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Biomedical subjects

J C Marsh

Publications and source records attributed to J C Marsh.

At least 55 records · Page 3Linked to original sources
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

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

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

Long-term bone marrow cultures in aplastic anaemia.

The long-term bone marrow culture (LTBMC) system provides an in vitro physiological model for the study of stromal cell mediated haemopoiesis in patients with aplastic anaemia. The two aspects of haemopoiesis--stromal and stem cell function--can be analysed separately using a modification of LTBMC with cross-over studies. Patients with aplastic anaemia universally demonstrate defective stem cell function in terms of reduced or absent marrow repopulating ability, reflecting a deficiency of long-term culture initiating cells. Defects in stromal cell function, as assessed by the ability of aplastic anaemia stroma to support normal generation of haemopoietic progenitors, are not common, but may conceal an isolated deficiency of a particular growth factor in some patients due to the overlapping nature of haemopoietic growth factor activities. The stem cell abnormality in aplastic anaemia reflects a deficiency in cell numbers, as well as dysfunction in certain cases. An increased level of apoptosis in aplastic anaemia marrow CD34+ cells exists, and this correlates well with disease severity. LTBMC studies demonstrate that more of the haemopoietic cells are nonviable (apoptotic and dead) compared with normal controls, and this correlates with reduced colony (CFU-GM) generation. An increase in apoptosis among primitive haemopoietic cells may contribute to the stem cell defect in aplastic anaemia. Haemopoietic growth factors such as G-CSF, when given after immunosuppressive therapy such as antilymphocyte globulin and cyclosporin for aplastic anaemia, may act partly by reducing the increased level of apoptosis, resulting in improved stem cell survival.

Anemia, Aplastic↗
PubMed1996

Campath-1G in vivo confers a low incidence of graft-versus-host disease associated with a high incidence of mixed chimaerism after bone marrow transplantation for severe aplastic anaemia using HLA-identical sibling donors.

We have evaluated the effect of in vivo Campath-1G on engraftment and GVHD in 23 patients with severe aplastic anaemia transplanted from HLA-identical sibling donors. In 14 patients Campath 1g was given pre-transplant for up to 9 days in an attempt to overcome graft rejection (group 1). In nine patients Campath-1G was given pre-transplant, but also continued post-transplant until day +5 to reduce GVHD (group 2). There were three patients with late graft failure in group I following initial neutrophil engraftment, and four cases of grade II+ GVHD. In group II, two patients had early graft failure (no take), and there were no cases of acute GVHD out of seven evaluable patients. One patient in group I developed chronic GVHD of the liver, and two patients (one in each group) had transient localised chronic GVHD. PCR of short tandem repeats was used to evaluate chimaeric status in 13 patients. Of 11 patients with initial neutrophil engraftment, only one had 100% donor haemopoiesis at all times. The remaining patients had either transient mixed chimaerism or persistence of recipient (< 20%) cells. We conclude that in vivo Campath-1G is associated with a high incidence of mixed chimaerism which tips the balance away from GVHD but towards graft rejection.

Adolescent↗
PubMed1996

Fatal autoimmune pancytopenia following bone marrow transplantation for aplastic anaemia.

We report a case of autoimmune pancytopenia 10 months after allogeneic bone marrow transplantation (BMT) for severe aplastic anaemia (SAA). The autoimmune haemolytic anaemia (AIHA) and immune thrombocytopenic purpura (ITP) were refractory to conventional immunosuppressive therapy which included steroids, azathioprine, vincristine and intravenous immunoglobulin. Splenectomy led to a recovery of the thrombocytopenia but the haemolysis continued despite further immunosuppressive therapy. Four months after the onset of haemolysis granulocyte-specific antibodies were detected. The patient subsequently received total lymph node irradiation (TLI) with a peripheral blood stem cell transplant (PBSCT) from his original donor, but died 9 days later from cerebral aspergillosis. The severe nature of autoimmune cytopenias and their lack of response to conventional treatment following allogeneic BMT is discussed further.

Adult↗
PubMed1995

Clonal hematopoiesis as defined by polymorphic X-linked loci occurs infrequently in aplastic anemia.

