Probes for INS

Human bocavirus 1 (HBoV1), a nonenveloped single-stranded DNA parvovirus, causes mild to life-threatening respiratory tract infections, acute otitis media, and encephalitis in young children. HBoV1 often persists in nasopharyngeal secretions for months, hampering diagnosis. It has also been shown to persist in pediatric palatine and adenoid tonsils, which suggests that lymphoid organs are reservoirs for virus spread; however, the tissue site and host cells remain unknown. Our aim was to determine, in healthy nonviremic children with preexisting HBoV1 immunity, the adenotonsillar persistence site(s), host cell types, and virus activity. We discovered that HBoV1 DNA persists in lymphoid germinal centers (GCs), but not in the corresponding tonsillar epithelium, and that the cell types harboring the virus are mainly naive, activated, and memory B cells and monocytes. Both viral DNA strands and both sides of the genome were detected, as well as infrequent mRNA. Moreover, we showed, in B-cell and monocyte cultures and ex vivo tonsillar B cells, that the cellular uptake of HBoV1 occurs via the Fc receptor (FcγRII) through antibody-dependent enhancement (ADE). This resulted in viral mRNA transcription, known to occur exclusively from double-stranded DNA in the nucleus, however, with no detectable productive replication. Confocal imaging with fluorescent virus-like particles moreover disclosed endocytosis. To which extent the active HBoV1 GC persistence has a role in chronic inflammation or B-cell maturation disturbances, and whether the virus can be reactivated, will be interesting topics for forthcoming studies.IMPORTANCE Human bocavirus 1 (HBoV1), a common pediatric respiratory pathogen, can persist in airway secretions for months hampering diagnosis. It also persists in tonsils, providing potential reservoirs for airway shedding, with the exact location, host cell types, and virus activity unknown. Our study provides new insights into tonsillar HBoV1 persistence. We observed HBoV1 persistence exclusively in germinal centers where immune maturation occurs, and the main host cells were B cells and monocytes. In cultured cell lines and primary tonsillar B cells, we showed the virus uptake to be significantly enhanced by HBoV1-specific antibodies, mediated by the cellular IgG receptor, leading to viral mRNA synthesis, but without detectable productive replication. Possible implications of such active viral persistence could be tonsillar inflammation, disturbances in immune maturation, reactivation, or cell death with release of virus DNA, explaining the long-lasting HBoV1 airway shedding.

To understand how sleep-wakefulness cycles are regulated, it is essential to disentangle structural and functional relationships between the preoptic area (POA) and lateral hypothalamic area (LHA), since these regions play important yet opposing roles in the sleep-wakefulness regulation. GABA- and galanin (GAL)-producing neurons in the ventrolateral preoptic nucleus (VLPO) of the POA (VLPOGABA and VLPOGAL neurons) are responsible for the maintenance of sleep, while the LHA contains orexin-producing neurons (orexin neurons) that are crucial for maintenance of wakefulness. Through the use of rabies virus-mediated neural tracing combined with in situ hybridization (ISH) in male and female orexin-iCre mice, we revealed that the vesicular GABA transporter (Vgat, Slc32a1)- and galanin (Gal)-expressing neurons in the VLPO directly synapse with orexin neurons in the LHA. A majority (56.3 ± 8.1%) of all VLPO input neurons connecting to orexin neurons were double-positive for Vgat and Gal Using projection-specific rabies virus-mediated tracing in male and female Vgat-ires-Cre and Gal-Cre mice, we discovered that VLPOGABA and VLPOGAL neurons that send projections to the LHA received innervations from similarly distributed input neurons in many brain regions, with the POA and LHA being among the main upstream areas. Additionally, we found that acute optogenetic excitation of axons of VLPOGABA neurons, but not VLPOGAL neurons, in the LHA of male Vgat-ires-Cre mice induced wakefulness. This study deciphers the connectivity between the VLPO and LHA, provides a large-scale map of upstream neuronal populations of VLPO→LHA neurons, and reveals a previously uncovered function of the VLPOGABA→LHA pathway in the regulation of sleep and wakefulness.SIGNIFICANCE STATEMENT We identified neurons in the ventrolateral preoptic nucleus (VLPO) that are positive for vesicular GABA transporter (Vgat) and/or galanin (Gal) and serve as presynaptic partners of orexin-producing neurons in the lateral hypothalamic area (LHA). We depicted monosynaptic input neurons of GABA- and galanin-producing neurons in the VLPO that send projections to the LHA throughout the entire brain. Their input neurons largely overlap, suggesting that they comprise a common neuronal population. However, acute excitatory optogenetic manipulation of the VLPOGABA→LHA pathway, but not the VLPOGAL→LHA pathway, evoked wakefulness. This study shows the connectivity of major components of the sleep/wake circuitry in the hypothalamus and unveils a previously unrecognized function of the VLPOGABA→LHA pathway in sleep-wakefulness regulation. Furthermore, we suggest the existence of subpopulations of VLPOGABA neurons that innervate LHA.

