Microbiome recovery in adult females with uncomplicated urinary tract infections in a randomised phase 2A trial of the novel antibiotic gepotidacin (GSK140944).

BACKGROUND: With increasing concerns about the impact of frequent antibiotic usage on the human microbiome, it is important to characterize the potential for such effects in early antibiotic drug development clinical trials. In a randomised Phase 2a clinical trial study that evaluated the pharmacokinetics of repeated oral doses of gepotidacin, a first-in-chemical-class triazaacenaphthylene antibiotic with a distinct mechanism of action, in adult females with uncomplicated urinary tract infections for gepotidacin (GSK2140944) we evaluated the potential changes in microbiome composition across multiple time points and body-sites ( ClinicalTrials.gov : NCT03568942). RESULTS: Samples of gastrointestinal tract (GIT), pharyngeal cavity and vaginal microbiota were collected with consent from 22 patients at three time points relative to the gepotidacin dosing regimen; Day 1 (pre-dose), Day 5 (end of dosing) and Follow-up (Day 28 ± 3 days). Microbiota composition was determined by DNA sequencing of 16S rRNA gene variable region 4 amplicons. By Day 5, significant changes were observed in the microbiome diversity relative to pre-dose across the tested body-sites. However, by the Follow-up visit, microbiome diversity changes were reverted to compositions comparable to Day 1. The greatest range of microbiome changes by body-site were GIT followed by the pharyngeal cavity then vagina. In Follow-up visit samples we found no statistically significant occurrences of pathogenic taxa. CONCLUSION: Our findings suggest that gepotidacin alteration of the human microbiome after 5 days of dosing is temporary and rebound to pre-dosing states is evident within the first month post-treatment. We recommend that future antibiotic drug trials include similar exploratory investigations into the duration and context of microbiome modification and recovery. TRIAL REGISTRATION: NCT03568942 . Registered 26 June 2018.

Lysine specific demethylase 1 inactivation enhances differentiation and promotes cytotoxic response when combined with all-trans retinoic acid in acute myeloid leukemia across subtypes.

Lysine specific demethylase 1 (LSD1) is a histone modifying enzyme that suppresses gene expression through demethylation of lysine 4 on histone H3. The anti-tumor activity of GSK2879552 and GSK-LSD1, potent, selective irreversible inactivators of LSD1, has previously been described. Inhibition of LSD1 results in a cytostatic growth inhibitory effect in a range of acute myeloid leukemia cell lines. To enhance the therapeutic potential of LSD1 inhibition in this disease setting, a combination of LSD1 inhibition and all-trans retinoic acid was explored. All-trans retinoic acid is currently approved for use in acute promyelocytic leukemia in which it promotes differentiation of abnormal blast cells into normal white blood cells. Combined treatment with all-trans retinoic acid and GSK2879552 results in synergistic effects on cell proliferation, markers of differentiation, and, most importantly, cytotoxicity. Ultimately the combination potential for LSD1 inhibition and ATRA will require validation in acute myeloid leukemia patients, and clinical studies to assess this are currently underway.

Airway host-microbiome interactions in chronic obstructive pulmonary disease.

BACKGROUND: Little is known about the interactions between the lung microbiome and host response in chronic obstructive pulmonary disease (COPD). METHODS: We performed a longitudinal 16S ribosomal RNA gene-based microbiome survey on 101 sputum samples from 16 healthy subjects and 43 COPD patients, along with characterization of host sputum transcriptome and proteome in COPD patients. RESULTS: Dysbiosis of sputum microbiome was observed with significantly increased relative abundance of Moraxella in COPD versus healthy subjects and during COPD exacerbations, and Haemophilus in COPD ex-smokers versus current smokers. Multivariate modeling on sputum microbiome, host transcriptome and proteome profiles revealed that significant associations between Moraxella and Haemophilus, host interferon and pro-inflammatory signaling pathways and neutrophilic inflammation predominated among airway host-microbiome interactions in COPD. While neutrophilia was positively correlated with Haemophilus, interferon signaling was more strongly linked to Moraxella. Moreover, while Haemophilus was significantly associated with host factors both in stable state and during exacerbations, Moraxella-associated host responses were primarily related to exacerbations. CONCLUSIONS: Our study highlights a significant airway host-microbiome interplay associated with COPD inflammation and exacerbations. These findings indicate that Haemophilus and Moraxella influence different components of host immune response in COPD, and that novel therapeutic strategies should consider targeting these bacteria and their associated host pathways in COPD.

Microbiome changes in healthy volunteers treated with GSK1322322, a novel antibiotic targeting bacterial peptide deformylase.

