Biomedical subjects

E C Gordon-Smith

Publications and source records attributed to E C Gordon-Smith.

At least 55 records · Page 3Linked to original sources

PubMed1997

Pilot study of HLA alloimmunization after transfusion with pre-storage leucodepleted blood products in aplastic anaemia.

We have performed a pilot study to examine the incidence of alloimmunization using pre-storage leucocyte-depleted blood products (PLDP) in 16 previously transfused aplastic anaemia (AA) patients with no detectable HLA antibodies. A further eight AA patients with HLA antibodies received HLA-matched PLDP. Leucodepleted apheresed platelets were obtained using either Cobe spectra or Haemonetics system with an integral pall filter. Pall BPF4 filters were used for red cell preparation. Patients' sera were tested for HLA antibodies using lymphocytotoxicity (LCT). Patients who were HLA antibody negative by LCT at study entry were further tested with enzyme-linked immunoassay (ELISA). Out of 16 patients, two (12%) formed anti-HLA antibodies with a median follow-up of 9 months (range 1-15), but did not display platelet refractoriness to random donor platelets. Two patients were inadvertently transfused with non-leucodepleted blood products when later referred back to their local hospital. Both subsequently demonstrated HLA antibodies by LCT and became platelet refractory. These results contrast with a 50% incidence of HLA alloimmunization in a control group of AA patients transfused prior to this study with non-PLDP. HLA antibodies could no longer be detected by LCT in follow-up of three out of eight patients with HLA antibodies at study entry. Only one patient experienced non-haemolytic febrile transfusion reactions (NHFTR). We conclude that PLDP reduce the risk of alloimmunization even in previously transfused AA patients, PLDP are associated with a low incidence of NHFTR, and all new AA patients should receive PLDP from diagnosis.

Adolescent↗

PubMed1997

The sensitivity of Fanconi anaemia group C cells to apoptosis induced by mitomycin C is due to oxygen radical generation, not DNA crosslinking.

Fanconi's anaemia (FA) is characterized by increased spontaneous and induced chromosome fragility. This has been widely regarded to be due to a defect in DNA crosslink repair, because of the sensitivity of cells to known DNA crosslinking agents such as mitomycin C (MMC) and diepoxybutane (DEB). Although Fanconi cells are also sensitive to molecular oxygen, and may be protected by antioxidants, this has generally been considered to be a secondary phenomenon. However, it has recently been demonstrated that the FAC protein, coded for by the Fanconi anaemia gene for complementation group C, is strictly cytoplasmic and does not enter the nucleus even after DNA damage, which seems inconsistent with a role in DNA repair. We have studied the effects of MMC and oxygen on apoptotic cell death in FA group C (FA-C) and normal lymphoblastoid cell lines. Hyperoxia alone failed to induce apoptosis in either FA-C or normal cells. At ambient oxygen, MMC is known to generate oxygen free radicals, whereas decreased oxygen tension facilitates the metabolic activation of MMC for DNA crosslinking. We therefore studied the effects of MMC at 20% and 5% oxygen to favour oxygen radical generation or DNA crosslinking respectively. FA-C cells showed increased sensitivity compared to normal cells for the induction of apoptosis by MMC at 20% oxygen. When cells were treated with MMC at 5% oxygen we found no increased sensitivity of Fanconi cells to MMC when compared to normal cells. These results imply a role for oxygen free radicals, but not for DNA crosslinking, in the sensitivity of FA cells to MMC.

Apoptosis↗

PubMed1997

Glycosylphosphatidyl-inositol (GPI)-linked protein deficiency on the platelets of patients with aplastic anaemia and paroxysmal nocturnal haemoglobinuria: two distinct patterns correlating with expression on neutrophils.

Deficiencies in glycosylphosphatidyl-inositol (GPI)-linked proteins on erythrocytes and leucocytes in patients with paroxysmal nocturnal haemoglobinuria (PNH) are well known; however, expression on platelets in these patients is less well documented. We have studied CD55 and CD59 on the platelets of PNH and aplastic anaemia (AA) patients using flow cytometry. In all cases of PNH, CD55 and CD59 negative populations of platelets were detected with single or bimodal distribution and these results showed close correlation with the CD55 and CD59 patterns of neutrophils. Previous published studies have not demonstrated this distribution. We suggest that our findings may be due to the methodology used.