We evaluated the methylation status of the X-linked gene phosphoglycerate kinase (PGK1) and the DXS 255 locus detected by probe M27 beta to study clonality in acquired aplastic anemia (AA). A total of 30 females were suitable for clonal analysis of peripheral blood polymorphonuclear cells (PMN) and mononuclear cells using a polymerase chain reaction-based procedure in 24 patients and Southern blotting in 9. Overall, 10 of 30 patients exhibited an imbalanced X-inactivation pattern. However, in 4 patients, analysis of constitutional DNA suggested a skewed methylation pattern and 2 further cases had to be excluded because of the lack of an appropriate control. A truly clonal pattern was thus established in 4 of 30 (13%) patients. In 7 patients who later developed clonal disorders of hematopoiesis, X-inactivation analysis did not predict this event in any case. In patients with a paroxysmal nocturnal hemoglobinuria phenotype, there was no correlation between the proportion of phosphatidylinositol glycan anchored protein (PIG-AP)-deficient blood cells and the corresponding X-inactivation pattern. X-inactivation analysis detected clonal hematopoiesis in only 3 of 10 patients with a deficiency in PIG-AP in the cell population under study, but sorting of nucleated cells on the basis of PIG-AP expression showed the clonal nature of PIG-AP-deficient cells. We conclude that the majority of patients with AA show polyclonal hematopoiesis using X-linked clonal analysis, but that minor clonal populations, such as PIG-AP-deficient cells, may not be detected unless sorted cell populations are separately analyzed.

Adolescent↗
PubMed1995

Comparison of purity and enrichment of CD34+ cells from bone marrow, umbilical cord and peripheral blood (primed for apheresis) using five separation systems.

Interest in the isolation and characterization of primitive hemopoietic cells in both the clinical and research fields has rapidly increased. In parallel, different purification systems have been developed to isolate these cells. We have compared five different methods for separation of CD34+ cells from human umbilical cord blood, normal bone marrow and apheresis harvests and analyzed purity, recovery, yield and enrichment of colony forming cells (CFC) for each individual system. Our results indicate that the most reliable methods of purification for all samples were fluorescence activated cell sorting (FACS) and magnetic activated cell sorting (MACS) which consistently yielded high purities (> 70%) and enrichment of CFC. In this respect the enrichment of CFC from the MACS was superior to all the other systems including FACS. Similar results (> 70%) for purity were obtained using avidin affinity columns and a biotinylated antibody but neither yield nor CFC enrichment approached the values achieved with MACS. On average CFC enrichment using these affinity columns was greater than that observed for FACS while the purity was comparable. Both CELLector flasks and immunomagnetic beads coated with CD34 antibodies were less effective in our hands in separating purified populations of progenitor cells. Both purity and CFC enrichment of CD34+ cells using these methods were at least 50% lower than obtained with either FACS, MACS or affinity columns.

Antigens, CD↗
PubMed1995

Biochemical modulation of 5-fluorouracil with brequinar: results of a phase I study.

Biochemical modulation can increase the efficacy of 5-fluorouracil (5-FU). Pizzorno et al. have previously shown that brequinar, a de novo pyrimidine synthesis inhibitor, enhances the antitumor effect of 5-FU in vivo [Cancer Res 52: 1660-1665, 1992]. On the basis of their data, we conducted a phase I study of brequinar in combination with 5-FU in patients with refractory solid tumors. The initial dose (100 mg/m2) of brequinar was raised in 100-mg/m2 increments in cohorts of three assessable patients. The initial dose of 5-FU was 500 mg/m2, but escalation was allowed in patients who showed no significant toxic reaction. Brequinar was administered over 1 h and 5-FU over 2 h starting 18-20 h after the initiation of infusion of brequinar. Treatments were repeated weekly. Responses were evaluated after 4 weeks (one course) and then every 8 weeks thereafter. Pharmacokinetics of brequinar and determination of plasma uridine levels were performed in at least three patients at each dose level. Of the 25 patients registered in the study, 21 were assessable for toxicity studies. The dose of brequinar was escalated up to 600 mg/m2. In addition, the dose of 5-FU was increased to 600 mg/m2 as a result of a lack of a significant toxic reaction in the first nine patients. No objective responses were observed. One patient developed grade 3 stomatitis, and one developed grade 3 esophagitis at the 400 and 600 mg/m2 dose of brequinar, respectively. Brequinar produced a dose-dependent decrease in plasma uridine levels at doses up to 500 mg/m2. No additional decrease in plasma uridine occurred with higher doses of brequinar, thus suggesting a plateau effect. This observation prompted us to terminate the study before reaching the maximum tolerated dose of brequinar. Our data indicate that brequinar in doses > or = 400 mg/m2 results in significant biochemical modulation. The lack of toxicity seen at these doses of brequinar suggests that the initial dose of the effector agent 5-FU should be increased in future studies.

