PubMed Health⌕ Search

Biomedical subjects

D R Sutherland

Publications and source records attributed to D R Sutherland.

At least 37 records · Page 2Linked to original sources
PubMed1996

Peripheral blood progenitor cell collections in multiple myeloma: predictors and management of inadequate collections.

Thirty-seven patients with previously treated multiple myeloma (MM) underwent peripheral blood progenitor cell (PBPC) collection following high-dose cyclophosphamide and GM-CSF or sequential IL-3 and GM-CSF. Patients with an inadequate collection were considered for a second or third collection. 25 patients underwent subsequent autotransplant. The only variable predictive of CFU-GM yield was the extent of prior melphalan therapy. All repeat collections were unsuccessful and patients infused with an autograft obtained from multiple sets of collections had a high incidence of delayed engraftment. We conclude that melphalan should be avoided or PBPC collection performed early in the disease course in patients who are potential transplant candidates.

Adult↗
PubMed1996

The ISHAGE guidelines for CD34+ cell determination by flow cytometry. International Society of Hematotherapy and Graft Engineering.

The increased use of Peripheral Blood Stem Cells (PBSC) to reconstitute hematopoiesis in autotransplant and, more recently, allotransplant settings has not been associated with a consensus means to quality control the PBSC product. Since the small population of cells that bear the CD34 antigen are thought to be responsible for multilineage engraftment, graft assessment by flow cytometric quantitation of CD34+ cells should provide a rapid, reliable, and reproducible assay. Unfortunately, although a number of flow cytometric assays for CD34 enumeration have been described, the lack of a standardized method has led to the generation of widely divergent data. Furthermore, none of these assays has been validated as to interlaboratory reproducibility and suitability for widespread clinical application. In early 1995, the International Society of Hematotherapy and Graft Engineering (ISHAGE) established a Stem Cell Enumeration Committee, the mandate of which was to validate a simple, rapid, and sensitive flow cytometric method to quantitate CD34+ cells in peripheral blood and apheresis products. We also sought to establish its utility on a variety of flow cytometers in clinical laboratories and its reproducibility between transplant centers. Here, we describe the four-parameter flow methodology adopted by ISHAGE for validation in a multicenter study in North America.

Antigens, CD34↗
PubMed1996

Increased levels of CD34+ hemopoietic progenitor cells in atopic subjects.

We investigated the contribution of hemopoietic progenitors to the accumulation of inflammatory cells in allergic airways disease. Using a multiparameter flow-cytometric method, the detection of peripheral blood (PB) and bone marrow (BM) cells expressing CD34, a progenitor cell marker, was explored. True CD34+ blast cells were detected as a discrete cluster exhibiting low intensity CD45 expression, low granularity, and low to intermediate cell size. A significantly greater number of CD34+ cells was detected in the PB of atopic individuals (1,438 +/- 347/10(6) nonadherent mononuclear cells [NAMNC], n = 19) compared with nonatopics (236 +/- 77/10(6) NAMNC, n = 13; P = 0.006). Similarly, in BM samples, a significantly greater number of CD34+ cells was detected in atopic (17,537 +/- 4,986/10(6) NAMNC, n = 7) compared with nonatopic subjects (6,422 +/- 1,853/10(6) NAMNC, n = 13, P = 0.02). Greater numbers of total colony-forming units (CFU) (granulocyte/macrophage [GM] and Eo/Baso) were present in cultures of PB NAMNC from atopics (24 +/- 5 CFU/10(6) NAMNC) cultured with recombinant human interleukin 5 (rhIL-5) (1 ng/ml) compared with nonatopics (5 +/- 2 CFU/10(6) NAMNC; P = 0.003). Analyses of colony subtypes showed significantly greater numbers of IL-5-responsive Eo/Baso-CFU in cultures from atopics (15 +/- 2 CFU/10(6) NAMNC) compared with nonatopics (5 +/- 2 CFU/10(6) NAMNC; P = 0.011). In contrast, no significant differences in colony counts were found between the two subject groups in cultures with rhIL-3 (1 ng/ml) or rhGM-CSF (10 ng/ml). A positive correlation was observed between PB CD34+ cell numbers and total CFU in cultures with rhIL-5 (r = 0.43, n = 32, P = 0.01) and rhGM-CSF (r = 0.45, n = 32, P = 0.009). Purging BM NAMNC with an anti-CD34 monoclonal antibody completely abrogated in vitro colony growth, supporting the view that a subset of CD34+ cells represents the relevant population of progenitors growing in culture. These data indicate that flow cytometric estimation of CD34+ cells is predictive of the colony-forming capacity of the sample and may be a useful alternative tool to clonogenic assays for enumerating progenitors. In addition, raised levels of CD34+ cells and IL-5-responsive Eo/Baso-CFU in atopics, including patients with atopic asthma, indicate a role for progenitors in allergic airways disease.

