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.     

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.     

Chicken thrombocytes in culture: lymphocyte-conditioned medium delays apoptosis.

Chicken thrombocytes are nucleated cells, analogs to mammalian platelets. These cells are involved in hemostasis, phagocytosis and secretion of specific products. Most of the properties of avian thrombocytes have been established in experiments that employed recently isolated blood cells. Attempts to cultivate these cells for a long period of time under optimal culture conditions for peripheral blood cells were unsuccessful; thrombocytes died after 24 h of cultivation unlike macrophages cocultured with them. Here we investigate the reasons and type of thrombocyte death in culture. Thrombocytes were separated from peripheral blood of roosters and cultured for 48 h. The influence of different culture conditions on thrombocyte viability was studied. Cells were cultured as adherent cell monolayers or under agitation (preventing adherence), in the presence or lack of lymphocytes or their soluble factors, and various concentrations of fetal bovine serum. After 24 h in standard culture thrombocytes displayed cytoplasm and chromatin condensation, DNA cleaved into oligonucleosomal fragments and unaltered mitochondria. These results strongly suggest that thrombocytes suffer an apoptotic cell death in culture. Apoptosis could be delayed by culturing thrombocytes in the presence of lymphocytes or their soluble factors.     

Erythrocyte senescence and haematological changes induced by starvation in the neotropical fish traíra, Hoplias malabaricus (Characiformes, Erythrinidae)

Adult specimens of traira (Hoplias malabaricus Bloch) were subjected to long-term starvation (30 to 240 days) and re-fed for 30 days after 90 and 240 days of food deprivation. Counting of immature erythrocytes in peripheral blood showed that erythropoiesis decreased significantly during the first 30 days of food deprivation. The results suggest that a process of senescence takes place in the pre-existent red blood cells and that the cells are not replaced during starvation. After 240 days of starvation, H. malabaricus had a significantly reduced number of red blood cells, causing changes in hematocrit and blood indices (mean corpuscular volume, mean corpuscular haemoglobin and mean corpuscular haemoglobin concentration). Furthermore, during this period, the fish presented leukopenia (lymphocytopenia) and thrombocytopenia. After re-feeding, the number of leukocytes and thrombocytes recovered, but the red blood cell number remained reduced and there was a significant increase in abnormal red cell nuclei.     

Cytogenetic effects of methotrexate on human cells in vivo: comparison between results obtained by chromosome studies on bone-marrow cells and blood lymphocytes and by the micronucleus test

Cytogenetic studies were performed in 22 patients treated with methotrexate (MTX). In some patients, metaphases from both bone-marrow cells and peripheral blood cells were studied. In the bone-marrow preparations an increased number of structural chromosomal aberrations was present, whereas abnormalities were not observed in the peripheral blood cells. An examination of the bone-marrow chromosomes must therefore be included in the study of the possible chromosome-breaking effect of chemical agents. The results obtained with the micronucleus test and chromosome studies were compared in 10 patients treated with MTX. The micronucleus test was more sensitive than the chromosome analysis as regards the clastogenic effect of MTX.     

Cell types in peripheral blood of the nurse shark: An approach to structure and function

Ultrastructural and functional studies were carried out on nurse shark (Ginglymostoma cirratum) peripheral blood cells in order to identify cells of definitive morphology and specific function. Along with erythrocytes and thrombocytes, four morphologically distinct leucocytes are recognized in peripheral blood: two types of granulocytes, the ‘eosiniphil’ and the ‘granulocyte’, and two mononuclear agranulocytic cells, one resembling mammalian macrophage and monocyte, the other resembling mammalian lymphocyte. Also present in peripheral circulation are blast-like cells and mitotic cells. In vitro phagocytosis was demonstrated by the monocyte-macrophage and the granulocyte while thrombocytes, eosinophils and lymphocytes showed no phagocytic activity in the system studied. It is stressed that care must be used in drawing functional analogies between blood cells of a mammal and an elasmobranch on the basis of morphological similarity alone.     

Characterization of a monoclonal antibody specific to rainbow trout thrombocytes.

A monoclonal antibody (MAb) specific for rainbow trout thrombocytes was produced and its reactivity was demonstrated by flow cytometry and immuno-electron microscopy. Flow cytometry analysis showed that this MAb (TTL-7D11) reacted positively with about 30% of the peripheral blood leucocytes (PBL) and about 1%, 2%, and 11% of the pronephros, mesonephros, and spleen cells, respectively. Electron microscopy using immunogold labeling demonstrated that this MAb reacted strongly with thrombocytes, where gold beads could be seen attached only to the membrane and canalicular system of these cells. Positive and negative leucocytes for this MAb were obtained by magnetic cell separation. In the positive fraction, 96% of the cells were thrombocytes, while in the negative fraction no more than 3% were, which clearly showed a high purity of the positive fraction. Aggregation studies showed that about 75% of the positive fraction cells aggregated after being mixed with U-46619 thromboxane-mimetic, whereas in the negative fraction only 10% of the cells did so. Thus, utilizing the TTL-7D11 we have succeeded in isolating a pure thrombocyte population, and this would facilitate further studies, particularly on their characteristics and function(s).     

