Short leucine-rich glycoproteins of the extracellular matrix display diverse patterns of complement interaction and activation

The extracellular matrix consists of structural macromolecules and other proteins with regulatory functions. An important family of the latter class of molecules found in most tissues is the small leucine-rich repeat proteins (SLRPs). We have previously shown that the SLRP fibromodulin binds directly to C1q and activates the classical pathway of complement. In the present study we further examine the interactions between SLRPs and complement. Osteoadherin, like fibromodulin, binds C1q and activates the classical pathway strongly while moderate activation is seen in the terminal pathway. This can be explained by the interaction of fibromodulin and osteoadherin with factor H, a major soluble inhibitor of complement. Also, chondroadherin was found to bind C1q and activate complement, albeit to a lesser extent. Chondroadherin also binds factor H. We confirm published data showing that biglycan and decorin bind C1q but do not activate complement. In this study a similar pattern is seen for lumican although its affinity for C1q is lower than for biglycan and decorin. Furthermore, using electron microscopy and radiolabeled SLRPs, we demonstrate two different classes of SLRP binding sites on C1q, to head and stalk respectively, where only binding to the head appears to be activating. We propose a role for SLRPs in the regulation of complement activation in diseases involving the extracellular matrix, particularly those characterized by chronic inflammation such as rheumatoid arthritis, atherosclerosis, osteoarthritis and chronic obstructive lung disease.     

Global DNA methylation in fetal human germ cells and germ cell tumours: association with differentiation and cisplatin resistance

Differences in the global methylation pattern, ie hyper‐ as well as hypo‐methylation, are observed in cancers including germ cell tumours (GCTs). Related to their precursor cells, GCT methylation status differs according to histology. We investigated the methylation pattern of normal fetal, infantile, and adult germ cells (n = 103) and GCTs (n = 251) by immunohistochemical staining for 5‐ cytidine. The global methylation pattern of male germ cells changes from hypomethylation to hypermethylation, whereas female germ cells remain unmethylated at all stages. Undifferentiated GCTs (seminomas, intratubular germ cell neoplasia unclassified, and gonadoblastomas) are hypomethylated, whereas more differentiated GCTs (teratomas, yolk sac tumours, and choriocarcinomas) show a higher degree of methylation. Embryonal carcinomas show an intermediate pattern. Resistance to cisplatin was assessed in the seminomatous cell line TCam‐2 before and after demethylation using 5‐azacytidine. Exposure to 5‐azacytidine resulted in decreased resistance to cisplatin. Furthermore, after demethylation, the stem cell markers NANOG and POU5F1 (OCT3/4), as well as the germ cell‐specific marker VASA, showed increased expression. Following treatment with 5‐azacytidine, TCam‐2 cells were analysed using a high‐throughput methylation screen for changes in the methylation sites of 14 000 genes. Among the genes revealing changes, interesting targets were identified: ie demethylation of KLF11, a putative tumour suppressor gene, and hypermethylation of CFLAR, a gene previously described in treatment resistance in GCTs. Copyright © 2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Clinical and genetic analysis of Korean patients with Marfan syndrome: possible ethnic differences in clinical manifestation

Yoo E‐H, Woo H, Ki C‐S, Lee HJ, Kim D‐K, Kang I‐S, Park P, Sung K, Lee CS, Chung T‐Y, Moon JR, Han H, Lee S‐T, Kim J‐W. Clinical and genetic analysis of Korean patients with Marfan syndrome: possible ethnic differences in clinical manifestation. Marfan syndrome (MFS) is an autosomal dominant disorder of the fibrous connective tissue caused by mutations in the fibrillin‐1 (FBN1) gene. Although clinical and genetic analyses have been performed in various populations, there have been few studies in Korea. The aim of this study was to investigate the clinical characteristics and genetic background of Korean patients with MFS. In 39 Korean patients with MFS who met the Ghent criteria, the most common clinical finding was aortic dilatation and/or dissection (94.9%), whereas only 35.9% of patients had ectopia lentis. The majority of MFS patients had fewer than four of the skeletal findings required to fulfill the major skeletal Ghent criterion for MFS. Only 21% of Korean patients had major skeletal abnormalities and most cases showed only minor skeletal involvement. FBN1 gene mutations were detected in 35 out of 39 patients (89.7%), which is similar to rates presented in the previous reports. These results suggest that some clinical features in Korean patients with MFS differed from those reported in Western MFS patients.     

