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Objectives: Oral immune tolerance (OT) is a complex process with unknown genetic regulation. Our aim is to explore possible genetic control of OT in patients with rheumatoid arthritis (RA)., Methods: RA patients with increased interferon γ production invitro when their isolated peripheral blood mononuclear cells (PBMC) were cultured with type II bovine collagen α1 chain [α1 (II)] were enrolled in this study and were randomly assigned to the "Low dose" type II collagen (CII) group (30 µg/day for 10 weeks, followed by 50 µg/day for 10 weeks, followed by 70 µg/day for 10 weeks) or "High dose" CII group (90 µg/day for 10 weeks, followed by 110 µg/day for 10 weeks, followed by 130 µg/day for 10 weeks). Heparinized blood was obtained at baseline and after each of the 10 weeks treatment for analysis of the invitro production of IFNγ by their PBMC stimulated by α1(II) . Single nucleotide polymorphism (SNP) analysis of the responders and non-responders to oral CII was conducted using GeneChip Mapping 10 K 2.0 Array., Results: The SNP A-15,737 was found to associate with the ability of CII to suppress IFNγ production by α1(CII)-stimulated RA PBMC. The potential for SNP A-15,737 to associate with the OT response for patients with another autoimmune disease [OT induced by oral type I bovine collagen (CI) in patients with diffuse cutaneous systemic sclersodid (dsSSc)] was also explored., Conclusions: The ROT1 region plays a role in the control of IFNγ production after oral dosing of auto-antigens, thereby determining if oral tolerance to that antigen will develop., Competing Interests: Declaration of competing interests Drs. Arnold E. Postlethwaite and Weikuan Gu are listed on a US Patent #10,450,610B2 owned by University of Tennessee Research Foundation. This work was supported by grant AR45252 (AEP) and AR9–2242 (AEP from NIAMS/NIH and grants from Department of Veterans Affairs (AEP, WG). The authors thank Ms. Angela Cody for excellent help in drafting this manuscript. This publication is dedicated to honoring Dr. Arnold E. Postlethwaite for his more 50 years of dedicated research and contribution on the immune disease and the field of biomedical sciences., (Copyright © 2024 Southern Society for Clinical Investigation. Published by Elsevier Inc. All rights reserved.)

Background: Non-pharmacological treatments based on collagen as a dietary supplement are emerging as a new area of interest to support preventive or therapeutic effects in patients with osteoarthritis (OA)., Aim: In a multicenter, prospective, double-blind, placebo-controlled, randomized study, to evaluate the effectiveness and safety of the use of the Artneo complex containing undenatured chicken collagen type II in patients with OA of the knee joints., Materials and Methods: The study enrolled 212 outpatients from 12 centers in the Russian Federation with knee OA, stages II and III according to the Kellgren-Lawrence classification. The participants included 171 women (80.7%) and 41 men (19.3%), with an average age of 60.2±9.0 years (range: 40 to 75 years). The study population was randomly allocated in equal proportions into two groups using an interactive web response system (IWRS). Group 1 (Artneo) consisted of 106 patients who took one capsule of the drug once daily for 180 days. Group 2 (Placebo) also had 106 patients, with the dosage form and regimen identical to Group 1. During the treatment period, the following outcomes were assessed: WOMAC index, KOOS, pain according to VAS, quality of life using the EQ-5D questionnaire, and the need for NSAIDs. All patients underwent a clinical blood test, general urine analysis, biochemical blood test, and ultrasound examination of the affected knee joint., Results: In a prospective, double-blind, placebo-controlled, randomized study, it was demonstrated that the Artneo combination, containing undenatured chicken collagen type II, has a positive effect on all clinical manifestations of OA: it effectively reduces pain, stiffness, and improves the functional state of joints and quality of life. It has a good safety profile and is superior to placebo in all parameters studied., Conclusion: The results of the study confirm the good effectiveness and safety of the Artneo combination in patients with OA of the knee joints.

Aim: To evaluate the efficacy of Artneo (AN) in comparison with a combination of glucosamine hydrochloride and chondroitin sulfate (GC) in patients with osteoarthritis (OA) of the knee joint (KJ)., Materials and Methods: 70 patients with stages I-III of primary knee OA were randomized into 2 groups. Participants in the 1st ( n =35) took AN 1 caps/day, in the 2nd ( n =35) GC according to the standard regimen. After 7, 30, 90, 180 days, the Lequesne index (severity of OA), pain when moving according to VAS, WOMAC score were assessed, after 1, 3, 6 months - quality of life SF-36 and morning stiffness, after 6 months - MRI with T2 mapping, laboratory safety indicators., Results: Over the course of 6 months of use, an improvement in the WOMAC index and a decrease in pain were observed without intergroup differences, and a greater decrease in stiffness in the AN group. After 3 months, the severity of OA decreased from moderate to mild in the AN group and was significantly lower compared to the GC group; quality of life (physical component of SF-36) was higher in the AN group. After 6 months, there was an improvement in cartilage ultrastructure (T2 relaxation time) in both groups and a more pronounced reduction of the synovitis area (MRI) in the AN group (2.95 and 1.37 times in the AN and GC group, respectively). There were no clinically significant adverse reactions observed in both groups., Conclusion: The use of AN in patients with stage I-III primary knee OA was not inferior in efficacy to the combination of GC. Further studies with greater statistical power (sample size) and follow-up period are warranted including in real clinical practice.

Chronic inflammation causes dysregulated expression of microRNAs. Aberrant microRNA expression is associated with endothelial dysfunction. In this study we determined whether TNF-α inhibition impacted the expression of miRNA-146a-5p and miRNA-155-5p, and whether changes in the expression of these miRNAs were related to inflammation-induced changes in endothelial function in collagen-induced arthritis (CIA). Sixty-four Sprague-Dawley rats were divided into control (n = 24), CIA (n = 24) and CIA+etanercept (n = 16) groups. CIA and CIA+etanercept groups were immunized with bovine type-II collagen, emulsified in incomplete Freund's adjuvant. Upon signs of arthritis, the CIA+etanercept group received 10mg/kg of etanercept intraperitoneally, every three days. After six weeks of treatment, mesenteric artery vascular reactivity was assessed using wire-myography. Serum concentrations of TNF-α, C-reactive protein, interleukin-6, vascular adhesion molecule-1 (VCAM-1) and pentraxin-3 (PTX-3) were measured by ELISA. Relative expression of circulating miRNA-146a-5p and miRNA-155-5p were determined using RT-qPCR. Compared to controls, circulating miRNA-155-5p, VCAM-1 and PTX-3 concentrations were increased, and vessel relaxation was impaired in the CIA (all p<0.05), but not in the CIA+etanercept (all p<0.05) groups. The CIA group had greater miRNA-146a-5p expression compared to the CIA+etanercept group (p = 0.005). Independent of blood pressure, miRNA-146a-5p expression was associated with increased PTX-3 concentrations (p = 0.03), while miRNA-155-5p expression was associated with impaired vessel relaxation (p = 0.01). In conclusion, blocking circulating TNF-α impacted systemic inflammation-induced increased expression of miRNA-146a-5p and miRNA-155-5p, which were associated with endothelial inflammation and impaired endothelial dependent vasorelaxation, respectively., Competing Interests: The authors have declared that no competing interests exist.

Tolerizing DNA vaccines encoding key autoantigens are one of emerging strategies for the treatment of rheumatoid arthritis (RA). Among these vaccines, the most representative is pcDNA-CCOL2A1, an antigen-specific DNA vaccine encoding chicken type Ⅱ collagen (CCⅡ) with significant therapeutic and prophylactic efficacy in collagen-induced arthritis (CIA) rat models. We compared the in situ expression levels of CCOL2A1-mRNA and CCⅡ protein and the protective efficacies against CIA after a single dose (300 μg/kg) of this vaccine via intramuscular (IM), subcutaneous (SC) and intravenous (IV) vaccinations. The IM vaccination routes resulted in good protective efficacies in terms of decreasing CIA incidence and severity and significantly improved radiographic and histopathologic findings and scores of joints. Furthermore, IM, SC, and IV vaccinations markedly decreased serum levels of anti-type Ⅱ collagen (CⅡ) IgG antibodies, but only IM vaccination significantly reduced serum levels of rheumatoid factor (RF) and anti-cyclic citrullinated peptide (anti-CCP) antibody. The vaccine exhibited a continuous CCOL2A1-mRNA expression in the tail and abdominal subcutaneous tissue injection sites, but no CCOL2A1-mRNA signal was observed in muscle. Strikingly, CCⅡ protein expression levels at the three injection sites were comparable with minimal variation. IM administration may be considered the preferred route for RA treatment in clinical practice., (Copyright © 2021 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

The current work aimed to examine the properties of oral supplementation of niacinamide and undenatured type II collagen (UCII) on the inflammation and joint pain behavior of rats with osteoarthritis (OA). Forty-nine Wistar rats were allocated into seven groups; control (no MIA), MIA as a non-supplemental group with monosodium iodoacetate (MIA)-induced knee osteoarthritis, MIA + undenatured type II collagen (UCII) at 4 mg/kg BW, MIA + Niacinamide at 40 mg/kg BW (NA40), MIA + Niacinamide at 200 mg/kg BW (NA200), MIA + UCII + NA40 and MIA + UCII + NA200. Serum IL-1β, IL-6, TNF-α, COMP, and CRP increased in rats with OA and decreased in UCII and NA groups (p < 0.05). Rats with osteoarthritis had greater serum MDA and knee joint MMP-3, NF-κB, and TGβ protein levels and decreased in treated groups with UCII and NA (p < 0.05). The rats with OA also bore elevated joint diameters with joint pain behavior measured as decreased the stride lengths, the paw areas, and the paw widths, and increased the Kellgren-Lawrence and the Mankin scores (p < 0.05) and decreased in UCII treated groups. These results suggest the combinations with the UCII + NA supplementation as being most effective and reduce the inflammation responses for most OA symptoms in rats., (© 2021. The Author(s).)

