Type 1 and type 2 cytokine-producing CD4+ and CD8+ T cells in primary antiphospholipid syndrome

Antiphospholipid syndrome (APS) is an autoimmune condition characterized by thrombosis and/or recurrent fetal loss as well as the presence of autoantibodies against epitopes present on phospholipid-binding proteins. The role of cellular immunity in the pathogenesis of the syndrome remains unclear. We studied the cellular phenotype and the production of type 1 [interferon (IFN)-, interleukin (IL)-2] and type 2 (IL-4, IL-10) cytokines by CD4+ and CD8+ T-lymphocyte subsets in 13 patients with untreated primary APS (PAPS) and in 32 healthy controls. The production of cytokines was determined in T cells after a 5-h culture with or without mitogenic stimulation using a flow cytometric method of intracellular cytokine staining. In six of the patients these studies were repeated 6 months later. In PAPS patients we found a reduced percentage of circulating CD4+CD45RA+ and an increased percentage and absolute number of CD8+HLA-DR+ cells. A type 1 response was observed in the patients unstimulated cells, indicated by an increase in IFN--producing CD8+, IL-2-producing CD4+ T cells, and a decrease in IL-4-producing CD4+ and CD8+ T cells. Similar results were obtained in the patients at follow-up. Taken together, these results suggest a chronic in vivo stimulation of CD4+ and CD8+ T cells in PAPS patients exhibiting a type 1 polarization. Changes of cellular immunity may contribute to the pathogenesis of the clinical manifestations of the syndrome and might be proven to be useful targets for therapeutic interventions in the future.     

Smoothelin is an indicator of reversible phenotype modulation of smooth muscle cells in balloon-injured rat carotid arteries

Restenosis is the major obstacle interfering with a successful long-term outcome of balloon angioplasty. Neointima formation following endothelial injury is the result of phenotype modulation and proliferation of smooth muscle cells (SMC). To characterize these time-dependent changes, a rat balloon injury model of carotid artery restenosis was assessed. We applied monoclonal antibodies recognizing desmin, sm-α-actin and smoothelin, a novel marker specific for the differentiated phenotype of SMC. Neointima formation could be seen from day 7 after injury onwards. During early phases, the number of smoothelin-positive cells in the media was decreased compared with uninjured controls. Smoothelin staining was absent in the neointima during formation. Increased levels of smoothelin in both media and neointima were observed at days 28 and 56, correlating with a decrease in proliferation as assessed by Ki-67 antigen staining. No such changes were observed for desmin and sm-α-actin. Following balloon injury, SMC in both the media and the neointima underwent an early, reversible dedifferentiation, followed by proliferation. The novel SMC-specific marker protein smoothelin can be used to monitor this SMC (de)differentiation in neointima and media. These findings support the pivotal role of SMC phenotype modulation in neointima formation and restenosis. Received: 29 January 2001, Returned for revision: 20 February 2001, Revision received: 26 June 2001, Accepted: 2 July 2001     

Phenotype-dependent alteration of pathways and networks reveals a pure synergistic mechanism for compounds treating mouse cerebral ischemia

Aim:

Our previous studies have showed that ursodeoxycholic acid (UA) and jasminoidin (JA) effectively reduce cerebral infarct volume in mice. In this study we explored the pure synergistic mechanism of these compounds in treatment of mouse cerebral ischemia, which was defined as synergistic actions specific for phenotype variations after excluding interference from ineffective compounds.

Methods:

Mice with focal cerebral ischemia were treated with UA, JA or a combination JA and UA (JU). Concha margaritifera (CM) was taken as ineffective compound. Cerebral infarct volume of the mice was determined, and the hippocampi were taken for microarray analysis. Particular signaling pathways and biological functions were enriched based on differentially expressed genes, and corresponding networks were constructed through Ingenuity Pathway Analysis.

Results:

In phenotype analysis, UA, JA, and JU significantly reduced the ischemic infarct volume with JU being superior to UA or JA alone, while CM was ineffective. As a result, 4 pathways enriched in CM were excluded. Core pathways in the phenotype-positive groups (UA or JA) were involved in neuronal homeostasis and neuropathology. JU-contributing pathways included all UA-contributing and the majority (71.7%) of JA-contributing pathways, and 10 new core pathways whose effects included inflammatory immunity, apoptosis and nervous system development. The functions of JU group included all functions of JA group, the majority (93.1%) of UA-contributing functions, and 3 new core functions, which focused on physiological system development and function.

Conclusion:

The pure synergism between UA and JA underlies 10 new core pathways and 3 new core functions, which are involved in inflammation, immune responses, apoptosis and nervous system development.     

