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Oxytocin (OT) is a neurohypophyseal peptide hormone containing a disulphide-bridged pseudocyclic conformation. The biomedical use of OT peptides is limited amongst others by disadvantageous pharmacokinetic parameters. To increase the stability of OT by replacing the disulphide bridge with the stable and more rigid [1,2,3]triazol-1-yl moiety, we employed the Cu2+-catalysed side chain-to-side chain azide-alkyne 1,3-cycloaddition. Here we report the design, synthesis, conformational analysis, and in vitro pharmacological activity of a homologous series of Cα1-to-Cα6 side chain-to-side chain [1,2,3]triazol-1-yl-containing OT analogues differing in the length of the bridge, location, and orientation of the linking moiety. Exploiting this macrocyclisation approach, it was possible to generate a systematic series of compounds providing interesting insight into the structure-conformation-function relationship of OT. Most analogues were able to adopt similar conformation to endogenous OT in water, namely, a type I β-turn. This approach may in the future generate stabilised pharmacological peptide tools to advance understanding of OT physiology.

The development of skin-care products is recently growing. Cosmetic formulas containing active ingredients with proven efficacy, namely cosmeceuticals, are based on various compounds, including peptides. Different whitening agents featuring anti-tyrosinase activity have been applied in the cosmeceutical field. Despite their availability, their applicability is often limited due to several drawbacks including toxicity, lack of stability, and other factors. In this work, we present the inhibitory effect on diphenolase activity of thiosemicarbazone (TSC)-peptide conjugates. Tripeptides FFY, FWY, and FYY were conjugated with three TSCs bearing one or two aromatic rings via amide bond formation in a solid phase. Compounds were then examined as tyrosinase and melanogenesis inhibitors in murine melanoma B16F0 cell line, followed by the cytotoxicity assays of these cells. In silico investigations explained the differences in the activity, observed among tested compounds. Mushroom tyrosinase was inhibited by TSC1-conjugates at micromolar level, with IC50 lower than this for kojic acid, a widely used reference compound. Up to now, this is the first report regarding thiosemicarbazones conjugated with tripeptides, synthesised for the purpose of tyrosinase inhibition.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has posed a great concern in human population. To fight coronavirus emergence, we have dissected the conserved amino acid region of the internal fusion peptide in the S2 subunit of Spike glycoprotein of SARS-CoV-2 to design new inhibitory peptides. Among the 11 overlapping peptides (9-23-mer), PN19, a 19-mer peptide, exhibited a powerful inhibitory activity against different SARS-CoV-2 clinical isolate variants in absence of cytotoxicity. The PN19 inhibitory activity was found to be dependent on conservation of the central Phe and C-terminal Tyr residues in the peptide sequence. Circular dichroism spectra of the active peptide exhibited an alpha-helix propensity, confirmed by secondary structure prediction analysis. The PN19 inhibitory activity, exerted in the first step of virus infection, was reduced after peptide adsorption treatment with virus-cell substrate during fusion interaction. Additionally, PN19 inhibitory activity was reduced by adding S2 membrane-proximal region derived peptides. PN19 showed binding ability to the S2 membrane proximal region derived peptides, confirmed by molecular modelling, playing a role in the mechanism of action. Collectively, these results confirm that the internal fusion peptide region is a good candidate on which develop peptidomimetic anti SARS-CoV-2 antivirals.

We report for the first time Antibody-Drug-Conjugates (ADCs) containing human (h) Carbonic Anhydrase (CA; EC 4.2.1.1) directed Monoclonal Antibodies (MAbs) linked to low molecular weight inhibitors of the same enzymes by means of hydrophilic peptide spacers. In agreement with the incorporated CA directed MAb fragments, in vitro inhibition data of the obtained ADCs showed sub-nanomolar KI values for the tumour associated CAs IX and XII which were up to 10-fold more potent when compared to the corresponding unconjugated MAbs. In addition, the introduction of the CA inhibitor (CAI) benzenesulfonamide allowed the ADCs to potently inhibit the housekeeping tumoral off-target human CA II isoform. Such results are supporting the definition of an unprecedented reported class of ADCs able to hit simultaneously multiple hCAs physiologically cooperative in maintaining altered cellular metabolic pathways, and therefore ideal for the treatment of chronic diseases such as cancers and inflammation diseases.

