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.

Peptide Amphiphiles for Pharmaceutical Applications.

During the last few decades, several efforts have been made towards developing biocompatible materials. Among them, peptide amphiphiles (PAs) constitute a novel nanotechnological strategy used in the field of biomedicine since they can provide tissue- specific binding and localization. PAs possess several regions combining hydrophobic and hydrophilic areas that are able to self-assemble in aqueous media, forming different tertiary nanostructures able to interact with cellular membranes. Moreover, these molecules can be tuned by incorporating collagen, lipids, or fluorescent markers. In addition, they can also be used as carriers in order to encapsulate active compounds for drug delivery showing promising features in this area. In this review, the self-assembled structures of PAs as well as their pharmacological applications have been summarized. Furthermore, their use as drug delivery systems has been highlighted and the latest advances in this field have been reviewed.

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.

Atorvastatin-loaded peptide amphiphiles against corneal neovascularization.

Background: Corneal neovascularization is a sight-threatening disease. It can be treated using antiangiogenic and anti-inflammatory compounds. Therefore, atorvastatin (ATV) constitutes a suitable candidate to be administered topically. To attain suitable efficacy, ATV can be encapsulated into custom-developed nanocarriers such as peptide amphiphiles. Methods: Three peptide amphiphiles bearing one, two or four C16-alkyl groups (mC16-Tat47-57, dC16-Tat47-57 and qC16-Tat47-57) were synthesized, characterized and loaded with ATV. Drug release and ocular tolerance were assessed as well as anti-inflammatory and antiangiogenic properties. Results: ATV-qC16-Tat47-57 showed higher encapsulation efficiency than mC16-Tat47-57 and dC16-Tat47-57 and more defined nanostructures. ATV-qC16-Tat47-57 showed ATV prolonged release with suitable ocular tolerance. Moreover, ATV-qC16-Tat47-57 was antiangiogenic and prevented ocular inflammation. Conclusion: ATV-qC16-Tat47-57 constitutes a promising topical medication against corneal neovascularization.

Corneal neovascularization is an eye disease that affects over 1 million people every year and can lead to blindness. It is caused by inflammation and the unwanted formation of blood vessels in the eye. Current treatments for this disease are not fully effective. Atorvastatin (ATV) is one drug that has been partially successful at treating corneal neovascularization, but it does not stay in the eye long enough and does not mix well with the water-based environment of the eye. To overcome this, ATV was combined with three specially designed nanocarriers. These nanocarriers were peptides, short stretches of protein. They were designed to be amphiphilic, meaning that one section is hydrophilic (literally meaning 'water loving') and one section is hydrophobic ('water hating'). These peptide nanocarriers allowed ATV to stay in the water-based environment of the eye longer. The peptide with the most hydrophobic chains (qC₁₆-Tat_47-57) was able to carry more ATV than the other peptides and produced particles of a desired shape. ATV-qC₁₆-Tat_47-57 nanocarriers were found to release slowly. These nanocarriers were also found to prevent the development of new blood vessels on a membrane in a hen's egg used to mimic the eye. There was also no sign of irritation on this membrane or in the eyes of New Zealand rabbits. These results show ATV-qC₁₆-Tat_47-57 has a prolonged therapeutic effect, prevents the formation of new blood vessels and is tolerated in the eye. ATV-qC₁₆-Tat_47-57 is therefore potentially a more effective alternative to ATV treatment alone.

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.

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.

Big dynorphin is a neuroprotector scaffold against amyloid ß-peptide aggregation and cell toxicity.

Amyloid ß-peptide (Aß) misfolding into ß-sheet structures triggers neurotoxicity inducing Alzheimer's disease (AD). Molecules able to reduce or to impair Aß aggregation are highly relevant as possible AD treatments since they should protect against Aß neurotoxicity. We have studied the effects of the interaction of dynorphins, a family of opioid neuropeptides, with Aß40 the most abundant species of Aß. Biophysical measurements indicate that Aß40 interacts with Big Dynorphin (BigDyn), lowering the amount of hydrophobic aggregates, and slowing down the aggregation kinetics. As expected, we found that BigDyn protects against Aß40 aggregates when studied in human neuroblastoma cells by cell survival assays. The cross-interaction between BigDyn and Aß40 provides insight into the mechanism of amyloid pathophysiology and may open up new therapy possibilities.

The synovial and blood monocyte DNA methylomes mirror prognosis, evolution, and treatment in early arthritis.