Adult↗
PubMed1995

Rapid progression of fibrosing alveolitis and thyrotoxicosis after antithymocyte globulin therapy for aplastic anemia.

Antithymocyte globulin (ATG) therapy is an established form of treatment for aplastic anaemia and has also been used as prophylaxis against graft rejection of bone marrow and renal allografts. Administration of ATG preparations has been associated with many mild clinical reactions, as have other forms of immunomodulatory therapy. However, serious adverse effects appear to be rare. We report a case of rapidly progressive fibrosing alveolitis and thyrotoxicosis in relation to ATG therapy, highlighting its potential toxicity and emphasising that its administration should be undertaken by experienced physicians in specialised centres.

Anemia, Aplastic↗
PubMed1995

Treatment of aplastic anaemia with antilymphocyte globulin and cyclosporin.

Improved survival of patients with aplastic anaemia (AA) has been reported over the last 20 years with immunosuppressive (IS) therapy using antilymphocyte globulin (ALG), and more recently cyclosporin (CSA). The antibody specificities of ALG have now been more clearly defined and are not only T cell directed but also include activities against B cells, NK cells and monocytes. Consequently, the effectiveness of ALG in AA may involve several different mechanisms, and may also help to explain the delayed response that occurs in AA. CSA increases the response rate to ALG in the first 3-6 months, but does not result in improved survival compared with ALG alone. Better supportive care has undoubtedly contributed to the improved survival of patients with time. Almost half the patients who do not respond to a first course of ALG can achieve a later response with a second course of ALG. Relapse occurs in 30% of patients, but up to 50% will respond again with a second course of ALG. Evaluation of the expression of (1) phosphatidylinositol-glycan (PIG)-anchored proteins on haemopoietic cells and (2) gamma-IFN in bone marrow mononuclear cells, may help to predict which patients are more likely to respond to IS therapy. Long term follow up of patients is required to assess the predictive value of X-inactivation DNA studies and PIG-protein expression for later clonal evolution.

Anemia, Aplastic↗
PubMed1995

A phase I/II trial of recombinant human interleukin-6 in patients with aplastic anaemia.

In a phase I/II study, 11 patients with marrow failure (10 with acquired aplastic anaemia and one with pancytopenic Fanconi anaemia) were treated with recombinant human interleukin-6 (rhIL-6) to assess the safety and tolerability of rhIL-6 and its effects on peripheral blood counts, bleeding complications and transfusion requirements. All patients with acquired aplastic anaemia were refractory to immunosuppressive treatment or had relapsed after immunosuppressive therapy and were not bone marrow transplantation candidates. Recombinant hIL-6 was to be given as a once-daily subcutaneous injection for 28 d at doses ranging from 0.5 to 5.0 micrograms/kg. After an observation period of 2 weeks, five patients received a second treatment course of 28 d. Only one patient had a sustained increase in platelet count from 18,000 to 72,000/microliters. Bleeding occurred in four patients and caused premature discontinuation of rhIL-6 therapy in three patients. A deterioration of pre-existing anaemia was observed in nine patients. No significant changes of leucocyte counts were observed during the first cycle. During the second cycle the peripheral blood monocyte counts decreased significantly. No significant changes in bone marrow cellularity were observed. Recombinant hIL-6 induced a dose-dependent increase in acute-phase reactants in all patients. Other adverse events included fever, headache, arthralgia, tachycardia and hypertension. In conclusion, rhIL-6 given alone at low doses does not increase platelet counts in the majority of patients with aplastic anaemia and can precipitate a sudden worsening of pre-existing anaemia and thrombocytopenia. This study was discontinued prematurely on account of the toxicity of rhIL-6 seen in patients with aplastic anaemia.

