The long-term immunosuppressive effects of disulfide-linked HLA-G dimer in mice with collagen-induced arthritis

HLA-G, a natural immunosuppressant present in the human placenta during pregnancy, prevents fetal destruction by the maternal immune system. The immunosuppressive effect of HLA-G is mediated by the immune cell inhibitory receptors, LILRB1 and LILRB2. HLA-G forms disulfide-linked dimers by natural oxidation, and the dimer associates with LILRB1/B2 much more strongly than the monomer. Furthermore, the dimer formation remarkably enhanced the LILRB-mediated signaling. In this report, we studied the in vivo immunosuppressive effect of the HLA-G dimer, using the collagen-induced arthritis model mouse. Mice were treated with the HLA-G monomer or dimer intracutaneously at the left foot joint, once or for 5 days, and the clinical severity was evaluated daily in a double-blind study. The HLA-G monomer and dimer both produced excellent anti-inflammatory effects with a single, local administration. Notably, as compared to the monomer, the dimer exhibited significant immunosuppressive effects at lower concentrations, which persisted for about two months. In accordance with this result, a binding study revealed that the HLA-G dimer binds PIR-B, the mouse homolog of the LILRBs, with higher affinity and avidity than the monomer. The HLA-G dimer is expected to be quite useful as an anti-rheumatoid arthritis agent, in small amounts with minimal side effects.     

The inter-observer reading variability in anti-nuclear antibodies indirect (ANA) immunofluorescence test: A multicenter evaluation and a review of the literature

Recently there has been an increase demand for Computer-Aided Diagnosis (CAD) tools to support clinicians in the field of Indirect ImmunoFluorescence (IIF), as the novel digital imaging reading approach can help to overcome the reader subjectivity. Nevertheless, a large multicenter evaluation of the inter-observer reading variability in this field is still missing. This work fills this gap as we evaluated 556 consecutive samples, for a total of 1679 images, collected in three laboratories with IIF expertise using HEp-2 cell substrate (MBL) at 1:80 screening dilution according to conventional procedures. In each laboratory, the images were blindly classified by two experts into three intensity classes: positive, negative, and weak positive. Positive and weak positive ANA-IIF results were categorized by the predominant fluorescence pattern among six main classes. Data were pairwise analyzed and the inter-observer reading variability was measured by Cohen's kappa test, revealing a pairwise agreement little further away than substantial both for fluorescence intensity and for staining pattern recognition (k = 0.602 and k = 0.627, respectively). We also noticed that the inter-observer reading variability decreases when it is measured with respect to a gold standard classification computed on the basis of labels assigned by the three laboratories. These data show that laboratory agreement improves using digital images and comparing each single human evaluation to potential reference data, suggesting that a solid gold standard is essential to properly make use of CAD systems in routine work lab.     

Anti-TNF therapy in patients with rheumatoid arthritis decreases Th1 and Th17 cell populations and expands IFN-γ-producing NK cell and regulatory T cell subsets

The aim of this work was to study the effect of anti-TNF treatment on CD4+ Th1, Th17 and regulatory T cells (Tregs), together with CD8+ T cells and NK cells from rheumatoid arthritis (RA) patients. For this purpose, 18 RA patients received adalimumab during 16 weeks and their peripheral blood lymphocytes were assessed by flow cytometry at the beginning and at the end of the study. We found that the proportion of Th17 cells was directly correlated with Th1 cells, but inversely correlated with IFN-γ-producing NK cells. A decrease was observed in Th1, Th17 cells and IFN-γ-producing CD8+ T cells by anti-TNF therapy. Conversely, the proportion of Tregs increased, as did the percentage of IFN-γ-producing NK cells. We postulate that a rise in IFN-γ production due to recovery of NK cells’ function, together with expanded Tregs, contribute to decrease the Th17 response in anti-TNF-treated RA patients.     

Psychiatry and political-institutional abuse from the historical perspective: the ethical lessons of the Nuremberg Trial on their 60th anniversary

