Costimulatory molecule-deficient dendritic cell progenitors (MHC class II⁺, CD80(dim), CD86^-) prolong cardiac allograft survival in nonimmunosuppressed recipients

We have shown previously that granulocyte-macrophage colony-stimulating factor-stimulated mouse bone marrow-derived MHC class II+ dendritic cell (DC) progenitors that are deficient in cell surface expression of the costimulatory molecules B7-1 (CD8O) and B7-2 (CD86) can induce alloantigen- specific T-cell anergy in vitro. To test the in vivo relevance of these findings, 2 x 106 B10 (H2(b)) mouse bone marrow-derived DC progenitors (NLDC 145+, MHC class II+, B7-1(dim), B7-2(-/dim)) that induced T-cell hyporesponsiveness in vitro were injected systemically into normal C3H (H2(k)) recipients. Seven days later, the mice received heterotopic heart transplants from B10 donors. No immunosuppressive treatment was given. Median graft survival time was prolonged significantly from 9.5 to 22 days. Median graft survival time was also increased, although to a lesser extent (16.5 days), in mice that received third-party (BALB/c; H2(d)) DC progenitors. Ex vivo analysis of host T-cell responses to donor and third-party alloantigens 7 days after the injection of DC progenitors (the time of heart transplant) revealed minimal anti-donor mixed leukocyte reaction and cytotoxic T lymphocyte reactivity. These responses were reduced substantially compared with those of spleen cells from animals pretreated with 'mature' granulocyte- macrophage colony-stimulating factor + interleukin-4-stimulated DC (MHC class II(bright), B7-1+, B7-2(bright)), many of which rejected their heart grafts in an accelerated fashion. Among the injected donor MHC class II+ DC progenitors that migrated to recipient secondary lymphoid tissue were cells that appeared to have up-regulated cell surface B7-1 and B7-2 molecule expression. This observation may explain, at least in part, the temporary or unstable nature of the hyporesponsiveness induced by the DC progenitors in nonimmunosuppressed recipients

Human CLEC9A antibodies deliver Wilms' tumor 1 (WT1) antigen to CD141+ dendritic cells to activate naïve and memory WT1‐specific CD8+ T cells

Objectives Vaccines that prime Wilms' tumor 1 (WT1)‐specific CD8+ T cells are attractive cancer immunotherapies. However, immunogenicity and clinical response rates may be enhanced by delivering WT1 to CD141+ dendritic cells (DCs). The C‐type lectin‐like receptor CLEC9A is expressed exclusively by CD141+ DCs and regulates CD8+ T‐cell responses. We developed a new vaccine comprising a human anti‐CLEC9A antibody fused to WT1 and investigated its capacity to target human CD141+ DCs and activate naïve and memory WT1‐specific CD8+ T cells. Methods WT1 was genetically fused to antibodies specific for human CLEC9A, DEC‐205 or β‐galactosidase (untargeted control). Activation of WT1‐specific CD8+ T‐cell lines following cross‐presentation by CD141+ DCs was quantified by IFNγ ELISPOT. Humanised mice reconstituted with human immune cell subsets, including a repertoire of naïve WT1‐specific CD8+ T cells, were used to investigate naïve WT1‐specific CD8+ T‐cell priming. Results The CLEC9A‐WT1 vaccine promoted cross‐presentation of WT1 epitopes to CD8+ T cells and mediated priming of naïve CD8+ T cells more effectively than the DEC‐205‐WT1 and untargeted control‐WT1 vaccines. Conclusions Delivery of WT1 to CD141+ DCs via CLEC9A stimulates CD8+ T cells more potently than either untargeted delivery or widespread delivery to all Ag‐presenting cells via DEC‐205, suggesting that cross‐presentation by CD141+ DCs is sufficient for effective CD8+ T‐cell priming in humans. The CLEC9A‐WT1 vaccine is a promising candidate immunotherapy for malignancies that express WT1

IKAP/Elp1 Is Required In Vivo for Neurogenesis and Neuronal Survival, but Not for Neural Crest Migration

Familial Dysautonomia (FD; Hereditary Sensory Autonomic Neuropathy; HSAN III) manifests from a failure in development of the peripheral sensory and autonomic nervous systems. The disease results from a point mutation in the IKBKAP gene, which encodes the IKAP protein, whose function is still unresolved in the developing nervous system. Since the neurons most severely depleted in the disease derive from the neural crest, and in light of data identifying a role for IKAP in cell motility and migration, it has been suggested that FD results from a disruption in neural crest migration. To determine the function of IKAP during development of the nervous system, we (1) first determined the spatial-temporal pattern of IKAP expression in the developing peripheral nervous system, from the onset of neural crest migration through the period of programmed cell death in the dorsal root ganglia, and (2) using RNAi, reduced expression of IKBKAP mRNA in the neural crest lineage throughout the process of dorsal root ganglia (DRG) development in chick embryos in ovo. Here we demonstrate that IKAP is not expressed by neural crest cells and instead is expressed as neurons differentiate both in the CNS and PNS, thus the devastation of the PNS in FD could not be due to disruptions in neural crest motility or migration. In addition, we show that alterations in the levels of IKAP, through both gain and loss of function studies, perturbs neuronal polarity, neuronal differentiation and survival. Thus IKAP plays pleiotropic roles in both the peripheral and central nervous systems

Formalising recall by genotype as an efficient approach to detailed phenotyping and causal inference.

