In Vitro Priming of Naïve T-cells with p-Phenylenediamine and Bandrowski's Base.

p-Phenylenediamine (PPD) is a component of hair dye formulations that is associated with T-cell mediated allergic contact dermatitis. Antigen-specific T-cells from allergic contact dermatitis patients are activated with either PPD or the oxidation product, Bandrowski's base. In nonallergic individuals, T-cells that are activated by Bandrowski's base, but not by PPD, are readily detectable. The aim of the current study was to use an in vitro T-cell priming assay to assess the activation of memory and naïve T-cells from healthy donors with PPD and Bandrowski's base, and to compare these responses to those observed from allergic patients. Both PPD and Bandrowski's base-responsive clones were generated from allergic patients. The majority of Bandrowski's base-responsive clones were CD4+ and displayed a lack of PPD reactivity. In contrast, CD4+ and CD8+ clones displaying PPD reactivity were detected. Approximately 25% of these displayed low levels of reactivity to Bandrowski's base. Clones from the allergic patients secreted a range of cytokines including IFN-γ, Il-13, and Il-22. In healthy donors, Bandrowski's base-specific T-cell proliferative responses and cytokine secretion were detected with both naïve and memory T-cells. T-cell clones generated from the Bandrowski's base-responsive cultures responded to Bandrowski's base but not PPD. PPD-specific naïve and memory T-cell responses were not detected from healthy donors. These data show that Bandrowski's base stimulates pre-existing memory T-cells isolated from healthy donors and primes naïve T-cells when the chemical is bound to autologous dendritic cells. Priming naïve T-cells against PPD failed, suggesting an important individual susceptibility factor is missing from the in vitro T-cell priming assay.

Nursing student placements in clinical research.

Research is part of the remit of the NHS and consequently many patients participate in research studies. Clinical nurses have a vital role in research, since they care for patients who participate in studies and act as patient advocates during research. However, nursing students are rarely provided with an opportunity to undertake a research placement. They therefore have a limited understanding of what being a research participant involves for patients. This article describes the type of research carried out in an NHS trust in northwest England, how research placements were set up in that trust and the beneficial effects on learning of such placements. The aim of the article is to encourage other research nurses to set up student placements and encourage nursing students to request research placements.

Electronic health records for biological sample collection: feasibility study of statin-induced myopathy using the Clinical Practice Research Datalink.

AIMS: Electronic healthcare records (EHRs) are increasingly used to store clinical information. A secondary benefit of EHRs is their use, in an anonymized form, for observational research. The Clinical Practice Research Datalink (CPRD) contains EHRs from primary care in the UK and, despite 1083 peer-reviewed research publications, has never been used to obtain pharmacogenetic samples. Using a statin-induced myopathy paradigm, we evaluated using the CPRD to obtain patient samples for a pharmacogenetic study targeting 250 cases and 500 controls from UK general practitioner (GP) practices. METHODS: The CPRD identified potential patients fitting specific case-definition criteria (active rhabdomyolysis or creatine phosphokinase > four times the upper limit of normal), and corresponding GP practices were asked to invite patient participation. Consenting patients were requested to provide either saliva or blood samples and to complete an ethnicity questionnaire. Control subjects were recruited from the same GP practice (saliva) or a small number of practices (blood). Samples were forwarded for DNA extraction. RESULTS: Thirty-six months of recruitment yielded DNA samples from 149 statin-induced myopathy cases and 587 tolerant controls. Data show that contacting patients through their GP is a reliable method for obtaining samples without compromising anonymity. Saliva collection directly from patients was considerably less effective than blood sampling. After 10 months of recruitment, saliva sampling was suspended in favour of blood sampling. CONCLUSIONS: We demonstrate the potential of EHRs for identifying accurately phenotyped cases and controls for pharmacogenetic studies. Recruitment was successful only because of the willingness of GP practices to participate and the existence of strong doctor-patient relationships. The present study provides a model that can be implemented in future genetic analyses using EHRs.

In silico analysis of HLA associations with drug-induced liver injury: use of a HLA-genotyped DNA archive from healthy volunteers.

BACKGROUND: Drug-induced liver injury (DILI) is one of the most common adverse reactions leading to product withdrawal post-marketing. Recently, genome-wide association studies have identified a number of human leukocyte antigen (HLA) alleles associated with DILI; however, the cellular and chemical mechanisms are not fully understood. METHODS: To study these mechanisms, we established an HLA-typed cell archive from 400 healthy volunteers. In addition, we utilized HLA genotype data from more than four million individuals from publicly accessible repositories such as the Allele Frequency Net Database, Major Histocompatibility Complex Database and Immune Epitope Database to study the HLA alleles associated with DILI. We utilized novel in silico strategies to examine HLA haplotype relationships among the alleles associated with DILI by using bioinformatics tools such as NetMHCpan, PyPop, GraphViz, PHYLIP and TreeView. RESULTS: We demonstrated that many of the alleles that have been associated with liver injury induced by structurally diverse drugs (flucloxacillin, co-amoxiclav, ximelagatran, lapatinib, lumiracoxib) reside on common HLA haplotypes, which were present in populations of diverse ethnicity. CONCLUSIONS: Our bioinformatic analysis indicates that there may be a connection between the different HLA alleles associated with DILI caused by therapeutically and structurally different drugs, possibly through peptide binding of one of the HLA alleles that defines the causal haplotype. Further functional work, together with next-generation sequencing techniques, will be needed to define the causal alleles associated with DILI.