Aloe QDM complex enhances specific cytotoxic T lymphocyte killing in vivo in metabolic disease mice.

We developed spontaneous diet-induced metabolic disease in mice by feeding them a high-fat diet for 23 weeks and administered Aloe QDM complex for 16 weeks to examine its restorative effect on immune disorders and metabolic syndrome. A series of immune functional assays indicated Aloe QDM complex enhanced lymphocyte proliferation and antigen-specific immunity as determined by the restored functions of cytotoxic T lymphocytes (CTL) and IgG production. The elevated serum TNF-α level was also regulated by Aloe QDM complex treatment, which suggested its complex therapeutic potential. As for metabolic phenotypes, oral administration of Aloe QDM complex significantly improved diabetic symptoms, including high fasting glucose levels and glucose tolerance, and distinctly alleviated lipid accumulation in adipose and hepatic tissue. The simultaneous restoration of Aloe QDM complex on metabolic syndrome and host immune dysfunction, especially on the specific CTL killing was first elucidated in our study.

Atorvastatin and rosuvastatin improve physiological parameters and alleviate immune dysfunction in metabolic disorders.

This study was designed to characterize the potential therapeutic effects of two statin drugs commonly used to treat dyslipidemia in inflammation-linked metabolic disorders related to type 2 diabetes. Atorvastatin (10 mg/kg/day) and rosuvastatin (3 mg/kg/day) were administered to mice with diet-induced obesity (DIO). The statins lowered serum total and LDL cholesterol levels, and improved the atherogenic index and cardiac risk index. Furthermore, the drugs decreased fasting glucose levels, improved glucose tolerance, and decreased fat tissue weight and adipocyte size; this was accompanied by an overall body weight loss tendency. The statins also improved antigen-specific immunity. The killing activity of cytotoxic T cells and exacerbation of IgG secretion levels were considerably normalized. Most importantly, serum tumor necrosis factor-α and interleukin 6 levels decreased, while their RNA expression levels in fat tissue were regulated by the statins as well. This study is the first to indicate that low doses of atorvastatin and rosuvastatin, the dosing regimen for which has been controversial, could significantly improve diabetes-related metabolic disorders, and could modulate pro-inflammatory cytokines, alleviating inflammation and simultaneously restoring overall humoral and cell-mediated immunity.

Modified Aloe Polysaccharide Restores Chronic Stress-Induced Immunosuppression in Mice.

Chronic stress generally experienced in our daily lives; is known to augment disease vulnerability by suppressing the host immune system. In the present study; the effect of modified Aloe polysaccharide (MAP) on chronic stress-induced immunosuppression was studied; this Aloe compound was characterized in our earlier study. Mice were orally administered with MAP for 24 days and exposed to electric foot shock (EFS; duration; 3 min; interval; 10 s; intensity; 2 mA) for 17 days. The stress-related immunosuppression and restorative effect of MAP were then analyzed by measuring various immunological parameters. MAP treatment alleviated lymphoid atrophy and body weight loss. The numbers of lymphocyte subsets were significantly normalized in MAP-treated mice. Oral administration of MAP also restored the proliferative activities of lymphocytes; ovalbumin (OVA)-specific T cell proliferation; antibody production; and the cell killing activity of cytotoxic T lymphocytes. In summary; oral administration of MAP ameliorated chronic EFS stress-induced immunosuppression.

Effect of metformin on metabolic improvement and gut microbiota.

Metformin is commonly used as the first line of medication for the treatment of metabolic syndromes, such as obesity and type 2 diabetes (T2D). Recently, metformin-induced changes in the gut microbiota have been reported; however, the relationship between metformin treatment and the gut microbiota remains unclear. In this study, the composition of the gut microbiota was investigated using a mouse model of high-fat-diet (HFD)-induced obesity with and without metformin treatment. As expected, metformin treatment improved markers of metabolic disorders, including serum glucose levels, body weight, and total cholesterol levels. Moreover, Akkermansia muciniphila (12.44%±5.26%) and Clostridium cocleatum (0.10%±0.09%) abundances increased significantly after metformin treatment of mice on the HFD. The relative abundance of A. muciniphila in the fecal microbiota was also found to increase in brain heart infusion (BHI) medium supplemented with metformin in vitro. In addition to the changes in the microbiota associated with metformin treatment, when other influences were controlled for, a total of 18 KEGG metabolic pathways (including those for sphingolipid and fatty acid metabolism) were significantly upregulated in the gut microbiota during metformin treatment of mice on an HFD. Our results demonstrate that the gut microbiota and their metabolic pathways are influenced by metformin treatment.

