Anti-Idiotype scFv Localizes an Autoepitope in the Globular Domain of C1q.

We addressed the issue of C1q autoantigenicity by studying the structural features of the autoepitopes recognized by the polyclonal anti-C1q antibodies present in Lupus Nephritis (LN) sera. We used six fractions of anti-C1q as antigens and selected anti-idiotypic scFv antibodies from the phage library "Griffin.1". The monoclonal scFv A1 was the most potent inhibitor of the recognition of C1q and its fragments ghA, ghB and ghC, comprising the globular domain gC1q, by the lupus autoantibodies. It was sequenced and in silico folded by molecular dynamics into a 3D structure. The generated 3D model of A1 elucidated CDR similarity to the apical region of gC1q, thus mapping indirectly for the first time a globular autoepitope of C1q. The VH CDR2 of A1 mimicked the ghA sequence GSEAD suggested as a cross-epitope between anti-DNA and anti-C1q antibodies. Other potential inhibitors of the recognition of C1q by the LN autoantibodies among the selected recombinant antibodies were the monoclonal scFv F6, F9 and A12.

Study of the Mechanism of the Antimicrobial Activity of Novel Water Soluble Ammonium Quaternary Benzanthrone on Model Membranes.

The increasing resistance of many pathogens to most of the common antimicrobials requires the development of new substances with more effective antimicrobial properties. In the present work, we investigated the mechanism of the antimicrobial activity of novel water soluble ammonium quaternary benzanthrone (Compound B) on model membranes, composed of dipalmitoylphosphatidylcholine, 1-palmitoyl-2-oleoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, 1-palmitoyl-2-oleoylphosphatidylglycerol, and dipalmitoylphosphatidylethanolamine (DPPE). The lipids were chosen to represent a model of a bacterial membrane. The changes in surface pressure of the model membranes, before and after the addition of Compound B, were studied by the Langmuir's monolayer method, and the compressional modulus for each monolayer was determined. In addition, the surface morphology of the lipid monolayers before and after injection of Compound B was monitored by Brewster Angle Microscopy. The results showed that Compound B penetrated all the monolayers studied. The most noticeable effects were found with the negatively charged phosphatidylglycerols and with DPPE leading to the conclusion that the electrostatic interactions between the compound and the lipid head groups and the possible formation of hydrogen bonds between the amino group of the ethanolamine and the keto groups in the structure of Compound B are of great importance. In addition, the penetration ability of the benzoquinone with all phospholipids studied was stable even at higher values of the surface pressure, i.e. thicker monolayers, due to the hydrophobic interaction, which plays also an important role for the antimicrobial activity of Compound B.

Rapid biophysical analyses of gastric aspirates from risk newborns for lung maturity assessment after corticosteroid therapy.

BACKGROUND: One of the main causes for the higher mortality among risk newborn children (including preterm infants) is neonatal respiratory distress syndrome (NRDS), which develops as a result of primary deficiency or secondary inactivation of alveolar surfactant (AS). Therefore, fast and early diagnostics of risk newborns lung maturity is crucial for their prompt therapy. MATERIALS AND METHODS: Gastric aspirates (GA) were collected from 77 infants divided into three groups: a control of 38 healthy full-term infants; 16 prematurely newborns with NRDS, and 23 prematurely born infants after in vitro fertilization and corticosteroid therapy (CST). Surface parameters: equilibrium (γeq), maximal (γmax) and minimal (γmin) surface tension, and the shape of hysteresis curves of GA monolayers were measured by axisymmetric drop shape analysis (ADSA) of a pending drop. In addition, the morphology of GA monolayers was studied by Brewster angle microscopy (BAM). RESULTS: Our results showed that only γmin values were reliable and were significantly lower in full-term infants, as compared to the risk neonates. The results obtained were proved by the shape of hysteresis curves of GA surface active films. BAM images of GA monolayers from NRDS group showed impaired surface morphology due to the surfactant insufficiency, as compared to the control group. Corticosteroid therapy improved both GA surface characteristics and monolayer morphology. CONCLUSIONS: GAs analyses by ADSA and BAM are fast and informative approaches for lung maturity assessment. In addition, the corticosteroid therapy applied improved all GAs surface parameters due to AS maturation.

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.

Recognition of Vipera ammodytes meridionalis neurotoxin vipoxin and its components using phage-displayed scFv and polyclonal antivenom sera.

Vipoxin is a potent postsynaptic heterodimeric neurotoxin isolated from the venom of the Bulgarian snake Vipera ammodytes meridionalis, whose snakebites cause different and strongly manifested pathophysiological effects (neurotoxic, hemolytic, anticoagulant, convulsant, hypotensive, hyperglycemic etc.). The neutralization of snake toxins calls for extensive research through the application of different approaches: antibodies, non-immunologic inhibitors, natural products derived from plants and animals, as well as synthetic drugs. In this study, we applied naive Tomlinson I + J (Cambridge, UK) libraries to obtain recombinant human scFv antibodies against the vipoxin's two subunits--basic and toxic phospholipase A2 (PLA2) and acidic, non-toxic component. We found that 33 of more than hundred tested clones were positive and recognized vipoxin and its subunits. Enriched scFv-phage samples (1.2 × 109 pfu/ml) were analyzed for their binding (ELISA) and enzyme-inhibiting abilities. Single chain Fv-phage clones--D12, E3, F6, D10 and G5 exhihest binding affinity for the toxic component. Clones A1, D12 and C12 recognized preferentially vipoxin's acidic component. Clones E3, G5 and H4 inhibited the enzymatic activity of both vipoxin and its purified and separated toxic subunit to the highest extent. Six of the selected clones (E3, G5, H4, C12, D10 and A11) inhibited direct hemolytic activity of vipoxin and its pure PLA2 subunit. The obtained specific scFv antibodies will be used for epitope mapping studies required to shed light on the role of the phospholipase A2 activity for the vipoxin toxicity and its effective neutralization.

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.