Anemia, Aplastic↗

PubMed1997

G-CSF-mobilized CD34 peripheral blood stem cells are significantly less apoptotic than unstimulated peripheral blood CD34 cells: role of G-CSF as survival factor.

The mechanism of release of CD34+ cells into the peripheral blood (PB) after mobilization treatment with chemotherapy and/or growth factors is not clearly understood. Growth factors may induce increased proliferation and self renewal within the stem cell compartment. It is possible that they alter adhesion molecule profiles or other progenitor:stroma interactions, to allow release of these cells into the periphery. However, CD34+ cells are present in the PB under steady-state conditions, albeit in low number. Growth factors such as granulocyte colony-stimulating factor (G-CSF) may promote the survival of CD34+ cells in the PB by suppressing apoptosis. In order to test this hypothesis, we have quantitated apoptotic cells in the CD34+ fraction of peripheral blood stem cell (PBSC) collections, using two-colour flow cytometry, after staining with anti-CD34 antibody and the fluorescent DNA binding agent, 7-amino actinomycin D (7AAD). 7AAD differentially stains live, apoptotic and dead cells, due to the altered accessibility of DNA in each subpopulation. We have shown a significant reduction in the proportion of apoptotic cells in the CD34+ population mobilized by G-CSF compared to CD34+ cells in unstimulated PB, consistent with the theory that G-CSF is acting, at least in part, by suppressing apoptosis. In addition, we found that G-CSF mobilized CD34+ cells are less apoptotic than CD34+ cells of unstimulated normal bone marrow, indicating that, at the doses used, G-CSF is significantly altering the survival capacity of the mobilized cells.

Adolescent↗

PubMed1997

Human immunodeficiency virus infection impairs hemopoiesis in long-term bone marrow cultures: nonreversal by nucleoside analogues.

Hematologic abnormalities are often seen in patients infected with human immunodeficiency virus (HIV). The effect of HIV infection of bone marrow stroma on support of uninfected CD34 progenitor cells in long-term bone marrow culture (LTBMC) was investigated. Results show that HIV-infected bone marrow stroma was unable to adequately support CD34 progenitor cells in vitro. Zidovudine or didanosine was added to cultures in an attempt to reverse the suppressive effects exerted by HIV and to determine whether such suppression was mediated by transfer of HIV infection to progenitor cells. Didanosine failed to reduce the suppressive effects of HIV, whereas zidovudine compounded the observed suppression. HIV infection of bone marrow stroma, while reducing the production of nonadherent cells, did not increase apoptosis and cell death in such cells. In contrast, zidovudine enhanced apoptosis and cell death in nonadherent cells produced by both HIV-infected and control LTBMC.

Anti-HIV Agents↗

PubMed1997

Frequency of the G6PD nt 1311 C/T polymorphism in English and Iranian populations: relevance to studies of X chromosome inactivation.

X chromosome inactivation is widely studied using DNA sequence polymorphisms and DNA methylation as a surrogate measure of inactivation, but the correlation of methylation with inactivation is not perfect. Thus, it may be better to study sequence polymorphisms expressed in the mRNA. A recent paper reported use of a silent C/T polymorphism at nt 1311 of the G6PD cDNA, and this polymorphism was reported to have a frequency of 40% in all ethnic groups. We have screened 218 English and 50 Iranian subjects by PCR and restriction digestion; 53/218 (24%) British and 22/50 (44%) Iranian subjects were heterozygous. Thus, X inactivation studies using this polymorphism may be useful in some populations, including Iran, but much less so in the UK.

Dosage Compensation, Genetic↗

PubMed1996

Downregulation of Ras gap expression in K562 cells correlates with increased differentiation to macrophages but does not affect cell proliferation or survival.

We have studied the role of Ras GTPase activating protein (GAP) in the chronic myeloid leukaemia cell line K562 by downregulating its expression using antisense RNA. This had no effect on cell proliferation and survival, suggesting that other effector molecules mediate these roles of Ras. Differentiation to macrophages following treatment with the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate was found to correlate with a significant increase in expression of GAP in K562 cells. When GAP expression was downregulated by antisense RNA, the degree of macrophage differentiation was increased, implicating GAP in the regulation of macrophage differentiation.

Cell Differentiation↗

PubMed1996

Phosphorothioate-capped antisense oligonucleotides to Ras GAP inhibit cell proliferation and trigger apoptosis but fail to downregulate GAP gene expression.

