Albumin/Globulin Ratio as Yin-Yang in Rheumatoid Arthritis and Its Correlation to Inflamm-Aging Cytokines.

PURPOSE: Inflamm-aging is a novel-concept in rheumatoid arthritis (RA) with accelerating aging process. We try to find a correlation between serum albumin/globulin (A/G) ratio and clinical biochemical parameters, incidence of aging-related diseases (ARDs) as well as inflammaging-related molecules. PATIENTS AND METHODS: Healthy controls (HC) and RA patients were compared with their clinical biochemical parameters including albumin and globulin levels, A/G ratio, and levels of serum lipids. Incidence of ARDs in RA was compared with A/G ratio, having a cut off value of 1.2. Expression levels of leptin and Trf2 genes in PBMCs, and inflammatory factors like IL-1ß, IL-6, IL-8 and TNF-ɑ between HC and RA patients were compared, and correlated with the A/G ratio. RESULTS: Compared to HC, RA patients had decreased levels of albumin, while globulin levels were found to be increased, which led to a significantly lower A/G ratio in RA patients. A/G ratio rather than ESR and CRP had significant correlation with dyslipidemia in RA patients. Patients with A/G <1.2 had a higher risk of ARDs than patients with A/G >1.2. The RR was 2.48 (95% CI: 1.79 to 3.64, p <0.0001). In addition, A/G ratio has positively correlated to leptin and Trf2 expression, while an inverse correlation was observed with the levels of inflamm-aging related cytokines like IL-6, IL-8 and TNF-ɑ. CONCLUSION: A decreased A/G ratio in RA patients has significantly correlated with dyslipidemia and ARDs, as well as inflammaging- related adipokine and pro-inflammatory cytokines. Thus, A/G ratio could be a reliable marker for evaluating the inflammaging process during clinical management in ARDs.

Potential treatment methods targeting 2019-nCoV infection.

The novel coronavirus, 2019-nCoV, has quickly spread across the world and pose serious threat to public health because it can infect people very easily. The major clinical symptoms of 2019-nCoV infection include fever, dry cough, myalgia, fatigue, and diarrhea. The 2019-nCoV belongs to the betacoronavirus family, and gene sequencing results demonstrate that it is a single-stranded RNA virus, closely related to Severe Acute Respiratory Syndrome CoV (SARS-CoV) and Middle East Respiratory Syndrome CoV (MERS-CoV). It has been observed that the virus invades human body mainly through binding to angiotensin-converting enzyme 2 (ACE2) receptors similar to SARS-CoV and the main protease (Mpro) acts as a critical protease for digesting the polyprotein into functional polypeptides during the replication and transcription process of 2019-nCoV. In this review, we summarized the real-time information of 2019-nCoV treatment methods and mainly focused on the chemical drugs including lopinavir/ritonavir, chloroquine, hydroxychloroquine, arbidol, remdesivir, favipiravir and other potential innovative active molecules. Their potential targets, activity, clinical status and side effects are described. In addition, Traditional Chinese Medicine (TCM), Convalescent plasma therapy (CPT) and biological reagents available, as well as the promising vaccine candidates against 2019-nCoV are also discussed.

Asperosaponin VI protects against bone destructions in collagen induced arthritis by inhibiting osteoclastogenesis.

BACKGROUND: Bone destructive diseases like rheumatoid arthritis (RA), osteoporosis and bone metastatic tumors are mainly mediated by over-activated osteoclasts. Asperosaponin VI (AVI), isolated from the rhizome of Dipsacus asper, belongs to triterpenoid saponins. It has multiple physiological activities but its effects on RA, especially on osteoclast differentiation and activation are still unclear. PURPOSE: Explore the protective role of AVI on collagen induced arthritis (CIA) in vivo and RANKL induced osteoclastogenesis in vitro. METHODS: The effects of AVI on cell viability and RANKL-induced osteoclastogenesis, actin ring formation, bone resorption activity as well as on osteoclast specific gene and protein expression were tested using bone marrow derived monocytes (BMMs). Paws from CIA mice were used for micro-CT, HE and TRAP staining, real-time PCR and western blot. Sera were used for cytokine analysis by ELISA. The signaling pathways were detected using western blot, real-time PCR and immunofluorescence assay. RESULTS: AVI significantly inhibited RANKL-induced osteoclast formation and bone resorption activity by suppressing the formation of actin ring. It also inhibited the expression of various osteoclatogenesis marker genes and signaling pathways. AVI protected arthritis in vivo by suppressing inflammation and bone loss. CONCLUSION: AVI exerts its anti-osteoclastogenic activity both in vitro and in vivo by inhibiting RANKL-induced osteoclast differentiation and function. Thus, our studies demonstrate a potential therapeutic role for AVI in preventing or inhibiting RANKL-mediated osteolytic bone diseases.

