Karim Zouaoui Boudjeltia

Alterations of red blood cell shape and sialic acid membrane content in septic patients

ObjectiveTo investigate the relationship between red blood cell (RBC) shape and modifications of RBC membrane protein content in critically ill patients with or without sepsis compared with healthy control volunteers. DesignProspective, observational in vitro study. SettingUniversity-affiliated cell biology laboratory. SubjectsHuman erythrocytes from healthy volunteers and nonseptic and septic intensive care unit patients. InterventionsSialic acid membrane content was measured on isolated RBC membrane proteins by high-performance liquid chromatography. RBC shape, estimated by the spherical index (M2/M1) or by the moment and effect of osmolality on RBC shape, was studied by flow cytometry at 25°C. Glycophorin A content was measured with antiglycophorin antibodies in flow cytometry. Measurements and Main ResultsSialic acid content was lower in the septic than in the nonseptic patients (1.98 ± 0.79, 2.20 ± 0.39 &mgr;g/100 &mgr;g membrane protein, respectively; p = .01) and than in the volunteers (2.71 ± 1.00 &mgr;g/100 &mgr;g membrane protein; p < .001). No significant difference was found in glycophorin A content between septic and nonseptic patients. RBCs from septic patients had a more spherical shape in isotonic solution than those of healthy volunteers, as assessed by a computed spherical index (M2/M1 ratio: 1.68 ± 0.34 vs. 1.95 ± 0.32; p = .001). Only the RBCs of septic patients failed to change their shape in hypo-osmolar solution (M2/M1 ratio: 1.68 ± 0.34 in iso-osmolar, 1.56 ± 0.28 in hypo-osmolar solution; p = .17). There was a significant correlation between the RBC shape evaluated by the spherical index or by the moment of the cytometric histogram and the sialic acid membrane content in all critically ill patients (septic and nonseptic patients) (r2 = .16, p = .01 for the moment, and r2 = .17, p = .01 for the spherical index, respectively). ConclusionsRBCs of septic patients are characterized by a more spherical shape, a decreased capacity of sphericity in hypo-osmolar solution, and a reduction in the sialic acid content of the RBC membrane. These modifications in RBC shape and membrane may contribute to the RBC rheologic abnormalities frequently described in sepsis.

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.

Benefits of napping and an extended duration of recovery sleep on alertness and immune cells after acute sleep restriction.

Understanding the interactions between sleep and the immune system may offer insight into why short sleep duration has been linked to negative health outcomes. We, therefore, investigated the effects of napping and extended recovery sleep after sleep restriction on the immune and inflammatory systems and sleepiness. After a baseline night, healthy young men slept for a 2-h night followed by either a standard 8-h recovery night (n=12), a 30-min nap (at 1 p.m.) in addition to an 8-h recovery night (n=10), or a 10-h extended recovery night (n=9). A control group slept 3 consecutive 8-h nights (n=9). Subjects underwent continuous electroencephalogram polysomnography and blood was sampled every day at 7 a.m. Leukocytes, inflammatory and atherogenesis biomarkers (high-sensitivity C-reactive protein, interleukin-8, myeloperoxidase, fibrinogen and apolipoproteins ApoB/ApoA), sleep patterns and sleepiness were investigated. All parameters remained unchanged in the control group. After sleep restriction, leukocyte and - among leukocyte subsets - neutrophil counts were increased, an effect that persisted after the 8-h recovery sleep, but, in subjects who had a nap or a 10-h recovery sleep, these values returned nearly to baseline. Inflammatory and atherogenesis biomarkers were unchanged except for higher myeloperoxidase levels after sleep restriction. The increased sleepiness after sleep restriction was reversed better in the nap and extended sleep recovery conditions. Saliva cortisol decreased immediately after the nap. Our results indicate that additional recovery sleep after sleep restriction provided by a midday nap prior to recovery sleep or a sleep extended night can improve alertness and return leukocyte counts to baseline values.

