Marie-Paule Carreno

Activation of the complement system at the interface between blood and artificial surfaces.

The interaction between blood and the artificial devices used in haemodialysis results in the activation of the immune system. Complement activation is known to play a key role in the production of inflammatory mediators. This article describes the complement system and the way in which it is triggered in the patient undergoing haemodialysis. It highlights those factors thought to be of particular importance in the pathogenesis of adverse reactions and targets them as obstacles to be overcome in the future design of biocompatible materials.

Influence of Temperature on Neutrophil Trafficking During Clinical Cardiopulmonary Bypass

BACKGROUND The adhesion of neutrophils to endothelial cells and their subsequent transendothelial migration play a major role in inflammatory damage elicited by cardiopulmonary bypass (CPB) because these events are linked to the release of cytotoxic proteases and oxidants. However, the patterns of neutrophil trafficking in relation to systemic temperature during clinical CPB have not yet been characterized. METHODS AND RESULTS Twenty case-matched patients undergoing warm (31.8 +/- 0.4 degrees C) or cold (26.3 +/- 0.5 degrees C, P < .0001 versus warm) bypass were studied. Blood samples were simultaneously collected from the right and left atria before, at the end of, and 30 minutes after CPB. Plasma levels of C3a, P- and E-selectins, elastase, and interleukin-8 were determined by immunoassays. The results demonstrate: (1) a rise in C3a, reflecting complement activation, (2) a fall in soluble E-selectin consistent with an increased adhesiveness of activated neutrophils, (3) a rise in soluble P-selectin expected to enhance endothelial adhesion of these neutrophils, (4) a rise in elastase, suggesting an adhesion-triggered neutrophil degranulation, and finally (5) a rise in interleukin-8 that is likely to promote transendothelial migration of adherent neutrophils. All of these changes occurred in the two groups of patients and were significant compared with prebypass values. However, in none of the groups was there a significant difference between right and left atrial values for any of the markers. The single difference between cold and warm bypass patients was a significant reduction of elastase release in the cold group (P < .001 versus the warm group). CONCLUSIONS Clinical CPB is associated with biological changes suggesting the occurrence of neutrophil trafficking. Hypothermia provides only partial protection through a reduced release of elastase. Overall, these results reinforce the rationale for the development of therapeutic strategies targeted at blunting the neutrophil-mediated component of bypass-induced inflammatory damage.

The ability of sephadex to activate human complement is suppressed in specifically substituted functional sephadex derivatives

The capacity of Sephadex and of chemically substituted Sephadex derivatives to activate human complement was examined by incubating polymer particles in normal human serum (NHS) under conditions that allow classical and/or alternative pathway activation, and by determining complement consumption or generation of C3a antigen in serum. Sephadex was found to activate complement in NHS, mainly through the alternative pathway. The complement-activating capacity of Sephadex was directly related to the surface area of polymer that could interact with serum. Substitution of hydroxyl groups of Sephadex with carboxymethyl (CM) groups suppressed the complement-activating capacity of the polymer in a dose-dependent fashion so that Sephadex bearing an average of one or more CM groups per saccharidic unit exhibited no complement-activating ability. Blocking of CM groups on CM sephadex with amide bonds did not restore a complement-activating capacity to the polymer, indicating that intact hydroxyl groups of the sugar units are required for complement activation by Sephadex. CM Sephadex was also found to adsorb C3adesArg which bound to the polymer with a calculated affinity of 1 x 10(6) l x M-1. Substitution of Sephadex with carboxymethyl and benzylamide sulphonate groups which confers to the polymer the capacity to catalyse thrombin inactivation on its surface also suppressed the complement-activating capacity of Sephadex. Sephadex derivatives that lack complement-activating properties and adsorb anaphylatoxins may provide useful models for the design of cellulosic membranes and biomaterials with blood compatible properties.

Neutrophil Reactive Oxygen Species Production during Hemodialysis: Role of Activated Platelet Adhesion to Neutrophils through P-Selectin

Platelet interaction with leukocytes can occur to a significant degree during hemodialysis, but it remains to be determined what pathophysiological consequences stem from the intradialytic formation of platelet-leukocyte coaggregates. By the use of flow cytometry techniques, this study was set out to analyze intradialytic platelet-neutrophil coaggregate formation and neutrophil hydrogen peroxide production from 10 end-stage renal disease patients each dialyzed with cuprophane and polyacrylonitrile membranes. Platelet-neutrophil coaggregates increased during dialysis with cuprophane, whereas no changes occurred with polyacrylonitrile membranes. Dialysis with cuprophane, unlike that with polyacrylonitrile, also resulted in a significant increase in neutrophil hydrogen peroxide production 10 min after dialysis initiation which persisted at significantly higher levels than predialysis values through the first 20 min. We found that the increased hydrogen peroxide production by neutrophils essentially occurred in concomitance with neutrophil-platelet coaggregation. Intracellular fluorescence representing hydrogen peroxide formation significantly increased through the first 20 min of cuprophane dialysis in neutrophils aggregated to platelets. By contrast, no change occurred in neutrophils not aggregated to platelets. Neutrophils which had formed aggregates with platelets produced higher hydrogen peroxide levels, as assessed by significantly higher fluorescence values, than non-aggregate-forming neutrophils at all time points tested. The phenomenon was duplicated in vitro when ADP-activated normal platelets were incubated with neutrophil cells but was largely inhibited when ADP-activated platelets were treated with anti-P-selectin antibody before incubation with neutrophils. These results strongly suggest that platelet-neutrophil aggregates occurring during hemodialysis, representing cell-cell interactions with pathophysiological effects, may serve as a new parameter to assess biocompatibility.

