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Anja Roos

Publications and source records attributed to Anja Roos.

46 records · Page 3Linked to original sources
PubMed2003

Functional characterization of the lectin pathway of complement in human serum.

Mannan-binding lectin (MBL) is a major initiator of the lectin pathway (LP) of complement. Polymorphisms in exon 1 of the MBL gene are associated with impaired MBL function and infections. Functional assays to assess the activity of the classical pathway (CP) and the alternative pathway (AP) of complement in serum are broadly used in patient diagnostics. We have now developed a functional LP assay that enables the specific quantification of autologous MBL-dependent complement activation in human serum. Complement activation was assessed by ELISA using coated mannan to assess the LP and coated IgM to assess the CP. Normal human serum (NHS) contains IgG, IgA and IgM antibodies against mannan, as shown by ELISA. These antibodies are likely to induce CP activation. Using C1q-blocking and MBL-blocking mAb, it was confirmed that both the LP and the CP contribute to complement activation by mannan. In order to quantify LP activity without interference of the CP, LP activity was measured in serum in the presence of C1q-blocking Ab. Activation of serum on coated IgM via the CP resulted in a dose-dependent deposition of C1q, C4, C3, and C5b-9. This activation and subsequent complement deposition was completely inhibited by the C1q-blocking mAb 2204 and by polyclonal Fab anti-C1q Ab. Evaluation of the LP in the presence of mAb 2204 showed a strong dose-dependent deposition of C4, C3, and C5b-9 using serum from MBL-wildtype (AA) but not MBL-mutant donors (AB or BB genotype), indicating that complement activation under these conditions is MBL-dependent and C1q-independent. Donors with different MBL genotypes were identified using a newly developed oligonucleotide ligation assay (OLA) for detection of MBL exon 1 polymorphisms. We describe a novel functional assay that enables quantification of autologous complement activation via the LP in full human serum up to the formation of the membrane attack complex. This assay offers novel possibilities for patient diagnostics as well as for the study of disease association with the LP.

Antibodies↗
PubMed2003

IgM-enriched human introvenous immunoglobulin strongly inhibits complement-dependent porcine cell cytotoxicity mediated by human xenoreactive antibodies.

Treatment with intravenous immunoglobulin preparations consisting of human IgG (IVIgG) prevents hyperacute rejection of pig xenografts transplanted into primates by inhibition of the classical complement pathway. Recent studies indicate that IVIg preparations mainly consisting of human IgM (IVIgM) have a stronger capacity than IVIgG to inhibit the complement system. IVIg preparations also contain xenoreactive antibodies (XAb) binding to pig cells. In the present study, we compared IVIgG and IVIgM for their capacity to inhibit xenogeneic complement activation, with special reference to the roles of IgG and IgM XAb present in these preparations. Xenogeneic complement activation was studied by exposure of pig cells (PK15) to human serum. For some experiments, IVIgG and IVIgM were depleted from XAb by immune absorption. Exposure of PK15 cells to human serum induced surface deposition of C4 and C3 and cytotoxicity, which could be inhibited in a dose-dependent fashion by both IVIgM and IVIgG. The efficacy of IVIgM was more than 10 times higher than that of IVIgG. IgG XAb were detected IVIgG and IVIgM whereas IgM XAb were only present in IVIgM. Depletion of XAb from the IVIg preparations did not modify the protective properties of IVIgG against cytotoxicity induced by human serum, whereas the IVIgM-mediated protection against xenogeneic cytotoxicity was only slightly improved. IgM-enriched IVIg is a potent inhibitor of xenogeneic complement activation and complement-dependent cytotoxicity of human serum to pig cells, irrespective of the presence of cytotoxic xenoreactive IgM antibodies in this preparation. Therefore, IVIgM has a promising therapeutic significance for the treatment of (hyper)acute xenograft rejection.

Animals↗
PubMed2002

The membrane attack complex of complement induces caspase activation and apoptosis.

