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PubMed1984

Analysis of pure and mixed murine mast cell colonies.

Mast cells have been proposed to originate from diverse sources, including connective tissues, macrophages, T lymphocytes, and hemopoietic cells. Evidence for a hemopoietic origin of mast cells includes the presence of mast cell precursors in spleen colonies and the presence of mast cells in hemopoietic colonies in culture. Here we report a detailed analysis of mouse spleen mixed hemopoietic colonies containing mast cells. All of the colonies in cultures plated at low cell densities were individually removed for analysis by May-Grunwald-Giemsa staining on day 15 of culture. Examination of five dishes which contained a total of 82 colonies showed 16 pure mast cell colonies and 36 mixed mast cell colonies. Sixteen different combinations of cell types were seen and were not distinguishable from each other in situ. The most diverse type of mixed colony contained macrophages (m), neutrophils (n), eosinophils (e), mast cells (Mast), megakaryocytes (M), erythroid cells (E), and blast cells. The clonal origin of mixed mast cell colonies was established by the replating of single cells obtained from blast cell colonies. Individual cells were removed with a micromanipulator, replated, and allowed to grow for 15 days. Cytospin preparations of 10 such colonies showed diverse combinations of cell lineages which were seen in the different types of mixed mast cell colonies described above. Replating studies of mixed mast cell colonies were carried out and a high incidence of replating was seen. Approximately one half of these colonies formed only mast cell colonies upon replating. Further studies showed that pure mast cell colonies could be serially replated four to five times. The replating efficiency of cells in the primary mast cell colonies varied over a wide range (2.5-44%) with an average replating efficiency of 13%. The data also revealed that cells containing metachromatic granules possess significant proliferative capacity. From these studies of pure and mixed mast cell colonies, we concluded that mast cells are in wide variety of types of mixed colonies and that the in situ identification of mixed colonies is unreliable, that mast cells are derived from pluripotent hemopoietic stem cells, and that mast cells with metachromatic granules can have a high proliferating ability.

Animals↗
PubMed1967

Hemopoietic spleen colony studies. II. Erythropoiesis.

The polycythemic repression of erythropoiesis and the restoration of erythropoiesis by specific stimulation were studied in the spleen colony system in irradiated mice. 1. A 5 day period of erythropoietin stimulation (exogenous erythropoietin) or repression (polycythemia) of the bone marrow donor only, does not significantly alter the number or type of colonies formed by the transplanted marrow cells. 2. Erythropoietin stimulation did not alter the number or type of endogenous colonies formed in mice receiving 580 R. Erythropoietin repression (polycythemia) markedly reduced the growth but not the number of erythroid colonies, while not affecting the other types of colonies formed endogenously. 3. Erythropoietin stimulation of the irradiated recipient during colony growth did not alter the number or type of spleen colonies formed by transplanted marrow. Erythropoietin repression by polycythemia during colony growth completely suppressed the appearance of morphologically erythroid colonies without significantly altering the incidence of the other colony types. This effect of polycythemia was completely prevented by exogenous erythropoietin. Irradiated mice are therefore presumed to be secreting sufficient erythropoietin for maximal erythroid colony development. 4. The erythroid colonies suppressed by polycythemia were recognizable as microscopic foci of undifferentiated cells. Exposure of these foci to erythropoietin stimulation at different periods in their development was manifested by different degrees of growth and differentiation, from which it is apparent that erythropoietin stimulates not only morphological differentiation but also rapid mitosis. Retransplantation of either erythroid or of neutrophilic primary spleen colonies gave rise to both erythroid and neutrophilic secondary spleen colonies. The percentage of erythroid secondary colonies was slightly but significantly higher among the progeny of transplanted erythroid primary colonies than among the progeny of transplanted neutrophilic primary colonies. On the basis of these and other results, a working hypothesis is proposed for factors controlling the growth and differentiation of spleen colonies from transplanted bone marrow. It is postulated that most but perhaps not all spleen colony-forming units are pluripotent hemopoietic stem cells. It is further postulated that hemopoietic-inductive microenvironments (HIM) of different kinds exist in both the spleen and the bone marrow, and that these determine the differentiation of pluripotent stem cells into each of the lines of hemopoietic differentiation. Erythropoietin therefore may "induce" erythroid differentiation of only those stem cells under the influence of an erythroid HIM. Alternatively erythropoietin may act only as a growth and function stimulant of those stem cells that have been "induced" by an erythroid HIM into a state of erythropoietin responsiveness. In the latter case morphological differentiation presumably results from the functional activity stimulated by ESF.

Animals↗
PubMed2005

[Roma colonies in Hungary--medical care of children and hygienic conditions].

