Exploration and Evaluation of Therapeutic Efficacy of Drug‐Free Macromolecular Therapeutics in Collagen‐Induced Rheumatoid Arthritis Mouse Model

Monoclonal antibodies (mAbs) against B cell antigens are extensively used in the treatment of rheumatoid arthritis (RA). The B cell depletion therapy prevents RA symptoms and/or alleviates existing inflammation. The previously established two‐step drug‐free macromolecular therapeutics (DFMT) is applied in the treatment of collagen‐induced rheumatoid arthritis in a collagen‐induced rheumatoid arthritis mouse model. DFMT is a B cell depletion strategy utilizing Fab′ fragment of anti‐CD20 mAb for biorecognition and receptor crosslinking to induce B cell apoptosis. DFMT is composed from two nanoconjugates: 1) bispecific engager, Fab′‐MORF1 (anti‐CD20 Fab′ fragment conjugated with morpholino oligonucleotide MORF1), and 2) a crosslinking (effector) component P‐(MORF2)_X (N‐(2‐hydroxypropyl)methacrylamide copolymer grafted with multiple copies of complementary morpholino oligonucleotide MORF2). The absence of Fc fragment has the potential to avoid development of resistance and infusion‐related reactions. DFMT produces B cell depletion, keeps the RA score low for more than 100 days, and shows minimal cartilage and bone erosion and inflammatory cell infiltration. Further improvements will be explored to optimize DFMT strategy in autoimmune disease treatment.     

Lipidome profile predictive of disease evolution and activity in rheumatoid arthritis

Lipid mediators are crucial for the pathogenesis of rheumatoid arthritis (RA); however, global analyses have not been undertaken to systematically define the lipidome underlying the dynamics of disease evolution, activation, and resolution. Here, we performed untargeted lipidomics analysis of synovial fluid and serum from RA patients at different disease activities and clinical phases (preclinical phase to active phase to sustained remission). We found that the lipidome profile in RA joint fluid was severely perturbed and that this correlated with the extent of inflammation and severity of synovitis on ultrasonography. The serum lipidome profile of active RA, albeit less prominent than the synovial lipidome, was also distinguishable from that of RA in the sustained remission phase and from that of noninflammatory osteoarthritis. Of note, the serum lipidome profile at the preclinical phase of RA closely mimicked that of active RA. Specifically, alterations in a set of lysophosphatidylcholine, phosphatidylcholine, ether-linked phosphatidylethanolamine, and sphingomyelin subclasses correlated with RA activity, reflecting treatment responses to anti-rheumatic drugs when monitored serially. Collectively, these results suggest that analysis of lipidome profiles is useful for identifying biomarker candidates that predict the evolution of preclinical to definitive RA and could facilitate the assessment of disease activity and treatment outcomes.Subject terms: Rheumatoid arthritis, Metabolomics     

Over-expression of extracellular superoxide dismutase in mouse synovial tissue attenuates the inflammatory arthritis

Oxidative stress such as reactive oxygen species (ROS) within the inflamed joint have been indicated as being involved as inflammatory mediators in the induction of arthritis. Correlations between extracellular- superoxide dismutase (EC-SOD) and inflammatory arthritis have been shown in several animal models of RA. However, there is a question whether the over-expression of EC-SOD on arthritic joint also could suppress the progression of disease or not. In the present study, the effect on the synovial tissue of experimental arthritis was investigated using EC-SOD over-expressing transgenic mice. The over-expression of EC- SOD in joint tissue was confirmed by RT-PCR and immunohistochemistry. The degree of the inflammation in EC-SOD transgenic mice was suppressed in the collagen-induced arthritis model. In a cytokine assay, the production of pro-inflammatory cytokines such as, IL-1β, TNFα, and matrix metalloproteinases (MMPs) was decreased in fibroblast-like synoviocyte (FLS) but not in peripheral blood. Histological examination also showed repressed cartilage destruction and bone in EC-SOD transgenic mice. In conclusion, these data suggest that the over-expression of EC-SOD in FLS contributes to the activation of FLS and protection from joint destruction by depressing the production of the pro-inflammatory cytokines and MMPs. These results provide EC-SOD transgenic mice with a useful animal model for inflammatory arthritis research.     