Antibodies, Monoclonal↗
PubMed1996

Fetal bone marrow CD34+CD41+ cells are enriched for multipotent hematopoietic progenitors, but not for pluripotent stem cells.

We have investigated the expression of CD41a (gpIIbIIIa) on a subpopulation of human fetal bone marrow (FBM) CD34+ progenitor cells. Human FBM CD34+Lin- cells were subfractionated into CD41a+ and CD41a- subpopulations by flow cytometry. All the megakaryocyte colony-forming cells (CFU-MK) and almost all the burst-forming units-megakaryocyte (BFU-MK) were found within the CD41a+ subpopulation. In addition, a 14-fold greater number of granulocyte-macrophage colony-forming units (CFU-GM) and a five-fold greater number of mixed lineage progenitor cells (CFU-mix) were observed within the CD34+Lin-CD41a+ subpopulation compared to the CD34+Lin-CD41a- subpopulation. The high proliferative potential of CD34+Lin-CD41a+ cells was demonstrated by their capacity to expand in in vitro culture containing human plasma and recombinant Mpl ligand (thrombopoietin [Tpo]) with production of over 80% CD41b+ (gpIIb+) MKs. However, in long-term bone marrow cultures, the CD34+Lin-CD41a- population contained a significantly higher frequency of cobblestone area-forming cells (CAFC) than the CD34+Lin-CD41a+ population, indicating the presence of a primitive hematopoietic stem cell (HSC) population within the CD34+Lin-CD41a- subset. These data suggest that fetal CD34+Lin-CD41a+ cells are enriched for MK progenitor cells (CFU-MK and BFU-MK), myeloid progenitors, and CFU-mix but do not contain the more primitive CAFC.

Antigens, CD34↗
PubMed1996

Identification of CD34+ subsets after glycoprotease selection: engraftment of CD34+Thy-1+Lin- stem cells in fetal sheep.

Epitopes on the CD34 molecule detected by some CD34 antibodies can be cleaved by a unique glycoprotease from Pasteurella haemolytica, which cleaves only glycoproteins rich in O-linked glycans. A method to isolate CD34+ cells from adult bone marrow was developed subsequently, in which CD34+ cells were isolated in high purity and yield following immunomagnetic bead selection and detachment with the glycoprotease. Using a variety of other cell-surface markers shown here to be insensitive to glycoprotease, committed progenitors of T lymphoid, B lymphoid, monomyeloid, megakaryoblastic, or erythroid lineages could be identified. Significantly, candidate hematopoietic stem cells (HSC) that are contained within a CD34+Lin- (CD2-, CD14-, CD15-, CD16-, CD19-) (or CD34+CD38-) subset expressing the Thy-1 antigen (CDw90), c-kit receptor (CD117), and CDw109 but lacking expression of CD71 and HLA-DR antigens also were detected. Functionally distinct subsets of glycoprotease-selected CD34+ cells were identified and subfractionated using flow cytometry and fluorescence-activated cell sorting (FACS). These subsets included candidate HSCs expressing the CD34+Thy-1+Lin- phenotype, which were sorted from a CD34+ fraction of a mobilized peripheral blood (MPB) sample. In a fetal sheep model, when CD34+Thy-1+Lin- cells were injected intraperitoneally, they were capable of homing to the marrow, where they generated long-term multilineage hematopoiesis and maintained human CD34+ cells, indicating that candidate HSC subsets of CD34+ cells selected with this highly specific enzyme were capable of engraftment in vivo. The ability to identify and purify virtually any phenotypically defined subset of glycoprotease-selected CD34+ stem/progenitor cells should facilitate the study of hematopoiesis in vitro and in animal models in vivo as well as the development of novel genetic techniques for the correction of specific blood cell disorders in humans.

