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R J Cooper

Publications and source records attributed to R J Cooper.

At least 55 records · Page 3Linked to original sources
PubMed1993

In vivo administration of recombinant methionyl human stem cell factor expands the number of human marrow hematopoietic stem cells.

A growing number of in vitro studies suggest that recombinant human stem cell factor (SCF) is capable of augmenting the proliferative capacity of human hematopoietic progenitor cells (HPC) and stem cells (HSC). We further evaluated this biologic effect by analyzing the response of bone marrow (BM) HPCs and HSCs to the administration of SCF in eight patients with locally advanced or metastatic breast cancer who were enrolled in an ongoing phase I study. SCF was administered for 14 days by daily subcutaneous injection at dosages of 10, 25, or 50 micrograms/kg/d. BM CD34+ HLA-DR+ and CD34+ HLA-DR- CD15- cells, previously shown by our laboratory to be enriched for various classes of differentiated and primitive HPCs, respectively, were quantitated in BM samples on day 0 (pretreatment) and day 15 (posttreatment). These CD34+ HLA-DR+ and CD34+ HLA-DR- CD15- cells were then isolated by cell-sorting and assayed for several classes of HPCs, including the high--proliferative potential colony-forming cell (HPP-CFC), the burst-forming unit--megakaryocyte (BFU-MK), and the long-term BM culture--initiating cell (LTBMC-IC). SCF administration resulted in a 3.3-fold (range, 1.4- to 18.8-fold; P = .018) increase in the absolute numbers of CD34+ cells, a 3.7-fold (range, 1.2- to 8.2-fold; P = .028) increase in the absolute numbers of CD34+ HLA-DR+ cells, and a 2.4-fold (range, 1.1- to 29.3-fold; P = .010) increase in the absolute numbers of CD34+ HLA-DR- CD15- cells. Following the infusion of SCF, a statistically significant increase in the absolute numbers of HPP-CFC (P = .018), BFU-MK (P = .046), CFU-granulocyte, erythrocyte, monocyte, megakaryocyte (CFU-GEMM: P = .043), BFU-erythrocyte (BFU-E; P = .043), CFU-granulocyte, macrophage (CFU-GM; P = .045), and CFU-megakaryocyte (CFU-MK; P = .028) per milliliter of marrow was observed. Stromal cell-free LTBMCs supplemented with SCF and interleukin-3 (IL-3), initiated with CD34+ HLA-DR- CD15- cells obtained on day 0, produced viable cells for 9.6 weeks, compared with 11.5 weeks for LTBMCs initiated with CD34+ HLA-DR- CD15- cells obtained on day 15. Cumulative cellular production by LTBMCs initiated with day 15 CD34+ HLA-DR- CD15- cells was statistically greater than that by day 0 LTBMCs (P = .031). These same cultures produced CFU-GM for 6.3 weeks (day 0) versus 9 weeks (day 15).(ABSTRACT TRUNCATED AT 400 WORDS)

Antigens, CD↗
PubMed1993

Basic fibroblast growth factor promotes the proliferation of human megakaryocyte progenitor cells.

Basic fibroblast growth factor (bFGF), a multifunctional growth factor produced by bone marrow stromal cells, is known to be a potent modulator of hematopoiesis. Because bFGF is present in both human megakaryocytes (MKs) and platelets, we have hypothesized that this growth factor might affect human megakaryocytopoiesis. To test this hypothesis, either low density bone marrow (BM) cells (LDBM), a human BM subpopulation (CD34+ DR+) enriched for the colony-forming unit megakaryocyte (CFU-MK) or a BM subpopulation (CD34+ DR-) enriched for the more primitive burst-forming unit megakaryocyte (BFU-MK) were assayed in the presence of this growth factor. The effect of bFGF on MK colony formation differed according to the cell population assayed. bFGF alone had on MK colony-stimulating activity (MK-CSA) when either CD34+ DR+ or CD34+ DR- BM cells were cloned, but exhibited MK-CSA equivalent to that of interleukin-3 (IL-3) when LDBM cells were used as the target cell population. The MK-CSA of bFGF was inhibited by the addition of neutralizing antisera to either IL-3 and/or granulocyte-macrophage colony-stimulating factor (GM-CSF) but not IL-6. The addition of excess amounts of either IL-3 or GM-CSF to cultures containing bFGF plus anti-IL-3 or anti-GM-CSF reversed the inhibition by the corresponding antisera. The addition of bFGF and IL-3 to assays containing CD34+ DR+ or CD34+ DR- cells increased the size of both CFU-MK- and BFU-MK-derived colonies, respectively, when compared with assays containing IL-3 alone. This increase in MK colony size mediated by bFGF was not affected by addition of either an anti-GM-CSF or anti-IL-6 neutralizing antisera. When LDBM cells were assayed, bFGF alone increased CFU-MK-derived colony size when compared with control values. However, this potentiation of MK colony size by bFGF could be reversed by the addition of either anti-IL-3 or anti-GM-CSF but not anti-IL-6 antisera. In addition, the effects of bFGF and IL-3 on the size of MK colonies cloned from LDBM were not additive. These results suggest that bFGF affects human megakaryocytopoiesis by directly promoting MK progenitor cell proliferation and stimulating BM accessory cells to release growth factor(s) with MK-CSA, such as IL-3 and GM-CSF. We conclude that bFGF, likely produced by cellular components of the BM microenvironment, plays an important role in the control of human megakaryocytopoiesis.

