Philippe Cauchie

Anti-hemostatic Effects of a Serpin from the Saliva of the Tick Ixodes ricinus

Serpins (serine protease inhibitors) are a large family of structurally related proteins found in a wide variety of organisms, including hematophagous arthropods. Protein analyses revealed that Iris, previously described as an immunomodulator secreted in the tick saliva, is related to the leukocyte elastase inhibitor and possesses serpin motifs, including the reactive center loop (RCL), which is involved in the interaction between serpins and serine proteases. Only serine proteases were inhibited by purified recombinant Iris (rIris), whereas mutants L339A and A332P were found devoid of any protease inhibitory activity. The highest Ka was observed with human leukocyte-elastase, suggesting that elastase-like proteases are the natural targets of Iris. In addition, mutation M340R completely changed both Iris substrate specificity and affinity. This likely identified Met-340 as amino acid P1 in the RCL. The effects of rIris and its mutants were also tested on primary hemostasis, blood clotting, and fibrinolysis. rIris increased platelet adhesion, the contact phase-activated pathway of coagulation, and fibrinolysis times in a dose-dependent manner, whereas rIris mutant L339A affected only platelet adhesion. Taken together, these results indicate that Iris disrupts coagulation and fibrinolysis via the anti-proteolytic RCL domain. One or more other domains could be responsible for primary hemostasis inhibition. To our knowledge, this is the first ectoparasite serpin that interferes with both hemostasis and the immune response.

Ir-CPI, a coagulation contact phase inhibitor from the tick Ixodes ricinus, inhibits thrombus formation without impairing hemostasis.

Blood coagulation starts immediately after damage to the vascular endothelium. This system is essential for minimizing blood loss from an injured blood vessel but also contributes to vascular thrombosis. Although it has long been thought that the intrinsic coagulation pathway is not important for clotting in vivo, recent data obtained with genetically altered mice indicate that contact phase proteins seem to be essential for thrombus formation. We show that recombinant Ixodes ricinus contact phase inhibitor (Ir-CPI), a Kunitz-type protein expressed by the salivary glands of the tick Ixodes ricinus, specifically interacts with activated human contact phase factors (FXIIa, FXIa, and kallikrein) and prolongs the activated partial thromboplastin time (aPTT) in vitro. The effects of Ir-CPI were also examined in vivo using both venous and arterial thrombosis models. Intravenous administration of Ir-CPI in rats and mice caused a dose-dependent reduction in venous thrombus formation and revealed a defect in the formation of arterial occlusive thrombi. Moreover, mice injected with Ir-CPI are protected against collagen- and epinephrine-induced thromboembolism. Remarkably, the effective antithrombotic dose of Ir-CPI did not promote bleeding or impair blood coagulation parameters. To conclude, our results show that a contact phase inhibitor is an effective and safe antithrombotic agent in vivo.

Sleep apnoea-hypopnoea index is an independent predictor of high-sensitivity C-reactive protein elevation.

Background: Recent reports have identified the apnoea and hypopnoea index (AHI) as an additional independent risk factor for cardiovascular morbidity and mortality. However, several studies reported contradictory results about the association between the serum C-reactive protein (CRP) level and the severity of apnoea. Objective: The purpose of this work is to study this association in patients referred to the sleep laboratory for clinical suspicion of sleep apnoea and presenting a wide range of AHI. Methods: Forty-nine consecutive patients were included in the study. The SigmaStat® software package (Jandle Scientific) was used. Multilinear regression analysis was tested using a stepwise backward selection of the explicative variables. The clinical characteristics (diabetes, hypertension, smoking habits, gender) were treated as dichotomous variables, while all other data (age, BMI, lipids, white blood cells) were continuous ones; high-sensitivity (hs)-CRP was the dependent variable. Results: In univariate analysis, AHI was correlated to hs-CRP: R = 0.43, p = 0.002. In multivariate analyses, we found an independent association between the AHI, adjusted for classical cardiovascular risk factors, and hs-CRP. Conclusion: In a sample of 49 patients, referred to the sleep laboratory for suspicion of sleep apnoea in routine practice, we observed an independent association between the AHI and hs-CRP.

Relationship between CRP and hypofibrinolysis: Is this a possible mechanism to explain the association between CRP and outcome in critically ill patients?

