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Biomedical subjects

R Schofield

Publications and source records attributed to R Schofield.

At least 19 recordsLinked to original sources
PubMed1992

Sustained reflow in dogs with coronary thrombosis with K2P, a novel mutant of tissue-plasminogen activator.

Coronary artery reocclusion after thrombolysis with human recombinant tissue-type plasminogen activator (rt-PA) is related to the short half-life of this agent in plasma. K2P, a mutant of rt-PA lacking the fibronectin fingerlike, epidermal growth factor-like and first kringle domains (amino acids 6 to 173) and having the glycosylation site Asn184 mutagenized to Gln, has been produced in Chinese hamster ovary cells. In this study we compared the thrombolytic effect of K2P and rt-PA in dogs with electrically induced coronary artery thrombosis. Both agents were given intravenously in equimolar amounts over 20 min after the occlusive thrombus was stable for 30 min; dogs were monitored for 1 h after reperfusion if flow occurred. Coronary blood flow was restored by rt-PA in 6 (60%) of 10 dogs. The restored flow lasted for 49 +/- 12 min and mean flow at 60 min from the start of reperfusion was 7 +/- 3 ml/min. The reocclusion rate was 50% (three of six dogs). Flow was restored in five (100%) of five dogs by K2P. The restored blood flow lasted during the entire 1-h observation period in all but one dog and mean flow at 60 min was 49 +/- 16 ml/min (p less than 0.02 vs. flow in rt-PA-treated dogs). Restored coronary blood flow showed marked cyclic flow variations in rt-PA-treated but not in K2P-treated dogs.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

TxA2 inhibition and ischemia-induced loss of myocardial function and reactive hyperemia.

To determine the contribution of thromboxane (Tx) A2 release in reperfusion injury, 17 dogs were subjected to total coronary occlusion for 1 h and reperfusion for 1 h. Eleven dogs were treated with saline, and six were treated with selective TxA2 synthetase inhibitor U63,557A (5 mg/kg iv) 30 min before coronary artery occlusion. In all saline-treated dogs, peak reactive hyperemia after 10-s total coronary artery occlusion was diminished (P less than 0.01) after reperfusion. Myocardial segmental shortening was also reduced (9.8 +/- 1.9 to -6.7 +/- 2.0%, P less than 0.01) in the reperfused region. Reperfusion was associated with 737 +/- 343 premature ventricular contractions (PVCs) per hour. Histology revealed extensive myocardial infiltration and capillary plugging by leukocytes in the reperfused region. Myeloperoxidase, an index of leukocyte infiltration, was also increased (P less than 0.02) in the reperfused region. In the U63,557A-treated animals, serum and plasma TxB2 levels were markedly (P less than 0.02) reduced. Decrease in myocardial shortening fraction was less in U63,557A- than in saline-treated animals (P less than 0.05). The frequency of reperfusion PVCs was also significantly reduced (10 +/- 5 PVCs/h, P less than 0.02 compared with saline-treated dogs). However, peak reactive hyperemia was reduced similar to that in saline-treated dogs. Myocardial infiltration and capillary plugging by leukocytes in the reperfused regions was also similar in the U63,557A- and saline-treated dogs. These results indicate that treatment with U63,557A decreases reperfusion arrhythmias and preserves myocardial function. However, coronary reperfusion-induced deterioration in reactive hyperemia is not affected.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

On the late seeding of CFU-S to the spleen: 8- vs 12-day CFU-S.

Marrow from 5-fluorouracil- or cyclophosphamide-treated mice, injected into lethally irradiated recipients, gives an increasing number of spleen colonies between days 7 and 14. It has been suggested that the later-forming colonies result from the more primitive spleen colony-forming units (CFU-S), which first seed into the marrow, only later to be recirculated and form colonies in the spleen. Strontium 89 (89Sr), a bone-seeking radionuclide, was injected into recipient mice to block such putative recirculation. A dose of 89Sr, which killed at least 99.8% of CFU-S in, or entering, the bone cavities, was incapable of preventing the increase in spleen colony numbers. Similarly, the splenic environment, modified by the presence of spleen colonies and able to provide a better bed for trapping CFU-S from the peripheral circulation, yielded the same number of further CFU-S, whether or not the animal had received 89Sr. Thus, it was concluded that the 12-day CFU-S does not seed initially into the marrow spaces. Direct observation of the quality of CFU-S initially seeding into the bone marrow and spleen showed, by retransplantation into secondary irradiated mice, that a similar spectrum of CFU-S types had seeded both organs.