Identifying predictive biomarkers at early stages of inflammatory arthritis is crucial for starting appropriate therapies to avoid poor outcomes. Monocytes (MOs) and macrophages, largely associated with arthritis, are contributors and sensors of inflammation through epigenetic modifications. In this study, we investigated associations between clinical features and DNA methylation in blood and synovial fluid (SF) MOs in a prospective cohort of patients with early inflammatory arthritis. DNA methylation profiles of undifferentiated arthritis (UA) blood MOs exhibited marked alterations in comparison with those from healthy donors. We identified additional differences both in blood and SF MOs after comparing patients with UA grouped by their future outcomes, i.e., good versus poor. Patient profiles in subsequent visits revealed a reversion toward a healthy level in both groups, those requiring disease-modifying antirheumatic drugs and those who remitted spontaneously. Changes in disease activity between visits also affected DNA methylation, which was partially concomitant in the SF of UA and in blood MOs of patients with rheumatoid arthritis. Epigenetic similarities between arthritis types allow a common prediction of disease activity. Our results constitute a resource of DNA methylation-based biomarkers of poor prognosis, disease activity, and treatment efficacy for the personalized clinical management of early inflammatory arthritis.

Development of Peptide Targeted PLGA-PEGylated Nanoparticles Loading Licochalcone-A for Ocular Inflammation.

Licochalcone-A is a natural compound with anti-inflammatory properties. However, it possesses low water solubility, making its application for the treatment of ocular inflammation difficult. To overcome this drawback, biodegradable nanoparticles incorporating Licochalcone-A have been developed. Additionally, to avoid fast clearance and increase cellular internalization into the ocular tissues, PLGA nanoparticles have been functionalized using PEG and cell penetrating peptides (Tet-1 and B6). To optimize the formulations, a factorial design was carried out and short-term stability of the nanoparticles was studied. Moreover, morphology was also observed by transmission electron microcopy and in vitro drug release was carried out. Ocular tolerance of the formulations was ensured in vitro and in vivo and anti-inflammatory therapeutic efficacy was also assessed. Surface functionalized nanoparticles loading Licochalcone-A were developed with an average size below 200 nm, a positive surface charge, and a monodisperse population. The formulations were non-irritant and showed a prolonged Licochalcone-A release. Despite the fact that both Licochalcone-A Tet-1 and B6 functionalized nanoparticles demonstrated to be suitable for the treatment of ocular inflammation, B6 targeted nanoparticles provided greater therapeutic efficacy in in vivo assays.

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.

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.

Palindromic Rheumatism: Just a Pre-rheumatoid Stage or Something Else?

Palindromic rheumatism (PR), a unique clinical entity, has a characteristic clinical presentation with a relapsing/remitting course. It is established that most patients with PR evolve to chronic disease, of which rheumatoid arthritis (RA) is by far the most common. The relationship between PR and RA is unclear, with similarities and differences between the two, and not all patients evolve to RA in the long-term. Therefore, PR is clearly a pre-RA stage for most, but not all, patients. Autoimmunity plays a substantial role in PR, with the same characteristic autoantibody profile observed in RA, although with some differences in the immune response repertoire. Autoinflammation may also be relevant in some cases of PR. Prognostic factors for RA progression are identified but their exact predictive value is not clear. There are several unmet needs in PR, such as the diagnostic criteria and clinical case definition, the pathogenic mechanisms involved in the unusual clinical course, and the evolution to RA, and our understanding of the therapeutic strategy that could best avoid progression to persistent and potentially destructive arthritis.

Interactions between rheumatoid arthritis antibodies are associated with the response to anti-tumor necrosis factor therapy.

BACKGROUND: Blocking of the Tumor Necrosis Factor (TNF) activity is a successful therapeutic approach for 50-60% of rheumatoid arthritis (RA) patients. However, there are yet no biomarkers to stratify patients for anti-TNF therapy. Rheumatoid factor (RF) and anti-cyclic-citrullinated antibodies (anti-CCP) have been evaluated as biomarkers of response but the results have shown limited consistency. Anti-carbamylated protein (anti-CarP) and anti-peptidylarginine deiminase type 4 (anti-PAD4) antibodies have been much less studied. Despite being linked to common immune processes, the interaction between these markers has not been evaluated yet. Our aim was to analyze the interaction between these four antibodies in relation to the response to anti-TNF therapy. METHODS: For this objective, a prospective cohort of n = 80 RA patients starting anti-TNF therapy was recruited. Serum determinations at baseline were performed for RF, anti-CCP, anti-CarP and anti-PAD4 antibodies using enzyme-linked immunosorbent assays (ELISA). The clinical response to anti-TNF therapy was determined at week 12 using the change in DAS28 score. Association was performed using multivariate linear regression adjusting for baseline DAS28, sex and age. RESULTS: The interaction between pairs of antibodies was tested by the addition of an interaction term. We found two highly significant antibody interactions associated with treatment response: anti-CarP with anti-PAD4 (p = 0.0062), and anti-CCP with RF (p = 0.00068). The latter antibody interaction was replicated in an independent retrospective cohort of RA patients (n = 199, p = 0.04). CONCLUSIONS: The results of this study suggest that antibody interaction effects are important factors in the response to anti-TNF therapy in RA.

