Antigen-Specific Tolerogenic Dendritic Cells Ameliorate the Severity of Murine Collagen-Induced Arthritis

Dendritic cells (DCs) play important roles in initiation of the pathogenic processes of autoimmune disorders, such as rheumatoid arthritis (RA). Tolerogenic dendritic cells (tolDCs) are generated from naïve DCs and induce T cell tolerance; thus, they represent a promising strategy for specific cellular therapy for autoimmune diseases. In this study, we generated green fluorescent protein (GFP)-labeled tolDCs and confirmed their phenotypes and biological functions. We found that tolDCs suppressed the memory lymphocyte response and exhibited strong tolerogenic potential; thus, these cells show promise for the treatment of autoimmune diseases. Additionally, a collagen-induced arthritis (CIA) mouse model was used to test the role of tolDCs in vivo. The results of a further mechanistic experiment revealed that tolDCs suppressed inflammatory arthritis at least partially by up-regulating regulatory T (Treg) cells. Collectively, our data suggest that tolDCs may be used as a promising alternative therapy for inflammatory arthritis.     

胶原诱导型关节炎大鼠的关节影像学特点

目的旨在分析CIA X线片四肢关节的破坏特点,揭示CIA大鼠关节破坏的规律,为规范评分方案提供依据。方法采用П型胶原和弗氏完全佐剂皮下注射清洁级Wistar大鼠,造模成功（每批10只,共3次）后第35天行全身X线钼靶照片,以正常组作为对照、每只大鼠评价96块骨破坏（erosion）和100个关节间隙（joint space narrowing,JSN）;处死动物,取左前肢和右后肢近端第3足趾关节苏木素-伊红（HE）染色,评价中性粒细胞、淋巴细胞、浆细胞浸润、滑膜增生和软骨破坏的情况。结果造模成功后CIA大鼠关节出现明显的红肿,活动受限;HE病理显示,CIA关节存在明显的中性粒细胞、淋巴细胞和浆细胞浸润,滑膜增生,纤维组织增生,软骨破坏;X线片分析结果显示：①广泛性骨质疏松,边缘性骨质侵蚀,关节间隙狭窄或增宽,部分踝关节间隙消失,关节相互融合甚至骨性强直。②67%的骨出现erosion,JSN影响为78%,关节破坏以中、重度为主;③远端、近端趾间关节和踝关节发病率高,损害严重,掌趾关节发病率低,破坏较轻。④后肢关节破坏重于前肢（P〈0.01）,左右肢没有显著性差异（P〉0.05）。结论①滑膜是CIA炎症反应启动的主要病灶,与骨交界的滑膜和血管翳造成了CIA的骨质破坏;②CIA影像学表现关节破坏严重,以远端、近端趾间关节和踝关节为主,这些关节可作为评价破坏程度的选择。本研究对于深入CIA关节破坏的病因病理和进一步规范X线片评分方案具有一定意义。     

Wistar大鼠胶原诱导的关节炎造模因素的分析

目的通过比较不同的造模方法,分析影响胶原诱导关节炎（collagen-induced arthritis,CIA）大鼠模型建立的因素。方法选用Wistar大鼠造模,通过改变性别、剂量、年龄、注射部位及免疫方式,来比较造模成功率。结果不同性别、剂量、年龄及免疫方式造模成功率不同。结论4～5周龄Wistar雌性大鼠,初次免疫注射乳剂4点共0.20 mL,加强免疫在14 d之后3点共0.15 mL的造模成功率最高。     

