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.

Synthesis and SAR Analysis of Novel 4-Hydroxytamoxifen Analogues Based on Their Cytotoxic Activity and Electron-Donor Character.

Utilizing McMurry reactions of 4,4'-dihydroxybenzophenone with appropriate carbonyl compounds, a series of 4-Hydroxytamoxifen analogues were synthesized. Their cytotoxic activity was evaluated in vitro on four human malignant cell lines (MCF-7, MDA-MB 231, A2058, HT-29). It was found that some of these novel Tamoxifen analogues show marked cytotoxicity in a dose-dependent manner. The relative ROS-generating capability of the synthetized analogues was evaluated by cyclic voltammetry (CV) and DFT modeling studies. The results of cell-viability assays, CV measurements and DFT calculations suggest that the cytotoxicity of the majority of the novel compounds is mainly elicited by their interactions with cellular targets including estrogen receptors rather than triggered by redox processes. However, three novel compounds could be involved in ROS-production and subsequent formation of quinone-methide preventing proliferation and disrupting the redox balance of the treated cells. Among the cell lines studied, HT-29 proved to be the most susceptible to the treatment with compounds having ROS-generating potency.

Influence of the Dabcyl group on the cellular uptake of cationic peptides: short oligoarginines as efficient cell-penetrating peptides.

Cell-penetrating peptides (CPPs) are promising delivery vehicles. These short peptides can transport wide range of cargos into cells, although their usage has often limitations. One of them is the endosomatic internalisation and thus the vesicular entrapment. Modifications which increases the direct delivery into the cytosol is highly researched area. Among the oligoarginines the longer ones (n > 6) show efficient internalisation and they are well-known members of CPPs. Herein, we describe the modification of tetra- and hexaarginine with (4-((4-(dimethylamino)phenyl)azo)benzoyl) (Dabcyl) group. This chromophore, which is often used in FRET system increased the internalisation of both peptides, and its effect was more outstanding in case of hexaarginine. The modified hexaarginine may enter into cells more effectively than octaarginine, and showed diffuse distribution besides vesicular transport already at low concentration. The attachment of Dabcyl group not only increases the cellular uptake of the cell-penetrating peptides but it may affect the mechanism of their internalisation. Their conjugates with antitumor drugs were studied on different cells and showed antitumor activity.

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.

Topoisomerases inhibition and DNA binding mode of daunomycin-oligoarginine conjugate.

Cancer is a major health issue adsorbing the attention of a biomedical research. To fight this disease, new drugs are developed, specifically tailored to target biological pathways or peculiar components of the tumour cells. Particularly interesting is the use of intercalating agents as drugs capable to bind DNA and inhibit enzymes involved in DNA metabolism. Anthracyclines are the most commonly used anticancer drugs. In particular, daunomycin is used to cancer treatment by exploiting its ability to intercalate DNA and inhibit the activity of DNA topoisomerases implicated in the replication processes. Unfortunately, clinical application of anthracyclines is limited by their side effects. The conjugation with specific carriers could affect the selectivity and reduce side effect by improving stability and/or cellular uptake properties. We here report the biochemical characterisation of a daunomycin oligopeptide conjugate containing six residues of arginine, by the analysis of its fluorescence properties, DNA interaction and topoisomerases inhibitory effects.

Novel Polycondensed Partly Saturated ß-Carbolines Including Ferrocene Derivatives: Synthesis, DFT-Supported Structural Analysis, Mechanism of Some Diastereoselective Transformations and a Preliminary Study of Their in vitro Antiproliferative Effects.

