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Biomedical subjects

B I Lord

Publications and source records attributed to B I Lord.

At least 91 records · Page 5Linked to original sources
PubMed1985

Enrichment of CFU-S proliferation inhibitor-producing cells based on their identification by the monoclonal antibody F4/80.

Cells capable of synthesizing a factor that can specifically inhibit the proliferation of haemopoietic spleen colony-forming units, CFU-S, carry many of the characteristics of macrophages. The monoclonal antibody, F4/80, which is specific for murine macrophages, has therefore been used to isolate macrophages from bone marrow. These macrophages were then assayed for their ability to produce the inhibitor. Low-density bone marrow cells were first separated by a density-cut procedure and then labelled with F4/80. Fluorescence-activated cell sorting was then used to select F4/80 positive and negative cell fractions. It was found that the F4/80 positive fraction contains inhibitor cells, enriched by at least 200-fold compared to unfractionated marrow. Subsequent culture of this fraction over a period of several weeks produced a further 15- to 20-fold increase in inhibitor-producing capacity. The cultured cells producing inhibitor were virtually 100% F4/80 positive, phagocytic and exhibited histochemical properties characteristic of macrophages. The macrophage-like character of the producer cells was thus confirmed and the removal of the majority of unwanted cells means that many of the impurities in the normal crude extracts have been excluded.

Animals↗
PubMed1984

The kinetics of hematopoietic stem cells during and after hypoxia. A model analysis.

A previously described mathematical model of the hematopoietic stem cell system has been extended to permit a detailed understanding of the data during and after hypoxia. The model includes stem cells, erythroid and granuloid progenitors and precursors. Concerning the intramedullary feedback mechanisms two basic assumptions are made: 1) The fraction "a" of CFU-S in active cell cycle is regulated. Reduced cell densities of CFU-S, progenitors or precursors lead to an accelerated stem cell cycling. Enlarged cell densities suppress cycling. 2) The self renewal probability "p" of CFU-S is also regulated. The normal steady state is described by p = 0.5, indicating that on statistical average each dividing mother stem cell is replaced by one daughter stem cell, while the second differentiates. Diminished cell densities of CFU-S or enlarged densities of progenitors and precursors induce a more intensive self renewal (p greater than 0.5), such that the stem cell number increases. The self renewal probability declines (p less than 0.5) if too many CFU-S or too few progenitors and precursors are present. The model reproduces bone marrow data for CFU-S, BFU-E, CFU-C, CFU-E, 59 Fe-uptake and nucleated cells in hypoxia and posthypoxia. Although the ratio of differentiation into the erythroid and granuloid cell lines is kept constant in the model, a changing ratio of CFU-E and CFU-C results. The model suggests that stem cells and progenitor cells are regulated by a regulatory interference of erythropoiesis and granulopoiesis.

Cell Differentiation↗
PubMed1984

Spatial organisation of CFU-S proliferation regulators in the mouse femur.

CFU-S proliferation can be regulated by an inhibitor and a stimulator which are produced locally in the bone marrow. The spatial distribution of cells elaborating these factors has been studied by separating marrow, zonally, into axial, marginal and bone-associated fractions and further separating the inhibitor and stimulator producing cells from these fractions by means of density cuts in bovine serum albumin. The inhibitor- and stimulator-producing cells are found respectively in bands of density less than 1.064 g/cm3 and 1.064-1.072 g/cm2. Factors synthesised by these individual fractions were assayed for their ability to inhibit or stimulate CFU-S proliferation by thymidine suicide measurements. Axial cells contain a high concentration of inhibitor-producing cells but the marginal and bone fractions contain relatively few. Stimulator-producing cells have a very sharp gradient in the opposite direction with an extremely high concentration in the vicinity of the bone surface. These distributions fit with those of CFU-S proliferation and of fibroblastoid-CFC suggesting that the inhibitor-producing cell is an integral part of the micro-environment which maintains the stem cell's capacities while the stimulator-producing cell may be more closely associated with the differentiation processes which take place closer to the bone surface.

Animals↗
PubMed1984

The cellular specificity of haemopoietic stem cell proliferation regulators.

