Biomedical subjects

C Dowding

Publications and source records attributed to C Dowding.

At least 19 recordsLinked to original sources

PubMed2000

Teaching palliative care principles to UK nursing home care assistants.

This paper describes the initiatives that led to a study day for Health Care Assistants in the UK, focusing on the principles and practice of palliative care for practitioners. Topics covered were 'What is palliative care?', 'How can you help patients with pain', 'Needs of the dying patient', 'I don't know what to say', 'Answering awkward questions', and 'Ways of relieving distressing symptoms'. Participants valued the study day and feedback showed that the Health Care Assistants, essentially assistants to qualified nursing staff with minimal or no training themselves, had many vocational and emotional needs that were not being met.

Education↗

PubMed1995

Selective elimination of malignant stem cells using photosensitizers followed by light treatment.

The pros and cons of purging of either bone marrow or peripheral blood stem cell preparations for autologous transplantation for cancer has been debated strongly over the past decade. Recent data implicating the role of minimal residual disease in autografted marrow in cancer relapse have renewed interest in this question. There is a considerable body of literature supporting the possibility that photosensitizer molecules in combination with light might provide a therapeutic window permitting selective elimination of malignant stem cells while sparing those of normal lineage. Molecules of this class are known to be taken up more actively by most malignant cells, and intracellular concentrations are critical in their cytotoxic effect when they are activated by light at an appropriate wavelength. The present paper reviews the observations made over the past decade on a variety of photosensitizers and their effects on hemopoietic progenitors.

Bone Marrow Purging↗

PubMed1993

Potential mechanisms of action of interferon-alpha in CML.

Treatment with interferon-alpha (IFN-alpha) adequately controls the leukemic cell mass in the majority of newly diagnosed patients with chronic myeloid leukemia (CML). However, the degree of response ranges from no 'hematologic' response to complete suppression of the leukemic clone. The mechanism(s) by which IFN-alpha elicits these responses is unknown, but in vitro studies have indicated that IFN-alpha might function by (1) selective toxicity against the leukemic clone, (2) enhancement of 'immune' regulation, and (3) modulation of bone marrow microenvironmental regulation of hematopoiesis. Using in vitro clonogenic assays we were unable to demonstrate that IFN-alpha selectively inhibited the proliferation of CML progenitor cells. We also found no difference in the expression of LFA-3 on normal or CML CD34+ cells. However, by panning and co-culturing hematopoietic cells on monolayers of bone marrow stromal cells, grown with and without IFN-alpha, we found that IFN-alpha enhanced the adhesion of CML progenitors to stromal cells, whereas adhesion by normal progenitor cells was essentially unaffected. This enhanced adhesion by CML progenitor cells was associated with a reduction in neuraminic acid levels in the extracellular matrix overlying stromal cells. Therefore, it is possible that one of the mechanisms by which IFN-alpha exerts its regulatory effect on the leukemic clone is through enhancement of hematopoietic cell-microenvironmental cell interactions.

Antigens, CD↗

PubMed1993

Phenotypic characterization of normal and CML CD34-positive cells: only the most primitive CML progenitors include Ph-neg cells.

We studied the sequence of acquisition of CD33, CD38 and HLA-DR antigens on CD34+ cells from marrow and blood of Ph-chromosome positive CML patients and normal marrow. We examined the Ph status of the various CML cell populations. The mean proportions of normal and CML CD34+ cells expressing CD33 and CD38 were not significantly different. However, a significantly greater proportion of CML CD34+ cells expressed HLA-DR antigens compared with normal CD34+ cells and the level of HLA-DR expression per CML cell was abnormally high. When the sequence of acquisition of these antigens on normal and CML CD34+ cells was evaluated using 3-colour fluorescence analysis, the results suggested that HLA-DR was expressed earlier than CD38 or CD33 and these findings were confirmed by following the acquisition of CD38 and CD34+/DR+/CD38-subpopulation during liquid culture. We performed cytogenetic studies on CD34+ subpopulations in 6 cases. In 4 cases there were some Ph-negative metaphases detectable in the CD34+/DR-subpopulation (range 12.5 to 60%). In the CD34+/DR+ fractions, however, all 6 patients had only Ph-positive metaphases and only 1/5 patients had detectable Ph-negative metaphases in the CD34+/CD38-subpopulation. We conclude that expression of HLA-DR antigens may precede the expression of CD38 on CD34+ cells during normal stem cell differentiation. In CML DR may be expressed aberrantly and Ph-negative cells are found predominantly in the DR negative subpopulation.

