Protein Citrullination Research Articles

Association between anti-P. gingivalis antibodies and anti-citrullinated vimentin antibodies in the patients with rheumatoid arthritis.

Abstract Objectives Anti-citrullinated-protein auto-antibodies (ACPA), especially that react to citrullinated vimentin (CV), are implicated in the pathogenesis of Rheumatoid arthritis (RA). P. gingivalis (Pg), a key pathogen of periodontitis, can citrullinate the host proteins by production of Pg-derived peptidylarginine deiminase (PPAD). This study evaluated the relationship among antibodies (Abs) that react cyclic citrullinated protein (CCP), CV and periodontal pathogens in the serum of RA patients or control subjects. Methods Blood serum of healthy subjects (n=10) and RA patients (n=26) were obtained from Precision for Medicine. Six distinctive peptide sequences of Vimentin (#1–#6) and corresponding CV (#1’–#6’) peptides were synthesized. ELISA was employed to detect IgG Ab titers for CCP, intact vimentin and CV peptides. Fixed periodontal pathogens, Pg 33277, F. nucleatum, P. intermedia, A. actinomycetemcomitans (Aa) Y4, S. mitis, S. gordonii, A. odontolyticus, and C. gingivalis were also used as IgG ELISA antigens. Results The levels of anti-CCP Ab were higher in RA patients with anti-Pg Ab than those without anti-Pg Ab. Since elevated anti-Pg Ab is hallmark of periodontitis, it is conceivable that RA patients with anti-Pg Ab would have periodontitis. However, there was no association between anti-CCP Ab and the Abs reactive to the other bacteria. Very interestingly, RA patients with anti-Pg Ab, but not those without anti-Pg Ab, showed significantly elevated Ab response to #6’ CV peptide compared to intact #6 vimentin peptide, among six peptide sequences tested. Conclusions Our findings indicated that periodontitis may be associated with the induction of ACPA via PPAD-mediated citrullination of host proteins. Supported by NIH NIDCR grants, DE-027851, DE-028715 and DE-331851

Citrullination modulates MHC class II antigen processing and presentation by revealing cryptic epitopes

Abstract Cryptic peptides, hidden from the immune system under physiologic conditions, are revealed by changes to MHC class II processing and can facilitate loss of tolerance to self-antigens. Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by immune responses to citrullinated self-antigens, in which arginine residues are converted to citrullines; however, the initiating events in RA remain poorly understood. We investigated the hypothesis that citrullination exposes cryptic epitopes by modifying protein structure and proteolysis in a manner sufficient to activate a previously ignorant repertoire of self-reactive T cells. We first utilized in vitro proteolytic mapping to define the effect of citrullination on four well-defined RA autoantigens-. We then used a natural antigen processing assay to elucidate the molecular mechanisms of citrullination-modulated processing and evaluated autoantigen-specific T cell responses against the unique peptide repertoire. We show that citrullination alters MHC class II processing and presentation of RA autoantigens, resulting in the destruction of dominant epitopes and the generation and presentation of cryptic epitopes, composed primarily of native peptide sequences. The citrullination-dependent repertoire stimulates T cells from RA patients with anti-citrullinated protein antibodies more robustly than the native repertoire and control T cells. The generation of this unique repertoire is achieved through altered protease affinity and protein destabilization, rather than direct presentation of citrulline-containing epitopes, suggesting a novel paradigm for the role of protein citrullination in the breach of immune tolerance in RA. Supported by a grant from the Rheumatology Research Foundation and by the Luke Evnin and Deann Wright Fellowship

Ability of α – II Spectrin Breakdown Products and Septin 5 as Predictors for Severity and Clinical Outcome in Rheumatoid Arthritis Patients

There is a continuous interesting in finding new biomarkers that can be used as diagnostic tools for severity of rheumatoid arthritis (RA). Septin-5, a protein that indirectly inhibit neurotransmitter release, Alpha-2 spectrin breakdown products 145 (SBDP145), a protein that increased in neuronal damage, and vitamin D (VitD) are measured in the sera of 120 RA patients and 60 healthy controls (HC). The results showed an increase in SBDP145 level and a decrease in the VitD and calcium levels in RA group compared to HC. While no significant difference in septin-5 and magnesium between the groups. The receiver operating characteristics study showed that VitD has a good sensitivity and specificity for differentiating RA from HC. While Septin-5 and SBDP145 have no significant sensitivity and specificity. There is an increase in the serum levels of erythrocyte sedimentation rate (ESR) and anticyclic citrullinated protein antibody (ACCPA) in RA patients compared with the control group. Also, there are high ratios of RA patients with positive results of C-reactive protein (CRP) and rheumatoid factor (RF). It can be concluded that RA patients are suffering from hypovitaminosis D and hypocalcemia.