INTRODUCTION: The hygienic conditions and the public health safety of the Hungarian Roma living in colonies are poorly known, the health care of Romany children is often subject to criticism. AIM: The authors aim was to identify the blank areas present in public health and to analyze the medical care of children living in Gipsy colonies. METHODS: Medical care of children living in Gipsy colonies in Hungary and the hygienic situation of these colonies have been analyzed. The examinations, performed by the staff of the National Public Health and Medical Officers' Service included all counties and the capital. Data were gathered from answers given to the questions in the circular sent by the Chief Medical Officer of State, by questionnaire-interview method as well as, by on site hygienic examination. Data from the series of examinations were completed by the data of the National Institute for Basic Medical Care. RESULTS: According to the hygienic definition of Romany colonies, there are 767 colonies in Hungary, scattered in 530 habitats, while according to the definition of availability of public utilities and structure of the colonies the number of colonies is 300-400. There are no Romany colonies in the capital, while most of them are located in the counties Hajdú-Bihar, Baranya, Szabolcs-Szatmár-Bereg and Borsod-Abaúj-Zemplén. A population of 3 million is living in the habitats with Romany colonies; 138000 (4,7%) of them are living in Romany colonies, or in colony-like communities with similar hygienic situation. In these habitats 5,8% and 16,3% of the population are children of 0-5 years old and 0-14 years old, respectively, while in the colonies the same numbers are 15,8% and 35,6%. Medical care is provided by 456 family pediatrist for the the habitats with Romany colonies. Consulting hours for the children population of 11,4% of the family doctors seems to be insufficient (< 15 hours/day/5 days/week). Only 60% of care for children is provided by specialist in pediatrics. 94% of emergency care for children is provided for family doctors of the adult population (this proportion is characteristic for the whole country). 12,5% of district nurses caring for the colonies provide care for more than 2 habitats; 147 of them (22% of all district nurses) provide care for more than 750 persons (experiences show that appropriate care can be ensured if the number of care recipients does not exceed 300 and the maximum number of settlements under care is two). Illegal waste deposits and animal carcase disposal site are located within 1000 m of 15% and 11% of the colonies, respectively. Most of the dwelling sites are messy, hygienically neglected, rodents and unvaccinated stray dogs are frequently met in their neighborhood. CONCLUSIONS: 1. sufficiently accurate data are not available on the Gipsy colonies (number, location, population, children population), partly due to the related different definitions and partly because this issue of public health importance is not treated uniformly. The repeated examinations of the National Public Health and Medical Officers' Service presented here only improved the relevant information; 2. child health care of the Gipsy colonies does not differ from that of the rest of the Hungarian children, but similarly to that, a lot of corrections are needed--especially regarding the emergency service; 3. the hygienic situation of the Gipsy colonies is not acceptable, it endangers the public health and epidemiological safety of not only their inhabitants, but the whole country.

Adolescent↗
PubMed1994

[Colony formation of mouse primitive hemopoietic progenitors with interleukin-6 and phorbol ester, and their signal transduction].

Colony formation of mouse primitive hemopoietic progenitors with interleukin-6 (IL-6) and 12-O-tetradecanoyl-phorbol-13-acetate (TPA), and their signal transduction were studied. Although IL-6 or TPA alone could not form colonies, their combination gave rise to significant number of colonies from Day-2 post 5-FU bone marrow cells. When colony numbers were compared with those supported by IL-3, IL-6+TPA gave rise to 86 + 47% of colonies formed with IL-3. Time course of colony formation with IL-6+TPA run parallel with that of IL-3. These colonies included not only granulocyte/macrophage (GM) colonies, but also granulocyte/erythrocyte/macrophage/megakaryocyte (GEMM) colonies and blast cell colonies. Delayed addition of IL-6 or TPA decreased colony numbers, suggesting that both IL-6 and TPA were needed from the start of cultures for maximal colony formation. When cultures were started with TPA, and IL-6 was added on Day 2 of culture or later, few colonies developed. These data suggested that IL-6 might be essential to the survival of the progenitors in culture. Chronic exposure of progenitors to TPA prior to the culture with IL-6+TPA suppressed colony formation. Addition of calphostin C, a specific protein kinase C (PKC) inhibitor or genistein and herbimycin A, specific tyrosine kinase (TK) inhibitors to the culture also decreased colony numbers formed with IL-6 and TPA. To clarify which effects of IL-6 or TPA on colony formation were blocked by the inhibitors, the inhibitors were added to preincubation of progenitors with IL-6. Both the PKC inhibitor and TK inhibitors blocked the increase of colonies resulted from a pre-incubation with IL-6. Although delayed addition of TPA enhanced IL-6-dependent colony formation, delayed addition of TPA with either the PKC inhibitor or TK inhibitors canceled the increase of colonies. These data suggested that both signals of IL-6 and TPA might be transduced via activation of PKC and TK, but further studies are needed to confirm that.

