%) who were primarily resistant to an anthracycline/cytarabine combination achieved a complete response (CR) (hemoglobin level not taken into account) with a median CR duration of 5 months and 2 months, respectively. The response rate was as high as 63% for patients at first or second relapse whether the remission was maintained or not. The median times to recovery of normal bone marrow cellularity, of blood granulocyte counts greater than 500/microL, and of platelets greater than 20,000/microL were 34, 27, and 22 days, respectively. Marked but reversible gastrointestinal toxicity was observed in 24% of the patients, and two patients died of infection during induction. The one-hour AMSA/continuous VP-16 combination is effective for patients with relapsing AML and shows no cross-resistance in a proportion of patients refractory to the standard anthracycline-cytarabine combination.