Antibodies↗
PubMed1993

Genome analysis of adenovirus 4 isolated over a six year period.

Genome analysis was carried out on 74 adenovirus 4 (Ad4) isolates from patients in Manchester between 1984 and 1989. Most of the isolates were associated with conjunctivitis. Of the 74 isolates studied, 51 were Ad4a and 10 were Ad4p (the prototype strain). The remaining isolates consisted of two new genome types we have designated Ad4a2 (10 isolates) and Ad4a3 (3 isolates). Most of the genome types co-circulated during the period of study. The Bst E II and Xho I restriction maps of the new variants are presented and compared with those of Ad4p. We are unable to associate genome types with particular clinical presentations.

Adenovirus Infections, Human↗
PubMed1993

The polymerase chain reaction for detecting adenovirus DNA in formalin-fixed, paraffin-embedded tissue obtained post mortem.

The polymerase chain reaction (PCR) was used to detect adenovirus DNA in formalin-fixed, paraffin-embedded tissue obtained post mortem. Adenovirus DNA was successfully amplified from specimens of lung and liver from two patients with disseminated adenovirus infection confirmed by virus isolation, electron microscopy and/or immunohistochemistry. Negative results were obtained for specimens of lung from two patients with cytomegalovirus pneumonia. The specificity of the adenovirus PCR was confirmed by means of a digoxigenin-labelled probe generated in a separate PCR. Detection of viral nucleic acid by PCR in tissues obtained post mortem has considerable diagnostic potential.

Adenovirus Infections, Human↗
PubMed1992

Recombinant GM-CSF/IL-3 fusion protein: its effect on in vitro human megakaryocytopoiesis.

An evaluation of the effectiveness of a genetically engineered recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF)/interleukin 3 (IL-3) fusion protein (FP) as a means of delivering cytokine combinations to megakaryocyte (MK) progenitor cells was performed, utilizing a serum-depleted clonal assay system and a long-term bone marrow culture system. The effects of the FP, alone and in combination with a variety of other cytokines, on the primitive MK progenitor cell, the megakaryocyte burst-forming unit (BFU-MK), and the more differentiated megakaryocyte colony-forming unit (CFU-MK) were assessed. Subpopulations of bone marrow cells (CD34+ DR- for BFU-MK and CD34+ DR+ for CFU-MK) served as sources of these two classes of MK progenitor cells. The FP was equivalent to a combination of optimal concentrations of GM-CSF and IL-3 in promoting both the number and size of BFU-MK-derived colonies. The GM-CSF/IL-3 combination, however, promoted the formation of far greater CFU-MK-derived colonies than did the FP alone. The size of MK colonies formed in the presence of the FP or GM-CSF/IL-3 was similar. The ability of the FP to stimulate BFU-MK- but not CFU-MK-derived colony formation was also further augmented by the addition of interleukin 1 alpha (IL-1 alpha). The addition of c-kit ligand (KL) increased both FP-stimulated CFU-MK- and BFU-MK-derived colony numbers but only BFU-MK-derived colony size. In addition, the FP alone sustained long-term megakaryocytopoiesis in vitro to a level equivalent to that of the GM-CSF/IL-3 combination and was superior in this regard to either GM-CSF or IL-3 alone. These data indicate that FP is capable of supporting various stages of human megakaryocytopoiesis. We conclude that such genetically engineered molecules as the FP may prove to be effective means of pharmacologically delivering the biological effects of specific cytokine combinations.

