Assessment of the value of prednisone test in differential diagnosis of neutropenic state.

The prednisone test revealed normal bone marrow reserve of neutrophils (BMR) in subjects with innocent neutropenias e.g. neutropenias not associated with any other disturbances in haemopoiesis or with increased incidence of infections, assumed to be an individual anomaly of the subject. On the other hand, diminution or absence of MBR in chronic hypoplastic neutropenias, transient post-chemotherapeutic neutropenias and neutropenias with increased destruction of neutrophils were observed. The magnitude of that diminution was not dependent of the cause of neutropenia. It is suggested, that the prednisone test (and probably tests with other BMR mobilizing agents) may serve as screening tests for exclusion (or confirmation) of relative granulopoietic insufficiency as the cause of neutropenia. Only after confirmation of reduced BMR another explanations of neutropenia should be sought using other method as muramidase level, adrenaline test or granulocyte kinetics with DF32P, 3HDFP, or 51Cr.     

Inhibition of human cytotoxic T lymphocyte activity in vitro by autologous peripheral blood granulocytes

Peripheral blood granulocytes from normal healthy donors were found to reproducibly inhibit the cytolytic effector function of specifically sensitized cytotoxic T lymphocytes in vitro when co-incubated with these effector cells and target cells in 8 hr 51Cr release assays. Inhibition required intact granulocytes, was proportional to the number of granulocytes present, and was independent of granulocyte adherence, phagocytic function, and viability. Equivalent numbers of enriched normal or leukemic peripheral T lymphocytes did not cause inhibition of 51Cr release, and preincubation of granulocytes with effectors did not significantly alter viability or cytotoxic function. Because granulocytes can inhibit natural killer cell function in vitro, these data indicate that granulocytes can regulate diverse antigen-specific and spontaneous cytotoxic functions in vitro, suggesting that circulating granulocytes may have the potential for in vivo regulation of these cytotoxic effectors.     

KINETICS OF NEUTROPHILIC GRANULOCYTES IN THE BLOOD OF RATS

The kinetics of neutrophilic granulocytes in the blood of rats were investigated using in vivo ³H-TdR labelling and autoradiography. The radioactive precursor was administered by single injection, repeated injections at 5 hr intervals and continuous infusion. The appearance of labelled granulocytes in the blood and in the sputum was recorded up to 120 hr after tracer application. In contrast to results after a single injection of ³H-TdR, complete labelling of the blood granulocyte pool was achieved when the DNA precursor was given by continuous infusion or four repeated injections at 5 hr intervals. In the latter experiments, an exponential replacement of unlabelled blood granulocytes by labelled granulocytes could be demonstrated, the mean intravascular half-life being 5·7 hr. This figure is in good agreement with values obtained by isotopic techniques in other mammalian species.     

The problem of the radiosensitivity of bone marrow cellular elements and assessments of the postradiation kinetics of myelopoiesis (an analysis based on data on the sequelae of the accident at the Chernobyl Atomic Electric Power Station]

On the basis of the data obtained from 61 cases of accidental exposure (0.1-12.5 Gy) at the Chernobyl A.P.S. the kinetics of acute radiation bone-marrow syndrome was analyzed and radiosensitivity of the entire spectrum of human granulocytic compartment cells was estimated. The radiosensitivity estimates were made by a "functional" criterion, developed by the authors, which was based on the comparative ability of irradiated and nonirradiated bone marrow cells of different maturity to produce peripheral blood neutrophils. Changes were found in physiology of myeloid cells during their maturation: the maturation mechanism, for cells of the committed pool, was "attached" to the division process, whereas these processes were independent for cells of the dividing and maturing pool. It is once again confirmed that the transit time of a maturing myeloid cell, to begin with the primarily committed one and to end with a peripheral blood neutrophil, is not constant and lasts normally for 32 days.     