We have studied the effects of an antisense oligonucleotide to Ras GAP in leukaemia cell lines. When terminal phosphorothioate linkages were introduced into this oligonucleotide, it caused major growth inhibition and apoptosis in the chronic myeloid leukaemia (CML) cell line K562, but had little effect on the promyelocytic leukaemia cell line HL60. Neither the expression of Ras GAP mRNA nor p120 GAP protein was downregulated by the antisense oligonucleotide, suggesting a non-antisense mechanism for growth inhibition. The antisense oligonucleotide contained GGC triplets which have previously been reported to inhibit the activity of p210bcr-abl both in vitro and in vivo. However, cellular phosphotyrosine levels were found to be unaffected, suggesting that the activity of p210bcr-abl was normal and that the antisense oligonucleotide may be interacting aptamerically with a different cellular protein. Since K562 is very resistant to apoptotic cell death, the identity of the putative target molecule would be of considerable interest.

Apoptosis↗

PubMed1996

Aplastic anemia: evidence for dysfunctional bone marrow progenitor cells and the corrective effect of granulocyte colony-stimulating factor in vitro.

We investigated the effects of granulocyte-macrophage colony-stimulating factor, interleukin-3, stem cell factor, interleukin-6, and granulocyte colony-stimulating factor (G-CSF) alone, and in combination, on the clonogenic potential of normal and aplastic anemia (AA) bone marrow mononuclear cells (BMMC and CD34+ cells. AA BMMC consistently produced a significantly lower absolute number of colonies than normal, but, when account was taken of the reduced proportion of CD34+ cells in AA BM, there was no significant difference in terms of cloning efficiency (CE). However, when removed from the influence of accessory cells, the CE of AA CD34+ cells decreased significantly more than normal, indicating a defect in their function, either in terms of dependence on accessory cell-derived factors or susceptibility to cell damage when sorted. Of the factors studied, G-CSF had the most significant effect on the response of CD34+ cells from both groups when removed from their accessory cells. This was particularly true for AA CD34+ cells, whose response to cytokine stimuli containing G-CSF enabled them to match the response of normal CD34+ cells.

Adult↗

PubMed1996

The use of 7-amino actinomycin D in identifying apoptosis: simplicity of use and broad spectrum of application compared with other techniques.

The detection and quantitation of apoptotic cells is becoming increasingly important in the investigation of the role of apoptosis in cellular proliferation and differentiation. The pathogenesis of hematologic disorders such as aplastic anemia and the development of neoplasia are believed to involve dysregulation of apoptosis. To quantitate accurately the proportion of apoptosis cells within different cell types of a heterogeneous cell population such as blood or bone marrow, a method is required that combines the analysis of large numbers of cells with concurrent immunophenotyping of cell surface antigens. In this study, we have evaluated such a method using the fluorescent DNA binding agent, 7-amino actinomycin D (7AAD), to stain three diverse human cell lines, induced to undergo apoptosis by three different stimuli. Flow cytometric analysis defines three populations on the basis of 7AAD fluorescence and forward light scatter. We have shown by cell sorting and subsequent morphological assessment and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling that the populations defined by 7AAD represent live, apoptotic, and late-apoptotic/dead cells. This method is quick, simple, reproducible, and cheap and will be a valuable tool in the investigation of the role of apoptosis in normal physiology and in disease states.

Apoptosis↗

PubMed1996

Macrophages are the major target cell for HIV infection in long-term marrow culture and demonstrate dual susceptibility to lymphocytotropic and monocytotropic strains of HIV-1.

Haematological abnormalities are often seen in patients infected with HIV. A number of mechanisms are thought to contribute to this bone marrow suppression, including impaired stromal function and direct infection of progenitor cells. Evidence suggests that both bone marrow progenitor cells and perhaps stromal cells are open to infection by HIV, which raises the possibility that bone marrow stromal cells may serve as a reservoir for HIV. This study investigated the cellular targets and kinetics of in vitro infection of stroma in long-term bone marrow culture (LTBMC) using both mono- and lymphocytotropic strains of HIV-1. p24 ELISA and reverse transcriptase (RT) assay demonstrated that stroma could be infected with HIV and release infectious virions. The target cells for infection were shown to be macrophages by immunohistochemistry (APAAP), dual immunofluorescence staining (using CD68 and p24) and electron microscopy. The data show that it was possible to infect stroma in LTBMC with HIV and that such infection was productive. The main target for infection was bone marrow macrophages. In contrast to peripheral blood derived macrophages, these cells were susceptible to both lymphocytotropic and monocytotropic strains of HIV-1. The data suggests that these bone marrow macrophages may act as a reservoir for HIV, Infection of bone marrow macrophages may affect haemopoiesis either by transmission of HIV infection to developing progenitor cells through direct cell-to-cell contact or by altering the ability of the stroma to support normal development.

