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Platelet (PLT) transfusions are potentially life saving for individuals with low PLT numbers; however, previous work revealed that PLT transfusions are associated with increased infection risk. During storage, PLT intended for transfusion continuously shed ectosomes (Ecto) from their surface, which express immunomodulatory molecules like phosphatidylserine or TGF-β1. Recently, PLT-Ecto were shown to reduce proinflammatory cytokine release by macrophages and to favor the differentiation of naive T cells toward regulatory T cells. Whether PLT-Ecto modify NK cells remains unclear. We exposed purified NK cells and full PBMCs from healthy donors to PLT-Ecto. We found a reduced expression of several activating surface receptors (NKG2D, NKp30, and DNAM-1) and decreased NK cell function, as measured by CD107a expression and IFN-γ production. Pretreatment of PLT-Ecto with anti–TGF-β1 neutralizing Ab restored surface receptor expression and NK cell function. We further observed a TGF-β1–mediated upregulation of miR-183, which, in turn, reduced DAP12, an important protein for stabilization and downstream signaling of several activating NK cell receptors. Again, these effects could antagonized, in part, when PLT-Ecto were preincubated with anti–TGF-β1 Ab. Erythrocyte Ecto did not affect NK cells. Polymorphonuclear cell Ecto expressed MHC class I and inhibited NK cell function. In addition, they induced the secretion of TGF-β1 by NK cells, which participated in an auto/paracrine manner in the suppressive activity of polymorphonuclear cell–derived Ecto. In sum, our study showed that PLT-Ecto could inhibit NK cell effector function in a TGF-β1–dependent manner, suggesting that recipients of PLT transfusions may experience reduced NK cell function.

ObjectivesGout is a highly inflammatory but self-limiting joint disease induced by the precipitation of monosodium urate (MSU) crystals. While it is well established that inflammasome activation by MSU mediates acute inflammation, little is known about the mechanism controlling its spontaneous resolution. The aim of this study was to analyse the role of neutrophil-derived microvesicles (PMN-Ecto) in the resolution of acute gout.MethodsPMN-Ecto were studied in a murine model of MSU-induced peritonitis using C57BL/6, MerTK−/−and C5aR−/−mice. The peritoneal compartment was assessed for the number of infiltrating neutrophils (PMN), neutrophil microvesicles (PMN-Ecto), cytokines (interleukin-1β, TGFβ) and complement factors (C5a). Human PMN-Ecto were isolated from exudates of patients undergoing an acute gouty attack and functionally tested in vitro.ResultsC5a generated after the injection of MSU primed the inflammasome for IL-1β release. Neutrophils infiltrating the peritoneum in response to C5a released phosphatidylserine (PS)-positive PMN-Ecto early on in the course of inflammation. These PMN-Ecto in turn suppressed C5a priming of the inflammasome and consequently inhibited IL-1β release and neutrophil influx. PMN-Ecto-mediated suppression required surface expression of the PS-receptor MerTK and could be reproduced using PS-expressing liposomes. In addition, ectosomes triggered the release of TGFβ independent of MerTK. TGFβ, however, was not sufficient to control acute MSU-driven inflammation in vivo. Finally, PMN-Ecto from joint aspirates of patients with gouty arthritis had similar anti-inflammatory properties.ConclusionsPMN-Ecto-mediated control of inflammasome-driven inflammation is a compelling concept of autoregulation initiated early on during PMN activation in gout.

Complement factor H (CFH) mutation is one of the causes of atypical haemolytic uraemic syndrome (aHUS). Patients with CFH mutation-associated aHUS progress often to end-stage renal disease despite plasma exchange therapy. When such patients are transplanted, aHUS recurs almost invariably and causes graft failure making the rationale of single kidney allograft transplantation questionable. Since CFH is synthesized mostly by the liver, combined liver-kidney transplantation has been recommended. However, fatal outcomes have been reported using this strategy. We report a case of successful single kidney allograft transplantation in a patient with a CFH gene mutation (R1210C), who had end-stage renal failure after three flares of aHUS treated with plasma exchange. He received peri- and postoperative infusions of fresh frozen plasma, which to date has prevented recurrence of the disease. He has preserved renal function 1-year post-transplant.

SummaryAccumulating evidence suggests an immune-modulatory role for platelets (PLT) and PLT-derived microvesicles. In particular, ectosomes, i.e. vesicles budding from PLT surface, have been shown to exert immunosuppressive activities on phagocytes. Here we investigated the effects mediated by PLT-derived ectosomes (PLT-Ecto) on CD4+ T cells. Exposure of activated CD4+ T cells to PLT-Ecto decreased their release of IFNγ, TNFα and IL-6, and increased the production of TGF-β1. Concomitantly, PLT-Ecto-exposed CD4+ T cells displayed increased frequencies of CD25high Foxp3+ cells. These phenomena were dose-dependent and PLT-Ecto specific, since they were not observed in the presence of polymorphonuclear- and erythrocyte-derived ectosomes. Analysis of specific T cell subsets revealed that PLT-Ecto induced differentiation of naïve T cells into Foxp3+ cells, but had no effect on predifferentiated Foxp3+ regulatory T cells (Tregs). Importantly, PLT-Ectoinduced Foxp3+ cells were as effective as peripheral blood Tregs in suppressing CD8+ T cell proliferation. PLT-Ecto-mediated effects were partly dependent on PLT-derived TGF-β1, as they were to some extent inhibited by PLT-Ecto pretreatment with TGF-β1-neutralising antibodies. Interestingly, ectosome-derived TGF-β1 levels correlated with Foxp3+ T cell frequencies in blood of healthy donors. In conclusion, PLT-Ecto induce differentiation of CD4+ T cells towards functional Tregs. This may represent a mechanism by which PLT-Ecto enhance peripheral tolerance.

