An easy to use tool for the analysis of subcellular mRNA transcript colocalisation in smFISH data.

Single molecule fluorescence in situ hybridisation (smFISH) has become a valuable tool to investigate the mRNA expression of single cells. However, it requires a considerable amount of programming expertise to use currently available open-source analytical software packages to extract and analyse quantitative data about transcript expression. Here, we present FISHtoFigure, a new software tool developed specifically for the analysis of mRNA abundance and co-expression in QuPath-quantified, multi-labelled smFISH data. FISHtoFigure facilitates the automated spatial analysis of transcripts of interest, allowing users to analyse populations of cells positive for specific combinations of mRNA targets without the need for computational image analysis expertise. As a proof of concept and to demonstrate the capabilities of this new research tool, we have validated FISHtoFigure in multiple biological systems. We used FISHtoFigure to identify an upregulation in the expression of Cd4 by T-cells in the spleens of mice infected with influenza A virus, before analysing more complex data showing crosstalk between microglia and regulatory B-cells in the brains of mice infected with Trypanosoma brucei brucei. These analyses demonstrate the ease of analysing cell expression profiles using FISHtoFigure and the value of this new tool in the field of smFISH data analysis.

Chemotaxis: Dendritic cells as trendsetters of the immune response.

A new study reports that dendritic cells actively shape the CCL19 chemokine gradient to which they respond and that the chemokine receptor CCR7 both senses CCL19 and mediates its internalisation. Generation of local changes in chemokines allows coordination of movement over longer distances than previous models could explain.

Cotransfer of antigen and contextual information harmonizes peripheral and lymph node conventional dendritic cell activation.

T cell responses against infections and cancer are directed by conventional dendritic cells (cDCs) in lymph nodes distant from the site of challenge. Migratory cDCs, which travel from the tissue to the lymph node, not only drive initial T cell activation but also transfer antigen to lymph node-resident cDCs. These resident cells have essential roles defining the character of the resulting T cell response; however, it is unknown how they can appropriately process and present antigens to suitably direct responses given their spatial separation. Here, using a novel strain of influenza A and a modified melanoma model, we show that tissue and lymph node cDC activation is harmonized and that this is driven by cotransfer of contextual cues. In the tumor, incomplete cDC activation in the tumor microenvironment is mirrored by lymph node-resident cDCs, whereas during influenza infection, pathogen-associated molecular patterns cotransferred with antigen drive TLR signaling in resident cDCs and their subsequent robust activation. This cotransfer mechanism explains how individual antigens can be handled distinctly by resident cDCs and how signals driving poor tumoral cDC activation further impact the lymph node. Our findings clarify how tissue context dictates antigenic and, consequently, T cell fate in the lymph node.

Chronic pain recruits hypothalamic dynorphin/kappa opioid receptor signalling to promote wakefulness and vigilance.

