Low-grade chronic inflammation in visceral adipose tissue and the intestines are important drivers of obesity associated insulin resistance. Bioactive compounds derived from plants are an important ...source of potential novel therapies for the treatment of chronic diseases. In search for new immune based treatments of obesity associated insulin resistance, we screened for tissue relevant anti-inflammatory properties in 20 plant-based extracts.
We screened 20 plant-based extracts to assess for preferential production of IL-10 compared to TNFα, specifically targetting metabolic tissues, including the visceral adipose tissue. We assessed the therapeutic potential of the strongest anti-inflammatory compound, indigo, in the C57BL/6J diet-induced obesity mouse model with supplementation for up to 16 weeks by measuring changes in body weight, glucose and insulin tolerance, and gut barrier function. We also utilized flow cytometry, quantitative PCR, enzyme-linked immunosorbent assay (ELISA), and histology to measure changes to immune cells populations and cytokine profiles in the intestine, visceral adipose tissue (VAT), and liver. 16SrRNA sequencing was performed to examine gut microbial differences induced by indigo supplementation.
We identifed indigo, an aryl hydrocarbon receptor (AhR) ligand agonist, as a potent inducer of IL-10 and IL-22, which protects against high-fat diet (HFD)-induced insulin resistance and fatty liver disease in the diet-induced obesity model. Therapeutic actions were mechanistically linked to decreased inflammatory immune cell tone in the intestine, VAT and liver. Specifically, indigo increased Lactobacillus bacteria and elicited IL-22 production in the gut, which improved intestinal barrier permeability and reduced endotoxemia. These changes were associated with increased IL-10 production by immune cells residing in liver and VAT.
Indigo is a naturally occurring AhR ligand with anti-inflammatory properties that effectively protects against HFD-induced glucose dysregulation. Compounds derived from indigo or those with similar properties could represent novel therapies for diseases associated with obesity-related metabolic tissue inflammation.
Control efforts of soil-transmitted helminthiases rely primarily on large scale administration of anthelminthic drugs. The assessment of drug efficacies and understanding of drug behavior is pivotal ...to the evaluation of treatment successes, both in preventive chemo-therapy programs as well as in research of novel treatment options. The current WHO guidelines recommend an interval of 14-21 days between the treatment and follow-up, yet no in-depth analysis of egg excretion patterns of Trichuris trichiura after treatment has been conducted to date.
Within the framework of a multi-country trial to assess the efficacy and safety of albendazole-ivermectin combination therapy vs albendazole monotherapy against T. trichiura infections, we conducted a study collecting daily stool samples over the period of 28 days post-treatment in 87 participants in Pak Khan, Lao PDR. Egg counts were derived by duplicate Kato-Katz on-site for T. trichiura, hookworm and Ascaris lumbricoides and stool sample aliquots were subsequently analyzed by qPCR for the detection of T. trichiura infections. Sensitivity and specificity was calculated for each day separately using data derived by Kato-Katz to determine the optimal timepoint at which to assess drug efficacy.
Egg excretion patterns varied across treatment arms. For T. trichiura, only the albendazole-ivermectin treatment led to a considerable reduction in mean egg counts, whereas both treatments reduced hookworm egg counts and A. lumbricoides were cleared in all participants after day 7. For T. trichiura, we found sensitivity to be highest at days 18 and 22 when using egg counts as outcome and days 19 and 24 when using qPCR. Specificity was high (>0.9) from day 14 onwards. For hookworm, the highest sensitivity and specificity were found at days 17 and 25, respectively.
Based on our study, the ideal time period to assess drug efficacy for soil-transmitted helminth infections would be between day 18 and 24. The current WHO recommendation of 14 to 21 days is likely to yield acceptable outcome measures for soil-transmitted helminth infections.
NCT03527732.