The range of specificity of the CFU-S proliferation inhibitor and stimulator which are produced endogenously in the bone marrow has been investigated by measuring their effects on the proportion of cells killed by tritiated thymidine in mixed colony- (CFC-mix), erythroid burst- (BFU-E) and granulocyte/macrophage colony- (GM-CFC) forming cells as well as spleen colony forming units (CFU-S). Both CFU-S and CFC-mix were triggered by the stimulator into DNA-synthesis but BFU-E and GM-CFC were unaffected. The range of activity of the inhibitor was confined solely to the CFU-S population. This defined the specificity of both inhibitor and stimulator for the multipotent cells. The differential sensitivity of CFU-S and CFC-mix to the inhibitor and the lack of it for the stimulator suggested (a) that the CFC-mix is a relatively mature subpopulation of the CFU-S compartment and (b) that the relative sensitivity of a CFU-S to these factors changes as it matures from the early stem cell stage (Inhibitor-sensitive) to the more mature stages (Stimulator-sensitive) before becoming committed to a specific line of differentiation. The specificity of the inhibitor for haemopoietic stem cells suggests its potential value during chemotherapeutic procedures.

Animals↗
PubMed1984

A comparison of low and high dose-rate radiation for recipient mice in spleen-colony studies.

Over the last 15 years, endogenous spleen-colony formation in our mice, following lethal irradiation, has increased to an unacceptable level. It has been found necessary, therefore, to introduce a new method of preparing recipient mice for spleen-colony studies. Irradiation with low dose-rate 60Cobalt gamma rays has been compared with high dose-rate linear accelerator electrons, and their effects on endogenous spleen colony formation compared with earlier X and gamma ray dose-response data. It was found that a large dose (13.5 Gy) of gamma rays results in fewer endogenous colonies than 8.5 Gy of electrons, yet because of its low dose rate (14.1 X 10(-3) Gy/min) it has a marked sparing of the intestinal tissue as measured by the intestinal microcolony technique. This in turn permits better survival and, therefore, a 'healthier' animal for spleen-colony work. Exogenous colony formation is also lower in the low dose-rate, gamma-irradiated recipients and this is shown to be due to a reduced spleen-seeding efficiency. It is concluded that very low dose-rate radiation is preferable for haemopoietic ablation, that a mouse colony requires constant monitoring for changes of endogenous spleen-colony formation and that the spleen-seeding efficiency of CFU-s depends on the irradiation technique used--there is no absolute value for a given strain of mouse.

Animals↗
PubMed1984

Haemopoietic stem cells and the problem of self-renewal.

Haemopoiesis occurs in association with a complex stromal cell network in which all levels of haemopoietic cell development can be found. In order to understand the interaction between stromal cells and growth factors with the processes of self-renewal and differentiation, we have carried out a series of experiments attempting to define the circumstances in which self-renewal occurs in long-term marrow cultures. We have found that highly purified (FACS sorted) CFU-S do not undergo significant self-renewal in vitro when inoculated onto marrow stromal cells that can support self-renewal of unfractionated CFU-S. We have examined the effects of expression of the src oncogene on self-renewal of CFU-S. We have found that, following infection of long-term cultures with a retrovirus carrying the src oncogene, there is expression of src in certain of the stromal cells. There is also a selection for CFU-S that have an extended self-renewal capacity in vivo and in vitro. These CFU-S are non-leukaemic and can reconstitute haempoiesis in irradiated mice. Cells from src-infected cultures can also be induced to proliferate and form cell lines in vitro in the presence of interleukin 3 (IL-3). The cell lines produced are multipotential and non-leukaemic. From such data we conclude that expression of the src oncogene has (directly or indirectly) permanently altered the stem cells in such a way that they can undergo extensive self-renewal in situations that are unfavourable for growth and self-renewal of normal stem cells.

Animals↗
PubMed1982

Stimulation of haemopoietic stem cell proliferation: characteristics of the stimulator-producing cells.

