Microbial-derived imidazole propionate links the heart failure-associated microbiome alterations to disease severity.

BACKGROUND: Interactions between the gut microbiota, diet, and host metabolism contribute to the development of cardiovascular disease, but a firm link between disease-specific gut microbiota alterations and circulating metabolites is lacking. METHODS: We performed shot-gun sequencing on 235 samples from 166 HF patients and 69 healthy control samples. Separate plasma samples from healthy controls (n = 53) were used for the comparison of imidazole propionate (ImP) levels. Taxonomy and functional pathways for shotgun sequencing data was assigned using MetaPhlAn3 and HUMAnN3 pipelines. RESULTS: Here, we show that heart failure (HF) is associated with a specific compositional and functional shift of the gut microbiota that is linked to circulating levels of the microbial histidine-derived metabolite ImP. Circulating ImP levels are elevated in chronic HF patients compared to controls and associated with HF-related gut microbiota alterations. Contrary to the microbiota composition, ImP levels provide insight into etiology and severity of HF and also associate with markers of intestinal permeability and systemic inflammation. CONCLUSIONS: Our findings establish a connection between changes in the gut microbiota, the presence, etiology, and severity of HF, and the gut-microbially produced metabolite ImP. While ImP appears promising as a circulating biomarker reflecting gut dysbiosis related to HF, further studies are essential to demonstrate its causal or contributing role in HF pathogenesis. TRIAL REGISTRATION: NCT02637167, registered December 22, 2015.

Gut Microbiota Alterations and Circulating Imidazole Propionate Levels Are Associated With Obstructive Coronary Artery Disease in People With HIV.

BACKGROUND: The impact of gut microbiota and its metabolites on coronary artery disease (CAD) in people with human immunodeficiency virus (PWH) is unknown. Emerging evidence suggests that imidazole propionate (ImP), a microbial metabolite, is linked with cardiometabolic diseases. METHODS: Fecal samples from participants of the Copenhagen Comorbidity in HIV infection (COCOMO) study were processed for 16S rRNA sequencing and ImP measured with liquid chromatography-tandem mass spectrometry. CAD severity was investigated by coronary computed tomography-angiography, and participants grouped according to obstructive CAD (n = 60), nonobstructive CAD (n = 80), or no CAD (n = 114). RESULTS: Participants with obstructive CAD had a gut microbiota with lower diversity and distinct compositional shift, with increased abundance of Rumiococcus gnavus and Veillonella, known producers of ImP. ImP plasma levels were associated with this dysbiosis, and significantly elevated in participants with obstructive CAD. However, gut dysbiosis but not plasma ImP was independently associated with obstructive CAD after adjustment for traditional and HIV-related risk factors (adjusted odds ratio, 2.7; 95% confidence interval, 1.1-7.2; P = .048). CONCLUSIONS: PWH with obstructive CAD displays a distinct gut microbiota profile and increased circulating ImP plasma levels. Future studies should determine whether gut dysbiosis and related metabolites such as ImP are predictive of incident cardiovascular events.

Increased Plasma Levels of Triglyceride-Enriched Lipoproteins Associate with Systemic Inflammation, Lipopolysaccharides, and Gut Dysbiosis in Common Variable Immunodeficiency.

PURPOSE: Triglycerides (TG) and their major transport lipoprotein in the circulation (VLDL) appear to be related to inflammation. Patients with common variable immunodeficiency (CVID) have inflammatory complications associated with gut microbial dysbiosis. We hypothesized that CVID patients have disturbed TG/VLDL profiles associated with these clinical characteristics. METHODS: We measured plasma concentrations of TGs, inflammatory markers, and lipopolysaccharide (LPS) in 95 CVID patients and 28 healthy controls. Additionally, in 40 CVID patients, we explored plasma lipoprotein profiling, fatty acid, gut microbial dysbiosis, and diet. RESULTS: TG levels were increased in CVID patients as compared to healthy controls (1.36 ± 0.53 mmol/l versus 1.08 ± 0.56 [mean, SD], respectively, P = 0.008), particularly in the clinical subgroup "Complications," characterized by autoimmunity and organ-specific inflammation, compared to "Infection only" (1.41 mmol/l, 0.71[median, IQR] versus [1.02 mmol/l, 0.50], P = 0.021). Lipoprotein profile analyses showed increased levels of all sizes of VLDL particles in CVID patients compared to controls. TG levels correlated positively with CRP (rho = 0.256, P = 0.015), IL-6 (rho = 0.237, P = 0.021), IL-12 (rho = 0.265, P = 0.009), LPS (r = 0.654, P = 6.59 × 10-13), CVID-specific gut dysbiosis index (r = 0.315, P = 0.048), and inversely with a favorable fatty acid profile (docosahexaenoic acid [rho = - 0.369, P = 0.021] and linoleic acid [rho = - 0.375, P = 0.019]). TGs and VLDL lipids did not appear to be associated with diet and there were no differences in body mass index (BMI) between CVID patients and controls. CONCLUSION: We found increased plasma levels of TGs and all sizes of VLDL particles, which were associated with systemic inflammation, LPS, and gut dysbiosis in CVID, but not diet or BMI.

