Prognostic value of C-reactive protein and cytokine assays for transplant-related mortality.
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Bone Marrow Transplantation↗
Biomedical subjects
A E Irvine
Publications and source records attributed to A E Irvine.
Fate of inflammatory neutrophils within the joint.
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Apoptosis↗
Peptide leucine arginine, a potent immunomodulatory peptide isolated and structurally characterized from the skin of the Northern Leopard frog, Rana pipiens.
On the basis of histamine release from rat peritoneal mast cells, an octadecapeptide was isolated from the skin extract of the Northern Leopard frog (Rana pipiens). This peptide was purified to homogeneity using reversed-phase high performance liquid chromatography and found to have the following primary structure by Edman degradation and pyridylethylation: LVRGCWTKSYPPKPCFVR, in which Cys(5) and Cys(15) are disulfide bridged. The peptide was named peptide leucine-arginine (pLR), reflecting the N- and C-terminal residues. Molecular modeling predicted that pLR possessed a rigid tertiary loop structure with flexible end regions. pLR was synthesized and elicited rapid, noncytolytic histamine release that had a 2-fold greater potency when compared with one of the most active histamine-liberating peptides, namely melittin. pLR was able to permeabilize negatively charged unilamellar lipid vesicles but not neutral vesicles, a finding that was consistent with its nonhemolytic action. pLR inhibited the early development of granulocyte macrophage colonies from bone marrow stem cells but did not induce apoptosis of the end stage granulocytes, i.e. mature neutrophils. pLR therefore displays biological activity with both granulopoietic progenitor cells and mast cells and thus represents a novel bioactive peptide from frog skin.
Adjuvants, Immunologic↗
High bax expression is a good prognostic indicator in acute myeloid leukaemia.
Most cytotoxic drugs kill cells by instigating the process of apoptosis and it has been suggested that apoptotic markers may provide an indication of tumour chemosensitivity. The aim of this study was to determine if such a relationship exists in acute myeloid leukaemia (AML). The levels of spontaneous apoptosis, bcl-2 and bax were evaluated in 56 newly diagnosed AML patients to determine if they correlated with a response to cytotoxic therapy. Spontaneous apoptosis was lower, but bcl-2, bax and the bcl-2/bax ratio were higher in AML compared with normal individuals. AML patients with high bax expression at diagnosis had significantly better prognosis for disease-free survival, event-free survival and overall survival (P = 0.016). In the standard risk group, high bax expression was in keeping with significantly improved survival. Multivariate analysis revealed bax to be an independent predictor of survival. There was a significant reduction in bcl-2 and bax expression when AML patients entered complete remission and also in relapsed AML patients who entered a second remission. This study suggests that bax is a useful prognostic indicator in AML and may assist with therapeutic decision-making for patients in the standard risk category.
Acute Disease↗
Discrimination of granulocyte colony-stimulating factor isoforms by high-performance capillary electrophoresis.
Granulocyte colony-stimulating factor (G-CSF) is a glycoprotein which acts primarily to stimulate the proliferation, differentiation and activation of committed progenitor cells of the neutrophil-granulocyte lineage into functionally mature neutrophils. The traditional biological assays employed to detect G-CSF are a myeloid bone marrow colony assay, a factor-dependent cell line specific for G-CSF and commercially available immunoassays. However, these methods will not distinguish between glycosylated and non-glycosylated forms of the molecule. In this study high-performance capillary electrophoresis (HPCE) was used to analyse glycosylated and non-glycosylated recombinant human granulocyte colony-stimulating factor (r-met-hG-CSF). Glycosylated G-CSF preparations contained human serum albumin (HSA), added as a protein carrier. Glycosylated and non-glycosylated G-CSFs were prepared in 40 mM Na2HPO4 buffer, pH 2.5, containing hydroxypropylmethylcellulose (HPMC) or 50 mM Na2HPO4 buffer, pH 9.0. Glycosylated G-CSF could be separated into two distinct glycoform populations at the lower pH studied. Differences in migration time and peak shape between glycosylated and non-glycosylated G-CSF were demonstrated. HPCE analysis of G-CSF produced using a baculovirus expression vector system revealed a further distinct G-CSF glycoform and demonstrated the resolving power of the technique.