The prevalence of thyroid dysfunction and thyroid autoantibodies among patients with rheumatoid arthritis

Background and objective: Rheumatoid arthritis is a chronic and inflammatory autoimmune systemic disease of unknown cause that may affect many tissues and organs. Rheumatoid arthritis and thyroid disorders may occur in the patient simultaneously. The main antigens that give rise to thyroid antibodies are thyroglobulin, thyroid peroxidase, anti-cyclic citrullinated protein, and thyroid hormone receptor. This study aimed to determine the frequency of thyroid dysfunction and seroprevalence of anti-thyroid antibody in patients with rheumatoid arthritis and its association with inflammatory marker C-reactive protein. Methods: A cross-sectional study was performed at Rizgari Teaching Hospital and CMC private hospital, Erbil, Iraq. From 15 January to15 October 2020. A hundred patients with rheumatoid arthritis were included in the study, in addition to 70 controls. The serum levels of biomarkers were determined by the chemiluminescent immunoassay method. Results: Patients with rheumatoid arthritis had 6% of thyroid dysfunctions. Regarding the prevalence of thyroid autoantibodies in patients with rheumatoid arthritis, 9% had positive anti-thyroid peroxidase, 13% had positive anti-thyroglobulin, and 6% were positive for a combination of both. There was a statistically significant (P <0.001) high level of serum anti-cyclic citrullinated protein and C-reactive protein in rheumatoid arthritis than in control. Euthyroid profiles were 73% inrheumatoid arthritis patients and 82.9% in control. Conclusion: The study delineated the co-existence of thyroid disorder in rheumatoid arthritis patients with or without autoimmune origin, besides the increased prevalence of auto-thyroid antibody among rheumatoid arthritis with thyroid dysfunction.

Antibodies to a Citrullinated Porphyromonas gingivalis Epitope Are Increased in Early Rheumatoid Arthritis, and Can Be Produced by Gingival Tissue B Cells: Implications for a Bacterial Origin in RA Etiology.

Based on the epidemiological link between periodontitis and rheumatoid arthritis (RA), and the unique feature of the periodontal bacterium Porphyromonas gingivalis to citrullinate proteins, it has been suggested that production of anti-citrullinated protein antibodies (ACPA), which are present in a majority of RA patients, may be triggered in the gum mucosa. To address this hypothesis, we investigated the antibody response to a citrullinated P. gingivalis peptide in relation to the autoimmune ACPA response in early RA, and examined citrulline-reactivity in monoclonal antibodies derived from human gingival B cells. Antibodies to a citrullinated peptide derived from P. gingivalis (denoted CPP3) and human citrullinated peptides were analyzed by multiplex array in 2,807 RA patients and 372 controls; associations with RA risk factors and clinical features were examined. B cells from inflamed gingival tissue were single-cell sorted, and immunoglobulin (Ig) genes were amplified, sequenced, cloned and expressed (n=63) as recombinant monoclonal antibodies, and assayed for citrulline-reactivities by enzyme-linked immunosorbent assay. Additionally, affinity-purified polyclonal anti-cyclic-citrullinated peptide (CCP2) IgG, and monoclonal antibodies derived from RA blood and synovial fluid B cells (n=175), were screened for CPP3-reactivity. Elevated anti-CPP3 antibody levels were detected in RA (11%), mainly CCP2+ RA, compared to controls (2%), p<0.0001, with a significant association to HLA-DRB1 shared epitope alleles, smoking and baseline pain, but with low correlation to autoimmune ACPA fine-specificities. Monoclonal antibodies derived from gingival B cells showed cross-reactivity between P. gingivalis CPP3 and human citrullinated peptides, and a CPP3+/CCP2+ clone, derived from an RA blood memory B cell, was identified. Our data support the possibility that immunity to P. gingivalis derived citrullinated antigens, triggered in the inflamed gum mucosa, may contribute to the presence of ACPA in RA patients, through mechanisms of molecular mimicry.

