Case Reports1. A Late Presentation of Loeys-Dietz Syndrome: Beware of TGFβ Receptor Mutations in Benign Joint Hypermobility

Background: Thoracic aortic aneurysms (TAA) and dissections are not uncommon causes of sudden death in young adults. Loeys-Dietz syndrome (LDS) is a rare, recently described, autosomal dominant, connective tissue disease characterized by aggressive arterial aneurysms, resulting from mutations in the transforming growth factor beta (TGFβ) receptor genes TGFBR1 and TGFBR2. Mean age at death is 26.1 years, most often due to aortic dissection. We report an unusually late presentation of LDS, diagnosed following elective surgery in a female with a long history of joint hypermobility. Methods: A 51-year-old Caucasian lady complained of chest pain and headache following a dural leak from spinal anaesthesia for an elective ankle arthroscopy. CT scan and echocardiography demonstrated a dilated aortic root and significant aortic regurgitation. MRA demonstrated aortic tortuosity, an infrarenal aortic aneurysm and aneurysms in the left renal and right internal mammary arteries. She underwent aortic root repair and aortic valve replacement. She had a background of long-standing joint pains secondary to hypermobility, easy bruising, unusual fracture susceptibility and mild bronchiectasis. She had one healthy child age 32, after which she suffered a uterine prolapse. Examination revealed mild Marfanoid features. Uvula, skin and ophthalmological examination was normal. Results: Fibrillin-1 testing for Marfan syndrome (MFS) was negative. Detection of a c.1270G > C (p.Gly424Arg) TGFBR2 mutation confirmed the diagnosis of LDS. Losartan was started for vascular protection. Conclusions: LDS is a severe inherited vasculopathy that usually presents in childhood. It is characterized by aortic root dilatation and ascending aneurysms. There is a higher risk of aortic dissection compared with MFS. Clinical features overlap with MFS and Ehlers Danlos syndrome Type IV, but differentiating dysmorphogenic features include ocular hypertelorism, bifid uvula and cleft palate. Echocardiography and MRA or CT scanning from head to pelvis is recommended to establish the extent of vascular involvement. Management involves early surgical intervention, including early valve-sparing aortic root replacement, genetic counselling and close monitoring in pregnancy. Despite being caused by loss of function mutations in either TGFβ receptor, paradoxical activation of TGFβ signalling is seen, suggesting that TGFβ antagonism may confer disease modifying effects similar to those observed in MFS. TGFβ antagonism can be achieved with angiotensin antagonists, such as Losartan, which is able to delay aortic aneurysm development in preclinical models and in patients with MFS. Our case emphasizes the importance of timely recognition of vasculopathy syndromes in patients with hypermobility and the need for early surgical intervention. It also highlights their heterogeneity and the potential for late presentation. Disclosures: The authors have declared no conflicts of interes

Comparison of Antigenic Regions Identified on IgG1Fc Using Bioinformatics vs Pepscan Analysis