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.     

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.     

The effect of temperature on non-specific defence parameters of three strains of juvenile Atlantic halibut (Hippoglossus hippoglossus L.)

The effect of temperature (8, 12, 15 and 18 degrees C) on a variety of non-specific defence and haematological parameters was examined in three geographically distinct reared strains (Canadian, Icelandic, Norwegian) of Atlantic halibut. The results indicate that temperature exerts a considerable influence on some blood parameters (packed cell volume and the percentage population of leucocytes in peripheral blood) and on some humoral parameters (serum lysozyme activity and serum protein levels) of halibut. A high temperature of 18 degrees C caused a decrease in the number of circulating blood cells and an increase in serum lysozyme levels; effects consistent with those reported within the literature for stress. The different strains of halibut exhibited differing responses with respect to differential counts of peripheral blood lymphocytes and thrombocytes, and to serum protein concentrations, serum lysozyme activity, serum iron content, unsaturated iron binding capacity of serum and O2- production by kidney macrophages.     

Temperature-mediated processes in teleost immunity: homeoviscous adaptation by channel catfish peripheral blood cells

1. Channel catfish peripheral blood erythrocyte, thrombocyte, T cell and B cell membranes were assayed by fluorescence depolarization using the fluorescent probe 1,6-diphenyl-1,3,5 hexatriene (DPH) to determine the effects of in vivo temperature acclimation on membrane viscosity and the kinetics of homeoviscous adaptation. 2. Erythrocyte membranes did not undergo homeoviscous adaptation during the 8 week time period studied and were more rigid compared with those of the other cell types. 3. The kinetics of homeoviscous adaptation exhibited by membranes from T cells, B cells and thrombocytes differed: B cells required 1-3 weeks while T cells and thrombocytes each required 3-5 weeks. Membranes from T cells, B cells and thrombocytes from fish acclimated for relatively short times (less than or equal to 3 weeks) exhibited similar membrane fluidities. 4. T cells from channel catfish appeared not only to be sensitive to temperature but also to a factor(s) independent of temperature but correlated to long term in vivo acclimation, i.e. T cell membranes underwent additional decreases in membrane viscosity between 3 and 5 weeks. 5. In conclusion, it appears that low temperature-mediated immunosuppression of T cell functions in channel catfish is probably not due to an inherent non-adaptability or rigid nature of the T cell membranes.     

Activation of purified human thymus-derived (T) cells by mitogens. II. Monocyte- macrophage potentiation of mitogen-induced DNA synthesis.

Thymus-derived (T) cells from peripheral blood were purified by rosette formation with neuraminidase-treated sheep red blood cells (SRBC) and centifugation on Ficoll-Hypaque. T cells recovered from the pellet were freed of SRBC by treatment with Tris-NH4Cl. T cells purified by this method showed a diminished ability to take up 3H-thymidine (3H-TdR) after mitogen stimulation when compared to the mitogenic response of an equal number of autologous peripheral blood mononuclear lymphocytes (PBL). Autologous monocytes restored the capacity of purified T cells to take up 3H-TdR in the presence of phytohemagglutinin (PHA) or Concanavalin A (Con A). The effect was proportional to the number of monocytes added. Similar restorative effects could be obtained with allogeneic or xenogeneic monocytes. These data suggest that the mitogenic stimulation of human PBL and Con A may reflect the participation of more than one cell type: the T cells and monocyte and that the genetic origin of the monocyte is not critical for augmentation of the mitogenic activation of human T cells.     

Composition of main haematopoietic compartments in normal and bled channel catfish

Differential cell counts showed that the head and trunk kidney of control and bled channel catfish Ictalurus punctatus had myeloid characteristics. They contained lymphoid and granuloid cells, thrombocytes, erythroid and agranular cells in decreasing order of abundance (%). Among the blast and precursor cells, the most numerous erythroid ones were followed by granuloid, lymphoid and agranular ones. The main changes after blood withdrawal were the decrease of thrombocytes and the increase of precursor cells in both kidney parts. In the group examined 7 days after bleeding the head kidney had a higher percentage of erythroid cells and lymphocyte precursors than the trunk kidney while the latter had more granuloid cells and their precursors. Basophils were present ( c . 1%) in both regions of the kidney of all groups. The spleen was predominantly a lymphatic organ. It contained c . 80% lymphoid cells, a higher incidence of granulated lymphocytes than in kidneys, 15% thrombocytes and 1.4% agranular cells. Blood withdrawal caused an increase of thrombocytes, a decrease of lymphoid cells and an increase of erythroid precursors in the spleen. The last probably stemmed from the circulation. While haematocrit values failed to indicate the anaemic state in the bled groups, the differential red blood cell count showed dramatic differences between the control and bled groups as well as between the two groups in different stages of recuperation from the blood loss.     

