In vitro differences in responsiveness of early (BFU-Mk) and late (CFU-Mk) murine megakaryocyte progenitor cells.
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Animals↗
Biomedical subjects
M W Long
Publications and source records attributed to M W Long.
Immune regulation of in vitro murine megakaryocyte development. Role of T lymphocytes and Ia antigen expression.
Mitogen-activated murine T lymphocytes or T cell hybridomas produce an activity (megakaryocyte [Mk] potentiator activity) that enhances the in vitro growth and development of Mk colonies. This activity was found in optimal concentrations (2.5%) in T cell hybridoma-conditioned medium, and was also produced by feeder layers of concanavalin A-activated T cells. A subpopulation of murine Mk progenitor cells (colony-forming units; CFU-Mk) bears the Ia antigen. Separate experiments indicated that T cell products stimulate CFU-Mk by increasing their basal levels of Ia expression as well as the frequency of cells actively synthesizing DNA. The hypothesis that the expression of this antigen was related to the cell cycle status of these progenitor cells was confirmed in studies that indicated that ablation of actively cycling cells in vivo abrogated the cytotoxic effects of anti-Ia monoclonal antibodies. The interdependence of T cell lymphokine regulation of both Ia expression and cell cycle status was also seen in in vitro experiments in which Ia+ progenitor cells were eliminated by complement-dependent cytotoxicity. The removal of Ia+ cells prevented 5-hydroxyurea-mediated inhibition of cells in S phase. We hypothesize that immune modulation of megakaryocytopoiesis occurs via soluble T cell products that augment Mk differentiation. Further, the mechanism of immune recognition/modulation may occur via Ia antigens present on the surface of these progenitor cells.
Animals↗
Phorbol diesters stimulate the development of an early murine progenitor cell. The burst-forming unit-megakaryocyte.
When murine (C57BL/6) bone marrow cells are cultivated with WEHI-3 conditioned media, a source of megakaryocyte-colony-stimulating activity (Mk-CSA), and phorbol myristate acetate (PMA), a previously undetected population of megakaryocyte (Mk) progenitor cells is observed. These new Mk colonies are reminiscent of erythroid bursts, in that they contain large numbers (40-500) of Mk and multiple foci (2-7) of development. These burst-forming units, Mk (BFU-Mk), are defined as having greater than or equal to 42 cells/colony and, at least, three foci of Mk development (colonies grown in soft agar cultures, all studies done at limiting dilutions; colonies detected by acetylcholinesterase [ACh-E] staining). CFU-Mk and BFU-Mk require two activities for optimal growth: Mk-CSA and PMA. However, the BFU-Mk require a tenfold greater concentration of PMA for optimal development (10(-6) vs. 10(-7) M). BFU-Mk detection is linear (over a range of 25-100 X 10(3) cells/ml), with the regression line passing through the origin. Bone marrow frequencies of these two progenitor cells are CFU-Mk, 36.7 +/- 2.5, and BFU-Mk, 7.3 +/- 0.7 per 10(5) total nucleated cells (mean +/- SEM; n = 28). The BFU-Mk have a restricted velocity sedimentation range (3.3-4.5 mmh-1 vs. 3.3-6.8 mmh-1 for CFU-Mk). Modal buoyant densities are 1.068 +/- 0.0002 and 1.070 +/- 0.002 for BFU-Mk and CFU-Mk, respectively. Thus, these cells are found among the smallest and less dense of the Mk progenitors, and are not clumps or clusters of CFU-Mk. Kinetic analysis indicates that CFU-Mk require 5-7 d for optimal growth, whereas BFU-Mk require 10-12 d. Examination of the proliferative potential (cells per colony) shows 19.3 +/- 1.5 cells per colony (n = 246 colonies) for day 10 CFU-Mk, vs. 118 +/- 6.0 for day 10 BFU-Mk (n = 163). Analysis of the cellularity/subcolony within each burst indicates 37.0 +/- 2.1 (n = 146) Mk/colony and 3.9 +/- 0.1 subcolonies/burst (n = 100). Finally, greater than 90% of the BFU-Mk contain only ACh-E positive cells, indicating that these are not mixed colonies. These results indicate that the BFU-Mk, compared with the CFU-Mk, require an increased amount of stimulation in order to differentiate, show delayed in vitro development, and have a higher proliferative potential. These data are consistent with the hypothesis that these cells are early progenitor cells in the Mk lineage that antedate the CFU-Mk.
