The Role of IgG Fc Region N-Glycosylation in the Pathomechanism of Rheumatoid Arthritis.

Anti-citrullinated protein antibodies (ACPAs) are involved in the pathogenesis of rheumatoid arthritis. N-glycosylation pattern of ACPA-IgG and healthy IgG Fc differs. The aim of this study is to determine the relative sialylation and galactosylation level of ACPAs and control IgG to assess their capability of inducing TNFα production, and furthermore, to analyze the correlations between the composition of Fc glycans and inflammatory markers in RA. We isolated IgG from sera of healthy volunteers and RA patients, and purified ACPAs on a citrulline-peptide column. Immunocomplexes (IC) were formed by adding an F(ab)2 fragment of anti-human IgG. U937 cells were used to monitor the binding of IC to FcγR and to trigger TNFα release determined by ELISA. To analyze glycan profiles, control IgG and ACPA-IgG were digested with trypsin and the glycosylation patterns of glycopeptides were analyzed by determining site-specific N-glycosylation using nano-UHPLC-MS/MS. We found that both sialylation and galactosylation levels of ACPA-IgG negatively correlate with inflammation-related parameters such as CRP, ESR, and RF. Functional assays show that dimerized ACPA-IgG significantly enhances TNFα release in an FcγRI-dependent manner, whereas healthy IgG does not. TNFα production inversely correlates with the relative intensities of the G0 glycoform, which lacks galactose and terminal sialic acid moieties.

Seropositivity and Antibody Profiling of Patients Are Dramatically Impacted by the Features of Peptides Used as Immunosorbents: A Lesson from Anti-Citrullinated Protein/Peptide Antibody.

Quantification of Abs toward a single epitope is critical to understanding immunobiological processes. In autoimmunity, the prognostic value of the serological profiles of patients draws much attention, but the detection of Abs toward a single epitope is not well controlled. Particularly, the rheumatoid arthritis (RA)-specific anti-citrullinated protein/peptide Abs (ACPA) are specific to a two-atom change on arginyl residues and are considered a heterogeneous family of Abs. As a model, we studied ACPA to decipher how peptide features used as immunosorbent impact Ab detection. We synthesized 30 peptides encompassing immunodominant epitopes of citrullinated fibrin differing by their length and biotin location and tested them using ELISA with 120 sera from RA and non-RA rheumatic disease controls, generating over 3000 experimental measurements. We showed that minor molecular changes in peptide chemical structure had dramatic consequences. Even when peptides exhibited the same epitope, measured Ab titers were extremely variable, and patients' seropositivity was discordant in up to 50% of cases. The distance between epitope and biotin was the most critical parameter for efficient Ab detection irrespective of biotin position or peptide length. Finally, we identified a 15-mer peptide bearing a single citrullinated epitope detecting almost all ACPA-positive sera, thus revealing a high degree of homogeneity in RA autoimmune response. This integrative analysis deciphers the dramatic impact of the molecular design of peptide-based technologies for epitope-specific Ab quantification. It provides a model for assay development and highlights that the studies using such technologies can give a wrong perception of biological processes and therefore that medical use of data must be cautious.

Affinity Purification and Comparative Biosensor Analysis of Citrulline-Peptide-Specific Antibodies in Rheumatoid Arthritis.

BACKGROUND: In rheumatoid arthritis (RA), anti-citrullinated protein/peptide antibodies (ACPAs) are responsible for disease onset and progression, however, our knowledge is limited on ligand binding affinities of autoantibodies with different citrulline-peptide specificity. METHODS: Citrulline-peptide-specific ACPA IgGs were affinity purified and tested by ELISA. Binding affinities of ACPA IgGs and serum antibodies were compared by surface plasmon resonance (SPR) analysis. Bifunctional nanoparticles harboring a multi-epitope citrulline-peptide and a complement-activating peptide were used to induce selective depletion of ACPA-producing B cells. RESULTS: KD values of affinity-purified ACPA IgGs varied between 10-6 and 10-8 M and inversely correlated with disease activity. Based on their cross-reaction with citrulline-peptides, we designed a novel multi-epitope peptide, containing Cit-Gly and Ala-Cit motifs in two-two copies, separated with a short, neutral spacer. This peptide detected antibodies in RA sera with 66% sensitivity and 98% specificity in ELISA and was recognized by 90% of RA sera, while none of the healthy samples in SPR. When coupled to nanoparticles, the multi-epitope peptide specifically targeted and depleted ACPA-producing B cells ex vivo. CONCLUSIONS: The unique multi-epitope peptide designed based on ACPA cross-reactivity might be suitable to develop better diagnostics and novel therapies for RA.

