Transcriptome changes of liver fluke Opisthorchis viverrini in diabetic hamsters.

A recent study in hamsters showed that infection with the liver fluke Opisthorchis viverrini in diabetic hosts worsens the severity of hepatobiliary disease. However, the effects of diabetes on the worm's phenotype and gene expression pattern remain unknown. This study investigated the impact of diabetes on the global gene expression and development of O. viverrini in diabetic hamsters. Parasitological parameters were assessed, and mRNA sequencing with bioinformatic analysis was performed. The study revealed that worm establishment rates in diabetic hamsters were directly correlated with fasting plasma glucose levels. Interestingly, worms collected from diabetic hosts exhibited stunted growth and reduced egg production. Transcriptomic analysis revealed significant alterations in gene expression, with 4314 and 567 differentially expressed genes at 21- and 35-days post-infection, respectively. Gene ontology enrichment analysis highlighted changes in biological processes related to stress response, metabolism, and cellular organization. Notably, genes associated with parasite virulence, including granulin, tetraspanins, and thioredoxins, showed significant upregulation in diabetic hosts. These findings demonstrate the profound impact of host diabetic status on O. viverrini development and gene expression, providing insights into the complex interplay between host metabolism and parasite biology, including molecular adaptations of O. viverrini in hosts. This study contributes to our understanding of opisthorchiasis in the context of metabolic disorders and may inform future strategies for disease management in diabetic human populations.

Title: Modifications du transcriptome de la douve du foie Opisthorchis viverrini chez les hamsters diabétiques. Abstract: Une étude récente sur les hamsters a montré que l'infection par la douve du foie Opisthorchis viverrini chez les hôtes diabétiques aggrave la gravité de la maladie hépatobiliaire. Cependant, les effets du diabète sur le phénotype et le profil d'expression génétique du ver restent inconnus. Cette étude a examiné l'impact du diabète sur l'expression génétique globale et le développement d'O. viverrini chez les hamsters diabétiques. Les paramètres parasitologiques ont été évalués et un séquençage de l'ARNm avec analyse bioinformatique a été effectué. L'étude a révélé que les taux d'établissement des vers chez les hamsters diabétiques étaient directement corrélés au taux de glucose plasmatique à jeun. Il est intéressant de noter que les vers récupérés auprès d'hôtes diabétiques présentaient une croissance retardée et une production d'œufs réduite. L'analyse transcriptomique a révélé des altérations significatives de l'expression génétique, avec 4 314 et 567 gènes exprimés de manière différentielle à 21 et 35 jours après l'infection, respectivement. L'analyse d'enrichissement de l'ontologie génétique a mis en évidence des changements dans les processus biologiques liés à la réponse au stress, au métabolisme et à l'organisation cellulaire. Notamment, les gènes associés à la virulence du parasite, en particulier la granuline, les tétraspanines et les thiorédoxines, ont montré une régulation positive significative chez les hôtes diabétiques. Ces résultats démontrent l'impact profond du statut diabétique de l'hôte sur le développement et l'expression génétique d'O. viverrini, offrant un aperçu de l'interaction complexe entre le métabolisme de l'hôte et la biologie du parasite, y compris les adaptations moléculaires d'O. viverrini chez les hôtes. Cette étude contribue à notre compréhension de l'opisthorchiase dans le contexte des troubles métaboliques et peut éclairer les futures stratégies de gestion de la maladie pour les populations humaines diabétiques.

Cannabidiol exhibits potent anti-cancer activity against gemcitabine-resistant cholangiocarcinoma via ER-stress induction in vitro and in vivo.