ADP-ribosyl Cyclase↗

PubMed1991

Interferon-alpha overrides the deficient adhesion of chronic myeloid leukemia primitive progenitor cells to bone marrow stromal cells.

Primitive blast colony-forming cells (BI-CFC) from chronic myeloid leukemia (CML) patients are defective in their attachment to bone marrow-derived stromal cells compared with normal BI-CFC. We investigated the effect of recombinant interferon-alpha 2a (IFN-alpha) on this interaction between hematopoietic progenitor cells and bone marrow-derived stromal cells by culturing normal stromal cells with IFN-alpha (50 to 5,000 U/mL). At 50 U/mL we found that: (1) the capacity of stromal cells to bind two types of CML primitive progenitor cells (BI-CFC and long-term culture-initiating cells) was increased; and (2) the amount of sulfated glycosaminoglycans (GAGs) in the stromal layer was increased. However, sulfated GAGs were not directly involved in binding CML BI-CFC, unlike binding by normal BI-CFC, which is sulfated GAG-dependent. Neuraminidase-treated control stromal cells bound an increased number of CML BI-CFC, reproducing the effect of IFN-alpha, whereas the binding to IFN-alpha-treated stromal cells was unaffected by neuraminidase treatment. Thus, the enhanced attachment by primitive CML progenitor cells to INF-alpha-treated stromal cells might be due to changes in the neuraminic acid composition in the stromal cell layer. Our in vitro evidence may provide insights into the mechanism of action of IFN-alpha in vivo. Prolonged administration may alter the marrow microenvironment in some patients such that it can restrain the aberrant proliferation of Philadelphia chromosome (Ph)-positive stem cells while permitting Ph-negative stem cells to function normally.

Bone Marrow↗

PubMed1991

Inactivation of the retinoblastoma susceptibility gene in a human high grade non-Hodgkin's lymphoma cell line.

We studied the expression of the retinoblastoma (RB) gene product (p105) in a B-cell line established from a patient with non-Hodgkin's lymphoma (large cell type). The karyotype of this cell line, named Ri-1, showed amongst other changes an apparent deletion of one chromosome 13 on band q14. No p105 could be detected by immunoprecipitation analysis and Western blotting in Ri-1 cells. Northern blotting revealed that RB mRNA is not expressed in Ri-1. Southern blotting confirmed the loss of one RB allele but showed a normal gross structure of the remaining allele. This suggests that the inactivation of the RB gene in Ri-1 cells is due to deletion of one allele and point mutations or small deletions in the other, as is often the case in retinoblastomas. Our findings imply that inactivation of the RB gene may play a role in the pathogenesis of high grade malignant lymphomas and that studies of RB in primary lymphoma samples would be of interest.

Cell Line↗

PubMed1991

The effects of interferon-alpha on the proliferation of CML progenitor cells in vitro are not related to the precise position of the M-BCR breakpoint.

