Lymphocytes as inducers of mitoses of human morphologically identifiable progenitor cells. Phase contrast observations.

Phase contrast time lapse observations of human bone marrow cells demonstrate the induction of progenitor cell mitoses or amitoses by the touch of locomotive lymphocytes on the cell surface. Interkinetic progenitor cells as well as precursor cells before mitosis were very rarely touched by lymphocytes. We suppose this cell-cell interaction is essential in the T-cell participation in hematopoietic stem cell proliferation.

No elevation of the frequencies of chromosomal alterations as a consequence of handling cytostatic drugs. Analyses with peripheral blood and urine of hospital personnel.

Peripheral blood lymphocytes of hospital personnel handling cytostatic drugs showed no elevation of structural chromosomal aberrations or sister-chromatid exchanges (SCE) over the baseline. The urine of the probands did not induce SCE in peripheral lymphocytes in vitro.

Associations of functional candidate genes derived from gene-expression profiles of prenatal porcine muscle tissue with meat quality and muscle deposition.

Ten genes (ANK1, bR10D1, CA3, EPOR, HMGA2, MYPN, NME1, PDGFRA, ERC1, TTN), whose candidacy for meat-quality and carcass traits arises from their differential expression in prenatal muscle development, were examined for association in 1700 performance-tested fattening pigs of commercial purebred and crossbred herds of Duroc, Pietrain, Pietrain x (Landrace x Large White), Duroc x (Landrace x Large White) as well as in an experimental F(2) population based on a reciprocal cross of Duroc and Pietrain. Comparative sequencing revealed polymorphic sites segregating across commercial breeds. Genetic mapping results corresponded to pre-existing assignments to porcine chromosomes or current human-porcine comparative maps. Nine of these genes showed association with meat-quality and carcass traits at a nominal P-value of < or = 0.05; PDGFRA revealed no association reaching the P < or = 0.05 threshold. In particular, HMGA2, CA3, EPOR, NME1 and TTN were associated with meat colour, pH and conductivity of loin 24 h postmortem; CA3 and MYPN exhibited association with ham weight and lean content (FOM) respectively at P-values of < 0.003 that correspond to false discovery rates of < 0.05. However, none of the genes showed significant associations for a particular trait across all populations. The study revealed statistical-genetic evidence for association of the functional candidate genes with traits related to meat quality and muscle deposition. The polymorphisms detected are not likely causal, but markers were identified that are in linkage disequilibrium with causal genetic variation within particular populations.

[Regulation of the human erythropoietic proliferation pool by the microenvironment (author's transl)]. / Studien zur humoralen Regulation des erythropoetischen Proliferationsspeichers beim Menschen.

The Effects of Various Substances in the Culture Microenvironment on the Proliferation of Erythroblasts in vitro were Studied and Evaluated by differential cell and mitosis counting in normal and pathologic bone marrow. The mitotic frequency and maturation correlated directly with the erythropoietin content of the medium and were potentiated on addition of either folic acid, etiocholanolon or cAMP. However, erythroblast proliferation was stimulated independently of erythropoietin concentration when either cobalt, and androgen or an anabolic substance was added to the medium. An iron-deficient medium prevented the maturation of erythroblasts to reticulocytes, thereby rendering erythropoiesis ineffective. The existence of an erythrocyte chalone ineffective. The existence of an erythrocyte chalone or an erythropoietin inhibitor could not be deduced from our experiments since the transformation of pluripotent to erythropoetin-sensitive stem cells is not included. A stimulation of the granulopoietic proliferation pool occurred when serum from patients with either polycythemia vera or after acute blood loss was added to the medium.

[Morphological characterization of the myelopoietic stem cell reserves in the human]. / Die morphologische Charakterisierung des myelopoetischen Stammzellspeichers beim Menschen.

