Comparison between upper and lower airway microbiome profiles in chronic rhinosinusitis patients.

BACKGROUND: Dysregulation of the airway microbiota is thought to contribute to airway inflammation in both chronic rhinosinusitis (CRS) and asthma. However, the relationship between the upper and lower airway microbiome remains unclear. METHODS: Sinus and lung brushes were collected from 29 CRS participants undergoing sinus surgery. DNA was extracted and submitted for 16s rRNA microbiome sequencing. Alpha and beta diversity metrics, taxonomic composition, and differences between individual taxa were compared for paired sinus and bronchial samples. RESULTS: Twenty-three out of 29 participants had sufficient samples for analysis. The mean (standard deviation) age was 51.59 (14.57) years, and 10 (44%) patients were female. Twelve (52%) patients had comorbid asthma. Sinus brushes had significantly higher alpha diversity indexes (Shannon and Faith) compared to bronchial brushes (p < 0.001). Beta diversity metrics were significantly different between the sinus and bronchial samples. Principal coordinate analysis showed no clustering of paired nasal and bronchial samples. Sinus brushes had significantly more Lawsonella, Corynebacterium, and Staphylococcus compared to bronchia brushes, while the latter were enriched in Tropheryma and Sphingomonas, among others (false discovery rate [FDR]-adjusted p < 0.01). Finally, CRS patients with comorbid asthma had significantly higher Pseudomonas and Peptoniphilus in sinus brushes and lower Prevotella in bronchial brushes when compared to non-asthmatics (FDR-adjusted p < 0.01). CONCLUSION: The sinus and bronchial bacterial microbiomes differ in important ways. Our study suggests that migration of bacteria from the sinus into the lower airways is unlikely in patients with CRS.

Single-cell RNA sequencing of bronchoscopy specimens: development of a rapid minimal handling protocol.

Single-cell RNA sequencing (scRNA-seq) is an important tool for understanding disease pathophysiology, including airway diseases. Currently, the majority of scRNA-seq studies in airway diseases have used invasive methods (airway biopsy, surgical resection), which carry inherent risks and thus present a major limitation to scRNA-seq investigation of airway pathobiology. Bronchial brushing, where the airway mucosa is sampled using a cytological brush, is a viable, less invasive method of obtaining airway cells for scRNA-seq. Here we describe the development of a rapid and minimal handling protocol for preparing single-cell suspensions from bronchial brush specimens for scRNA-seq. Our optimized protocol maximizes cell recovery and cell quality and facilitates large-scale profiling of the airway transcriptome at single-cell resolution.

Methionine-producing tumor micro(be) environment fuels growth of solid tumors.

BACKGROUND: Recent studies have uncovered the near-ubiquitous presence of microbes in solid tumors of diverse origins. Previous literature has shown the impact of specific bacterial species on the progression of cancer. We propose that local microbial dysbiosis enables certain cancer phenotypes through provisioning of essential metabolites directly to tumor cells. METHODS: 16S rDNA sequencing of 75 patient lung samples revealed the lung tumor microbiome specifically enriched for bacteria capable of producing methionine. Wild-type (WT) and methionine auxotrophic (metA mutant) E. coli cells were used to condition cell culture media and the proliferation of lung adenocarcinoma (LUAD) cells were measured using SYTO60 staining. Further, colony forming assay, Annexin V Staining, BrdU, AlamarBlue, western blot, qPCR, LINE microarray and subcutaneous injection with methionine modulated feed were used to analyze cellular proliferation, cell-cycle, cell death, methylation potential, and xenograft formation under methionine restriction. Moreover, C14-labeled glucose was used to illustrate the interplay between tumor cells and bacteria. RESULTS/DISCUSSION: Our results show bacteria found locally within the tumor microenvironment are enriched for methionine synthetic pathways, while having reduced S-adenosylmethionine metabolizing pathways. As methionine is one of nine essential amino acids that mammals are unable to synthesize de novo, we investigated a potentially novel function for the microbiome, supplying essential nutrients, such as methionine, to cancer cells. We demonstrate that LUAD cells can utilize methionine generated by bacteria to rescue phenotypes that would otherwise be inhibited due to nutrient restriction. In addition to this, with WT and metA mutant E. coli, we saw a selective advantage for bacteria with an intact methionine synthetic pathway to survive under the conditions induced by LUAD cells. These results would suggest that there is a potential bi-directional cross-talk between the local microbiome and adjacent tumor cells. In this study, we focused on methionine as one of the critical molecules, but we also hypothesize that additional bacterial metabolites may also be utilized by LUAD. Indeed, our radiolabeling data suggest that other biomolecules are shared between cancer cells and bacteria. Thus, modulating the local microbiome may have an indirect effect on tumor development, progression, and metastasis.