Media conditioned by regenerating murine bone marrow cells contain a stimulator of haemopoietic stem cell proliferation. Fractionated cell populations have been examined for production of this stimulatory activity in order to characterize its cellular source. The stimulator is produced by adherent, phagocytic, radioresistant, Thy 1.2-, Fc+ cells in a population concentrated in a density range of 1.064-1.072 g/ml. The results indicate that the producer cells reside in the heterogenous mononuclear phagocytic population of the bone marrow.

Animals↗
PubMed1982

Interaction of inhibitor and stimulator in the regulation of CFU-S proliferation.

An inhibitor and stimulator of CFU-S proliferation can be obtained from haemopoietic tissue containing, respectively, relatively quiescent CFU-S (e.g. normal bone marrow) and proliferating CFU-S (e.g. regenerating bone marrow). In this paper we explore the capacity for inhibitor and stimulator production in relation to changes in the proliferative status of the CFU-S and their mode of interaction. The increase in proliferative activity of CFU-S following a concentrated regime of phenylhydrazine injections is paralleled by an increase in the capacity for stimulator production and a decrease in the capacity for inhibitor production. These processes are reversed as the normal low proliferative activity returns. Using density fractionated marrow populations, dose-response measurements show that while inhibitor- and stimulator-producing cells persist throughout the changes in CFU-S proliferation, their magnitude of factor production moderates in relation to those changes. The inhibitor and stimulator are not mutually destructive: the two factors retain their activities after co-incubation and re-separation. On the other hand, the presence of either factor blocks the synthesis of the other by the appropriate producer cells. A model for the regulation of CFU-S proliferation by the inhibitor and stimulator is thus suggested in which the relative spatial distributions of CFU-S in a microenvironment containing inhibitor- and stimulator-producing cells are an important feature. It also implies the existence of a signal from the CFU-S compartment which determines the appropriate inhibitor or stimulator production.

Animals↗
PubMed1982

The relative spatial distribution of erythroid progenitor cells (BFUe and CFUe) in the normal mouse femur.

Femoral mouse bone marrow cells were separated into axial and marginal fractions, in order to investigate the relative concentration of erythroid progenitor cells (BFUe and CFUe) with respect to their location across the diameter of the femur. Two areas of high incidence of early progenitor cells (BFUe) were identified: one lying near the bone surface with a peak at about 410 micrometers radial distance from the axis of the bone; the other nearer the centre of the bone with a peak at about 270 micrometers. The more immature BFUe were found in higher proportion in the marginal peak. In contrast, CFUe, apart from very low concentration values in the vicinity of the bone surface, demonstrated a fairly uniform distribution throughout the marrow. The present results indicate that the distribution of erythroid progenitor cells within the bone marrow is not random. The haemopoietic tissue seems to exhibit a well-defined structure that may be relevant in regulating proliferation and differentiation processes.

Animals↗
PubMed1981

The relationship of G0 to the cell cycle of haemopoietic spleen colony-forming cells.

Normal haemopoietic stem cells, defined here as spleen colony-forming units (CFUs), are slowly proliferating and are generally considered to spend most of their time in the non-proliferative G0-state. A series of experiments using various combinations of the stem cell proliferation inhibitor (NBME-IV) and stimulator (RBME-III) together with vinblastine as a mitotic blocking agent was designed to determine the location of the G0-state relative to the cell cycle of the CFUs. From a knowledge of the effects of these agents, the expected results from three different G0-cell cycle models were charted and compared with the observed proliferative behaviour of the CFUs following these treatments. It was concluded that the out-of-style G0-state is located at the end of the G1-phase of the cycle, so that on receiving a stimulatory signal, the CFUs can rapidly enter the DNA-synthesis phase.

Animals↗
PubMed1980

Self-maintenance capacity of CFU-S.

The numbers of CFU-S which developed in spleen colonies were measured 11 days after injection of irradiated mice with marrow from normal mice or mice which had been treated in one of a variety of ways. The broad spread of CFU-S numbers, seen by other authors, in colonies derived from normal marrow was confirmed. However, the range and distribution of CFU-S per colony was generally different in colonies derived from the marrow of mice which were recovering or had recovered from some form of depopulation. From the data obtained, the mean CFU-S/colony, M1, and the probability of self-renewal, p, of the CFU-S were calculated. These values are used to calculate the number of cell cycles undergone during development of the colony and, by making certain assumptions, the cell cycle time of the CFU-S. The plot of p against log M for the various samples measured should be linear if all CFU-S proliferate at the same rate in a growing colony. It is not linear, however, so that CFU-S obtained under different experimental conditions do not all undergo the same number of cycles. In general, treatments given to the mice result in a lowering of the capacity for self-renewal of their CFU-S and also to a shortening of their cell cycle time. Some of the possible implications of these findings are discussed.

