Routine techniques for the isolation of human peripheral blood mononuclear cells (PBMCs) include density centrifugation with Ficoll-Paque and isolation by cell preparation tubes (CPTs) and SepMate tubes with Lymphoprep. In a series of experiments, these three PBMC isolation techniques were compared for cell recovery and viability, PBMC population composition, and cell functionality, aiming to provide a starting basis for the selection of the most appropriate method of PBMC isolation for a specific downstream application. PBMCs were freshly isolated from venous blood of healthy male donors, applying the different techniques in parallel. Cell recovery and viability were assessed using a hemacytometer and trypan blue. Immunophenotyping was performed by flow cytometry. Cell functionality was assessed in stimulated (100 ng/mL staphylococcal enterotoxin B [SEB]) and unstimulated 24 hours PBMC cultures, with cytokine production and lactate dehydrogenase (LDH) release as readout measures. PBMC isolation by SepMate and CPT resulted in a 70% higher recovery than Ficoll isolation. CPT-isolated populations contained more erythrocyte contamination. Cell viability, assessed by trypan blue exclusion, was 100% for all three isolation techniques. SepMate and CPT isolation gave higher SEB-induced cytokine responses in cell cultures, for IFNγ and for secondary cytokines. IL-6 and IL-8 release in unstimulated cultures was higher for CPT-isolated PBMCs compared to Ficoll- and SepMate-isolated PBMCs. LDH release did not differ between cell isolation techniques. In addition to criteria such as cost and application practicalities, these data may support selection of a specific PBMC isolation technique for downstream analysis.
Variation in metabolite levels reflects individual differences in genetic and environmental factors. Here, we investigated the role of these factors in urinary metabolomics data in children. We examined the effects of sex and age on 86 metabolites, as measured on three metabolomics platforms that target amines, organic acids, and steroid hormones. Next, we estimated their heritability in a twin cohort of 1300 twins (age range: 5.7-12.9 years). We observed associations between age and 50 metabolites and between sex and 21 metabolites. The monozygotic (MZ) and dizygotic (DZ) correlations for the urinary metabolites indicated a role for non-additive genetic factors for 50 amines, 13 organic acids, and 6 steroids. The average broad-sense heritability for these amines, organic acids, and steroids was 0.49 (range: 0.25-0.64), 0.50 (range: 0.33-0.62), and 0.64 (range: 0.43-0.81), respectively. For 6 amines, 7 organic acids, and 4 steroids the twin correlations indicated a role for shared environmental factors and the average narrow-sense heritability was 0.50 (range: 0.37-0.68), 0.50 (range; 0.23-0.61), and 0.47 (range: 0.32-0.70) for these amines, organic acids, and steroids. We conclude that urinary metabolites in children have substantial heritability, with similar estimates for amines and organic acids, and higher estimates for steroid hormones.
Plasma levels of lipoprotein(a) — Lp(a) — are associated with cardiovascular risk (Danesh et al., 2000) and were long believed to be influenced by the LPA locus on chromosome 6q27 only. However, a recent report of Broeckel et al. (2002) suggested the presence of a second quantitative trait locus on chromosome 1 influencing Lp(a) levels. Using a two-locus model, we found no evidence for an additional Lp(a) locus on chromosome 1 in a linkage study among 483 dizygotic twin pairs.
The exclusion of deep venous thrombosis (DVT) in the elderly is hampered by low specificity in clinical decision of D-dimer assays in older patients. To reduce false-positive results, we evaluated specific fibrin(ogen) degradation product assays for their contribution in the diagnosis of DVT. In a post-hoc study with outpatients suspected for DVT, we evaluated the elastase-specific fibrinogen (fibrinogen elastase degradation product, FgEDP) and D-E-specific fibrin (fibrin degradation product, FbDP) degradation product assays in relation to DVT. Results were combined with five D-dimer assays as ratio, with a specific focus on age-dependency. In 437 patients (DVT prevalence 39%), FgEDP correlated with D-dimer in DVT-negative patients (P<0.001), but not in DVT-positive patients (P > 0.55). FbDP results correlated with D-dimer in both groups (P<0.001). The values of the area under the curve of the receiver operating characteristic curve for both assays were lower than D-dimer. Using the ratios, only in the fourth age quartile D-dimer/FgEDP ratios had diagnostic value with lower number needed to test (1.8-12.7) as compared to D-dimer less than 500 μg/l alone (5.4-102). The D-dimer/FbDP ratios in DVT-negative elderly patients increased to a plateau by increasing D-dimer. In DVT-positive patients, these ratios were near constant for increasing values of D-dimer. In elderly patients, the D-dimer/FgEDP ratios may improve the number of patients in whom DVT could be excluded. The D-dimer/FbDP ratios showed differences in composition of fibrin degradation products between DVT-negative and DVT-positive patients and between young and old DVT-negative patients.
