Serotonin transporter polymorphisms predict response inhibition in healthy volunteers.

Serotoninergic transmission is reliably implicated in inhibitory control processes. The aim of this study was to test the hypothesis if serotonin transporter polymorphisms mediate inhibitory control in healthy people. 141 healthy subjects, carefully screened for previous and current psychopathology, were genotyped for the 5-HTTLPR and rs25531 polymorphisms. Inhibitory control was ascertained with the Stop Signal Task (SST) from the Cambridge Neuropsychological Test Automated Battery (CANTAB). The triallelic gene model, reclassified and presented in a biallelic functional model, revealed a dose-dependent gene effect on SST performance with Individuals carrying the low expressive allele had inferior inhibitory control compared to high expressive carriers. This directly implicates serotonin transporter polymorphisms (5-HTTLPR plus rs25531) in response inhibition in healthy subjects.

Anticoagulant effects of an antidiabetic drug on monocytes in vitro.

INTRODUCTION: Monocyte- and microparticle (MP)-associated tissue factor (TF) is upregulated in diabetes. Lipopolysaccharide (LPS) induces expression of TF and alternatively spliced TF (asTF) and increases MP release from monocytes. Using LPS-stimulated TF-bearing human monocytes, we examined whether glibenclamide, a sulfonylurea used to treat diabetes type 2, might possess anticoagulant properties. METHODS: We studied the effects of glibenclamide on cell- and supernatant-associated procoagulant activity (Factor Xa-generating assay and clot formation assay), on expression of TF and asTF (flow cytometry, RT-qPCR, western blot) and on cell viability and MP release (flow cytometry). RESULTS: Glibenclamide dose-dependently decreased procoagulant activity of cells and supernatants. The reduction in cellular procoagulant activity coincided with reduced expression of TF and asTF in cells, whereas cell viability remained almost unchanged. The glibenclamide-induced reduction in procoagulant activity of supernatants appeared to be associated with a decreased number of released MPs. CONCLUSIONS: Reduction of monocyte- and supernatant-associated procoagulant activity by glibenclamide is associated with decreased expression of TF and asTF and possibly with a reduced MP number. Our data indicate that glibenclamide reduces the prothrombotic state in LPS-stimulated monocytes in vitro. Glibenclamide might therefore also have an anticoagulant effect in vivo, but this needs to be further evaluated.

Circulating lipopolysaccharides in the blood from "bioprotein" production workers.

BACKGROUND: Workers producing bacterial single-cell protein (BSCP), "bioprotein," are exposed to organic dust containing high levels of endoxins (lipopolysaccharides, LPS). Workers in this industry have complained of episodes of fever, fatigue, chest tightness, skin dryness and rubor. The aim of the present study was to quantify LPS and inflammatory mediators in plasma among the workers and non-exposed control subjects. METHODS: We included eight non-smoking production workers, aged 32-51 (median 38), and eight non-smoking, non-exposed controls, aged 30-51 (median 39). Airborne and plasma endotoxin concentrations were measured, as well as plasma hsCRP and different cytokines, chemokines and metalloproteinases. RESULTS: The workers who did not use personal respiratory protection were exposed to varying airborne levels of endotoxin, 430 (75-15 000) EU/m3 (median, range). The level of plasma LPS was significantly elevated (p = 0.01) among the workers compared to the non-exposed controls. The workers also had elevated levels of MCP-1 (p = 0.02), MIP-1alpha (p = 0.05) and MMP-3 (p = 0.04). IL-6 and hsCRP were also elevated among the exposed group, but not significantly (p = 0.10 and p = 0.07, respectively). CONCLUSIONS: In this study, we detected LPS in plasma of individuals exposed to high levels of LPS at their workplace. This finding is supported by elevated levels of several inflammatory cytokines among the workers, significantly exceeding that of the non-exposed control group. To the best of our knowledge, this is the first time that plasma LPS, together with increased inflammatory markers in plasma, has been detected in an occupational setting.

Tumour necrosis factor alpha and its promoter polymorphisms' role in the phenotypic expression of hemochromatosis.

