Protein Citrullination Research Articles

Small molecule activates citrullination through targeting PAD2.

Citrullination is a post-translational modification catalysed by peptidyl arginine deiminase (PAD) enzymes, and dysregulation of protein citrullination is involved in various pathological disorders. During the past decade, a panel of citrullination inhibitors has been developed, while small molecules activating citrullination have rarely been reported so far. In this study, we screened citrullination activator using an antibody against citrullinated histone H3 (cit-H3), and a natural compound demethoxycurcumin (DMC) significantly activated citrullination. The requirement of PAD2 for DMC-activated citrullination was confirmed by a loss of function assay. Notably, DMC directly engaged with PAD2, and showed binding selectivity among PAD family enzymes. Point mutation assay indicated that residue E352 is essential for DMC targeting PAD2. Consistently, DMC induced typical phenotypes of cells with dysregulation of PAD2 activity, including citrullination-associated cell apoptosis and DNA damage. Overall, our study not only presents a strategy for rationally screening citrullination activators, but also provides a chemical approach for activating protein citrullination. This article is part of the Theo Murphy meeting issue 'The virtues and vices of protein citrullination'.

Co-expression of PADI isoforms during progenitor differentiation enables functional diversity.

Protein isoforms, generated through alternative splicing or promoter usage, contribute to tissue function. Here, we characterize the expression of predicted Padi3α and Padi3β isoforms in hair follicles and describe expression of Padi2β, a hitherto unknown PADI2 isoform, in the oligodendrocyte lineage. Padi2β transcription is initiated from a downstream intronic promoter, generating an N-terminally truncated, unstable, PADI2β. By contrast to the established role of the canonical PADI2 (PADI2α) (Falcao et al. 2019 Cell Rep. 27, 1090-1102.e10. (doi:10.1016/j.celrep.2019.03.108)), PADI2β inhibits oligodendrocyte differentiation, suggesting that PADI2 isoforms exert opposing effects on oligodendrocyte lineage progression. We localize Padi3α and Padi3β to developing hair follicles and find that both transcripts are expressed at low levels in progenitor cells, only to increase in expression concomitant with differentiation. When expressed in vitro, PADI3α and PADI3β are enriched in the cytoplasm and precipitate together. Whereas PADI3β protein stability is low and PADI3β fails to induce protein citrullination, we find that the enzymatic activity and protein stability of PADI3α is reduced in the presence of PADI3β. We propose that PADI3β modulates PADI3α activity by direct binding and heterodimer formation. Here, we establish expression and function of Padi2 and Padi3 isoforms, expanding on the mechanisms in place to regulate citrullination in complex tissues. This article is part of the Theo Murphy meeting issue 'The virtues and vices of protein citrullination'.

Citrullination of matrisomal proteins in health and diseases.

Proteins once translated are subjected to post-translational modifications (PTMs) that can critically modify their characteristics. Citrullination is a unique type of PTM that is catalysed by peptidylarginine deiminase (PAD) enzymes, which regulate a multitude of physiological functions such as apoptosis, gene expression and immune response by altering the structure and function of cellular proteins. However, emerging data have unravelled compelling evidence to support that PAD-mediated citrullination is not exclusive to cellular proteins; rather citrullination of extracellular matrix (ECM) proteins also plays a major contributing role in various physiological/pathological conditions. Here, we discuss putative mechanisms for citrullination-induced alterations in the function of ECM proteins. Further, we put emphasis on influential roles of ECM citrullination in various pathological scenarios to underscore the clinical potential of its manipulation in human diseases. This article is part of the Theo Murphy meeting issue 'The virtues and vices of protein citrullination'.

An unbiased proteomic analysis of PAD4 in human monocytes: novel substrates, binding partners and subcellular localizations.

