ABSTRACT
Statins possess anti-inflammatory effects that may contribute to their ability to slow atherogenesis, whereas nitric oxide (NO) also influences inflammatory cell adhesion. This study aimed to determine whether a novel NO-donating pravastatin derivative, NCX 6550 [(1S-[1alpha(betaS*,deltaS*),2alpha,6alpha,8beta-(R*),8a alpha]]-1,2,6,7,8,8a-hexahydro-beta,delta,6-trihydroxy-2-methyl-8-(2-methyl-1-oxobutoxy)-1-naphthalene-heptanoic acid 4-(nitrooxy)butyl ester)], has greater anti-inflammatory properties compared with pravastatin in normal and atherosclerotic apolipoprotein E receptor knockout (ApoE-/-) mice. C57BL/6 and ApoE-/- mice were administered pravastatin (40 mg/kg), NCX 6550 (48.5 mg/kg), or vehicle orally for 5 days. Ex vivo studies assessed splenocyte adhesion to arterial segments and splenocyte reactive oxygen species (ROS) generation. NCX 6550 significantly reduced splenocyte adhesion to artery segments in both C57BL/6 (8.8 +/- 1.9% versus 16.6 +/- 6.7% adhesion; P < 0.05) and ApoE-/- mice (9.3 +/- 2.9% versus 23.4 +/- 4.6% adhesion; P < 0.05) concomitant with an inhibition of endothelial intercellular adhesion molecule-1 expression. NCX 6550 also significantly reduced phorbol 12-myristate 13-acetate-induced ROS production that was enhanced in isolated ApoE-/- splenocytes. Conversely, pravastatin had no significant effects on adhesion in normal or ApoE-/- mice but reduced the enhanced ROS production from ApoE-/- splenocytes. In separate groups of ApoE-/- mice, NCX 6550 significantly enhanced endothelium-dependent relaxation to carbachol in aortic segments precon-tracted with phenylephrine (-logEC(50), 6.37 +/- 0.37) compared with both vehicle-treated (-logEC50, 5.81 +/- 0.15; P < 0.001) and pravastatin-treated (-logEC50, 5.57 +/- 0.45; P < 0.05) mice. NCX 6550 also significantly reduced plasma monocyte chemoattractant protein-1 levels (648.8 pg/ml) compared with both vehicle (1191.1 pg/ml; P < 0.001) and pravastatin (847 +/- 71.0 pg/ml; P < 0.05) treatment. These data show that NCX 6550 exerts superior anti-inflammatory actions compared with pravastatin, possibly through NO-related mechanisms.
Subject(s)
Atherosclerosis/metabolism , Nitrates/pharmacology , Nitric Oxide Donors/pharmacology , Pravastatin/analogs & derivatives , Reactive Oxygen Species/metabolism , Spleen/drug effects , Animals , Apolipoproteins E/physiology , Cell Adhesion/drug effects , Chemokine CCL2/blood , Cholesterol/blood , Endothelium, Vascular/drug effects , Endothelium, Vascular/physiology , Female , Intercellular Adhesion Molecule-1/analysis , Male , Mice , Mice, Inbred C57BL , Pravastatin/pharmacology , Spleen/cytology , Spleen/metabolism , Thrombin/pharmacologyABSTRACT
BACKGROUND AND PURPOSE: Protease-activated receptor-2 (PAR-2) is expressed on lymphocytes and endothelial cells, and plays a significant role in inflammatory reactions. Since leukocyte-endothelial cell interaction and reactive oxygen species (ROS) generation are hallmarks of the development of inflammation, the effects of PAR-2 activation by trypsin on lymphocyte adhesion and ROS generation was examined utilising PAR-2 wild type and knockout (PAR-2-/-) mice. EXPERIMENTAL APPROACH: Lymphocyte adhesion to the luminal surface of mouse isolated aortae was measured using 51Cr-labelled leukocytes and ROS generation from isolated lymphocytes was quantified using chemiluminescence. KEY RESULTS: Trypsin induced adhesion of lymphocytes when added exogenously to the endothelial surface of the aorta for 30 min. Similarly, increased lymphocyte adhesion was also observed when mice were injected with trypsin intravenously 24 h prior to the adhesion assay, an effect which was partly ICAM-1 mediated. Trypsin also increased ROS generation from isolated mouse lymphocytes in a dose-dependent manner. The increase in lymphocyte adhesion and ROS production in response to trypsin were abolished in PAR-2-/- mice indicating a PAR-2 dependent mechanism. Superoxide dismutase had a greater inhibitory effect in PAR-2-/- mice compared to wild type mice when lymphocytes were stimulated with PMA but not trypsin. CONCLUSIONS AND IMPLICATIONS: The present study indicates that activation of PAR-2 may be an important factor in modulating lymphocyte adhesion and ROS generation. The results have implications for developing anti-inflammatory strategies.