Background-Endothelial cell dysfunction may be implicated in the development of multiple organ failure (MOF) by a number of mechanisms. Among these, altered fibrinolysis promotes fibrin deposition, which may create microvascular alterations during inflammation. Elevated concentrations of C-reactive protein (CRP), especially when these persist over time, are correlated with an increased risk of MOF and death. CRP may inhibit fibrinolysis by inducing plasminogen activator inhibitor-1 (PAI-1) release from human aortic endothelial cells. Moreover, the administration of recombinant CRP in volunteers may increase circulating PAI-1 levels.In this study, we tested the hypothesis that CRP is associated with hypofibrinolysis in intensive care patients with and without sepsis.Methods-We studied the association of inflammation and abnormal fibrinolysis in intensive care unit (ICU) patients with (n = 11) and without (n = 21) sepsis. The inflammatory response was assessed by serum concentration of C-reactive protein (CRP), a marker of the acute phase reaction, which increase rapidly in the inflammatory response, and the plasma fibrinolytic capacity was evaluated by the Euglobulin Clot Lysis Time (ECLT), determined by a new semi-automatic method.Results-ECLT was significantly higher in septic than non-septic patients (1104 ± 439 vs 665 ± 275 min; p = 0.002) and was significantly correlated with CRP concentration (R2 = 0.45; p < 0.001). In a multivariate analysis, CRP was the strongest predictor of ECLT (R2 = 0.51, F = 25.6, p < 0.001). In addition, the overall ICU length of stay was significantly correlated with CRP (R2 = 0.264, p = 0.003) and ECLT (R2 = 0.259, p = 0.003).Conclusion-In critically ill patients a significant correlation thus exists between plasma fibrinolytic capacity and serum CRP levels. Our data were obtained in the first 24 hours of ICU admission or of sepsis, thus, the relation between CRP and hypofibrinolysis appeared very quickly. This finding is compatible with a link between inflammation and abnormal fibrinolysis, and may explain the negative prognostic value of CRP in critically ill patients.

Plasma fibrinolysis is related to the degree of organ dysfunction but not to the concentration of von Willebrand Factor in critically ill patients

BackgroundEndothelial cell dysfunction, by promoting fibrin deposition, has been implicated in the development of multiple organ failure. Altered fibrinolysis during inflammation may participate in microvascular alterations. We sought to determine whether plasma fibrinolysis was related to the severity of organ dysfunction and/or to the levels of von Willebrand factor (vWF antigen), as a marker of endothelium dysfunction, in critically ill patients.MethodsForty-nine consecutive patients admitted to an adult medico-surgical intensive care unit (ICU) with (18) or without sepsis (31) were included. C-reactive protein and vWF levels were measured on ICU admission and plasma fibrinolysis was assessed by the Euglobulin Clot Lysis Time (ECLT). The sequential organ failure assessment (SOFA) score and the simplified acute physiology score (SAPS) II were calculated on admission.ResultsECLT was significantly longer in septic than in non-septic patients [1033 min (871–1372) versus 665 min (551–862), p = 0.001]. There were significant correlations between ECLT and C-reactive protein (CRP) concentrations (r = 0.78, p < 0.001) and the Sequential Organ Failure Assessment (SOFA) score (r = 0.39, p = 0.006). The level of vWF was not correlated with the ECLT (r = -0.06, p = 0.65) or the SOFA score (r = -0.02, p = 0.88).ConclusionECLT measurement at admission could be a marker of organ dysfunction and a prognostic indicator in critically ill patients.

Monocyte-platelet complexes on CD14/CD16 monocyte subsets: relationship with ApoA-I levels. A preliminary study

The adhesion of the monocytes to the endothelium and their extravasation into the intima are key steps in atherogenesis. Studies showed the essential role of L-selectin (CD62-L), expressed by the monocytes, and the platelets by forming complexes with monocytes. The delipided apolipoprotein (Apo) A or high-density lipoprotein (HDL) has antiinflammatory effects on monocytes and can bind platelets (monocyte-platelet complexes [MPCs]). The aim of this study was to identify a possible relationship between the MPCs, the monocyte subset, and ApoA-I/HDL serum levels in vivo. Platelet-monocyte complexes were estimated by flow cytometry in 16 volunteers. Monocyte-platelet interaction was characterized by the percentage of monocytes coexpressing the constitutive platelet marker, glycocalicin gpIb-alpha (CD42b; CD42b+monocytes in %, MPC%). Monocytes were divided into four subsets based on lipopolysaccharide receptor (CD14) and FcgammaIII receptor (CD16) expression (CD14++/CD16-, G1; CD14++/CD16+, G2; CD14+/CD16-, G3; and CD14+/CD16+, G4). HDL and ApoA-I levels were measured by routine laboratory techniques. MPC% in the different subsets were G1=8.1+/-3.4%, G2=21.2+/-14%, G3=18+/-12.6%, and G4=22.3+/-14.3% (analysis of variance: P<.001). MPC% in the entire monocyte population was negatively correlated to ApoA-I (R=-0.71, P=.001). The relationship between ApoA-I and MPC% was found mainly in the subsets G1 (R=-0.67, P=.001) and G2 (R=-0.61, P=.01). MPC% was not correlated with any other lipids or lipoprotein or high-sensitivity C-reactive protein. When whole blood was incubated with HDL/ApoA-I, no modification of platelet CD42b fluorescence was observed, indicating that there is no direct interaction between the HDL/ApoA-I and the CD42b fluorescence. Among the monocytes, the G2 subset appeared to have the highest extravasation potential. Indeed, we previously showed that those cells overexpressed CD62-L, and we observed in this work that they were coated with platelets more than the G1 cells. The G2 subset could be more directly involved in the development of atherosclerotic lesions.

