Biomedical subjects

S A Burstein

Publications and source records attributed to S A Burstein.

At least 37 records · Page 2Linked to original sources

PubMed1995

Demonstration that thiazole-orange-positive platelets in the dog are less than 24 hours old.

Approximately 6% of dog platelets are positive for staining with thiazole orange, a dye frequently used to stain ribonucleic acid. In this report, thiazole-orange positivity is shown to mark platelets that are less than 24 hours old. Dog platelets were derivatized in vivo with N-hydroxysuccinimido biotin such that greater than 95% of all platelets were biotinylated. Newly synthesized, nonbiotinylated platelets were then monitored by flow cytometry for their ability to bind thiazole orange. After biotinylation, the percentage of biotin-negative, thiazole-orange-positive platelets increased gradually from 0.72% at 30 minutes to 5.44% at 24 hours. These data indicate that thiazole-orange staining does label newly synthesized platelets.

Animals↗

PubMed1994

Fluorescein derivatization of fibrinogen for flow cytometric analysis of fibrinogen binding to platelets.

Dog and human fibrinogen were derivatized with N-hydroxysuccinimido-fluorescein and utilized for flow cytometric estimation of fibrinogen binding to activated platelets. Fluorescein-fibrinogen binding fulfilled the criteria for specific binding to platelets; the binding was saturable, dependent on agonist activation, and inhibited by unlabeled fibrinogen. In addition, EDTA and barbourin, a KGD-containing peptide, were found to inhibit the binding of fluorescein-fibrinogen. Fluorescein-fibrinogen bound to dog platelets with an apparent affinity of 0.31 microM after stimulation with either adenosine-5'-diphosphate (ADP) or plateletactivating factor. The labeled fibrinogen was also used to study the fibrinogen binding capacity of aged, biotinylated platelets. Aged platelets were indistinguishable from young platelets with regard to fibrinogen binding in response to ADP. These studies document that direct derivatization of fibrinogen with fluorescein generates a useful probe for analyzing fibrinogen binding to platelets with flow cytometry.

Adenosine Diphosphate↗

PubMed1994

Alteration of platelet function in dogs mediated by interleukin-6.

To determine if interleukin-6 (IL-6) administration influences platelet function, platelet activation was analyzed sequentially in IL-6-treated (80 micrograms/kg/d) and control dogs. Platelet activation was determined in whole blood by flow cytometry by quantitating the binding of a monoclonal antibody to platelet surface P-selectin after stimulation with graded doses of thrombin. Administration of IL-6 resulted in a twofold decrease in the thrombin concentration required for induction of half-maximal P-selectin expression (ED50) compared with control animals. The ED50 returned to normal after cessation of IL-6 administration. As measured by P-selectin expression, enhanced responsiveness to the strong agonist platelet activating factor (PAF) was also observed in the IL-6-treated dogs. IL-6 had no effect on the susceptibility of platelets to thrombin activation when incubated with anticoagulated dog blood. The data show that, in addition to augmenting the platelet count in normal dogs, IL-6 enhances the sensitivity of platelets to activation in response to thrombin and PAF.

Amino Acid Sequence↗

PubMed1994

Aged platelets have an impaired response to thrombin as quantitated by P-selectin expression.

After the intravenous infusion of N-hydroxysuccinimido biotin into dogs, 80.6% +/- 9.7% (n = 5) of platelets were covalently labeled with biotin. The in vivo survival of the biotinylated platelets was monitored by flow cytometry and was normal as compared with previous reports for dog platelets. The ability of the biotinylated platelets to be activated was analyzed by measuring the expression of cell-surface P-selectin after incubation with graded concentrations of thrombin. When P-selectin expression was examined 3 hours after labeling, biotinylated platelets were indistinguishable from the nonlabeled population of platelets, indicating that biotinylation did not adversely affect the cells. On consecutive days after biotinylation, the thrombin dose-response curves for biotinylated and nonbiotinylated platelets were repeated, and as the biotinylated-platelets aged, they became less responsive to thrombin. On days 3, 4, and 5, the thrombin EC50 for the aged, biotinylated platelets as compared with the total population of platelets was 136%, 150%, and 178%, respectively. Increasing age clearly impairs the reactivity of platelets towards thrombin as quantitated by the expression of cell-surface P-selectin.

