[Establishment of a risk model for severe adenovirus pneumonia and prospective study of the timing of intravenous immunoglobulin therapy in children]. / å„¿ç«¥é‡ç—‡è ºç— æ¯’æ€§è‚ºç‚Žé£Žé™©æ¨¡åž‹çš„æž„å»ºåŠé™è„‰æ³¨å°„å ç–«çƒè›‹ç™½æ²»ç–—æ—¶æœºçš„å‰çž»æ€§ç ”ç©¶.

OBJECTIVES: To develop a risk prediction model for severe adenovirus pneumonia (AVP) in children, and to explore the appropriate timing for intravenous immunoglobulin (IVIG) therapy for severe AVP. METHODS: Medical data of 1 046 children with AVP were retrospectively analyzed, and a risk prediction model for severe AVP was established using multivariate logistic regression. The model was validated with 102 children with AVP. Then, 75 children aged ≤14 years who were considered at risk of developing severe AVP by the model were prospectively enrolled and divided into three groups (A, B and C) in order of visit, with 25 children in each group. Group A received symptomatic supportive therapy only. With the exception of symptomatic supportive therapy, group B received IVIG treatment at a dose of 1g/(kg·d) for 2 consecutive days, before progressing to severe AVP. With the exception of symptomatic supportive therapy, group C received IVIG treatment at a dose of 1 g/(kg·d) for 2 consecutive days after progressing to severe AVP. Efficacy and related laboratory indicators were compared among the three groups after treatment. RESULTS: Age<18.5 months, underlying diseases, fever duration >6.5 days, hemoglobin level <84.5 g/L, alanine transaminase level >113.5 U/L, and co-infection with bacteria were the six variables that entered into the risk prediction model for severe AVP. The model had an area under the receiver operating characteristic curve of 0.862, sensitivity of 0.878, and specificity of 0.848. The Hosmer-Lemeshow test showed good consistency between the predicted values and the actual observations (P>0.05). After treatment, group B had the shortest fever duration and hospital stay, the lowest hospitalization costs, the highest effective rate of treatment, the lowest incidence of complications, the lowest white blood cell count and interleukin (IL)-1, IL-2, IL-6, IL-8, IL-10 levels, and the highest level of tumor necrosis factor alpha (P<0.05). CONCLUSIONS: The risk model for severe AVP established in this study has good value in predicting the development of severe AVP. IVIG therapy before progression to severe AVP is more effective in treating AVP in children.

Evidence that synergism between potassium and nitrate enhances the alleviation of ammonium toxicity in rice seedling roots.

Ammonium toxicity in plants is considered a global phenomenon, but the primary mechanisms remain poorly characterized. Here, we show that although the addition of potassium or nitrate partially alleviated the inhibition of rice seedling root growth caused by ammonium toxicity, the combination of potassium and nitrate clearly improved the alleviation, probably via some synergistic mechanisms. The combined treatment with potassium and nitrate led to significantly improved alleviation effects on root biomass, root length, and embryonic crown root number. The aberrant cell morphology and the rhizosphere acidification level caused by ammonium toxicity, recovered only by the combined treatment. RNA sequencing analysis and weighted gene correlation network analysis (WGCNA) revealed that the transcriptional response generated from the combined treatment involved cellulose synthesis, auxin, and gibberellin metabolism. Our results point out that potassium and nitrate combined treatment effectively promotes cell wall formation in rice, and thus, effectively alleviates ammonium toxicity.

Clinical features of NK/T-cell EBV-associated LPD manifested as gastrointestinal symptoms in patients with normal immunity: a case report and literature review.

BACKGROUND: Epstein-Barr virus (EBV)-associated NK/T-cell lymphoproliferative disorder (LPD) involving the gastrointestinal tract is rarely observed in individuals with normal immunity. The atypical clinical, colonoscopic manifestations often confuse clinicians, leading to misdiagnosis and delays in the treatment. CASE PRESENTATION: Herein, we reported on a single case of a patient with gastrointestinal symptoms. Several colonoscopies showed multiple irregular ulcerations, while biopsies showed colitis with infiltration of neutrophils or lymphocytes. After 2 months follow-up, the patient was diagnosed with the extranodal NK/T-cell lymphoma, nasal type, and was treated with thalidomide. Later on, a second check was performed on his first pathological sample. Immunohistochemistry revealed EBV associated NK/T-cell LPD. CONCLUSIONS: Multiple, multiform, and segmental gastrointestinal ulcers should be an indication for EBV infection, regardless of the presence of fever, lymphadenopathy, and hepatosplenomegaly. If EBV-associated NK/T-cell LPD is considered, serum EBV-DNA should be measured, and the tissue obtained by biopsy should be carefully analyzed for a positive expression of the EBER marker.

