PIMS-TS, the New Paediatric Systemic Inflammatory Disease Related to Previous Exposure to SARS-CoV-2 Infection-"Rheumatic Fever" of the 21st Century?

Paediatric inflammatory multisystem syndrome temporally associated with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (PIMS-TS) is a new systemic inflammatory disease that mainly affects children. Its course in many features resembles that of acute rheumatic fever (ARF). Therefore, it is interesting that the experiences with ARF can be used in the management of patients with PIMS-TS. The aim of the article is to analyse the current data on PIMS-TS in relation to ARF. PIMS-TS and ARF are associated with an abnormal immune response to specific pathogens (SARS-CoV-2 and group A streptococcus, respectively). The main symptoms of both diseases are fever and cardiac involvement. Current therapy for PIMS-TS is based on anti-inflammatory treatment: intravenous immunoglobulin (first-line), intravenous glucocorticoids (second-line), or biological therapy (third-line; including interleukin [IL]-1 antagonists, IL-6 receptor blockers, and anti-tumour necrosis factor agents). Vaccination might be good prophylaxis, but the efficacy and safety of the vaccines against SARS-CoV-2 have not yet been established in children. Interesting insights may be gained by considering PIMS-TS in light of what is known of ARF due to their similar courses, but there are still many unanswered questions surrounding this disease and its pathogenesis.

Genetic variants in rheumatic fever and rheumatic heart disease.

Genetic association studies in rheumatic heart disease (RHD) have the potential to contribute toward our understanding of the pathogenetic mechanism, and may shed light on controversies about RHD etiology. Furthermore, genetic association studies may uncover biomarkers that can be used to identify susceptible individuals, and contribute toward developing vaccine and novel therapeutic targets. Genetic predisposition to rheumatic fever and RHD has been hypothesized by findings from familial studies and observed associations between genes located in the human leukocyte antigens on chromosome 6p21.3 and elsewhere in the genome. We sought to summarize, from published Genetic association studies in RHD, evidence on genetic variants implicated in RHD susceptibility. Using HuGENet™ systematic review methods, we evaluated 66 studies reporting on 42 genes. Existing meta-analyses of candidate gene studies suggest that TGF-ß1 [rs1800469], and IL-1ß [rs2853550] single nucleotide polymorphisms (SNPs) contribute to susceptibility to RHD, whereas the TNF-α [rs1800629 and rs361525], TGF-ß1 [rs1800470 and rs4803457], IL-6 [rs1800795], IL-10 [rs1800896] were not associated with RHD. However, candidate gene studies in RF/RHD are relatively small, thus lacking statistical power to identify reliable and reproducible findings, emphasizing the need for large-scale multicenter studies with different populations.

The Role of Thiocyanate in Modulating Myeloperoxidase Activity during Disease.

Thiocyanate (SCN-) is a pseudohalide anion omnipresent across mammals and is particularly concentrated in secretions within the oral cavity, digestive tract and airway. Thiocyanate can outcompete chlorine anions and other halides (F-, Br-, I-) as substrates for myeloperoxidase by undergoing two-electron oxidation with hydrogen peroxide. This forms their respective hypohalous acids (HOX where X- = halides) and in the case of thiocyanate, hypothiocyanous acid (HOSCN), which is also a bactericidal oxidative species involved in the regulation of commensal and pathogenic microflora. Disease may dysregulate redox processes and cause imbalances in the oxidative profile, where typically favoured oxidative species, such as hypochlorous acid (HOCl), result in an overabundance of chlorinated protein residues. As such, the pharmacological capacity of thiocyanate has been recently investigated for its ability to modulate myeloperoxidase activity for HOSCN, a less potent species relative to HOCl, although outcomes vary significantly across different disease models. To date, most studies have focused on therapeutic effects in respiratory and cardiovascular animal models. However, we note other conditions such as rheumatic arthritis where SCN- administration may worsen patient outcomes. Here, we discuss the pathophysiological role of SCN- in diseases where MPO is implicated.