BACKGROUND: Failure of treatment with gemcitabine in most cholangiocarcinoma (CCA) patients is due to drug resistance. The therapeutic potential of natural plant secondary compounds with minimal toxicity, such as cannabidiol (CBD), is a promising line of investigation in gemcitabine-resistant CCA. We aim to investigate the effects of CBD on gemcitabine-resistant CCA (KKU-213BGemR) cells in vitro and in vivo. MATERIALS: In vitro, cell proliferation, colony formation, apoptosis and cell cycle arrest were assessed using MTT assay, clonogenicity assay and flow cytometry. The effect of CBD on ROS production was evaluated using the DCFH-DA fluorescent probe. The mechanism exerted by CBD on ER stress-associated apoptosis was investigated by western blot analysis. A gemcitabine-resistant CCA xenograft model was also used and the expression of PCNA and CHOP were evaluated by immunohistochemical analysis. RESULTS: The IC50 values of CBD for KKU-213BGemR cells ranged from 19.66 to 21.05 µM. For a non-cancerous immortalized fibroblast cell line, relevant values were 18.29 to 19.21 µM. CBD suppressed colony formation by KKU-213BGemR cells in a dose-dependent manner in the range of 10 to 30 µM. CBD at 30 µM significantly increased apoptosis at early (16.37%) (P = 0.0024) and late (1.8%) stages (P < 0.0001), for a total of 18.17% apoptosis (P = 0.0017), in part by increasing ROS production (P < 0.0001). Multiphase cell cycle arrest significantly increased at G0/G1 with CBD 10 and 20 µM (P = 0.004 and P = 0.017), and at G2/M with CBD 30 µM (P = 0.005). CBD treatment resulted in increased expression of ER stress-associated apoptosis proteins, including p-PERK, BiP, ATF4, CHOP, BAX, and cytochrome c. In xenografted mouse, CBD significantly suppressed tumors at 10 and 40 mg/kg·Bw (P = 0.0007 and P = 0.0278, respectively), which was supported by an increase in CHOP, but a decrease in PCNA expression in tumor tissues (P < 0.0001). CONCLUSION: The results suggest that CBD exhibits potent anti-cancer activity against gemcitabine-resistant CCA in vitro and in vivo, in part via ER stress-mediated mechanisms. These results indicate that clinical explorative use of CBD on gemcitabine-resistant CCA patients is warranted.

Curcumin synergistically enhances the efficacy of gemcitabine against gemcitabine-resistant cholangiocarcinoma via the targeting LAT2/glutamine pathway.

Cholangiocarcinoma (CCA) is often diagnosed late, leading to incomplete tumor removal, drug resistance and reduced chemotherapy efficacy. Curcumin has the potential for anti-cancer activity through various therapeutic properties and can improve the efficacy of chemotherapy. We aimed to investigate the synergistic effect of a combination of curcumin and gemcitabine against CCA, targeting the LAT2/glutamine pathway. This combination synergistically suppressed proliferation in gemcitabine-resistant CCA cells (KKU-213BGemR). It also resulted in a remarkable degree of CCA cell apoptosis and cell cycle arrest, characterized by a high proportion of cells in the S and G2/M phases. Knockdown of SLC7A8 decreased the expressions of glutaminase and glutamine synthetase, resulting in inhibited cell proliferation and sensitized CCA cells to gemcitabine treatment. Moreover, in vivo experiments showed that a combination curcumin and gemcitabine significantly reduced tumor size, tumor growth rate and LAT2 expression in a gemcitabine-resistant CCA xenograft mouse model. Suppression of tumor progression in an orthotopic CCA hamster model provided strong support for clinical application. In conclusion, curcumin synergistically enhances gemcitabine efficacy against gemcitabine-resistant CCA by induction of apoptosis, partly via inhibiting LAT2/glutamine pathway. This approach may be an alternative strategy for the treatment of gemcitabine-resistant in CCA patients.

Metagenomics and metaproteomics alterations are associated with kidney disease in opisthorchiasis hamsters fed a high-fat and high-fructose diet.

