Optimized high-throughput screening of non-coding variants identified from genome-wide association studies.

The vast majority of disease-associated single nucleotide polymorphisms (SNP) identified from genome-wide association studies (GWAS) are localized in non-coding regions. A significant fraction of these variants impact transcription factors binding to enhancer elements and alter gene expression. To functionally interrogate the activity of such variants we developed snpSTARRseq, a high-throughput experimental method that can interrogate the functional impact of hundreds to thousands of non-coding variants on enhancer activity. snpSTARRseq dramatically improves signal-to-noise by utilizing a novel sequencing and bioinformatic approach that increases both insert size and the number of variants tested per loci. Using this strategy, we interrogated known prostate cancer (PCa) risk-associated loci and demonstrated that 35% of them harbor SNPs that significantly altered enhancer activity. Combining these results with chromosomal looping data we could identify interacting genes and provide a mechanism of action for 20 PCa GWAS risk regions. When benchmarked to orthogonal methods, snpSTARRseq showed a strong correlation with in vivo experimental allelic-imbalance studies whereas there was no correlation with predictive in silico approaches. Overall, snpSTARRseq provides an integrated experimental and computational framework to functionally test non-coding genetic variants.

[The causes and coping strategies for refractory vertigo].

Refractory vertigo is a disease entity characterized by uncontrollable recurrent vertigo and/or persistent dizziness instability, which can be caused by various diseases. The main pathogenesis may be related to recurrent episodes of the primary disease and compensatory dysfunction of the vestibular system. Understanding the common causes and pathological mechanisms of refractory vertigo, and comprehensively analyzing the relevant factors that cause symptoms, can facilitate accurate diagnosis and effective differentiation, and then provide comprehensive treatment targeting various factors such as etiology, symptoms, functional status, and psychological problems, ultimately achieving the goal of controlling the occurrence and development of refractory vertigo. Based on the characteristics of symptoms, this article focuses on analyzing possible mechanisms, relative factors, diagnosis and differential diagnosis of common diseases that lead to refractory vertigo, effective coping strategies, key issues that need attention, and future prospects, in order to improve clinical diagnostic accuracy and treatment effectiveness.

Lung pericytes as mediators of inflammation.

Pericytes are microvascular mural cells that directly contact endothelial cells. They have long been recognized for their roles in vascular development and homeostasis, but more recently have been identified as key mediators of the host response to injury. In this context, pericytes possess a surprising degree of cellular plasticity, behaving dynamically when activated and potentially participating in a range of divergent host responses to injury. Although there has been much interest in the role of pericytes in fibrosis and tissue repair, their involvement in the initial inflammatory process has been understudied and is increasingly appreciated. Pericytes mediate inflammation through leukocyte trafficking and cytokine signaling, respond to pathogen-associated molecular patterns and tissue damage-associated molecular patterns, and may drive vascular inflammation during human SARS-CoV-2 infection. In this review, we highlight the inflammatory phenotype of activated pericytes during organ injury, with an emphasis on novel findings relevant to pulmonary pathophysiology.

Nephronectin is required to maintain right lung lobar separation during embryonic development.

Nephronectin (NPNT) is a basement membrane (BM) protein and high-affinity ligand of integrin α8ß1 that is required for kidney morphogenesis in mice. In the lung, NPNT also localizes to BMs, but its potential role in pulmonary development has not been investigated. Mice with a floxed Npnt allele were used to generate global knockouts (KOs). Staged embryos were obtained by timed matings of heterozygotes and lungs were isolated for analysis. Although primary and secondary lung bud formation was normal in KO embryos, fusion of right lung lobes, primarily the medial and caudal, was first detected at E13.5 and persisted into adulthood. The lung parenchyma of KO mice was indistinguishable from wild-type (WT) and lobe fusion did not alter respiratory mechanics in adult KO mice. Interrogation of an existing single-cell RNA-seq atlas of embryonic and adult mouse lungs identified Npnt transcripts in mesothelial cells at E12.5 and into the early postnatal period, but not in adult lungs. KO embryonic lungs exhibited increased expression of laminin α5 and deposition of collagen IV in the mesothelial BM, accompanied by abnormalities in collagen fibrils in the adjacent stroma. Cranial and accessory lobes extracted from KO embryonic lungs fused ex vivo when cultured in juxtaposition, with the area of fusion showing loss of the mesothelial marker Wilms tumor 1. Because a similar pattern of lobe fusion was previously observed in integrin α8 KO embryos, our results suggest that NPNT signaling through integrin α8, likely in the visceral pleura, maintains right lung lobe separation during embryogenesis.

miR-145 as a Potential Biomarker and Therapeutic Target in Patients with Non-Small Cell Lung Cancer.