Electrophoresis, Capillary↗
Apoptosis and its clinical significance for bladder cancer therapy.
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Apoptosis↗
Increased apoptotic peripheral blood neutrophils in systemic lupus erythematosus: relations with disease activity, antibodies to double stranded DNA, and neutropenia.
OBJECTIVE: To quantify the percentage of apoptotic peripheral blood neutrophils in systemic lupus erythematosus (SLE) and to determine the relations with disease activity and neutropenia. METHODS: Neutrophil apoptosis in SLE patients (n =50) was assessed by flow cytometry using annexin V binding and fluorescent labelled anti-fas. Rheumatoid arthritis (RA, n =20) and inflammatory bowel disease patients (IBD, n =20) were studied as disease controls. RESULTS: The percentage of apoptotic neutrophils, determined by annexin V binding, was increased in peripheral blood of SLE patients (median = 3.25%) compared with normal healthy donors (n =20, median = 1.20%) and disease controls (RA: median = 1.15%) (IBD: median = 1.15%). SLE neutrophil apoptosis correlated positively with lupus disease activity measured by SLAM score. SLE patients with increased antibodies to dsDNA (>10 mg/ml) had increased apoptotic neutrophils. Eight of 14 neutropenic SLE patients had increased apoptotic neutrophils. Increased neutrophil fas expression compared with normal controls was observed in SLE, RA, and IBD. CONCLUSION: Neutrophil fas expression is increased non-specifically in inflammatory disease. Increased circulating apoptotic neutrophils in SLE correlate positively with disease activity (SLAM) and may contribute to autoantigen excess including dsDNA.
Antibodies, Antinuclear↗
Long-term bone marrow culture profiles in patients with myelodysplastic syndromes are not explicable by defective apoptosis.
It has been suggested that increased intramedullary apoptosis may explain the paradox between peripheral blood cytopenias and the hyper- or normo-cellular bone marrow observed in the myelodysplastic syndromes (MDS). We wished to see if culture performance could be related to the presence of apoptotic cells in a group of patients with MDS (12 patients) and other patients with peripheral blood cytopenias (six patients) which caused diagnostic difficulty. There was no correlation between LTBMC or adherent cell growth and the presence of apoptotic cells in the original marrow sample. A variable degree of apoptosis was observed in both groups of patients. LTBMC profiles correlated well with diagnosis but were unrelated to the extent of intramedullary apoptosis. This suggests that apoptosis is a much more ubiquitous process in disease than previously thought.
Apoptosis↗
Spontaneous intramedullary apoptosis is present in disorders other than myelodysplasia.
Myelodysplastic syndrome (MDS) is a group of hematopoietic disorders characterized by peripheral cytopenias in the presence of normo- or hypercellular dysplastic marrow. It has been suggested that premature intramedullary apoptosis may contribute to this phenomenon. We used terminal dUTP nick-end labeling (TUNEL) of bone marrow biopsy specimens and cytocentrifuge preparations from patients with MDS and a variety of other hematopoietic disorders to determine whether there is increased intramedullary apoptosis in MDS and whether any such effect is specific to MDS. TUNEL labeling of bone marrow from 24 patients with MDS revealed significant positivity in 10 of 11 patients with refractory anemia (RA), five of seven with RA and excess of blasts (RAEB), all three patients with RAEB in transformation (RAEB-t), and all three patients with RA with ring sideroblasts (RARS). The percent of positive cells ranged from 5 to 50% but showed no apparent correlation with morphological subtype. In a series of 29 patients with acute leukemia, 17 showed significant positivity (13 of 13 with myeloid disease: three M1, seven M2, one M3, two M4; four of 16 patients with lymphoid disease: one Burkitt-type lymphoma, two null acute leukemia, and one common acute lymphoid leukemia). Intramedullary apoptosis was associated with myeloid or early committed progenitor cells and was highest in secondary acute myeloid leukemia (AML). Normal bone marrow samples from 12 individuals showed no evidence of apoptosis. Our results suggest that an increased level of intramedullary apoptosis is apparent in both patients with MDS and those with AML; those with secondary AML have the highest levels. The relative absence of such findings in lymphoid malignancy suggests that the apoptotic pathways are different in this lineage.