Animals↗

PubMed1994

Dog platelets accumulate intracellular fibrinogen as they age.

The alpha granules of circulating platelets are dynamic structures that acquire endogenous and exogenous components by synthesis and uptake, respectively. The uptake of exogenous components is a result of either receptor-mediated endocytosis or fluid-phase pinocytosis. Despite many detailed studies on the function and content of alpha-granules, little is known of the impact of platelet age on these organelles. In this report, we describe the use of platelet biotinylation to identify and isolate aged platelets for the analysis of alpha-granule contents. When aged platelets were permeabilized and examined by flow cytometry utilizing fluorescently labeled antibodies, two exogenously acquired proteins, fibrinogen and immunoglobulin G, were found to increase significantly with platelet age. The levels of intracellular fibrinogen were found to be elevated relative to control, 114 +/- 2% and 119 +/- 5% on days 4 and 5 postbiotinylation, respectively; the life span of dog platelets is 6.0 days. Intracellular immunoglobulin G content increased similarly. Levels of two endogenously synthesized proteins, thrombospondin and P-selectin, were not elevated in aged platelets. Confirmation of the flow cytometric data was obtained by isolating aged, biotinylated platelets by fluorescence-activated cell sorting and quantitating the fibrinogen levels with an ELISA assay. For platelets averaging 4.6 days of age, the fibrinogen level was elevated to 128 +/- 23% of the level for the entire platelet population. These data demonstrate that age-dependent changes in exogenously acquired alpha-granule proteins do occur and that the uptake mechanism for these proteins is active throughout the platelet life span.

Animals↗

PubMed1994

Effects of interleukin 6 on megakaryocytes and on canine platelet function.

The multifunctional cytokine interleukin 6 (IL-6) is a potent promoter of megakaryocytic maturation in vitro. In vivo, IL-6 has similar effects on the maturation of megakaryocytes, as shown by enhancing size, ploidy and platelet production. IL-6 is capable of augmenting the platelet count in both normal animals and those with reduced megakaryocyte mass; ongoing clinical trials suggest a similar thrombocytopoietic effect in man. Moreover, IL-6 alters platelet function, rendering them more sensitive to activation by thrombin and platelet activating factor. Finally, IL-6 promotes increases in plasma fibrinogen and von Willebrand factor, and a decrease in free protein S concentration. These modifications of the platelet and coagulant phases of the clotting mechanism may result in an overall prohemostatic tendency, which may prove beneficial for the amelioration of bleeding propensity following chemotherapy. However, additional investigation will be required to determine if IL-6-mediated alterations of hemostasis may lead to pathologic thrombosis.

Animals↗

PubMed1994

Platelets and cytokines.

Cytokines that are capable of stimulating megakaryocytopoiesis provide new approaches to ameliorate clinical thrombocytopenia. Administration of non-lineage-specific factors that promote megakaryocytic progenitor cell proliferation has been shown to increase platelet counts following chemotherapy. Preclinical and preliminary clinical studies have shown that cytokines that enhance maturation of megakaryocytes also accelerate platelet recovery. Combinations of factors may prove even more efficacious and novel approaches to enhance platelet recovery using mobilized peripheral blood stem cells have been introduced. Lineage-specific cytokines may appear in the near future. The notion that platelet function might be enhanced with cytokines has been proposed; whether this will translate into clinical utility is not clear. Despite this recent progress in the development of thrombopoietic cytokine therapy, an important but unresolved matter is the establishment of precise clinical indications for such treatment.

Blood Platelets↗

PubMed1993

Biotinylated platelets: a new approach to the measurement of platelet life span.

