Upregulation of cytokine mRNA in circulating leukocytes during human endotoxemia.

Endotoxemia induces pronounced changes in leukocyte count and enhances the release of many cytokines. However, the molecular regulation of this cytokine release is poorly characterized in humans. The time course of mRNA expression of 24 cytokines in circulating leukocytes was studied in a well-standardized model of human endotoxemia (2 ng/kg). Real-time polymerase chain reaction (RT-PCR) was used to quantify the lipopolysaccharide (LPS)-inducible mRNA levels of leukocytes from 16 healthy volunteers in a randomized, placebo-controlled trial. Baseline mRNA levels of interleukins including IL-1α, IL-3, IL-5, IL-6, IL-12p40, IL-13, IL-15, IL-17, granulocyte colony-stimulating factor (G-CSF) and granulocyte monocyte CSF (GM-CSF) were below detectable levels in normal blood of the healthy participants. After 2 h, LPS infusion increased median mRNA levels of IL-1α by >1100-fold and IL-1ß and IL-8 by 33-fold and 46-fold, respectively. In contrast, levels of tumor necrosis factor (TNF-α) and IL-10 mRNA increased by only 7-fold, whereas changes in mRNA expression of other cytokines showed either a more than two fold increase or were undetectable. In vitro incubation of whole blood with 50 pg/mL LPS for 2 h enhanced transcription levels of IL-1α mRNA by >10,000-fold, IL-6 and IL-12p40 by >1000-fold, IL-1ß by 400-fold, TNF-α by 100-fold, IL-8, IL-18, interferon γ (IFN-γ) and G-CSF by >10-25-fold, and IL-10, IL-12p35, TNF-ß, and IL-13 by 10-25-fold. Only half of the 24 evaluated cytokines were expressed at the mRNA level in circulating leukocytes under basal conditions and after an LPS challenge. Only IL-1α, IL-1ß, IL-10, IL-8, and TNF-α were upregulated in the circulating leukocytes, whereas several other cytokines (including IL-6 and G-CSF), were expressed on the mRNA level following in vitro incubation of blood with LPS. In addition, IL-1α and IL-1ß might be potential diagnostic targets in inflammatory diseases.

Mean platelet volume may represent a predictive parameter for overall vascular mortality and ischemic heart disease.

OBJECTIVE: An increased mean platelet volume (MPV), as an indicator of larger, more reactive platelets resulting from an increased platelet turnover, may represent a risk factor for overall vascular mortality, including myocardial infarction. We intended to identify patients at higher risk of dying from vascular disease in a large, hospital-based cohort. METHODS AND RESULTS: A total of 206 554 first-ever admissions to the Allgemeines Krankenhaus Wien for determination of MPV between January 1996 and July 2003 were included. Primary end points were overall vascular mortality and death due to ischemic heart disease. Multivariate Cox regression adjusted for sex, age, and platelet count was applied for analysis. MPV values were categorized into quintiles, with the lowest quintile serving as the reference category. Compared with individuals with lower MPV (<8.7 fL), hazard ratios for overall vascular mortality gradually increased to 1.5 in the highest category (≥11.01 fL). The relationship of MPV to ischemic heart disease was even stronger and increased from 1.2 (8.71 to 9.60 fL category) to 1.8 in the highest category (≥11.01 fL). CONCLUSIONS: Our results indicate that patients with an increased MPV (≥11.01 fL) are at higher risk of death due to ischemic heart disease, with hazard ratios comparable to those reported for obesity or smoking.

Effects of aspirin and NO-aspirin (NCX 4016) on platelet function and coagulation in human endotoxemia.

