Biliary Microbiota, Gallstone Disease and Infection with Opisthorchis felineus.

BACKGROUND: There is increasing interest in the microbiome of the hepatobiliary system. This study investigated the influence of infection with the fish-borne liver fluke, Opisthorchis felineus on the biliary microbiome of residents of the Tomsk region of western Siberia. METHODOLOGY/PRINCIPAL FINDINGS: Samples of bile were provided by 56 study participants, half of who were infected with O. felineus, and all of who were diagnosed with gallstone disease. The microbiota of the bile was investigated using high throughput, Illumina-based sequencing targeting the prokaryotic 16S rRNA gene. About 2,797, discrete phylotypes of prokaryotes were detected. At the level of phylum, bile from participants with opisthorchiasis showed greater numbers of Synergistetes, Spirochaetes, Planctomycetes, TM7 and Verrucomicrobia. Numbers of \u3e 20 phylotypes differed in bile of the O. felineus-infected compared to non-infected participants, including presence of species of the genera Mycoplana, Cellulosimicrobium, Microlunatus and Phycicoccus, and the Archaeans genus, Halogeometricum, and increased numbers of Selenomonas, Bacteroides, Rothia, Leptotrichia, Lactobacillus, Treponema and Klebsiella. CONCLUSIONS/SIGNIFICANCE: Overall, infection with the liver fluke O. felineus modified the biliary microbiome, increasing abundance of bacterial and archaeal phylotypes

Hemozoin "knobs" in Opisthorchis felineus infected liver

Background Hemozoin is the pigment produced by some blood-feeding parasites. It demonstrates high diagnostic and therapeutic potential. In this work the formation of co-called hemozoin “knobs” – the bile duct ectasia filled up by hemozoin pigment - in Opisthorhis felineus infected hamster liver has been observed. Methods The O. felineus infected liver was examined by histological analysis and magnetic resonance imaging (MRI). The pigment hemozoin was identified by Fourier transform infrared spectroscopy and high resolution electrospray ionization mass spectrometry analysis. Hemozoin crystals were characterised by high resolution transmission electron microscopy. Results Hemozoin crystals produced by O. felineus have average length 403 nm and the length-to-width ratio equals 2.0. The regurgitation of hemozoin from parasitic fluke during infection leads to formation of bile duct ectasia. The active release of hemozoin from O. felineus during in vitro incubation has also been evidenced. It has been shown that the hemozoin knobs can be detected by magnetic resonance imaging. Conclusions In the paper for the first time the characterisation of hemozoin pigment extracted from liver fluke O. felineus has been conducted. The role of hemozoin in the modification of immune response by opisthorchiasis is assumed

GADD34 Ablation Exacerbates Retinal Degeneration in P23H RHO Mice

The UPR is sustainably activated in degenerating retinas, leading to translational inhibition via p-eIF2α. Recent findings have demonstrated that ablation of growth arrest and DNA damage-inducible protein 34 (GADD34), a protein phosphatase 1 regulatory subunit permitting translational machinery operation through p-eIF2α elevation, does not impact the rate of translation in fast-degenerating rd16 mice. The current study aimed to validate whether P23H RHO mice degenerating at a slower pace manifest translational attenuation and whether GADD34 ablation impacts the rate of retinal degeneration via further suppression of retinal protein synthesis and apoptotic cell death. For this study, mice were examined with ERG and histological analyses. The molecular assessment was conducted in the naïve and LPS-challenged mice using Western blot and qRT-PCR analyses. Thus, this study demonstrates that the P23H RHO retinas manifest translational attenuation. However, GADD34 ablation resulted in a more prominent p-eIF2a increase without impacting the translation rate. GADD34 deficiency also led to a reduction in scotopic ERG amplitudes and an increased number of TUNEL-positive cells. Molecular analysis revealed that GADD34 deficiency reduces the expression of p-STAT3 and Il-6 while increasing the expression of Tnfa. Overall, the data indicate that GADD34 plays a multifunctional role. Under chronic UPR activation, GADD34 acts as a feedback player, dephosphorylating p-eIF2a, although this role does not seem to be critical. Additionally, GADD34 controls cytokine expression and STAT3 activation. Perhaps these molecular events are particularly important in controlling the pace of retinal degeneration

Plasma metabolomics of the time resolved response to Opisthorchis felineus infection in an animal model (golden hamster, Mesocricetus auratus).

BACKGROUND:Opisthorchiasis is a hepatobiliary disease caused by flukes of the trematode family Opisthorchiidae. Opisthorchiasis can lead to severe hepatobiliary morbidity and is classified as a carcinogenic agent. Here we investigate the time-resolved metabolic response to Opisthorchis felineus infection in an animal model. METHODOLOGY:Thirty golden hamsters were divided in three groups: severe infection (50 metacercariae/hamster), mild infection (15 metacercariae/hamster) and uninfected (vehicle-PBS) groups. Each group consisted of equal number of male and female animals. Plasma samples were collected one day before the infection and then every two weeks up to week 22 after infection. The samples were subjected to 1H Nuclear Magnetic Resonance (NMR) spectroscopy and multivariate statistical modelling. PRINCIPAL FINDINGS:The time-resolved study of the metabolic response to Opisthorchis infection in plasma in the main lines agrees with our previous report on urine data. The response reaches its peak around the 4th week of infection and stabilizes after the 10th week. Yet, unlike the urinary data there is no strong effect of the gender in the data and the intensity of infection is presented in the first two principal components of the PCA model. The main trends of the metabolic response to the infection in blood plasma are the transient depletion of essential amino acids and an increase in lipoprotein and cholesterol concentrations. CONCLUSIONS:The time resolved metabolic signature of Opisthorchis infection in the hamster's plasma shows a coherent shift in amino acids and lipid metabolism. Our work provides insight into the metabolic basis of the host response on the helminth infection