Antigens, CD↗
PubMed1991

Effect of c-kit ligand on in vitro human megakaryocytopoiesis.

An evaluation of the effects of a recombinant, soluble form of the c-kit ligand alone and in combination with either granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin-3 (IL-3) on the regulation of human megakaryocytopoiesis was performed using a serum-depleted clonal assay system and a long-term bone marrow culture system. The effects of the c-kit ligand on the primitive megakaryocyte (MK) progenitor cell, the burst-forming unit-megakaryocyte (BFU-MK), and the more differentiated colony-forming unit-megakaryocyte (CFU-MK) were determined. The c-kit ligand alone had no megakaryocyte colony-stimulating activity (MK-CSA) but was capable of augmenting the MK-CSA of both GM-CSF and IL-3. The range of synergistic interactions of c-kit ligand varied with the class of MK progenitor cell assayed. In the case of the BFU-MK, the c-kit ligand synergistically augmented the numbers of colonies formed in the presence of IL-3, but not GM-CSF, but increased the size of BFU-MK-derived colonies cloned in the presence of both of these cytokines. However, at the level of the CFU-MK, c-kit ligand synergized with both GM-CSF and IL-3 by increasing both colony numbers and size. Although the c-kit ligand alone exhibited limited potential in sustaining long-term megakaryocytopoiesis in vitro, it synergistically augmented the ability of IL-3, but not GM-CSF, to promote long-term megakaryocytopoiesis. These data indicate that multiple cytokines are necessary to optimally stimulate the proliferation of both classes of MK progenitor cells and that the c-kit ligand plays a significant role in this process by amplifying the MK-CSA of both GM-CSF and IL-3.

Antigens, CD↗
PubMed1991

Further examination of the effects of recombinant cytokines on the proliferation of human megakaryocyte progenitor cells.

The effect of several recombinant cytokines, including interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-6, and IL-1 alpha, on megakaryocyte (MK) colony formation by a normal human bone marrow subpopulation (CD34+ DR+), enriched for the MK colony-forming unit (CFU-MK), was studied using a serum-depleted, fibrin clot culture system. IL-3 and GM-CSF, but not IL-6 or IL-1 alpha, stimulated MK colony formation by CD34+ DR+ cells. However, the addition of IL-1 alpha to CD34+ DR+ cultures containing IL-6 resulted in the appearance of CFU-MK-derived colonies, suggesting that IL-6 requires the presence of IL-1 alpha to exhibit its MK colony-stimulating activity (MK-CSA). Addition of neutralizing antibodies to IL-3 and GM-CSF, but not to IL-6 and IL-1 alpha, specifically inhibited the MK-CSA of IL-3 and GM-CSF, respectively. The addition of either anti-IL-6, anti-IL-1 alpha, or anti-IL-3 antisera to cultures containing both IL-6 and IL-1 alpha totally abolished the MK-CSA of the IL-6/IL-1 alpha combination. However, neither anti-IL-3 nor anti-GM-CSF antisera could totally neutralize the additive effect of the combination of IL-3 and GM-CSF on MK colony formation, indicating that these two cytokines act by affecting distinct effector pathways. These results suggest that while IL-3 and GM-CSF can directly affect CFU-MK-derived colony formation, IL-1 alpha and IL-6 act in concert to promote de novo elaboration of IL-3 and thereby promote CFU-MK proliferative capacity.

Antibodies↗
PubMed1987

Effect of ketamine on local cerebral glucose utilization in fetal sheep.

Ketamine hydrochloride was infused intravenously in eight near-term fetal sheep, in doses ranging from 4.6 to 62.2 mg/kg. Local cerebral glucose utilization was determined by the [14C]deoxyglucose method. Heterogeneity in density of autoradiographs, a prominent feature in untreated fetal brains, was greatly diminished in animals receiving greater than 18 mg/kg, with differences between cerebral cortical gray and white matter virtually disappearing at high doses. In striking contrast to the pale, washed-out appearance in autoradiographs of most cerebral, cerebellar, and brain stem structures was a relatively high density of entorhinal cortex, hippocampus, lateral olfactory tract, and olfactory bulbs. Local cerebral glucose utilization of most gray and white matter structures was elevated in one ketamine-treated fetus experiencing a marked drop in PO2 during the study.