Gut microbiota composition during hospitalization is associated with 60-day mortality after severe COVID-19.

BACKGROUND: Gut microbiota alterations have been reported in hospitalized COVID-19 patients, with reduced alpha diversity and altered microbiota composition related to respiratory failure. However, data regarding gut microbiota and mortality are scarce. METHODS: Rectal swabs for gut microbiota analyses were collected within 48 h after hospital admission (baseline; n = 123) and three-month post-admission (n = 50) in a subset of patients included in the Norwegian SARS-CoV2 cohort study. Samples were analysed by sequencing the 16S rRNA gene. Gut microbiota diversity and composition at baseline were assessed in relation to need for intensive care unit (ICU) admission during hospitalization. The primary objective was to investigate whether the ICU-related gut microbiota was associated with 60-day mortality. RESULTS: Gut microbiota diversity (Shannon index) at baseline was lower in COVID-19 patients requiring ICU admission during hospitalization than in those managed in general wards. A dysbiosis index representing a balance of enriched and reduced taxa in ICU compared with ward patients, including decreased abundance of butyrate-producing microbes and enrichment of a partly oral bacterial flora, was associated with need of ICU admission independent of antibiotic use, dexamethasone use, chronic pulmonary disease, PO2/FiO2 ratio, C-reactive protein, neutrophil counts or creatinine levels (adjusted p < 0.001). The ICU-related dysbiosis index at baseline correlated with systemic inflammation and was associated with 60-day mortality in univariate analyses (Hazard ratio 3.70 [2.00-8.6], p < 0.001), as well as after separate adjustment for covariates. At the three-month follow-up, the dysbiosis index remained elevated in ICU patients compared with ward patients (adjusted p = 0.007). CONCLUSIONS: Although our data should be regarded as exploratory due to low number of clinical end points, they suggest that gut microbiota alterations during hospitalization could be related to poor prognosis after severe COVID-19. Larger studies of gut involvement during COVID-19 in relation to long-term clinical outcome are warranted. Trial registration NCT04381819 . Retrospectively registered May 11, 2020.

Network-based multi-omics integration reveals metabolic at-risk profile within treated HIV-infection.

Multiomics technologies improve the biological understanding of health status in people living with HIV on antiretroviral therapy (PWH). Still, a systematic and in-depth characterization of metabolic risk profile during successful long-term treatment is lacking. Here, we used multi-omics (plasma lipidomic, metabolomic, and fecal 16 S microbiome) data-driven stratification and characterization to identify the metabolic at-risk profile within PWH. Through network analysis and similarity network fusion (SNF), we identified three groups of PWH (SNF-1-3): healthy (HC)-like (SNF-1), mild at-risk (SNF-3), and severe at-risk (SNF-2). The PWH in the SNF-2 (45%) had a severe at-risk metabolic profile with increased visceral adipose tissue, BMI, higher incidence of metabolic syndrome (MetS), and increased di- and triglycerides despite having higher CD4+ T-cell counts than the other two clusters. However, the HC-like and the severe at-risk group had a similar metabolic profile differing from HIV-negative controls (HNC), with dysregulation of amino acid metabolism. At the microbiome profile, the HC-like group had a lower α-diversity, a lower proportion of men having sex with men (MSM) and was enriched in Bacteroides. In contrast, in at-risk groups, there was an increase in Prevotella, with a high proportion of MSM, which could potentially lead to higher systemic inflammation and increased cardiometabolic risk profile. The multi-omics integrative analysis also revealed a complex microbial interplay of the microbiome-associated metabolites in PWH. Those severely at-risk clusters may benefit from personalized medicine and lifestyle intervention to improve their dysregulated metabolic traits, aiming to achieve healthier aging.

