Rheumatoid arthritis sera react with a phage-displayed peptide selected by a monoclonal antibody to type II collagen that has homology to EBNA-1

Antibodies to type II collagen, and to Epstein Barr virus nuclear antigen-1 (EBNA-1) have been associated with rheumatoid arthritis (RA). In studies involving probing of phage-displayed random peptide libraries with an antibody to type II collagen, CII-C1, we observed that among 17 phagotopes selected 5 expressed peptides with homology with the sequence of EBNA-1. The residues in common were RLPFG. Hence we tested sera from 50 patients with RA, of whom 26 had antibodies to native type II collagen, and 43 healthy controls, for reactivity by ELISA with a phagotope selected 4 times, which expressed the peptide RRLPFGSQM. Eight RA sera (16%) but no normal sera reacted with the phagotope (p = 0.025). This reactivity could not be correlated with reactivity of RA sera with EBNA-1 by semi-quantitative western blot, with which reactivity occurred in 78% of RA patients and 81% of controls. Evidence for molecular mimicry was not found insofar as the phagotope did not inhibit reactivity of RA sera with EBNA-1 and CII-C1 was not reactive with EBNA-1. We conclude that the reactivity of the RA sera with the phagotope is most likely due to the phagotope being a mimic of an epitope of type II collagen for a proportion of RA sera.

Mimotopes identified by phage display for the monoclonal antibody CII- C1 to type II collagen

The characterization of B cell epitopes has been advanced by the use of random peptide libraries displayed within the coat protein of bacteriophage. This technique was applied to the monoclonal antibody (mAb) C1 to type II collagen (CII-C1). CII-C1 is known to react with a conformational epitope on type II collagen that includes residues 359-363. Three rounds of selection were used to screen two random nonameric phage libraries and 18 phagotopes were isolated. CII-C1 reacted by ELISA with 17 of the 18 phagotopes: one phagotope contained a stop codon. Of the eight most reactive phage, seven inhibited the reactivity by ELISA of CII-C1 with type II collagen. Of the 18 phage isolated, 11 encoded the motif F-G-x-Q with the sequence F-G-S-Q in 6, 2 encoded F-G-Q, and one the reverse motif Q-x-y-F. Most phagotopes that inhibited the reactivity of CII-C1 encoded two particular motifs consisting of two basic amino acid residues and a hydrophobic residue in the first part of the insert and the F-G-x-Q or F-G-Q motif ill the second part; phagotopes which contained only one basic residue in the first part of the sequence were less reactive. These motifs are not represented in the linear sequence of type II collagen and thus represent mimotopes of the epitope for CII-C1 on type II collagen. There were five phagotopes with peptide inserts containing the sequence RLPFG occurring in the Epstein-Barr virus nuclear antigen, EBNA- 1. This is of interest because EBV has been implicated in the initiation of rheumatoid arthritis (RA) by reason of increased reactivity to EBNA-1 in RA sera. In conclusion, the phage display technique disclosed mimotopes for a conformational epitope of type II collagen, and revealed an interesting homology with a sequence of the EBNA-1 antigen from Epstein Barr virus.

Antibody reactivity to the transmembrane protein of the caprine arthritis encephalitis virus correlates with severity of arthritis: No evidence for the involvement of epitope mimicry

Serum and synovial antibody reactivities of caprine arthritis encephalitis virus (CAEV) infected goats were assessed by Western blotting against purified CAEV antigen and the greatest intensity of reactivity in the serum of arthritic goats was to the gp45 transmembrane protein (TM). The extracytoplasmic domain of the TM gene was cloned into a pGEX vector and expressed in Escherichia coil as a glutathione S transferase fusion protein (GST-TM). This clone was found to be 90.5 and 89.2% homologous to published sequences of CAEV TM gene. Serum of 16 goats naturally infected with CAEV were examined by Western blotting for reactivity to the fusion protein. Antibody reactivity to the GST-TM correlated with clinically detectable arthritis (R = 0.642, P ≤ 0.007). The hypothesis that the immune response to the envelope proteins of the CAEV contributes to the severity of arthritis in goats naturally infected with CAEV via epitope mimicry was tested. Antibodies from 5 CAEV infected goats were affinity purified against the GST-TM fusion protein and tested for cross-reactivity with a series of goat synovial extracts and proteogylcans. No serum antibody response or cross-reactivity of affinity purified antibodies could be detected. Peptides of the CAEV SU that were predicted to be linear epitopes and a similar heat shock protein 83 (HSP) peptide identified by database searching, were synthesized and tested for reactivity in CAEV goats using ELISA, in vitro lymphocyte proliferation and delayed type hypersensitivity (DTH) assays. Peripheral blood lymphocytes from 10 of 17 goats with long term natural CAEV infections proliferated in vitro in response to CAEV and in vivo 3 of 7 CAEV infected goats had a DTH reaction to CAEV antigen. However, none of the peptides elicited significant cell mediated immune responses from CAEV infected goats. No antibody reactivity to the SU peptides or HSP peptide was found. We observed that the antibody reactivity to the CAEV TM protein associated with severity of arthritis however epitope mimicry by the envelope proteins of CAEV is unlikely to be involved.

