Serum Levels of Calreticulin in Correlation with Disease Activity in Patients with Rheumatoid Arthritis

Objective

The aim of our study was to investigate the contribution of serum calreticulin (CRT) in the assessment of disease activity in rheumatoid arthritis (RA).

Methods

Serum CRT levels were measured by ELISA in 70 patients with established RA, 30 systemic lupus erythematosus (SLE), 25 other autoimmune diseases, 20 osteoarthritis (OA), and 35 of healthy controls (HC). Correlations of CRT serum levels with disease activity [Disease Activity Score for 28 joints (DAS28)], erythrocyte sedimentation rate(ESR) and C-reactive protein (CRP) were assessed. Serum CRT levels were also detected in RA patients whose RF, anti-CCP and anti- MCV antibodies were positive and negative.

Results

Serum CRT levels in RA patients (4.817?±?2.425 ng/ml) was significantly higher (P <0.05) compared with those in the serum of OA (3.574?±?0.942 ng/ml), SLE (4.013?±?1.536 ng/ml), other autoimmune diseases (3.882?±?0.837 ng/ml) and HC (3.726?±?0.627 ng/ml). Significant positive correlation of CRT with DAS28, ESR and CRP was found in RA patients. Furthermore, RA patients whose anti-CCP and anti-MCV antibodies were positive had higher levels of CRT (P?<?0.01).

Conclusion

Serum CRT levels were increased in patients with RA compared with those controls. Moreover, a significant correlation was observed between serum CRT levels and disease activity in RA. It might be used as a potential biomarker for clinical diagnosis and provide additional information regarding disease activity along with the traditional indices such as ESR and CRP.     

Molecular Identification of Bacterial DNA in the Chorioretinal Scars of Chronic Granulomatous Disease

Purpose

Chronic granulomatous disease (CGD) is an inherited disorder characterized by defects in phagocyte-derived nicotinamide adenine dinucleotide phosphate oxidase. It is typically diagnosed in childhood and leads to severe, recurrent bacterial or fungal infections. Chorioretinal lesions are the most common ocular manifestation. We sought to determine whether there are infectious agents in CGD-associated chorioretinopathy.

Methods

Medical records and ocular histopathology from CGD cases from January 1983 to January 2012 at the National Institutes of Health were retrospectively reviewed. Chorioretinal cells from normal and lesional tissues of the same eye were microdissected. Primers for Staphylococcus epidermidis, Staphylococcus aureus, Pseudomonas aeruginosa, Burkholderia sp., and a panbacterial 16S ribosomal DNA were used for polymerase chain reaction.

Results

Seventeen CGD patients had ocular tissues (16 autopsied cases and 1 chorioretinal biopsy) examined. Of these 17, 8 demonstrated CGD-associated chorioretinal lesions in at least one eye on histopathology. Of these 8, 7 showed amplification of 16S ribosomal DNA within the lesion; of these 7, two also amplified S. epidermidis and one P. aeruginosa. One had no bacterial DNA amplified. Importantly, no microbial DNA was amplified from the normal, non-lesional ocular tissues of these 8 cases. Furthermore, only 1 of the 9 eyes without chorioretinopathy had amplified Burkholderia DNA, that patient had a history of Burkholderia infection.

Conclusions

We detected bacterial DNA in 7 of 8 (88 %) cases with CGD-associated chorioretinopathy and only in 1 normal ocular tissue of 17 CGD cases. Bacterial infection may play a role in the pathogenesis of CGD-associated chorioretinal lesions.     

Repairing Defect and Preventing Collapse of Canine Femoral Head Using Titanium Implant Enhanced by Autogenous Bone Graft and rhBMP-2

There is a direct relationship between mechanical stress and the progressive collapse of the necrotic region in osteonecrosis of the femoral head. The titanium implant combined with autogenous bone graft and recombinant human bone morphogenetic protein (rhBMP)-2 to repair the defect and prevent collapse of the femoral head was investigated. The femoral head defects were made by the trapdoor procedure and then the defects were filled, respectively, with the titanium implant combined with autogenous bone graft and rhBMP-2, with autogenous bone graft and rhBMP-2, and with autogenous bone graft alone. Roentgenographic and histological examinations were performed at various times postoperatively. The defects were repaired completely and the titanium implant was integrated with the surrounding bone tissues. The defects healed faster than did without rhBMP-2. No trapdoor cartilage collapsed and joint space narrowed. The titanium implant combined with autogenous bone graft and rhBMP-2 can enhance the repairing procedure and prevent the collapse of the femoral head.     