Our previous study found that miR-145 was downregulated in non-small cell lung cancer (NSCLC) tissues and that it could inhibit the cell proliferation in transfected NSCLC cells. In this study, we found that miR-145 was downregulated in NSCLC plasma samples compared to healthy controls. A receiver operating characteristic curve analysis indicated that plasma miR-145 expression was correlated with NSCLC in patient samples. We further revealed that the transfection of miR-145 inhibited the proliferation, migration, and invasion of NSCLC cells. Most importantly, miR-145 significantly delayed the tumor growth in a mouse model of NSCLC. We further identified GOLM1 and RTKN as the direct targets of miR-145. A cohort of paired tumors and adjacent non-malignant lung tissues from NSCLC patients was used to confirm the downregulated expression and diagnostic value of miR-145. The results were highly consistent between our plasma and tissue cohorts, confirming the clinical value of miR-145 in different sample groups. In addition, we also validated the expressions of miR-145, GOLM1, and RTKN using the TCGA database. Our findings suggested that miR-145 is a regulator of NSCLC and it plays an important role in NSCLC progression. This microRNA and its gene targets may serve as potential biomarkers and novel molecular therapeutic targets in NSCLC patients.

Effect of lung pericyte-like cell ablation on the bleomycin model of injury and repair.

We previously showed that pericyte-like cells derived from the FoxD1-lineage contribute to myofibroblasts following bleomycin-induced lung injury. However, their functional significance in lung fibrosis remains unknown. In this study, we used a model of lung pericyte-like cell ablation to test the hypothesis that pericyte-like cell ablation attenuates lung fibrosis in bleomycin-induced lung injury. Lung fibrosis was induced by intratracheal instillation of bleomycin. To ablate pericyte-like cells in the lung, diphtheria toxin (DT) was administered to Foxd1-Cre;Rosa26-iDTR mice at two different phases of bleomycin-induced lung injury. For early ablation, we coadministered bleomycin with DT and harvested mice at days 7 and 21. To test the effect of ablation after acute injury, we delivered DT 7 days after bleomycin administration. We assessed fibrosis by lung hydroxyproline content and semiquantitative analysis of picrosirius red staining. We performed bronchoalveolar lavage to determine cell count and differential. We also interrogated mRNA expression of fibrosis-related genes in whole lung RNA. Compared with DT-insensitive littermates where pericyte-like cells were not ablated, DT-sensitive animals exhibited no difference in fibrosis at day 21 both in the early and late pericyte ablation models. However, early ablation of pericytes reduced acute lung inflammation, as indicated by decreased inflammatory cells. Our data confirm a role for pericytes in regulating pulmonary inflammation in early lung injury.

Pericyte-like cells undergo transcriptional reprogramming and distinct functional adaptations in acute lung injury.

We previously reported on the role of pericyte-like cells as functional sentinel immune cells in lung injury. However, much about the biological role of pericytes in lung injury remains unknown. Lung pericyte-like cells are well-positioned to sense disruption to the epithelial barrier and coordinate local inflammatory responses due to their anatomic niche within the alveoli. In this report, we characterized transcriptional responses and functional changes in pericyte-like cells following activation by alveolar components from injured and uninjured lungs in a mouse model of acute lung injury (ALI). Purified pericyte-like cells from lung digests using PDGFRß as a selection marker were expanded in culture as previously described (1). We induced sterile acute lung injury in mice with recombinant human Fas ligand (rhFasL) instillation followed by mechanical ventilation (1). We then collected bronchoalveolar lavage fluid (BALF) from injured and uninjured mice. Purified pericyte-like cells in culture were exposed to growth media only (control), BALF from uninjured mice, and BALF from injured mice for 6 and 24 hours. RNA collected from these treatment conditions were processed for RNAseq. Targets of interest identified by pathway analysis were validated using in vitro and in vivo assays. We observed robust global transcriptional changes in pericyte-like cells following treatment with uninjured and injured BALF at 6 hours, but this response persisted for 24 hours only after exposure to injured BALF. Functional enrichment analysis of pericytes treated with injured BALF revealed the activation of pro-inflammatory, cell migration, and angiogenesis-related pathways, whereas processes associated with tissue development and cell differentiation were down-regulated. We validated select upregulated targets in the inflammatory, angiogenic, and cell migratory pathways using functional biological assays in vitro and in vivo. We conclude that lung pericyte-like cells are highly responsive to alveolar compartment content from both uninjured and injured lungs, but injured BALF elicits a more sustained response. The inflammatory, angiogenic, and migratory changes exhibited by activated pericyte-like cells underscore the phenotypic plasticity of these specialized stromal cells in the setting of acute lung injury.