Joint pain and disease affects more than one in four adults in the United States. We conducted a double-blind, randomized, placebo-controlled trial to investigate the efficacy of a hydrolyzed chicken collagen type II (HCII) supplement in reducing joint-related discomfort such as pain and stiffness, and in improving mobility. We enrolled adults aged 40-65 (65.5% were women) who had joint discomfort, but had no co-morbidities, and who were not taking pain medications. The participants were randomized to receive either the HCII supplement ( n = 47) or a placebo ( n = 43) for eight weeks. At the baseline, and at week 4 and week 8, we administered the Western Ontario and McMaster Universities Arthritis Index (WOMAC) survey with three additional wrist-related questions and the Visual Analog Scale for assessments of joint-related symptoms. In the WOMAC stiffness and physical activity domains and in the overall WOMAC score, the HCII group had a significant reduction in joint-related discomforts compared with the placebo group. For example, at week 4, the HCII group had a 36.9% reduction in the overall WOMAC score, compared with a 14.3% reduction in the placebo group ( p = 0.027). This HCII product is effective in reducing joint pain and stiffness and in improving joint function among otherwise healthy adults.

Osteoarthritis (OA) has been scarcely researched among patients with diabetes mellitus. This study aims to confirm the preventive and therapeutic effects of undenatured type II collagen (UC II) on OA in aging db/db mice and in patients with T2DM. Firstly, aging db/db mice were randomly assigned to three groups: the UC II intervention (UC II) group, old model (OM) group and positive control group. Meanwhile db/m mice and young db/db mice were used as the normal control and young control groups, respectively. Secondly, fifty-five T2DM patients diagnosed with knee OA were randomly assigned to two groups: UC-II and placebo control groups. After a three-month intervention in both mice and T2DM patients, the subjects' gait and physical activities were assessed and the serum biomarkers including inflammatory cytokines, oxidative stress factors and matrix metalloproteinases (MMPs) were measured. Compared with the OM group mice, those in the UC II group showed a significantly greater superiority in terms of motor functions including the movement trajectories area (163.25 ± 20.3 vs. 78.52 ± 20.14 cm 2 ), the tremor index (0.42 vs. 1.23), standing time (left hind: 0.089 ± 0.03 vs. 0.136 ± 0.04 s), swing (right front: 0.12 ± 0.02 vs. 0.216 ± 0.02 s), stride length (right hind: 7.2 ± 0.9 vs. 5.7 ± 1.1 cm), step cycle (right hind: 0.252 ± 0.05 vs. 0.478 ± 0.11 s) and cadence (14.12 ± 2.7 vs. 7.35 ± 4.4 steps per s). In addition, the levels of IL-4, IL-10, CTX- II and TGF-β in the UC II group were 1.74, 2.23, 1.67 and 1.84 times higher than those in the OM group, respectively, while the levels of MMP-3 and MMP-13 in the UC II group were half those in the OM group. Correspondingly, UC II intervention significantly decreased the scores of pain, stiffness and physical function (p < 0.05), whereas the 6 MWT and total MET distances in the UC II group increased remarkably (p < 0.05). After a three-month period of intervention, the varus angle significantly decreased from 4.6 ± 2.0° to 3.0 ± 1.4° and the knee flexion range obviously increased from 57.9 ± 14.0° to 66.9 ± 10.4°. Importantly, the declining trend in the levels of hs-CRP and MDA and the incremental trend in the SOD level were consistent in the db/db mice and OA patients following UC II administration.

The development of collagen type II (CII)-induced arthritis (CIA), a model of rheumatoid arthritis, in rats housed in cages with bedding composed of Celliant fibres containing ceramic particles, which absorb body heat and re-emit the energy back to the body in the form of infrared radiation (+IRF rats), and those housed in cages with standard wooden shaving bedding (-IRF control rats) was examined. The appearance of the first signs of CIA was postponed, while the disease was milder (judging by the arthritic score, paw volume, and burrowing behaviour) in +IRF compared with -IRF rats. This correlated with a lower magnitude of serum anti-CII IgG antibody levels in +IRF rats, and lower production level of IL-17, the Th17 signature cytokine, in cultures of their paws. This could be partly ascribed to impaired migration of antigen-loaded CD11b + dendritic cells and their positioning within lymph nodes in +IRF rats reflecting diminished lymph node expression of CCL19 /CCL21. Additionally, as confirmed in rats with carrageenan-induced paw inflammation (CIPI), the infrared radiation from Celliant fibres, independently from immunomodulatory effects, exerted anti-inflammatory effects (judging by a shift in pro-inflammatory mediator to anti-inflammatory/immunoregulatory mediator ratio towards the latter in paw cultures) and ameliorated burrowing behaviour in CIA rats.

The aim of this study was to evaluate the effect of an oral preparation containing a naturally occurring matrix of hydrolyzed collagen type II, chondroitin sulfate (CS), and hyaluronic acid (HA), and bioactive oligopeptides of natural hydrolyzed keratin (K) in patients affected by knee OA through the evaluation of synovial fluid (SF) and clinical changes before and after treatment. Thirty patients with knee OA and swollen joint were included in the study and submitted to arthrocentesis. Patients were randomized in two groups: 1) the treatment group (N.15) took a dietary supplement containing 120 mg HA, 240 mg CS and 300 mg K once a day for 4 weeks; 2) the control group (N.15) was only submitted to arthrocentesis. Patient symptoms were evaluated at the beginning and at the end of the study by the WOMAC self-assessment questionnaire, the Lequesne algofunctional index, and the VAS forms. SF changes were evaluated by measuring local inflammatory indices, cytokines IL-1β, IL-8, IL-6, IL-10 and GM-CSF. The group of patients treated with the oral supplement showed an improvement in the clinical indices WOMAC (p<0.01), Lequesne (p=0.014) and VAS pain (p<0.01). On the contrary, no significant changes were found in the control group. The SF collected from the treated group showed a reduction of IL-8 (p=0.015), IL-6 and IL-10 levels, while no changes in cytokines were observed in the control group. This pilot study suggests that an oral administration of a preparation containing a combination of HA, CS and K can improve some clinical parameters and affect cytokine concentrations in SF in patients with knee OA.

To compare human adipose tissue mesenchymal stem cells (AT-MSCs) and etanercept as immunomodulatory agents for collagen-induced arthritis (CIA). CIA was induced by rats' immunization with collagen type II (CII) in complete Freund's adjuvant in days 0 and 7. Before the onset of CIA, prevention group received five doses of AT-MSCS intraperitoneally. After establishment of arthritis, rats received either five doses of AT-MSCs or phosphate-buffered saline (PBS) intraperitoneally or six doses of etanercept subcutaneously. Clinical and histopathological evaluation were performed in all groups; serum levels of tumor necrosis factor-α (TNF-α), interleukin-10 (IL-10), and anti-collagen II were assessed by enzyme-linked immunosorbent assay (ELISA). A total percent of autoreactive T and regulatory T (Treg) cells were quantified using spleen immune histochemical analysis. AT-MSCs were able to delay the onset of CIA, suppress the ongoing clinical and histopathological signs, decrease serum levels of TNF-α and anti-collagen type II, and downregulate the autoreactive T cells as etanercept. AT-MSCs were more potent in Treg cells upregulation, producing high serum levels of IL10. AT-MSCs might have a therapeutic effect in CIA via their potency in immune cell education, representing an effective new promising approach in rheumatoid arthritis in human.

Securidaca inappendiculata is a xanthone rich medicinal plant that has been used in the treatment of inflammation and autoimmune diseases like rheumatoid arthritis (RA) for centuries; however, the material base and mechanism of action responsible for its anti-arthritis effect still remains elusive. The objective of this study is to evaluate the therapeutic effects of xanthone-enriched extract of the plant against collagen-induced arthritis (CIA) in rats and explore the underlying mechanisms. The xanthone-deprived fraction (XDF) and xanthone-rich fraction (XRF) were obtained by using a resin adsorption coupled with acid-base treatment method, and their chemical composition difference was characterized by UPLC-MS/MS analysis. Effects of the two on CIA were analyzed using radiographic, histological, and immunohistochemical analyses. The results indicated that XRF alleviated joint structures destructions with the higher efficacy than XDF, and decreased levels of TNF-α, IL-6, and anti-cyclic citrullinated peptide antibody in CIA rats significantly. Furthermore, XRF inhibited nicotinamide phosphoribosyl transferase (NAMPT) mediated fat biosynthesis and utilization indicated by clinical evidences and metabonomics analysis, which thereby disrupted energy-metabolism feedback. In addition, Toll-like Receptor 4 and High Mobility Group Protein 1 expressions were downregulated in XRF-treated CIA rats. Collective evidences suggest NAMPT could be an ideal target for RA treatments and reveal a novel antirheumatic mechanism of S. inappendiculata by regulating NAMPT controlled fat metabolism., (© 2020 The Authors. The Kaohsiung Journal of Medical Sciences published by John Wiley & Sons Australia on behalf of Kaohsiung Medical University.)

