InlA Promotes Dissemination of Listeria monocytogenes to the Mesenteric Lymph Nodes during Food Borne Infection of Mice

Intestinal Listeria monocytogenes infection is not efficient in mice and this has been attributed to a low affinity interaction between the bacterial surface protein InlA and E-cadherin on murine intestinal epithelial cells. Previous studies using either transgenic mice expressing human E-cadherin or mouse-adapted L. monocytogenes expressing a modified InlA protein (InlA^m) with high affinity for murine E-cadherin showed increased efficiency of intragastric infection. However, the large inocula used in these studies disseminated to the spleen and liver rapidly, resulting in a lethal systemic infection that made it difficult to define the natural course of intestinal infection. We describe here a novel mouse model of oral listeriosis that closely mimics all phases of human disease: (1) ingestion of contaminated food, (2) a distinct period of time during which L. monocytogenes colonize only the intestines, (3) varying degrees of systemic spread in susceptible vs. resistant mice, and (4) late stage spread to the brain. Using this natural feeding model, we showed that the type of food, the time of day when feeding occurred, and mouse gender each affected susceptibility to L. monocytogenes infection. Co-infection studies using L. monocytogenes strains that expressed either a high affinity ligand for E-cadherin (InlA^m), a low affinity ligand (wild type InlA from Lm EGDe), or no InlA (ΔinlA) showed that InlA was not required to establish intestinal infection in mice. However, expression of InlA^m significantly increased bacterial persistence in the underlying lamina propria and greatly enhanced dissemination to the mesenteric lymph nodes. Thus, these studies revealed a previously uncharacterized role for InlA in facilitating systemic spread via the lymphatic system after invasion of the gut mucosa.     

CED-10/Rac1 regulates endocytic recycling through the RAB-5 GAP TBC-2

Rac1 is a founding member of the Rho-GTPase family and a key regulator of membrane remodeling. In the context of apoptotic cell corpse engulfment, CED-10/Rac1 acts with its bipartite guanine nucleotide exchange factor, CED-5/Dock180-CED-12/ELMO, in an evolutionarily conserved pathway to promote phagocytosis. Here we show that in the context of the Caenorhabditis elegans intestinal epithelium CED-10/Rac1, CED-5/Dock180, and CED-12/ELMO promote basolateral recycling. Furthermore, we show that CED-10 binds to the RAB-5 GTPase activating protein TBC-2, that CED-10 contributes to recruitment of TBC-2 to endosomes, and that recycling cargo is trapped in recycling endosomes in ced-12, ced-10, and tbc-2 mutants. Expression of GTPase defective RAB-5(Q78L) also traps recycling cargo. Our results indicate that down-regulation of early endosome regulator RAB-5/Rab5 by a CED-5, CED-12, CED-10, TBC-2 cascade is an important step in the transport of cargo through the basolateral recycling endosome for delivery to the plasma membrane.     

Study protocol of a mixed-methods evaluation of a cluster randomized trial to improve the safety of NSAID and antiplatelet prescribing: data-driven quality improvement in primary care

ABSTRACT: BACKGROUND: Trials of complex interventions are criticized for being 'black box', so the UK Medical Research Council recommends carrying out a process evaluation to explain the trial findings. We believe it is good practice to pre-specify and publish process evaluation protocols to set standards and minimize bias. Unlike protocols for trials, little guidance or standards exist for the reporting of process evaluations. This paper presents the mixed-method process evaluation protocol of a cluster randomized trial, drawing on a framework designed by the authors.Methods/designThis mixed-method evaluation is based on four research questions and maps data collection to a logic model of how the data-driven quality improvement in primary care (DQIP) intervention is expected to work. Data collection will be predominately by qualitative case studies in eight to ten of the trial practices, focus groups with patients affected by the intervention and quantitative analysis of routine practice data, trial outcome and questionnaire data and data from the DQIP intervention. DISCUSSION: We believe that pre-specifying the intentions of a process evaluation can help to minimize bias arising from potentially misleading post-hoc analysis. We recognize it is also important to retain flexibility to examine the unexpected and the unintended. From that perspective, a mixed-methods evaluation allows the combination of exploratory and flexible qualitative work, and more pre-specified quantitative analysis, with each method contributing to the design, implementation and interpretation of the other.As well as strengthening the study the authors hope to stimulate discussion among their academic colleagues about publishing protocols for evaluations of randomized trials of complex interventions.Data-driven quality improvement in primary care trial registrationClinicalTrials.gov: NCT01425502.     

Multi-Tissue Microarray Analysis Identifies a Molecular Signature of Regeneration

The inability to functionally repair tissues that are lost as a consequence of disease or injury remains a significant challenge for regenerative medicine. The molecular and cellular processes involved in complete restoration of tissue architecture and function are expected to be complex and remain largely unknown. Unlike humans, certain salamanders can completely regenerate injured tissues and lost appendages without scar formation. A parsimonious hypothesis would predict that all of these regenerative activities are regulated, at least in part, by a common set of genes. To test this hypothesis and identify genes that might control conserved regenerative processes, we performed a comprehensive microarray analysis of the early regenerative response in five regeneration-competent tissues from the newt Notophthalmus viridescens. Consistent with this hypothesis, we established a molecular signature for regeneration that consists of common genes or gene family members that exhibit dynamic differential regulation during regeneration in multiple tissue types. These genes include members of the matrix metalloproteinase family and its regulators, extracellular matrix components, genes involved in controlling cytoskeleton dynamics, and a variety of immune response factors. Gene Ontology term enrichment analysis validated and supported their functional activities in conserved regenerative processes. Surprisingly, dendrogram clustering and RadViz classification also revealed that each regenerative tissue had its own unique temporal expression profile, pointing to an inherent tissue-specific regenerative gene program. These new findings demand a reconsideration of how we conceptualize regenerative processes and how we devise new strategies for regenerative medicine.     