Time Course of CD64, A Leukocyte Activation Marker, During Cardiopulmonary Bypass Surgery.

ABSTRACT Distinction between inflammation secondary to surgery, especially coronary artery bypass graft with cardiopulmonary bypass (CPB), and inflammation due to infection is difficult in surgical intensive care unit (ICU) patients. Development of biomarkers of infection could help clinicians in the early identification and thus treatment of sepsis in these patients. We compared the time course of the neutrophil CD64 index, a high affinity immunoglobulin FC &ggr; receptor I whose expression is increased in bacterial infection, in 39 patients undergoing cardiac surgery with CPB and 11 patients admitted to the ICU with severe sepsis or septic shock. The CD64 index was significantly more elevated in septic patients than in patients who had CPB except at day 5. The CD64 index increased moderately on day 1 after cardiac surgery but the value remained lower than in septic patients. The duration for which the CD64 index was greater than 1.0 was longer in septic than in CPB patients. Receiver operating curves to differentiate CPB from sepsis on day 1 were not significantly different between C-reactive protein (CRP) concentrations and CD 64 index. Nevertheless, combination of low CD64 index with low CRP concentrations on day 1 ruled out sepsis except in three patients. There were no correlations between the CD64 index and cytokine levels (tumor necrosis factor [TNF]-&agr;, interferon [IFN]&ggr;, interleukin [IL]-6, IL-10, IL-8, IL-12) measured in subpopulations. In conclusion, CD64 index only in combination with CRP concentrations could be used to discriminate inflammation due to surgery from that due to infection in this particular population.

Surface Membrane Immunoglobulin Expression in Chronic Lymphocytic Leukemia

Dr. D. Brohée, Department of Internal Medicine, CHU Vésale, rue de Gozée, 706, B-6110 Montigny-le-Tilleul (Belgium) Chronic lymphocytic leukemias (CLL) can be classified on morphological grounds as classical (less than 11% of prolymphocytes), mixed cell types (CLL/PL, 11 to 55% of prolymphocytes), and prolymphocytic (PL, more than 55% of prolymphocytes) [1, 2]. It is currently presented that PL lymphocytes express surface membrane immuno-globulins (Sm Ig) very brightly in contrast to the weak expression on CLL lymphocytes [1-7]. In most of the cases, Sm IgM alone or, less frequently, Sm IgM and IgD are expressed [5, 6]. It has been suggested that kappa chains were expressed more often [1,4,5,8]. However, analysis of positivity has relied mainly on fluorescence microscopy and subjective quantitation. We used a FACScanTM 488 nm argon flow cytometer (Becton Dickinson) to determine antigen density on a semiquantitative basis, in 20 consecutive patients (M/F 13:7, mean age 74 years) observed in our community hospital. The data were acquired in list-mode files using the SimultestTM software after automatic setting of the cytometer with the AutoCompTM software. Further analyses were performed with the LysisTM software. Gate accuracy was checked with Simultest LeucoGATETM anti-CD45 and CD14 monoclonal antibodies. The negative control was determined using Simultest Control γl/γ2aTM irrelevant antibodies. For Sm Ig determination, the following antibodies were used: Simultest Anti-Kappa (FITC)⁄Anti-Lambda (PE)TM after Fcreceptor blocking with rabbit serum, FITC-labelled antihuman IgG, IgA, IgD and IgM, and in some selected cases, PE or FITC-labelled anti-kappa or anti-lambda Fab’2 rabbit fragments (DAKO). Fluorescence intensity was quantified in arbitrary units (AV) on a scale from 1 to 10,000 (256 channels). The number of positive cells was expressed as the percentage of lymphocytes with a fluorescence intensity above the negative control (typically above 10 AU). Raw data are displayed in table 1. In only one case was no Ig determinant found (5%). In contrast with previous studies [1,4,5,8], but in good keeping with a recent one [9], we observed a lambda predominance (55%). No Sm IgG or IgA were detected. Heavy chain expression was remarkable, in contrast with the current opinion, showing IgM alone in 10%, IgD alone in 25%, both IgD and IgM in 45%, and none in 20%. If we applied a threshold of 20% for positivity, the corresponding figures would be 5, 40,15 and 40%. To our knowledge, this is the first report demonstrating that Sm IgD are the most frequently expressed im-munoglobulins (at least in 55%, and 70% without threshold restriction). It should be noticed that the percentages of positive cells