Chemokine CCL6 Plays Key Role in the Inhibitory Effect of Vitamin A on Norovirus Infection.

Norovirus (NoV) is the most common viral cause of acute gastroenteritis worldwide. Vitamin A has demonstrated the potential to protect against gastrointestinal infections. However, the effects of vitamin A on human norovirus (HuNoV) infections remain poorly understood. This study aimed to investigate how vitamin A administration affects NoV replication. We demonstrated that treatment with retinol or retinoic acid (RA) inhibited NoV replication in vitro based on their effects on HuNoV replicon-bearing cells and murine norovirus-1 (MNV-1) replication in murine cells. MNV replication in vitro showed significant transcriptomic changes, which were partially reversed by retinol treatment. RNAi knockdown of CCL6, a chemokine gene that was downregulated by MNV infection but upregulated by retinol administration, resulted in increased MNV replication in vitro. This suggested a role of CCL6 in the host response to MNV infections. Similar gene expression patterns were observed in the murine intestine after oral administration of RA and/or MNV-1.CW1. CCL6 directly decreased HuNoV replication in HG23 cells, and might indirectly regulate the immune response against NoV infection. Finally, relative replication levels of MNV-1.CW1 and MNV-1.CR6 were significantly increased in CCL6 knockout RAW 264.7 cells. This study is the first to comprehensively profile transcriptomes in response to NoV infection and vitamin A treatment in vitro, and thus may provide new insights into dietary prophylaxis and NoV infections.

Elevated post-transfusion serum transaminase values associated with a highly significant trend for increasing prevalence of anti-Vesivirus antibody in Korean patients.

A highly significant increase in anti-Vesivirus (family Caliciviridae) antibody prevalence, along the axis from healthy blood donors; donors with elevated transaminase; patients with clinical hepatitis; and patients with post-transfusion/dialysis hepatitis, has been reported in human sera from the USA and Europe. Asian samples have now been tested retrospectively using serology and enzyme immunoassay (EIA) with a Vesivirus partial-capsid antigen expressed as a fusion protein. Anti-vesiviral antibodies were measured by optical densities (OD(650)) and compared in patients separated by age, gender and Groups A-F as follows: Control Group A, an Experimental Group B, which was divided further into Group C, patients with elevated enzymes (alanine transaminase (ALT), aspartate transaminase (AST), and γ-glutamyl transpeptidase (γ-GT); Group D, patients receiving transfused blood; Group E, patients with high enzyme levels after transfusion; and Group F, hepatitis B and C positive patients. Using multivariate logistic regression analyses, a significantly greater proportion of patients receiving transfusion(s), were positive for anti-Vesivirus antibody compared with non-transfused patients (P = 0.008; OR: 3.86, 95% CI: 1.43-10.43). Also, anti-Vesivirus antibody was significantly associated with elevated biochemical liver function tests: ALT ≥ 20 IU or AST ≥ 120 IU (P = 0.017; OR: 4.23, 95% CI: 1.30-13.80). In the blood transfusion group, anti-Vesivirus antibody was significantly correlated with high enzyme levels (ALT, P = 0.018; AST, P = 0.010; γ-GT, P = 0.020). These data provide serologic evidence of vesiviral transfusion-transmission-associated disease, which could include infection of any organ system where cytopathology resulted in high levels of either ALT or AST.

A Novel Bacterium, Butyricimonas virosa, Preventing HFD-Induced Diabetes and Metabolic Disorders in Mice via GLP-1 Receptor.