Subject(s)
CD4-Positive T-Lymphocytes/metabolism , Reactive Oxygen Species/metabolism , Receptor, PAR-2/physiology , Animals , Aorta, Thoracic/cytology , Aorta, Thoracic/drug effects , Aorta, Thoracic/metabolism , B-Lymphocytes/cytology , B-Lymphocytes/drug effects , B-Lymphocytes/metabolism , CD11b Antigen/analysis , CD4-Positive T-Lymphocytes/cytology , CD4-Positive T-Lymphocytes/drug effects , Cell Adhesion/drug effects , Cell Adhesion/physiology , Dose-Response Relationship, Drug , Female , Flow Cytometry , In Vitro Techniques , Intercellular Adhesion Molecule-1/metabolism , Luminescent Measurements/methods , Male , Mice , Mice, Knockout , Myeloid Cells/cytology , Myeloid Cells/drug effects , Myeloid Cells/metabolism , P-Selectin/metabolism , Receptor, PAR-2/genetics , Receptor, PAR-2/metabolism , Spleen/cytology , Spleen/drug effects , Spleen/metabolism , Superoxide Dismutase/pharmacology , Tetradecanoylphorbol Acetate/pharmacology , Trypsin/pharmacologyABSTRACT
Myelodysplastic syndromes (MDS) are characterized by a clonal disorder of haemopoiesis with defective growth in vitro. The long-term culture system was used to examine aspects of stromal function in MDS patients. Primary long-term cultures of MDS bone marrow showed poor myelopoiesis with progenitors being detected for a median 3.5 weeks (n = 12) compared with 18 weeks in cultures of normal marrow (n = 10; P < 0.0001). The haemopoietic function of adherent layers was assessed in secondary co-cultures seeded with 5 x 10(6) cord blood mononuclear cells on irradiated normal (n = 27; aged 38-82 years) or MDS (n = 32; aged 41-86 years) adherent layers (> 60% confluent). The median myeloid progenitor number/cord blood co-culture was 135 in 5-week-old cultures with normal adherent layers and 22 in those with MDS layers (P < 0.0001). Myeloid colonies were detectable for a median 11 weeks with normal adherent layers and 6 weeks with MDS adherent layers (P < 0.0001); erythroid colonies were detectable for 7 weeks (normal) compared with 5 weeks (MDS) (P < 0.01). The differences in granulocyte-macrophage colony forming unit (CFU-GM) generation were not related to patient age. Cells from adherent layers of at least half of the primary normal (n = 48) and MDS (n = 26) long-term cultures expressed cytokines [interleukin (IL)-3, IL-1 beta, thrombopoietin (Tpo) and erythropoietin (Epo)] and receptors for retinoic acid (RAR alpha) [IL-2, IL-3, macrophage colony stimulating factor (M-CSF) (Fms) and Tpo (Mpl)]. Only IL-1 beta expression was reduced in week-5 MDS cultures compared with those from normal marrows (P < 0.05). There was also a highly significant decline in IL-1 beta expression in normal (but not MDS) adherent layers between week 5 and week 10. Thus, the adherent layers in cultures grown from MDS patients were haemopoietically defective and showed abnormal IL-1 beta expression.
