Human megakaryocyte ploidy.

We reviewed the literature concerning the history of determination of the ploidy of human megakaryocytes and its relationship with diseases. The ploidy of rabbit megakaryocytes was analyzed by microspectrophotometry in 1964, and the analysis of the ploidy in human megakaryocytes was first performed in 1968. Presently, microphotometry and flow cytometry are the primary methods for the evaluation of the ploidy, but they have their merits and demerits. In the ploidy of human megakaryocytes, a peak has often been reported at 16N in healthy individuals, and the next peaks have been observed at 32N and 8N. The results of ploidy analyses have been reported by many investigators to be comparable between patients with idiopathic thrombocytopenic purpura and normal subjects, but various shifts of the peaks have also been documented. The ploidy is often reported to shift to a larger ploidy class in polycythemia vera and essential thrombocythemia, but it has invariably been reported to shift to a smaller class in chronic myelogenous leukemia. In reactive thrombocytosis, the ploidy pattern was reported to be the same as that in normal individuals by some investigators but to shift to a larger ploidy by others. These differences are considered to be due to heterogeneity of the subjects. In myelodysplastic syndrome, the ploidy shifts mostly to a smaller class, but it may show various patterns. We also reviewed the ploidy in other rare hematological disorders, the relationships of the ploidy with diabetes mellitus and atherosclerotic disorders, and its changes in the ontogeny. Details of the mechanism of polyploidization and its biological significance remain unknown, and further advances in the studies of these topics are anticipated.     

The relationship between megakaryocyte ploidy and platelet volume

The origins and biologic significance of platelet heterogeneity in general, and platelet volume heterogeneity in particular, have been controversial scientific issues during the past decade. Although it has generally been held that specific megakaryocyte properties, especially ploidy level, are important determinants of platelet volume, the precise relationship between megakaryocyte properties and platelet properties is not well defined. The physiologic processes that specifically determine the relationship between megakaryocyte ploidy and platelet volume are unclear, and understanding of these processes has been further complicated due to the multiplicity of experimental and clinical models used to study the problem. Although it is generally true that increases in megakaryocyte ploidy are associated with increases in megakaryocyte volume, it is not well established that platelet volume is also increased during normal or abnormal thrombopoiesis as a direct result of a change in the ploidy level. Reexamination of earlier studies and some recent investigations suggest that changes in platelet volume and megakaryocyte ploidy are in fact dissociated in response to experimental thrombocytopenia. Critical review of the literature concerning the relationship between megakaryocyte ploidy and platelet volume reveals a limited number of conclusions that are well substantiated and emphasizes the relative lack of understanding about the events governing the complex process of platelet production and platelet heterogeneity.     

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.     

The control of megakaryocyte ploidy and platelet production: biology and pathology

Following experimental platelet destruction in animals, large platelets, which are more hemostatically active, are produced before any change in bone marrow megakaryocyte DNA content. When platelet production is stimulated by administration of i.v. vincristine in rats, megakaryocyte ploidy is increased, but mean platelet volume is unchanged. When platelet production and destruction are both stimulated by chronic hypoxia or administration of anti-platelet serum, mean platelet volume and megakaryocyte DNA content are both increased. Since platelet volume is determined primarily at thrombopoiesis, these results imply that mean platelet volume and megakaryocyte DNA content are under separate hormonal control. Therefore, it has been postulated that changes in mean platelet volume occur following changes in platelet production rate, whereas changes in megakaryocyte ploidy are associated with an increased rate of platelet production. In myocardial infarction, platelets have increased mean volume and reduced bleeding time more than in controls. In addition, men with myocardial infarction have increased megakaryocyte size and increased DNA content when compared to controls. These changes are similar to those observed in rabbits following cholesterol feeding. If megakaryocyte polyploidy and mean platelet volume are under separate hormonal control, this suggests that in myocardial infarction, both hormones are active--one stimulating an increased platelet size, the other stimulating the increased megakaryocyte DNA content. In contrast, patients with lymphoma exhibiting a secondary thrombocytosis have no change in mean platelet volume. However, these subjects also have larger bone marrow megakaryocytes when compared to controls. The relation between megakaryocyte size and ploidy implies that the DNA content of these cells is increased in lymphoma.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interferon-alfa corrects thrombocytosis in patients with myeloproliferative disorders