Microbial dysbiosis and the host airway epithelial response: insights into HIV-associated COPD using multi'omics profiling.

BACKGROUND: People living with HIV (PLWH) are at increased risk of developing Chronic Obstructive Pulmonary Disease (COPD) independent of cigarette smoking. We hypothesized that dysbiosis in PLWH is associated with epigenetic and transcriptomic disruptions in the airway epithelium. METHODS: Airway epithelial brushings were collected from 18 COPD + HIV + , 16 COPD - HIV + , 22 COPD + HIV - and 20 COPD - HIV - subjects. The microbiome, methylome, and transcriptome were profiled using 16S sequencing, Illumina Infinium Methylation EPIC chip, and RNA sequencing, respectively. Multi 'omic integration was performed using Data Integration Analysis for Biomarker discovery using Latent cOmponents. A correlation > 0.7 was used to identify key interactions between the 'omes. RESULTS: The COPD + HIV -, COPD -HIV + , and COPD + HIV + groups had reduced Shannon Diversity (p = 0.004, p = 0.023, and p = 5.5e-06, respectively) compared to individuals with neither COPD nor HIV, with the COPD + HIV + group demonstrating the most reduced diversity. Microbial communities were significantly different between the four groups (p = 0.001). Multi 'omic integration identified correlations between Bacteroidetes Prevotella, genes FUZ, FASTKD3, and ACVR1B, and epigenetic features CpG-FUZ and CpG-PHLDB3. CONCLUSION: PLWH with COPD manifest decreased diversity and altered microbial communities in their airway epithelial microbiome. The reduction in Prevotella in this group was linked with epigenetic and transcriptomic disruptions in host genes including FUZ, FASTKD3, and ACVR1B.

Systemic and Airway Epigenetic Disruptions Are Associated with Health Status in COPD.

Epigenetic modifications are common in chronic obstructive pulmonary disease (COPD); however, their clinical relevance is largely unknown. We hypothesized that epigenetic disruptions are associated with symptoms and health status in COPD. We profiled the blood (n = 57) and airways (n = 62) of COPD patients for DNA methylation (n = 55 paired). The patients' health status was assessed using the St. George's Respiratory Questionnaire (SGRQ). We conducted differential methylation analyses and identified pathways characterized by epigenetic disruptions associated with SGRQ scores and its individual domains. 29,211 and 5044 differentially methylated positions (DMPs) were associated with total SGRQ scores in blood and airway samples, respectively. The activity, impact, and symptom domains were associated with 9161, 25,689 and 17,293 DMPs in blood, respectively; and 4674, 3730 and 5063 DMPs in airways, respectively. There was a substantial overlap of DMPs between airway and blood. DMPs were enriched for pathways related to common co-morbidities of COPD (e.g., ageing, cancer and neurological) in both tissues. Health status in COPD is associated with airway and systemic epigenetic changes especially in pathways related to co-morbidities of COPD. There are more blood DMPs than in the airways suggesting that blood epigenome is a promising source to discover biomarkers for clinical outcomes in COPD.

Exposure to diesel exhaust alters the functional metagenomic composition of the airway microbiome in former smokers.

The lung microbiome plays a crucial role in airway homeostasis, yet we know little about the effects of exposures such as air pollution therein. We conducted a controlled human exposure study to assess the impact of diesel exhaust (DE) on the human airway microbiome. Twenty-four participants (former smokers with mild to moderate COPD (N = 9), healthy former smokers (N = 7), and control healthy never smokers (N = 8)) were exposed to DE (300 µg/m3 PM2.5) and filtered air (FA) for 2 h in a randomized order, separated by a 4-week washout. Endobronchial brushing samples were collected 24 h post-exposure and sequenced for the 16S microbiome, which was analyzed using QIIME2 and PICRUSt2 to examine diversity and metabolic functions, respectively. DE exposure altered airway microbiome metabolic functions in spite of statistically stable microbiome diversity. Affected functions included increases in: superpathway of purine deoxyribonucleosides degradation (pathway differential abundance 743.9, CI 95% 201.2 to 1286.6), thiazole biosynthesis I (668.5, CI 95% 139.9 to 1197.06), and L-lysine biosynthesis II (666.5, CI 95% 73.3 to 1257.7). There was an exposure-by-age effect, such that menaquinone biosynthesis superpathways were the most enriched function in the microbiome of participants aged >60, irrespective of smoking or health status. Moreover, exposure-by-phenotype analysis showed metabolic alterations in former smokers after DE exposure. These observations suggest that DE exposure induced substantial changes in the metabolic functions of the airway microbiome despite the absence of diversity changes.