Antibody treatments of inflammatory arthritis

Inflammatory arthropathies such as rheumatoid arthritis, ankylosing spondylitis, and psoriatic arthritis are extremely common in the community, with a prevalence of up to 5%, and they cause substantial morbidity. The development of anti-TNF agents for use initially in rheumatoid arthritis, and subsequently more broadly in inflammatory arthritis, represents the biggest advance in management of these conditions since the introduction of corticosteroid agents, and is a major vindication of public funded arthritis research. However, there are limitations of even these highly effective agents. A significant minority of patients with inflammatory arthritis do not respond to these anti-TNF agents, they are associated with substantial risk of toxicity, require parenteral administration, and are extremely expensive. New antibody treatments in development can be divided into anti-cytokine agents, cell-targeted therapies, co-stimulation inhibitors, and treatments aimed at preventing joint erosion consequent on inflammation. This review discusses the state of the art in the development of these agents for management of this common group of diseases.

Multiple primary cancers associated with Merkel cell carcinoma in Queensland, Australia, 1982-2011

The relatively high incidence of Merkel cell carcinoma (MCC) in Queensland provides a valuable opportunity to examine links with other cancers. A retrospective cohort study was performed using data from the Queensland Cancer Registry. Standardized incidence ratios (SIRs) were used to approximate the relative risk of being diagnosed with another primary cancer either following or prior to MCC. Patients with an eligible first primary MCC (n=787) had more than double the expected number of subsequent primary cancers (SIR=2.19, 95% confidence interval (CI)=1.84–2.60; P<0.001). Conversely, people who were initially diagnosed with cancers other than MCC were about two and a half times more likely to have a subsequent primary MCC (n=244) compared with the general population (SIR=2.69, 95% CI=2.36–3.05; P<0.001). Significantly increased bi-directional relative risks were found for melanoma, lip cancer, head and neck cancer, lung cancer, myelodysplastic diseases, and cancer with unknown primary site. In addition, risks were elevated for female breast cancer and kidney cancer following a first primary MCC, and for subsequent MCCs following first primary colorectal cancer, prostate cancer, non-Hodgkin lymphoma, or lymphoid leukemia. These results suggest that several shared pathways are likely for MCC and other cancers, including immunosuppression, UV radiation, and genetics.

Role of enhanced vector transmission of a new West Nile virus strain in an outbreak of equine disease in Australia in 2011

Background In 2011, a variant of West Nile virus Kunjin strain (WNVKUN) caused an unprecedented epidemic of neurological disease in horses in southeast Australia, resulting in almost 1,000 cases and a 9% fatality rate. We investigated whether increased fitness of the virus in the primary vector, Culex annulirostris, and another potential vector, Culex australicus, contributed to the widespread nature of the outbreak. Methods Mosquitoes were exposed to infectious blood meals containing either the virus strain responsible for the outbreak, designated WNVKUN2011, or WNVKUN2009, a strain of low virulence that is typical of historical strains of this virus. WNVKUN infection in mosquito samples was detected using a fixed cell culture enzyme immunoassay and a WNVKUN- specific monoclonal antibody. Probit analysis was used to determine mosquito susceptibility to infection. Infection, dissemination and transmission rates for selected days post-exposure were compared using Fisher’s exact test. Virus titers in bodies and saliva expectorates were compared using t-tests. Results There were few significant differences between the two virus strains in the susceptibility of Cx. annulirostris to infection, the kinetics of virus replication and the ability of this mosquito species to transmit either strain. Both strains were transmitted by Cx. annulirostris for the first time on day 5 post-exposure. The highest transmission rates (proportion of mosquitoes with virus detected in saliva) observed were 68% for WNVKUN2011 on day 12 and 72% for WNVKUN2009 on day 14. On days 12 and 14 post-exposure, significantly more WNVKUN2011 than WNVKUN2009 was expectorated by infected mosquitoes. Infection, dissemination and transmission rates of the two strains were not significantly different in Culex australicus. However, transmission rates and the amount of virus expectorated were significantly lower in Cx. australicus than Cx. annulirostris. Conclusions The higher amount of WNVKUN2011 expectorated by infected mosquitoes may be an indication that this virus strain is transmitted more efficiently by Cx. annulirostris compared to other WNVKUN strains. Combined with other factors, such as a convergence of abundant mosquito and wading bird populations, and mammalian and avian feeding behaviour by Cx. annulirostris, this may have contributed to the scale of the 2011 equine epidemic.