Peptidylarginine deiminase IV (PAD4) post-translationally converts arginine residues in proteins to citrullines and is implicated in playing a central role in the pathogenesis of several diseases. Although PAD4 was historically thought to be a nuclear enzyme, recent evidence has revealed a more complex localization of PAD4 with evidence of additional cytosolic and cell surface localization and activity. However, the mechanisms by which PAD4, which lacks conventional secretory signal sequences, traffics to extranuclear localizations are unknown. In this study, we show that PAD4 was enriched in the organelle fraction of monocytes with evidence of citrullination of organelle proteins. We also demonstrated that PAD4 can bind to several cytosolic, nuclear and organelle proteins that may serve as binding partners for PAD4 to traffic intracellularly. Additionally, cell surface expression of PAD4 increased with monocyte differentiation into monocyte-derived dendritic cells and co-localized with several endocytic/autophagic and conventional secretory pathway markers, implicating the use of these pathways by PAD4 to traffic within the cell. Our results suggest that PAD4 is expressed in multiple subcellular localizations and may play previously unappreciated roles in physiological and pathological conditions. This article is part of the Theo Murphy meeting issue 'The virtues and vices of protein citrullination'.

Citrulline is not a major determinant of autoantibody reactivity to neutrophil extracellular traps.

One of the main strategies of neutrophils in responding to microbial infections is the formation of neutrophil extracellular traps (NETs). NETs are web-like structures of decondensed chromatin associated with antimicrobial proteins. Citrullination plays an important role during NET formation and a substantial fraction of NET-associated proteins appeared to be citrullinated. The release of citrullinated intracellular proteins from netting neutrophils led to the hypothesis that the production of anti-citrullinated protein autoantibodies by autoimmune patients, in particular patients with rheumatoid arthritis, might be initiated when citrullinated NET components are not properly cleared and are exposed to the immune system. Here, we discuss the processes that lead to NET formation, including the role of peptidylarginine deiminase activation and our current knowledge on citrullinated NET-associated proteins. Citrulline-dependent epitopes do not appear to play a major role in the recognition of NETs by autoantibodies from rheumatoid arthritis and systemic lupus erythematosus patients, even though anti-NET autoantibodies are frequently observed in sera from these patients. The neutrophil proteases associated with NETs have a major impact on the integrity of NET-associated proteins when NET formation is induced by activating isolated human neutrophils. Cleavage/degradation of these proteins also resulted in a strong reduction of the reactivity with autoantibodies. This article is part of the Theo Murphy meeting issue 'The virtues and vices of protein citrullination'.

Transcriptomic analysis of PADI4 target genes during multi-lineage differentiation of embryonic stem cells.

During mammalian embryo development, pluripotent epiblast cells diversify into the three primary germ layers, which will later give rise to all fetal and adult tissues. These processes involve profound transcriptional and epigenetic changes that require precise coordination. Peptidylarginine deiminase IV (PADI4) is a transcriptional regulator that is strongly associated with inflammation and carcinogenesis but whose physiological roles are less well understood. We previously found that Padi4 expression is associated with pluripotency. Here, we examined the role of PADI4 in maintaining the multi-lineage differentiation potential of mouse embryonic stem (ES) cells. Using bulk and single-cell transcriptomic analyses of embryoid bodies (EBs) derived from Padi4 knock-out (Padi4-KO) mouse ES cells, we find that PADI4 loss impairs mesoderm diversification and differentiation of cardimyocytes and endothelial cells. Additionally, Padi4 deletion leads to concerted downregulation of genes associated with polarized growth, sterol metabolism and the extracellular matrix (ECM). This study indicates a requirement for Padi4 in the specification of the mesodermal lineage and reports the Padi4 associated transcriptome, providing a platform for understanding the physiological functions of Padi4 in development and homeostasis. This article is part of the Theo Murphy meeting issue 'The virtues and vices of protein citrullination'.

Neutrophil attachment via Mac-1 (αMβ2; CD11b/CD18; CR3) integrins induces PAD4 deimination of profilin and histone H3.