The in vivo life span of dog platelets was determined by derivatizing whole blood with N-hydroxysuccinimido biotin, reinfusing the biotinylated blood and subsequently monitoring the survival of the biotinylated platelets with flow cytometry. We found that the biotinylated platelets had a mean life span of 6.0 +/- 1.1 d as determined by curve-fitting the platelet disappearance data to gamma functions. These data are in good agreement with literature values of platelet life span for canine platelets labelled with either 111Indium-oxine or 51Chromium. Biotinylated platelets were analysed after reinfusion and found to aggregate normally in response to the agonists adenosine diphosphate and phorbol myristate acetate. These experiments demonstrate that biotinylated platelets survive normally in vivo and that this labelling method can be used for determining platelet life spans.

Animals↗

PubMed1992

Thrombocytopoiesis in normal and sublethally irradiated dogs: response to human interleukin-6.

The response of megakaryocytes and platelets to the administration of recombinant human interleukin-6 (IL-6) was investigated in normal and sublethally irradiated dogs. IL-6 was administered for 2 weeks at doses of 10 to 160 micrograms/kg/d to normal animals to assess dose-response and toxicity. Subsequently, 40, 80, or 160 micrograms/kg/d for 2 weeks was administered to animals treated with 200 cG total body irradiation. Analysis of normal dogs showed a significant increment in the platelet count detectable approximately 11 days after initiation of IL-6 at all administered doses. Large platelets greater than 6.3 microns in diameter were observed 1 day after beginning IL-6, progressively increasing to as many as 19.1% of the total circulating platelets by day 10. The ploidy distribution of the marrow megakaryocytes did not differ from the normal at doses of less than or equal to 80 micrograms/kg/d, but at 160 micrograms/kg/d, a shift toward higher ploidy cells was noted. No change in total white count was noted; however, a decrease in hematocrit was seen at all doses. In the irradiated animals, the platelet count recovered earlier in the IL-6-treated dogs than in the controls, but no consistent change in the ploidy distribution was observed irrespective of dose. Large platelets were also noted in the treated animals, comprising up to 6.9% of the total platelet count. Fibrinogen levels were elevated to greater than 4 times normal. A significant decrease in hematocrit was seen in all animals, while no consistent change was noted in the white count. Elevations in serum cholesterol, triglycerides, and alkaline phosphatase, together with a decline in serum albumin were observed in all the treated animals (both normal and irradiated), but clinical symptoms were observed only in the dogs receiving greater than or equal to 80 micrograms/kg/d. The data show that IL-6 alone is capable of enhancing platelet recovery in dogs with bone marrow suppression.

Animals↗

PubMed1992

Leukemia inhibitory factor and interleukin-11 promote maturation of murine and human megakaryocytes in vitro.

The growth-promoting activities of optimally stimulating concentrations of leukemia inhibitory factor (LIF) and interleukin-11 (IL-11), a stromal cell-derived cytokine, on megakaryocytes in liquid marrow cultures were compared to interleukin-6 (IL-6), a known megakaryocytes maturation factor. Maximally stimulating concentrations of LIF (25 ng/ml), IL-11 (10 ng/ml), or IL-6 alone (10 ng/ml) promoted an 81, 157, and 153% increase, respectively, in acetylcholinesterase (AchE) activity in murine serum-free cultures compared with controls (n = 5). In combination with 25 U/ml murine interleukin-3 (IL-3), LIF, IL-6, and IL-11 showed increases, respectively, of 35%, 49%, and 174% in AchE activity compared with IL-3 alone (n = 4). Flow cytometric analysis of 4-day-old cultures showed that LIF alone had minimal effect on megakaryocytic ploidy, whereas IL-11 and IL-6 alone markedly augmented high ploidy cells. Enumeration of cells stained for AchE showed that IL-11 increased the numbers of Mks in comparison to LIF, IL-6 or controls by up to 59%. Moreover, a twofold increment in Mk number was noted when IL-11 was used in combination with IL-3 (compared with either IL-3 alone of IL-3+IL-6). Flow cytometric ploidy analysis of 8-day-old human liquid marrow cultures showed that either LIF, IL-11, or IL-6 alone markedly augmented the percentage of 32N cells compared with cultures containing only human IL-3. The data suggest that LIF and IL-11 promote murine and human Mk maturation in vitro, although the effect of IL-11 exceeds that of LIF in mice. Despite the comparable influence of IL-11 and IL-6 on Mk ploidy, IL-11 has the additional characteristic of enhancing the number of Mks, particularly in combination with IL-3.