Gram-Positive and Gram-Negative Bacteria Do Not Trigger Monocytic Cytokine Production through Similar Intracellular Pathways

ABSTRACT Toll-like receptors (TLRs) are involved in human monocyte activation by lipopolysaccharide (LPS) and Staphylococcus aureus Cowan (SAC), suggesting that gram-positive and gram-negative bacteria may trigger similar intracellular events. Treatment with specific kinase inhibitors prior to cell stimulation dramatically decreased LPS-induced cytokine production. Blocking of the p38 pathway prior to LPS stimulation decreased interleukin-1α (IL-1α), IL-1ra, and tumor necrosis factor alpha (TNF-α) production, whereas blocking of the ERK1/2 pathways inhibited IL-1α, IL-1β, and IL-1ra but not TNF-α production. When cells were stimulated by SAC, inhibition of the p38 pathway did not affect cytokine production, whereas only IL-1α production was decreased in the presence of ERK kinase inhibitor. We also demonstrated that although LPS and SAC have been shown to bind to CD14 before transmitting signals to TLR4 and TLR2, respectively, internalization of CD14 occurred only in monocytes triggered by LPS. Pretreatment of the cells with SB203580, U0126, or a mixture of both inhibitors did not affect internalization of CD14. Altogether, these results suggest that TLR2 signaling does not involve p38 mitogen-activated protein kinase signaling pathways, indicating that divergent pathways are triggered by gram-positive and gram-negative bacteria, thereby inducing cytokine production.

Binding of LFA-1 (CD11a) to Intercellular Adhesion Molecule 3 (ICAM-3; CD50) and ICAM-2 (CD102) Triggers Transmigration of Human Immunodeficiency Virus Type 1-Infected Monocytes through Mucosal Epithelial Cells

ABSTRACT Transmigration of human immunodeficiency virus (HIV)-infected mononuclear cells through the genital mucosa is one of the possible mechanisms of sexual transmission of HIV. Here, we investigated the transmigration of cell-associated R5-tropic HIV type 1 (HIV-1) through a tight monolayer of human epithelial cells in vitro. We show that this process is dependent on an initial interaction between αLβ2 integrin CD11a/CD18 on infected monocytic cells and intercellular adhesion molecule 2 (ICAM-2; CD102) and ICAM-3 (CD50) on the apical membrane of epithelial cells. The CD50 and CD102 ligands were overexpressed on epithelial cells when the cells were activated by proinflammatory cytokines in the cellular microenvironment. An accumulation of proviral DNA was found in the transmigrated cells, clearly reflecting the preferential transepithelial migration of HIV-1-infected cells under proinflammatory conditions. Our observations provide new insights supporting the hypothesis that HIV-infected mononuclear cells contained in genital secretions from infected individuals may cross the epithelial genital mucosa of an exposed receptive sexual partner, particularly under inflammatory conditions of damaged genital tissue. Understanding the fundamental aspects of the initial HIV entry process during sexual transmission remains a critical step for preventing human infection and developing further vaccinal strategies and virucidal agents.

Detection of Y Chromosome DNA as Evidence of Semen in Cervicovaginal Secretions of Sexually Active Women

ABSTRACT The detection of traces of semen in cervicovaginal secretions (CVS) from sexually active women practicing unprotected sex is a prerequisite for the accurate study of cervicovaginal immunity. Two semen markers, the prostatic-specific antigen (PSA) and the Y chromosome, were detected in parallel in CVS obtained by a standardized vaginal washing of consecutive women attending the principal medical center for sexually transmitted diseases of Bangui, Central African Republic. PSA was detected by immunoenzymatic capture assay in the cell-free fraction of CVS, and the Y chromosome was detected by a single PCR assay of DNA extracted by silica from the cell fraction (Y PCR). Fifty (19%) cell-free fractions of the 264 β-globin-positive CVS samples were positive for PSA, and 100 (38%) cell fractions of the CVS samples were positive for the Y chromosome. All the 50 (19%) PSA-containing CVS samples were also positive for the Y chromosome. Fifty (19%) CVS samples were positive only for the Y chromosome, with no detectable PSA. The remaining 164 (62%) CVS samples were both PSA and Y chromosome negative. These findings demonstrate that CVS from sexually active women may contain cell-associated semen residues unrecognized by conventional immunoenzymatic assays used to detect semen components. The detection of cell-associated male DNA with a highly sensitive and specific procedure such as Y PCR constitutes a method of choice to detect semen traces in female genital secretions.