Sixty years ago at the Nuremberg Trials, 23 Nazi leaders were tried as war criminals, in what was known as "The Doctors' Trial". This trial exposed a perverse system of the criminal use of medicine in the fields of public health and human research. These practices, in which racial hygiene constituted one of the fundamental principles and euthanasia programmes were the most obvious consequence, violated the majority of known bioethical principles. Psychiatry played a central role in these programmes, and the mentally ill were the principal victims. The aim of the present work is to review, from the historical perspective, the antecedents of the shameful euthanasia programmes for the mentally ill, the procedures involved in their implementation and the use of mentally ill people as research material. The Nuremberg Code, a direct consequence of the Doctors' Trial, is considered to be the first international code of ethics for research with human beings, and represented an attempt to prevent any repeat of the tragedy that occurred under Nazism. Nevertheless, the last 60 years have seen continued government-endorsed psychiatric abuse and illegitimate use of psychoactive drugs in countries such as the Soviet Union or China, and even in some with a long democratic tradition, such as the United States. Even today, the improper use of psychiatry on behalf of governments is seen to be occurring in numerous parts of the globe: religious repression in China, enforced hospitalization in Russia, administration of psychoactive drugs in immigrant detention centres in Australia, and the application of the death penalty by lethal injection and psychiatric participation in coercive interrogation at military prisons, in relation to the USA. The Declaration of Madrid in 1996 constituted the most recent attempt to eradicate, from the ethical point of view, these horrendous practices. Various strategies can be used to combat such abuses, though it is uncertain how effective they are in preventing them.     

Expression and localization analyses of the cholinergic anti-inflammatory pathway and α7nAchR in different tissues of rats with rheumatoid arthritis

Rheumatoid arthritis (RA) is a complicated chronic multisystem autoimmune disease, wherein the inflammatory cascade leads to vasospasm and osteoclastogenesis, which ultimately results in bone and cartilage destruction. In this study, we investigated the expression and localization of the alpha-7 nicotinic receptor (α7nAchR) gene CHRNA7 in the heart, liver, spleen, lung, kidney, and joints of the collagen-induced arthritis (CIA) rat model. The CHRNA7 mRNA and protein expression levels in these tissues of rats from CIA and normal groups were analyzed via real-time quantitative polymerase chain reaction (RT-qPCR) and western blotting, respectively. The cellular localization of CHRNA7 protein was determined via immunohistochemistry (IHC) assays. CHRNA7 was expressed at varying levels in different tissues of rats from the groups, among which joints showed significantly higher CHRNA7 expression levels than other tissues (P < 0.05). CIA rats had significantly higher CHRNA7 expression levels in the spleen and joints than the control group rats (P < 0.05). Positive expression signals for CHRNA7 were detected in various tissues of CIA and control group rats, among which strong positive signals were detected in joint fibroblast-like synoviocytes (FLSs), endothelial cells, stromal cells, and macrophages. Our results further confirmed the involvement of the CAP in the onset and development of inflammatory responses in RA, suggesting that CHRNA7 may be a new therapeutic target for RA. This study is of great clinical and theoretical significance for understanding the differential expression of CHRNA7 in various tissues and cholinergic anti-inflammatory pathway (CAP)-targeted treatment of RA.     

Therapeutic effect of long‐interval repeated intravenous administration of human umbilical cord blood‐derived mesenchymal stem cells in DBA/1 mice with collagen‐induced arthritis

Rheumatoid arthritis (RA) is a common inflammatory chronic disease. It has been reported that mesenchymal stem cells (MSCs) have the effect of immune suppression in collagen‐induced arthritis (CIA) mice model. However, the in vivo therapeutic effect from the long‐interval repeated intravenous administration of human umbilical cord blood‐derived (hUCB)‐MSCs had not been investigated in CIA mice model. This study was undertaken to investigate the effects of long‐interval repeated intravenous administration of hUCB‐MSCs at different doses in CIA mice model. Mice were intravenously injected with three different doses of hUCB‐MSCs once every 2 weeks for three times. RA severity was assessed by clinical joint score and histologic analysis including hematoxylin and eosin staining, safranin‐O staining, and toluidine blue staining. We used real‐time polymerase chain reaction and flow cytometry to quantify differences in inflammatory cytokines and Tregs. Mice treated with hUCB‐MSCs showed significant improvement in clinical joint score. Histologic analysis revealed that hUCB‐MSCs definitely reduced joint inflammation, cartilage damage, and formation of pannus in multimedium and multihigh groups. These hUCB‐MSCs also significantly decreased IL‐1 beta protein levels in multimedium and multihigh groups and IL‐6 protein levels in all hUCB‐MSCs‐treated groups. Furthermore, mRNA levels of IL‐1 beta and IL‐6 were decreased significantly in all hUCB‐MSCs‐treated groups, whereas the expression of anti‐inflammatory cytokine IL‐10 was increased in the multihigh group. Tregs known as suppressor T cells were also significantly increased in the multihigh group. Our findings suggest that long‐interval repeated intravenous administration of hUCB‐MSCs has therapeutic effects by improving symptoms of RA in CIA mice model in a dose‐dependent manner.     