Increased vigilance in settings of potential threats or in states of vulnerability related to pain is important for survival. Pain disrupts sleep and conversely, sleep disruption enhances pain, but the underlying mechanisms remain unknown. Chronic pain engages brain stress circuits and increases secretion of dynorphin, an endogenous ligand of the kappa opioid receptor (KOR). We therefore hypothesized that hypothalamic dynorphin/KOR signalling may be a previously unknown mechanism that is recruited in pathological conditions requiring increased vigilance. We investigated the role of KOR in wakefulness, non-rapid eye movement (NREM) sleep and rapid eye movement (REM) sleep in freely moving naïve mice and in mice with neuropathic pain induced by partial sciatic nerve ligation using EEG/EMG recordings. Systemic continuous administration of U69,593, a KOR agonist, over 5 days through an osmotic minipump decreased the amount of NREM and REM sleep and increased sleep fragmentation in naïve mice throughout the light-dark sleep cycle. We used KORcre mice to selectively express a Gi-coupled designer receptor activated by designer drugs (Gi-DREADD) in KORcre neurons of the hypothalamic paraventricular nucleus, a key node of the hypothalamic-pituitary-adrenal stress response. Sustained activation of Gi-DREADD with clozapine-N-oxide delivered in drinking water over 4 days, disrupted sleep in these mice in a similar way as systemic U69,593. Mice with chronic neuropathic pain also showed disrupted NREM and total sleep that was normalized by systemic administration of two structurally different KOR antagonists, norbinaltorphimine and NMRA-140, currently in phase II clinical development, or by CRISPR/Cas9 editing of paraventricular nucleus KOR, consistent with endogenous KOR activation disrupting sleep in chronic pain. Unexpectedly, REM sleep was diminished by either systemic KOR antagonist or by CRISPR/Cas9 editing of paraventricular nucleus KOR in sham-operated mice. Our findings reveal previously unknown physiological and pathophysiological roles of dynorphin/KOR in eliciting arousal. Physiologically, dynorphin/KOR signalling affects transitions between sleep stages that promote REM sleep. Furthermore, while KOR antagonists do not promote somnolence in the absence of pain, they normalized disrupted sleep in chronic pain, revealing a pathophysiological role of KOR signalling that is selectively recruited to promote vigilance, increasing chances of survival. Notably, while this mechanism is likely beneficial in the short-term, disruption of the homeostatic need for sleep over longer periods may become maladaptive resulting in sustained pain chronicity. A novel approach for treatment of chronic pain may thus result from normalization of chronic pain-related sleep disruption by KOR antagonism.

Building a healthy mouse model ecosystem to interrogate cancer biology.

In a recent study, Sargent et al. characterise several novel Rag1-/- mouse strains and demonstrate that genetic background strongly influences xenograft development and phenotype. Here, we discuss this work within the broader context of cancer mouse modelling. We argue that new technologies will enable insights into how specific models align with human disease states and that this knowledge can be used to develop a diverse ecosystem of complementary mouse models of cancer. By utilising these diverse, well-characterised models to provide multiple perspectives on specific cancers, it should be possible to reduce the inappropriate attrition of sound hypotheses while protecting against false positives. Furthermore, careful re-introduction of biological variation, be that through outbred populations, environmental diversity or including animals of both sexes, can ensure that results are more broadly applicable and are less impacted by particular traits of homogeneous experimental populations. Thus, careful characterisation and judicious use of an array of mouse models provides an opportunity to address some of the issues surrounding both the reproducibility and translatability crises often referenced in pre-clinical cancer research.

Analysis of Prostate Cancer Tumor Microenvironment Identifies Reduced Stromal CD4 Effector T-cell Infiltration in Tumors with Pelvic Nodal Metastasis.

BACKGROUND: Pelvic nodal metastasis in prostate cancer impacts patient outcome negatively. OBJECTIVE: To explore tumor-infiltrating immune cells as a potential predictive tool for regional lymph node (LN) metastasis. DESIGN SETTING AND PARTICIPANTS: We applied multiplex immunofluorescence and targeted transcriptomic analysis on 94 radical prostatectomy specimens in patients with (LN+) or without (LN-) pelvic nodal metastases. Both intraepithelial and stromal infiltrations of immune cells and differentially expressed genes (mRNA and protein levels) were correlated with the nodal status. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The identified CD4 effector cell signature of nodal metastasis was validated in a comparable independent patient cohort of 184 informative cases. Patient outcome analysis and decision curve analysis were performed with the CD4 effector cell density-based signature. RESULTS AND LIMITATIONS: In the discovery cohort, both tumor epithelium and stroma from patients with nodal metastasis had significantly lower infiltration of multiple immune cell types, with stromal CD4 effector cells highlighted as the top candidate marker. Targeted gene expression analysis and confirmatory protein analysis revealed key alteration of extracellular matrix components in tumors with nodal metastasis. Of note, stromal CD4 immune cell density was a significant independent predictor of LN metastasis (odds ratio [OR] = 0.15, p = 0.004), and was further validated as a significant predictor of nodal metastasis in the validation cohort (OR = 0.26, p < 0.001). CONCLUSIONS: Decreased T-cell infiltrates in the primary tumor (particularly CD4 effector cells) are associated with a higher risk of LN metastasis. Future evaluation of CD4-based assays on prostate cancer diagnostic biopsy materials may improve selection of at-risk patients for the treatment of LN metastasis. PATIENT SUMMARY: In this report, we found that cancer showing evidence of cancer metastasis to the lymph nodes tends to have less immune cells present within the tumor. We conclude that the extent of immune cells present within a prostate tumor can help doctors determine the most appropriate treatment plan for individual patients.