Acetylsalicylic acid (ASA) prevents thromboembolic events by inhibiting platelet function through blocking of cyclooxygenase type 1 (COX-1). A nitroderivate of ASA, 2-(acetyloxy)benzoic acid 3-(nitrooxymethyl)-phenyl ester (NCX 4016) was synthesized, which additionally acts through nitric oxide release. In various in vitro and animal studies NCX 4016 exhibited antithrombotic and anti-platelet properties. We used the standardized model of endotoxin infusion into human volunteers to compare the effects of NCX 4016 and ASA on platelet function and TF-induced coagulation activation. The trial consisted of two parts. In the first part, 10 healthy male volunteers were included in a randomized, open cross-over trial to find a NCX formulation with optimal tolerability and pharmacokinetic data were obtained. The second part was a randomized, double blind placebo controlled clinical trial consisting of 30 healthy male volunteers in three parallel groups (n = 10 per group). Volunteers received either NCX 4016 (800 mg b.i.d.), ASA (425 mg b.i.d.) or placebo for 7 days, before infusion of 2 ng/kg endotoxin on day 8. ASA attenuated the endotoxin-induced platelet plug formation (measured by PFA-100) significantly better than NCX 4016 and placebo (p < 0.004), while there was no difference in soluble P-selectin or VWF-levels. Urine 11-dehydro-thromboxane B(2) levels were significantly lower in the ASA and NCX 4016 groups as compared to placebo (p < 0.05). Neither ASA nor NCX 4016 significantly changed prothrombin fragment(1 + 2), D-Dimer or tissue factor (TF)-mRNA levels. In summary, NCX 4016 had no effect on VWF release, platelet activation as measured by soluble P-selectin or TF gene expression. NCX 4016, at the dose tested, unlike ASA, had no effect on platelet collagen/epinephrine induced plug formation under high shear rates.

Coagulation factor VIII levels are associated with long-term survival - interactions with gender in a large hospital-based cohort.

INTRODUCTION: Elevated coagulation factor VIII activity has been associated with increased risk for both venous and arterial thrombosis. The current study evaluated the influence of Factor VIII levels and interactions with gender on all cause mortality in a large Austrian cohort. PATIENTS AND METHODS: During 1991 and 2003, 11203 individuals, first ever request for laboratory analyses of FVIII: C, age > or =18 years, were included in this study. The median observation period was 5 years covering a total of 46000 person-years. The death rate was 17.1%. RESULTS: Compared to individuals within the reference category (FVIII: C <94%) hazard ratios gradually increased from 1.4 (95% CI: 1.1-1.8) in the 152-170% category (5th decile) to finally 4.4 (95% CI: 3.5-5.5) in the >313% category (highest decile, all p < 0.05). The association between FVIII: C levels and mortality remained essentially unchanged when considering non-cancer mortality, all cause vascular mortality or mortality due to ischemic heart disease. Compared to males females with elevated FVIII: C had a worse outcome resulting in higher hazard ratios reaching 6.8 (95% CI: 4.6-9.9) within the highest decile compared to males (HR: 3.4 (95% CI: 2.6-4.5)). CONCLUSIONS: In our large patient cohort we might be able to demonstrate for the first time that FVIII: C plasma activity is strongly associated with all cause mortality. Additionally, FVIII: C appears to interact with gender. Especially in women FVIII: C might help identifying high-risk cohorts, which might benefit from individualized prevention strategies.

The EGF 61A/G polymorphism - a predictive marker for recurrence of liver metastases from colorectal cancer.

BACKGROUND: Epidermal growth factor (EGF) plays an important role in tumorigenesis. Variations in the DNA sequence of the gene EGF can lead to alterations in EGF activity, which is suspected to influence tumor progression. This retrospective study aimed to investigate the influence of EGF 61A/G polymorphism on the recurrence of liver metastases after hepatic surgery in patients with colorectal cancer. METHODS: EGF 61A/G polymorphism was determined in 268 consecutive patients (175 [65%] men and 93 [35%] women, mean age 62 +/- 10.3 years) who had liver metastases at primary diagnosis and were treated by surgery with curative intent (R0) for liver metastases from colorectal cancer. RESULTS: Overall, 81 of 268 (30%) patients exhibited wild-type EGF 61 A/A, 137 (51%) were heterozygous EGF 61 A/G and 50 (19%) were homozygous EGF 61 G/G. After adjusting for age, sex, UICC stage and tumor location, we observed a trend-wise 1.6-fold increased risk for hepatic recurrence (HR 1.6; 95% CI 1.0-2.5, P = 0.06) in individuals with the G/G genotype compared with carriers of the A-allele. The effect was much more pronounced in younger patients (or= 65 years). Interestingly, male patients with EGF G/G had a 1.6-fold higher risk of recurrence (HR 1.6; 95% CI 1.0-2.5, P = 0.07). A significant correlation (P = 0.033) was detected between Dukes classification and the homozygous 61 G/G genotype. CONCLUSION: Despite the limitations of our study, the retrospective results indicate that carriers of the EGF polymorphism might be at higher risk of developing liver recurrences. If confirmed in subsequent studies, genotyping for the EGF A/G variant might help in identification of patients at high risk of recurrence of liver metastases.

