Development of In Situ Hybridisation for Cytokines in Rejecting and Tolerant Cardiac Allografts

An In situ Hybridisation method was developed and utilised to evaluate and pinpoint the cellular presence of IL2 and IL4 mRNA transcripts produced by Th 1 and Th2 subsets respectively of activated T cells in frozen tissue sections of Lewis heart allografts from rejecting DA recipients and from recipients tolerised by Cyclosporin, Blood Transfusion and Antibody therapy regimens. A panel of monoclonal antibodies was used on the tissue sections initially and confirmed the presence of the activated T cells known to express the cytokines being studied. The Polymerase Chain Reaction was used next on tissue homogenates from the same samples to establish the presence of IL2 and IL4 mRNA transcripts and showed that IL2 mRNA sequences were present in all rejecting samples, but not in all tolerised samples, whereas with the exception of some day 2 grafts, IL4 transcripts were present in all other rejecting grafts, and also in CsA and Transfused grafts but not in all Antibody treated grafts. As the presence of cytokine transcripts had been established by PCR, the ISH method was utilised to attempt to pinpoint specific cellular production of the IL2 and IL4 transcripts in the frozen sections of the tissue. All rejecting grafts and grafts from Transfused recipients were found to be positive for IL2 transcripts whereas CsA treated recipients grafts were negative, and Antibody treated recipients grafts found to be variable. IL4 transcripts were found in 50% of the rejecting grafts only, suggesting that ISH is not sensitive enough to record low levels of mRNA expression and that the use of both the ISH and PCR methods in tandem would be more beneficial in intragraft cytokine transcription detection

Pathfinder cells provide a novel therapeutic intervention for acute kidney injury

Pathfinder cells (PCs) are a novel class of adult-derived cells that facilitate functional repair of host tissue. We used rat PCs to demonstrate that they enable the functional mitigation of ischemia reperfusion (I/R) injury in a mouse model of renal damage. Female C57BL/6 mice were subjected to 30 min of renal ischemia and treated with intravenous (i.v.) injection of saline (control) or male rat pancreas-derived PCs in blinded experimentation. Kidney function was assessed 14 days after treatment by measuring serum creatinine (SC) levels. Kidney tissue was assessed by immunohistochemistry (IHC) for markers of cellular damage, proliferation, and senescence (TUNEL, Ki67, p16ink4a, p21). Fluorescence in situ hybridization (FISH) was performed to determine the presence of any rat (i.e., pathfinder) cells in the mouse tissue. PC-treated animals demonstrated superior renal function at day 14 post-I/R, in comparison to saline-treated controls, as measured by SC levels (0.13 mg/dL vs. 0.23 mg/dL, p<0.001). PC-treated kidney tissue expressed significantly lower levels of p16ink4a in comparison to the control group (p=0.009). FISH analysis demonstrated that the overwhelming majority of repaired kidney tissue was mouse in origin. Rat PCs were only detected at a frequency of 0.02%. These data confirm that PCs have the ability to mitigate functional damage to kidney tissue following I/R injury. Kidneys of PC-treated animals showed evidence of improved function and reduced expression of damage markers. The PCs appear to act in a paracrine fashion, stimulating the host tissue to recover functionally, rather than by differentiating into renal cells. This study demonstrates that pancreatic-derived PCs from the adult rat can enable functional repair of renal damage in mice. It validates the use of PCs to regenerate damaged tissues and also offers a novel therapeutic intervention for repair of solid organ damage in situ

Comparing subjective and objective neighbourhood deprivation and their association with health over time among older adults in England

The importance of neighbourhood on individual health is widely documented. Less is known about the relative role of objective and subjective reports of neighbourhood conditions, how their effect on health changes as people age, and whether they moderate each other’s impact on health. This study uses the English Longitudinal Study of Ageing (ELSA) to determine whether older adults report worse self-rated health as they age, and whether this differs between objective and subjective measures of neighbourhood. ELSA data contain 53,988 person-years across six waves collected biannually between 2002 and 03 and 2012 and 13. Objective neighbourhood conditions are measured by the 2004 Index of Multiple Deprivation, and subjective neighbourhood conditions are captured by a summative neighbourhood dissatisfaction score. We find both objective and subjective neighbourhood composite scores independently predict poor health. There is no change over time in the probability of reporting poor health by baseline objective or subjective neighbourhood scores, suggesting neighbourhood effects do not compound as older adults age. There is no moderating effect of area dissatisfaction on the relationship between neighbourhood deprivation and health. The findings provide little support for causal neighbourhood effects operating in later life and indicate different causal pathways through which objective and subjective neighbourhood deprivation impact on health