Bone Marrow↗

PubMed1996

In vitro progenitor analysis in a Diamond Blackfan anaemia patient who responded once but not twice to interleukin-3 therapy, using short-term and long-term cultures and c-kit analysis.

Interleukin-3 (IL-3) therapy as a treatment for Diamond Blackfan anaemia (DBA) patients has been largely disappointing despite early hope it would be suitable for stimulating arrested erythropoiesis. Initial hope came from in vitro discoveries that IL-3 (+EPO) generated well-haemoglobinized BFU-E colonies in some patients, but was soon tempered by the realization that in vitro and in vivo IL-3 response did not, in the majority of cases, correlate. Nevertheless in vitro testing has been the main focus in analysing the abnormality in the stem and progenitor cell compartment in DBA. Here we report in vitro analysis of a DBA patient who responded once to IL-3 therapy, but not a second time following relapse, using short-term culture, long-term culture and c-kit analysis. Progenitor numbers before and after the first therapy were in the high normal range, but after relapse were much reduced below normal levels. Long-term cultures suggested some arrested progenitors had been reactivated into normal cycle by the first therapy, but may not have been replaced by more immature progenitors. c-kit analysis revealed increased expression in all tested cell populations. These results imply that the first IL-3 therapy reactivated some erythroid progenitors but left the progenitor pool depleted when more immature cells remained arrested.

Cell Count↗

PubMed1996

Diamond Blackfan anaemia: differential pattern of in vitro progenitor response to macrophage inflammatory protein 1-alpha.

The congenital disorder of erythropoiesis Diamond Blackfan anaemia (DBA) exhibits a defect in the stem/progenitor cell compartment, located at the erythroid progenitor level (CFU-GEMM, BFU-E, CFU-E). Treatment of DBA with interleukin-3 (IL-3) has had limited effect, despite in vitro studies suggesting that progenitor cells were capable of responding to IL-3. Whether IL-3 is not reaching the appropriate defective target cell, the cells cannot respond, or the marrow humoral inhibitory system is overriding it, is not clear. To investigate humoral inhibitory activities we examined the response of 15 DBA bone marrows in vitro to the inhibitory chemokine macrophage inflammatory protein 1-alpha (MIP1-alpha) in the presence of the stimulatory cytokines erythropoietin, granulocyte-macrophage colony-stimulating factor, IL-3, and stem cell factor. In vitro data agreed with our previous work showing that our patients formed three statistically different groups in response to stimulatory cytokines (type I DBA erythroid colony numbers approximately normal > type II DBA > type III DBA). Addition of MIP1-alpha to cultures caused average erythroid and myeloid suppression, which sequentially increased with DBA type (type I inhibition < type II < type III). The differential level of inhibition shown by MIP1-alpha in these DBA patients lends further evidence for the presence of distinct subgroups in this disorder.

Adolescent↗

PubMed1996

Myelodysplasia following aplastic anaemia-paroxysmal nocturnal haemoglobinuria syndrome after treatment with immunosuppression and G-CSF: evidence for the emergence of a separate clone.

Myelodysplasia (MDS) and aplastic anaemia-paroxysmal nocturnal haemoglobinuria (AA/PNH) syndrome developed in a severe aplastic anaemia (AA) patient after treatment with immunosuppressive (IS) therapy. Glycosylphosphatidyl inositol (GPI)-linked proteins were determined, and during the AA/PNH phase, a high proportion of neutrophils were found to be negative, without clinical evidence of haemolysis. However, MDS developed with cytogenetic abnormalities of monosomy 7,9q- and a rearranged chromosome 6; the GPI-linked protein negative cells were completely replaced by positively expressing cells. This represents the emergence of a GPI-linked protein positive myelodysplasia clone arising separately from an AA/PNH clone.

Aged↗

PubMed1996

Diamond-Blackfan anaemia in the U.K.: analysis of 80 cases from a 20-year birth cohort.

