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Biomedical subjects

E M Mazur

Publications and source records attributed to E M Mazur.

At least 19 recordsLinked to original sources

Collaboration between an internal medicine residency program and a federally qualified health center: Norwalk Hospital and the Norwalk Community Health Center.

In 1999, Norwalk Hospital and an independent, community-based board collaboratively developed the Norwalk Community Health Center (the NCHC). The objectives of the affiliation were to (1) create a new, free-standing, high-quality community health center, (2) optimize grant and clinical revenue, (3) create an ideal venue for ambulatory care training for residents, and (4) replace the traditional and increasingly inefficient hospital-based primary care clinics. The hospital transferred all of its primary care clinical activity to the new community health center and provides an ongoing financial subsidy of the NCHC operations via a forgivable loan. In exchange, the NCHC granted Norwalk Hospital 24% of the seats on its board of directors and purchases all primary care provider services from the hospital. For adult medicine, the contract providers are exclusively Norwalk Hospital internal medicine residents and faculty. Contract charges are based not upon actual staffing but upon a standard formula relating full-time-equivalent providers to patient visits. The new 10,000 square-foot NCHC contains 2,500 square feet of additional integrated space, rented from the NCHC by Norwalk Hospital, which supports the residency education program. The NCHC opened in April 1999 and received FQHC status in November 1999. Adult medicine volume increased 30%, from 36.8 daily visits in the old hospital-based clinics to 48.0 at the NCHC. Resident and patient satisfaction are high. The NCHC now receives cost-based visit reimbursement from Medicaid and has received $1.8 million in state, federal, and local grants.

Adult↗

Medical management of a large aortic thrombus in a young woman with essential thrombocythemia.

Aortic thrombus formation is rare in the patients with essential thrombocytosis (ET); therefore, no guidelines for its management have been established. Embolism from ET-associated large vessel thrombi is potentially lethal and has been managed surgically in a few reported cases. We describe herein a 45-year-old black woman with ET found to have a 3.5-cm, pedunculated intra-aortic thrombus at the thoracoabdominal junction. How to treat this potentially devastating aortic thrombus was a management dilemma. We believed, based on the patient's diagnosis of ET and the histology of similar thrombi in 1 reported series, that the aortic thrombus was a "white thrombus" consisting primarily of aggregated platelets with a minimal fibrin network and almost no entrapped erythrocytes. The patient was treated with aspirin, 325 mg daily, as a platelet antiaggregating agent and hydroxyurea, 1,500 mg daily, to reduce the platelet count to less than 450 x 10(9)/L. The thrombus resolved without severe thromboembolic events. To our knowledge, this is the first reported case of a large intra-aortic thrombosis associated with ET that has been successfully managed with medical therapy alone.

Aorta, Abdominal↗

Plasminogen activator inhibitor-1 mRNA is expressed in platelets and megakaryocytes and the megakaryoblastic cell line CHRF-288.

Plasminogen activator inhibitor-1 (PAI-1) is present in the platelet alpha-granule and is released on platelet activation. Platelet PAI-1 could either be synthesized by the megakaryocyte or taken up from the plasma. In this report we confirm the presence of PAI-1 protein in human megakaryocytes by Western blot analysis and show its synthesis in guinea pig megakaryocytes by metabolic labeling. We document the presence of PAI-1 mRNA in human platelets and show a 3-kb mRNA species on Northern blot analysis of guinea pig megakaryocytes. Neither untreated CHRF-288 cells, a megakaryoblastic cell line, nor human erythroleukemia (HEL) cells expressed PAI-1 mRNA. Phorbol ester (phorbol 12-myristate 13-acetate, 160 nM) treatment of CHRF-288 and HEL cells for 4 days induced PAI-1 mRNA expression in CHRF-288 cells but not in HEL cells. These studies show that PAI-1 is synthesized by megakaryocytes. Megakaryocytes most likely determine the PAI-1 content of platelets and thereby establish the antifibrinolytic potential of the platelet.

Animals↗

Analysis of the mechanism of anagrelide-induced thrombocytopenia in humans.