The inflammatory response is influenced by FXIII VAL 34 LEU polymorphism in a human LPS model.

BACKGROUND: In sepsis, activation of coagulation and inhibition of fibrinolysis lead to microvascular thrombosis. The Val34Leu polymorphism affects the function of FXIII by increasing the rate of FXIII activation by thrombin, which results in an increased and faster rate of fibrin stabilization. Sepsis and multi-organ failure cause disturbance of the normal balance of inflammation and coagulation, one of the most frequent causes of death in ICU patients. Research in polymorphism has shown the possible influence of FXIII in coagulation and inflammation. METHODS: We analyzed the influence of the common FXIII Val34Leu polymorphism on inflammatory and coagulation parameters in human experimental endotoxinemia. Healthy volunteers (n = 62) received 2 ng endotoxin (LPS) per kg body weight as a bolus infusion over 2 min. We developed a new mutagenic separated PCR assay for determination of the FXIII promoter polymorphism. RESULTS: FXIII levels were higher for homozygous carriers of the FXIII polymorphism in comparison with wild-type 34 Val/Val and heterozygous 34 Val/Leu. Interestingly, persons homozygous for the FXIII Val34Leu polymorphism had lower monocyte and neutrophil counts throughout the observation period, yet prothrombin fragment 1+2 and D-dimer levels did not differ after LPS challenge. CONCLUSION: Our findings indicate that the common FXIII Val34Leu polymorphism is associated with differences in monocyte and neutrophil cell counts in response to systemic LPS infusion in humans.

Multi-hit inhibition of circulating and cell-associated components of the toll-like receptor 4 pathway by oxidized phospholipids.

OBJECTIVE: Oxidized phospholipids (OxPLs) that are abundant in atherosclerotic lesions are increasingly recognized as context-dependent lipid mediators demonstrating both pro- and antiinflammatory activities. Molecular mechanisms of their effects are largely unknown. Here we present novel information on the mechanisms whereby OxPLs modulate activation of TLR4 by lipopolysaccharide (LPS). METHODS AND RESULTS: We show, using several cell types and various inflammatory genes as readouts, that different classes and molecular species of OxPLs do not stimulate TLR4 but exert prominent inhibitory effects on LPS-induced reactions. Our data demonstrate that binding of OxPLs to the LPS-binding protein (LBP) and CD14 prevents recognition of LPS by these proteins, thus impairing activation of TLR4. In addition, OxPLs inhibited LBP- and CD14-independent activation of TLR4 by the synthetic TLR4 agonist E6020 indicating that in parallel with LBP and CD14, OxPLs target cell-associated steps in TLR4 cascade. CONCLUSIONS: Our data suggest that OxPLs inhibit action of LPS via a multi-hit mechanism. These results support the notion that OxPLs are endogenous inhibitors of TLR4 produced in response to oxidative stress.

Differentiation of nonalcoholic from alcoholic steatohepatitis: are routine laboratory markers useful?