Use of a Pictet-Spengler reaction of tryptamine and l-tryptophan methyl ester and subsequent reduction of the nitro group followed by further cyclocondensation with aryl aldehydes and formyl-substituted carboxylic acids, including ferrocene-based components, furnished a series of diastereomeric 6-aryl-substituted 5,6,8,9,14,14b-hexahydroindolo[2',3':3,4]pyrido[1-c]-quinazolines and 5,5b,17,18-tetrahydroindolo[2',3':3,4]pyrido[1,2-c]isoindolo[2,1-a]quinazolin-11-(15bH)-ones with the elements of central-, planar and conformational chirality. The relative configuration and the conformations of the novel polycyclic indole derivatives were determined by 1H- and 13C-NMR methods supplemented by comparative DFT analysis of the possible diastereomers. The structure of one of the pentacyclic methyl esters with defined absolute configuration "S" was also confirmed by single crystal X-ray diffraction measurement. Accounting for the characteristic substituent-dependent diastereoselective formation of the products multistep mechanisms were proposed on the basis of the results of DFT modeling. Preliminary in vitro cytotoxic assays of the products revealed moderate-to-significant antiproliferative effects against PANC-1-, COLO-205-, A-2058 and EBC-1 cell lines that proved to be highly dependent on the stereostructure and on the substitution pattern of the pending aryl substituent.

The effect of the branched chain polypeptide carrier on biodistribution of covalently attached B-cell epitope peptide (APDTRPAPG) derived from mucin 1 glycoprotein.

In order to establish structure-function relationship for the design of a new group of oligopeptide antigen-macromolecule conjugate, multiple copies of mucin-1 B-cell epitope peptide, APDTRPAPG were conjugated with branched chain polymeric polypeptides possessing poly[L-Lys] backbone. By the synthesis, radiolabeling (125I) and in vivo treatment of BALB/c mice with epitope conjugates containing XiK/XAK type carrier, where X = Glu (EiK or EAK) or Leu (LAK), the influence of the polypeptide structure on the blood clearance profile and on tissue distribution profile concerning the epitope delivery to relevant organs (e.g. immunocompetent or involved in excretion) were investigated. We observed significant differences in the blood clearance profiles for the conjugates, the respective polypeptide carriers and free epitope peptide. All conjugates, regardless of their charge properties exhibited longer presence in the circulation than the free oligopeptide. Tissue distribution data also showed that the structural properties (e.g. amino acid composition, charge) of the carrier polypeptide have marked influence on the tissue accumulation of the epitope peptide conjugates. In contrast to conjugates with linear (K) or branched chain (LAK) polycationic polymers exhibiting rapid blood clearance and high spleen/liver uptake, amphoteric epitope peptide conjugates with different branches, but similar charge properties (EiK or EAK) had extended blood survival and generally lower tissue accumulation. The results on this systematic investigation suggest that further studies on the immune response induced by these epitope conjugates would be needed to provide correlation between biodistribution properties (presence in the blood, level of tissue accumulation) and the capacity of these conjugates to elicit antibody production.

Mapping the tandem mass spectrometric characteristics of citrulline-containing peptides.

RATIONALE: Protein citrullination (deimination) is a post-translational modification of proteins converting arginine(s) into citrulline(s). "Overcitrullination" could be associated with severe pathological conditions. Mass spectrometric analysis of modified proteins is hindered by several problems. A comprehensive study of the fragmentation of deiminated peptides is not yet available. In this paper we have made an attempt to describe the characteristics of these processes, based on the studies of epitope model oligopeptides derived from clinically relevant proteins. METHODS: Solutions of purified model peptides containing either one or two citrulline residues as well as their native variants were injected directly into the electrospray source of a high accuracy and resolution quadrupole-time-of-flight instrument and were analysed by tandem mass spectrometry using low-energy collision-induced dissociation. RESULTS: Loss of isocyanic acid from citrulline residues is a preferred fragmentation route for deiminated peptides, which yields ornithine residues in the sequence. However, simultaneous detection of both the isocyanic acid loss and sequence fragments is often compromised. A preferential cleavage site was observed between citrulline and any other following amino acids yielding intensive complementary b- and y-type ions. Also, citrulline positioned at the C-termini displays a preferential cleavage N-terminal to this residue yielding characteristic y1 ions. These phenomena are described here for the first time and are referred to as the "citrulline effect". CONCLUSIONS: We found that the citrulline effect is very pronounced and could be used as a complementary tool for the confirmation of modification sites in addition to losses of isocyanic acids from the protonated molecules or from fragment ions. Low collision energy applied to peptide ions having partially mobile protons reveals the site of modification by generating specific and intensive fragments of the sequence. On the other hand, fragmenting precursor ions with mobile protons usually allows full sequence coverage, although citrulline-specific fragments may exhibit lower intensities compared to other fragments.