Metagenomic Exploration of Viruses throughout the Indian Ocean

The characterization of global marine microbial taxonomic and functional diversity is a primary goal of the Global Ocean Sampling Expedition. As part of this study, 19 water samples were collected aboard the Sorcerer II sailing vessel from the southern Indian Ocean in an effort to more thoroughly understand the lifestyle strategies of the microbial inhabitants of this ultra-oligotrophic region. No investigations of whole virioplankton assemblages have been conducted on waters collected from the Indian Ocean or across multiple size fractions thus far. Therefore, the goals of this study were to examine the effect of size fractionation on viral consortia structure and function and understand the diversity and functional potential of the Indian Ocean virome. Five samples were selected for comprehensive metagenomic exploration; and sequencing was performed on the microbes captured on 3.0-, 0.8- and 0.1 µm membrane filters as well as the viral fraction (<0.1 µm). Phylogenetic approaches were also used to identify predicted proteins of viral origin in the larger fractions of data from all Indian Ocean samples, which were included in subsequent metagenomic analyses. Taxonomic profiling of viral sequences suggested that size fractionation of marine microbial communities enriches for specific groups of viruses within the different size classes and functional characterization further substantiated this observation. Functional analyses also revealed a relative enrichment for metabolic proteins of viral origin that potentially reflect the physiological condition of host cells in the Indian Ocean including those involved in nitrogen metabolism and oxidative phosphorylation. A novel classification method, MGTAXA, was used to assess virus-host relationships in the Indian Ocean by predicting the taxonomy of putative host genera, with Prochlorococcus, Acanthochlois and members of the SAR86 cluster comprising the most abundant predictions. This is the first study to holistically explore virioplankton dynamics across multiple size classes and provides unprecedented insight into virus diversity, metabolic potential and virus-host interactions.     

Mathematical Modelling of Mycobacterium tuberculosis VNTR Loci Estimates a Very Slow Mutation Rate for the Repeats

Minisatellites are highly variable tandem repeats used for over 20 years in humans for DNA fingerprinting. In prokaryotes fingerprinting techniques exploiting VNTR (variable number of tandem repeats) polymorphisms have become widely used recently in bacterial typing. However although many investigations into the mechanisms underlying minisatellite variation in humans have been performed, relatively little is known about the processes that mediate bacterial minisatellite polymorphism. An understanding of this is important since it will influence how the results from VNTR experiments are interpreted. The minisatellites of Mycobacterium tuberculosis are well characterized since they are some of the few polymorphic loci in what is otherwise a very homogeneous organism. Using VNTR results from a well-defined and characterized set of M. tuberculosis strains we show that the repeats at a locus are likely to evolve by stepwise contraction or expansion in the number of repeats. A stochastic continuous-time population mathematical model was developed to simulate the evolution of the repeats. This allowed estimation of the tendency of the repeats to increase or decrease and the rate at which they change. The majority of loci tend to lose rather than gain repeats. All of the loci mutate extremely slowly, with an average rate of 2.3 x 10(-8), which is 350 times slower than that of a set of VNTR repeats with similar diversity observed experimentally in Escherichia coli. This suggests that the VNTR profile of a strain of M. tuberculosis will be indicative of its clonal lineage and will be unlikely to vary in epidemiologically-related strains.     

Immunoproteasome responds to injury in the retina and brain

It is well known that immunoproteasome generates peptides for MHC Class I occupancy and recognition by cytotoxic T lymphocytes (CTL). The present study focused on evidence for alternative roles for immunoproteasome. Retina and brain were analyzed for expression of immunoproteasome subunits using immunohistochemistry and western blotting under normal conditions and after injury/stress induced by CTL attack on glia (brain) or neurons (retina). Normal retina expressed substantial levels of immunoproteasome in glia, neurons, and retinal pigment epithelium. The basal level of immunoproteasome in retina was two-fold higher than in brain; CTL-induced retinal injury further up-regulated immunoproteasome expression. Immunoproteasome up-regulation was also observed in injured brain and corresponded with expression in Purkinje cells, microglia, astrocytes, and oligodendrocytes. These results suggest that the normal environment of the retina is sufficiently challenging to require on-going expression of immunoproteasome. Further, immunoproteasome up-regulation with retinal and brain injury implies a role in neuronal protection and/or repair of damage.     

Cutting edge: progesterone regulates IFN-alpha production by plasmacytoid dendritic cells

Use of the progesterone (Pg) birth control depot medroxyprogesterone acetate (DMPA) increases a woman's risk for sexually transmitted infection with HIV or HSV-2 via unknown mechanisms. Plasmacytoid dendritic cells (pDCs) are circulating and tissue-resident sentinels capable of making large quantities of IFN-alpha upon recognizing viruses through TLRs 7 and 9. In this study, we show that Pg inhibits TLR9-induced IFN-alpha production by human and mouse pDCs and that DMPA impairs TLR9- and virus-induced IFN-alpha production by pDCs in mice, providing a potential explanation for how DMPA impairs innate antiviral immunity in women. Pg failed to inhibit the Mda-5 pathway of IFN-alpha induction in dendritic cells, suggesting that Pg regulates select antiviral DC programs. This may occur through selective blockade of IFN regulatory factor-7 activation, a novel steroid action. Thus, through inhibition of TLR-mediated IFN-alpha production by pDCs, Pg may regulate antiviral immunity.