Animals↗
Role of phorbol diesters in in vitro murine megakaryocyte colony formation.
In vitro megakaryocyte differentiation is regulated by two activities: a megakaryocyte colony-stimulating activity (Mk-CSA), which is required for proliferation, and an auxiliary factor, megakaryocyte potentiating activity, which plays a role in later differentiation events. Tumor-promoting phorbol esters alter many cellular differentiation-related events. Thus, it was hypothesized that phorbol esters may bring about megakaryocyte differentiation in vitro. 4 beta-Phorbol 12-myristate 13-acetate (PMA), when co-cultured with a source of Mk-CSA, stimulated a threefold increase in colony numbers. Co-culture of PMA and megakaryocyte potentiator activity did not stimulate colony formation, thus eliminating any action of PMA as an Mk-CSA. The direct effect of PMA on the formation of megakaryocyte colonies was established by (a) the function of PMA as a megakaryocyte potentiator in serum-free experiments, (b) the ability of PMA to stimulate megakaryocyte colony formation using bone marrow cells depleted of populations known to produce potentiating activity, (c) the inability of bone marrow adherent cells previously treated with phorbol, 12,13-dibutyrate (PDBu) to augment megakaryocyte colony formation, and (d) the ability of PMA to induce the growth of immature megakaryocytes into large single megakaryocytes. Structure:activity experiments resulted in equivalent activities for PMA and PDBu, whereas the nontumor promoter phorbol 12,13-diacetate and phorbol itself lacked activity. The observations in this study indicate that phorbol esters can bring about megakaryocyte differentiation, and during colony formation, can induce effects identical to those brought about by biological sources of megakaryocyte potentiator activity.
Animals↗
Six-months isoniazid-rifampin therapy for pulmonary tuberculosis. Report of a United States Public Health Service Cooperative Trial.
In a multicenter trial of 2 regimens for treatment of pulmonary tuberculosis, all patients received 300 mg of isoniazid (INH) and 600 mg of rifampin (RIF) daily for 6 months (the Initial Phase). During the next 9 months (the Maintenance Phase) patients received either daily INH (300 mg) and ethambutol (EMB) (15 mg per kg body weight) or matching placebos. Of the 672 patients who met the admission criteria, only 309 (46%) completed the Initial and Maintenance Phases. Approximately 20% of the patients failed to keep their appointments. Adverse drug reaction, most commonly hepatotoxicity, accounted for the withdrawal of 37 patients (5.5%). No visual toxicity caused by EMB was observed. During the Maintenance Phase, 3 patients who were taking INH and EMB, and 16 who were taking placebos, developed relapses, i.e., 2 or more positive cultures. The significant difference in relapse rate between regimens (Fisher's exact test, p less than 0.001) demonstrates the inadequacy of INH-RIF given alone for only 6 months.
Adult↗
Hepatotoxicity from isoniazid and rifampin among children treated for tuberculosis.
To estimate rates of hepatotoxicity in the United States among children treated for tuberculosis, we conducted a retrospective survey of health departments and individual practitioners. We received 874 reports suitable for analysis of children treated during 1977 to 1979. A total of 16 hepatotoxic reactions were reported; 14/430 (3.3%) children receiving isoniazid and rifampin had a hepatotoxic reaction, which approximates the rate seen in adults taking these drugs. Half of the reactions occurred during the first month of therapy, and all of the well-documented reactions were noted during the first 10 weeks. Because the likelihood of hepatotoxicity may be increased with higher drug doses, limiting the dose of isoniazid to 10 mg/kg and that of rifampin to 15 mg/kg may help minimize hepatotoxic reactions. Because more serious disease, especially disseminated tuberculosis, may further increase the risk of hepatotoxicity, close monitoring of such children receiving isoniazid and rifampin should help minimize serious hepatotoxicity. Routine biochemical monitoring may not be necessary for all children, eg, those with mild forms of disease and those with normal pretreatment liver function who are treated with lower drug doses.