Apoptosis↗
Analysis of the effect of prior therapy on progenitor cell yield: use of a chemotherapy scoring system.
A quantitative analysis of peripheral blood stem cell (PBSC) yield, measuring absolute numbers of CD34+ cells x 10(6)/kg and CFU-C x 10(4)/kg was performed in 74 consecutive patients. The interval or 'gap' from the end of previous chemotherapy to the date of priming was recorded in weeks. Geometric mean CD34 and CFU-C values were significantly higher in patients with a score of < or = 60 compared to those with score > 60 (P = 0.003 and 0.02, respectively) and a significant difference in CD34 values was also found when scores of < or = 38 were compared with scores > 38 (P = 0.003), with the difference in CFU-C values approaching significance (P = 0.08). Patients exposed to toxicity factor 4 drugs had significant lowering of both CD34 and CFU-C values (P < 0.001 and P = 0.038) and this emerged as the only independent factor when analysed using linear regression. No significant difference in the geometric mean CD34 or CFU-C values of patients was found in any of the gap categories analysed. Prior exposure to toxicity factor 4 drugs had a significant adverse effect on stem cell yield and should be avoided or minimized prior to stem cell harvesting.
Adolescent↗
Measurement of colony-stimulating factors in synovial fluid: potential clinical value.
In this study, 100 synovial fluid (SF) samples from patients with a variety of arthritides were assayed for levels of colony-stimulating factors (CSFs) using a human bone-marrow bioassay and enzyme immunoassays for granulocyte (G-) and granulocyte-macrophage (GM-) CSFs. GM-CSF was found more frequently in samples from rheumatoid arthritis (RA) subjects (49%) than in non-RA samples (29%). Absence of GM- but not G- or bioassay CSFs characterised samples from subjects with psoriatic arthritis and ankylosing spondylitis (n = 14). There was strong evidence of an antagonistic relationship between levels of G- and GM-CSFs in samples from RA patients, an effect independent of drug treatment. However, treatment with non-steroidal anti-inflammatory agents (NSAIDs) may affect reported CSF concentrations: G-CSF levels were significantly lower in samples from subjects not taking NSAIDs. These results suggest that SF-CSF estimations using commercially available assays could provide useful diagnostic clues for clinicians, but careful interpretation is warranted particularly in patients on long-term NSAID treatment.
Arthritis, Rheumatoid↗
Human blood and synovial fluid neutrophils cultured in vitro undergo programmed cell death which is promoted by the addition of synovial fluid.
OBJECTIVE: To assess the influence of inflammatory synovial fluid (SF) on apoptosis of joint and blood neutrophils with particular reference to levels of colony stimulating factors (CSF) contained therein. METHODS: Neutrophils were separated from fresh synovial fluid and from peripheral blood by density gradient centrifugation. Apoptosis was assayed by light microscope morphology and DNA degradation. CSFs were assayed using bone marrow bioassay and enzyme linked immunosorbent assays for granulocyte (G-) and granulocyte macrophage (GM-) CSF. Separated neutrophils were cultured in vitro and exposed to: varying concentrations of SF in which CSF levels were measured, recombinant G-CSF and GM-CSF, and hyaluronic acid control solutions. Numbers of apoptotic neutrophils and CSF levels were also measured in fresh SF samples. RESULTS: The addition of autologous or heterologous inflammatory SF to blood or joint cavity neutrophils cultured in vitro caused a significant dose dependent increase in the percentage of cells becoming apoptotic with time as measured morphologically and confirmed by DNA degradation. The effect bore no relationship to levels of CSF in joint fluid, despite our finding that GM-CSF produced inhibition of neutrophil apoptosis in vitro. CONCLUSION: These data suggest that SF contains a factor or factors capable of directly or indirectly promoting neutrophil apoptosis and normally powerful enough to overcome the apoptosis inhibiting effects of cytokines such as GM-CSF at concentrations usually found in inflammatory synovial fluids.
