Gut microbiota interacts with intrinsic brain activity of patients with amnestic mild cognitive impairment.

AIMS: To explore the potential relationships among gut microbiota (GM), local brain spontaneous activity, and neuropsychological characteristics in amnestic mild cognitive impairment (aMCI) patients. METHODS: Twenty aMCI and 22 healthy control (HC) subjects were recruited. The GM composition was determined by 16S ribosomal RNA gene sequencing. Resting-state functional magnetic resonance imaging scans were performed, and fractional amplitude of low-frequency fluctuations (fALFF) was calculated across different frequencies. The Spearman or Pearson correlation analysis was used to analyze the relationship between spontaneous brain activity and cognitive function, and GM composition. RESULTS: aMCI patients had altered GM state and local spontaneous brain activity as compared with HC subjects. Correlation analysis showed that aMCI and HC groups had different "GM-intrinsic brain activity interaction" patterns. In aMCI group, at the typical band (0.01-0.08 Hz), the relative abundance (RA) of Bacteroides from phylum to genus level was negatively correlated with fALFF value of cerebellar vermis IV-V, and the Ruminococcaceae RA was negatively correlated with fALFF values of left lenticular nucleus and pallidum. The Clostridiaceae RA and Blautia RA were positively correlated with the left cerebellum lobules IV-V at the slow-4 band (0.027-0.073 Hz). The Veillonellaceae RA was positively correlated with fALFF values of left precentral gyrus at the slow-5 band (0.073-0.08 Hz). Correlation analysis showed that Clostridium members (Lachnospiraceae and Blautia) were positively, while Veillonellaceae was negatively, correlated with cognition test. Bacteroides was positively correlated with attention and computation, and negatively correlated with the three-stage command score. CONCLUSIONS: aMCI patients have a specific GM-intrinsic brain activity-cognitive function interaction pattern.

Fecal Microbiome Data Distinguish Liver Recipients With Normal and Abnormal Liver Function From Healthy Controls.

Emerging evidence suggests that altered intestinal microbiota plays an important role in the pathogenesis of many liver diseases, mainly by promoting inflammation via the "intestinal microbiota-immunity-liver" axis. We aimed to investigate the fecal microbiome of liver recipients with abnormal/normal liver function using 16S rRNA gene sequencing. Fecal samples were collected from 90 liver recipients [42 with abnormal liver function (Group LT_A) and 48 with normal liver function (Group LT_N)] and 61 age- and gender-matched healthy controls (HCs). Fecal microbiomes were analyzed for comparative composition, diversity, and richness of microbial communities. Principal coordinates analysis successfully distinguished the fecal microbiomes of recipients in Group LT_A from healthy subjects, with the significant decrease of fecal microbiome diversity in recipients in Group LT_A. Other than a higher relative abundance of opportunistic pathogens such as Klebsiella and Escherichia/Shigella in all liver recipients, the main difference in gut microbiome composition between liver recipients and HC was the lower relative abundance of beneficial butyrate-producing bacteria in the recipients. Importantly, we established a fecal microbiome index (specific alterations in Staphylococcus and Prevotella) that could be used to distinguish Group LT_A from Group LT_N, with an area under the receiver operating characteristic curve value of 0.801 and sensitivity and specificity values of 0.771 and 0.786, respectively. These findings revealed unique gut microbial characteristics of liver recipients with abnormal and normal liver functions, and identified fecal microbial risk indicators of abnormal liver function in liver recipients.

Efficacy of glycyrrhizin combined with cyclosporine in the treatment of non-severe aplastic anemia.