We investigated the effects of brief (2 h) and continuous exposure to recombinant interferon-alpha (2a) (rIFN-alpha) on the proliferation of primitive (blast colony-forming cells, Bl-CFC) and committed myeloid progenitor cells (BFU-E and GM-CFC) derived from blood and bone marrow of patients with chronic myeloid leukaemia (CML) and normal subjects. In all three clonogenic assays, rIFN-alpha suppressed colony formation in a dose-dependent manner. No differences were detected in the proliferation of CML or normal Bl-CFC and GM-CFC exposed to rIFN-alpha. Erythroid colony formation by normal, but not by CML BFU-E, was inhibited by relatively low concentrations (100 U/ml) of rIFN-alpha. However, in patients whose blood or marrow contained a mixture of Philadelphia chromosome (Ph)-positive and Ph-negative BFU-E, cytogenetic analysis of individual erythroid colonies showed no differential inhibition by rIFN-alpha. We found no difference in the sensitivity to rIFN-alpha of GM-CFC from patients whose leukaemic cells expressed BCR/ABL mRNA with the b2a2 junction and that of GM-CFC from patients with the b3a2 mRNA. We conclude that (1) rIFN-alpha does not have a significant leukaemia-specific effect on the progenitor cells detected in these assays, and (2) the sensitivity of CML GM-CFC to rIFN-alpha is independent of the type of BCR/ABL message present in the cells. The clinical efficacy of rIFN-alpha could be due to selective toxicity to cells not assayed in this study, to effects on accessory cells or to alterations induced in progenitor cell/stromal cell interactions.

Blotting, Southern↗

PubMed1991

Interferon-alpha alters the distribution of CFU-GM between the adherent and nonadherent compartments in long-term cultures of chronic myeloid leukemia marrow.

We studied the effect of recombinant interferon-alpha 2a (IFN alpha) on the interaction between stromal cells and granulocyte-macrophage colony-forming units (CFU-GM) from the marrow of normal individuals and chronic myeloid leukemia (CML) patients in chronic phase in long-term bone marrow cultures using preformed stromal layers. These stromal layers were established with marrow cells from normal allogeneic donors and grown to confluence in the presence or absence of IFN alpha at low concentration (100 U/ml). The number of CML CFU-GM localized within IFN alpha-treated stromal layers was significantly greater than the corresponding number localized within control stromal layers. Conversely, the distribution of normal CFU-GM between the adherent and nonadherent compartments was unaffected by IFN alpha treatment of the stromal layer. Preincubation of CML marrow cells with IFN alpha did not alter this distribution, so the observed effect of IFN alpha must be due to a primary action on stromal layers. Thus, in addition to its well known antiproliferative effect, IFN alpha enhances the capacity of marrow stromal cells to bind and/or retain CML progenitor cells, and the resulting restoration of normal regulatory control may be the basis for the selectivity of IFN alpha in CML.

Bone Marrow↗

PubMed1990

The position of the M-BCR breakpoint does not predict the duration of chronic phase or survival in chronic myeloid leukemia.

It has been reported that patients with chronic myeloid leukemia (CML) with 5' breakpoints within the major breakpoint cluster region (M-BCR) of the BCR gene have somewhat better prognoses than those with 3' breakpoints. We studied the position of the breakpoint in 67 patients with CML in chronic phase using conventional Southern blotting. Using restriction enzymes BglII, BamHI and HindIII and two genomic probes, a 0.6 kb (3' M-BCR) probe hybridizing to a part of the intron between exons b3 and b4 and a 2.0 kb (5' M-BCR) probe hybridizing to sequences including exon b1, we localized the breakpoint in M-BCR as occurring 5' (n = 38) or 3' (n = 28) of the HindIII restriction site located just downstream of exon b3. We failed to localize the breakpoint in one patient. The median durations of chronic phase (37 versus 44 months respectively) and of survival (50 versus 51 months respectively) for patients with 5' and 3' breakpoints were not significantly different. When we analysed only patients whose DNA was collected within 4 weeks of diagnosis (5' breakpoints, n = 30; 3' breakpoints, n = 19), there was again no significant difference in duration of chronic phase or survival. The median survivals of patients divided into good, intermediate and poor prognosis categories in accordance with the prognostic index developed by Sokal and colleagues were 54, 50 and 26 months respectively. This study confirms the value of the Sokal prognostic index but provides no support for the notion that the precise genomic position of the breakpoint in M-BCR correlates with prognosis.