Summary During previous therapeutic trials with interferon, decreased levels of peripheral platelet counts have been observed. Taking advantage of this effect, we investigated the efficacy of recombinant interferon (rec-IFN) in the treatment of thrombocytosis in myeloproliferative diseases. A total of 15 patients with polycythemia vera, essential thrombocytosis, or chronic myeloid leukemia received rec-IFN-alfa at initial doses of 25–70×10⁶ units/week; maintenance therapy following week 8 of treatment consisted of 20–35×10⁶ units/week rec-IFN. Observation periods ranged from 24 to 48 weeks. Significant reductions in the number of platelets were noted in all cases; 12/15 patients achieved platelet counts below 440×10⁹/1 and maintained those normal values for at least 4 weeks. The number of bone marrow megakaryocytes, which had been increased prior to treatment, diminished during rec-IFN therapy, while the previously shortened platelet half-life further decreased with rec-IFN treatment. During rec-IFN-induced remission, the plasma levels of platelet factors, the activity of natural killer cells, and platelet aggregation showed changes between slight improvement and normal values. Severe side effects were only observed with the highest rec-IFN doses; dosage adjustments were effective in improving or eliminating all treatment-related symptoms. Rec-IFN may prove to be a valuable therapeutic alternative to cytostatic treatment of thrombocytosis in myeloproliferative disorders.This study was supported in part by the Austrian Research Grant: P4999 and the Ludwig Boltzmann Institute for Gerontology, Vienna, Austria     

Kinetic analysis of megakaryocyte numbers and ploidy levels in developing colonies from mouse bone marrow cells

Abstract. The kinetics of megakaryocyte formation from mouse bone marrow cells in semi-solid medium was studied directly in the culture dish by staining the cells for acetylcholinesterase after drying the cultures. A WEHI-3 cell-conditioned medium (WEHI-3 CM) was used as a general source of stimulus for megakaryocyte colony formation. The addition of peritoneal exudate supernatant as well as WEHI-3 CM increased the frequency of megakaryocyte colonies detected. Colonies containing acetylcholinesterase-positive cells were first detected at day 3. Maximum numbers of 25–40 megakaryocyte colonies per 10⁵ nucleaet mouse bone marrow cells were observed from days 7 to 11. The mean number of cells within each colony increased progressively with time of culture, and a modal range of 11–20 cells was obtained by day 7. Between 3 and 200 cells per colony were generally detected. A continuous distribution of the number of megakaryocytes per colony suggests that the clonable precursor cells are not synchronized either with respect to maturation stage or with respect to their capability to undergo nuclear endoreduplication. The addition of peritoneal exudate supernatant to the cell cultures increased the DNA levels of megakaryocytes grown in the presence of WEHI-3 CM but did not affect the number of cells per colony. The DNA content of colony megakaryocytes was measured after staining the cells with Feulgen reagent. A modal DNA value of 8 N was observed between days 4 and 7 for megakaryocytes stimulated with WEHI-3 CM. In the presence of both WEHI-3 CM and peritoneal exudate supernatant, the DNA content of megakaryocytes increased with the time of cell culture. Modal DNA values increased from 8 N at days 4 and 5, to 16 N by day 6. In these optimally stimulated cultures, 44% of colony megakaryocytes were 32 N or greater, a proportion higher than in normal bone marrow, but similar to that seen in the marrow of acutely thrombocytopenic animals. It is concluded that megakaryocytopoiesis in cell cultures is not a strictly controlled process with respect to cell division and endomitosis and that when certain culture conditions are employed, megakaryocyte development in vitro might reflect that seen in a stressed animal condition.     