Anagrelide is a new therapeutic compound recently demonstrated to have a rapid and selective thrombocytopenic effect in humans. The effects of anagrelide were evaluated in plasma clot and liquid suspension cultures of optimally stimulated normal human peripheral blood megakaryocyte progenitors in order to determine the mechanism of its thrombocytopenic activity. In plasma clot cultures, at clinically relevant, therapeutic concentrations (5 to 50 ng/mL), anagrelide exerted no significant inhibitory effect on megakaryocyte colony numbers or colony size. Only at anagrelide concentrations of 10 to 500 times therapeutic doses did anagrelide inhibit megakaryocyte colony development: an anagrelide concentration of 5 micrograms/mL reduced colony numbers by 57% and colony size by 31%. In contrast, lower, therapeutic anagrelide concentrations exerted profound effects in liquid culture on megakaryocyte cytoplasmic maturation, size, and DNA content. When present for the entire 12-day culture duration, anagrelide induced left-shifted megakaryocyte maturation and reduced both megakaryocyte ploidy and megakaryocyte diameter. Anagrelide, at concentrations of 5 to 50 ng/mL, shifted the modal cultured megakaryocyte morphologic stage from III to II, reduced the model ploidy value from 16N to 8N, and decreased the mean megakaryocyte diameter by up to 22%, from 27.6 microns to 21.6 microns. Megakaryocyte diameter was significantly reduced in most instances, even when analyzed as a function of morphologic stage. When anagrelide was added to the cultures after 6- and 9-day delays (during the final 6 and 3 days, respectively, of culture), similar inhibitory effects on megakaryocyte maturation stage and ploidy distribution were observed. However, the magnitude of the left-shift in ploidy appeared to be less as the duration of anagrelide exposure was reduced. Conversely, megakaryocyte diameter was not significantly affected by the shorter 3- and 6-day anagrelide exposures. These data indicate that therapeutic concentrations of anagrelide influence primarily the postmitotic phase of megakaryocyte development, decreasing platelet production by reducing megakaryocyte size and ploidy, as well as by disrupting full megakaryocyte maturation. Inhibition of megakaryocyte diameter appears to require more prolonged anagrelide exposure than inhibition of maturation stage and ploidy. The molecular mechanisms responsible for the inhibitory effects of anagrelide on megakaryocytopoiesis remain to be defined.

Cell Aggregation↗

Megakaryocytes and megakaryocyte progenitors in human cord blood.

Thrombocytopenia contributes significantly to morbidity in the sick term or preterm infant. However, few data exist on newborn's megakaryocytes and megakaryocyte progenitor cells (CFU-MK). We therefore studied CFU-MK in term and preterm infant cord blood and compared the results with data on CFU-MK from adult bone marrow and adult peripheral blood in a plasma clot culture with postirradiated aplastic canine serum (PIACS) as a source of megakaryocyte colony-stimulating activity. The number of CFU-MK and the number of cells per CFU-MK were counted with an immunofluorescent method at day 12. The effect of T-lymphocyte depletion on cord blood cultures for CFU-MK was studied with PIACS and a partially purified product of PIACS. We also studied individual megakaryocytes from newborns. The number and sizes of circulating megakaryocytes, isolated from adult peripheral blood and term venous cord blood by elutriation, were compared. Term and preterm cord blood contained more CFU-MK than adult peripheral blood. The numbers of CFU-MK in preterm cord blood were comparable to those in adult bone marrow. When the number of cells per colony were compared, cord blood contained significantly more cells than adult marrow CFU-MK. The depletion of T lymphocytes did not significantly change the growth of CFU-MK compared to nondepleted cultures. A substantial number of circulating megakaryocytes were obtained from venous cord blood, though they were significantly smaller than adult peripheral blood megakaryocytes. Since cord blood is easily obtained and contains large numbers of megakaryocytes and CFU-MK, it may provide a convenient model for studying the regulation of fetal megakaryocytopoiesis.

Animals↗

Isolation of large numbers of enriched human megakaryocytes from liquid cultures of normal peripheral blood progenitor cells.

