Microbial intestinal dysbiosis drives long-term allergic susceptibility by sculpting an ILC2-B1 cell-innate IgE axis.

BACKGROUND: The abundance and diversity of intestinal commensal bacteria influence systemic immunity with impact on disease susceptibility and severity. For example, loss of short chain fatty acid (SCFA)-fermenting bacteria in early life (humans and mice) is associated with enhanced type 2 immune responses in peripheral tissues including the lung. OBJECTIVE: Our goal was to reveal the microbiome-dependent cellular and molecular mechanisms driving enhanced susceptibility to type 2 allergic lung disease. METHODS: We used low-dose vancomycin to selectively deplete SCFA-fermenting bacteria in wild-type mice. We then examined the frequency and activation status of innate and adaptive immune cell lineages with and without SCFA supplementation. Finally, we used ILC2-deficient and signal transducer and activator of transcription 6 (STAT6)-deficient transgenic mouse strains to delineate the cellular and cytokine pathways leading to enhanced allergic disease susceptibility. RESULTS: Mice with vancomycin-induced dysbiosis exhibited a 2-fold increase in lung ILC2 primed to produce elevated levels of IL-2, -5, and -13. In addition, upon IL-33 inhalation, mouse lung ILC2 displayed a novel ability to produce high levels of IL-4. These expanded and primed ILC2s drove B1 cell expansion and IL-4-dependent production of IgE that in turn led to exacerbated allergic inflammation. Importantly, these enhanced lung inflammatory phenotypes in mice with vancomycin-induced dysbiosis were reversed by administration of dietary SCFA (specifically butyrate). CONCLUSION: SCFAs regulate an ILC2-B1 cell-IgE axis. Early-life administration of vancomycin, an antibiotic known to deplete SCFA-fermenting gut bacteria, primes and amplifies this axis and leads to lifelong enhanced susceptibility to type 2 allergic lung disease.

The role of anticipatory and reflexive compensatory muscle activation in catching errors under load uncertainty.

This experiment investigated the role of anticipatory and reflexive compensatory neuromotor control in catching errors occurring under load uncertainty. Participants performed 64 trials of a one-handed ball catching task using visually identical balls of four different weights without knowing the weight of the ball on each trial. Anticipatory and reflexive compensatory muscle activation were recorded in five muscles (anterior deltoid, biceps brachii, wrist flexors group, triceps brachii, lumbar erector spinae) using the EMG integral. In each muscle, the anticipatory and reflexive compensatory muscle activation were compared between successful catches and catching errors for the lightest ball and the heaviest ball. Anticipatory muscle activation was not implicated in errors made with the lightest ball. However, reflexive compensatory muscle activation in the anterior deltoid, biceps brachii, and wrist flexors were implicated in errors made with the lightest ball. Specifically, catching errors with the lightest ball were characterized by elevated reflexive compensatory muscle activation. In the case of the heaviest ball, both anticipatory (anterior deltoid, wrist flexors) and reflexive compensatory muscle activation (anterior deltoid, biceps brachii, wrist flexors) were implicated in catching errors. That is, catching errors with the heaviest ball were characterized by lower anticipatory and reflexive compensatory muscle activation. Results are considered in the context of the likely influence of limb compliance in catching under load uncertainty.

Real-timing processing of fiber bundle endomicroscopy images in Python using PyFibreBundle.

Fiber imaging bundles allow the transfer of optical images from place-to-place along narrow and flexible conduits. Traditionally used extensively in medical endoscopy, bundles are now finding new applications in endoscopic microscopy and other emerging techniques. PyFibreBundle is an open-source Python package for fast processing of images acquired through imaging bundles. This includes detection and removal of the fiber core pattern by filtering or interpolation, and application of background and flat-field corrections. It also allows images to be stitched together to create mosaics and resolution to be improved by combining multiple shifted images. This paper describes the technical implementation of PyFibreBundle and provides example results from three endomicroscopy imaging systems: color transmission, monochrome transmission, and confocal fluorescence. This allows various processing options to be compared quantitatively and qualitatively, and benchmarking demonstrates that PyFibreBundle can achieve state-of-the-art performance in an open-source package. The paper demonstrates core removal by interpolation and mosaicing at over 100 fps, real-time multi-frame resolution enhancement and the first demonstration of real-time endomicroscopy image processing, including core removal, on a Raspberry Pi single board computer. This demonstrates that PyFibreBundle is potentially a valuable tool for the development of low-cost, high-performance fiber bundle imaging systems.