Adult↗
PubMed1995

Microbial biotransformation of the angiotensin II antagonist GR117289 by Streptomyces rimosus to identify a mammalian metabolite.

Screening a range of microorganisms incubated with the angiotensin II antagonist GR117289 resulted in the use of Streptomyces rimosus to generate five related biotransformation products. These comprised three compounds hydroxylated on the aliphatic side chain, one further oxidized to a ketone, and one hydroxylated on the phenyl ring. These microbial metabolites were used as standards to identify a human metabolite detected in plasma and urine, but present in insufficient quantities for full structural characterisation. This further demonstrates how the use of microbial biotransformation systems at an early stage of drug metabolism studies can act as a valuable tool in facilitating identification of minor human metabolites.

Angiotensin Receptor Antagonists↗
PubMed1995

Optimizing the CD34+ and CD34+Thy-1+ stem cell content of peripheral blood collections.

We have previously described a sensitive and specific CD34 enumeration assay and report here a prospective analysis of 25 myeloma patients undergoing PBSC mobilization using this assay to determine the optimal days for collection of CD34+ and CD34+Thy-1+ cells after chemotherapy and growth factor mobilization. Correlations between frequency of peripheral blood CD34+ cells, circulating white blood cell (WBC) count, apheresis CD34+ cell count, nucleated cell count (NCC), and numbers of apheresis colony-forming units granulocyte/macrophage (CFU-GM) were determined. To assess levels of the more primitive subsets of CD34+ cells in the PBSC collections, coexpression of the Thy-1 antigen (CDw90) on CD34+ cells was also assessed. Marked heterogeneity was noted between patients with apheresis samples containing a median NCC of 4.2 x 10(8)/kg (range 1.3-8.1), median CFU-GM 17 x 10(4)/kg (range 0.15-32 x 10(4)/kg), and median CD34+ cell count of 1.39 x 10(6)/kg (range 0.02-6.6). The frequency of CD34+ cells in PBSC collections coexpressing Thy-1 was also heterogenous (6.2-50% of CD34+ cells), median 21.6%, mean 24.7 +/- 2%. The apheresis CD34+ cell count correlated with the peripheral blood CD34+ cell percentage (r = 0.71, p < 0.0001) but only weakly with the peripheral WBC. Apheresis CD34+Thy-1+ cell numbers correlated strongly with the circulating CD34+ cell numbers (r = 0.80), but no correlation was noted between these candidate stem cells and the peripheral WBC. In contrast, apheresis CFU-GM levels correlated most strongly with the peripheral WBC count (r = 0.61, p < 0.0001). The apheresis CD34+ cell count correlated with apheresis CFU-GM (r = 0.75, p < 0.0001) but not with the apheresis NCC. Apheresis CD34+Thy-1+ counts significantly correlated only with the apheresis CD34+ cell count and not with the apheresis CFU-GM or NCC. A higher percentage of circulating and apheresis CD34+ cells expressing Thy-1 were found on day 1 of collection, and the percentage of CD34+ cells expressing Thy-1 decreased on each subsequent day of measurement: median of 22% day 1 vs. 16.6% day 4, p = 0.04. This study therefore confirms that accurate quantitation of circulating CD34+ cells best predicts the optimal day for apheresis collection of CD34+ and CD34+Thy-1+ cells and is superior to the WBC count in this regard. Furthermore, the candidate stem cell (CD34+Thy-1+) subset is most prevalent during the earliest phases of CD34+ cell mobilization.

Blood Component Removal↗
PubMed1994

Tools to cleave glycoproteins.