Investigations linking human megakaryocyte development and cell biology have been hindered by an inability to obtain large, relatively pure megakaryocyte cell preparations from in vitro stem cell cultures. We report here that such preparations can be generated from liquid cultures of normal human peripheral blood mononuclear cells stimulated by a serum source of megakaryocyte colony stimulating activity (Meg-CSA, the 0% to 60% ammonium sulfate protein fraction of aplastic canine serum). Adherent-depleted peripheral blood mononuclear cells are suspended at 5 x 10(5) to 10(6) cells/mL in supplemented liquid culture medium, platelet-poor human plasma 20% (vol/vol) and 1 to 2 mg/mL serum Meg-CSA protein. After 12 to 14 days of incubation, megakaryocytes constitute 3.0 +/- 2.9% (mean +/- SD, n = 8) of the unseparated cultured cell population. Megakaryocytes can be enriched by counterflow centrifugal elutriation to a purity of 58 +/- 14% (+/- SD) with a recovery of 13 +/- 7% and a viability of 67 +/- 19%. This algorithm results in the average isolation of approximately 3 x 10(5) enriched megakaryocytes from a 100-mL starting volume of peripheral blood. Cultured megakaryocytes exhibit normal light and ultrastructural morphology by Wright-Giemsa staining and electron microscopic analysis. After a 12-day culture interval, enriched megakaryocyte preparations exhibit morphologic stage distributions that are similar to normal human marrow. Stage distributions move rightward with culture duration indicating partial synchrony of megakaryocyte maturation. On cytospin preparations, megakaryocyte diameter averages 30.2 +/- 1.5 microns and increases with maturation stage. Flow cytometric analyses demonstrate the expression of platelet glycoproteins (GP) Ib and IIb/IIIa by the cultured megakaryocytes. The modal ploidy of the enriched cells at day 12 of culture is 16N and most remaining megakaryocytes are 8N or 32N. Liquid culture of serum Meg-CSA-stimulated human peripheral blood mononuclear cells represents a valuable investigative tool that should permit studies of human megakaryocyte biology that have not been possible in the past.

Algorithms↗

Human serum megakaryocyte colony-stimulating activity appears to be distinct from interleukin-3, granulocyte-macrophage colony-stimulating factor, and lymphocyte-conditioned medium.

Sera from patients with bone marrow megakaryocyte aplasia are a rich source of megakaryocyte colony-stimulating activity (Meg-CSA). Other biologic materials exhibiting Meg-CSA include phytohemagglutinin-stimulated human lymphocyte-conditioned medium (PHA-LCM), recombinant interleukin-3 (IL-3), and recombinant granulocyte macrophage colony-stimulating factor (GM-CSF). Neutralizing antisera to both recombinant IL-3 and GM-CSF were used to evaluate the relationship among these sources of Meg-CSA. Varying dilutions of IL-3 and GM-CSF antisera were tested in plasma clot cultures of normal human peripheral blood megakaryocyte progenitors optimally stimulated by either IL-3 (1 U/mL), GM-CSF (1 U/mL), PHA-LCM (2.5% to 5% vol/vol), or aplastic human serum (10% vol/vol). IL-3 antiserum at dilutions up to 1/2,000 totally abrogated megakaryocyte colony growth stimulated by IL-3. A 1/500 dilution of GM-CSF antiserum completely eliminated GM-CSF-induced megakaryocyte colony development. A combination of anti-IL-3 and anti-GM-CSF, each at a 1/500 dilution, inhibited all megakaryocyte colony growth stimulated by optimal concentrations of IL-3 and GM-CSF together. There was no neutralizing crossreactivity between the IL-3 and GM-CSF antisera. At maximally neutralizing concentrations, IL-3 antiserum inhibited 66% of the megakaryocyte colony growth stimulated by PHA-LCM. Residual megakaryocyte colony growth was eliminated by the addition of a 1/500 dilution of anti-GM-CSF.(ABSTRACT TRUNCATED AT 250 WORDS)

Anemia, Aplastic↗

Aplastic presentation of acute lymphoblastic leukemia: evidence for cellular inhibition of normal hematopoietic progenitors.