Effects of different diets on intestinal microbiota and nonalcoholic fatty liver disease development.

AIM: To study the effects of different diets on intestinal microbiota and nonalcoholic fatty liver disease (NAFLD) development at the same caloric intake. METHODS: Thirty male Sprague-Dawley rats were randomized into five groups (six rats each). The control diet (CON) group and free high-fat diet (FFAT) group were allowed ad libitum access to a normal chow diet and a high-fat diet, respectively. The restrictive high-fat diet (RFAT) group, restrictive high-sugar diet (RSUG) group, and high-protein diet (PRO) group were fed a high-fat diet, a high-sugar diet, and a high-protein diet, respectively, in an isocaloric way. All rats were killed at 12 wk. Body weight, visceral fat index (visceral fat/body weight), liver index (liver/body weight), insulin resistance, portal lipopolysaccharide (LPS), serum alanine aminotransferase (ALT), serum aspartate aminotransferase (AST), and liver triglycerides were measured. The intestinal microbiota in the different groups of rats was sequenced using high-throughput sequencing technology. RESULTS: The FFAT group had higher body weight, visceral fat index, liver index, peripheral insulin resistance, portal LPS, serum ALT, serum AST, and liver triglycerides compared with all other groups (P < 0.05). Taking the same calories, the RFAT and RSUG groups demonstrated increased body weight, visceral fat index, peripheral insulin resistance and liver triglycerides compared with the PRO group (P < 0.05). The RFAT group also showed increased portal LPS compared with the PRO group (P < 0.05). Unweighted UniFrac principal coordinates analysis of the sequencing data revealed that the intestinal microbiota structures of the CON, FFAT, RSUG and PRO groups were roughly separated away from each other. Taxon-based analysis showed that, compared with the CON group, the FFAT group had an increased abundance of Firmicutes, Roseburia and Oscillospira bacteria, a higher ratio of Firmicutes to Bacteroidetes, and a decreased abundance of Bacteroidetes, Bacteroides and Parabacteroides bacteria (P < 0.05). The RFAT group showed an increased abundance of Firmicutes and decreased abundance of Parabacteroides bacteria (P < 0.05). The RSUG group showed an increased abundance of Bacteroidetes and Sutterella bacteria, higher ratio of Bacteroidetes to Firmicutes, and a decreased abundance of Firmicutes (P < 0.05). The PRO group showed an increased abundance of Bacteroidetes, Prevotella, Oscillospira and Sutterella bacteria, and a decreased abundance of Firmicutes (P < 0.05). Compared with the FFAT group, the RFAT group had an increased abundance of Bacteroidetes, higher ratio of Bacteroidetes to Firmicutes, and decreased abundance of Firmicutes and Oscillospira bacteria (P < 0.05). CONCLUSION: Compared with the high-protein diet, the NAFLD-inducing effects of high-fat and high-sugar diets are independent from calories, and may be associated with changed intestinal microbiota.

Transgenic Rice Expressing Ictb and FBP/Sbpase Derived from Cyanobacteria Exhibits Enhanced Photosynthesis and Mesophyll Conductance to CO2.

To find a way to promote the rate of carbon flux and further improve the photosynthetic rate in rice, two CO2-transporting and fixing relevant genes, Ictb and FBP/Sbpase, which were derived from cyanobacteria with the 35SCaMV promotor in the respective constructs, were transformed into rice. Three homologous transgenic groups with Ictb, FBP/Sbpase and the two genes combined were constructed in parallel, and the functional effects of these two genes were investigated by physiological, biochemical and leaf anatomy analyses. The results indicated that the mesophyll conductance and net photosynthetic rate were higher at approximately 10.5-36.8% and 13.5-34.6%, respectively, in the three groups but without any changes in leaf anatomy structure compared with wild type. Other physiological and biochemical parameters increased with the same trend in the three groups, which showed that the effect of FBP/SBPase on improving photosynthetic capacity was better than that of ICTB and that there was an additive effect in ICTB+FBP/SBPase. ICTB localized in the cytoplasm, whereas FBP/SBPase was successfully transported to the chloroplast. The two genes might show a synergistic interaction to promote carbon flow and the assimilation rate as a whole. The multigene transformation engineering and its potential utility for improving the photosynthetic capacity and yield in rice were discussed.