Suppression of collagen-induced arthritis with a serine proteinase inhibitor (serpin) derived from myxoma virus

Many viruses encode virulence factors to facilitate their own survival by modulating a host's inflammatory response. One of these factors, secreted from cells infected with myxoma virus, is the serine proteinase inhibitor (serpin) Serp-1. Because Serp-1 had demonstrated anti-inflammatory properties in arterial injury models and viral infections, it was cloned and evaluated for therapeutic efficacy in collagen-induced arthritis (CIA). Clinical severity was significantly lower in the Serp-1 protocols (p < 0.0001) and blinded radiographs indicated that the Serp-1 group had significantly less erosions than the controls (p < 0.01). Delayed-type hypersensitivity was lower in the Serp-1 group but antibody titers to type II collagen were not significantly altered. Recipients had minimal histopathologic synovial changes and did not develop neutralizing antibodies to Serp-1. These results indicate that Serp-1 impedes the pathogenesis of CIA and suggests that the therapeutic potential of serine proteinase inhibitors in inflammatory joint diseases, such as rheumatoid arthritis, should be investigated further.     

中性粒细胞NETs介导类风湿关节炎疾病的进展研究

目的: 探索下调中性粒细胞能否缓解胶原诱导关节炎(collagen induced arthritis,CIA)小鼠疾病程度,结合测序分析类风湿关节炎(rheumatoid arthritis,RA)患者中性粒细胞外捕获网(neutrophil extratrapping networks,NETs)形成后的转录组表达差异,为NETs影响RA疾病进展的后续研究提供一定的理论基础。方法: 利用DBA1小鼠构建CIA模型,在二次胶原强化免疫后利用抗Ly6G单克隆抗体敲减CIA小鼠体内中性粒细胞(1次/周),持续干预8周后,处死小鼠采集肺、脾、小鼠后肢进行组织病理学观察;磁珠分选法收集4例RA患者外周血中性粒细胞,DAPI染料对中性粒细胞核进行染色,室温避光静置15 min后,镜下观察中性粒细胞形态特征;利用不同浓度(8 nmol/L、16 nmol/L、32 nmol/L)佛波醇-12-肉豆蔻酸酯-13-乙酸酯(phorbol-12-myristate-13-acetate,PMA)刺激后培养2 h并进行NETs标志物染色,荧光倒置显微镜下观察刺激中性粒细胞形成NETs的最佳浓度;利用PMA激活中性粒细胞后收集样本进行转录组测序,分析RA中性粒细胞NETs形成后的转录组表达变化。结果: 中性粒细胞表达下调部分缓解CIA小鼠关节滑膜、肺及脾的病理改变;8 nmol/L PMA刺激RA中性粒细胞放置2 h、37 ℃、5 % CO₂培养箱中形成NETs的形态最佳;差异分析筛选出2 742个差异基因(P≤0.05),其中包括1 579个上调差异基因[log₂(RA_control/RA_PMA＞0)]和1 163个下调差异基因[log₂(RA_control/RA_PMA＜0],从中筛选出340个显著差异基因(log₂|RA_control/RA_PMA|≥2),进行PPI网络构建进行展示;340个差异基因的GO、KEGG和GSEA KEGG通路分析分别富集较多为免疫回应和信号受体活性的调控、细胞因子之间相互调控通路、细胞因子受体互作、凋亡和Toll受体信号通路中。结论: 下调中性粒细胞可缓解CIA小鼠关节滑膜、肺及脾的病理改变;RA的NETs形成与免疫调控、凋亡以及细胞因子之间相互作用通路相关,表明NETs的形成可能参与以上免疫过程,对RA疾病进展起重要的作用。     

复方丹参片对类风湿性关节炎大鼠胫骨形态计量学静态参数的影响

目的研究复方丹参片对大鼠类风湿相关骨质疏松的对抗作用。方法利用皮下注射牛Ⅱ型胶原诱导SD大鼠发病,建立胶原诱导性关节炎（collagen induced arthritis,CIA）大鼠模型,设立正常对照组、实验对照组和实验组（复方维生素D组及不同剂量的复方丹参组）,分别给予灭菌用水、复方维生素D及不同剂量的复方丹参处理2月后,比较各组大鼠胫骨的干重、湿重、长度、周径以及骨组织形态计量学静态参数。结果实验各组及实验对照组中大鼠胫骨的湿重、干重及骨组织形态计量学静态参数显著低于正常对照组,而实验各组大鼠胫骨的上述参数显著高于实验对照组。结论复方丹参能部分抵抗类风湿疾病引起的骨质破坏和骨质疏松。