There are a variety of enzymes available that are able to cleave glycoproteins, including enzymes that are specific for carbohydrate-carbohydrate linkages, carbohydrate-protein bonds and the peptide backbone. Such enzymes are useful for determining the sites of glycosylation within proteins, and for releasing glycan structures for subsequent carbohydrate analysis. One protease has been identified as being specific for O-sialoglycoproteins and can be used to identify such molecules and their epitope regions. The lack of cytotoxicity and the narrow specificity of this enzyme provides an improved method for the immunomagnetic selection of human bone-marrow stem-cells.

Animals↗
PubMed1994

Sensitive detection and enumeration of CD34+ cells in peripheral and cord blood by flow cytometry.

Peripheral blood stem cell autografts are increasingly used to reconstitute hematopoiesis after intensive, potentially marrow-ablative therapy. Assessment of autograft adequacy by enumeration of hematopoietic progenitors in colony-forming assays is handicapped by lack of reproducibility and prolonged assay time. Alternative approaches of graft assessment by flow-cytometric enumeration of stem/progenitor cells bearing the CD34 antigen can be hampered by low specificity and sensitivity. Here, we report a rapid and reliable multiparameter flow-cytometric approach to accurately enumerate CD34+ cells in peripheral blood (PB) mononuclear cells (MNCs). Total nucleated white blood cells (WBCs) are quantified by staining with fluorescein isothiocyanate (FITC)-conjugated CD45 antibody. Simultaneous staining by phycoerythrin (PE)-conjugated CD34 antibody defines an approximate number for the CD34+ progenitor/stem cell subfraction. When starting CD34+ cell numbers are low (0.01-0.5%), other nonspecifically stained leukocytes make accurate enumeration impossible. However, when the CD34+ fraction is analyzed for CD45 expression vs. side scatter (granularity), true CD34+ blast cells form a discrete cluster exhibiting low-density CD45 expression and low side-scatter characteristics. Cells within this "blast region" can be readily distinguished from lymphocytes, monocytes, granulocytes, and other events that can contaminate the CD34+ population. Here, we used this sensitive procedure to enumerate CD34+ cells in steady-state PB samples (0.03-0.09%), normal bone marrow (BM) aspirates, and umbilical cord blood collections (0.33-1.98%). This approach thus provides a means to analyze CD34+ cells in specimens from patients who have been extensively treated with chemotherapy and those undergoing PB stem cell mobilization with cytokines. Additionally, it is useful for assessment of CD34+ cells in a variety of clinical samples exhibiting perturbations of the hematopoietic progenitor/stem cell compartments.

Antigens, CD↗
PubMed1993

CD34 antigen: molecular features and potential clinical applications.

Despite the wide variety of functions exhibited by mature peripheral blood cells, all are derived from a small pool (1-3%) of primitive precursor cells in the bone marrow (BM) that bear a unique surface glycoprotein, CD34. Isolated CD34+ cells are capable of reconstituting all hematopoietic lineages, both in experimental animals and in humans following intensive therapy. CD34+ cells capable of reconstituting hematopoiesis are also found at low frequency in peripheral blood (PB), a frequency which can be dramatically increased by combinations of chemotherapy and recombinant cytokines. In some cases, PB "stem cells" (PBSC) can be used to augment or even replace conventional BM autografts. The availability of CD34 antibodies has greatly aided the development of techniques for the enrichment of primitive progenitor cells, thus allowing studies of the hematopoietic potential of stem cells in vitro. Additionally, the use of CD34 antibodies for the "positive selection" of hematopoietic stem/progenitor cells from tumor-contaminated marrow may possibly represent an alternative "purging" strategy prior to transplantation. The availability of pure populations of the most primitive hematopoietic progenitor cells will also facilitate study of genetic manipulation as a practical therapeutic modality.

Antigens, CD↗
PubMed1993

Role of aberrant sialylation of chronic myeloid leukemia granulocytes on binding and signal transduction by chemotactic peptides and colony stimulating factors.