Abstract Immune response to biologics treatment, while widely reported, yet fails to correlate with clinical outcomes and assay to assay comparison is often not possible. Hence, we developed a new peptide based-detection assay to stratify pediatric patients with juvenile idiopathic arthritis (JIA) or chronic non-infectious uveitis (CNU) and monitor anti-drug antibodies (ADAbs) formed as part of an immune response to treatment with the fully human monoclonal therapeutic antibody Adalimumab. Adalimumab derived synthetic peptides were optimized for maximum immunogenicity and were tested by SP-ELISA on a development cohort of 18 JIA and CNU treated patients. The two best performing peptides able to differentiate patient groups were selected for evaluation with a larger scale ELISA testing on a total of 29 sera from pediatric patients with JIA or CNU. The results of this peptide-based assay were compared to an in-house developed SPR biosensor ADAbs assay and a commercially available bridging ELISA. The first peptide, termed HC3, was able to positively detect ADAbs in 7 out of the 29 sera, while the second peptide, called LC3, was able to detect ADAbs in 11 out of 29 sera in the evaluation group. Following statistical data evaluation, it has been found that the detection of ADAbs using the peptide-based ELISA assay positively correlates with disease progression and remission. Two synthetic peptides derived from Adalimumab may provide a beneficial tool to clinicians for monitoring patient response to such treatment and taking informed decisions for treatment alternatives.

The peptide hormone relaxin (RLX), also available as clinical-grade recombinant protein (serelaxin), holds great promise as a cardiovascular and anti-fibrotic agent but is limited by the pharmacokinetic issues common to all peptide drugs. In this study, by a computational modelling chemistry approach, we have synthesized and tested a set of low molecular weight peptides based on the putative receptor-binding domain of the B chain of human H1 RLX isoform, with the objective to obtain RLX analogues with improved pharmacokinetic features. Some of them were stabilized to induce the appropriate 3-D conformation by intra-chain tri-azolic staples, which should theoretically enhance their resistance to digestive enzymes making them suited for oral administration. Despite these favourable premises, none of these H1 peptides, either linear or stapled, revealed a sufficient affinity to the specific RLX receptor RXFP1. Moreover, none of them was endowed with any RLX-like biological effects in RXFP1-expressing THP-1 human monocytic cells and mouse NIH-3T3-derived myofibroblasts in in vitro culture, in terms of significantly relevant cAMP elevation and ERK1/2 phosphorylation, which represent two major signal transduction events downstream RXFP1 activation. This was at variance with authentic serelaxin, which induced a clear-cut, significant activation of both these classical RLX signaling pathways. Albeit negative, the results of this study offer additional information about the structural requirements that new peptide therapeutics shall possess to effectively behave as RXFP1 agonists and RLX analogues.

The involvement of Myelin Basic Protein (MBP) in Multiple Sclerosis (MS) has been widely discussed in the literature. This intrinsically disordered protein has an interesting α-helix motif, which can be considered as a conformational epitope. In this work we investigate the importance of the helical structure in antibody recognition by MBP peptides of different lengths. Firstly, we synthesized the peptide MBP (81–106) (1) and observed that its elongation at both N- and C-termini, to obtain the peptide MBP (76–116) (2) improves IgM antibody recognition in SP-ELISA, but destabilizes the helical structure. Conversely, in competitive ELISA, MBP (81–106) (1) is recognized more efficiently by IgM antibodies than MBP (76–116) (2), possibly thanks to its more stable helical structure observed in CD and NMR conformational experiments. These results are discussed in terms of different performances of peptide antigens in the two ELISA formats tested.

The currently devastating pandemic of severe acute respiratory syndrome known as coronavirus disease 2019 or COVID-19 is caused by the coronavirus SARS-CoV-2. Both the virus and the disease have been extensively studied worldwide. A trimeric spike (S) protein expressed on the virus outer bilayer leaflet has been identified as a ligand that allows the virus to penetrate human host cells and cause infection. Its receptor-binding domain (RBD) interacts with the angiotensin-converting enzyme 2 (ACE2), the host-cell viral receptor, and is, therefore, the subject of intense research for the development of virus control means, particularly vaccines. In this work, we search for smaller fragments of the S protein able to elicit virus-neutralizing antibodies, suitable for production by peptide synthesis technology. Based on the analysis of available data, we selected a 72 aa long receptor binding motif (RBM436-507) of RBD. We used ELISA to study the antibody response to each of the three antigens (S protein, its RBD domain and the RBM436-507 synthetic peptide) in humans exposed to the infection and in immunized mice. The seroreactivity analysis showed that anti-RBM antibodies are produced in COVID-19 patients and immunized mice and may exert neutralizing function, although with a frequency lower than anti-S and -RBD. These results provide a basis for further studies towards the development of vaccines or treatments focused on specific regions of the S virus protein, which can benefit from the absence of folding problems, conformational constraints and other advantages of the peptide synthesis production.