Acute-Phase Reaction↗
PubMed1995

Erythrocyte aplasia and systemic lupus erythematosus.

Pure erythrocyte aplasia is a recognised feature of systemic lupus erythematosus (SLE); here we report two cases, one predating the onset of SLE, the other following a long period of disease quiescence. One case demonstrates the typical features of this disorder and was successfully treated with prednisolone. The second case is unusual in being resistant to immunosuppressive treatment. Bone marrow culture from the second patient revealed an inhibition of BFU-E colony formation in the presence of the patient's serum, indicating that a serum inhibitor of haemopoiesis was present. Furthermore, following T cell depletion of this patient's marrow, there was an increase in BFU-E, CFU-G and CFU-GM colony growth implicating, in addition, a possible T cell-mediated inhibition of marrow haemopoiesis. This is a novel observation and may explain the resistance shown by this patient to standard treatment.

Adult↗
PubMed1995

The novel monoclonal antibody By114 helps detect the early emergence of a paroxysmal nocturnal hemoglobinuria clone in aplastic anemia.

The newly described monoclonal antibody By114 has been used with flow cytometry to investigate the status of the 90-kD glycosylphosphatidyl-inositol (GPI)-anchored component of CD66 (CD66c) on neutrophils from nine patients with paroxysmal nocturnal hemoglobinuria (PNH), seven with aplastic anemia/PNH, and 63 with aplastic anemia (AA) and a negative Ham's test. We have found that By114 is a sensitive indicator for recognizing patients with PNH and has helped delineate a group of nine patients with aplastic anemia and a negative Ham's test who have evidence of a larger PNH clone than indicated by other monoclonal antibodies (mAbs). By114 is a valuable marker for detecting the emergence of a PNH clone before the Ham's test becomes positive and is a more sensitive detector of deficient GPI-anchored proteins than other mAbs.

Adolescent↗
PubMed1995

Increased apoptosis in aplastic anemia bone marrow progenitor cells: possible pathophysiologic significance.

We have quantitated apoptotic cells by flow cytometry in human bone marrow (BM) and peripheral blood (PB) from normal donors and aplastic anemia (AA) patients, using the fluorescent DNA-binding dye 7-amino actinomycin D (7AAD). No significant difference was found in baseline percent apoptosis between normal and AA samples. Serum deprivation induced cell death to a greater degree in AA samples than in normal samples, but this was not significant. Using dual staining with anti CD34 antibody and 7AAD, we have shown that CD34+ progenitors in normal PB are significantly more apoptotic than those in normal BM. AA BM CD34+ cells contain a significantly greater proportion of apoptotic cells than normal BM CD34+ cells. Those AA patients with the lowest absolute number of CD34+ cells showed the highest proportion of apoptotic CD34+ cells. This appears to be related to clinical severity (transfusion dependence) at the time of study. We conclude that apoptosis is accelerated in AA BM progenitors and that this may contribute to the stem cell deficiency characteristic of this disorder.

Adolescent↗
PubMed1994

Haemopoietic growth factors in aplastic anaemia: a cautionary note. European Bone Marrow Transplant Working Party for Severe Aplastic Anaemia.

We are concerned about the inappropriate use of haemopoietic growth factors in patients with severe aplastic anaemia (SAA). The treatment of choice for this disorder is bone-marrow transplantation from an HLA-identical sibling donor if the patient is younger than 45 years, but it must be done soon after onset before the patient becomes sensitised by multiple red-cell and platelet transfusions. Other patients should receive immunosuppressive therapy with antithymocyte globulin alone or with cyclosporin or oxymetholone. Haemopoietic growth factors may have a role in stimulation of granulopoiesis after immunosuppressive therapy, but there is no evidence that they can correct the underlying stem-cell defect in SAA, and therefore no justification for their use alone in newly diagnosed SAA. Such treatment is harmful because it delays bone-marrow transplantation, or immunosuppressive therapy in older patients and those without suitable donors, thus reducing the chances of a successful outcome.

Age Factors↗