Novel antiviral activity of PAD inhibitors against human beta-coronaviruses HCoV-OC43 and SARS-CoV-2

The current SARS-CoV-2 pandemic, along with the likelihood that new coronavirus strains will appear in the nearby future, highlights the urgent need to develop new effective antiviral agents. In this scenario, emerging host-targeting antivirals (HTAs), which act on host-cell factors essential for viral replication, are a promising class of antiviral compounds. Here we show that a new class of HTAs targeting peptidylarginine deiminases (PADs), a family of calcium-dependent enzymes catalyzing protein citrullination, is endowed with a potent inhibitory activity against human beta-coronaviruses (HCoVs). Specifically, we show that infection of human fetal lung fibroblasts with HCoV-OC43 leads to enhanced protein citrullination through transcriptional activation of PAD4, and that inhibition of PAD4-mediated citrullination with either of the two pan-PAD inhibitors Cl-A and BB-Cl or the PAD4-specific inhibitor GSK199 curbs HCoV-OC43 replication. Furthermore, we show that either Cl-A or BB-Cl treatment of African green monkey kidney Vero-E6 cells, a widely used cell system to study beta-CoV replication, potently suppresses HCoV-OC43 and SARS-CoV-2 replication. Overall, our results demonstrate the potential efficacy of PAD inhibitors, in suppressing HCoV infection, which may provide the rationale for the repurposing of this class of inhibitors for the treatment of COVID-19 patients.

Peptidylarginine deiminase 2 promotes T helper 17-like T cell activation and activated T cell-autonomous death (ACAD) through an endoplasmic reticulum stress and autophagy coupling mechanism

Peptididylarginine deiminase type 2 (PADI2) catalyzes the conversion of arginine residues to citrulline residues on proteins. We demonstrate that PADI2 induces T cell activation and investigate how PADI2 promotes activated T cell autonomous death (ACAD). In activated Jurkat T cells, overexpression of PADI2 significantly increases citrullinated proteins and induces endoplasmic reticulum (ER) stress and unfolded protein response (UPR) signaling, ultimately resulting in the expression of autophagy-related proteins and autophagy. PADI2 promoted autophagy and resulted in the early degradation of p62 and the light chain 3B (LC3B)-II accumulation. In Jurkat T cells, silencing the autophagy-related gene (Atg) 12 protein inhibits PADI2-mediated autophagy and promotes ER stress and apoptosis, whereas overexpression of Atg12 decreased ER stress and prolonged autophagy to promote cell survival. Additionally, PADI2 regulates T cell activation and the production of Th17 cytokines in Jurkat T cells (interleukins 6, IL-17A, IL-17F, IL-21, and IL-22). In Jurkat T cells, silencing IL-6 promotes autophagy mediated by PADI2 and inhibits PADI2-induced apoptosis, whereas silencing Beclin-1 increases the activation and survival of Th17-like T cells while decreasing autophagy and apoptosis. PADI2 silencing alleviates ER stress caused by PADI2 and decreases cytokine expression associated with Th17-like T cell activation and ACAD. We propose that PADI2 was involved in Th17 lymphocyte ACAD via a mechanism involving ER stress and autophagy that was tightly regulated by PADI2-mediated citrullination. These findings suggest that inhibiting Th17 T cell activation and the development of severe autoimmune diseases may be possible through the use of novel antagonists that specifically target PADI2.

