Prognosis in aplastic anemia

In catamnestic examinations of patients with aplastic anaemia 11 cases had to be excluded from a primarily diagnosed total number of 112 because these proved to be cases of preleukaemia. In the rest of 101 patients there was a highly significant positive correlation (p less than 0.001) of the survival time to the bone-marrow cellularity and to the average values of corrected reticulocytes and granulocytes calculated from findings of 0,4, and 8 weeks. For thrombocytes, however, a slightly significant positive correlation (p less than 0.05) could only be identified in the present material for the 4-weeks value. The fact that the prognosis is deteriorated by a rapid development of the disease could be made probable by means of a positive correlation between the time of the first symptom and diagnosis (p less than 0.01). With patients falling below 2 or 3 limiting values of the peripheral blood cells they were classified to the SAA group according to the proposals made by Camitta, however, by using the average values described above The survival rate which remained constant after 3 years amounted to 16% for SAA patients classified in this way, 44% for non-SAA cases with constant values of 39% beginning from the fourth year. Patients with average values of all three cell parameters falling below those limiting areas had a six months survival time of only 11%. No patients were alive after one year. The latter was also true if reticulocytes and granulocytes were affected by a diminution of only 2 parameters. Those patients, however, who had an average of reticulocytes and thrombocytes only, but no granulocytes below the limiting area within the first 8 weeks showed a mortality curve which did not differ from that of non-SAA patients. Therefore, a classification of these cases can only be made with great caution allowing for a prognosis-oriented therapy, such as indication for bone-marrow transplantation.     

Granulocyte kinetics with radioactive diisopropylfluorophosphate (DF32P) and radiochrome (51Cr)]

Determining granulocyte kinetics with DF32P allows various parameters to be gained during the in-vitro marking, such as the total blood granulocyte pool, circulating granulocyte pool, marginal granulocyte pool, daily granulocyte exchange rate and half decay period of granulocytes. The half decay period of granulocytes, bone-marrow reserve in myelocytes, metamyelocytes and band cells as well as polymorphonuclear neutrophils can be determined by in-vitro marking, with DF32P being intravenously injected. The combination of both procedures with DF32P will reveal the half decay period, pool sizes and exchange rates of the proliferating myelocyte compartiment in bone-marrow and mature blood granulocytes. If 51Cr is used for determining granulocyte kinetics the surface activities of various organs, such as heart, liver, spleen, and lungs, can mainly be determined in addition to the half-life of leucocytes, indicating the degradation or storage of cells in certain areas of the body. In addition to normal values those findings are principally presented which were obtained with in-vitro marking by DF32P and 51Cr in chronic myeloid leukaemia, osteomyelofibrosis or osteomyelosclerosis respectively and in hypersplenism.     

Composition and ultrastructure of elasmobranch granulocytes. II. Rays (Rajiformes)

The blood granulocyte composition of seven species of ray is given together with ultrastructural observations made on the epigonal organ and blood of Pavoraja spinifera and the spleen of a deepwater rajid skate. Under the light microscope three granulocyte types, eosinophils, eosinophilic granulocytes and neutrophilic granulocytes could be distinguished. At the EM level two granulocyte types were apparent, one with elongated granules containing longitudinal fibrils that consolidated to form an axial rod-like inclusion, and the other with large, spherical, uniformly electron-dense granules. Correlation of light and electron microscope observations indicated that the neutrophilic granulocytes with weakly basophilic, elongated granules become weakly eosinophilic, as eosinophilic granulocytes, and these in turn develop to eosinophils with granules containing axial rods. The other granulocyte type forms another population of eosinophils with spherical granules.
The inter-relationship of these granulocytes, the identification of eosinophilic granulocytes, or heterophils, as immature eosinophils, and the co-existence of two morphologically distinct eosinophil forms are discussed.     

Augmentation of host resistance to Candida albicans infection in ascites tumor-bearing mice.