BACKGROUND: Cyclosporine A (CsA) has been widely used in the treatment of aplastic anemia (AA), but the application of CsA was limited in patients who had liver diseases or abnormal liver function due to its liver toxicity. Glycyrrhizin has long been used in China in the treatment of various liver diseases to lower transaminases. In this study, we observed the efficacy and safety of glycyrrhizic acid combined with CsA in the treatment of newly diagnosed patients with non-severe AA (NSAA). METHODS: A total number of 76 patients with newly diagnosed NSAA were enrolled into the study at our hospital between July 2005 and June 2010. The patients were divided randomly into two groups: the glycyrrhizin-treatment group (group A) and the control group (group B) with 38 patients in each group. All patients received 3 - 5 mg×kg(-1)×d(-1) CsA for at least 4 months and were treated either with or without glycyrrhizin for 4 months. RESULTS: sixty-eight patients were eligible for evaluation. In the control group, 9.09% patients (n = 3) achieved a complete response while 51.52% (n = 17) attained a partial response. The overall response rate was 60.61% (n = 20). The remaining 13 patients (39.39%) did not have any response. In the glycyrrhizin-treatment group, complete response rate was 20% (n = 7) and partial response rate was 62.86% (n = 22). The overall response rate was 82.86% (n = 29) and the non-response rate was 17.14% (n = 6). Response rate was significantly increased with the addition of glycyrrhizin to CsA compared with CsA alone (P < 0.05). CONCLUSION: The combination of glycyrrhizin and cyclosporine regimen was an effective treatment for NSAA in terms of improvement of response rate, reduction in CsA-related liver injury, and attenuation of severity of nausea and other adverse events in the treatment of patients with NSAA.

Metabolomic analyses of faeces reveals malabsorption in cirrhotic patients.

BACKGROUND: The study of faeces offers a unique opportunity to observe cooperation between the microbiome and the metabolism of mammalian hosts, an essential element in the study of the human metabolome. In the present study, a global metabolomics approach was used to identify metabolites differentially excreted in the faeces of cirrhotic patients compared to controls. METHODS: Seventeen cirrhotic patients and 24 healthy individuals were recruited. Faecal metabolites were detected through non-targeted reversed-phase ultra-performance liquid chromatography coupled to electrospray ionization quadrupole time-of-flight mass spectrometry. RESULTS: A total of 9215 peaks were detected. Using unequal variance t-tests, 2393 peaks were observed with P≤0.05, approximately 74.0% of which were due to decreased faecal metabolite concentrations in liver cirrhosis vs. healthy controls. Integrating multivariate data analyses, we identified six major groups of metabolites. Relative levels of identified metabolites were as follows: strong increase in lysophosphatidylcholines, aromatic amino acids, fatty acids, and acylcarnitines, and a dramatic decrease in bile acids and bile pigments. CONCLUSION: With severe hepatic injury in patients with liver cirrhosis, malabsorption occurs along with disorders of fatty acid metabolism, potentially due to changes in gut microflora.

Expression of H5N1 influenza virus hemagglutinin protein fused with protein transduction domain in an alphavirus replicon system.

Alphavirus replicons, in which structural protein genes are replaced by heterologous genes, express high levels of the heterologous proteins. On the basis of the potencies of replicons to self-replicate and express foreign proteins and the remarkable intercellular transport property of VP22, a novel alphavirus Semliki Forest virus (SFV) replicon system of VP22 fused with a model antigen, hemagglutinin (HA), of the human-avian H5N1 influenza virus, was explored in this study. Further, replicon particles expressing HA, VP22, and enhanced green fluorescent protein (EGFP) individually were used as controls. By flow cytometry based on the analysis of transfection efficiency, SFV-EGFP replicon particle titer was 1.13 x 10(7)transducing units (TU)/ml. The titers of SFV-HA, SFV-VP22 and SFV-VP22-HA replicon particles, which were titrated by using SFV-EGFP replicon particles, were 1.42 x 10(7), 3.23 x 10(7), and 1.01 x 10(7)TU/ml, respectively. HA and VP22-HA expression was observed in SFV-HA- and SFV-VP22-HA-transfected BHK-21 cells, respectively. Immunofluorescence staining revealed that the fluorescence intensity in the SFV-VP22-HA-transfected BHK-21 cells was more than that in the SFV-HA-transfected BHK-21 cells. Both SFV-VP22-HA and SFV-HA replicon particles presented a promising approach for developing vaccines against human-avian influenza. VP22-HA fusion protein with similar trafficking properties may also enhance vaccine potency.

Construction and cellular immune response induction of HA-based alphavirus replicon vaccines against human-avian influenza (H5N1).

