Implications of Post-Translational Modifications in Autoimmunity with Emphasis on Citrullination, Homocitrullination and Acetylation for the Pathogenesis, Diagnosis and Prognosis of Rheumatoid Arthritis.

Post-translational modifications (PTMs) influence cellular processes and consequently, their dysregulation is related to the etiologies of numerous diseases. It is widely known that a variety of autoimmune responses in human diseases depend on PTMs of self-proteins. In this review we summarize the latest findings about the role of PTMs in the generation of autoimmunity and, specifically, we address the most relevant PTMs in rheumatic diseases that occur in synovial tissue. Citrullination, homocitrullination (carbamylation) and acetylation are responsible for the generation of Anti-Modified Protein/Peptide Antibodies (AMPAs family), autoantibodies which have been implicated in the etiopathogenesis, diagnosis and prognosis of rheumatoid arthritis (RA). Synthetic peptides provide complete control over the exact epitopes presented as well as the specific positions in their sequence where post-translationally modified amino acids are located and are key to advancing the detection of serological RA biomarkers that could be useful to stratify RA patients in order to pursue a personalized rheumatology. In this review we specifically address the latest findings regarding synthetic peptides post-translationally modified for the specific detection of autoantibodies in RA patients.

Peptide Amphiphilic-Based Supramolecular Structures with Anti-HIV-1 Activity.

In a previous work, we defined a novel HIV-1 fusion inhibitor peptide (E1P47) with a broad spectrum of activity against viruses from different clades, subtypes, and tropisms. With the aim to enhance its efficacy, in the present work we address the design and synthesis of several peptide amphiphiles (PAs) based on the E1P47 peptide sequence to target the lipid rafts of the cell membrane where the cell-cell fusion process takes place. We report the synthesis of novel PAs having a hydrophobic moiety covalently attached to the peptide sequence through a hydrophilic spacer of polyethylene glycol. Characterization of self-assembly in condensed phase and aqueous solution as well as their interaction with model membranes was analyzed by several biophysical methods. Our results demonstrated that the length of the spacer of polyethylene glycol, the position of the peptide conjugation as well as the type of the hydrophobic residue determine the antiviral activity of the construct. Peptide amphiphiles with one alkyl tail either in C-terminus (C-PAmonoalkyl) or in N-terminus (N-PAmonoalkyl) showed the highest anti-HIV-1 activities in the cellular model of TZM-bl cells or in a preclinical model of the human mucosal tissue explants.

Peptides Bearing Multiple Post-Translational Modifications as Antigenic Targets for Severe Rheumatoid Arthritis Patients.

Rheumatoid arthritis (RA) is characterized by the presence of autoantibodies that are of paramount importance for the diagnosis and prognosis of the disease and have been implicated in its pathogenesis. Proteins resulting from post-translational modifications (PTMs) are capable of triggering autoimmune responses important for the development of RA. In this work, we investigate serum antibody reactivity in patients with an established RA against a panel of chimeric peptides derived from fibrin and filaggrin proteins and bearing from one to three PTMs (citrullination, carbamylation and acetylation) by home-designed ELISA tests (anti-AMPA autoantibodies). The role of anti-AMPAs as biomarkers linked to the presence of a more severe RA phenotype (erosive disease with radiological structural damage) and to the presence of interstitial lung disease (ILD), a severe extra-articular manifestation in RA patients entailing a high mortality, was also analyzed. In general, the association with the clinical phenotype of RA was confirmed with the different autoantibodies, and especially for IgA and IgM isotypes. The prevalence of severe joint damage was only statistically significant for the IgG isotype when working with the peptide bearing three PTMs. Furthermore, the median titers were significantly higher in patients with RA-ILD, a finding not observed for the IgG isotype when working with the single- and double-modified peptides.

Design, Characterization, and Biopharmaceutical Behavior of Nanoparticles Loaded with an HIV-1 Fusion Inhibitor Peptide.