Acetylcholinesterase↗

PubMed1992

Serum interleukin-6 levels in patients with thrombocytosis.

Interleukin-6 (IL-6) has been shown to increase platelet counts in several animal models and to enhance megakaryocytopoiesis in vitro. In order to investigate the possible relationship between IL-6 and thrombocytosis, serum IL-6 levels in patients with platelet counts > or = 6 x 10(5)/microliters were measured using an IL-6-responsive bioassay. A cohort of healthy volunteers with normal platelet counts was used to establish a control mean serum IL-6 level [2.19 U/ml +/- 1.08 SD (range 0-5.5)]. Patients with primary thrombocytosis had a mean serum IL-6 level not significantly different from controls. In comparison, serum IL-6 levels of patients with reactive thrombocytosis were significantly greater than controls (38.3 U/ml +/- 94.6; range 0-933; P < 0.001). Although no significant correlation was observed between the degree of serum IL-6 elevation and the height of the platelet count in any individual, elevated serum IL-6 was highly correlated with reactive thrombocytosis.

Hematopoiesis↗

PubMed1992

Characteristics of a novel rat anti-mouse platelet monoclonal antibody: application to studies of megakaryocytes.

Using murine platelets as an immunogen, a rat monoclonal antibody (designated 4A5) that recognizes only murine blood platelets and marrow megakaryocytes was developed. The extent of binding of 4A5 to platelets was dependent upon their state of activation. Following phorbol ester, ionophore, or thrombin stimulation of resting platelets, a decrease of > 50% in the binding of 4A5 was observed by flow cytometry. This decrease in antibody binding to the platelets was accompanied by an increase in antibody released into the platelet-free supernatant following platelet activation. When platelets were first radioiodinated, followed by activation and incubation of the platelet-free supernatant with 4A5-derivatized beads, no precipitable counts were observed compared with control resting platelets. This suggests that antibody release was related to an activation-dependent conformational change in the 4A5 epitope. Following solubilization of biotinylated platelets, 4A5 bound to an 80-kd membrane protein. Immunohistochemical studies with 4A5 showed that megakaryocytes could be identified both in vitro and ex vivo. When marrow was first stained histochemically with 4A5 followed by staining for acetylcholinesterase, the distribution of stained cells was similar. Flow cytometric analysis using 4A5 and propidium iodide showed that the antibody could be used to identify megakaryocytes for ploidy analysis in vivo or in vitro. 4A5 was capable of inducing a moderate thrombocytopenia in mice compared with polyclonal anti-platelet serum. When bound to plastic or to magnetic beads, 4A5 could be used to purify murine megakaryocytes to homogeneity. The data suggest that monoclonal antibody 4A5 will be useful in quantitative studies of murine platelets and megakaryocytes.

Animals↗

PubMed1991

Megakaryocytic maturation in murine long-term bone marrow culture: role of interleukin-6.