Synthetic Peptide-Based ELISA and ELISpot Assay for Identifying Autoantibody Epitopes.

Enzyme-linked immunosorbent assay (ELISA) is an invaluable diagnostic tool to detect serum autoantibody binding to target antigen. To map the autoantigenic epitope(s), overlapping synthetic peptides covering the total sequence of a protein antigen are used. A large set of peptides synthesized on the crown of pins can be tested by Multipin ELISA for fast screening. Next, to validate the results, the candidate epitope peptides are resynthesized by solid-phase synthesis, coupled to ELISA plate directly, or in a biotinylated form, bound to neutravidin-coated surface and the binding of autoantibodies from patients' sera is tested by indirect ELISA. Further, selected epitope peptides can be applied in enzyme-linked immunospot assay to distinguish individual, citrullinated peptide-specific autoreactive B cells in a pre-stimulated culture of patients' lymphocytes.

Bioactivity studies on atypical natural opioid hexapeptides processed from proenkephalin (PENK) precursor polypeptides.

Endogenous opioids are derived from four related polypeptide precursors: proenkephalin (PENK), prodynorphin (PDYN), pronociceptin (PNOC) and proopiomelanocortin (POMC). In mammals PENK encodes for four copy of Met-enkephalin, one octapeptide Met-enkephalin-Arg-Gly-Leu, one heptapeptide Met-enkephalin-Arg-Phe and a single copy of Leu-enkephalin. Our detailed bioinformatic search on the existing PENK sequences revealed several atypical hexapeptide Met-enkephalins in different vertebrate animals. They are located either in the second enkephalin unit or in the seventh enkephalin core position at the C-terminus. Altogether four different hexapeptide sequences were obtained representing eleven animal species: Met-enkephalin-Arg(6) (YGGFMR) in the bird zebra finch, Met-enkephalin-Asp(6) (YGGFMD), Met-enkephalin-Ile(6) (YGGFMI) in zebrafish; and Met-enkephalin-Ser(6) (YGGFMS) in two pufferfish species. All novel peptides were chemically synthesized and studied in receptor binding and G-protein activation assays performed on rat brain membranes. The four novel enkephalins were equipotent in stimulating G-proteins. Affinities of the peptides determined by equilibrium competition assays in receptor binding experiments were statistically different. At the MOP receptors the highest affinity (Ki 4nM) was obtained with the zebra finch peptide Met-enkephalin-Arg(6). The pufferfish Met-enkephalin-Ser(6) exhibited the highest affinity (Ki 6.7nM) at the DOP receptor. Phylogenetic neuropeptide libraries, defined here as a collection of mutationally different species variants of orthologous and paralogous peptide sequences, represent the natural molecular diversity of the neuropeptides. Such libraries can provide a wide range of structural information establishing comparative functional analyses. Since DNA sequencing data are rapidly increasing, more development in the natural peptide library concept is expected.

Role of N- or C-terminal biotinylation in autoantibody recognition of citrullin containing filaggrin epitope peptides in rheumatoid arthritis.

Here, we report on the synthesis, conformational analysis, and autoantibody binding properties of new sets of rheumatoid arthritis (RA) specific biotin-peptide conjugates derived from filaggrin epitope peptides. The biotin with or without a linker was attached to the Cit or Arg containing epitope core ((311)TXGRS(315)) or epitope region ((306)SHQESTXGXSXGRSGRSGS(324)) peptide (where X = Cit), through an amide bond at the N- or C-terminal of the epitopes. Antibody binding was detected by indirect enzyme-linked immunosorbent assay (ELISA) using sera from RA, Systemic lupus erythematosus (SLE) patients, as well as healthy individuals, and the secondary structure of conjugates was investigated by electronic circular dichroism (ECD). We found that autoantibodies from RA patients recognize specifically both filaggrin epitope region ((306)SHQESTXGXSXGRSGRSGS(324)) and short epitope core ((311)TXGRS(315)) peptides. Our data also indicate that the positioning of the biotin label within a peptide sequence can markedly influence the antibody binding, but the length of the linker incorporated has essentially no effect on the recognition. ECD experiments demonstrate that the Arg/Cit change does not influence the solution conformation of the peptide conjugates. However, the presence and position of the biotin moiety has a pronounced effect on the conformation of the 5-mer epitope core peptides, while it does not alter the secondary structure of the 19-mer epitope region peptides.