Male frequent attenders of general practice and their help seeking preferences

Background: Low rates of health service usage by men are commonly linked to masculine values and traditional male gender roles. However, not all men conform to these stereotypical notions of masculinity, with some men choosing to attend health services on a frequent basis, for a variety of different reasons. This study draws upon the accounts of male frequent attenders of the General Practitioner's (GP) surgery, examining their help-seeking preferences and their reasons for choosing services within general practice over other sources of support. Methods: The study extends thematic analysis of interview data from the Self Care in Primary Care study (SCinPC), a large scale multi-method evaluation study of a self care programme delivered to frequent attenders of general practice. Data were collected from 34 semi-structured interviews conducted with men prior to their exposure to the intervention. Results: The ages of interviewed men ranged from 16 to 72 years, and 91% of the sample (n= 31) stated that they had a current health condition. The thematic analysis exposed diverse perspectives within male help-seeking preferences and the decision-making behind men's choice of services. The study also draws attention to the large variation in men's knowledge of available health services, particularly alternatives to general practice. Furthermore, the data revealed some men's lack of confidence in existing alternatives to general practice. Conclusions: The study highlights the complex nature of male help-seeking preferences, and provides evidence that there should be no 'one size fits all' approach to male service provision. It also provides impetus for conducting further studies into this under researched area of interest. © 2011 WPMH GmbH

Environmentalism, performance and applications: uncertainties and emancipations

This introductory article for a themed edition on environmentalism provides a particular context for those articles that follow, each of which engages with different aspects of environmentalism and performance in community-related settings. Responding to the proposition that there is a lacuna in the field of applied drama and environmentalism (Bottoms, 2010), we suggest that the more significant lack is that of ecocriticism. As the articles in this journal testify, there are many examples of applied theatre practice; what is required is sustained and rigorous critical engagement. It is to the gap of ecocriticism that we address this issue, signalling what we hope is the emergence of a critical field. One response to the multiple challenges of climate change is to more transparently locate the human animal within the environment, as one agent amongst many. Here, we seek to transparently locate the critic, intertwining the personal – ourselves, human actants – with global environmental concerns. This tactic mirrors much contemporary writing on climate change and its education, privileging personal engagement – a shift we interrogate as much as we perform. The key trope we anchor is that of uncertainty: the uncertainties that accompany stepping into a new research environment; the uncertainties arising from multiple relations (human and non-human); the uncertainties of scientific fact; the uncertainties of forecasting the future; and the uncertainties of outcomes – including those of performance practices. Having analysed a particular turn in environmental education (towards social learning) and the failure to successfully combine ‘art and reality’ in recent UK mainstream theatre events, such uncertainties lead to our suggestion for an ‘emancipated’ environmentalism. In support of this proposal, we offer up a reflection on a key weekend of performance practice that brought us to attend to the small – but not insignificant – and to consider first hand the complex relationships between environmental ‘grand narratives’ and personal experiential encounters. Locating ourselves within the field and mapping out some of the many conceptual challenges attached to it serves to introduce the territories which the following journal articles expand upon

Critical implications of IVDR for innovation in diagnostics: input from the BioMed alliance diagnostics task force

With the implementation of Regulation (European Union [EU]) 2017/746 on in vitro diagnostic medical devices (IVDR), from May 26, 2022, onwards, the development and use of diagnostic tests will be governed by a vastly expanded and upgraded EU regulatory framework. We provide here an overview of the amended transition timelines, the role of notified bodies, EU reference laboratories, expert panels, and the Medical Device Coordination Group (MDCG). We also describe the implications of the IVDR for innovative laboratory medicine by explaining the exemption for in-house devices (IH-IVDs). Two key challenges faced by the academic diagnostic sector are: (1) the stipulation on equivalence of tests (article 5.5d), which poses a new condition for the use of IH-IVDs and (2) the gray area between CE marked in vitro diagnostics (CE-IVDs), modified CE-IVDs, Research Use Only (RUO) tests, and IH-IVDs. Furthermore, the results of a questionnaire on current diagnostic practice conducted by European medical societies collaborating in the BioMed Alliance indicate widespread use of IH-IVDs in diagnostic laboratories across Europe and emphasize the need for support and guidance to comply with the IVDR. Diagnostic equivalents of the European Reference Networks (ERNs) for rare diseases could help ensure affordable and equal access to specialized diagnostics across the EU. Concerted action by clinical and laboratory disciplines, regulators, industry, and patient organizations is needed to support the efficient and effective implementation of the IVDR in a way that preserves innovation and safeguards the quality, safety, and accessibility of innovative diagnostics.Peer reviewe