Syndecan-2 regulates PAD2 to exert antifibrotic effects on RA-ILD fibroblasts

Rheumatoid arthritis (RA)-associated interstitial lung disease (RA-ILD) is the most common pulmonary complication of RA, increasing morbidity and mortality. Anti-citrullinated protein antibodies have been associated with the development and progression of both RA and fibrotic lung disease; however, the role of protein citrullination in RA-ILD remains unclear. Here, we demonstrate that the expression of peptidylarginine deiminase 2 (PAD2), an enzyme that catalyzes protein citrullination, is increased in lung homogenates from subjects with RA-ILD and their lung fibroblasts. Chemical inhibition or genetic knockdown of PAD2 in RA-ILD fibroblasts attenuated their activation, marked by decreased myofibroblast differentiation, gel contraction, and extracellular matrix gene expression. Treatment of RA-ILD fibroblasts with the proteoglycan syndecan-2 (SDC2) yielded similar antifibrotic effects through regulation of PAD2 expression, phosphoinositide 3-kinase/Akt signaling, and Sp1 activation in a CD148-dependent manner. Furthermore, SDC2-transgenic mice exposed to bleomycin-induced lung injury in an inflammatory arthritis model expressed lower levels of PAD2 and were protected from the development of pulmonary fibrosis. Together, our results support a SDC2-sensitive profibrotic role for PAD2 in RA-ILD fibroblasts and identify PAD2 as a promising therapeutic target of RA-ILD.

Molecular Dynamics Study of Citrullinated Proteins Associated with the Development of Rheumatoid Arthritis.

Biological activity regulation by protein post-translational modification (PTM) is critical for cell function, development, differentiation, and survival. Dysregulation of PTM proteins is present in various pathological conditions, including rheumatoid arthritis (RA). RA is a systemic autoimmune disease that primarily affects joints, and there are three main types of protein PTMs associated with the development of this disease, namely, glycosylation, citrullination, and carbamylation. Glycosylation is important for the processing and presentation of antigen fragments on the cell surface and can modulate immunoglobulin activity. The citrullination of autoantigens is closely associated with RA, as evidenced by the presence of antibodies specific to citrullinated proteins in the serum of patients. Carbamylation and dysregulation have recently been associated with RA development in humans.In this study, we performed an overview analysis of proteins with post-translational modifications associated with the development of RA adverted in peer-reviewed scientific papers for the past 20 years. As a result of the search, a list of target proteins and corresponding amino acid sequences with PTM in RA was formed. Structural characteristics of the listed modified proteins were extracted from the Protein Data Bank. Then, molecular dynamics experiments of intact protein structures and corresponding structures with PTMs were performed regarding structures in the list announced in the ProtDB service. This study aimed to conduct a molecular dynamics study of intact proteins and proteins, including post-translational modification and protein citrullination, likely associated with RA development. We observed another exhibition of the fundamental physics concept, symmetry, at the submolecular level, unveiled as the autonomous repetitions of outside the protein structural motif performance globule corresponding to those in the whole protein molecule.

12-Plex DiLeu Isobaric Labeling Enabled High-Throughput Investigation of Citrullination Alterations in the DNA Damage Response.

Protein citrullination is a key post-translational modification (PTM) that leads to the loss of positive charge on arginine and consequent protein structural and functional changes. Though it has been indicated to play critical roles in various physiological and pathological processes, effective analytical tools are largely limited due to a few challenges such as the small mass shift induced by this PTM and its low-abundance nature. Recently, we developed a biotin thiol tag, which enabled large-scale profiling of protein citrullination from complex biological samples via mass spectrometry. However, a high-throughput quantitative approach is still in great need to further improve the understanding of this PTM. In this study, we report an efficient pipeline using our custom-developed N,N-dimethyl leucine isobaric tags to achieve a multiplexed quantitative analysis of citrullination from up to 12 samples for the first time. We then apply this strategy to investigating citrullination alterations in response to DNA damage stress using human cell lines. We unveil important biological functions regulated by protein citrullination and observe hypercitrullination on RNA-binding proteins and DNA repair proteins, respectively. Our results reveal the involvement of citrullination in DNA damage pathways and may provide new insights into DNA-damage-related disease pathogenesis.

Multiple infections with hepatitis A virus and development of rheumatoid arthritis among waste water treatment plant workers.