Despite the global interest and the unprecedented number of scientific studies triggered by the COVID-19 pandemic, few data are available from developing and low-income countries. In these regions, communities live under the threat of various transmissible diseases aside from COVID-19, including malaria. This study aims to determine the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) seroreactivity of antibodies from COVID-19 and pre-COVID-19 samples of individuals in Mali (West Africa). Blood samples from COVID-19 patients (n = 266) at Bamako Dermatology Hospital (HDB) and pre-COVID-19 donors (n = 283) from a previous malaria survey conducted in Dangassa village were tested by ELISA to assess IgG antibodies specific to the full-length spike (S) protein, the receptor-binding domain (RBD), and the receptor-binding motif (RBM436–507). Study participants were categorized by age, gender, treatment duration for COVID-19, and comorbidities. In addition, the cross-seroreactivity of samples from pre-COVID-19, malaria-positive patients against the three antigens was assessed. Recognition of the SARS-CoV-2 proteins by sera from COVID-19 patients was 80.5% for S, 71.1% for RBD, and 31.9% for RBM (p < 0.001). While antibody responses to S and RBD tended to be age-dependent, responses to RBM were not. Responses were not gender-dependent for any of the antigens. Higher antibody levels to S, RBD, and RBM at hospital entry were associated with shorter treatment durations, particularly for RBD (p < 0.01). In contrast, higher body weights negatively influenced the anti-S antibody response, and asthma and diabetes weakened the anti-RBM antibody responses. Although lower, a significant cross-reactive antibody response to S (21.9%), RBD (6.7%), and RBM (8.8%) was detected in the pre-COVID-19 and malaria samples. Cross-reactive antibody responses to RBM were mostly associated (p < 0.01) with the absence of current Plasmodium falciparum infection, warranting further study.

This study illustrates the sensing and wound healing properties of silk fibroin in combination with peptide patterns, with an emphasis on the printability of multilayered grids, and envisions possible applications of these next-generation silk-based materials. Functionalized silk fibers covalently linked to an arginine–glycine–aspartic acid (RGD) peptide create a platform for preparing a biomaterial ink for 3D printing of grid-like piezoresistors with wound-healing and sensing properties. The culture medium obtained from 3D-printed silk fibroin enriched with RGD peptide improves cell adhesion, accelerating skin repair. Specifically, RGD peptide-modified silk fibroin demonstrated biocompatibility, enhanced cell adhesion, and higher wound closure rates at lower concentration than the neat peptide. It was also shown that the printing of peptide-modified silk fibroin produces a piezoresistive transducer that is the active component of a sensor based on a Schottky diode harmonic transponder encoding information about pressure. We discovered that such biomaterial ink printed in a multilayered grid can be used as a humidity sensor. Furthermore, humidity activates a transition between low and high conductivity states in this medium that is retained unless a negative voltage is applied, paving the way for utilization in non-volatile organic memory devices. Globally, these results pave the way for promising applications, such as monitoring parameters such as human wound care and being integrated in bio-implantable processors.

Despite human recombinant H2 relaxin or serelaxin holding promise as a cardiovascular drug, its actual efficacy in chronic treatment of heart failure patients was hampered by the need to be administered by multiple daily IV injections for a long time, with obvious drawbacks in terms of patients’ compliance. This in vitro study aimed at exploring the molecular background for a possible administration of the peptide hormone relaxin by the oral route. Serelaxin and purified porcine relaxin (pRLX) were subjected to simulated intestinal fluid (SIF) enzymatic digestion in vitro to mimic the behavior of gastroprotective formulations. The digestion time course was studied by HPLC, and the relative bio-potency of the intact molecules and their proteolytic fragments was assessed by second messenger (cAMP) response in RXFP1 relaxin receptor-bearing THP-1 human monocytic cells. Both intact proteins (100 ng/mL) induced a significant cAMP rise in THP-1 cells. Conversely, SIF-treated serelaxin showed a brisk (30 s) bioactivity decay, dropping down to the levels of the unstimulated controls at 120 s, whereas SIF-treated pRLX retained significant bioactivity for up to 120 s. After that, it progressively declined to the levels of the unstimulated controls. HPLC analysis indicates that this bioactivity could be ascribed to a minor component of the pRLX sample more resistant to proteolysis. When identified and better characterized, this peptide could be exploited for the development of synthetic relaxin agonists suitable for oral formulations.

Peptidomimetics play a fundamental role in drug design due to their preferential properties regarding natural peptides. In particular, compounds possessing nitrogen-containing heterocycles have been intensively studied in recent years. The triazolyl moiety incorporation decreases the molecule susceptibility to enzymatic degradation, reduction, hydrolysis, and oxidation. In fact, peptides containing triazole rings are a typical example of peptidomimetics. They have all the advantages over classic peptides. Both efficient synthetic methods and biological activity make these systems an interesting and promising object of research. Peptide triazole derivatives display a diversity of biological properties and can be obtained via numerous synthetic strategies. In this review, we have highlighted the importance of the triazole-modified peptidomimetics in the field of drug design. We present an overview on new achievements in triazolyl-containing peptidomimetics synthesis and their biological activity as inhibitors of enzymes or against cancer, viruses, bacteria, or fungi. The relevance of above-mentioned compounds was confirmed by their comparison with unmodified peptides.