BACKGROUND/AIMS: Specific markers for differentiation of nonalcoholic (NASH) from alcoholic steatohepatitis (ASH) are lacking. We investigated the role of routine laboratory parameters in distinguishing NASH from ASH. METHODS: Liver biopsies performed at our hospital over a 10-year period were reviewed, 95 patients with steatohepatitis identified and their data prior to biopsy reevaluated. The diagnosis NASH or ASH was assigned (other liver diseases excluded) on the basis of the biopsy and history of alcohol consumption (< 140 g/week). Logistic regression models were used for analysis. RESULTS: NASH was diagnosed in 58 patients (61%; 30 f) and ASH in 37 (39%; 9 f). High-grade fibrosis (59% vs. 19%, P < 0.0001) and an AST/ALT ratio > 1 (54.1% vs 20.7%, P = 0.0008) were more common in ASH. The MCV was elevated in 53% of ASH patients and normal in all NASH patients (P < 0.0001). Multivariate analysis identified the MCV (P = 0.0013), the AST/ALT ratio (P = 0.011) and sex (P = 0.0029) as relevant regressors (aROC = 0.92). The AST/ALT ratio (P < 0.0001) and age (P = 0.00049) were independent predictors of high-grade fibrosis. Differences in MCV were more marked in high-grade fibrosis. CONCLUSIONS: Higher MCVs and AST/ALT ratios in ASH reflect the severity of underlying liver disease and do not differentiate NASH from ASH. Instead, these biomarkers might prove useful in guiding selection of patients for liver biopsy and in targeting therapy.

Genetic variation in heme oxygenase 1 (HMOX1) and the risk of recurrent venous thromboembolism.

BACKGROUND/OBJECTIVE: Products of heme oxygenase 1 (HO1) possess antithrombotic properties, and impairment of HO1 activity may contribute to thrombus formation. Transcriptional activity of long GT-repeat alleles in HO1 gene (HMOX1) is lower as compared with short alleles. We hypothesize that these long alleles are associated with decreased HO1 anticoagulant activity and, thus, an increased risk of thrombosis.. DESIGN/METHODS: In a prospective cohort study, we followed 860 patients with a first VTE, and investigated the impact of a promoter GT-dinucleotid length polymorphism in HOMX1 on the risk of recurrent venous thromboembolism (VTE). RESULTS: Allele groups short (S), medium (M) and long (L) of the promoter GT-dinucleotide length polymorphism were distinguished. L-alleles, but not M- or S-alleles, were found to be more frequent among patients with recurrence. Heterozygous carriers of L-alleles had a two-fold higher relative risk of recurrence [(RR 2.2 (95% CI: 1.4-3.4)] as compared to wild type, which was independent of other thrombotic risk factors. At five years, the cumulative probability of recurrence was 18% (95% CI: 15%-22%) in patients without an L-allele compared to 32% (95% CI: 19%-46%) in patients heterozygous for the L-allele (P = .001). CONCLUSION: Patients with first VTE and long GT-repeat alleles in HMOX1 have an increased risk of recurrence. Genetically determined alterations in HO1 function may represent a new pathomechanism in VTE.

C-reactive protein and all-cause mortality in a large hospital-based cohort.

BACKGROUND: C-reactive protein (CRP), an acute-phase protein, is a sensitive systemic marker of inflammation and acute-phase reactions. Testing CRP concentrations at hospital admission may provide information about disease risk and overall survival. METHODS: All first-ever transmittals to the department of medical and chemical laboratory diagnostics for determination of low-sensitivity CRP (n = 274 515, 44.5% male, median age 51 years) between January 1991 and July 2003 were included [median follow-up time: 4.4 years (interquartile range, 2.3-7.4 years)]. The primary endpoint was all-cause mortality. Multivariate Cox regression adjusted for sex and age was applied for analysis. RESULTS: Compared to individuals within the reference category (CRP <5 mg/L), hazard ratios (HR) for all-cause mortality increased from 1.4 (5-10 mg/L category) to 3.3 in the highest category (>80 mg/L, all P <0.001). CRP was associated with various causes of death. The relation of CRP to cancer death was stronger than to vascular death. Younger patients with increased CRP had relatively far worse outcome than older patients (maximal HR: < or =30 years: 6.7 vs >60 years: 1.7-3.7). Interestingly, both short- and long-term mortality were associated with increasing CRP concentrations (>80 mg/L: HR 22.8 vs 1.4). CONCLUSION: Measurement of low-sensitivity CRP at hospital admission allowed for the identification of patients at increased risk of unfavorable outcome. Our findings indicate that close attention should be paid to hospitalized patients with high CRP not only because of very substantial short-term risk, but also long-term excess risk, the basis for which needs to be determined.