Learned adaptive multiphoton illumination microscopy for large-scale immune response imaging.

Multiphoton microscopy is a powerful technique for deep in vivo imaging in scattering samples. However, it requires precise, sample-dependent increases in excitation power with depth in order to generate contrast in scattering tissue, while minimizing photobleaching and phototoxicity. We show here how adaptive imaging can optimize illumination power at each point in a 3D volume as a function of the sample's shape, without the need for specialized fluorescent labeling. Our method relies on training a physics-based machine learning model using cells with identical fluorescent labels imaged in situ. We use this technique for in vivo imaging of immune responses in mouse lymph nodes following vaccination. We achieve visualization of physiologically realistic numbers of antigen-specific T cells (~2 orders of magnitude lower than previous studies), and demonstrate changes in the global organization and motility of dendritic cell networks during the early stages of the immune response. We provide a step-by-step tutorial for implementing this technique using exclusively open-source hardware and software.

The "broken escalator" phenomenon: Vestibular dizziness interferes with locomotor adaptation.

BACKGROUND: Although vestibular lesions degrade postural control we do not know the relative contributions of the magnitude of the vestibular loss and subjective vestibular symptoms to locomotor adaptation. OBJECTIVE: To study how dizzy symptoms interfere with adaptive locomotor learning. METHODS: We examined patients with contrasting peripheral vestibular deficits, vestibular neuritis in the chronic stable phase (n = 20) and strongly symptomatic unilateral Meniere's disease (n = 15), compared to age-matched healthy controls (n = 15). We measured locomotor adaptive learning using the "broken escalator" aftereffect, simulated on a motorised moving sled. RESULTS: Patients with Meniere's disease had an enhanced "broken escalator" postural aftereffect. More generally, the size of the locomotor aftereffect was related to how symptomatic patients were across both groups. Contrastingly, the degree of peripheral vestibular loss was not correlated with symptom load or locomotor aftereffect size. During the MOVING trials, both patient groups had larger levels of instability (trunk sway) and reduced adaptation than normal controls. CONCLUSION: Dizziness symptoms influence locomotor adaptation and its subsequent expression through motor aftereffects. Given that the unsteadiness experienced during the "broken escalator" paradigm is internally driven, the enhanced aftereffect found represents a new type of self-generated postural challenge for vestibular/unsteady patients.

Kappa opioid receptor antagonists: A possible new class of therapeutics for migraine prevention.

Background Stress is the most commonly reported migraine trigger. Dynorphin, an endogenous opioid peptide acting preferentially at kappa opioid receptors (KORs), is a key mediator of stress responses. The aim of this study was to use an injury-free rat model of functional cephalic pain with features of migraine and medication overuse headache (MOH) to test the possible preventive benefit of KOR blockade on stress-induced cephalic pain. Methods Following sumatriptan priming to model MOH, rats were hyper-responsive to environmental stress, demonstrating delayed cephalic and extracephalic allodynia and increased levels of CGRP in the jugular blood, consistent with commonly observed clinical outcomes during migraine. Nor-binaltorphimine (nor-BNI), a long-acting KOR antagonist or CYM51317, a novel short-acting KOR antagonist, were given systemically either during sumatriptan priming or immediately before environmental stress challenge. The effects of KOR blockade in the amygdala on stress-induced allodynia was determined by administration of nor-BNI into the right or left central nucleus of the amygdala (CeA). Results KOR blockade prevented both stress-induced allodynia and increased plasma CGRP. Stress increased dynorphin content and phosphorylated KOR in both the left and right CeA in sumatriptan-primed rats. However, KOR blockade only in the right CeA prevented stress-induced cephalic allodynia as well as extracephalic allodynia, measured in either the right or left hindpaws. U69,593, a KOR agonist, given into the right, but not the left, CeA, produced allodynia selectively in sumatriptan-primed rats. Both stress and U69,593-induced allodynia were prevented by right CeA U0126, a mitogen-activated protein kinase inhibitor, presumably acting downstream of KOR. Conclusions Our data reveal a novel lateralized KOR circuit that mediated stress-induced cutaneous allodynia and increased plasma CGRP in an injury-free model of functional cephalic pain with features of migraine and medication overuse headache. Selective, small molecule, orally available, and reversible KOR antagonists are currently in development and may represent a novel class of preventive therapeutics for migraine.