Schistosomiasis affects over 200 million people worldwide but only praziquantel is available for treatment and control. Drug discovery is often based on phenotypic drug screening, involving different ...parasite stages retrieved from infected mice. Aiming to reduce animal use, we validated an in vitro growth method for juvenile Schistosoma mansoni for the purpose of drug sensitivity assays.
We compared inter-batch variability of serum, worm size and organ development, gender distribution, and drug sensitivity between in vitro and in vivo grown worms over different life stages. In vitro developed S. mansoni in Hybridoma medium supplemented with 20% human serum were similar in size as in vivo worms until 28 days of incubation (males 1.4 ± 0.2 mm, females 1.1 ± 0.5 mm long). qPCR analysis revealed similar gender distribution both on newly transformed schistosomula and worms grown for 21 days. Worms developed in vitro and in vivo were similarly sensitive to praziquantel from 7 to 35 days of development with the exception of 21 days of development, where a slightly lower activity was observed for the in vitro grown worms (IC50: 0.54 μM in vitro, 0.14 μM in vivo 72 hours post-incubation). The evaluation of five additional drugs revealed a similar sensitivity on worms developed for 21 days, with the exception of mefloquine, where we observed a 10-fold lower sensitivity on in vitro developed schistosomes when compared to in vivo grown (IC50: 4.43 μM in vitro, 0.48 μM in vivo).
A large number of juvenile S. mansoni worms can be grown in vitro, which show similar drug sensitivity, gender distribution, size and morphology as the worms recovered from rodents, supporting the use of this method in drug screening efforts.
Soil-transmitted helminth infections represent a large burden with over a quarter of the world's population at risk. Low cure rates are observed with standard of care (albendazole); therefore, a more ...effective combination therapy (albendazole and ivermectin) is being investigated but showed variable treatment efficacies without evidence of intrinsic parasite resistance. Here, we analyzed the microbiome of Trichuris trichiura and hookworm-infected patients and found an association of different enterotypes with treatment efficacy. 80 T. trichiura-infected patients with hookworm co-infections from Pak-Khan, Laos, received either albendazole (n = 41) or albendazole and ivermectin combination therapy (n = 39). Pre-/post-treatment stool samples were collected to monitor treatment efficacy and microbial communities were profiled using 16S rRNA gene sequencing, qPCR, and shotgun sequencing. We identified three bacterial enterotypes and show that pre-treatment enterotype is associated with efficacy of the combination treatment for both T. trichiura (CR
= 5.8%; CR
= 16.6%; CR
= 68.8%) and hookworm (CR
= 31.3%; CR
= 16.6%; CR
= 78.6%). This study shows that pre-treatment enterotype enables predicting treatment outcome of combination therapy for T. trichiura and hookworm infections.Trial registration: ClinicalTrials.gov, NCT03527732. Registered 17 May 2018, https://clinicaltrials.gov/ct2/show/NCT03527732 .
The composition and function of the intestinal microbiota are major determinants of human health and are strongly influenced by diet, antibiotic treatment, lifestyle and geography. Nevertheless, we ...currently have only little data on microbiomes of non-westernized communities. We assess the stool microbiota composition in 59 children aged 2-5 years from the Adadle district of Ethiopia, Somali Regional State. Here, milk and starch-rich food are predominant components of the local diet, where the inhabitants live a remote, traditional agropastoral lifestyle. Microbiota composition, function and the resistome were characterized by both 16S rRNA gene amplicon and shotgun metagenomic sequencing and compared to 1471 publicly available datasets from children living in traditional, transitional, and industrial communities with different subsistence strategies. Samples from the Adadle district are low in Bacteroidaceae, and Prevotellaceae, the main bacterial representatives in the feces of children living in industrialized and non-industrialized communities, respectively. In contrast, they had a higher relative abundance in Streptococcaceae, Bifidobacteriaceae and Erysipelatoclostridiaceae. Further, genes involved in degradation pathways of lactose, D-galactose and simple carbohydrates were enriched. Overall, our study revealed a unique composition of the fecal microbiota of these agropastoral children, highlighting the need to further characterize the fecal bacterial composition of human populations living different lifestyles.
