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Biomedical subjects

S Ebbe

Publications and source records attributed to S Ebbe.

At least 19 recordsLinked to original sources
PubMed1996

Uptake of indium-111-labeled platelets and indium-111 oxine by murine kidneys after total-body irradiation.

Radiation nephropathy is a well-known late manifestation of renal irradiation in human beings and experimental animals. Its pathogenesis is unclear, but vascular injury may play a role. Endothelial cells have been demonstrated to manifest a variety of abnormalities within hours of exposure to radiation. In the present experiments mice were exposed to lethal doses of whole-body radiation, and the distribution of 111In-labeled platelets was evaluated during the first week after irradiation. The purpose was to determine if early abnormalities of endothelial cells would be manifested by altered sequestration of platelets in kidneys and other organs. It was found that the indium accumulated in the kidneys of irradiated mice to a greater extent than in nonirradiated mice, supporting the possibility of early vascular injury. In control experiments, administration of 111In-oxine was also followed by excessive accumulation of radioactivity in kidneys of irradiated mice, but the pattern of accumulation differed from that seen after injection of radiolabeled platelets. Renal hyperemia was not demonstrable with 51Cr-labeled red cells, renal vascular permeability was not detected with 125I-labeled albumin, and the pattern of renal uptake of plasma proteins labeled with 59Fe or 111In did not coincide with that seen from 111In administered as labeled platelets or oxine. Renal uptake of 111In-oxine was not associated with alterations in urinary or fecal excretion or an increase in total-body retention of the radioisotope. The findings are consistent with the notion that renal vascular injury at the time of irradiation results in accumulation of platelets or platelet constituents during the first week after total-body irradiation of mice.

Animals↗
PubMed1996

Evaluation of ploidy of mature canine megakaryocytes, using Feulgen staining and microspectrophotometry.

OBJECTIVE: To evaluate megakaryocyte size and ploidy, using Feulgen staining and microspectrophotometry, in adult dogs with normal platelet count. ANIMALS: Group A contained 8 and group B contained 11 adult dogs. PROCEDURE: Megakaryocytes were evaluated by light microscopy and staged according to maturation status. Stage-III megakaryocytes were measured and mapped for future relocation. Bone marrow aspirates were destained and restained, using the Feulgen method. Previously identified stage-III megakaryocytes were measured for DNA content, using microspectrophotometry. RESULTS: Megakaryocyte size correlated with ploidy values, and mean sizes within ploidy groups were significantly (P < 0.05) different from each other for both groups. The model ploidy value of stage-III megakaryocytes, which represented 18% of the total megakaryocyte population of the combined groups, was 32N. This is in contrast to results of flow cytometric studies, which indicated that the modal ploidy value for all canine megakaryocytes was 16N. CONCLUSIONS: Reasons for the disparate results between microspectrophotometric techniques and flow cytometry include maturation stage of the megakaryocyte population evaluated and percentage of megakaryocytes within that maturation stage. Flow cytometric methods, which evaluate all megakaryocytes detectable by antibody, may include cells still capable of DNA synthesis, resulting in a shift in the observed modal ploidy value. Recognition of the difference between canine and human megakaryocyte ploidy distribution is important, particularly in studies in which the dog is used as an animal model for human megakaryocytopoiesis.

Analysis of Variance↗
PubMed1994

Erythropoiesis, but not thrombocytopoiesis, is affected by the presence of a large amount of subcutaneously implanted bone marrow.