Gonadal hormones and sex chromosome complement differentially contribute to ethanol intake, preference, and relapse-like behaviour in four core genotypes mice.

Alcohol use and high-risk alcohol drinking behaviours among women are rapidly rising. In rodent models, females typically consume more ethanol (EtOH) than males. Here, we used the four core genotypes (FCG) mouse model to investigate the influence of gonadal hormones and sex chromosome complement on EtOH drinking behaviours. FCG mice were given access to escalating concentrations of EtOH in a two-bottle, 24-h continuous access drinking paradigm to assess consumption and preference. Relapse-like behaviour was measured by assessing escalated intake following repeated cycles of deprivation and re-exposure. Twenty-four-hour EtOH consumption was greater in mice with ovaries (Sry-), relative to those with testes, and in mice with the XX chromosome complement, relative to those with XY sex chromosomes. EtOH preference was higher in XX versus XY mice. For both consumption and preference, the influences of the Sry gene and sex chromosomes were concentration dependent. Escalated intake following repeated cycles of deprivation and re-exposure emerged only in XX mice (vs. XY). Mice with ovaries (Sry- FCG mice and C57BL/6J females) were also found to consume more water than mice with testes. These results demonstrate that aspects of EtOH drinking behaviour may be independently regulated by sex hormones and chromosomes and inform our understanding of the neurobiological mechanisms which contribute to EtOH dependence in male and female mice. Future investigation of the contribution of sex chromosomes to EtOH drinking behaviours is warranted. We used the FCG mouse model to investigate the influence of gonadal hormones and sex chromosome complement on EtOH drinking behaviours, including the alcohol deprivation effect. Escalated intake following repeated cycles of deprivation and re-exposure emerged only in XX mice (vs. XY). These results demonstrate that aspects of EtOH drinking behaviour may be independently regulated by sex hormones and chromosomes.

Podocalyxin is required for maintaining blood-brain barrier function during acute inflammation.

Podocalyxin (Podxl) is broadly expressed on the luminal face of most blood vessels in adult vertebrates, yet its function on these cells is poorly defined. In the present study, we identified specific functions for Podxl in maintaining endothelial barrier function. Using electrical cell substrate impedance sensing and live imaging, we found that, in the absence of Podxl, human umbilical vein endothelial cells fail to form an efficient barrier when plated on several extracellular matrix substrates. In addition, these monolayers lack adherens junctions and focal adhesions and display a disorganized cortical actin cytoskeleton. Thus, Podxl has a key role in promoting the appropriate endothelial morphogenesis required to form functional barriers. This conclusion is further supported by analyses of mutant mice in which we conditionally deleted a floxed allele of Podxl in vascular endothelial cells (vECs) using Tie2Cre mice (PodxlΔTie2Cre). Although we did not detect substantially altered permeability in naïve mice, systemic priming with lipopolysaccharide (LPS) selectively disrupted the blood-brain barrier (BBB) in PodxlΔTie2Cre mice. To study the potential consequence of this BBB breach, we used a selective agonist (TFLLR-NH2) of the protease-activated receptor-1 (PAR-1), a thrombin receptor expressed by vECs, neuronal cells, and glial cells. In response to systemic administration of TFLLR-NH2, LPS-primed PodxlΔTie2Cre mice become completely immobilized for a 5-min period, coinciding with severely dampened neuroelectric activity. We conclude that Podxl expression by CNS tissue vECs is essential for BBB maintenance under inflammatory conditions.

IL-4Rα blockade reduces influenza-associated morbidity in a murine model of allergic asthma.