The combination of pharmacologic and endoscopic therapies is the gold standard for treating intestinal failures. The possibility of chemical solubility in water is mandatory for intelligent capsules. Functionalised silk fibroin with peptides and covalently linking different molecular entities to its structure make this protein a platform for preparing gels dissolving in the small and large intestine for drug delivery. In the present study, we linked a peptide containing the cell-adhesive motif Arginine–Glycine–Aspartic acid (RGD) to degummed silk fibres (DSF). Regenerated silk fibroin (RS) films obtained by dissolving functionalised DSF in formic acid were used to prepare composite gelatin. We show that such composite gelatin remains stable and elastic in the simulated gastric fluid (SGF) but can dissolve in the small and large intestines’ neutral-pH simulated intestine fluid (SIF). These findings open up the possibility of designing microfabricated and physically programmable scaffolds that locally promote tissue regeneration, thanks to bio-enabled materials based on functionalised regenerated silk.

Diagnosis of Latent Autoimmune Diabetes in Adults (LADA) is based on the adult-age, anti-islet autoantibodies, and temporary insulin-independence. As in Type-1-Diabetes (T1DM), autoimmunity may trigger LADA and enteroviruses-infections can play a role. Anti-human Glutamic-Acid-Decarboxylase (hGAD) autoantibodies are accepted clinical biomarkers, but do not discriminate LADA vs. T1DM. The hypothesis is that protein antigens detecting anti-hGAD antibodies do not expose epitopes specific for different disease forms.We investigated the diagnostic value of autoantibodies in LADA vs. T1DM to peptides of hGAD65/67 isoforms, and Enterovirus-Coxsackie-B4 (CVB4), as antigens sharing the epitope PEVKXK (X: E/T) included in CD8 T-cell CVB4 epitope restricted by diabetes-associated HLA-A2.1. Statistically significant differences of IgM and/or IgG in LADA and T1DM vs. controls were identified. In LADA IgMs to GAD65/67 peptides are diagnostics, IgGs to GAD65/67 peptides correlate with anti-CVB4 peptide antibodies. IgM and/or IgG to all tested peptides can predict LADA, monitoring CVB4 infected patients, improving LADA vs. T1DM stratification. • A customized SP-ELISA based on synthetic peptides Ac-hGAD65(250-273)-NH2 (1), Ac-hGAD67(258-281)-NH2 (2), and Ac-CVB4P2C(28-50)-NH2 (3) is described. • The method was designed to detect specific IgM and/or IgG in LADA, T1DM, vs. controls • Final aim is improvement of LADA vs. T1DM patient stratification.

Two isoforms of the glutamate decarboxylase (GAD) enzyme exist, GAD65 and GAD67, which are associated with type 1 diabetes (T1D) and stiff-person syndrome (SPS), respectively. Interestingly, it has been reported that T1D patients seldom develop SPS, whereas patients with SPS occasionally develop T1D. In addition, coxsackievirus B4 (CVB4) has previously been proposed to be involved in the onset of T1D through molecular mimicry. On this basis, we aimed to examine antibody cross-reactivity between a specific region of GAD65 and GAD67, which has high sequence homology to the nonstructural P2C protein of CVB4 to determine potential correlations at antibody level. Monoclonal peptide antibodies generated in mice specific for a region with high similarity in all three proteins were screened for reactivity along with human sera in immunoassays. In total, six antibodies were generated. Two of the antibodies reacted to both GAD isoforms. However, none of the antibodies were cross-reactive to CVB, suggesting that antibody cross-reactivity between GAD65 and CVB, and GAD67 and CVB may not contribute to the onset of T1D and SPS, respectively.

Rheumatoid arthritis (RA) is a chronic disease which causes joint inflammation and, ultimately, erosion of the underlying bone. Diagnosis of RA is based on the presence of biomarkers, such as anti-citrullinated protein antibodies (ACPA) and rheumatoid factors, along with clinical symptoms. Much evidence points to a link between the Epstein-Barr virus and RA. In this study, we analyzed ACPA reactivity to citrullinated peptides originating from Epstein-Barr nuclear antigens (EBNA1, EBNA2, and EBNA3) in order to elaborate the diagnostic potential of citrullinated EBNA peptides. Moreover, ACPA cross-reactivity to citrullinated peptides from myelin basic protein (MBP) was analyzed, as citrullinated MBP recently was described to be associated with multiple sclerosis, and some degree of sequence homology between MBP and citrullinated EBNA exists. A peptide from EBNA2, (EBNA2-A, GQGRGRWRG-Cit-GSKGRGRMH) reacted with approximately 70% of all RA sera, whereas only limited reactivity was detected to EBNA1 and EBNA3 peptides. Moreover, screening of ACPA reactivity to hybrid peptides of EBNA3-A (EPDSRDQQS-Cit-GQRRGDENRG) and EBNA2-A and peptides containing citrulline close to the N-terminal confirmed that ACPA sera contain different populations of ACPAs. No notable ACPA reactivity to MBP peptides was found, confirming that ACPAs are specific for RA, and that other factors than the presence of a central Cit-Gly motif are crucial for antibody binding. Collectively, these findings illustrate that citrullinated EBNA2 is an optimal candidate for ACPA detection, supporting current evidence that EBV is linked to RA onset.