Acetylcholine is an important modulator of striatal activity and it is vital to controlling striatal-dependent behaviors, including motor and cognitive functions. Despite this significance, the mechanisms determining how acetylcholine impacts striatal signaling are still not fully understood. In particular, little is known about the role of nicotinic acetylcholine receptors (nAChRs) expressed by striatal interneurons. In the present study, we used fluorescent in situ hybridization (FISH) to determine which neuronal types express the most prevalent beta2 nicotinic subunit in the mouse striatum. Our data support a common view that nAChR expression is mostly restricted to striatal interneurons. Surprisingly though, cholinergic interneurons (CINs) were identified as a population with the highest expression of beta2 nicotinic subunit. To investigate the functional significance of beta2-containing nAChRs in striatal interneurons, we deleted them by injecting the AAV-Cre vector into the striatum of beta2-flox/flox male mice. The deletion led to alterations in several behavioral domains, namely to an increased anxiety-like behavior, decrease in sociability ratio, deficit in discrimination learning and increased amphetamine-induced hyperlocomotion and c-Fos expression in mice with beta2 deletion. Further colocalization analysis showed that the increased c-Fos expression was present in both medium spiny neurons and presumed striatal interneurons. The present study concludes, that despite being relatively rare, beta2-containing nAChRs are primarily expressed in striatal neurons by CINs and play a significant role in behavior.SIGNIFICANCE STATEMENTA large variety of nicotinic acetylcholine receptors are expressed in the striatum, a brain region that is crucial in the control of behavior. The complexity of receptors with different functions is hindering our understanding of mechanisms through which striatal acetylcholine modulates behavior. We focused on the role of a small population of beta2-containing nicotinic acetylcholine receptors. We identified neuronal types expressing these receptors and determined their impact in the control of explorative behavior, anxiety-like behavior, learning and sensitivity to stimulants. Additional experiments showed that these alterations were associated with an overall increased activity of striatal neurons. Thus, the small population of nicotinic receptors represents an interesting target for a modulation of response to stimulant drugs and other striatal-based behavior.

Background: Inhaled antibiotics control chronic airway infection and maintain respiratory health in cystic fibrosis (CF). Given variation in patient responses to inhaled antibiotics, the ability to identify distinct responder phenotypes would facilitate delivery of personalized care. Previously a 10- gene panel was identified, measured directly from blood leukocytes, that predicted response to intravenous antibiotic treatment during pulmonary exacerbations. In the current study, we tested whether the same panel predicted clinical response in subjects receiving a month of inhaled antibiotic therapy with aztreonam lysine for inhalation (AZLI). Methods: A cohort of CF subjects infected with P. aeruginosa were enrolled before initiating 11 month’s treatment with AZLI using the Altera nebulizer system. Eighteen CF subjects underwent blood leukocyte gene panel measurements, sputum quantitative microbiology, spirometry, and CRP measurement before onset and at completion of 4 weeks of AZLI therapy. Results: ppFEV1 improvement was 3%. Significant reductions in sputum bacterial colony counts were detected with treatment. C-reactive protein (CRP) increased after treatment. Although no single gene within the panel changed significantly after treatment, multivariate analysis demonstrated that genes significantly predicted reduction in bacterial load (PLXND1 and HCA112) and improvement in FEV1 (HCA112). Hierarchical clustering based on gene expression yielded 2 distinctive molecular clusters before and after AZLI therapy. Based on overall expression patterns, subjects were identified as Pauci-inflammatory and inflammatory. In the analysis of pretreatment gene expression, the inflammatory group manifested greater systemic and airway inflammation, based on CRP and sputum neutrophil elastase. Consistent with sputum and systemic inflammatory variables, 4 genes (ADAM9, CSPG2, HCA112, HPSE) were more highly expressed in the inflammatory cluster at pre- and post-AZLI time points. In comparison, in the analysis of posttreatment molecular clusters, neutrophil elastase and CRP values were not significantly different between the 2 groups. Conclusion: Whole-blood gene leukocyte expression identifies distinct populations of CF subjects before inhaled antibiotic therapy with AZLI. Molecular quantification of systemic inflammation may indicate subgroups of CF subjects with baseline differences and with variable clinical responses to inhaled antibiotics. Application of a molecular panel may thus be valuable in sub-phenotyping patients before inhaled treatment. A goal of future, larger studies would be to validate whether inflammatory differences define phenotypes of responders to AZLI and to inhaled antibiotics in general, especially given challenges that will be present in future studies of antimicrobial agents in the setting of CFTR modulating agents. Clinical Trials.gov Identifier: NCT01736839.