Molecular cloning and characterization of two manganese superoxide dismutases from Miscanthus × giganteus.

KEY MESSAGE: Six MnSOD genes were isolated from five Miscanthus species. MgMnSOD1 functions in mitochondria and MgMnSOD1 seems to be the main MnSOD gene involved in stress response of M. × giganteus. Miscanthus × giganteus is a promising biomass energy crop with advantages of vigorous growth, high yield, low fertilizer and pesticide inputs. However, poor overwinter ability limits its widespread cultivation. Moreover, narrow genetic base may increase the risk of susceptibility to diseases and pests. Manganese superoxide dismutase (MnSOD), an important antioxidant enzyme involved in stress tolerance is able to protect plant cells from accumulated reactive oxygen species by converting superoxide to peroxide and oxygen. In many plants, overexpression of MnSOD has shown the ability to enhance the resistance to various stresses. This article describes the studies performed in an attempt to elucidate the molecular and enzymatic properties of MnSODs in M. × giganteus. MnSOD genes from M. × giganteus (MgMnSOD1, MgMnSOD2), M. lutarioriparia (MlMnSOD), M. sacchariflora (MsaMnSOD), M. sinensis (MsiMnSOD), and M. floridulus (MfMnSOD) were cloned and sequenced. The sequence analysis and expression patterns of MgMnSOD1 and MgMnSOD2 suggest that they were orthologous genes which were inherited from the two parents, M. sacchariflora and M. sinensis, respectively. In addition, MgMnSOD1 is predicted to be the main MnSOD gene involved in stress response of M. × giganteus. The activity of purified recombinant MgMnSOD1 was 1854.79 ± 39.98 U mg(-1) (mean ± SD). Further enzymatic assays revealed that the protein exhibited an outstanding thermal stability. MgMnSOD1 is predicted to be targeted to mitochondria and involved in removing the superoxide radical generated by respiration. The presence and sequences of other SOD isozymes transcripts were also investigated in this study.

Evaluation of an individual-donation nucleic acid amplification testing algorithm for detecting hepatitis B virus infection in Chinese blood donors.

BACKGROUND: This multicenter study was performed to evaluate the efficiency of a multiplex individual-donation nucleic acid amplification technology (ID-NAT) and discriminatory testing algorithm for detecting hepatitis B virus (HBV) infection in Chinese blood donors. STUDY DESIGN AND METHODS: A total of 1,205,796 hepatitis B surface antigen (HBsAg)-nonreactive donations from 10 blood centers were tested by ID-NAT using the Ultrio assay. Multiplex Ultrio-reactive donations were tested in the discriminatory tests as well as in quantitative polymerase chain reaction (qPCR) and in supplemental electrochemiluminescence immunoassays for HBsAg, hepatitis B surface antibody (anti-HBs), hepatitis B e antigen, and antibody to hepatitis B core antigen (anti-HBc). Meanwhile, a control group of 4317 Ultrio-nonreactive donations was tested for anti-HBc and anti-HBs. RESULTS: Of all donations, 2033 (0.17%) were reactive in the multiplex Ultrio assay. Among 1776 further tested samples, 548 (30.9%) were HBV discriminatory assay (dHBV)-reactive, while 1214 (68.4%) were nonreactive. Of 472 Ultrio+ and dHBV+ samples 86.2% were qPCR positive compared to 15.0% in 1046 Ultrio+ and dHBV- samples. The proportion of anti-HBc+ and anti-HBs- (potentially infectious) donations was higher in 409 Ultrio+ and dHBV+ than in 1028 Ultrio+ and dHBV- samples (51.3% vs. 31.1%, p < 0.001). The yield rate of Ultrio+, dHBV+, and qPCR+ donations was estimated at 1 in 2500, but at 1 in 1100 when all supplemental tests were taken into account assuming that 44% of detected donations by Ultrio were false reactive. CONCLUSIONS: A quarter of HBsAg-negative Ultrio+ and dHBV- donations in China are likely given by potentially infectious low-viral-load occult carriers. Although this has no implication for blood safety, the testing algorithm needs to be redesigned to more efficiently discriminate between true and false NAT reactivity.