Activation of the terminal pathway of the complement system leads to insertion of terminal complement complexes (C5b-9) into the cell membrane, which may induce cytolysis. Recent data indicate that the terminal complement pathway can also result in apoptosis in vivo. To further define the cell death pathway induced by complement, we examined induction of apoptosis by complement in vitro. Rat mesangial cells opsonized with a complement-activating antibody and exposed to rat serum as a complement source underwent apoptotic cell death in a time- and dose-dependent fashion, as demonstrated by membrane exposure of phosphatidylserine and fragmentation of nuclei. No significant apoptosis was detected in either cultures treated with C6-deficient serum or in control cultures. The pan-caspase-inhibitor zVAD-fmk inhibited complement-induced apoptosis completely. In line with this observation, complement induced cleavage and activation of caspase 3. Importantly, cellular exposure to purified cytolytically inactive C5b-9, in the absence of antibody and early complement components, also resulted into caspase activation and apoptosis. Together, these results indicate that C5b-9 is involved in induction of apoptosis via a caspase-dependent pathway. Apoptosis as a consequence of complement-mediated cell damage may provide an explanation for the presence of apoptosis in inflammatory processes, for instance in hyperacute xenograft rejection.

Amino Acid Chloromethyl Ketones↗
PubMed2002

Direct binding of C1q to apoptotic cells and cell blebs induces complement activation.

Deficiency of early components of the classical pathway of complement, particularly C1q, predisposes to the development of systemic lupus erythematosus. Several studies have suggested an association between the classical complement pathway and the clearance of apoptotic cells. Mice with a targeted deletion of the C1q gene develop a lupus-like renal disease, which is associated with the presence of multiple apoptotic bodies in the kidney. In the present study we demonstrate that highly purified C1q binds to apoptotic cells and isolated blebs derived from these apoptotic cells. Binding of C1q to apoptotic cells occurs via the globular heads of C1q and induces activation of the classical complement pathway, as shown by the deposition of C4 and C3 on the surface of these cells and on cell-derived blebs. In addition, for the first time, we demonstrate that surface-bound C1q is present on a subpopulation of microparticles isolated from human plasma. Taken together, these observations demonstrate that C1q binds directly to apoptotic cells and blebs derived therefrom and support a role for C1q, possibly in concert with C4 and C3, in the clearance of apoptotic cells and blebs by the phagocytic system.

Apoptosis↗
PubMed2002

Antibody-mediated activation of the classical complement pathway in xenograft rejection.

Transplant rejection is a multifactorial process involving complex interactions between components of the innate and the acquired immune system. In view of the shortage of donor organs available for transplantation, xenotransplantation of pig organs into man has been considered as a potential solution. However, in comparison to allografts, xenografts are subject to extremely potent rejection processes that are currently incompletely defined. Consequently, an appropriate and safe treatment protocol ensuring long-term graft survival is not yet available. The first barrier that has to be taken for a xenograft is hyperacute rejection, a rapid process induced by the binding of pre-formed antibodies from the host to the graft endothelium, followed by activation of the classical complement pathway. The present review concentrates on the role of antibodies and complement in xenograft rejection as well as on the approaches for treatment that target these components. The first part focuses on porcine xenoantigens that are recognized by human xenoreactive antibodies and the different treatment strategies that aim on interference in antibody binding. The second part of the review deals with complement activation by xenoreactive antibodies, and summarizes the role of complement in the induction of endothelial cell damage and cell activation. Finally, various options that are currently under development for complement inhibition are discussed, with special reference to the specific inhibition of the classical complement pathway by soluble complement inhibitors.

Animals↗
PubMed2002

Activation of complement pathways in xenotransplantation: an in vitro study.

Pig-to-human xenotransplantation faces the problem of hyperacute graft rejection due to the presence of human naturally occurring antibodies against the disaccharide Galalpha1-3Gal (anti-Gal antibodies) expressed on pig endothelium. Antibody-mediated complement activation is usually referred to as classical pathway activation. In this study we examined if the alternative complement pathway is also directly activated through anti-Gal antibodies or if the classical pathway is indispensable. We therefore developed a hemolysis test with rabbit erythrocytes (E), which have an activating surface for the alternative complement pathway and express abundant amounts of Galalpha1-3Gal, and used this assay in addition to the standard complement tests CH50 and AP50. In this rabbit E CH50 (RECH50) assay we were able to study activation of both major complement pathways simultaneously. FACS analysis was used to trace complement and antibody deposition on rabbit E. Anti-Gal depletion of human serum by immunoabsorption revealed a 65% reduction of rabbit E hemolysis in the RECH50 test (value before absorption: 28 +/- 5.8, after absorption: 9.9 +/- 2.8, P<0.001), but only a 35% reduction of lysis in the AP50 test (AP50 before 11.3 +/- 2.1, after 7.4 +/- 2.0, P<0.002). Repletion with purified anti-Gal fully restored hemolysis in both assays. Serum depleted of Clq showed a reduced lysis of rabbit E as compared to normal human serum; this effect increased with higher serum dilutions. The reciprocal picture, i.e. less effect on hemolysis with increasing dilution, was seen with factor D depleted serum. Comparison of the RECH50 values with the AP50 values revealed an 8.4-fold increase of lysis in the RECH50 test, in which both complement pathways are running. By FACS analysis, complement deposition on rabbit E was determined and components of the classical pathway were found, especially in sera where the alternative pathway was disrupted. We conclude that in our model anti-Gal induce lysis via both classical and alternative complement pathways, but that the alternative pathway activation is of minor importance. In addition, we saw that with higher serum dilutions, the classical pathway (i.e. anti-Gal-mediated lysis) takes a predominant role in lysing the rabbit E. As anti-Gal-mediated activation of the alternative complement cascade seems of minor importance based on our results, and as there are only few surfaces in transplanted organs that would favor the alternative pathway to be executed, the specific inhibition of early steps of the classical pathway appears as a realistic strategy in pig-to-primate xenotransplantation that-to the benefit of the patient-leaves the mainly anti-bacterial defense by the alternative pathway intact.