The concept that megakaryocytopoiesis is regulated in part by the number of megakaryocytes themselves, separately from its regulation by the number of platelets, is referred to as autoregulation. Its occurrence has been implied from animal models that demonstrate compensated megakaryocytopenia. In that condition, normal platelet production can occur even though numbers of megakaryocytes are substantially reduced, and individual megakaryocytes exhibit changes consistent with their being stimulated. The present experiments were undertaken to determine whether the converse situation, namely suppression of megakaryocytopoiesis by an excess of megakaryocytes, could be created. The experimental model consisted of C57Bl mice in which as many as 10 isogeneic femurs were implanted subcutaneously 10 weeks before analysis. The implanted femurs contained hematopoietic marrow morphologically. They incorporated radioiron, but only about 40% as much as an equal number of normal femurs, so hematopoiesis did not regenerate to the full potential of the implanted marrows. After implantation of nine or 10 femurs, thrombocytopoiesis was normal as judged by platelet counts, mean platelet volumes, numbers of megakaryocytes in tibial marrow, and megakaryocyte sizes. Hematocrits were increased, but plasma erythropoietin levels were normal. The implants showed a 15- to 20-hour incorporation of radioiron of 9.4 +/- 0.5%. Iron incorporation into endogenous bones and spleen was reduced, demonstrating that erythropoiesis was redistributed. The total incorporation of iron into all hematopoietic tissue was slightly increased, but red-cell iron incorporation was normal, implying that there was more ineffective erythropoiesis than normal. Leukocyte counts, differentials, and tibial cellularity were normal. After implantation of three or six femurs, no abnormalities of hematopoiesis were detected, even though the implants incorporated iron in proportion to their number. Thus, erythropoiesis was adjusted by the excess of total marrow caused by nine or 10 subcutaneously implanted femurs. This is about the maximum of implanted marrow that is feasible with this model, and the failure to observe suppression of megakaryocytopoiesis may have been due to an inability to achieve a large enough number of megakaryocytes to elicit a detectable response. Alternatively, autoregulation of megakaryocytes may depend more on the marrow concentration of megakaryocytes than on the total body content.

Animals↗
PubMed1992

Microcytic thrombocytosis, small megakaryocytes, platelet lipids and hyperreactivity to collagen, lymphocytopenia, eosinophilia, and low blood volume in genetically hyperlipidemic rabbits.

Three- to 15-month-old rabbits with Watanabe heritable hyperlipidemia (WHHL) were tested to determine if hematological abnormalities would accompany the known hyperlipidemia and deficiency of receptors for low-density lipoprotein; the findings were compared to those of New Zealand white (NZW) rabbits of the same ages. WHHL plasma cholesterol and triglyceride levels were always greater than or equal to 6x those of NZW, and both were lower in older than in younger WHHL rabbits. From age 7 to 18 months, WHHL platelet counts were higher than those of age-matched NZW; the average for all WHHL was 1.45x that for NZW (p = 0.001). Average WHHL mean platelet volume (MPV) was 0.94x that for NZW (p = 0.025), with a tendency for greater microcytosis to occur at more advanced ages; electron microscopy supported the small size of WHHL platelets. WHHL platelet mass per microliter of blood (platelet count x MPV) was 1.39x that of NZW (p = 0.001), with differences occurring after the age of 6 months. The average WHHL blood volume was 18.3% less than in NZW (33.5 vs 41.0 ml/kg body weight) (p = 0.00005), so platelet count and mass per kilogram of body weight were similar in the two strains. The predominant ploidy of mature megakaryocytes from each strain was 32N; megakaryocytes were smaller in WHHL than NZW due to a smaller size of 32N cells (p = 0.002). Total leukocyte counts were the same in WHHL and NZW rabbits, but eosinophils were 32% higher (p = 0.037) and lymphocytes 34% lower (p = 0.008) in WHHL. Hematocrits and reticulocytes did not differ. Platelet-free cholesterol was 1.2x, esterified cholesterol 11.3x, phospholipids 0.9x, triglyceride 2.4x, and free cholesterol/phospholipid molar ratio 1.29x corresponding values in NZW platelets. WHHL platelets released more serotonin in response to a small dose (5 micrograms/ml) of collagen than did NZW platelets. These findings suggest that: 1) megakaryocytopoiesis and leukopoiesis are affected by lipid metabolism and/or LDL receptors, and 2) platelet production may be regulated more by the total mass of platelets than by their concentration in the blood.

Animals↗
PubMed1991

Regulation of murine megakaryocyte size and ploidy by non-platelet-dependent mechanisms in radiation-induced megakaryocytopenia.