Chronic myelogenous leukemia (CML) granulocytes exhibit a number of characteristics attributable to immature granulocytes, including marked increases in cell surface sialylation of glycoproteins which may be due, at least in part, to an increased activity of cytidine 5'-monophosphate-N-acetylneuraminic acid:Ga1 beta 1-3Ga1NAc alpha(2-3)-sialyltransferase (EC 2.4.99.4), and perhaps to altered activity of other glycosyltransferases and sialidases. This aberrant sialylation of CML granulocytes contributes to the decreased binding of the synthetic chemotactic peptide, formyl Met Leu Phe (fMLP), to the surface of CML granulocytes which leads to a rapid, transient increase in cytosolic free calcium ([Ca2+]i), an integral step in the biochemical cascade leading to cell activation. To determine if the decrease in binding of fMLP to CML granulocytes translates into a functional deficit, we measured fMLP-induced increases in [Ca2+]i. Compared to normal granulocytes, fMLP-induced increases in [Ca2+]i were markedly decreased in CML granulocytes. After sialidase treatment, a significant augmentation in fMLP-induced increases in [Ca2+]i was noted in CML granulocytes, indicating that the decreased signalling may be a consequence of aberrant sialylation. To determine if the effects of aberrant sialylation also alters the binding of endogenous polypeptide mediators, we determined the effect of desialylation of CML and normal granulocytes on binding of the colony stimulating factor for granulocytes and monocytes (GM-CSF), which plays a role in differentiation and proliferation of myeloid-lineage cells. As with fMLP binding, we also showed that the binding of GM-CSF to CML granulocytes, but not normal granulocytes, was markedly increased after sialidase treatment. Similarly, binding of GM-CSF to undifferentiated HL-60 cells was markedly increased after sialidase treatment. Therefore, we have demonstrated that aberrant sialylation of CML granulocytes not only alters the binding of fMLP and GM-CSF to their receptor(s), but may also alter signal transduction. Thus, aberrant glycosylation of CML granulocytes may reduce the binding of hematopoietic growth factors, which in turn may be responsible for the immature phenotype of CML granulocytes.

Calcium↗
PubMed1992

Cleavage of the cell-surface O-sialoglycoproteins CD34, CD43, CD44, and CD45 by a novel glycoprotease from Pasteurella haemolytica.

The study of structural/functional characteristics of the cell-surface glycoproteins of leukocytes has led to a better understanding of the differentiation and maturation of hematopoietic cells. We have assessed the ability of a unique metalloprotease that is secreted by the bovine fibrinous pneumonia pathogen Pasteurella haemolytica, to cleave cell-surface glycoproteins expressed on human leukocytes. Biochemical analysis shows that the O-glycosylated cell surface Ag CD34, CD43 (leukosialin), CD44 (hyaluronic acid receptor), and CD45 (leukocyte common Ag), are all cleaved by this protease. Although these enzyme-sensitive structures contain N-linked glycans, they are all extensively glycosylated with O-linked carbohydrates, which are especially abundant on CD34 and CD43. In contrast, the glycoproteins CD18/11a,b,c (leukocyte integrins), CD71 (transferrin receptor), HLA class I, and 8A3 Ag, which contain N-linked glycans but no O-sialo-glycans, were resistant to the action of the enzyme. Inasmuch as previous studies using glycophorin A had indicated that the substrate specificity of this enzyme may be uniquely restricted to the cleavage of O-sialoglycoproteins, we have designated this activity, P. haemolytica glycoprotease. Immunofluorescence analysis with a variety of antibodies to different epitopes of the P. haemolytica glycoprotease-sensitive structures indicate that this enzyme may have widespread applications in epitope-mapping studies, and represents a novel tool with which to study structure/function relationships for O-sialoglycosylated cell-surface proteins. However, most significantly these results suggest that the P. haemolytica glycoprotease may be of use in the affinity purification and recovery of clinically important leukocyte subsets, such as primitive hematopoietic progenitors that express CD34.

Animals↗
PubMed1992

The CD34 antigen: structure, biology, and potential clinical applications.