In the present work, we studied the association between multiple exposure of waste water treatment plant workers to infection with existing hepatitis A virus in waste water and development of rheumatoid arthritis, taking in consideration number of working years as an indicator for frequency of exposure to infection, compared to non waste water treatment plant workers. A total of 105 waste water treatment plant workers and 48 NWWTPWs were included in the study. Exclusion criteria were positivity for HBV and/or HCV IgG, negativity to HAV IgG and suffering from rheumatic diseases other than rheumatoid arthritis. 96.2% of waste water treatment plant workers were anti-HAV-IgG positive, of whom 5 had high antibody titer indicating ongoing infection and were anti-HAV-IgM negative excluding primary infection. These 5 samples were further subjected to quantification of liver enzymes, glutamate oxaloacetate trasaminase and glutamate pyruvate transaminase and HAV-RT-PCR to check viremia and results showed increase of glutamate oxaloacetate trasaminase and glutamate pyruvate transaminase as well as viremea in all of them. Rheumatoid arthritis diagnosis was carried out by detection of C-reactive protein, rheumatoid factor and anti-cyclic citrullinated protein. Rheumatoid arthritis development was 19% in the waste water treatment plant workers with >10 working years and 8% for < 10 working years. Also, disease development started earlier (Age 30-40 years) among the waste water treatment plant workers compared to non waste water treatment plant workers (age: 40-50 years). Multiple exposures of waste water treatment plant workers to HAV might be one of the etiological stimuli of rheumatoid arthritis.

Belimumab Decreases Autophagy and Citrullination in Peripheral Blood Mononuclear Cells from Patients with Systemic Lupus Erythematosus.

Belimumab (BLM) is a B lymphocyte stimulator (BLyS) inhibitor approved for the treatment of systemic lupus erythematosus (SLE). Autophagy is a cell survival mechanism involved in the pathogenesis of SLE. Citrullination is a post-translational modification catalyzed by peptidylarginine deiminase (PAD) enzymes. Autophagy and citrullination may generate neoepitopes, evoking an autoimmune response. No previous studies have investigated the connection of these processes, and how BLM could affect them, in SLE. Ex vivo autophagy and protein citrullination were analyzed by western blot in lysates from 26 SLE patients’ PBMCs at baseline and after 2, 4, and 12 weeks of BLM administration, and from 16 healthy donors’ PBMCs. Autophagic PBMCs were identified by the immunofluorescent detection of the autophagy-associated proteins LC3B (LC3 puncta) and LAMP-1. Autophagosome accumulation was evaluated in CD14− (PBLs) and CD14+ (monocytes) SLE cells. The presence of the BLyS receptors BAFF-R, BCMA, and TACI on SLE CD4+, CD8+ T cells and monocytes, as well as serum IL-18 levels, was also assessed. Following BLM administration, we observed a decrease in autophagy and citrullination, with a lowering of LC3-II, citrullinated vimentin, and PAD4 expression levels in PBMCs from SLE patients. LC3-II levels showed a correlation with the SLE Disease Activity Index 2000 (SLEDAI-2K) after 12 weeks of therapy. The LC3B/LAMP-1 analysis confirmed the reduction in autophagy. A lesser autophagosome accumulation occurred in PBLs and monocytes which, in turn, seemed to be the main cellular populations contributing to autophagy. A reduction in patients’ serum IL-18 concentrations occurred. CD4+ and CD8+ cells weakly expressed BAFF receptors; monocytes expressed only BAFF-R. BLM could impact on autophagy and citrullination, offering an opportunity for a deeper understanding of these mechanisms in SLE, and a possible tool for the clinical management of SLE.

Aggregatibacter actinomycetemcomitans as the Aetiological Cause of Rheumatoid Arthritis: What Are the Unsolved Puzzles?

Leukotoxin A (LtxA) is the major virulence factor of an oral bacterium known as Aggregatibacter actinomycetemcomitans (Aa). LtxA is associated with elevated levels of anti-citrullinated protein antibodies (ACPA) in rheumatoid arthritis (RA) patients. LtxA targets leukocytes and triggers an influx of extracellular calcium into cytosol. The current proposed model of LtxA-mediated hypercitrullination involves the dysregulated activation of peptidylarginine deiminase (PAD) enzymes to citrullinate proteins, the release of hypercitrullinated proteins through cell death, and the production of autoantigens recognized by ACPA. Although model-based evidence is yet to be established, its interaction with the host’s immune system sparked interest in the role of LtxA in RA. The first part of this review summarizes the current knowledge of Aa and LtxA. The next part highlights the findings of previous studies on the association of Aa or LtxA with RA aetiology. Finally, we discuss the unresolved aspects of the proposed link between LtxA of Aa and RA.