Megakaryocytic macrocytosis was evaluated in mice after irradiation with 6.5 Gy 60Co gamma rays. During the second and third months after sublethal irradiation, one or more of the following abnormalities of thrombocytopoiesis was present: thrombocytopenia, megakaryocytopenia, macromegakaryocytosis, a shift to higher ploidies, and enlargement of cells within ploidy groups. After transfusion-induced thrombocytosis, reductions in megakaryocyte size were delayed or absent relative to non-irradiated mice, and there was more of a tendency to shift to lower values for megakaryocyte ploidy. Mice with radiation-induced megakaryocytopenia failed to show rebound thrombocytosis during recovery from immunothrombocytopenia, in spite of further increases in megakaryocyte size and ploidy. The findings support the hypotheses that numbers of megakaryocytes may influence the regulation of megakaryocytopoiesis even when there is an excess of platelets and that ploidy distribution is not the sole determinant of the average size of a population of megakaryocytes. After irradiation, persistent megakaryocytopenia may not severely affect platelet production under steady-state conditions, but the ability of the marrow to respond to homeostatic regulation is compromised.

Animals↗
PubMed1990

Biochemical and functional abnormalities in hypercholesterolemic rabbit platelets.

This study was designed to elucidate changes in rabbit platelet lipids induced by a cholesterol rich diet and to explore the possible correlation of these lipid changes with platelet abnormalities. Pronounced biochemical alterations were observed when serum cholesterol levels of 700-1000 mg% were reached. Hypercholesterolemic (HC) platelets contained 37% more neutral lipids and 16% less phospholipids than the controls. Lysolecithin, cholesterol esters and phosphatidylinositol (PI) levels were increased in HC platelets, and the levels of phosphatidylcholine (PC) were decreased. The cholesterol/phospholipid molar ratio of lipidemic platelets increased from 0.55 +/- 0.011 to 0.89 +/- 0.016 (P less than 0.01) in eight weeks. HC platelets had 90% more arachidonic acid (AA) in the PI than normal platelets. No significant changes in AA of PC were observed. Platelet function was monitored by the uptake and release of [14C]serotonin in platelet rich plasma (PRP), using varying concentrations of collagen as an aggregating agent. The uptake of [14C]serotonin in HC and normal platelets ranged from 78-94%. The percent of [14C]serotonin released from normal and HC platelets was proportional to the concentration of collagen. However, lipidemic platelets were hyperreactive to low concentrations of collagen. Incorporation of 50 microM acetylsalicylic acid into the aggregating medium suppressed the release of [14C]serotonin in normal PRP by more than 90%, but had only a partial effect on lipidemic PRP.

Animals↗
PubMed1989

Serum erythropoietin and hemoglobin affinity for oxygen in patients phlebotomized for polycythemia vera.

Serum erythropoietin (Ep) was measured by radioimmunoassay before and 24 hours after therapeutic phlebotomies in patients with polycythemia vera (PV) and in normal subjects before and after phlebotomies of comparable volumes. In addition the in vivo oxygen affinity of hemoglobin (P50) was calculated, and red cell indices and 2,3 DPG values were measured. Paired t tests determined whether the differences between pre- and post--phlebotomy values were statistically significant. Blood hemoglobin (Hb) levels declined after phlebotomy, and generally continued to be at or above normal levels. Serum Ep increased after phlebotomy in both groups of subjects. The in vivo P50 value for patients with PV (29.4 +/- 0.4 mmHg) was significantly (p less than 0.005) greater than the normal value (27.2 +/- 0.5). Seven of the PV patients (5 males, 2 females) were restudied. Their Hb and hematocrit values were either normal or slightly higher than normal. The MCV for 4 males and 1 female was below normal. The MCHC was slightly lower than normal and reticulocytosis was not present. The male PV patients had greater than normal 2,3 DPG values and most had right-shifted P50 values. There was no correlation between 2,3 DPG values and P50 values. The female patients did not have 2,3 DPG values consistently greater than normal and their P50 values were not right shifted. These results showed that serum EP increased in response to small reductions in Hb even when subnormal Hb values were not produced. The reduced affinity of Hb in PV patients may explain earlier observations that patients with PV have lower levels of urinary or plasma Ep than normals with the same hematocrits.

