393 research outputs found

    Development of techniques for the isolation and characterisation of human monoclonal antibodies from Hepatitis C virus infected individuals

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    Infection with hepatitis C virus (HCV) is cleared spontaneously in only 20% of cases with the majority of individuals developing a chronic infection. This discrepancy in disease outcome is incompletely understood but current understanding of the immune response to HCV suggests that rapid induction of a broadly neutralising antibody (nAb) response leads to resolution of acute infection. The majority of nAb identified target the envelope glycoproteins, particularly E2, and most appear to inhibit binding of E2 to the cellular receptor CD81. Antibodies targeting other interactions, such as those with the receptor CLDN or the fusion determinant, are underrepresented in the repertoire of anti-HCV antibodies. However, the antibody discovery process may have been biased by the nature of the assays used. Therefore new assays are needed to enable the discovery and characterisation of antibodies in an unbiased manner. To facilitate this, a novel insect cell display library was developed for mapping antibody-binding epitopes. Cells expressing specific E2 mutants provided the necessary proof-of-principle that loss of antibody binding could be detected in this system before a library expressing randomly mutated E2 was developed. Sorting experiments demonstrated that single cells could be isolated and enriched based on loss of antibody binding. Secondly, a method for characterising the immunoglobulin (Ig) genes of HCV infected patients was developed; Ig genes were isolated from small numbers of B cells and the sequences analysed. Finally, a patient cohort was studied with a view to investigating the evolution of the antibody response during early infection. The unreliable nature of the samples prevented such analysis; however a DNA fingerprinting method of testing the origin and relatedness of serum samples was developed. This will improve the reliability of future studies. Together these methods provide a work model for the assessment of samples and the isolation and characterisation of antibodies

    Development of techniques for the isolation and characterisation of human monoclonal antibodies from Hepatitis C virus infected individuals

    Get PDF
    Infection with hepatitis C virus (HCV) is cleared spontaneously in only 20% of cases with the majority of individuals developing a chronic infection. This discrepancy in disease outcome is incompletely understood but current understanding of the immune response to HCV suggests that rapid induction of a broadly neutralising antibody (nAb) response leads to resolution of acute infection. The majority of nAb identified target the envelope glycoproteins, particularly E2, and most appear to inhibit binding of E2 to the cellular receptor CD81. Antibodies targeting other interactions, such as those with the receptor CLDN or the fusion determinant, are underrepresented in the repertoire of anti-HCV antibodies. However, the antibody discovery process may have been biased by the nature of the assays used. Therefore new assays are needed to enable the discovery and characterisation of antibodies in an unbiased manner. To facilitate this, a novel insect cell display library was developed for mapping antibody-binding epitopes. Cells expressing specific E2 mutants provided the necessary proof-of-principle that loss of antibody binding could be detected in this system before a library expressing randomly mutated E2 was developed. Sorting experiments demonstrated that single cells could be isolated and enriched based on loss of antibody binding. Secondly, a method for characterising the immunoglobulin (Ig) genes of HCV infected patients was developed; Ig genes were isolated from small numbers of B cells and the sequences analysed. Finally, a patient cohort was studied with a view to investigating the evolution of the antibody response during early infection. The unreliable nature of the samples prevented such analysis; however a DNA fingerprinting method of testing the origin and relatedness of serum samples was developed. This will improve the reliability of future studies. Together these methods provide a work model for the assessment of samples and the isolation and characterisation of antibodies

    Coagulation Profiles in Humans Exposed to Exertional Hypobaric Decompression Stress Determined by Calibrated Automated Thrombogram

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    Citation: Madden, L.A.; Vince, R.V.; Edwards, V.C.; Lee, V.M.; Connolly, D.M. Coagulation Profiles in Humans Abstract: The blood coagulation response to decompression stress in humans has yet to be fully investigated. Here we utilised calibrated automated thrombogram (CAT) on samples from healthy volunteers exposed to decompression stress to investigate real-time thrombin generation. To induce decompression stress, fifteen apparently healthy males (age 20-50 yr) were exposed to two consecutive ascents to 25,000 ft for 60 min (1st ascent) and then 90 min (2nd ascent) while breathing 100% oxygen. Citrated blood samples were taken prior to exposure (T0), following the 2nd ascent (T8) and at 24 h (T24). Thrombin generation curves were obtained using Thrombinoscope TM. Parameters determined were lag time (LAG), time to peak (TTP), peak thrombin (PEAK), endogenous thrombin potential (ETP) and velocity index (VEL). Of the 15 subjects, 12 had validated coagulation profiles. TTP and ETP showed no significant differences. However, there was a significant increase in VEL from T0 to T8 (p = 0.025) and from T8 to T24 (p = 0.043). A non-significant trend of an overall increase in PEAK was also observed from T0 to T8 (p = 0.069) and from T8 to T24 (p = 0.098). PEAK and VEL were found to be correlated. Taken together, these two parameters suggest an overall shift towards a more procoagulant profile following hypobaric stress

    Use of short-tandem repeat (STR) fingerprinting to validate sample origins in hepatitis C virus molecular epidemiology studies