Anti-Arthritic Effect of Chebulanin on Collagen-Induced Arthritis in Mice

Rheumatoid arthritis is a chronic degenerative autoimmune disease characterized by persistent inflammation of synovial membranes, which leads to cartilage destruction and bone erosion. To date, there are no effective therapies to slow the progress of this degenerative condition. Here, we evaluate the anti-arthritic effect of chebulanin, an abundant anti-inflammatory agent isolated from Terminalia chebula, in collagen induced arthritis in DBA/1 mice by intragastric administration. Arthritic severity was scored by performing histopathological evaluation of the joints and measuring the expression of inflammatory cytokines and relative enzymes by immunohistochemical staining. In parallel, bone destruction and erosion were confirmed by micro-CT. Our data revealed that chebulanin significantly improved the severity of arthritis. Specifically, the histopathological characteristics of the tissues were improved and expression of TNF-α, IL-6, MMP-3 and COX-2 in the paws and joints of the treated mice decreased in a dose-dependent manner compared with control mice. Furthermore, micro-CT analysis revealed that chebulanin induced a dose-dependent reduction in cartilage destruction and bone erosion. Taken together, our findings suggest that chebulanin suppresses the expression of inflammatory mediators and prevents cartilage destruction and bone erosion in mice. Therefore, chebulanin is a strong therapeutic alternative for the treatment of RA.     

Prophylactic Injection of Recombinant Alpha-Enolase Reduces Arthritis Severity in the Collagen-Induced Arthritis Mice Model

Objective

To evaluate the ability of the glycolytic enzyme alpha-enolase (ENO1) or its immunodominant peptide (pEP1) to reduce the severity of CIA in DBA/1 mice when injected in a prophylactic way.

Methods

Mice were treated with mouse ENO1 or pEP1 one day prior to collagen II immunization. Clinical assessment was evaluated using 4 parameters (global and articular scores, ankle thickness and weight). Titers of serum anti-ENO1, anti-cyclic citrullinated peptides (anti-CCP) and anti-CII (total IgG and IgG1/IgG2a isotypes) antibodies were measured by ELISA at different time-points. Disease activity was assessed by histological analysis of both anterior and hind paws at the end of experimentation.

Results

Prophylactic injection of 100 μg of ENO1 reduced severity of CIA. Serum levels of anti-CII antibodies were reduced in ENO1-treated mice. Concordantly, ENO1-treated mice joints presented less severe histological signs of arthritis. ENO1 did not induce a shift toward a Th2 response since IgG1/IgG2a ratio of anti-CII antibodies remained unchanged and IL-4 serum levels were similar to those measured in the control group.

Conclusions

Pre-immunization with ENO1 or its immunodominant peptide pEP1 reduces CIA severity at the clinical, immunological and histological levels. Effects of pEP1 were less pronounced. This immunomodulatory effect is associated with a reduction in anti-CII antibodies production but is not due to a Th1/Th2 shift.     

Age-Related Differences in Collagen-Induced Arthritis: Clinical and Imaging Correlations