2,3-Diphosphoglycerate↗
PubMed1989

Megakaryocytopenia in W/Wv mice is accompanied by an increase in size within ploidy groups and acceleration of maturation.

Megakaryocytopoiesis was evaluated in W/Wv mice and their normal +/+ littermates to analyze the mechanisms by which normal platelet production is maintained in W/Wv mice even though numbers of megakaryocytes are low. Relative sizes of megakaryocytes, and their nuclei and cytoplasm, were measured microscopically in bone marrow smears, and the ploidy of the same cells was measured by two-wavelength microspectrophotometry. Maturation rate of megakaryocytes was estimated after they were labeled with tritiated thymidine. W/Wv megakaryocytes were macrocytic: average cell size was increased in each ploidy group. The increase in cytoplasmic area exceeded that of the nucleus. Further analysis of the predominant 16N ploidy group revealed that the increase in average cell size was due to depletion of cells of small size. Megakaryocytes matured more rapidly than normal in W/Wv mice. These results showed that megakaryocyte size and ploidy can be regulated separately. They suggest that alterations in cell growth and maturation may be mechanisms by which the organism can compensate for a deficiency in numbers of megakaryocytes, but they do not define the mechanism by which the deficiency may be sensed or by which the compensatory changes may be mediated. This is a US government work. There are no restrictions on its use.

Animals↗
PubMed1989

5-fluorouracil-induced thrombocytosis in mice is independent of the spleen and can be partially reproduced by repeated doses of cytosine arabinoside.

Experiments were done to characterize the pronounced and prolonged thrombocytosis that develops in mice during recovery from the marrow hypoplastic effects of a single injection of 5-fluorouracil (5-FU). Measurements were made of platelet and megakaryocyte numbers, size of mature megakaryocytes, marrow cellularity, hematocrit, and reticulocytes. Mice that had been splenectomized a month before receiving 5-FU displayed the same pattern of thrombocytosis as intact mice, so a role for the spleen in its genesis or persistence could not be identified. During recovery from two or three doses of cytosine arabinoside (Ara-C), given at intervals of 2 days, thrombocytosis and megakaryocytosis of similar magnitudes to those seen after 5-FU occurred. Bone marrow cellularity, hematocrits, and reticulocytes were identical after three doses of Ara-C or one dose of 5-FU. Megakaryocytes were initially macrocytic during recovery from the hypoplastic thrombocytopenia produced by 5-FU or Ara-C. These similarities to effects of repeated doses of Ara-C suggested that some of the effects of 5-FU were due to its prolonged action in vivo, which could cause sequential killing of proliferating cells. Abnormalities of megakaryocytes and platelets reversed promptly when normal or increased numbers of megakaryocytes and/or platelets were produced after Ara-C, but they persisted in mice that received 5-FU. This difference suggested that regulation of megakaryocyte progenitors was perturbed during recovery from 5-FU.

Animals↗
PubMed1988

Morphological and kinetic abnormalities of platelets in hypercholesterolemic rabbits.

Hypercholesterolemia (HC = hypercholesterolemia or hypercholesterolemic) was produced in rabbits by feeding them diets supplemented with cholesterol and peanut oil. Platelet counts and volumes, white cell counts, reticulocyte counts, and hematocrits were determined at intervals for 8-12 weeks in blood from HC animals and controls on a normal rabbit diet. Microthrombocytosis was a consistent occurrence in the presence of HC, developing as early as 2 weeks into the diet. Microthrombocytosis was generally associated with normal platelet counts, but mild thrombocytosis occurred late in the diet at the time of the highest levels of serum cholesterol (greater than 1300 mg/dl). Platelets from HC rabbits were morphologically normal by transmission electron microscopy. Survivals of 51Cr-labeled platelets from HC and non-HC rabbits were measured in HC and non-HC recipients. The results identified an intrinsic defect in the ability of HC platelets to survive in the circulation. They also confirmed previous findings of an environmental defect in HC that causes shortened platelet survival.

Animals↗
PubMed1988

Megakaryocytes increase in size within ploidy groups in response to the stimulus of thrombocytopenia.

