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.

We investigated expression of several antigens on neutrophils and monocytes, involved in cell adhesion, from patients hemodialyzed with cellulosic and polyacrylonitrile membranes. Among the antigens tested only the expression of CD15s and CD11b was significantly increased on neutrophils and monocytes in patients dialyzed with cellulosic membranes. No changes occurred with polyacrylonitrile membranes. Leukocyte counts from patients dialyzed with cuprophane membranes decreased at the same time as expression of cellular CD15s increased, resulting in a significant negative correlation at all time points tested. No correlation was found between the drop of monocytes and their expression of CD11b. When CD15s expression increased on neutrophils and monocytes, we observed a concomitant increase of CD62P, a specific selectin of activated platelets. When whole blood cells were incubated with complement activated serum both antigens increased but not when cells were incubated with hrC5a. We also observed that CD61, a platelet phenotypic antigen, was present on leukocytes incubated with complement activated serum. At the time when platelet-leukocyte coaggregates decreased, CD62P expression remained stable on leukocytes, suggesting that both neutrophils and monocytes are able to trap either CD62P shed by activated platelets or soluble CD62P present in normal human serum. The present study documents a major role of P-selectin (CD62P)/sialyl-Lewis x (CD15s) interaction in the transient leukocyte margination during hemodialysis.

We have investigated the effects of intravenous immunoglobulin (IVIg), a therapeutic preparation of normal human polyspecific IgG, on the synthesis and release of cytokines by peripheral blood monocytes. IVIg was found to selectively induce gene transcription and secretion of interleukin-1 receptor antagonist (IL-1ra) and IL-8 in cultures of normal human monocytes. The addition of IVIg to cultures of purified monocytes induced a dose-dependent secretion of IL-1ra and IL-8 without stimulating the production of IL-1 alpha, IL-1 beta, tumor necrosis factor-alpha or IL-6. The effects of IVIg required both the Fc and F(ab')2 portions of IgG. IVIg-induced production of IL-8 by monocytes was enhanced by lipopolysaccharide (LPS), although LPS inhibited the secretion of IL-1ra, suggesting that IVIg and LPS stimulate distinct intracellular pathways in monocytes. Induction of IL-1ra and IL-8 by IVIg was enhanced in the presence of autologous T lymphocytes. Our observations document the selectivity of the effects of IVIg on the synthesis of cytokines and cytokine antagonists by human monocytes. Induction of IL-1ra and IL-8 by IVIg may contribute to the anti-inflammatory effects of immunoglobulin therapy in patients with autoimmune and systemic inflammatory disorders.

Toll-like receptors (TLRs) are involved in human monocyte activation by lipopolysaccharide (LPS) andStaphylococcus 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-1␣ (IL-1␣), IL-1ra, and tumor necrosis factor alpha (TNF-␣) production, whereas blocking of the ERK1/2 pathways inhibited IL-1␣, IL-1␤, and IL-1ra but not TNF-␣ production. When cells were stimulated by SAC, inhibition of the p38 pathway did not affect cytokine production, whereas only IL-1␣ 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.Invasive infection by gram-positive and gram-negative bacteria in humans results in septic shock and death. The early cellular response to both groups of microorganisms is still unclear. Several investigators have proposed that lipopolysaccharides (LPS) from gram-negative bacteria, as well as several components from gram-positive bacteria, trigger monocytic cytokine production following interaction with membrane-bound CD14 (mCD14) and activation of Toll-like receptors (TLRs; the human homologues of Drosophila Toll) which are present on the cell membrane (23,30,43,61). Several lines of evidence suggest that after binding to mC14, LPS initiates intracellular signaling pathways activating a number of tyrosine kinases as an initial step. Various ␣ subunits of heterodimeric G proteins and Src kinases, physically associated with membrane-bound CD14 molecules in LPS-stimulated normal human monocytes, induce p38 mitogen-activated protein (MAP) kinase activation, which is involved in cytokine synthesis (44). Other proteins that are phosphorylated upon LPS stimulation include p42 and p44 MAP kinases, which are encoded by the erk2 and erk1 genes, respectively (10, 25). However, the upstream events that occur during ERK1, ERK2, and p38 kinase phosphorylation remain unclear. There is some evidence that LPS activates Ras and consequently Raf, resulting in MEK-1 and MAP kinase phosphorylation, but the precise pathways are not fully understood (8). In addition to MEK and MAP kinase activation, Syk molecules are phosphorylated upon LPS stimulation of macrophages. However, several studies have indicated that neither Syk, Src family tyrosine kinases, Hck, Lyn, nor Fgr is indispe...