Neutrophil adhesion to endothelia, entry into tissues and chemotaxis constitute essential steps in the immune response to infections that drive inflammation. Neutrophils bind to other cells and migrate via adhesion receptors, notably the αMβ2 integrin dimer (also called Mac-1, CR3 or CD11b/CD18). Here, the response of neutrophils to integrin engagement was examined by monitoring the activity of peptidylarginine deiminase 4 (PAD4). Histone H3 deimination was strongly stimulated by manganese, an integrin-activating divalent cation, even in the absence of additional inflammatory stimuli. Manganese-induced cell attachment resulted in neutrophil swarm formation that paralleled histone deimination, whereas antibodies that impair integrin binding prevented both cell adhesion and histone deimination. Manganese treatment led to putative deimination of profilin, a protein that functions as an actin-organizing hub, as detected by two-dimensional gel electrophoresis and citrulline immunoblotting. Cl-amidine, a covalent inhibitor of PAD4, and GSK484, a specific PAD4 inhibitor, blocked profilin deimination. Neutrophil migration toward leukotriene B4 and toward synovial fluid from a rheumatoid arthritis patient were inhibited by chloramidine, thus supporting the contribution of deimination to chemotaxis. The data, based on a simplified system for integrin activation, imply a mechanism whereby integrin attachment coordinates neutrophil responses to inflammation and orchestrates deimination of nuclear and cytoskeletal proteins. This article is part of the Theo Murphy meeting issue 'The virtues and vices of protein citrullination'.

Characterizing citrullination by mass spectrometry-based proteomics.

Citrullination is an important post-translational modification (PTM) of arginine, known to play a role in autoimmune disorders, innate immunity response and maintenance of stem cell potency. However, citrullination remains poorly characterized and not as comprehensively understood compared to other PTMs, such as phosphorylation and ubiquitylation. High-resolution mass spectrometry (MS)-based proteomics offers a valuable approach for studying citrullination in an unbiased manner, allowing confident identification of citrullination modification sites and distinction from deamidation events on asparagine and glutamine. MS efforts have already provided valuable insights into peptidyl arginine deaminase targeting along with site-specific information of citrullination in for example synovial fluids derived from rheumatoid arthritis patients. Still, there is unrealized potential for the wider citrullination field by applying MS-based mass spectrometry approaches for proteome-wide investigations. Here we will outline contemporary methods and current challenges for studying citrullination by MS, and discuss how the development of neoteric citrullination-specific proteomics approaches still may improve our understanding of citrullination networks. This article is part of the Theo Murphy meeting issue 'The virtues and vices of protein citrullination'.

PADI6: What we know about the elusive fifth member of the peptidyl arginine deiminase family.

Peptidyl arginine deiminase 6 (PADI6) is a maternal factor that is vital for early embryonic development. Deletion and mutations of its encoding gene in female mice or women lead to early embryonic developmental arrest, female infertility, maternal imprinting defects and hyperproliferation of the trophoblast. PADI6 is the fifth and least well-characterized member of the peptidyl arginine deiminases (PADIs), which catalyse the post-translational conversion of arginine to citrulline. It is less conserved than the other PADIs, and currently has no reported catalytic activity. While there are many suggested functions of PADI6 in the early mouse embryo, including in embryonic genome activation, cytoplasmic lattice formation, maternal mRNA and ribosome regulation, and organelle distribution, the molecular mechanisms of its function remain unknown. In this review, we discuss what is known about the function of PADI6 and highlight key outstanding questions that must be answered if we are to understand the crucial role it plays in early embryo development and female fertility. This article is part of the Theo Murphy meeting issue 'The virtues and vices of protein citrullination'.

Deimination in epidermal barrier and hair formation.

Peptidylarginine deiminases (PADs) transform a protein arginine residue into the non-standard amino acid citrulline. This calcium-dependent post-translational modification of proteins is called citrullination or deimination. As described in this special issue, PADs play a role in various physiological processes, and PAD deregulations are involved in many human diseases. Three PADs are expressed in the epidermis, where their roles begin to be deciphered. PAD1 and PAD3 are involved in keratinocyte differentiation, particularly in the epidermal barrier function, keratins, filaggrin and filaggrin-related proteins being the most abundant deiminated epidermal proteins. Reduced amounts of deiminated proteins and PAD1 expression may be involved in the pathogenesis of psoriasis and atopic dermatitis, two very frequent and chronic skin inflammatory diseases. The trichohyalin/PAD3/transglutaminase three pathway is important for hair shaft formation. Mutations of the PADI3 gene, leading to a decreased activity or abnormal localization of the corresponding isotype, are the cause of a rare hair disorder called uncombable hair syndrome, and are associated with the central centrifugal cicatricial alopecia, a frequent alopecia mainly affecting women of African ancestry. This article is part of the Theo Murphy meeting issue 'The virtues and vices of protein citrullination'.