Arthritis is among the most common chronic diseases in both children and adults. Although intraarticular inflammation is the feature common among all patients with chronic arthritis there are, in addition to age at onset, clinical characteristics that further distinguish the disease in pediatric and adult populations. In this study, we aimed to demonstrate the utility of microCT (µCT) and ultrasonography in characterizing pathologic age-related differences in a collagen-induced arthritis (CIA) rat model. Juvenile (35 d old) and young adult (91 d old) male Wistar rats were immunized with bovine type II collagen and incomplete Freund adjuvant to induce polyarthritis. Naïve male Wistar rats served as controls. All paws were scored on a scale of 0 (normal paw) to 4 (disuse of paw). Rats were euthanized at 14 d after the onset of arthritis and the hindpaws imaged by µCT and ultrasonography. Young adult rats had more severe signs of arthritis than did their juvenile counterparts. Imaging demonstrated that young adult CIA rats exhibited more widespread and severe skeletal lesions of the phalanges, metatarsals, and tarsal bones, whereas juvenile CIA rats had more localized and less proliferative and osteolytic damage that was confined predominantly to the phalanges and metatarsals. This report demonstrates the utility of imaging modalities to compare juvenile and young adult rats with CIA and provides evidence that disease characteristics and progression differ between the 2 age groups. Our observations indicate that the CIA model could help discern age-related pathologic processes in inflammatory joint diseases.Abbreviations: μCT, microCT, CIA, collagen-induced arthritisArthritis is among the most common diseases in both children and adults. In children, growth, hormonal changes, and neuroimmune responsiveness and plasticity might confer influences on arthritic processes not seen in adults. Age-dependent outcomes have been demonstrated by using animal models of osteoarthritis;^5,6,11 however, there is limited information about how pathogenic processes vary among different age groups with experimental inflammatory arthritis in general^7,10 and in the collagen-induced arthritis (CIA) model of arthritis in particular.⁸ Studying and comparing features of the CIA model in growing compared with mature rats can be undertaken in ways that are not possible in humans and could help to understand age-related pathologic differences in children and adults with inflammatory joint diseases. Therefore, we undertook to compare clinical and imaging features of CIA in juvenile (growing) compared with young adult (mature) rats.In some animal models, collagen immunization results in a monophasic, polyarticular, inflammatory arthritis that is mediated by an autoimmune response¹⁴ and that is histopathologically similar to rheumatoid arthritis.^3,9,13 CIA predominantly affects the peripheral appendicular joints and is characterized by intense synovitis and pannus formation, with consequent erosions of cartilage and subchondral bone.^12,15Here we report that juvenile and young adult rats differ in their clinical responses to collagen immunization and that, according to findings from microCT (µCT) and ultrasonography, juvenile and young adult rats differ in their responses to collagen immunization with regard to disease pathology.     

A Metabolically-Stabilized Phosphonate Analog of Lysophosphatidic Acid Attenuates Collagen-Induced Arthritis

Rheumatoid arthritis (RA) is a destructive arthropathy with systemic manifestations, characterized by chronic synovial inflammation. Under the influence of the pro-inflammatory milieu synovial fibroblasts (SFs), the main effector cells in disease pathogenesis become activated and hyperplastic while releasing a number of signals that include pro-inflammatory factors and tissue remodeling enzymes. Activated RA SFs in mouse or human arthritic joints express significant quantities of autotaxin (ATX), a lysophospholipase D responsible for the majority of lysophosphatidic acid (LPA) production in the serum and inflamed sites. Conditional genetic ablation of ATX from SFs resulted in attenuation of disease symptoms in animal models, an effect attributed to diminished LPA signaling in the synovium, shown to activate SF effector functions. Here we show that administration of 1-bromo-3(S)-hydroxy-4-(palmitoyloxy)butyl-phosphonate (BrP-LPA), a metabolically stabilized analog of LPA and a dual function inhibitor of ATX and pan-antagonist of LPA receptors, attenuates collagen induced arthritis (CIA) development, thus validating the ATX/LPA axis as a novel therapeutic target in RA.     

Neurostimulation of the Cholinergic Anti-Inflammatory Pathway Ameliorates Disease in Rat Collagen-Induced Arthritis

Introduction

The inflammatory reflex is a physiological mechanism through which the nervous system maintains immunologic homeostasis by modulating innate and adaptive immunity. We postulated that the reflex might be harnessed therapeutically to reduce pathological levels of inflammation in rheumatoid arthritis by activating its prototypical efferent arm, termed the cholinergic anti-inflammatory pathway. To explore this, we determined whether electrical neurostimulation of the cholinergic anti-inflammatory pathway reduced disease severity in the collagen-induced arthritis model.

Methods

Rats implanted with vagus nerve cuff electrodes had collagen-induced arthritis induced and were followed for 15 days. Animals underwent active or sham electrical stimulation once daily from day 9 through the conclusion of the study. Joint swelling, histology, and levels of cytokines and bone metabolism mediators were assessed.

Results

Compared with sham treatment, active neurostimulation of the cholinergic anti-inflammatory pathway resulted in a 52% reduction in ankle diameter (p = 0.02), a 57% reduction in ankle diameter (area under curve; p = 0.02) and 46% reduction overall histological arthritis score (p = 0.01) with significant improvements in inflammation, pannus formation, cartilage destruction, and bone erosion (p = 0.02), accompanied by numerical reductions in systemic cytokine levels, not reaching statistical significance. Bone erosion improvement was associated with a decrease in serum levels of receptor activator of NF-κB ligand (RANKL) from 132±13 to 6±2 pg/mL (mean±SEM, p = 0.01).