Rheumatoid Arthritis Synovial Inflammation Quantification Using Computer Vision.

ObjectiveWe quantified inflammatory burden in rheumatoid arthritis (RA) synovial tissue by using computer vision to automate the process of counting individual nuclei in hematoxylin and eosin images.MethodsWe adapted and applied computer vision algorithms to quantify nuclei density (count of nuclei per unit area of tissue) on synovial tissue from arthroplasty samples. A pathologist validated algorithm results by labeling nuclei in synovial images that were mislabeled or missed by the algorithm. Nuclei density was compared with other measures of RA inflammation such as semiquantitative histology scores, gene‐expression data, and clinical measures of disease activity.ResultsThe algorithm detected a median of 112,657 (range 8,160‐821,717) nuclei per synovial sample. Based on pathologist‐validated results, the sensitivity and specificity of the algorithm was 97% and 100%, respectively. The mean nuclei density calculated by the algorithm was significantly higher (P < 0.05) in synovium with increased histology scores for lymphocytic inflammation, plasma cells, and lining hyperplasia. Analysis of RNA sequencing identified 915 significantly differentially expressed genes in correlation with nuclei density (false discovery rate is less than 0.05). Mean nuclei density was significantly higher (P < 0.05) in patients with elevated levels of C‐reactive protein, erythrocyte sedimentation rate, rheumatoid factor, and cyclized citrullinated protein antibody.ConclusionNuclei density is a robust measurement of inflammatory burden in RA and correlates with multiple orthogonal measurements of inflammation.

Amidst the chaos of late rheumatoid arthritis with no treatment

A 78-year-old man with 24 years history of Rheumatoid Arthritis (RA) came with neck pain. He always refused any treatment except for NSAIDs. From 2 years ago, he was bedridden. He was chronic cigarette smoker. On examination, he had rheumatoid nodules, severe both hands deformity, elbows flexion contracture and knee joints limitation of motion. His lab tests showed high titer of rheumatoid factor and anti cyclic citrullinated protein antibody, high ESR, CRP and vitamin D deficiency. Radiologic studies showed multiple vertebral osteoporotic fractures. Cervical spine CT scan (a and b) showed destruction of the odontoid process and fusion of facet joints of the C2, C3 and C4. MRI of cervical spine (c) showed destruction of odontoid process surrounding by inflammatory synovitis. CT scan of lung (e and f) showed multiple upper lobeand sub pleural nodules along fibrotic changes in base of right lobe. X-ray of knee and hip (d and g) showed severe osteoarthritis andsite of previouship nailing. The patient was diagnosed with advanced erosive nodular RA, severe secondary osteoporosis, osteoarthritis and rheumatoid lung. Standard treatment was recommended, although he again refused. RA can result in progressive joint destruction, deformity and disability [1]. In the spine, it has a predilection for the cervical area, leads to odontoid erosion, spinal instability, and subluxation. In the era of importance of early treatment, these complications are rarely seen, however despite biologic therapy, it has been shown that 15-30% of RA patients can still have preclinical cervical spine abnormalities [2]. The Osteoporosis (OP) is more frequent in patients with high disease activity, bone erosions, &gt;10 years disease duration, and high titers of anti-CCP and RF positivity [3]. Our patient with multiple poor prognostic factors for erosive RA came with extra articular and complications of this disease due refusing treatment.

The effects of vasopressors with and without dobutamine on haemodynamics, metabolism and gut injury during endotoxic shock in rabbits. Acontrolled study.