The effect of conjugation on antitumor activity of vindoline derivatives with octaarginine, a cell-penetrating peptide.

Some Vinca alkaloids (eg, vinblastine, vincristine) have been widely used as antitumor drugs for a long time. Unfortunately, vindoline, a main alkaloid component of Catharanthus roseus (L.) G. Don, itself, has no antitumor activity. In our novel research program, we have prepared and identified new vindoline derivatives with moderate cytostatic activity. Here, we describe the effect of conjugation of vindoline derivative with oligoarginine (tetra-, hexa-, or octapeptides) cell-penetrating peptides on the cytostatic activity in vitro and in vivo. Br-Vindoline-(l)-Trp-OH attached to the N-terminus of octaarginine was the most effective compound in vitro on HL-60 cell line. Analysis of the in vitro activity of two isomer conjugates (Br-vindoline-(l)-Trp-Arg8 and Br-vindoline-(d)-Trp-Arg8 suggests the covalent attachment of the vindoline derivatives to octaarginine increased the antitumor activity significantly against P388 and C26 tumour cells in vitro. The cytostatic effect was dependent on the presence and configuration of Trp in the conjugate as well as on the cell line studied. The configuration of Trp notably influenced the activity on C26 and P388 cells: conjugate with (l)-Trp was more active than conjugate with the (d)-isomer. In contrast, conjugates had very similar effect on both the HL-60 and MDA-MB-231 cells. In preliminary experiments, conjugate Br-vindoline-(l)-Trp-Arg8 exhibited some inhibitory effect on the tumor growth in P388 mouse leukemia tumor-bearing mice. Our results indicate that the conjugation of modified vindoline could result in an effective compound even with in vivo antitumor activity.

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.

Ferrocene-Containing Impiridone (ONC201) Hybrids: Synthesis, DFT Modelling, In Vitro Evaluation, and Structure⁻Activity Relationships.

Inspired by the well-established clinical evidence about the interplay between apoptotic TRAIL (tumour necrosis factor-related apoptosis-inducing ligand) mechanism and reactive oxygen species (ROS)-mediated oxidative stress, a set of novel ONC201 hybrids containing the impiridone core and one or two differently positioned ferrocenylalkyl groups were synthesised in our present work. These two types of residues have been implicated in the aforementioned mechanisms associated with cytotoxic activity. A straightforward, primary amine-based synthetic approach was used allowing the introduction of a variety of N-substituents into the two opposite regions of the heterocyclic skeleton. Reference model compounds with benzyl and halogenated benzyl groups were also synthesised and tested. The in vitro assays of the novel impiridones on five malignant cell lines disclosed characteristic structure-activity relationship (SAR) featuring significant substituent-dependent activity and cell-selectivity. A possible contribution of ROS-mechanism to the cytotoxicity of the novel metallocenes was suggested by density functional theory (DFT)studies on simplified models. Accordingly, unlike the mono-ferrocenylalkyl-substituted products, the compounds containing two ferrocenylalkyl substituents in the opposite regions of the impiridone core display a much more pronounced long-term cytotoxic effect against A-2058 cell line than do the organic impiridones including ONC201 and ONC212. Furthermore, the prepared bis-metallocene derivatives also present substantial activity against COLO-205- and EBC-1 cell lines.