Therapeutic effects of artesunate on lupus-prone MRL/lpr mice are dependent on T follicular helper cell differentiation and activation of JAK2-STAT3 signaling pathway.

BACKGROUND: Anti-malarial drug artesunate (ART), a semi-synthetic derivative of artemisnin, has immunosuppressive effects on several autoimmune diseases, including Systemic lupus erythematosus (SLE), Rheumatoid arthritis (RA), and Colitis. However, molecular mechanisms of ART, especially on follicular helper T cells (Tfh), central players in SLE pathology, are far from clear. PURPOSE: The object for this work is to investigate the therapeutic effect of ART on lupus-prone MRL/lpr mice and its regulatory function on Tfh cells. STUDY DESIGN AND METHODS: MRL/lpr mice were used to explore therapeutic effects of ART on lupus-prone MRL/lpr mice and its regulatory functions on Tfh cells. Then, experiments of renal function were accomplished using the biochemical kits. Effects of ART on histopathology of kidneys, inflammatory factors and autoantibodies were examined using H&E staining, ELISA and real-time PCR. Flow cytometry and western blot analysis were used to examine effects of ART on Tfh differentiation and Jak2-Stat3 signaling pathway. RESULTS: Upon oral administration, ART significantly prolonged the survival of MRL/lpr mice, ameliorated the lupus nephritis symptoms, decreased the levels of anti-dsDNA antibodies deposited in the kidney, and the levels of pathogenic cytokines (IL-6, IFN-γ and IL-21). After ART treatment, T-cell compartment in the spleen of MRL/lpr mice was restored in terms of reduction in the number of Tfh cells and in the maintenance of the ratio of Tfr to follicular regulatory T cells (Tfh). In addition, ART has significantly inhibited the phosphorylation levels of Jak2 and Stat3 in the MRL/lpr mice. CONCLUSION: ART showed therapeutic effects on lupus-prone MRL/lpr mice by inhibiting the differentiation of Tfh cells as well as altering the activation status of Jak2-Stat3 signaling cascade.

Protective effects of Wang-Bi tablet on bone destruction in collagen-induced arthritis by regulating osteoclast-osteoblast functions.

ETHNOPHARMACOLOGICAL RELEVANCE: Wang-bi tablet (WB) consists of 17 traditional Chinese medicines and has been used for treating rheumatoid arthritis (RA) in China for many years, however, its pharmacologic mechanism is not clear. AIM OF STUDY: The aim of this study was to investigate the therapeutic effect of WB on collagen-induced mouse arthritis and explored the underlying mechanism. MATERIALS AND METHODS: DBA/1 mice were used to establish a type II collagen-induced arthritis (CIA) model. From the day of arthritis onset, mice were treated daily by gavage with either total glucosides of paeony (TGP, 0.37  g/kg/d) or WB at a lower (1.11  g/kg/d, WBL) or higher dose of (3.33  g/kg/d, WBH) for 8 weeks. The severity of arthritis, levels of cytokines and the activation of signaling pathways were determined. RESULTS: Our results revealed that WB treatment effectively alleviated inflammatory symptoms and prevented bone erosions and joint destructions. It obviously decreased the serum concentration of pro-inflammatory cytokines TNF-α, IL-6 and IL-17α, while increased the concentration of anti-inflammatory cytokine IL-10. Interestingly, the proportion of splenic Treg cells were increased significantly. In vitro experiments showed that WB inhibited the differentiation of osteoclasts. Consistently, the mRNA levels of tartrate-resistant acid phosphatase (TRAP) and cathepsin K (CtsK), and the activation of NF-κB and JAK-STAT3 signaling pathways in the paws of CIA mice were inhibited by WB treatment. On the other hand, up-regulation of osteogenic genes Runx2, Osterix mRNA, and activation of Wnt/ß-catenin signaling pathway along with a decreased receptor activator of nuclear factor κB ligand (RANKL) expression were found in WB treated mice. CONCLUSION: Our results suggest that the therapeutic effect of Wang-bi tablet could be attributed to its inhibitory activity on NF-κB and STAT3 signaling pathway-mediated osteoclast differentiation, and its enhancement on Wnt/ß-catenin signaling pathway-mediated osteoblast functions.