GSK1322322 is a novel antibacterial agent under development, and it has known antibacterial activities against multidrug-resistant respiratory and skin pathogens through its inhibition of the bacterial peptide deformylase. Here, we used next-generation sequencing (NGS) of the bacterial 16S rRNA genes from stool samples collected from 61 healthy volunteers at the predosing and end-of-study time points to determine the effects of GSK1322322 on the gastrointestinal (GI) microbiota in a phase I, randomized, double-blind, and placebo-controlled study. GSK1322322 was administered either intravenously (i.v.) only or in an oral-i.v. combination in single- and repeat-dose-escalation infusions. Analysis of the 16S rRNA sequence data found no significant changes in the relative abundances of GI operational taxonomic units (OTUs) between the prestudy and end-of-study samples for either the placebo- or i.v.-only-treated subjects. However, oral-i.v. treatment resulted in significant decreases in some bacterial taxa, the Firmicutes and Bacteroidales, and increases in others, the Betaproteobacteria, Gammaproteobacteria, and Bifidobacteriaceae. Microbiome diversity plots clearly differentiated the end-of-study oral-i.v.-dosed samples from all others collected. The changes in genome function as inferred from species composition suggest an increase in bacterial transporter and xenobiotic metabolism pathways in these samples. A phylogenetic analysis of the peptide deformylase protein sequences collected from the published genomes of clinical isolates previously tested for GSK1322322 in vitro susceptibility and GI bacterial reference genomes suggests that antibiotic target homology is one of several factors that influences the response of GI microbiota to this antibiotic. Our study shows that dosing regimen and target class are important factors when considering the impact of antibiotic usage on GI microbiota. (This clinical trial was registered at the GlaxoSmithKline Clinical Study Register under study identifier PDF 113376.).

A Hidradenitis Suppurativa molecular disease signature derived from patient samples by high-throughput RNA sequencing and re-analysis of previously reported transcriptomic data sets.

Hidradenitis suppurativa (HS) is a common, debilitating inflammatory skin disease linked to immune dysregulation and abnormalities in follicular structure and function. Several studies have characterized the transcriptomic profile of affected and unaffected skin in small populations. In this study of 20 patients, RNA from lesional and matching non-lesional skin biopsies in 20 subjects were used to identify an expression-based HS disease signature. This was followed by differential expression and pathway enrichment analyses, as well as jointly reanalyzing our findings with previously published transcriptomic profiles. We establish an RNA-Seq based HS expression disease signature that is mostly consistent with previous reports. Bulk-RNA profiles from 104 subjects in 7 previously reported data sets identified a disease signature of 118 differentially regulated genes compared to three control data sets from non-lesional skin. We confirmed previously reported expression profiles and further characterized dysregulation in complement activation and host response to bacteria in disease pathogenesis. Changes in the transcriptome of lesional skin in this cohort of HS patients is consistent with smaller previously reported populations. The findings further support the significance of immune dysregulation, in particular with regard to bacterial response mechanisms. Joint analysis of this and previously reported cohorts indicate a remarkably consistent expression profile.

Gut microbiome differences between metformin- and liraglutide-treated T2DM subjects.

INTRODUCTION: Metformin and glucagon-like peptide-1 (GLP-1) agonists are widely used for treating type two diabetes mellitus (T2DM). While recent studies suggest these drugs might modify the gastrointestinal tract (GIT) microbiome, further confirmation is required from human clinical trials. MATERIALS AND METHODS: Here, we compare, in patients with T2DM, the effects of metformin (n = 18 subjects) and liraglutide (n = 19), a GLP-1 agonist, on their GIT microbiomes over a 42 day period (n = 74 samples) using 16S ribosomal RNA (rRNA) sequencing. RESULTS: We found that these drugs had markedly different effects on the microbiome composition. At both baseline and Day 42, subjects taking metformin had a significant increase (Baseline adj. P = .038, Day 42 adj. P = .041) in the relative abundance of the bacterial genus Sutterella, whereas liraglutide dosing is associated with a significant increase (Baseline adj. P = .048, Day 42 adj. P = .003) in the genus Akkermansia, a GIT bacteria positively associated with gut barrier homoeostasis. Bacteroides and Akkermansia relative abundances were also significantly associated with duration of subject diabetes (adj P < .05). Specifically, there was a significantly higher abundance of Akkermansia in subjects with short and medium durations than those with long duration of diabetes. DISCUSSION: To our knowledge, this is the first report of GLP-1 agonist-associated changes in the human microbiome and its differentiating effects to metformin. Our study suggests that modulation of the GIT microbiome is a potentially important component in the mechanism of action of these drugs.

Tumor-immune profiling of murine syngeneic tumor models as a framework to guide mechanistic studies and predict therapy response in distinct tumor microenvironments.