[Standardized vestibular rehabilitation should be emphasized after surgical treatment involving vestibular system].

Surgical treatment involving vestibular system includes intractable vertigo surgery, inner ear surgery and vestibular tumor surgery. These operations often lead to the weakening or even loss of vestibular function, or further aggravation of the original dysfunction. If there is no standardized treatment after operation, patients are prone to recurrent vertigo, or long-term blurred vision and/or imbalance, as well as other symptoms. Through the use of medicines to promote vestibular compensation and standardized non-invasive vestibular rehabilitation training, symptoms can be eliminated as soon as possible, the duration of vestibular compensation can be shortened, stable vestibular compensation could be rapidly established, and thus the postoperative quality of life for those patients could be improved. Therefore, we should pay attention to standardize the vestibular rehabilitation after surgical treatment involving vestibular system.

[Direct projection pathway between medial vestibular nucleus and vestibular efferent neurons in rats and its electrophysiological characteristics].

Objective: To confirm the direct projection pathway between the medial vestibular nucleus (MVN) and vestibular efferent (VE) neurons and explore its electrophysiological characteristics. Methods: Newborn [(9±1) day-old] male and female Wistar rats were used in the study. The postsynaptic currents of VE were recorded after stimulating neurons in MVN by the whole-cell patch clamp recording technique. The action potentials (APs) of the afferent neurons in MVN were recorded retrogradely after stimulating the area of VE neurons distribution medial to genu of facial nerve (g7), and the position and shape of the recorded neurons were determined by biocytin staining. Results: The resting membrane potentials of VE neurons located medial to g7 ranged between -70 mV and -55 mV in current clamp recordings. Excitatory postsynaptic currents (EPSCs) were recorded in the VE neurons medial to the g7 evoked by single-pulse (0.08 mA, 0.1 Hz, 100 µs) electrical stimulation of MVN. The mean values of amplitude and duration were (195.6±23.7) pA and (23.9±5.9) ms, respectively. APs were recorded in MVN after stimulating the distribution area of VE neurons. The mean amplitude of the action potentials was (62.0±4.3) mV, and the mean duration was (94.9±4.7) ms. Biocytin staining indicated that the recorded neurons located in MVN and the axons' terminals went into the area medial to g7 in which VE neurons located. Conclusions: There is a direct excitatory pathway projecting from MVN to VE neurons medial to g7. Its physiological function may be related to the feedback regulation of vestibular center to peripheral vestibular afferent signals.

Pericytes in the Lung.

The lung has numerous roles, including gas exchange, immune surveillance, and barrier function. Being a highly vascularized organ, the lung receives dual blood supply from both the pulmonary and bronchial circulation. Therefore, pericytes likely play a prominent role in lung physiology given their localization in the perivascular niche. New genetic approaches have increased our understanding of the origin and the diverse functions of lung pericytes. Lung pericytes are myofibroblast progenitors, contributing to development of fibrosis in mouse models. Lung pericytes are also capable of responding to danger signals and amplify the inflammatory response through elaboration of cytokines and adhesion molecules. In this chapter, we describe the molecular, anatomical, and phenotypical characterization of lung pericytes. We further highlight their potential roles in the pathogenesis of lung diseases including pulmonary fibrosis, asthma, and pulmonary hypertension. Finally, current gaps in knowledge and areas of ongoing investigation in lung pericyte biology are also discussed.

Characterization of human PDGFR-ß-positive pericytes from IPF and non-IPF lungs.

Pericytes are key regulators of the microvasculature through their close interactions with the endothelium. However, pericytes play additional roles in tissue homeostasis and repair, in part by transitioning into myofibroblasts. Accumulation of myofibroblasts is a hallmark of fibrotic diseases such as idiopathic pulmonary fibrosis (IPF). To understand the contribution and role of pericytes in human lung fibrosis, we isolated these cells from non-IPF control and IPF lung tissues based on expression of platelet-derived growth factor receptor-ß (PDGFR-ß), a common marker of pericytes. When cultured in a specialized growth medium, PDGFR-ß+ cells retain the morphology and marker profile typical of pericytes. We found that IPF pericytes migrated more rapidly and invaded a basement membrane matrix more readily than control pericytes. Exposure of cells to transforming growth factor-ß, a major fibrosis-inducing cytokine, increased expression of α-smooth muscle actin and extracellular matrix genes in both control and IPF pericytes. Given that pericytes are uniquely positioned in vivo to respond to danger signals of both systemic and tissue origin, we stimulated human lung pericytes with agonists having pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs). Both control and IPF lung pericytes increased expression of proinflammatory chemokines in response to specific PAMPs and DAMPs released from necrotic cells. Our results suggest that control and IPF lung pericytes are poised to react to tissue damage, as well as microbial and fibrotic stimuli. However, IPF pericytes are primed for migration and matrix invasion, features that may contribute to the function of these cells in lung fibrosis.