Conclusions

The severity of collagen-induced arthritis is reduced by neurostimulation of the cholinergic anti-inflammatory pathway delivered using an implanted electrical vagus nerve stimulation cuff electrode, and supports the rationale for testing this approach in human inflammatory disorders.     

HFJ和Wistar大鼠脂肪肝及胰岛素抵抗动物模型的比较

目的建立近交系HFJ大鼠和封闭群Wistar大鼠脂肪肝胰岛素抵抗动物模型及比较其生物学特性。方法雄性HFJ大鼠和Wistar大鼠,分别随机分为模型组和正常组,模型组喂养高脂饮食,正常组喂养普通饮食,两组脂肪分别占摄入能量的44.2%和19.2%,共饲养12周。每周称体质量,测定血糖、甘油三酯、胆固醇、ALT、AST、HDLC、LDLC和血胰岛素水平。实验期末处死动物摘取肝脏并称质量,计算肝指数;鼠肝脏用10%甲醛固定,石蜡包埋切片,HE染色,光镜下评估肝脂肪变性和炎症活动情况。结果镜下可见,HFJ和Wistar大鼠模型组肝细胞均呈现弥漫性脂肪变性,小叶内可见炎症细胞浸润,HFJ比Wistar脂肪变性较重,对照组肝脏均未见异常。两种动物的模型组ALT、AST、肝指数、HOMA-IR指数均显著高于其正常组,HFJ和Wistar种系间各指标的差异无显著性;HFJ大鼠模型组体重和正常组体重具有显著性差异(P<0.01),而Wistar大鼠模型组体重与正常组体重间无显著差异(P>0.05);HFJ大鼠TG和TC含量均显著高于Wistar大鼠。结论通过高脂饮食喂养成功建立了HFJ大鼠和Wistar大鼠脂肪肝与胰岛素抵抗疾病动物模型,与Wistar大鼠比较,HFJ大鼠具有自发性高血脂特征,造模更易成功,可为胰岛素抵抗和脂肪肝的发病机制研究、防治高血脂药物筛选提供一种新的实验动物。     

Sympathetic Neurotransmitters Modulate Osteoclastogenesis and Osteoclast Activity in the Context of Collagen-Induced Arthritis

Excessive synovial osteoclastogenesis is a hallmark of rheumatoid arthritis (RA). Concomitantly, local synovial changes comprise neuronal components of the peripheral sympathetic nervous system. Here, we wanted to analyze if collagen-induced arthritis (CIA) alters bone marrow-derived macrophage (BMM) osteoclastogenesis and osteoclast activity, and how sympathetic neurotransmitters participate in this process. Therefore, BMMs from Dark Agouti rats at different CIA stages were differentiated into osteoclasts in vitro and osteoclast number, cathepsin K activity, matrix resorption and apoptosis were analyzed in the presence of acetylcholine (ACh), noradrenaline (NA) vasoactive intestinal peptide (VIP) and assay-dependent, adenylyl cyclase activator NKH477. We observed modulation of neurotransmitter receptor mRNA expression in CIA osteoclasts without affecting protein level. CIA stage-dependently altered marker gene expression associated with osteoclast differentiation and activity without affecting osteoclast number or activity. Neurotransmitter stimulation modulated osteoclast differentiation, apoptosis and activity. VIP, NA and adenylyl cyclase activator NKH477 inhibited cathepsin K activity and osteoclastogenesis (NKH477, 10^-6M NA) whereas ACh mostly acted pro-osteoclastogenic. We conclude that CIA alone does not affect metabolism of in vitro generated osteoclasts whereas stimulation with NA, VIP plus specific activation of adenylyl cyclase induced anti-resorptive effects probably mediated via cAMP signaling. Contrary, we suggest pro-osteoclastogenic and pro-resorptive properties of ACh mediated via muscarinic receptors.     