Comparative analysis of internalisation, haemolytic, cytotoxic and antibacterial effect of membrane-active cationic peptides: aspects of experimental setup.

Cationic peptides proved fundamental importance as pharmaceutical agents and/or drug carrier moieties functioning in cellular processes. The comparison of the in vitro activity of these peptides is an experimental challenge and a combination of different methods, such as cytotoxicity, internalisation rate, haemolytic and antibacterial effect, is necessary. At the same time, several issues need to be addressed as the assay conditions have a great influence on the measured biological effects and the experimental setup needs to be optimised. Therefore, critical comparison of results from different assays using representative examples of cell penetrating and antimicrobial peptides was performed and optimal test conditions were suggested. Our main goal was to identify carrier peptides for drug delivery systems of antimicrobial drug candidates. Based on the results of internalisation, haemolytic, cytotoxic and antibacterial activity assays, a classification of cationic peptides is advocated. We found eight promising carrier peptides with good penetration ability of which Penetratin, Tat, Buforin and Dhvar4 peptides showed low adverse haemolytic effect. Penetratin, Transportan, Dhvar4 and the hybrid CM15 peptide had the most potent antibacterial activity on Streptococcus pneumoniae (MIC lower than 1.2 µM) and Transportan was effective against Mycobacterium tuberculosis as well. The most selective peptide was the Penetratin, where the effective antimicrobial concentration on pneumococcus was more than 250 times lower than the HC50 value. Therefore, these peptides and their analogues will be further investigated as drug delivery systems for antimicrobial agents.

Pitfalls in the synthesis of fluorescent methotrexate oligopeptide conjugates.

Methotrexate (MTX) conjugates with poly[Lys(DL-Alam)] based polymeric polypeptides are efficient against Leishmania donovani parasite infection, but the mechanism of the effect is not known yet. We prepared therefore the 5(6)-carboxyfluorescein (Cf) labeled oligopeptide [Cf-K(AaAa)] (a: D-alanine, A: L-alanine) and the corresponding MTX conjugates [Cf-K(MTX-AaAa)] as model compounds for structure-activity experiments. The conjugate aimed to be synthesized with solid phase methodology on MBHA resin with Boc strategy, using Fmoc-Lys(Boc)-OH. However, various side reactions were identified. Here we report three problems observed during the synthesis as well as solutions developed by us: (1) unexpected cyclopeptide-formation with the lactone-carboxylic group of the Cf was detected, when Cf was attached to the α-amino group of the Lys residue on solid phase. This was avoided by changing the order of Cf incorporation with using Fmoc/Dde strategy. Alternatively, we have built the peptide with Fmoc strategy on solid phase first and performed the labeling with Cf-OSu subsequently in solution. (2) During HF cleavage of the protected conjugates, MTX was demonstrated to form adducts with anisole and p-cresol scavengers, and the TMSOTf cleavage methodology was also found to be inadequate due to the large number of side products formed. We report here that using Fmoc/Dde strategy is an appropriate method to circumvent the cleavage with HF or TMSOTf. (3) During the coupling of MTX with oligopeptide, structural and stereo isomers are formed. We have described here the suitable conditions of HPLC separation of these products.

Synthesis, structure and in vitro cytostatic activity of ferrocene-Cinchona hybrids.

Exploring copper(I)- and ruthenium(II)-catalyzed azide-alkyne cycloadditions and a Sonogashira protocol, novel cytostatic ferrocene-cinchona hybrids were synthetized displaying significant in vitro activity on HepG-2 and HT-29 cells. Preliminary SAR studies disclosed that compounds incorporating linkers with 1,2,3-triazole and chalchone residues can be considered as promising lead structures. According to the best of our knowledge this is the first letter on the incorporation of ferrocene nucleus in the reputed cinchona family via triazole and chalcone linkers with established pharmaceutical profile.

