Monica Schaller

Autoantibodies to GPI in rheumatoid arthritis: linkage between an animal model and human disease

In K/BxN T cell receptor–transgenic mice, spontaneous inflammatory arthritis exhibiting many of the features of human rheumatoid arthritis (RA) is initiated by T cells, but is almost entirely sustained by antibodies to the self-antigen glucose-6-phosphate isomerase (GPI). The relevance of these observations to human disease has been questioned. Here we show that 64% of humans with RA, but not controls, had increased concentrations of anti-GPI immunoglobulin G (IgG) in serum and synovial fluid. In addition, the concentrations of soluble GPI in the sera and synovial fluids of RA patients were also elevated, which led to immune complex formation. Using phage-display methods, we cloned a panel of specific high-affinity human monoclonal anti-GPI IgGs from a patient with RA. These antibodies were highly somatically mutated, which was indicative of an affinity-matured response that was antigen driven. Immunohistochemistry of RA synovium showed high concentrations of GPI on the surface of the synovial lining and on the endothelial cell surface of arterioles; this indicated a mechanism by which antibodies to GPI may precipitate joint disease. The results indicate that the immunological events that lead to the development of autoimmune disease in the K/BxN mouse model may also occur in human RA. This data may be used to develop new strategies for therapeutic intervention.

Toward Molecular Dissection of PrPC-PrPSc Interactions

Direct interaction between endogenous cellular prion protein (PrPC) and misfolded, disease-associated (PrPSc) conformers is a key event in prion propagation, which precedes templated conversion of PrPC into nascent PrPSc and prion infectivity. Although almost none of the molecular details of this pivotal process are understood, the persistence of individual prion strains suggests that assembly of the prion replicative complex is mechanistically precise. To systematically map defined regions of PrPC sequence that bind tightly to PrPSc, we have generated a comprehensive panel of over 45 motif-grafted antibodies containing overlapping peptide grafts collectively spanning PrP residues 19–231. Grafted antibody binding experiments, performed under stringent conditions, clearly identified only three distinct and independent high affinity PrPSc recognition motifs. The first of these binding motifs lies at the very N-terminal region of the mature PrP molecule within PrP-(23–33); the second motif lies within PrP-(98–110); and the third is contained within PrP-(136–158). Mutational analyses of these PrPSc-binding regions revealed that reactivity of the 23–33 and 98–110 segments are largely dependent upon the presence of multiple positively charged amino acid residues. These studies yield new insight into critical peptidic components composing one side of the prion replicative interface.

Autoantibodies against Complement C1q Specifically Target C1q Bound on Early Apoptotic Cells

Autoantibodies against complement C1q (anti-C1q) are frequently found in patients with systemic lupus erythematosus (SLE). They strongly correlate with the occurrence of severe lupus nephritis, suggesting a pathogenic role in SLE. Because anti-C1q are known to recognize a neoepitope on bound C1q, but not on fluid-phase C1q, the aim of this study was to clarify the origin of anti-C1q by determining the mechanism that renders C1q antigenic. We investigated anti-C1q from serum and purified total IgG of patients with SLE and hypocomplementemic urticarial vasculitis as well as two monoclonal human anti-C1q Fab from a SLE patient generated by phage display. Binding characteristics, such as their ability to recognize C1q bound on different classes of Igs, on immune complexes, and on cells undergoing apoptosis, were analyzed. Interestingly, anti-C1q did not bind to C1q bound on Igs or immune complexes. Neither did we observe specific binding of anti-C1q to C1q bound on late apoptotic/necrotic cells when compared with binding in the absence of C1q. However, as shown by FACS analysis and confocal microscopy, anti-C1q specifically targeted C1q bound on early apoptotic cells. Anti-C1q were found to specifically target C1q bound on cells undergoing apoptosis. Our observations suggest that early apoptotic cells are a major target of the autoimmune response in SLE and provide a direct link between human SLE, apoptosis, and C1q.