Globule Leukocyte And Mast Cell in Bile Ducts of Cattle Naturally Infected with Liver Flukes

In n previous report dealing with the pathology of bovine fascio-liasis the author described an unknown cell type in the epithelium of bile ducts. The histological and histochemical investigations published in this paper suggest that the cell may be considered a globule leukocyte. Globule leukocytes are rare in uninfected livers but are occurring in abundance in main bile ducts of cattle with spontaneous fascioliasis and also in small perilobular ducts in dicrocoeliasis. Liver fluke infection causes an increase in the population of subepithelial mast cells. Mast cell and globule leukocyte present similarities In their cytochemical properties. However, at low pH toluldinc blue shows a stronger but Alcian blue a weaker affinity for mast cells than for globule leukocytes.     

Cinnamic Acid and Cinnamaldehyde Ameliorate Cisplatin‐Induced Splenotoxicity in Rats

Cinnamic acid (CA) and cinnamaldehyde (CD) are major constituents of cinnamon species. They possess various pharmacological properties of which their antioxidant activity is a prime one. This study aims to investigate potential protective effects against cisplatin (CP)‐induced splenotoxicity in rats. A single dose of CP (5 mg/kg) injected i.p. caused a significant decrease in hemoglobin content (18%), total leucocytic count (46%), neutrophils (78%), and catalase (CAT) splenic activity (64%) with a marked increase in lymphocytes (26%) and splenic content of malondialdehyde (68%) and TNF‐α (69%) as compared with the control group. Contrarily, CA (50 mg/kg, p.o.) or CD (40 mg/kg, p.o.) administration for 7 days before CP ameliorated CP‐induced splenotoxicity as indicated by mitigation of the biochemical parameters and histopathological changes. These results revealed the promising protective effects of CA and CD on CP‐induced splenotoxicity in rats; an effect that might be attributed to antioxidant and anti‐inflammatory activities.     

Berberine Ameliorate Oxidative Stress and Astrogliosis in the Hippocampus of STZ-Induced Diabetic Rats

Diabetes mellitus increases the risk of central nervous system (CNS) disorders such as stroke, seizures, dementia, and cognitive impairment. Berberine, a natural isoquinoline alkaloid, is reported to exhibit beneficial effect in various neurodegenerative and neuropsychiatric disorders. Moreover, astrocytes are proving critical for normal CNS function, and alterations in their activity and impaired oxidative stress could contribute to diabetes-related cognitive dysfunction. Metabolic and oxidative insults often cause rapid changes in glial cells. Key indicators of this response are increased synthesis of glial fibrillary acidic protein (GFAP) as an astrocytic marker. Therefore, we examined the effects of berberine on glial reactivity of hippocampus in streptozotocin (STZ)-induced diabetic rats, using GFAP immunohistochemistry. Lipid peroxidation, superoxide dismutase (SOD) activity, and nitrite levels were assessed as the parameters of oxidative stress. Eight weeks after diabetes induction, we observed increased numbers of GFAP⁺ astrocytes immunostaining associated with increased lipid peroxidation, decreased superoxide dismutase activity, and elevated nitrite levels in the hippocampus of STZ-diabetic rats. In contrast, chronic treatment with berberine (50 and 100 mg/kg p.o. once daily) lowered hyperglycemia, reduced oxidative stress, and prevented the upregulation of GFAP in the brain of diabetic rats. In conclusion, the present study demonstrated that the treatment with berberine resulted in an obvious reduction of oxidative stress and GFAP-immunoreactive astrocytes in the hippocampus of STZ-induced diabetic rats.