Megakaryocytic maturation was analyzed in long-term bone marrow cultures in the absence of added growth factors. Megakaryocytes could be observed for periods of up to 13 weeks in both the supernatant and stromal layer of these cultures. Using acetylcholinesterase staining for enumeration and sizing of megakaryocytes, and a novel rat antimurine platelet monoclonal antibody (MoAb) that detects only megakaryocytes in bone marrow, the number, volume, and ploidy of these cells were assessed microscopically and by flow cytometry. Correlation of these measurements with ambient interleukin-6 (IL-6) levels showed no relationship between IL-6 bioactivity and megakaryocyte number. Conversely, the relatively high IL-6 bioactivity present during the first 2 weeks of culture was correlated with increased megakaryocytic size and ploidy, while the relatively lower IL-6 bioactivity present after week 3 corresponded to decreased megakaryocytic size and ploidy. Addition of neutralizing anti-IL-6 MoAb decreased megakaryocytic size and ploidy at times when ambient IL-6 levels were relatively high, while the addition of exogenous IL-6 increased size and ploidy at times when endogenous IL-6 concentrations were low. The data show that long-term bone marrow cultures can be used as a means to evaluate megakaryocytic maturation in vitro, and suggest that, to some extent, IL-6 plays a role in the maturation process in this system.

Animals↗

PubMed1991

Interleukin-6 mRNA and protein increase in vivo following induction of acute thrombocytopenia in mice.

Induction of experimental thrombocytopenia in rodents results in the enhancement of megakaryocytic growth and differentiation. Interleukin-3 (IL-3) and IL-6, cytokines with a broad spectrum of biologic activities, stimulate megakaryocytopoiesis in vitro. To determine if expression of these factors might increase in response to experimental thrombocytopenia, we measured steady-state levels of IL-3 and IL-6 mRNA following rabbit antiplatelet serum (APS) injection. Groups of mice were injected intravenously with 0.2 mL APS while control animals received rabbit antilymphocyte serum (ALS), normal rabbit serum (NRS), or phosphate-buffered saline (PBS). At various times up to 72 hours after injection mice were exsanguinated and splenectomized. Platelet counts in the experimental animals were less than 12% of controls. Splenic RNA was hybridized in solution to 32P-UTP-labeled cRNA probes for IL-3 and IL-6. RNase-resistant hybrids were resolved on denaturing gels and visualized autoradiographically. IL-3 hybrids were undetectable at all time points tested, irrespective of the film exposure time or specific activity of the probe. Conversely, IL-6 hybrids were easily visualized and showed peak expression at 1.5 to 2.0 hours. By 3 hours, IL-6 mRNA had returned almost to the level of the controls. Similar results were observed in the bone marrow, although maximal IL-6 mRNA in that tissue was observed 4 hours following APS administration. To determine if this mRNA increment was associated with a concomitant increase in bioactive protein, serum was tested for its ability to stimulate IL-6-dependent B9 cells. At 1.75 hours following injection, experimental animals showed a small but significant increment in IL-6 activity compared with controls (200 +/- 30 U/mL IL-6 compared with 129 +/- 17 U/mL in ALS-injected controls, 106 +/- 17 U/mL in NRS-injected controls and 84 +/- 17 U/mL in PBS-injected controls). The data show that IL-6 mRNA and bioactive protein increase in response to acute immunothrombocytopenia, while no increment in IL-3 is detectable. These results suggest that IL-6 may play a role in the physiologic response to acute immunothrombocytopenia.

Acute Disease↗

PubMed1991

Elevated serum interleukin-6 levels in patients with reactive thrombocytosis.

Interleukin-6 (IL-6) is known to promote megakaryocytopoiesis in vitro and raise platelet counts in vivo. To determine if there is a relationship between circulating IL-6 and thrombocytosis in man, we measured bioactive IL-6 in the serum of 13 patients with myeloproliferative disorders and 143 patients with reactive thrombocytosis having platelet counts greater than or equal to 600 x 10(9)/l. IL-6 activity was assayed using the IL-6-responsive B9 cell line. Seventy-one controls with normal platelet counts had a mean IL-6 level of 2.19 U/ml +/- 1.08 (SD). None of the 13 patients with myeloproliferative disorders had elevated IL-6 levels (1.56 U/ml +/- 1.2). In contrast, serum IL-6 levels of 143 patients (158 samples) with reactive thrombocytosis were significantly greater than controls (38.3 U/ml +/- 94.6; P less than 0.001), with 83% of the samples showing elevated serum IL-6. No significant correlation was observed between serum IL-6 levels and platelet counts in the reactive thrombocytosis group. We conclude that elevated IL-6 is associated with reactive thrombocytosis, and hypothesize that the increased platelet count in many cases is causally related to elevated IL-6.