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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 ( Mucosal immunity of the female genital tract has recently gained special attention as an important factor that could modulate the transmission of many sexually transmitted infections (STIs), including human immunodeficiency virus (HIV) infection. Furthermore, current concepts of designing vaccines against viral infections acquired through sexual portals focus on the potential interest in inducing specific mucosal immunity at the sites of sexual exposure in association with systemic and cellular immune responses.Mucosal immunity is investigated by collecting cervicovaginal secretions (CVS), either by vaginal washing (1) or by a vaginal or cervical swab further treated with collecting buffer (2). One potential methodological pitfall when sampling CVS of sexually active women is the presence of contaminating semen in the vaginal fluid that will bias the immunological characterization of the collected genital fluid. Female participants in clinical studies are generally asked to avoid sexual intercourse and intravaginal medications for 3 (10, 11) to 5 (4) days before sampling of CVS. However, semen residues may be detected in the lower female genital tract up to 5 days after sexual intercourse (12), and CVS collected from women at high risk for sexually transmitted diseases have frequently been found to contain traces of semen (13). Thus, ensuring that vaginal fluid is free of semen is essential to avoid misinterpretation of the data and accurately assess the immune response in the female genital tract. Similar precautionary measures should be undertaken when analyzing genital shedding of HIV in infected women. Finally, sensitive methods to detect traces of semen may be required in forensic medicine.The presence of semen in CVS is assessed by microscopic observation of motile spermatozoids (3), determination of acid phosphatase activity in CVS (9), and the detection of semen components, including prostatic acid phosphatase, prostaticspecific antigen (PSA) (6), and seminal vesicle-specific antigen (5). The latter methods, including those based on the immunochemical detection of semen-derived molecules by immunocapture assays, may lack specificity and sensitivity. The present study was undertaken to assess the validity of using a highly sensitive PCR assay for the Y chromosome (designated Y PCR) in the cellular fraction of CVS for detecting contaminating semen in female genital fluids. MATERIALS AND METHODSStudy population. Two hundred seventy-four unselected women attending the National Reference Center for Sexually Transmissible Diseases and AIDS in Bangui, Central African Republic, participated in the study. The Center offers multipurpose reproductive health services, including STI services, and operates as the main voluntary HIV testing and counselling site in Bangui. We follo...

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Sephadex [alpha(1----6) cross-linked dextran] activates the human alternative pathway of complement. Substitution of hydroxyl groups of Sephadex with carboxymethyl groups (CM) results in a dose-dependent decrease of the activating capacity of the polymer in normal human serum. Sephadex bearing one CM group/glycosyl unit (CM-Seph 0.95) exhibited no activating capacity. CM groups did not interfere with the ability of the polymer to covalently bind C3b in the presence of purified alternative pathway proteins nor with the capacity of bound-C3b to form a C3 convertase in the absence of regulatory proteins. C3b that was bound to CM-Seph 0.95 was more susceptible to inactivation by factors H and I in serum than C3b bound to Sephadex. Binding studies using 125I-labeled H demonstrated that H bound with a similar affinity to the activating particle Sephadex, to Sephadex bearing C3b and to the nonactivating particle CM-Seph 0.95. However, factor H bound with a 5- to 7-fold higher affinity to CM-Seph 0.95 bearing C3b. These results demonstrate a requirement for both CM groups and C3b molecules in order for H to bind with high affinity to C3b on the non-activating surface, and indicate that H formed a ternary complex with surface-bound C3b and CM groups on CM-Seph 0.95. Using a chemically defined model system, the present study provides a molecular basis for the enhanced interaction between surface-bound C3b and factor H on nonactivators of the human alternative pathway.