Modelling the Impact of NETosis During the Initial Stage of Systemic Lupus Erythematosus.

The development of autoimmune diseases often takes years before clinical symptoms become detectable. We propose a mathematical model for the immune response during the initial stage of Systemic Lupus Erythematosus which models the process of aberrant apoptosis and activation of macrophages and neutrophils. NETosis is a type of cell death characterised by the release of neutrophil extracellular traps, or NETs, containing material from the neutrophil's nucleus, in response to a pathogenic stimulus. This process is hypothesised to contribute to the development of autoimmunogenicity in SLE. The aim of this work is to study how NETosis contributes to the establishment of persistent autoantigen production by analysing the steady states and the asymptotic dynamics of the model by numerical experiment.

Galectin-3 - A novel ligand of complement protein C1q.

In the present study we report a novel interaction of human C1q, a primary activator of the Complement system, with human Galectin-3 (Gal-3). We investigated the potential recognition between C1q and Gal-3 on a solid hydrophobic surface by ELISA, by fluorescence spectroscopy, molecular docking and molecular dynamics (MD). The data showed that C1q and Gal-3 had a pronounced affinity for protein-protein interaction and supramolecular binding, locating the binding sites within the globular domains of C1q (gC1q) and on the backside of the carbohydrate recognition domain (CRD) of Gal-3. Fluorescence spectroscopy gave quantitative assessment of the recognition with KD value of 0.04 µM. MD analysis showed that when the active AAs of the two proteins interacted, electrostatic attraction, aided by a large number of hydrogen bonds, was dominant for the stabilization of the complex. When the contact of C1q and Gal-3 was not limited to active residues, the complex between them was stabilized mainly by Van der Waals interactions and smaller in number but stronger hydrogen bonds. This is the first report analyzing the interaction of Gal-3 with C1q, which could open the way to new applications of this protein-protein complex.

Uncovering the Interrelation between Metabolite Profiles and Bioactivity of In Vitro- and Wild-Grown Catmint (Nepeta nuda L.).

Nepeta nuda L. is a medicinal plant enriched with secondary metabolites serving to attract pollinators and deter herbivores. Phenolics and iridoids of N. nuda have been extensively investigated because of their beneficial impacts on human health. This study explores the chemical profiles of in vitro shoots and wild-grown N. nuda plants (flowers and leaves) through metabolomic analysis utilizing gas chromatography and mass spectrometry (GC-MS). Initially, we examined the differences in the volatiles' composition in in vitro-cultivated shoots comparing them with flowers and leaves from plants growing in natural environment. The characteristic iridoid 4a-α,7-ß,7a-α-nepetalactone was highly represented in shoots of in vitro plants and in flowers of plants from nature populations, whereas most of the monoterpenes were abundant in leaves of wild-grown plants. The known in vitro biological activities encompassing antioxidant, antiviral, antibacterial potentials alongside the newly assessed anti-inflammatory effects exhibited consistent associations with the total content of phenolics, reducing sugars, and the identified metabolic profiles in polar (organic acids, amino acids, alcohols, sugars, phenolics) and non-polar (fatty acids, alkanes, sterols) fractions. Phytohormonal levels were also quantified to infer the regulatory pathways governing phytochemical production. The overall dataset highlighted compounds with the potential to contribute to N. nuda bioactivity.

Exploring the Phytochemical Composition and Biological Potential of Balkan Endemic Species Stachys scardica Griseb.

Stachys scardica Griseb. is a Balkan endemic species listed in The Red Data Book of Bulgaria with the conservation status "endangered". Successful micropropagation was achieved on MS medium supplemented with 1.5 mg/L benzyladenine (BA), followed by a subsequent ex vitro adaptation in an experimental field resulting in 92% regenerated plants. Using nuclear magnetic resonance (NMR), phenylethanoid glycosides (verbascoside, leucosceptoside A), phenolic acids (chlorogenic acid), iridoids (allobetonicoside and 8-OAc-harpagide), and alkaloids (trigonelline) were identified, characteristic of plants belonging to the genus Stachys. High antioxidant and radical scavenging activities were observed in both in situ and ex vitro acclimated S. scardica plants, correlating with the reported high concentrations of total phenols and flavonoids in these variants. Ex vitro adapted plants also exhibited a well-defined anti-inflammatory potential, demonstrating high inhibitory activity against the complement system. Employing a disk diffusion method, a 100% inhibition effect was achieved compared to positive antibiotic controls against Staphylococcus epidermidis and Propionibacterium acnes, with moderate activity against Bacillus cereus. The induced in vitro and ex vitro model systems can enable the conservation of S. scardica in nature and offer future opportunities for the targeted biosynthesis of valuable secondary metabolites, with potential applications in the pharmaceutical and cosmetic industries.