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.     

DNA ploidy and survival in breast cancer patients

Flow cytometric DNA ploidy measurements using frozen or deparaffinized tumor specimens were performed on 565 primary breast cancers from patients treated in the period 1975-1984. Twenty-nine percent of the cases were diploid, 61% had a single aneuploid stemline, and 10% were multiploid. Aneuploid tumors more often had negative estrogen receptor values than diploid tumors, but no significant correlation was found between ploidy class and TNM stage. Patients with more than ten positive axillary lymph nodes had predominantly aneuploid tumors. Overall and distant relapse-free survival were higher for patients with diploid tumors and low-aneuploid tumors. Stratification of the patients according to degree of lymph node involvement, TNM stage, and menopausal stage showed that the prognostic effect of aneuploidy was apparent predominantly in patients with locally advanced disease. Postmenopausal node-positive patients with diploid tumors had a significantly better prognosis than those with aneuploid tumors, but this difference was not found for the comparable premenopausal group. Multivariate analysis with the Cox proportional hazards model indicated that ploidy is an additional, independent prognostic factor in postmenopausal patients.     

Complement-dependent cytotoxic factor to megakaryocyte progenitors in sera from patients with idiopathic thrombocytopenic purpura

The influence of sera from patients with idiopathic thrombocytopenic purpura (ITP) was examined on colony formation from megakaryocyte (M) progenitors. Though incubation of marrow cells in Iscove's modified Dulbecco's medium (IMDM) containing 50% sera from several ITP patients stimulated M-colony formation in 8 of 13 cases, incubation in the sera from the patients and in baby rabbit serum as a source of complement significantly suppressed the colony formation. Experiments showed that sera of immunoglobulin G from ITP patients had significant complement-dependent cytotoxicity to M-progenitors in normal marrow cells or in the marrow cells from corresponding patients, but not to CFU-e, BFU-e or CFU-gm. Cytospin preparations of individually collected M-colonies from marrow cells treated with ITP patients' sera and complement revealed a reduction of megakaryocyte colonies containing cells of multilineages. These results indicate that autoantibodies detected in ITP patients can bind not only to platelets and megakaryocytes, but may also bind to M-progenitors.     

Monkeying around with ploidy

Inferences of whole genome duplication (WGD) events accompany the annotation of every newly sequenced plant genome, but much remains unknown about the evolutionary processes and pathways relating to WGD (Soltis et al. 2010). What ecological, biogeographical and genetic factors cause WGD to occur in nature? How does WGD affect gene expression? How do genomes evolve after WGD? New species that have arisen recently through WGD are good places to seek answers to such questions. These could be relatively common in nature, but reliably demonstrating their recent origin requires documentary evidence, which can be very hard to come by. Thus far, records of species introductions and meticulous botanizing have demonstrated six new natural allopolyploids in just four genera: Tragopogon miscellus and T. mirus, Senecio cambrensis and S. eboracensis, Spartina anglica and Cardamine schultzii (Abbott & Rieseberg 2012; Ainouche et al. 2009; Soltis & Soltis 2009). It is risky to generalize about a universal feature of plant evolution from such a small sample; more examples are needed, in different genera. It is therefore of considerable interest that Mario Vallejo‐Marin of University of Stirling has this year named a new allopolyploid species of monkey flower, Mimulus peregrinus, and presented evidence that it is <140 years old (Vallejo‐Marin 2012). This discovery is particularly timely as the monkey flower genus is developing rapidly as a model system for ecological genetics (Wu et al. 2008), and in the current issue of Molecular Ecology, Jennifer Modliszewski and John Willis of Duke University present new data showing high genetic diversity in another recently discovered monkey flower allopolyploid, M. sookensis (Modliszewski & Willis 2012).     