Altered Polarization and Impaired Phagocytic Activity of Lung Macrophages in People With Human Immunodeficiency Virus and Chronic Obstructive Pulmonary Disease.

BACKGROUND: People with human immunodeficiency virus (PWH) have an increased risk of developing chronic obstructive pulmonary disease (COPD). METHODS: We phenotyped lung macrophages in 4 subgroups-M1 (CD40+CD163-), M2 (CD40-CD163+), double positives (CD40+CD163+), and double negatives and (CD40-CD163-)-and we determined their phagocytic capacity in PWH with and without COPD. RESULTS: People with human immunodeficiency virus with COPD have more double-negative macrophages (84.1%) versus PWH without (54.3%) versus controls (23.9%) (P=.004) and reduced phagocytosis (P=.012). Double-negative macrophages had the worst phagocytic capacity (P<.001). CONCLUSIONS: People with human immunodeficiency virus with COPD have an abundance of nonpolarized macrophages, which have poor phagocytic capacity and therefore predispose PWH to increased risk of disease progression.

Macrophages with reduced expressions of classical M1 and M2 surface markers in human bronchoalveolar lavage fluid exhibit pro-inflammatory gene signatures.

The classical M1/M2 polarity of macrophages may not be applicable to inflammatory lung diseases including chronic obstructive pulmonary disease (COPD) due to the complex microenvironment in lungs and the plasticity of macrophages. We examined macrophage sub-phenotypes in bronchoalveolar lavage (BAL) fluid in 25 participants with CD40 (a M1 marker) and CD163 (a M2 marker). Of these, we performed RNA-sequencing on each subtype in 10 patients using the Illumina NextSeq 500. Approximately 25% of the macrophages did not harbor classical M1 or M2 surface markers (double negative, DN), and these cells were significantly enriched in COPD patients compared with non-COPD patients (46.7% vs. 14.5%, p < 0.001). 1886 genes were differentially expressed in the DN subtype compared with  all other subtypes at a 10% false discovery rate. The 602 up-regulated genes included 15 mitochondrial genes and were enriched in 86 gene ontology (GO) biological processes including inflammatory responses. Modules associated with cellular functions including oxidative phosphorylation were significantly down-regulated in the DN subtype. Macrophages in the human BAL fluid, which were negative for both M1/M2 surface markers, harbored a gene signature that was pro-inflammatory and suggested dysfunction in cellular homeostasis. These macrophages may contribute to the pathogenesis and manifestations of inflammatory lung diseases such as COPD.

Effects of sex and chronic cigarette smoke exposure on the mouse cecal microbiome.

RATIONALE: Chronic smoke exposure is associated with weight loss in patients with Chronic Obstructive Pulmonary Disease (COPD). However, the biological contribution of chronic smoking and sex on the cecal microbiome has not been previously investigated. METHODS: Adult male, female and ovariectomized mice were exposed to air (control group) or smoke for six months using a standard nose-only smoke exposure system. DNA was extracted from the cecal content using the QIAGEN QIAamp® DNA Mini Kit. Droplet digital PCR was used to generate total 16S bacterial counts, followed by Illumina MiSeq® analysis to determine microbial community composition. The sequencing data were resolved into Amplicon Sequence Variants and analyzed with the use of QIIME2®. Alpha diversity measures (Richness, Shannon Index, Evenness and Faith's Phylogenetic Diversity) and beta diversity (based on Bray-Curtis distances) were assessed and compared according to smoke exposure and sex. RESULTS: The microbial community was different between male and female mice, while ovariectomy made the cecal microbiome similar to that of male mice. Chronic smoke exposure led to significant changes in the cecal microbial community in both male and female mice. The organism, Alistipes, was the most consistent bacteria identified at the genus level in the cecal content that was reduced with chronic cigarette exposure and its expression was positively related to the whole-body weight of these mice. CONCLUSION: Chronic smoke exposure is associated with changes in the cecal content microbiome; these changes may play a role in the weight changes that are observed in cigarette smokers.