Palindromic rheumatism: a unique and enigmatic entity with a complex relationship with rheumatoid arthritis.

INTRODUCTION: Palindromic rheumatism (PR) is a form of relapsing/remitting arthritis that may evolve to chronic rheumatic disease, mainly rheumatoid arthritis (RA). The exact nature of PR is unclear, as it may be considered a disease in itself, an abortive form of RA or just a pre-RA stage. AREAS COVERED: The authors review the most relevant epidemiological and clinical aspects of PR, especially the pathogenetic role of autoimmunity in PR, with most patients having a characteristic autoantibody profile similar to that observed in RA. The role of autoinflammation is also discussed. A literature review on the rate of RA progression and its prognostic factors was analyzed. Data on the efficacy of drug therapies used to treat PR are presented. PubMed was searched using the terms 'palindromic rheumatism' OR 'palindromic arthritis'. EXPERT OPINION: PR is a disease entity with a close but unclear relationship with RA. In PR there is an unmet need, which is to clarify the clinical spectrum and elucidate the risk factors for evolution to RA. The role of autoimmunity and the autoinflammatory component should be investigated. Since most patients evolve to RA, PR may display a unique therapeutic opportunity to avoid this evolution.

Nanotechnology-Based Platforms for Vaginal Delivery of Peptide Microbicides.

BACKGROUND: Vaginal drug delivery offers an opportunity for effective microbicide treatments. However, the physiological characteristics of the vagina represent a challenge when preparing microbicide formulations for local delivery that avoid systemic effects. OBJECTIVE: To review recent advances in vaginal microbicides placing special emphasis on nanocarriers that encapsulate peptide microbicides. The review includes an overview of local treatment and prevention strategies for bactericidal, fungal, protozoal and viral vaginal infections that use peptide microbicides. METHODS: Scientific literature and clinical trials published in the PubMed database since 2015 are analyzed. RESULT: Peptide microbicides are used for vaginal drug delivery as a means of prevention and treatment of several infectious diseases, especially sexually transmitted infections, and it is clear that such peptide-based microbicides constitute a suitable strategy. Their combination with nanotechnological drug delivery systems improves local peptide administration while avoiding their degradation and adverse effects. To date, only a few polymeric and lipid-based nanocarriers have been used for peptide microbicide vaginal delivery. CONCLUSION: The combination of nanotechnology and peptide microbicides has emerged over recent years, and different platforms are being used for vaginal peptide delivery. Nanostructured systems for peptide delivery via vaginal rings or hydrogels are increasingly used as platforms capable of providing suitable drug delivery. However, there is still a lack of knowledge about nanocarriers aimed at encapsulating peptides for local microbicide delivery.

Anti-carbamylated protein antibody isotype pattern differs between palindromic rheumatism and rheumatoid arthritis.

BACKGROUND: A restricted response against citrullinated peptides/proteins, with less isotype usage, has been found in palindromic rheumatism (PR) in comparison with rheumatoid arthritis (RA). We hypothesized that this different antibody response may be observed for other post-translational modified proteins. We compared the prevalence and isotype usage of two specificities of anti-carbamylated peptide/protein antibodies (Anti-CarP) in patients with PR and RA. METHODS: Cross-sectional study including 54 patients with pure PR and 53 patients with RA, matched by sex, age, disease duration and ACPA. Anti-CarP specificities were determined by home-made enzyme-linked immunosorbent assay tests using a synthetic chimeric fibrin/filaggrin homocitrullinated peptide (CFFHP) and fetal calf serum (FCS) homocitrullinated protein as antigens. IgG, IgA and IgM isotypes were measured. RESULTS: Anti-CarP were positive (CFFHP or FCS) in 24% and 64% of patients with PR and RA, respectively (p < 0.005). All Anti-CarP isotype proportions were significantly lower in PR than in RA: Anti-CarP-IgG (24% versus 51%), Anti-CarP-IgA (7% versus 34%) and Anti-CarP-IgM (7% versus 36%). Mean titers of Anti-CarP isotypes were also lower in PR. In Anti-CarP positive patients, the isotype distribution differed between PR and RA: IgG Anti-CarP was used in all PR patients and in 79% of RA patients. By contrast, a significantly lower isotype usage of both IgA (31% versus 53%) and IgM (31% versus 56%) was observed in PR patients. No significant differences in clinical or demographic characteristics were observed according to Anti-CarP status in PR patients, except for a higher prevalence of ACPA and higher mean titers of ACPA and rheumatoid factor in Anti-CarP positive patients. CONCLUSION: Anti-CarP are found in patients with PR but in a lower proportion and with a different isotype usage from in RA, suggesting a distinct B cell response to homocitrullinated antigens in PR.

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.