A sphingosine 1-phosphate receptor 2 selective allosteric agonist.

Molecular probe tool compounds for the Sphingosine 1-phosphate receptor 2 (S1PR2) are important for investigating the multiple biological processes in which the S1PR2 receptor has been implicated. Amongst these are NF-κB-mediated tumor cell survival and fibroblast chemotaxis to fibronectin. Here we report our efforts to identify selective chemical probes for S1PR2 and their characterization. We employed high throughput screening to identify two compounds which activate the S1PR2 receptor. SAR optimization led to compounds with high nanomolar potency. These compounds, XAX-162 and CYM-5520, are highly selective and do not activate other S1P receptors. Binding of CYM-5520 is not competitive with the antagonist JTE-013. Mutation of receptor residues responsible for binding to the zwitterionic headgroup of sphingosine 1-phosphate (S1P) abolishes S1P activation of the receptor, but not activation by CYM-5520. Competitive binding experiments with radiolabeled S1P demonstrate that CYM-5520 is an allosteric agonist and does not displace the native ligand. Computational modeling suggests that CYM-5520 binds lower in the orthosteric binding pocket, and that co-binding with S1P is energetically well tolerated. In summary, we have identified an allosteric S1PR2 selective agonist compound.

Reversal of pancreatitis-induced pain by an orally available, small molecule interleukin-6 receptor antagonist.

Pancreatic pain resulting from chronic inflammation of the pancreas is often intractable and clinically difficult to manage with available analgesics reflecting the need for more effective therapies. The mechanisms underlying pancreatitis pain are not well understood. Here, the possibility that interleukin-6 (IL-6) may promote pancreatitis pain was investigated with TB-2-081 (3-O-formyl-20R,21-epoxyresibufogenin, EBRF), a small molecule IL-6 receptor antagonist that was semi-synthetically derived from natural sources. The potential activity and mechanism of TB-2-081 were investigated following the induction of persistent pancreatitis using dibutyltin dichloride (DBTC) in rats. TB-2-081 displaces the binding of IL-6 to the human recombinant soluble IL-6 receptor with apparent high affinity and inhibits IL-6 mediated cell growth. Systemic or oral, but not intrathecal, administration of TB-2-081 reversed DBTC-induced abdominal hypersensitivity in a dose- and time-dependent manner. IL-6 levels were significantly up-regulated in the dorsal root ganglia (DRG) of rats with pancreatitis on day 6 after DBTC injection. IL-6-enhanced capsaicin-evoked release of calcitonin gene-related peptide from cultured DRG neurons was blocked by TB-2-081. Our data demonstrate that TB-2-081 acts as a systemically available and orally active small molecule IL-6 receptor antagonist. TB-2-081 effectively reduces pancreatitis-induced pain through peripheral mechanisms that are likely due to (a) increased expression of IL-6 in the DRG and (b) IL-6-mediated sensitization of nociceptive neurons. The activity of TB-2-081 implicates an important role for IL-6 in sustaining pancreatitis pain. Strategies targeting IL-6 actions through small molecule antagonists may offer novel approaches to improve the therapy of chronic pancreatitis and other chronic pain states.

'Reverse' alpha-ketoamide-based p38 MAP kinase inhibitors.

We have identified a second series of potent p38 inhibitors. As with our first generation series, these compounds are based on an alpha-ketoamide scaffold. The reversal of the ketoamide order, however, introduces more chemical flexibility and in addition results in improve potencies against p38.

