Functional Characterization of Autoantibodies against Complement Component C3 in Patients with Lupus Nephritis.

Lupus nephritis (LN) is a complication of the autoimmune disease systemic lupus erythematosus. Because the complement system plays a critical role in orchestrating inflammatory and immune responses as well as in the clearance of immune complexes, autoreactivity to complement components may have considerable pathological consequences. Autoantibodies against the central complement component C3 have been reported in systemic lupus erythematosus, but their molecular mechanism and functional relevance are not well understood. The objective of this study was to evaluate the frequency and the functional properties of the anti-C3 autoantibodies. Anti-C3 autoantibodies were measured in plasma of 39 LN patients, and identification of their epitopes on the C3 molecule was performed. By using surface plasmon resonance, we analyzed the influence of patient-derived IgG antibodies on the interaction of C3b with Factor B, Factor H, and complement receptor 1. The capacity of these antibodies to dysregulate the C3 convertase on the surface of endothelial cell was measured by flow cytometry. Here we report that the frequency of anti-C3 autoantibodies in LN is ∼30%. They inhibited interactions of the negative complement regulators Factor H and complement receptor 1 with C3b. An enhanced C3 deposition was also observed on human endothelial cells in the presence of C3 autoantibodies. In addition, anti-C3 autoantibody levels correlated with disease activity. In conclusion, the anti-C3 autoantibodies in LN may contribute to the autoimmune pathology by their capacity to overactivate the complement system.

Effects of renal insufficiency on the elimination of fluorescein lisicol (NRL972), an investigational marker of hepatic transporter function.

BACKGROUND: NRL972 (Fluorescein Lisicol), a fluorescent-labelled bile salt, is an investigational marker of hepatic biliary transporter function. OBJECTIVE: To investigate the pharmacokinetics (PK) of NRL972 in patients with severe (SRI: creatinine clearance (CLCr)<30 mL/min per 1.73 m2 body surface area (BSA)) and mild-to-moderate renal insufficiency (MRI: 30≤CLCr<80 mL/min) relative to matched controls (CON: CLCr≥90 mL/min). METHODS: The plasma and urinary PK of NRL972 were determined after single 2-mg doses of NRL972 administered by 15-second intravenous (IV) injection. The PK were derived noncompartmentally using all data points up to 6 hours after dosing or only using the concentrations at 10 and 30 minutes after injection. RESULTS: 17, 22, and 16 subjects were enrolled in the SRI, MRI, and CON group, respectively. NRL972 was hardly quantifiable in urine in any of the subjects groups. The plasma concentrations of NRL972 declined rapidly after dosing in mostly monoexponential fashion. The decline tended to be faster in patients with renal insufficiency: in SRI patients, the point and 95% confidence interval (CI) estimates of the group ratios (SRI/CON) were 0.691 (CI: 0.517 to 0.925) for the C(30):C(10) concentration ratio, 0.785 (CI: 0.634 to 0.970) for t1/2, and 1.344 (CI: 1.028 to 1.757) for bodyweight normalized clearance (CL/BW); in MRI patients, the effect was slightly less. CONCLUSION: Renal insufficiency does not impair the elimination of NRL972; instead, there is a trend of enhanced NRL972 disposition in patients with compromised renal function. Concomitant renal impairment is unlikely to have confounding effects on the evaluation of hepatic function by NRL972 testing.

Effects of advanced age and renal dysfunction on the single- and repeated-dose pharmacokinetics of modified-release flupirtine.

