Structural basis for complement factor H-linked age-related macular degeneration

This is the final version of the article. Available from the publisher via the DOI in this record.Nearly 50 million people worldwide suffer from age-related macular degeneration (AMD), which causes severe loss of central vision. A single-nucleotide polymorphism in the gene for the complement regulator factor H (FH), which causes a Tyr-to-His substitution at position 402, is linked to approximately 50% of attributable risks for AMD. We present the crystal structure of the region of FH containing the polymorphic amino acid His402 in complex with an analogue of the glycosaminoglycans (GAGs) that localize the complement regulator on the cell surface. The structure demonstrates direct coordination of ligand by the disease-associated polymorphic residue, providing a molecular explanation of the genetic observation. This glycan-binding site occupies the center of an extended interaction groove on the regulator's surface, implying multivalent binding of sulfated GAGs. This finding is confirmed by structure-based site-directed mutagenesis, nuclear magnetic resonance-monitored binding experiments performed for both H402 and Y402 variants with this and another model GAG, and analysis of an extended GAG-FH complex.B. Prosser is funded by the Wellcome Trust Structural Biology Training Program (075415/Z/04/Z). S. Johnson and P. Roversi were funded by grants to S.M. Lea from the Medical Research Council (MRC) of the United Kingdom (grants G0400389 and G0400775). D. Uhrin and P.N. Barlow were funded by the Wellcome Trust (078780/ Z/05/Z). S.J. Clark was funded by an MRC Doctoral Training Account (G78/7925), and R.B. Sim and A.J. Day were funded by MRC core funding to the MRC Immunochemistry Unit

Detection of Mycolactone A/B in Mycobacterium ulcerans–Infected Human Tissue

Skin infection with bacteria called Mycobacterium ulcerans causes Buruli ulcer, a disease common in West Africa and mainly affecting children. M. ulcerans is the only mycobacterium to cause disease by production of a toxin. This lipid molecule called mycolactone diffuses from the site of infection, killing surrounding cells and, at low concentration, suppressing the immune response. The aim of this study was to show that mycolactone can be detected among lipids extracted from human M. ulcerans lesions in order to study its role in the pathogenesis of M. ulcerans disease. Lipids were extracted from skin biopsies and tested for the presence of mycolactone using thin layer chromatography and mass spectrometry. The extracts were shown to kill cultured cells in a cytotoxicity assay. Mycolactone was detected in both pre-ulcerative and ulcerative forms of the disease and also in lesions during antibiotic treatment but with reduced bioactivity, suggesting a lower concentration compared to untreated lesions. These findings indicate that there is mycolactone in affected skin at all stages of M. ulcerans disease and it could be used as a biomarker for monitoring the clinical response to antibiotic treatment

Glycan composition of serum alpha-fetoprotein in patients with hepatocellular carcinoma and non-seminomatous germ cell tumour

Although estimation of serum alpha-fetoprotein (AFP) is widely used in the diagnosis of hepatocellular carcinoma (HCC) and non-seminomatous germ cell tumours (NSGCT), the clinical usefulness of this test is limited by a low specificity. However, there exist glycoforms of AFP which may be more specific for particular tumours. Previously, detailed analysis has been prevented by the low levels of AFP in human serum. We report here the application of fluorescence labelling, sequential exoglycosidase digestion, high-performance liquid chromatography and matrix-assisted laser desorption ionization in time-of-flight mass spectrometry, to determine the glycan structures of purified serum AFP from patients with HCC and NSGCT. Eleven major glycans were found, of which seven were N-linked, and four were O-linked, to the protein backbone. The structure of the N-linked glycans (all of bi-antennary complex-type with varying degrees of sialylation, fucosylation and galactosylation) were consistent with those previously reported. The O-linked glycans (three mucin O-GalNAc type glycans with variable degrees of sialylation, one O-HexNAc monosaccharide glycan) have not previously been reported. The finding of mucin O-GalNAc type glycans was supported by the prediction of potential O-GalNAc glycosylation sites on the protein backbone by analysis of the AFP structure by molecular modelling. With knowledge of these structures it may be possible to develop more specific assays for the detection of HCC and NSGCT. © 1999 Cancer Research Campaign © 1999 Cancer Research Campaig

Identification of a new European rabbit IgA with a serine-rich hinge region

<div><p>In mammals, the most striking IgA system belongs to Lagomorpha. Indeed, 14 IgA subclasses have been identified in European rabbits, 11 of which are expressed. In contrast, most other mammals have only one IgA, or in the case of hominoids, two IgA subclasses. Characteristic features of the mammalian IgA subclasses are the length and amino acid sequence of their hinge regions, which are often rich in Pro, Ser and Thr residues and may also carry Cys residues. Here, we describe a new IgA that was expressed in New Zealand White domestic rabbits of <i>IGHV</i>a1 allotype. This IgA has an extended hinge region containing an intriguing stretch of nine consecutive Ser residues and no Pro or Thr residues, a motif exclusive to this new rabbit IgA. Considering the amino acid properties, this hinge motif may present some advantage over the common IgA hinge by affording novel functional capabilities. We also sequenced for the first time the IgA14 CH2 and CH3 domains and showed that IgA14 and IgA3 are expressed.</p></div