Animals↗
PubMed1987

The radiation sensitivity of the haemopoietic microenvironment--effect of dose rate on ectopic ossicle formation.

The haemopoietic microenvironment (HM) consists of a complex mixture of cellular types and extra-cellular matrix. It is essential for prolonged haemopoiesis in both the normal situation and after bone marrow transplantation. The competence of the HM can be assessed by ectopic grafting of femoral marrow. A complete haemopoietic organ develops at the site of implantation. Stem cells (CFU-S) which inhabit the ossicle formed after ectopic implantation can be measured, to assess the function of the engrafted HM to support haemopoiesis. Using this functional endpoint we have examined the radiation sensitivity of the HM at both high and low dose rates, and conclude that high doses of gamma-irradiation delivered at 4 Gy/min or 0.016 Gy/min have widely different effects on the HM, the former proving much more damaging than the latter.

Animals↗
PubMed1987

Marrow repopulation in mice treated with busulphan or isopropyl methane sulphonate and bone marrow.

By using karyotypic analysis of female mice treated with busulphan or isopropyl methane sulphonate (IMS), and injected with male bone marrow the donor contribution to both total marrow cellularity and spleen colony forming cells (CFU-S) was assessed for up to 6 months after transplant. In the mice treated with busulphan the marrow cells yielded metaphases of which between 40% and 83% were of donor type. Between 60% and 97% of metaphases in spleen colonies formed in irradiated mice were of donor type during the 24-week study period. In contrast, mice prepared for the transplant with IMS showed no cells of donor type at any time after transplant, neither did they possess CFU-S of donor type. We were therefore led to conclude that the donor cells made no contribution to longterm engraftment in mice prepared with IMS, whilst in those prepared with busulphan they were the predominantly active haemopoietic cells. These results are consistent with a model of haemopoiesis in which the most primitive cells reside in a 'niche' where they are resistant to the effects of IMS but susceptible to the action of busulphan. Busulphan may vacate some niches to allow engraftment by transplanted marrow, whilst IMS yields no unoccupied niches for grafted cells to occupy, and cannot therefore lead to a stable chimaerism.

Alkylating Agents↗
PubMed1987

Standardization of procedures for ectopic marrow grafting. II. Influence on recipients of radiation dose and field size.

The ectopic implantation of mouse marrow to the kidney capsule offers considerable scope as an assay of the hemopoietic microenvironment. Our previous work has shown that whole-body irradiation of the graft recipient prior to implantation results in superior ossicle formation in the kidney of the host. Here we report that a range of irradiation doses over a 4-Gy threshold are equivalent with respect to conditioning the graft recipient. We also show that two distinct and separable influences affect graft growth in the irradiated recipient, namely, a local effect brought about in the irradiated kidney (and restricted to it) and secondly, a systemic effect resulting from irradiation of sites other than the kidney, which nevertheless affects ossicle growth in the shielded renal capsule.

Animals↗
PubMed1987

Ectopic implantation studies using Sl/Sld marrow and recipients.

Marrow from Sl/Sld mice (in which the hemopoietic stromal microenvironment is defective), when implanted beneath the renal capsule of a normal littermate, produces an ectopic marrow site containing the same number of stem cells (CFU-S) and nearly as many GM-CFC as that obtained by implanting marrow from a normal littermate. On the other hand, a marrow plug from an Sl/Sld donor implanted beneath the renal capsule of an Sl/Sld littermate produces less than half the number of CFU-S and about 10% of the number of GM-CFC. This suggests that the recipient of the ectopic implant can contribute in some way to the stromal environment of the grafted marrow.

Animals↗
PubMed1987

Hematopoietic effects of TCNU in mice.

Carmustine has been in clinical use since the 1960s and has proved efficacious in many treatment protocols. It has, however, been limited in its applications because of delayed hematopoietic toxicity which results in curtailment of treatment. A new derivative based on taurine, 1-(2-chloroethyl)-3-/2-(dimethylaminosulfonyl)ethyl-1-nitrosourea, has been developed and we have investigated its effects with a view to assessing the possibility of its being a causative agent for long-term marrow damage. We have found that while this new compound is less hematoxic than carmustine, it still demonstrates significant residual impairment of blood cell formation after application to mice. This impairment is noted in the CFU-S (stem cell) numbers and in the microenvironmental populations responsible for forming an ectopic site of hemopoiesis, persists for at least 180 days after the cessation of treatment, and may therefore be considered irreversible.