BACKGROUND: Asthma was identified as the most common comorbidity in hospitalized patients during the 2009 H1N1 influenza pandemic. We determined using a murine model of allergic asthma whether these mice experienced increased morbidity from pandemic H1N1 (pH1N1) viral infection and whether blockade of interleukin-4 receptor α (IL-4Rα), a critical mediator of Th2 signalling, improved their outcomes. METHODS: Male BALB/c mice were intranasally sensitized with house dust mite antigen (Der p 1) for 2 weeks; the mice were then inoculated intranasally with a single dose of pandemic H1N1 (pH1N1). The mice were administered intraperitoneally anti-IL-4Rα through either a prophylactic or a therapeutic treatment strategy. RESULTS: Infection with pH1N1 of mice sensitized to house dust mite (HDM) led to a 24% loss in weight by day 7 of infection (versus 14% in non-sensitized mice; p < .05). This was accompanied by increased viral load in the airways and a dampened anti-viral host responses to the infection. Treatment of HDM sensitized mice with a monoclonal antibody against IL-4Rα prior to or following pH1N1 infection prevented the excess weight loss, reduced the viral load in the lungs and ameliorated airway eosinophilia and systemic inflammation related to the pH1N1 infection. CONCLUSION: Together, these data implicate allergic asthma as a significant risk factor for H1N1-related morbidity and reveal a potential therapeutic role for IL-4Rα signalling blockade in reducing the severity of influenza infection in those with allergic airway disease.

The Transcription Factor RORα Preserves ILC3 Lineage Identity and Function during Chronic Intestinal Infection.

Innate lymphoid cells (ILCs) are critical for host defense and tissue repair but can also contribute to chronic inflammatory diseases. The transcription factor RORα is required for ILC2 development but is also highly expressed by other ILC subsets where its function remains poorly defined. We previously reported that Rorasg/sg bone marrow chimeric mice (C57BL/6J) were protected from Salmonella-induced intestinal fibrosis due to defective ILC3 responses. In this study, single-cell RNA analysis of ILCs isolated from inflamed tissues indicates that RORα perturbation led to a reduction in ILC3 lineages. Furthermore, residual Rorasg/sg ILC3s have decreased expression of key signature genes, including Rorc and activating cytokine receptors. Collectively, our data suggest that RORα plays a key role in preserving functional ILC3s by modulating their ability to integrate environmental cues to efficiently produce cytokines.

Inline holographic microscopy through fiber imaging bundles.

Fiber imaging bundles are widely used as thin, passive image conduits for miniaturized and endoscopic microscopy, particularly for confocal fluorescence imaging. Holographic microscopy through fiber bundles is more challenging; phase conjugation approaches are complex and require extensive calibration. This paper describes how simple inline holographic microscopy can be performed through an imaging bundle using a partially coherent illumination source from a multimode fiber. The sample is imaged in transmission, with the intensity hologram sampled by the bundle and transmitted to a remote camera. The hologram can then be numerically refocused for volumetric imaging, achieving a resolution of approximately 6 µm over a depth range of 1 mm. The scheme does not require any complex prior calibration and hence is insensitive to bending.

Information for anticipatory neuromotor control in catching under load uncertainty.

Humans employ anticipatory muscle activation when catching under conditions of load uncertainty. Questions addressed were (a) on what information referent do catchers base their anticipatory neuromotor control when catching balls of unknown weight?, and (b) how do catchers use this functional referent? Thirty-six participants caught visually identical balls dropped from 0.75 m. Participants performed 40 trials, half with knowledge of ball weight and half without. Group L caught balls with a large weight range, while group S caught balls with a smaller range of weights. EMG integrals were computed for the ball flight period in five muscles. Anticipatory EMG integrals in the unknown weight condition were normalized to anticipatory EMG integrals for the maximum, minimum and average ball weights in the known ball weight condition. We assumed participants would base anticipatory control in the unknown weight condition on similar information, regardless of group. Therefore, differences in normalized EMG integrals between groups L and S would suggest that the specific referent tested (e.g., minimum possible ball weight) was not used to scale anticipatory muscle activation under load uncertainty. Independent sample t tests ascertained differences in normalized EMG integrals between groups L and S. The results suggested that the information referent participants used to catch balls of an unknown weight was knowledge of the maximum ball weight. Participants used this referent to generate a submaximal level of anticipatory muscle activation, i.e., about 93.2% of that used to catch the heaviest ball when ball weight was known in advance.

The first identified heterozygous nonsense mutations in podocalyxin offer new perspectives on the biology of podocytopathies.