BACKGROUND: The majority of hemochromatosis patients are homozygous for the HFE-C282Y mutation. However, less than half of C282Y homozygous subjects identified by population screening studies actually develop the disease. The cytokine TNF-alpha is implicated in the regulation of iron metabolism at different levels. Our aim was to study the role of TNF-alpha and its promoter polymorphisms in the phenotypic expression of hemochromatosis in individuals with and without the C282Y mutation. METHODS: We studied 4 groups of 10 subjects each: (1) C282Y homozygotes without clinical hemochromatosis; (2) C282Y homozygotes with hemochromatosis; (3) secondary hemochromatosis (without C282Y mutation); and (4) controls. Groups were age-matched and sex-matched. Peripheral blood mononuclear cells (PBMC) were stimulated with lipopolysaccharide (LPS) and the release of TNF-alpha was measured. Additionally, the G/A polymorphisms at position -238 and -308 of the TNF-alpha, gene were determined by PCR and RFLP analysis in 178 hemochromatosis patients and 41 controls. RESULTS: TNF-alpha production from PBMC at 8 and 24 h after increasing concentrations of LPS stimulation were similar in the four groups. The prevalence of TNF-alpha polymorphisms was similar in patients and controls. The prevalences of cirrhosis, siderosis, median s-ferritin and median ALT values were similar in patients with and without the TNF-alpha polymorphisms. CONCLUSIONS: Neither TNF-alpha, released from PBMC nor the presence of TNF-alpha polymorphisms seem to be associated with disease manifestation in hemochromatosis.

Flow cytometric evaluation of apoptosis, necrosis and recovery when culturing monocytes.

After developing and applying a method for cryopreserving monocytes, we found a substantial cell loss when culturing these cells. Monocytes were isolated from blood donors by density gradient centrifugation, purified by elutriation and cryopreserved. Thawed cells were cultured in ultra low attachment wells and studied with Annexin V, Propidium iodide, Dihexyloxacarbocyanine (DiOC(6)(3)), bromolated deoxyuridine triphosphate nucleotides (Br-dUTP), DNA ploidy and DNA ladder methodologies. The main cell loss was within the first 24 h and recovery on day 7 was 35-40%. The first 2-6 h of culture were found to be crucial for determining which cells survive. Initially (2-4 h), apoptosis was the main feature but after 6 h, necrosis dominated. Two populations of cells developed after 24 h: "A" consisting of larger cells with low levels of apoptosis and necrosis signals and population "B" comprising smaller cells with a high expression of necrotic but low levels of apoptotic signals. Signs of DNA fragmentation were slight. These early, dynamic changes may be important for the interpretation of experimental results when investigating monocytes in culture.

Non-mannose-capped lipoarabinomannan stimulates human peripheral monocytes to expression of the "early immediate genes" tissue factor and tumor necrosis factor-alpha.

In the present study, we have shown that stimulation of cryopreserved, human peripheral blood monocytes with the cell wall components from Gram-negative bacteria, lipopolysaccharide (LPS), and from rapid-growing Mycobacterium sp., non-mannose-capped lipoarabinomannan (AraLAM), both induce expression of the "early immediate genes" tissue factor (TF) and tumor necrosis factor-alpha (TNF-alpha). This was demonstrated both at the protein and the mRNA levels. Antibodies against the CD14 receptor could block the stimulating effects. AraLAM was a significantly weaker inducer than LPS, and we speculate that this may reside in the number of the fatty acids in the part of the molecule that interacts with the CD14/Toll-like receptors (TLR). Finally, both LPS and AraLAM activated the "early immediate genes" through translocation of the transcription factor proteins NF-kappaB/Rel and increasing the binding activity of AP-1.

Aspirin potentiates LPS-induced fibrin formation (FPA) and TNF-alpha-synthesis in whole blood.

The effect of aspirin on LPS-incubation of whole blood was investigated. Aspirin induced a concentration dependent increase (2.5-5-fold at 5 mM aspirin) in LPS-induced appearance of TNF-alpha and fibrinopeptide A (FPA) in plasma, despite the concomitant increase in the inhibitory cytokine IL-100. Aspirin substantially raised the levels of LPS-induced TF-mRNA and TNFalpha-mRNA in monocytes isolated from whole blood. The median ratio for TF-/beta-actin mRNA increased from 1.5 +/- 0.44 in the presence of LPS-alone, to 2.5 +/- 0.51 when 5 mM aspirin was added. The TNFalpha/beta-actin mRNA ratios were 1.8 +/- 0.4 and 5.5 +/- 2.7 respectively. Addition of exogenous PGE2 before incubation nearly abrogated the effect of aspirin on TNF-alpha, substantiating the role of PGE2 as a regulator of TNF-alpha synthesis, whereas the effect on FPA was small. Thus, in the presence of LPS in this whole blood model, aspirin apparently had a pro-inflammatory rather than an anti-inflammatory effect.