Intra-arterial vitamin C prevents endothelial dysfunction caused by ischemia-reperfusion.

OBJECTIVE: Ischemia-reperfusion (IR) injury causes tissue injury and endothelial dysfunction. There is evidence that oxidative stress plays an important role. METHODS: We tested if IR-induced endothelial dysfunction could be prevented by administration of the antioxidant vitamin C. Twenty-six healthy male subjects and eight male patients with peripheral arterial disease (PAD) were enrolled in this randomised placebo-controlled study. Forearm blood flow (FBF) measurements in response to the vasodilators acetylcholine (ACh; endothelium-dependent agonist) or nitroglycerin (NTG; endothelium-independent) were performed before and after forearm ischemia for 20 min. FBF responses were reassessed during reperfusion with intra-arterial co-administration of 24 mg/min vitamin C or placebo. In six volunteers responses to the NO-synthase inhibitor N-monomethyl-L-arginine (L-NMMA) were also assessed before and after ischemia with and without vitamin C. RESULTS: ACh-induced vasodilation was blunted in subjects receiving placebo after reperfusion (p<0.05 versus baseline). Administration of vitamin C completely prevented impaired responsiveness. NTG-induced vasodilation was not affected by reperfusion or vitamin C. This finding was consistent in patients with PAD and impaired endothelial function, where local vitamin C infusion restored FBF reactivity to ACh before and after IR injury (p<0.05 versus baseline). Again, NTG-induced vasodilation was not affected. Blunted L-NMMA responses seen during reperfusion could be completely reversed by vitamin C. CONCLUSIONS: Our data indicate that IR-induced vascular injury can be prevented by administration of antioxidants.

S-ibuprofen effectively inhibits thromboxane B2 levels and platelet function in an experimental model of lipopolysaccharide-stimulated and non-stimulated whole blood.

OBJECTIVE: The present study aimed at testing the effect of S- and R-ibuprofen on thromboxane B(2) (TXB(2)), collagen-epinephrine closure time (CEPI-CT) and collagen-adenosine 5'-diphosphate closure time (CADP-CT) in lipopolysaccharide (LPS)-stimulated and non-stimulated human whole blood. MATERIALS AND METHODS: Whole blood was incubated with S- or R-ibuprofen with and without prior stimulation with LPS. To verify ibuprofen's potential effects on TXB(2), varying ratios of concentrations of S- and R-ibuprofen ranging from 0 to 100% were used. TXB(2) levels were measured by ELISA. The effects of S- and R-ibuprofen enantiomers on platelet aggregability were tested utilizing a PFA-100 apparatus. RESULTS: In non-stimulated and LPS-stimulated whole blood, S-ibuprofen markedly decreased TXB(2) levels at concentrations ranging from 10 to 200 microg/ml. R-ibuprofen showed its inhibiting effect at concentrations >100 microg/ml. In inflammatory and non-inflammatory conditions, CEPI-CT was prolonged at concentrations of 12.5 and 75 microg/ml for S-ibuprofen and at a concentration of 150 microg/ml of combined R- and S-ibuprofen. S-ibuprofen was significantly more effective than R-ibuprofen (p < 0.05). The combined use of S- and R-ibuprofen did not additively or synergistically prolong CEPI-CTs. CADP-CTs remained unaffected by both enantiomers. CONCLUSIONS: S-ibuprofen was more effective than the R-ibuprofen enantiomer in inhibiting TXB(2) plasma levels and aggregability of thrombocytes in non-inflammatory and inflammatory conditions.

Daptomycin does not exert immunomodulatory effects in an experimental endotoxin model of human whole blood.