Knowledge of the impact of the gut microbiota on human health has increased, and modulation of the bacterial community is now considered a therapeutic target for various diseases. Certain novel bacterial species have probiotic properties associated with improvement in obesity and related metabolic disorders. The relative abundance of Butyricimonas spp. is correlated with metabolic parameters; however, the physiological role of Butyricimonas in metabolic improvement is unclear. In this study, live and heat-killed Butyricimonas virosa were administered to mice with high-fat diet (HFD)-induced obesity. Both live and heat-killed B. virosa ameliorated HFD-impaired body weight, serum glucose level, insulin resistance, and liver steatosis. Moreover, activation of the glucagon-like peptide-1 receptor (GLP-1R) and peroxisome proliferator-activated receptor α (PPARα) was observed in the liver, and the expression levels of insulin receptor substrate (IRS)-1, IRS-2, Toll-like receptor 5 (TLR5), and zonula occludens-1 (ZO-1) were upregulated in the ileum. Finally, we demonstrated that the effect of B. virosa treatment on glucose regulation may be linked to the upregulation of GLP-1R in the liver and is not a result of colonization of the gut by B. virosa or B. virosa-produced butyrate. Our results provide a rationale for the development of Butyricimonas spp.-based therapeutics and prophylactics for hyperglycemia.

Evaluation of electropositive filtration for recovering norovirus in water.

The virus adsorption-elution (VIRADEL) technique has been widely used in the recovery of various enteric viruses in water, and an electropositive filter such as 1 MDS has been commonly applied. However, effective methods of monitoring waterborne norovirus (NoV) have not yet been well characterized and optimized. Hence, in this study, the VIRADEL technique was evaluated and optimized for effectively detecting NoV in water by two commonly used electropositive filters (1MDS and NanoCeram filter). Various elution and concentration methods were evaluated by using both murine norovirus (MNV) and human NoV. Among the tested elution buffers, the most effective was 1.5% beef extract plus 0.01% Tween 80 for both 1MDS (67.5%) and NanoCeram (85.7%) microfilters. The recovery rate of GII-4 human NoV was higher by organic flocculation (86.6%) than by polyethylene glycol (PEG) precipitations (11.6~73.6%). When both 1MDS and NanoCeram filters were tested to detect NoV in surface and groundwater, the sensitivity of NoV recovered by these filters appeared to depend on the types and conditions of environmental water. The results of this study will help to set a standard of detection method for NoV in water.

Modulation of Pro-inflammatory and Anti-inflammatory Cytokines in the Fat by an Aloe Gel-based Formula, QDMC, Is Correlated with Altered Gut Microbiota.

Abnormal inflammatory responses are closely associated with intestinal microbial dysbiosis. Oral administration of Qmatrix-diabetes-mellitus complex (QDMC), an Aloe gel-based formula, has been reported to improve inflammation in type 2 diabetic mice; however, the role of the gut microbiota in ameliorating efficacy of QDMC remains unclear. We investigated the effect of QDMC on the gut microbiota in a type 2 diabetic aged mouse model that was administered a high-fat diet. Proinflammatory (TNF-α and IL-6) and anti-inflammatory (IL-4 and IL-10) cytokine levels in the fat were normalized via oral administration of QDMC, and relative abundances of Bacteroides, Butyricimonas, Ruminococcus, and Mucispirillum were simultaneously significantly increased. The abundance of these bacteria was correlated to the expression levels of cytokines. Our findings suggest that the immunomodulatory activity of QDMC is partly mediated by the altered gut microbiota composition.

Molecular characterization of bacteriophages for microbial source tracking in Korea.

We investigated coliphages from various fecal sources, including humans and animals, for microbial source tracking in South Korea. Both somatic and F+-specific coliphages were isolated from 43 fecal samples from farms, wild animal habitats, and human wastewater plants. Somatic coliphages were more prevalent and abundant than F+ coliphages in all of the tested fecal samples. We further characterized 311 F+ coliphage isolates using RNase sensitivity assays, PCR and reverse transcription-PCR, and nucleic acid sequencing. Phylogenetic analyses were performed based on the partial nucleic acid sequences of 311 F+ coliphages from various sources. F+ RNA coliphages were most prevalent among geese (95%) and were least prevalent in cows (5%). Among the genogroups of F+ RNA coliphages, most F+ coliphages isolated from animal fecal sources belonged to either group I or group IV, and most from human wastewater sources were in group II or III. Some of the group I coliphages were present in both human and animal source samples. F+ RNA coliphages isolated from various sources were divided into two main clusters. All F+ RNA coliphages isolated from human wastewater were grouped with Qbeta-like phages, while phages isolated from most animal sources were grouped with MS2-like phages. UniFrac significance statistical analyses revealed significant differences between human and animal bacteriophages. In the principal coordinate analysis (PCoA), F+ RNA coliphages isolated from human waste were distinctively separate from those isolated from other animal sources. However, F+ DNA coliphages were not significantly different or separate in the PCoA. These results demonstrate that proper analysis of F+ RNA coliphages can effectively distinguish fecal sources.