Experiments were done to determine if sizes of megakaryocytes within a defined maturation stage were strictly determined by amount of nuclear DNA. Normal mice, mice recovering from an acute episode of thrombocytopenia induced by a single injection of heterologous antiplatelet serum (APS), and mice with sustained thrombocytopenia from daily injections of APS were examined. Areas of mature megakaryocytes were measured in bone marrow smears stained with polychromatic stains. The nuclear DNA content of the same cells was then measured microspectrophotometrically after staining by the Feulgen reaction. Normal megakaryocytes showed a trimodal, lognormal distribution of nuclear chromophore, corresponding to 8n, 16n, and 32n with smaller numbers of 4n and 64n cells; 16n was the predominant ploidy class. In response to thrombocytopenia, ploidy values shifted: the proportions of 8n and 16n cells decreased; 32n and 64n cells increased; 128n megakaryocytes occasionally appeared. These shifts were accompanied by an increase in the average size of all megakaryocytes. In addition to shifts to higher ploidy values, megakaryocytes within ploidy groups became larger than normal megakaryocytes of the same ploidy especially in the mice with sustained thrombocytopenia. These findings show that megakaryocyte size in thrombocytopenic mice is influenced by factors other than the ploidy and maturity of the cell.

Anemia↗
PubMed1988

Survival of rabbit platelets labeled with gallium 67.

The viability of rabbit platelets labeled with radioactive gallium was determined to analyze the feasibility of using platelets labeled with gallium 68 as an imaging reagent for positron emission tomography. Platelets were labeled with a complex of the longer lived gallium 67 and mercaptopyridine-N-oxide (MPO) or with sodium chromate Cr 51. Their survival after transfusion was measured. Labelling efficiency of 67Ga-MPO was 6.5% to 45.8% (26.8% +/- 2.8%) when platelets were suspended in saline solution, but was much lower (1.6% +/- 0.8%) in plasma. Platelets labeled with either radioisotope in a saline medium survived as well as platelets labeled with 51Cr in plasma. Recovery values 1 hour after transfusion and mean platelet survivals were 68.6% +/- 4.9% and 3.4 +/- 0.2 days for 67Ga in saline solution, 76.5% +/- 6.8% and 3.8 +/- 0.5 days for 51Cr in saline solution, and 73.7% +/- 7.4% and 3.6 +/- 0.5 days for 51Cr in plasma. Labeled platelet concentrates always contained extra radioactivity not firmly bound to viable platelets. A postlabeling wash in saline solution did not reduce this contamination and resulted in reduction of the number of viable platelets. The results showed that rabbit platelets labeled with 67Ga-MPO survived in the circulation as well as those labeled by a standard protocol with sodium chromate Cr 51.

Animals↗
PubMed1987

Thrombocytopoietic response to immunothrombocytopenia in nude mice.

Thrombocytopoiesis was evaluated in T cell-deficient nu/nu mice and in T cell-replete nu/+ controls to determine if abnormalities would be associated with the deficiency of T cells. Mice were studied in the unperturbed steady state and after acute immunothrombocytopenia was induced by an injection of guinea pig antimouse platelet serum (APS). The state of thrombocytopoiesis was determined from platelet counts, megakaryocyte size, megakaryocyte number, and numbers of Meg-CFC. Splenic lymphocytes were evaluated by response to the mitogens bacterial lipopolysaccharide (LPS), phytohemagglutinin (PHA), and concanavalin A (Con A). Hematocrits, reticulocyte counts, leukocyte counts, marrow cellularity, GM-CFC, and BFU-E also were measured. Steady state thrombocytopoiesis was identical in nu/nu and nu/+ mice. In response to an injection of APS, acute thrombocytopenia was followed by macromegakaryocytosis and rebound thrombocytosis in mice of both genotypes. Splenic Meg-CFC increased in nude mice after APS or an injection of normal guinea pig serum (NGpS), and splenic GM-CFC increased after APS. Neither Meg-CFC nor GM-CFC increased in the spleens of nu/+ mice, but they showed early transient increases in bone marrow that did not occur in nu/nu mice. Sporadic, but weak, mitogenic responses to PHA or Con A were occasionally observed with nu/nu spleen cells, but these did not correlate with the state of thrombocytopoiesis. The results demonstrated that platelet production was normal in nu/nu mice and that megakaryocytopoiesis and platelet production responded to the stimulus imposed by acute immunothrombocytopenia. Increases in megakaryocyte size and platelet production occurred independently of changes in numbers of Meg-CFC, GM-CFC, or BFU-E. A normal complement of T cells appears to be unnecessary for normal platelet production and its augmentation in response to the stimulus of acute immunothrombocytopenia in vivo.