Clinical Medicine: Arthritis and Musculoskeletal Disorders is an international, open access, peer reviewed journal.Epitope mapping allowed the location of antigenic determinants on a protein macromolecule to be identified. In particular, pepscan techniques that utilize a series of overlapping peptides, help detect key amino acid residues that are important in antibody recognition and binding. In a previous study, we employed 15-mer peptides spanning the entire length of IgG1Fc to ascertain successfully the target epitopes of isotypic/allotypic monoclonal reagents. As an extension to this work we have used these peptides to evaluate the location of epitope targets of five IgM rheumatoid factor antibodies (RFAbs). Overall, 2 antibodies, RFAb TS2 and TS1, detected a similar epitope within the CH3 domain (360-KNQVSLTCLVKGFYP-374), whilst 1 (RFAb SJ1) recognised an epitope in the CH2 domain (294- EQYNSTYRVVSVLTV-308). In contrast, 2 RFAbs, PRSJ2 and PRTS1 detected four and five epitopes respectively within the Fc region. RFAb PRSJ2 recognised epitopes detected by RFAB TS2 and TS1 but also further epitopes in the CH2 domain (256-TPEVTCVVVDVSHED-270) and CH3 domain (418-QQGNVFSCSVMHEAL-432). Similarly, RFAb PRTS1 detected all four epitopes plus a fifth in the CH3 domain (382-ESNGQPENNYKTTPP-396). In essence there was a consensus of target epitopes identified by these rheumatoid factor antibodies. Interestingly, two epitopes (256–270, CH2 domain and 360–374, CH3 domain) were novel in that they had not been identified in previous pepscan studies. The other epitopes recognised, either overlapped or were immediately adjacent to previous epitopes detected by poly/monoclonal rheumatoid factor antibodies. Molecular modelling (PCImdad) of IgG1Fc showed that all five epitopes were exposed and surface accessible for antibody interaction. In addition, a bioinformatics analysis of the Fc region using ExPASy was employed to identify key antigenic determinants. This ‘in silico’ approach may provide a means of determining key regions without the need to develop overlapping peptides spanning the entire length of a macromolecule

In: Abstracts from the World Federation of Neuro-Oncology Second Quadrennial Meeting and the Sixth Meeting of the European Association for Neuro-Oncology: May 5–8, 2005, Edinburgh, UK. No.174

Abstracts from Neuro-Oncology are provided here courtesy of Society for Neuro-Oncology and Duke University Press.Human endogenous retroviruses (HERVs) belong to the family of transposable elements that make up 8% of the human genome. Unlike exogenous retrovirus (e.g., HIV and HTLV), HERVs are inherited in a Mendelian manner. More than 22 families of HERVs have been identified over the past two decades. Importantly, some HERVs have been found to possess large open reading frames and produce viral like particles. More latterly, these viruses have been linked with certain autoimmune diseases and cancers. Indeed, HERVs may contribute toward carcinogenesis through retrotransposition, promoter insertion, immunomodulation, disruption of normal HERV-related functions, recombination, or by the production of fusion proteins. Of importance, HERV-K, HERV-W, and HERV-H have the potential to be transcriptionally active in the brain. We have developed robust RT-PCR systems using primers/probes specific to HERV-K and HERV-W to assess mRNA expression in conjunction with the house keeping gene, histidyl tRNA synthetase. In employing a gel-documentation system, we are able to provide semiquantitative levels of HERV expression in cell lines. Pilot data shows markedly enhanced expression of HERV-K in the cell line U251-MG (derived from a glioblastoma multiforme; WHO grade IV astrocytoma) as compared to a control cell line SW480 (colon adenocarcinoma): RT-PCR values; 1.0 and 0.42, respectively. This observation raises an intriguing possibility that HERV-K expression may be elevated in malignant brain tumors. In addition, this approach provides a useful approach to optimize primers and probes prior to using real-time quantitative PCR

In: The 33rd Congress of the Czech Society of Pathologists, 2nd Satellite Symposium & Workshop on Molecular Pathology, Regional Centre Olomouc & Faculty of Medicine, Palacky University Olomouc, May 4–6, 2006, 29-30.