Growth, delayed fluorescence and pigment composition of four Prorocentrum minimum strains growing at two salinities

Prorocentrum minimum is a potentially harmful and widely distributed marine dinoflagellate. Several P. minimum strains have already been studied, showing phylogenetical relations of strains isolated from the same geographical regions. Similarity among the strains was further examined on the basis of their physiology. Pigment composition and concentration, as well as delayed fluorescence (DF) decay kinetics and intensity, were measured in four P. minimum strains isolated from the Baltic and Adriatic Seas. The strains were grown at two salinities characteristic of the Baltic (8 PSU) and North Adriatic Seas (32 PSU). Strain differences in DF decay kinetics and growth did not always follow their genetic relations. While two strains showed similarities to the previously described strains from the Baltic and Adriatic Seas in DF parameters, the other two strains seemed to be specific. The differences among strains isolated from the same sea could stem from adaptations to conditions in the specific habitats. Cluster analysis based on the ratio of individual carotenoid pigments concentrations to the chlorophyll a concentration or to total carotenoids were not conclusive in showing relations among the strains. Among the measured pigments, only peridinin concentration depended on salinity in all st.     

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.     

A mathematical model of canine granulocytopoiesis

Summary The granulocyte cell renewal system of the dog is represented by a mathematical model consisting of the following compartments: The pool of pluripotential stem cells, the committed stem cell pool, divided into a blood and a bone marrow compartment, the proliferation pool, the maturation pool, the reserve pool and the blood pool of functional granulocytes. This chain of compartments is described by a system of non-linear differential equations. Cell losses anyplace in the system provoke increased production in all pools containing cells capable to divide. A reduced number of granulocytes in the blood pool stimulates production of a granulocyte releasing factor which mobilizes a rising number of cells to transit from the marrow reserve into the blood pool.The model was simulated on a digital computer. It was found to be capable to reproduce the steady state conditions and it also fits the data of two distinct experimental perturbations of the system both equally well. These perturbations are a loss of proliferating cells as it occurs after the administration of cytostatic drugs and losses of functional cells as they are induced by leukapheresis experiments of differing leukapheresis rates.This study was supported by the Deutsche Forschungsgemeinschaft (SFB 112)     

Antibody-dependent cellular cytotoxicity (AAZZ) against leukemia cells--K-cell activity of human leukocytes

The activity of human leukocytes from the peripheral blood (PB) and bone-marrow (BM) to take the function as K-cells in the antibody dependent cellular cytotoxicity (ADCC) was tested in a 51Cr-test against mouse leukaemic cells and ALL cells covered with specific heterologous antibodies. Mononuclear PB-leukocytes and granulocytes of healthy donors and patients with leukemia and lymphoma in remission lysed murine and human leukaemic cells in the presence of specific antibodies. There was no lowering of K-cell activity of mononuclear PB-leukocytes of patients with leukaemia and lymphoma in remission under chemotherapy as compared with healthy donors and patients in remission without chemotherapy. There was a good correlation between the percentage of K-cell active mononuclear leukocytes in PB and BM. Attempts of fractionation with peripheral blood leukocytes of healthy donors resulted in the non-adherent mononuclear PB-leukocytes (lymphocytes) and granulocytes being effector cells in ADCC. To a high degree K-cell active lymphocytes could be identified in the non-B-fraction and only slightly in the fraction forming E rosettes.     

NK and K cell activity in children with leukemias and aplastic anemias before and after allogeneic bone marrow transplantation

Mononuclear leukocytes from the peripheral blood and bone-marrow of children affected with aplastic anemia and leukemia were investigated for K-cell activity (antibody-dependent cellular cytotoxicity) and NK-cell activity before and after allogenous bone-marrow transplantation. 51Cr liberation test against murine Graffi erythroblast leukemic cells covered with xenoantibodies and K-562 cells were used for identification. Strongly lowered NK- and K-cell activities could be found in aplastic anemia prior to bone-marrow transplantation. However, NK-cell activity was only lowered significantly in leukemic patients with indication of bone-marrow transplantation. K-cell and NK-cell activities normalised after bone-marrow transplantation. K-cell and NK-cell activities could be observed to be reconstituted very early after bone-marrow transplantation.     