Animals↗
PubMed2000

Autogenous Aedes togoi sub-colony (Chanthaburi, Thailand strain), an efficient laboratory vector in study of filariasis.

Comparative filarial susceptibility and biology between stock colony and selectively autogenous Aedes. togoi sub-colony were carried out to determine the laboratorial vector-capacity and viability of autogenous sub-colony. The results of susceptibility revealed that the selectively autogenous Ae. togoi sub-colony yielded higher susceptibility than the stock colony, ie Dirofilaria immitis: susceptibility rates=80.00% [Exp1(F8)] and 76.19% [Exp2(F17)] (autogenous sub-colony), 53.33% (Exp1) and 71.43% (Exp2) (stock colony); Brugia malayi: susceptibility rates=83.33% [Exp1(F17)] and 84.38% [Exp2(F19)] (autogenous sub-colony), 81.25% (Exp1) and 75.00% (Exp2) (stock colony), but not at the level of statistically significant differences except the Exp1 of D. immitis, which was significant difference. In addition, the average No. L3 per infected mosquito in the selective autogenous sub-colony (D. immitis: Exp1=3.37, Exp2=3.19; B. mallayi: Exp1=8.80, Exp2=3.37) was also higher than in stock colony (D. immitis: Exp1=2.44, Exp2=2.73; B. malayi: Exp1=7.85, Exp2=3.02), but not at the level of statistically significant difference. The results of comparisons on some biological aspects demonstrated that most of the cases they have similar biology except the average egg deposition per gravid female of stock colony (130.17+/-43.33) was significantly more than selectively autogenous sub-colony(F9) (94.33+/-13.69), egg length x width 575.62+/-18.06 microm x 186.15+/-9.35 microm of stock colony was significantly larger than selectively autogenous sub-colony(F9) 560.49+/-18.96 microm x 177.99+/-8.40 microm, and mean longevity of adult female of stock colony [41.60(6-61)] was significantly longer than selectively autogenous sub-colony(F9) [35.00(5-39)]. The selectively autogenous sub-colony was established and twenty-two successive generations have been colonized.

Aedes↗
PubMed1983

Growth and differentiation of spermatogenetic colonies in the mouse testis after irradiation with fission neutrons.

The longitudinal outgrowth of spermatogenetic colonies arising from stem cells that survived neutron doses of 150, 300, and 350 rad was studied up to 30 weeks in histological sections of CBA mouse testes. Two methods were used: (1) the repopulation index (RI) as a measure of the length of total colonies per testis and (2) measurement of the individual length of all colonies in serially sectioned testes 4 and 15 weeks after 300 rad and 15 weeks after 350 rad. The mean initial growth of the colonies is linear up to 8, 15, and 20 weeks after 150, 300, and 350 rad, respectively. Although after these doses the mean initial colony growth rate did not differ significantly (about 27 microns/day), both methods showed that the colonies grow about 20% slower after 350 rad. Screening of individual colonies revealed a great variation in colony length per testis and a higher frequency of short colonies with higher neutron doses. Counting of colonies after 300 rad showed that all surviving stem cells had started to form a colony within 4 weeks after irradiation. The development of spermatogenetic cells to mature spermatozoa was studied after 100, 150, 300, and 350 rad in sections of repopulating tubules used for RI determination as well as in serial sections of individual colonies. Although after 300 and 350 rad spermatogenetic cell types beyond the stage of young spermatocytes reappeared 1 week late, we found no great disturbances in the regular reappearance of the successive spermatogenetic cell types after irradiation. However, from the study of individual colonies it appeared that colonies differ widely in their development even within one testis. Moreover, the frequency of less developed colonies was higher after 350 rad than after 300 rad. Our data suggest that this retardation in the reappearance of further developed cells is caused by a delay in the production of developed cells in spermatogonia in an increasing fraction of the colonies after higher neutron doses. Even in fully developed colonies the production of differentiating spermatogenetic cell types was subnormal after 300 and 350 rad. This was caused by an extensive cell degeneration in the colonies as well as by a tendency of the undifferentiated and/or A1-spermatogonial population to increase its own number at the cost of the production of further developed cells.

Animals↗
PubMed1987

Erythroid differentiation of K562 cells: mixed colonies as an index of delayed expression of commitment.