Detailed phenotyping is required to deepen our understanding of the biological mechanisms behind genetic associations. In addition, the impact of potentially modifiable risk factors on disease requires analytical frameworks that allow causal inference. Here, we discuss the characteristics of Recall-by-Genotype (RbG) as a study design aimed at addressing both these needs. We describe two broad scenarios for the application of RbG: studies using single variants and those using multiple variants. We consider the efficacy and practicality of the RbG approach, provide a catalogue of UK-based resources for such studies and present an online RbG study planner

Microneedle Array Design Determines the Induction of Protective Memory CD8+ T Cell Responses Induced by a Recombinant Live Malaria Vaccine in Mice

BACKGROUND: Vaccine delivery into the skin has received renewed interest due to ease of access to the immune system and microvasculature, however the stratum corneum (SC), must be breached for successful vaccination. This has been achieved by removing the SC by abrasion or scarification or by delivering the vaccine intradermally (ID) with traditional needle-and-syringes or with long microneedle devices. Microneedle patch-based transdermal vaccine studies have predominantly focused on antibody induction by inactivated or subunit vaccines. Here, our principal aim is to determine if the design of a microneedle patch affects the CD8(+) T cell responses to a malaria antigen induced by a live vaccine. METHODOLOGY AND FINDINGS: Recombinant modified vaccinia virus Ankara (MVA) expressing a malaria antigen was percutaneously administered to mice using a range of silicon microneedle patches, termed ImmuPatch, that differed in microneedle height, density, patch area and total pore volume. We demonstrate that microneedle arrays that have small total pore volumes induce a significantly greater proportion of central memory T cells that vigorously expand to secondary immunization. Microneedle-mediated vaccine priming induced significantly greater T cell immunity post-boost and equivalent protection against malaria challenge compared to ID vaccination. Notably, unlike ID administration, ImmuPatch-mediated vaccination did not induce inflammatory responses at the site of immunization or in draining lymph nodes. CONCLUSIONS/SIGNIFICANCE: This study demonstrates that the design of microneedle patches significantly influences the magnitude and memory of vaccine-induced CD8(+) T cell responses and can be optimised for the induction of desired immune responses. Furthermore, ImmuPatch-mediated delivery may be of benefit to reducing unwanted vaccine reactogenicity. In addition to the advantages of low cost and lack of pain, the development of optimised microneedle array designs for the induction of T cell responses by live vaccines aids the development of solutions to current obstacles of immunization programmes

Scrub typhus ecology: a systematic review of Orientia in vectors and hosts

Abstract Scrub typhus, caused by Orientia tsutsugamushi, is an important and neglected vector-borne zoonotic disease with an expanding known distribution. The ecology of the disease is complex and poorly understood, impairing discussion of public health interventions. To highlight what we know and the themes of our ignorance, we conducted a systematic review of all studies investigating the pathogen in vectors and non-human hosts. A total of 276 articles in 7 languages were included, with 793 study sites across 30 countries. There was no time restriction for article inclusion, with the oldest published in 1924. Seventy-six potential vector species and 234 vertebrate host species were tested, accounting for over one million trombiculid mites (‘chiggers’) and 83,000 vertebrates. The proportion of O. tsutsugamushi positivity was recorded for different categories of laboratory test and host species. Vector and host collection sites were geocoded and mapped. Ecological data associated with these sites were summarised. A further 145 articles encompassing general themes of scrub typhus ecology were reviewed. These topics range from the life-cycle to transmission, habitats, seasonality and human risks. Important gaps in our understanding are highlighted together with possible tools to begin to unravel these. Many of the data reported are highly variable and inconsistent and minimum data reporting standards are proposed. With more recent reports of human Orientia sp. infection in the Middle East and South America and enormous advances in research technology over recent decades, this comprehensive review provides a detailed summary of work investigating this pathogen in vectors and non-human hosts and updates current understanding of the complex ecology of scrub typhus. A better understanding of scrub typhus ecology has important relevance to ongoing research into improving diagnostics, developing vaccines and identifying useful public health interventions to reduce the burden of the disease.</jats:p

FFPE breast tumour blocks provide reliable sources of both germline and malignant DNA for investigation of genetic determinants of individual tumour responses to treatment