Screening for granulopoiesis inhibitors (chalones) by different assays.

The suitability of various granulocyte chalone sources was examined; for this purpose rat ascites fluid and the conditioned media of ascites and bone marrow cells were fractionated by ultrafiltration and Sephadex gel filtration. To evaluate different assay systems, the ability of the fractions to inhibit the growth of granulocytic and T-lymphocytic colonies in agar capillaries, as well as to inhibit the uptake of [3H]thymidine in bone-marrow cells, T- and B-lymphocytes, was tested and compared. Three granulocyte colony inhibiting fractions were obtained that contained apparent chalone activities, but showed different elution parameters with molecular weights well below 10 000. Comparison of the test systems revealed that the granulocyte colony assay may detect inhibitors different from those found by the [3H]thymidine bone-marrow assay; the validity of the latter test is seriously questioned, however. The need for precisely defined assays to screen for the apparently various inhibitors is emphasized by these studies.     

Isolation of blood granulocytes from golden hamsters for the study of chemotaxis.

Since the number of granulocytes is low in the circulating pool of adult golden hamsters, intraperitoneal injection of oyster glycogen was used to mobilize white blood cells from the non-circulating pool. The percentage of blood granulocytes increased from 7 to 49%. Mobilized granulocytes, purified by a discontinuous Percoll gradient, showed adherence to polycarbonate filters only when coated by collagen. Random migration was high using the modified Boyden chamber technique. While endotoxin-activated serum caused chemotactic response, it was absent when the tripeptide N-formyl-methionyl-leucyl-phenylalanine was applied at concentrations of 10(-7) or 10(-8) M. The true chemotactic response corresponded to the proportion of migrated cells versus the total cell number adherent to both sides of the filter. It is concluded that the golden hamster is a good model for the study of granulocyte sequestration.     

Role of circulating granulocytes in sheep lung injury produced by phorbol myristate acetate

Phorbol myristate acetate (PMA) and endotoxin cause pulmonary granulocyte sequestration and alteration in lung fluid and solute exchange in awake sheep that are felt to be analogous to the adult respiratory distress syndrome in humans. The basic hypothesis that PMA causes lung injury by activating circulating granulocytes has never been tested. The effects of infused PMA on lung mechanics and the cellular constituents of lung lymph have also not been reported. We therefore characterized the effects of intravenous PMA, 5 micrograms/kg, on lung mechanics, pulmonary hemodynamics, lung fluid and solute exchange, pulmonary gas exchange, blood and lymph leukocyte counts, and plasma and lymph cyclooxygenase products of arachidonate metabolism in 10 awake sheep with normal granulocyte counts and after granulocyte depletion with hydroxyurea. PMA significantly altered lung mechanics from base line in both nongranulocyte depleted and granulocyte-depleted sheep. Dynamic compliance decreased by over 50% and resistance to airflow across the lungs increased over threefold acutely following PMA infusion in both sets of experiments. Changes in lung mechanics, pulmonary hemodynamics, lung fluid and solute exchange, pulmonary gas exchange, and plasma and lymph arachidonate metabolites were not significantly affected by greater than 99% depletion of circulating granulocytes. We conclude that the lung injury caused by PMA in chronically instrumented awake sheep probably is not a result of activation of circulating granulocytes.     