K562 cells demonstrate commitment, defined as the clonal expression of a differentiated phenotype coupled with a limitation in proliferation. Upon exposure to certain agents, K562 cells are induced to synthesize hemoglobin, detectable by benzidine staining. If plated in semisolid medium, they produce benzidine-positive colonies, benzidine-negative colonies, and mixed colonies, the latter containing both positive and negative cells. To test whether or not mixed colonies represent a delay in the expression of commitment, we conducted two types of experiments. The first type showed that, following inducer removal, a delay in plating causes not only a decline in the number of mixed colonies, but also a rise in the proportion of negative colonies, with no change in the proportion of positive colonies. To explain this result, we propose that a plating delay can conceal a negative cell producing a positive cell if that cell division has occurred before plating. Instead of one mixed colony, one observes one positive colony and one other colony, either negative or mixed (depending on subsequent negative-to-positive events). Thus delay does not change the proportion of positive colonies, presumably because they breed true. But delay causes an increase in negative colonies to balance the decrease in mixed colonies due to concealment of negative-to-positive events and provides evidence that the converse, positive-to-negative events, do not occur. The second type of experiment utilized cordycepin, which inhibits commitment. We predicted that, if mixed colonies represent a delay in the expression of commitment, the addition of cordycepin to cells already exposed to thymidine should increase the percentage of mixed colonies. We found that cordycepin does indeed preferentially increase the proportion of mixed colonies. These two types of experiments provide evidence that mixed colonies represent a delay in expression of commitment. Such an inducible system, in which the commitment event and its expression can be separated in time by a generation or more, may provide an opportunity to more fully characterize the commitment process.

Cell Differentiation↗
PubMed1986

Analysis of growth fractions and stem cell compartments in transformed rat tracheal epithelial cell colonies.

The purpose of the studies described here was to define the biological behavior of the various clonally transformed colonies observed in cultures of carcinogen-exposed rat tracheal epithelial cells. As described in the preceding paper (H. Kitamura et al., Cancer Res., 46: 4631-4641, 1986), these colonies fall into four morphologically distinct categories. In the studies reported here we found that type I colonies had the smallest growth fraction (7%) and contained the lowest frequency of clonogenic cells (approximately 10(-3)). Colonies of types II to IV had mean growth fractions of 21 to 28%, and the frequency of clonogenic cells was 2 to 5 X 10(-2) when measured under growth-permissive conditions (3T3 feeders). When the clonogenic cell assays were performed under selective conditions to identify cell variants which can grow without feeder support, the average frequency of such clonogenic cells in type I colonies was less than 4 X 10(-5) and in colonies of types II to IV, between 5 X 10(-4) and 10(-2). In type IV colonies, the total number of cells per colony increased 8-fold between 5 and 12 wk postcarcinogen, but the clonogenic cell compartment increased 42-fold; the compartment of variant clonogenic cells, which are able to replicate on plastic, increased 139-fold during the same period of time. This indicated that major changes in the self-renewal capacity of the clonogenic cells were taking place during this early stage of transformation. Examination of the daughter colonies produced by replating colonies of types I to IV revealed that clonogenic cells with different growth potential existed within the same parent colony. Comparison of transformed colonies of the same type showed a marked degree of heterogeneity in the sizes of growth fractions and clonogenic cell fractions. These studies further indicated that, within all colonies, including the most advanced transformants, the majority of the cells were nonreplicating, terminal cells, suggesting that, at least during early stages of transformation, the transformed characteristics were not transferred from parent to daughter cells. With the exception of type I colonies, most of the colonies recognizable at 5 wk after carcinogen exposure progressed with time and acquired the morphological characteristics of type IV colonies, which were the most transformed phenotype. We conclude that transformation of rat tracheal epithelial cells is an asynchronous process and that the morphologically distinct types of rat tracheal epithelial cell colonies represent different stages of the clonal evolution of transformants.

Animals↗
PubMed1990

Modulation of tartrate-resistant acid phosphatase expression by calcitriol in CSF-induced macrophage colonies.

The osteoclast is thought to be a hemopoietically derived cell, but questions exist about which hemopoietic growth factors are responsible for proliferation of osteoclast precursors. Experiments were thus performed to see if recombinant human colony-stimulating factors (CSFs) influenced the expression of tartrate-resistant acid phosphatase (TRAP), an osteoclast marker enzyme, by monkey bone marrow colonies in vitro. In addition, the effect of 1,25-dihydroxyvitamin D3 (calcitriol) on CSF-induced colony growth and TRAP expression was also determined. Bone marrow was obtained from a single Macaca nemestrina monkey, kept frozen in liquid nitrogen, and aliquots of frozen cells were thawed and placed at 10(5) cells per plate in a standard cell colony-forming unit (CFU-C) assay. The recombinant human CSFs (granulocyte-macrophage CSF, GM-CSF; macrophage CSF, M-CSF; interleukin 3, IL-3; and granulocyte CSF, G-CSF) were added to the cultures at 50 U/ml, and calcitriol was titrated for each CSF from 0.1 to 100 nM. Day-14 colonies were stained to demonstrate TRAP-positive cells in individual colonies. GM-CSF caused an increase (193%, p less than 0.0004) in total colony numbers that was only partially inhibited by calcitriol. IL-3 and M-CSF had less effect, and G-CSF had no effect. GM-CSF also caused a large increase in TRAP-positive macrophage (M) colonies (326%, p less than 0.0001) and changed the relative proportion of TRAP-positive M colonies from 39% to 62% of all M colonies. M-CSF caused less increase in numbers of TRAP-positive M colonies and had no effect on the proportion of TRAP-positive colonies. When GM-CSF was present, calcitriol caused a maximum number of TRAP-positive colonies to appear at 1 nM, and it caused a drastic decrease in TRAP-positive colonies at higher doses. Calcitriol at 10 nM caused TRAP-negative colonies to increase in number and proportion when GM-CSF was present, but in the presence of M-CSF, the same dose of calcitriol caused a decline in numbers of TRAP-negative colonies. These results suggest that GM-CSF may be important in the replication of TRAP-positive mononuclear cells that resemble osteoclast precursors and that myeloid cell development may be weighted toward TRAP-positive or TRAP-negative progeny depending on whether GM-CSF or M-CSF predominates. They further suggest that calcitriol concentration may be critical in this process.