Pathogenesis of Henoch-Schönlein purpura nephritis

The severity of renal involvement is the major factor determining the long-term outcome of children with Henoch-Schönlein purpura (HSP) nephritis (HSPN). Approximately 40% children with HSP develop nephritis, usually within 4 to 6 weeks after the initial onset of the typical purpuric rashes. Although the pathogenetic mechanisms are still not fully delineated, several studies suggest that galactose-deficient IgA1 (Gd-IgA1) is recognized by anti-glycan antibodies, leading to the formation of the circulating immune complexes and their mesangial deposition that induce renal injury in HSPN

Current trends in cannulation and neuroprotection during surgery of the aortic arch in Europe†‡

OBJECTIVES To conduct a survey across European cardiac centres to evaluate the methods used for cerebral protection during aortic surgery involving the aortic arch. METHODS All European centres were contacted and surgeons were requested to fill out a short, comprehensive questionnaire on an internet-based platform. One-third of more than 400 contacted centres completed the survey correctly. RESULTS The most preferred site for arterial cannulation is the subclavian-axillary, both in acute and chronic presentation. The femoral artery is still frequently used in the acute condition, while the ascending aorta is a frequent second choice in the case of chronic presentation. Bilateral antegrade brain perfusion is chosen by the majority of centres (2/3 of cases), while retrograde perfusion or circulatory arrest is very seldom used and almost exclusively in acute clinical presentation. The same pumping system of the cardio pulmonary bypass is most of the time used for selective cerebral perfusion, and the perfusate temperature is usually maintained between 22 and 26°C. One-third of the centres use lower temperatures. Perfusate flow and pressure are fairly consistent among centres in the range of 10-15 ml/kg and 60 mmHg, respectively. In 60% of cases, barbiturates are added for cerebral protection, while visceral perfusion still receives little attention. Regarding cerebral monitoring, there is a general tendency to use near-infrared spectroscopy associated with bilateral radial pressure measurement. CONCLUSIONS These data represent a snapshot of the strategies used for cerebral protection during major aortic surgery in current practice, and may serve as a reference for standardization and refinement of different approache

A human neural crest model reveals the developmental impact of neuroblastoma-associated chromosomal aberrations

Early childhood tumours arise from transformed embryonic cells, which often carry large copy number alterations (CNA). However, it remains unclear how CNAs contribute to embryonic tumourigenesis due to a lack of suitable models. Here we employ female human embryonic stem cell (hESC) differentiation and single-cell transcriptome and epigenome analysis to assess the effects of chromosome 17q/1q gains, which are prevalent in the embryonal tumour neuroblastoma (NB). We show that CNAs impair the specification of trunk neural crest (NC) cells and their sympathoadrenal derivatives, the putative cells-of-origin of NB. This effect is exacerbated upon overexpression of MYCN, whose amplification co-occurs with CNAs in NB. Moreover, CNAs potentiate the pro-tumourigenic effects of MYCN and mutant NC cells resemble NB cells in tumours. These changes correlate with a stepwise aberration of developmental transcription factor networks. Together, our results sketch a mechanistic framework for the CNA-driven initiation of embryonal tumours

Basic science232. Certolizumab pegol prevents pro-inflammatory alterations in endothelial cell function

Background: Cardiovascular disease is a major comorbidity of rheumatoid arthritis (RA) and a leading cause of death. Chronic systemic inflammation involving tumour necrosis factor alpha (TNF) could contribute to endothelial activation and atherogenesis. A number of anti-TNF therapies are in current use for the treatment of RA, including certolizumab pegol (CZP), (Cimzia ®; UCB, Belgium). Anti-TNF therapy has been associated with reduced clinical cardiovascular disease risk and ameliorated vascular function in RA patients. However, the specific effects of TNF inhibitors on endothelial cell function are largely unknown. Our aim was to investigate the mechanisms underpinning CZP effects on TNF-activated human endothelial cells. Methods: Human aortic endothelial cells (HAoECs) were cultured in vitro and exposed to a) TNF alone, b) TNF plus CZP, or c) neither agent. Microarray analysis was used to examine the transcriptional profile of cells treated for 6 hrs and quantitative polymerase chain reaction (qPCR) analysed gene expression at 1, 3, 6 and 24 hrs. NF-κB localization and IκB degradation were investigated using immunocytochemistry, high content analysis and western blotting. Flow cytometry was conducted to detect microparticle release from HAoECs. Results: Transcriptional profiling revealed that while TNF alone had strong effects on endothelial gene expression, TNF and CZP in combination produced a global gene expression pattern similar to untreated control. The two most highly up-regulated genes in response to TNF treatment were adhesion molecules E-selectin and VCAM-1 (q 0.2 compared to control; p > 0.05 compared to TNF alone). The NF-κB pathway was confirmed as a downstream target of TNF-induced HAoEC activation, via nuclear translocation of NF-κB and degradation of IκB, effects which were abolished by treatment with CZP. In addition, flow cytometry detected an increased production of endothelial microparticles in TNF-activated HAoECs, which was prevented by treatment with CZP. Conclusions: We have found at a cellular level that a clinically available TNF inhibitor, CZP reduces the expression of adhesion molecule expression, and prevents TNF-induced activation of the NF-κB pathway. Furthermore, CZP prevents the production of microparticles by activated endothelial cells. This could be central to the prevention of inflammatory environments underlying these conditions and measurement of microparticles has potential as a novel prognostic marker for future cardiovascular events in this patient group. Disclosure statement: Y.A. received a research grant from UCB. I.B. received a research grant from UCB. S.H. received a research grant from UCB. All other authors have declared no conflicts of interes