Adolescent↗

PubMed1990

The role of the MDR-1/P-170 mechanism in the development of multidrug resistance in chronic myeloid leukemia.

We studied blood and bone marrow cells from 42 patients with Ph-chromosome positive chronic myeloid leukemia (CML) and 20 normal subjects for amplification of the multidrug resistance gene (MDR-1) by Southern blotting and for overexpression of P-glycoprotein (P-170) by immunocytochemistry on intact cells with the monoclonal antibody C219. No P-170 could be detected in normal bone marrow or buffy coat. Overexpression of P-170 without amplification of MDR-1 was found in four of 11 patients with chronic phase CML at diagnosis, seven of 16 patients treated with busulfan or hydroxyurea in chronic phase and four of 15 patients in blast crisis. The P-170 overexpression involved only cells of the granulocyte lineage and varied from weak to strong in individual patients. It did not correlate with duration of or response to treatment during chronic phase. In transformation P-170 expression was seen in differentiated cells of the granulocyte lineage but not in blast cells, although three patients had been treated intensively with lipophilic and other cytotoxic drugs to which they had become resistant. We conclude that resistance to busulfan and hydroxyurea in chronic phase and resistance of blast cells to other cytotoxic drugs in transformation are not mediated primarily through the MDR-1/P-170 pathway.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1989

Autografting for patients with chronic myeloid leukaemia in chronic phase: peripheral blood stem cells may have a finite capacity for maintaining haemopoiesis.

We treated 14 patients with Ph-chromosome-positive chronic myeloid leukaemia still in chronic phase by autografting with blood-derived haemopoietic stem cells. Eleven patients were autografted electively after cytoreductive treatment with busulphan (16 mg/kg) and melphalan (60 mg/m2) and three were autografted after marrow cells from HLA-identical sibling donors had failed to engraft. In 13 patients haemopoiesis recovered; one failed to engraft and died 114 d after autografting. Two other patients became pancytopenic and received further stem cell transfusions at 3 and 40 months respectively after first autografting. One patient entered lymphoid transformation and died 14 months after autografting. Twelve patients survive at a median of 41 months (range 24-53) after autografting. Nine of the survivors have required further chemotherapy after autografting and four of the nine were electively autografted on a second occasion. Three patients surviving after autografting for 28, 43 and 53 months respectively have not required further chemotherapy. In two of these patients haemopoiesis is now predominantly Ph-negative. We conclude that autografting in chronic phase might prolong survival in some cases by reducing the size of the leukaemic stem cell population. The fact that initially successful grafts failed in two patients suggests that blood-derived stem cells may have a finite potential for self-replication.

Adult↗

PubMed1989

The effect of cytomegalovirus on hemopoiesis: in vitro evidence for selective infection of marrow stromal cells.

We studied the effects of adding cytomegalovirus (CMV) in vitro to normal human bone marrow mononuclear cells (BM-MNCs), committed myeloid progenitor cells, primitive myeloid blast-colony forming cells, and pre-formed marrow stromal cell monolayers in order to shed light on the mechanism by which hemopoiesis is suppressed in patients who acquire systemic CMV infection after allogeneic bone marrow transplantation. Incubation of BM-MNCs or committed progenitor cells with laboratory strain AD169 or wild strain CMV had no significant effect on total colony numbers or the morphology of component cells. CMV mRNA was not identified by in situ hybridization. In contrast, incubating marrow stromal monolayers with CMV produced specific cytopathic effects in fibroblasts and adipocytes and reduced the capacity of the stromal layers to support the proliferation of primitive myeloid progenitor cells. We conclude that CMV infection may impair hemopoiesis in vivo by a direct effect on the cellular components of the marrow stroma.

Bone Marrow↗

PubMed1989

Recurrent graft failure following syngeneic bone marrow transplantation for aplastic anaemia.