Animals↗
PubMed1986

Postirradiation thrombocytopoiesis: suppression, recovery, compensatory states, and macromegakaryocytosis.

Two unusual features of the regulation of megakaryocytopoiesis have been found in irradiated mice. The first is that the response to thrombocytopenia loses its specificity for thrombocytopoiesis when the thrombocytopenia is induced at the time of exposure to sublethal doses of radiation. Under these conditions, there is stimulation of both thrombocytopoiesis and erythropoiesis. The second unusual feature is that a completely or partially compensated hypomegakaryocytic state may develop after "recovery" from the earlier severe postirradiation myelodepression. Occurrence of this condition is not dependent on the presence or absence of the spleen. It is characterized by a dissociation between platelet and megakaryocyte numbers, with platelets being relatively higher. There is an associated increase in mean megakaryocyte size. Both the megakaryocytopenia and macromegakaryocytosis are due to a deficiency of smaller megakaryocytes in the marrow. The postirradiation abnormality of megakaryocyte size distribution can not be accounted for by irradiation-induced abnormalities of either hemopoietic or stromal cells. The degree of megakaryocytic macrocytosis does not correlate with the platelet or megakaryocyte count after recovery from sublethal irradiation or after recovery from lethal irradiation and rescue with normal bone marrow cells. Megakaryocytic macrocytosis, as identified by an increase in average size of mature cells, occurs in response to thrombocytopenia and in several hypomegakaryocytic states in which thrombocytopenia is absent or is mild in degree. Comparison of size distribution curves, analysis of sizes of immature megakaryocytes, and determination of the stability of the megakaryocyte count indicate that different mechanisms probably prevail. The presence or absence of thrombopoietin or other megakaryocyte growth factors in these conditions may provide clues about the mechanisms.

Animals↗
PubMed1986

Functionally abnormal stromal cells and megakaryocyte size, ploidy, and ultrastructure in Sl/Sld mice.

The first goal of the present studies was to determine if Sl/Sld megakaryocytes have features in common with the macrocytic megakaryocytes that genetically normal mice produce in response to acute platelet depletion. The second was to test the hypothesis that megakaryocyte abnormalities in Sl/Sld mice are due to genetically determined hemopoietic stromal cell abnormalities. Sizes and ploidies of mature Sl/Sld megakaryocytes were measured. Macrocytosis and a shift to higher ploidy values were found compared with normal. Within ploidy groups 16N-64N, Sl/Sld megakaryocytes were larger than normal megakaryocytes of the same ploidy. Transmission electron microscopy revealed that Sl/Sld megakaryocyte nuclei contain more and larger nucleoli, and the chromatin was more dispersed than in normal megakaryocyte nuclei of comparable maturity. Asynchronous megakaryocyte cytoplasmic maturation was found. Sl/Sld macrophages were also ultrastructurally abnormal. Megakaryocytic macrocytosis was reproduced in long-term bone marrow cultures in which the adherent layer was formed by Sl/Sld cells. It was the same if cultures were recharged with Sl/Sld or +/+ hemopoietic cells. Previously reported ambiguities in mixed cell cultures were avoided by recharging the adherent layers with only a million cells. These results were correlated with previously published observations. Sl/Sld megakaryocytes have features in common with megakaryocytes from acutely thrombocytopenic animals. One feature, macrocytosis, appears to be due to abnormal Sl/Sld stromal cells that are reproduced as adherent layer cells in long-term cultures. The responsible stromal cells in Sl/Sld mice may be counterparts of megakaryocytopoietic regulatory cells in the marrow stroma of normal animals.

Animals↗