Several approaches are being taken worldwide to develop vaccines against H5N1 viruses; most of them, however, pose both practical and immunological challenges. One potential strategy for improving the immunogenicity of vaccines involves the use of alphavirus replicons and VP22, a herpes simplex type 1 (HSV-1) protein. In this study, we analysed the antigenic peptides and homogeneity of the HA sequences (human isolates of the H5N1 subtype, from 1997 to 2003) and explored a novel alphavirus replicon system of VP22 fused with HA, to assess whether the immunogenicity of an HA-based replicon vaccine could be induced and augmented via fusion with VP22. Further, replicon particles expressing VP22, and enhanced green fluorescent protein (EGFP) were individually used as controls. Cellular immune responses in mice immunised with replicons were evaluated by identifying specific intracellular cytokine production with flow cytometry (FCM). Animal-based experimentation indicated that both the IL-4 expression of CD4(+) T cells and the IFN-gamma expression of CD8(+) T cells were significantly increased in mice immunised with VPR-HA and VPR-VP22/HA. A dose titration effect vis-à-vis both IL-4 expression and IFN-gamma expression were observed in VPR-HA- and VPR-VP22/HA-vaccinated mice. Our results revealed that both VPR-VP22/HA and VPR-HA replicon particles presented a promising approach for developing vaccines against human-avian influenza, and VP22 could enhance the immunogenicity of the HA antigens to which it is fused.

[Influence of 1.8 GHz microwave on DNA damage induced by ultraviolet C ray].

OBJECTIVE: To study whether 1.8 GHz microwaves (MW) (SAR, 3 W/kg) exposure can influence DNA damage induced by ultraviolet ray (UV). METHODS: The lymphocytes were obtained from three young healthy donors. The cells were exposed to 254 nm UV at the doses of 0.25, 0.50, 0.75, 1.00, 1.50 and 2.00 J/m(2). The lymphocytes were also exposed to 1.8 GHz MW (SAR, 3 W/kg) for 0, 1.5 and 4.0 h. The combination exposure of UV plus MW was conducted. The treated cells were incubated for 0, 1.5 and 4.0 h. Finally, comet assay was used to detect DNA damage of above treated lymphocytes. RESULTS: The difference of DNA damage induced between MW group and control group was not significant (P>0.05). the MTLs induced by UV were (1.71+/-0.09), (2.02+/-0.08), (2.27+/-0.17), (2.27+/-0.06), (2.25+/-0.12), (2.24+/-0.11)microm, respectively, which were significantly higher than that of control [(0.96+/-0.05) microm], (P<0.01). MTLs of some sub-groups in combination exposure groups at 1.5 h incubation were significantly lower than those of corresponding UV sub-groups (P<0.01 or P<0.05. However, MTLs of some sub-groups in combination exposure groups at 4.0 h incubation were significantly higher than those of corresponding UV sub-groups (P<0.01 or P<0.05). CONCLUSION: The exposure to 1.8 GHz (SAR, 3 W/kg) MW for 1.5 and 4.0 h can not enhance significantly human lymphocyte DNA damage. But MW can reduce or enhance DNA damage of lymphocytes induced by UV at 1.5 h and 4.0 h incubation in comet assay in vitro, respectively.

[Influence of 1.8 GHz microwave on DNA damage induced by 4 chemical mutagens].

OBJECTIVE: To observe the influence of 1.8 GHz microwave (MW) specific absorption rate (SAR, 3 W/kg) on human lymphocytes DNA damage induced by 4 chemical mutagens [mitomycin C (MMC), bleomycin (BLM), methyl methanesulfonate (MMS), and 4-nitroquinoline 1-oxide (4NQO)]. METHODS: Comet assay in vitro was used to detect human lymphocyte DNA damage induced by 1.8 GHz MW, 4 chemical mutagens, and MW plus 4 chemicals 0 h and 21 h respectively after exposure. The time exposed to MW or mutagens was 2 h or 3 h respectively. The results were showed by tail length (TL) and tail moment (TM). RESULTS: The difference of DNA damage between MW group and control group was not statistically significant (P > 0.05). DNA damages in MW plus MMC groups and MW plus 4NQO groups were significantly greater than those in the corresponding concentrations of MMC groups and 4NQO groups (P < 0.01 or P < 0.05). However, MW did not enhance DNA damage induced by MMS and BLM (P > 0.05). CONCLUSION: Exposure to 1.8 GHz (SAR, 3 W/kg) microwave may not induce human lymphocyte DNA damage, but could enhance DNA damage induced by MMC and 4NQO.

Assessment of human DNA repair (NER) capacity with DNA repair rate (DRR) by comet assay.