Objective— Transfusion of aged blood has been associated with increased morbidity and mortality in critically ill patients. During storage, erythrocytes release increasing numbers of microvesicles (red blood cell–derived microvesicles [RBC-MV]). We hypothesized that RBC-MV mediate some of the deleterious effects of aged blood transfusions. Approach and Results— We established a murine transfusion model using RBC-MV purified from aged mouse erythrocytes. Injection of RBC-MV into healthy mice had no effect. However, they aggravated pulmonary leukocyte sequestration and peripheral blood leukopenia induced by lipopolysaccharides. Lipopolysaccharide-induced proinflammatory cytokines were significantly increased in plasma after RBC-MV injection. These effects were not seen in C5aR-deficient mice. In vitro, RBC-MV bound C3 fragments after incubation with plasma but failed to bind immunoglobulins, C1q, or mannose-binding lectin. Preventing thrombin generation inhibited complement activation in vitro and in vivo and reversed the proinflammatory effects of RBC-MV in lipopolysaccharide-primed mice. Finally, the RBC-MV–induced phenotype was recapitulated using phosphatidylserine-expressing liposomes, suggesting that surface expression of phosphatidylserine by RBC-MV was mechanistically involved. Conclusions— These results point toward a thrombin-dependent mechanism of complement activation by RBC-MV independent of the classical, lectin, or alternative pathway. Besides identifying RBC-MV as potential mediators of transfusion-related morbidity, our findings may be relevant for other inflammatory disorders involving intravascular microvesicle release, for example, sickle cell disease or thrombotic microangiopathy.

Urine results from a coordinated activity of glomerular and tubular compartments of the kidney. As a footprint of these cellular functional processes, urinary exosomes, and 40-80 nm membrane vesicles released after fusion with the plasma membrane into the extracellular environment by renal epithelial cells, are a source for identification of proteins and investigation of their role in the kidney. The aim of the present study was the identification of podocyte exosome proteins based on urine immunoabsorption using podocyte-specific CR1-immunocoated beads followed by proteomic analysis using LC MS/MS techniques. This methodology allowed the identification of 1195 proteins. By using a bioinformatic approach, 27 brain-expressed proteins were identified, in which 14 out of them were newly demonstrated to be expressed in the kidney at a mRNA level, and, one of them, the COMT protein, was demonstrated to be expressed in podocytes at a protein level. These results, attesting the reliability of the methodology to identify podocyte proteins, need now to be completed by further experiments to analyze more precisely their biological function(s) in the podocytes.

Ectosomes are vesicles shed directly from the cell surface. Human polymorphonuclear neutrophils release ectosomes (PMN-Ect) upon their activation. PMN-Ect expose phosphatidylserine (PS) on the outer leaflet of the plasma membrane, and down-modulate the inflammatory response of human macrophages and dendritic cells exposed to TLR-2 and -4 ligands. This down-modulation is mediated by PS via the engagement and activation of the Mer receptor tyrosine kinase (MerTK). In the present study, we demonstrate that exposure of macrophages to PMN-Ect activates directly 2 additional pathways, an immediate Ca(2+) flux and a rapid release of TGF-beta1. As expected, the Ca(2+) flux was necessary for the activation of TLR-2 pathway with the release of cytokines. However, MerTK blockade with antibodies did not modify the Ca(2+) flux, indicating an independent activation of Ca(2+) by PMN-Ect. Striking was that the rapid release of TGF-beta1 was independent of the MerTK pathway and did not require a Ca(2+) flux. TGF-beta1 was present in cytosolic storage pools, which were depleted after exposure of the macrophages to PMN-Ect, and no increase in TGF-beta1 mRNA could be detected in the 3 first hours when maximal release had occurred. The release of TGF-beta1 by macrophages was seen only for PMN-Ect and not for PS-liposomes or erythrocyte ectosomes, which express PS. However, blocking the PS of PMN-Ect inhibited TGF-beta1 release, suggesting that PS expression was necessary although not sufficient for this release. Interestingly, the effects of PMN-Ect pre-exposure were lasting for 24h with the macrophages being less receptive to TLR-2 activation and TGF-beta1 stores remaining low. In sum, PMN-Ect induce several signaling pathways in resting and stimulated macrophages, which include independently the MerTK pathway, Ca(2+) flux and the release of stored TGF-beta1, and each might influence the immunomodulatory effects of macrophages.

Summary One approach to fight against schistosomiasis is to develop an efficient vaccine. Schistosoma mansoni tetraspanning orphan receptor (SmTOR) might be a vaccine candidate, as it is a tegument membrane protein expressed most highly in cercariae. In this study we characterized the recombinant first extracellular domain of SmTOR (rSmTORed1) as having the expected property to bind C2 of complement similarly to a smaller peptide of the same domain, and to produce specific and high-titre antibodies in BALB/c mice immunized using complete Freund's adjuvant/incomplete Freund's adjuvant (CFA/IFA). Immunization was protective against parasite infection, as demonstrated by a significant decrease in worm burden in immunized BALB/c mice versus the control groups over two independent trials [64 and 45% reduction for mean adult worm burden in immunized versus phosphate-bufferd saline (PBS) injected mice]. Interestingly, infection by itself did not lead to the generation of anti-rSmTORed1 antibodies, corresponding to the low frequency of specific anti-rSmTORed1 antibodies detected in the sera of patients infected with S. mansoni (2/20; 10%). These data suggest that, as opposed to the natural infection during which SmTOR induces antibodies only rarely, immunization with its smaller first extracellular domain might be more efficient.

Microparticles (MP) shed by platelets (PLT) during storage have procoagulant activities, but little is known about their properties to modify inflammation or immunity. In this study, we studied the capacity of MP present in PLT concentrates to alter the function of macrophages and dendritic cells (DC). The size of the purified MP was between 100 and 1000 nm, and they expressed phosphatidylserine; surface proteins of PLT (CD61, CD36, CD47), including complement inhibitors (CD55, CD59), but not CD63; and proteins acquired from plasma (C1q, C3 fragments, factor H). These characteristics suggest that the MP shed by PLT are formed by budding from the cell surface, corresponding to ectosomes. The purified PLT ectosomes (PLT-Ect) reduced the release of TNF-α and IL-10 by macrophages activated with LPS or zymosan A. In addition, PLT-Ect induced the immediate release of TGF-β from macrophages, a release that was not modified by LPS or zymosan A. Macrophages had a reduced TNF-α release even 24 h after their exposure to PLT-Ect, suggesting that PLT-Ect induced a modification of the differentiation of macrophages. Similarly, the conventional 6-d differentiation of monocytes to immature DC by IL-4 and GM-CSF was modified by the presence of PLT-Ect during the first 2 d. Immature DC expressed less HLA-DP DQ DR and CD80 and lost part of their phagocytic activity, and their LPS-induced maturation was downmodulated when exposed to PLT-Ect. These data indicate that PLT-Ect shed by stored PLT have intrinsic properties that modify macrophage and DC differentiation toward less reactive states.