Cytochemical and functional characterization of blood and inflammatory cells from the lizard Ameiva ameiva

The fine structure and differential cell count of blood and coelomic exudate leukocytes were studied with the aim to identify granulocytes from Ameiva ameiva, a lizard distributed in the tropical regions of the Americas. Blood leukocytes were separated with a Percoll cushion and coelomic exudate cells were obtained 24 h after intracoelomic thioglycollate injection. In the blood, erythrocytes, monocytes, thrombocytes, lymphocytes, plasma cells and four types of granulocytes were identified based on their morphology and cytochemistry. Types I and III granulocytes had round intracytoplasmic granules with the same basic morphology; however, type III granulocyte had a bilobued nucleus and higher amounts of heterochromatin suggesting an advance stage of maturation. Type II granulocytes had fusiformic granules and more mitochondria. Type IV granulocytes were classified as the basophil mammalian counterpart based on their morphology and relative number. Macrophages and granulocytes type III were found in the normal coelomic cavity. However, after the thioglycollate injection the number of type III granulocyte increased. Granulocytes found in the coelomic cavity were related to type III blood granulocyte based on the morphology and cytochemical localization of alkaline phosphatase and basic proteins in their intracytoplasmic granules. Differential blood leukocyte counts showed a predominance of type III granulocyte followed by lymphocyte, type I granulocyte, type II granulocyte, monocyte and type IV granulocyte. Taken together, these results indicate that types I and III granulocytes correspond to the mammalian neutrophils/heterophils and type II to the eosinophil granulocytes.     

Delayed fluorescence emitted from light harvesting complex II and photosystem II of higher plants in the 100 ns-5 micros time domain

This study presents the first report on delayed fluorescence (DF) emitted from spinach thylakoids, D1/D2/Cytb-559 preparations and solubilized light harvesting complex II (LHCII) in the ns time domain after excitation with saturating laser flashes. The use of a new commercially available multichannel plate with rapid gating permitted a sufficient suppression of detector distortions due to the strong prompt fluorescence. The following results were obtained: (a) in dark-adapted thylakoids, the DF amplitudes at 100 ns and 5 micros after each flash of a train of saturating actinic pulses exhibit characteristic period four oscillations of opposite sign: the DF amplitudes at 100 ns oscillate in the same manner as the quantum yield of prompt fluorescence, whereas those at 5 micros resemble the oscillation of the micros kinetics of P680(.) reduction in samples with an intact water oxidizing complex, (b) the quantum yield of total DF emission in the range up to a few micros is estimated to be <10(-4) for thylakoids, (c) the DF of D1/D2/Cytb-559 exhibits a monophasic decay with tau approximately 50 ns, (d) DF emission is also observed in isolated LHCII with biphasic decay kinetics characterized by tau values of 65 ns and about 800 ns, (e) in contrast to thylakoids, the amplitudes of DF in D1/D2/Cytb-559 preparations and solubilized LHCII do not exhibit any oscillation pattern and (f) all spectra of DF from the different sample types are characteristic for emission from the lowest excited singlet state of chlorophyll a. The implications of these findings and problems to be addressed in future research are briefly discussed.     

Cryopreservation of granulocytes for transfusion: Studies on human granulocyte isolation,the effect of glycerol on lysosomes,kinetics of glycerol uptake and cryopreservation with dimethyl sulfoxide and glycerol

Existing methods for the cryopreservation of granulocytes employ primarily dimethyl sulfoxide (Me₂SO) rather than glycerol as the cryoprotective additive of choice. Although Me₂SO has been demonstrated to be an effective cryoprotective additive for granulocyte preservation to yield viable cells (dye exclusion, phagocytosis, etc.), the inherent toxicity and clinical objections of Me₂SO as a cryoprotective additive for granulocyte preservation preclude its extensive and routine use in patients. Therefore, glycerol, with its important advantage of nontoxicity, has been investigated for its potential usefulness as a cryoprotective additive for preserving human granulocytes for transfusion.Granulocyte preparations were isolated from impure leukocyte concentrates obtained from the buffy coats of human whole blood. Studies on the isolation and purification of the granulocytes involved separation by sedimentation with dextran, removal of red cells by hypotonic shock with water, resuspension with Plasmatein and further purification by centrifugation. Intact viable granulocytes were obtained with a purity in excess of 90%.Lysosomes were studied as indicators of cryoinjury in granulocytes using β-glucuronidase as the key marker enzyme. This enzyme has been characterized as a sensitive indicator of damage to lysosomes and a direct linear relationship has been established between damage to granulocytes by freezing and amount of lysosomal enzyme released. Addition or presence of the cryoprotectant, glycerol, did not appear to have any adverse effect on lysosomes of intact granulocytes.Studies on the permeation kinetics of glycerol in granulocytes indicated that the additive was freely permeable and did not cause any potentially damaging osmotic changes in cell volume. Granulocytes in various concentrations of glycerol were then frozen at slow, moderate, and rapid cooling rates. Based on the small amount of β-glucuronidase released, good preservation of granulocyte lysosomes has been obtained with a slow cooling rate of 5 °C/min and a concentration of 15% glycerol. Further studies now are necessary to define those conditions of cooling rate and glycerol concentration required to develop a simple method for optimal preservation of granulocytes based on additional functional criteria of viability.     