Analysis of ploidy in a planarian by flow cytometry

Flow cytometry (flow microfluorimetry) provides a quick means for analysis of ploidy in planarians. Nuclei from homogenized tissues of the freshwater planarian Dugesia japonica japonica Ichikawa et Kawakatsu were stained with propidium iodide and measured with an argon-laser flow cytometer to produce histograms of DNA content. Tissues from sexually mature individuals produced histograms with a 1n (haploid) peak but no 3n peak (triploid peak), whereas those from asexual individuals showed a 2n peak or a 3n peak or both, but no 1n peak. Thus, the 1n peak distinguished sexual individuals. Mixoploid individuals, i.e., mosaics with both diploid and triploid tissues, were identified by the presence of both a 2n peak and a 3n peak. The ratios of the heights of the 2n and 3n peaks from tissues in different parts of a single mixoploid individual were similar, suggesting that the diploid and triploid cells are homogeneously distributed.     

In vitro stimulation of megakaryocyte maturation by megakaryocyte stimulatory factor

Counterflow centrifugal elutriation and Percoll density gradient centrifugation were employed to prepare cell populations from rat bone marrow that were selectively enriched in the cytoplasmically immature megakaryocytes and depleted of the most mature megakaryocytes. The incorporation of [14C]leucine into the platelet-specific alpha-granule protein, platelet factor 4, as well as the incorporation of [35S]sulfate into platelet proteoglycans synthesized by the maturing megakaryocytes were monitored as markers of cytoplasmic maturation. Rat platelet factor 4 was specifically isolated and characterized by its high affinity for heparin-Sepharose and its amino-terminal sequence homology to human and rabbit platelet factor 4. The [35S]sulfate-labeled proteoglycans were primarily composed of chondroitin 4-sulfate glycosaminoglycans and were identified as platelet granule components by their ability to be secreted by megakaryocytes in response to thrombin or A23187. The production of both components was increased as much as 3-fold in a dose-dependent manner by the addition of picomolar concentrations of purified megakaryocyte stimulatory factor, without a concomitant increase in general protein synthesis. The above results suggest that the megakaryocyte stimulatory factor may regulate the synthesis of platelet granule components by megakaryocytes and hence control the rate and/or extent of cytoplasmic maturation during megakaryocyte development.     

产甘油假丝酵母(Candida glycerinogenes)染色体倍性分析

摘要：【目的】产甘油假丝酵母作为一株优良高产甘油菌株,已成功应用于工业生产15年。近年来由于产甘油假丝酵母染色体倍性尚不明确,限制了对其进行遗传改造的研究进展,因而我们对产甘油假丝酵母染色体倍性研究,分析确定其染色体倍性。【方法】选用酿酒酵母细胞进行生孢,制备酿酒酵母单倍体细胞作对照,并选用热带假丝酵母作为二倍体酵母细胞对照,利用血球计数板得到热带假丝酵母、产甘油假丝酵母、单倍体及二倍体酿酒酵母细胞数,提取染色体,通过二苯胺检测法测定DNA含量。由于在相同紫外照射条件下单倍体细胞比二倍体细胞更容易死亡,因     

Alpha-actinin arcs in megakaryocyte spreading

Cultured megakaryocytes, isolated from guinea pig bone marrow, were treated with buffer or adenosine diphosphate (ADP) (10 microM) on plain or coated glass surfaces. Control cells were rounded and non-adherent. The nucleotide induced the cells to spread to several times the initial diameter, and to become flattened and markedly adherent to the substratum. 'Cytoskeletons' were examined by transmission electron microscopy (TEM). Those from unspread cells contained only rare microfilaments and no filament bundles; those from spread cells contained large numbers of microfilaments in nets and many filament bundles, which were largely oriented circumferentially. Interference reflection microscopy demonstrated that the spread cells were attached to the substratum in arc-shaped regions, which corresponded to arcs containing alpha-actinin as seen by specific immunofluorescence of the same cells. However, other arcs of alpha-actinin staining did not correspond to arcs of tight attachment. We conclude that fibrous arcs, which appear to assemble as part of the spreading process, play a role in what are probably transient surface attachment sites. A survey of observations of spreading in other cell types suggests that similar arcs are more widespread than has been realized.     