Effect of SXWS/WSXWS peptides on chemotaxis and adhesion of the macrophage-like cell line J774.

WSXWS motif is a conserved amino acid sequence that is present in type I cytokine receptors. This motif that can be found both in the ligand binding chains and signal transducer molecule of the receptors with different amino acids at the position "X" plays a role in the receptor folding, ligand binding and signal transduction as well. Structural analysis proved that WSEWS motif of IL-6R is located in a highly accessible location in the protein. Structural properties and chemotaxis of a tetrapeptide library with SXWS sequence, where X was the 19 proteinogenic amino acids except cystein were systematically studied earlier. It has been proved that C-terminal amidation and the identity of amino acid X had a pronounced influence on the chemotactic properties but less of the structure of the peptides. Here, we present our findings on the effect of a tetrapeptide and a pentapeptide library with the sequence of SXWS and WSXWS on the chemotaxis and adhesion of J774 murine macrophage cell line. We studied the effect of the presence/absence of N-terminal tryptophan and the different amino acids at the X position on these physiological responses. Results indicated that amino acid X had a marked influence on chemotaxis, adhesion as well as on proliferation induced by (W)SXWS peptides. Elongation of SXWS sequence with a tryptophan at the N terminus also altered pronouncedly all the physiological responses of the cells studied. A good correlation could be observed between the chemotaxis and the proliferation and physicochemical parameters of the amino acid X.

Characterization of desmoglein-3 epitope region peptides as synthetic antigens: analysis of their in vitro T cell stimulating efficacy, cytotoxicity, stability, and their conformational features.

Desmoglein-3 (Dsg3) adhesion protein is the main target of autoantibodies and autoreactive T cells in Pemphigus vulgaris (PV) autoimmune skin disorder. Several mapping studies of Dsg3 T cell epitope regions were performed, and based on those data, we designed and synthesized four peptide series corresponding to Dsg3 T cell epitope regions. Each peptide series consists of a 17mer full-length peptide (Dsg3/189-205, Dsg3/206-222, Dsg3/342-358, and Dsg3/761-777) and its N-terminally truncated derivatives, resulting in 15 peptides altogether. The peptides were prepared on solid phase and were chemically characterized. In order to establish a structure-activity relationship, the solution conformation of the synthetic peptides has been investigated using electronic circular dichroism spectroscopy. The in vitro T cell stimulating efficacy of the peptides has been determined on peripheral blood mononuclear cells isolated from whole blood of PV patients and also from healthy donors. After 20 h of stimulation, the interferon (IFN)-γ content of the supernatants was measured by enzyme-linked immunosorbent assay. In the in vitro conditions, peptides were stable and non-cytotoxic. The in vitro IFN-γ production profile of healthy donors and PV patients, induced by peptides as synthetic antigens, was markedly different. The most unambiguous differences were observed after stimulation with 17mer peptide Dsg3/342-358, and three truncated derivatives from two other peptide series, namely, peptides Dsg3/192-205, Dsg3/763-777, and Dsg3/764-777. Comparative analysis of in vitro activity and the capability of oligopeptides to form ordered or unordered secondary structure showed that peptides bearing high solvent sensibility and backbone flexibility were the most capable to distinguish between healthy and PV donors.

Recognition of new citrulline-containing peptide epitopes by autoantibodies produced in vivo and in vitro by B cells of rheumatoid arthritis patients.