Animals↗
PubMed1986

Comparison of haemopoiesis in young and old mice.

Haemopoietic status and functions have been compared in young (2-3-month-old) and old (2-2.5-year-old) BDF1 mice. The parameters measured include total marrow cellularity, CFU-S, CFU-mix, GM-CFC, BFU-E and CFU-F. In all cases the numbers of these cells in the femoral marrow of the old mice was equal to or greater than those in the femoral marrow of young mice. In addition to these parameters we have compared the ability of marrow from young and old mice to repopulate the marrow of recipient mice whose marrow had been eliminated by radiation; to grow in long-term bone marrow cultures; to produce ectopic grafts of marrow beneath the renal capsule of normal recipients; and to supply inhibitor and stimulator of stem cell proliferation in the marrow and to resynthesise these substances. We could detect no differences in any of these functions with the exception of that of resynthesis of the stem cell regulator substances, which appears to be somewhat slower in the old mice. This, however, does not impose any limitation upon the ability of the marrow to function either under normal conditions or in conditions requiring rapid proliferation. Therefore we can find no evidence whatsoever to suggest that aging of the haemopoietic system plays any part in aging of the individual or influencing the life-span.

Aging↗
PubMed1986

Effects of plutonium-239 on haemopoiesis. I. Quantitative and qualitative changes in CFU-S in different regions of the mouse femur and vertebrae.

Mice were injected with plutonium-239 (960 Bq/mouse) and, over a period of four months, the response of haemopoietic tissue and the self-renewal capacity of its stem cells was monitored. Cellularity, CFU-S concentration and self-renewal capacity were measured in five different regions of bone and marrow--axial and marginal marrow of the femoral shaft, femur shaft, proximal and distal ends of the femur shaft and vertebrae. Cellularities were little affected by plutonium but CFU-S were reduced in all regions, most severely in the bone shaft and marginal marrow due to the initial deposition of plutonium on the bone surface, by four days. The reduction in axial CFU-S, however, was due probably to a relatively long plasma half-life resulting from the tendency of plutonium to combine with plasma proteins. The capacity of CFU-S for self-renewal was reduced and remained low in all zones. Thus, although the highly self-renewing axial CFU-S were depleted, and remained so, due probably to a longer term redistribution of plutonium throughout the marrow, additional proliferation of the more mature CFU-S in the other zones kept their self-renewal low while replenishing their numbers and maintaining a normal cell output.

Alpha Particles↗
PubMed1986

Standardization of procedures for ectopic marrow grafting: I. Influence of sex of recipient.

Bone marrow plugs implanted beneath the renal capsule of a normal syngeneic mouse recipient develop, within a few weeks, into a shell of marrow-containing bone. The marrow stromal microenvironment of the implant is reported to be of donor origin and therefore the technique has the potential for development into a quantitative assay of the stroma, or stroma-forming, capacity of the implanted marrow. A surprising difference has been shown, however, in that the female mouse does not permit the development of such an ectopic implant to the same extent as does a male recipient. The suppression of development of the implant is particularly dramatic when marrow from a male donor is implanted into a female recipient, but is strongly operative even upon donor marrow from a syngeneic female. The effect is partly strain dependent, being more pronounced in C57B1/6 and B6D2F1 mice than in DBA/2 or Balb/c. Castration and ovariectomy do not abrogate or modify the suppression. On the other hand, exposure of recipients to 6 Gy 137Cs gamma-radiation before implantation results in bigger implants developing in male recipients, and the suppressive effect of the female recipient upon the graft is reduced considerably or eliminated altogether. Marrow plugs were implanted into chimeras made by transplanting marrow from syngeneic male or female donors, i.e., into heavily irradiated B6D2F1 mice of the same or opposite sex. In female mice repopulated with marrow cells from male donors, the ectopic implants contained 2-3 times as many spleen colony-forming units (CFU-S) as did those in female mice populated by female marrow cells. Ectopic implants into male mice repopulated with female marrow cells contained fewer CFU-S than implants into male recipients having a male marrow, though the differences are smaller than those found in female recipients and may not be significant.

Animals↗
PubMed1986

Development of spleen CFU-S colonies from day 8 to day 11: relationship to self-renewal capacity.