According to microkinomatographic observations made on single cells up to ten days in vitro, there are the following laws of growth in haematopoiesis: Small cells will increase in growth up to five times in size, with their morphologic and kinetic properties being preserved, the blood lymphocyte will grow to immunoblasts, the small pluripotent stem cell to bone-marrow histiocytes. When growing, the myelopoietic stem cell may be gradually deviated into all myelopoietic cell lines. Instead of bone-marrow histiocytes it may differentiate to promyelocytes, promonocytes or proerythroblasts, all having an equal nucleus size, when it is induced by serum factors. Apart from histiocytes these large cells are capable of differentiating to a clon of blood cells, such as granulocytes, monocytes or reticulocytes, by several successive divisions of maturity. Contrary to the stimulated lymphocyte, symmetric mitoses will frequently occur, when small pluripotent stem cells are growing to bone-marrow histiocytes to be no further differentiated. Occasionally, asymmetric divisions may also be observed, i.e. one of the daughter cells will differentiate into one of the myelopoietic lines, whereas the other one will remain a progenitor cell. Moreover, there are various pathological mitoses in all progenitor cell sizes, such as endomitosis, cytoplasm fusion after mitosis, nucleus fusion after cytoplasm conjunction or amitotic nucleus division without cytokinesis. They produce megacaryoblasts further differentiating to megacaryocytes by corresponding pathological mitoses. According to our vital observations the pluripotency of the haematopoetic stem cell is being lost step by step.

[Effect of different glucocorticoids on the in vitro cell kinetics of human hematopoiesis represented by a bone marrow culture and by phytohemagglutinin- and pokeweed mitogen-stimulated blood cultures]. / Die Wirkung verschiedener Glukokortikoide auf die Zellkinetik der menschlichen Hämatopoese in vitro, dargestellt an der Knochenmarkkultur und an der mit Phytohämagglutinin und Pokeweed Mitogen stimulierten Blutkultur.

The effect of nine glucocorticoids, DHEA, cyclohexanol and dextran sulphate on the hematopoietic proliferation was investigated. Three glucocorticoids inhibited the proliferation of normal bone marrow cells, the others enhanced it. The T-cell stimulation was not inhibited by fluorcortolone contrary to prednisolone. Specially the proliferation of acute myeloid leukemic blasts was inhibited by fluorcortolone hemisulfate, which significantly stimulated the proliferation of normal bone cells.

Interactions between lymphocytes and hematopoietic progenitor cells in normal and leukemic human bone marrow (phase contrast observations).

Single cell observations of normal and of leukemic human bone marrow cells demonstrated cell-cell interactions of lymphocytes with hematopoietic progenitor cells. In all cases lymphocytes and target cells were from the same individual. Lymphocyte-target cell interactions occurred more frequently with normal committed progenitor cells and leukemic blast cells from acute myeloid leukemia than with precursor cells of the proliferative cell pool, including myeloblasts, promonocytes, erythroblasts and megakaryocytes. Both induction of mitosis and degeneration of the progenitor cells occurred after cell-cell interaction with almost the same frequency. Acute myeloid leukemic blast cells degenerated after contact with lymphocytes with the same frequency as normal progenitor cells (i. e. in 16% of cell contacts), but especially during mitosis. In contrast, normal and regenerating bone marrow progenitor cells from myeloproliferative diseases demonstrated no degeneration after cell-cell interaction with lymphocytes during mitosis. Otherwise the induction of mitoses by lymphocyte-target cell interactions was more frequently observed in normal progenitor cells than in leukemic blasts.

[Different effects of cytostatics on cell kinetics of normal and leukemic human bone marrow in vitro]. / Unterschiedliche Wirkungen von Zytostatika auf die Zellkinetik des normalen und leukämischen menschlichen Knochenmarkes in vitro.

The kinetics of the erythropoietic and of the granulocytopoietic pool of normal und leukemic human bone marrow cells were investigated in vitro under the influence of 23 cytostatic drugs. Clot cultures were evaluated up to 72 hours by differential and mitotic counting in smears stained according to Pappenheim. Based on mitotic index, caryological curve and percentage of proliferative cells different patterns of proliferation were observed: spindle poisons enhanced the mitotic index, all other investigated cytostatic drugs diminished it, but in a different degree the one of the erythroblasts, of the granuloblasts and of the leukemic blasts. The decrease of the percentage of proliferative precursors did not always correlate with the decrease of the mitotic index, because sometimes an ineffective granulocytopoiesis arised or leukemic blasts entered the Go-phase of the cell cycle. 1-asparaginase was the only drug which increased erythroblasts. Chloramphenicol was the only out of 18 drugs tested on leukemic bone marrow that decreased only the leukemic proliferation and had no influence on the normal one.