Apoptosis↗
Drug-induced neutropenia due to direct effects on CFU-C--ten years of culture experience.
Seventeen patients with suspected drug-induced neutropenia were referred to our laboratory for investigation within a 10-year period. In each case, the suspected drugs were incorporated separately into in vitro cultures of the patients' bone marrow. The cultures were performed in triplicate, using multiple controls. In 10 of these patients a drug-induced inhibition of CFU-C was demonstrated in vitro. The in vitro culture technique is a valuable investigation in patients with suspected drug-induced neutropenia, as it can help identify the causative agent, especially in cases of multidrug administration. It also has a useful application in allowing the clinician to predict drugs to which a patient may be unduly sensitive, and prescribe accordingly.
Adult↗
Granulocyte and granulocyte-macrophage colony-stimulating factors in cord and maternal serum at delivery.
Impaired neutrophil responses contribute to the neonate's increased susceptibility to infection. Because granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) enhance granulocyte and macrophage number and function, their use in the management of neonatal sepsis may be beneficial. Little is known about the endogenous levels of G-CSF and GM-CSF. In adults, raised values for G-CSF, but not GM-CSF, have been demonstrated in patients with infection, and conflicting data has emerged regarding CSF levels in neonates. We have used an ELISA to measure maternal and cord serum G-CSF and GM-CSF at the time of delivery, with gestational age between 25 and 42 wk. In mothers, an inverse linear relationship between gestational age and GM-CSF levels (p = 0.049) was found, but no association with G-CSF levels was observed. In neonates, a quadratic association was found between GM-CSF levels and gestational age (p = 0.019), whereas G-CSF levels showed an inverse linear association (p = 0.015). In addition, an association was found between maternal and cord GM-CSF (p = 0.007) but not G-CSF levels in paired samples. The effect of gestational age on the cytokine levels could not be explained by the white cell count, the absolute neutrophil count, pregnancy-induced hypertension, or the presence of infection.(ABSTRACT TRUNCATED AT 250 WORDS)
Delivery, Obstetric↗
Polyethylene glycol (PEG) modification of granulocyte-macrophage colony stimulating factor (GM-CSF) enhances neutrophil priming activity but not colony stimulating activity.
PEG-modified proteins have numerous advantages over their unmodified counterparts (increased half life, reduced antigenicity, improved solubility), but almost without exception, they show a modest to marked reduction in biological or enzymatic activity. However, while investigating a new protocol for the preparation of PEG-proteins, we compared PEG-modified and unmodified GM-CSF with respect to their polymorphonuclear neutrophil granulocyte (PMN) priming activities. PEG-GM-CSF was unexpectedly more active than GM-CSF in its ability to prime neutrophils to respond to the synthetic peptide n-formyl-methionyl-leucyl-phenylalanine (FMLP) with an oxidative burst (assessed both by nitroblue tetrazolium reduction and ferricytochrome c reduction). These results were in contrast to the findings for colony stimulating activity and with GM-CSF induced thymidine uptake, where the biological activity was unchanged or reduced. The enhanced neutrophil priming activity of PEG-GM-CSF was confirmed using FPLC fractionated PEG-modified GM-CSF. This showed changes in the bioactivity profile consistent with both the shift in protein elution profile and enhanced activity of the PEG-modified material (reflected in the increased area under the bioactivity curve). We also excluded a neutrophil priming action for PEG-modified fetal calf serum proteins, carrier proteins and 'irrelevant' cytokine, erythropoietin. The dissociation of the two bioactivities was confirmed using individual FPLC fractions. These results suggest the presence of differences in either binding, receptor/ligand processing or signal transduction for neutrophils versus progenitors, that are differentially affected by PEG-modification of GM-CSF. The demonstration that PEG-modification can partially dissociate two biological activities suggests the feasibility of using PEG-modification to produce proteins with subtly altered spectra of biological activity and hence new ranges of clinical applications.