Childhood acute lymphoblastic leukemia (ALL) may rarely present with blood and bone marrow findings suggestive of aplastic anemia. Although numerous examples of ALL presenting with this phenomenon have been reported, there is no accepted explanation for the pathogenesis of this preleukemic hypoplasia. We report a case of a child with ALL whose initial presentation was characterized by pancytopenia and bone marrow hypoplasia and who had repeated episodes of pancytopenia at times of systemic relapse. In vitro coculture experiments demonstrated that the leukemic cells from this patient were inhibitory for the growth of myeloid, erythroid, and megakaryocytic progenitor cells from normal peripheral blood. This inhibitory effect exhibited a dose-dependent relationship with the number of added lymphoblasts and persisted when the lymphoblasts were irradiated to prevent leukemic cell growth. Inhibitory activity was not present in media conditioned by the growth of the patient's lymphoblasts, nor was it present in lymphoblasts from three other children with ALL with similar immunophenotype but without marrow aplasia. These data suggest that the aplastic presentation of ALL may be attributable to inhibitory properties intrinsic to the leukemic cells rather than to other host factors.

Anemia, Aplastic↗

Recombinant gibbon interleukin-3 stimulates megakaryocyte colony growth in vitro from human peripheral blood progenitor cells.

Gibbon interleukin-3 (rIL-3) has recently been cloned and found to have a high degree of homology with the human IL-3 molecule. In this investigation, we evaluated the effects of gibbon rIL-3 on normal human peripheral blood megakaryocyte progenitor cell growth in vitro. Gibbon rIL-3 exhibited substantial megakaryocyte colony stimulatory activity (Meg-CSA), supporting peak colony numbers at a concentration of 1 U/ml. Megakaryocyte colony growth induced by rIL-3 reached 58% of the maximum achieved with the active, Meg-CSA-containing protein fraction of aplastic canine serum. Increasing gibbon rIL-3 concentrations also stimulated a 4-5-fold increase in megakaryocyte colony size and resulted in a decrease in geometric mean megakaryocyte ploidy. Ploidy values fell from 8.5N +/- 1.4 (+/- SEM) at an rIL-3 concentration of 0.1 U/ml to a minimum of 2.9N +/- 0.3 at 10 U/ml. In the presence of rIL-3 at 1.0 U/ml, megakaryocyte colony growth was linear with cell plating density and the regression line passed approximately through the origin. The effects of rIL-3 on megakaryocyte colony growth were independent of the presence of T-lymphocytes in the cultures. Cross-species evaluation of murine and gibbon IL-3 indicated that its bioactivity is species restricted. Murine IL-3 did not support colony growth from human megakaryocyte progenitors and gibbon rIL-3 showed no activity in stimulating acetylcholinesterase production by murine bone marrow cells. Gibbon rIL-3 is a potent stimulator of the early events of human megakaryocyte progenitor cell development promoting predominantly mitosis and early megakaryocytic differentiation.

Animals↗

Defective megakaryocytopoiesis in the syndrome of thrombocytopenia with absent radii.

The syndrome of thrombocytopenia with absent radii (TAR) is a hereditary condition whose pathogenesis is poorly understood. In this investigation we evaluated a female infant with TAR and her parents using in vitro haematopoietic colony forming assays and an antiserum against platelet membrane glycoproteins (PGP) to label smears of her bone marrow. Megakaryocyte colony growth in vitro was virtually absent in optimally stimulated cultures of the patient's bone marrow progenitors. In contrast, erythroid and myeloid colony growth from the TAR infant's marrow cells was preserved. Staining of the patient's bone marrow smears with PGP antiserum detected no immature, small megakaryocyte precursors. A high level of megakaryocyte colony stimulating activity was detected in serum from the TAR infant, activity comparable to that present in sera from adults with aplastic anaemia. The elevated serum activity decreased by 6 months of age at which time partial platelet recovery had occurred. Evaluation of both peripheral blood haematopoietic progenitor cells and sera from the TAR infant's parents demonstrated no significant abnormalities. We conclude that the principle haematopoietic defect in this patient with TAR syndrome is the absence or arrested development of the committed megakaryocyte progenitor cell. Humoral regulation of megakaryocytopoiesis appears intact and is responsive to the degree of megakaryocytic hypoplasia.

Colony-Forming Units Assay↗

Growth characteristics of circulating hematopoietic progenitor cells from patients with essential thrombocythemia.