Dysregulated IL-1ß-GM-CSF Axis in Acute Rheumatic Fever That Is Limited by Hydroxychloroquine.

BACKGROUND: Acute rheumatic fever (ARF) and rheumatic heart disease are autoimmune consequences of group A streptococcus infection and remain major causes of cardiovascular morbidity and mortality around the world. Improved treatment has been stymied by gaps in understanding key steps in the immunopathogenesis of ARF and rheumatic heart disease. This study aimed to identify (1) effector T cell cytokine(s) that might be dysregulated in the autoimmune response of patients with ARF by group A streptococcus, and (2) an immunomodulatory agent that suppresses this response and could be clinically translatable to high-risk patients with ARF. METHODS: The immune response to group A streptococcus was analyzed in peripheral blood mononuclear cells from an Australian Aboriginal ARF cohort by a combination of multiplex cytokine array, flow cytometric analysis, and global gene expression analysis by RNA sequencing. The immunomodulatory drug hydroxychloroquine was tested for effects on this response. RESULTS: We found a dysregulated interleukin-1ß-granulocyte-macrophage colony-stimulating factor (GM-CSF) cytokine axis in ARF peripheral blood mononuclear cells exposed to group A streptococcus in vitro, whereby persistent interleukin-1ß production is coupled to overproduction of GM-CSF and selective expansion of CXCR3+CCR4-CCR6- CD4 T cells. CXCR3+CCR4-CCR6- CD4 T cells are the major source of GM-CSF in human CD4 T cells and CXCL10, a CXCR3 ligand and potent T helper 1 chemoattractant, was elevated in sera from patients with ARF. GM-CSF has recently emerged as a key T cell-derived effector cytokine in numerous autoimmune diseases, including myocarditis, and the production of CXCL10 may explain selective trafficking of these cells to the heart. We provide evidence that interleukin-1ß amplifies the expansion of GM-CSF-expressing CD4 T cells, which is effectively suppressed by hydroxychloroquine. RNA sequencing showed shifts in gene expression profiles and differentially expressed genes in peripheral blood mononuclear cells derived from patients at different clinical stages of ARF. CONCLUSIONS: Given the safety profile of hydroxychloroquine and its clinical pedigree in treating autoimmune diseases such as rheumatoid arthritis, where GM-CSF plays a pivotal role, we propose that hydroxychloroquine could be repurposed to reduce the risk of rheumatic heart disease after ARF.

BOB.1 controls memory B-cell fate in the germinal center reaction.

During T cell-dependent (TD) germinal center (GC) responses, naïve B cells are instructed to differentiate towards GC B cells (GCBC), high-affinity long-lived plasma cells (LLPC) or memory B cells (Bmem). Alterations in the B cell-fate choice could contribute to immune dysregulation leading to the loss of self-tolerance and the initiation of autoimmune disease. Here we show that mRNA levels of the transcription regulator BOB.1 are increased in the lymph node compartment of patients with rheumatoid arthritis (RA), a prototypical autoimmune disease caused by the loss of immunological tolerance. Investigating to what extent levels of BOB.1 impact B cells during TD immune responses we found that BOB.1 has a crucial role in determining the B cell-fate decision. High BOB.1 levels promote the generation of cells with phenotypic and functional characteristics of Bmem. Mechanistically, overexpression of BOB.1 drives ABF1 and suppresses BCL6, favouring Bmem over LLPC or recycling GCBC. Low levels of BOB.1 are sufficient for LLPC but not for Bmem differentiation. Our findings demonstrate a novel role for BOB.1 in B cells during TD GC responses and suggest that its dysregulation may contribute to the pathogenesis of RA by disturbing the B cell-fate determination.

A novel approach for characterizing variations in serum peptides in rheumatic heart disease.