A Rheumatoid arthritis study using Raman spectroscopy

Rheumatoid arthritis (RA) is characterized by chronic inflammation of the joints and can lead to a progressive destruction of articular cartilage and bone. In this study, the specificity and sensitivity of the RA diagnostic methods based on the receiver-operating characteristic curves for monitoring C-reactive protein (CRP) and rheumatoid factor (RF) were compared with the Raman spectroscopic diagnostic method developed in this work. Sera from 24 patients with rheumatoid arthritis and from 16 healthy individuals were analyzed to assess the biochemical composition and presence of inflammatory activity by the aforementioned methods. By comparing with the clinical results for specificity and sensitivity from the RF and CRP tests, we show that the overall results from the newly developed Raman method were significantly better, with a specificity of 96%, a sensitivity of 88%, and correctly identifying 92% of the RA and healthy individuals, while the RF test gave a specificity of 100% and a sensitivity of 54%, and the CRP test gave a specificity of 87% and a sensitivity of 58%, respectively.     

Calorimetric properties of degenerative human shoulder joint hyaline cartilage

The glenohumeral joint is not a classical mass bearing joint, the treatment of primary osteoarthritis is conservative. In all other cases, when the arthritis is associated with unbalance of the soft tissues, the treatment solution of this pathology is arthroplasty. The purpose of this study was to examine the altered metabolism in human degenerated cartilage of the shoulder joint. With the rise of temperature an endothermic reaction was observed in all cases. The use differential scanning calorimetry as part of thermal analysis was a reliable method for differentiating normal hyaline cartilage from degenerated samples.     

Identification of novel rheumatoid arthritis-associated MiRNA-204-5p from plasma exosomes

Rheumatoid arthritis (RA) is an autoimmune disease characterized by infiltration of immune cells in the synovium. However, the crosstalk of immune cells and synovial fibroblasts is still largely unknown. Here, global miRNA screening in plasma exosomes was carried out with a custom microarray (RA patients vs. healthy controls = 9:9). A total of 14 exosomal miRNAs were abnormally expressed in the RA patients. Then, downregulated expression of exosomal miR-204-5p was confirmed in both the replication (RA patients vs. healthy controls = 30:30) and validation groups (RA patients vs. healthy controls = 56:60). Similar to the findings obtained in humans, a decreased abundance of exosomal miR-204-5p was observed in mice with collagen-induced arthritis (CIA). Furthermore, Spearman correlation analysis indicated that plasma exosomal miR-204-5p expression was inversely correlated with disease parameters of RA patients, such as rheumatoid factor, erythrocyte sedimentation rate, and C-reactive protein. In vitro, our data showed that human T lymphocytes released exosomes containing large amounts of miR-204-5p, which can be transferred into synovial fibroblasts, inhibiting cell proliferation. Overexpression of miR-204-5p in synovial fibroblasts suppressed synovial fibroblast activation by targeting genes related to cell proliferation and invasion. In vivo assays found that administration of lentiviruses expressing miR-204-5p markedly alleviated the disease progression of the mice with CIA. Collectively, this study identified a novel RA-associated plasma exosomal miRNA-204-5p that mediates the communication between immune cells and synovial fibroblasts and can be used as a potential biomarker for RA diagnosis and treatment.Subject terms: Diagnostic markers, Non-coding RNAs     

Modeling EBV infection and pathogenesis in new-generation humanized mice

The development of highly immunodeficient mouse strains has allowed the reconstitution of functional human immune system components in mice. New-generation humanized mice generated in this manner have been extensively used for modeling viral infections that are exclusively human tropic. Epstein–Barr virus (EBV)-infected humanized mice reproduce cardinal features of EBV-associated B-cell lymphoproliferative disease and EBV-associated hemophagocytic lymphohistiocytosis (HLH). Erosive arthritis morphologically resembling rheumatoid arthritis (RA) has also been recapitulated in these mice. Low-dose EBV infection of humanized mice results in asymptomatic, persistent infection. Innate immune responses involving natural killer cells, EBV-specific adaptive T-cell responses restricted by human major histocompatibility and EBV-specific antibody responses are also elicited in humanized mice. EBV-associated T-/natural killer cell lymphoproliferative disease, by contrast, can be reproduced in a distinct mouse xenograft model. In this review, recent findings on the recapitulation of human EBV infection and pathogenesis in these mouse models, as well as their application to preclinical studies of experimental anti-EBV therapies, are described.     