Functional inactivation of UDP-N-acetylglucosamine pyrophosphorylase 1 (UAP1) induces early leaf senescence and defence responses in rice.

Plant leaf senescence and defence responses are important biological processes, but the molecular mechanisms involved are not well understood. This study identified a new rice mutant, spotted leaf 29 (spl29). The SPL29 gene was identified by map-based cloning, and SPL29 was confirmed as UDP-N-acetylglucosamine pyrophosphorylase 1 (UAP1) by enzymatic analysis. The mutant spl29 lacks UAP activity. The biological phenotypes for which UAP is responsible have not previously been reported in plants. The spl29 mutant displayed early leaf senescence, confirmed by chlorophyll loss and photosystem II decline as physiological indicators, chloroplast degradation as a cellular characteristic, and both upregulation of senescence transcription factors and senescence-associated genes, and downregulation of photosynthesis-related genes, as molecular evidence. Defence responses were induced in the spl29 mutant, shown by enhanced resistance to bacterial blight inoculation and upregulation of defence response genes. Reactive oxygen species, including O2 (-) and H2O2, accumulated in spl29 plants; there was also increased malondialdehyde content. Enhanced superoxide dismutase activity combined with normal catalase activity in spl29 could be responsible for H2O2 accumulation. The plant hormones jasmonic acid and abscisic acid also accumulated in spl29 plants. ROS and plant hormones probably play important roles in early leaf senescence and defence responses in the spl29 mutant. Based on these findings, it is suggested that UAP1 is involved in regulating leaf senescence and defence responses in rice.

Ulcerative colitis as a polymicrobial infection characterized by sustained broken mucus barrier.

To reduce medication for patients with ulcerative colitis (UC), we need to establish the etiology of UC. The intestinal microbiota of patients with inflammatory bowel disease (IBD) has been shown to differ from that of healthy controls and abundant data indicate that it changes in both composition and localization. Small intestinal bacterial overgrowth is significantly higher in IBD patients compared with controls. Probiotics have been investigated for their capacity to reduce the severity of UC. The luminal surfaces of the gastrointestinal tract are covered by a mucus layer. This normally acts as a barrier that does not allow bacteria to reach the epithelial cells and thus limits the direct contact between the host and the bacteria. The mucus layer in the colon comprises an inner layer that is firmly adherent to the intestinal mucosa, and an outer layer that can be washed off with minimal rinsing. Some bacteria can dissolve the protective inner mucus layer. Defects in renewal and formation of the inner mucus layer allow bacteria to reach the epithelium and have implications for the causes of colitis. In this review, important elements of UC pathology are thought to be the intestinal bacteria, gut mucus, and the mucosa-associated immune system.

Nitrite promotes the growth and decreases the lignin content of indica rice calli: a comprehensive transcriptome analysis of nitrite-responsive genes during in vitro culture of rice.

As both major macronutrients and signal molecules, nitrogen metabolites, such as nitrate and nitrite, play an important role in plant growth and development. In this study, the callus growth of indica rice cv. 9311 was significantly enhanced by nitrite, whereas the soluble protein content remained unchanged. The deep RNA sequencing technology (RNA-seq) showed that the transcriptional profiles of cv. 9311 calli were significantly changed after adding nitrite to the nitrate-free medium, and these nitrite-responsive genes were involved in a wide range of plant processes, particularly in the secondary metabolite pathways. Interestingly, most of the genes involved in phenylpropanoid-related pathways were coordinately down-regulated by nitrite, such as four cinnamoyl-CoA reductase, and these in turn resulted in the decrease of lignin content of indica calli. Furthermore, several candidate genes related to cell growth or stress responses were identified, such as genes coding for expansins, SMALL AUXIN UP RNA (SAUR) and HSP20s, and these suggested that nitrite could probably serve as a transcriptome signal to enhance the indica calli growth by regulation of various downstream genes expression. This study contributes to a better understanding of the function of nitrite during the process of plant tissue culture and could aid in the application of this technology to improved indica genetic transformation efficiency.

Efficacy profiles for different concentrations of Lactobacillus acidophilus in experimental colitis.