Acid Phosphatase↗
PubMed1983

Patterns of tumor colony development over time in soft-agar culture.

Human tumors were cultured by the two-layer soft-agar technique and the time course of tumor colony development was evaluated during periods of up to 6 weeks in culture. All colony counting was performed with an automated tumor colony counter (Omnicon; Bausch and Lomb, Inc, Rochester, NY, USA). This instrument provided colony counts per culture plate in six size categories from greater than 60 microns diameter colonies to greater than 149 microns diameter colonies. Six to 24 culture plates were used for each "growth curve", generally 24. Control (non-drug-treated) cultures were obtained from 117 tumors, of which 25 also provided enough cells to allow evaluation of the time course of colony development after exposure to cytostatic agents. The development of colonies in non-drug-treated plates usually demonstrated a lag phase, a logarithmic growth phase to maximum colony development and a subsequent deterioration of colonies. In spite of clumps seeded into the agar, real colony growth could be recognized by frequent colony counting of culture dishes, although the temporal patterns of growth were sometimes different if pure single-cell suspensions were compared with suspensions containing clumps from the same tumor. Drug pre-incubation caused changes in the temporal pattern of colony growth as well as in the total number of colonies. Some cultures showed drug sensitivity when evaluated at certain time points while evaluation at later time points showed only borderline drug effect or none at all. The potential utility of tumor colony growth curves in the clinical applications of tumor colony cultures is discussed.

Agar↗
PubMed1985

Colony growth and self renewal of plasma cell precursors in multiple myeloma.

Culture conditions that support the growth of multi-and single-lineage hemopoietic colonies are also able to give rise to large myeloma colonies from bone marrow and peripheral blood samples of some patients with multiple myeloma. The culture system was used to determine the frequency of hemopoietic precursors and clonogenic myeloma progenitors in 71 patients with multiple myeloma studied in various clinical phases of the disease. The frequency of normal hemopoietic precursors in patients with benign monoclonal gammopathy and smoldering myeloma were indistinguishable from normal controls. Myeloma colonies were not observed in these subgroups. In contrast, patients with active disease showed significantly reduced hemopoietic colony formation, even before the initiation of therapy. A further reduction was demonstrated for patients with acute phase disease. A correlation between the frequency of hemopoietic colonies and the concentration of plasma cells in the plated sample was not observed. Large myeloma colonies with recloning potential were identified in cultures of specimens derived from 14 of the studied patients. These colonies were most frequently (ten cases) obtained from patients who had entered the acute phase of the disease. These patients manifested marrow failure (pancytopenia) and their marrow had a limited capacity to generate normal hemopoietic colonies. Three of the patients that formed myeloma colonies were studied in chronic phase following chemotherapy and one patient was examined at diagnosis. The myeloma colonies were composed exclusively of cells characterized by the same M protein as the patient. Some of the cells within the colonies retained their ability to self renew extensively, as demonstrated by serial recloning studies. Colonies derived from six of the patients are now propagated in semisolid and liquid medium for as many as nine to 34 generations. Patients that form myeloma colonies under these culture conditions represent a high-risk group with significantly shorter survival than patients not able to give rise to myeloma colonies. A Cox proportional hazards model was fitted to the data to determine the prognostic role of myeloma colony growth in culture after accounting for the influence of other well established risk factors, such as concentration of plasma cells and disease status. The analysis indicated that myeloma colony growth in culture serves as a strong and independent predictive indicator of poor clinical prognosis.

Actuarial Analysis↗
PubMed1986

Cell kinetic study on GM colonies using autoradiography and collagen gel culture with special reference to unique proliferation of eosinophils.