BACKGROUND: Opisthorchis viverrini (O. viverrini, Ov) infection and consumption of high-fat and high-fructose (HFF) diet exacerbate liver and kidney disease. Here, we investigated the effects of a combination of O. viverrini infection and HFF diet on kidney pathology via changes in the gut microbiome and host proteome in hamsters. METHODOLOGY/PRINCIPAL FINDINGS: Twenty animals were divided into four groups; 1) fed a normal diet not infected with O. viverrini (normal group), 2) fed an HFF diet and not infected with O. viverrini (HFF), 3) fed a normal diet and infected with O. viverrini (Ov), and 4) fed an HFF diet and infected with O. viverrini (HFFOv). DNA was extracted from fecal samples and the V3-V4 region of the bacterial 16S rRNA gene sequenced on an Illumina MiSeq sequencing platform. In addition, LC/MS-MS analysis was done. Histopathological studies and biochemical assays were also conducted. The results indicated that the HFFOv group exhibited the most severe kidney injury, manifested as elevated KIM-1 expression and accumulation of fibrosis in kidney tissue. The microbiome of the HFFOv group was more diverse than in the HFF group: there were increased numbers of Ruminococcaceae, Lachnospiraceae, Desulfovibrionaceae and Akkermansiaceae, but fewer Eggerthellaceae. In total, 243 host proteins were identified across all groups. Analysis using STITCH predicted that host proteome changes may lead to leaking of the gut, allowing molecules such as soluble CD14 and p-cresol to pass through to promote kidney disease. In addition, differential expression of TGF-beta-activated kinase 1 and MAP3K7-binding protein 2 (Tab2, involving renal inflammation and injury) are predicted to be associated with kidney disease. CONCLUSIONS/SIGNIFICANCE: The combination of HFF diet and O. viverrini infection may promote kidney injury through alterations in the gut microbiome and host proteome. This knowledge may suggest an effective strategy to prevent kidney disease beyond the early stages.

Overexpression of microRNA-205-5p promotes cholangiocarcinoma growth by reducing expression of homeodomain-interacting protein kinase 3.

The microRNA miR-205-5p has diverse effects in different malignancies, including cholangiocarcinoma (CCA), but its effects on CCA progression is unclear. Here we investigated the role and function of miR-205-5p in CCA. Three CCA cell lines and human serum samples were found to have much higher expression levels of miR-205-5p than seen in typical cholangiocyte cell lines and healthy controls. Inhibition of miR-205-5p suppressed CCA cell motility, invasion and proliferation of KKU-213B whereby overexpression of miR-205-5p promoted cell proliferation and motility of KKU-100 cells. Bioinformatics tools (miRDB, TargetScan, miRWalk, and GEPIA) all predicted various miR-205-5p targets. Experiments using miR-205-5p inhibitor and mimic indicated that homeodomain-interacting protein kinase 3 (HIPK3) was a potential direct target of miR-205-5p. Overexpression of HIPK3 using HIPK3 plasmid cloning DNA suppressed migration and proliferation of KKU-100 cells. Notably, HIPK3 expression was lower in human CCA tissues than in normal adjacent tissues. High HIPK3 expression was significantly associated with longer survival time of CCA patients. Multivariate regression analysis indicated tissue HIPK3 levels as an independent prognostic factor for CCA patients. These findings indicate that overexpression of miR-205-5p promotes CCA cells proliferation and migration partly via HIPK3-dependent way. Therefore, targeting miR-205-5p may be a potential treatment approach for CCA.

Slight Changes in the Gut Microbiome in Early-stage Chronic Kidney Disease of Unknown Etiology.

Gut dysbiosis and changes in short-chain fatty acids (SCFAs) occur in end-stage chronic kidney disease (CKD); however, the degree of these changes in the gut microbiome and serum SCFA profiles in the early stages of CKD,| |particularly in| |CKD| |of unknown etiology (CKDu), is unclear. We herein investigated the gut microbiome and SCFA profiles of early-stage CKD patients (CKD stages 1-3) in a community in Khon Kaen Province, Thailand. Seventy-two parasite-free participants were distributed among a healthy control group (HC, n=18) and three patient groups (an underlying disease group [UD, n=18], early-stage CKD with underlying disease [CKD-UD, n=18], and early-stage CKD of unknown etiology, [CKDu, n=18]). Fecal DNA was individually extracted and pooled for groups of six individuals (three pools in each group) to examine the composition of the gut microbiome using next-generation sequencing. A SCFA ana-lysis was performed on serum samples from each individual using gas chromatography-mass spectrometry. The results revealed that microbial abundance differed between the healthy group and all patient groups (UD, CKD-UD, and CKDu). [Eubacterium]_coprostanoligenes_group was more abundant in the CKDu group than in the HC and CKD-UD groups. Furthermore, serum concentrations of acetate, a major SCFA component, were significantly lower in all patient groups than in the HC group. The present results indicate that minor changes in the gut microbiome and a significant decrease in serum acetate concentrations occur in early-stage CKDu, which may be important for the development of prevention strategies for CKD patients.

Strongyloides stercoralis infection induces gut dysbiosis in chronic kidney disease patients.