The proinflammatory cytokine IFN-γ, which is chronically elevated in multiple sclerosis, induces pathological quiescence in human oligodendrocyte progenitor cells (OPCs) via upregulation of the transcription factor PRRX1. In this study using animals of both sexes, we investigated the role of heparan sulfate proteoglycans in the modulation of IFN-γ signaling following demyelination. We found that IFN-γ profoundly impaired OPC proliferation and recruitment following adult spinal cord demyelination. IFN-γ-induced quiescence was mediated by direct signaling in OPCs as conditional genetic ablation of IFNγR1 (Ifngr1) in adult NG2+ OPCs completely abrogated these inhibitory effects. Intriguingly, OPC-specific IFN-γ signaling contributed to failed oligodendrocyte differentiation, which was associated with hyperactive Wnt/Bmp target gene expression in OPCs. We found that PI-88, a heparan sulfate mimetic, directly antagonized IFN-γ to rescue human OPC proliferation and differentiation in vitro and blocked the IFN-γ mediated inhibitory effects on OPC recruitment in vivo Importantly, heparanase modulation by PI-88 or OGT2155 in demyelinated lesion rescued IFN-γ mediated axonal damage and demyelination. In addition to OPC-specific effects, IFN-γ augmented lesions were characterized by increased size, reactive astrogliosis and proinflammatory microglial/macrophage activation along with exacerbated axonal injury and cell death. Heparanase inhibitor treatment rescued many of the negative IFN-γ-induced sequalae suggesting a profound modulation of the lesion environment. Together, these results suggest that the modulation of the heparanome represents a rational approach to mitigate the negative effects of proinflammatory signaling and rescuing pathological quiescence in the inflamed and demyelinated human brain.SIGNIFICANCE STATEMENT: The failure of remyelination in multiple sclerosis contributes to neurological dysfunction and neurodegeneration. The activation and proliferation of oligodendrocyte progenitor cells (OPCs) is a necessary step in the recruitment phase of remyelination. Here, we show that the proinflammatory cytokine interferon-gamma directly acts on OPCs to induce pathological quiescence and thereby limit recruitment following demyelination. Heparan sulfate is a highly structured sulfated carbohydrate polymer that is present on the cell surface and regulates several aspects of the signaling microenvironment. We find that pathological interferon-gamma can be blocked by modulation of the heparanome following demyelination using either a heparan mimetic or by treatment with heparanase inhibitor. These studies establish the potential for modulation of heparanome as a regenerative approach in demyelinating disease.