Peripheral blood mononuclear cells from five patients with essential thrombocythemia (ET) were cultured in vitro to evaluate restricted megakaryocytic (CFU-Meg), myeloid (CFU-GM), and erythroid (BFU-E) progenitor cell development. Varying concentrations of aplastic canine serum served as the source of megakaryocyte colony-stimulating activity, and cultured megakaryocyte ploidy distributions were determined by Feulgen staining and microfluorometry. Megakaryocyte colony growth was strikingly abnormal in all five patients evaluated. Four of the 5 had a marked expansion in the concentration of circulating CFU-Meg and 3 of 4 manifested abnormalities in cultured megakaryocyte colony size (2 unusually large and 1 small). Unstimulated megakaryocyte colony growth was substantially increased in three patients. However, the fraction of megakaryocyte progenitors in cell cycle was near or below normal in all instances. Endomitotic megakaryocyte development was disordered in each of the four ET patients in whom it was evaluable. In normal subjects, mean megakaryocyte ploidy values vary biphasically with aplastic canine serum concentration and peak at 13.2 N following 12 to 15 days of culture. In contrast, day 12 mean ploidy values in cultures from the ET patients remained low at all aplastic canine serum concentrations and reached a maximum averaging only 8.4 N. Three patients were evaluated serially at extended culture durations of up to 21 days. The cultured megakaryocyte ploidy was unchanged during this interval for two of the patients. For the third patient, ploidy increased steadily, reaching abnormally high ploidy values by day 21. Progenitor cell expansion was limited to the megakaryocyte line in three patients. However, two patients had substantial increases in CFU-GM, one of whom also had a marked increase in BFU-E. There was no significant unstimulated colony growth by either CFU-GM or BFU-E. These data indicate that ET is usually characterized by an expansion in the concentration of circulating CFU-Meg in vivo which manifest both disordered replication and endoreduplication in vitro.

Cell Division↗

Evaluation of bone marrow megakaryocyte ploidy distributions in persons with normal and abnormal platelet counts.

Using bone marrow smears of the type prepared routinely in clinical practice, we determined megakaryocyte ploidy distributions in five normal persons, eight patients with both normal platelet counts and normal bone marrow morphology, and 18 patients with quantitative platelet disorders. To include 2N and 4N megakaryocytes in the ploidy distribution histograms, all megakaryocytes were identified by serial immunologic labelling with rabbit antiserum to human platelet glycoproteins and rhodamine-conjugated goat anti-rabbit igG. Cell nuclei were concurrently Feulgen stained with bis-aminophenyl-oxdiazole, and the nuclear fluorescent signals were quantified photometrically. A modal megakaryocyte ploidy value of 32N was seen in 10 of the 13 persons with normal platelet counts, and geometric mean megakaryocyte ploidy values averaged 24.9N +/- 7.0N (arithmetic mean +/- SD). In these normal control individuals, 2N and 4N megakaryocytes accounted for 11.1% of all megakaryocytes, and 2.6% of the megakaryocytes were 128N. Shifts to a higher mean ploidy were observed in five of seven patients with idiopathic thrombocytopenic purpura, resulting from increased percentages of 64N and 128N megakaryocytes at the expense of 4N, 8N, and 16N cells. Shifts to lower ploidy were demonstrated in two patients with acute myelogenous leukemia and one patient each with thrombotic thrombocytopenic purpura and isoimmune thrombocytopenia. Four of five patients with essential thrombocythemia had strikingly abnormal megakaryocyte ploidy histograms characterized by the presence of unusually high ploidy 256N and 512N megakaryocytes. These 256N and 512N cells were virtually unique to the patients with essential thrombocythemia.(ABSTRACT TRUNCATED AT 250 WORDS)

Bone Marrow↗

Epstein-Barr virus-associated malignancies.

The infectious aspects of cancer in humans were epidemiologically pioneered by Dr. David Burkitt through his observations of lymphomatous tumors seen in children in equatorial Africa. Years, later, the Epstein-Barr virus (EBV) was shown to be intimately associated with such tumors and is now recognized as a component of some B-cell lymphomas and nasopharyngeal carcinoma. Still the questions of an active, passive, or accessory role persist. The ability of this virus to cause immunosuppressive hemopoietic disturbances in individuals infected with EBV but not developing cancer raise questions about host susceptibility, host immune response, and possible coconspiring, infectious, oncogenic agents. Recent associations of EBV antibody found in diseases, such as squamous cell carcinoma of the head and neck and acquired immunodeficiency syndromes, point to its possible accessory role as an immunosuppressive agent. The ability of EBV to spread by extracellular and intracellular mechanisms demonstrates its variable infectious potential. Numerous EBV-transformed human cell lines attest to its ability to confer "immortality" with uncontrolled growth patterns. This review critically examines the association of EBV with various malignancies, the type of evidence which links it there, and the implications for further investigations and therapy.