BACKGROUND: Strongyloides stercoralis infection typically causes severe symptoms in immunocompromised patients. This infection can also alter the gut microbiota and is often found in areas where chronic kidney disease (CKD) is common. However, the relationship between S. stercoralis and the gut microbiome in chronic kidney disease (CKD) is not understood fully. Recent studies have shown that gut dysbiosis plays an important role in the progression of CKD. Hence, this study aims to investigate the association of S. stercoralis infection and gut microbiome in CKD patients. METHODOLOGY/PRINCIPAL FINDINGS: Among 838 volunteers from Khon Kaen Province, northeastern Thailand, 40 subjects with CKD were enrolled and divided into two groups (S. stercoralis-infected and -uninfected) matched for age, sex and biochemical parameters. Next-generation technology was used to amplify and sequence the V3-V4 region of the 16S rRNA gene to provide a profile of the gut microbiota. Results revealed that members of the S. stercoralis-infected group had lower gut microbial diversity than was seen in the uninfected group. Interestingly, there was significantly greater representation of some pathogenic bacteria in the S. stercoralis-infected CKD group, including Escherichia-Shigella (P = 0.013), Rothia (P = 0.013) and Aggregatibacter (P = 0.03). There was also a trend towards increased Actinomyces, Streptococcus and Haemophilus (P > 0.05) in this group. On the other hand, the S. stercoralis-infected CKD group had significantly lower representation of SCFA-producing bacteria such as Anaerostipes (P = 0.01), Coprococcus_1 (0.043) and a non-significant decrease of Akkermansia, Eubacterium rectale and Eubacterium hallii (P > 0.05) relative to the uninfected group. Interesting, the genera Escherichia-Shigella and Anaerostipes exhibited opposing trends, which were significantly related to sex, age, infection status and CKD stages. The genus Escherichia-Shigella was significantly more abundant in CKD patients over the age of 65 years and infected with S. stercoralis. A correlation analysis showed inverse moderate correlation between the abundance of the genus of Escherichia-Shigella and the level of estimated glomerular filtration rate (eGFR). CONCLUSIONS/SIGNIFICANCE: Conclusion, the results suggest that S. stercoralis infection induced gut dysbiosis in the CKD patients, which might be involved in CKD progression.

Curcumin-loaded nanocomplexes ameliorate the severity of nonalcoholic steatohepatitis in hamsters infected with Opisthorchis viverrini.

BACKGROUND: Comorbidity of Opisthorchis viverrini (OV) infection and nonalcoholic fatty-liver disease (NAFLD) enhances NAFLD progression to nonalcoholic steatohepatitis (NASH) by promoting severe liver inflammation and fibrosis. Here, we investigated the effect of supplementation with curcumin-loaded nanocomplexes (CNCs) on the severity of NASH in hamsters. METHODOLOGY: Hamsters were placed in experimental groups as follows: fed standard chow diet (normal control, NC); fed only high-fat and high-fructose (HFF) diet; O. viverrini-infected and fed HFF diet (HFFOV); group fed with blank nanocomplexes (HFFOV+BNCs); groups fed different doses of CNCs (25, 50 and 100 mg/kg body weight: HFFOV+CNCs25; HFFOV+CNCs50; HFFOV+CNCs100, respectively) and a group given native curcumin (HFFOV+CUR). All treatment were for three months. RESULTS: The HFF group revealed NAFLD as evidenced by hepatic fat accumulation, ballooning, mild inflammation and little or no fibrosis. These changes were more obvious in the HFFOV group, indicating development of NASH. In contrast, in the HFFOV+CNCs50 group, histopathological features indicated that hepatic fat accumulation, cell ballooning, cell inflammation and fibrosis were lower than in other treatment groups. Relevantly, the expression of lipid-uptake genes, including fatty-acid uptake (cluster of differentiation 36), was reduced, which was associated with the lowering of alanine aminotransferase, total cholesterol and triglyceride (TG) levels. Reduced expression of an inflammation marker (high-mobility group box protein 1) and a fibrosis marker (alpha smooth-muscle actin) were also observed in the HFFOV+CNCs50 group. CONCLUSION: CNCs treatment attenuates the severity of NASH by decreasing hepatic steatosis, inflammation, and fibrosis as well as TG synthesis. CNCs mitigate the severity of NASH in this preclinical study, which indicates promise for future use in patients.