Summary Vesicles released by cells have been described using various names, including exosomes, microparticles, microvesicles and ectosomes. Here we propose to differentiate clearly between ectosomes and exosomes according to their formation and release. Whereas exosomes are formed in multi-vesicular bodies, ectosomes are vesicles budding directly from the cell surface. Depending upon the proteins expressed, exosomes activate or inhibit the immune system. One of the major properties of exosomes released by antigen-presenting cells is to induce antigen-specific T cell activation. Thus, they have been used for tumour immunotherapy. By contrast, the major characteristics of ectosomes released by various cells, including tumour cells, polymorphonuclear leucocytes and erythrocytes, are the expression of phosphatidylserine and to have anti-inflammatory/immunosuppressive activities similarly to apoptotic cells.

Several clinical studies have suggested that blood transfusions are immunosuppressive. Whereas there have been reports describing immunosuppression induced by leukocytes or fragments thereof, the possibility that microparticles, released by erythrocytes during storage, are also involved was not investigated. We present evidence here that such microparticles have all the properties of ectosomes including size, the presence of a lipid membrane, and the specific sorting of proteins. These erythrocyte-derived ectosomes (E-ecto) fixed C1q, which was followed by activation of the classical pathway of complement with binding of C3 fragments. Similarly to ectosomes released by PMN, they express phosphatidylserine on their surface membrane, suggesting that they may react with and down-regulate cells of the immune system. In vitro, they were taken up by macrophages, and they significantly inhibited the activation of these macrophages by zymosan A and LPS, as shown by a significant drop in TNF-α and IL-8 release (respectively, 80% and 76% inhibitions). In addition, the effect of E-ecto was not transient but lasted for at least 24 h. In sum, E-ecto may interfere with the innate immune system/inflammatory reaction. Therefore, E-ecto transfused with erythrocytes may account for some of the immunosuppressive properties attributed to blood transfusions.

Podocytes (glomerular visceral epithelial cells) release vesicles into urine. Podocyte vesicle-enriched fractions from normal and pathological human urine samples were prepared for proteomic analysis. An immunoadsorption method was applied and enrichment of podocyte vesicles was assessed. We identified 76 unique proteins. One protein, serum paraoxonase/arylesterase 1 (PON-1), was newly identified in normal human urine sample. We confirmed this result and showed PON-1 expression in normal human kidney. These results demonstrated the potential for using the urine samples enriched in podocyte vesicles as a starting material in studies aimed at discovery of biomarkers for diseases.

Die Assoziation zwischen Komplementdefizienzen, insbesondere von Komponenten der terminalen Kaskade (C5–C9), und dem Auftreten von Meningokokkeninfekten und bakteriellen Meningitiden ist gut beschrieben. In dem vorliegenden Fallbericht wird dabei erstmals ein kombiniert-heterozygoter Defekt im C7-Gen beschrieben, der noch eine Restproduktion von C7 erlaubte. Diese Restproduktion reichte jedoch nicht aus, um vor rezidivierenden Meningitiden zu schutzen. Der Fallbericht zeigt erneut den Stellenwert der Komplementdiagnostik bei Patienten mit Meningokokkeninfekt und die Notwendigkeit, auch Patienten mit reduzierter, aber noch messbarer Komplementaktivitat einer weiteren Abklarung auf eine Komplementdefizienz zuzufuhren.

SummaryAutoantibodies against C1q have been described in many immune-complex diseases including hypocomplementaemic urticarial vasculitis and systemic lupus erythematosus (SLE). No study has focused on the role of anti-C1q antibodies in hepatitis C virus (HCV) infection. The aim of this study was (i) to evaluate the prevalence of anti-C1q antibodies in HCV infection; and (ii) to analyse the association of anti-C1q antibodies with clinical and biological features of HCV–mixed cryoglobulinaemia (MC) vasculitis. We searched for anti-C1q antibodies using an enzyme-linked immunosorbent assay (ELISA) test in 111 HCV patients (75 had cryoglobulin and 23 systemic vasculitis), 60 SLE patients and 109 blood donors. Anti-C1q antibodies were detected in 26% of HCV patients compared to 10% of healthy donors (P

Complement C2 receptor inhibitor trispanning (CRIT) inhibits the classical pathway (CP) C3 convertase formation by competing with C4b for the binding of C2. The C-terminal 11-amino-acid of the first CRIT-extracellular domain (CRIT-H17) has a strong homology with a sequence in the C4beta chain, which is responsible for the binding of C2. Since the CP and alternative pathway (AP) C3 convertases have many functional and structural similarities, we further investigated the effects of CRIT-H17 on the AP. The factor D-mediated cleavage of factor B (FB) was blocked by CRIT-H17. By ELISA and immunoblot, CRIT-H17 was shown to bind FB. CRIT-H17 had no decay activity on the C3bBb complex as compared to decay-accelerating factor. Binding of CRIT-H17 to FB did not interfere with the assembly of C3bB complex. In a haemolytic assay using C2-deficient serum, CRIT-H17 interfered with AP complement activation.