Tatanan A from the Acorus calamus L. root inhibited dengue virus proliferation and infections.

BACKGROUND: Acorus calamus l. (Acoraceae) is a well-known traditional Chinese medicinal plant, whose root are historically mainly used to treat neurodegenerative diseases, and for cholera treatment. This datum strongly indicates the antimicrobial activity of A. calamus. PURPOSE: Our goal is to find the active constituents of A. calamus to treat dengue virus (DENV) infections, and to study the effects and mechanisms of these active substances. METHODS: The root of A. calamus was extracted by ethanol. Mosquito larva C6/36 cells were used for DENV2 replication and transfection host. Mouse kidney fibroblast cells (BHK-21) were used as a host cell to study the infection ability of the virus. DENV2-induced cytopathic effect (CPE) and plaque assay were used to evaluate the inhibitory effect of A. calamus extracts on DENV2 infectivity inhibition. The levels of E and NS1 protein expression were measured by real-time PCR and western blot assays. RESULTS: 12 compounds were isolated from ethanol extract of A. calamus root, tatanan A showed the best anti-DENV ability among these 12 compounds, which significantly alleviated DENV2-induced CPE and cytotoxicity effects, with an EC50 of 3.9 µM. In addition, RNA replication assay further confirmed the antivirus ability of tatanan A. Time-addition assay showed that tatanan A affected the early stage of viral RNA replication, which in turn inhibited mRNA and protein levels of DENV2. CONCLUSIONS: These results demonstrated the anti-DENV2 effect of tatanan A, in inhibiting DENV2 RNA replication and infections. In summary, tatanan A was found to be a novel natural DENV inhibitor and a potential candidate for the treatment of DENV infectious disease.

Targeting IgG in Arthritis: Disease Pathways and Therapeutic Avenues.

Rheumatoid arthritis (RA) is a polygenic and multifactorial syndrome. Many complex immunological and genetic interactions are involved in the final outcome of the clinical disease. Autoantibodies (rheumatoid factors, anti-citrullinated peptide/protein antibodies) are present in RA patients' sera for a long time before the onset of clinical disease. Prior to arthritis onset, in the autoantibody response, epitope spreading, avidity maturation, and changes towards a pro-inflammatory Fc glycosylation phenotype occurs. Genetic association of epitope specific autoantibody responses and the induction of inflammation dependent and independent changes in the cartilage by pathogenic autoantibodies emphasize the crucial contribution of antibody-initiated inflammation in RA development. Targeting IgG by glyco-engineering, bacterial enzymes to specifically cleave IgG/alter N-linked Fc-glycans at Asn 297 or blocking the downstream effector pathways offers new avenues to develop novel therapeutics for arthritis treatment.

Inhibition of dengue viral infection by diasarone-I is associated with 2'O methyltransferase of NS5.