A model of Ph' positive chronic myeloid leukemia-blast crisis cell line growth in immunodeficient SCID mice.

A human Philadelphia-chromosome positive chronic myeloid leukemia-blast crisis (CML-BC) cell line BV173 proliferated in the hematopoietic tissues, infiltrated various organs and caused the death of immunodeficient SCID mice. Leukemia spreading was assessed with diminished number of bone marrow cells and caused splenomegaly. The leukemic colonies grew from single cell suspension of bone marrow, spleen and peripheral blood. Bcr-abl m-RNA was detectable in bone marrow, spleen, peripheral blood, liver, lungs and brain. Dying mice demonstrated severely hypoplastic bone marrow, splenomegaly and massive metastases in the liver and kidneys. The survival time of animals was dependent on the number of inoculated leukemia cells.     

Changes in mouse erythropoiesis during continuous irradiation with a daily dose of 957 mGy

Quantitative and qualitative changes in the blood producing organs and in the peripheral blood of mice are evaluated in this paper. The animals were irradiated for 42 days continually with a daily dosage of 957 mGy. Until the 7th day of irradiation a significant diminution of the cellularity of the bone-marrow and of the cellularity as well as the mass of the spleen could be observed. After the 14th day or irradiation a temporary stabilization of the cell number in the bone-marrow could be found until the 28th day, after that time there was a moderately strong decrease. The cellularity and the mass of the spleen increased temporarily until the 28th day of irradiation because of erythropoiesis and myelopoiesis increasing from 20% to 50%. The most significant changes in the peripheral blood could be observed in not granulated cells as a kind of sudden and permanent decrease. The diminution of the granulocyte and reticulocyte number proceeded somewhat more slowly, temporarily revealing an increasing tendency on the 28th day of irradiation. The erythrocyte numbers as well as the haematocrit and haemoglobin values decreased continually beginning from the 7th day of irradiation until the death of the test animals.     

Autoantibodies to the thrombocytes and erythrocytes in HIV-infected patients

In 48 patients with HIV infection were tested for the presence of autologous and allogenic antibodies to red blood cells with the use of Coombs' direct and indirect tests. 18 HIV-infected patients had IgG antibodies to thrombocytes, circulating in the blood (detected by the method of EIA) and bound to thrombocytes (detected by the method of RIA). In 5 out of 48 patients Coombs' direct test yielded positive results with red blood cells. 6 out of 18 examined patients had an elevated content of thrombocyte-bound antibodies. The presence of cross reactions between gp120 of human immunodeficiency virus and gp3a thrombocytes led to the formation of antithrombocyte antibodies and, consequently, to a decrease in the number of thrombocytes.     

Chicken TREM-B1, an Inhibitory Ig-Like Receptor Expressed on Chicken Thrombocytes

Triggering receptors expressed on myeloid cells (TREM) form a multigene family of immunoregulatory Ig-like receptors and play important roles in the regulation of innate and adaptive immunity. In chickens, three members of the TREM family have been identified on chromosome 26. One of them is TREM-B1 which possesses two V-set Ig-domains, an uncharged transmembrane region and a long cytoplasmic tail with one ITSM and two ITIMs indicating an inhibitory function. We generated specific monoclonal antibodies by immunizing a Balb/c mouse with a TREM-B1-FLAG transfected BWZ.36 cell line and tested the hybridoma supernatants on TREM-B1-FLAG transfected 2D8 cells. We obtained two different antibodies specific for TREM-B1, mab 7E8 (mouse IgG1) and mab 1E9 (mouse IgG2a) which were used for cell surface staining. Single and double staining of different tissues, including whole blood preparations, revealed expression on thrombocytes. Next we investigated the biochemical properties of TREM-B1 by using the specific mab 1E9 for immunoprecipitation of either lysates of surface biotinylated peripheral blood cells or stably transfected 2D8 cells. Staining with streptavidin coupled horse radish peroxidase revealed a glycosylated monomeric protein of about 50 kDa. Furthermore we used the stably transfected 2D8 cell line for analyzing the cytoplasmic tyrosine based signaling motifs. After pervanadate treatment, we detected phosphorylation of the tyrosine residues and subsequent recruitment of the tyrosine specific protein phosphatase SHP-2, indicating an inhibitory potential for TREM-B1. We also showed the inhibitory effect of TREM-B1 in chicken thrombocytes using a CD107 degranulation assay. Crosslinking of TREM-B1 on activated primary thrombocytes resulted in decreased CD107 surface expression of about 50–70%.     