Fecal level of butyric acid, a microbiome-derived metabolite, is increased in patients with severe carotid atherosclerosis.

The short-chain fatty acid (SCFA) butyric acid maintains a healthy gut barrier and vascular endothelium. We aimed to investigate the association between fecal butyric acid, carotid atherosclerosis and risk factors for ischemic stroke. Patients with severe carotid atherosclerosis (i.e. ≥ 50% stenosis) (n = 43) were compared with healthy controls (n = 38). We analyzed fecal SCFAs by gas chromatography, microbiota composition by 16S rRNA sequencing, markers of gut barrier damage and inflammasome activation by immunoassay, and plasma SCFAs by ultra-high performance liquid chromatography-tandem mass spectroscopy. Patients had higher fecal butyric acid level (p = 0.024), along with increased functional potential of microbial butyric acid production (p = 0.031), compared with controls. Dietary fiber intake was comparable. Patients had higher levels of gut barrier damage markers CCL25 and IFABP, and the inflammasome activation marker IL-18, whereas plasma level of butyric was similar. Increased fecal butyric acid was associated with higher BMI, waist-hip ratio, HbA1c, CRP and leukocyte count. Contrary to our hypothesis, patients with severe carotid atherosclerosis had higher fecal butyric acid level, and increased microbial production, compared with controls. Gut barrier damage in patients might indicate decreased absorption of butyric acid and hence contribute to the higher fecal level.

Reduced gut microbial diversity in familial hypercholesterolemia with no effect of omega-3 polyunsaturated fatty acids intervention - a pilot trial.

Individuals with familial hypercholesterolemia (FH) undergo an aggressive treatment with cholesterol-lowering drugs to prevent coronary heart disease. Recent evidence suggests an interplay between the gut microbiota, blood lipid levels and lipid-lowering drugs, but this has yet to be studied in individuals with FH. The objective of the study was to characterize the gut microbiota of individuals with familial hypercholesterolemia and examine if effects of omega-3 polyunsaturated fatty acids (PUFAs) on blood lipids act through modification of the gut microbiome. The gut microbiota composition of individuals with FH (N = 21) and healthy controls (N = 144) was analyzed by extracting DNA from stool samples and sequencing of the V3-V4 region of the 16S rRNA gene. A subgroup (n = 15) of the participants received omega-3 polyunsaturated fatty acids (PUFAs) supplementation or placebo in a crossover manner, and the effect of PUFAs on the gut microbiota was also investigated. Individuals with FH had a different gut microbiota composition compared to healthy controls, characterized by reduced richness (p = .001) and reduction of several genera belonging to Clostridia and Coriobacteriia. Patients using ezetimibe in addition to statins appeared to have lower richness compared to those only using statins (p = .01). Intervention with omega-3 PUFAs had negligible impact on the microbiota composition. Positive effects on blood lipids after intervention with omega-3 PUFA were not associated with baseline gut microbiota composition or gut microbial changes during treatment. Further, patients with FH have an altered gut microbiota compared to healthy controls, possibly driven by the use of ezetimibe.

Respiratory dysfunction three months after severe COVID-19 is associated with gut microbiota alterations.

BACKGROUND: Although coronavirus disease 2019 (COVID-19) is primarily a respiratory infection, mounting evidence suggests that the gastrointestinal tract is involved in the disease, with gut barrier dysfunction and gut microbiota alterations being related to disease severity. Whether these alterations persist and are related to long-term respiratory dysfunction remains unknown. METHODS: Plasma was collected during hospital admission and after 3 months from the NOR-Solidarity trial (n = 181) and analyzed for markers of gut barrier dysfunction and inflammation. At the 3-month follow-up, pulmonary function was assessed by measuring the diffusing capacity of the lungs for carbon monoxide (DLCO ). Rectal swabs for gut microbiota analyses were collected (n = 97) and analyzed by sequencing the 16S rRNA gene. RESULTS: Gut microbiota diversity was reduced in COVID-19 patients with respiratory dysfunction, defined as DLCO below the lower limit of normal 3 months after hospitalization. These patients also had an altered global gut microbiota composition, with reduced relative abundance of 20 bacterial taxa and increased abundance of five taxa, including Veillonella, potentially linked to fibrosis. During hospitalization, increased plasma levels of lipopolysaccharide-binding protein (LBP) were strongly associated with respiratory failure, defined as pO2 /fiO2 (P/F ratio) <26.6 kPa. LBP levels remained elevated during and after hospitalization and were associated with low-grade inflammation and respiratory dysfunction after 3 months. CONCLUSION: Respiratory dysfunction after COVID-19 is associated with altered gut microbiota and persistently elevated LBP levels. Our results should be regarded as hypothesis generating, pointing to a potential gut-lung axis that should be further investigated in relation to long-term pulmonary dysfunction and long COVID.