We found that the number of peripheral blood polymorphonuclear leukocytes (PMN) dramatically increased in both sarcoma 180 (S-180) and MM-46 mammary carcinoma (MM-46) ascites tumor-bearing mice, and mice required a remarkable resistance to Candida albicans infection via intravenous route. When the resistance was determined by number of cells of C. albicans in the kidney, a significant decrease in the number of fungal cells was observed in the kidneys of infected ascites tumor-bearing mice. An increase of active oxygen levels of PMN from ascites tumor-bearing mice was observed, suggesting that this factor is important in developing of resistance in ascites tumor-bearing mice. Additionally, a culture supernatant of tumor cells co-cultivated with bone-marrow cells in vitro increased the number of granulocytes and macrophages differentiated from the bone-marrow cells.     

Hydrocortisone acetate-induced eosinopenia in mice: independence from the thymus.

The eosinopenia-inducing effect of the adrenal-cortical steroid, hydrocortisone acetate (HCA), was studied with respect to the involvement of thymus-dependent lymphocytes. Thymectomized, irradiated, bone-marrow-reconstituted male B6D2F1 mice experienced as extensive an eosinopenic effect of HCA as did sham-operated, irradiated, bone-marrow reconstituted control mice. Femoral bone marrow appeared unaffected during the times studied. A similar decrease in circulating eosinophils could be induced in congenitally athymic nude (nu/nu) mice. The results argue against a role for the thymus in hydrocortisone-induced eosinopenia.     

The enzyme cytochemistry and composition of elasmobranch granulocytes

Peroxidase, alkaline phosphatase, acid phosphatase, β-glucuronidase, α-naphthyl acetate esterase (ANAE), α-naphthyl butyrate esterase, naphthol AS-D chloroacetate esterase, acetyl-L-tyrosine-α-naphthyl esterase (ATNE), tosyl-L-lysine-α-naphthyl esterase (TLNE) and periodic acid-Schiff (PAS) were studied in 17 species of elasmobranchs in which granulocytes had previously been identified at the ultrastructural level.
Eosinophils, eosinophilic and neutrophilic granulocytes contained variable acid phosphatase, esterases and PAS, but they were strongest in neutrophilic granulocytes; particularly ANAE. Esterases were released into surrounding plasma and therefore probably function as ectoenzymes. In eosinophils and some neutrophilic granulocytes there were indications of weak peroxidase, but this could not be conclusively demonstrated. Alkaline phosphatase was diffuse between granules in some eosinophils of Pavoraja , and (β-glucuronidase was diffuse in neutrophilic granulocytes of Etmopterus baxteri , otherwise granulocytes lacked these enzymes. Neutrophilic granulocytes stained moderately to strongly for ATNE and weakly and inconsistently for TLNE in Squalus acanthias and Dalatias licha . with a similar reaction in granular lymphocytoid and thrombocytoid cells of Galeorhinus ausiralis and Raja nasuta . The enzyme composition of these granulocytes is discussed.     

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.     

A novel cell surface antigen, 4C8, is expressed on human eosinophils

BACKGROUND: A novel monoclonal antibody, anti-4C8, reacted with human peripheral lymphocytes and monocytes but not with neutrophils. In this study, we investigated whether the 4C8 antigen is expressed on human peripheral eosinophils. METHODS: Expression of the 4C8 antigen on eosinophils was analyzed by flow cytometry and molecular analysis of the antigen was performed with eosinophils by Western blotting. RESULTS: Among human peripheral granulocytes, the 4C8 antigen was expressed on CD16-negative cells but not on CD16-positive cells. The 4C8 antigen also appeared to be expressed on eosinophils. To confirm the latter finding, eosinophils were purified from peripheral blood. On flow cytometric analysis, anti-4C8 antibody reacted with purified eosinophils. On Western blotting analysis, anti-4C8 reacted with a single band of 80 kDa in lysates from purified eosinophils. The correlation between the percentage of eosinophils determined by May-Giemsa staining and the percentage of 4C8-positive/CD16-negative cells among granulocytes was good (r = 0.91, P < 0.0001). CONCLUSIONS: Only a few cell surface antigens are available to distinguish human peripheral eosinophils from neutrophils. The novel cell surface antigen, 4C8, is a useful new marker of human eosinophils.     