BACKGROUND: Recent studies have shown that distinct classes of antimicrobial agents might exert immunomodulatory effects in experimental settings. Daptomycin is the first member of the class of cyclic lipopeptide antibiotics, which exert their antimicrobial activity via a unique mode of action on the bacterial cytoplasmic membrane. Thus, we tested its ability to influence pro-inflammatory cytokines by use of an established experimental model of human endotoxemia. METHODS: A controlled experimental study design with 4 parallel groups was used. Whole blood from 10 healthy male volunteers was incubated either with saline (negative control), daptomycin (40 microg/ml, control), lipopolysaccharide (LPS; 50 pg/ml, positive control), or the combination of daptomycin plus LPS for 4 h. Real-time polymerase chain reaction was utilized for the measurement of selected pro-inflammatory cytokines, namely IL-1 beta, IL-6 (high sensitivity) and TNF-alpha on the mRNA level. Protein concentrations of these respective cytokines were measured in the supernatant using a commercially available ELISA. RESULTS: Incubation of whole blood with LPS significantly increased protein and mRNA levels of cytokines compared to baseline (p < 0.05). However, the combination of daptomycin plus LPS did not exert any significant effect on mRNA and protein levels of IL-1 beta, IL-6 (high sensitivity) and TNF-alpha after 2 and 4 h of incubation compared to LPS incubation alone. CONCLUSION: Daptomycin does not affect pro-inflammatory cytokines in the early phase of endotoxemia. This is most likely due to the unique mode of action of daptomycin, its low potential to penetrate into human cells and its high affinity to bacterial cytoplasmic membranes.

Immunomodulatory effects of fosfomycin in experimental human endotoxemia.

To evaluate the effect of fosfomycin on proinflammatory cytokines, a bolus of 2 ng of bacterial lipopolysaccharide/kg of body weight was injected intravenously into healthy volunteers. After 2 h, subjects received 8 g of fosfomycin or placebo in a randomized crossover study design. The resulting concentrations of tumor necrosis factor alpha, interleukin-1beta (IL-1beta), and IL-6 expressed as protein and mRNA levels were almost identical with and without fosfomycin.

Gamma glutamyltransferase and long-term survival: is it just the liver?

BACKGROUND: Increased gamma glutamyltransferase (GGT) is associated with cardiovascular disease. To date, however, few studies with sufficient sample size and follow-up have investigated the association of GGT with all-cause mortality. METHODS: The relation of GGT to the risk of death was examined in a cohort of 283 438 first attendants (inpatients or outpatients) of the Vienna General Hospital with request for GGT analysis as part of a routine screening panel and was monitored for up to 13 years. To evaluate GGT as a predictor, Cox proportional hazards models were calculated, which were adjusted for age and sex. RESULTS: In both men and women, GGT above the reference category (GGT > or = 9 U/L in women, > or = 14 U/L in men) was significantly (P <0.001) associated with all-cause, cancer, hepatobiliary, and vascular mortalities. Hazard ratios (HRs) for men and women were similar in all categories. Among patients who presented with GGT above the reference category, those younger than 30 years had higher all-cause mortality rates than did older individuals (HR 1.5-3.3 vs HR 1-1.3 >80 years, respectively). CONCLUSIONS: GGT is associated with mortality in both men and women, especially in patients younger than 30 years, and even high-normal GGT is a risk factor for all-cause mortality.

The fibrinogen -148 C/T polymorphism influences inflammatory response in experimental endotoxemia in vivo.

INTRODUCTION: The acute phase reactant fibrinogen plays a critical role in the coagulation system and inflammation. Recently several polymorphisms have been described regulating basal and peak fibrinogen expression. We evaluated the role of a frequent promoter polymorphism in the beta chain of the fibrinogen gene (-148 C/T) in a human in vivo model of experimental endotoxemia. MATERIAL AND METHODS: Healthy volunteers received 2 ng/kg endotoxin (LPS, n=73) as a bolus infusion over 2 min. Blood samples were collected by venipunctures into EDTA anticoagulated vacutainer tubes before LPS infusion. For determination of the fibrinogen promoter polymorphism, we developed a new mutagenic separated polymerase chain reaction assay. RESULTS: Carriers of the -148 T allele had significantly lower TNFalpha expression throughout the whole time course of LPS stimulation and Interleukin-6 levels were trendwise lower, however only basal levels reached statistical significance. No effects were observed on markers of coagulation activation (D-Dimer, Prothrombin F(1+2)). CONCLUSION: Our findings indicate, that the common -148 C/T polymorphism is associated with differences in the TNFalpha release in response to systemic LPS infusion in humans, and add to current evidence that gene-sequence changes in beta-fibrinogen locus can alter the ability of the host to respond to endotoxin.