Plasma extracellular vesicles in people living with HIV and type 2 diabetes are related to microbial translocation and cardiovascular risk.

HIV and type 2 diabetes (T2D) are both associated with gut microbiota alterations, low-grade endotoxemia and increased cardiovascular risk. We investigated the potential role of plasma extracellular vesicles (EVs) in relation to these processes. Plasma EVs were isolated by size exclusion chromatography in fasting individuals with HIV and T2D (n = 16), T2D only (n = 14), HIV only (n = 20) or healthy controls (n = 19), and characterized by transmission electron microscopy, western blot, nanoparticle tracking analysis and quantitative proteomics. The findings were compared to gut microbiota alterations, lipopolysaccharide levels and cardiovascular risk profile. Individuals with concomitant HIV and T2D had higher plasma EV concentration, which correlated closely with plasma lipopolysaccharides, triglycerides and Framingham score, but not with gut microbiota alterations. Proteomic analyses identified 558 human proteins, largely related to cardiometabolic disease genes and upstream regulation of inflammatory pathways, including IL-6 and IL-1ß, as well as 30 bacterial proteins, mostly from lipopolysaccharide-producing Proteobacteria. Our study supports that EVs are related to microbial translocation processes in individuals with HIV and T2D. Their proteomic content suggests a contributing role in low-grade inflammation and cardiovascular risk development. The present approach for exploring gut-host crosstalk can potentially identify novel diagnostic biomarkers and therapeutic targets.

NEIL3-deficiency increases gut permeability and contributes to a pro-atherogenic metabolic phenotype.

Atherosclerosis and its consequences cause considerable morbidity and mortality world-wide. We have previously shown that expression of the DNA glycosylase NEIL3 is regulated in human atherosclerotic plaques, and that NEIL3-deficiency enhances atherogenesis in Apoe-/- mice. Herein, we identified a time point prior to quantifiable differences in atherosclerosis between Apoe-/-Neil3-/- mice and Apoe-/- mice. Mice at this age were selected to explore the metabolic and pathophysiological processes preceding extensive atherogenesis in NEIL3-deficient mice. Untargeted metabolomic analysis of young Apoe-/-Neil3-/- mice revealed significant metabolic disturbances as compared to mice expressing NEIL3, particularly in metabolites dependent on the gut microbiota. 16S rRNA gene sequencing of fecal bacterial DNA indeed confirmed that the NEIL3-deficient mice had altered gut microbiota, as well as increased circulating levels of the bacterially derived molecule LPS. The mice were challenged with a FITC-conjugated dextran to explore gut permeability, which was significantly increased in the NEIL3-deficient mice. Further, immunohistochemistry showed increased levels of the proliferation marker Ki67 in the colonic epithelium of NEIL3-deficient mice, suggesting increased proliferation of intestinal cells and gut leakage. We suggest that these metabolic alterations serve as drivers of atherosclerosis in NEIL3-deficient mice.

Uptake of circulating extracellular vesicles from rectal cancer patients and differential responses by human monocyte cultures.

Extracellular vesicles (EVs) released by tumor cells can directly or indirectly modulate the phenotype and function of the immune cells of the microenvironment locally or at distant sites. The uptake of circulating EVs and the responses by human monocytes in vitro may provide new insights into the underlying biology of the invasive and metastatic processes in cancer. Although a mixed population of vesicles is obtained with most isolation techniques, we predominantly isolated exosomes (small EVs) and microvesicles (medium EVs) from the SW480 colorectal cancer cell line (established from a primary adenocarcinoma of the colon) by sequential centrifugation and ultrafiltration, and plasma EVs were prepared from 22 patients with rectal adenoma polyps or invasive adenocarcinoma by size-exclusion chromatography. The EVs were thoroughly characterized. The uptake of SW480 EVs was analyzed, and small SW480 EVs were observed to be more potent than medium SW480 EVs in inducing monocyte secretion of cytokines. The plasma EVs were also internalized by monocytes; however, their cytokine-releasing potency was lower than that of the cell line-derived vesicles. The transcriptional changes in the monocytes highlighted differences between adenoma and adenocarcinoma patient EVs in their ability to regulate biological functions, whereas the most intriguing changes were found in monocytes receiving EVs from patients with metastatic compared with localized cancer.