Investigation of gut microbiota and short-chain fatty acids in Strongyloides stercoralis-infected patients in a rural community.

Intestinal parasitic infections can change gut microbiota and short-chain fatty acids (SCFAs). We aimed to study the interaction among Strongyloides stercoralis, human gut microbiota, and serum SCFAs in a community. Fifty-two subjects in Donchang sub-district, Khon Kaen Province, northeastern Thailand, were included based on specific inclusion and exclusion criteria. Characteristics of the participants were matched between those positive for S. stercoralis infection alone (no other intestinal parasites; Ss+, n=26) and uninfected controls (infection status confirmed by polymerase chain reaction (PCR); Ss-, n=26). Serum short-chain fatty acids were evaluated by gas chromatography-mass spectrometry. DNA was extracted from individual faecal samples and then pooled into two groups (Ss+ and Ss-) for amplification and sequencing of the V3-V4 region of the 16S gene with next-generation technology. We explored the impact of infection with S. stercoralis on the faecal microbiota: individuals infected with this parasite exhibited increased alpha diversity of bacteria. At the genus level, gut microbiota in Ss+ patients showed high abundances of Escherichia-Shigella and Bacteroides but low abundances of the genera Bifidobacterium, Lactobacillus, and Blautia. PCR of individual samples to identify certain species of interest gave results consistent with those from next-generation sequencing of pooled samples and showed that significantly more Ss+ samples contained Bacteroides fragilis. Intriguingly, a major SCFA, acetic acid, was significantly decreased in S. stercoralis infection. In conclusion, S. stercoralis infection caused an imbalance of gut microbiota and decreased acetic acid in serum. This information adds to the knowledge concerning the effect of intestinal nematode-related chronic diseases.

Opisthorchis viverrini infection induces metabolic disturbances in hamsters fed with high fat/high fructose diets: Implications for liver and kidney pathologies.

A combination of Opisthorchis viverrini infection and high fat/high fructose diets (HFa/HFr) intake is likely to enhance fatty liver and kidney pathologies. Here we investigated the combined effects of chronic O. viverrini infection and HFa/HFr intake on liver and kidney pathologies, metabolism, and gut microbiome in hamsters. Animals were infected with O. viverrini and fed with either standard chow (OV group) or HFa/HFr diet (OH group) and non-infected hamsters were fed with either standard chow (NC) or HFa/HFr diet (HF) for 8 months. The OH group exhibited dyslipidemia and the highest severity of fatty liver. Tubular damage, inflammatory cell infiltration, and tubular fibrosis were the most prominently observed in this group, supported by increased expression of KIM-1, HMGB-1, and MCP-1. Urinary 1H NMR metabolic profiles revealed that tauro-ß-muricholic acid level was increased in the OV and OH groups, whereas metabolites involved in the TCA cycle and gut microbiota-associated metabolites (phenylacetylglycine, trimethylamine, and trimethylamine-N-oxide) were lower in OV, HF and OH groups compared to the NC group. Gut microbial profiles of the OH group were also different from other groups. In conclusion, O. viverrini infection and HFa/HFr diet-induced disturbance of metabolites and gut microbiota associated with concurrent liver and kidney pathologies in hamsters.

Opisthorchis viverrini Infection Induces Metabolic and Fecal Microbial Disturbances in Association with Liver and Kidney Pathologies in Hamsters.