Background Hyperglycaemia is a common finding in diabetic and non-diabetic patients presenting with ACS and is a powerful predictor of prognosis and mortality. The role of hyperglycaemia in ischemia-reperfusion injury (IRI) is not fully understood, and whether the Sodium Glucose Co-Transporter 1 (SGLT1) plays a role in infarct augmentation, before and/or after reperfusion, remains to be elucidated. However, diabetes clinical trials have shown SGLT inhibition improves cardiovascular outcomes, yet the mechanism is not fully understood. Purpose (1) Characterise the expression of SGLT1 in the myocardium, (2) investigate if SGLT1 is involved in a glucotoxicity injury during IRI, and (3) whether inhibiting SGLT1 with an SGLT inhibitor may reduce infarct size. Methods RT-PCR and in-situ hybridization (RNAScope) combined with Immunoflurescence integrated co detection with different cell marker techniques were used to detect SGLT1 mRNA expression in Sprague-Dawley whole myocardium and isolated primary cardiomyocytes. An Ex-vivo Langendorff ischemia-reperfusion perfusion model was used to study the effect of high glucose (22 mmol) on myocardium at reperfusion and Canagliflozin (CANA) a non-selective SGLT inhibitor (1000 nmol/L to block both the SGLT1 receptor and SGLT 2 receptor and 5 nmol/L to block the SGLT2 receptor only) was introduced following ischaemia at two different concentrations, at reperfusion and its effect on infarct size measured using triphenyltetrazolium chloride (TTC) staining. Results RT-PCR found SGLT1 mRNA is expressed in whole myocardium and in individual cardiac chambers. RNAscope detected SGLT1 mRNA is expressed homogenously within intact myocardium, particularly evident within the vasculature. Importantly, hyperglycaemia (22 mmol) at reperfusion increased infarct size (51.80 ± 3.52% vs 40.80 ± 2.89%; p-value: 0.026) compared to normoglycaemia, low dose CANA (5 nmol/L) did not attenuate infarct size in low glucose or high glucose, whereas high CANA concentration (1μmoL/L) significantly reduced infarct size in high glucose (22 mM) when administered at reperfusion (P value = 0.0047). Conclusion We have shown that SGLT1 is present in the myocardium. Hyperglycaemia appears augment myocardial infarction and inhibition of SGLT1 attenuates this increase.

Stress-induced release of dynorphins (Dyn) activates kappa opioid receptors (KOR) in serotonergic neurons to produce dysphoria and potentiate drug reward; however, the circuit mechanisms responsible for this effect are not known. In male mice, we found that conditional deletion of KOR from Slc6a4 (SERT)-expressing neurons blocked stress-induced potentiation of cocaine conditioned place preference (CPP). Within the dorsal raphe nucleus (DRN), two overlapping populations of KOR-expressing neurons: Slc17a8 (VGluT3) and SERT, were distinguished functionally and anatomically. Optogenetic inhibition of these SERT+ neurons potentiated subsequent cocaine CPP, whereas optical inhibition of the VGluT3+ neurons blocked subsequent cocaine CPP. SERT+/VGluT3- expressing neurons were concentrated in the lateral aspect of the DRN. SERT projections from the DRN were observed in the medial nucleus accumbens (mNAc), but VGluT3 projections were not. Optical inhibition of SERT+ neurons produced place aversion, whereas optical stimulation of SERT+ terminals in the mNAc attenuated stress-induced increases in forced swim immobility and subsequent cocaine CPP. KOR neurons projecting to mNAc were confined to the lateral aspect of the DRN, and the principal source of dynorphinergic (Pdyn) afferents in the mNAc was from local neurons. Excision of Pdyn from the mNAc blocked stress-potentiation of cocaine CPP. Prior studies suggested that stress-induced dynorphin release within the mNAc activates KOR to potentiate cocaine preference by a reduction in 5-HT tone. Consistent with this hypothesis, a transient pharmacological blockade of mNAc 5-HT1B receptors potentiated subsequent cocaine CPP. 5-HT1B is known to be expressed on 5-HT terminals in NAc, and 5-HT1B transcript was also detected in Pdyn+, Adora2a+ and ChAT+ (markers for direct pathway, indirect pathway, and cholinergic interneurons, respectively). Following stress exposure, 5-HT1B transcript was selectively elevated in Pdyn+ cells of the mNAc. These findings suggest that Dyn/KOR regulates serotonin activation of 5HT1B receptors within the mNAc and dynamically controls stress response, affect, and drug reward.