Vasopressors increase arterial pressure but they may have deleterious effects on mesenteric blood flow. We aimed to evaluate the response of gut biomarkers and superior mesenteric blood flow to different vasopressors with and without dobutamine. Thirty New Zealand rabbits were included and randomly allocated to 5 groups: group A- sham group; group B - norepinephrine; group C - norepinephrine plus dobutamine; group D - vasopressin; and group E - vasopressin plus dobutamine. Mean arterial pressure (MAP) target was greater than 60 mmHg. Endotoxic shock was induced by intra-venous injection of lipopolysaccharide (LPS) in four of the five groups. Aortic blood flow (Qao), superior mesenteric artery flow (QSMA) and lactate were measured after LPS injection. Enterocyte damage was evaluated by measurements of serum citrulline and intestinal fatty acid-binding protein (I-FABP) after 4 h. The largest reduction in Qao occurred in group D (64 ± 17.3 to 38 ± 7.5 mL min-1; P = 0.04). QSMA also declined significantly in groups D and E and remained lower than in the other groups over 4 h (group D - baseline: 65 ± 31; 1 h: 37 ± 10; 2 h: 38 ± 10; 3 h: 46 ± 26; and 4 h: 48 ± 15 mL min-1; P < 0.005; group E - baseline: 73 ± 14; 1 h: 28 ± 4.0; 2 h: 37 ± 6.4; 3 h: 40 ± 11; and 4 h: 48 ± 11; P < 0.005; all in mL min-1). Serum citrulline was significantly lower in groups D (P = 0.014) and E (P = 0.019) in comparison to group A. The fluid administration regimen was similar in all groups. Vasopressin seems to negatively impact gut enterocyte function during endotoxic shock despite the association of an inodilator and adequate fluid replacement.

The Effects of Non-Surgical Periodontitis Therapy on the Clinical Features and Serological Parameters of Patients Suffering from Rheumatoid Arthritis as Well as Chronic Periodontitis.

ObjectiveThe present study aims to evaluate the effects of basic periodontal disease therapy on the general condition and serum inflammatory indicators of patients with rheumatoid arthritis (RA) combined with chronic periodontitis (CP).MethodsForty patients with RA were enrolled in the study and, based on the results of an oral examination and in line with the 2018 periodontitis diagnostic criteria, they were divided into a group with CP (the RA + CP group) and a group without CP (the RA group). Twenty-nine patients with CP who attended the periodontal department of our hospital were recruited as a group with only CP (the CP group), and 20 volunteers without any systemic or periodontal disease were recruited as a healthy control group (the H group). The periodontal and joint conditions of the subjects in the four groups were recorded; anti-cyclic citrullinated protein antibodies, interleukin 6 (IL-6), C-reactive protein (CRP), the erythrocyte sedimentation rate (ESR), and rheumatoid factor levels, which reflect the severity of the RA, were detected, and the differences between the groups were analyzed. The probing depth (PD), clinical attachment loss, and sulcus bleeding index (SBI), which reflect the severity of the periodontitis, were correlated with the factor levels. The RA + CP and CP groups received therapeutic intervention, and the differences in each indicator before and six weeks after the treatment were compared, and their data were compared with those of patients in the RA group and H groups.ResultsCompared with the RA group, the serum expressions of ESR, CRP, and IL-6 were significantly higher in the RA + CP group. There were significant differences in the levels of PD, SBI, IL-6, and CRP in the patients receiving basic periodontal disease therapy before and after the treatment.ConclusionA relatively large proportion of patients with RA have chronic periodontitis, and the local inflammatory state of CP might exacerbate the systemic inflammatory response in RA. Basic periodontal disease therapy may improve the oral condition of patients with RA and reduce the serum levels of the inflammatory factors.

Inhibition of Porphyromonas gingivalis peptidyl arginine deiminase, a virulence factor, by antioxidant-rich Cratoxylum cochinchinense: In vitro and in silico evaluation