Recruitment of leukocytes is critical for controlling infections. However, chronic leukocyte activity is deleterious in a variety of pathological conditions. As such, leukocyte behavior is an attractive pharmacological target, although knowledge is lacking on the exact triggers driving leukocyte migration. We explored the contribution of coagulation pathways versus TLR4 signaling on the leukocyte recruitment by mimicking non-sterile and sterile leukocyte activation. A sterile inflammatory milieu was generated by spontaneous ex-vivo coagulation of human whole blood, a non-sterile milieu by incubating human whole blood with LPS (at the maximal effective dose), inducing TLR-4 mediated cytokines. The chemoattractant activity of supernatant obtained from these conditions was compared by assessment of primary human leukocyte recruitment in transwell cultures, microscopically and by flow cytometry. Human recombinant IL-8 (100 ng/mL) was used as control chemoattractant. Ex-vivo coagulation resulted in a strong release of IL-8 (119±58 ng/mL), being 5-fold higher than LPS-induced IL-8 release. Leukocyte recruitment (mainly identified as granulocytes) towards the sterile milieu was greater than TLR4-driven chemoattraction. Comparison of recombinant IL-8-induced and coagulation-induced chemotaxis indicated that chemotaxis towards the coagulated milieu was driven by more factors than IL-8 alone. Additional experiments indicated that fibrinogen cleavage product fibrinopeptide B may be involved, as this dose-dependently increased leukocyte migration. Coagulation activation drives substantial neutrophil recruitment, primarily mediated by IL-8. For this ex-vivo coagulation model, a stronger and differently mediated leukocyte response was observed compared to TLR4-driven chemoattraction. The model can be used to study the involvement of extracellular coagulation pathways in specific inflammatory pathways that differ distinctly from pathways involved in non-sterile inflammatory conditions. This model, when further optimized, may be helpful as a methodological tool for early evaluation of new treatment modalities for chronic inflammatory diseases.
Dense maps of short-tandem-repeat polymorphisms (STRPs) have allowed genome-wide searches for genes involved in a great variety of diseases with genetic influences, including common complex diseases. Generally for this purpose, marker sets with a 10 cM spacing are genotyped in hundreds of individuals. We have performed power simulations to estimate the maximum possible inter-marker distance that still allows for sufficient power. In this paper we further report on modifications of previously published protocols, resulting in a powerful screening set containing 229 STRPs with an average spacing of 18·3 cM. A complete genome scan using our protocol requires only 80 multiplex PCR reactions which are all carried out using one set of conditions and which do not contain overlapping marker allele sizes. The multiplex PCR reactions are grouped by sets of chromosomes, which enables on-line statistical analysis of a set of chromosomes, as sets of chromosomes are being genotyped. A genome scan following this modified protocol can be performed using a maximum amount of 2·5 μg of genomic DNA per individual, isolated from either blood or from mouth swabs.
Vascular smooth muscle (VSM) cell migration and proliferation play a major role in the development of atherosclerotic lesions, graft occlusion, and restenosis after angioplasty. Cell migration implies the digestion of the surrounding extracellular matrix. Cell-associated proteolysis has been extensively studied in neoplastic and inflammatory cells, but very little is known about the proteolytic properties of VSM. We have evaluated the ability of rat cultured VSM cells to solubilize [3H]amino acid-labeled extracellular matrices produced by bovine VSM. When plated at a density of 30,000 cells per well in 24 multiwell plates, VSM cells were able to solubilize 63.3 +/- 7.0% of the extracellular matrix after 10 days in culture. Extracellular matrix digestion occurred also when the cells were cultured in plasminogen-depleted serum but was higher in the presence of 10 micrograms/ml purified plasminogen (net percent digestion after the subtraction of the appropriate control, 8.6 +/- 3.0% versus 21.2 +/- 3.5% after 3 days in culture, p less than 0.005, respectively). The involvement of other enzymes in addition to plasmin is confirmed by the ability of VSM cells to degrade extracellular matrices from which the plasmin-sensitive component was removed with plasmin pretreatment. Rat VSM cells were able to solubilize 52.3 +/- 2.0% of this residual extracellular matrix-associated radioactivity after 6 days in culture versus 26.1 +/- 1.5% in the control dishes (p less than 0.01, n = 5). Cell contact was required for extracellular matrix degradation: cell-conditioned medium did not have any effect on extracellular matrix digestion.(ABSTRACT TRUNCATED AT 250 WORDS)
A pedigree-based maximum likelihood method developed by Lange et al. (12) was used to study the contribution of a newly defined di-allelic polymorphism in histidine-rich glycoprotein (HRG) to the plasma levels of HRG. In four families (n = 99) and 20 volunteers we found a heritability of 70%, an age effect of 3% and an effect of individual environmental factors of 27%. These results are remarkably similar to the results found in a previous parent-twin study in which a heritability of 69% and an effect of random environment of 31% was found. The overall genetic influence in the present study can be subdivided into an effect of 59% by the HRG phenotype and 11% by residual genetic factors. The influence of the HRG phenotype of 59% can entirely be explained by adding up the effect of the two alleles that make up the phenotype. These results indicate a codominant inheritance pattern of HRG levels in which the genetic influences can almost completely be ascribed to the additive effect of the di-allelic HRG locus whereas only a small part is due to other loci.