Native, amyloid fibrils and beta-oligomers of the C-terminal domain of human prion protein display differential activation of complement and bind C1q, factor H and C4b-binding protein directly

Prion protein (PrP) is an endogenous protein involved in the pathogenesis of bovine spongiform encephalopathy and Creutzfeldt-Jakob disease. Murine PrP has been reported to bind C1q and activate the classical pathway of complement in a copper-dependent manner. Here we show that various conformational isoforms (native, amyloid fibrils, and beta-oligomers) of recombinant human PrP (90-231 and 121-231) bind C1q and activate complement. PrP binds both the globular head and collagenous stalk domains of C1q. Native, beta-oligomeric and amyloid fibrils of PrP all activate the classical and alternative pathways of complement to different extent. However, they do not trigger the lectin pathway. Of the tested PrP conformational isoforms we find that beta-oligomers bind C1q and activate complement most strongly. Membrane attack complex formation initiated by PrP is subdued in comparison to deposition of early complement components. This is most likely attributed to the interaction between human PrP and complement inhibitors factor H and C4b-binding protein. Accordingly, PrP-triggered complement activation in the terminal pathway was increased in serum lacking C4b-binding protein. Taken together the present study indicates that complement activation may be an important factor in human prion diseases, suggesting that complement induced activities may prove relevant therapeutic targets.     

Work-Home Interference,Perceived Total Workload,and the Risk of Future Sickness Absence Due to Stress-Related Mental Diagnoses Among Women and Men: a Prospective Twin Study

Purpose

Work-home interference has been proposed as an important explanation for sickness absence (SA). Previous studies show mixed results, have not accounted for familial factors (genetics and shared everyday environment), or investigated diagnosis specific SA. The aim was to study whether work-home interference and perceived total workload predict SA due to stress-related mental diagnoses, or SA due to other mental diagnoses, among women and men, when adjusting for various confounders and familial factors.

Methods

This study included 11,916 twins, 19–47 years (49% women). Data on work-to-home and home-to-work conflicts, perceived total workload, and relevant confounders were derived from a 2005 survey, and national register data on SA spells until 2013 were obtained. Odds ratios (ORs) with 95% confidence intervals (CIs) were calculated. Discordant twin pair design was applied to adjust for familial factors.

Results

Each one unit increase in work-to-home and home-to-work conflicts, and perceived total workload was associated with higher odds for SA due to stress-related mental diagnoses and to SA due to other mental diagnoses among women, when adjusting for sociodemographic factors (ORs 1.15–1.31). Including health or familial factors, no associations remained. For men, each one unit increase in work-to-home conflicts was associated with higher odds for SA due to stress-related diagnoses (ORs 1.23–1.35), independently of confounders.

Conclusion

Work-to-home conflict was independently associated with future SA due to stress-related diagnoses among men only. Health- and work-related factors seem to be important confounders when researching work-home interference, perceived total workload, and SA. Not including such confounders involves risking drawing incorrect conclusions. Further studies are needed to confirm sex differences and whether genetic factors are important for the associations studied.

     

IL‐25 exhibits disparate roles during Th2‐cell differentiation versus effector function

A keenly sought therapeutic approach for the treatment of allergic disease is the identification and neutralization of the cytokine that regulates the differentiation of T helper 2 (Th2) cells. Th2 cells are exciting targets for asthma therapies. Recently, the cytokine IL‐25 has been shown to enhance Th2‐type immune activity and play important roles in mediating allergic inflammatory responses. To investigate this further, we crossed IL‐25^?/? C57BL/6 mice with G4 IL‐4 C57BL/6 reporter mice and developed an assay for in vitro and in vivo IL‐4‐independent Th2‐cell differentiation. These assays were used to determine whether IL‐25 was critical for the formation of Th2 cells. We found there was no physiological role for IL‐25 in either the differentiation of Th2 cells or their development to effector or memory Th2‐cell subsets. Importantly, this data challenges the newly found and growing status of the cytokine IL‐25 and its proposed role in promoting Th2‐cell responses.     

The Ariadne principles: how to handle multimorbidity in primary care consultations

Multimorbidity is a health issue mostly dealt with in primary care practice. As a result of their generalist and patient-centered approach, long-lasting relationships with patients, and responsibility for continuity and coordination of care, family physicians are particularly well placed to manage patients with multimorbidity. However, conflicts arising from the application of multiple disease oriented guidelines and the burden of diseases and treatments often make consultations challenging. To provide orientation in decision making in multimorbidity during primary care consultations, we developed guiding principles and named them after the Greek mythological figure Ariadne. For this purpose, we convened a two-day expert workshop accompanied by an international symposium in October 2012 in Frankfurt, Germany. Against the background of the current state of knowledge presented and discussed at the symposium, 19 experts from North America, Europe, and Australia identified the key issues of concern in the management of multimorbidity in primary care in panel and small group sessions and agreed upon making use of formal and informal consensus methods. The proposed preliminary principles were refined during a multistage feedback process and discussed using a case example. The sharing of realistic treatment goals by physicians and patients is at the core of the Ariadne principles. These result from i) a thorough interaction assessment of the patient’s conditions, treatments, constitution, and context; ii) the prioritization of health problems that take into account the patient’s preferences – his or her most and least desired outcomes; and iii) individualized management realizes the best options of care in diagnostics, treatment, and prevention to achieve the goals. Goal attainment is followed-up in accordance with a re-assessment in planned visits. The occurrence of new or changed conditions, such as an increase in severity, or a changed context may trigger the (re-)start of the process. Further work is needed on the implementation of the formulated principles, but they were recognized and appreciated as important by family physicians and primary care researchers.Please see related article: http://www.biomedcentral.com/1741-7015/12/222.     