Release of microparticles (MPs) from blood cells may occur upon various activation signals. MPs from neutrophil and platelet have been studied in systemic infectious diseases and cardiovascular diseases, respectively. They are here investigated in common nephropathies including vasculitis and dialysis, two conditions characterized by neutrophil activation. Flow cytometry analysis of neutrophil-derived (CD66b-positive) and platelet-derived (CD41a-positive) MPs was performed on 213 plasma samples from patients with various nephropathies, including 46 patients with vasculitis and 40 hemodialysis patients. MPs released ex vivo, during neutrophil activation in whole blood, were also measured in these patients. Correlations with clinical parameters and creatinine clearance were evaluated. The results show that MPs present in plasma from patients or healthy controls are from various origins: platelet-derived (38+/-22%), neutrophil-derived (2.8+/-3.8%) MPs, mixed aggregates of neutrophil/platelet MPs (28+/-15%) or neither from neutrophil or platelet (null) 31+/-20%. Acute vasculitis showed the highest level of all types of MPs, while other nephropathies did not result in significant changes of MP levels. A significant increase was observed during hemodialysis sessions. In patients with renal failure, no correlation was seen between MP levels and creatinine clearance. In conclusion, neutrophil and platelet MP levels are non-specific markers of neutrophil activation during vasculitis acute phase and dialysis-induced inflammation. Circulating aggregates of neutrophil/platelet MPs co-express adhesion molecules of both cell types and may be thus endowed with inflammation and coagulation- thus modulating properties.

Complement C2 receptor inhibitor trispanning (CRIT) is a receptor for the second component of complement and is found in various tissues and hemopoietic cells. On binding to CRIT, C2 cannot be activated to potentially form a variant-C3 convertase as it is rendered non-cleavable by C1s. CRIT thus limits the amount of C3 convertase formed on the cell surface. In this study we have shown, using flow cytometry and immunofluorescence microscopy, that human CRIT undergoes endocytosis from the plasma membrane. The endocytosis, possibly ligand mediated, occurs via clathrin-coated pits as it can be inhibited by prior incubation of cells in hypertonic medium or with chlorpromazine, at 37 degrees C. However, inhibition of endocytosis was not possible after treatment with nystatin, or filipin, inhibitors of caveolae/raft-dependent endocytosis. In the presence of C2 alone, CRIT associates with the adapter protein, beta-arrestin-2, and whether in association with C2 or not, then appears in the perinuclear region, but does not appear to be translocated into the nucleus. Apart from the C3aR and C5aR that internalize the anaphylatoxic peptides, this is the first report of the internalization via the clathrin pathway of a receptor for a complement serum protein.

CRIT (complement C2 receptor inhibitor trispanning) is a newly described transmembrane molecule that is capable of binding C2 via its first extracellular domain (ed1). CRIT competes with C4b for the binding of C2. Previous experiments have suggested that a major binding site for C2 is located on short, almost identical peptide sequences of CRIT-ed1 and the beta-chain of C4. The C2 domains involved in binding, however, remain unknown. We cloned the vWFA (von Willebrand factor-A) domain of C2, as it is a region likely to be involved in interactions with other proteins, and were able to functionally express the 25 kDa human complement C2 vWFA domain (amino acids 224-437). The recombinant vWFA protein fixed on MagneHis Ni-Particles bound C4 in normal human serum. The C4 alpha, beta and gamma chains were separated by SDS/PAGE and purified separately by electro-elution. The purified C4 chains were then used in a sandwich ELISA, which showed the vWFA to bind C4 only via the C4beta chain. In a haemolytic assay, the recombinant vWFA protein inhibited complement activation by the classical pathway in a dose-dependent manner by competing with native C2 for binding to C4b. vWFA bound the ed1 peptide of CRIT as well, and specifically to the 11-amino-acid peptide fragment of ed1 that is known to interact with whole C2. These findings show that the vWFA domain is centrally involved in the C2-CRIT and C2-C4b bindings. The cloned vWFA domain will allow us to dissect out the fine interactions between C2 and CRIT or C4b.

The release of cell surface-derived microparticles, or ectosomes, has now been described for many different cell types. In various diseases characterized by systemic inflammation, the numbers of ectosomes released from specific cell-types are found increased manifold in the circulation. Their pro-inflammatory and pro-coagulant functions make them potentially important actors in disease establishment and/or progression. Until now, ectosomes have been believed to be free in the circulation. Herein, we provide evidence for sequestration of ectosomes derived from human polymorphonuclear neutrophils to erythrocytes, similarly to immune complexes. We show that ectosomes activate and bind complement in vitro. In whole blood, opsonization of ectosomes by complement mediated their immune adherence to erythrocytes through complement receptor 1. Taken together, our data suggest an important role for complement and erythrocytes in the sequestration, and possibly clearance, of blood-borne ectosomes stemming from neutrophils. The immune adherence described here may modify the biological activity and function of ectosomes.

The complement system presents a powerful defense against infection and is tightly regulated to prevent damage to self by functionally equivalent soluble and membrane regulators. We describe complement C2 receptor inhibitor trispanning (CRIT), a novel human complement regulatory receptor, expressed on hemopoietic cells and a wide range of tissues throughout the body. CRIT is present in human parasites through horizontal transmission. Serum complement component C2 binds to the N-terminal extracellular domain 1 of CRIT, which, in peptide form, blocks C3 convertase formation and complement-mediated inflammation. Unlike C1 inhibitor, which inhibits the cleavage of C4 and C2, CRIT only blocks C2 cleavage but, in so doing, shares with C1 inhibitor the same functional effect, of preventing classical pathway C3 convertase formation. Ab blockage of cellular CRIT reduces inhibition of cytolysis, indicating that CRIT is a novel complement regulator protecting autologous cells.

A fraction of HIV is associated with erythrocytes even when the virus becomes undetectable in plasma under antiretroviral therapy. The aim of the present work was to further characterize this association in vitro. We developed an in vitro model to study the factors involved in the adherence of HIV-1 to erythrocytes. Radiolabeled HIV-1 (HIV) and preformed HIV-1/anti-HIV immune complexes (HIV-IC) were opsonized in various human sera, purified using sucrose density gradient ultracentrifugation, and incubated with human erythrocytes. We observed that, when opsonized in normal human serum, not only HIV-IC, but also HIV, bound to erythrocytes, although the adherence of HIV was lower than that of HIV-IC. The adherence was abolished when the complement system was blocked, but was maintained in hypogammaglobulinemic sera. Complement-deficient sera indicated that both pathways of complement were important for optimal adherence. No adherence was seen in C1q-deficient serum, and the adherence of HIV was reduced when the alternative pathway was blocked using anti-factor D Abs. The adherence could be inhibited by an mAb against complement receptor 1. At supraphysiological concentrations, purified C1q mediated the binding of a small fraction of HIV and HIV-IC to erythrocytes. In conclusion, HIV-IC bound to erythrocytes as other types of IC do when exposed to complement. Of particular interest was that HIV alone bound also to erythrocytes in a complement/complement receptor 1-dependent manner. Thus, erythrocytes may not only deliver HIV-IC to organs susceptible to infection, but free HIV as well. This may play a crucial role in the progression of the primary infection.