Label-free Electrochemical Immunosensor for Sensitive Detection of Rheumatoid Arthritis Biomarker Anti-CCP-ab

The accurate and sensitive analysis of rheumatoid arthritis(RA) trace biomarkers is essential for early warning and diagnosis of RA, while anti-cyclic citrullinated peptide antibody (anti-CCP-ab) is a specific one. In this study, a simple label-free electrochemical immunosensor for the detection of anti-CCP-ab was constructed with nitrogen-doped graphene (N−G) and gold nanoparticles (AuNPs). The proposed non-enzyme immunosensor had exhibited high specificity, selectivity, and stability in the linear calibration curve range from 0.125∼2000 pg mL-1 and has been successfully used in the detection of anti-CCP-ab in human serum, providing a new idea for the early diagnosis of RA.     

Biological regulation on synovial fibroblast and the treatment of rheumatoid arthritis by nobiletin-loaded tetrahedral framework nucleic acids cargo tank

The hyperplasia and destruction of synovial tissue have an important impact on the development of rheumatoid arthritis (RA), the abnormal proliferation and migration of synovial fibroblast in synovial tissue is similar to tumor cells. Targeting anomalous synovial fibroblast and designing a high bioavailability nano drug delivery system can reduce the dosage for the treatment of rheumatoid arthritis and it is of great significance to reduce toxic and side effects and improve curative effect. In this experiment, the nobiletin-loaded tetrahedral framework nucleic acids cargo tank was established, carrying anti-inflammatory small molecule monomer drug nobiletin with minimal bioavailability. Both in vitro cell experiments and in vivo animal studies proved the nano cargo tank enhance the role of nobiletin in reducing the invasiveness of pathological synovial fibroblast and promote their apoptosis, effectively alleviate the disease development of rheumatoid arthritis.     

Novel calorimetric investigation of different degenerative disorders of the human hyaline cartilage

The purpose of this investigation was to further elucidate calorimetric properties of cartilage samples from femoral head necrosis and osteoarthritis from live surgeries. The natural course of this disease is one of steady progression with eventual collapse of the femoral head, followed by secondary osteoarthritis in the hip joint. All samples showed a clear denaturation peak on the calorimetric curve. Cartilage obtained from necrotic femoral head required the lowest amount of energy for decomposition. The use differential scanning calorimetry as part of thermal analysis was a reliable method for differentiating.     

Study of the human plasma proteome of rheumatoid arthritis

In this study, we report a combined proteomic and peptidomic analysis of human plasma from patients with rheumatoid arthritis (RA) and controls. We used molecular weight cut-off filters (MWCO: 10 kDa) to enrich low-molecular-weight (LMW) peptides from human plasma. The peptide fraction was analyzed without trypsin digestion by capillary reversed-phase high-performance liquid chromatography (HPLC) coupled to a linear ion trap–FT-MS system, which identified 771 unique peptides in the peptidome study (from 145 protein progenitors). An anti-albumin/anti-IgG column was used to remove albumin and immunoglobulin G (IgG) from the human plasma. After that, the albumin/IgG-depleted sample was fractionated into a bound fraction and an unbound fraction on a multi-lectin affinity column (M-LAC). LC–MS analysis of the corresponding tryptic digests identified 308 proteins using the M-LAC approach. Relative differences in the following protein classifications were observed in the RA human plasma samples compared with controls: structural proteins, immuno-related proteins, protease inhibitors, coagulation proteins, transport proteins and apolipoproteins. While some of these proteins/peptides have been previously reported to be associated with RA disease such as calgranulin A, B, C and C-reactive protein, several others were newly identified (such as thymosin β4, actin, tubulin, and vimentin), which may further the understanding of the disease pathogenesis. Moreover, we have found that the peptidomic and glycoproteomic approaches were complementary and allow a more complete picture of the human plasma proteome which can be valuable in studies of disease etiology.     