Animals↗
PubMed1987

The assay, purification and properties of vaccinia virus-induced uncoating protein.

Vaccinia virus cores prepared in vitro can be uncoated by incubation with extracts of cells infected with vaccinia virus, as determined by the conversion of the genome to DNase susceptibility. This uncoating activity had all the characteristics of the corresponding in vivo activity and of the agent responsible for non-genetic reactivation. Thus, it was not induced by heat-inactivated virus, nor was it produced in the presence of inhibitors of RNA or protein synthesis. The uncoating protein induced by cowpox virus will uncoat vaccinia virus cores. The uncoating protein was concentrated from infected cell extracts by ultrafiltration and purified by gel filtration and ion-exchange and affinity chromatography. It was characterized as a trypsin-like protease with a mol. wt. of 23,050. Cores treated with the purified uncoating protein had an altered sedimentation rate but no differences between treated and untreated cores were detected by electron microscopy or polyacrylamide gel electrophoresis.

Carrier Proteins↗
PubMed1984

The inhibition of HeLa cell RNA synthesis following infection with vaccinia virus.

Vaccinia virus WR induces an immediate and rapid inhibition of HeLa S3 cell RNA synthesis as determined by pulse-labelling with [3H]uridine. The inhibition was independent of the purity of the infecting virus preparation and the multiplicity of infection over the range of 4 to 200 pk.f.u./cell. Inhibition was not evident in cells pre-treated with cycloheximide or following infection with u.v.- or heat-inactivated virus, suggesting that viral protein synthesis was required. There was no apparent selective inhibition of any particular species of RNA. Following infection, the uptake of [3H]uridine into cellular pools and the subsequent biosynthesis of UTP proceeded at the same rate as in mock-infected control cells. The rate of degradation of pre-labelled RNA was not enhanced in infected cells compared to controls. Analysis of the nuclear DNA-dependent RNA polymerase (EC 2.7.7.6) activities revealed a progressive and eventually total loss of RNA polymerase B activity, no obvious effect on RNA polymerase A and the presence of a viral RNA polymerase, the possible significance of which is discussed.

DNA-Directed RNA Polymerases↗
PubMed1984

The DNA polymerase activity of vaccinia virus 'virosomes': solubilization and properties.

Intracellular DNA-protein complexes ('virosomes') of vaccinia virus have been isolated. The solubilization of the 'virosome'-bound DNA polymerase activity was attempted by a variety of high-salt extraction procedures. The most efficient of these used 0.3 M-ammonium sulphate followed by brief sonication. The solubilized DNA polymerase activity from the 'virosomes', together with the DNA polymerases from 100000 g supernatant fluids from the cytoplasm of infected and uninfected cells were chromatographed on DEAE-cellulose and their properties compared. The 'virosome' DNA polymerase activity differed from the soluble vaccinia virus-induced DNA polymerase activity in its requirements for divalent cations and in respect of pH optimum, Km for the deoxyribonucleoside triphosphates and the effect of N-ethylmaleimide.

Chlorides↗
PubMed1980

An assessment by progesterone assay of the accuracy of oestrus detection in dairy cows.

The use of a milk progesterone assay in determining the extent to which dairy cows are presented for insemination at the wrong time is reported. Milk samples were taken from cows at the time of insemination and were subjected to plasma progesterone assay. In a preliminary study samples were obtained from 96 cows in an experimental herd at insemination. The calving rate of these cows was compared with the milk progesterone level at insemination. Eight cows which had levels of progesterone much above the normal oestrus level 9 more than 0.3 ng per ml) failed to conceive. As the progesterone level at insemination rose, calving rate declined. A further 1177 milk samples taken from cows being inseminated in commercial dairy herds were also examined. Ninety-one animals (7.7%) had levels of milk progesterone in excess of those observed at normal oestrus. A negative correlation was confirmed between non-return rate and milk progesterone level at insemination. These investigations show that the proportion of cows from the national herd which have high progesterone levels when presented for insemination is similar to that found under experimental conditions and suggests that the problem of heat detection may be a relatively constant factor in reduced conception to artificial insemination.

Animals↗