Anti-citrullinated peptide/protein antibodies (ACPAs) are highly sensitive and specific markers of rheumatoid arthritis (RA). Identification of peptide epitopes that may detect different subgroups of RA patients might have diagnostic and prognostic significance. We have investigated citrulline- and arginine-containing peptide pairs derived from filaggrin, collagen or vimentin, and compared this citrulline-peptide panel with the serological assays conventionally used to detect ACPAs. Furthermore, we studied if the same citrulline-peptides identify antibody-secreting cells in in vitro cultures of RA B cells. Recognition of citrulline- and arginine-containing filaggrin, vimentin and collagen peptide epitopes were tested by Multipin ELISA system, by indirect ELISA and by a peptide-specific microarray. B cells were purified from blood by negative selection; antibody-producing cells were enumerated by ELISPOT assay. The panel composed of citrulline-peptide epitopes of filaggrin, collagen and vimentin was recognized by RA sera with a sensitivity and specificity comparable with the currently used tests. Moreover, the combined citrulline-peptide panel including the new short epitope peptide of filaggrin, fil311-315, also identified nearly one-third of RA cases that were negative for antibodies against cyclic citrullinated peptides, mutated citrullinated vimentin or for rheumatoid factor. The results with the peptide-specific microarray have shown that although most ACPAs recognizing the four citrulline peptides are IgG, some of them specifically recognizing citrulline-containing filaggrin peptides (fil311-315 and fil306-326) are IgM, and so may be produced either by newly formed activated B cells or by unswitched B memory cells. Furthermore, the citrulline-peptides of filaggrin and vimentin detect ACPA-producing cells, and so could also be applied to study the B cells of RA patients.

Nanoparticle encapsulated lipopeptide conjugate of antitubercular drug isoniazid: in vitro intracellular activity and in vivo efficacy in a Guinea pig model of tuberculosis.

Considering that Mycobacterium tuberculosis (Mtb) can survive in host phagocytes for decades and currently applied drugs are largely ineffective in killing intracellular Mtb, novel targeted delivery approaches to improve tuberculosis chemotherapy are urgently needed. In order to enhance the efficacy of a clinically used antitubercular agent (isoniazid, INH) a novel lipopeptide carrier was designed based on the sequence of tuftsin, which has been reported as a macrophage-targeting molecule. The conjugate showed relevant in vitro activity on Mtb H37Rv culture with low cytotoxicity and hemolytic activity on human cells. The conjugate directly killed intracellular Mtb and shows much greater efficacy than free INH. To improve bioavailability, the conjugate was encapsulated into poly(lactide-co-glycolide) (PLGA) nanoparticles and tested in vivo in a guinea pig infection model. External clinical signs, detectable mycobacterial colonies in the organs, and the histopathological findings substantiate the potent chemotherapeutic effect of orally administered conjugate-loaded nanoparticles.

The effect of glycosylation on the antibody recognition of a MUC2 mucin epitope.

MUC2 glycoprotein, produced by the epithelium of the colon and built up mainly of repeat units of (1) PTTTPITTTTTVTPTPTPTGTQT(23) , can be overexpressed or underglycosylated in gastrointestinal diseases, e.g. in case of colon carcinoma. We have been studying the epitope structure of the MUC2 by focusing on the repeat unit with the mucin peptide specific MAb 996 monoclonal antibody. This antibody recognizes the (18) PTGTQ(22) sequence as minimal, and (16) PTPTGTQ(22) as optimal epitope within the underglycosylated glycoprotein. In this article, we aim to clarify the effect of glycosylation of the epitope on MAb 996 antibody binding including its correlation with the secondary structure of the modified peptides: glycosylation in the epitope core and in the flank. For this we have prepared the (16) PTPTGTQ(22) peptide glycosylated with N-acetylgalactoseamine (Tn antigen) in position 17, 19, 21, or on all three threonines. The MAb 996 antibody binding properties of the peptides were characterized in competitive ELISA experiments, and their solution secondary structure was studied by circular dichroism spectroscopy in water and in the ordered structure promoting trifluoroethanol. Our results show that glycosylation in position 19 (peptide (16) PTPT(GalNAcα)GTQ(22) ) resulted in enhanced antibody recognition and significantly altered secondary structure, while glycosylation in position 21 completely demolished the binding. These findings could be useful in determining the nature of antigen-antibody interaction, and perhaps designing synthetic peptide vaccines for tumor therapy.

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.