Among the many aspects that contribute to the wellness of each individual, healthy and younger-looking skin play a relevant role, as clearly shown by the important growth of the skin-care products market observed in recent years. In this scenario, the field of cosmeceuticals appears particularly promising, being based on cosmetic products containing active ingredients. Among these, several peptides were proposed for cosmeceutical applications, thanks to their specific interaction with biological targets. In this mini-review, we report some of the most investigated and used peptides for cosmetic formulations, taking into account that cosmeceutical peptides are basically divided into three main categories (i.e., neurotransmitter inhibitors, carriers, and signal peptides). Special attention was payed to the scientific studies supporting the claimed biological activity of these peptides, as a fundamental aspect that should underpin the growth of this field in the framework of a sustainable wellness economy.

Rheumatoid arthritis (RA) is an autoimmune disease affecting approximately 1–2% of the world population. In addition to the first discovered serologic markers for RA, the rheumatoid factors (RFs), anti-citrullinated protein antibodies (ACPAs) are even more specific for the disease compared to RFs and are found in 70–80% of RA patient sera. RA etiopathogenesis still needs to be elucidated, as different factors are proposed to be involved, such as Epstein–Barr virus infection. Hence, understanding the interaction between ACPAs and their citrullinated peptide targets is relevant for a better knowledge of RA pathophysiology and for diagnostic purposes. In this study, a cohort of RA sera, healthy control sera and multiple sclerosis sera were screened for reactivity to a variety of citrullinated peptides originating from α-enolase, pro-filaggrin, proteoglycan and Epstein–Barr nuclear antigen-2 by enzyme-linked immunosorbent assay. ACPA reactivity to citrullinated α-enolase peptides was found to depend on peptide length and peptide conformation, favouring cyclic (disulfide bond) conformations for long peptides and linear peptides for truncated ones. Additional investigations about the optimal peptide conformation for ACPA detection, employing pro-filaggrin and EBNA-2 peptides, confirmed these findings, indicating a positive effect of cyclization of longer peptides of approximately 20 amino acids. Moreover, screening of the citrullinated peptides confirmed that ACPAs can be divided into two groups based on their reactivity. Approximately 90% of RA sera recognize several peptide targets, being defined as cross-reactive or overlapping reactivities, and whose reactivity to the citrullinated peptide is considered primarily to be backbone-dependent. In contrast, approximately 10% recognize a single target and are defined as nonoverlapping, primarily depending on the specific amino acid side-chains in the epitope for a stable interaction. Collectively, this study contributed to characterize epitope composition and structure for optimal ACPA reactivity and to obtain further knowledge about the cross-reactive nature of ACPAs.

Collagen fibres degradation is a complex process involving a variety of enzymes. Fibrillar collagens, namely type I, II, and III, are the most widely spread collagens in human body, e.g., they are responsible for tissue fibrillar structure and skin elasticity. Nevertheless, the hyperactivity of fibrotic process and collagen accumulation results with joints, bone, heart, lungs, kidneys or liver fibroses. Per contra, dysfunctional collagen turnover and its increased degradation leads to wound healing disruption, skin photoaging, and loss of firmness and elasticity. In this review we described the main enzymes participating in collagen degradation pathway, paying particular attention to enzymes degrading fibrillar collagen. Therefore, collagenases (MMP-1, -8, and -13), elastases, and cathepsins, together with their peptide and peptidomimetic inhibitors, are reviewed. This information, related to the design and synthesis of new inhibitors based on peptide structure, can be relevant for future research in the fields of chemistry, biology, medicine, and cosmeceuticals.