Ablation of Pericyte-Like Cells in Lungs by Oropharyngeal Aspiration of Diphtheria Toxin.

We demonstrated previously that FoxD1-derived cells in the lung are enriched in pericyte-like cells in mouse lung. These cells express the common pericyte markers and are located adjacent to endothelial cells. In this study, we demonstrate the feasibility of administering diphtheria toxin (DT) by oropharyngeal aspiration as an approach to ablating FoxD1-derived cells. We crossed mice expressing Cre-recombinase under the FoxD1 promoter to Rosa26-loxP-STOP-loxP-iDTR mice and generated a bitransgenic line (FoxD1-Cre;Rs26-iDTR) in which FoxD1-derived cells heritably express simian or human diphtheria toxin receptor and are sensitive to DT. We delivered low-dose (0.5 ng/g) and high-dose (1ng/g × 2) to FoxD1-Cre;Rs26-iDTR mice and littermate control mice by oropharyngeal aspiration and evaluated ablation by flow cytometry and immunohistochemistry. FoxD1-Cre mice showed a 40-50% reduction in PDGFRß+ cells by flow cytometry at Days 2 and 7 after DT administration, with a return of PDGFRß+ cells at Day 28. Confocal microscopy revealed an observable reduction in pericyte markers. Bronchoalveolar lavage fluid analysis revealed no significant differences in total protein, bronchoalveolar lavage fluid red blood cell, or white blood cell counts at low dose. However, at high-dose DT, there was a proinflammatory effect in the control mice and increased mortality associated with systemic toxicity in Cre+ mice. Low-dose DT reduced lung PDGFRß+ stromal cells in the FoxD1-Cre;iDTR transgenic model without a differential effect on lung inflammation in DT-sensitive and DT-insensitive animals. Low-dose DT is a viable method for transient lineage-specific stromal cell ablation in the lung that minimizes systemic toxicity.

Lung pericyte-like cells are functional interstitial immune sentinel cells.

Pericytes are perivascular PDGF receptor-ß+ (PDGFRß+) stromal cells required for vasculogenesis and maintenance of microvascular homeostasis in many organs. Because of their unique juxtaposition to microvascular endothelium, lung PDGFRß+ cells are well situated to detect proinflammatory molecules released following epithelial injury and promote acute inflammatory responses. Thus we hypothesized that these cells represent an unrecognized immune surveillance or injury-sentinel interstitial cell. To evaluate this hypothesis, we isolated PDGFRß+ cells from murine lung and demonstrated that they have characteristics consistent with a pericyte population (referred to as pericyte-like cells for simplicity hereafter). We showed that pericyte-like cells expressed functional Toll-like receptors and upregulated chemokine expression following exposure to bronchoalveolar lavage fluid (BALF) collected from mice with sterile lung injury. Interestingly, BALF from mice without lung injury also induced chemokine expression in pericyte-like cells, suggesting that pericyte-like cells are primed to sense epithelial injury (permeability changes). Following LPS-induced lung inflammation, increased numbers of pericyte-like cells expressed IL-6, chemokine (C-X-C motif) ligand-1, chemokine (C-C motif) ligand 2/ monocyte chemotactic protein-1, and ICAM-1 in vivo. Sterile lung injury in pericyte-ablated mice was associated with decreased inflammation compared with normal mice. In summary, we found that pericyte-like cells are immune responsive and express diverse chemokines in response to lung injury in vitro and in vivo. Furthermore, pericyte-like cell ablation attenuated inflammation in sterile lung injury, suggesting that these cells play an important functional role in mediating lung inflammatory responses. We propose a model in which pericyte-like cells function as interstitial immune sentinels, detecting proinflammatory molecules released following epithelial barrier damage and participating in recruitment of circulating leukocytes.

Effects of diabetes mellitus on the clinical presentation and treatment response in tuberculosis.