Using a newly developed technique of autoradiography and collagen gel culture, a kinetic study on human GM colonies was attempted. Colonies of immature cells appeared first on day 5. The number of mixed colonies (mixture of immature cells, neutrophils, and/or monocyte-macrophages) and neutrophil colonies attained a maximum on days 8 to 10 and a broad peak of monocyte-macrophage colonies was observed on days 11 to 16. Eosinophil colonies appeared first on day 12, reached a maximum on day 18, and then gradually decreased. A detailed analysis of the order of appearance of the colonies suggests that mixed, neutrophil and monocyte-macrophage colonies originate from immature cell colonies or clusters, while eosinophil colonies do not. An autoradiographic study was designed to study the proliferation characteristics of each colony. Labeling indices (LI) with 3H-TdR of the cells in immature cell colonies were always high. LI of the cells in differentiated colonies such as neutrophil, monocyte-macrophage, and mixed colonies were low throughout the observation period. In contrast, LI of the cells in eosinophil colonies were constantly high regardless of the size of cell aggregates and the duration of the culture period. Both mitotic indices and mean grain counts on the nuclei of eosinophils were similar to those of immature cells. These results suggest that eosinophil colonies develop from their own small clusters and that eosinophils retain a fairly good proliferative capacity even when differentiated to the level in which specific granules appear in the cytoplasm.

Autoradiography↗
PubMed2005

Pre-B-cell colony-enhancing factor is a secreted cytokine-like protein from the human amniotic epithelium.

OBJECTIVE: The purpose of this study was to determine whether pre-B-cell colony-enhancing factor is a secreted cytokine in the human amnion and to study its chemotaxic and antiapoptotic properties. STUDY DESIGN: Pre-B-cell colony-enhancing factor secretion was studied from amniotic epithelial-like WISH cells and primary amniotic epithelial cells that were seeded on squares of immobilon-P membrane and stimulated with lipopolysaccharide or tumor necrosis factor-alpha, respectively. The pre-B-cell colony-enhancing factor protein was detected both intracellularly and after secretion, as bound to the membrane, by immunostaining and densitometry. Medium and cell lysates that were obtained from WISH cells that were treated with lipopolysaccharide alone or together with a pre-B-cell colony-enhancing factor antisense oligonucleotide to block pre-B-cell colony-enhancing factor translation were also analyzed for secreted pre-B-cell colony-enhancing factor by Western blotting and densitometry. A chemotaxic effect of pre-B-cell colony-enhancing factor on human neutrophils was compared with the chemoattractants interleukin-8 and N-Formyl-Met-Leu-Phe methyl ester in a rapid fluorescence-based neutrophil migration assay. Apoptosis was induced in primary amniotic epithelial cells and fibroblasts by actinomycin D (1 microg/mL); the antiapoptotic effects of pre-B-cell colony-enhancing factor on early apoptosis were measured by the annexin V assay, and the late effects were determined by measurement of nuclear matrix protein in the media. RESULTS: Treatment of amnion cells that adhered to immobilon-P membrane to induce the secretion of pre-B-cell colony-enhancing factor showed significantly (P<.05) more pre-B-cell colony-enhancing factor protein surrounding the cells compared with the controls. Although the addition of lipopolysaccharide to cultured WISH cells caused the secretion of pre-B-cell colony-enhancing factor into the medium, co-treatment with an antisense oligonucleotide to pre-B-cell colony-enhancing factor obliterated it. Analysis of the cell lysates showed no significant change, which suggests that most of the pre-B-cell colony-enhancing factor protein had been secreted. No significant chemotaxic effects of pre-B-cell colony-enhancing factor were observed; however, pre-B-cell colony-enhancing factor treatment (100 ng/mL), together with actinomycin D, cancelled the early induction of apoptosis, although there was a dose-dependent and significant late antiapoptotic effect on primary amnion epithelial cells (P<.001) and fibroblasts (P<.01). CONCLUSION: Pre-B-cell colony-enhancing factor is a secreted protein from amniotic epithelial cells. Although it had no chemotaxic effects, it was antiapoptotic for both amniotic epithelial cells and fibroblasts and may protect these cells against apoptosis that is induced by chronic distension, labor, or infection.

Amnion↗
PubMed1980

Lemna paucicostata Hegelm. 6746: LIFE CYCLE AND CHARACTERIZATION OF THE COLONY TYPES IN A POPULATION.