Alterations in Gut Microbiota by Statin Therapy and Possible Intermediate Effects on Hyperglycemia and Hyperlipidemia.

Dysbiosis of the gut microbiota is a contributing factor for obesity-related metabolic diseases such as hyperglycemia and hyperlipidemia. Pharmacotherapy for metabolic diseases involves the modulation of gut microbiota, which is suggested to be a potential therapeutic target. In this study, the modulation of gut microbiota by statins (cholesterol-lowering drugs: atorvastatin and rosuvastatin) was investigated in an aged mouse model of high-fat diet-induced obesity, and the association between gut microbiota and immune responses was described. Atorvastatin and rosuvastatin significantly increased the abundance of the genera Bacteroides, Butyricimonas, and Mucispirillum. Moreover, the abundance of these genera was correlated with the inflammatory response, including levels of IL-1ß and TGFß1 in the ileum. In addition, oral fecal microbiota transplantation with fecal material collected from rosuvastatin-treated mouse groups improved hyperglycemia. From these results, the effect of statins on metabolic improvements could be explained by altered gut microbiota. Our findings suggest that the modulation of gut microbiota by statins has an important role in the therapeutic actions of these drugs.

Downregulation of IL-18 Expression in the Gut by Metformin-induced Gut Microbiota Modulation.

IL-18 is a crucial pro-inflammatory cytokine that mediates chronic intestinal inflammation. Metformin, an anti-diabetic drug, was reported to have ameliorative effects on inflammatory bowel disease. Recently, the mechanism of action of metformin was explained as a modulation of gut microbiota. In this study, fecal microbiota transplantation (FMT) using fecal material from metformin-treated mice was found to upregulate the expression of GLP-1 and pattern-recognition receptors TLR1 and TLR4 for the improvement in hyperglycemia caused by a high-fat diet. Further, FMT downregulated the expression of the inflammatory cytokine IL-18. Within the genera Akkermansia, Bacteroides, and Butyricimonas, which were promoted by metformin therapy, Butyricimonas was found to be consistently abundant following FMT. Our findings suggest that modulation of gut microbiota is a key factor for the anti-inflammatory effects of metformin which is used for the treatment of hyperglycemia.

Modulation of the gut microbiota by metformin improves metabolic profiles in aged obese mice.

The gut microbiota is a contributing factor in obesity-related metabolic disorders. The effect of metformin on the gut microbiota has been reported; however, the relationship between the gut microbiota and the mechanism of action of metformin in elderly individuals is unclear. In this study, the effect of metformin on the gut microbiota was investigated in aged obese mice. The abundance of the genera Akkermansia, Bacteroides, Butyricimonas, and Parabacteroides was significantly increased by metformin in mice fed a high-fat diet. Metformin treatment decreased the expression of IL-1ß and IL-6 in epididymal fat, which was correlated with the abundance of various bacterial genera. In addition, both fecal microbiota transplantation from metformin-treated mice and extracellular vesicles of Akkermansia muciniphila improved the body weight and lipid profiles of the mice. Our findings suggest that modulation of the gut microbiota by metformin results in metabolic improvements in aged mice, and that these effects are associated with inflammatory immune responses.

Multifaceted Effect of Rubus Occidentalis on Hyperglycemia and Hypercholesterolemia in Mice with Diet-Induced Metabolic Diseases.