Subject(s)
Hematopoietic Stem Cells/pathology , Myelodysplastic Syndromes/pathology , Myeloid Progenitor Cells/pathology , Adult , Aged , Aged, 80 and over , Case-Control Studies , Cell Adhesion , Coculture Techniques , Erythropoietin/metabolism , Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Hematopoietic Stem Cells/immunology , Humans , Interleukin-1/metabolism , Interleukin-3/metabolism , Linear Models , Middle Aged , Myelodysplastic Syndromes/immunology , Myeloid Progenitor Cells/immunology , Receptors, Cytokine/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Statistics, Nonparametric , Thrombopoietin/metabolism , Time FactorsABSTRACT
Primary long-term bone marrow cultures grown in 40 mM HEPES-buffered McCoy's 5A medium produced granulocyte-macrophage colony-forming units (CFU-GM) for a median of 9 weeks compared with 7 weeks with CO2/bicarbonate-buffered cultures. Reducing the medium glucose concentration (from 12.5 to 2.75 mM) extended the culture longevity to 17 weeks. The median period of erythroid colony detection increased from 6 to 8 weeks. Secondary cultures (5 x 106 cord blood mononuclear cells seeded on irradiated stroma) showed statistically similar myeloid and erythroid longevity to primary cultures. Improved control of medium pH significantly improved the capacity of long-term stromal layers to maintain stem cells in vitro.
Subject(s)
Bone Marrow Cells/cytology , Hematopoiesis/drug effects , Bone Marrow Cells/drug effects , Buffers , Cell Differentiation , Cells, Cultured , Colony-Forming Units Assay , Culture Media , Glucose/pharmacology , Humans , Hydrogen-Ion Concentration , Time FactorsABSTRACT
A novel colony stimulating factor-1 (CSF-1) binding factor present in the serum from a patient in remission from lymphoma is described. Radioimmunoassay (RIA) repeatedly failed to detect circulating levels of CSF-1 in the peripheral blood system of this patient. Molecular analysis showed a normal CSF-1 gene structure by Southern blot analysis and a 46,XX karyotype by cytogenetic analysis. CSF-1 mRNA expression in peripheral blood leucocytes was confirmed using reverse transcriptase polymerase chain reaction analysis. Morphological analysis of bone marrow cells was normal and peripheral blood progenitor cell colony assays showed a pattern of growth within the normal range in response to CSF-1 alone and in combination with other cytokines. Analysis of the patient's plasma and conditioned media prepared from peripheral blood mononuclear and granulocytic cell fractions for their ability to bind 125Iodine-labelled CSF-1 revealed the presence of a plasma CSF-1 binding factor. This binding factor was not present in the patient's urine, because CSF-1 was detected by RIA and production of the binding factor by the patients peripheral blood white cells could not be demonstrated in vitro. To our knowledge, this is the first reported case of a soluble CSF-1 binding factor.
Subject(s)
Colony-Stimulating Factors/blood , Hodgkin Disease/drug therapy , Antineoplastic Agents/therapeutic use , Base Sequence , Blotting, Southern , Cells, Cultured , Colony-Stimulating Factors/genetics , Culture Media, Conditioned , Female , Gene Expression , Humans , Middle Aged , Molecular Sequence Data , Polymerase Chain Reaction , RNA, Messenger/genetics , Remission InductionABSTRACT
Patients successfully treated for a malignancy with cytotoxic therapy have an increased risk of developing secondary myelodysplasia (MDS) and acute myeloid leukemia (AML). We report a patient in remission from Hodgkin's disease (HD) who remains hematologically normal 4 years after combination chemotherapy, but who has biological and genetic abnormalities characteristic of myelodysplasia. X-inactivation analysis using a 5' phosphoglycerate kinase (PGK) probe demonstrates polyclonal hematopoiesis, but cytogenetic analysis reveals a clonal population with a minority of metaphases having a 7q-deletion. NRAS mutations are not detectable 1 year after treatment, but are present in two separate clones (at codons 12 and 15) analyzed by single-stranded conformational polymorphism (SSCP), followed by cloning and sequencing 4 years after treatment. The presence of an activated NRAS with the same codon 12 mutation was independently confirmed by the nude mouse tumorigenicity assay. In vitro peripheral blood granulocyte-macrophage colony-forming units (CFU-GM) have changed from normal to undetectable levels while erythroid burst forming units (BFU-E) were significantly reduced on two occasions during the period of observation. These abnormalities are characteristic of MDS. Continued clinical follow-up will determine whether these evolving genetic and biological abnormalities pre-date the onset of clinical and morphological features of MDS.