High serum galectin-3 in advanced melanoma: preliminary results

Background.  Galectin‐3 (Gal‐3) is a member of the family of β‐galactoside‐binding mammalian lectins, and has been implicated in tumour invasion and metastatic process in vitro and in vivo.

Use of capillary zone electrophoresis for differentiating excessive from moderate alcohol consumption

BACKGROUND The poorly sialylated transferrin isoforms in serum were analyzed by capillary zone electrophoresis (CZE) to differentiate moderate from heavy alcohol consumption. METHODS We enrolled 614 volunteers, classified after interviews, self-reported drinking habits, and AUDIT scores as alcohol abusers (consuming >50 g/day ethanol for the previous 3 months or longer; n = 413) or moderate drinkers (<30 g/day ethanol; n = 201). Serum transferrin isoforms were separated at 28 kV and monitored at 214 nm on a P/ACE 5500 CZE with use of fused-silica capillaries and the related CEofix CDT reagent set. Immunosubtraction by anti-human transferrin and electrophoretic migration times identified the isoforms. Previous markers of alcohol abuse and an assay combining anion-exchange minicolumn chromatography with immunoturbidimetry (%CDT) were included in the study. Sensitivities and specificities were compared by ROC analysis. RESULTS The asialylated isoform was missing in 95% of moderate drinkers but present in 92% of alcohol misusers. Disialotransferrin had a specificity and sensitivity of 0.75 at a cutoff of 0.7% of total transferrin, whereas the sum (asialo- + disialotransferrin) at a threshold of 1.2% had a sensitivity of 0.73 and a specificity of 0.92. Trisialotransferrin values did not distinguish between the two populations. Sensitivities and specificities of %CDT averaged 0.77 and 0.74, respectively, at a 2.6% cutoff; 0.67 and 0.83 at 2.8%; and 0.63 and 0.90 at 3%. CDT data were more sensitive and specific for males. Conventional biomarkers appeared less discriminating. CONCLUSIONS Asialotransferrin detected by CZE in sera of alcohol abusers offers the highest discrimination between excessive and moderate drinking.

Microarray analyses of the effects of NF-kappaB or PI3K pathway inhibitors on the LPS-induced gene expression profile in RAW264.7 cells: synergistic effects of rapamycin on LPS-induced MMP9-overexpression.

Lipopolysaccharide (LPS) activates a broad range of signalling pathways including mainly NF-kappaB and the MAPK cascade, but recent evidence suggests that LPS stimulation also activates the PI3K pathway. To unravel the specific roles of both pathways in LPS signalling and gene expression profiling, we investigated the effects of different inhibitors of NF-kappaB (BAY 11-7082), PI3K (wortmannin and LY294002) but also of mTOR (rapamycin), a kinase acting downstream of PI3K/Akt, in LPS-stimulated RAW264.7 macrophages, analyzing their effects on the LPS-induced gene expression profile using a low density DNA microarray designed to monitor the expression of pro-inflammatory genes. After statistical and hierarchical cluster analyses, we determined five clusters of genes differentially affected by the four inhibitors used. In the fifth cluster corresponding to genes upregulated by LPS and mainly affected by BAY 11-7082, the gene encoding MMP9 displayed a particular expression profile, since rapamycin drastically enhanced the LPS-induced upregulation at both the mRNA and protein levels. Rapamycin also enhanced the LPS-induced NF-kappaB transactivation as determined by a reporter assay, phosphorylation of the p38 and Erk1/2 MAPKs, and counteracted PPAR activity. These results suggest that mTOR could negatively regulate the effects of LPS on the NF-kappaB and MAPK pathways. We also performed real-time RT-PCR assays on mmp9 expression using rosiglitazone (agonist of PPARgamma), PD98059 (inhibitor of Erk 1/2) and SB203580 (inhibitor of p38(MAPK)), that were able to counteract the rapamycin mediated overexpression of mmp9 in response to LPS. Our results suggest a new pathway involving mTOR for regulating specifically mmp9 in LPS-stimulated RAW264.7 cells.