The tumor necrosis factor alpha -308 G/A polymorphism does not influence inflammation and coagulation response in human endotoxemia.

Tumor necrosis factor (TNF)-alpha plays a major role in the immune system. Release of proinflammatory cytokines, such as TNF-alpha and interleukin 6, by macrophages and other cells occurs in response to bacterial products. It has been reported that the TNF-alpha -308 G/A polymorphism in the TNF-alpha gene determines basal TNF-alpha levels. We hypothesized that it may also be associated with the degree of inflammatory response in a well-standardized model of systemic inflammation. Eighty-seven young men (age range, 19-35 years) received 2 ng/kg i.v. endotoxin (lipopolysaccharide [LPS]). The TNF-alpha promoter genotype was analyzed on a TaqMan genomic analyzer. Inflammation markers (interleukin 6, TNF-alpha), temperature, and coagulation markers (prothrombin fragment F1+2, D-dimer) were measured at 0, 2, 6, and 24 h after LPS infusion. Tumor necrosis factor-alpha plasma concentrations increased from a baseline 1.3 ng/L (range, 0.8-3.1 ng/L) before LPS infusion to a peak of 57.5 ng/L (range, 10.8-131.4 ng/L) at 2 h after LPS and then decreased continually to 10.8 ng/L (range, 4.7-16.5 ng/L) after 6 h and returned to baseline values after 24 h (1.9 ng/L [range, 1.1-3.9 ng/L]). We observed no significant differences in TNF-alpha baseline levels or in response to LPS after stratification of the data according to TNF-alpha genotype. Basal and peak values of selected inflammatory and coagulation markers were not different between wild-type TNF-alpha -308 individuals (GG) and carriers of the TNF-alpha -308 mutant allele (GA and AA). The TNF-alpha -308 G/A polymorphism does not contribute significantly to the individual variability of systemic TNF-alpha plasma concentrations after endotoxin challenge. Thus, if any, the impact of the TNF-alpha -308 G/A polymorphism on systemic endotoxin-triggered inflammation seems to be limited.

Immunomodulatory effects of fosfomycin in an endotoxin model in human blood.

OBJECTIVES: Although a wide range of therapeutic strategies have been developed to improve the outcome of severe sepsis, a convincing reduction in mortality is lacking. Recently, increasing attention has been paid to immunomodulatory effects of antimicrobials. This study set out to explore the immunomodulatory effects of fosfomycin, a broad-spectrum antibiotic frequently used in septic patients, at the protein and molecular levels in vitro. METHODS: Whole blood from 11 healthy volunteers was incubated with 50 pg/mL endotoxin and 100 microg/mL fosfomycin or physiological sodium chloride for 4 h. Real-time RT-PCR was performed for various pro- and anti-inflammatory cytokines. Concentrations of tumour necrosis factor (TNF)-alpha and interleukin (IL)-6 in the supernatant were measured using a commercially available ELISA. RESULTS: Incubation of human leucocytes with endotoxin increased messenger RNA (mRNA) levels of cytokines several thousand fold compared with baseline. The addition of fosfomycin significantly inhibited mRNA levels of pro-inflammatory cytokines such as IL-1-alpha, IL-6 and TNF-alpha after 2 h (P < 0.01), while no significant reduction was observed for the anti-inflammatory cytokines IL-4, IL-10 and IL-13 (P = 0.26). At the protein level, the concentrations of IL-6 and TNF-alpha increased approximately 3000- and 600-fold after 4 h of incubation with lipopolysaccharide as compared with baseline, respectively. Addition of fosfomycin significantly reduced cytokine levels by 56% and 73% for IL-6 and TNF-alpha, respectively. CONCLUSIONS: Fosfomycin extensively decreased mRNA levels and release of pro-inflammatory cytokines in human blood. The broad antimicrobial coverage of fosfomycin and its immunosuppressive effects could be clinically useful in patients with sepsis.