TALEN-Mediated Gene Editing of HBG in Human Hematopoietic Stem Cells Leads to Therapeutic Fetal Hemoglobin Induction.

Elements within the γ-hemoglobin promoters (HBG1 and HBG2) function to bind transcription complexes that mediate repression of fetal hemoglobin expression. Sickle cell disease (SCD) subjects with a 13-bp deletion in the HBG1 promoter exhibit a clinically favorable hereditary persistence of fetal hemoglobin (HPFH) phenotype. We developed TALENs targeting the homologous HBG promoters to de-repress fetal hemoglobin. Transfection of human CD34+ cells with TALEN mRNA resulted in indel generation in HBG1 (43%) and HBG2 (74%) including the 13-bp HPFH deletion (∼6%). Erythroid differentiation of edited cells revealed a 4.6-fold increase in γ-hemoglobin expression as detected by HPLC. Assessment of TALEN-edited CD34+ cells in vivo in a humanized mouse model demonstrated sustained presence of indels in hematopoietic cells up to 24 weeks. Indel rates remained unchanged following secondary transplantation consistent with editing of long-term repopulating stem cells (LT-HSCs). Human γ-hemoglobin expressing F cells were detected by flow cytometry approximately 50% more frequently in edited animals compared to mock. Together, these findings demonstrate that TALEN-mediated indel generation in the γ-hemoglobin promoter leads to high levels of fetal hemoglobin expression in vitro and in vivo, suggesting that this approach can provide therapeutic benefit in patients with SCD or ß-thalassemia.

Younger Men With Prostate Cancer Have Lower Risk of Upgrading While on Active Surveillance: A Meta-analysis and Systematic Review of the Literature.

Active surveillance has become a popular option for patients with low risk prostate cancer. Our objective was to examine the correlation between age and the risk of Gleason upgrading and biopsy progression. A systematic search was conducted. Eight studies met our eligibility criteria including 6522 patients with a median age of 65.8 (41-86) years. Per decade decrease in age, the pooled odds ratio and hazard ratio (CI 95%) for Gleason upgrading were 0.83 (0.73-0.94) and 0.87 (0.82-0.92), and for biopsy progression were 0.80 (0.74-0.86) and 0.88 (0.79-0.99), respectively. Overall, younger patients have a lower risk of GS upgrading and biopsy progression.

Decreased microbiome diversity in the HIV small airway epithelium.

BACKGROUND: Persons living with human immunodeficiency virus (PLWH) face an increased burden of chronic obstructive pulmonary disease (COPD). Repeated pulmonary infections, antibiotic exposures, and immunosuppression may contribute to an altered small airway epithelium (SAE) microbiome. METHODS: SAE cells were collected from 28 PLWH and 48 HIV- controls through bronchoscopic cytologic brushings. DNA extracted from SAE cells was subjected to 16S rRNA amplification and sequencing. Comparisons of alpha and beta diversity between HIV+ and HIV- groups were performed and key operational taxonomic units (OTUs) distinguishing the two groups were identified using the Boruta feature selection after Random Forest Analysis. RESULTS: PLWH demonstrated significantly reduced Shannon diversity compared with HIV- volunteers (1.82 ± 0.10 vs. 2.20 ± 0.073, p = 0.0024). This was primarily driven by a reduction in bacterial richness (23.29 ± 2.75 for PLWH and 46.04 ± 3.716 for HIV-, p < 0.0001). Phyla distribution was significantly altered among PLWH, with an increase in relative abundance of Proteobacteria (p = 0.0003) and a decrease in Bacteroidetes (p = 0.0068) and Firmicutes (p = 0.0002). Six discriminative OTUs were found to distinguish PLWH from HIV- volunteers, aligning to Veillonellaceae, Fusobacterium, Verrucomicrobiaceae, Prevotella, Veillonella, and Campylobacter. CONCLUSIONS: Compared to HIV- controls, PLWH's SAE microbiome is marked by reduced bacterial diversity and richness with significant differences in community composition.

High Efficiency CRISPR/Cas9-mediated Gene Editing in Primary Human T-cells Using Mutant Adenoviral E4orf6/E1b55k "Helper" Proteins.