Absorption, excretion and retention of 51Cr from labelled Cr-(III)-picolinate in rats

The bioavailability of chromium from Cr-picolinate (CrPic₃) and Cr-chloride (CrCl₃) was studied in rats using ⁵¹Cr-labelled compounds and whole-body-counting. The intestinal absorption of Cr was twice as high from CrPic₃ (1.16% vs 0.55%) than from CrCl₃, however most of the absorbed ⁵¹Cr from CrPic₃ was excreted into the urine within 24 h. After i.v. or i.p. injection, the whole-body retention curves fitted well to a multiexponential function, demonstrating that plasma chromium is in equilibrium with three pools. For CrPic₃, a large pool exists with a very rapid exchange (T _1/2 = <0.5 days), suggesting that CrPic₃ is absorbed as intact molecule, from which the main part is directly excreted by the kidney before degradation of the chromium complex in the liver can occur. CrCl₃ is less well absorbed but the rapid exchange pool is much smaller, resulting in even higher Cr concentrations in tissue such as muscle and fat. However, 1–3 days after application, the relative distribution of ⁵¹Cr from both compounds was similar in all tissues studied, indicating that both compounds contribute to the same storage pool. In summary, the bioavailability of CrPic₃ in rats is not superior compared to CrCl₃.     

基于电子回流理论的植物延迟荧光来源理论与实验研究

延迟荧光(DF)技术在植物学、农业和环境等领域有着广泛的应用前景,延迟荧光来源的机理研究对其应用起着重要的作用。基于广泛接受的电子回流理论由一系列的光化学反应方程推导得出多指数衰减的延迟荧光动力学模型,首次提出了一个简化的三指数衰减近似模型,描述了光合电子传递链上的三个最重要成分的电子回流。为了验证近似模型的准确性,以实验室培养的相似的8周大的Jin Dan No.39玉米叶片为模型样品,利用实验室自制的延迟荧光探测系统,得到延迟荧光衰减动力学曲线。分别用三种电子阻断剂:DCMU、DB-MIB和Paraquat处理后的衰减动力学的实验与理论模型进行了对比研究,通过曲线特征来确定不同成分的存在及依赖关系。结果表明延迟荧光衰减动力学三指数拟合非常好(R2=0.99909),而且这三种成分是相互独立的,分别来源于QA,QB和光系统Ⅰ环式电子流。简化模型的提出为延迟荧光的应用题提供一个平台。     

Lymphocyte and granulocyte migration across the endothelial layer of bovine pulmonary artery intimal explants towards lymphocyte conditioned medium