In the last two decades, our understanding of the genetic underpinnings of inherited podocytopathies has advanced immensely. By sequencing the genomes of a large pool of families affected by focal segmental glomerulosclerosis (FSGS), researchers have identified a common theme: familial podocytopathies are frequently caused by genes selectively expressed in podocytes. Podocalyxin is a podocyte-specific surface sialomucin that has long been known to play important roles in podocyte morphogenesis and function. Few studies, however, have shown a conclusive link between mutations in the gene and FSGS complemented by functional evidence. In a fascinating new paper published in Clinical Science, Lin et al. identify two unrelated pedigrees in which dominant loss-of-function mutations in PODXL lead to adult-onset FSGS. Nonsense-mediated decay of the mutated PODXL transcripts leads to protein insufficiency, which in turn cause podocyte dysfunction through defects in motility and cytoskeletal organization. This is the first study to date that demonstrates, mechanistically, how autosomal dominant mutations in podocalyxin can lead to FSGS and renal insufficiency. Here, we summarize the experimental findings of this manuscript and propose, perhaps, a more controversial hypothesis: down-regulation of podocalyxin protein expression from podocytes is a critical turning point in the progression of most podocytopathies and may be mechanistically relevant to glomerulopathies in which podocyte damage is not necessarily induced by genetic lesions.

Gene-Edited Human Kidney Organoids Reveal Mechanisms of Disease in Podocyte Development.

A critical event during kidney organogenesis is the differentiation of podocytes, specialized epithelial cells that filter blood plasma to form urine. Podocytes derived from human pluripotent stem cells (hPSC-podocytes) have recently been generated in nephron-like kidney organoids, but the developmental stage of these cells and their capacity to reveal disease mechanisms remains unclear. Here, we show that hPSC-podocytes phenocopy mammalian podocytes at the capillary loop stage (CLS), recapitulating key features of ultrastructure, gene expression, and mutant phenotype. hPSC-podocytes in vitro progressively establish junction-rich basal membranes (nephrin+ podocin+ ZO-1+ ) and microvillus-rich apical membranes (podocalyxin+ ), similar to CLS podocytes in vivo. Ultrastructural, biophysical, and transcriptomic analysis of podocalyxin-knockout hPSCs and derived podocytes, generated using CRISPR/Cas9, reveals defects in the assembly of microvilli and lateral spaces between developing podocytes, resulting in failed junctional migration. These defects are phenocopied in CLS glomeruli of podocalyxin-deficient mice, which cannot produce urine, thereby demonstrating that podocalyxin has a conserved and essential role in mammalian podocyte maturation. Defining the maturity of hPSC-podocytes and their capacity to reveal and recapitulate pathophysiological mechanisms establishes a powerful framework for studying human kidney disease and regeneration. Stem Cells 2017;35:2366-2378.

Loss of Vascular CD34 Results in Increased Sensitivity to Lung Injury.

Survival during lung injury requires a coordinated program of damage limitation and rapid repair. CD34 is a cell surface sialomucin expressed by epithelial, vascular, and stromal cells that promotes cell adhesion, coordinates inflammatory cell recruitment, and drives angiogenesis. To test whether CD34 also orchestrates pulmonary damage and repair, we induced acute lung injury in wild-type (WT) and Cd34-/- mice by bleomycin administration. We found that Cd34-/- mice displayed severe weight loss and early mortality compared with WT controls. Despite equivalent early airway inflammation to WT mice, CD34-deficient animals developed interstitial edema and endothelial delamination, suggesting impaired endothelial function. Chimeric Cd34-/- mice reconstituted with WT hematopoietic cells exhibited early mortality compared with WT mice reconstituted with Cd34-/- cells, supporting an endothelial defect. CD34-deficient mice were also more sensitive to lung damage caused by influenza infection, showing greater weight loss and more extensive pulmonary remodeling. Together, our data suggest that CD34 plays an essential role in maintaining vascular integrity in the lung in response to chemical- and infection-induced tissue damage.

Dendritic-cell expression of Ship1 regulates Th2 immunity to helminth infection in mice.