Animals↗
PubMed2002

Therapeutic inhibition of the early phase of complement activation.

The complement system is a key component of innate immunity against invading pathogens. However, undesired activation of complement is involved in inflammation and associated tissue damage in a number of pathological conditions, such as ischemia/reperfusion injury, autoimmune diseases, and rejection of allo- and xenografts. During recent years, various therapeutically active complement inhibitors have been developed. In vivo studies using these inhibitors underscored the value of complement inhibition in the prevention of tissue damage. The currently available complement inhibitors mainly target the effector phase of the complement system that is common to all three activation pathways. Such a complete block of complement activation breaks the innate anti-microbial barrier, thereby increasing the risk for infection. Therefore, the development of potent complement inhibitors that interfere in the recognition phase of a specific complement activation pathway will generate important novel possibilities for treatment. The present review is focused on molecules that are able to inhibit the function of C1q and MBL, the recognition units of the classical pathway and the lectin pathway of complement, respectively. The potential value of these molecules for the development of therapeutically active complement inhibitors is discussed.

Animals↗
PubMed2000

Strong and selective glomerular localization of CD134 ligand and TNF receptor-1 in proliferative lupus nephritis.

CD134 (OX40) is a member of the tumor necrosis factor (TNF) receptor (TNFR) family that can be expressed on activated T lymphocytes. Interaction between CD134 and its ligand (CD134L) is involved in costimulation of T and B lymphocyte activation, and in T cell adhesion to endothelium. To examine the possible role of this interaction in the pathogenesis of systemic lupus erythematosus (SLE), expression of CD134 and CD134L on peripheral blood leukocytes was studied, and no significant differences between SLE patients and control individuals were found. Immunohistology on renal biopsies from patients with lupus nephritis or other renal disorders, using a recombinant human CD134-containing chimeric molecule to detect CD134L, demonstrated the abundant presence of CD134L in all cases of proliferative lupus nephritis in a granular distribution predominantly along the epithelial side of the glomerular capillary wall. Confocal laser scanning microscopy indicated colocalization with subepithelial immune deposits. In none of the other renal disorders examined, including nonproliferative forms of lupus nephritis, was glomerular staining for CD134L detected in a similar pattern. Endothelial CD134L expression was frequently observed in different types of vasculitis. CD134 was detected on perivascular infiltrating leukocytes and on part of the tubular epithelium, but not on glomerular resident cells. Immunohistology for several other TNF(R) family members revealed in proliferative lupus nephritis a similar distribution for TNFR1 as was observed for CD134L. In contrast, glomerular expression of TNFR2 was similar in all cases examined. The glomerular presence of CD134L and TNFR1 in proliferative lupus nephritis in association with subepithelial immune deposits may be of pathogenetic significance and have diagnostic value.

Adult↗
PubMedDate not supplied

Role of complement in innate and autoimmunity.

The complement system is a major driver of our innate immune response and plays an important role in defence against foreign pathogens. It is composed of a large number of proteins that are found in the circulation, in tissues and other body fluids in pro-enzymatic form. The many biologic activities of the complement system are uncovered following activation by three known pathways. Together with phagocytic cells the complement system is a powerful tool to defend the host against foreign attack. However the complement system, under certain conditions, can be unfavorable to the host namely in diseases such as IgA nephropathy, systemic lupus erythematosus and in diseases where minor changes have occurred in the integrity of tissue such as in ischemia reperfusion. This review describes the many faces of complement in the field of innate and acquired immunity and takes the reader on a journey of the role of complement in health and disease.

Animals↗