The influence of alpha1-acid glycoprotein on collagenase-3 activity in early rheumatoid arthritis

The concentration and glycosylation of alpha(1)-acid glycoprotein (AGP) alter significantly during inflammation. A definitive physiological role for AGP remains elusive and is the subject of extensive investigation. This study investigated the influence of AGP on the activity of collagenase-3, an important mediator of cartilage destruction in rheumatoid arthritis. AGP was isolated from normal and rheumatoid plasma. Fucosylation was determined by high pH anion-exchange chromatography; sialylation was assessed following enzymatic digest. Rheumatoid AGP displayed elevated fucosylation and sialylation compared with normal. The influence of each sample on collagenase-3 activity was measured fluorometrically. AGP influenced collagenase-3 catalysis and collagen binding, with catalytic activity correlating with fucosylation. Rheumatoid AGP exhibited less efficient inhibition than normal plasma AGP. It is hypothesized that AGP within rheumatoid synovial fluid may be inadequate to prevent excessive cartilage destruction and hence may exacerbate the disease process.     

Bioinorganic and medicinal chemistry: aspects of gold(I)-protein complexes

Gold(I)-based drugs have been used successfully for the treatment of rheumatoid arthritis (RA) for several years. Although the exact mechanism of action of these gold(I) drugs for RA has not been clearly established, the interaction of these compounds with mammalian enzymes has been extensively studied. In this paper, we describe the interaction of therapeutic gold(I) compounds with mammalian proteins that contain cysteine (Cys) and selenocysteine (Sec) residues. Owing to the higher affinity of gold(I) towards sulfur and selenium, gold(I) drugs rapidly react with the activated cysteine or selenocysteine residues of the enzymes to form protein-gold(I)-thiolate or protein-gold(I)-selenolate complexes. The formation of stable gold(I)-thiolate/selenolate complexes generally lead to inhibition of the enzyme activity. The gold-thiolate/selenolate complexes undergo extensive ligand exchange reactions with other nucleophiles and such ligand exchange reactions alter the inhibitory effects of gold(i) complexes. Therefore, the effect of gold(I) compounds on the enzymatic activity of cysteine- or selenocysteine-containing proteins may play important roles in RA. The interaction of gold(I) compounds with different enzymes and the biochemical mechanism underlying the inhibition of enzymatic activities may have broad medicinal implications for the treatment of RA.     

Anti-inflammatory activity of Gentiana striata Maxim

The anti-inflammatory activity and the mechanism of action of Gentiana striata Maxim. has been investigated. The most active phase, the ethyl acetate extract of Gentiana striata Maxim. (EGS), displayed potent inhibitory activity on feet oedema of rheumatoid arthritis (RA) inflicted rats. This anti-inflammatory activity might be partly based on the notable reduction of prostaglandin E2 (PGE?) and nitric oxide (NO) levels. Six further compounds isolated from EGS have previously been reported as having anti-inflammatory activity.     

Functional Analysis of Autoantibody Signatures in Rheumatoid Arthritis

For the identification of antigenic protein biomarkers for rheumatoid arthritis (RA), we conducted IgG profiling on high density protein microarrays. Plasma IgG of 96 human samples (healthy controls, osteoarthritis, seropositive and seronegative RA, n = 24 each) and time-series plasma of a pristane-induced arthritis (PIA) rat model (n = 24 total) were probed on AIT’s 16k protein microarray. To investigate the analogy of underlying disease pathways, differential reactivity analysis was conducted. A total of n = 602 differentially reactive antigens (DIRAGs) at a significance cutoff of p < 0.05 were identified between seropositive and seronegative RA for the human samples. Correlation with the clinical disease activity index revealed an inverse correlation of antibodies against self-proteins found in pathways relevant for antigen presentation and immune regulation. The PIA model showed n = 1291 significant DIRAGs within acute disease. Significant DIRAGs for (I) seropositive, (II) seronegative and (III) PIA were subjected to the Reactome pathway browser which also revealed pathways relevant for antigen presentation and immune regulation; of these, seven overlapping pathways had high significance. We therefore conclude that the PIA model reflects the biological similarities of the disease pathogenesis. Our data show that protein array analysis can elucidate biological differences and pathways relevant in disease as well be a useful additional layer of omics information.     