In mouse models of infection with the gastrointestinal parasite Trichuris muris, appropriate dendritic-cell (DC) Ag sampling, migration, and presentation to T cells are necessary to mount a protective Th2-polarized adaptive immune response, which is needed to clear infection. SH2-containing inositol 5'-phosphatase 1 (SHIP-1) has been shown to be an important regulator of DC function in vitro through the negative regulation of the phosphoinositide 3-kinase (PI3K) pathway, but its role in vivo is relatively unexplored. In the current work, mice with a specific deletion of SHIP-1 in DCs (Ship1(ΔDC) ) were infected with the parasite T. muris. Ship1(ΔDC) mice were susceptible to infection due to ineffective priming of Th2-polarized responses. This is likely due to an increased production of interleukin (IL) 12p40 by SHIP-1-deficient DCs, as in vivo antibody blockade of IL-12p40 was able to facilitate the clearing of infection in Ship1(ΔDC) mice. Our results describe a critical role for SHIP-1 in regulating the ability of DCs to efficiently prime Th2-type responses.

Chronic Trichuris muris infection alters hematopoiesis and causes IFN-γ-expressing T-cell accumulation in the mouse bone marrow.

Proinflammatory cytokines produced during immune responses to infectious stimuli are well-characterized to have secondary effects on the function of hematopoietic progenitor cells in the BM. However, these effects on the BM are poorly characterized during chronic infection with intestinal helminth parasites. In this study, we use the Trichuris muris model of infection and show that Th1 cell-associated, but not acute Th2 cell-associated, responses to chronic T. muris infection cause a major, transient expansion of CD48- CD150- multipotent progenitor cells in the BM that is dependent on the presence of adaptive immune cells and IFN-γ signaling. Chronic T. muris infection also broadly stimulated proliferation of BM progenitor cells including CD48- CD150+ hematopoietic stem cells. This shift in progenitor activity during chronic T. muris infection correlated with a functional increase in myeloid colony formation in vitro as well as neutrophilia in the BM and peripheral blood. In parallel, we observed an accumulation of CD4+ , CD8+ , and CD4- CD8- (double negative) T cells that expressed IFN-γ, displaying activated and central memory-type phenotypes in the bone marrow during chronic infection. Thus, these results demonstrate that Th1 cell-driven responses in the intestine during chronic helminth infection potently influence upstream hematopoietic processes in the BM via IFN-γ.

Does load uncertainty affect adaptation to catch training?

Catching relies on anticipatory and compensatory control processes. Load uncertainty increases anticipatory and compensatory neuromotor effort in catching. This experiment tested the effect of load uncertainty in plyometric catch/throw training on elbow flexion reaction time (RT), movement time (MT) and peak torque, as well as the distribution of anticipatory and compensatory neuromotor effort in catching. We expected load uncertainty training to be superior to traditional training for improving elbow flexion MT and peak torque, as well as for reallocating neuromotor effort from compensatory to anticipatory control in catching. Three groups of men (mean age = 21), load knowledge training (K) (n = 14), load uncertainty training (U) (n = 13) and control (C) (n = 14), participated. Groups K and U trained three times/week for 6 weeks using single-arm catch/throw exercises with 0.45-4.08 kg balls. Sets involved 16 repetitions of four different ball masses presented randomly. Group K had knowledge of ball mass on every repetition, whereas group U never did. Change scores were analyzed using Kruskal-Wallis tests and follow-up Wilcoxon rank-sum tests. Group K improved both RT and MT (by 6.2 and 12 %, respectively), whereas group U did not. Both groups K and U improved peak eccentric elbow flexion torque. Group K reallocated neuromotor effort from compensatory to anticipatory processes in the biceps, triceps and the all muscle average, whereas group U did so in the triceps only. In sum, plyometric catch/throw training caused a reallocation of neuromotor effort from compensatory to anticipatory control in catching. However, load uncertainty training did not amplify this effect and in fact appeared to inhibit the reallocation of neuromotor effort from compensatory to anticipatory control.

Lineage-specific regulation of allergic airway inflammation by the lipid phosphatase Src homology 2 domain-containing inositol 5-phosphatase (SHIP-1).