Cell-associated adhesion molecules as early markers of bioincompatibility

BACKGROUND Transient nature of adhesive interactions occurring during cell margination is mainly dependent on expression of selectins which are shed by activated cells. This shedding in the circulation may play an important role as anti-inflammatory mediator. Haemodialysis is also associated with P-selectin (CD62P)/sialyl-Lewis(x) (CD15s) interactions which mediate platelet-leukocyte coaggregation. We further investigated the mechanisms underlying leukocyte margination during haemodialysis. METHODS CD15s, CD11b and CD61 expression on circulating leukocytes from patients dialysed on synthetic membranes (modified polyacrylonitrile (SPAN), polysulphone (PS), and polyacrylonitrile (AN69) was assessed by cytofluorometry in a prospective crossover trial. We measured plasma levels C3a/C3a desArg, soluble CD62P, and CD62E molecules obtained from patients and healthy individuals. RESULTS Expression of CD11b and CD15s was upregulated on neutrophils from patients dialysed with SPAN and PS membranes during the dialysis session. A significant negative correlation was found between the expression of CD11b or CD15s molecules and neutrophil counts as well as between CD15s expression and monocyte counts during haemodialysis. As assessed by CD61 expression on leukocytes, we observed that platelets bound significantly onto both neutrophils and monocytes during dialysis with both membranes. A significant positive correlation was found between the expression of CD11b molecules and the percentage of CD61+ monocytes counts during SPAN and PS dialysis. We found a significant increase of soluble CD62P in plasma samples obtained from haemodialysed patients before the dialysis session as compared to the levels detected in plasma from healthy individuals. CONCLUSIONS This study documents a major role of CD15s, CD11b, CD61, CD62P molecules in the transient leukocytes activation and margination during haemodialysis on synthetic membranes despite their low complement-activating properties.

Activation of the complement system by polysaccharidic surfaces bearing carboxymethyl, carboxymethylbenzyl-amide and carboxymethylbenzylamide sulphonate groups

Substituted Sephadex derivatives bearing carboxymethyl (CM), CM-benzylamide (CMB), CM-propylamide (CMP) and CMB-sulphonate (CMBS) groups are used as models of polysaccharidic surfaces to measure the effects of substituting OH groups on the complement activating capacity (CAC) of the modified surfaces in normal human serum. CM substitution decreases and can suppress the CAC of Sephadex. Low CMB substitution also decreases the CAC, whereas high CMB or CMP substitutions increase it again after a minimum. In addition to C3 cleavage occurring at high substitution with CMB or CMP groups, the presence of CMB induces consumption of a protein, limiting CH50 measurements. The CAC variations could be due to rearrangements of the polymer surfaces at the aqueous interface with proteins. Highly substituted CMB-bearing surfaces could activate complement-like polystyrene surfaces. The presence of CMBS groups does not reduce the CAC of the surface. Such polymer surfaces, which are heparin-like concerning coagulation, are not heparin-like concerning complement inhibition.

Quantitation of Human Immunodeficiency Virus Type 1 (HIV-1) RNA in Cell-Free Cervicovaginal Secretions: Comparison of Reverse Transcription-PCR Amplification (AMPLICOR HIV-1 MONITOR 1.5) with Enhanced-Sensitivity Branched-DNA Assay (Quantiplex 3.0)

ABSTRACT Two commercially available hypersensitive assays for human immunodeficiency virus type 1 (HIV-1) RNA quantitation, AMPLICOR HIV-1 Monitor Test 1.5 and Quantiplex HIV RNA 3.0, were compared to detect and quantify HIV-1 RNA in the cell-free fraction of cervicovaginal secretions collected by vaginal washing. Three panel specimens were used: pooled cervicovaginal secretions spiked with HIV-1 subtype A or HIV-1 subtype B and cervicovaginal lavages from HIV-positive and HIV-negative women. Compared to the AMPLICOR HIV-1 Monitor Test 1.5 assay, the Quantiplex HIV-1 3.0 assay yielded higher estimates of HIV-1 RNA concentrations in several tested samples spiked with HIV-1 RNA subtype A, as well as subtype B, particularly samples containing low amounts of HIV-1 RNA. The sensitivity and specificity of the AMPLICOR HIV-1 Monitor Test 1.5 assay were 93 and 100%, respectively; the sensitivity and specificity of the Quantiplex HIV RNA 3.0 assay were 97 and 50%, respectively. In conclusion, in quantifying HIV-1 RNA in cervicovaginal secretions, the Quantiplex HIV RNA 3.0 may lack specificity, and the AMPLICOR HIV-1 Monitor Test 1.5 assay, although highly specific, may lack sensitivity.