Rheumatoid arthritis is a chronic systemic autoimmune disease that causes joint swelling and cartilage damage. The objective of the present work was to develop a temperature-sensitive hydrogel (D-NGel) containing nanoparticles (D-NPs), which could simultaneously deliver combination indomethacin and methotrexate. D-NPs were formed by multiple non-covalent interactions between PEI-SS and the carboxyl-containing hydrophobic small molecule drugs IND and MTX, which were then loaded into a temperature-sensitive hydrogel matrix. The T sol/gel of the temperature-sensitive hydrogel matrix composed of 27% F127 and 10% F68 was 33 °C and the gelation time was less than 15 s. The resultant D-NGel was injected into the articular cavity of collagen-induced arthritis rats and quickly transformed in situ into gels which slowly released drug in the joint fluid for up to 72 h. The D-NGel effectively reduced joint swelling, bone erosion and expression of inflammatory cytokines in the ankle fluid and knee joint fluid. In addition, liver and kidney function tests and histopathological examination indicated there was a good biological safety for D-NGel. In conclusion, this work has demonstrated the great potential of the D-NGel for sustained co-delivery of IND and MTX for the synergistic treatment of rheumatoid arthritis, treating both the symptoms and the root causes of rheumatoid arthritis.

Rheumatoid arthritis (RA) has a negative impact on muscle mass, and reduces patient's mobility and autonomy. Furthermore, RA is associated with metabolic comorbidities, notably in lipid homeostasis by unknown mechanisms. To understand the links between the loss in muscle mass and the metabolic abnormalities, arthritis was induced in male Sprague Dawley rats (n = 11) using the collagen-induced arthritis model. Rats immunized with bovine type II collagen were compared to a control group of animals (n = 11) injected with acetic acid and complete Freund's adjuvant. The clinical severity of the ensuing arthritis was evaluated weekly by a semi-quantitative score. Skeletal muscles from the hind limb were used for the histological analysis and exploration of mitochondrial activity, lipid accumulation, metabolism and regenerative capacities. A significant atrophy in tibialis anterior muscle fibers was observed in the arthritic rats despite a non-significant decrease in the weight of the muscles. Despite moderate inflammation, accumulation of triglycerides (P < 0.05), reduced mitochondrial DNA copy number (P < 0.05) and non-significant dysfunction in mitochondrial cytochrome c oxidase activity were found in the gastrocnemius muscle. Concomitantly, our results suggested an activation of the muscle specific E3 ubiquitin ligases MuRF-1 and MAFbx. Finally, the adipose tissue from the arthritic rats exhibited decreased PPARγ mRNA suggesting reduced adipogenic capacities. In conclusion, the reduced adipose tissue adipogenic capacity and skeletal muscle mitochondrial capacity are probably involved in the activation of protein catabolism, inhibition of myogenesis, accumulation of lipids and fiber atrophy in the skeletal muscle during RA., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Huai Qi Huang (HQH) exerts great effects in clinic, such as anti-inflammation, immune-regulation, anti-cancer, and so on. However, the mechanism by which HQH protects juvenile idiopathic arthritis (JIA) is obscure. Thus, we explored deeply the protective mechanisms in juvenile collagen-induced arthritis (CIA) rat model. Pyroptosis is Gasdermin D (GSDMD)-dependent programmed cell death, involved in many diseases, such as sepsis. We investigated whether GSDMD-induced pyroptosis take part in mechanisms of juvenile CIA arthritis. Juvenile Wistar rats (3-4 weeks) were injected intradermally with fully emulsified bovine type II collagen and complete Freund's adjuvant to establish CIA rat models. Later, the CIA rats received oral administration of HQH (4.16 g/kg) once a day from the day 21 of modeling, with the treatment lasting for 28 days. Varieties of indicators were measured for evaluation of anti-inflammation effect of HQH, including hind paw swelling, arthritis scores, micro CT, and histopathological changes and the level of pro-inflammatory cytokines in the serum, including tumor necrosis factor alpha (TNF-±) and interleukin-18 (IL-18). The expression of GSDMD and caspase-1 in the joint synovial tissues was detected. The results demonstrated that the expression of the pyroptotic protein GSDMD and its upstream caspase-1 was significantly increased in the synovial tissues of CIA rats. The treatment of HQH ameliorated the symptoms in CIA rats, reduced levels of pro-inflammatory cytokines and hind paw swelling, down-regulated the expression of GDSMD and caspase-1. GSDMD-induced pyroptosis participated in the pathogenesis of CIA rats. The study supported that HQH can effectively improve joints inflammation of juvenile collagen-induced arthritis rats by inhibiting pyroptosis pathway in the joint synovial tissues.

Rheumatoid arthritis (RA)-associated interstitial lung disease (ILD), a primary cause of mortality in patients with RA, has limited treatment options. A previously established RA model in D1CC transgenic mice aberrantly expressed major histocompatibility complex class II genes in joints, developing collagen II-induced polyarthritis and anti-cyclic citrullinated peptide antibodies and interstitial pneumonitis, similar to those in humans. Molecular hydrogen (H 2 ) is an efficient antioxidant that permeates cell membranes and alleviates the reactive oxygen species-induced injury implicated in RA pathogenesis. We used D1CC mice to analyse chronic lung fibrosis development and evaluate H 2 treatment effects. We injected D1CC mice with type II collagen and supplied them with H 2 -rich or control water until analysis. Increased serum surfactant protein D values and lung densities images were observed 10 months after injection. Inflammation was patchy within the perilymphatic stromal area, with increased 8-hydroxy-2'-deoxyguanosine-positive cell numbers and tumour necrosis factor-α, BAX, transforming growth factor-β, interleukin-6 and soluble collagen levels in the lungs. Inflammatory and fibrotic changes developed diffusely within the perilymphatic stromal area, as observed in humans. H 2 treatment decreased these effects in the lungs. Thus, this model is valuable for studying the effects of H 2 treatment and chronic interstitial pneumonia pathophysiology in humans. H 2 appears to protect against RA-ILD by alleviating oxidative stress., (© 2019 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Context: Dietary supplement manufacturers claim cutaneous anti-aging properties for their products; however, research supporting these claims remains sparse., Objectives: The study intended to determine if a correlation existed between the effects of a collagen dietary supplement and changes associated with skin aging., Design: The study was a 12-week, double-blind, placebo-controlled trial., Setting: The study took place at a clinical facility specializing in dermatological testing that could perform biophysical, instrumental analysis on the effects of proprietary supplement on human skin., Participants: Participants were 128 females, aged 39-59 (50.57 ± 5.55)., Intervention: Participants were randomly assigned to an intervention or a placebo. The intervention consisted of twice daily oral administration of a supplement containing 500 mg BioCell Collagen, a chicken sternal cartilage derived dietary ingredient composed of a naturally-occurring matrix of hydrolyzed collagen type-II (≥300 mg), chondroitin sulfate (≥100 mg), hyaluronic acid (≥50 mg)., Outcome Measures: The primary parameters included transepidermal water loss, viscoelasticity, hydration, (indirect) collagen content, chromophore (melanin) content and hemoglobin level, and photographic analysis. An expert visually graded participants' skin to determine the intervention's efficacy, measuring facial lines and wrinkles, crow's feet lines and wrinkles, skin texture and smoothness, and skin tone. The presence of erythema and/or dryness determined tolerance. Secondary outcome measures were tolerance and incidence of adverse events, and the participant's perception of the supplement's value., Results: For the 113 participants completing the study, the dietary supplementation compared to a placebo: (1) significantly reduced facial lines and wrinkles (P = .019) and crow's feet lines and wrinkles (P = .05), (2) increased skin elasticity (P = .008) and cutaneous collagen content (P < .001) by 12%, (3) improved indicators associated with a more youthful skin appearance based on visual grading and wrinkle width (P = .046), and (4) decreased skin dryness and erythema. No difference existed between the supplement and the placebo for skin-surface water content or retention. The supplement was well tolerated, with no reported adverse reactions., Conclusions: Dietary supplementation with chicken, sternal cartilage extract supports the accumulation of types-I/III collagen in skin to promote increased elasticity and reduced skin wrinkling.

Rheumatoid arthritis (RA) is a systemic autoimmune disease primarily affecting joints and is featured by chronic synovial inflammation and angiogenesis. We employed a bovine type-II collagen (BIIC)-induced Sprague-Dawley rat arthritis model and an in vitro RA model based on interleukin (IL)-1β-stimulated rat synovial cells (RSC-364) to explore the preventive effect of resveratrol on RA and the underlying mechanisms. We found that resveratrol ameliorated BIIC-elicited synovitis and RA-related pathological hallmarks such as inflammatory cell infiltration and angiogenesis in the synovial tissue. Also, BIIC-stimulated rats displayed increased serum levels of proinflammatory cytokines and reactive oxygen species (ROS), as manifested by elevated serum malonaldehyde contents combined with reduced superoxide dismutase activity. It is noteworthy that resveratrol abolished BIIC-induced ROS and inflammation, confirming the antioxidative and anti-inflammatory actions of resveratrol in the context of RA. Furthermore, immunoblotting indicated that resveratrol downregulated the increase in the levels of hypoxia-inducible factor-1α (HIF-1α) and that of the activated phosphorylation of p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase in IL-1β-stimulated RSC-364 cells. Moreover, we observed that resveratrol-treated RSC-364 cells displayed both G 0 /G 1 cell-cycle arrest and enhanced levels of apoptosis. Altogether, the present evidence established the preventive role of resveratrol in RA progression. Mechanistically, resveratrol inhibits MAPK signaling pathways, likely by reducing ROS accumulation, to suppress the inflammatory response and cell proliferation and to provoke cell apoptosis in the synovial tissue, along with mitigation of HIF-1α-mediated angiogenesis. Thus resveratrol appears to hold great potential for clinical translation as a novel RA therapeutic.