BACKGROUND: Inpp5d (Src homology 2 domain-containing inositol-5-phosphatase [Ship1])-deficient mice experience spontaneous airway inflammation and have enhanced sensitivity to allergen-induced airway inflammation. OBJECTIVE: We hypothesized that lineage-specific deletion of Ship1 expression in cells known to be crucial for adaptive TH2 responses would uncover distinct roles that could either positively or negatively regulate susceptibility to allergic airway inflammation (AAI). METHODS: Ship1 expression was deleted in B cells, T cells, or dendritic cells (DCs), and the resulting Ship1(ΔB cell), Ship1(ΔT cell), Ship1(ΔDC), or Ship1(F/F) (wild-type) control mice were evaluated in a model of house dust mite (HDM)-induced AAI. RESULTS: Unlike germline panhematopoietic Ship1 deletion, deletion of Ship1 selectively in either the B-cell, T-cell, or DC lineages did not result in spontaneous airway inflammation. Strikingly, although loss of Ship1 in the B-cell lineage did not affect HDM-induced AAI, loss of Ship1 in either of the T-cell or DC lineages protected mice from AAI by skewing the typical TH2 immune response toward a TH1 response. CONCLUSIONS: Although panhematopoietic deletion of Ship1 leads to spontaneous lung inflammation, selective deletion of Ship1 in T cells or DCs impairs the formation of an adaptive TH2 response and protects animals from HDM-induced AAI.

Podocalyxin enhances breast tumor growth and metastasis and is a target for monoclonal antibody therapy.

INTRODUCTION: Podocalyxin (gene name PODXL) is a CD34-related sialomucin implicated in the regulation of cell adhesion, migration and polarity. Upregulated expression of podocalyxin is linked to poor patient survival in epithelial cancers. However, it is not known if podocalyxin has a functional role in tumor progression. METHODS: We silenced podocalyxin expression in the aggressive basal-like human (MDA-MB-231) and mouse (4T1) breast cancer cell lines and also overexpressed podocalyxin in the more benign human breast cancer cell line, MCF7. We evaluated how podocalyxin affects tumorsphere formation in vitro and compared the ability of podocalyxin-deficient and podocalyxin-replete cell lines to form tumors and metastasize using xenogenic or syngeneic transplant models in mice. Finally, in an effort to develop therapeutic treatments for systemic cancers, we generated a series of antihuman podocalyxin antibodies and screened these for their ability to inhibit tumor progression in xenografted mice. RESULTS: Although deletion of podocalyxin does not alter gross cell morphology and growth under standard (adherent) culture conditions, expression of PODXL is required for efficient formation of tumorspheres in vitro. Correspondingly, silencing podocalyxin resulted in attenuated primary tumor growth and invasiveness in mice and severely impaired the formation of distant metastases. Likewise, in competitive tumor engraftment assays where we injected a 50:50 mixture of control and shPODXL (short-hairpin RNA targeting PODXL)-expressing cells, we found that podocalyxin-deficient cells exhibited a striking decrease in the ability to form clonal tumors in the lung, liver and bone marrow. Finally, to validate podocalyxin as a viable target for immunotherapy, we screened a series of novel antihuman podocalyxin antibodies for their ability to inhibit tumor progression in vivo. One of these antibodies, PODOC1, potently blocked tumor growth and metastasis. CONCLUSIONS: We show that podocalyxin plays a key role in the formation of primary tumors and distant tumor metastasis. In addition, we validate podocalyxin as potential target for monoclonal antibody therapy to inhibit primary tumor growth and systemic dissemination.

Imaging breast cancer morphology using probe-based confocal laser endomicroscopy: towards a real-time intraoperative imaging tool for cavity scanning.