The C-reactive protein (+)1444C/T alteration modulates the inflammation and coagulation response in human endotoxemia.

BACKGROUND: C-reactive protein (CRP) plays a major role in the immune system and is an independent risk marker of cardiovascular disease. However, CRP's role in atherogenesis as innocent bystander, causative, or even protective agent, remains unresolved. The (+)1444C/T alteration in the CRP gene has been reported to determine basal CRP concentrations. We hypothesized that this alteration may also be associated with the degree of inflammatory response and coagulation activation in a well-standardized model of systemic inflammation. METHODS: We administered 2 ng/kg endotoxin [Escherichia coli bacterial lipopolysaccharide (LPS)] intravenously to stimulate inflammation in 91 healthy young Caucasian male paid volunteers (age range, 19-40 years). Participants were confined to bed rest and fasted for 8.5 h after LPS infusion. We collected blood samples before LPS infusion and at 0, 2, 6, and 24 h after LPS infusion to measure inflammation markers [interleukin 6 (IL6), tumor necrosis factor-alpha (TNFalpha)], temperature, and coagulation markers (prothrombin fragment F(1+2), D-dimer). We analyzed the CRP 3' untranslated variant with a mutagenic separated PCR assay. RESULTS: Basal concentrations of high-sensitivity CRP were approximately 40% lower in (+)1444CC alteration carriers than in T homozygous (TT) allele carriers (P = 0.04). In contrast, basal IL6 concentrations were 2-fold higher in wild-type C homozygous (CC) than in TT individuals (P = 0.01). In response to the LPS challenge, CC individuals had 4-fold higher peak TNFalpha concentrations (P <0.01), >2.5-fold higher peak IL6 concentrations (P <0.01), and increased temperature (P <0.01). Twenty-four hours after LPS challenge, prothrombin fragment F(1+2) concentrations were 75% higher and D-dimer concentrations 50% higher in CC than in TT individuals (P <0.05). CONCLUSIONS: Genetic factors regulating CRP concentrations also modulate the individual response to endotoxin-stimulated inflammation.

Homozygosity in the single nucleotide polymorphism Ser128Arg in the E-selectin gene associated with recurrent venous thromboembolism.

BACKGROUND: The single nucleotide polymorphism (SNP) Ser128Arg in the E-selectin gene is overrepresented in certain patient populations with atherosclerosis or restenosis. As this SNP enhances tissue factor-triggered coagulation in humans during systemic inflammation, we hypothesized that it may also predispose for the development of recurrent venous thromboembolism (VTE). METHODS: A total of 585 patients were prospectively observed after first VTE for recurrent, objectively documented, symptomatic VTE. Patients with secondary VTE, homozygous factor V Leiden, natural inhibitor deficiencies, lupus anticoagulant, or long-term anticoagulation therapy were excluded. The S128R SNP was genotyped by mutagenically separated polymerase chain reaction. RESULTS: A total of 102 patients (17%) were heterozygous, and 11 were homozygous (2%) for the Ser128Arg mutation. Ninety patients (15%) had recurrent VTE during follow-up. Homozygosity for the Ser128Arg SNP increased the cumulative likelihood, particularly for early recurrent VTE (log rank test, P<.05) and was an independent predictor of recurrent VTE (hazard ratio [HR], 4.1; 95% confidence interval [CI], 1.5-11.4) in a multivariate Cox regression model. In contrast, heterozygosity for the polymorphism was associated with an unaltered HR (HR, 1.1; 95% CI, 0.6-1.9) for recurrent VTE. CONCLUSIONS: Homozygosity for the S128R E-selectin allele appears to increase the risk for recurrent VTE several fold. If these findings are confirmed, this may represent a novel risk factor for recurrent VTE. These results also expand our knowledge on the association of this SNP with thrombotic disorders.