Biomolecular interaction monitoring of autoantibodies by scanning surface plasmon resonance microarray imaging

A new commercial surface plasmon resonance (SPR) imaging analysis system with a novel SPR dip angle scanning principle allows the measurement, without the need for labeling, of the exact SPR dip angle. With this system hundreds of biomolecular interactions can be monitored on microarrays simultaneously and with great precision. The potency of this system is demonstrated by automatically monitoring the interactions between citrullinated peptides and serum autoantibodies of 50 rheumatoid arthritis (RA) patients and 29 controls in a single step. The smallest antibody concentration that could be measured in this experimental setup was 0.5 pM.     

5-Methylcytosine content of DNA in blood, synovial mononuclear cells and synovial tissue from patients affected by autoimmune rheumatic diseases

The percentage of 5-methylcytosine (m5Cyt) has been determined in peripheral blood, synovial mononuclear cells and synovial tissue from patients affected by various rheumatic autoimmune diseases. The determination was performed by reversed-phase high-performance liquid chromatography. Fifteen controls were compared to twenty-one patients affected by rheumatoid arthritis and to nine patients affected by systemic lupus erythematosus. The mean percentage of m5Cyt in normal individuals was significantly higher than in the rheumatoid arthritis and systemic lupus erythematosus patients. In addition, patients with active disease showed lower values than patients in remission. This finding is in agreement with the hypothesis that DNA hypomethylation may play a role in the pathogenesis of the autoimmune diseases, resulting in altered oncogene expression. Therapy with cyclosporin A led to a decrease in the percentage of m5Cyt in three rheumatoid arthritis patients, but a rebound was observed when the cyclosporin A was suspended. The percentage of m5Cyt in the DNA of synovial tissue from four rheumatoid arthritis patients and five patients with osteoarthritis was similar; this observation confirms that, in addition to disease-specific and disease activity-specific variations, the percentage of m5Cyt may also show tissue-specific variations.     

Thermal analysis of the osteoarthritic human hyaline cartilage

Summary Arthritis of major joints, especially osteoarthritis of the knee is a very frequent disease of human beings mainly in the developed countries. The pathology of osteoarthritis has been subject of many publications before, using a wide spectrum of different methods to evaluate degenerative changes of hyaline cartilage. The authors examined osteoarthritic human knee joint hyaline cartilage with differential scanning calorimetry. The different stages of cartilage degeneration have been verified by histological examinations. The research group demonstrated thermal differences between various stages of osteoarthritis. Besides explaining possible causes for experienced thermodynamic effects, the authors reflect upon future research ways and the possibilities of applying the method in practice.     

Nanotechnology for diagnosis and therapy of rheumatoid arthritis: Evolution towards theranostic approaches

Rheumatoid arthritis(RA),as a chronic autoimmune disease,damages the bone and cartilage of patients,and even leads to disability.Therefore,the diagnosis and treatment of RA is particularly important.However,due to the complexity of RA,it is difficult to make effective early diagnosis of RA,which is detrimental to RA treatment.Besides,long-term intake of anti-RA drugs can also cause damage to patients' organs.The emergence of nanotechnology provides the new train of thoughts for the diagnosis and treatment of RA.And the combination of diagnosis and therapy is an ideal method to solve the problem of disease management of RA patients.In this review,we summarize the mechanism and microenvironment of RA,discuss the commonly used diagnostic techniques and therapeutic drugs for RA,and review their advantages and disadvantages.New nanotherapy strategies such as drug-carrying nanoparticles,PTT,PDT are listed,and their applications in RA treatment are also summarized.In addition,multimodal imaging,combined therapy and responsive diagnosis and treatment are also summarized as important contents.At last,we also review typical nanocarriers that can be used in the integration of diagnosis and therapy,and discussed their potential applications in RA theranostics.