BACKGROUND: Flupirtine is a nonopioid, central analgesic without antipyretic or antiphlogistic properties. Flupirtine-MR is an oral modified-release formulation with a 100 mg fast-input and a 300 mg portion with slow protracted release. METHODS: Single- (D01) and repeated-dose (D03-D09) pharmacokinetics of 400 mg flupirtine-MR were investigated in patients with severe renal dysfunction (REN: N: 12; 21 50 years of age; creatinine clearance (CLCr)≤30 mL/min per 1.73 m2 body surface area (BSA)) and healthy older subjects (EN1: N: 8; 60-69 years; CLCr≥80 mL/min and EN2: N: 8; ≥70 years, CLCr≥60 mL/min) vs. young healthy control subjects (YN: N: 12; 21-40 years; CLCr≥90 mL/min). RESULTS: Renal dysfunction led to a relatively small average increase in systemic exposure to flupirtine: on D09, the REN : YN-ratios were 1.37 (95% confidence interval (CI): 1.03-1.82), 1.21 (CI: 1.01-1.45), and 1.34 (CI: 1.09-1.64) for Css,0, Css,max, and Css,av, respectively. A similar increase in exposure was observed in older subjects: the respective EN1:YN-ratios were 1.30 (CI: 0.95-1.79), 1.23 (CI: 1.01-1.49), and 1.23 (CI: 0.98-1.54); the EN2:YN-ratios were 1.50 (CI: 1.10-2.04), 1.16 (CI: 0.85-1.41), and 1.41 (CI: 1.12-1.79), respectively. Neither age nor renal function was a predominant factor of pharmacokinetic variability. Single and repeated doses of flupirtine-MR were very well tolerated. CONCLUSIONS: The average renal and age effects were small, but the use of a lower starting dose (1/2 tablet) is recommended since some of these subjects might have relatively high exposure levels.

Autoantigenicity of human C1q is associated with increased hydrophobicity due to conformational transitions in the globular heads.

We analyzed the structural features of C1q that underlie its autoantigenicity by means of a model system using the amphiphilic polyzwitterion (PZ), poly(ethylene oxide-b-N,N-dimethyl(methacryloyloxyethyl) ammonium propanesulfonate) in the process of C1q immobilization. The source of anti-C1q autoantibodies was human sera from patients with Lupus Nephritis (LN). Both analyzed concentrations of PZ, 25 mM and 50 mM, were found to be applicable for inducing conformational transitions which resulted in increased recognition of C1q and the globular domain of its B polypeptide chain, designated ghB, by the LN autoantibodies. The registered conformational transitions displayed a hydrophobic enhancement of the protein microenvironment due to the presence of hydrophobic binding sites in ghB which consequently affected the autoantigenicity of the whole C1q molecule.

Biochemical analysis of the epitope specificities of anti-C1q autoantibodies accompanying human lupus nephritis reveals them as a dynamic population in the course of the disease.

We analyzed the epitope specificities of the polyclonal anti-C1q antibodies, present in human LN sera, searching to deduce the structural characteristics of C1q associated with its transition to an autoantigen. We screened 78 serum samples from LN patients distributed in three clinical groups - non-active, moderately active and severely active. We found three classes of C1q autoepitopes: (a) neo-epitopes, exposed upon immobilization due to conformational changes; (b) epitopes formed by sequences that are brought together by the conformation of the whole molecule; (c) cryptic epitopes that become exposed only after fragmentation of C1q. The latter suggest that the immunogen involved in the initiation of anti-C1q autoantibodies might be an extrinsic molecule that shares some degree of structural similarity to C1q. None of the tested epitope specificities was associated with active LN. We found a prevalence of anti-gC1q antibodies among the non-active LN patients suggesting that they might be the fraction of the polyclonal anti-C1q, preceding the initiation of autoimmunity to C1q, or alternatively, preceding LN flare.

Anti-C1q autoantibodies specific against the globular domain of the C1qB-chain from patient with lupus nephritis inhibit C1q binding to IgG and CRP.