BACKGROUND Osteoprotegerin (OPG) inhibits bone resorption and binds with strong affinity to receptor activator of NF κB ligand (RANKL), thereby preventing RANKL from binding to its receptor RANK. Osteoclasts have documented effects on bone erosion of rheumatoid arthritis (RA). The aim of this study was to examine the role of miR-145-5p in the regulation of RA osteoclast differentiation and bone erosion. MATERIAL AND METHODS Expression of microRNA-145-5p in human peripheral blood mononuclear cells (PBMC) and synovial tissue was assayed by real-time polymerase chain reaction (RT-PCR). OPG, RANK, and RANKL expression in RAW-264.7 cells was examined by RT-PCR and Western blot analysis. Osteoclast formation was detected by tartrate-resistant acid phosphatase (TRAP) staining. The effect of miR-145-5p on predicted target mRNAs was examined by luciferase reporter assays. Collagen-induced arthritis (CIA) was induced by injecting DBA/1 mice with bovine type II collagen (CII), and miR-145-5p agomir was administered by intravenous injection. Morphological changes in the CIA joint were assessed by micro-computed tomography (CT) and histopathology. RESULTS miR-145-5p levels significantly increased in RA PBMC and synovial tissue compared with normal PBMC and osteoarthritis (OA) tissue. After transfection of RAW-264.7 cells with miR-145-5p, RANK and RANKL expression increased significantly, while OPG expression decreased significantly. TRAP staining results showed osteoclast numbers increased. Micro-CT analysis of the arthritic joints showed that the miR-145-5p agomir caused bone erosion in mice, and histopathological analysis revealed that miR-145-5p agomir aggravates cartilage erosion. CONCLUSIONS Our findings indicate that administration of miR-145-5p aggravates joint erosion in CIA mice. This suggests that miR-145-5p is a potential target for the treatment of RA.

The cornerstone of treatment for rheumatoid arthritis is low dose methotrexate (MTX), but its use is limited by concerns regarding its potential for hepatotoxicity. Allylpyrocatechol (APC), a phytoconstituent sourced from leaves of Piper betle demonstrated antioxidant, anti-inflammatory, and antiarthritic properties. The present study aimed to evaluate the combined effect of APC and MTX on limiting progression of lipopolysaccharide accelerated collagen-induced arthritis, along with reduction of MTX-induced hepatic damage. A collagen-induced arthritis (CIA) model was established by immunising Sprague-Dawley rats with bovine collagen type II (CII) and lipopolysaccharide, followed by a booster dose of CII on day 15. Rats from days 11-27 were administered APC (20 mg/kg), methotrexate (1.5 mg/kg), or a combination of MTX and APC. The combinatorial therapy of APC and MTX significantly improved the parameters of arthritis as evident from the reduction in paw oedema and arthritic score and was endorsed by radiological and histopathological changes. This combination prevented the rise in levels of proinflammatory cytokines, tumour necrosis factor (TNF-α), and interleukin 6 (IL-6). Furthermore, unlike MTX-monotherapy, the APC-MTX combination decreased the associated cachexia, splenomegaly, and oxidative stress. Importantly, the hepatic damage mediated by MTX monotherapy was effectively attenuated by the inclusion of APC. Taken together, antioxidants such as APC when combined with MTX not only potentiated the antiarthritic effect but importantly alleviated the MTX-induced hepatic damage, thus endorsing its effectiveness in preventing progression of articular diseases such as rheumatoid arthritis.

Objective: The aim of this study was to determine the ability of undenatured native chicken type II collagen (UC-II) to prevent excessive articular cartilage deterioration in a rat model of osteoarthritis (OA)., Methods: Twenty male rats were subjected to partial medial meniscectomy tear (PMMT) surgery to induce OA. Immediately after the surgery 10 rats received vehicle and another 10 rats oral daily dose of UC-II at 0.66 mg/kg for a period of 8 weeks. In addition 10 naïve rats were used as an intact control and another 10 rats received sham surgery. Study endpoints included a weight-bearing capacity of front and hind legs, serum biomarkers of bone and cartilage metabolism, analyses of subchondral and cancellous bone at the tibial epiphysis and metaphysis, and cartilage pathology at the medial tibial plateau using histological methods., Results: PMMT surgery produced moderate OA at the medial tibial plateau. Specifically, the deterioration of articular cartilage negatively impacted the weight bearing capacity of the operated limb. Immediate treatment with the UC-II preserved the weight-bearing capacity of the injured leg, preserved integrity of the cancellous bone at tibial metaphysis and limited the excessive osteophyte formation and deterioration of articular cartilage., Conclusion: Study results demonstrate that a clinically relevant daily dose of UC-II when applied immediately after injury can improve the mechanical function of the injured knee and prevent excessive deterioration of articular cartilage., (Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: In part 1 of this study it was shown that liposuctioned fat could be a sufficient source of autologous collagen for use as a filler or in reconstruction. The collagen composition in liposuctioned fat was shown to form a cross-linked helical matrix composed of types II, III, and IV. Additionally, viable adipocytes and fibroblasts among other cells were found., Objectives: The purpose of this research was to study the biology of this matrix after subsequent implantation compared to Juvederm (Allergan, Parsippany, NJ) common soft tissue filler., Methods: Fat was obtained from individuals undergoing routine liposuction and was processed by a two-step process to obtain a connective tissue matrix. The matrix was then cryo-frozen for a minimum of 4 weeks after which it was thawed and implanted in 46 nude mice. Juvederm Ultra was used as the control article and the animals followed for one year., Results: Liposuctioned fat was obtained from 10 individuals and processed as previously described. Mice were harvested at 3, 6, 9, and 12 months and histology obtained. There were no adverse effects from either article and the bio-reactivity rating was 0. The implanted collagen compared favorably to Juvederm at all stages and was found to be replaced by new collagen and fat., Conclusions: A collagen matrix with viable cells for autologous use can be obtained from liposuctioned fat which has been processed and cryo-frozen. The material lasts at least one year and is slowly replaced by new collagenand fat., Level of Evidence: 5., (© 2017 The American Society for Aesthetic Plastic Surgery, Inc. Reprints and permission: journals.permissions@oup.com)

Phospholipids occurring in cell membranes and lipoproteins are converted into oxidized phospholipids (OxPL) by oxidative stress promoting atherosclerotic plaque formation. Here, OxPL were characterized as novel targets in acute and chronic inflammatory pain. Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (OxPAPC) and its derivatives were identified in inflamed tissue by mass spectrometry and binding assays. They elicited calcium influx, hyperalgesia and induced pro-nociceptive peptide release. Genetic, pharmacological and mass spectrometric evidence in vivo as well as in vitro confirmed the role of transient receptor potential channels (TRPA1 and TRPV1) as OxPAPC targets. Treatment with the monoclonal antibody E06 or with apolipoprotein A-I mimetic peptide D-4F, capturing OxPAPC in atherosclerosis, prevented inflammatory hyperalgesia, and in vitro TRPA1 activation. Administration of D-4F or E06 to rats profoundly ameliorated mechanical hyperalgesia and inflammation in collagen-induced arthritis. These data reveal a clinically relevant role for OxPAPC in inflammation offering therapy for acute and chronic inflammatory pain treatment by scavenging OxPAPC.

Objectives: Zaocys type II collagen is an active collagen extracted from Zaocys that has been used to treat rheumatoid arthritis in China for over 1000 years. However, the mechanism still remains unknown. Therefore, we set out to investigate the inhibitory effect and possible mechanism of action of zaocys type II collagen on collagen-induced arthritis., Methods: Collagen-induced arthritis was induced in C57BL/6 mice by immunisation with type II collagen. After immunisation, the mice were treated with Zaocys type II collagen. Clinical and histological scores were assessed and the cytokine levels in the serum and lymphocytes supernatant from the spleen and mesenteric lymph node were determined by enzyme-linked immune sorbent assay. The T-helper 17 cell and regulatory-T cell frequencies were analysed by flow cytometry and the expression of interest markers was examined by direct immuno-fluorescence., Results: The arthritis score and severity of histological inflammation and cartilage destruction were dose-dependently reduced after treatment. The analysis results indicated that Zaocys type II collagen significantly increased the proportion of regulatory-T cells and lowered the T-helper 17 cells, it also increased the number of regulatory-T cells and conversely decreased the T-helper 17 cells in synovial tissue compared with the model group. Treatment also caused a higher level of transforming growth factor-β and a decreased production of interleukin -17A., Conclusions: The oral administration of Zaocys type II collagen potently suppressed the severity of collagen-induced arthritis by repairing the imbalance between regulatory-T cells and T-helper 17 cells, suggesting that it might be a promising candidate for the treatment of rheumatoid arthritis.