[Morphological phase-contrast cinematographic studies on the behavior of bone marrow cells in vitro. X. Myeloblasts and monoblasts]. / Morphologische Phasenkontrast-kinematographische Studien zum Verhalten von Knochenmarkzellen in vitro

The myeloblast is nearly of the same size as the lymphozyte and has the same nucleus cytoplasma ratio of 0.7 but differs from it in its morphology, its high frequency of multiplication, and by its kinetic behaviour. By phase contrast observation the transformation of the myeloblast into the promyelocyte has been seen several times. The myeloblast like the lymphocyte is able to move although at a much slower speed. By locomotion the myeloblast, and likewise the lymphocyte can move between blood and bone marrow. Of the granulocytopoietic series only the promyelocyte and the myelocyte with their nuclear cytoplasm relation of 0.3 are found in the bone marrow. The monoblast but is about twice its size, and moves less often. Apart from its transformation into promonocyte, monocyte and histiocyte, the monoblast may evalue in another way: it grows into a cell of doubled size with the same nucleus cytoplasm ratio 0.7 in whose cytoplasm are seen a lot of big granules. The cell may now be characterized as a tissue macrophage or mastcell. In consequence a frequently multiplying basophilic cell with a coarse nuclear structure and nucleoli, grows from a diameter of 6.5 to one of 13.0 mum without marked morphologic change apart from becoming alpha-NA-Esterase positive and acquiring big lysosomes. When this haemocytoblast is small (K 1/4 to K 1/2), it can be triggered into the granulocytopoietic series, when it is bigger (K 1/2 to K1) into the monocytopoietic series, or in the size K1 to K2 by erythropoietin into the erythropoietic series. If no triggering takes place, the cell degenerates after having granulated. The lymphopoietic system is not connected to this system of stem cells committed to the granulocytopoietic, the monocytopoietic and the erythropoietic series. Therefore we postulate a dual haematopoietic system. In acute leukemias the spread of size of cells and nuclei is wider and kinetically active stem cells are rarer. Only leukemic promyelocytes are in locomotion vividly, in contrast to the normal ones. In leukemic myeloblasts there are fewer mitoses than in normal ones, but in leukemic promyelocytes and monoblasts the mitotic frequency is not reduced.

Origin of the early megakaryopoietic progenitor cell and further polyploidization in the thrombopoietic cell series. Phase-contrast observations of human bone marrow.

Continuous observation of living cells by phase-contrast time lapse microcinematography revealed different origins of multinuclear cells and cells with lobed nuclei in the hematopoietic progenitor cell series, which must be classified as megakaryoblasts, the determined progenitor cells of the megakaryopoietic series. Hematopoietic progenitor cells with single nuclei of nuclear diameter 5-13 microns, the promegakaryoblasts, may become polyploid by various means: by postmitotic cytoplasmic fusion, by consecutive nuclear fusion, by endomitosis, or by nuclear amitosis or lobe formation presumably after endoreduplication and nuclear growth. Including amitotic nuclear division in the mitotic rate of normal progenitor cells nearly the half of all these cells in G2 phase (0.4) produce in vitro octoploid megakaryoblasts, respectively osteoclasts. Thrombopoietin-rich sera in the medium enhance the mitotic index of the hematopoietic progenitor cell line from 0.1 to 0.25, but do not change the proportion of polyploid mitoses.

Documentation of normal and leukemic myelopoietic progenitor cells with high-resolution phase-contrast time-lapse cinematography.