Dengue virus (DENV) is the most prevalent mosquito borne viral pathogen worldwide. However, antiviral drugs against this infection are not available. To identify novel anti-DENV compound from traditional Chinese medicine, we discovered the ethanol extract of Acorus tatarinowii Schott containing potent anti-DENV activity and diasarone-I was isolated from this extract. Diasarone-I has antiviral effect with half maximal effective concentration (EC50) of 4.5µM and half maximal cytotoxicity concentration (CC50) of >80µM. Time of drug addition assay suggested that this compound inhibited at RNA replication step in the DENV life cycle. Further, in silico analysis indicated that diasarone-I might act as an inhibitor of 2'O Methyltransferase of NS5. Diasarone-I has also decreased the DENV2-induced STAT1 phosphorylation and ISGs. In summary, we suggest that diasarone-I may be a 2'O Methyltransferase inhibitor and might serve as a potential candidate for the treatment of DENV2 infections.

System A amino acid transporters regulate glutamine uptake and attenuate antibody-mediated arthritis.

Proliferation of rapidly dividing bone marrow-derived cells is strongly dependent on the availability of free glutamine, whose uptake is mediated through different amino acid transporters. The sodium-coupled neutral amino acid transporter (SNAT) family was previously reported to be associated with the development of collagen-induced arthritis in mice. Here, we tested the hypothesis whether impairment of SNAT proteins influences immune cell function and in turn alters arthritis development. The 2-(methylamino)isobutyric acid (MeAIB), a SNAT-specific substrate, was used to modulate the function of SNAT proteins. We demonstrate that glutamine uptake by murine naive lymphocytes, and consequent cell proliferation, is strongly associated with system A transporters. Physiological impairment of SNAT proteins reduced the antibody-initiated effector phase of arthritis, mainly by affecting the levels of circulating monocytes and neutrophils. MeAIB was also shown to affect the proliferation of immortalized cells, through trans-inhibition of SNAT proteins. Based on our observations, we conclude that SNAT proteins regulate the initial stages of lymphocyte activation by regulating glutamine uptake, and that the effector phase of arthritis can be affected by non-metabolized SNAT substrates. Most probably, metabolically active cells within both the adaptive and the innate immune systems are regulated by SNAT proteins and play a role in modifying arthritis development.

A dominant suppressive MHC class II haplotype interacting with autosomal genes controls autoantibody production and chronicity of arthritis.

OBJECTIVE: To investigate the genetic control of chronic arthritis and collagen epitope specific antibody responses in an experimental model for rheumatoid arthritis. METHODS: The chronic collagen induced arthritis (CCIA) model was used, induced with collagen type II (CII) in mineral oil lacking mycobacterium in BALB/c (n=24), B10.Q (n=44), (BALB/c × B10.Q) F1 (n=85) and B10.Q × (BALB/c × B10.Q) N2 (n=684) mice. Genome-wide genotyping for 190 N2 mice was performed with extreme phenotypes: chronic arthritis that persisted for 4 months or non-affected. Statistical and linkage analysis were performed with R/qtl software using arthritis and serum subphenotypes. RESULTS: (BALB/c × B10.Q) F1 mice were highly prone to develop a chronic relapsing arthritis (66%), whereas both parental strains were relatively resistant: BALB/c (H-2(d); 0%) and B10.Q (H-2(q); 4.5%). CCIA experiments were performed on 684 mice backcrossed to B10.Q; 38% of the mice developed arthritis and more than half of them developed chronic arthritis phenotype. Genome-wide genotyping revealed mainly the major histocompatibility complex (MHC) locus that had an independent and dominant influence on the chronicity. Interestingly, the H2(d) allele had a dominant suppressive effect. This effect overrode the role of other loci as interaction analysis, after conditioning MHC, revealed additional loci, controlling arthritis and autoantibody phenotypes. CONCLUSIONS: A dominant negative influence of specific MHC haplotype (H2(d)) on CCIA was identified. Further, loci controlling the autoantibody response to different CII epitopes were also identified, and it has been shown that these are dependent on MHC and non-MHC genes.

Rabeximod reduces arthritis severity in mice by decreasing activation of inflammatory cells.