Treatment of thrombocytosis in myeloproliferative disorders with interferon alpha-2a

In an open prospective pilot trial, we tested the effect of recombinant interferon alpha-2 a (rIFN alpha-2 a) on thrombocytosis in myeloproliferative disorders (MPD). Since October 1986, 13 patients with MPD (4 with chronic granulocytic leukemia, 4 with polycythemia vera, 3 with essential thrombocythemia and 2 with myeloid metaplasia) were treated with rIFN alpha-2 a. Platelet counts decreased in all treated patients within 2 to 10 weeks from a median value of 1,050 x 10(9)/l (range 610-1,940 x 10(9)/l) to 340 x 10(9)/l (range 230-495 x 10(9)/l). The response was dose-dependent. In 11 patients we observed a simultaneous reduction of the white blood cell count. Six patients still continue the IFN alpha-2 a therapy. In 7 treatment was discontinued, because of chronic side effects in 3, and because of noncompliance in one. In these patients, thrombocytosis recurred after discontinuation of the therapy. These results show that rIFN alpha-2 a is effective in controlling thrombocytosis in MPD. However, the long-term benefit of interferon in these disorders remains to be established.     

Evaluation of platelet function in postsplenectomy thrombocytosis

The usually transient post-splenectomy thrombocytosis has no well defined effect on the development of thromboembolism. We studied 14 patients submitted to splenectomy for different causes: portal vein thrombosis (PVT), idiopathic thrombocytopenia purpura (ITP) and lymphoma. The mean platelet count in PVT patients was significantly higher than in all other subjects. In PVT group we demonstrated various alterations in platelet aggregation curves and a frequent increase of platelet 5-HT levels. In effect, we noted that all the PVT patients showed a myeloproliferative disease (MPD) before the surgical procedure. We therefore conclude that the increased incidence of thromboembolism in the patients who underwent splenectomy is probably caused by a preexisting MPD rather than the post-splenectomy thrombocytosis.     

DNA ploidy as a prognostic factor in rhabdomyosarcoma. Analysis of 35 cases with image cytometry

OBJECTIVE: To correlate DNA ploidy in rhabdomyosarcoma (RMS) with other prognostic factors and patient survival and to search for possible reasons for inconsistent conclusions in similar, published studies. STUDY DESIGN: DNA content was measured in archival specimens obtained from 35 patients (23 children and 12 adults) with RMS. Cell suspensions were prepared by the modified Hedley technique, stained by the modified Feulgen-thionin method and analyzed by automated high-resolution image cytometry. DNA ploidy was assessed on the basis of DNA index values. We used the chi 2 test to correlate DNA ploidy with other prognostic factors, Kaplan-Meier procedure to estimate overall survival in terms of individual prognostic factors, log-rank test to calculate differences in survival between groups and Cox multivariate regression analysis to determine the independence of variables in relation to survival. RESULTS: A statistically significant correlation was found only between DNA ploidy and histologic subtype of RMS, patient sex and patient age. A hyperdiploid DNA pattern predominated among patients with embryonal RMS, and a tetraploid pattern dominated among patients with alveolar RMS. The highest 5-year survival rate was seen among patients with hyperdiploid RMS, followed by those with diploid, tetraploid and hypertetraploid RMS. Although DNA ploidy was a significant prognostic factor in univariate analysis, it did not retain its independent prognostic value in multivariate analysis, in which patient age, tumor size and histologic subtype were the only significant factors. We found 12 articles reporting on the association between DNA ploidy and survival of patients with RMS: 6 found a correlation, and 6 did not. The main reasons for the discrepancies seem to be the inclusion of chemotherapy-treated and nontreated patients, low number of patients and differences in grouping DNA histograms. CONCLUSION: The precise prognostic value of DNA ploidy in RMS remains equivocal. Larger, cooperative studies could give statistically more reliable results.