Antibodies against myelin oligodendrocyte glycoprotein (MOG) are associated to several disorders, and their occurrence in patients presenting an acquired demyelinating disease affects a higher proportion of paediatric subjects, as compared to adults. Despite heterogeneity in clinical presentation, few connexions have been reported between the progressive neurodevelopmental disorder affecting child’s brain development and cognitive ability, i.e. Rett syndrome (RTT), and a demyelination process. In order to identify the possible target of humoral autoimmune response in RTT patients, we set-up a home-made solid-phase ELISA, using the recombinant extracellular portion of human MOG(1-117) as an antigen. The screening to evaluate anti-MOG antibodies in RTT patient sera, compared to other relative non-RTT pervasive developmental disorders (non-RTT PDD), including mainly autism, and a healthy control group gave uncertain results. In fact, Student t-test and Mann-Whitney unpaired t test showed that differences in both IgG and IgM antibody titres between the different patient populations, were not statistically significant. We can conclude that the absence of anti-MOG antibody recognition in RTT has possibly to be ascribed to a different relevant protein folding and/or to the lack of a relevant aberrant post-translational modification, such as N-glucosylation, that we previously demonstrated, for the first time, fundamental to recognize antibodies in RTT.

Diagnostics of Multiple Sclerosis (MS) are essentially based on the gold standard magnetic resonance imaging. Few alternative simple assays are available to follow up disease activity. Considering that the disease can remain elusive for years, identification of antibodies fluctuating in biological fluids as relevant biomarkers of immune response is a challenge. In previous studies, we reported that anti-N-glucosylated (N-Glc) peptide antibodies that can be easily detected in Solid-Phase Enzyme-Linked ImmunoSorbent Assays (SP-ELISA) on MS patients’ sera preferentially recognize hyperglucosylated adhesin of non-typeable Haemophilus Influenzae. Since multivalency can be useful for diagnostic purposes to allow an efficient coating in ELISA, we report herein the development of a collection of Multiple N-glucosylated Peptide Epitopes (N-Glc MEPs) to detect anti-N-Glc antibodies in MS. To this aim, a series of N-Glc peptide antigens to be represented in the N-GlcMEPs were tested in competitive ELISA. We confirmed that the epitope recognized by antibodies shall contain at least 5-mer sequences including the fundamental N-Glc moiety. Using a 4-branched dendrimeric lysine scaffold, we selected the N-Glc MEP 24, carrying the minimal epitope Asn(Glc) anchored to a polyethylene glycol-based spacer (PEG) containing a 19-atoms chain, as an efficient multivalent probe to reveal specific and high affinity anti-N-Glc antibodies in MS.

We report herein a novel ChemMatrix® Rink resin functionalised with two phenylboronate (PhB) moieties linked on the N-α and N-ε amino functions of a lysine residue to specifically capture deoxyfructosylated peptides, compared to differently glycosylated peptides in complex mixtures. The new PhB-Lys(PhB)-ChemMatrix® Rink resin allows for exploitation of the previously demonstrated ability of cis diols to form phenylboronic esters. The optimised capturing and cleavage procedure from the novel functionalised resin showed that only the peptides containing deoxyfructosyl-lysine moieties can be efficiently and specifically detected by HR-MS and MS/MS experiments. We also investigated the high-selective affinity to deoxyfructosylated peptides in an ad hoc mixture containing unique synthetic non-modified peptides and in the hydrolysates of human and bovine serum albumin as complex peptide mixtures. We demonstrated that the deoxyfructopyranosyl moiety on lysine residues is crucial in the capturing reaction. Therefore, the novel specifically-designed PhB-Lys(PhB)-ChemMatrix® Rink resin, which has the highest affinity to deoxyfructosylated peptides, is a candidate to quantitatively separate early glycation peptides from complex mixtures to investigate their role in diabetes complications in the clinics.

The interaction of lipid environments with the type I’ β-turn peptide structure called CSF114 and its N-glucosylated form CSF114(Glc), previously developed as a synthetic antigenic probe recognizing specific autoantibodies in a subpopulation of multiple sclerosis patients’ serum, was investigated by fluorescence spectroscopy and electrochemical experiments using large unilamellar vesicles, mercury supported lipid self-assembled monolayers (SAMs) and tethered bilayer lipid membranes (tBLMs). The synthetic antigenic probe N-glucosylated peptide CSF114(Glc) and its unglucosylated form interact with the polar heads of lipid SAMs of dioleoylphosphatidylcholine at nonzero transmembrane potentials, probably establishing a dual electrostatic interaction of the trimethylammonium and phosphate groups of the phosphatidylcholine polar head with the Glu5 and His9 residues on the opposite ends of the CSF114(Glc) β-turn encompassing residues 6-9. His9 protonation at pH 7 eliminates this dual interaction. CSF114(Glc) is adsorbed on top of SAMs of mixtures of dioleoylphosphatidylcholine with sphingomyelin, an important component of myelin, whose proteins are hypothesized to undergo an aberrant N-glucosylation triggering the autoimmune response. Incorporation of the type I’ β-turn peptide structure CSF114 into lipid SAMs by potential scans of electrochemical impedance spectroscopy induces defects causing a slight permeabilization toward cadmium ions. The N-glucopeptide CSF114(Glc) does not affect tBLMs to a detectable extent.