Biological Activity and NMR-Fingerprinting of Balkan Endemic Species Stachys thracica Davidov.

Stachys thracica Davidov is a Balkan endemic species distributed in Bulgaria, Greece, and Turkey. In Bulgaria, it is classified as "rare" and is under the protection of the Bulgarian biodiversity law. The aim of our study was to develop an efficient protocol for ex situ conservation of S. thracica and to perform comparative NMR-based metabolite profiling and bioactivity assays of extracts from in situ grown, in vitro cultivated, and ex vitro acclimated plants. Micropropagation of S. thracica was achieved by in vitro cultivation of mono-nodal segments on basal MS medium. Ex vitro adaptation was accomplished in the experimental field with 83% survival while conserved genetic identity between in vitro and ex vitro plants as shown by the overall sequence-related amplified polymorphism marker patterns was established. Verbascoside, chlorogenic acid, and trigonelline appeared the main secondary metabolites in in situ, in vitro cultivated, and ex vitro acclimated S. thracica. High total phenolic and flavonoid content as well as antioxidant and radical scavenging activity were observed in in situ and ex vitro plants. Further, the anti-inflammatory activity of S. thracica was tested by hemolytic assay and a high inhibition of the complement system was observed. Initiated in vitro and ex vitro cultures offer an effective tool for the management and better exploitation of the Stachys secondary metabolism and the selection of lines with high content of bioactive molecules and nutraceuticals.

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.

Autoinduction as Means for Optimization of the Heterologous Expression of Recombinant Single-Chain Fv (scFv) Antibodies.

The monoclonal antibodies and the recombinant antibody fragments are widely used in the biotechnology studies and in medicine as a powerful therapeutic and diagnostic tool. The most commonly used recombinant antibody fragments are single-chain fragment variable (scFv) because of their small size and minimal immunogenicity while still retaining high-affinity antigen binding. A wide range of expression systems such as bacterial and eukaryotic cell systems enable the sufficient production of scFv antibodies. However, their stable expression in soluble form and correct protein folding are often insufficient. In the present study, we present the autoinduction as a key element of the optimized scheme for heterologous expression of human monoclonal scFv antibodies (clones A1 and A12) in Escherichia coli HB2151, which resulted in two-fold increase of the total protein yield in 24 h.

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.

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.

Chaperone-like effect of polyzwitterions on the interaction of C1q with IgG.

The amphiphilic polyzwitterion (PZ) poly(ethylene oxide-b-N,N-dimethyl(methacryloyloxyethyl)ammonium propanesulfonate), zwitterionic surfactant (ZS) n-dodecyl-N,N-dimethyl-3-ammonium-1-propanesulfonate, and zwitterionic monomer (ZM) N,N-dimethyl(methacryloyloxyethyl)ammonium propanesulfonate were analyzed for their suggested chaperone-like effect on the interaction of C1q and IgG. Our results proved that the PZ retarded the C1q interaction with IgG, demonstrating a specific protein-folding helper effect. The ZS enhanced this interaction, when the ZS concentration was lower than the critical micelle concentration (CMC), and retarded it, when the ZS concentration was above the CMC. The ZM, with no self-assembling ability, did not influence this interaction. These results support the hypothesis of a hydrophobic interaction between Pts and hydrophobic domains of partly denatured protein molecules. The amphiphilic self-assemblies, formed by polyzwitterionic macromolecules or zwitterionic surfactants, have the ability to transform the hydrophobic domains of the protein molecules into hydrophilic ones, covering them with their hydrophilic parts.

Generation of recombinant antibodies against orchardgrass Acidic nsLTP-like proteins.

Embryogenic and non-embryogenic suspension cultures of orchardgrass (Dactylis glomerata L.) secreted into the culture medium a set of proteins, among which low molecular mass (11/12 kDa) proteins were found. However, only the 11/12 kDa proteins from the embryogenic suspension cultures reacted specifically with an antiserum raised against the carrot EP2 non-specific lipid transfer protein (nsLTP). Two-dimensional (2-D) electrophoretic analysis revealed that the extracellular nsLTP-like proteins from the embryogenic lines were acidic proteins, with pI values ranging between 4.3 and 6.4, and the 11/12 kDa proteins of the non-embryogenic lines were basic ones (pI 8-9.3). This is only the second case to report on the accumulation of extracellular acidic nsLTP-like proteins in the culture medium during somatic embryogenesis. A naive phage display Griffin1. library was used to select single-chain phage antibodies, which specifically bind to acidic nsLTP-like proteins. Nine phage clones were selected after four rounds of biopanning of the target proteins blotted on a nitrocellulose membrane. Three soluble monoclonal single-chain phage antibodies, expressed in the non-suppressor E. coli strain HB2151, were purified by metal affinity chromatography and found to be highly specific for the acidic nsLTP-like proteins from the embryogenic suspension cultures. The application of the selected monoclonal antibodies for localization and elucidation of the role of the acidic nsLTP-like proteins in vivo is discussed.