High and long‐term von Willebrand factor expression after Sleeping Beauty transposon‐mediated gene therapy in a mouse model of severe von Willebrand disease

Essentials

• von Willebrand disease (VWD) is the most common inherited bleeding disorder.
• Gene therapy for VWD offers long‐term therapy for VWD patients.
• Transposons efficiently integrate the large von Willebrand factor (VWF) cDNA in mice.
• Liver‐directed transposons support sustained VWF expression with suboptimal multimerization.

Summary

Background

Type 3 von Willebrand disease (VWD) is characterized by complete absence of von Willebrand factor (VWF). Current therapy is limited to treatment with exogenous VWF/FVIII products, which only provide a short‐term solution. Gene therapy offers the potential for a long‐term treatment for VWD.

Objectives

To develop an integrative Sleeping Beauty (SB) transposon‐mediated VWF gene transfer approach in a preclinical mouse model of severe VWD.

Methods

We established a robust platform for sustained transgene murine VWF (mVWF) expression in the liver of Vwf^?/? mice by combining a liver‐specific promoter with a sandwich transposon design and the SB100X transposase via hydrodynamic gene delivery.

Results

The sandwich SB transposon was suitable to deliver the full‐length mVWF cDNA (8.4 kb) and supported supra‐physiological expression that remained stable for up to 1.5 years after gene transfer. The sandwich vector stayed episomal (~60 weeks) or integrated in the host genome, respectively, in the absence or presence of the transposase. Transgene integration was confirmed using carbon tetrachloride‐induced liver regeneration. Analysis of integration sites by high‐throughput analysis revealed random integration of the sandwich vector. Although the SB vector supported long‐term expression of supra‐physiological VWF levels, the bleeding phenotype was not corrected in all mice. Long‐term expression of VWF by hepatocytes resulted in relatively reduced amounts of high‐molecular‐weight multimers, potentially limiting its hemostatic efficacy.

Conclusions

Although this integrative platform for VWF gene transfer is an important milestone of VWD gene therapy, cell type‐specific targeting is yet to be achieved.

     

Resampling to Address the Winner's Curse in Genetic Association Analysis of Time to Event

The “winner's curse” is a subtle and difficult problem in interpretation of genetic association, in which association estimates from large‐scale gene detection studies are larger in magnitude than those from subsequent replication studies. This is practically important because use of a biased estimate from the original study will yield an underestimate of sample size requirements for replication, leaving the investigators with an underpowered study. Motivated by investigation of the genetics of type 1 diabetes complications in a longitudinal cohort of participants in the Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications (DCCT/EDIC) Genetics Study, we apply a bootstrap resampling method in analysis of time to nephropathy under a Cox proportional hazards model, examining 1,213 single‐nucleotide polymorphisms (SNPs) in 201 candidate genes custom genotyped in 1,361 white probands. Among 15 top‐ranked SNPs, bias reduction in log hazard ratio estimates ranges from 43.1% to 80.5%. In simulation studies based on the observed DCCT/EDIC genotype data, genome‐wide bootstrap estimates for false‐positive SNPs and for true‐positive SNPs with low‐to‐moderate power are closer to the true values than uncorrected naïve estimates, but tend to overcorrect SNPs with high power. This bias‐reduction technique is generally applicable for complex trait studies including quantitative, binary, and time‐to‐event traits.     

河南省某农村地区中老年女性人群高甘油三酯腰围患病率及其危险因素

目的：分析农村中老年女性人群中高甘油三酯腰围（ HTGW）的患病率及其危险因素。方法：于2013年7～8月,采用整群抽样的方法,对河南省新安县某镇4573名40岁及以上的常住女性人群进行横断面调查,调查内容包括人口学特征、行为危险因素、人体测量及空腹脂质谱检测。结果：1642人（35．91％）患有HTGW。在调整了年龄、受教育程度和婚姻状况后,用logistic回归分析筛选出的HTGW患病危险因素有超重、肥胖和高血压,其OR （95％CI）分别是5．27（4．46～6．23）、8．89（7．37～10．72）和1．23（1．07～1．42）,而重体力活动为保护性因素,其OR（95％CI）为0．84（0．73～0．96）。结论：该农村中老年女性人群具有较高的HTGW患病率,超重、肥胖和高血压为其主要危险因素。     

1例罕见的B(A)亚型的鉴定

B(A)亚型是一种罕见的ABO亚型,其分子机制为B等位基因在正常B基因序列的基础上发生单碱基突变,使其具有编码双功能活性酶的能力。B(A)亚型在血清学检测中除表现B抗原特异性外,还表现少量A抗原特异性,从而干扰血型鉴定及配血工作。B(A)型如误定为AB型,输入AB型红细胞可引起溶血性输血反应。国内已报道7个等位基因,其中B(A)02型发生率为0.78/10万。现将报道1例工作中遇到的B(A)O2型。