Neutrophil peptidylarginine deiminase 4 is essential for detrimental age-related cardiac remodelling and dysfunction in mice.

Mice fully deficient in peptidylarginine deiminase 4 (PAD4) enzyme have preserved cardiac function and reduced collagen deposition during ageing. The cellular source of PAD4 is hypothesized to be neutrophils, likely due to PAD4's involvement in neutrophil extracellular trap release. We investigated haematopoietic PAD4 impact on myocardial remodelling and systemic inflammation in cardiac ageing by generating mice with Padi4 deletion in circulating neutrophils under the MRP8 promoter (Ne-PAD4-/-), and ageing them for 2 years together with littermate controls (PAD4fl/fl). Ne-PAD4-/- mice showed protection against age-induced fibrosis, seen by reduced cardiac collagen deposition. Echocardiography analysis of structural and functional parameters also demonstrated preservation of both systolic and diastolic function with MRP8-driven PAD4 deletion. Furthermore, cardiac gene expression and plasma cytokine levels were evaluated. Cardiac genes and plasma cytokines involved in neutrophil recruitment were downregulated in aged Ne-PAD4-/- animals compared to PAD4fl/fl controls, including decreased levels of C-X-C ligand 1 (CXCL1). Our data confirm PAD4 involvement from circulating neutrophils in detrimental cardiac remodelling, leading to cardiac dysfunction with old age. Deletion of PAD4 in MRP8-expressing cells impacts the CXCL1-CXCR2 axis, known to be involved in heart failure development. This supports the future use of PAD4 inhibitors in cardiovascular disease. This article is part of the Theo Murphy meeting issue 'The virtues and vices of protein citrullination'.

Citrullination and the protein code: crosstalk between post-translational modifications in cancer.

Post-translational modifications (PTMs) of proteins are central to epigenetic regulation and cellular signalling, playing an important role in the pathogenesis and progression of numerous diseases. Growing evidence indicates that protein arginine citrullination, catalysed by peptidylarginine deiminases (PADs), is involved in many aspects of molecular and cell biology and is emerging as a potential druggable target in multiple diseases including cancer. However, we are only just beginning to understand the molecular activities of PADs, and their underlying mechanistic details in vivo under both physiological and pathological conditions. Many questions still remain regarding the dynamic cellular functions of citrullination and its interplay with other types of PTMs. This review, therefore, discusses the known functions of PADs with a focus on cancer biology, highlighting the cross-talk between citrullination and other types of PTMs, and how this interplay regulates downstream biological events. This article is part of the Theo Murphy meeting issue 'The virtues and vices of protein citrullination'.

Peptidylarginine deiminase 4 and ADAMTS13 activity in Staphylococcus aureus bacteraemia.

Staphylococcus aureus infection is associated with increased levels of neutrophil extracellular traps (NETs) and von Willebrand factor (VWF), and with reduced activity of ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type 1 motifs, member 13). Peptidylarginine deiminase 4 (PAD4) contributes to NET formation and inactivates ADAMTS13 in vitro. The role of PADs in the dynamics of NETs, VWF and ADAMTS13 has not yet been studied. We thus aimed to assess the longitudinal evolution of NETs, PADs, VWF and ADAMTS13 activity in S. aureus infection. Plasma samples from S. aureus bacteraemia patients were longitudinally collected and analysed for NETs, PAD4/PAD2, VWF and ADAMTS13 activity. Correlation analyses with clinical data were performed. Recombinant PAD4 and S. aureus were assessed in vitro for their potential to modulate ADAMTS13 activity. Sixty-seven patients were included. Plasma levels of NETs, VWF, PAD4 and PAD2 were increased and ADAMTS13 activity was decreased. Levels of PADs were negatively correlated with ADAMTS13 activity. NETs were positively correlated with PADs, and negatively with ADAMTS13 activity. In vitro, recombinant PAD4 but not S. aureus reduced ADAMTS13 activity in plasma. Levels of PAD4 and PAD2 correlate with reduced ADAMTS13 activity, with neutrophils as the likely source of PAD activity in S. aureus bacteraemia. This article is part of the Theo Murphy meeting issue 'The virtues and vices of protein citrullination'.