Vitamin D deficiency in patients with malignancy in Brisbane

PURPOSE: This study aims to investigate the prevalence and factors predictive of vitamin D deficiency in patients with malignancy in Brisbane, Australia (latitude 27° S). METHODS: This is a prospective cross-sectional study measuring serum levels of 25-hydroxyvitamin D (25-OHD) in 100 subjects with non-haematological cancer at least 18 years of age not taking vitamin D supplements attending a day oncology unit and oncology/palliative care inpatient ward in Brisbane, Australia. RESULTS: Thirty-seven per cent of outpatient and 49 % of inpatient subjects respectively were vitamin D deficient. Functional status was predictive of low vitamin D levels. CONCLUSION: There was a high prevalence of vitamin D deficiency in patients with cancer in Brisbane, Australia.

Archiving primary data: Solutions for long-term studies

The recent trend for journals to require open access to primary data included in publications has been embraced by many biologists, but has caused apprehension amongst researchers engaged in long-term ecological and evolutionary studies. A worldwide survey of 73 principal investigators (Pls) with long-term studies revealed positive attitudes towards sharing data with the agreement or involvement of the PI, and 93% of PIs have historically shared data. Only 8% were in favor of uncontrolled, open access to primary data while 63% expressed serious concern. We present here their viewpoint on an issue that can have non-trivial scientific consequences. We discuss potential costs of public data archiving and provide possible solutions to meet the needs of journals and researchers.

Removal of organic content from diesel exhaust particles alters cellular responses of primary human bronchial epithelial cells cultured at an air-liquid interface

Background Exposure to air pollutants, including diesel particulate matter, has been linked to adverse respiratory health effects. Inhaled diesel particulate matter contains adsorbed organic compounds. It is not clear whether the adsorbed organics or the residual components are more deleterious to airway cells. Using a physiologically relevant model, we investigated the role of diesel organic content on mediating cellular responses of primary human bronchial epithelial cells (HBECs) cultured at an air-liquid interface (ALI). Methods Primary HBECs were cultured and differentiated at ALI for at least 28 days. To determine which component is most harmful, we compared primary HBEC responses elicited by residual (with organics removed) diesel emissions (DE) to those elicited by neat (unmodified) DE for 30 and 60 minutes at ALI, with cigarette smoke condensate (CSC) as the positive control, and filtered air as negative control. Cell viability (WST-1 cell proliferation assay), inflammation (TNF-α, IL-6 and IL-8 ELISA) and changes in gene expression (qRT-PCR for HO-1, CYP1A1, TNF-α and IL-8 mRNA) were measured. Results Immunofluorescence and cytological staining confirmed the mucociliary phenotype of primary HBECs differentiated at ALI. Neat DE caused a comparable reduction in cell viability at 30 or 60 min exposures, whereas residual DE caused a greater reduction at 60 min. When corrected for cell viability, cytokine protein secretion for TNF-α, IL-6 and IL-8 were maximal with residual DE at 60 min. mRNA expression for HO-1, CYP1A1, TNF-α and IL-8 was not significantly different between exposures. Conclusion This study provides new insights into epithelial cell responses to diesel emissions using a physiologically relevant aerosol exposure model. Both the organic content and residual components of diesel emissions play an important role in determining bronchial epithelial cell response in vitro. Future studies should be directed at testing potentially useful interventions against the adverse health effects of air pollution exposure.

