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T L Holyoake

Publications and source records attributed to T L Holyoake.

46 records · Page 3Linked to original sources
PubMed1996

Cytokines at the research-clinical interface: potential applications.

Developments in the characterization of growth factors and the recognition of their potential for clinical use has advanced through a number of stages. The development of clonogenic haemopoietic colony assays in the 1960s led to the discovery of colony-stimulating activity in the conditioned medium produced by certain cell lines. This activity was then purified and the colony-stimulating factors were identified. With rapid progress in molecular biology techniques in the 1980s, many further growth factors were cloned and produced on an industrial scale. Although erythropoietin, interferons, G-CSF, GM-CSF and IL-2 were all introduced into clinical practice as single agents, cytokines have more recently been investigated for use either in combination, or sequentially. Clinical trials are currently in progress to examine the optimum combinations and timing of administration. Current clinical applications include optimization of methods for mobilization of peripheral blood progenitor cells and amelioration of cytopenias following chemotherapy and bone-marrow transplantation. In the future, cytokines will be employed to expand stem and progenitor cells ex vivo, to improve gene transduction strategies, possibly to protect the gastrointestinal epithelium and as immunomodulators, both in vivo and in vitro. This review will focus on recently characterized growth factors including c-kit ligand/stem cell factor, flt3 ligand, c-mpl ligand/thrombopoietin and interleukins-11, 4, 7, 10, 12 and 13.

Animals↗
PubMed1996

The CD34 antigen: potential clinical advantages of CD34 selection.

The availability of monoclonal antibodies directed towards the haemopoietic cell surface antigen CD34 has facilitated accurate measurement, by flow cytometry, of CD34 positive cell frequencies in bone marrow and peripheral blood. In addition, a range of CD34 selection techniques, to purify peripheral blood progenitor cells or bone marrow prior to transplantation, have been developed. CD34 positive stem and progenitor cells may be selected with final purities in excess of 90%. Such pure populations of CD34 positive stem cells may be useful in several clinical areas, including tumour cell purging and T-cell depletion, and as a basis for gene therapy and stem cell expansion.

Antigens, CD34↗
PubMed1996

CD34-positive cells isolated from cryopreserved peripheral-blood progenitor cells can be expanded ex vivo and used for transplantation with little or no toxicity.

PURPOSE: The objectives of this phase I study were to assess the feasibility of using cryopreserved peripheral-blood progenitor cells (PBPC) for large-scale CD34 selection and subsequent expansion, and the safety of their use for reinfusion following chemoradiotherapy. PATIENTS AND METHODS: For 10 patients with nonmyeloid malignancy, an aliquot from a PBPC harvest was recovered from liquid nitrogen, and CD34 selected using the Isolex system (Baxter Healthcare, Newbury, United Kingdom) and expanded for 8 days ex vivo in a medium free of animal proteins but supplemented with autologous serum, stemcell factor (SCF), interleukin-1 beta (IL-1 beta), IL-3, IL-6, and erythropoietin. RESULTS: The mean increase for cell number was 21-fold, for colony-forming units-granulocyte/macrophage (CFU-GM) 139-fold, and for burst-forming units-erythroid (BFU-E) 114-fold. The expanded cells were reinfused in tandem with unmanipulated material (> or = 25 x 10(4) CFU-GM/kg). The patients did not experience any adverse effects immediately on cell infusion or within 48 hours. The 10 index patients were compared with 10 historical controls for parameters of myelosuppressive morbidity. In this small study, there were no differences in either neutrophil or platelet recovery between the patients who received expanded cells and historical controls. CONCLUSION: These data demonstrate that CD34 cells can successfully be selected from cryopreserved material, expanded ex vivo on a large scale, and safely reinfused following myeloablative conditioning regimens.

Adolescent↗
PubMed1994

Efficient isolation of human CD34 positive hemopoietic progenitor cells by immune panninga.

In this study we have assessed the use of soybean agglutinin (SBA) and CD34 microcellector devices for the selection of CD34 positive hemopoietic progenitor cells. Burst forming unit-erythroid (BFU-E), colony forming unit-granulocyte/macrophage (CFU-GM) and the recently developed multipotential human colony forming unit-type A (CFU-A) clonogenic assays were used to measure progenitor numbers in the starting mononuclear cell (MNC), the SBA negative, the nonadherent CD34 negative and the adherent CD34 positive fractions during panning. CFU-A progenitors were present at a relatively high incidence in the MNC fraction (220 per 10(5) MNC) and were enriched 15-fold in the adherent CD34 positive fraction. This progenitor incidence and enrichment were similar to those of CFU-GM and BFU-E. The mean recovery for CD34 positive cells was 2.3 x 10(6) cells per marrow aspirate. Analyses by flow cytometry demonstrated that 1-5% of input MNC were CD34 positive, that the purity of the CD34 fraction was approximately 80% and that the calculated recovery for CD34 positive cells was 61%. Recoveries for CFU-GM, BFU-E and CFU-A were between 18 and 40%. CFU-A progenitors were found exclusively in the adherent CD34 positive fraction, whereas a significant proportion of both CFU-GM and BFU-E were present in the nonadherent CD34 negative fraction. We propose that the Applied Immune Sciences (AIS) flasks preferentially bind the cells which express CD34 most strongly and that this is reflected in the finding of primitive CFU-A only in the CD34 positive fraction, with lineage-restricted progenitors found in both CD34 positive and negative fractions. This hypothesis is strengthened by data on long-term bone marrow cultures in which the CD34 positive fraction is better able to maintain output of CFU-GM compared with the CD34 negative fraction. In conclusion, relatively pure populations of CD34 positive cells may be rapidly and efficiently isolated from bone marrow samples with good recovery. The isolated cells show enhanced colony forming capacity in standard clonogenic assays and in the multipotential CFU-A assay.