Abstract is provided here courtesy of Palacky University, Olomouc.Human endogenous retroviruses (HERVs) are a group of integrated RNA viruses within our human genome. Whilst many are regarded as defective, a number possess the potential to generate retroviral products. Indeed HERVs such as those belonging to the HERV-K family produce retroviral particles in the teratocarcinoma cell line GH and the breast cancer cell line T47D. It has been argued that some retroelements may be beneficial to the human host, perhaps conferring a selective advantage, whereas others may be harmful. Furthermore certain HERVs might be involved in the pathogenesis of autoimmune diseases. The precise mechanisms in diseases such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) may include molecular mimicry and superantigen motifs that evoke and augment unwarranted immune responses. The precise mechanisms in diseases such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) may include molecular mimicry and superantigen motifs that evoke and augment unwarranted immune responses. In the RA joint, tissue destruction is evident over time with recruitment of lymphoid and other cells plus the presence of rheumatoid factor that exhibits increased affinity and change in isotype; evidence of an antigen-driven immune response. The precise trigger of course, remains unknown although certain HERVs have been implicated. In a previous study we found evidence for increased expression of HERV-K10 mRNA in patients with RA. Here we have extended this work by investigating the serological expression to HERV-K10 in patients with RA, SLE, osteoarthritis, normals and other inflammatory disease groups. The study utilised a novel peptide ELISA immunoassay using segments of HERV-K10 identified through bioinformatic analysis. In particular, biotinylation of peptides was necessary for serological discrimination between patients. Overall a significant difference (p<0.05) was found for RA patients in terms of antibody activity to HERV-K10. There was also an increased level of antibodies to HERV-K10 in patients with renal lupus although this was below the level of significance. It is possible that HERV-K10 could act as a trigger in RA/SLE through regions of similarity to host proteins. In this case, the immune response to HERV-K10 could lead to collateral damage and pathogenesis of disease

Characterization of anti-myosin monoclonal antibodies.

The characterization of monoclonal antibodies (MAbs) with regard to reactivity and specificity is important for the successful application as a molecular probe and/or diagnostic reagent. Furthermore, it is recognized that some monoclonal reagents perform well in some assay systems but not others. In this study, the reactivity profiles of two anti-myosin MAbs (H1 and DH2, raised against human cardiac myosin) were evaluated in enzyme-linked immunosorbent assay (ELISA), slot-blotting, and immunocytochemistry. Both antibodies performed well in slot-blotting against myosin heavy chain preparations from cardiac and skeletal muscle and from non-human sources. In general, MAb H1 demonstrated strong to moderate reactivity in all assay systems, whilst MAb DH2 faired poorly in ELISA. MAb H1 also showed reactivity to synthetic peptides of myosin, one of which possessed a motif (ERRDA, single amino acid code) that was found in other human and nonhuman myosin protein sequences that could explain its cross-reactive profile. Intriguingly, this motif was found on viral and other pathogenic agents associated with myocarditis. Hence, it is speculated that this region could give some credence to the mechanism of molecular mimicry associated with some cardiac diseases. Overall, MAb H1 may serve as a useful probe of myosin structure

A novel multiplex RT-PCR system detects human endogenous retrovirus-K in breast cancer

Metadata only.Human endogenous retrovirus HERV-K like-sequences have been implicated in certain cancers. We developed a novel multiplex RT-PCR system for HERV-K that yielded a 533bp product together with a smaller sized product (319bp) of the house keeping gene, histidyl tRNA synthetase (HtRNAS). The latter spanned an intron that also served to validate target cDNA. PCR amplicons of HERV-K and HtRNAS were visualised using a gel documentation system and the pixel intensity used to derive semi-quantitative levels of viral expression. Our data showed that HERV-K10 was significantly elevated in MCF-7 cells treated with estrogen. Interestingly, HERV-K expression was higher in MCF-7 cells selected with adriamycin. RT-PCR combined with Southern blotting also detected HERV-K from breast cancer tissue using laser capture microscopy. This study highlights the presence of HERV-K in the breast cancer cell lines MCF-7 and MCF-7 ADR and confirms HERV-K10 transcripts in the cell line T47D. We believe this study to be a novel approach in determining levels of HERV-K expression and for detecting this virus in cancer cell lines and tissues

Demystified. Human endogenous retroviruses.

Human endogenous retroviruses (HERVs) are a family of viruses within our genome with similarities to present day exogenous retroviruses. HERVs have been inherited by successive generations and it is possible that some have conferred biological benefits. However, several HERVs have been implicated in certain cancers and autoimmune diseases. This article demystifies these retroviruses by providing an insight into HERVs, their means of classification, and a synopsis of HERVs implicated in cancer and autoimmunity. Furthermore, the biological roles of HERVs are explored