Uterine natural killer cells are regulated via surface inhibitory receptors for IL15 and galectin-9 (LGALS9) secreted by endometrial stromal cells (ESCs). However, the mechanism that regulates LGALS9 mRNA levels in ESCs is unclear. The aim of this study is to clarify the transcriptional regulation of LGALS9 in ESCs. Here, LGALS9 mRNA expression levels significantly decreased in the endometrial tissue in the early- to mid-secretory phase, and recovered in the mid- to late-secretory phase, compared to that in the proliferative phase. In ESCs, LGALS9 mRNA expression significantly decreased following estradiol + medroxyprogesterone acetate treatment for 1 day and increased after 12 days compared to that in the control. The transcriptional activity of the LGALS9 upstream region was up-regulated by heart and neural crest derivatives expressed 2 (HAND2) and down-regulated by forkhead box O1 (FOXO1). In ESCs, HAND2 expression significantly increased throughout the 12 days treatment with steroid hormones, whereas FOXO1 expression significantly increased on day 1, reached a plateau, and significantly increased again after 6 days of treatment. Levels of FOXO1 phosphorylation (pFOXO1) remained unchanged after 3-day treatment of ESCs with steroid hormones, but significantly increased following a 12-day treatment. pFOXO1 could not bind to the DNA and was thus unable to directly suppress LGALS9 transcription. Therefore, expression level of HAND2 and phosphorylation status of FOXO1 may determine LGALS9 mRNA expression. This study provides a novel molecular mechanism underlying the transcriptional regulation of LGALS9 mRNA in ESCs, which could be valuable in the treatment of diseases associated with decidualization failure.

Systemic treatment with resiniferatoxin (RTX) induces small-fiber sensory neuropathy by damaging TRPV1-expressing primary sensory neurons and causes distinct thermal sensory impairment and tactile allodynia, which resemble the unique clinical features of postherpetic neuralgia. However, the synaptic plasticity associated with RTX-induced tactile allodynia remains unknown. In this study, we found that RTX-induced neuropathy is associated with α2δ-1 upregulation in the dorsal root ganglion (DRG) and increased physical interaction between α2δ-1 and GluN1 in the spinal cord synaptosomes. RNAscope _in situ_ hybridization showed that RTX treatment significantly increased α2δ-1 expression in DRG neurons labeled with CGRP, IB4, NF200, and tyrosine hydroxylase. Electrophysiological recordings revealed that RTX treatment augmented the frequency of miniature excitatory postsynaptic currents (mEPSCs) and the amplitude of evoked EPSCs in spinal dorsal horn neurons, and these effects were reversed by blocking of NMDA receptors with AP5. Inhibiting α2δ-1 with gabapentin, genetically ablating α2δ-1, or targeting α2δ-1–bound NMDA receptors with α2δ-1Tat peptide largely normalized the baseline frequency of mEPSCs and the amplitude of evoked EPSCs potentiated by RTX treatment. Furthermore, systemic treatment with memantine or gabapentin and intrathecal injection of AP5 or Tat-fused α2δ-1 C terminus peptide reversed allodynia in RTX-treated rats and mice. In addition, RTX-induced tactile allodynia was attenuated in α2δ-1 knockout mice and in mice in which GluN1 was conditionally knocked out in DRG neurons. Collectively, our findings indicate that α2δ-1–bound NMDA receptors at presynaptic terminals of sprouting myelinated afferent nerves contribute to RTX-induced potentiation of nociceptive input to the spinal cord and tactile allodynia.

Methamphetamine (METH) is a highly addictive psychostimulant. The continuous use of METH may lead to its abuse and neurotoxicity that have been associated with METH-induced increases in release of dopamine (DA) and glutamate in the brain. METH action in DA has been shown to be mediated by redistribution of DA from vesicles into cytoplasm via vesicular monoamine transporter 2 (VMAT2) and the subsequent reversal of membrane DA transporter (DAT), while little is known about the mechanisms underlying METH-induced glutamate release. Recent studies indicate that a subpopulation of midbrain DA neurons co-expresses VMAT2 and vesicular glutamate transporter 2 (VGLUT2). Therefore, we hypothesized that METH-induced glutamate release may in part originate from such a dual phenotype of DA neurons. To test this hypothesis, we used Cre-LoxP techniques to selectively delete VGLUT2 from midbrain DA neurons, and then examined nucleus accumbens (NAc) DA and glutamate responses to METH using in vivo brain microdialysis between DA-VGLUT2-KO mice and their VGLUT2-HET littermates. We found that selective deletion of VGLUT2 from DA neurons did not significantly alter basal levels of extracellular DA and glutamate, but attenuated METH-induced increases in extracellular levels of DA and glutamate. In addition, DA-VGLUT2-KO mice also displayed lower locomotor response to METH than VGLUT2-HET control mice. These findings, for the first time, suggest that cell-type specific VGLUT2 expression in DA neurons plays an important role in the behavioral and neurochemical effects of METH. Glutamate corelease from DA neurons may in part contributes to METH-induced increase in NAc glutamate release.