Rosuvastatin alters the genetic composition of the human gut microbiome.

The gut microbiome contributes to the variation of blood lipid levels, and secondary bile acids are associated with the effect of statins. Yet, our knowledge of how statins, one of our most common drug groups, affect the human microbiome is scarce. We aimed to characterize the effect of rosuvastatin on gut microbiome composition and inferred genetic content in stool samples from a randomized controlled trial (n = 66). No taxa were significantly altered by rosuvastatin during the study. However, rosuvastatin-treated participants showed a reduction in the collective genetic potential to transport and metabolize precursors of the pro-atherogenic metabolite trimethylamine-N-oxide (TMAO, p < 0.01), and an increase of related metabolites betaine and γ-butyrobetaine in plasma (p < 0.01). Exploratory analyses in the rosuvastatin group showed that participants with the least favorable treatment response (defined as < median change in high-density/low-density lipoprotein (HDL/LDL) ratio) showed a marked increase in TMAO-levels compared to those with a more favorable response (p < 0.05). Our data suggest that while rosuvastatin has a limited effect on gut microbiome composition, it could exert broader collective effects on the microbiome relevant to their function, providing a rationale for further studies of the influence of statins on the gut microbiome.

Low fibre intake is associated with gut microbiota alterations in chronic heart failure.

AIMS: Recent reports have suggested that patients with heart failure (HF) have an altered gut microbiota composition; however, associations with diet remain largely uninvestigated. We aimed to explore differences in the gut microbiota between patients with HF with reduced ejection fraction and healthy controls, focusing on associations with diet and disease severity. METHODS AND RESULTS: The microbiota composition of two cross-sectional cohorts (discovery, n = 40 and validation, n = 44) of patients with systolic HF and healthy controls (n = 266) was characterized by sequencing of the bacterial 16S rRNA gene. The overall microbial community (beta diversity) differed between patients with HF and healthy controls in both cohorts (P < 0.05). Patients with HF had shifts in the major bacterial phyla, resulting in a lower Firmicutes/Bacteroidetes (F/B) ratio than controls (P = 0.005). Patients reaching a clinical endpoint (listing for heart transplant or death) had lower bacterial richness and lower F/B ratio than controls (P < 0.01). Circulating levels of trimethylamine-N-oxide were associated with meat intake (P = 0.016), but not with gut microbiota alterations in HF. Low bacterial richness and low abundance of several genera in the Firmicutes phylum were associated with low fibre intake. CONCLUSIONS: The gut microbiota in HF was characterized by decreased F/B ratio and reduced bacterial diversity associated with clinical outcome. The gut microbiota alterations in HF were partly related to low fibre intake, emphasizing the importance of diet as a covariate in future studies. Our data could provide a rationale for targeting the gut microbiota in HF with high-fibre diet.

Impact of Human Immunodeficiency Virus-Related Gut Microbiota Alterations on Metabolic Comorbid Conditions.

BACKGROUND: We aimed to identify a human immunodeficiency virus (HIV)-related microbiota signature, independent of sexual preferences and demographic confounders, in order to assess a possible impact of the microbiome on metabolic comorbid conditions. METHODS: Bacterial 16S ribosomal RNA analyses were performed on stool samples from 405 HIV-infected and 111 uninfected participants of the Copenhagen Comorbidity in HIV Infection (COCOMO) study. Individuals were stratified according to sexual behavior (men who have sex with men [MSM] vs non-MSM). RESULTS: After excluding MSM-associated microbiota traits and adjusting for confounders, we identified an HIV-related microbiota signature, consisting of lower biodiversity, increased relative abundance of the bacterial clades Gammaproteobacteria and Desulfovibrionaceae and decrease in several Clostridia. This microbiota profile was associated with a 2-fold excess risk of metabolic syndrome, driven by increase in Desulfovibrionaceae and decrease in Clostridia (Butyrivibrio, Coprococcus 2, Lachnospiraceae UCG-001 and CAG-56). This association was accentuated (5-fold excess risk) in individuals with previous severe immunodeficiency, which also modified the association between HIV-related microbiota signature and visceral adipose tissue (VAT) area (P for interaction = .01). Accordingly, HIV-related microbiota was associated with 30-cm2 larger VAT in individuals with history of severe immunodeficiency, but not in those without. CONCLUSION: The HIV-related microbiota was associated with increased risk of metabolic syndrome and VAT accumulation, particularly in individuals with previous severe immunodeficiency, driven by increased Desulfovibrionaceae and lower abundance of several Clostridia. Our findings suggest a potential interplay between HIV-related microbiota, immune dysfunction and metabolic comorbid conditions. Interventions targeting the gut microbiome may be warranted to reduce cardiovascular risk, particularly in individuals with previous immunodeficiency.