BACKGROUND & OBJECTIVES: Acute rheumatic fever and rheumatic heart disease (RHD) are important public health problems in developing countries. In this study, peptidomic analyses on RHD patients and healthy individuals were performed to characterize variations in serum peptide levels using label-free quantitation approaches. METHODS: Blood samples were obtained from 160 healthy controls and 160 RHD patients. Of the 448 identified peptides, 272 were analyzed by two label-free mass spectrometry methods, the spectral count and spectral index. RESULTS: There were 38 proteins and 95 peptides with significant (adjusted P<0.001) differences in the abundance of peptides between healthy controls and RHD patients, including multiple peptides derived from histone H2B, villin-like protein, complement C4-B and motile sperm domain containing protein-2. The levels of 10 peptides were upregulated, and 85 peptides were downregulated in patients compared to controls. In addition, in patients, the levels of four proteins were upregulated and 34 were downregulated compared to controls. INTERPRETATION & CONCLUSIONS: This study shows that detection of significant changes in serum peptides reflects the difference between RHD patients and healthy controls. This label-free method may be helpful for clinicians to treat RHD patients during the perioperative period.

Association of rheumatic fever & rheumatic heart disease with plausible early & late-stage disease markers.

BACKGROUND & OBJECTIVES: Rheumatic fever (RF) and rheumatic heart disease (RHD) are the autoimmune sequelae caused by Group A Streptococcus. RHD still remains a major concern in the developing countries due to its poor diagnosis, lack of vaccines and social awareness among population. This study was aimed to identify the plausible early- and late-stage disease markers associated with RF/RHD. METHODS: A total of 84 patients with confirmed pharyngitis (n=18), RF (n=23) and RHD (n=43) were included in the comparative analysis of different factors involved in host-pathogen interaction during RF/RHD pathogenesis. RESULTS: This study revealed high titre of serum antistreptolysin O (ASO) antibody in pharyngitis compared to RF and RHD patients, whereas procollagen type 1 C-peptide (PICP) level was elevated in RHD which showed an inverse correlation with serum ASO titre. The significant elevation of serum anti-peptide associated with RF (PARF) antibody in RF patients was correlated as a probable stage-specific determinant. In addition, pro-inflammatory cytokine profile revealed high levels of interleukin-12 (IL-12)/IL-23p40, IL-17A in RF, whereas IL-6 concentration was higher in RHD compared to healthy controls. INTERPRETATION & CONCLUSIONS: The overall assessment of the factors/ disease markers involved in host-pathogen interaction in RF/RHD may be suggestive of plausible disease marker in different groups of patients. Further studies with larger sample need to be done to better understand RF/RHD pathogenesis.

[Fibroblasts as pathogenic cells in rheumatic inflammation]. / Fibroblasten als pathogene Zellen in der rheumatischen Entzündung.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by synovitis, synovial hyperplasia and progressive degeneration of affected joints. These processes are mediated by cells of the immune system as well as by synovial fibroblasts (RASF) originating from the lining layer of the synovium. In this scenario RASFs display an activated phenotype: they show an altered expression of adhesion molecules which allows attachment to articular cartilage and by synthesis of proteases they mediate progressive cartilage and bone destruction. Furthermore, they produce various cytokines and chemokines, which are essential for promoting the inflammatory response. In recent years it has become evident that RASFs not only passively respond to the proinflammatory milieu in the joints of RA patients but also actively contribute by the overproduction of several cytokines and chemokines. These proinflammatory cytokines trigger the transformation of RASFs into an aggressive and invasive phenotype. Additionally, the primarily altered genuine RASFs are actively involved in the recruitment and activation of immune cells. Taken together, they are key players in the development of the well-known chronic, destructive inflammatory response in joints affected by RA.

Optimal use of MRI in clinical trials, clinical care and clinical registries of patients with rheumatoid arthritis.