DAF is a GPI-anchored protein expressed on all blood cells and most other cell types. This complement regulatory protein functions intrinsically in cell membranes to protect host cells from autologous complement attack. A soluble form is found in body fluids. In human urine, two forms of DAF have been described. Here we report that these two forms correspond to soluble DAF and to DAF bound onto urinary vesicles. With a newly established, highly sensitive ELISA, the proportion of these two forms could be quantified in healthy individuals and patients having renal disorders. This ELISA allowed us to measure DAF on blood cells and also in plasma. In contrast to urine, human plasma contained only soluble DAF when the plasma was thoroughly depleted of platelets. The concentration of soluble DAF in human serum was always lower than in the corresponding plasma, suggesting that this form might adhere to the clot during the coagulation process.

C3 nephritic factor (C3NeF) is an autoantibody against the C3 convertase which stabilizes this otherwise inherently labile neoenzyme and induces a continuous activation of the alternative pathway with C3 depletion. NeF is found in patients with membranoproliferative glomerulonephritis and/or partial lipodystrpohy. NeF activity is usually detected in plasma by hemolytic tests. In order to obtain reproducible data for the functional activity of purified C3NeF IgG a solid phase assay was developed. C3 convertase was generated on immobilized C3b by incubation with factors B and D in the presence of Ni(2+). Convertase sites were left to decay in the presence of normal IgG or NeF IgG. Residual convertase activity was measured by adding 125I-C3 and capturing nascent 125I-C3b on the plate surface via covalently coupled NH2-Glu-Tyr dipeptide. In the presence of factor H during C3 convertase decay, a dose dependent stabilizing activity was shown for NeF IgG including NeF IgG purified from urine. A second format of the assay was developed in which C3 convertase was assembled on C3b(2)-IgG complexes in the presence of Mg(2+). Since these complexes are more efficient as convertase precursors the signal was five-fold higher than with C3b. Convertase decay, on the other hand, was not influenced by the nature of the precursor and in both systems the stabilizing activity of NeF IgG was similar.

Obwohl fieberhafte Erkrankungen in der allgemeinen klinischen Praxis sehr häufig sind, fehlen heutzutage immer noch gute und zuverlässige Parameter, um die Ursache des Fiebers besser diagnostizieren zu können. Immer noch können wir für viele Erkrankungen keine exakte Aussage über die Notwendigkeit, die Dauer und den Zeitpunkt der Therapieeinleitung anhand von Laborwerten geben. Biomarker wie Procalcitonin, IL-6, IL-8 zeigen erste Lösungsansätze, allerdings gibt es weiterhin viele ungelöste Fragen.

Cryoproteins are defined as proteins precipitating at low temperature. Most frequently, the precipitate contains immunoglobulins (Igs), and are therefore called cryoglobulins. Three types of cryoglobulins have been described: type I contains a single monoclonal Ig, whereas type II is a mixture of a monoclonal Ig with polyclonal Igs, and type III is a mixture of polyclonal Igs of different isotypes, most frequently IgG and IgM. Type II and type III are also called mixed cryoglobulins. A new type of cryoglobulins, containing polyclonal IgG associated with a mixture of polyclonal and monoclonal IgM has recently been described after two-dimensional polyacrylamide gel electrophoresis (2-DE). This type of cryoglobulin has been called type II-III cryoglobulin. In this study, we report on 2-DE analysis of 335 cryoproteins from patients with heterogeneous clinical conditions. In 69 out of 335 samples (20.7%), 2-DE revealed patterns that were inadequate to characterize the cryoproteins. Out of 335 (79.3%) cryoproteins, 266 were identified according to their two-dimensional patterns: 265 samples contained Igs and were diagnosed as cryoglobulins, and one sample consisted of fibrinogen, and was identified as cryofibrinogen. Among the 265 cryoglobulins, types I, II, and III were observed in 9 (3.4%), 69 (26%), and 116 (43.8%) cases, respectively, whereas type II-III was detected in 71 (26.8%) cases. Eleven of the latter consisted of oligoclonal Igs (IgM in 10 cases, IgA in 1 case) mixed with traces of polyclonal IgG. These cryoproteins were tentatively named type II-IIIvariant cryoglobulins. Taken together, our result clearly show that 2-DE is a suitable technique to analyze cryoproteins.

Clusterin is a widely expressed, well conserved, secreted glycoprotein, which is highly induced in tissues regressing as a consequence of apoptotic cell death in vivo. It has recently been shown that clusterin expression is only confined to surviving cells following the induction of apoptosis in vitro, suggesting that it is involved in cell survival rather than death. In the hypothesis that clusterin may be implicated in cellular responses to stress, clusterin gene expression was analyzed in the A431 human epidermoid cancer cell line following heat shock and oxidative stress. Our results show that both a transient heat shock (20 min at 42 degrees C) and various oxidative stresses, including hydrogen peroxide, superoxide anion, hyperoxia and UVA exposure, induce a strong increase in clusterin mRNA levels as assessed by northern blot. Nuclear run-on analysis suggests that transcriptional activation is involved in inducing clusterin mRNA in response to heat shock. Using pulse-chase analysis of control and heat shocked cells, it is shown that clusterin mRNA is translated and secreted, thus resulting in increased extracellular levels of the protein following heat shock. To investigate the function of clusterin in response to these stresses, clusterin anti-sense transfectants that stably express virtually no clusterin at the mRNA and protein level were generated in A431 cells. These anti-sense transfectants are shown to be highly sensitive to apoptotic cell death induced by heat shock or oxidative stress compared with wild-type A431 cells or control transfectants. Taken together, our results show that clusterin gene expression is induced in response to heat shock and oxidative stress in human A431 cells, and confers cellular protection against heat shock and oxidative stress.