Expression of Interleukin‐22 in Myasthenia Gravis

IL‐17 and IL‐22 are implicated in the pathogenesis of autoimmune diseases. The roles of IL‐22 in the pathophysiology of myasthenia gravis (MG) remain unsettled. The aim of this study was to investigate the possible relationship between serum IL‐22, IL‐17 levels, anti‐acetylcholine receptor antibody (anti‐AChR Ab) titres and clinical parameters in patients with MG. The serum IL‐22, IL‐17 levels and anti‐AChR Ab titres were tested by enzyme‐linked immunosorbent assay (ELISA), while the expression of IL‐22 and IL‐17 mRNAs in peripheral blood mononuclear cells (PBMC) from healthy and MG subjects were detected by quantitative real‐time PCR (qRT‐PCR). Furthermore, PBMC from 12 patients with generalized MG were purified and treated with recombinant human IL‐22 (rhIL‐22), the IL‐17 levels of supernatant were detected by ELISA. We found that the IL‐17 levels were significantly increased, but IL‐22 levels were significantly decreased in the serum of patients with MG compared with healthy controls. Consistantly, a significant decrease in IL‐22 mRNA levels and an increase in IL‐17 mRNA levels were detected in PBMC collected from patients with MG, compared with healthy controls. A negative correlation between IL‐22 mRNA in PBMC, serum IL‐22 and serum anti‐AChR Ab levels was found in patients with MG. Moreover, in cultured MG PBMC treated with recombinant human IL‐22 (rhIL‐22), the IL‐17 levels were decreased in a dose‐dependent manner. Our findings indicated a possible role of IL‐22 as a protective factor in MG.     

In vitro biocompatibility evaluation of bioresorbable copolymers prepared from l-lactide, 1, 3-trimethylene carbonate,and glycolide for cardiovascular applications

PLLA-TMC-GA terpolymer was prepared by ring-opening polymerization of l-lactide, 1, 3-trimethylene carbonate (TMC), and glycolide (GA). The biocompatibility of terpolymer was evaluated in comparison with PLLA and PLLA-TMC with the aim of assessing their potential in the development of bioresorbable cardiovascular stents. Various aspects of in vitro biocompatibility were considered, including MTT assay, hemolytic test, dynamic clotting time, platelet adhesion, platelet activation, protein adsorption, plasma recalcification time and release of cytokines. The results revealed that the terpolymer presents good cytocompatibility and hemocompatibility. Moreover, no significant increase in the release of cytokines was detected. It is thus concluded that these polymers, in particular PLLA-TMC-GA terpolymer present good biocompatibility for cardiovascular applications.     

Inhibition of EGF Signaling Protects the Diabetic Retina from Insulin-Induced Vascular Leakage