Magnetic resonance imaging (MRI) clearly is more sensitive than clinical examination and conventional radiography (x-ray) for detection of inflammation (synovitis, bone marrow oedema (osteitis) and tenosynovitis) and damage (bone erosion and cartilage loss/joint space narrowing) in patients with rheumatoid arthritis (RA). The question is when and how MRI should be used. The present article reviews our knowledge about, and provides suggestions for, the use of MRI in clinical trials, clinical care and clinical registries. In clinical trials, the OMERACT RA MRI scoring system (RAMRIS) is a thoroughly validated method which in less time and with fewer patients than x-ray can discriminate between different therapies regarding structural damage progression, and which on top of this offers detailed assessment of upstream inflammatory drivers of damage. In routine clinical care, MRI can contribute to an earlier diagnosis of RA, can reveal subclinical disease activity, e.g. in the synovium (synovitis) and bone (osteitis), and can provide information of strong prognostic significance for the subsequent disease course, which may be useful when deciding the treatment strategy. Future studies will clarify the benefits of including MRI in treat-to-target strategies. The benefits of incorporating MRI into clinical registries are not yet known, but may include improved knowledge about the real-life advantages of MRI, as well as opportunities to develop better clinical and laboratory composite measures to monitor and predict the disease course in RA. In conclusion, MRI has well-documented relevance in several settings in clinical trials and care, but not yet in clinical registries.

New onset of dermatomyositis/polymyositis during anti-TNF-α therapies: a systematic literature review.

We performed a systematic search of databases from 1990 to 2013 to identify articles concerning the new onset of dermatomyositis/polymyositis (DM/PM) in patients treated with anti-TNF-α therapy. We retrieved 13 publications describing 20 patients where the new onset of DM/PM after anti-TNF-α therapy was recorded. 17 patients were affected by rheumatoid arthritis (RA), one by Crohn's disease, one by ankylosing spondilytis, and one by seronegative arthritis. In 91% of the cases antinuclear autoantibodies were detected after the introduction of anti-TNF-α therapy. In 6 patients antisynthetase antibodies were detected and other clinical findings as interstitial lung disease (ILD) were recorded. Improvement of DM/PM after anti-TNF suspension (with the concomitant use of other immunosuppressors) was recorded in 94% of cases. The emergence of DM/PM and antisynthetase syndrome seem to be associated with the use of anti-TNF-α agents, especially in patients with chronic inflammatory diseases (mainly RA) with positive autoantibodies before therapy initiation. In particular, physicians should pay attention to patients affected by RA with positive antisynthetase antibodies and/or history of ILD. In those cases, the use of the TNF-α blocking agents may trigger the onset of PM/DM or antisynthetase syndrome or may aggravate/trigger the lung disease.

KCa1.1 potassium channels regulate key proinflammatory and invasive properties of fibroblast-like synoviocytes in rheumatoid arthritis.

Fibroblast-like synoviocytes (FLS) play important roles in the pathogenesis of rheumatoid arthritis (RA). Potassium channels have regulatory roles in many cell functions. We have identified the calcium- and voltage-gated KCa1.1 channel (BK, Maxi-K, Slo1, KCNMA1) as the major potassium channel expressed at the plasma membrane of FLS isolated from patients with RA (RA-FLS). We further show that blocking this channel perturbs the calcium homeostasis of the cells and inhibits the proliferation, production of VEGF, IL-8, and pro-MMP-2, and migration and invasion of RA-FLS. Our findings indicate a regulatory role of KCa1.1 channels in RA-FLS function and suggest this channel as a potential target for the treatment of RA.

Protective effect of allograft inflammatory factor-1 on the apoptosis of fibroblast-like synoviocytes in patients with rheumatic arthritis induced by nitro oxide donor sodium nitroprusside.