Efficient extracellular vesicle isolation by combining cell media modifications, ultrafiltration, and size-exclusion chromatography.

Extracellular vesicles (EVs) are a heterogeneous population of biological particles released by cells. They represent an attractive source of potential biomarkers for early detection of diseases such as cancer. However, it is critical that sufficient amounts of EVs can be isolated and purified in a robust and reproducible manner. Several isolation methods that seem to produce distinct populations of vesicles exist, making data comparability difficult. While some methods induce cellular stress that may affect both the quantity and function of the EVs produced, others involve expensive reagents or equipment unavailable for many laboratories. Thus, there is a need for a standardized, feasible and cost-effective method for isolation of EVs from cell culture supernatants. Here we present the most common obstacles in the production and isolation of small EVs, and we suggest a combination of relatively simple strategies to avoid these. Three distinct cell lines were used (human oral squamous cell carcinoma (PE/CA-PJ49/E10)), pancreatic adenocarcinoma (BxPC3), and a human melanoma brain metastasis (H3). The addition of 1% exosome-depleted FBS to Advanced culture media enabled for reduced presence of contaminating bovine EVs while still ensuring an acceptable cell proliferation and low cellular stress. Cells were gradually adapted to these new media. Furthermore, using the Integra CELLine AD1000 culture flask we increased the number of cells and thereby EVs in 3D-culture. A combination of ultrafiltration with different molecular weight cut-offs and size-exclusion chromatography was further used for the isolation of a heterogeneous population of small EVs with low protein contamination. The EVs were characterized by nanoparticle tracking analysis, immunoaffinity capture, flow cytometry, Western blot and transmission electron microscopy. We successfully isolated a significant amount of small EVs compatible with exosomes from three distinct cell lines in order to demonstrate reproducibility with cell lines of different origin. The EVs were characterized as CD9 positive with a size between 60-140 nm. We conclude that this new combination of methods is a robust and improved strategy for the isolation of EVs, and in particular small EVs compatible with exosomes, from cell culture media without the use of specialized equipment such as an ultracentrifuge.

Effect of Lactobacillus rhamnosus GG Supplementation on Intestinal Inflammation Assessed by PET/MRI Scans and Gut Microbiota Composition in HIV-Infected Individuals.

BACKGROUND: Alterations in the gut microbiome have been associated with inflammation and increased cardiovascular risk in HIV-infected individuals. The aim of this study was to investigate the effects of the probiotic strain Lactobacillus rhamnosus GG (LGG) on intestinal inflammation, gut microbiota composition, and systemic markers of microbial translocation and inflammation in HIV-infected individuals. METHODS: This prospective, clinical interventional trial included 45 individuals [15 combination antiretroviral treatment (cART) naive and 30 cART treated] who ingested LGG twice daily at a dosage of 6 × 109 colony-forming units per capsule for a period of 8 weeks. Intestinal inflammation was assessed using F-2-fluoro-2-deoxy-D-glucose positron emission tomography/magnetic resonance imaging (F-FDG PET/MRI) scans in 15 individuals. Gut microbiota composition (V3-V4 region of the 16s rRNA gene) and markers of microbial translocation and inflammation (lipopolysaccharide, sCD14, sCD163, sCD25, high-sensitive CRP, IL-6, and tumor necrosis factor-alpha) were analyzed at baseline and after intervention. RESULTS: At baseline, evidence of intestinal inflammation was found in 75% of the participants, with no significant differences between cART-naive and cART-treated individuals. After LGG supplementation, a decrease in intestinal inflammation was detected on PET/MRI (-0.3 mean difference in the combined activity grade score from 6 regions, P = 0.006), along with a reduction of Enterobacteriaceae (P = 0.018) and Erysipelotrichaceae (P = 0.037) in the gut microbiome, with reduced Enterobacteriaceae among individuals with decreased F-FDG uptake on PET/MRI (P = 0.048). No changes were observed for soluble markers of microbial translocation and inflammation. CONCLUSIONS: A decrease in intestinal inflammation was found in HIV-infected individuals after ingestion of LGG along with a reduced abundance of Enterobacteriaceae, which may explain the local anti-inflammatory effect in the gut.