Animals↗
PubMed1980

The regulation of hemopoiesis in long-term bone marrow cultures. II. Stimulation and inhibition of stem cell proliferation.

The isolation of a DNA synthesis inhibitor (NBME fraction IV) and stimulator (RBME fraction III) specific for the hemopoietic stem cell (CFU-s) from freshly isolated normal adult and regenerating murine bone marrow, respectively, has been well documented. We have utilized long-term liquid bone marrow cultures in a further analysis of the role of these factors in the regulation of CFU-s proliferation. Our results show that shortly after feeding, at a time when the cultured CFU-s are actively proliferating, high levels of the hemopoietic stem cell proliferation stimulator fraction III can be isolated from the culture medium. In contrast, the presence of essentially noncycling CFU-s found in cultures fed 8-10 days previously correlates with high levels of the hemopoietic stem cell inhibitor fraction IV. These results suggest that a certain balance between these factors determines CFU-s proliferation in the long-term cultures. In support of this, DNA synthesis in actively cycling CFU-s in the long-term cultures is inhibited for at least 3 days by the addition of excess NBME fraction IV (inhibitor). Furthermore, DNA synthesis in noncycling cultured CFU-s is stimulated for at least 5 days by the addition of RBME fraction III (stimulator).

Animals↗
PubMed1980

Sources of haemopoietic stem cell proliferation: stimulators and inhibitors.

Based on earlier findings that haemopoietic tissue contains extractable factors which are capable of specifically inhibiting or stimulating the movement of CFU-S into DNA synthesis, a series of preliminary experiments has now been carried out to investigate their cellular source(s), their activity in vivo, and their applicability to human problems. In vivo treatment of mice, in which femoral CFU-S are proliferating rapidly, with the inhibitory factor reduces the proportion of CFU-S in DNA synthesis to non-significant proportions. In addition, the inhibitor is capable of reducing the number of CFU-S induced to enter S following treatment with hydroxyurea, thus protecting CFU-S from the lethal effects of S-phase cytotoxic agents. Removal of specific types of marrow cells shows that both inhibitor and stimulator are adherent, phagocytic and, in the case of inhibitor, Thy--1-. These results suggest that the producer cells probably reside somewhere in the heterogeneous macrophage complex though their different densities suggest they are probably different cell types. Fresh human bone marrow is found to contain a very similar inhibitor and long-term cultures are also found to produce it continuously. The isolation of the producer cells may thus contribute to the understanding of normal physiological stem cell regulation and, by in vivo application, its eventual manipulation and protection.

Animals↗
PubMed1979

Mechanisms of haemopoietic stem cell proliferation control.

The control of stem cell (CFU-S) proliferation is mediated by short-range acting factors which can be detected by the proliferation modifying activities present in media conditioned by haemopoietic cells. A specific inhibitor of stem cell proliferation is obtained from haemopoietic tissue containing minimally proliferating CFU-S, whilst stimulatory material is obtained from cell suspensions containing rapidly proliferating CFU-S. Used competitively, these factors, which are detected in different molecular weight range fractions, manipulate the rate of CFU-S proliferation in a manner compatible with a physiological control mechanism. In addition, a long-term bone marrow culture system has been shown to provide an in vitro model of stem cell control. Fractionation of cell populations from haemopoietic tissues reveals marked concentration differences of the CFU-S proliferation modifying activities depending on the proliferative state of the CFU-S. However, irrespective of whether the tissue contains stem cells that are actively or minimally proliferating, both stimulatory and inhibitory activities are detected. From dose-response studies it is concluded that stem cell proliferation is controlled by an appropriate balance of stimulatory and inhibitory factors which, however, are not produced by the stem cells themselves.

Animals↗