Fine mapping and replication of genetic risk Loci in primary sclerosing cholangitis

Background and aims. Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease characterized by progressive inflammation and fibrosis of the bile ducts eventually leading to biliary cirrhosis. Recent genetic studies in PSC have identified associations at 2q13, 2q35, 3p21, 4q27, 13q31 and suggestive association at 10p15. The aim of this study was to further characterize and refine the genetic architecture of PSC. Methods. We analyzed previously reported associated SNPs at four of these non-HLA loci and 59 SNPs tagging the IL-2/IL-21 (4q27) and IL2RA (10p15) loci in 992 UK PSC cases and 5162 healthy UK controls. Results. The most associated SNPs identified were rs3197999 (3p21 (MST1), p = 1.9 × 10 -6, OR A vs G = 1.28, 95% CI (1.16-1.42)); rs4147359 (10p15 (IL2RA), p = 2.6 × 10 -4, OR A vs G = 1.20, 95% CI (1.09-1.33)) and rs12511287 (4q27 (IL-2/IL-21), p = 3.0 × 10 -4, OR A vs T = 1.21, 95% CI (1.09-1.35)). In addition, we performed a meta-analysis for selected SNPs using published summary statistics from recent studies. We observed genome-wide significance for rs3197999 (3p21 (MST1), P combined = 3.8 × 10 -12) and rs4147359 (10p15 (IL2RA), P combined = 1.5 × 10 -8). Conclusion. We have for the first time confirmed the association of PSC with genetic variants at 10p15 (IL2RA) locus at genome-wide significance and replicated the associations at MST1 and IL-2/IL-21 loci in a large homogeneous UK population. These results strongly implicate the role of IL-2/IL2RA pathway in PSC and provide further confirmation of MST1 association. © Informa Healthcare.

Risedronate in adults with osteogenesis imperfecta type I: Increased bone mineral density and decreased bone turnover, but high fracture rate persists

Summary Bisphosphonates can increase bone mineral density (BMD) in children with osteogenesis imperfecta (OI). In this study of adults with OI type I, risedronate increased BMD at lumbar spine (but not total hip) and decreased bone turnover. However, the fracture rate in these patients remained high. Introduction Intravenous bisphosphonates given to children with OI can increase BMD and reduce fracture incidence. Oral and/or intravenous bisphosphonates may have similar effects in adults with OI. We completed an observational study of the effect of risedronate in adults with OI type I. Methods Thirty-two adults (mean age, 39 years) with OI type I were treated with risedronate (total dose, 35 mg weekly) for 24 months. Primary outcome measures were BMD changes at lumbar spine (LS) and total hip (TH). Secondary outcome measures were fracture incidence, bone pain, and change in bone turnover markers (serum procollagen type I aminopropeptide (P1NP) and bone ALP). A meta-analysis of published studies of oral bisphosphonates in adults and children with OI was performed. Results Twenty-seven participants (ten males and seventeen females) completed the study. BMD increased at LS by 3.9% (0.815 vs. 0.846 g/cm 2, p=0.007; mean Z-score, -1.93 vs. -1.58, p=0.002), with no significant change at TH. P1NP fell by 37% (p=0.00041), with no significant change in bone ALP (p=0.15). Bone pain did not change significantly (p=0.6). Fracture incidence remained high, with 25 clinical fractures and 10 major fractures in fourteen participants (0.18 major fractures per person per year), with historical data of 0.12 fractures per person per year. The meta-analysis did not demonstrate a significant difference in fracture incidence in patients with OI treated with oral bisphosphonates. Conclusions Risedronate in adults with OI type I results in modest but significant increases in BMD at LS, and decreased bone turnover. However, this may be insufficient to make a clinically significant difference to fracture incidence.