Subject(s)
Hodgkin Disease/therapy , Myelodysplastic Syndromes/genetics , Adult , Amino Acid Sequence , Animals , Combined Modality Therapy , Dosage Compensation, Genetic , Female , Genes, ras/genetics , Humans , Mice , Mice, Nude , Molecular Sequence Data , Neoplasms, Second Primary/genetics , Point MutationABSTRACT
Myeloid colony growth from the peripheral blood of myelodysplastic (MDS) patients was assessed for abnormal in vitro response to haemopoietic growth factors (granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-1 (IL-1), interleukin-3 (IL-3)). Abnormal colony growth, increased or reduced, was observed with each of the factors. No specific growth pattern was related to any of the French-American-British classification (FAB) types of disease. MDS patients who had survived greater than 600 days after diagnosis (n = 34) showed significantly fewer abnormalities than patients assayed at the time of diagnosis (n = 37), the major difference being less frequent stimulation of colony growth. These findings indicate that the time of sampling relative to diagnosis needs to be considered when interpreting the in vitro response to growth factors of myeloid colonies from MDS patients.
Subject(s)
Bone Marrow/pathology , Cytokines/pharmacology , Myelodysplastic Syndromes/pathology , Stem Cells/pathology , Cell Division/drug effects , Cytogenetics , Humans , Myelodysplastic Syndromes/diagnosis , Myelodysplastic Syndromes/mortality , Stem Cells/physiology , Survival Analysis , Time FactorsABSTRACT
Circulating myeloid progenitors were assayed in 172 normal subjects and 147 patients with myelodysplastic syndrome (MDS). Patients whose cultures had colony/cluster ratios (CCR) less than 0.3 had significantly shorter survival periods than comparable patients with CCR greater than 0.3. A second prognostic indicator, which complemented CCR, was identified in patients with less than 5% blasts. Median survival was significantly reduced in patients with greater than 15 clusters/ml blood despite colony and cluster numbers being predominantly within the normal range. Characteristic differences were found in three FAB groups large enough to allow statistical analysis. Survival amongst patients with refractory anaemia with excess of blasts (RAEB) was related to CCR and was independent of cluster number. Amongst sideroblastic patients (SA) survival related only to cluster number. Refractory anaemia (RA) patients included individuals in both high-risk groups with only three patients out of 64 showing both features. Amongst all the MDS patients, those with CCR greater than 0.3 and less than 15 clusters/ml blood formed a low-risk group (n = 60) with a relatively good prognosis of whom 85% survived the study period (median duration 938 d) including 94% of those in this group with less than 5% marrow blast cells.