Cryoproteins are proteins precipitating at low temperature. Usually, the precipitate contains immunoglobulins (Igs), and are therefore called cryoglobulins. Very rarely, Igs do not precipitate, but, upon cooling, form a gel. Here, we report a case of cryogel observed in a patient presenting with Waldenström's disease. Using proteomic tools, a monoclonal IgM was identified as being the cause of the gel formation. Furthermore, addition of H(2)O before incubation at 4 degrees C demonstrated that the monoclonal IgM was precipitable as a type I cryoglobulin (hypocryoglobulin).

Objective. To investigate synovial fluid (SF) for the presence of CR1 and to study its relationship to SF leukocytes and to serum levels of soluble CR1 (sCR1) in patients with rheumatic diseases. Methods. Synovial fluids were collected from 35 patients with rheumatoid arthritis (RA) and 26 patients with other inflammatory joint diseases. Total CR1 in the SF and serum were measured with a sandwich enzyme-linked immunosorbent assay (ELISA) that recognized both soluble and transmembrane forms of CR1. The characteristics of CR1 in SF were analyzed by ultracen-trifugation and by a second ELISA specific for trans-membrane CR1. Results. CR1 was found in all SF samples tested (range 5-281 ng/ml). SF CR1 was higher in patients with RA (mean ± SD 81 ± 66 ng/ml) than in those with other inflammatory joint diseases (31.8 ± 23.8 ng/ml) (P < 0.001). Serum sCR1 was not significantly increased in the patients compared with the normal subjects. There was no correlation between serum sCR1 and SF CR1. In 44% of the patients, the SF CR1 level was higher than the serum sCR1 level. A fraction (30–80%) of SF CR1 was pelleted by ultracentrifugation and, unlike serum sCR1, it reacted in an ELISA specific for transmembrane CR1. Thus, SF contained 2 forms of CR1: a membrane-associated and a soluble form, which was confirmed by sucrose density-gradient ultracentrifugation. SF CR1 levels correlated directly with the number of SF total leukocytes and polymorphonuclear leukocytes (PMN). These 2 forms of CR1 were also found in the supernatant of in vitro—activated PMN from normal subjects. SF CR1 exhibited the capacity to act as a cofactor for the factor I degradation of C3b. Conclusion. CR1 is found in the SF of patients with joint inflammation. The data suggest that SF CR1 originates from the infiltrating leukocytes, which shed both a soluble and a membrane-associated form. Whether SF CR1 participates in the local regulation of complement activation remains to be examined.

Induction of plasminogen activator inhibitor type 1 in murine lupus-like glomerulonephritis. Three major components of the plasminogen activators (PA)/plasmin system are synthesized physiologically in glomeruli, and can be involved in glomerular proteolysis and extracellular matrix metabolism: tissue-type PA (tPA), urokinase (uPA) and PA inhibitor type 1 (PAI-1). To explore the possible role of a dysregulation of the plasmin protease system in the development and progression of lupus-like glomerulonephritis, we studied the expression of the renal plasmin protease components during the course of the disease, either acute, induced by IgG3 monoclonal cryoglobulins, or chronic, occurring spontaneously in three different lupus-prone mice: (NZBxNZW)F1, BXSB and MRL- lpr / lpr . RNase protection assays and in situ hybridizations revealed a marked glomerular induction of PAI-1 mRNA abundance without any significant changes in renal tPA and uPA mRNA levels in the two different types of lupus-like glomerulonephritis. The overexpression of PAI-1 mRNA occurred in parallel with a significant decrease in glomerular tPA-catalyzed enzymatic activity as determined by zymographic analysis. In addition, a concomitant increase in glomerular expression of transforming growth factor β1 (TGF-β1) mRNA was observed. The demonstration of a close correlation between the PAI-1 and TGF-β1 mRNA levels and the severity of lupus-like glomerular lesions suggests that a perturbation of the glomerular PA/PAI balance, resulting from a marked TGF-β1-mediated induction of PAI-1 gene expression, plays an important role in the progression of lupus-like glomerular lesions, leading to glomerulosclerosis.

Cryoglobulinemia induces an immune complex-mediated glomerulonephritis that is characterized by the presence of large immune deposits, including complement C3 and C5b-9, marked macrophage influx and mesangial cell proliferation. The precise role of complement in cryoglobulin-induced glomerulonephritis in humans remains unclear, whereas in mice there has been evidence that complement activation might be a central factor favoring glomerular inflammation, particularly by the recruitment of neutrophils. We report on an exceptional case of cryoglobulin-induced glomerulonephritis in a patient with mixed essential cryoglobulinemia type II. The clinical features included relapsing proteinuria and renal function impairment that were controlled by plasmapheresis. Complement was low in plasma and two renal biopsies at 1-year interval showed prominent immunoglobulin and complement deposits, with unusual high numbers of neutrophils. In a 1-patient clinical trial, we tested whether the monoclonal anti-C5 antibody eculizumab would be sufficient to control renal function at the time of a relapse. Although during the initial weeks renal function was stabilized, slow increase in creatinine could not be controlled by this treatment, so that plasmapheresis was reinstituted. This result suggests that despite evidence for a role of complement in enhancing renal damage in this patient, other inflammatory processes dominated.

Study of patients with complement deficiency has supplied important insights into the physiological importance of this component of the innate immune system. The most surprising finding to emerge from the study of such patients is the strong link between deficiencies of classical pathway proteins and susceptibility to SLE. This observation has stimulated many studies into the relationship between the complement system and processing mechanisms for immune complexes, which we have reviewed in this chapter. Although it is clear that complement deficiency is associated with several abnormalities of immune complex processing in vivo, the challenge still remains to provide a convincing link between these and the development of SLE.