Genetic risk and a primary role for cell-mediated immune mechanisms in multiple sclerosis

Multiple sclerosis is a common disease of the central nervous system in which the interplay between inflammatory and neurodegenerative processes typically results in intermittent neurological disturbance followed by progressive accumulation of disability. Epidemiological studies have shown that genetic factors are primarily responsible for the substantially increased frequency of the disease seen in the relatives of affected individuals, and systematic attempts to identify linkage in multiplex families have confirmed that variation within the major histocompatibility complex (MHC) exerts the greatest individual effect on risk. Modestly powered genome-wide association studies (GWAS) have enabled more than 20 additional risk loci to be identified and have shown that multiple variants exerting modest individual effects have a key role in disease susceptibility. Most of the genetic architecture underlying susceptibility to the disease remains to be defined and is anticipated to require the analysis of sample sizes that are beyond the numbers currently available to individual research groups. In a collaborative GWAS involving 9,772 cases of European descent collected by 23 research groups working in 15 different countries, we have replicated almost all of the previously suggested associations and identified at least a further 29 novel susceptibility loci. Within the MHC we have refined the identity of the HLA-DRB1 risk alleles and confirmed that variation in the HLA-A gene underlies the independent protective effect attributable to the class I region. Immunologically relevant genes are significantly overrepresented among those mapping close to the identified loci and particularly implicate T-helper-cell differentiation in the pathogenesis of multiple sclerosis. © 2011 Macmillan Publishers Limited. All rights reserved.

Dietary and exercise interventions to improve quality of life, metabolic risk factors and androgen deficiency symptoms in men with prostate cancer undergoing androgen deprivation therapy

Introduction Lifestyle interventions might be useful in the management of adverse effects of androgen deprivation therapy (ADT) in men with prostate cancer. Objectives To examine the effects of dietary and exercise interventions on quality of life (QoL), metabolic risk factors and androgen deficiency symptoms in men with prostate cancer undergoing ADT. Methods CINAHL, Cochrane library, Medline and PsychINFO were searched to identify randomised controlled trials published from January, 2004 to October, 2014. Data extraction and methodological quality assessment was independently conducted by two reviewers. Meta-analysis was conducted using RevMan® 5.3.5. Results Of 2183 articles retrieved, 11 studies met the inclusion criteria and had low risk of bias.Nine studies evaluated exercise (resistance and/or aerobic and/or counselling) and three evaluated dietary supplementation. Median sample size =79 (33–121) and median intervention duration was 12 weeks (12–24). Exercise improved QoL measures (SMD 0.26, 95%CI −0.01 to 0.53) but not body composition, metabolic risk or vasomotor symptoms. Qualitative analysis indicated soy (or isoflavone) supplementation did not improve vasomotor symptoms; however, may improve QoL. Conclusions Few studies have evaluated the efficacy of lifestyle interventions in the management of adverse effects of ADT. We found inconclusive results for exercise in improving QoL and negative results for other outcomes. For soy-based products, we found negative results for modifying vasomotor symptoms and inconclusive results for improving QoL. Future work should investigate the best mode of exercise for improving QoL and other interventions such as dietary counselling should be investigated for their potential to modify these outcomes.

Positive psychological strengths and school engagement in primary school children

A sizeable body of research has investigated the impact of specific character strengths or traits on significant outcomes. Some recent research is beginning to consider the effects of groups of strengths, combined as a higher order variable, and termed covitality. This study investigated the combined influence of four positive character traits: gratitude, optimism, zest and persistence, upon school engagement, within a sample of 112 Australian primary school students. The combined effect of these four traits, in defining covitality as a higher or second order factor within a path analysis, was found to predict relatively higher levels of school engagement and pro-social behaviour.

Immunization of male bonnet monkeys (M-radiata) with a recombinant FSH receptor preparation affects testicular function and fertility

Immunization of proven fertile adult male monkeys (n = 3) with a recombinant FSH receptor protein preparation (oFSHR-P) (representing amino acids 1-134 of the extracellular domain of the receptor Mr similar to 15KDa) resulted in production of receptor blocking antibodies. The ability of the antibody to bind a particulate FSH receptor preparation and receptors in intact granulosa cells was markedly (by 30-80%) inhibited by FSH. Serum T levels and LH receptor function following immunization remained unchanged. The immunized monkeys showed a 50% reduction (p<0.001) in transformation of spermatogonia(2C) to primary spermatocytes (4C) as determined by flow cytometry and the 4C:2C ratio showed a correlative change (R 0.81, p<0.0007) with reduction in fertility index (sperm counts X motility score). Breeding studies indicated that monkeys became infertile between 242-368 days of immunization when the fertility index was in the range of 123+/-76 to 354+/-42 (compared to a value of 1602+/-384 on day 0). As the effects observed ate near identical to that seen following immunization with FSH it is suggestive that oFSHR-P can substitute for FSH in the development of a contraceptive vaccine.