The state of hemopoiesis under conditions of long-term bone marrow irradiation in residents of the Techa riverside villages

The paper presents the results of a 50-year study on the state of hemopoiesis in the Techa riverside residents chronically exposed to radiation in the range from low to intermediate doses. The highest bone marrow doses were attributable to intakes of osteotropic 9OSr with drinking water and local food products. During the period of maximum radiation exposures (1951-1953) exposed residents were manifesting decreased counts of peripheral blood leukocytes (neutrophils and lymphocytes) and thrombocytes. Normal counts of erythrocytes were maintained owing to the effect of sufficient compensatory mechanisms, including accelerated rates of erythrocaryocyte proliferation and maturation. The development of peripheral blood granulocytopenia was influenced by the delay in the differentiation of neutrophilic bone-marrow granulocytes at the myelocyte phase, a marked increase in the frequency of lethal abnormalities in bone-marrow neutrophils, and pathological mitoses. The period of normalization of the blood cell composition was significantly variable for different blood cell series, and was noted to depend on exposure dose rate, extent of the primary hemopoiesis inhibition and specific physiological characteristics of exposed individuals.     

Prostaglandin E2 and dexamethasone regulate eosinophil differentiation and survival through a nitric oxide- and CD95-dependent pathway.

Apoptosis, involving both CD95/CD95L interactions and their modulation by nitric oxide (NO), is central to regulation of mature eosinophil numbers. However, its role in regulating eosinophil production from bone-marrow precursors is unknown. We examined the effects of prostaglandin E2 (PGE2) and dexamethasone on eosinophil differentiation and survival in murine bone-marrow cultures, and their relationship to: NO production as well as CD95/CD95L-dependent apoptosis. Bone-marrow cultures were established with IL-5, alone or in association with PGE2, dexamethasone or both. PGE2 (10(-7)M) inhibited eosinophil differentiation by selectively inducing apoptosis in developing eosinophils. Dexamethasone (10(-7)M) protected developing eosinophils from PGE2-induced apoptosis. Since dexamethasone prevents induction of nitric oxide synthase (NOS), we evaluated the role of NO in the effects of both PGE2 and dexamethasone. NO donors (SNAP and SNP) down-modulated eosinophil precursor responses to IL-5. SNAP induced apoptosis through a dexamethasone-resistant mechanism. The NOS inhibitors, Nomega-nitro-L-arginine and aminoguanidine, blocked the effects of PGE2 on developing eosinophils. PGE2 was ineffective in bone-marrow from knockout mice lacking inducible NOS. PGE2 up-regulated CD95 and CD95L expression in developing eosinophils. Neither PGE2 nor SNAP were effective in cultures from CD95L-deficient gld mice. These data suggest that PGE2 induces apoptosis in developing eosinophils through inducible NOS, leading to NO-dependent activation of the CD95L/CD95 pathway, while dexamethasone antagonizes the effects of PGE2 on the same targets.     

Myeloperoxidase-deficient polymorphonuclear leucocytes. Longitudinal study during the preremission--and the remission phase in acute myeloid leukaemia. Comparison to neutrophil alkaline phosphatase (NAP) activity

Serial determinations of MPO and NAP activities in granulocytes were performed during the preremission phase and the remission phase in patients with AML. Of 18 patients examined during the preremission period, 9 showed an increased number of MPO deficient PMN. Complete remission was attained in 4 of these, in 3 the number of abnormal granulocytes changed to normal 7, 7 and 14 days before and in 1 simultaneously with the attainment of complete remission. In the other patients no changes in granulocyte MPO activity occurred during the preremission period. All 20 patients examined during complete remission showed a normal MPO activity in granulocytes. Of eight patients, who at diagnosis had shown abnormal granulocyte MPO activity, three developed relapse. In two of these, an increased number of MPO deficient PMN reappeared two and eight months prior to and in one simultaneous with clinical and laboratory suspicion of relapse. A statistically significant relation between low NAP scores and an increased number of MPO deficient PMN was found (P = 0.011). Serial determinations of MPO activities in PMN, although restricted to cases of AML with initially abnormal values, may prove helpful in predicting achievement of complete remission and may furthermore prove to be useful as an indicator of early relapse.     