OBJECTIVES: Inadequate apoptosis of rheumatic arthritis (RA) fibroblast-like synoviocytes (FLS) plays a crucial role in the immunopathogenesis of RA. Allograft inflammatory factor-1 (AIF-1) is a novel member of the cytokine network that has been found to be involved in the immunological process underlying RA. This study was undertaken to investigate the potential effects of AIF-1 on nitric oxide donor (NO) sodium nitroprusside (SNP)-induced RA-FLS apoptosis, and the possible molecular mechanisms underlying these effects. METHOD: FLS obtained from patients with active RA were cultured in vitro and treated with SNP in the present or absence of AIF-1. RA-FLS viability was tested by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. RA-FLS apoptosis was analysed by flow cytometry and terminal dUTP nick-end labeling (TUNEL). The levels of phospho-Akt (p-Akt) and phospho-BAD (p-BAD) protein were detected by Western blot. RESULTS: A 24-h AIF-1 pretreatment at concentrations ranging from 10 to 100 ng/mL increased the viability of RA-FLS and prevented RA-FLS apoptosis in a dose-dependent manner in the presence of SNP. AIF-1 induced phosphorylation of Akt and BAD in a time- and concentration-dependent manner. The effect was reversed by treatment with the PI3K inhibitor LY2940042 (LY) and the nuclear factor kappa B (NF-κB) inhibitor pyrrolidine dithiocarbamate (PDTC). CONCLUSIONS: AIF-1 can protect RA-FLS from apoptosis induced by NO by upregulating the expression of p-Akt and p-BAD.

Protective role of systemic furin in immune response-induced arthritis.

OBJECTIVE: Rheumatoid arthritis (RA) is an autoimmune joint disease associated with chronic inflammation of the synovium that causes profound damage of joints. Inflammation results in part from the influx of immune cells secreting inflammatory cytokines and the reduction in the number of Treg cells. We undertook this study to assess the effect of furin, a proteinase implicated in the proteolytic activity of various precursor proteins and involved in the regulation of both proteinase maturation and immune cells, in an experimental model of RA. METHODS: The effect of furin and its inhibitor α1-PDX was tested in mice with collagen-induced arthritis (CIA). Joints were processed for histology and protein expression. Levels of cytokines were measured in joint tissue, and Treg cell numbers were measured in spleens. RESULTS: Furin expression and activity were high in the synovial pannus in RA patients and mice with CIA. Systemic administration of furin prevented increases in the arthritis score, joint destruction, and bone loss, in contrast to systemic administration of the furin inhibitor α1-PDX, which enhanced these parameters. By preventing the development of synovial pannus, furin reduced the expression of metalloproteinases in the joints. In contrast, α1-PDX enhanced synovial proliferation and the expression and activity of matrix metalloproteinases. Furthermore, furin reversed the local Th1/Th2 balance and restored the number of Treg cells in the spleen, indicating mediation by immune cells. CONCLUSION: These findings show the protective role of exogenous furin against RA, mediated by an immune response. The data suggest the potential therapeutic use of furin or its derivatives in autoimmune diseases including RA.

Rheumatic fever & rheumatic heart disease: the last 50 years.

Rheumatic fever (RF) and rheumatic heart disease (RHD) continue to be a major health hazard in most developing countries as well as sporadically in developed economies. Despite reservations about the utility, echocardiographic and Doppler (E&D) studies have identified a massive burden of RHD suggesting the inadequacy of the Jones' criteria updated by the American Heart Association in 1992. Subclinical carditis has been recognized by E&D in patients with acute RF without clinical carditis as well as by follow up of RHD patients presenting as isolated chorea or those without clinical evidence of carditis. Over the years, the medical management of RF has not changed. Paediatric and juvenile mitral stenosis (MS), upto the age of 12 and 20 yr respectively, severe enough to require operative treatement was documented. These negate the belief that patients of RHD become symptomatic ≥20 years after RF as well as the fact that congestive cardiac failure in childhood indicates active carditis and RF. Non-surgical balloon mitral valvotomy for MS has been initiated. Mitral and/or aortic valve replacement during active RF in patients not responding to medical treatment has been found to be life saving as well as confirming that congestive heart failure in acute RF is due to an acute haemodynamic overload. Pathogenesis as well as susceptibility to RF continue to be elusive. Prevention of RF morbidity depends on secondary prophylaxis which cannot reduce the burden of diseases. Primary prophylaxis is not feasible in the absence of a suitable vaccine. Attempts to design an antistreptococcal vaccine utilizing the M-protein has not succeeded in the last 40 years. Besides pathogenesis many other questions remain unanswered.