Detection of autoantibodies against the globular domain of human C1q in the sera of systemic lupus erythematosus patients.

The anti-C1q antibodies present in systemic lupus erythematosus (SLE) patients' sera are associated with renal involvement and the titer of these autoantibodies correlates with the clinical activity of the disease. It has previously been shown that anti-C1q antibodies bind neo-epitopes within the collagen region of human C1q. Evidence that these polyclonal autoantibodies recognize epitopes within the globular domain (gC1q) of the molecule has not been documented. In this study, we screened, using ELISA, a number of sera from SLE patients 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. The recombinant proteins were used as test antigens to determine the levels of autoantibodies directed against ghA, ghB and ghC. SLE sera, containing high levels of anti-C1q antibodies, showed differentially increased binding towards ghA and ghB, which suggested that the gC1q domain can also be target of anti-C1q antibodies generated in SLE patients. Such antibodies can have severe pathophysiological consequences since these are likely to further impair the ability of C1q to clear immune complexes.

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.

Mutational analyses of the recombinant globular regions of human C1q A, B, and C chains suggest an essential role for arginine and histidine residues in the C1q-IgG interaction.

The first step in the activation of the classical complement pathway by immune complexes involves the binding of the globular domain (gC1q) of C1q to the Fc regions of aggregated IgG or IgM. Each gC1q domain is a heterotrimer of the C-terminal halves of one A (ghA), one B (ghB), and one C (ghC) chain. Our recent studies have suggested a modular organization of gC1q, consistent with the view that ghA, ghB, and ghC are functionally autonomous modules and have distinct and differential ligand-binding properties. Although C1q binding sites on IgG have been previously identified, the complementary interacting sites on the gC1q domain have not been precisely defined. The availability of the recombinant constructs expressing ghA, ghB, and ghC has allowed us, for the first time, to engineer single-residue substitution mutations and identify residues on the gC1q domain, which are involved in the interaction between C1q and IgG. Because C1q is a charge pattern recognition molecule, we have sequentially targeted arginine and histidine residues in each chain. Consistent with previous chemical modification studies and the recent crystal structure of gC1q, our results support a central role for arginine and histidine residues, especially Arg(114) and Arg(129) of the ghB module, in the C1q-IgG interaction.

Complement inhibiting properties of dragon's blood from Croton draco.

The latex of Croton draco, its extracts and several latex components have been investigated for their influence on both classical (CP) and alternative (AP) activation pathways of the complement system using a hemolytic assay. The best inhibition was found for the classical pathway. The latex, ethyl acetate and ethyl ether extracts exhibited extremely high inhibition on the CP (94, 90 and 77%, respectively) at a concentration of 1 mg/ml. The flavonoid myricitrin, the alkaloid taspine and the cyclopeptides P1 and P2 showed high inhibition on CP (83, 91, 78 and 63%, respectively) at a concentration of 0.9 mM.

Localization of ligand-binding sites on human C1q globular head region using recombinant globular head fragments and single-chain antibodies.

As a charge pattern recognition molecule, human C1q can bind a range of immunoglobulin and non-immunoglobulin ligands via its carboxy-terminal globular domain and activate the classical complement pathway. Each globular domain has a heterotrimeric organization, composed of the carboxy-terminal halves of one A (ghA), one B (ghB), and one C (ghC) chain. Recently, we have found that the recombinant forms of individual ghA, ghB and ghC bind differentially to IgG, IgM, gp41 peptide 601-613 of human immunodeficiency virus-1 (HIV-1), gp21 peptide 400-429 of human T cell lymphotrophic virus-I (HTLV-I), beta-amyloid peptide, and apoptotic cells, suggesting a modular organization of the globular domain. This paper examines the interaction of ghA, ghB and ghC with two known C1q ligands: Klebsiella pneumoniae porin OmpK36 and salivary agglutinin. In addition, we have used a panel of recombinant single-chain antibodies (scFv) specific for ghA, ghB and ghC in order to map sites on the heterotrimeric globular domain which are likely to interact with IgG1, IgG3, IgM, OmpK36, salivary agglutinin and gp41 loop peptide. The combined use of recombinant ghA, ghB, ghC and single-chain antibodies has revealed at least three ligand-binding sites on the globular domain of C1q: one is IgG- and OmpK36-specific, the second (IgM-binding site) is most likely overlapping with IgG/OmpK36 binding site, and the third (the gp41-binding site) seems to be located at the junction between the collagen and globular domains.