Background: Bio-banked formalin-fixed paraffin-embedded (FFPE) tissues provide an excellent opportunity for translational genomic research. Historically matched blood has not always been collected as a source of germline DNA. This project aimed to establish if normal FFPE breast tissue could be used as an alternative to blood. Methods: Exome sequencing was carried out on matched tumour tissue, normal breast tissue and blood on five patients in the START trial. Retrieved samples had been archived at different centres for at least 13 years. Following tissue macro-dissection and DNA extraction, targeted exome capture was performed using SureSelect Human All Exome v5 reagents (Agilent). Illumina paired-end libraries were prepared from the captured target regions and sequenced on a HiSeq2500 (Illumina) acquiring 2 × 75 bp reads. Somatic variants were called using the MuTect software analysis tool and copy number abnormalities (CNA) were identified using CNVkit. Targeted sequencing and droplet digital PCR were used to validate somatic variants and CNA, respectively. Results: Overlap of somatic variants and CNA called on tumour versus blood and tumour versus normal breast tissue was good. Agreement in somatic variant calling ranged from 76.9 to 93.6%. Variants with an allele frequency lower than 10% were more difficult to validate irrespective of the type of germline DNA used. Pearson’s correlation coefficients for paired comparisons of CNA using blood or normal tissue as reference ranged from 0.70 to 0.94. Conclusions: There is good correlation between the somatic mutations and CNA called using archived blood or normal breast tissue as germline reference material

Marking their own homework: The pragmatic and moral legitimacy of industry self-regulation

When is industry self-regulation (ISR) a legitimate form of governance? In principle, ISR can serve the interests of participating companies, regulators and other stakeholders. However, in practice, empirical evidence shows that ISR schemes often under-perform, leading to criticism that such schemes are tantamount to firms marking their own homework. In response, this paper explains how current management theory on ISR has failed to separate the pragmatic legitimacy of ISR based on self-interested calculations, from moral legitimacy based on normative approval. The paper traces three families of management theory on ISR and uses these to map the pragmatic and moral legitimacy of ISR schemes. It identifies tensions between the pragmatic and moral legitimacy of ISR schemes, which the current ISR literature does not address, and draws implications for the future theory and practice of ISR

A Field Guide to Pandemic, Epidemic and Sporadic Clones of Methicillin-Resistant Staphylococcus aureus

In recent years, methicillin-resistant Staphylococcus aureus (MRSA) have become a truly global challenge. In addition to the long-known healthcare-associated clones, novel strains have also emerged outside of the hospital settings, in the community as well as in livestock. The emergence and spread of virulent clones expressing Panton-Valentine leukocidin (PVL) is an additional cause for concern. In order to provide an overview of pandemic, epidemic and sporadic strains, more than 3,000 clinical and veterinary isolates of MRSA mainly from Germany, the United Kingdom, Ireland, France, Malta, Abu Dhabi, Hong Kong, Australia, Trinidad & Tobago as well as some reference strains from the United States have been genotyped by DNA microarray analysis. This technique allowed the assignment of the MRSA isolates to 34 distinct lineages which can be clearly defined based on non-mobile genes. The results were in accordance with data from multilocus sequence typing. More than 100 different strains were distinguished based on affiliation to these lineages, SCCmec type and the presence or absence of PVL. These strains are described here mainly with regard to clinically relevant antimicrobial resistance- and virulence-associated markers, but also in relation to epidemiology and geographic distribution. The findings of the study show a high level of biodiversity among MRSA, especially among strains harbouring SCCmec IV and V elements. The data also indicate a high rate of genetic recombination in MRSA involving SCC elements, bacteriophages or other mobile genetic elements and large-scale chromosomal replacements

A Field Guide to Pandemic, Epidemic and Sporadic Clones of Methicillin-Resistant Staphylococcus aureus

In recent years, methicillin-resistant Staphylococcus aureus (MRSA) have become a truly global challenge. In addition to the long-known healthcare-associated clones, novel strains have also emerged outside of the hospital settings, in the community as well as in livestock. The emergence and spread of virulent clones expressing Panton-Valentine leukocidin (PVL) is an additional cause for concern. In order to provide an overview of pandemic, epidemic and sporadic strains, more than 3,000 clinical and veterinary isolates of MRSA mainly from Germany, the United Kingdom, Ireland, France, Malta, Abu Dhabi, Hong Kong, Australia, Trinidad & Tobago as well as some reference strains from the United States have been genotyped by DNA microarray analysis. This technique allowed the assignment of the MRSA isolates to 34 distinct lineages which can be clearly defined based on non-mobile genes. The results were in accordance with data from multilocus sequence typing. More than 100 different strains were distinguished based on affiliation to these lineages, SCCmec type and the presence or absence of PVL. These strains are described here mainly with regard to clinically relevant antimicrobial resistance- and virulence-associated markers, but also in relation to epidemiology and geographic distribution. The findings of the study show a high level of biodiversity among MRSA, especially among strains harbouring SCCmec IV and V elements. The data also indicate a high rate of genetic recombination in MRSA involving SCC elements, bacteriophages or other mobile genetic elements and large-scale chromosomal replacements