Biochemical analysis of TssK, a core component of the bacterial Type VI secretion system, reveals distinct oligomeric states of TssK and identifies a TssK–TssFG subcomplex

Gram-negative bacteria use the Type VI secretion system (T6SS) to inject toxic proteins into rival bacteria or eukaryotic cells. However, the mechanism of the T6SS is incompletely understood. In the present study, we investigated a conserved component of the T6SS, TssK, using the antibacterial T6SS of Serratia marcescens as a model system. TssK was confirmed to be essential for effector secretion by the T6SS. The native protein, although not an integral membrane protein, appeared to localize to the inner membrane, consistent with its presence within a membrane-anchored assembly. Recombinant TssK purified from S. marcescens was found to exist in several stable oligomeric forms, namely trimer, hexamer and higher-order species. Native-level purification of TssK identified TssF and TssG as interacting proteins. TssF and TssG, conserved T6SS components of unknown function, were required for T6SS activity, but not for correct localization of TssK. A complex containing TssK, TssF and TssG was subsequently purified in vitro, confirming that these three proteins form a new subcomplex within the T6SS. Our findings provide new insight into the T6SS assembly, allowing us to propose a model whereby TssK recruits TssFG into the membrane-associated T6SS complex and different oligomeric states of TssK may contribute to the dynamic mechanism of the system

Down-regulation of mechanisms involved in cell transport and maintenance of mucosal integrity in pigs infected with Lawsonia intracellularis

Lawsonia intracellularis is an obligate intracellular bacterium, responsible for the disease complex known as proliferative enteropathy (PE). L. intracellularis is associated with intestinal crypt epithelial cell proliferation but the mechanisms responsible are yet to be defined. Microarray analysis was used to investigate the host-pathogen interaction in experimentally infected pigs to identify pathways that may be involved. Ileal samples originating from twenty-eight weaner pigs experimentally challenged with a pure culture of L. intracellularis (strain LR189/5/83) were subjected to microarray analysis. Microarray transcriptional signatures were validated using immunohistochemistry and quantitative real time PCR of selected genes at various time points post challenge. At peak of infection (14 days post challenge) 86% of altered transcripts were down regulated, particularly those involved in maintenance of mucosal integrity and regulation of cell transport. Among the up-regulated transcripts, CD163 and CDK1 were novel findings and considered to be important, due to their respective roles in innate immunity and cellular proliferation. Overall, targeted cellular mechanisms included those that are important in epithelial restitution, migration and protection; maintenance of stable inter-epithelial cell relationships; cell transport of nutrients and electrolytes; innate immunity; and cell cycle

The MNI data-sharing and processing ecosystem

AbstractNeuroimaging has been facing a data deluge characterized by the exponential growth of both raw and processed data. As a result, mining the massive quantities of digital data collected in these studies offers unprecedented opportunities and has become paramount for today's research. As the neuroimaging community enters the world of “Big Data”, there has been a concerted push for enhanced sharing initiatives, whether within a multisite study, across studies, or federated and shared publicly. This article will focus on the database and processing ecosystem developed at the Montreal Neurological Institute (MNI) to support multicenter data acquisition both nationally and internationally, create database repositories, facilitate data-sharing initiatives, and leverage existing software toolkits for large-scale data processing

Is telomere length socially patterned? Evidence from the West of Scotland Twenty-07 study

Lower socioeconomic status (SES) is strongly associated with an increased risk of morbidity and premature mortality, but it is not known if the same is true for telomere length, a marker often used to assess biological ageing. The West of Scotland Twenty-07 Study was used to investigate this and consists of three cohorts aged approximately 35 (N = 775), 55 (N = 866) and 75 years (N = 544) at the time of telomere length measurement. Four sets of measurements of SES were investigated: those collected contemporaneously with telomere length assessment, educational markers, SES in childhood and SES over the preceding twenty years. We found mixed evidence for an association between SES and telomere length. In 35-year-olds, many of the education and childhood SES measures were associated with telomere length, i.e. those in poorer circumstances had shorter telomeres, as was intergenerational social mobility, but not accumulated disadvantage. A crude estimate showed that, at the same chronological age, social renters, for example, were nine years (biologically) older than home owners. No consistent associations were apparent in those aged 55 or 75. There is evidence of an association between SES and telomere length, but only in younger adults and most strongly using education and childhood SES measures. These results may reflect that childhood is a sensitive period for telomere attrition. The cohort differences are possibly the result of survival bias suppressing the SES-telomere association; cohort effects with regard different experiences of SES; or telomere possibly being a less effective marker of biological ageing at older ages