Adolescent↗
Immature megakaryocytes in the mouse: in vitro relationship to megakaryocyte progenitor cells and mature megakaryocytes.
An assay describing conditions for the maturation of single immature megakaryocytes in vitro is reported. Enriched populations of small, relatively immature megakaryocytes have been found to develop into single, mature megakaryocytes by 60 hours in semisolid agar cultures. Continued incubation of these cells did not lead to the formation of colonies within 5-7 days. Maturation was indicated by increasing cell size and cytoplasmic and acetylcholinesterase content. Factors stimulating the development of immature megakaryocytes were found in preparations of human embryonic kidney cell-conditioned media (a source of in vivo Thrombopoietic Stimulatory Factor), peritoneal exudate cell-conditioned medium, lung-conditioned medium, or bone marrow cellular sources of activity (adherent cells or cells that sediment at 5-6 mm hr-1). Immature megakaryocytes cultured serum free responded to sources of an auxiliary megakaryocyte potentiating activity by developing into single, large megakaryocytes but did not respond to a megakaryocyte colony-stimulating factor devoid of detectable potentiator activity present in WEH1-3-conditioned medium. In contrast, serum-free proliferation of the megakaryocyte progenitor cell required both megakaryocyte colony-stimulating factor and the auxiliary potentiator activity. In the presence of megakaryocyte colony-stimulating factor alone, progenitor cells did not form colonies of easily detectable megakaryocytes. However, groups of cells comprised entirely of small acetylcholinesterase containing immature megakaryocytes were observed, thus establishing that megakaryocyte colony development passes through a stage of immature cells prior to detectable megakaryocyte development and that some acetylcholinesterase-containing cells can undergo cellular division.
Animals↗
Differences in the regulation of megakaryocytopoiesis in the murine bone marrow and spleen.
Murine splenic megakaryocytopoiesis has been analysed and compared to that of the bone marrow. Quantification of megakaryocytes by acetylcholinesterase staining indicated a reduction in the total numbers of megakaryocytes in the spleen, with the largest decrease being in the total numbers of immature megakaryocytes. On a per organ basis, the spleen also contained a lower number of the megakaryocyte progenitor cells (CFU-Mk) than the bone marrow. The splenic and bone marrow progenitor cells had similar in vitro responses to megakaryocyte colony-stimulating activities. However, the splenic progenitor cells developed a lower number of megakaryocytes per colony, compared to bone marrow. This lower number of cell divisions was not compensated by increased endomitotic activity, since the splenic colony megakaryocytes had a similar distribution of DNA to those derived from marrow megakaryocytes. Cell cycle analysis indicated that, in contrast to marrow cells, splenic megakaryocyte progenitor cells are a rapidly-cycling population. This change in cell cycle status, together with altered proportions of progenitor cells, immature and mature megakaryocytes, suggests that the regulation and kinetics of megakaryocyte development are different in spleen and bone marrow.
Animals↗
Successful intermittent treatment of smear-positive pulmonary tuberculosis in six months: a cooperative study in Poland.
One hundred nineteen patients 15 to 70 yr of age, all with smear-positive, previously untreated, pulmonary tuberculosis, were treated with a 6-month regimen containing isoniazid and rifampin, supplemented during the initial 2 months with streptomycin and pyrazinamide. The 4 drugs were administered daily in the hospital during the initial 2 months, followed by isoniazid and rifampin administered twice weekly on an outpatient basis during the next 4 months. Adverse reactions to the drugs were seen in 19 patients, but only 6 had toxic reactions requiring withdrawal of drugs for 7 days or more. Three of the toxic reactions were attributed to streptomycin, 2 to rifampin, 1 to isoniazid, and none to pyrazinamide. Eighty-five (71%) of the 119 eligible patients completed treatment. After the first 2 months of therapy, 91% of these patients had negative sputum cultures, and all of them had negative cultures by the end of the third month of treatment. No relapses have occurred among the 84 patients observed for 18 months after therapy was completed.