New therapeutic alternatives to fight against the spread of HIV-1 are based on peptides designed to inhibit the early steps of HIV-1 fusion in target cells. However, drawbacks, such as bioavailability, short half-life, rapid clearance, and poor ability to cross the physiological barriers, make such peptides unattractive for the pharmaceutical industry. Here we developed, optimized, and characterized polymeric nanoparticles (NPs) coated with glycol chitosan to incorporate and release an HIV-1 fusion inhibitor peptide (E1) inside the vaginal mucosa. The NPs were prepared by a modified double emulsion method, and optimization was carried out by a factorial design. In vitro, ex vivo, and in vivo studies were carried out to evaluate the optimized formulation. The results indicate that the physicochemical features of these NPs enable them to incorporate and release HIV fusion inhibitor peptides to the vaginal mucosa before the fusion step takes place.

Comparative study of the diagnostic and prognostic value of antibodies against chimeric citrullinated synthetic peptides and CCP3/CCP3.1 assays.

BACKGROUND: The objective of the study was to compare the diagnostic yield of home-made ELISA tests based on synthetic chimeric fibrin/filaggrin citrullinated peptides (CFFCPs) with CCP3 and CCP3.1 commercial tests to detect anti-citrullinated protein/peptide antibodies (ACPAs) in rheumatoid arthritis (RA) patients. The prognostic value is also studied in a cohort of patients with early RA. Moreover, we transfer immunological assays from microtiter plates to microarray formats to allow the simultaneous analysis of several peptide sequences and reduce the volume of serum from patients. METHODS: The diagnostic study includes: 100 RA patients who fulfilled the 1987 ACR criteria; 100 healthy blood donors; 35 patients with SLE according ACR criteria; 35 patients with PsA fulfilling the Wright and Moll criteria and 30 patients with HCV infection. The prognostic value study includes 50 patients with early RA with follow-up data available. All samples are from outpatients attending the Rheumatology Department of the Hospital Clinic of Barcelona. RESULTS: Similar sensitivity, specificity and predictive values for the diagnosis of RA of CCFCPs compared to CCP3/CCP3.1 were obtained. Although a high concordance is observed between anti-CFFCPs and anti-CCP3/CCP3.1 in the early patients that rendered Larsen radiographic progression, CFFCPs could be a better marker of radiographic outcome. Strong correlations between the microarray and ELISA results were found for individual CFFCPs peptides. CONCLUSIONS: The development of multiplexing techniques combining a different spectrum of markers in a single analysis, including CFFCP peptides, could allow a more detailed analysis of the autoantibodies reactivity found in the sera of patients suffering of this heterogeneous disease.

Neutrophilic Activity Biomarkers (Plasma Neutrophil Extracellular Traps and Calprotectin) in Established Patients with Rheumatoid Arthritis Receiving Biological or JAK Inhibitors: A Clinical and Ultrasonographic Study.

