Background-It has been suggested by clinical, epidemiological, and experimental in vitro studies that homocysteine potentiates thrombin generation. This prothrombotic effect however has not previously been demonstrated in patients presenting with acute coronary syndromes (ACS). Methods and Results-Patients with ACS (n ϭ117) presenting with confirmed acute myocardial infarction (MI) (n ϭ57) or unstable angina pectoris (UAP) (n ϭ60) were consecutively recruited together with patients (n ϭ18) in whom the presenting chest pain was not of cardiac origin (NCP), included as controls. Plasma samples were collected on admission and before clinical intervention. Homocysteine was assayed by high performance liquid chromatography, and both Factor VIIa and prothrombin fragment F1ϩ2 were analyzed by ELISA. There were significant elevations in F1ϩ2 in MI (PϽ0.001) and UAP (Pϭ0.003), and modest elevations in Factor VIIa in UAP (PϽ0.05) compared with NCP but no differences in homocysteine levels among those groups. On dividing patients with ACS into quartiles of homocysteine, there was a stepwise increase in F1ϩ2 (PϽ0.0001) and of Factor VIIa (PϽ0.05). There were significant correlations in ACS between homocysteine and F1ϩ2 (rϭ0.46, PϽ0.0001), homocysteine and Factor VIIa (rϭ0.24, PϽ0.01), and F1ϩ2 and Factor VIIa (rϭ0.41, PϽ0.0001). There was no correlation between homocysteine and either F1ϩ2 (rϭϪ0.15, Pϭ0.57) or Factor VIIa (rϭ0.22, Pϭ0.37) in the NCP patients. Conclusions-Elevated plasma homocysteine is associated with and may cause elevated Factor VIIa and thrombin generation in patients presenting with ACS. These findings suggest an explanation for the prothrombotic effect of homocysteine in ACS.
SummaryThere is growing evidence that the tissue factor/factor VIIa pathway of coagulation is enhanced during cardiopulmonary bypass. Hitherto, available evidence has suggested that upregulated monocyte bound tissue factor is made available, either in the blood collected from the site of surgery or on circulating cells. However, cellular upregulation is slow, while generation of factor VIIa in blood collected from the pericardial cavity is rapid. We have therefore investigated the possibility of an alternative source of tissue factor, plasma (as opposed to cellular) tissue factor in blood samples taken from the central vein catheter (systemic circulation) and collected from the pericardial cavity during cardiopulmonary bypass. Six patients undergoing first time cardiopulmonary bypass grafting were studied. Tissue factor antigen was found to be rapidly elevated (by 15 min) in the pericardial plasma, ∼5-fold above systemic levels (p <0.004). Similar elevations were found in markers of coagulation activation, factor VIIa antigen (p = 0.066), prothrombin fragment F1+2 (p <0.003) and thrombin-antithrombin complex (p <0.03). To explore whether plasma tissue factor was (or had been) functionally active, factor VIIa was measured also with the soluble tissue factor functional assay after removal of heparin. Functional factor VIIa activity fell significantly in the systemic circulation, probably due to the heparin-induced increase (∼15-fold) in tissue factor pathway inhibitor (TFPI), but was elevated in pericardial blood compared with that taken from the central line catheter (p <0.006). These results demonstrate that both components of the activation complex for the extrinsic pathway of coagulation are rapidly generated in pericardial blood during bypass.
Summary Despite its role in cancer surveillance, adoptive immunotherapy using γδ T cells has achieved limited efficacy. To enhance trafficking to bone marrow, circulating Vγ9Vδ2 T cells are expanded in serum-free medium containing TGF-β1 and IL-2 (γδ[T2] cells) or medium containing IL-2 alone (γδ[2] cells, as the control). Unexpectedly, the yield and viability of γδ[T2] cells are also increased by TGF-β1, when compared to γδ[2] controls. γδ[T2] cells are less differentiated and yet display increased cytolytic activity, cytokine release, and antitumor activity in several leukemic and solid tumor models. Efficacy is further enhanced by cancer cell sensitization using aminobisphosphonates or Ara-C. A number of contributory effects of TGF-β are described, including prostaglandin E 2 receptor downmodulation, TGF-β insensitivity, and upregulated integrin activity. Biological relevance is supported by the identification of a favorable γδ[T2] signature in acute myeloid leukemia (AML). Given their enhanced therapeutic activity and compatibility with allogeneic use, γδ[T2] cells warrant evaluation in cancer immunotherapy.
Ciliopathies are an expanding group of rare conditions characterised by multiorgan involvement, that are caused by mutations in genes encoding for proteins of the primary cilium or its apparatus. Among these genes, CEP290 bears an intriguing allelic spectrum, being commonly mutated in Joubert syndrome and related disorders (JSRD), Meckel syndrome (MKS), Senior-Loken syndrome and isolated Leber congenital amaurosis (LCA). Although these conditions are recessively inherited, in a subset of patients only one CEP290 mutation could be detected. To assess whether genomic rearrangements involving the CEP290 gene could represent a possible mutational mechanism in these cases, exon dosage analysis on genomic DNA was performed in two groups of CEP290 heterozygous patients, including five JSRD/MKS cases and four LCA, respectively. In one JSRD patient, we identified a large heterozygous deletion encompassing CEP290 C-terminus, that resulted in marked reduction of mRNA expression. No copy number alterations were identified in the remaining probands. The present work expands the CEP290 genotypic spectrum to include multiexon deletions. Although this mechanism does not appear to be frequent, screening for genomic rearrangements should be considered in patients in whom a single CEP290 mutated allele was identified.
We report the development of an enzyme-linked immunosorbent assay (ELISA) that is specific for factor VIIa (FVIIa). This assay uses a neoantigen specific capture antibody directed to the amino acid peptide sequence N terminal to the FVII cleavage activation site. The antibody exhibits ∼3,000-fold greater reactivity to FVIIa than FVII on a molar basis. Experiments using plasma with added (exogenous) human FVIIa gave quantitative recovery in the ELISA over a range of 0.20 to 3.2 ng/mL of FVIIa. The intra- and inter-assay coefficient of variation (CVs) of the ELISA are 4.5% and 9.8%, respectively. The ELISA shows excellent correlation (r = .99) with a functional assay (using recombinant soluble tissue factor) in detecting FVIIa added to plasma over the range 0.05 to 18.0 ng/mL. However, a major discrepancy exists between the two assays when normal endogenous plasma concentrations of FVIIa are measured. Using normal plasma (n = 14) the functional assay reported 3.10 ± 0.30 ng/mL (mean ± SE) whereas only 0.025 ± 0.010 ng/mL was detected in the same samples by the immunoassay. Patients (n = 43) presenting with acute coronary syndromes (myocardial infarction and unstable angina) exhibited elevations (P < .05) in immunologically detected FVIIa, 0.093 ± 0.013 ng/mL (mean ± SE) compared to patient controls (n = 20) contemporaneously admitted with noncardiac chest pain, 0.048 ± 0.007 ng/mL (mean ± SE). These elevations in the acute coronary syndromes were accompanied by increased (P < .05) and correlating prothrombin fragment F1 + 2 levels (Spearman correlation coefficient rs = .4, P < .01), demonstrating that thrombin generation is certainly associated with, and may even be caused by, extrinsic pathway activation.
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