The influence of vascular risk factors on cognitive decline in patients with Alzheimer's Disease

Introduction

The influence of vascular risk factors (VRFs) on the rate of cognitive decline in patients with established dementia is unclear. This study aims to examine the association between VRFs and the rate of cognitive decline in patients with Alzheimer's disease (AD).

Methods

Data were obtained from patients visiting a memory clinic between 2004 and 2012. VRFs were determined at baseline and included hypertension, hypercholesterolemia, diabetes mellitus, overweight and smoking. Continuous values of blood pressure, total cholesterol, glucose level and body mass index were also obtained. Mini-Mental State Exam (MMSE) scores were obtained at baseline and during follow-up visits. The association between VRFs and the annual change in MMSE scores was analysed with a multivariable linear mixed model adjusted for age, sex and the aforementioned VRFs.

Results

From 174 patients (mean age 78.3 years), with a follow-up time up to 5.8 years (mean 1.1 year), in total 447 MMSE scores were obtained. The multivariable analyses showed an association between age as well as systolic blood pressure and a decline in annual rates of change in MMSE scores of −0.05 (95% confidence interval (CI): −0.09 to 0.00) and −0.01 (CI: −0.03 to 0.00), respectively. For all other VRFs, including sex, patients did not show a significant difference.

Conclusion

This study did not find an association between preventable vascular risk factors and cognitive decline in patients with AD, except for systolic blood pressure. As the association between systolic blood pressure and decline in MMSE was small, clinical relevance may be limited.     

Postprandial ghrelin responses are associated with the intermeal interval in time-blinded normal weight men,but not in obese men

ObjectiveTo investigate the relation between ghrelin responses and meal initiation and the effects of BMI and energy status on this.DesignThe experiment had a randomised, cross-over design.Setting and subjectsNine normal-weight (age: 33.2 ± 4.8 y, BMI: 23.2 ± 0.5 kg/m²) and eleven obese (age: 40.8 ±4.7 y, BMI: 33.2 ± 0.8 kg/m²) healthy men were recruited from a pool of volunteers and by advertisements.InterventionsSubjects followed a three-day energy restrictive and a three-day energy balanced diet separated by one month. Each diet was followed by a time-blinded (overnight) stay at the research facility. Subjects received a breakfast (preload) and were instructed to ask for lunch when they felt hungry. Ghrelin, insulin, glucose, free fatty acids, appetite, IMI and energy intake during lunch were assessed.ResultsPostprandial decreases in ghrelin (r = ? 0.54; p < 0.05) and the AUC of the ghrelin response (r = ? 0.57, p = 0.01) were associated with the intermeal interval, independent of diet, but in normal weight subjects only. Lunch request was preceded by an increase in ghrelin, reaching at least 93% of fasting values. These preprandial increases in ghrelin were correlated with IMI, after energy restriction only. Ghrelin concentrations but not changes in ghrelin were correlated with appetite.ConclusionMeal-related changes in ghrelin are correlated with the IMI in normal weight subjects only, independent of diet. Ghrelin concentrations may need to reach a certain threshold level before the next meal is initiated.SponsorshipSupported by Dutch Ministry of Education, Culture and Science, Dutch Ministry of Health, Welfare and Sport and Danone Research.     

Molecular characterization of the gene encoding H antigen in Escherichia coli and development of a PCR-restriction fragment length polymorphism test for identification of E. coli O157:H7 and O157:NM.

Recent outbreaks of disease caused by Escherichia coli O157:H7 have focused much attention on this newly emerged pathogen. Identification of the H7 flagellar antigen is critical for the confirmation of E. coli O157:H7; however, clinical isolates are frequently nonmotile and do not produce detectable H antigen. To further characterize nonmotile isolates (designated NM), we developed a PCR-restriction fragment length polymorphism (PCR-RFLP) test to identify and characterize the gene encoding the H antigen (fliC) in E. coli. The entire coding sequence of fliC was amplified by PCR, the amplicon was restricted with RsaI, and the restriction fragment pattern was examined after gel electrophoresis. Two hundred eighty E. coli isolates representing serotypes O157:H7 and O157:NM, flagellar antigen H7 groups associated with other O serogroups, and all other flagellar antigen groups were analyzed. A single restriction pattern (pattern A) was identified for O157:H7 isolates, O157:NM isolates that produced Shiga toxin (formerly Shiga-like toxin or verotoxin), and 16 of 18 O55:H7 isolates. Flagellar antigen group H7 isolates of non-O157 serotypes had one of three banding patterns distinct from pattern A. A wide variety of patterns were found among isolates of the other 52 flagellar antigen groups; however, none was identical to the O157:H7 pattern. Thirteen of 15 nonmotile strains that did not produce the A pattern had patterns that matched those of other known H groups. The PCR-RFLP in conjunction with O serogroup determination will be useful in identifying E. coli O157:H7 and related strains that do not express immunoreactive H antigen and could be expanded to include other clinically important E. coli strains.