1H-NMR analysis of feces: new possibilities in the helminthes infections research

Abstract Background Analysis of the stool samples is an essential part of routine diagnostics of the helminthes infections. However, the standard methods such Kato and Kato-Katz utilize only a fraction of the information available. Here we present a method based on the nuclear magnetic resonance spectroscopy (NMR) which could be auxiliary to the standard procedures by evaluating the complex metabolic profiles (or phenotypes) of the samples. Method The samples were collected over the period of June-July 2015, frozen at −20 °C at the site of collection and transferred within four hours for the permanent storage at −80 °C. Fecal metabolites were extracted by mixing aliquots of about 100 mg thawed stool material with 0.5 mL phosphate buffer saline, followed by the homogenization and centrifugations steps. All NMR data were recorded using a Bruker 600 MHz AVANCE II spectrometer equipped with a 5 mm triple resonance inverse cryoprobe and a z-gradient system. Results Here we report an optimized method for NMR based metabolic profiling/phenotyping of the stools samples. Overall, 62 metabolites were annotated in the pool sample using the 2D NMR spectra and the Bruker Biorefcode database. The compounds cover a wide range of the metabolome including amino acids and their derivatives, short chain fatty acids (SCFAs), carboxylic acids and their derivatives, amines, carbohydrates, purines, alcohols and others. An exploratory analysis of the metabolic profiles reveals no strong trends associated with the infection status of the patients. However, using the penalized regression as a variable selection method we succeeded in finding a subset of eleven variables which enables to discriminate the patients on basis of their infections status. Conclusions A simple method for metabolic profiling/phenotyping of the stools samples is reported and tested on a pilot opisthorchiasis cohort. To our knowledge this is the first report of a NMR-based feces analysis in the context of the helminthic infections

1H-NMR analysis of feces: new possibilities in the helminthes infections research

Backgroun

Biliary Microbiota and Bile Acid Composition in Cholelithiasis

Background. A functional interplay between BAs and microbial composition in gut is a well-documented phenomenon. In bile, this phenomenon is far less studied, and with this report, we describe the interactions between the BAs and microbiota in this complex biological matrix. Methodology. Thirty-seven gallstone disease patients of which twenty-one with Opisthorchis felineus infection were enrolled in the study. The bile samples were obtained during laparoscopic cholecystectomy for gallstone disease operative treatment. Common bile acid composition was measured by LC-MS/MS. Gallbladder microbiota were previously analyzed with 16S rRNA gene sequencing on Illumina MiSeq platform. The associations between bile acid composition and microbiota were analyzed. Results. Bile acid signature and Opisthorchis felineus infection status exert influence on beta-diversity of bile microbial community. Direct correlations were found between taurocholic acid, taurochenodeoxycholic acid concentrations, and alpha-diversity of bile microbiota. Taurocholic acid and taurochenodeoxycholic acid both show positive associations with the presence of Chitinophagaceae family, Microbacterium and Lutibacterium genera, and Prevotella intermedia. Also, direct associations were identified for taurocholic acid concentration and the presence of Actinomycetales and Bacteroidales orders, Lautropia genus, Jeotgalicoccus psychrophilus, and Haemophilus parainfluenzae as well as for taurochenodeoxycholic acid and Acetobacteraceae family and Sphingomonas genus. There were no differences in bile acid concentrations between O. felineus-infected and noninfected patients. Conclusions/Significance. Associations between diversity, taxonomic profile of bile microbiota, and bile acid levels were evidenced in patients with cholelithiasis. Increase of taurochenodeoxycholic acid and taurocholic acid concentration correlates with bile microbiota alpha-diversity and appearance of opportunistic pathogens

Carbonyl stress phenomena during chronic infection with Opisthorchis felineus.

Infection with the fish borne liver fluke Opisthorchis felineus is common in the Eastern Europe (Ukraine, European part of Russia), Northern Asia (Siberia) and Central Asia (Northern Kazakhstan). Better understanding of the molecular pathogenesis of the biliary tract and liver during chronic opisthorchiasis can be expected to improve protection against and management of complications of this disease. We hypothesize that infection with O. felineus associates with formation of methylglyoxal and carbonyl stress in the liver and hence here we investigated the glyoxalase system and the receptor for advanced glycated end products (RAGE) in the liver of hamsters infected with this liver fluke. Expression of mRNA encoding glyoxalase 1 decreased at 8 weeks of the infection and catalytic activity as well decreased at 8 and 12 weeks after infection, and the expression of the glyoxalase 2 decreased until 36 week post infection, which associated with the decreasing activity of the enzyme at 8, 12 week post infection. Glutathione levels in infected livers had decreased at week 8, whereas up-regulation of RAGE at mRNA levels was seen for the extended duration of the experimental infection of the hamsters. This outcome supported the notion of hepatic dicarbonyl stress during chronic opisthorchiasis. The inhibition of the glyoxalase system and accumulation of methylgyloxal at the early stages of the infection may underpin development of insulin resistance during opisthorchiasis