Humans↗

PubMed1991

Regulation of interleukin 6 expression in murine bone marrow stromal cells.

The regulation of interleukin 6 (IL-6) expression in the B-lymphocyte-supporting murine stromal cell line BMS2 has been examined in response to exogenous cytokines and chemical agents. Kinetic analyses of IL-6 mRNA induction and decay are presented together with analysis of the IL-6 biological activity. The cytokines tumor necrosis factor, interleukin 1 (alpha and beta), and transforming growth factor beta, as well as forskolin and dibutyryl cyclic AMP, all induce a transient rise in the steady-state level of IL-6 mRNA and an increased release of IL-6 protein. To study its regulation at the chromatin level, the murine IL-6 genomic gene has been cloned. Induction of IL-6 expression correlates with increased DNA nicking, consistent with increased topoisomerase I and endogenous nuclease activity. This finding is supported by kinetic analyses using camptothecin, a topoisomerase I inhibitor. We conclude that IL-6 regulation in murine stromal cells capable of supporting B-lymphopoiesis is comparable to that observed in human diploid fibroblasts.

Animals↗

PubMed1991

Canine megakaryocytopoiesis: analysis utilizing a monoclonal antibody to a 140-kd dog platelet protein.

The dog, a convenient and relatively large animal whose size permits repetitive blood and marrow sampling, marrow biopsy, and mechanical apheresis, would be a suitable experimental model for the study of in vivo megakaryocytopoiesis. A monoclonal antibody to a 140-kd dog platelet membrane protein has been developed that reacts with canine megakaryocytes. Using the fluoresceinated derivative of this antibody to identify megakaryocytes and propidium iodide staining to measure relative DNA content, the DNA distribution of megakaryocytes in dog bone marrow or in cultured dog marrow cells could be rapidly assessed by flow cytometry. Normal dogs showed a modal ploidy of 16N (54%), with 17% 8N and 16% 32N. In contrast, dogs made thrombocytopenic by plateletpheresis showed a shift in distribution to higher ploidy cells (36% 32N). The data show that use of a specific marker of megakaryocytes in combination with flow cytometric analysis is an accurate and reproducible method of assessing megakaryocytopoiesis in a convenient and easily manipulable animal model.

Animals↗

PubMed1990

Interleukin 6 is a differentiation factor for human megakaryocytes in vitro.

The response of cells of the megakaryocytic lineage to interleukin 6 (IL6), a cytokine with multiple biological activities, was studied by adding the factor to human bone marrow (BM) cultures. IL6 alone had no effect on megakaryocytic colony formation in methylcellulose; however, in the presence of maximally stimulating concentrations of IL 3, almost a twofold increment in colony formation was observed. Tritiated thymidine suicide studies of BM incubated for 2 h with growth factors showed that almost one-half of megakaryocytic progenitors (CFU-Mk) preincubated with IL3 or IL3 plus IL6 were in S phase, whereas BM incubated with IL6 alone was similar to control (approximately 24% of CFU-Mk in S phase). When greater than or equal to 1 ng/ml of IL6 was added to liquid suspension cultures of BM, the size of individual megakaryocytes was significantly augmented compared with that seen in control cultures. Moreover, the DNA content of megakaryocytes grown in the presence of IL6 was increased (modal ploidy 16N) compared with cultures grown with IL3 (modal ploidy 8N). To determine if the effect of IL6 could be direct rather than mediated via accessory BM cells, the factor was added to cultures of isolated single megakaryocytes. Seventy-one percent of cells increased in diameter in the presence of IL6, while only 27% increased in size in the absence of the factor. The data show that IL6 is a direct maturation factor for human megakaryocytes, promoting increments in size and ploidy of these cells.

Cell Cycle↗