An intravenous infusion of endotoxin into sheep results in accumulation of equal numbers of lymphocytes and granulocytes in the pulmonary microcirculation. The role of the sequestered lymphocytes in acute lung injury is not known. The present study examines whether lymphocyte migration through pulmonary endothelium contributes to endothelial damage and also examines the effect of lymphokines on granulocyte migration. Bovine pulmonary artery intimal explants were mounted in Boyden chambers and conditioned media, prepared from bovine peripheral blood lymphocytes, was used as the chemoattractant. The rate of 51Cr labelled bovine granulocyte lymphocyte migration into intimal explants was determined over a 3 hr incubation period. Permeability changes were assessed by adding trace amounts of 14C-sucrose and 3H-water to the upper well and following their rate of equilibration with the lower well. 6-Keto-PGF1 alpha was measured in the upper well. Lymphocyte conditioned media was found to be chemotactic for both lymphocytes and granulocytes (lymphocyte migration at 60 min: lymphocyte conditioned media = 18.5 +/- 2.3%, mean +/- s.e. RPMI control = 12.5 +/- 1.5; granulocyte migration at 120 min: conditioned media = 36.1 +/- 5.7, RPMI control = 18.2 +/- 3.0). Ultrastructural examination revealed leukocyte migration followed an orderly sequence during which the leukocytes maintained close contact with the adjacent endothelial cells. No structural evidence of endothelial cell damage was seen at any time examined. Granulocyte migration was associated with an increased rate of 14C-sucrose equilibration after 2 hr of incubation (lower well counts/upper well counts at 2 hr, RPMI control = 0.18 +/- 0.02; lymphocyte conditioned medium = 0.30 +/- 0.04) indicating alteration in the endothelial barrier function. Leukocyte migration, particularly lymphocyte migration, was accompanied by a marked increase in prostacyclin accumulation (3 hr: no leukocytes, 188 +/- 17 ng/ml; lymphocytes, 560 +/- 104). These in vitro findings suggest that lymphocytes and lymphokines may be involved in acute lung injury and also that permeability changes associated with granulocyte migration may depend on the chemoattractant.     

Kinetics of neutrophil-releasing activity of filtration leukapheresis and postleukapheresis plasma

A rat model was used to study the effect of filtration leukapheresis on the kinetics of granulocyte mobilization in normal rat donors and in recipients of plasma obtained from these donors. Our earlier observations showed that infusion of postpheresis plasma (PPP) into normal homologous recipients is consistently capable of inducing a marked granulocytosis. The effects of such a transfusion is dose related and duration dependent. This study shows that the granulocytosis occurring subsequent to administration of PPP was directly related to the duration of pheresis in the donor. Its maximum effect occurred 2–3 hr after transfusion. Animals with a low granulocyte count were less capable of mobilizing granulocytes during filtration leukapheresis than animals with a higher prepheresis count, possibly due to low endogenous stores of granulopoietin. Additionally, rats < 300 g were capable of mobilizing fewer granulocytes per volume of circulating blood than were larger (older?) animals. The maximum ability of PPP to increase circulating granulocyte numbers was found when postpheresis plasma was injected along with concurrent removal of circulating granulocytes by filtration leukapheresis.     

Granulocyte and macrophage proliferation after injection of antitumor active and inactive strains of Corynebacterium parvum

Summary Corparvax, a strain of Corynebacterium parvum with strong antitumor activity, had a greater and more prolonged effect of increasing the production of granulocytes and macrophages than did a weak antitumor strain, CN5888. Following the injection of Coparvax to mice, there was a prompt and sustained increase in serum granulocyte/macrophage colony-stimulating activity, an increase in the number of spleen granulocyte/macrophage progenitor cells, an increased rate of proliferation of the bone marrow granulocyte/macrophage progenitor cells and an increase in the number of blood granulocytes and monocytes. The time courses of the increased rates of proliferation of granulocyte/macrophage progenitor cells following the injection of Coparvax were different in the bone marrow and the spleen, suggesting that local microenvironmental factors were also important.If immunostimulants such as C. parvum are to be used in chemoimmunotherapy programs, the kinetics of the increased proliferative rate of the granulocyte/macrophage progenitor cells may be important, since the more rapidly proliferating cells will be more affected by cell cycle-active chemotherapeutic agents.with the technical assistance of Beverly M. Dunne and L. Atherton