Adolescent↗
Immature megakaryocytes in the mouse: physical characteristics, cell cycle status, and in vitro responsiveness to thrombopoietic stimulatory factor.
The heterogeneity among immature megakaryocytes has been examined by physical properties, cell cycle status, and responsiveness to thrombopoietic stimulatory factor. Three types of immature megakaryocytes exist that can be recognized by acetylcholinesterase staining, nuclear shape, high nucleus/cytoplasm ratio, and small size (8--18 mu) with respect to mature megakaryocytes (greater than 18 mu). These three acetylcholinesterase-containing cell types are distinguished by their nuclear configuration: a round, indented, and lobed nucleus. The lobed cell type was found to overlap with and enhance detection of megakaryoblasts (stage I megakaryocytes). These cells had a sedimentation velocity range of 3.5--19.0 mm hr-1 and a density range of 1.072--1.095 g cm-3. Separation of these three classes of immature megakaryocytes was achieved by equilibrium density centrifugation with modal buoyant densities of 1.079 g cm-3 (round), 1.084 g cm-3 (indented), and 1.089 g cm-3 (lobed). In the presence of thrombopoietic stimulatory factor, the round nucleated cells, but not the indented or lobed nuclei morphology, were observed to develop into large mature megakaryocytes in 60-hr semisolid cell cultures. Development of two cell groups, or colonies of megakaryocytes, was not observed during this in vitro incubation period. In vivo treatment with hydroxyurea indicated that 57.5% +/- 19% of the round nucleus form were actively synthesizing DNA. No reduction in the numbers of indented or lobed nucleus forms were observed following hydroxyurea treatment. The data in this report strongly support the concept that these three types of immature megakaryocytes reflect the early maturation stages occurring in megakaryocyte differentiation.
Acetylcholinesterase↗
[Early and late results of 6-month treatment of pulmonary tuberculosis with various drug combinations].
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Adolescent↗
Short-course (6-month) cooperative tuberculosis study in Poland: results 30 months after completion of treatment.
This final report compared the efficacy of four 6-month chemotherapy regimens 30 months after the completion of treatment. All patients received isoniazid, rifampin, and ethambutol daily in hospital for the first 8 wk of treatment. One group continued to receive these 3 drugs daily as outpatients for an additional 18 wk; a second group received the same drugs twice-weekly, and a third group received the 3 drugs once-weekly during the 18-wk "continuation phase." A fourth group of patients received 2 drugs, isoniazid and rifampin, twice-weekly during the continuation phase. Drug toxicity was not a major problem; drugs were permanently discontinued in only 1% of the patients. All 4 regimens were highly effective in achieving sputum negativity. However, the relapse rate was found to be relatively high for all regimens (range, 8 to 22%).
Adolescent↗
Isoniazid-rifampin regimens for tuberculosis.
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Drug Therapy, Combination↗
Immature Megakaryocytes in the Mouse: Morphology and quantitation by acetylcholinesterase staining.
Three types of immature megakaryocytes, detected by their morphological properties, have been characterized in bone marrow of normal C57BL/6 mice. Morphological classification of these cells was carried out by determining (1) presence and relative amount of acetylcholinesterase, (2) cell size, (3) nuclear/cytoplasm ratio, and (4) nuclear shape. The immature megakaryocytes were classified as: (A) cells distinguished by a round nucleus (10.6 +/- 1.1 mu diameter; mean +/- SEM), which had the highest nucleus / cytoplasm ratio and lowest content of acetylcholinesterase; (B) cells with an indented nucleus (13.0 +/- 1.9 mu diameter), which had increased acetylcholinesterase content and reduced nucleus/cytoplasm ratio compared to the round-nucleus cell type; and (C) lobed-nucleus cells (14.5 +/- 2.9 mu diameter), which showed further increase in acetylcholinesterase content and reduction in nucleus/cytoplasm ratio. Increased numbers of immature megakaryocytes were detected, indicating that a proportion of these cells are undetected using conventional staining techniques. Based on the observed alterations in size, acetylcholinesterase content, and nuclear complexity, it was concluded that these cells constitute part of a progressive maturation sequence intermediate between the progenitor cell (CFU-Mk) and mature easily recognizable megakaryocytes.