INTRODUCTION: This study assesses the accuracy of neutrophil activation markers, including neutrophil extracellular traps (NETs) and calprotectin, as biomarkers of disease activity in patients with established rheumatoid arthritis (RA). We also analyse the relationship between NETs and various types of therapies as well as their association with autoimmunity. METHODS: Observational cross-sectional study of patients with RA receiving treatment with biological disease-modifying antirheumatic drugs or Janus kinase inhibitors (JAK-inhibitors) for at least 3 months. Plasma calprotectin levels were measured using an enzyme-linked immunosorbent assay test kit and NETs by measuring their remnants in plasma (neutrophil elastase-DNA and histone-DNA complexes). We also assessed clinical disease activity, joint ultrasound findings and autoantibody status [reumatoid factor (RF), anti-citrullinated peptide/protein antibodies (ACPAs) and anti-carbamylated protein (anti-CarP)]. Associations between neutrophilic biomarkers and clinical or ultrasound scores were sought using correlation analysis. The discriminatory capacity of both neutrophilic biomarkers to detect ultrasound synovitis was analysed through receiver-operating characteristic (ROC) curves. RESULTS: One hundred fourteen patients were included. Two control groups were included to compare NET levels. The active control group consisted of 15 patients. The second control group consisted of 30 healthy subjects. Plasma NET levels did not correlate with clinical disease status, regardless of the clinic index analysed or the biological therapy administered. No significant correlation was observed between NET remnants and ultrasound synovitis. There was no correlation between plasma NET and autoantibodies. In contrast, plasma calprotectin positively correlated with clinical parameters (swollen joint count [SJC] rho = 0.49; P < 0.001, Clinical Disease Activity Index [CDAI] rho = 0.30; P < 0.001) and ultrasound parameters (rho > 0.50; P < 0.001). Notably, this correlation was stronger than that observed with acute phase reactants. CONCLUSION: While NET formation induced by neutrophils may play a role in RA pathogenesis, our study raises questions about the utility of NET remnants in peripheral circulation as a biomarker for inflammatory activity. In contrast, this study strongly supports the usefulness of calprotectin as a biomarker of inflammatory activity in patients with RA.

Contribution of the Tyr-1 in Plantaricin149a to disrupt phospholipid model membranes.

Plantaricin149a (Pln149a) is a cationic antimicrobial peptide, which was suggested to cause membrane destabilization via the carpet mechanism. The mode of action proposed to this antimicrobial peptide describes the induction of an amphipathic α-helix from Ala7 to Lys20, while the N-terminus residues remain in a coil conformation after binding. To better investigate this assumption, the purpose of this study was to determine the contributions of the Tyr1 in Pln149a in the binding to model membranes to promote its destabilization. The Tyr to Ser substitution increased the dissociation constant (KD) of the antimicrobial peptide from the liposomes (approximately three-fold higher), and decreased the enthalpy of binding to anionic vesicles from -17.2 kcal/mol to -10.2 kcal/mol. The peptide adsorption/incorporation into the negatively charged lipid vesicles was less effective with the Tyr1 substitution and peptide Pln149a perturbed the liposome integrity more than the analog, Pln149S. Taken together, the peptide-lipid interactions that govern the Pln149a antimicrobial activity are found not only in the amphipathic helix, but also in the N-terminus residues, which take part in enthalpic contributions due to the allocation at a lipid-aqueous interface.

Cell penetrating peptides-functionalized Licochalcone-A-loaded PLGA nanoparticles for ocular inflammatory diseases: Evaluation of in vitro anti-proliferative effects, stabilization by freeze-drying and characterization of an in-situ forming gel.

Licochalcone-A (Lico-A) PLGA NPs functionalized with cell penetrating peptides B6 and Tet-1 are proposed for the treatment of ocular anti-inflammatory diseases. In this work, we report the in vitro biocompatibility of cell penetrating peptides-functionalized Lico-A-loaded PLGA NPs in Caco-2 cell lines revealing a non-cytotoxic profile, and their anti-inflammatory activity against RAW 264.7 cell lines. Given the risk of hydrolysis of the liquid suspensions, freeze-drying was carried out testing different cryoprotectants (e.g., disaccharides, alcohols, and oligosaccharide-derived sugar alcohol) to prevent particle aggregation and mitigate physical stress. As the purpose is the topical eye instillation of the nanoparticles, to reduce precorneal wash-out, increase residence time and thus Lico-A bioavailability, an in-situ forming gel based on poloxamer 407 containing Lico-A loaded PLGA nanoparticles functionalized with B6 and Tet-1 for ocular administration has been developed. Developed formulations remain in a flowing semi-liquid state under non-physiological conditions and transformed into a semi-solid state under ocular temperature conditions (35 °C), which is beneficial for ocular administration. The pH, viscosity, texture parameters and gelation temperature results met the requirements for ophthalmic formulations. The gel has characteristics of viscoelasticity, suitable mechanical and mucoadhesive performance which facilitate its uniform distribution over the conjunctiva surface. In conclusion, we anticipate the potential clinical significance of our developed product provided that a synergistic effect is achieved by combining the high anti-inflammatory activity of Lico-A delivered by PLGA NPs with B6 and Tet-1 for site-specific targeting in the eye, using an in-situ forming gel.