Cells, Cultured↗
Polyethylene glycol (PEG)-modified granulocyte-macrophage colony-stimulating factor (GM-CSF) with conserved biological activity.
Polyethylene glycol (PEG) modification improves the pharmacological properties of proteins, usually extending plasma half-life and concomitantly increasing in vivo bioactivity, reducing both antigenicity and immunogenicity, and increasing solubility and resistance to proteolysis. Despite these established benefits, few PEG proteins are in use. Current coupling methods are either traumatic for the protein or involve lengthy and difficult procedures to activate monomethoxyPEG (MPEG). We have applied a new coupling method that allows coupling of MPEG directly to proteins under physiological conditions. Using this method with recombinant human (rh)granulocyte-macrophage colony-stimulating factor (GM-CSF) we were able to construct biologically active PEG-GM-CSF. Fast protein liquid chromatography (FPLC) and phase-partitioning confirmed the presence of PEG modification, and the former was used to fractionate modified and unmodified material. Bioactivity was measured in colony assays of normal human bone marrow cells and by tritiated thymidine uptake (of chronic myeloid leukemia cells and TF-1 cells). With both uptake and colony assays, using unfractionated material, we observed only a modest reduction in biological activity. Assays of FPLC-fractionated material confirmed that much of the bioactivity of the PEG-GM-CSF preparations was due to the modified species and any residual unmodified GM-CSF. Species uncontaminated by tresylmonomethoxyPEG (TMPEG; which was somewhat inhibitory in the thymidine uptake assay and eluted over a broad region of the FPLC profile) had no significant reduction in activity, but we cannot rule out the possibility that PEG-GM-CSF species eluting elsewhere in the profile had modest reduction of activity. Subcutaneous injection into mice confirmed the anticipated improved half-life in vivo and demonstrated a longer uptake from the injection site. This is, as far as we are aware, the first successful construction of PEG-GM-CSF with conserved biological activity.
Animals↗
Normal unstimulated lymphocytes produce granulopoietic inhibitory activity.
Normal unstimulated lymphocytes cocultured with normal bone marrow will inhibit day-7 colony- forming units in culture. We have shown that this phenomenon has a molecular basis and attempted to characterize it further. Using specific assays and neutralizing monoclonal antibodies we have found that this granulopoietic inhibitory activity (GIA) is not due to alpha- or gamma-interferon, tumor necrosis factor, or acidic or basic isoferritins. Biochemical studies suggest that it is a glycoprotein with a molecular weight greater than 100,000 daltons. It appears to act on cells in S phase, although it may not be S-phase specific. GIA represents a novel inhibitor that merits further investigation.
Bone Marrow↗
Dissociation of the proliferation and differentiation stimuli of granulocyte colony-stimulating factor (G-CSF).
One proposed therapeutic application of granulocyte colony-stimulating factor (G-CSF) is in differentiation induction therapy of myelodysplastic states (MDS) or acute myeloid leukemia (AML). G-CSF however has a substantial growth including effect which limits its potential as a differentiation inducing agent. We have therefore made a systematic search for agents which might restrain the proliferative effects of G-CSF whilst retaining the differentiation stimulus. Of all the agents we have tested on human bone marrow progenitor cells: (6-thioguanine, all-trans retinoic acid, vincristine, recombinant human alpha-2b and gamma-interferon) only the latter abolished the stimulation of cell growth and retained, or possibly increased, the differentiation effect of G-CSF. The antiproliferative drugs 6-thioguanine and vincristine both antagonized the neutrophil-granulocyte differentiation inducing action of G-CSF. Retinoic acid and alpha-2b interferon both had weak effects on proliferation and failed to enhance differentiation. These results suggest that it may be possible, by combining G-CSF with a suitable second agent, to utilize its substantial differentiation inducing effect without incurring the potentially hazardous effects of increased leukemic cell growth.
Cell Differentiation↗