Acetylcholinesterase↗
The influence of disseminated intravascular coagulation on renal function after experimental hemorrhagic shock.
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Acute Kidney Injury↗
Rifampin-isoniazid therapy of alcoholic and nonalcoholic tuberculous patients in a U.S. Public Health Service Cooperative Therapy Trial.
A history of alcoholism is often regarded as a relative contraindication to the use of isoniazid and rifampin in patients with tuberculosis. To test the validity of this assumption the outcome of 6 months of rifampin-isoniazid therapy was analyzed for the first 531 eligible patients enrolled in a U.S. Public Health Service Cooperative Trial of Short-Course Chemotherapy of Pulmonary Tuberculosis. In this study, data were available to classify a patient as an alcoholic in the following 2 ways: (1) patient's statement that he was a moderate, heavy, or excessive user of alcohol, or (2) patient's score of 6 or more on a Brief Michigan Alcoholism Screening Test (MAST). Based on their statements, 58% of the patients were classified as alcoholic, whereas only 17.9% were thus classified by their MAST scores. Although alcoholics had more abnormal concentrations of aspartate aminotransferase (AST) before and during therapy, there was no significant difference between the alcoholics and non-alcoholics in the incidence of adverse reactions, including hepatotoxic reactions, including hepatotoxic reactions, attributed to the drugs. We concluded that in the absence of clinically significant and persistent pretreatment abnormalities of hepatic function tests, rifampin and isoniazid are not contraindicated in patients categorized as alcoholic by our 2 commonly used methods.
Alcoholism↗
Short-course (6 month) cooperative tuberculosis study in Poland: results 18 months after completion of treatment.
This study compared the efficacy and toxicity of 4 drug regimens containing rifampin, isoniazid, and ethambutol administered both daily and intermittently for a total duration of 6 months (26 wk). There were 411 patients with newly diagnosed, previously untreated, pulmonary tuberculosis admitted to the study. All patients received isoniazid, rifampin, and ethambutol daily in a hospital for the first 8 wk of treatment. One group continued to receive these 3 drugs daily as outpatients for an additional 18 wk; a second group received the same drugs twice weekly, and a third group received the 3 drugs once weekly during the 18-wk "continuation phase." A fourth group of patients received 2 drugs, isoniazid and rifampin, twice weekly during the continuation phase. Drug toxicity was not a major problem; drugs were permanently discontinued in only 1% of the patients. All 4 regimens were highly effective in achieving sputum negativity. By the fifty month, 100% of the patients had become culture negative. However, the relapse rate was found to be relatively high for all regimens (range, 7 to 20%). Patients with extensive disease, large cavities, heavy growth on pretreatment cultures, slow sputum conversion, persistent cavities, heavy use of alcohol, and concomitant diseases were more likely to relapse. In order to achieve relapse rates acceptable in developed countries, regimens containing rifampin and isoniazid must either be given for longer than 6 months or strengthened by the addition of supplemental drugs during the initial phase.
Adolescent↗
Somatostatin-induced changes in the hemostasis of rats.
Somatostatin (SRIF) given intravenously, either as a single bolus or as a 2 hr infusion caused a significant prolongation of partial thromboplastin time (PTT) and depressed platelet counts and platelet aggregation in the rat. Following daily injections of protamin-zinc SRIF for 2 weeks the platelet count returned to normal, PTT remained prolonged and platelet aggregation was enhanced. The doses of SRIF used in this work were adequate to suppress the secretion of insulin and glucagon by the isolated pancreatic islets of treated animals.
Animals↗