Antigens, CD↗
PubMed1994

CD34+ positive haemopoietic cells: biology and clinical applications.

CD34+ is a heavily glycosylated surface antigen which is preferentially expressed on haemopoietic stem/progenitor cells. No definitive function has been attributed to CD34+, but it appears to play a role in cell to cell adhesion and may be involved in signal transduction to regulate the expression of other haemopoiesis-associated genes. A number of monoclonal antibodies to CD34+ have been raised and these have allowed the identification and characterization of a whole range of haemopoietic progenitor cells. CD34+ is expressed most strongly on the most primitive cells and is progressively lost as cells differentiate. The restricted expression of CD34+ to haemopoietic stem/progenitor cells has been exploited for transplantation studies. Several techniques have been developed to select cells expressing CD34+ from haemopoietic tissues. Successful sustained engraftment can be achieved using such positively selected cells. Alternatively, CD34+ cells may be expanded in vitro by incubation with synergistic cytokine combinations before being re-infused. An exciting new development has been the use of purified populations of CD34+ cells as the targets for gene marking and gene therapy protocols.

Antigens, CD↗
PubMed1993

Contrasting effects of rh-MIP-1 alpha and TGF-beta 1 on chronic myeloid leukemia progenitors in vitro.

In chronic myeloid leukemia (CML) an abnormality at the stem cell level results in unregulated expansion of myeloid progenitors. The mechanism underlying this uncontrolled proliferation remains unclear. An in vitro clonogenic assay which detects the human counterpart of the murine colony forming unit (CFU) CFU-A/CFU-S day 12 was described in a report of our recent findings. CML bone marrow samples were found to proliferate in the CFU-A assay, producing colonies morphologically indistinguishable from normal controls. The bcr/abl transcripts were sought in the RNA from individual colonies using the polymerase chain reaction (PCR). For the five CML samples tested to date, the majority of CFU-A colonies at diagnosis or in early chronic phase were found to be bcr/abl positive. For normal controls both macrophage inflammatory protein-1 alpha (MIP-1 alpha) and transforming growth factor-beta 1 (TGF-beta 1) inhibited the proliferation of CFU-A colonies when directly added to the assay. In contrast, CML progenitors responded normally to TGF-beta 1, but showed no response to MIP-1 alpha. In suicide assays, for five normal bone marrow samples, CFU-A progenitors induced into S-phase returned to a quiescent state after treatment with MIP-1 alpha. CML progenitors demonstrated inherently high cycle status which showed no definite response to MIP-1 alpha. However, TGF-beta 1 resulted in quiescence of CML progenitor cycling. In conclusion, the primitive progenitors from CML samples were inhibited normally by TGF-beta 1 but showed no response to MIP-1 alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Division↗
PubMed1993

Use of plasma ferritin concentration to diagnose iron deficiency in elderly patients.

AIMS: To determine a concentration of ferritin below which the possibility of iron deficiency should be considered in elderly patients. METHODS: Consecutive new referrals to a geriatric unit (n = 472) were studied prospectively. Full blood count, ferritin, serum vitamin B12 and red cell folate were measured for all patients. A blood film was assessed independently by three haematologists for features of iron deficiency. For those with ferritin of 12-45 ng/ml, bone marrow aspirates were performed and examined for the presence of stainable iron. When possible, a trial of oral iron was given to those with ferritin of < or = 45 ng/ml and response was determined by re-measurement of full blood count and ferritin after a minimum of three weeks of treatment. RESULTS: Bone marrow examination was performed in 32 patients with ferritin of 12-45 ng/ml, of whom 27 (84%) had absent stainable iron, suggesting that most elderly patients with ferritin in this range have iron deficiency. Compared with those with ferritin of 100-299 ng/ml, in whom iron stores were presumed to be normal, patients with ferritin of 12-45 ng/ml had a significantly lower mean haemoglobin and mean red blood cell volume. Furthermore, patients with ferritin up to 75 ng/ml had a significantly higher mean red cell distribution width, and were more likely to have an iron deficient blood film. CONCLUSION: Iron deficient erythropoiesis can occur in elderly patients with ferritin up to 75 ng/ml. This is much higher than the lower limit of the "normal" range usually quoted for younger subjects; this difference should be taken into account when ferritin concentrations are interpreted in elderly patients.

Aged↗
PubMed1993

Mixed colony formation in vitro by the heterogeneous compartment of multipotential progenitors in human bone marrow.

The physiology of the human haemopoietic primitive progenitor populations can be studied in normal and disease states by clonal in vitro cultures in which the primitive progenitor cells proliferate and differentiate to form mixed colonies. For many applications it is essential that such assays detect a high proportion of primitive progenitor cells. We describe an in vitro assay which detects a high incidence of human CD34+ multipotential progenitor cells. Bone marrow mononuclear cells (MNC) or selected CD34+ cells were plated at low cell concentrations in semisolid agar cultures with synergizing growth factor combinations. The optimum growth factor combination of conditioned medium from Mia PaCa-2 cells (Mia-CM), recombinant granulocyte-macrophage colony-stimulating factor and recombinant stem cell factor (SCF) supported the formation of macroscopic (> or = mm) colonies (97% of which were multilineage), at an average incidence of 250/10(5) MNC. The colony-forming cells (human colony-forming unit, type A) detected, showed a low cycling status (7.3%) and the macroscopic colonies had a high replating efficiency (46%), reflecting their probable primitive nature. This assay should prove invaluable, for studies on the regulation of proliferation of the multipotential compartment and in studies involving the assessment of these cells in transplantation and neoplastic disease.

Antigens, CD↗