The sequence of frond emergence and the intervals required for daughter colony separation have been determined for Lemna paucicostata Hegelm. 6746 growing under standardized conditions. After separation of a new mother colony, the first daughter colony is produced from the left meristematic pocket and separates after approximately 60 hours, the second daughter, produced from the right pocket, separates after a further 30 hours, and the third daughter, again from the left, after a further 40 hours. The pattern of alternating longer and shorter intervals for separation of daughters continues throughout the life of the mother colony.Protein contents of fronds and whole colonies were determined either by chemical methods or by labeling to isotopic equilibrium with (35)SO(4) (2-). The smallest fronds measured were 5 to 6% of the final area they would attain when fully expanded and contained 10% of the protein they would finally contain. Thus, most protein accumulation occurred during the phase of rapid frond expansion rather than in an earlier primordial stage. The specific rates of protein accumulation were 0.027 to 0.030 micrograms per microgram protein per hour during rapid frond expansion and relatively constant thereafter at 0.014 to 0.019 for whole colonies until at least four daughter colonies had separated.A substantial amount of (35)S is transferred directly from the mother frond to its attached daughter fronds. The results are consistent with the transfer of soluble compounds arising through turnover of protein in the mother frond.Tree diagrams and a mathematical treatment were utilized to relate the distribution of colony types and the over-all doubling time in a large population of Lemna colonies to the doubling times and modes of division of the individual colonies within that population. It was shown that a population with the growth properties described increases exponentially and that within a relatively few generations reaches an equilibrium population distribution which is independent of the initial proportions of the members of the population. The measured distribution of colony types producing odd-numbered as compared to those producing even-numbered daughters was the same as that predicted by the theoretical analyses. Once a mass culture of Lemna paucicostata has attained its equilibrium distribution of colony types, under our standard conditions the proportions of colonies in which the indicated daughter will be next to separate will be: 60%, first; 15%, second; 11%, third; 5%, fourth; 8% fifth or higher. Thus, about 91% of colonies will have produced no more than four daughter colonies. For at least this period the specific rate of protein accumulation is relatively constant and senescent changes in mother fronds are rare. In the equilibrium distribution, 39% of colonies will themselves be first daughters, 25% will be second daughters, 14% will be third daughters, 9% fourth, and 13% greater than fourth daughters. These combined results suggest that obtaining representative samples of colonies from such a population for biochemical studies should be relatively simple.

Journal Article↗
PubMed1962

Phenotypic alterations in the colonial morphology of Brucella abortus due to a bacteriophage carrier state.

Jones, Lois M. (University of Wisconsin, Madison), C. R. McDuff, and J. B. Wilson. Phenotypic alterations in the colonial morphology of Brucella abortus due to a bacteriophage carrier state. J. Bacteriol. 83:860-866. 1962.-In the course of examining a number of Brucella cultures with a brucellaphage, it was observed that B. abortus cultures of intermediate colonial morphology, which had a blue-gray colonial appearance, were not lysed within 24 hr; in 48 hr they had developed sticky white growth in the area of the phage drop. When this growth was streaked on agar plates, both white and blue-gray colonies developed. White colonies which were sticky always carried phage and upon restreaking always gave rise to both white and blue-gray colonies. White colonies which were not sticky were rough and phage resistant. Blue-gray colonies produced only blue-gray colonies, did not carry phage, and were similar to the parent in their response to phage. When sticky white colonies were incubated for 6 hr or more in phage antiserum, all phage was eliminated and only blue-gray colonies developed. It was believed that the sticky white colonies were carrier clones in which lysis was delayed until after cell division, thus resulting in the establishment of a colony containing some phage-free progeny. With the accumulation of phage, the colony became sticky. This effect may be caused by the action of bacteriophage enzymes on the cell walls. Brucellaphage had an extremely slow rate of adsorption on a culture of intermediate colonial morphology. A phage mutant which was more strongly lytic for cultures of intermediate colonial morphology was selected from the original phage. The adsorption rate of this phage was more rapid and the latent period shorter. A serological difference between phages could not be demonstrated.

Adsorption↗
PubMed1976

Tumor colony formation by Friend virus-infected cells in immunosuppressed mice.

We have investigated the role of host immunological factors in the formation of "tumor colonies" in the spleens of unirradiated C57BL/6 X C3Hf/Bi FI mice 9 days after i.v. injection of spleen cells from Friend virus (FV)-infected C3Hf/Bi donors. Pretreatment of hosts with antilymphocyte serum (ATS) increased the number of tumor colonies. Pretreatment with formalinized FV-infected cells had the opposite effect, and ATS diminished the inhibitory effect of preimmunization. Cell suspensions from 11 individual FV-infected donors were examined. The suspensions differed with respect to their behavior on transplantation into untreated and ATS-pretreated F1 hybrid hosts. With several suspensions, the number of tumor colonies produced was approximately proportional to the number of cells injected; in all of these, ATS increased the slope of the line relating colony number to cell number. With most of the suspensions, tumor colony-forming efficiencies in untreated hosts strikingly decreased with increasing number of cells injected; ATS induced an increase in the number of tumor colonies and rendered the colony-forming response more nearly proportional to cell number. With two suspensions, few or no colonies developed; pretreatment with ATS had no significant effect. When the 11 cell suspensions were considered together, a proportional relation was found between the magnitude of the ATS effect (i.e., colony number in the presence of ATS minus colony number in the absence of ATS) and the colony-forming efficiency in ATS-treated mice. The ATS effect on the average was equivalent to a 2-fold increase in tumor colony-forming efficiency. We interpret these findings to indicate that two factors interact to determine the number of tumor colonies produced by spleen cells from FV-infected C3H donors in untreated F1 hybrid hosts. One is a property of the FV-infected cell population and includes its frequency of tumor colony-forming units; this factor varies widely among different cell suspensions. The other is a property of the tumor colony-forming units-host interrelationship and includes the vulnerability of tumor colony-forming units to the host immune response elicited by the injected cells; this factor appears to be constant with different cell suspensions. The present results show that the two factors can be dissociated in immunosuppressed hosts.