It is now possible to comprehensively characterize the microbiota of the lungs using culture-independent, sequencing-based assays. Several sample types have been used to investigate the lung ...microbiota, each presenting specific challenges for preparation and analysis of microbial communities. Bronchoalveolar lavage fluid (BALF) enables the identification of microbiota specific to the lower lung but commonly has low bacterial density, increasing the risk of false-positive signal from contaminating DNA. The objectives of this study were to investigate the extent of contamination across a range of sample densities representative of BALF and identify features of contaminants that facilitate their removal from sequence data and aid in the interpretation of BALF sample 16S sequencing data.
Using three mock communities across a range of densities ranging from 8E+ 02 to 8E+ 09 16S copies/ml, we assessed taxonomic accuracy and precision by 16S rRNA gene sequencing and the proportion of reads arising from contaminants. Sequencing accuracy, precision, and the relative abundance of mock community members decreased with sample input density, with a significant drop-off below 8E+ 05 16S copies/ml. Contaminant OTUs were commonly inversely correlated with sample input density or not reproduced between technical replicates. Removal of taxa with these features or physical concentration of samples prior to sequencing improved both sequencing accuracy and precision for samples between 8E+ 04 and 8E+ 06 16S copies/ml. For the lowest densities, below 8E+ 03 16S copies/ml BALF, accuracy and precision could not be significantly improved using these approaches. Using clinical BALF samples across a large density range, we observed that OTUs with features of contaminants identified in mock communities were also evident in low-density BALF samples.
Relative abundance data and community composition generated by 16S sequencing of BALF samples across the range of density commonly observed in this sample type should be interpreted in the context of input sample density and may be improved by simple pre- and post-sequencing steps for densities above 8E+ 04 16S copies/ml.
The reuse of domestic and industrial wastewater in urban settings of the developing world may harm the health of people through direct contact or via contaminated urban agricultural products and ...drinking water. We assessed chemical and microbial pollutants in 23 sentinel sites along the wastewater and faecal sludge management and reuse chain of Kampala, Uganda. Water samples were examined for bacteria (thermotolerant coliforms (TTCs),
Escherichia coli
and
Salmonella
spp.) and helminth eggs. Physico-chemical parameters were determined. Water, sediment and soil samples and edible plants (yams and sugar cane) were tested for heavy metals. Water samples derived from the Nakivubo wetland showed mean concentrations of TTCs of 2.9 × 10
5
colony-forming units (CFU)/100 mL. Mean
E. coli
was 9.9 × 10
4
CFU/100 mL. Hookworm eggs were found in 13.5 % of the water samples. Mean concentrations of iron (Fe), copper (Cu) and cadmium (Cd) were 21.5, 3.3 and 0.14 mg/L, respectively. In soil samples, we found a mean lead (Pb) concentration of 132.7 mg/L. In yams, concentrations of Cd, chromium (Cr) and Pb were 4.4, 4.0 and 0.2 mg/L, while the respective concentrations in sugar cane were 8.4, 4.3 and 0.2 mg/L. TTCs and
E. coli
in the water, Pb in soil, and Cd, Cr and Pb in the plants were above national thresholds. We conclude that there is considerable environmental pollution in the Nakivubo wetland and the Lake Victoria ecosystem in Kampala. Our findings have important public health implications, and we suggest that a system of sentinel surveillance is being implemented that, in turn, can guide adequate responses.
Oral and gut microbiomes have emerged as potential biomarkers in cancer. We characterised the oral and gut microbiomes in a prospective observational cohort of HPV+ oropharyngeal squamous cell ...carcinoma (OPSCC) patients and evaluated the impact of chemoradiotherapy (CRT).
Saliva, oropharyngeal swabs over the tumour site and stool were collected at baseline and post-CRT. 16S RNA and shotgun metagenomic sequencing were used to generate taxonomic profiles, including relative abundance (RA), bacterial density, α-diversity and β-diversity.