Haematological and haematopoietic responses to starvation in an air-breathing fish Channapunctatus Bloch

Changes in haematological values (RBC numbers, haemoglobin content, haematocrit value, MCV, MCH, MCHC, TLC and DLC) based on weekly samples from a group of starved fish were investigated. After 8 weeks of starvation, the effects of restoration to a normal diet was evaluated. Parallel studies on haematopoietic tissues were also made. Changes in some biochemical values such as blood glucose, liver and muscle glycogen were also examined to correlate biochemical effects with those of haematological changes. Erythrocytes, thrombocytes and neutrophils were found to be most sensitive to starvation. The initial response to deprivation of food was an increase in RBCs and related values and in total leukocyte population. However, from week 5 onwards a sharp decline in these cell populations was noted. The leukocytes and thrombocytes showed a change parallel to RBC and the total leukocyte counts. However, neutrophils were observed to show a consistent increase throughout the starvation period. A blood glucose level below SOmglOOmh¹ appeared critical in relation to blood cell population. Haematopoietic studies revealed that reticulocytes and mesomyelocytes were unable to keep pace with the changing peripheral blood picture. Other stages in development responded to the changes in the peripheral blood.     

Differential spontaneous killing of human and murine tumour cell lines by leucocyte subpopulations from carp peripheral blood leucocytes

Cell populations from carp (Cyprinus carpio L.) peripheral blood leucocytes (PBLs) were examined for nonspecific cytotoxicities. By using monoclonal antibodies (MAbs) against carp thrombocytes (TCL-HB8) and both neutrophils and monocytes (TCL-BE8), PBLs with a density of 1.08 g ml-1 were separated into three fractions: thrombocytes, a mixture of neutrophils and monocytes, and other cells (mainly lymphocytes), and the separated cells were tested for cytotoxic activities against mammalian tumour cell lines (K562, HeLa, P815 and Yac-1 cell). Consequently, the mixture of neutrophils and monocytes exhibited cytolysis against these target cells, whereas the lymphocyte-rich and thrombocyte fractions did not show any cytolysis. To isolate only neutrophils, which do not contain monocytes, the MAb (TCL-BE8) positive cells from PBLs with a density of 1.08-1.09 g ml-1 were separated. Pure isolated neutrophils showed cytotoxic activities against K562 cells, but not P815 cells. Furthermore, analysis of the cytolytic mechanisms indicated that killing of these cells depended on H2O2 or HOCl. These results suggest that both neutrophils and monocytes are effectors for nonspecific cytotoxicity in carp PBLs, and neutrophils may be distinct from monocytes in their reactivity in cytolysis, including target cell selectivity and/or target cell sensitivity, and the cytolytic pathway. In carp, cytotoxicity of target cells can be mediated by several populations of their leucocytes which have cytotoxic capacities with various recognition and cytolytic mechanisms.     

Chronic alcohol consumption impairs distribution and compromises circulation of B cells in B16BL6 melanoma-bearing mice

Accumulating research indicates that B cells are involved in anti-tumor immunity. Chronic alcohol consumption is associated with decreased survival of cancer patients. The effect of alcohol consumption on B cells in tumor-bearing hosts is unknown. Results in melanoma-bearing mice showed that chronic alcohol consumption did not alter the percentage and number of B cells in bone marrow, spleen, and lymph nodes but dramatically decreased B cells in the peripheral blood. Alcohol consumption did not alter the development of B cells in the bone marrow and did not affect follicular B cells in the spleen; however, it increased T1 B cells and decreased marginal zone B cells in the spleen. Alcohol consumption also decreased mature B cells in the blood. It did not alter the chemotactic capacity of plasma to facilitate migration of splenocytes or the chemotactic response of splenocytes to CXCL13 and CCL21. However, the response of splenocytes to sphingosine-1-phosphate was impaired in alcohol-consuming, melanoma-bearing mice. The expression of sphingosine-1-phosphate receptor-1 (S1PR1) and sphingosine-1-phosphate lyase-1 (SPL1) in splenocytes was downregulated. Taken together, these results indicate that chronic alcohol consumption decreases peripheral blood B cells by compromising B cell egress from the spleen. The downregulation of S1PR1 and SPL1 expression in alcohol-consuming, melanoma-bearing mice could be associated with compromised egress of B cells from the spleen.