Chemotactic effect of odorants and tastants on the ciliate Tetrahymena pyriformis.

Naturally occurring aroma compounds are able to elicit physiological and migratory responses such as chemotaxis even at nano to femtomolar concentrations in organisms at different levels of phylogeny. Despite the amazing chemical variety of these substances the apparatus by which they can be detected i.e. the chemosensory receptors and the signaling pathways seem to be rather uniform and evolutionary well-conserved. The intracellular signaling process is supposed to be mediated by either cAMP or inositol 1,4,5-trisphosphate. The present work aimed to investigate the chemotactic behavior of 11 odorants that occur naturally in foods and are also used by the industry as additives, on the eukaryotic ciliate Tetrahymena pyriformis. Intracellular signaling pathways that might be activated by these compounds were also investigated. Activation of the phospholipase C (PLC) was measured by FACS and the stimulation of inositol-1,4,5-trisphosphate 3-kinases (IP3K) was measured using two specific inhibitors, wortmannin and LY294002. The strongest chemoattractant character was observed for isoamyl acetate (10(⁻6) M), propyl isobutyrate (10(⁻8) M), isobutyl propionate (10(⁻6) M). The strongest repellent action was exerted by benzyl acetate (10(⁻8) M), furfuryl thioacetate (10(⁻12) M). Our results suggest that Tetrahymena responds in a very sensitive way to slight changes in the molecular structure. According to our study, tracer amounts of solvents do not contribute significantly to the chemotactic profile of the respective odorants. No significant activation of PLC or PI3K could be observed following stimulation with attractant odorants which implies that some other pathways may be involved, hence further investigation is needed.

Specific expression of PAD4 and citrullinated proteins in lung cancer is not associated with anti-CCP antibody production.

Anti-citrullinated protein antibodies (ACPAs), produced against citrullinated proteins, are diagnostic and prognostic markers of rheumatoid arthritis (RA). The underlying mechanism that explains the connection of smoking, citrullination [catalyzed by peptidyl arginine deiminases (PADs)] and ACPAs is still unclarified in RA. Thus, we searched for a non-arthritic model in which an increased cell death allows the formation of autoantibodies. Data supporting that lung cancer might be a good candidate are as follows: (i) smoking plays a role in its pathogenesis, (ii) the disease is frequently accompanied by paraneoplastic syndrome, (iii) smoking increases citrullination in the lung, (iv) various types of malignancies are associated with increased citrullination and (v) lung cancer tissue shows similarities with RA synovium. Serum PAD4, rheumatoid factor (RF) and ACPA levels were measured in 42 lung cancer patients; expression of cytokeratin 7 (CK7), PAD4 and citrullinated proteins was visualized in 113 lung cancer tissues. All parameters were analyzed in correlation with smoking history. None of the patients had polyarthritis or autoimmune disease. Significantly increased RF levels were associated with higher PAD4 levels in smoker lung cancer patients compared with non-smokers. Both PAD4 and citrullination immunostaining strongly correlated with that of CK7 in lung cancer, however, did not differ according to smoking history. Two of 30 smoker lung cancer patients had high anti-cyclic citrullinated peptide levels. In conclusion, PAD4 and citrullination may be helpful in distinguishing lung cancer from healthy tissue. Smoking, abnormal serum PAD4 and RF levels may not be sufficient for the production of ACPAs and development of autoimmunity.

New daunomycin-oligoarginine conjugates: synthesis, characterization, and effect on human leukemia and human hepatoma cells.

In this article, the synthesis, a novel chromatographic procedure and characteristics of a new class of daunomycin (Dau)-oligoarginine conjugates are described. In these compounds oligoarginine with 6 or 8 residues (Arg(n), n = 6, 8) is attached to Dau by different covalent bond: squaric amide (Dau- square-Arg(n)), oxime (Dau=N-O-CH2-CO-Arg(n)), or hydrazone (H-Glu(Arg(n))-NH-N=Dau). Conjugates were characterized by RP-HPLC and mass spectrometry. We report also on our findings concerning chemical and biological properties of Dau-conjugates as a function of covalent linkage, site of conjugation and length of the oligoarginine moiety. Stability, fluorescent properties as well as cytostatic effect and cellular uptake of these compounds were studied. Dau-conjugates with squaric amide or oxime linkage were stable, but continuous release of free Dau was observed from the hydrazone conjugate in solution. We found that some spectral characteristics (e.g., the amplitude of the emission spectrum) of conjugates could be sensitive for the site of coupling (amino vs. oxo function). Cytostasis and cellular uptake of conjugates were investigated both on human leukemia (HL-60) and human hepatoma (HepG2) cell lines by MTT assay and flow cytometry We found that cytostatic effect and uptake properties of Dau-conjugates were dependent on the acid stability of the linkage (hydrazone vs. oxime/amide) applied and more markedly on the cell line studied.

