No evidence of pathogenic involvement of cathelicidins in patient cohorts and mouse models of lupus and arthritis.

Apart from their role in the immune defence against pathogens evidence of a role of antimicrobial peptides (AMPs) in autoimmune diseases has accumulated in the past years. The aim of this project was to examine the functional impact of the human cathelicidin LL-37 and the mouse cathelicidin-related AMP (CRAMP) on the pathogenesis of lupus and arthritis. Serum LL-37 and anti-LL-37 levels were measured by ELISA in healthy donors and patients with Systemic Lupus Erythematosus (SLE) and Rheumatoid arthritis (RA). Pristane-induced lupus was induced in female wild type (WT) and cathelicidin-deficient (CRAMP-/-) mice. Serum levels of anti-Sm/RNP, anti-dsDNA, and anti-histone were determined via ELISA, cytokines in sera and peritoneal lavages were measured via Multiplex. Expression of Interferon I stimulated genes (ISG) was determined by real-time PCR. Collagen-induced arthritis (CIA) was induced in male WT and CRAMP-/- mice and arthritis severity was visually scored and analysed histomorphometrically by OsteoMeasure software. Serum levels of anti-LL-37 were higher in SLE-patients compared to healthy donors or patients with RA. However, no correlation to markers of disease activity or organ involvement was observed. No significant differences of autoantibody or cytokine/chemokine levels, or of expression of ISGs were observed between WT and CRAMP-/- mice after pristane-injection. Furthermore, lung and kidney pathology did not differ in the absence of CRAMP. Incidence and severity of CIA and histological parameters (inflammation, cartilage degradation, and bone erosion) were not different in WT and CRAMP-/- mice. Although cathelicidins are upregulated in mouse models of lupus and arthritis, cathelicidin-deficiency did not persistently affect the diseases. Also in patients with SLE, autoantibodies against cathelicidins did not correlate with disease manifestation. Reactivity against cathelicidins in lupus and arthritis could thus be an epiphenomenon caused by extensive overexpression in blood and affected tissues. In addition, other cationic AMPs could functionally compensate for the deficiency of cathelicidins.

Direct measurement of the interactions of glycosaminoglycans and a heparin decasaccharide with the malaria circumsporozoite protein.

Circumsporozoite (CS) protein is a predominant surface antigen of malaria sporozoites, the infective form of the parasite, and has been used for making anti-malaria vaccines. For the first time we have examined the interaction of CS protein with various glycosaminoglycans in real time using surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). Heparin was the best binder among the glycosaminoglycans tested and bound to CS protein with nanomolar affinity. Using purified and structurally defined small heparin oligosaccharides, we identified a decasaccharide to be the minimum sized CS protein-binding sequence. In an indirect competition assay, this decasaccharide blocked the CS protein interaction with HepG2 cells with an ID(50) of less than 60 nM. The decasaccharide has a structure commonly found in hepatic heparan sulfate, and the same sequence has recently been shown to bind specifically to apolipoprotein E. Examination of porcine liver heparan sulfate in this indirect competition assay showed that it and heparin were the only glycosaminoglycans that could effectively block CS protein interaction with HepG2 cells in culture. These data support the hypothesis that the invasion of liver cells by the parasite shares a common mechanism with the hepatic uptake of lipoprotein remnants from the blood.

Affinity purification of secreted alkaline phosphatase produced by baculovirus expression vector system.

Human secreted alkaline phosphatase (SEAP) was produced in a stably-transformed Spodoptera frugiperda Sf-9 insect cell line (Sfb4GalT) following infection with a recombinant Autographa californica multiple nuclear polyhedrovirus containing the SEAP gene under control of the polyhedrin promoter. An affinity chromatographic column prepared by linking 4-amino-benzylphosphonic acid to histidyl-expoxy-Sepharose was used to isolate SEAP from the cell supernatant following removal of cells and virus and 10-fold concentration through ultrafiltration. We found that the binding of SEAP on the affinity matrix follows the Langmuir isotherm model. In addition, either recycling SEAP sample through the column for 24 h or loading high SEAP concentrations resulted in a high-purity product. Some nonspecific binding of protein on the matrix occurred when low concentrations of SEAP sample were loaded. Finally, we found that SEAP binding occurs rapidly, i.e., within 30 min of adding the SEAP sample to the affinity matrix.

Modulation of adhesion of lymphocytes to murine brain endothelial cells in vitro: relation to class II major histocompatibility complex expression.

Adhesion of lymphocytes to mouse brain endothelial cells was studied after treatment of the endothelium with 1000 U/ml gamma interferon (IFN-gamma) for 1 h to 2 days. Adhesion was not significantly different from controls after 1 h but at 4 h and thereafter, adhesion increased in a time-related manner. IFN-gamma also increased the expression of class II major histocompatibility complex (MHC) and murine intercellular adhesion molecule-1 (ICAM-1) molecules on the endothelial cells. The level of expression of class II MHC molecules was related to the length of exposure to IFN-gamma. MAb blocking studies suggested that class II molecules were responsible for the IFN-gamma-induced increase in lymphocyte-endothelial cell adhesion. Transfection of a murine lung endothelial cell line with cDNA for the class II MHC molecule also produced a significant increase in lymphocyte-endothelial cell adhesion, suggesting that the class II MHC molecule may have a role in adhesion which is distinct from antigen presentation.