Background: The purpose of this study was to investigate the therapeutic efficacy of intravenously administered immunoselected STRO-3 + mesenchymal precursor cells (MPCs) on clinical scores, joint pathology and cytokine production in an ovine model of monoarthritis., Methods: Monoarthritis was established in 16 adult merino sheep by administration of bovine type II collagen into the left hock joint following initial sensitization to this antigen. After 24 h, sheep were administered either 150 million allogeneic ovine MPCs (n = 8) or saline (n = 8) intravenously (IV). Lameness, joint swelling and pain were monitored and blood samples for leukocytes and cytokine levels were collected at intervals following arthritis induction. Animals were necropsied 14 days after arthritis induction and gross and histopathological evaluations were undertaken on tissues from the arthritic (left) and contralateral (right) joints., Results: MPC-treated sheep demonstrated significantly reduced clinical signs of lameness, joint pain and swelling compared with saline controls. They also showed decreased cartilage erosions, synovial stromal cell activation and angiogenesis. This was accompanied by decreased infiltration of the synovial tissues by CD4 + lymphocytes and CD14 + monocytes/macrophages. Over the 3 days following joint arthropathy induction, the numbers of neutrophils circulating in the blood and plasma concentrations of activin A were significantly reduced in animals administered MPCs., Conclusions: The results of this study have demonstrated the capacity of IV-administered MPCs to mitigate the clinical signs and some of the inflammatory mediators responsible for joint tissue destruction in a large animal model of monoarthritis.

Biomaterials that provide signals present in the native extracellular matrix have been proposed as scaffolds to support improved cartilage regeneration. This study harnesses the biological activity of collagen type II and the superior mechanical properties of collagen type I by characterizing gels made of collagen type I and II blends. The collagen blend hydrogels were able to incorporate both types of collagen and retained chondroitin sulfate and hyaluronic acid. Cryo-scanning electron microscopy images showed that the 3:1 ratio of collagen type I to type II gels had a lower void space percentage (36.4%) than the 1:1 gels (46.5%). The complex modulus was larger for the 3:1 gels (G* = 5.0 Pa) compared to the 1:1 gels (G* = 1.2 Pa). The 3:1 blend consistently formed gels with superior mechanical properties compared to the other blends and has the potential to be implemented as a scaffold for articular cartilage engineering.

Rheumatoid arthritis is characterised by synovial inflammation and proliferation of fibroblast-like synoviocytes. The induction of apoptosis has long been proposed as a target for proliferative autoimmune diseases, and has further been shown to act as a successful treatment of experimental models of arthritis, such as collagen-induced arthritis. Here we examined the effects of specific oral small-molecule inhibitors of the transcription regulating cyclin-dependent kinase 9 on the development and progression of collagen-induced arthritis. DBA/1 mice were immunised with bovine collagen type II and treated orally with specific CDK9 inhibitors. The effects of CDK9 inhibition on RNA levels and protein expression, apoptosis induction, caspase activation and lymphocyte phenotype were further analysed. Mice showed a significant delay in disease onset and a reduction in disease severity following treatment with CDK9 inhibitors. Inhibiting CDK9 activity in peripheral blood mononuclear cells resulted in the loss of Mcl-1 expression at both the protein and RNA levels, along with a subsequent increase in apoptosis. CDK9 specific inhibitors may be a potential alternative treatment not only of cancer, but also for autoimmune- and inflammatory diseases. Taken together, these results show that transient inhibition of CDK9 induces apoptosis in leukocyte subsets and modulates the immune response.

TGF-β3 is enzymatically immobilized by transglutaminase-2 action to poly(l-lactic acid) microparticles coated with collagen II. Microparticles are then encapsulated with stem cells inside liquified spherical compartments enfolded with a permselective shell through layer-by-layer adsorption. Magnetic nanoparticles are electrostatically bound to the multilayered shell, conferring magnetic-response ability. The goal of this study is to engineer a closed environment inside which encapsulated stem cells would undergo a self-regulated chondrogenesis. To test this hypothesis, capsules are cultured in chondrogenic differentiation medium without TGF-β3. Their biological outcome is compared with capsules encapsulating microparticles without TGF-β3 immobilization and cultured in normal chondrogenic differentiation medium containing soluble TGF-β3. Glycosaminoglycans quantification demosntrates that similar chondrogenesis levels are achieved. Moreover, collagen fibrils resembling the native extracellular matrix of cartilage can be observed. Importantly, the genetic evaluation of characteristic cartilage markers confirms the successful chondrogenesis, while hypertrophic markers are downregulated. In summary, the engineered capsules are able to provide a suitable and stable chondrogenesis environment for stem cells without the need of TGF-β3 supplementation. This kind of self-regulated capsules with softness, robustness, and magnetic responsive characteristics is expected to provide injectability and in situ fixation, which is of great advantage for minimal invasive strategies to regenerate cartilage., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Here, we investigate induction of immunological tolerance by lentiviral based gene therapy in a mouse model of rheumatoid arthritis, collagen II-induced arthritis (CIA). Targeting the expression of the collagen type II (CII) to antigen presenting cells (APCs) induced antigen-specific tolerance, where only 5% of the mice developed arthritis as compared with 95% of the control mice. In the CII-tolerized mice, the proportion of Tregs as well as mRNA expression of SOCS1 (suppressors of cytokine signaling 1) increased at day 3 after CII immunization. Transfer of B cells or non-B cell APC, as well as T cells, from tolerized to naïve mice all mediated a certain degree of tolerance. Thus, sustainable tolerance is established very early during the course of arthritis and is mediated by both B and non-B cells as APCs. This novel approach for inducing tolerance to disease specific antigens can be used for studying tolerance mechanisms, not only in CIA but also in other autoimmune diseases.

Collagen-induced arthritis (CIA) is a widely used mouse model for studying inflammatory arthritis (IA). However, CIA induction protocols differ between laboratories, and direct comparison between protocol variations has not been reported. To address this issue, DBA/1 mice housed in conventional and specific-pathogen free (SPF) facilities were administered various combinations of two doses of collagen type II (CII) in complete (CFA) or incomplete Freund's adjuvant (IFA); some mice were also injected with lipopolysaccharide (LPS) and/or additional CII at specific intervals. Mice were evaluated for IA over the subsequent 2 months. Depending directly on the combination of CII, CFA, IFA, and LPS used, the incidence of IA ranged between 20%-100%, and severity extended from mild to severe even in an SPF environment. Our results demonstrate for the first time in head-to-head comparisons that specific variations in the use of CII, CFA, IFA, and LPS can induce a range of arthritic disease intensity and severity in an SPF facility. Thus, distinct experimental settings can be designed for robust assessment of factors that either exacerbate or inhibit arthritis pathogenesis. Furthermore, by achieving 100% incidence in an SPF facility, the protocols provide a practical and humane benefit by reducing the number of mice necessary for experimental assessment.

Background: Undenatured type II collagen (UC-II) is a nutritional supplement derived from chicken sternum cartilage. The purpose of this study was to evaluate the efficacy and tolerability of UC-II for knee osteoarthritis (OA) pain and associated symptoms compared to placebo and to glucosamine hydrochloride plus chondroitin sulfate (GC)., Methods: One hundred ninety one volunteers were randomized into three groups receiving a daily dose of UC-II (40 mg), GC (1500 mg G & 1200 mg C), or placebo for a 180-day period. The primary endpoint was the change in total Western Ontario McMaster Universities Osteoarthritis Index (WOMAC) from baseline through day 180 for the UC-II group versus placebo and GC. Secondary endpoints included the Lequesne Functional Index (LFI), the Visual Analog Scale (VAS) for pain and the WOMAC subscales. Modified intent-to-treat analysis were performed for all endpoints using analysis of covariance and mixed model repeated measures, while incremental area under the curve was calculated by the intent-to-treat method., Results: At day 180, the UC-II group demonstrated a significant reduction in overall WOMAC score compared to placebo (p = 0.002) and GC (p = 0.04). Supplementation with UC-II also resulted in significant changes for all three WOMAC subscales: pain (p = 0.0003 vs. placebo; p = 0.016 vs. GC); stiffness (p = 0.004 vs. placebo; p = 0.044 vs. GC); physical function (p = 0.007 vs. placebo). Safety outcomes did not differ among the groups., Conclusion: UC-II improved knee joint symptoms in knee OA subjects and was well-tolerated. Additional studies that elucidate the mechanism for this supplement's actions are warranted., Trial Registration: CTRI/2013/05/003663 ; CTRI/2013/02/003348 .