Assessment of anti-malondialdehyde-acetaldehyde antibody frequencies in rheumatoid arthritis with new data from two independent cohorts, meta-analysis, and meta-regression.

BACKGROUND: Autoantibodies are critical elements in RA pathogenesis and clinical assessment. The anti-malondialdehyde-acetaldehyde (anti-MAA) antibodies are potentially useful because of their claimed high sensitivity for all RA patients, including those lacking RF and anti-CCP antibodies. Therefore, we aimed to replicate these findings. METHODS: We independently attempted replication in Santiago and Barcelona using sera from 517 and 178 RA patients and 272 and 120 healthy controls, respectively. ELISA protocols for anti-MAA antibodies included five antigens (human serum albumin in three formulations, fibrinogen, and a synthetic peptide) and assays for the IgG, IgM, and IgA isotypes. We integrated our results with information found by searching the Web of Science for reports of anti-MAA antibodies in RA. The available patients (4989 in 11 sets) were included in a meta-analysis aimed at heterogeneity between studies. Factors accounting for heterogeneity were assessed with meta-regression. RESULTS: The sensitivity of anti-MAA antibodies in our RA patients was low, even in seropositive patients, with the percentage of positives below 23% for all ELISA conditions. Our results and bibliographic research showed IgG anti-MAA positive patients ranging from 6 to 92%. The extreme between-studies heterogeneity could be explained (up to 43%) in univariate analysis by sex, African ethnicity, the site of study, or recruitment from the military. The best model, including African ancestry and smoking, explained a high heterogeneity fraction (74%). CONCLUSION: Anti-MAA antibody sensitivity is extremely variable between RA patient collections. A substantial fraction of this variability cannot be attributed to ELISA protocols. On the contrary, heterogeneity is determined by complex factors that include African ethnicity, smoking, and sex.

Synthetic peptides derived from an N-terminal domain of the E2 protein of GB virus C in the study of GBV-C/HIV-1 co-infection.

Synthetic peptides derived from GB virus C (GBV-C) have previously been studied in our group for the development of new systems capable of diagnosing diseases caused by this humanotropic virus. We also recently described specific peptide domains of the E2 envelop protein of GBV-C that have the capacity to interfere with the HIV-1 fusion peptide, produce a notable decrease in cellular membrane fusion, and perturb HIV-1 infectivity in a dose-dependent manner. The present work discloses the design and synthesis of both linear and cyclic branched peptides based on a previously reported N-terminal sequence of the GBV-C E2 protein. Immunoassays and cell-cell fusion assays were performed to evaluate their diagnostic value to detect anti-GBV-C antibodies in HIV-1 patients, as well as their putative anti-HIV-1 activity as entry inhibitors. Our results showed that chemical modifications of the selected E2(7-26) linear peptide to afford cyclic architecture do not result in an enhanced inhibition of gp41 HIV-1-mediated cell-cell fusion nor improved sensitivity in the detection of GBV-C antibodies in HIV-1 co-infected patients. Thus, the ELISA data reinforce the potential utility of linear versions of the E2(7-26) region for the development of new peptide-based immunosensor devices for the detection of anti-GBV-C antibodies in HIV-1 co-infected patients.

Assessment of synthetic chimeric multiple antigenic peptides for diagnosis of GB virus C infection.