Mouse syngeneic tumor models are widely used tools to demonstrate activity of novel anti-cancer immunotherapies. Despite their widespread use, a comprehensive view of their tumor-immune compositions and their relevance to human tumors has only begun to emerge. We propose each model possesses a unique tumor-immune infiltrate profile that can be probed with immunotherapies to inform on anti-tumor mechanisms and treatment strategies in human tumors with similar profiles. In support of this endeavor, we characterized the tumor microenvironment of four commonly used models and demonstrate they encompass a range of immunogenicities, from highly immune infiltrated RENCA tumors to poorly infiltrated B16F10 tumors. Tumor cell lines for each model exhibit different intrinsic factors in vitro that likely influence immune infiltration upon subcutaneous implantation. Similarly, solid tumors in vivo for each model are unique, each enriched in distinct features ranging from pathogen response elements to antigen presentation machinery. As RENCA tumors progress in size, all major T cell populations diminish while myeloid-derived suppressor cells become more enriched, possibly driving immune suppression and tumor progression. In CT26 tumors, CD8 T cells paradoxically increase in density yet are restrained as tumor volume increases. Finally, immunotherapy treatment across these different tumor-immune landscapes segregate into responders and non-responders based on features partially dependent on pre-existing immune infiltrates. Overall, these studies provide an important resource to enhance our translation of syngeneic models to human tumors. Future mechanistic studies paired with this resource will help identify responsive patient populations and improve strategies where immunotherapies are predicted to be ineffective.

Characterization of Gene Expression Phenotype in Amyotrophic Lateral Sclerosis Monocytes.

Importance: Amyotrophic lateral sclerosis (ALS) is a common adult-onset neurodegenerative disease characterized by selective loss of upper and lower motor neurons. Patients with ALS have persistent peripheral and central inflammatory responses including abnormally functioning T cells and activated microglia. However, much less is known about the inflammatory gene profile of circulating innate immune monocytes in these patients. Objective: To characterize the transcriptomics of peripheral monocytes in patients with ALS. Design, Setting, and Participants: Monocytes were isolated from peripheral blood of 43 patients with ALS and 22 healthy control individuals. Total RNA was extracted from the monocytes and subjected to deep RNA sequencing, and these results were validated by quantitative reverse transcription polymerase chain reaction. Main Outcomes and Measures: The differential expressed gene signatures of these monocytes were identified using unbiased RNA sequencing strategy for gene expression profiling. Results: The demographics between the patients with ALS (mean [SD] age, 58.8 [1.57] years; 55.8% were men and 44.2% were women; 90.7% were white, 4.65% were Hispanic, 2.33% were black, and 2.33% were Asian) and control individuals were similar (mean [SD] age, 57.6 [2.15] years; 50.0% were men and 50.0% were women; 90.9% were white, none were Hispanic, none were black, and 9.09% were Asian). RNA sequencing data from negative selected monocytes revealed 233 differential expressed genes in ALS monocytes compared with healthy control monocytes. Notably, ALS monocytes demonstrated a unique inflammation-related gene expression profile, the most prominent of which, including IL1B, IL8, FOSB, CXCL1, and CXCL2, were confirmed by quantitative reverse transcription polymerase chain reaction (IL8, mean [SE], 1.00 [0.18]; P = .002; FOSB, 1.00 [0.21]; P = .009; CXCL1, 1.00 [0.14]; P = .002; and CXCL2, 1.00 [0.11]; P = .01). Amyotrophic lateral sclerosis monocytes from rapidly progressing patients had more proinflammatory DEGs than monocytes from slowly progressing patients. Conclusions and Relevance: Our data indicate that ALS monocytes are skewed toward a proinflammatory state in the peripheral circulation and may play a role in ALS disease progression, especially in rapidly progressing patients. This increased inflammatory response of peripheral immune cells may provide a potential target for disease-modifying therapy in patients with ALS.

Genetic variants in the epithelial sodium channel associate with oedema in type 2 diabetic patients receiving the peroxisome proliferator-activated receptor gamma agonist farglitazar.

Peroxisome proliferator-activated receptor gamma (PPARgamma) agonists are highly effective in the treatment of type 2 diabetes. In some patients, PPARgamma ligands are associated with fluid retention/oedema, for which the mechanism is not fully understood. A pharmacogenetic study was undertaken to investigate effects of variations in 21 candidate genes related to epithelial sodium channel (ENaC) pathways on oedema. This study used DNA samples collected from type 2 diabetes phase III clinical trials of the PPARgamma agonist farglitazar (administered alone or in combination with insulin or glyburide) and investigated oedema reported as an adverse event as phenotype. Initial case-control analysis of oedema identified candidate gene single nucleotide polymorphisms with significant associations. These included three polymorphisms in ENaCbeta subunit (SCNN1B) that showed significant associations (P<0.05) with the two combination treatments in discrete regions of the gene, but not farglitazar treatment alone. Sequencing of SCNN1B in 207 Caucasian participants receiving farglitazar plus insulin or glyburide combination therapies, identified additional polymorphisms that were also significantly associated with oedema (P<0.0005) and maintained the treatment-regional associations. Further covariate analysis accounting for clinical factors influencing oedema supported these observations. One of the SCNN1B polymorphisms, at position -405 of the 5' flanking region (rs34241435), was predicted to modify transcriptional interactions and in a transfected COS cell luciferase reporter gene assay exhibited higher promoter activity. These exploratory studies provide clinical pharmacogenetic and functional genomic evidence to support a pivotal role for ENaC regulation in PPARgamma-induced oedema and provide insight into mechanisms and possible management of this side effect.