We present the case of a 60-year-old woman with drug-induced aplastic anaemia with a healthy monozygotic twin. Proof of monozygosity was confirmed by studies using the hypervariable minisatellite probe to obtain identical DNA fingerprints in donor and recipient. In vitro co-culture studies performed showed no evidence of a recipient-derived cellular or humoral inhibitor of donor haemopoiesis. Despite this, there was no engraftment following simple marrow infusion without preconditioning. A second syngeneic transplant following high dose cyclophosphamide therapy produced trilineage engraftment but severe thrombocytopenia developed at 3 months, followed later by pancytopenia with generalized marrow failure. Following a third syngeneic transplant with cyclophosphamide and total lymphoid irradiation there was good initial engraftment but graft failure occurred at 14 weeks. A fourth transplant using Campath 1G as preconditioning resulted in no engraftment and the patient died of septicaemia 8 weeks following her fourth transplant. We suggest that the cause of the recurrent aplastic anaemia in this case was a defect of marrow stroma as neither an inhibitor of donor haemopoiesis nor an intrinsic defect of donor stem cell growth could be demonstrated in vitro.

Anemia, Aplastic↗

PubMed1987

Complete remission after autografting for chronic myeloid leukaemia.

A previously untreated 31-yr old female with Ph-positive chronic myeloid leukaemia (CML) received busulphan and melphalan at high dosage followed by an autograft of peripheral blood stem cells collected 4 weeks earlier. Though recovering haemopoiesis was at first mainly Ph-positive, Ph-negative haemopoiesis predominated at 12 months and has persisted until the most recent study (24 months post-autograft). Her haemopoietic cells now show no rearrangement of the bcr gene, unlike the cells collected at diagnosis. Autografting carried out soon after diagnosis could be valuable for obtaining cytogenetic remissions in other patients with CML.

Adult↗

PubMed1986

Purification of haemopoietic progenitor cells from patients with chronic granulocytic leukaemia using percoll density gradients and elutriation.

Purification of haemopoietic progenitor cells from chronic granulocytic leukaemia buffy coat preparations requires a multistep approach using complementary cell separation techniques. In this study Percoll density gradient centrifugation and centrifugal elutriation were used to isolate large numbers of immature progenitor cells. Percoll density gradients were valuable as a first separation step: CFU-GM and CFU-GEMM could be enriched 75-fold in a light density fraction of d less than 1.056 g/ml and the technique could be adapted to cope with more than 10(10) buffy coat leucocytes. Progenitors cells were concentrated 3-fold by elutriation used as single method to separate buffy coat cells or when used to purify further light density Percoll fractions. When Percoll gradients and elutriation were used sequentially, undifferentiated mononuclear cells were enriched to more than 90% purity and between 5% and 40% of these cells formed CFU-GM or BFU-E colonies consisting of more than 40 cells. The enriched fractions were further characterized with monoclonal antibodies. The density and elutriation profiles of these colony forming cells resembled corresponding profiles of cells that reacted with the monoclonal antibody BI-3C5, which recognizes an antigen on primitive haemopoietic progenitor cells. Physical separation methods are a valuable first stage in the attempt to procure relatively pure myeloid progenitor cell populations, whose characteristics can then be further studied at a cellular or molecular level.

Cell Separation↗

PubMed1986

Antigenic determinants on myeloid leukaemia colony-forming cells resemble those of normal myeloid progenitor cells and differ from those of circulating blast cells.