Background: This work aimed to evaluate the effects of genistein treatment in Collagen Induced Arthritis (CIA) mouse model., Methods: CIA was elicited in DBA/1 Mice by an intradermal injection of 100 μL of an emulsion of bovine type II collagen (CII) in isovolumic incomplete Freund's adjuvant (IFA) at the base of the tail. Twenty-one days later, a second injection of CII in IFA was administered at the base of the tail. After the symptoms of arthritis showed in mouse model, we divided animals into two groups according to their clinical symptom scores. The treatment group was intraperitoneally injected daily with genistein (based on the pre-experiment data and literature reported, 5 mg/kg dose was selected and tested) for 12 days, while the control group was injected with phosphate buffered saline. Inflammatory cytokines titer, radiological, and histological observations were completed at different time's points after treatment. CT analysis was conducted 3 months after the treatment to observe the articular structures. Immunohistochemical analysis was performed to investigate the expression and distribution of VEGF in joint tissues., Results: Genistein suppressed the expressions of IL-1β, IL-6 and TNF-α in the serum. Radiological results showed that bone degradation was inhibited by the treatment. Moreover, hematoxylin and eosin staining showed that the degree of inflammation was relieved. In the cartilage area, TRAP stain-positive cells were detected, which was notably reduced in the treatment group compared to the control group. Micro-CT 3D images clearly exhibited that the joint adhered and structures destroyed in the control group with less destruction in the treatment group. Furthermore, genistein suppressed VEGF expression, and blocked angiogenesis in the synovial tissue., Conclusion: Our work provides further data regarding the effects of genistein as a potential treatment drug for RA, as well as the role of genistein in the anti-inflammatory pathway in RA therapy., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Dendritic cells (DCs) are professional antigen presenting cells that have the dual ability to stimulate immunity and maintain tolerance. However, the signalling pathways mediating tolerogenic DC function in vivo remain largely unknown. The β-catenin pathway has been suggested to promote a regulatory DC phenotype. The aim of this study was to unravel the role of β-catenin signalling to control DC function in the autoimmune collagen-induced arthritis model (CIA). Deletion of β-catenin specifically in DCs was achieved by crossing conditional knockout mice with a CD11c-Cre transgenic mouse line. Bone marrow-derived DCs (BMDCs) were generated and used to study the maturation profile of these cells in response to a TLR2 or TLR4 ligand stimulation. CIA was induced by intra-dermal immunization with 100 μg chicken type II collagen in complete Freund's adjuvant on days 0 and 21. CIA incidence and severity was monitored macroscopically and by histology. The T cell profile as well as their cytokine production were analysed by flow cytometry. Lack of β-catenin specifically in DCs did not affect the spontaneous, TLR2- or TLR4-induced maturation and activation of BMDCs or their cytokine production. Moreover, no effect on the incidence and severity of CIA was observed in mice lacking β-catenin in CD11c+ cells. A decreased frequency of splenic CD3+CD8+ T cells and of regulatory T cells (Tregs) (CD4+CD25highFoxP3+), but no changes in the frequency of splenic Th17 (CCR6+CXCR3-CCR4+), Th2 (CCR6-CXCR3-CCR4+) and Th1 (CCR6-CXCR3+CCR4-) cells were observed in these mice under CIA condition. Furthermore, the expression of IL-17A, IL-17F, IL-22, IL-4 or IFNγ was also not affected. Our data indicate that ablation of β-catenin expression in DCs did not alter the course and severity of CIA. We conclude that although deletion of β-catenin resulted in a lower frequency of Tregs, this decrease was not sufficient to aggravate the onset and severity of CIA.

Introduction: Diabetic neuropathy secondary to diabetes mellitus type 1 (DM1) is responsible for retrograde ejaculation (RE) in 5-18% of cases. Medical treatment of RE is based either on increasing the sympathetic tone of the bladder or on decreasing the parasympathetic activity. However, the onset of side effects and the lack of response should be considered., Aims: The aim of this study was to analyze long-term outcome of endourethral injection of volume-forming material (VFM) of collagen type 2 into bladder neck submucosa in patients with RE secondary to DM1., Methods: Twenty-four patients with complete RE refractory to imipramine and DM1 were included in the study. Patients were single-blinded randomized according to a computer-generated random sequence with a 1:1 ratio in two treatment groups, namely group A (endourethral collage type 2 injection) and group B (endourethral saline water injection). New technique includes an endoscopic injection of VFM such as collagen (Correcting MIT®, Ltd. minimally invasive technologies, Moscow, Russia) into bladder neck submucosa. Primary endpoint of the study was the reduction of semen antegrade volume (mL). Secondary endpoints were considered as the changes of antegrade count (millions/mL), antegrade total motility (%), antegrade progressive motility (%), State-Trait Anxiety Inventory, Beck Depression Questionnaire and International Index of Erectile Function (IIEF-5). Pregnancy rate was calculated in each group., Results: Twenty-three patients completed the study. In group A, significant differences from baseline to 12 months were observed relative to antegrade volume (mL) (mean difference: 0.71, P < 0.05), antegrade count (millions/mL) (mean difference: 45.6, P < 0.05), antegrade total motility (%) (mean difference: 15.4, P < 0.05) and antegrade progressive motility (%) (mean difference: 8.4, P < 0.05). In group A, we observed significant differences in terms State-Trait Anxiety Inventory (mean difference: -20.5, P < 0.05) and Beck Depression Inventory (mean difference: -8.4, P < 0.05) with significant differences compared with group B. We observed significant improvements in group A vs. group B when considering primary and secondary endpoints of the study, but not for the IIEF-5., Conclusion: Correction of RE in DM1 patients could be achieved with endourethral injection of collagen type 2., (© 2015 International Society for Sexual Medicine.)

Purpose: To evaluate the therapeutic potential of Col-Treg, a collagen II-specific type 1 regulatory T-cell immunotherapy for the treatment of noninfectious uveitis (NIU)., Methods: Col-Treg cells were produced from collagen II-specific T cell receptor (TCR) transgenic mice or peripheral blood of healthy donors. Phenotypic characterization was performed by flow cytometry, and cytokine secretion was evaluated with Flowcytomix or ELISA. In vitro functional characterization included ATP hydrolysis, cytotoxicity, and contact-independent T-cell suppression and plasticity assays. Col-Treg migration was assessed by quantitative PCR specific to Col-Treg TCR. Col-Treg cells were administered intravenously in mice displaying experimental autoimmune uveitis (EAU) induced by interphotoreceptor retinoid-binding protein (IRBP) immunizations. Efficacy of Col-Treg was assessed by ophthalmology, histology, and immunohistochemistry., Results: Mice Col-Treg cells displayed identity features of type 1 Treg cells with expression of CD25, FoxP3, low surface expression of CD127, and cytokine secretion profile (IL-10(high), IL-4(low), IFN-γ(int)). In vitro functional assays demonstrated Col-Treg suppressive capacity via soluble factor-dependent immunosuppression, cytotoxicity, and ATP hydrolysis. Col-Treg cells expressed granzyme B, CD39, and glucocorticoid-induced TNF-related protein (GITR). Administration of Col-Treg in EAU mice inhibited clinical and morphologic signs of uveitis and decreased ocular leukocyte infiltration. Col-Treg cells homed in the ocular tissues 24 hours after intravenous injection. Human Col-Treg cells were comparable to mice Col-Treg cells in identity and function and did not show the capacity to differentiate into Th17 cells in vitro., Conclusions: These results demonstrate the therapeutic potential of Col-Treg cells as a targeted approach for the treatment of NIU and the feasibility of translating this approach to the human clinical setting.

Background: The cuff tendon that is most prone to full-thickness rotator cuff tears is the supraspinatus (SSP). Arthroscopic SSP repair ensures good to satisfactory mid- to long-term clinical outcomes. However, the intense postoperative pain reduces rehabilitation compliance and is cause of patient dissatisfaction. Many natural compounds act by inhibiting inflammatory pathways in a similar way to anti-inflammatory drugs, Materials and Methods: This was a prospective randomized trial designed to assess the analgesic effect of a dietary supplement (DS) containing Boswellia serrata and Curcuma longa in a population of subjects with full-thickness SSP tendon tear treated by arthroscopy. Three weeks before surgery, patients were randomized to receive Tendisulfur(®) (group T) or a placebo (group P) for 2 months. The primary outcome measure was subjective VAS pain. Secondary outcomes measures were Constant-Murley score simple shoulder test, and patient global assessment (PGA) scores. Patients were assessed immediately at baseline and subsequently at 1, 2, 4, 6, 8, 12, and 24 weeks., Results: Stratification of pain scores and subscores demonstrated significantly lower overall pain scores in group T versus group P at 1 week (p = 0.0477), and lower but not significantly different scores on week 2 (p = 0.0988); at subsequent time points, differences were not significant (p > 0.05). PGA scores were good in all subjects., Conclusions: In conclusion, this study provides objective data on the effect of a DS containing natural substances, added to standard analgesics, on postoperative RC pain. DS alleviated short and partially mid-term pain, while long-term pain was unchanged. This limitation can probably be addressed by a dosage increase over the first 4 weeks and by extending treatment by 1 or 2 months.

Background: We have shown previously, in an animal model of multiple sclerosis and in TNBS-induced colitis, that epicutaneous (EC) immunization with protein antigen induces T suppressor cells that strongly inhibit the inflammatory response in contact hypersensitivity reactions., Methods: EC immunization was performed by applying to the shaved skin of the mouse dorsum a gauze patch soaked with a solution containing various amounts of type II collagen (COLL II) in a volume of 100 µl of PBS on days 0 and 4. On day 7 the patches were removed and mice were intradermally (i.d.) immunized with COLL II to induce collagen-induced arthritis (CIA)., Results: Our study shows that EC immunization with 100 or 30 μg of COLL II reduces disease severity, whereas lower doses (10 or 3 μg) do not affect CIA. Decreased disease severity observed after EC immunization with COLL II correlates with reduced myeloperoxidase activity in joint tissue and with reduced production of anti-citrullinated protein and anti-COLL II IgG2a antibodies. Transfer experiments show that EC immunization with COLL II induces suppressor cells that belong to the population of TCRαβ lymphocytes and that EC-induced suppression declines with time. Both in vitro and in vivo experiments show that IL-17A plays an important role in EC-induced suppression of CIA. EC application of COLL II at the first signs of CIA also results in disease suppression., Conclusions: The suppression of inflammatory responses by T suppressor cells induced through EC immunization of a protein antigen may become an attractive noninvasive therapeutic method for a variety of clinical situations., (© 2015 S. Karger AG, Basel.)