The use of synthetic peptides of both structural and nonstructural proteins of GB virus C (GBV-C) has been studied for the development of new systems to diagnose infection caused by this virus. In an attempt to increase the antigenicity of linear peptide sequences, chimeric multiple antigenic peptides (MAPs) containing epitopes from E2, NS4, and NS5 GBV-C proteins have been synthesized. The synthetic constructs were evaluated by ELISA to establish whether the epitopes in chimeric branched peptides are more efficiently recognized by the specific antibodies compared to the monomeric linear sequences. Moreover, we have investigated the application of a commercial biosensor instrument for the detection of antibodies against the GBV-C in human serum samples. The results of the immunoassays reported in this work highlight the usefulness of synthetic tetrameric branched peptides containing sequences from envelope and nonstructural GBV-C proteins for the diagnosis of GBV-C infection. The potential clinical value of the MAP(4)(E2-NS5a) for the serodiagnosis of GBV-C infection was demonstrated, thus providing the basis for performing prevalence studies of the infection among the hemodialyzed and hepatitis C virus (HCV)-infected population.

Importance of structure-based studies for the design of a novel HIV-1 inhibitor peptide.

Based on the structure of an HIV-1 entry inhibitor peptide two stapled- and a retro-enantio peptides have been designed to provide novel prevention interventions against HIV transmission. The three peptides show greater inhibitory potencies in cellular and mucosal tissue pre-clinical models than the parent sequence and the retro-enantio shows a strengthened proteolytic stability. Since HIV-1 fusion inhibitor peptides need to be embedded in the membrane to properly interact with their viral target, the structural features were determined by NMR spectroscopy in micelles and solved by using restrained molecular dynamics calculations. Both parent and retro-enantio peptides demonstrate a topology compatible with a shared helix-turn-helix conformation and assemble similarly in the membrane maintaining the active conformation needed for its interaction with the viral target site. This study represents a straightforward approach to design new targeted peptides as HIV-1 fusion inhibitors and lead us to define a retro-enantio peptide as a good candidate for pre-exposure prophylaxis against HIV-1.

Carotid intima-media thickness as a predictor of response to cholinesterase inhibitors in Alzheimer's disease: an open-label trial.

BACKGROUND: Cholinesterase inhibitors are modestly effective in treating patients with Alzheimer's disease. However, there may be important inter-individual variations ranging from no improvement at all to significant improvement and long periods of stabilization. Carotid atherosclerosis is associated with cognitive decline in elderly people. OBJECTIVE: The objective of this study was to investigate whether carotid intima-media thickness (IMT) predicts response to cholinesterase inhibitors in Alzheimer's disease. PATIENTS AND METHODS: A series of 54 patients with mild to moderate Alzheimer's disease were enrolled consecutively in an open-label trial. At baseline, all patients were assessed on the following clinical scales: Mini-Mental State Examination, Clinical Dementia Rating, the Hachinski Ischemic Scale, Blessed Dementia Rating Scale, Alzheimer's Disease Assessment Scale-cognitive subscale (ADAS-cog), Neuropsychiatric Inventory (NPI) and a daily-living activities scale (Disability Assessment for Dementia [DAD]). Investigations included magnetic resonance imaging of the brain and a colour echo-Doppler scan of the carotid arteries to measure the maximum IMT. Patients were then commenced on galantamine treatment for 6 months, after which scores on the ADAS-cog, NPI and DAD scales were reassessed. RESULTS: A total of 50 patients completed the study. Their mean age was 77.78 years (SD 6.51 years); 34 patients were female. Galantamine treatment decreased the mean NPI score from 17.68 to 13.86 points, but this difference was not statistically significant (p=0.07). On the ADAS-cog scale, a modest and nonsignificant mean difference of -0.4 points (p=0.7) was observed. A weak (correlation coefficient r=0.4) but significant correlation between IMT and changes in clinical scale score was found, with low carotid IMT being shown to be a predictor of response on both the ADAS-cog (p=0.003) and NPI (p=0.006) scales; these findings were corroborated in multivariate analysis. For men, the correlation was stronger (r=0.7 and 0.8 for the ADAS-cog and NPI scales, respectively). CONCLUSION: Although the magnitude of effect was moderate, carotid IMT could be a significant predictor of clinical response to cholinesterase inhibitors in patients with Alzheimer's disease.