BACKGROUND AND OBJECTIVE: With the colliding global epidemics of diabetes mellitus (DM) and tuberculosis (TB), we studied the effects of DM on the presentation of TB and its response to treatment. METHODS: Consecutive TB patients from 2006 to 2010 in a territory-wide treatment programme offering 9-month extended treatment for TB patients with DM were examined and followed up prospectively to assess their treatment response. Successful treatment completers were tracked through the TB registry and death registry for relapse, death or till 31 December 2014, whichever was the earliest. RESULTS: DM was independently associated with more chest symptoms (adjusted OR (AOR): 1.13) and systemic symptoms (AOR: 1.30) but less with other site-specific symptoms (AOR: 0.58) at TB presentation. There was more frequent pulmonary involvement (AOR: 1.69), with more extensive lung lesion (AOR: 1.25), lung cavity (AOR: 2.00) and positive sputum smear (AOR: 1.83) and culture (AOR: 1.38), but no difference in the proportion of retreatment cases or isoniazid and/or rifampicin resistance. After treatment initiation, there was higher overall incidence (AOR: 1.38) of adverse effects (mainly gastrointestinal symptoms, renal impairment and peripheral neuropathy but less fever and skin hypersensitivity reactions), more smear non-conversion (AOR: 1.59) and culture non-conversion (AOR: 1.40) at 2 months, and lower combined cure/treatment completion rate at 12 months (AOR: 0.79), but no difference in the relapse rate after having successfully completed treatment. CONCLUSION: DM adversely affected the clinical presentation and treatment response of TB, but there was no difference in the drug resistance and relapse rates.

Allergic rhinitis and allergy are risk factors for otitis media with effusion: A meta-analysis.

OBJECTIVE/HYPOTHESIS: We systematically reviewed the associations between allergic rhinitis or allergy and otitis media with effusion, by reference to published data. STUDY DESIGN: A meta-analysis of case-controlled studies. DATA SOURCE: Five databases (Pubmed, Highwire, Medline, Wanfang, and China National Knowledge Infrastructure) were searched for relevant studies in the English language published prior to November 12, 2015. STUDIES CHOSEN: Studies with clearly defined experimental and control groups, in which the experimental groups had otitis media with effusion together with allergic rhinitis or allergy, were selected. METHODS: We performed a meta-analysis on data from the identified cross-sectional and case-controlled studies using fixed- or random-effects models (depending on heterogeneity). We used Reviewer Manager 5.3 software to this end. RESULTS: Seven studies met the inclusion criteria. The prevalence of allergic rhinitis in patients with otitis media with effusion and the control groups differed significantly in three studies (P<0.00001), as did the prevalence of allergy (in six studies; P=0.003). CONCLUSION: Allergic rhinitis and allergy appear to be risk factors for otitis media with effusion.

Comparative efficacy of whole-brain radiotherapy with and without elemene liposomes in patients with multiple brain metastases from non-small-cell lung carcinoma.

PURPOSE: We explored and compared the clinical effects of whole-brain radiotherapy (wbrt) with and without elemene liposomes in patients with multiple brain metastases from non-small-cell lung carcinoma (nsclc). METHODS: We retrospectively analyzed 62 patients with multiple brain metastases from nsclc who received wbrt (30 Gy in 10 fractions) at Shengjing Hospital of China Medical University from January 2012 to May 2013. In 30 patients, elemene liposomes (400 mg) were injected intravenously via a peripherally inserted central catheter for 21 consecutive days from the first day of radiotherapy. Overall survival (os) and nervous system progression-free survival (npfs) for the two groups were compared by Kaplan-Meier analysis. Factors influencing npfs were examined by Cox regression analysis. Chi-square or Fisher exact tests were used for group comparisons. RESULTS: The median os was 9.0 months in the wbrt plus elemene group and 7.8 months in the wbrt-alone group (p = 0.581); the equivalent median npfs durations were 5.2 months and 3.7 months (p = 0.005). Patient treatment plan was an independent factor associated with npfs (p = 0.002). Tumour response and disease-control rates in the wbrt plus elemene group were 26.67% and 76.67% respectively; they were 18.75% and 62.5% in the wbrt group (p = 0.452). Compared with the patients in the wbrt-alone group, significantly fewer patients in the wbrt plus elemene group developed headaches (p = 0.04); quality of life was also significantly higher in the wbrt plus elemene group both at 1 month and at 2 months (p = 0.021 and p = 0.001 respectively). CONCLUSIONS: The addition of elemene liposomes to wbrt might prolong npfs in patients with multiple brain metastases from nsclc, while also reducing the incidence of headache and improving patient quality of life.