OBJECTIVE: Alkaline comet assay was used to evaluate DNA repair (nucleotide excision repair, NER) capacity of human fresh lymphocytes from 12 young healthy non-smokers (6 males and 6 females). METHODS: Lymphocytes were exposed to UV-C (254 nm) at the dose rate of 1.5 J/m2/sec. Novobiocin (NOV) and aphidicolin (APC), DNA repair inhibitors, were utilized to imitate the deficiency of DNA repair capacity at the incision and ligation steps of NER. Lymphocytes from each donor were divided into three grougs: UVC group, UVC plus NOV group, and UVC plus APC group. DNA single strand breaks were detected in UVC irradiated cells incubated for 0, 30, 60, 90, 120, 180, and 240 min after UVC irradiation. DNA repair rate (DRR) served as an indicator of DNA repair capacity. RESULTS: The results indicated that the maximum DNA damage (i.e. maximum tail length) in the UVC group mainly appeared at 90 min. The ranges of DRRs in the UVC group were 62.84%-98.71%. Average DRR value was 81.84%. The DRR difference between males and females was not significant (P < 0.05). However, the average DRR value in the UVC plus NOV group and the UVC plus APC group was 52.98% and 39.57% respectively, which were significantly lower than that in the UVC group (P < 0.01). CONCLUSION: The comet assay is a rapid, simple and sensitive screening test to assess individual DNA repair (NER) capacity. It is suggested that the time to detect DNA single strand breaks in comet assay should include 0 (before UV irradiation), 90 and 240 min after exposure to 1.5 J x m(-2) UVC at least. The DRR, as an indicator, can represent the individual DNA repair capacity in comet assay.

[Investigating genetic damage of workers occupationally exposed to methotrexate].

OBJECTIVE: To study genetic damage of workers alone occupationally exposed to methotrexate (MTX) with three end-points. METHODS: The blood samples from 21 workers exposed to MTX and 21 controls were detected with micronucleus test, comet assay, hprt gene mutation test and TCR gene mutation test. RESULTS: The mean micronuclei rate (MNR) and mean micronucleated cells rate (MCR) in 21 workers were 10.10 per thousand +/- 0.95 per thousand and 8.05 per thousand +/- 0.75 per thousand, respectively, which were significantly higher than those (5.48 per thousand +/- 0.82 per thousand and 4.38 per thousand +/- 0.58 per thousand) in control (P < 0.01). The mean tail length (MTL) of 21 workers and 21 controls were (1.30 +/- 0.06) microm and (0.07 +/- 0.01) microm, respectively, there was significant difference between workers and controls (P < 0.01). But the difference between workers and controls for mean tail moment (MTM) was not significant (P > 0.05). The average mutation frequency (Mf-hprt) of hprt and (Mf-TCR) of TCR in workers were 1.00 per thousand +/- 0.02 per thousand and (6.87 +/- 0.52) x 10(-4), respectively, which were significantly higher than those [0.86 per thousand +/- 0.01 per thousand and (1.67 +/- 0.14) x 10(-4)] in control (P < 0.01). CONCLUSION: The genetic damage to some extent appeared in workers occupationally exposed to methotrexate.

[Detecting DNA repair capacity of human lymphocytes exposed to ultraviolet C with comet assay].

OBJECTIVE: To assess DNA repair capacity of human lymphocytes with comet assay. METHODS: Fresh lymphocytes form twelve 26-year old donors (6 males, 6 females) were exposed to ultraviolet C (UVC, 254 nm) at the dose rate of 1.5 J/m(2). The lymphocytes of each donor were divided into three parts: UVC group, UVC + aphidicolin (APC) group, UVC + novobiocin (NOV) group. DNA single strand breaks were detected with comet assay in UVC-irradiated cells and unirradiated cells incubated for 30, 60, 90, 120, 180 and 240 min. DNA repair rate (DRR) was calculated and served as an indicator of DNA repair capacity. RESULTS: The maximum average comet tail length (MTL) in three groups appeared 90 min after UVC exposure. The DRR range of UVC group was 81.84% (62.84% - 98.71%); There was no significant difference in DRR between males and females (P > 0.05). However, the average DRRs of UVC + NOV group and UVC + APC group (52.98% and 39.57% respectively) were significantly lower than that of UVC group (P < 0.01). CONCLUSION: Comet assay is a rapid and simple screening test to assess DNA repair capacity. DRR, as an indicator, may express the individual DNA repair capacity.