OBJECTIVES: The novel small molecule 9-chloro-2,3-dimethyl-6-(N,N-dimethylaminoethylamino-2-oxoethyl)-6H-indolo[2,3-b] quinoxaline (Rabeximod) reduces severity of arthritis in rodent models of rheumatoid arthritis (RA) and multiple sclerosis (MS). This study aimed to investigate the cellular target in vivo. METHODS: Collagen antibody-induced arthritis (CAIA) is induced by monoclonal collagen type II antibodies and enhanced by lipopolysaccharide. It was investigated how and when Rabeximod operates on inflammatory cells after stimulation of either Toll-like receptor (TLR)4 (lipopolysaccharide) or TLR2 (lipomannan) in mice lacking functional signalling through TLR4 due to a spontaneous deletion of the Tlr4 gene. RESULTS: Rabeximod efficiently prevented arthritis during the time window when TLR2 or TLR4 ligands activate inflammatory macrophages. The effect operated downstream of TLR activation as Rabeximod was highly therapeutic in CAIA enhanced through TLR2 stimuli in TLR4 deficient mice. In addition, it was found that the arthritis ameliorating effect of Rabeximod was time dependent, since inhibition of tumour necrosis factor alpha production from macrophages in vitro was more pronounced if administered close to stimulation. CONCLUSIONS: Rabeximod suppresses arthritis by preventing activation of inflammatory cells, most likely macrophages, in a time dependent fashion, downstream of TLR2 and TLR4 stimulation.

Monoclonal antibody production using a new supermacroporous cryogel bioreactor.

A supermacroporous cryogel bioreactor has been developed to culture hybridoma cells for long-term continuous production of monoclonal antibodies (mAb). Hybridoma clone M2139, secreting antibodies against J1 epitope (GERGAAGIAGPK; amino acids, 551-564) of collagen type II, are immobilized in the porous bed matrix of a cryogel column (10 mL bed volume). The cells got attached to the matrix within 48 h after inoculation and grew as a confluent sheet inside the cryogel matrix. Cells were in the lag phase for 15 days and secreted mAb into the circulation medium. Glucose consumption and lactic acid production were also monitored, and during the exponential phase (approximately 20 days), the hybridoma cell line consumed 0.75 mM day-1 glucose, produced 2.48 mM day-1 lactic acid, and produced 6.5 microg mL-1 day-1 mAb during the exponential phase. The mAb concentration reached 130 microg mL-1 after continuous run of the cryogel column for 36 days. The yield of the mAb after purification was 67.5 mg L-1, which was three times greater than the mAb yield obtained from T-flask batch cultivation. Even after the exchange of medium reservoir, cells in the cryogel column were still active and had relatively stable mAb production for an extended period of time. The bioreactor was operated continuously for 55 days without any contamination. The results from ELISA as well as arthritis experiments demonstrate that the antibodies secreted by cells grown on the cryogel column did not differ from antibodies purified from the cells grown in commercial CL-1000 culture flasks. Thus, supermacroporous cryogels can be useful as a supporting material for productive hybridoma cell culture. Cells were found to be viable inside the porous matrix of the cryogel during the study period and secreted antibodies continuously. The antibodies thus obtained from the cryogel reactor were found to be functionally active in vivo, as demonstrated by their capacity to induce arthritis in mice.

Ethanol prevents development of destructive arthritis.

Environmental factors are thought to play a major role in the development of rheumatoid arthritis. Because the use of ethanol is widespread, we assessed the role of ethanol intake on the propensity to develop chronic arthritis. Collagen type II-immunized mice were given water or water containing 10% (vol/vol) ethanol or its metabolite acetaldehyde. Their development of arthritis was assessed, as well as the impact of ethanol on leukocyte migration and activation of intracellular transcription factors. Mice exposed daily to this dose of ethanol did not display any liver toxicity, and the development of erosive arthritis was almost totally abrogated. In contrast, the antibody-mediated effector phase of collagen-induced arthritis was not influenced by ethanol exposure. Also, the major ethanol metabolite, acetaldehyde, prevented the development of arthritis. This antiinflammatory and antidestructive property of ethanol was mediated by (i) down-regulation of leukocyte migration and (ii) up-regulation of testosterone secretion, with the latter leading to decreased NF-kappaB activation. We conclude that low but persistent ethanol consumption delays the onset and halts the progression of collagen-induced arthritis by interaction with innate immune responsiveness.