Prevalence of and risk factors for chronic arthralgia and rheumatoid-like polyarthritis more than 2 years after infection with chikungunya virus.

OBJECTIVES: Chikungunya virus (CHIKV), transmitted to humans from infected mosquitoes, causes acute fever, arthralgia and rash. There is increasing evidence that it also causes longer-term rheumatic symptoms. In a circumscribed part of Mauritius where infectivity was high, a cohort of inhabitants was surveyed with the objectives of assessing the prevalence of and risk factors for chronic musculoskeletal symptoms and for a rheumatoid arthritis-like condition at 27.5 months after initial infection. METHODS: Participants were recruited May-November 2008 and invited to complete a questionnaire. CHIKV was diagnosed clinically. The primary outcomes for the analyses were (a) self-reported ongoing musculoskeletal symptoms and (b) fulfilment of modified diagnostic criteria for rheumatoid arthritis. Risk factors for these outcomes were explored in univariate analyses using logistic regression. Subsequently, multivariate logistic regression was used to identify factors that were independently associated with the outcomes. RESULTS: 173 individuals were identified with CHIKV, of whom 136 (78.6%) reported persisting musculoskeletal symptoms 27.5 months after infection. Persistent symptoms were associated with older age at time of infection, female gender and baseline symmetrical distribution of joint symptoms. We found that 5% of those infected with CHIKV fulfilled a modified version of the American College of Rheumatology criteria for rheumatoid arthritis 27.5 months after infection. CONCLUSIONS: CHIKV is associated with a high prevalence of persistent rheumatic symptoms. Physicians need to be aware of CHIKV as a cause of acute and chronic rheumatic symptoms.

Efficacy of radiosynovectomy in rheumatoid arthritis.

In this retrospective study, we evaluated the effect of radiosynovectomy of patients with rheumatoid arthritis. Radiosynovectomy was performed in 577 joints of 137 rheumatoid patients. We applied 185 MBq yttrium-90 in knees (n = 58), 74-111 MBq rhenium-186 colloids in ankle (n = 50), wrists (n = 43) and shoulders (n = 35), and 15 to 37 MBq in finger (n = 298) and toe joints (n = 46). The effect of radiosynovectomy was scored in 4 subjective categories: excellent response (no symptoms); good response (significant reduction of symptoms); moderate response (slight decrease); and bad response (no change or worsening), of pain and/or swelling in treated joint 3 months after the procedure. Excellent or good response was observed in 57% of treated knees, 63% of shoulders, 60% of wrists, 64% of ankles, 54% of thumb bases, 55% of MCP's, 54% of PIP's, 53% of DIP's, and 54% of MTP's. Side effects associated to the RSO, i.e., swelling or transient increase of pain, were recorded in 7% of the patients that resolved within 1 month. No patient had any non-reversible skin alteration after treatment, only slight erythema was observed in 5 patients. Radiosynovectomy is effective and safe in the treatment of rheumatoid arthritis.

Rheumatic immune-related adverse events induced by immune checkpoint inhibitors.