Burkitt Lymphoma↗

Modest stimulatory effect of recombinant human GM-CSF on colony growth from peripheral blood human megakaryocyte progenitor cells.

Recombinant human granulocyte-macrophage colony-stimulating factor (rGM-CSF) has been previously demonstrated to stimulate colony formation from human myeloid, erythroid, and multipotential stem cells. In this investigation, we evaluated the effects of rGM-CSF on colony growth by human megakaryocyte progenitors (CFU-Meg). rGM-CSF was tested at concentrations of 0.1-100 U/ml in plasma clot cultures of adherent-depleted normal peripheral blood mononuclear cells. Control cultures were concurrently prepared containing either no stimulator or megakaryocyte colony-stimulating factor (Meg-CSF) partially purified from aplastic canine serum. rGM-CSF increased megakaryocyte colony numbers from a baseline of 4.3 +/- 1.4 (+/- SEM) in the unstimulated cultures to a maximum of 21.0 +/- 5.3 colonies at an rGM-CSF concentration of 1.0 U/ml. Corresponding megakaryocytic colony size increased from 4.4 to 8.3 cells/colony. Further increasing the rGM-CSF concentration resulted in decreasing megakaryocyte colony growth, reaching 6.8 +/- 2.9 colonies at 100 U/ml. The maximum number of megakaryocyte colonies stimulated by rGM-CSF was only 23.3% of that achieved in the control cultures containing optimal concentrations of serum-derived Meg-CSF protein (2.0 mg/ml). Megakaryocyte colonies stimulated by rGM-CSF consisted of predominantly low ploidy cells approximately equally distributed in 2N, 4N, and 8N ploidy classes. There was no increase in ploidy with any rGM-CSF concentration. These data indicate that rGM-CSF has modest activity in stimulating human megakaryocyte colony growth that is substantially less than that present in serum-derived Meg-CSF. rGM-CSF appears to primarily affect the early mitotic phase of megakaryocyte colony development with little influence on megakaryocyte endoreduplication.

Cell Division↗

Interrelationship between mitosis and endomitosis in cultures of human megakaryocyte progenitor cells.

Sera from dogs rendered aplastic by total-body irradiation stimulate human bone marrow megakaryocyte progenitors to form megakaryocyte colonies in plasma clot cultures. In this investigation, we evaluated the effects of varying concentrations of such sera on both the mitotic and endomitotic phases of human megakaryocyte development in vitro. When low concentrations of aplastic canine sera (2.5% to 5.0% [vol/vol]) were added to cultures of human peripheral blood mononuclear cells in place of normal AB serum, megakaryocyte colony formation was augmented fivefold, cell numbers per colony increased approximately 2.5-fold, and the geometric mean megakaryocyte ploidy almost doubled. Further increasing the aplastic canine serum concentration from 10% to 30% (vol/vol) stimulated no additional colony formation. However, there was a further augmentation of cell numbers per colony associated with a progressive decrease in the mean megakaryocyte ploidy. Megakaryocyte cultures were harvested after 7, 12, 15, and 19 days of incubation, and these demonstrated that the lower mean ploidy values found at the higher concentrations of aplastic canine serum did not result from delayed endoreduplication. At all aplastic serum concentrations evaluated, there existed a strong correlation between nuclear ploidy and cell diameter. We conclude that both the mitotic and endomitotic events in human megakaryocytopoiesis may be influenced by a factor or factors present in aplastic canine serum. At lower in vitro concentrations, such sera stimulate both mitosis and endomitosis, which promotes the development of megakaryocyte colonies composed of larger cells with a higher mean ploidy. With increasing aplastic serum concentrations, colony formation plateaus and mitosis is favored over endomitosis. This results in colonies composed of more numerous but smaller megakaryocytes with a lower mean ploidy. Our data suggest that the size and extent of polyploidization that can be achieved by a developing megakaryocyte may be influenced by the mitotic prior history of its immediate precursor cell.

Adult↗