Lupus nephritis is one of the most severe manifestations of systemic lupus erythematosus. Higher titers of serum anti-C1q autoantibodies correlate with disease activity in patients with lupus nephritis. Anti-C1q autoantibodies have been shown to bind neo-epitopes within the collagen region of human C1q. In a preliminary study, we recently reported that the anti-C1q autoantibodies could also recognize epitopes within the globular domain (gC1q) of the C1q molecule. Here, 38 sera from patients with renal biopsy-proven lupus nephritis were screened for the presence of anti-gC1q autoantibodies, using recombinant globular head regions of individual A (ghA), B (ghB) and C (ghC) chains of human C1q. We isolated anti-gC1q autoantibodies from three selected patients. Human C1q was pre-incubated with increasing concentrations of the isolated anti-ghA, anti-ghB or anti-ghC autoantibodies and its binding to different C1q target molecules such as IgG and CRP was then evaluated. Anti-ghB, but not anti-ghA and anti-ghC autoantibodies, markedly inhibited C1q interaction with IgG as well as CRP. These results appear to suggest that the anti-ghB autoantibodies may partially induce acquired functional C1q deficiency and thus may interfere with the biological function of C1q.

New insight into the autoimmunogenicity of the complement protein C1q.

C1q along with its physiological role in maintenance of homeostasis and normal function of the immune system is involved in pathological conditions associated with repetitive generation of anti-C1q autoantibodies. The time and events that cause their first appearance are still unknown. We addressed this issue by analyzing the immunogenicity of C1q in two target groups-one of non-diseased humans and the other of lupus nephritis (LN) patients whose autoimmune disorder is associated with high titers of anti-C1q autoantibodies. The non-diseased humans were represented by pregnant women because the sex hormones are thought to be involved in triggering autoimmune pathologies by their ability to tip the balance of female adaptive immune response to production of antibodies. We screened, using ELISA, 31 sera from healthy pregnant women for the presence of IgM and IgG classes of autoantibodies, recognizing epitopes within the native C1q molecule, its collagen-like region (CLR) and globular head fragment (gC1q). The latter was represented by recombinant analogs of the three globular fragments of A, B and C chains, comprising C1q-ghA, ghB and ghC. We did not find IgM antibodies for all test-antigens which suggest that the natural IgM antibodies are not involved in triggering autoimmunity to C1q. Still more, we did not detect anti-CLR antibodies which have been proved pathogenic in already manifested LN. We completed the analysis with comparative epitope mapping of gC1q and we found similar immunogenic behavior in both target groups-ghA and ghC contained the immunodominant epitopes. This implies that the initial immune response to C1q might occur when the molecule has interacted with its ligands via ghB as part of gC1q. The presence of anti-gC1q in both healthy and diseased humans also implies that these antibodies, unlike anti-CLR, may have a contribution to an onset of autoimmunity.

Effects of endoxan on oxidative processes in model systems in vitro.

Endoxan (cyclophosphamide) is a cyclic propylene phosphamide ester of nitrogen mustard. Endoxan--main advantage of chemotherapy is complete penetration of the tissues, reaching the most widely spread malignant cells. It is one of the most useful cytotoxics available today. Endoxan is a "transport form" and as such it has a selective tumour affinity. Endoxan is used for active treatment of all neoplastic diseases of the reticulo-endothelial system, e.g. lymphomas, lymphosarcomas, reticular-sarcomas, Hodgkin's disease, chronic lymphatic leukaemias, multiple myelomas. In our experiments Endoxan is equivalent with mean therapeutic the concentration used in chemotherapy in tumours, we suppose that at these levels Endoxan interact with renal cells and probably induce or inhibits new generation of superoxide free forms in the same tissue. Endoxan was tested at renal supernatant and free enzyme model systems, for superoxide-scavenging and antioxidante activity. The ability of Endoxan to interact with the superoxide radical, to influence their generation and probably to change the levels of lipid peroxidation in model systems were investigated. The ability of Endoxane to affect Fe2+-induced lipid peroxidation in a renal supernatant was studied. The results show that Endoxan in a concentration range of 10(-4); 10(-5) M has small but significant effect. The values for the control samples without Endoxan, are compared. We found a dose-dependent superoxide-scavenging effect of the drug in xanthite/xanthine oxidase system for generation of superoxide. According to obtained results lndoxan could be used in insertion in a liposomes and this could impact lndoxan tissue penetration.