Subject(s)
Hematopoietic Stem Cells/pathology , Myelodysplastic Syndromes/blood , Cell Aggregation/physiology , Colony-Forming Units Assay , Humans , Myelodysplastic Syndromes/mortality , Prognosis , Reference Values , Risk FactorsABSTRACT
The clonal growth of myeloid colonies from peripheral blood was maximal when cultures were established with an initial osmolality of 220 mosmol/kg which increased during incubation as a result of partial drying. When osmolality was stabilized by secondary humidification, the optimum osmolality was 270 mosmol/kg, but growth was always two- to fivefold less than similar cultures established at low osmolality and incubated on an open shelf. Cultures established at 270 mosmol/kg or above were statistically similar whether or not drying was eliminated. Maximum colonies were apparent after 14 days incubation under both conditions; addition of conditioned medium did not alter the pattern of growth. The greater sensitivity of cultures established at 220 mosmol/kg is advantageous when assaying circulating progenitors in pathological conditions where a low number of granulocyte/macrophage colony-forming units is common.
Subject(s)
Colony-Forming Units Assay/methods , Culture Media/pharmacology , Hematopoiesis/drug effects , Hypotonic Solutions/pharmacology , Culture Techniques/methods , Humans , Humidity , Incubators , Osmolar Concentration , Stimulation, ChemicalABSTRACT
There was no overall increase in PB-CFU-GM from normal subjects, or MDS patients, when exogenous CSA (5637-CM) was added to the culture medium. However, there was a sub-group of MDS patients (seven of 35) whose PB-CFU-GM numbers were significantly stimulated by 5637-CM. In addition, there were 11 (out of 48) MDS patients with undetectable PB-CFU-GM in assays without exogenous CSA but only two when 5637-CM was added (p less than 0.01). This sub-group is of particular interest as it is known that those without detectable PB-CFU-GM tend to have significantly shorter survival times than others. The mechanism of the functional abnormality is yet to be determined.
Subject(s)
Colony-Stimulating Factors/pharmacology , Hematopoietic Stem Cells/drug effects , Myelodysplastic Syndromes/blood , Aged , Cell Division/drug effects , Cells, Cultured , Culture Media , Female , Granulocytes/physiology , Hematopoietic Stem Cells/physiology , Humans , Leukocyte Count , Macrophages/physiology , Male , Middle Aged , PrognosisABSTRACT
The survival of MDS patients without detectable circulating CFU-GM (median = 188 days) was significantly lower than those in whose peripheral blood CFU-GM were detected (median greater than 1000 days) (p less than 0.01). About half of those with detectable PB-CFU-GM died within 2 yr whilst the remainder survived more than ca 3 yr. There was no significant difference in the distribution of patients having 0-5% and greater than 5% marrow blast cells within the three groups.
Subject(s)
Anemia, Refractory, with Excess of Blasts/mortality , Anemia, Refractory/mortality , Granulocytes/pathology , Hematopoietic Stem Cells/pathology , Macrophages/pathology , Anemia, Refractory/blood , Anemia, Refractory, with Excess of Blasts/blood , Humans , Leukocyte Count , PrognosisABSTRACT
Peripheral blood (PB) granulocyte-macrophage progenitor (CFU-GM) growth was measured in 47 normal subjects and, together with bone marrow CFU-GM and blast cell numbers, in 45 newly diagnosed patients with myelodysplastic syndromes (MDS). Both PB colony and cluster numbers were significantly reduced in MDS patients. In patients with greater than 5% marrow blasts there was a negative correlation between blast cell numbers and PB colony growth. Bone marrow and PB colony growth were also well correlated in this group. Poor growth of PB CFU-GM appears to be more closely related to prognosis than does bone marrow progenitor growth.
Subject(s)
Granulocytes/cytology , Hematopoietic Stem Cells/pathology , Macrophages/cytology , Myelodysplastic Syndromes/pathology , Bone Marrow Cells , Colony-Forming Units Assay , Female , Humans , Leukocyte Count , Male , Middle Aged , Myelodysplastic Syndromes/mortalityABSTRACT
The effect of medium osmolality was examined in primary, continuous bone-marrow cultures established from TO strain mice. The non-adherent cell population increased exponentially between weeks 2 and 5 and thereafter declined steadily. The number of CFU-GM followed a similar pattern but showed greater variability. The optimum osmolality in 4 week old cultures was found to be about 345 mosmol/kg which was higher than the plasma osmolality (n = 20; mean = 323.3 mosmol/kg; range = 313-331). Maximum non-adherent cell numbers were found at about 345 mosmol/kg (better than half-maximum between 320 and 370 mosmol/kg). CFU-GM numbers in the culture supernatant were maximal at about 355 mosmol/kg (better than half-maximum between 320 and 400 mosmol/kg). An adherent layer developed over a wider range of osmolality than supported granulopoiesis (better than half-maximum between 258 and 402 mosmol/kg). It was necessary to increase the osmolality of Fischer's medium in order to obtain maximum growth.