Size and concentration analyses of extracellular vesicles by nanoparticle tracking analysis: a variation study.

Current methods for characterisation of extracellular vesicles (EVs) need further standardisation in order to obtain an acceptable level of data comparability. Size and concentration of EVs can be determined by nanoparticle tracking analysis (NTA). However, both the heterogeneity of EVs and the choice of instrument settings may cause an appreciable analytical variation. Intra-assay (within-day, n = 6) and inter-assay (day-to-day, n = 6) variations (coefficient of variation, % CV) of different preparations of EVs and artificial vesicles or beads were determined using two NanoSight NS500 instruments, located at different laboratories. All analyses were performed by the same operator. The effect of applying identical software settings or instrument-optimised settings for each sample type and instrument was also evaluated. Finally, the impact of different operators and the use of two different software versions were investigated. The intra-assay CVs were 1-12% for both EVs and artificial samples, measured on the same instrument. The overall day-to-day variation was similar for both instruments, ranging from 2% to 25%. However, significantly different results were observed between the two instruments using identical software settings. The effect of applying instrument-optimised settings reduced the mismatch between the instruments, resulting in little to no significant divergences. The impact of using different operators and software versions when analysing silica microspheres and microvesicles from monocytes using instrument-optimised settings on the same instrument did not contribute to significant variation compared to the overall day-to-day variation of one operator. Performance differences between two similar NTA instruments may display significant divergences in size and concentration measurements when analysing EVs, depending on applied instrument settings and technical conditions. The importance of developing a streamlined and standardised execution of analysis, as well as monitoring longitudinal variation parameters on both biological and synthetic samples, should be highlighted.

Identification of potential saliva and tear biomarkers in primary Sjögren's syndrome, utilising the extraction of extracellular vesicles and proteomics analysis.

BACKGROUND: There is a long-lasting need for non-invasive, more accurate diagnostic techniques when evaluating primary Sjögren's syndrome (pSS) patients. Incorporation of additional diagnostics involving screening for disease-specific biomarkers in biological fluid is a promising concept that requires further investigation. In the current study we aimed to explore novel disease biomarkers in saliva and tears from pSS patients. METHODS: Liquid chromatography-mass spectrometry (LC-MS) was performed on stimulated whole saliva and tears from 27 pSS patients and 32 healthy controls, and salivary and tear proteomic biomarker profiles were generated. LC-MS was also combined with size exclusion chromatography to isolate extracellular vesicles (EVs) from both fluids. Nanoparticle tracking analysis was conducted on joint fractions from the saliva and tears to determine size distribution and concentration of EVs. Further EV characterisation was performed by immunoaffinity capture of CD9-positive EVs using magnetic beads, detected by flow cytometry. The LC-MS data were analysed for quantitative differences between patient and control groups using Scaffold, and the proteins were further analysed using the Database for Annotation, Visualization and Integrated Discovery (DAVID), for gene ontology overrepresentation, and the Search Tool for the Retrieval of Interacting Genes/Proteins for protein-protein interaction network analysis. RESULTS: Upregulation of proteins involved in innate immunity (LCN2), cell signalling (CALM) and wound repair (GRN and CALML5) were detected in saliva in pSS. Saliva EVs also displayed biomarkers critical for activation of the innate immune system (SIRPA and LSP1) and adipocyte differentiation (APMAP). Tear analysis indicated overexpression of proteins involved in TNF-α signalling (CPNE1) and B cell survival (PRDX3). Moreover, neutrophil gelatinase-associated lipocalin was upregulated in saliva and tears in pSS. Consistently, DAVID analysis demonstrated pathways of the adaptive immune response in saliva, of cellular component assembly for saliva EVs, and of metabolism and protein folding in tears in pSS patients. CONCLUSIONS: LC-MS of saliva and tears from pSS patients, solely and in combination with size-exclusion chromatography allowed screening for possible novel biomarkers encompassing both salivary and lacrimal disease target organs. This approach could provide additional diagnostic accuracy in pSS, and could possibly also be applied for staging and monitoring the disease.