Does ceftazidime or latamoxef affect the phagocytic capabilities of human granulocytes?

In comparative studies made in patients with pneumonia we have treated 14 cases with Latamoxef and 16 cases with Ceftazidime. We have focused attention on the side effects of these drugs and on the phagocytic capacity of granulocytes. In our patients we have noted a decrease of bactericidal activity of plasma and granulocytes and an elevated NBT reduction capability of resting granulocytes. The other tested function of the granulocytes exhibited only small variations. The applied therapy caused the bactericidal activity to be increased in granulocytes and plasma. Both drugs had no harmful effect on the phagocytic activities of granulocytes.     

Thrombocyte kinetics before and after gold therapy in rheumatoid arthritis]

Before and after performing a basic therapy with sanocrysin, thromboytic kinetic examinations were carried out in 12 patients suffering from rheumatoid arthritis. The thrombocyte survival time, maximal recovery and thrombocyte turnover were determined. The surface activity values were given in the form of spleen-heart quotients and liver-heart quotients as well as by surplus impulses over these organs. The findings evaluated statistically reveal that even the untreated rheumatoid arthritis will have thrombocytic kinetics deviating from the norm. By influencing RES the gold treatment will lead to an reduction of the enhanced platelet decomposition. The behaviour of single parameters before and after gold therapy are discussed. The examination of thrombocyte kinetic could not identify a damage of thrombopoiesis in the bone-marrow and in the periphery caused by sanocrysin.     

Immunological detection of residual leukaemic disease in the bone marrow of children with acute lymphoblastic leukaemia

Peripheral blood lymphocytes incubated with tumour cells or extracts may undergo blastogenesis. This is the basis of a technique studied in children with acute lymphoblastic leukaemia (ALL) in childhood in an attempt to predict relapse. Samples of peripheral blood and bone marrow from 82 children with varying degrees of ALL were analysed. Cultures were prepared by incubating a lymphocyte suspension with an autologous bone-marrow suspension. Final ratios of lymphocytes to bone-marrow cells (L: BM) were 1: 1 and 2: 1. Control wells received bone-marrow or lymphocyte suspension only. Cultures were incubated for 72, 96, and 120 hours. All were pulse-labelled with ³H-TdR and radioactivity was measured by scintillation counting. Results were expressed as the stimulation index, calculated by dividing the mean counts per minute (cpm) of wells containing both lymphocytes and bone-marrow cells by the sum of the mean cpm for control wells. If the stimulation index exceeded 1 at 72, 96, or 120 hours at either L: BM ratio a positive response was recorded.Seventy-six children were in clinical remission at the time of testing (group A) and six were in clinical relapse (group B). In group A 24 patients showed stimulation and relapsed later at a mean time of 3·8 months (21 with marrow disease, two with testicular infiltration, and one with lung infiltration). Sixteen patients showed stimulation and had up to 4% blasts in their bone marrow but remained in remission. Nineteen other patients showed a positive response and several factors may have contributed to this: two underwent a “rebound” lymphocytosis after stopping treatment, nine had current or intercurrent infections, two had persistent unexplained bone-marrow lymphocytosis, but six had no causative symptoms and thus their responses were “true false-positives.” Seventeen patients from group A showed no response and remained in remission for a mean of 22·9 months after testing. None of the six children in group B responded, and at testing had 17-85% blasts in their bone marrow.During the study no patient relapsed who had not shown a positive response. The technique merits further study as a guide to the presence of leukaemic cells.     