The high-resolution phase-contrast, time-lapse cinematography using oil immersion lenses and 16-mm film demonstrates the kinetic cell events as maturation, locomotion, mitosis, and apoptosis of cells cultivated at 37 degrees C for up to 10 days. 0.5 v/v frozen-thawed sera with presumably high cytokine concentrations were added to the plasma or agar clot. Vital progenitor cells from human bone marrow and blood have a large, bright, unstructured nucleus with a large nucleolus and a narrow rim of cytoplasm (nuclear/cytoplasmic volume ratio = 0.7). Their nuclei are 6-14 micrometer in diameter and double their volume within 8 h. Many (70%) move at a mean speed of 2 micrometer/min, and many (30%) multiply with alpha-2alpha mitoses, generating progenitor cell families. Various disturbances during the course of mitosis lead to the formation of polyploid cells, thereby yielding the megakaryocytic cell line. Some of the progenitor cells undergo asymmetric alpha-alphan mitoses: One of the two initially identical daughter cells remains a progenitor cell in the morphological sense, whereas the other daughter cell - depending on the size of its mother cell - matures in the same culture medium to form a granulocytopoietic, monocytopoietic or erythrocytopoietic cell line. - In acute myeloid leukemias (AML), the blasts and their nuclei are slightly larger than the corresponding progenitor cells and move faster (5 micrometer/min). Symmetric alpha-2alpha mitoses permit unlimited multiplication of the leukemic blasts if contact with cytotoxic lymphocytes does not render them apoptotic. This results in more stromal cells than normal. Granulocytopenia, monocytopenia, and anemia occur due to the genetic impairment of signaling control for asymmetric alpha-alphan mitoses, and thrombocytopenia occurs due to the reduction in polyploidization.

A long-term clinical trial of interferon alpha-therapy in essential thrombocythemia.

In a prospective clinical trial involving six patients suffering from essential thrombocythemia (ET), recombinant human interferon alpha 2b significantly and consistently lowered highly elevated peripheral platelet numbers over long time periods. One patient has now been on continuous treatment for 4 years. During the treatment phase none of the patients suffered from bleeding episodes, thrombosis or disturbances of the microcirculation. The interferon maintenance dose varies considerably from patient to patient, but it is usually much lower than the induction dose. One of the patients had to be withdrawn from the study due to interferon-specific chronic toxicity concomitant with the development of non-neutralizing interferon antibodies. With the exception of one patient, stopping interferon treatment led to an increase in peripheral platelet numbers of up to one million cells per microliter of blood within 4 to 12 weeks. We conclude that interferon alpha can correct peripheral thrombocytosis in selected patients with essential thrombocythemia over a period of years and prevent morbidity attributable to this disease.

[Quantitative determination of human bone marrow proliferation kinetics during three culture days]. / Quntitative Bestimmung der Proliferationskinetik des menschlichen Knochenmarks während drie Kulturtagen

In order to obtaon of human bone marrow cells, fresh bioptic material was homogenized and the cell suspensions were incubated for 72 hs in a fluid medium. After 24, 48 and 72 hs of incubation the total cell number of the culture was determined. At the same time differential counts of stained smears were performed. Both, erythrocytopoiesis and granulocytopoiesis showed regeneration, maturation, and an absolute increase of the number of precursors and of mature cells. The quantitative data obtained in vitro during 24 hs correspond with our data of kinetics obtained by observed mitotic duration and cell differential countings in vivo. However, after a longer cultivation time we found a diminution of divisible precursors, and an increase of mature erythroblasts as well as an excessibe survival of the PMNs.

[PHA- and PWM-stimulation in acute leukaemia (author's transl)]. / Antigenstimulation peripherer Leukozyten von Patienten mit akuten Leukämien.

Peripheral blood cells from patients with acute leukaemia were stimulated in RPMI-medium with Phytohaemagglutinin (PHA) and/or Pokeweed-Mitogen (PWM). Samples of the cultures were taken at 3, 5 and 7 day intervals for cytological characterization. From 119 cultures from 65 patients 76 cultures from 47 patients could be analysed. Cultures stimulated with PHA and/or PWM showed in 43,4% a higher stimulation rate in comparison with cultures from normal persons. These findings are discussed in relation to the increased agar-colony growth growth of leukaemic cells after in vitro PHA-stimulation and transformation of blast cells.

Maturation and proliferation capacity of blood cells from untreated acute myeloid leukemia and its prognostic significance.

Blood cells from 68 patients with untreated acute myeloid luekemia were cultured in RPMI-medium without stimulating factors up to ten days. The cultures showed in part maturation and proliferation to monocytes-macrophages, in part to promyelocytes, myelocytes and Pelger-like cells, in part we did not find any differentiation or the cultures were degenerated during the first days. Retrospectively we found that in the 16 blood cell cultures with capacity to differentiation into the monocyte-macrophages-system 5 patients had a smouldering leukemia. Our preliminary evidences suggest that the diagnosis "smouldering leukemia" is to be found with out in vitro culture system. Further analysis suggest that patients with acute leukemia whose blood cells have the capacity for maturation to monocytes-macrophages or to promyelocytes, myelocytes and Pelger-like cells have a better chance of achieving a complete remission and a longer median survival time.