The U.K. Diamond-Blackfan Anaemia (DBA) Registry was established with the aim of providing a representative database for studies on the aetiology, pathophysiology and treatment of DBA. We have analysed retrospective data from 80 cases (33 male, 47 female) born in the U.K. in a 20-year period (1975-94), representing an annual incidence of 5 per million live births. Ten children from seven families had an apparently familial disorder. 13% were anaemic at birth, and 72.5% had presented by the age of 3 months. 67% had macrocytosis at presentation. 72% responded initially to steroids, and at the time of study 61% were transfusion-independent (45% steroid-dependent) and 39% required regular transfusions. Unequivocal physical anomalies, predominantly craniofacial, were present in 37%, and were more likely in boys (52%) than girls (25%). 18% had thumb abnormalities. Height was below the third centile for age in 28%, and 31% had neither short stature nor physical anomalies. Four children without physical abnormalities had normal red cell indices, and achieved steroid-independent remission, suggesting transient erythroblastopenia of childhood rather than DBA. The birth month distribution of children with sporadic DBA and craniofacial dysmorphism showed a possible seasonality, consistent with a viral aetiology.

Age of Onset↗

PubMed1996

A novel approach to investigating the erythroid lineage, using both receptor analysis and haemoglobin detection.

Progenitor cell failure in the erythroid lineage is a particular problem in bone marrow failure. To provide insight into early erythopoietic development we used sensitive techniques to examine the effects of SCF, IL-3 and MIP-1 alpha on two developmentally arrested progenitor cell lines, HEL and K562. Quantitative flowcytometric analysis showed that both expressed receptors (SCF > MIP-1 alpha > IL-3). Qualitative analysis revealed HEL cells expressed more receptors than K562 cells. Clonogenic assays with sensitive haemoglobin detection showed that SCF and IL-3 did not support HEL development and reduced haemoglobin production. MIP-1 alpha reduced partially developed HEL colonies and haemoglobin in developed colonies. SCF increased development, but not haemoglobin in K562 cells, with IL-3 being more effective in both. MIP-1 alpha increased the proportion of well-developed K562 colonies but not haemoglobin. This suggests SCF, IL-3 and MIP-1 alpha all have a role to play in early erythroid cellular development, with differing actions depending on the stage of development.

Cell Differentiation↗

PubMed1996

Serum thrombopoietin levels in patients with aplastic anaemia.

Endogenous serum thrombopoietin (TPO) levels were measured in 31 patients with aplastic anaemia (AA) using an enzyme immunoassay with a sensitivity of 20 pg/ ml. The median platelet count for all AA patients was 30 +/- 29 x 10(9)/l (range 5-102) compared with a median of 284 +/- 59 x 10(9)/l (range 148-538) for normal controls. Serum TPO levels were significantly elevated in all patients compared with normals (1706 +/- 1114.2, range 375-5000 v 78 +/- 54, range 16.5-312.9, P < 0.0001). There was no correlation between serum TPO levels and the degree of thrombocytopenia in AA patients, but TPO levels were significantly higher in patients who were platelet transfusion dependent than in patients who were transfusion independent (P < 0.01). There was a trend for higher TPO levels in patients with severe AA compared with non-severe AA patients. Clinical trials of TPO and a related truncated, pegylated molecule, megakaryocyte growth and development factor (PEG-rHuMGDF), are awaited to determine whether treatment with these drugs will result in increased platelet counts in patients with AA.

Adult↗

PubMed1996

The use of recombinant SCF protein for rapid determination of c-kit expression in normal and abnormal erythropoiesis.

Stem cell factor (SCF) is the ligand for the dimeric c-kit tyrosine kinase receptor. Binding of SCF to c-kit is a crucial element in the developmental stimulus of late stem cells and early progenitor cells. In the erythroid lineage the SCF stimulus is important not only for proliferation and differentiation, but is also known to enhance later haemoglobin production. In an earlier report we described a rapid non-radioactive technique using the extended ester-attached labelled SCF protein itself for detecting c-kit expression in marrow and peripheral blood mononuclear populations. In the present study we have taken this a step further to analyse c-kit expression in developing erythroid cells in vitro, principally using normal donor samples. This was designed for use as a foundation for the comparison of haematological disorders. In this case we tested 4 patients with the congenital disorder of erythropoiesis, Diamond-Blackfan anaemia (DBA), finding that in all cases DBA c-kit expression was elevated over normal, in 1 case as high as 348% of the normal average. This may be indicative of the reduced state of progenitor development in these patients. These results show that the described technique is beneficial for analysis in the stem and progenitor compartment.

Adult↗