Many future therapeutic applications of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 and related RNA-guided nucleases are likely to require their use to promote gene targeting, thus necessitating development of methods that provide for delivery of three components-Cas9, guide RNAs and recombination templates-to primary cells rendered proficient for homology-directed repair. Here, we demonstrate an electroporation/transduction codelivery method that utilizes mRNA to express both Cas9 and mutant adenoviral E4orf6 and E1b55k helper proteins in association with adeno-associated virus (AAV) vectors expressing guide RNAs and recombination templates. By transiently enhancing target cell permissiveness to AAV transduction and gene editing efficiency, this novel approach promotes efficient gene disruption and/or gene targeting at multiple loci in primary human T-cells, illustrating its broad potential for application in translational gene editing.

The transcription factor ST18 regulates proapoptotic and proinflammatory gene expression in fibroblasts.

Suppression of tumorigenicity 18 (ST18) and the homologues neural zinc-finger protein-3 (NZF3) and myelin transcription factor 3 (Myt3) are transcription factors with unknown function. Previous studies have established that they repress transcription of a synthetic reporter construct consisting of the consensus sequence AAAGTTT linked to the thymidine kinase promoter. In addition, ST18 exhibits significantly reduced expression in breast cancer and breast cancer cell lines. We report here for the first time evidence that ST18 mediates tumor necrosis factor (TNF) -alpha induced mRNA levels of proapoptotic and proinflammatory genes in fibroblasts by mRNA profiling and silencing with ST18 small interfering RNA (siRNA). Gene set enrichment analysis and mRNA profiling support this conclusion by identifying several apoptotic and inflammatory pathways that are down-regulated by ST18 siRNA. In addition, ST18 siRNA reduces TNF-induced fibroblast apoptosis and caspase-3/7 activity. Fibroblasts that overexpress ST18 by transient transfection exhibit significantly increased apoptosis and increased expression of TNF-alpha, interleukin (IL) -1alpha, and IL-6. In addition, cotransfection of ST18 and a TNF-alpha or IL-1alpha reporter construct demonstrates that ST18 overexpression in fibroblasts significantly enhanced promoter activity of these genes. Taken together, these studies demonstrate that the transcription factor ST18/NZF3 regulates the mRNA levels of proapoptotic and proinflammatory genes in revealing a previously unrecognized function.

A survey of PDA use by palliative medicine practitioners.

This paper describes the results of a Web survey on the use of personal digital assistants (PDAs) by physicians across Canada involved with the delivery of palliative medicine in different settings. Seventy-two physicians responded to the survey from April to July 2005. The survey revealed 58.3% of respondents currently use PDAs on a daily basis, mostly to organize their practice and to look up medical references. Some use their PDAs to store patient information and to access a central electronic patient record (EPR). In terms of potential PDA use in palliative medicine, six thematic areas are suggested: medical references, EPR, staying connected, personal productivity, clinical research, and issues/concerns. For implications, healthcare organizations should consider mobile technology as part of their information systems strategy. The feasibility of a portable EPR for palliative medicine should be explored, and an information-based approach can help advance palliative medicine research in Canada.

The slow and long-lasting blockade of dopamine transporters in human brain induced by the new antidepressant drug radafaxine predict poor reinforcing effects.

BACKGROUND: (2S,3S)-2-(3-Chlorophenyl)-3,5,5,-trimethyl-2-morpholinol hydrochloride (radafaxine) is a new antidepressant that blocks dopamine transporters (DAT). A concern with drugs that block (DAT) is their potential reinforcing effects and abuse liability. Using positron emission tomography (PET) we have shown that for DAT-blocking drugs to produce reinforcing effects they must induce >50% DAT blockade and the blockade has to be fast (within 15 minutes). This study measures the potency and kinetics for DAT blockade by radafaxine in human brain. METHODS: PET and [11C]cocaine were used to estimate DAT blockade at 1, 4, 8, and 24 hours after radafaxine (40 mg p.o.) in 8 controls. Plasma pharmacokinetics and behavioral and cardiovascular effects were measured in parallel. RESULTS: DAT blockade by radafaxine was slow, and at 1 hour, it was 11%. Peak blockade occurred at about 4 hours and was 22%. Blockade was long lasting: at 8 hours 17%, and at 24 hours 15%. Peak plasma concentration occurred about 4 to 8 hours. No behavioral or cardiovascular effects were observed. CONCLUSIONS: The relatively low potency of radafaxine in blocking DAT and its slow blockade suggests that it is unlikely to have reinforcing effects. This is consistent with preclinical studies showing no self-administration. This is the first utilization of PET to predict abuse liability of a new antidepressant in humans based on DAT occupancy and pharmacokinetics.