Varying course of pancytopenia after busulfan treatment of chronic myelocytic leukemia (CML)]

In 84 patients with chronic myeloid leukaemia receiving a cytostatic monotherapy with busulfan, an aplastic syndrome developed which was confirmed by a biopsy of the pelvis crest and examination of the sternal marrow. The time interval until pancytopenia was detected varied considerably in each case, ranging between 6 and 126 months. There are no correlations to the initial doses of busulfan. 3 patients died of the immediate effects of the bone-marrow damage caused by busulfan. In 4 from 6 of the following pancytopenic patients the leukocyte values lay between 12,800/microliter and 80,400 microliter when busulfan adminstration was interrupted. Thus, it is scarcely possible to give any reliable informations about a leukocyte limit value as a standard for an interruption of therapy in order to prevent bone-marrow aplasia. Taking this into account, the conclusion may be drawn that relatively short control intervals have to be made in this monochemotherapy of CML which often can be used successfully for many years.     

Schistosoma mansoni: control of extramedullar eosinophil myelopoiesis in chronically infected mice by inflammatory macrophages

Extramedullar proliferation of eosinophil granulocytes can be induced in mice chronically infected with Schistosoma mansoni, by intraperitoneal implants of glass coverslips. Immature eosinophils are located in discrete foci on glass implants; they are not correlated with the eosinophil population of the peritoneal cavity, where only mature eosinophils can be observed. The same induction of eosinophil proliferation can be obtained in normal mice, by the transfer of macrophages elicited by glass implants in mice with chronic schistosomiasis. This induction could not be done with cells mobilized in normal mice, either after transplant into normal mice or into schistosome infected ones. Stimulated macrophages of mice with chronic schistosomiasis have a capacity to induce peripherical proliferation of eosinophil granulocytes. This capacity is independent of the quality of the intraperitoneal environment. It can be expressed after transferring macrophages elicited in schistosome infected mice into normal mice.     

Mitotic index in the bone marrow of children during the clinical course of acute lymphoblastic leukemia (ALL)]

From 31 children with acute lymphoblastic leukaemia the mitosis index in the bone-marrow was determined before the onset of therapy and during the clinical progress. Initially, the mean white mitosis index lay with 3.4% below that of the normal test persons, it rose significantly in the hematologic full remission and showed a decreasing tendency with a great range of dispersion in the recidive. The most lowered mitosis index was found in the final stage. Strong shifts in the kariologic distributions make a remaining in the prophase of the mitosis evident. The influence of polychemotherapy on the mitosis index and the phases of mitosis is discussed. Correlations between the mitosis index and clinical as well as paraclinical parameters were only to be found with respect to granulocytes and lymphoblasts. The considerable ranges of fluctuations of the mitosis index and the lack of congruity with the clinical progress of ALL allow no ensured assertions to be made for the single patient. It seems to be important in eosinophilia and in leukaemoid reactions.     

An immunofluorescent method for a specific demonstration of granulocytes and some of their proteins (ECP and CCP)

Using p-benzoquinone as a fixative the non-specific fluorescence of granulocytes and especially the eosinophils is removed for both FITC and TRITC. In this way we have been able to detect the eosinophil cationic protein (ECP) and the chymotrypsin-like cationic protein (CCP) in human lung tissue and by double immunofluorescent labelling shown that these two proteins very likely are related to eosinophils respectively the neutrophils.     

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.     

Recovery of hematopoiesis in mice following a continuous irradiation with a dose rate of 0.957 Gy/d and a total accumulated dose of 19.14 Gy. II. Peripheral blood

This paper deals with quantitative and qualitative changes in the peripheral blood of mice after continuous irradiation with a dose rate of 0.957 Gy/day and with a total accumulated dose of 19.14 Gy. During irradiation a significant diminution of nongranular leukocytes, a granulocytopenia and a decrease in erythrocytes to about 70% of the control values could be observed. Erythrocytes recovered their original state until the 14th day after the end of irradiation, granulocytes until the 21st day and agranulocytes until the 60th post-irradiation day. The leukocyte number decreased significantly again until the 80th day after the end of irradiation. Leukocytes with intense morphological anomalies could be observed in the peripheral blood during the whole period of examination.