Nocodazole sensitivity, age-related aneuploidy, and alterations in the cell cycle during maturation of mouse oocytes.

To detect age-related alterations in the formation and function of the spindle apparatus, we examined in vitro maturing oocytes obtained from young (2-4 mo) and aged (greater than 9 mo) diestrous CBA/Ca mice. Observation of cells processed for antitubulin immunofluorescence revealed that oocytes from aged females progress faster through first maturation division than those from young animals. They are also more prone to nondisjunction, as shown by a significantly higher level of aneuploidy in C-banded cells arrested at metaphase II. The ability of oocytes to recover from treatment with a microtubule inhibitor, nocodazole, and the effect of the drug on spindle integrity and chromosome segregation were also studied. In both age groups, treatment of metaphase I oocytes with 10 microM nocodazole caused rapid and complete microtubule depolymerization and chromosome scattering. Upon recovery, oocytes from both age groups were able to reestablish a spindle apparatus, proceed through anaphase, and extrude a first polar body. However, nocodazole treatment led to a dramatic increase of aneuploidy. Unexpectedly, the relative rise in hyperploids was greater in oocytes from young mice than in those from aged mice, so that the absolute percentage of hyperploid metaphase II cells was similar in both age groups after drug treatment. Concomitantly, nocodazole exposure abolished or, at least, diminished intrinsic differences in the cell cycle and anaphase trigger present in the controls (e.g., the earlier onset of chromosome separation in oocytes from aged females). It shortened the period available for spindle formation before chromosome segregation in all oocytes. Therefore, our study implies that temporal differences in the progression of oocytes through maturation, in particular, the shortening of the time available for alignment of bivalents before chromosome separation occurs in oocytes of old females, are mainly responsible for age-related rises in aneuploidy. There is no indication that (1) the spindle apparatus of oocytes from aged mammals is more labile or susceptible to disturbances than the spindle apparatus of oocytes from young individuals or that (2) an increase in the number of univalents makes oocytes from aged mammals particularly prone to nondisjunction.

[Cytoplasmic vacuoles and kinetics of bone marrow cells in alcoholism, liver cirrhosis and anemia]. / Zytoplasmavakuolen und Kinetik von Knochenmarkzellen bei Akloholismus, Leberzirrhose und Anämien.

Bone marrow smears show vacuoles in the cytoplasm of haematopoietic cells due to alcoholism, liver cirrhosis and anemias different origin, for example in acute leukemias, in proportion to the decreased content of haemoglobin. The cell doubling rat in vivo is not obviously impaired by the vacuolisation of the cytoplasm exactly as is the case with proliferation in vitro, although the vacuolation increases in vitro. Phase contrast observation makes visible the vacuoles without membranes and content as they appear transitorily in pinocytosis, and not as secondary lysosomes. The common cause for the formation of the vacuoles without membranes in the cytoplasm in all these pathological states could be intracellular hypoxia and acidosis.

Kinetic and morphological changes induced in human blood leucocytes by cytochalasin D and E.

The effect of increasing concentrations of cytochalasin D and E, up to toxicity, on the velocity of blood leucocytes from normal subjects was measured in vitro using a high-resolution objective and phase-contrast time-lapse photography. The dose-response effect for the two different cytochalasins differed in accordance with the different cell specificity of their membrane binding. The average velocity of granulocytes was reduced at cytochalasin D concentrations above 5 x 10(-7)M and cytochalasin E concentrations above 5 x 10(-5)M. The effect on monocytes and eosinophils was similar. In contrast the velocity of lymphocytes was not affected until cytotoxic concentrations were reached. The concentration ranges which inhibited locomotion corresponded well with the concentration ranges of the cytochalasins which have an in vitro effect on microfilaments. The concentrations which induced additional morphological changes in lymphocytes also correlate well with the concentrations found to inhibit cross-linking in vitro, as well as those known to induce morphological changes in, for example, fibroblasts in vivo. Cytotoxic effects were first observed with ten-fold higher concentrations of cytochalasin E than of cytochalasin D.