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    Sequence analysis is used to define the molecular epidemiology and evolution of the hepatitis C virus. Whilst most studies have shown that individual patients harbour viruses that are derived from a limited number of highly related strains, some recent reports have shown that some patients can be co-infected with very distinct variants whose frequency can fluctuate greatly. Whilst co-infection with highly divergent strains is possible, an alternative explanation is that such data represent contamination or sample mix-up. In this study, we have shown that DNA fingerprinting techniques can accurately assess sample provenance and differentiate between samples that are truly exhibiting mixed infection from those that harbour distinct virus populations due to sample mix-up. We have argued that this approach should be adopted routinely in virus sequence analyses to validate sample provenance

    Improving Social Norms and Actions to Prevent Sexual and Intimate Partner Violence: A Pilot Study of the Impact of Green Dot Community on Youth

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    Sexual violence (SV) and intimate partner violence (IPV), which often cooccur with bullying, are serious public health issues underscoring the need for primary prevention. The purpose of this study was to examine the impact of a community-building SV and IPV prevention program, Green Dot Community, on adolescents’ perceptions of community social norms and their propensity to intervene as helpful actionists using two independent data sources. Green Dot Community takes place in towns and aims to influence all town members to prevent SV and IPV by addressing protective factors (i.e., collective efficacy, positive prevention social norms, and bystander helping, or actionism). In the current study, one town received Green Dot Community (the prevention-enhanced town), and two towns received prevention as usual (i.e., awareness and fundraising events by local IPV and SV advocacy centers). The program was evaluated using a two-part method: (a) A cross-sectional sample of high school students from three rural communities provided assessment of protective factors at two time points (Time 1, n = 1,187; Time 2, n = 877) and (b) Youth Risk Behavior Survey data from the state Department of Health were gathered before and after program implementation (Time 1, n = 2,034; Time 2, n = 2,017) to assess victimization rates. Youth in the prevention-enhanced town reported higher collective efficacy and more positive social norms specific to helping in situations of SV and IPV over time but did not differ on bystander behaviors or on victimization rates. Community-based prevention initiatives may be helpful in changing community norms to prevent SV/IPV

    Sprouty2 loss‐induced IL6 drives castration‐resistant prostate cancer through scavenger receptor B1

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    Metastatic castration‐resistant prostate cancer (mCRPC) is a lethal form of treatment‐resistant prostate cancer and poses significant therapeutic challenges. Deregulated receptor tyrosine kinase (RTK) signalling mediated by loss of tumour suppressor Sprouty2 (SPRY2) is associated with treatment resistance. Using pre‐clinical human and murine mCRPC models, we show that SPRY2 deficiency leads to an androgen self‐sufficient form of CRPC. Mechanistically, HER2‐IL6 signalling axis enhances the expression of androgen biosynthetic enzyme HSD3B1 and increases SRB1‐mediated cholesterol uptake in SPRY2‐deficient tumours. Systemically, IL6 elevated the levels of circulating cholesterol by inducing host adipose lipolysis and hepatic cholesterol biosynthesis. SPRY2‐deficient CRPC is dependent on cholesterol bioavailability and SRB1‐mediated tumoral cholesterol uptake for androgen biosynthesis. Importantly, treatment with ITX5061, a clinically safe SRB1 antagonist, decreased treatment resistance. Our results indicate that cholesterol transport blockade may be effective against SPRY2‐deficient CRPC

    The role of neutralizing antibodies in hepatitis C virus infection

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    Hepatitis C virus (HCV) is a blood-borne virus estimated to infect around 170 million people worldwide and is, therefore, a major disease burden. In some individuals the virus is spontaneously cleared during the acute phase of infection, whilst in others a persistent infection ensues. Of those persistently infected, severe liver diseases such as cirrhosis and primary liver cancer may develop, although many individuals remain asymptomatic. A range of factors shape the course of HCV infection, not least host genetic polymorphisms and host immunity. A number of studies have shown that neutralizing antibodies (nAb) arise during HCV infection, but that these antibodies differ in their breadth and mechanism of neutralization. Recent studies, using both mAbs and polyclonal sera, have provided an insight into neutralizing determinants and the likely protective role of antibodies during infection. This understanding has helped to shape our knowledge of the overall structure of the HCV envelope glycoproteins -the natural target for nAb. Most nAb identified to date target receptor-binding sites within the envelope glycoprotein E2. However, there is some evidence that other viral epitopes may be targets for antibody neutralization, suggesting the need to broaden the search for neutralization epitopes beyond E2. This review provides a comprehensive overview of our current understanding of the role played by nAb in HCV infection and disease outcome and explores the limitations in the study systems currently used. In addition, we briefly discuss the potential therapeutic benefits of nAb and efforts to develop nAb-based therapies. Š 2012 SGM

    Establishing a distributed national research infrastructure providing bioinformatics support to life science researchers in Australia

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    EMBL Australia Bioinformatics Resource (EMBL-ABR) is a developing national research infrastructure, providing bioinformatics resources and support to life science and biomedical researchers in Australia. EMBL-ABR comprises 10 geographically distrib- uted national nodes with one coordinating hub, with current funding provided through Bioplatforms Australia and the University of Melbourne for its initial 2-year development phase. The EMBL-ABR mission is to: (1) increase Australia’s capacity in bioinformatics and data sciences; (2) contribute to the development of training in bioinformatics skills; (3) showcase Australian data sets at an international level and (4) enable engagement in international programs. The activities of EMBL-ABR are focussed in six key areas, aligning with comparable international initiatives such as ELIXIR, CyVerse and NIH Commons. These key areas—Tools, Data, Standards, Platforms, Compute and Training—are described in this article
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