Essential requirement for sphingosine kinase 2 in a sphingolipid apoptosis pathway activated by FTY720 analogues.

The clinical immunosuppressant FTY720 is a sphingosine analogue that, once phosphorylated by sphingosine kinase 2 (Sphk2), is an agonist of multiple receptor subtypes for sphingosine 1-phosphate. Short exposures to FTY720 afford long term protection in lymphoproliferative and autoimmune disease models, presumably by inducing apoptosis in subsets of cells essential for pathogenesis. Sphingosine itself is pro-apoptotic, and apoptosis induced with FTY720 or sphingosine is thought to proceed independently of their phosphorylation. Following chemical mutagenesis of Jurkat cells we isolated mutants that are selectively resistant to FTY720 analogue AAL(R), as well as natural sphingolipid bases, including sphingosine. Cells lacking functional Sphk2 were resistant to apoptosis induced with AAL(R), indicating that apoptosis proceeds through AAL(R) phosphorylation. Phosphorylation of AAL(R) was also required for induction of lymphocyte apoptosis in mice, as apoptosis was not induced with the non-phosphorylatable chiral analogue, AAL(S). Apoptosis was induced in the spleen but not the thymus of mice administered 1 mg/kg AAL(R), correlating with levels of AAL(R)-phosphate (AFD(R)) in organ extracts. AFD(R) did not induce apoptosis when added to the cell culture medium, indicating that it induces apoptosis through an intracellular target. NBD-labeled AAL(R) localized to the endoplasmic reticulum, and AAL(R) treatment resulted in elevated cytosolic calcium, Bax redistribution from cytosol to mitochondrial and endoplasmic reticulum membranes, and caspase-independent mitochondrial permeabilization in Jurkat cells. We therefore describe an apoptotic pathway triggered by intracellular accumulation of sphingolipid base phosphates and suggest that sphingoid base substrates for Sphk2 acting intracellularly could be useful in the treatment of lymphoproliferative diseases.

pH-induced conformational change of the rotavirus VP4 spike: implications for cell entry and antibody neutralization.

The rotavirus spike protein, VP4, is a major determinant of infectivity and neutralization. Previously, we have shown that trypsin-enhanced infectivity of rotavirus involves a transformation of the VP4 spike from a flexible to a rigid bilobed structure. Here we show that at elevated pH the spike undergoes a drastic, irreversible conformational change and becomes stunted, with a pronounced trilobed appearance. These particles with altered spikes, at a normal pH of 7.5, despite the loss of infectivity and the ability to hemagglutinate, surprisingly exhibit sialic acid (SA)-independent cell binding in contrast to the SA-dependent cell binding exhibited by native virions. Remarkably, a neutralizing monoclonal antibody that remains bound to spikes throughout the pH changes (pH 7 to 11 and back to pH 7) completely prevents this conformational change, preserving the SA-dependent cell binding and hemagglutinating functions of the virion. A hypothesis that emerges from the present study is that high-pH treatment triggers a conformational change that mimics a post-SA-attachment step to expose an epitope recognized by a downstream receptor in the rotavirus cell entry process. This process involves sequential interactions with multiple receptors, and the mechanism by which the antibody neutralizes is by preventing this conformational change.

Structures of rotavirus reassortants demonstrate correlation of altered conformation of the VP4 spike and expression of unexpected VP4-associated phenotypes.

Numerous prior studies have indicated that viable rotavirus reassortants containing structural proteins of heterologous parental origin may express unexpected phenotypes, such as changes in infectivity and immunogenicity. To provide a structural basis for alterations in phenotypic expression, a three-dimensional structural analysis of these reassortants was conducted. The structures of the reassortants show that while VP4 generally maintains the parental structure when moved to a heterologous protein background, in certain reassortants, there are subtle alterations in the conformation of VP4. The alterations in VP4 conformation correlated with expression of unexpected VP4-associated phenotypes. Interactions between heterologous VP4 and VP7 in reassortants expressing unexpected phenotypes appeared to induce the conformational alterations seen in VP4.