Opisthorchis viverrini (Ov) infection causes hepatobiliary diseases and is a major risk factor for cholangiocarcinoma. While several omics approaches have been employed to understand the pathogenesis of opisthorchiasis, effects of Ov infection on the host systemic metabolism and fecal microbiota have not been fully explored. Here, we used a 1H NMR spectroscopy-based metabolic phenotyping approach to investigate Ov infection-induced metabolic disturbances at both the acute (1 month postinfection, 1 mpi) and chronic (4 mpi) stages in hamsters. A total of 22, 3, and 4 metabolites were found to be significantly different in the liver, serum, and urine, respectively, between Ov+ and Ov- groups. Elevated levels of hepatic amino acids and tricarboxylic acid (TCA)-cycle intermediates (fumarate and malate) were co-observed with liver injury in acute infection, whereas fibrosis-associated metabolites (e.g., glycine and glutamate) increased at the chronic infection stage. Lower levels of lipid signals ((CH2)n and CH2CH2CO) and higher levels of lysine and scyllo-inositol were observed in serum from Ov+ hamsters at 1 mpi compared to Ov- controls. Urinary levels of phenylacetylglycine (a host-bacterial cometabolite) and tauro-ß-muricholic acid were higher in the Ov+ group, which coexisted with hepatic and mild kidney fibrosis. Furthermore, Ov+ animals showed higher relative abundances of fecal Methanobrevibacter (Archaea), Akkermansia, and Burkholderia-Paraburkholderia compared to the noninfected controls. In conclusion, along with liver and kidney pathologies, O. viverrini infection resulted in hepatic and mild renal pathologies, disturbed hepatic amino acid metabolism and the TCA cycle, and induced changes in the fecal microbial composition and urinary host-microbial cometabolism. This study provides the initial step toward an understanding of local and systemic metabolic responses of the host to O. viverrini infection.

CagA+ Helicobacter pylori infection and N-nitrosodimethylamine administration induce cholangiocarcinoma development in hamsters.

BACKGROUND: Helicobacter pylori (HP) has been detected in the hepatobiliary tract of cholangiocarcinoma (CCA) patients in regions both endemic and non-endemic for Opisthorchis viverrini (OV) infection. However, whether H. pylori infection promotes CCA development remains unknown. We investigated CCA development in hamsters induced by a combination of infection with H. pylori and administration of N-nitrosodimethylamine (NDMA) and compared findings with those in an OV plus NDMA group. MATERIALS AND METHODS: Eighty-five hamsters were divided into four groups: (1) normal, (2) administered NDMA, (3) infected with cagA+ H. pylori and administered NDMA (HN group), and (4) infected with OV and administered NDMA (ON group). Animals were euthanized at 3 and 6 months post-infection. Histopathological changes of liver and the expression of markers associated with carcinogenesis were studied. RESULTS: At 3 months post-infection (p.i.), cholangitis and lymphoid follicles without tumor appearance were noted in the HN group, whereas extensive fibrosis was seen in members of the ON group, 10% of which had developed tumors. At 6 months p.i., 10% of hamsters administered NDMA alone had developed CCA, whereas in the HN and ON groups, 20% and 60% of hamsters, respectively, had developed CCA. Cytokeratin-19 (CK19) expression was observed in the CCA tissues of both the HN and the ON groups, confirming the bile duct origin of the CCA cells. CCA development in the HN group might be inflammation-mediated, as suggested by overexpression of HMGB1, PCNA, IL-8, and 8-OxodG in CCA tissues. CONCLUSION: cagA+ H. pylori infection and carcinogen intake can induce CCA development with slow progression.

A combination of monosodium glutamate and high-fat and high-fructose diets increases the risk of kidney injury, gut dysbiosis and host-microbial co-metabolism.

Consumption of either monosodium glutamate (MSG) or high-fat and high-fructose (HFF) diets changes the gut microbiome and hence contributes to development of several diseases. In this study, with an emphasis on kidney injury, hamsters were divided into 4 groups as follows: (1) hamsters fed with standard diet (control); (2) hamsters fed with standard diet and MSG in drinking water (MSG); (3) hamsters fed with high-fat and high-fructose diets (HFF), and (4) animals fed MSG+HFF. After 8 months, the animals were used for the study. Despite showing normal kidney function, hamsters fed with MSG+HFF exhibited signs of kidney damage as demonstrated by the highest expression levels of high-mobility group box-1 and kidney injury molecule-1 in kidney tissues, while slight changes of histopathological features in H&E-stained sections and normal levels of creatinine were observed, indicating possible early stages of kidney injury. Sequencing of the microbial 16S rRNA gene revealed that animals fed with the MSG+HFF diet had a higher ratio of gut Firmicutes/Bacteroidetes along with marked changes in abundance and diversity of gut microbiome compared to hamsters fed with MSG or HFF alone. In addition, 1H Nuclear magnetic resonance spectroscopy showed an elevation of urine p-cresol sulfate levels in the MSG+HFF group. These results indicate that consumption of both MSG and HFF increases the risk of kidney injury, induces gut dysbiosis and an increase in the amount of p-cresol sulfate in hamsters.