Immune checkpoint inhibitors (ICIs) block the major inhibitory pathways in T cells, resulting in an augmented antitumor response. Immune-related adverse events (irAEs) are a new class of side effects caused by ICIs and tend to be more prevalent in patients with preexisting autoantibodies and autoimmune diseases. The rheumatic subset of irAEs mainly includes arthralgia, arthritis, myalgia, myositis, vasculitis, sicca syndrome, scleroderma and systemic lupus erythematosus. The most common classification system for AEs, the Common Terminology Criteria for Adverse Events, is of limited use for irAEs, especially rheumatic irAEs. Therapy with glucocorticoid and temporary or permanent discontinuation of ICIs are the cornerstones of irAE treatment, and can be complemented with immunosuppressants (e.g., methotrexate), biologic agents (e.g., tumor necrosis factor inhibitors and interleukin-6 receptor antagonists), intravenous immunoglobin and plasma exchange. Thus, the evaluation and treatment of rheumatic irAEs require multidisciplinary cooperation among physicians. Here, we review the most prevalent ICI-associated rheumatic irAEs.

[Acute interstitial pneumonia in a patient treated with methotrexate: Late-onset with neutrophilia and eosinophilia in bronchoalveolar lavage fluid]. / Pneumopathie interstitielle aiguë chez une patiente traitée par méthotrexate : atteinte tardive avec neutrophilie et éosinophilie dans le lavage bronchoalvéolaire.

INTRODUCTION: Methotrexate-induced pneumonitis is a rare but potentially fatal side effect. It is a diagnosis of exclusion. There are early and late forms and different cell patterns in the bronchoalveolar lavage (BAL). CASE REPORT: We present a case of acute interstitial lung disease in a 54-year-old patient who had been taking methotrexate for a year and a half for rheumatoid arthritis. After excluding other causes and based on the diagnostic criteria of Searles and McKendry, we could reasonably identify methotrexate as the cause of the lung disease. It was of late onset and the BAL showed neutrophilia and eosinophilia. CONCLUSION: Methotrexate-induced pneumonitis is a diagnosis of exclusion. A late onset combined with the predominance of neutrophils and eosinophils in BAL is rare in the literature, demonstrating the wide heterogeneity of methotrexate-related interstitial lung disease.

Mitigation of Rheumatic Arthritis in a Rat Model via Transdermal Delivery of Dapoxetine HCl Amalgamated as a Nanoplatform: In vitro and in vivo Assessment.

PURPOSE: Dapoxetine HCl (DH), a selective serotonin reuptake inhibitor, may be useful for the treatment of rheumatic arthritis (RA). The purpose of this study was to investigate the therapeutic efficacy of transdermal delivery of DH in transethosome nanovesicles (TENVs). This novel delivery of DH may overcome the drawbacks associated with orally administered DH and improve patient compliance. METHODS: DH-TENV formulations were prepared using an injection- sonication method and optimized using a 33 Box-Behnken-design with Design Expert® software. The TENV formulations were assessed for entrapment efficiency (EE-%), vesicle size, zeta potential, in vitro DH release, and skin permeation. The tolerability of the optimized DH-TENV gel was investigated using a rat skin irritation test. A pharmacokinetic analysis of the optimized DH-TENV gel was also conducted in rats. Moreover, the anti-RA activity of the optimized DH-TENV gel was assessed based on the RA-specific marker anti-cyclic cirtullinated peptide antibody (anti-CCP), the cartilage destruction marker cartilage oligomeric matrix protein (COMP) and the inflammatory marker interleukin-6 (IL-6). Level of tissue receptor activator of nuclear factor kappa-Β ligand (RANKL) were also assessed. RESULTS: The optimized DH-TENV formulation involved spherical nanovesicles that had an appropriate EE- % and skin permeation characteristic. The DH-TENV gel was well tolerated by rats. The pharmacokinetics analysis showed that the optimized DH-TENV gel boosted the bioavailability of the DH by 2.42- and 4.16-fold compared to the oral DH solution and the control DH gel, respectively. Moreover, it significantly reduced the serum anti-CCP, COMP and IL-6 levels and decreased the RANKL levels. Furthermore, the DH-TENV gel attenuated histopathological changes by almost normalizing the articular surface and synovial fluid. CONCLUSION: The results indicate that DH-TENVs can improve transdermal delivery of DH and thereby alleviate RA.