Porphyromonas gingivalis, the cause of periodontitis, is also linked to many systemic disorders due to its citrullination capability from a unique peptidyl arginine deiminase (PPAD). Protein citrullination is able to trigger an autoimmune response, increasing the severity of rheumatoid arthritis. The main objective of this study is to evaluate the inhibitory activity of Cratoxylym cochinchinense leaves extract towards the PPAD in vitro and in silico. Methanolic extract of Cratoxylum cochinchinense (CCM) was tested for total phenolic and flavonoid contents along with antioxidative assays. Inhibition of PPAD activities was conducted thereafter using recombinant PPAD in cell lysate. Phytocompounds postulated present in the CCM such as mangiferin, vismiaquinone A, δ-tocotrienol and α-tocotrienol and canophyllol were used as ligands in a simulated docking study against PPAD. Results obtained indicated high antioxidant potential in CCM while recording abundant phenolic (129.0 ± 2.5495 mg GA/g crude extract) and flavonoid (159.0 ± 2.1529 mg QE/g crude extract) contents. A dose-dependent inhibition of PPAD was observed when CCM was evaluated at various concentrations. CCM at 1 mg/mL exhibited citrulline concentration of 24.37 ± 3.25 mM which was 5 times lower than the negative control (114.23 ± 3.31 mM). Molecular docking simulation revealed that mangiferin and vismiaquinone A engaged in H-bonding and pi-pi interactions with important active site residues (Asp130, Arg152, Arg154 and Trp127) of PPAD and could be the potential phytochemicals that accounted for the inhibitory activities observed in the methanolic leaves extract. As such, CCM could be further explored for its therapeutic properties not only for periodontitis, but also for other systemic diseases like rheumatoid arthritis.

A Hairy Cituation – PADIs in Regeneration and Alopecia

In this Review article, we focus on delineating the expression and function of Peptidyl Arginine Delminases (PADIs) in the hair follicle stem cell lineage and in inflammatory alopecia. We outline our current understanding of cellular processes influenced by protein citrullination, the PADI mediated posttranslational enzymatic conversion of arginine to citrulline, by exploring citrullinomes from normal and inflamed tissues. Drawing from other stem cell lineages, we detail the potential function of PADIs and specific citrullinated protein residues in hair follicle stem cell activation, lineage specification and differentiation. We highlight PADI3 as a mediator of hair shaft differentiation and display why mutations in PADI3 are linked to human alopecia. Furthermore, we propose mechanisms of PADI4 dependent fine-tuning of the hair follicle lineage progression. Finally, we discuss citrullination in the context of inflammatory alopecia. We present how infiltrating neutrophils establish a citrullination-driven self-perpetuating proinflammatory circuitry resulting in T-cell recruitment and activation contributing to hair follicle degeneration. In summary, we aim to provide a comprehensive perspective on how citrullination modulates hair follicle regeneration and contributes to inflammatory alopecia.

Abstract 11607: Protein Citrullination Landscape of Human Coronary Atherosclerosis

Introduction: Recently, an irreversible enzymatic post-translational modification, citrullination, has been shown to be present in the cardiovascular system (e.g. aorta). Citrullination, in which arginine is converted to citrulline, can alter protein structure and function, and produce autoantigens Hypothesis: Emerging evidence suggest a potential role for autoimmunity in the pathogenesis of atherosclerosis. However, it remains unknown whether of post-translational citrullination of arterial proteins contributes to local inflammatory processes that initiate or accelerate the atherosclerotic process. The aim of this study was to investigate changes in protein citrullination in human left anterior descending (LAD) coronary artery specimens with and without early atherosclerosis obtained from autopsied young adults with no clinical manifestations of coronary disease. Methods: Segments of the mid-LAD from 74 autopsied individuals (age range:15-55 yrs., 75% male, 67% White) were graded by a pathologist for percent of surface area involved with fatty streaks (FS) or fibrous plaque (FP). The frozen samples were homogenized and digested with Lys-C for subsequent proteomic mass-spectrometry using a data-independent acquisition (DIA) proteomic strategy with a hyper-citrullinated spectral library approach and our published CitFinder software. Results: We found 133 differentially expressed proteins (FDR&lt;0.05) between control, FS and FP groups. Among them, 19 citrullinated proteins with 26 modified amino acid residues were identified. Citrullinated peptides from fibrinogen alpha chain (R84) and transgelin (R146) were upregulated in FP samples in comparison to FS and normal samples. Similarly, the citrullination site (R269) on alpha-enolase was significantly increased in the FP samples compared to normal. Interestingly, citrullination (R110) on myosin light polypeptide 6 was downregulated in FP samples. Conclusions: These data represent the most comprehensive interrogation of citrullinated proteins in human arterial samples to-date and suggest a possible association between this modification of several key proteins involved in tight junction, vascular smooth muscle contraction and inflammation and early atherosclerosis.