Inhibiting MEK1 R189 citrullination enhances the chemosensitivity of docetaxel to multiple tumour cells.

Drug resistance is still a big challenge for cancer patients. We previously demonstrated that inhibiting peptidylarginine deiminase 2 (PADI2) enzyme activity with Cl-amine increases the efficacy of docetaxel (Doc) on tamoxifen-resistant breast cancer cells with PADI2 expression. However, it is not clear whether this effect applies to other tumour cells. Here, we collected four types of tumour cells with different PADIs expression and fully evaluated the inhibitory effect of the combination of PADIs inhibitor (BB-Cla) and Doc in vitro and in vivo on tumour cell growth. Results show that inhibiting PADIs combined with Doc additively inhibits tumour cell growth across the four tumour cells. PADI2-catalysed citrullination of MEK1 Arg 189 exists in the four tumour cells, and blocking the function of MEK1 Cit189 promotes the anti-tumour effect of Doc in these tumour cells. Further analysis shows that inhibiting MEK1 Cit189 decreases the expression of cancer cell stemness factors and helps prevent cancer cell stemness maintenance. Importantly, this combined treatment can partially restore the sensitivity of chemotherapy-resistant cells to docetaxel or cisplatin in tumour cells. Thus, our study provides an experimental basis for the combined therapeutic approaches using docetaxel- and PADIs inhibitors-based strategies in tumour treatment. This article is part of the Theo Murphy meeting issue 'The virtues and vices of protein citrullination'.

Periodontal health status, Porphyromonas gingivalis and anti-cyclic citrullinated peptide antibodies among rheumatoid arthritis patients

Porphyromonas gingivalis (P. gingivalis) is the primary periodontal pathogen involved in protein citrullination, which triggers the production of anti-cyclic citrullinated peptide (anti-CCP) antibodies, exacerbating rheumatoid arthritis (RA). This study aims to evaluate the amount of P. gingivalis and its association with anti-CCP antibodies in RA patients with periodontitis. This cross-sectional study involves 100 RA patients with a mean age of 52.36 (SD 13.90) years. Smokers and patients with other uncontrolled systemic diseases were excluded. Disease Activity Score-28 (DAS-28) was used to determine RA disease severity. Periodontal parameters were examined to determine periodontal status. Subsequently, plaque samples were collected from the subgingival periodontal pocket for assessment of P. gingivalis bacterial load using the loop-mediated isothermal amplification method. Blood samples (5 ml) were obtained from all participants to analyse anti-CCP antibody levels. Data was analysed by using SPSS version 24.0. Most participants were female (85.0%) and had low RA disease severity (62%). The mean RA disease duration was 7.77 (SD 6.3) years, with a mean DAS-28 of 3.17 (SD 1.0). Forty-seven per cent of participants had periodontitis, but all periodontal parameters were not associated with RA disease activity (P = 0.38). P. gingivalis bacterial load ranged from 10 to 109 copies/μl. Fifty-five per cent of the collected samples showed positive anti-CCP antibody levels, but no significant association was observed with the P. gingivalis bacterial load (P = 0.58). Considering the study's limitations, although periodontitis is prevalent among RA patients, there is a lack of association between P. gingivalis bacterial load and anti-CCP antibody levels, which should be investigated further.