Pathologic Prion Protein Infects Cells by Lipid-Raft Dependent Macropinocytosis

Transmissible spongiform encephalopathies, including variant-Creutzfeldt-Jakob disease (vCJD) in humans and bovine spongiform encephalopathies in cattle, are fatal neurodegenerative disorders characterized by protein misfolding of the host cellular prion protein (PrPC) to the infectious scrapie form (PrPSc). However, the mechanism that exogenous PrPSc infects cells and where pathologic conversion of PrPC to the PrPSc form occurs remains uncertain. Here we report that similar to the mechanism of HIV-1 TAT-mediated peptide transduction, processed mature, full length PrP contains a conserved N-terminal cationic domain that stimulates cellular uptake by lipid raft-dependent, macropinocytosis. Inhibition of macropinocytosis by three independent means prevented cellular uptake of recombinant PrP; however, it did not affect recombinant PrP cell surface association. In addition, fusion of the cationic N-terminal PrP domain to a Cre recombinase reporter protein was sufficient to promote both cellular uptake and escape from the macropinosomes into the cytoplasm. Inhibition of macropinocytosis was sufficient to prevent conversion of PrPC to the pathologic PrPSc form in N2a cells exposed to strain RML PrPSc infected brain homogenates, suggesting that a critical determinant of PrPC conversion occurs following macropinocytotic internalization and not through mere membrane association. Taken together, these observations provide a cellular mechanism that exogenous pathological PrPSc infects cells by lipid raft dependent, macropinocytosis.

Antinucleosome Antibodies as a Marker of Active Proliferative Lupus Nephritis

BACKGROUND Antinucleosome autoantibodies were previously described to be a marker of active lupus nephritis. However, the true prevalence of antinucleosome antibodies at the time of active proliferative lupus nephritis has not been well established. Therefore, the aim of this study is to define the prevalence and diagnostic value of autoantibodies against nucleosomes as a marker for active proliferative lupus nephritis. STUDY DESIGN Prospective multicenter diagnostic test study. SETTING & PARTICIPANTS 35 adult patients with systemic lupus erythematosus (SLE) at the time of the renal biopsy showing active class III or IV lupus nephritis compared with 59 control patients with SLE. INDEX TEST Levels of antinucleosome antibodies and anti-double-stranded DNA (anti-dsDNA) antibodies. REFERENCE TEST Kidney biopsy findings of class III or IV lupus nephritis at the time of sampling in a study population versus clinically inactive or no nephritis in a control population. RESULTS Increased concentrations of antinucleosome antibodies were found in 31 of 35 patients (89%) with active proliferative lupus nephritis compared with 47 of 59 control patients (80%) with SLE. No significant difference between the 2 groups with regard to number of positive patients (P = 0.2) or antibody concentrations (P = 0.2) could be found. The area under the receiver operating characteristic curve as a marker of the accuracy of the test in discriminating between proliferative lupus nephritis and inactive/no nephritis in patients with SLE was 0.581 (95% confidence interval, 0.47 to 0.70; P = 0.2). Increased concentrations of anti-dsDNA antibodies were found in 33 of 35 patients (94.3%) with active proliferative lupus nephritis compared with 49 of 58 control patients (84.5%) with SLE (P = 0.2). In patients with proliferative lupus nephritis, significantly higher titers of anti-dsDNA antibodies were detected compared with control patients with SLE (P < 0.001). The area under the receiver operating characteristic curve in discriminating between proliferative lupus nephritis and inactive/no nephritis in patients with SLE was 0.710 (95% confidence interval, 0.60 to 0.82; P < 0.001). CONCLUSIONS Antinucleosome antibodies have a high prevalence in patients with severe lupus nephritis. However, our data suggest that determining antinucleosome antibodies is of limited help in the distinction of patients with active proliferative lupus nephritis from patients with SLE without active renal disease.

Autoantibodies against C1q in systemic lupus erythematosus are antigen-driven

Autoantibodies against complement C1q (anti-C1q Abs) were shown to strongly correlate with the occurrence of severe nephritis in patients with systemic lupus erythematosus (SLE), suggesting a potential pathogenic role by interfering with the complement cascade. To analyze the humoral immune response against C1q at the molecular level, we screened a bone marrow-derived IgGκ/IgGλ Fab phage display library from a SLE patient with high anti-C1q Ab titer against purified human C1q. Six Fabs that exhibited strong binding to C1q in ELISA were isolated. The anti-C1q Fabs recognized neoepitopes that were only exposed on bound C1q and not present on soluble C1q mapping to different regions of the collagen-like region of C1q. Analysis of the genes encoding the variable H and L chains of the IgG-derived anti-C1q Fab revealed that all the variable H and L chain regions were highly mutated, with nucleotide and amino acid homologies to the closest germline in the range of 71–97% (average 85 ± 4) and 72–92% (average 88 ± 6), respectively. In addition, the variable region of the Fabs exhibited high replacement to silent ratios. The six anti-C1q Fabs were shown to be of high affinity, with a Kd ranging from of 8.4 × 10−8 M to 1.4 × 10−7 M, comparable to an antiviral immune response. Our data underlines the notion that the development of anti-C1q Abs in SLE is the consequence of an Ag-driven, affinity-matured immune response. Those anti-C1q Fabs are unique tools to address how complement C1q is implicated in the pathogenesis of SLE.