Diabetes mellitus is a disease with considerable morbidity and mortality worldwide. Breakdown of the blood–retinal barrier and leakage from the retinal vasculature leads to diabetic macular edema, an important cause of vision loss in patients with diabetes. Although epidemiologic studies and randomized clinical trials suggest that glycemic control plays a major role in the development of vascular complications of diabetes, insulin therapies for control of glucose metabolism cannot prevent long-term retinal complications. The phenomenon of temporary paradoxical worsening of diabetic macular edema after insulin treatment has been observed in a number of studies. In prospective studies on non–insulin-dependent (type 2) diabetes mellitus patients, a change in treatment from oral drugs to insulin was often associated with a significant increased risk of retinopathy progression and visual impairment. Although insulin therapies are critical for regulation of the metabolic disease, their role in the retina is controversial. In this study with diabetic mice, insulin treatment resulted in increased vascular leakage apparently mediated by betacellulin and signaling via the epidermal growth factor (EGF) receptor. In addition, treatment with EGF receptor inhibitors reduced retinal vascular leakage in diabetic mice on insulin. These findings provide unique insight into the role of insulin signaling in mediating retinal effects in diabetes and open new avenues for therapeutics to treat the retinal complications of diabetes mellitus.Diabetic maculopathy, an important cause of vision loss in patients with type 2 diabetes, is characterized by hyperpermeability of retinal blood vessels and subsequent formation of macular edema and hard exudates. Although the increase in retinal vascular permeability occurs both diffusely and in focal regions, the basic physiological defect that causes retinal vascular leakage is unknown. The blood–retinal barrier (BRB) isolates the retina from the bloodstream, establishing a favorable environmental milieu with the regulation of ionic balance, nutrient availability, and blockage of potentially toxic molecules that allows for optimal retinal function. The BRB consists of an inner BRB, formed by endothelial cells lining the retinal blood vessels and the outer BRB formed by the retinal pigment epithelium (RPE), a layer of epithelial cells between the retina and the non-neuronal choroid.^1,2 Disruption of the BRB is an important feature of diabetic retinopathy.Based on data from the Wisconsin Epidemiologic Study of Diabetic Retinopathy (WESDR), a prospective population-based cohort study of patients with type 1 and 2 diabetes mellitus, the prevalence of clinically significant macular edema is 5.9% for type 1 and 7.5% for type 2 diabetes.³ Although epidemiologic studies and randomized clinical trials suggest that glycemic control plays a major role in the development of vascular complications of diabetes,⁴ insulin therapies for control of glucose metabolism may not prevent long-term complications.^5,6 Even though both laser photocoagulation and anti-VEGF therapies have shown significant promise in the treatment of proliferating vessels in proliferative diabetic retinopathy, diabetic macular edema (DME) appears to be more resistant to these treatment approaches, suggesting that other factors might contribute to this complication. We have recently reported the potential role of betacellulin (Btc) in inducing retinal vascular permeability in diabetes.⁷ Clinical trials and other studies have determined that initiation of acute intensive insulin therapy in patients with long-standing poor glycemic control results in a transient worsening of diabetic retinopathy.^8–13 A change in treatment from oral drugs to insulin in patients with non–insulin-dependent (type 2) diabetes mellitus was associated with a significantly increased risk of retinopathy progression and visual impairment.^14–16 In addition, it has been reported that patients who undergo total pancreatectomy for cancer develop severe diabetes because of the complete absence of insulin but rarely if ever develop proliferative diabetic retinopathy,¹⁷ even when they survive for more than one or two decades. These reports led us to model and evaluate the pathophysiological effects of insulin on the retinal vasculature and the potential crosstalk between insulin and Btc in the regulation of retinal vascular permeability.     

腺相关病毒注射稀疏标记神经元方法优化研究

目的 探究利用腺相关病毒(AAV)稀疏标记策略对神经元进行标记时,不同稀释倍数和注射剂量条件下荧光标记神经元的数量、形态及范围差别,为实现神经元投射纤维的精细结构追踪提供参考.方法 利用携带Cre/LoxP控制系统的AAV,采用小鼠颅内微量注射方式进行表达.在梯度稀释编码Cre重组酶的AAV之后,将携带有DIO表达元件的AAV按照1∶1混合比例注射到目标脑区,注射4周后心脏灌流取脑切片,观察荧光表达情况.结果 在稀释20 000倍50 nl注射剂量条件下可提供较为稀疏且范围较小的标记效果,稀释20 000倍150 nl注射剂量则可实现范围更广且较稀疏的标记,但在稀释10000倍150 nl注射剂量条件下,在注射位点中央区域会出现较为密集的标记.结论 通过比较不同AAV稀释倍数和注射剂量条件下神经元标记情况,发现高剂量较低浓度稀释的AAV可达到更佳的表达效果.该研究为利用稀疏标记病毒开展神经环路和神经元树突棘形态的研究提供了实验依据.     

Rbfox-1 contributes to CaMKIIα expression and intracerebral hemorrhage-induced secondary brain injury via blocking micro-RNA-124

RNA-binding protein fox-1 homolog 1 (Rbfox-1), an RNA-binding protein in neurons, is thought to be associated with many neurological diseases. To date, the mechanism on which Rbfox-1 worsens secondary cell death in ICH remains poorly understood. In this study, we aimed to explore the role of Rbfox-1 in intracerebral hemorrhage (ICH)-induced secondary brain injury (SBI) and to identify its underlying mechanisms. We found that the expression of Rbfox-1 in neurons was significantly increased after ICH, which was accompanied by increases in the binding of Rbfox-1 to Ca²⁺/calmodulin-dependent protein kinase II (CaMKIIα) mRNA and the protein level of CaMKIIα. In addition, when exposed to exogenous upregulation or downregulation of Rbfox-1, the protein level of CaMKIIα showed a concomitant trend in brain tissue, which further suggested that CaMKIIα is a downstream-target protein of Rbfox-1. The upregulation of both proteins caused intracellular-Ca²⁺ overload and neuronal degeneration, which exacerbated brain damage. Furthermore, we found that Rbfox-1 promoted the expression of CaMKIIα via blocking the binding of micro-RNA-124 to CaMKIIα mRNA. Thus, Rbfox-1 is expected to be a promising therapeutic target for SBI after ICH.