Treatment of human cardiac fibroblasts with the protein arginine deiminase inhibitor BB-Cl-amidine activates the Nrf2/HO-1 signaling pathway

BackgroundCardiac fibrosis contributes to end-stage extracellular matrix remodeling and heart failure (HF). Cardiac fibroblasts (CFs) differentiate into myofibroblasts (myoFbs) to preserve the structural integrity of the heart; however, the molecular mechanisms regulating CF transdifferentiation remain poorly understood. Protein arginine deiminase (PAD), which converts arginine to citrulline, has been shown to play a role in myocardial infarction, fibrosis, and HF. This study aimed to investigate the role of PAD in CF differentiation to myoFbs and identify the citrullinated proteins that were associated with phenotypic changes in CFs. ResultsGene expression analysis showed that PAD1 and PAD2 isoforms, but not PAD4 isoforms, were abundant in both CFs and myoFbs, and PAD1 was significantly upregulated in myoFbs. The pan-PAD inhibitor BB-Cl-amidine (BB-Cl) downregulated the mRNA expression of PAD1 and PAD2 as well as the protein expression of the fibrosis marker COL1A1 in CFs and myoFbs. Interestingly, a proteomic approach pointed to the activation of the Nrf2/HO-1 signaling pathway upon BB-Cl treatment in CFs and myoFbs. BB-Cl administration resulted in the upregulation of HO-1 at both the gene and protein levels in CFs and myoFbs. Importantly, the protein citrullination landscape of CFs consisting of 86 novel citrullination sites associated with focal adhesion (FN1(R1054)), inflammation (TAGLN(R12)) and DNA replication (EEF2(R767)) pathways was identified. ConclusionsIn summary, we revealed that BB-Cl treatment resulted in increased HO-1 expression via the Nrf2 pathway, which could prevent excessive tissue damage, thereby leading to substantial clinical benefits for the treatment of cardiac fibrosis.

Molecular Factors in PAD2 (PADI2) and PAD4 (PADI4) Are Associated with Interstitial Lung Disease Susceptibility in Rheumatoid Arthritis Patients.

Around 50% of rheumatoid arthritis (RA) patients show some extra-articular manifestation, with the lung a usually affected organ; in addition, the presence of anti-citrullinated protein antibodies (ACPA) is a common feature, which is caused by protein citrullination modifications, catalyzed by the peptidyl arginine deiminases (PAD) enzymes. We aimed to identify single nucleotide variants (SNV) in PADI2 and PADI4 genes (PAD2 and PAD4 proteins, respectively) associated with susceptibility to interstitial lung disease (ILD) in RA patients and the PAD2 and PAD4 levels. Material and methods: 867 subjects were included: 118 RA-ILD patients, 133 RA patients, and 616 clinically healthy subjects (CHS). Allelic discrimination was performed in eight SNVs using qPCR, four in PADI2 and four in PADI4. The ELISA technique determined PAD2 and PAD4 levels in serum and bronchoalveolar lavage (BAL) samples, and the population structure was evaluated using 14 informative ancestry markers. Results: The rs1005753-GG (OR = 4.9) in PADI2 and rs11203366-AA (OR = 3.08), rs11203367-GG (OR = 2.4) in PADI4 are associated with genetic susceptibility to RA-ILD as well as the ACTC haplotype (OR = 2.64). In addition, the PAD4 protein is increased in RA-ILD individuals harboring the minor allele homozygous genotype in PADI4 SNVs. Moreover, rs1748033 in PADI4, rs2057094, and rs2076615 in PADI2 are associated with RA susceptibility. In conclusion, in RA patients, single nucleotide variants in PADI4 and PADI2 are associated with ILD susceptibility. The rs1748033 in PADI4 and two different SNVs in PADI2 are associated with RA development but not ILD. PAD4 serum levels are increased in RA-ILD patients.

Citrullination of myelin basic protein induces a Th17-cell response in healthy individuals and enhances the presentation of MBP85-99 in patients with multiple sclerosis