The Anabolic Androgenic Steroid Nandrolone Decanoate Disrupts Redox Homeostasis in Liver,Heart and Kidney of Male Wistar Rats

The abuse of anabolic androgenic steroids (AAS) may cause side effects in several tissues. Oxidative stress is linked to the pathophysiology of most of these alterations, being involved in fibrosis, cellular proliferation, tumorigenesis, amongst others. Thus, the aim of this study was to determine the impact of supraphysiological doses of nandrolone decanoate (DECA) on the redox balance of liver, heart and kidney. Wistar male rats were treated with intramuscular injections of vehicle or DECA (1 mg.100 g⁻¹ body weight) once a week for 8 weeks. The activity and mRNA levels of NADPH Oxidase (NOX), and the activity of catalase, glutathione peroxidase (GPx) and total superoxide dismutase (SOD), as well as the reduced thiol and carbonyl residue proteins, were measured in liver, heart and kidney. DECA treatment increased NOX activity in heart and liver, but NOX2 mRNA levels were only increased in heart. Liver catalase and SOD activities were decreased in the DECA-treated group, but only catalase activity was decreased in the kidney. No differences were detected in GPx activity. Thiol residues were decreased in the liver and kidney of treated animals in comparison to the control group, while carbonyl residues were increased in the kidney after the treatment. Taken together, our results show that chronically administered DECA is able to disrupt the cellular redox balance, leading to an oxidative stress state.     

莫诺苷对大鼠局灶性脑缺血再灌注损伤神经功能的影响

目的观察中药山茱萸的有效成分莫诺苷对大鼠局灶性脑缺血再灌注后神经功能的影响。方法Wistar雄性大鼠随机分为假手术组、模型组、莫诺苷小剂量组（30 mg/kg）、莫诺苷中剂量组（90 mg/kg）、莫诺苷大剂量组（270 mg/kg）、维生素E（VE）（35 mg/kg）,采用线栓法制作大鼠局灶性脑缺血再灌注模型,缺血30 min后再灌注3 d,应用Zea Longa法、爬网格、平行木、吊绳、Ludmila Belayev 12分评分法,观察莫诺苷对神经功能缺损的改善作用。结果与模型组比较,莫诺苷给药组（小、中、大剂量）Zea Longa法评分均差异极显著（P〈0.01）;与模型组比较,莫诺苷给药组（小、中、大剂量）吊绳法评分均差异极显著（P〈0.01）;Ludmila Belayev 12分评分法评分与模型组比较,莫诺苷大剂量组差异极显著（P〈0.001）,中剂量组差异极显著（P〈0.01）。结论莫诺苷对局灶性脑缺血再灌注模型大鼠有改善行为学评分的作用。     

Genome Edited Sirt1-Overexpressing Human Mesenchymal Stem Cells Exhibit Therapeutic Effects in Treating Collagen-Induced Arthritis

Even though mesenchymal stem cells (MSCs) are known for cartilage regeneration, their therapeutic efficacy needs to be enhanced. In the present study, we produced genome-edited silent information regulator 2 type 1 (Sirt1)-overexpressing MSCs, and evaluated their therapeutic potential in a damaged cartilage mouse liver fibrosis model. The Sirt1 gene was successfully inserted into a ‘safe harbor’ genomic locus in amniotic mesenchymal stem cells (AMMs), and the chondrogenic properties of the Sirt1 gene overexpressing AMMs (AMM/S) were characterized using quantitative PCR and histology. Therapeutic potentials were investigated in a collagen-induced arthritis (CIA) mouse model. Chondrocyte-differentiated AMM/S expressed cartilage-specific genes and were positive for Safranin O staining. Transplantation of AMM/S attenuated CIA progression and suppressed T helper (Th)-17 cell activation while increasing the Treg cell population in CIA mice. Pro-inflammatory factors, such as interleukin (IL)-1β, IL-6, monocyte chemoattractant protein (MCP)-1, and tumor necrosis factor (TNF)-α were significantly decreased in AMM/S-injected joint tissues. In conclusion, genome-edited AMM/S may represent a safe and alternative therapeutic option for the treatment and repair of damaged cartilage, or in inflammatory joint arthritis.