A total of 132 samples from 22 patients were analysed. Baseline saliva and swabs had similar taxonomic composition (R
= 0.006; p = 0.827). Oropharyngeal swabs and stool taxonomic composition varied significantly by stage, with increased oral RA of Fusobacterium nucleatum observed in stage III disease (p < 0.05). CRT significantly reduced the species richness and increased the RA of gut-associated taxa in oropharyngeal swabs (p < 0.05), while it had no effect in stool samples. These findings remained significant when adjusted by stage, smoking status and antibiotic use.
Baseline oral and gut microbiomes differ by stage in this HPV+ cohort. CRT caused a shift towards a gut-like microbiome composition in oropharyngeal swabs. Stage-specific features and the transitions in oral microbiome might have prognostic and therapeutic implications.
It remains unclear how gastroesophageal reflux disease (GERD) affects allograft microbial community composition in lung transplant recipients and its impact on lung allograft inflammation and ...function.
Our objective was to compare the allograft microbiota in lung transplant recipients with or without clinically diagnosed GERD in the first year after transplant and assess associations between GERD, allograft microbiota, inflammation, and acute and chronic lung allograft dysfunction (ALAD and CLAD).
A total of 268 BAL samples were collected from 75 lung transplant recipients at a single transplant center every 3 months after transplant for 1 year. Ten transplant recipients from a separate transplant center provided samples before and after antireflux Nissen fundoplication surgery. Microbial community composition and density were measured using 16S ribosomal RNA gene sequencing and quantitative polymerase chain reaction, respectively, and inflammatory markers and bile acids were quantified.
We observed a range of allograft community composition with three discernible types (labeled community state types CSTs 1-3). Transplant recipients with GERD were more likely to have CST1, characterized by high bacterial density and relative abundance of the oropharyngeal colonizing genera
and
. GERD was associated with more frequent transitions to CST1. CST1 was associated with lower inflammatory cytokine concentrations than pathogen-dominated CST3 across the range of microbial densities observed. Cox proportional hazard models revealed associations between CST3 and the development of ALAD/CLAD. Nissen fundoplication decreased bacterial load and proinflammatory cytokines.
GERD was associated with a high bacterial density,
and
dominated CST1. CST3, but not CST1 or GERD, was associated with inflammation and early development of ALAD and CLAD. Nissen fundoplication was associated with a reduction in microbial density in BAL fluid samples, especially the CST1-specific genus,
.
Viruses belonging to the Flaviviridae and Bunyaviridae families show considerable genetic diversity. However, this diversity is not necessarily taken into account when developing diagnostic assays, ...which are often based on the pairwise alignment of a limited number of sequences. Our objective was to develop and evaluate a bioinformatics workflow addressing two recurrent issues of molecular assay design: (i) the high intraspecies genetic diversity in viruses and (ii) the potential for cross-reactivity with close relatives.
The workflow developed herein was based on two consecutive BLASTn steps; the first was utilized to select highly conserved regions among the viral taxon of interest, and the second was employed to assess the degree of similarity of these highly-conserved regions to close relatives. Subsequently, the workflow was tested on a set of eight viral species, including various strains from the Flaviviridae and Bunyaviridae families.
The genetic diversity ranges from as low as 0.45% variable sites over the complete genome of the Japanese encephalitis virus to more than 16% of variable sites on segment L of the Crimean-Congo hemorrhagic fever virus. Our proposed bioinformatics workflow allowed the selection-based on computing scores-of the best target for a diagnostic molecular assay for the eight viral species investigated.
Our bioinformatics workflow allowed rapid selection of highly conserved and specific genomic fragments among the investigated viruses, while considering up to several hundred complete genomic sequences. The pertinence of this workflow will increase in parallel to the number of sequences made publicly available. We hypothesize that our workflow might be utilized to select diagnostic molecular markers for higher organisms with more complex genomes, provided the sequences are made available.
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