Type II collagen (CII) is a cartilage structural protein that plays important roles in joint function, arthritis and ageing. In studying the ability of CII to induce eye-mediated specific immune tolerance, we have recently proven that CII is capable of inducing anterior chamber-associated immune deviation (ACAID) in Balb/c mice. Here, we study the ability of CII to induce eye-mediated immune tolerance in strains of mice that are prone to the induction of rheumatoid arthritis. Thus, we hypothesized that CII induces ACAID in DBA/1 mice and in C57BL/6 mice through the AC route (direct injection) or the intravenous route (adoptive transfer of in vitro-generated CII-specific ACAID macrophages or of CII-specific in vitro-generated T regulatory cells). Specific immune tolerance induction was assessed using both delayed-type hypersensitivity (DTH) and local adoptive transfer (LAT) assays. Results indicated the ability of CII to generate CII-specific ACAID-mediated immune tolerance in vivo and in vitro in both DBA/1 mice and C57BL/6 mice. These findings could be beneficial in studies of immune tolerance induction using CII., (© 2014 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Purpose: This study assesses and compares the clinical outcomes of the arthroscopic matrix-induced autologous chondrocyte implant (MACI) and autologous matrix-induced chondrogenesis (AMIC) techniques for the treatment of acetabular chondral defects between 2 and 4 cm(2) consequent to femoral acetabular impingement., Methods: Fifty-seven consecutive patients were treated with the MACI (n = 26) or AMIC (n = 31) technique. Patients were assessed pre-operatively and up to five years using the modified Harris Hip Score (mHHS) to compare outcomes., Results: In both the MACI and AMIC groups, significant hip score improvements were measured over baseline levels at six months post-op (81.2 ± 8.4 for MACI, 80.3 ± 8.3 for AMIC, both p < 0.001). The mHHS continued to improve up to three years post-op and remained stable over time until the final five year follow-up. Statistically significant differences between the groups were not observed. The mean mHHS improvement at the five year follow-up with respect to preoperative level was 37.8 ± 5.9 and 39.1 ± 5.9 in patients who underwent MACI and AMIC, respectively (NS). Subgroup analysis of both MACI and AMIC treatment outcomes for patients with an initial chondral defect larger than 3 cm(2) yielded comparable results at each time point., Conclusions: This study suggests that both arthroscopic MACI and AMIC are valid procedures to repair medium-sized chondral defects on the acetabular side of the hip found during treatment of femoroacetabular impingement. Due to its high sustainability and minimal invasiveness, the single-stage AMIC procedure can reduce total treatment time and minimise morbidity while providing the same beneficial effects as the two-stage MACI intervention.

The aim of this work was to study the proteolytic activity of IgGs purified from blood serum of Wistar rats at experimental rheumatoid arthritis (ERA) induced by an injection of bovine collagen of type II. Twenty rats were immunized with a preparation of bovine collagen II (Sigma-Aldrich, USA) in the presence of complete Freund's adjuvant. ERA development was determined by inflammation in limbs of treated animals. IgG preparations were isolated from blood serum of immunized and non-immunized animals by precipitation of antibodies with 33% ammonium sulfate followed by chromatography on the Protein G-Sepharose column. Human histone H1, bovine collagen II, calf thymus histones, myelin basic protein (MBP), bovine serum albumin (BSA), and bovine casein were used as substrates of the proteolytic activity of IgGs. It was found that IgG preparations from blood serum of rats with ERA were capable of cleaving histone H1 and MBP, however, they were catalytically inactive towards collagen II, casein, BSA, and core histones. IgGs from blood serum of non-immunized rats were proteolytically inactive towards all used protein substrates. Thus, we demonstrated that immunization of rats with bovine collagen II induced IgG-antibodies possessing the proteolytic activity towards histone H1 and MBP. This activity might be associated with the development of inflammatory processes in the immunized rats.

Mast cells are implicated in the pathogenesis of inflammatory and autoimmune diseases. However, this notion based on studies in mast cell-deficient mice is controversial. We therefore established an in vivo model for hyperactive mast cells by specifically ablating the NF-κB negative feedback regulator A20. While A20 deficiency did not affect mast cell degranulation, it resulted in amplified pro-inflammatory responses downstream of IgE/FcεRI, TLRs, IL-1R, and IL-33R. As a consequence house dust mite- and IL-33-driven lung inflammation, late phase cutaneous anaphylaxis, and collagen-induced arthritis were aggravated, in contrast to experimental autoimmune encephalomyelitis and immediate anaphylaxis. Our results provide in vivo evidence that hyperactive mast cells can exacerbate inflammatory disorders and define diseases that might benefit from therapeutic intervention with mast cell function., Competing Interests: The authors have declared that no competing interests exist.

Earlier studies have reported the efficacy of type II collagen (C II) in treating rheumatoid arthritis (RA). However, a few studies have investigated the ability of the antigenic collagen to induce oral tolerance, which is defined as active nonresponse to an orally administered antigen. We hypothesized that water-soluble undenatured C II had a similar effect as C II in RA. The present study was designed to examine the oral administration of a novel, water-soluble, undenatured C II (commercially known as NEXT-II) on collagen-induced arthritis (CIA) in mice. In addition, the underlying mechanism of NEXT-II was also identified. After a booster dose (collagen-Freund's complete adjuvant), mice were assigned to control CIA group, or NEXT-II treatment group, to which saline and NEXT-II were administered, respectively. The arthritis index in the NEXT-II group was significantly lower compared with the CIA group. Serum IL-6 levels in the NEXT-II group were significantly lower compared with the CIA group, while serum IL-2 level was higher. Furthermore, oral administration of NEXT-II enhanced the proportion of CD4+CD25+T (Treg) cells, and gene expressions of stimulated dendritic cells induced markers for regulatory T cells such as forkhead box p3 (Foxp3), transforming growth factor (TGF)-β1, and CD25. These results demonstrated that orally administered water-soluble undenatured C II (NEXT-II) is highly efficacious in the suppression of CIA by inducing CD4+CD25+ Treg cells.

Objective: Functional cross-talk exists between sympathetic nerve fibers and cytokine-producing splenic cells in early collagen type II-induced arthritis (CIA) (day 32). These earlier experiments demonstrated exclusively neuronal sympathetic regulation of IFN-γ, CXCL1, IL-6, and TGF-β. However, in late arthritis, the sympathetic influence might change due to loss of sympathetic nerve fibers and appearance of neurotransmitter-producing cells. We aimed to investigate neurotransmitter-dependent regulation of IFN-γ, CXCL1, IL-6, and TGF-β in murine spleen in late CIA., Methods: Spleens were removed when animals reached day 58 (46-68) after immunization to generate 0.35 mm-thick spleen slices, which were transferred to superfusion microchambers to electrically induce release of neurotransmitters. Using respective neurotransmitter antagonists, effects of released neurotransmitters on cytokine secretion were investigated., Results: There was electrically induced inhibition of IFN-γ, CXCL1, and IL-6, and stimulation of TGF-β, which was much less pronounced than in early CIA. There existed β adrenergic inhibition of IFN-γ, IL-6, and TGF-β (and stimulation of CXCL1) independent of electrical stimulation (interpreted as non-neuronal). However, there was a neuronal α1/2 adrenergic stimulation of IFN-γ, CXCL1, and IL-6 and, we observed neuronal A1-adenosinergic stimulation of TGF-β., Conclusions: In the late phase of CIA, non-neuronal modulation of cytokine secretion increases while neuronal regulation strikingly decreases. Particularly, β-adrenergic effects are non-neuronal while α1/2-adrenergic effects are clearly neuronal. We suggest that alterations in sympathetic innervation of the spleen fundamentally change the functional neuroimmune interplay in the spleen of arthritic mice., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Background: Osteoarthritis is the most widespread joint-affecting disease. Patients with osteoarthritis experience pain and impaired mobility resulting in marked reduction of quality of life. A progressive cartilage loss is responsible of an evolving disease difficult to treat. The characteristic of chronicity determines the need of new active disease modifying drugs. Aim of the present research is to evaluate the role of low doses of native type II collagen in the rat model of osteoarthritis induced by sodium monoiodoacetate (MIA)., Methods: 1, 3 and 10 mg kg-1 porcine native type II collagen were daily per os administered for 13 days starting from the day of MIA intra-articular injection., Results: On day 14, collagen-treated rats showed a significant prevention of pain threshold alterations induced by MIA. Evaluation were performed on paws using mechanical noxious (Paw pressure test) or non-noxious (Electronic Von Frey test) stimuli, and a decrease of articular pain was directly measured on the damaged joint (PAM test). The efficacy of collagen in reducing pain was as higher as the dose was lowered. Moreover, a reduced postural unbalance, measured as hind limb weight bearing alterations (Incapacitance test), and a general improvement of motor activity (Animex test) were observed. Finally, the decrease of plasma and urine levels of CTX-II (Cross Linked C-Telopeptide of Type II Collagen), a biomarker of cartilage degradation, suggests a collagen-dependent decrease of structural joint damage., Conclusions: These results describe the preclinical efficacy of low dosages of native type II collagen as pain reliever by a mechanism that involves a protective effect on cartilage.