Metabolic syndrome is characterized by a combination of several metabolic disorders, including obesity, hyperglycemia, and hyperlipidemia. A simultaneous occurrence is one of the most crucial features of metabolic syndrome; therefore, we selected an animal model in which this would be reflected. We fed C57BL/6N mice a high-fat diet for 23 weeks to develop metabolic syndrome and examined the efficacy of Rubus occidentalis (RO) for hyperglycemia and hypercholesterolemia. Oral administration of RO for 16 weeks improved hyperglycemia as indicated by significantly decreased fasting glucose levels and a glucose tolerance test. Improvements were also observed in hypercholesterolemia, in which significant decreases in serum total cholesterol, non-high-density lipoprotein (non-HDL) cholesterol, apolipoprotein A-1, and apolipoprotein B levels were observed. The time comparison of major biomarkers, observed at the initiation and termination of the experimental period, consistently supported the beneficial effects of RO on each metabolic phenotype. In addition, RO treatment attenuated the excessive fat accumulation in hepatic and adipose tissue by decreasing the size and number of lipid droplets. These results suggested that RO simultaneously exerted antihyperglycemic and antihyperlipidemic effects in mice with diet-induced metabolic syndrome.

New perspectives regarding the antiviral effect of vitamin A on norovirus using modulation of gut microbiota.

Gut microbiota has been revealed to play an important role in various health conditions, and recent studies have suggested the modulation of gut microbiota as a therapeutic strategy. There is no effective treatment of norovirus infection, though vitamin A has been suggested to have an antiviral effect in an epidemiological study. We demonstrated that vitamin A significantly inhibited murine norovirus replication. Vitamin A supplementation significantly increased the abundance of Lactobacillus sp. during norovirus infection, which played a crucial role in antiviral efficacy, inhibiting murine norovirus. Therefore, we elaborated the antiviral effect of vitamin A via modulation of gut microbiota. Furthermore, we suggest a novel strategy, using potential probiotics, as having a protective and therapeutic effect on noroviral infection.

Restoration of Declined Immune Responses and Hyperlipidemia by Rubus occidenalis in Diet-Induced Obese Mice.

Hyperlipidemia, which is closely associated with a fatty diet and aging, is commonly observed in the western and aged society. Therefore, a novel therapeutic approach for this disease is critical, and an immunological view has been suggested as a novel strategy, because hyperlipidemia is closely associated with inflammation and immune dysfunction. In this study, the effects of an aqueous extract of Rubus occidentalis (RO) in obese mice were investigated using immunological indexes. The mice were fed a high-fat diet (HFD) to induce hyperlipidemia, which was confirmed by biochemical analysis and examination of the mouse physiology. Two different doses of RO and rosuvastatin, a cholesterol synthesis inhibitor used as a control, were orally administered. Disturbances in immune cellularity as well as lymphocyte proliferation and cytokine production were significantly normalized by oral administration of RO, which also decreased the elevated serum tumor necrosis factor (TNF)-α level and total cholesterol. The specific immune-related actions of RO comprised considerable improvement in cytotoxic T cell killing functions and regulation of antibody production to within the normal range. The immunological evidence confirms the significant cholesterol-lowering effect of RO, suggesting its potential as a novel therapeutic agent for hyperlipidemia and associated immune decline.

Antiviral effect of vitamin A on norovirus infection via modulation of the gut microbiome.

The effect and underlying mechanism of vitamin A on norovirus infection are largely unknown. This study aimed to investigate how vitamin A administration affects the gut microbiome after norovirus infection. Here, we demonstrate that treatment with either retinol or retinoic acid (RA) inhibits murine norovirus (MNV) replication using both in vitro and in vivo models. Compositional changes in the gut microbiome associated with RA administration and/or norovirus infection were also investigated. Oral administration of RA and/or MNV significantly altered intestinal microbiome profiles. Particularly, bacterial species belonging to the Lactobacillaceae families were remarkably increased by MNV inoculation and RA administration, suggesting that the antiviral effects of RA occur via the modulation of specific microbiota. The antiviral causal effect of Lactobacillus was identified and demonstrated using in vitro models in RAW264.7 cells. The antiviral immune response to MNV was mediated by IFN-ß upregulation. This study represents the first comprehensive profiling of gut microbiota in response to RA treatment against norovirus infection. Moreover, we conclude that the abundance of Lactobacillus through gut microbiota modulation by RA is at least partially responsible for norovirus inhibition.