Mutations of the cyclin-dependent kinase-like 5 (CDKL5) gene cause severe neurodevelopmental disorders characterized by intractable epilepsy, intellectual disability, and autism. Multiple mouse models generated for mechanistic studies have exhibited phenotypes similar to some human pathological features, but none of the models has developed one of the major symptoms affecting CDKL5 deficiency disorder (CDD) patients: intractable recurrent seizures. As disrupted neuronal excitation/inhibition balance is closely associated with the activity of glutamatergic and γ-aminobutyric acidergic (GABAergic) neurons, our aim was to study the effect of the loss of CDKL5 in different types of neurons on epilepsy. Using the Cre-LoxP system, we generated conditional knockout (cKO) mouse lines allowing CDKL5 deficiency in glutamatergic or GABAergic neurons. We employed noninvasive video recording and in vivo electrophysiological approaches to study seizure activity in these Cdkl5 cKO mice. Furthermore, we conducted Timm staining to confirm a morphological alteration, mossy fiber sprouting, which occurs with limbic epilepsy in both human and mouse brains. Finally, we performed whole-cell patch clamp in dentate granule cells to investigate cell-intrinsic properties and synaptic excitatory activity. We demonstrate that Emx1- or CamK2α-derived Cdkl5 cKO mice manifest high-frequency spontaneous seizure activities recapitulating the epilepsy of CDD patients, which ultimately led to sudden death in mice. However, Cdkl5 deficiency in GABAergic neurons does not generate such seizures. The seizures were accompanied by typical epileptic features including higher amplitude spikes for epileptiform discharges and abnormal hippocampal mossy fiber sprouting. We also found an increase in spontaneous and miniature excitatory postsynaptic current frequencies but no change in amplitudes in the dentate granule cells of Emx1-cKO mice, indicating enhanced excitatory synaptic activity. Our study demonstrates that Cdkl5 cKO mice, serving as an animal model to study recurrent spontaneous seizures, have potential value for the pathological study of CDD-related seizures and for therapeutic innovation.

High-level disinfection protects tens-of-millions of patients from the transmission of viruses on reusable medical devices. The efficacy of high-level disinfectants for preventing human papillomavirus (HPV) transmission has been called into question by recent publications, which if true, would have significant public health implications. Evaluation of the clinical relevance of these published findings required the development of novel methods to quantify and compare: (i) Infectious titres of lab-produced, clinically-sourced, and animal-derived papillomaviruses, (ii) The papillomavirus dose responses in the newly developed in vitro and in vivo models, and the kinetics of in vivo disease formation, and (iii) The efficacy of high-level disinfectants in inactivating papillomaviruses in these systems. Clinical virus titres obtained from cervical lesions were comparable to those obtained from tissue (raft-culture) and in vivo models. A mouse tail infection model showed a clear dose-response for disease formation, that papillomaviruses remain stable and infective on fomite surfaces for at least 8 weeks without squames and up to a year with squames, and that there is a 10-fold drop in virus titre with transfer from a fomite surface to a new infection site. Disinfectants such as ortho-phthalaldehyde and hydrogen peroxide, but not ethanol, were highly effective at inactivating multiple HPV types in vitro and in vivo. Together with comparable results presented in a companion manuscript from an independent laboratory, this work demonstrates that high-level disinfectants inactivate HPV and highlights the need for standardized and well-controlled methods to assess HPV transmission and disinfection. Advanced Sterilization Products, UK-MRC (MR/S024409/1 and MC-PC-13050) and Addenbrookes Charitable Trust.