The diversity of function of mature circulating blood cells is reflected in their respective complements of cell-surface molecules and receptors. Although monoclonal antibodies have been instrumental in the identification and characterization of many cell-surface molecules on mature hematopoietic cells, the CD34 antigen represents to date, the only molecule, similarly identified, whose expression within the blood system is restricted to a small number of primitive progenitor cells in the bone marrow. Although its precise function remains unknown, the pattern of expression of the CD34 structure suggests that it plays an important role in early hematopoiesis. The availability of CD34 antibodies has greatly aided the development of techniques for the enrichment of primitive progenitor cells for studies of hematopoiesis in vitro. Additionally, the use of CD34 antibodies for the 'positive selection' of hematopoietic stem/progenitor cells represents and alternative strategy to 'negative selection' or purging for the large-scale manipulation of bone marrow cells prior to transplantation. The availability of pure populations of the most primitive hematopoietic progenitor cells may also facilitate the development of genetic techniques for the repair of specific blood cell disorders. In this article, we review the biology of the CD34 molecule and assess some of the roles for CD34 antibodies in immunopathology and for progenitor/stem cell purification in clinical applications.

Animals↗
PubMed1992

Activated protein kinase C directly phosphorylates the CD34 antigen in acute lymphoblastic leukemia cells.

The precursors of all blood cell lineages are contained within the 1-3% of bone marrow cells which express the CD34 antigen, and this population can reconstitute the hematopoietic system of lethally irradiated animals and humans. A potential regulatory role for the CD34 antigen in progenitor cell function and differentiation was indicated by our recent findings that the CD34 antigen can be phosphorylated in vivo to high stoichiometry in primitive CD34+ cell-lines by activated protein kinase C. To exclude the possibility that these effects were restricted to cell-lines, we have performed similar experiments on fresh cells from a patient with drug-resistant acute lymphoblastic leukemia. Similar to our previous findings, we found the CD34 antigen to be hyperphosphorylated in lymphoblasts labeled in the presence of active phorbols. The same peptides which were hyperphosphorylated in phorbol-stimulated cell-lines were also phosphorylated in phorbol-stimulated lymphoblasts. These data indicate that CD34 is a substrate molecule for PKC in fresh CD34+ lymphoblasts and underline the role of modulators of PKC activity in the biology of primitive leucocytes.

Adult↗
PubMed1992

Differential sensitivity of CD34 epitopes to cleavage by Pasteurella haemolytica glycoprotease: implications for purification of CD34-positive progenitor cells.

Our previous studies have shown that a unique glycoprotease from Pasteurella haemolytica specifically cleaves only proteins containing sialylated O-linked glycans. The hematopoietic progenitor cell antigen, CD34, which is heavily glycosylated with both N- and O-linked glycans, is readily cleaved by this protease. In this study, we demonstrate that the epitopes detected by five of the seven CD34 monoclonal antibodies are removed by the glycoprotease. The differential sensitivity of the CD34 epitopes to cleavage with either neuraminidase and/or glycoprotease establishes three classes of epitopes: 1) (class I) those identified by MY10, B1.3C5, 12.8, and ICH3 that are differentially affected by neuraminidase and removed by the glycoprotease; 2) (class II) the epitope detected by QBEND 10 that is removed only by the glycoprotease; and 3) (class III) those identified by TUK3 and 115.2 that are not removed by either enzyme. Cleavage of the 110-kd CD34 structure by the glycoprotease generates a major cell-bound fragment of about 75 kd, identified by the class III antibodies. We have also used the enzyme to improve the rapid recovery of CD34+ cells selected by immunomagnetic affinity techniques. In a preclinical model, we separated CD34+ KG1 cells with high yield (90%-95%) and high purity (94%-98%) from sham mixtures containing 50% CD34- cells. We also separated CD34+ blast cells from a patient in megakaryoblastic crisis of chronic myelogenous leukemia. In this case, the purity and yield were 93% and 94%, respectively. Enzyme treatment had no detrimental effect on cell viability, and the treated cells showed a normal quantitative expression and distribution of CD34 antigen as assessed with class III antibodies. We conclude that the P. haemolytica glycoprotease has potential to improve the isolation, from human bone marrow, of primitive hematopoietic cells that carry the CD34 antigen.

Antigens, CD↗