Synthesis of enzymatically resistant nociceptin-related peptides containing a carbamic acid residue.

Nociceptin, a 17-amino acid peptide (FGGFTGARKSARKLANQ, N/OFQ), is the endogenous ligand of the nociceptin/orphanin FQ (NOP) receptor. This receptor-ligand system is involved in various physiological as well as pathophysiological mechanisms, but owing to the peptidic structure, it is rapidly degraded by enzymes. The enzymatic digestion of nociceptin involves mainly aminopeptidases and yields Noc(2-17)-OH and other smaller fragments. We aimed at increasing the enzymatic stability against aminopeptidases in the case of peptide Noc(1-13)-NH(2), which possesses the minimum sequence capable of interacting with the NOP receptor. Therefore we developed a new procedure for the synthesis of peptides with the carbamic acid residue [...-NH-CH(R)-CO-NH-CO-NH-CH(Q)-CO-.]. A set of four carbamic acid-nociceptin derivatives were produced. The carbamic acid residue was incorporated into the inner part of the peptides, building on solid phase, by using a suitable dipeptide fragment with carbamic acid residue produced by a simple and efficient three-step solution phase procedure. Enzymatic stability of carbamic acid peptides was studied in the presence of aminopeptidase M (AP-M) and in rat brain membrane homogenate. The receptor-binding properties were also studied by radioligand binding assay on crude rat brain membranes and the activity of the ligands were analyzed on isolated mouse vas deferens (MVD) tissues. We found that incorporation of the carbamic acid residue into the N-terminal part of nociceptin significantly increases the resistance against AP-M. We observed the decrease of binding affinities to the NOP receptor in case of the peptides modified in the N-terminal portion. Consequently, the incorporation of the carbamic acid residue into peptides can be proposed as a promising and reasonable tool for increasing enzymatic stability, where the native molecule is less sensitive for carbamic acid residue-related structural change.

[How do Hungarian family physicians think about Helicobacter pylori? Results of national survey]. / Hogyan viszonyulnak a magyar háziorvosok a Helicobacter pylori kérdésköréhez? Egy országos felmérés eredményei.

UNLABELLED: INTRODUCTION, AIMS: In spring of 2000 a national survey was conducted on the diagnostic and therapeutic attitudes of Hungarian family practitioners concerning Helicobacter pylori. RESULTS: Based on responses, endoscopy, urea breath test and serology are easily available to 98, 42 and 48% of family practitioners respectively. Only 45% of responders performs Helicobacter pylori testing him/herself, while 82% prescribes eradication therapy without a gastroenterologist. In the second part of the questionnaire responders had to decide in 15 routine clinical cases, whether to perform Helicobacter pylori testing, and if so, whether to be satisfied with noninvasive testing or also to perform an endoscopy. Responses comply with international and Hungarian national recommendations, reflecting at the same time the uncertainties inherent in medical literature. Five responses of Hungarian family practitioners differed from the recommendations of literature. Two questions were related to screening Helicobacter pylori: in a complaint-free patient family practitioners are not inspired to perform a screening either in the case of familiarity of gastric cancer or in that of duodenal ulcer known from the individual case history. Helicobacter pylori testing is often superfluously suggested to young patients suffering from gastro-esophageal reflux disease. An empirical approach is revealed by the fact, that in cases of Helicobacter pylori positive duodenal ulcer, when eradication eliminates complaints, 41% of family practitioners would not test result of eradication. At the same time it is considered a mistake, that in case of young patients with a Helicobacter pylori positive gastric ulcer, being free of complaints after eradication, 66% of physicians refrains from endoscopic control. CONCLUSIONS: The possibilities, education, diagnostic and therapeutic skills as to diseases due to Helicobacter pylori of Hungarian family practitioners seems to be appropriate. In education emphasis should be primarily placed on screening and post-eradication tasks. Guidelines should be offered to family practitioners concerning controversary issues (gastro-esophageal reflux disease, dyspepsia, administration of non-steroid anti-inflammatory drugs).