Rapid alterations in transferrin sialylation during sepsis

The inflammatory process is associated with alterations in iron metabolism. Transferrin, an acute-phase N-glycosylated glycoprotein, plays an important role in iron transport. Human serum transferrin contains two biantennary glycans, each consisting of 0 to 4 molecules of sialic acid (SA); its SA content is heterogeneous with high concentration of tetrasialotransferrin (4SA) and low amounts of disialo-, trisialo-, penta-, and hexasialotransferrin. The hepatic uptake of iron is greater for desialylated transferrin isoforms (disialotransferrin) than for the other forms. We hypothesized that serum levels of carbohydrate-deficient transferrin (CDT, disialotransferrin) may increase rapidly in septic patients. Blood samples were obtained from critically ill patients with (n = 15) and without (n = 14) documented sepsis and compared with healthy volunteers. The different forms of transferrin were studied by capillary zone electrophoresis; SA concentrations were measured by enzymatic colorimetric assay. There was a significant increase in the proportion of CDT in septic compared with nonseptic patients and volunteers (18.3% [1.3-30.5] vs. 0.7% [0.5-0.9]; P < 0.01 and 0.9% [0.5-1.1]; P < 0.05). Conversely, tri- and tetrasialotransferrin levels were lower in septic patients. Total and free SA concentrations were significantly higher in septic patients than in healthy volunteers. In a sheep model of septic shock secondary to peritonitis, serum free SA was already increased after 15 h. Sepsis is associated with decreased SA content on circulating transferrin and with an increase in blood free SA concentrations. In view of these rapid modifications and the long half-life of transferrin, the most likely explanation is degradation of transferrin by neuraminidase. Further studies including measurement of blood neuraminidase concentration and activity are needed to understand the process and exact role of SA decrease in septic patients.

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.

Glycosylation Pattern of Mature Dimeric Leukocyte and Recombinant Monomeric Myeloperoxidase: GLYCOSYLATION IS REQUIRED FOR OPTIMAL ENZYMATIC ACTIVITY*

The involvement of myeloperoxidase (MPO) in various inflammatory conditions has been the scope of many recent studies. Besides its well studied catalytic activity, the role of its overall structure and glycosylation pattern in biological function is barely known. Here, the N-glycan composition of native dimeric human MPO purified from neutrophils and of monomeric MPO recombinantly expressed in Chinese hamster ovary cells has been investigated. Analyses showed the presence of five N-glycans at positions 323, 355, 391, 483, 729 in both proteins. Site by site analysis demonstrated a well conserved micro- and macro-heterogeneity and more complex-type N-glycans for the recombinant form. Comparison of biological functionality of glycosylated and deglycosylated recombinant MPO suggests that glycosylation is required for optimal enzymatic activity. Data are discussed with regard to biosynthesis and the three-dimensional structure of MPO.

Exosites mediate the anti-inflammatory effects of a multifunctional serpin from the saliva of the tick Ixodes ricinus.

Serine protease inhibitors (serpins) are a structurally related but functionally diverse family of ubiquitous proteins. We previously described Ixodes ricinus immunosuppressor (Iris) as a serpin from the saliva of the tick I. ricinus displaying high affinity for human leukocyte elastase. Iris also displays pleotropic effects because it interferes with both the immune response and hemostasis of the host. It thus inhibits lymphocyte proliferation and the secretion of interferon‐γ or tumor necrosis factor‐α by peripheral blood mononuclear cells, and also platelet adhesion, coagulation and fibrinolysis. Its ability to interfere with coagulation and fibrinolysis, but not platelet adhesion, depends on the integrity of its antiproteolytic reactive center loop domain. Here, we dissect the mechanisms underlying the interaction of recombinant Iris with peripheral blood mononuclear cells. We show that Iris binds to monocytes/macrophages and inhibits their ability to secrete tumor necrosis factor‐α. Recombinant Iris also has a protective role in endotoxemic shock. The anti‐inflammatory ability of Iris does not depend on its antiprotease activity. Moreover, we pinpoint the exosites involved in this activity.