Introduction: Autologous Matrix-Induced Chondrogenesis (AMIC) is an innovative treatment for localized full-thickness cartilage defects combining the well-known microfracturing with collagen I/III scaffold. The purpose of this analysis was to evaluate the medium-term results of this enhanced microfracture technique for the treatment of chondral lesions of the knee., Methods and Materials: Patients treated with AMIC (Chondro-Gide, Geistlich Pharma, Switzerland) were followed using the AMIC Registry, an internet-based tool to longitudinally track changes in function and symptoms by the Lysholm score and VAS., Results: A series of 57 patients was enrolled. The average age of patients (19 females, 38 males) was 37.3 years (range 17-61 years). The mean defect size of the chondral lesions was 3.4 cm(2) (range 1.0-12.0 cm(2)). All defects were classified as grade III (n = 20) or IV (n = 37) according to the Outerbridge classification. Defects were localized at the medial (n = 32) or lateral (n = 6) condyle, at the trochlea (n = 4) and at the patella (n = 15). The follow-up period was 2 years. The majority of patients were satisfied with the postoperative outcome, reporting a significant decrease of pain (mean VAS preop = 7.0; 1 year postop = 2.7; 2 years postop = 2.0). Significant improvement of the mean Lysholm score was observed as early as 1 year after AMIC and further increased values were noted up to 2 years postoperatively (preop. 50.1, 1 year postop. 79.9, 2 year postop. 85.2)., Conclusions: AMIC is an effective and safe method of treating symptomatic chondral defects of the knee. However, further studies with long-term follow-up are needed to determine if the grafted area will maintain structural and functional integrity over time., Level of Evidence: Prognostic study, Level IV.

Introduction: Citrullinated self-proteins are thought to be involved in the onset/progression of rheumatoid arthritis (RA). Numerous studies have been performed to look for the self-antigen that becomes citrullinated and induces RA. Importantly, these studies have been performed using citrullinated self-antigens injected into an animal model in the presence of a strong adjuvant in order to derive the response. However, to date no studies have been performed to determine if these phenotypes can be induced in the absence of an adjuvant., Methods: To investigate this possibility, mice were immunized with citrullinated or non-citrullinated mouse Type II collagen (Cit-Col or Col) in the presence or absence of Freund's Complete Adjuvant (FCA)., Results: An autoimmune-like RA response was observed in mice immunized with Cit-Col in the absence of FCA; by the increase in caliper score, visual observation, and micro-CT analysis of bone erosions. Antibody and T-cell responses were increased in the Cit-Col injected mice to Cit-Col as well as antibody to Anti-Citrullinated Peptide Antigens (ACPA) as determined by a commercially available test kit., Conclusions: Therefore, the use of citrullinated mouse collagen induces an autoimmune-like RA in the absence of an adjuvant. These data also suggest that citrullinate self-proteins may be potential molecular adjuvants that assist in driving an inflammatory response, that increases the production of PAD in joint tissue, resulting in the citrullination of other self-proteins to exacerbate the disease., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background: Localized scleroderma (LS) is a disfiguring inflammatory autoimmune disease of the skin and underlying tissue. As in systemic sclerosis, a key feature is the presence of T cells in inflammatory lesions., Aim: To evaluate the effect of polymerized type I collagen vs. methylprednisolone (MP) in LS, and to determine the influence of this polymerized collagen (PC) on CD4+ peripheral T cells expressing interleukin (IL)-4, IL-17A, interferon-γ and Forkhead box protein (Foxp)3, and on cells expressing transforming growth factor (TGF)-β1, IL-17A, IL-22 and Foxp3 in the skin., Methods: In total, 16 patients with LS were treated for 3 months with monthly subcutaneous intralesional injections of 0.1 mL MP (giving a total dose of 20 mg/mL each month) and 15 patients were treated, with weekly subcutaneous intralesional injections of PC, ranging from 0.2 mL (equivalent to 1.66 mg collagen) for a lesion of 50 mm in size, up to a maximum of 1.0 mL (8.3 mg collagen) for a lesion > 100 mm in size, and followed up for a further 6 months. Skin biopsies were obtained from lesions at baseline (before treatment) and 9 months later (6 months after treatment end). Tissue sections were evaluated by histology and immunohistochemistry (IL-17A, IL-22, TGF-β1 and Foxp3). CD4+ T-cell subsets were determined in peripheral blood by flow cytometry., Results: Abnormal tissue architecture was seen in the biopsies taken from patients treated with MP, whereas the PC treatment restored normal skin architecture. PC downregulated pro-inflammatory/profibrotic cytokine expression in peripheral cells, and upregulated the number of regulatory T cells (Tregs) in skin. PC was safe and well tolerated., Conclusions: PC is not only an antifibrotic/fibrolytic agent but also an immunomodulator biodrug that restores the balance between T helper (Th)1, Th2, Th17 and Tregs, downregulates production of pro-inflammatory or profibrogenic cytokines (IL-17A, IL-22 and TGF-β1), and renews skin architecture, without adverse effects., (© The Author(s). CED © 2012 British Association of Dermatologists.)

White fat cells secrete adipokines that induce inflammation and obesity has been reported to be characterized by high serum levels of inflammatory cytokines such as IL-6 and TNF-α. Rheumatoid arthritis (RA) is a prototype of inflammatory arthritis, but the relationship between RA and obesity is controversial. We made an obese inflammatory arthritis model: obese collagen-induced arthritis (CIA). C57BL/6 mice were fed a 60-kcal high fat diet (HFD) from the age of 4 weeks and they were immunized twice with type II collagen (CII). After immunization, the obese CIA mice showed higher arthritis index scores and histology scores and a more increased incidence of developing arthritis than did the lean CIA mice. After treatment with CII, mixed lymphocyte reaction also showed CII-specific response more intensely in the obese CIA mice than lean CIA. The anti-CII IgG and anti-CII IgG2a levels in the sera of the obese CIA mice were higher than those of the lean CIA mice. The number of Th17 cells was higher and the IL-17 mRNA expression of the splenocytes in the obese CIA mice was higher than that of the lean CIA mice. Obese CIA mice also showed high IL-17 expression on synovium in immunohistochemistry. Although obesity may not play a pathogenic role in initiating arthritis, it could play an important role in amplifying the inflammation of arthritis through the Th1/Th17 response. The obese CIA murine model will be an important tool when we investigate the effect of several therapeutic target molecules to treat RA.

Skin aging and its clinical manifestation is associated with altered molecular metabolism in the extracellular matrix of the dermis. In a pilot open-label study, we investigated the effect of a dietary supplement, BioCell Collagen(®) (BCC), which contains a naturally occurring matrix of hydrolyzed collagen type II and low-molecular-weight hyaluronic acid and chondroitin sulfate, in 26 healthy females who displayed visible signs of natural and photoaging in the face. Daily supplementation with 1 g of BCC for 12 weeks led to a significant reduction of skin dryness/scaling (76%, P = 0.002) and global lines/wrinkles (13.2%, P = 0.028) as measured by visual/tactile score. Additionally, a significant increase in the content of hemoglobin (17.7%, P = 0.018) and collagen (6.3%, P = 0.002) in the skin dermis was observed after 6 weeks of supplementation. At the end of the study, the increase in hemoglobin remained significant (15%, P = 0.008), while the increase in collagen content was maintained, but the difference from baseline was not significant (3.5%, P = 0.134). This study provides preliminary data suggesting that dietary supplementation with BCC elicits several physiological events which can be harnessed to counteract natural photoaging processes to reduce visible aging signs in the human face. A controlled study is necessary to verify these observations.

Objective: To assess the therapeutic potential of a P2X purinergic receptor antagonist, namely, periodate oxidized ATP, in collagen-induced arthritis (CIA)., Methods: Arthritis was induced in male DBA/1J mice by immunization with type II collagen (CII). Animals showing digit inflammation and paw swelling were treated intraperitoneally with 100 μl of 3 mM oxidized ATP daily for 10 days. At the end of the treatment period, animals were killed and paws were removed for histologic analysis and evaluation of T cell infiltration. Humoral response to CII was analyzed, and specific serum autoantibody levels were correlated with the clinical scores observed in the different treatment groups., Results: Treatment with oxidized ATP resulted in a sustained reduction in disease activity, which was associated with a significant decrease in CD3+ T cell infiltration in arthritic lesions and a significant amelioration of cartilage erosion. Peripheral Treg cells were significantly increased upon P2X blockade in mouse lymph nodes. Moreover, a marked reduction in circulating autoantibodies directed against mouse CII was detected. There was a significant correlation between serum autoantibody levels and the clinical efficacy of oxidized ATP., Conclusion: Our findings indicate that P2X receptor antagonism has important therapeutic potential in chronic inflammatory rheumatic disorders. Taken together, our results underscore the value of the P2X receptor signaling pathway as a potential pharmacologic target for the modulation of adaptive immunity in CIA., (Copyright © 2011 by the American College of Rheumatology.)