To investigate the persistence of spleen colonies from day 8 to day 11 of their development, we injected low numbers of marrow cells in order to obtain single colonies on the spleens of irradiated mice. Colonies were isolated on either half of the spleen on the eighth day. The position of day-11 colonies, determined relative to the ligature, indicated where novel colonies appear between those times. The results showed no evidence of the persistence of colonies from day 8 to day 11. The self-reproduction capacity of CFU-S that survive various cytotoxic drugs depends on the specific subpopulations that are affected by the drug. Using cyclophosphamide, busulphan, or BCNU, the self-renewal capacity of surviving CFU-S was manipulated. The results show that after cytotoxic treatments, a high day-11-day-8 ratio is not necessarily a reflection of a high self-renewal capacity of the CFU-S population that forms the day-11 colonies.

Animals↗
PubMed1986

An analysis of haemopoietic and microenvironmental populations of mouse bone marrow after treatment with busulphan.

The effects of the cytotoxin busulphan (myleran) have been investigated in order to ascertain the unique nature of the lesion which it induces. It is one of only few compounds which can cause marked residual marrow dysfunction and the only drug reported to induce a marrow lesion of such magnitude that after a prolonged interval mice may die of the effects of pancytopaenia resulting from hypoplastic marrow failure. We have found that busulphan has a major microenvironmental effect as assessed by the ability of the marrow to form a complete organ in an ectopic site, which confirms existing evidence from a range of putative stromal assays. The effects upon CFU-S are known to include a selective action against certain subpopulations. Our investigation of the dose relationship of busulphan effects have shown that induction of the prolonged marrow lesion is not dependent on the dose of drug administered, or the fractionation regime employed, and is not dependent therefore on the number of CFU-S removed by the drug. The unique action of busulphan probably rests therefore in its ability to damage the microenvironment and the most primitive stem cells which are closely associated with it, if not entirely dependent on it, for their existence.

Animals↗
PubMed1986

Radiosensitivity of murine hemopoietic colony-forming units assayed in situ in the rib and in other marrow sites.

The radiosensitivity of murine hemopoietic colony-forming cells, which produce colonies in situ and which were counted at Day 8 after irradiation in sections of the femur, humerus, sternum, and spleen, is characterized by a D0 value of 91 +/- 9 cGy. The radiosensitivity of such cells in the rib was assessed using a new technique measuring regeneration or ablation of marrow in transverse sections of ribs observed at Day 8 after irradiation. The mean D0 value over a range determined using several different criteria was 108 cGy. These results provide evidence for the common assumptions that radiosensitivity measured using conventional transplantation assays reflects radiosensitivity in situ, and that the radiosensitivity of stem cells in different medullary marrow sites is similar. The techniques could be used with other species where assays for stem cells are not available.

Animals↗
PubMed1985

Studies on the self-renewal ability of CFU-S which have been serially transferred in long-term culture or in vivo.

The progressive decline in the repopulating ability of bone marrow serially-transferred through a succession of recipients is well documented. A similar series of transfers onto successive long-term culture adherent layers has been carried out using as 'donor' cells both adherent layer cells and cells from the culture supernatant. For as long as the in vitro serial transfer regime can be maintained the decline in self-renewal ability ('quality') of the CFU-S parallels the similar decline observed in vivo and occurs irrespective of the quality of CFU-S transferred. In both the in vivo and in vitro transfer regimes there is little or no loss of quality of CFU-S as a result of one 'transfer' although there may be a reduction in the total numbers of CFU-S in the mouse. However, a second and third transfer in vivo or in vitro leads to a rapid decline in the quality (self-renewal ability) of the CFU-S. Furthermore, despite the fact that the cells are transferred in vitro to a new adherent layer there is no recovery of the quality lost in the second transfer of the CFU-S. This fact implies that self-renewal potential of the CFU-S is a property intrinsic to the cell. The data presented here appear to exclude mitotic history and proliferative stress as factors determining the loss of self-renewal in CFU-S. They also fail to implicate stromal involvement in the decline. It may be that the dilution of an accessory cell or simply the disaggregation of the marrow may be major factors. The work presented indicates that the loss of self-renewal and repopulating ability of haemopoietic stem cells as a result of marrow transplantation may be studied using the long-term marrow culture and yield results relevant to in vivo marrow transplantation.

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↗