The clonality of immunoglobulins (Igs) in cryoprecipitates (n = 41) was studied by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Our series included 24 cryoglobulins characterized by immunofixation electrophoresis (IF), 12 'trace amount' cryoglobulins, defined by a protein content in the precipitate of less than 0.05 mg/ml of serum, and five cryoglobulins of undetermined protein composition by IF. 2-D PAGE analysis showed polyclonal IgG associated either with monoclonal Igs (type II cryoglobulins; n = 14) or with polyclonal IgM (type III cryoglobulins; n = 14). In ten cryoprecipitates (two 'trace amount' cryoglobulins as well as seven of 19 type II and as one of five type III cryoglobulins by IF) polyclonal IgG were associated with a mixture of polyclonal and monoclonal IgM. These cryoglobulins were tentatively named type II-III cryoglobulins. A monoclonal IgM was observed in one cryoprecipitate (type I cryoglobulins). Two cryoglobulins presented unexpected 2-D patterns, characterized by the presence of oligoclonal IgM, with trace amounts of Igs of different isotypes (tentatively named type II-III(variant) cryoglobulins). A peptide of 44 kDa with a pI of 5.45 was observed in all cryoglobulins containing IgM (n = 40). This peptide was also present in purified monoclonal or polyclonal IgM fractions. N-terminal microsequencing (12 amino acid residues) revealed that this IgM-associated peptide was an unknown protein. Our results highlight the role of 2-D PAGE as an aid in the analysis of cryoglobulins.

Clusterin is a serum glycoprotein endowed with cell aggregating, complement inhibitory, and lipid binding properties, and is also considered as a specific marker of dying cells, its expression being increased in various tissues undergoing programmed cell death (PCD). However, no study has so far directly shown that cells expressing clusterin in these tissues are actually apoptotic as defined by morphological and biochemical criteria. We have studied cellular clusterin gene expression in vitro using three different models of PCD: (a) ultraviolet B (UV-B) irradiation of human U937, HeLa, and A431 cell lines, (b) in vitro aging of human peripheral blood neutrophils (PMNs), and (c) dexamethasone-induced cell death of the human lymphoblastoid cell line CEM-C7. In all three models, the classical morphological and biochemical features of PCD observed did not correlate with an increase, but with either a marked decrease or an absence of clusterin gene expression as assessed by Northern blot analysis. In situ hybridization of U937 and A431 cells after UV-B irradiation revealed, in addition, that only morphologically normal cells that are surviving continue to express the clusterin gene. Our results demonstrate that in the human myeloid, lymphoid, and epithelial cell types studied, clusterin gene expression is not a prerequisite to their death by apoptosis. In addition, and most interestingly, in situ hybridization of U937 and A431 cells revealed that only surviving cells express the clusterin gene after the induction of PCD, thus providing novel evidence suggesting that clusterin may be associated with cell survival within tissues regressing as a consequence of PCD.

Prophylactic oral anticoagulation in nephrotic patients with idiopathic membranous nephropathy. Whether the high incidence of thromboembolic events in nephrotic patients with membranous nephropathy justifies prophylactic administration of oral anticoagulants remains controversial. We used a Markov-based decision analysis model, explicitly considering the consequences of recurrent embolic and bleeding events to quantify the risk-benefit trade-offs of: (1) prophylactic therapy, in which oral anticoagulation was started at the time of diagnosis of nephrotic syndrome (before any thromboembolic event); and (2) anticoagulant therapy, in which treatment was started after the first clinical thromboembolic event. We assumed that anticoagulant therapy was discontinued if there was remission of the nephrotic syndrome. The overall number of fatal emboli prevented by prophylactic anticoagulants exceeded the one of fatal bleeding events for all clinically meaningful ranges of the following parameters: nephrotic syndrome duration, incidence of thromboembolic events, likelihood of embolization, and mortality rates of embolic and bleeding events. For a hypothetical 50-year-old patient who remained nephrotic for 2 years, prophylactic anticoagulation yielded a gain representing 2.5 months of quality-adjusted life expectancy. We conclude that for nephrotic patients with membranous nephropathy, the benefits of prophylactic administration or oral anticoagulants outweigh the risks.

One of the characteristics of systemic lupus erythematosus (SLE) is the large inter- and intra-individual variability of the clinical course. Lupus nephritis is no exception. Some patients with kidney involvement may show rapid progression to renal failure, while others may enter complete and stable remission after adequate therapy. More difficult to manage are the large number of patients who have similar clinical and histological patterns at presentation, but alternate periods of clinical quiescence with renal relapses of different severity. It is still uncertain which, if any, immunologic parameters may help to diagnose a renal flare. The increase in anti double-stranded DNA (dsDNA) titre or hypocomplementaemia related to classical pathway activation provides no indication as to whether a relapse includes the kidney. Here we review the evidence, which has accumulated over the last few years and appears to indicate that antiC1q autoantibodies (antiC1q Ab) may help in distinguishing a renal from a non-renal relapse under certain circumstances.

Cryoglobulinemia was identified and named many years ago by clinicians who related the in vitro precipitation of globulins from serum incubated in the cold with diseases. One major point was that there was no evidence for a known disease in many patients with “mixed” cryoglobulins. Thus, the terminology “mixed essential cryoglobulinemia” became accepted. The discovery of HCV as the causative agent of mixed essential cryoglobulinemia allowed not only the introduction of a new nomenclature (largely removing the designation “essential”), but studies of the relationship between this viral disease and the emergence of pathological clones of B cells.

The contact of blood with some biomaterials results in complement activation, primarily by the alternative pathway (AP). Insoluble polystyrene derivatives bearing isolated sulphonate groups (PSSO3) deplete complement, whereas identical surfaces substituted with both sulphonate and hydroxymethyl groups (PSCH2OH-SO3) are non-activators. Polystyrene sulphonate derivatives possess high adsorptive properties, particularly for serine proteases of the coagulation cascade. Thus, we studied the interactions between polystyrene derivatives and factor D, an enzyme essential for AP activation. C3 was activated when normal human serum (NHS) was incubated with PSSO3, whereas PSCH2OH-SO3 did not induce any specific C3 activation. Both polymers adsorbed factor D from serum, as shown by the loss of haemolytic factor D from NHS incubated with the polymers and by the specific adsorption of radiolabelled factor D. When bound to the polymers, factor D was not functional. The disappearance of factor D was in contradiction to the observed complement activation induced by PSSO3. When other AP components were studied, it was evident that PSSO3 adsorbed factor H even more rapidly and efficiently than factor D. Thus, the net effect was an immediate deregulation of the AP resulting in C3 activation, followed by inhibition of the AP when factor D was finally depleted. Pre-exposure of PSSO3 to NHS prevented any complement activation because the polymer was saturated with factor H, but still adsorbed factor D. Such properties could be beneficial during haemodialysis with membranes for uremic patients who have increased levels of factor D in their serum.