Identification of human and animal fecal contamination after rainfall in the Han River, Korea.

We investigated the effect of rainfall on the levels and sources of microbial contamination in the Han River, Korea. Thirty-four samples were collected at two sampling sites located upstream and downstream in the river from July 2010 to February 2011. Various fecal indicator microorganisms, including total coliform, fecal coliform, Escherichia coli, Enterococcus spp., somatic and male-specific (F+) coliphage, and four major enteric viruses were analyzed. Rainfall was positively correlated with the levels of fecal coliform and norovirus at both sampling sites. Additionally, rainfall was positively correlated with the levels of total coliform, E. coli, Enterococcus spp., and F+ coliphage at the upstream site. To identify the source of fecal contamination, microbial source tracking (MST) was conducted using both male-specific (F+) RNA coliphage and the Enterococcus faecium esp gene as previously described. Our results clearly indicated that the majority of fecal contamination at the downstream Han River site was from a human source. At the upstream sampling site, contamination from human fecal matter was very limited; however, fecal contamination from non-point animal sources increased following rainfall. In conclusion, our data suggest that rainfall significantly affects the level and source of fecal contamination in the Han River, Korea.

Association of the vaginal microbiota with human papillomavirus infection in a Korean twin cohort.

Human papillomavirus (HPV) is the most important causative agent of cervical cancers worldwide. However, our understanding of how the vaginal microbiota might be associated with HPV infection is limited. In addition, the influence of human genetic and physiological factors on the vaginal microbiota is unclear. Studies on twins and their families provide the ideal settings to investigate the complicated nature of human microbiota. This study investigated the vaginal microbiota of 68 HPV-infected or uninfected female twins and their families using 454-pyrosequencing analysis targeting the variable region (V2-V3) of the bacterial 16S rRNA gene. Analysis of the vaginal microbiota from both premenopausal women and HPV-discordant twins indicated that HPV-positive women had significantly higher microbial diversity with a lower proportion of Lactobacillus spp. than HPV-negative women. Fusobacteria, including Sneathia spp., were identified as a possible microbiological marker associated with HPV infection. The vaginal microbiotas of twin pairs were significantly more similar to each other than to those from unrelated individuals. In addition, there were marked significant differences from those of their mother, possibly due to differences in menopausal status. Postmenopausal women had a lower proportion of Lactobacillus spp. and a significantly higher microbiota diversity. This study indicated that HPV infection was associated with the composition of the vaginal microbiota, which is influenced by multiple host factors such as genetics and menopause. The potential biological markers identified in this study could provide insight into HPV pathogenesis and may represent biological targets for diagnostics.

F+ RNA coliphage-based microbial source tracking in water resources of South Korea.

We previously demonstrated that genotyping followed by proper statistical analyses of F plus (F+)-specific RNA coliphages can effectively represent fecal origins of either humans or animals. Here, we performed microbial source tracking (MST) using F+ RNA coliphages as a target MST microorganism for identifying fecal sources contaminating ground and surface water in metropolitan Seoul and Gyeonggi Province in South Korea. In total, 71 groundwater and 5 surface water samples were collected and screened for the presence of F+ RNA coliphages. More than 124 F+ coliphages were isolated from six groundwater and five surface water samples by the single agar layer method. F+ RNA coliphages were predominant in both waters (100% and 91%, respectively). Genotyping of 118 F+ RNA coliphages revealed that most (51/60) of the groundwater F+ RNA coliphages belonged to group I, whereas both groups I (25/58) and IV (31/58) were predominantly observed in surface water. Further comparison of phage isolates from human and animal (pig, cow, goose, and chicken) fecal sources using nucleic acid sequencing and principal coordinate analysis showed that groundwater samples formed clusters associated with cow feces, whereas surface waters formed clusters related to chicken and human feces. These results indicate the potential of the F+ RNA coliphage-based MST for identifying fecal contamination sources, which may be further exploited and validated in different geographical regions of the world.