Anti-C1q autoantibodies do not correlate with the occurrence or severity of experimental lupus nephritis

BACKGROUND In systemic lupus erythematosus patients, a strong association between the occurrence of antibodies against complement C1q (anti-C1q) and lupus nephritis can be observed. However, the predictive value of anti-C1q titres for a renal flare remains to be determined. Increasing titres of anti-C1q before the occurrence of clinical apparent nephritis might not only serve as a clinical parameter but also indicate a direct pathogenic mechanism of anti-C1q. METHODS The aim of this study was to analyse the occurrence of anti-C1q before the onset of experimental lupus nephritis in MRL/MpJ +/+ mice and to correlate anti-C1q titres with the type and severity of glomerulonephritis (GN) developing at advanced age. RESULTS As judged by a number of morphological and immunological analyses, GN in MRL/MpJ +/+ mice resembled human lupus nephritis and occurred in variable degrees of severity. We also observed an abundant and early presence of anti-C1q. However, anti-C1q neither correlated with overall survival nor with any histological marker of severity of GN. CONCLUSIONS The absence of a correlation between the presence of anti-C1q and the occurrence of experimental lupus nephritis contradicts the hypothesis that anti-C1q are pathogenic. However, different pathogenic mechanisms of experimental lupus nephritis and human proliferative lupus nephritis cannot be excluded.

Mononuclear cells ingest E. coli opsonized by investigational intravenous immunoglobulin preparations in the absence of complement more efficiently than polymorphonuclear phagocytes.

OBJECTIVE The increasing use of intravenous immunoglobulins (IVIg) to treat systemic inflammatory, mostly autoimmune, diseases raises the concern that their competition for FcgammaReceptor (FcgammaR) binding with immune complexes might interfere with the removal of opsonized microbial agents more than it favoured their opsonophagocytosis. METHODS Using a sensitive FACS-based phagocytosis assay allowing for the identification of the phagocytosing cell-type, we have tested the relative capacity of Pentaglobin and two investigational IVIg preparations, IVIgG and IVIgM, to promote E. coli phagocytosis. The plasma IL-8 levels 4 h after phagocytosis were measured by ELISA. RESULTS Heat inactivated, complement (C) depleted serum allowed about 25% polymorphonuclear leukocytes (PMNLs) and 15% monocytes (MNs) of 16 donors to phagocyte E. coli. The addition of IVIg favoured mean phagocytosis by MNs to 55%, but by PMNLs only to 25%. The addition of fresh autologous plasma as a C source resulted in a boost of phagocytosing capacity to reach >90% of the PMNLs and >72% of the MNs. Blockade of FcgammaRII and FcgammaRI using monoclonal antibodies (mAb) reduced phagocytosis and IL-8 release. CONCLUSION We conclude that excess IVIgG/M preparations added to a phagocytic system of PMNLs and MNs, without preopsonization of E. coli, promotes phagocytosis as expected, but MNs rather than PMNLs do this more thoroughly on their own.

In-process cell counts during preparation of platelet concentrates from buffy coats.

To ensure the quality of platelet concentrates (PCs), we studied in-process recoveries of blood cell counts in pooled PCs derived from four or five buffy coats (BCs) from Biopack Compoflex Systems in Bern (PC-BC/4 or PC-BC/5) and from five BCs from Optipac (Baxter) in Zurich (PC-BC/5). BCs were pooled employing a sterile connecting device and flushing them with 300 mL of platelet additive solution. The pools were centrifuged for 12 min at 500 g at 20 degrees C and filtered with PALLs Auto-Stop BC-leukocyte removal filter. Automated platelet counting was performed on whole blood donation, on single BC, on pooled BC and in the final product. Four out of 10 PC-BC/4 (= 40%) and 29 out of 30 PC-BC/5 (= 97%) had a total platelet count of > 200 x 10(9) platelets. Average percentage recoveries in PC compared to the pre-centrifugation BC pools were similar with the Biopack Compoflex and the Optipac systems, 62% and 57% respectively, whereby the absolute platelet count per one donation was similar, i.e. 49.5 x 10(9), 55 x 10(9) and 53 x 10(9) in PC-BC/4 and PC-BC/5 from Bern and PC-BC/5 from Zurich. There was a significant positive correlation between the inital number of BCs taken for pooling and the final platelet counts in the PCs. In order to recover a minimal platelet content of 200 x 10(9) platelets per pooled unit, it is safer to start out with five rather than with four donations unless recoveries during the production steps can be improved.