Cardiovascular risk with SCORE system in patients with different degree of renal function impairment.

A risk prediction system, Systematic Coronary Risk Evaluation, that is based on European studies has been developed and recommended to define absolute 10-year risk of a fatal cardiovascular event and mortality. The aim of the study was to compare cardiovascular risk calculated with SCORE system at patients with different degree of renal impairment. The study included 90 patients divided in 4 groups: 1st group=30 patients without renal failure, 2nd group=25 patients with CRF in predialysis stage, 3rd group=19 hemodialysis non-diabetic patients and 4th group=16 hemodialysis diabetics patients. SCORE was calculated from age, sex, systolic blood pressure, smoking and cholesterol levels. There were no significant differences in age and blood pressure in four examined groups. The incidence of smokers and cholesterol level were higher in predialysis patients. The highest SCORE was calculated in predialysis patients: 1st group: 2.5+/-1.8; 2nd group: 5.3+/-4.3, 3rd group: 3.7+/-1.1 and 4th group: 4.06+/-4. We supposed that traditional risk factors from SCORE risk system are suitable to explain the cardiovascular risk and mortality in all population but underestimates cardiovascular risk of high-risk groups like patients with chronic renal disease.

Lupus nephritis sera contain autoantibodies that recognize epitopes within the globular fragment of C1q.

High levels of autoantibodies to some complement proteins are detected in the sera of SLE patients. Anti-C1q autoantibodies make a great part of them. Their presence is associated with renal involvement, in particular with lupus nephritis (LN) and the titre of these autoantibodies correlates with the clinical activity of the disease. We analysed by ELISA 18 SLE sera with biopsy-proved LN for the presence of autoantibodies against the globular fragment of C1q using recombinant globular head regions of A, B and C chains of human C1q (ghA, ghB and ghC, respectively). For reference we analysed the sera from 62 healthy volunteers. The recombinant proteins were used as test-antigens to evaluate the levels of autoantibodies specific for ghA, ghB and ghC. LN sera, containing high levels of anti-C1q antibodies, showed differential increased binding to ghA, ghB and ghC.

The efficacy of an individual treatment schedule in patients with vasculitis.

The aim of the study was to determine the efficacy of an individual treatment schedule in patients with systemic vasculitis. Clinical, laboratory, morphological and immunological data before and after treatment were followed in 18 patients: eight with microscopic polyangiitis, two with Wegener's granulomatosis, four with leukocytoclastic vasculitis and four with necrotizing/crescentic glomerulonephritis. Patients received individual treatment for 14.89+/-13.9 months according to the disease activity. Methylprednisolone pulse therapy (PT) was given to 16 patients, mean 1.94+/-0.4 PT/patient, followed by a slowly tapered oral dose (0.7-1 mg/kg). Cyclophosphamide PT was received by 15 patients, mean 3.13+/-0.9/patient in doses of 8-10 mg/kg, followed by an oral dose of 1 mg/kg for 9.17+/-1.9 months. Four additional patients were treated with cyclosporin A for 3 months. Eleven patients received heparin for 30 days. Plasmapheresis was provided in seven patients. Two patients were treated with azathioprine and one patient with mycophenolate mofetil. There were no significant changes in serum creatinine and creatinine clearance during the observation period. Proteinuria and haematuria improved after treatment. Kidney function improved or became stable in 66.67% of patients. No patient required haemodialysis. Haematuria was no longer observed at the end of the study in nine of 11 patients. Thirteen patients (72.22%) had clinical remission. Relapses occurred in five patients. Kidney re-biopsies showed a decrease in morphological changes in 57.2%. In conclusion, individual treatment is more flexible and controls the disease activity better.