Synthetic peptides of hepatitis G virus (GBV-C/HGV) in the selection of putative peptide inhibitors of the HIV-1 fusion peptide.

The GB virus C or hepatitis G virus (GBV-C/HGV) is a single-stranded positive sense RNA virus that belongs to the Flaviviridae family. Recent years have seen the publication of numerous works in which coinfection with GBV-C/HGV and HIV has been associated with slower progression of the illness and a higher survival rate of patients once AIDS has developed. The mechanism by which the GBV-C/HGV virus has a "protective effect" in patients with HIV has still not been defined. Study of the interaction of the GBV-C/HGV and HIV viruses could lead to the development of new therapeutic agents for the treatment of AIDS. Given that the mechanism responsible for the beneficial effect exercised by the GBV-C/HGV virus in the course of HIV infection has not been defined, the present work is intended as a study of the structure and interactions between the fusion peptide of HIV-1, gp41(1-23), and synthetic peptide sequences of the E2 envelope protein of GBV-C/HGV using biophysical techniques. Our results highlight that the E2(269-286) sequence interacts with the target fusion peptide of HIV-1 and modifies its conformation.

Effective In Vivo Topical Delivery of siRNA and Gene Silencing in Intact Corneal Epithelium Using a Modified Cell-Penetrating Peptide.

Autosomal dominantly inherited genetic disorders such as corneal dystrophies are amenable to allele-specific gene silencing with small interfering RNA (siRNA). siRNA delivered to the cornea by injection, although effective, is not suitable for a frequent long-term treatment regimen, whereas topical delivery of siRNA to the cornea is hampered by the eye surface's protective mechanisms. Herein we describe an attractive and innovative alternative for topical application using cell-penetrating peptide derivatives capable of complexing siRNA non-covalently and delivering them into the cornea. Through a rational design approach, we modified derivatives of a cell-penetrating peptide, peptide for ocular delivery (POD), already proved to diffuse into the corneal layers. These POD derivatives were able to form siRNA-peptide complexes (polyplexes) of size and ζ-potential similar to those reported able to undergo cellular internalization. Successful cytoplasmic release and gene silencing in vitro was obtained when an endosomal disruptor, chloroquine, was added. A palmitoylated-POD, displaying the best delivery properties, was covalently functionalized with trifluoromethylquinoline, an analog of chloroquine. This modified POD, named trifluoromethylquinoline-palmitoyl-POD (QN-Palm-POD), when complexed with siRNA and topically applied to the eye in vivo, resulted in up to 30% knockdown of luciferase reporter gene expression in the corneal epithelium. The methods developed within represent a valid standardized approach that is ideal for screening of a range of delivery formulations.

Peptide Assembly on the Membrane Determines the HIV-1 Inhibitory Activity of Dual-Targeting Fusion Inhibitor Peptides.

Novel strategies in the design of HIV-1 fusion/entry inhibitors are based on the construction of dual-targeting fusion proteins and peptides with synergistic antiviral effects. In this work we describe the design of dual-targeting peptides composed of peptide domains of E2 and E1 envelope proteins from Human Pegivirus with the aim of targeting both the loop region and the fusion peptide domains of HIV-1 gp41. In a previous work, we described the inhibitory role of a highly conserved fragment of the E1 protein (domain 139-156) which interacts with the HIV-1 fusion peptide at the membrane level. Here, two different dual-targeting peptides, where this E1 peptide is located on the N- or the C-terminus respectively, have been chemically synthesized and their antiviral activities have been evaluated with HIV pseudotyped viruses from different clades. The study of the functional behaviour of peptides in a membranous environment attending to the peptide recognition of the target sites on gp41, the peptide conformation as well as the peptide affinity to the membrane, demonstrate that antiviral activity of the dual-targeting peptides is directly related to the peptide affinity and its subsequent assembly into the model membrane. The overall results point out to the necessity that fusion inhibitor peptides that specifically interfere with the N-terminal region of gp41 are embedded within the membrane in order to properly interact with their viral target.