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PubMed1986

Morphological characterization of transformed colonies in rat tracheal epithelial cell cultures exposed to carcinogen.

The purpose of the studies described in this and the accompanying paper (D.J. Fitzgerald et al., Cancer Res., 46:4642-4649, 1986) was to define the transformed colonies of the rat tracheal epithelial cell transformation system (see P. Nettesheim and J.C. Barrett, CRC Crit. Rev. Toxicol., 12: 215-239, 1984) in terms of differentiation and growth characteristics. Exposure of low-density primary rat tracheal epithelial cell cultures to N-methyl-N'-nitro-N-nitrosoguanidine results in the development of growth-altered colonies of different sizes and morphologies, which can be readily scored 5 wk after treatment. These colonies were classified into four morphological types (I to IV) based on their light microscopic and ultrastructural features. Colonies designated as types I and II were small, had a low cell density, and were composed principally of large, pale-staining (Giemsa) flattened cells with a low nuclear/cytoplasmic ratio. Examination of the fine structure of these colonies revealed a monolayer of extremely attenuated cells containing well-developed Golgi complexes, endoplasmic reticulum, numerous secondary lysosomes, but few tonofilaments and desmosomes. Colonies of types III and IV were large, basophilic (Giemsa), high-density colonies, consisting primarily of closely packed, small, round cells with high nuclear/cytoplasmic ratio. Analysis of the fine structure of these colonies revealed two to four stratified layers of poorly differentiated cells and cells having features of keratinocyte differentiation, as evidence by abundant tonofilament bundles, well-developed desmosomes, and occasional keratohyaline granules. Differential cell counts were carried out at the ultrastructural level on pooled cells from each colony type (except type I); approximately 90% of the cells from type II colonies were large, electron-lucent cells, while this cell type comprised only 20 to 30% of the cells of colonies of types III and IV. The predominant cell type (approximately 60%) in colonies of types III and IV showed clear signs of keratinocyte differentiation. Type IV colonies contained a significantly larger proportion (greater than 20%) of small, poorly differentiated cells than type III (6%) and type II (1%) colonies. A combined autoradiographic and ultrastructural study revealed that the small, poorly differentiated cells were most active in synthesizing DNA and had a labeling index of 60%. Other cell types were far less frequently labeled, particularly the large, electron-lucent cells which had a labeling index of only 10%. Morphometric measurements on fixed and stained colonies disclosed that each colony type differs markedly in cell population size.(ABSTRACT TRUNCATED AT 400 WORDS)

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PubMed1992

Derivation of osteoclasts from hematopoietic colony-forming cells in culture.

The osteoclast is known to be derived from the hematopoietic stem cell, but its lineage remains controversial. There is evidence that osteoclastic differentiation is induced through a contact-dependent interaction between bone marrow stromal cells and hematopoietic precursors. To analyze osteoclastic lineage, colonies were generated in semisolid medium from mouse spleen cells in the presence of Wehi-conditioned medium, interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), or macrophage colony-stimulating factor (M-CSF) with or without erythropoietin (epo). After 5-8 days colonies were picked and phenotyped and incubated with 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] on bone slices or coverslips with bone marrow-derived cell lines (ts8 or ST2) that induce osteoclastic differentiation. Cells of osteoclastic phenotype [as judged by calcitonin receptor (CTR) expression or bone resorption] were observed only in multilineage colonies. The ability of cells that generate macrophage colonies (CFU-M) to generate osteoclasts was tested by incubating alveolar or peritoneal macrophages on ts8 or ST2 cells. Despite colony formation, no osteoclastic differentiation was detectable. Last, individual cells from blast cell colonies were incubated (1 cell per culture well) on ts8 or ST2 cells in the presence of 1,25-(OH)2D3 and epo (to expose the lineage potential of the plated cell). We found CTR-positive (CTRP) cells in 6 of 66 macrophage colonies, 7 of 12 granulocyte-macrophage (GM) colonies, and 49 of 50 colonies containing multiple lineages other than GM colonies. No single-lineage CTRP colonies were observed. Although most macrophage colonies did not contain CTRP, no CTRP were observed in colonies from which macrophages were absent. These results suggest that osteoclasts are derived from a multilineage precursor rather than from CFU-M.

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