Subject(s)
Bone Marrow/pathology , Histological Techniques , Animals , Cell Division , Cells, Cultured , Culture Media , Female , Mice , Osmolar ConcentrationABSTRACT
Total parenteral nutrition for 48 h with Aminoplex solutions and a calorie source led to statistically similar, severe depression of the serum folate concentration (by 63-77%) irrespective of whether the calorie source contained ethanol, sorbitol, lipid or glucose. Folate levels were also depressed following infusion of other ethanol-free regimens (Freamine and glucose, 59%; Vamin and glucose, 44%). Least depression (31%0 followed use of Aminosol and glucose, a regimen based on casein hydrolysate. It seems important, therefore, to give prophylactic folate to all patients receiving parenteral nutrition, regardless of whether the calorie source is ethanol.
Subject(s)
Folic Acid/blood , Ethanol , Folic Acid/administration & dosage , Folic Acid Deficiency/etiology , Humans , Parenteral Nutrition/adverse effects , Preoperative CareABSTRACT
Aminoacid solutions were given to cholecystectomy patients for a period of 48 h starting 24 h after the operation. Infusion of the aminoacid component of parenteral nutrition regimens, without calorie source, frequently led to an acute fall in the serum folate concentration. The degree of depression varied with different commercial aminoacid solutions. (Aminoplex 14 = c.60%, Vamin = 30%, Aminosol = c.0%). The folate depression by Vamin infusion was unaffected by supplementation with L-methione but was alleviated by the addition of glycine. As a consequence of surgical operation itself, folate values fell by 37% during the 24 h following cholecystectomy. This seems likely to increase the risk of haematological toxicity (i.e. acute leukopenia and thrombocytopenia) in patients given folate-free parenteral nutrition after a surgical operation.
Subject(s)
Amino Acids/administration & dosage , Cholecystectomy , Folic Acid/blood , Parenteral Nutrition , Folic Acid/administration & dosage , Folic Acid Deficiency/etiology , Humans , Parenteral Nutrition/adverse effects , Postoperative CareABSTRACT
A randomized controlled double-blind trial was undertaken in south Wales to prevent the recurrence of neural-tube defects in women who had had one child with a neural-tube defect. Sixty women were allocated before conception to take 4 mg of folic acid a day before and during early pregnancy and 44 complied with these instructions. Fifty-one women were allocated to placebo treatment. There were no recurrences among the compliant mothers but two among the non-compliers and four among the women in the placebo group. Thus there were no recurrences among those who received supplementation and six among those who did not; this difference is significant (p = 0.04). It is concluded that folic acid supplementation might be a cheap, safe, and effective method of primary prevention of neural-tube defects but that this must be confirmed in a large, multicentre trial.
Subject(s)
Fertilization , Folic Acid/therapeutic use , Neural Tube Defects/prevention & control , Clinical Trials as Topic , Diet , Double-Blind Method , Female , Folic Acid/blood , Humans , Pregnancy , Random Allocation , RecurrenceABSTRACT
Contamination of folic acid assay medium with oleate led to a lag phase when Lactobacillus casei cultures were grown on folic acid but not when whole serum provided the folate source. This led to high assay values when using 20-hour incubation periods but satisfactory results were obtained after 48 hours' incubation. The addition of Tween 80 to the medium eliminated the lag phase and the assay results were consistent after 13, 24, and 48 hours' incubation.