The post-translational modification citrullination has been proposed to play a role in the pathogenesis of multiple sclerosis (MS). Myelin basic protein (MBP) is a candidate autoantigen which is citrullinated to a minor extent under physiological conditions and hypercitrullinated in MS. We examined immune cell responses elicited by hypercitrullinated MBP (citMBP) in cultures of mononuclear cells from 18 patients with MS and 42 healthy donors (HDs). The immunodominant peptide of MBP, MBP85-99, containing citrulline in position 99, outcompeted the binding of native MBP85-99 to HLA-DR15, which is strongly linked to MS. Moreover, using the monoclonal antibody MK16 as probe, we observed that B cells and monocytes from HLA-DR15+ patients with MS presented MBP85-99 more efficiently after challenge with citMBP than with native MBP. Both citMBP and native MBP induced proliferation of CD4+ T cells from patients with MS as well as TNF-α production by their B cells and CD4+ T cells, and citrullination of MBP tended to enhance TNF-α secretion by CD4+ T cells from HLA-DR15+ patients. Unlike native MBP, citMBP induced differentiation into Th17 cells in cultures from HDs, while neither form of MBP induced Th17-cell differentiation in cultures from patients with MS. These data suggest a role for citrullination in the breach of tolerance to MBP in healthy individuals and in maintenance of the autoimmune response to MBP in patients with MS.

Effects of Selenium Supplementation on the Indices of Disease Activity, Inflammation and Oxidative Stress in Patients with Rheumatoid Arthritis: a Randomized Clinical Trial.

The aim of study was to evaluate the effect of selenium supplementation on disease activity, inflammation, and oxidative stress in patients with rheumatoid arthritis (RA).This study was a randomized double-blind placebo-controlled trial on 59 patients with RA. Participants were randomly divided to receive 200μg/day of selenium or a placebo for 12weeks. The disease activity score (DAS.CRP and DAS.ESR), erythrocyte sedimentation rate (ESR), serum levels of C-reactive protein (CRP), fasting blood glucose, lipids, antibodies to cyclic citrullinated protein (anti-CCP), nitric oxide, glutathione, and total antioxidant capacity were assessed. The mean of DAS.CRP and DAS.ESR decreased significantly within both study groups after the intervention. However, the between-group comparisons revealed no significant differences. The CRP levels decreased significantly in the selenium group, and this decrease was near the significance level compared to the placebo (P = 0.05). However, after adjusting for baseline values, the observed difference between groups did not remain significant. In addition, the values of ESR and anti-CCP decreased significantly within the selenium group. Although, between-group comparison did not statistically significant, the change in ESR and anti-CCP in the selenium group was small clinically relevant compared to the placebo [the effect size (95% CI) for ESR: 0.38 (- 0.14, 0.89), and for anti-CCP: 0.32 (- 0.2, 0.83)]. Our study showed that selenium caused a small clinically relevant improvement in some RA biomarkers such as ESR and anti-CCP. Future studies that evaluate the effects of novel forms of supplements such as selenium nanoparticles on the clinical symptoms and biomarkers of RA are suggested.Trial Registration: At www.irct.ir as IRCT20190924044869N1 on 2020-06-14.

Prevalence of types of arthritis and its association with biochemical parameters among pre- and post-menopausal women in different cities of Pakistan.

Introduction: The prevalence of arthritis among both pre and post menopausal is alarmingly high. Old age, gender, obesity, unhealthy life style, poor nutrition, less physical activity, genetics, lack of knowledge about disease, lack if education are the factors which are strongly associated with onset of arthritis.
 Objective: To document prevalence of different types of arthritis and its association with biochemical parameters among pre- and post-menopausal women in different cities of Pakistan.
 Methodology: A total of 300 females’ patients who were diagnosed as case of arthritis from different aetiology, during April 2022 to September 2022, aged above 40 years were enrolled for this cross-sectional observational questionnaire and laboratory-based study. Their demographic and disease-related biochemical parameters were recorded and analysed.
 Results: Over all prevalence of rheumatoid arthritis was high (39%) among both pre- and post-menopausal females. Biochemical laboratory analysis showed mean low (11.90 ± 2.18) level of haemoglobin. 66.6% and 60.6 % of the female patients were seropositive for Rheumatoid factor (RF) and Anti Cyclic Citrullinated Protein (Anti-CCP) which is clinical characteristics of Arthritis patients.
 Conclusion: Women with different type of arthritis have border line mean levels of biochemical parameters which indicate co-morbidities like diabetes, cardiovascular and renal diseases, hypertension, obesity and osteoporosis.
 
 Keywords: Post-menopausal, rheumatoid, psoriatic, osteoarthritis, biochemical parameters.