Current techniques for assessing the adequacy of tumour excision during breast conserving surgery do not provide real-time direct cytopathological assessment of the internal cavity walls within the breast. This study investigates the ability of probe-based confocal laser endomicroscopy (pCLE), an emerging imaging tool, to image the morphology of neoplastic and non-neoplastic breast tissues, and determines the ability of histopathologists and surgeons to differentiate these images. Freshly excised tumour samples and adjacent non-diseased sections from 50 consenting patients were stained with 0.01 % acriflavine hydrochloride and imaged using pCLE. All discernible pCLE features were cross-examined with conventional histopathology. Following pattern recognition training, 17 histopathologists and surgeons with no pCLE experience interpreted 50 pCLE images independently whilst blinded to histopathology results. Three-hundred and fifty pCLE image mosaics were analysed. Consistent with histopathology findings, the glandular structures, adipocytes and collagen fibres of normal breast were readily visible on pCLE images. These were distinguishable from the morphological architecture exhibited by invasive and non-invasive carcinoma. The mean accuracy of pCLE image interpretation for histopathologists and surgeons was 94 and 92 %, respectively. Overall, inter-observer agreement for histopathologists was 'almost perfect', κ = 0.82; and 'substantial' for surgeons, κ = 0.74. pCLE morphological features of neoplastic and non-neoplastic breast tissues are readily visualized and distinguishable with high accuracy by both histopathologists and surgeons. Further research is required to investigate a potential role for the use of pCLE intraoperatively for in situ detection of residual cancerous foci, thereby guiding operating decision-making based on real-time breast cavity scanning.

SIGIRR, a negative regulator of TLR/IL-1R signalling promotes Microbiota dependent resistance to colonization by enteric bacterial pathogens.

Enteric bacterial pathogens such as enterohemorrhagic E. coli (EHEC) and Salmonella Typhimurium target the intestinal epithelial cells (IEC) lining the mammalian gastrointestinal tract. Despite expressing innate Toll-like receptors (TLRs), IEC are innately hypo-responsive to most bacterial products. This is thought to prevent maladaptive inflammatory responses against commensal bacteria, but it also limits antimicrobial responses by IEC to invading bacterial pathogens, potentially increasing host susceptibility to infection. One reason for the innate hypo-responsiveness of IEC is their expression of Single Ig IL-1 Related Receptor (SIGIRR), a negative regulator of interleukin (IL)-1 and TLR signaling. To address whether SIGIRR expression and the innate hypo-responsiveness of IEC impacts on enteric host defense, Sigirr deficient (-/-) mice were infected with the EHEC related pathogen Citrobacter rodentium. Sigirr -/- mice responded with accelerated IEC proliferation and strong pro-inflammatory and antimicrobial responses but surprisingly, Sigirr -/- mice proved dramatically more susceptible to infection than wildtype mice. Through haematopoietic transplantation studies, it was determined that SIGIRR expression by non-haematopoietic cells (putative IEC) regulated these responses. Moreover, the exaggerated responses were found to be primarily dependent on IL-1R signaling. Whilst exploring the basis for their susceptibility, Sigirr -/- mice were found to be unusually susceptible to intestinal Salmonella Typhimurium colonization, developing enterocolitis without the typical requirement for antibiotic based removal of competing commensal microbes. Strikingly, the exaggerated antimicrobial responses seen in Sigirr -/- mice were found to cause a rapid and dramatic loss of commensal microbes from the infected intestine. This depletion appears to reduce the ability of the microbiota to compete for space and nutrients (colonization resistance) with the invading pathogens, leaving the intestine highly susceptible to pathogen colonization. Thus, SIGIRR expression by IEC reflects a strategy that sacrifices maximal innate responsiveness by IEC in order to promote commensal microbe based colonization resistance against bacterial pathogens.

Improved resolution in fiber bundle inline holographic microscopy using multiple illumination sources.

Recent work has shown that high-quality inline holographic microscopy images can be captured through fiber imaging bundles. Speckle patterns arising from modal interference within the bundle cores can be minimized by use of a partially-coherent optical source such as an LED delivered via a multimode fiber. This allows numerical refocusing of holograms from samples at working distances of up to approximately 1 mm from the fiber bundle before the finite coherence begins to degrade the lateral resolution. However, at short working distances the lateral resolution is limited not by coherence, but by sampling effects due to core-to-core spacing in the bundle. In this article we demonstrate that multiple shifted holograms can be combined to improve the resolution by a factor of two. The shifted holograms can be rapidly acquired by sequentially firing LEDs, which are each coupled to their own, mutually offset, illumination fiber. Following a one-time calibration, resolution-enhanced images are created in real-time at an equivalent net frame rate of up to 7.5 Hz. The resolution improvement is demonstrated quantitatively using a resolution target and qualitatively using mounted biological slides. At longer working distances, beyond 0.6 mm, the improvement is reduced as resolution becomes limited by the source spatial and temporal coherence.