The cerebrovascular pathology in Alzheimer's disease and its influence on clinical variables.

Vascular pathology is frequently found in the brains of patients with Alzheimer's disease (AD). The aim of this study is to assess the frequency of vascular pathology in the brain in AD patients in a systematic manner and its clinical significance at presentation. A series of 51 patients with mild to moderate AD were consecutively enrolled. At baseline, every patient underwent the following clinical scales: Mini-Mental, Clinical Dementia Rating Scale, Ischemic Scale, Blessed Dementia Rating Scale, Alzheimer's Disease Assessment Scale Cognitive Subscale, Neuropsychiatric Inventory, and an Activities of Daily Living Scale (Disability Assessment for Dementia). We also carried out magnetic resonance imaging of the brain and color echo Doppler of carotids to measure the intima-media thickness. White matter hyperintensities were quantitatively evaluated with the Wahlund scale. We did not find correlation between intima-media thicknesses of carotids and clinical scales and between the Wahlund scale and clinical scales. The presence or absence of both microinfarctions and hypertension had no influence in the scores of the clinical scales. We conclude that the vascular component is common in AD but only as coincident pathology.

Membrane-transferring sequences of the HIV-1 Gp41 ectodomain assemble into an immunogenic complex.

The membrane-proximal stem region of gp41 has been postulated to host the two conserved membrane-transferring domains that promote HIV-1 fusion: the amino-terminal fusion peptide (FP) and the highly aromatic pre-transmembrane sequence. Our results confirm that the hydrophobic FP and membrane-proximal sequences come into contact and form structurally defined complexes. These complexes are immunogenic and evoke responses in rabbits that compete with the recognition of native functional gp41 by the 2F5 monoclonal antibody. We conclude that co-assembly of the FP and the pre-transmembrane sequences might exert a constraint that helps maintain a gp41 stem region pre-fusion structure.

Antibodies to citrullinated human fibrinogen synthetic peptides in diagnosing rheumatoid arthritis.

Since aggressive therapy given early in the rheumatoid arthritis (RA) disease course has the greatest therapeutic potential, early diagnostic tests with both high specificity and sensitivity are desirable. Rheumatoid sera were found to contain antibodies against citrullinated peptides, which are considered to be highly specific markers of RA. In the present work several analogues of the alpha- and beta-chains of fibrin peptides containing different degrees of citrullination have been synthesized and analyzed by ELISA using 111 sera from RA patients. In addition, we have also investigated the synergistic effects of different presentation formats of the synthetic constructs. We have designed chimeric and cyclic peptides that bear different peptide sequences within the same molecule. Our results indicate that the synthesis of peptides bearing fibrinogen and filaggrin domains could be a robust method for the design of useful diagnostic strategies in RA.

Hexapeptides that interfere with HIV-1 fusion peptide activity in liposomes block GP41-mediated membrane fusion.

Upon receptor-mediated activation, the gp41 hydrophobic, conserved fusion peptide inserts into the target membrane and promotes the kind of perturbations required for the progression of the HIV-cell fusion reaction. Using a synthetic combinatorial library we have identified all d-amino acid hexapeptide sequences that inhibited the fusion peptide capacity of perturbing model membranes. Two hexapeptides that effectively inhibited the fusion peptide in these systems were subsequently shown to inhibit cell-cell fusion promoted by gp41 expressed at cell surfaces. These observations might be of importance for understanding the mechanisms underlying fusion peptide activity and suggest new strategies for screening compounds that target these viral sequences.