Surface plasmon resonance technique is particularly interesting in immunology because it has the potential to visualize label-free antigen-antibody interactions in real-time, thus enabling antibody detection and monitoring. Herein we release the guidelines for the correct use of a method to detect specific antibodies directly in Multiple Sclerosis patients’ sera using a glucopeptide-based label-free biosensor. The protocol describes the strategy employed for the immobilization of glucopeptide antigen onto a gold sensor chip and the evaluation of the specific binding of serum antibodies to the immobilized antigen. • Label-free method for the real time screening of disease-specific antibodies within a few minutes; • The described protocol employs small quantities of glucopeptide antigen and blood serum samples saving method-cost; • Stability of the immobilized glucopeptide antigen guarantees the regeneration of the surface allowing re-use the immunosensor with high automated throughput. The antibodies detected using the described methodology can be evaluated as biomarkers of Multiple Sclerosis. The SPR detection system is able to characterize antibodies significantly different from those evaluated in the classical enzyme-linked immunosorbent assays (ELISA).

Characterization of multiple antibody epitopes has revealed the necessity of specific groups of amino acid residues for reactivity. This applies to the majority of antibody−antigen interactions, where especially charged and hydrophilic amino acids have been reported to be essential for antibody reactivity. This study describes thorough characterization of glutamic acid decarboxylase (GAD) 65 antigenic epitopes, an immunodominant autoantigen in type 1 diabetes (T1D). As linear epitopes are sparsely described for GAD65 in T1D, we aimed to identify and thoroughly characterize two GAD65 antibodies using immunoassays. A monoclonal antibody recognized an epitope in the N-terminal domain of GAD65, 8FWSFGSE14, whereas a polyclonal antibody recognized two continuous epitopes in the C-terminal domain, corresponding to amino acids 514RTLED518 and 549PLGDKVNF556. Hydrophobic amino acids were essential for antibody reactivity, which was verified by competitive inhibition assays. Moreover, the epitopes were located in flexible linker regions and turn structures. These findings confirm the versatile nature of antibody−antigen interactions and describe potential continuous epitopes related to T1D, which predominantly have been proposed to be of discontinuous nature.

Systemic lupus erythematosus is a chronic autoimmune disease with multifactorial ethiopathogenesis. The complement system is involved in both the early and late stages of disease development and organ damage. To better understand autoantibody mediated complement consumption we examined ex vivo immune complex formation on autoantigen arrays. We recruited patients with SLE (n = 211), with other systemic autoimmune diseases (n = 65) and non-autoimmune control subjects (n = 149). Standard clinical and laboratory data were collected and serum complement levels were determined. The genotype of SNP rs1143679 in the ITGAM gene was also determined. Ex vivo formation of immune complexes, with respect to IgM, IgG, complement C4 and C3 binding, was examined using a functional immunoassay on autoantigen microarray comprising nucleic acids, proteins and lipids. Complement consumption of nucleic acids increased upon binding of IgM and IgG even when serum complement levels were decreased due to consumption in SLE patients. A negative correlation between serum complement levels and ex vivo complement deposition on nucleic acid autoantigens is demonstrated. On the contrary, complement deposition on tested protein and lipid autoantigens showed positive correlation with C4 levels. Genetic analysis revealed that the non-synonymous variant rs1143679 in complement receptor type 3 is associated with an increased production of anti-dsDNA IgG antibodies. Notwithstanding, homozygous carriers of the previously reported susceptible allele (AA) had lower levels of dsDNA specific IgM among SLE patients. Both the non-synonymous variant rs1143679 and the high ratio of nucleic acid specific IgG/IgM were associated with multiple organ involvement. In summary, secondary complement deficiency in SLE does not impair opsonization of nucleic-acid-containing autoantigens but does affect other antigens and potentially other complement dependent processes. Dysfunction of the receptor recognizing complement opsonized immune complexes promotes the development of class-switched autoantibodies targeting nucleic acids.

Our study tested the hypothesis that immunoglobulins differ in their ability to activate the nuclear factor-κB pathway mediated cellular responses. These responses are modulated by several properties of the immune complex, including the ratio of antibody isotypes binding to antigen. Immunoassays allow the measurement of antigen specific antibodies belonging to distinct immunoglobulin classes and subclasses but not the net biological effect of the combination of these antibodies. We set out to develop a biosensor that is suitable for the detection and characterization of antigen specific serum antibodies. We genetically modified the monocytoid U937 cell line carrying Fc receptors with a plasmid encoding NF-κB promoter-driven GFP. This clone, U937-NF-κB, was characterized with respect to FcR expression and response to solid-phase immunoglobulins. Human IgG3, IgG4 and IgG1 induced GFP production in a time- and dose-dependent manner, in this order of efficacy, while IgG2 triggered no activation at the concentrations tested. IgA elicited no response alone but showed significant synergism with IgG3 and IgG4. We confirmed the importance of activation via FcγRI by direct stimulation with monoclonal antibody and by competition assays. We used citrullinated peptides and serum from rheumatoid arthritis patients to generate immune complexes and to study the activation of U937-NF-κB, observing again a synergistic effect between IgG and IgA. Our results show that immunoglobulins have distinct pro-inflammatory potential, and that U937-NF-κB is suitable for the estimation of biological effects of immune-complexes, offering insight into monocyte activation and pathogenesis of antibody mediated diseases.