Mouse adipsin is a serine protease secreted mainly by adipocytes. Similarly to factor D of human complement, it cleaves factor B. That adipsin is the equivalent of human factor D in the mouse is further suggested by their structural homology. Specific antisera against recombinant mouse adipsin (r-adipsin) were produced in rabbits. Anti-r-adipsin IgG was shown to bind to radiolabeled r-adipsin and to inhibit its hemolytic activity. In vitro, these antibodies Ab and Fab fragments thereof inhibited the adipsin/factor D hemolytic activity of mouse serum. They also blocked C3 activation induced by cobra venom factor (CVF), but did not interfere with classical pathway function. After intravenous injection of anti-r-adipsin Fab into BALB/c mice, the adipsin/factor D hemolytic activity of serum was abolished during a 4-h period. The C3 depleting effect of CVF injected intravenously was significantly delayed in BALB/c mice which had been pretreated with anti-r-adipsin Fab. These experiments demonstrate that mouse adipsin is the only form of mouse factor D and that anti-r-adipsin antibody can be used to produce a specific inhibition of the alternative pathway in vivo.

Despite the fact that the appearance of fever is related to certain diseases, in general medical practice there are still no good and reliable parameters to specify the underlying cause of fever. There is still a lot of insecurity regarding urgency, necessity, duration and form of treatment for many diseases because of the lack of reliable markers.Biomarkers like Procalcitonin, IL-6 and IL-8 show first solutions but there are still many unanswered questions.

Considerable progress has been made in recognizing microvesicles as important mediators of intercellular communication rather than irrelevant cell debris. Microvesicles released by budding directly from the cell membrane surface (i.e., ectocytosis) either spontaneously or in response to various stimuli are called shed vesicles or ectosomes. Ectosomes are rightside-out vesicles with cytosolic content, and they expose phosphatidylserine in the outer leaflet of their membrane. Depending on their cellular origin, ectosomes have been associated with a broad spectrum of biological activities. In the light of recent findings, we now know that ectosomes derived from polymorphonuclear leukocytes, erythrocytes, platelets, and tumor cells have profound effects on the innate immune system, as well as on the induction of the adaptive immunity, globally reprogramming cells such as macrophages or dendritic cells toward an immunosuppressive and possibly tolerogenic phenotype. Although the effects observed in the circulation are mainly procoagulant and pro-inflammatory, ectosomes might be anti-inflammatory/immunosuppressive in local inflammation.

Atypical hemolytic uremic syndrome (aHUS) is a rare disease of microangiopathic hemolytic anemia, thrombocytopenia, and predominant renal impairment. It is characterized by the absence of Shiga toxin-producing bacteria as a triggering factor. During the last decade, aHUS has been demonstrated to be a disorder of the complement alternative pathway dysregulation, as there is a growing list of mutations and polymorphisms in the genes encoding the complement regulatory proteins that alone or in combination may lead to aHUS. Approximately 60% of aHUS patients have so-called ‘loss-of-function’ mutations in the genes encoding the complement regulatory proteins, which normally protect host cells from complement activation: complement factor H (CFH), factor I (CFI) and membrane cofactor protein (MCP or CD46), or have ‘gain-of-function’ mutations in the genes encoding the complement factor B or C3. In addition, approximately 10% of aHUS patients have a functional CFH deficiency due to anti-CFH antibodies. Recent advances in understanding the pathogenesis of aHUS have led to a revised classification of the syndrome. Normal plasma levels of CFH and CFI do not preclude the presence of a mutation in these genes. Further, genotype-phenotype correlations of aHUS have clinical significance in predicting renal recovery and transplant outcome. Therefore, it is important to make a comprehensive analysis and perform genetic screening of the complement system in patients with aHUS to allow a more precise approach, especially before transplantation. This may also provide opportunities for more specific treatments in the near future, as complement inhibition could represent a therapeutic target in these patients who have a considerably poor prognosis in terms of both mortality and progression to end-stage renal disease and a great risk of disease recurrence after transplantation.

Clusterin is a multifunctional protein endowed with cell-aggregating, complement-inhibitory, and lipid-binding properties. Since several studies have demonstrated highly increased clusterin gene expression in epithelial and nervous tissues regressing as a consequence of tissue involution and apoptotic cell death, clusterin is also considered as a specific marker of dying cells. To determine whether clusterin expression is also upregulated during thymocyte death occurring during the negative selection process we analyzed the cellular distribution of clusterin mRNA and protein by in situ hybridization and immunocytochemistry in the human thymus. We observed that the expression of clusterin mRNA was confined to cells present in the thymic medulla, concentrated mainly around Hassal's bodies. Immunostaining of adjacent sections with antikeratin Ab revealed that cells containing clusterin mRNA were predominantly epithelial. By contrast no clusterin mRNA was found in thymocytes by in situ hybridization and Northern blot analysis of total RNA from purified thymocyte populations. Clusterin protein colocalized with the membrane attack complex of complement and vitronectin in the center of the largest Hassal's bodies, but was not detectable by immunocytochemistry in or at the surface of epithelial cells. Our results demonstrate that clusterin gene expression does not take place in apoptotic thymocytes, and therefore that clusterin synthesis by the dying cell is probably not a prerequisite to its death. However, synthesis of clusterin by medullary epithelial cells may be related to their terminal differentiation, and, furthermore, its presence in Hassal's bodies raises the possibility that the secreted protein is involved in the disposal of cell debris resulting from thymocyte apoptosis.