We studied the antigenic characteristics of leukaemic colony-forming cells (CFU-L) from the blood of patients with chronic granulocytic leukaemia (CGL) in blastic transformation (BT) and acute myeloid leukaemia (AML) by in vitro culture techniques after complement-mediated lysis with one anti-DR and 10 selected myeloid monoclonal antibodies (McAbs), all of which were cytotoxic in the presence of complement. At the same time we studied the antigenic characteristics of the circulating blast cells from the same patients using in addition one non-complement fixing antibody (BI.3C5) with standard immunofluorescence and immunoalkaline phosphatase techniques. We also used myeloid progenitor cell assays in conjunction with cytotoxic McAbs to investigate the antigenic determinants on Day 7 CFU-GM, Day 14 CFU-GM and BFU-E from the blood of patients with CGL in chronic phase (CP) and from normal bone marrow. We found that two of the McAbs, S4-7 and WGHS29.1, recognized a higher proportion of CFU-L from the blood of AML patients than from patients with CGL-BT. However, the patterns of reactivity for CFU-L from CGL-BT and AML patients with the other McAbs quite closely resembled those observed in CFU-GM and BFU-E from normal individuals and patients with CGL in CP. A McAb with DR specificity and one of the myeloid McAbs, 54/39, recognized both CFU-L from CGL-BT and AML and reacted also with circulating blast cells from the same patients. In contrast, six of the other myeloid McAbs that recognized CFU-L failed to label the corresponding blast cells. We conclude that the antigenic properties of CFU-L in CGL-BT and AML are very similar to, but perhaps not identical with, those of normal CFU-GM and BFU-E. There was a major discrepancy in the antigenic profiles of CFU-L and of the blast cells predominating in the blood.

Antibodies, Monoclonal↗

PubMed1986

Rearrangement of the bcr gene in Philadelphia chromosome-negative chronic myeloid leukemia.

We studied the clinical, hematologic, cytogenetic, and molecular biologic features of seven patients with Philadelphia (Ph1) chromosome-negative chronic myeloid leukemia (CML). In five cases the hematologic findings were indistinguishable from those of patients with classical Ph1-positive disease. Myeloid cells were studied by chromosome-banding techniques. One patient had a masked Ph1 chromosome (with translocation t(4;9;22)), one had a deletion involving chromosome 16, and one had a small minority population of 22q- cells without 9q+ but otherwise normal metaphases; metaphases from the other four patients were entirely normal. DNA prepared from the myeloid cells was digested with the restriction enzymes EcoRI, HindIII, BamHI and BglII. Southern analysis using a 0.6-kb fragment of the breakpoint cluster region (bcr) gene showed the presence in each patient's DNA of a germline fragment together with a rearranged fragment or fragments with at least one of the restriction enzymes. We conclude that genomic changes in the bcr gene characteristic of CML can be present in the absence of a Ph1 chromosome.

Adult↗

PubMed1986

Decrease in helper (T4+) lymphocytes following cimetidine treatment for duodenal ulcer.

We studied the possible in vivo influence of cimetidine on peripheral blood lymphocyte (PBL) subpopulations defined with monoclonal antibodies (MoAb) in eight haematologically normal patients with uncomplicated duodenal ulcers before cimetidine treatment, 1 week after the start, and finally 1 week following cessation of therapy. After cimetidine had been given for 3-5 weeks there was a significant decrease, compared with pretreatment numbers, in the proportion of T3+ cells (64.6 +/- 8.1 (mean +/- s.d.) v 51.0 +/- 7.8%) and in T4+ cells (47.3 +/- 4.3 v 30.8 +/- 4.7%). The number of T8+ cells was not affected (20.3 +/- 4.3 v 20.1 +/- 6.7%). These changes resulted in a significant reduction in the T4/T8 ratio (2.46 +/- 0.8 v 1.67 +/- 0.6). The total numbers of lymphocytes and monocytes as well as the percentage of B1+ lymphocytes did not change significantly. The observed decrease in T4/T8 ratio after cimetidine treatment is explained by a reduction in the number of T4+ cells and the appearance of a new subpopulation of T3-, T4-, T8-, B1-lymphocytes. The underlying mechanism, however, is not clear. Cimetidine does not seem to have a direct receptor-modulating effect, since in vitro exposure of normal lymphocytes to the drug did not change the proportions of the T cell subsets.

Adult↗