In multiple sclerosis (MS) the gold standard for the diagnosis and prognosis is, up to now, the use of magnetic resonance imaging markers. No alternative simpler assays proven of use, except for cerebrospinal fluid analysis, have been provided in MS diagnosis. Therefore, there is a need to develop non-invasive, sensitive, simple new techniques for the clinical routine. Herein we present the evaluation of the feasibility of a glycopeptide-based biosensor to detect MS specific antibodies in sera using the surface plasmon resonance technology. The previously described glycopeptide antigen CSF114(Glc) has been immobilized on a gold sensor chip and the method has been optimized for real-time specific autoantibody detection directly in sera. A population of 60 healthy blood donors and 61 multiple sclerosis patients has been screened. The receiver operating characteristic (ROC)-based analysis has established the optimal diagnostic cut-off value for the method obtaining a sensitivity of 36% and a specificity of 95%. Sample sera have been also screened with a previously validated ELISA.

Rett syndrome (RTT), a neurodevelopmental disorder affecting exclusively (99%) female infants, is associated with loss-of-function mutations in the gene encoding methyl-CpG binding protein 2 (MECP2) and, more rarely, cyclin-dependent kinase-like 5 (CDKL5) and forkhead box protein G1 (FOXG1). In this study, we aimed to evaluate the function of the immune system by measuring serum immunoglobulins (IgG and IgM) in RTT patients (n=53) and, by comparison, in age-matched children affected by non-RTT pervasive developmental disorders (non-RTT PDD) (n=82) and healthy age-matched controls (n=29). To determine immunoglobulins we used both a conventional agglutination assay and a novel ELISA based on antibody recognition by a surrogate antigen probe, CSF114(Glc), a synthetic N-glucosylated peptide. Both assays provided evidence for an increase in IgM titer, but not in IgG, in RTT patients relative to both healthy controls and non-RTT PDD patients. The significant difference in IgM titers between RTT patients and healthy subjects in the CSF114(Glc) assay (P=0.001) suggests that this procedure specifically detects a fraction of IgM antibodies likely to be relevant for the RTT disease. These findings offer a new insight into the mechanism underlying the Rett disease as they unveil the possible involvement of the immune system in this pathology.

ASDs (autism spectrum disorders) are a complex group of neurodevelopment disorders, still poorly understood, steadily rising in frequency and treatment refractory. Extensive research has been so far unable to explain the aetiology of this condition, whereas a growing body of evidence suggests the involvement of environmental factors. Phthalates, given their extensive use and their persistence, are ubiquitous environmental contaminants. They are EDs (endocrine disruptors) suspected to interfere with neurodevelopment. Therefore they represent interesting candidate risk factors for ASD pathogenesis. The aim of this study was to evaluate the levels of the primary and secondary metabolites of DEHP [di-(2-ethylhexyl) phthalate] in children with ASD. A total of 48 children with ASD (male: 36, female: 12; mean age: 11 ± 5 years) and age- and sex-comparable 45 HCs (healthy controls; male: 25, female: 20; mean age: 12 ± 5 years) were enrolled. A diagnostic methodology, based on the determination of urinary concentrations of DEHP metabolites by HPLC-ESI-MS (HPLC electrospray ionization MS), was applied to urine spot samples. MEHP [mono-(2-ethylhexenyl) 1,2-benzenedicarboxylate], 6-OH-MEHP [mono-(2-ethyl-6-hydroxyhexyl) 1,2-benzenedicarboxylate], 5-OH-MEHP [mono-(2-ethyl-5-hydroxyhexyl) 1,2-benzenedicarboxylate] and 5-oxo-MEHP [mono-(2-ethyl-5-oxohexyl) 1,2-benzenedicarboxylate] were measured and compared with unequivocally characterized, pure synthetic compounds (>98%) taken as standard. In ASD patients, significant increase in 5-OH-MEHP (52.1%, median 0.18) and 5-oxo-MEHP (46.0%, median 0.096) urinary concentrations were detected, with a significant positive correlation between 5-OH-MEHP and 5-oxo-MEHP ( r s =0.668, P