AIM: To determine the efficacy profiles of different concentrations of Lactobacillus acidophilus (L. acidophilus) for treating colitis using an experimental murine model. METHODS: Colitis was established in 64 BALB/c mice by adding 5% dextran sodium sulfate (DSS) to the drinking water and allowing ad libitum access for 7 d. The mice were then randomly divided into the following control and experimental model groups (n = 8 each; day 0): untreated model control; negative-treatment model control (administered gavage of 1 mL/10 g normal saline); experimental-treatment models C4-C8 (administered gavage of 10(4), 10(5), 10(6), 10(7), or 10(8) CFU/10 g L. acidophilus, respectively); positive-treatment model control (administration of the anti-inflammatory agent prednisone acetate at 45 µg/10 g). Eight mice given regular water (no DSS) and no subsequent treatments served as the normal control group. Body weight, fecal traits, and presence of fecal occult blood were assessed daily. All animals were sacrificed on post-treatment day 7 to measure colonic length, perform histological scoring, and quantify the major bacteria in the proximal and distal colon. Intergroup differences were determined by one-way ANOVA and post-hoc Student-Newman-Keuls comparison. RESULTS: All treatments (L. acidophilus and prednisone acetate) protected against colitis-induced weight loss (P < 0.05 vs model and normal control groups). The extent of colitis-induced colonic shortening was significantly reduced by all treatments (prednisone acetate > C4 > C5 > C7 > C8 > C6; P < 0.05 vs untreated model group), and the C6 group showed colonic length similar to that of the normal control group (P > 0.05). The C6 group also had the lowest disease activity index scores among the model groups. The bacterial profiles in the proximal colon were similar between all of the experimental-treatment model groups (all P > 0.05). In contrast, the bacterial profile in the distal colon of the C6 group showed the distinctive features (P < 0.05 vs all other experimental-treatment model groups) of Lactobacillus sp. and Bifidobacterium sp. being the most abundant bacteria and Staphylococcus aureus being the least abundant bacteria. CONCLUSION: The most therapeutically efficacious concentration of L. acidophilus (10(6) CFU/10 g) may exert its effects by modulating the bacterial profile in the distal colon.

Calcitonin gene-related peptide is a promising marker in ulcerative colitis.

BACKGROUND: Assessment of the severity and extent of disease activity continues to present challenges for physicians in the treatment of ulcerative colitis. Standard markers that can objectively reflect disease activity are useful for physicians to both evaluate the course of ulcerative colitis and monitor the effectiveness of therapy for any given patient. AIMS: We hypothesize that calcitonin gene-related peptide (CGRP) can reflect the activity and severity of ulcerative colitis and be used as a marker to assess the effectiveness of various therapies. METHODS: We examined the expression levels of CGRP by reverse transcription polymerase chain reaction (RT-PCR) and semi-quantitative immunohistochemisty in mucosal biopsies from 38 patients with UC and 18 controls. Levels of CGRP mRNA and protein expression were compared between patients and controls with the clinical activity index (CAI) and the endoscopic activity index (EAI) for various levels of UC severity. RESULTS: Our results showed that the levels of CGRP mRNA and protein expression were significantly reduced in UC patients compared to controls. This effect was more pronounced in patients with more severe cases of UC. There is a statistically significant negative correlation between levels of CGRP mRNA expression and CAI/EAI scores. A statistically significant negative correlation was also found between levels of CGRP protein expression and CAI/EAI scores. Overall, high CAI and EAI scores were accompanied by low CGRP mRNA and protein expression levels. CONCLUSION: Levels of CGRP protein and mRNA expression in the colonic mucosa of patients are closely associated with UC severity and corroborate traditional indices used to assess the disease.

Lactobacillus crispatus M206119 exacerbates murine DSS-colitis by interfering with inflammatory responses.

AIM: To investigate the role of Lactobacillus crispatus (L. crispatus) strain China Center for Type Culture Collection (CCTCC) M206119 in intestinal inflammation. METHODS: Forty 8-wk-old Balb/c mice (20 ± 2 g) were divided into four groups of 10 mice each. Three groups that had received dextran sulfate sodium (DSS) were administered normal saline, sulfasalazine or CCTCC M206119 strain, and the fourth group received none of these. We assessed the severity of colitis using a disease activity index, measured the colon length and weight, collected stools and mesenteric lymph nodes for bacterial microflora analysis. One centimeter of the proximal colon, middle colon and distal colon were collected and fixed in 10% buffered formalin, dehydrated in ethanol, and embedded in paraffin. Interleukin (IL)-1ß, IL-6 and tumor necrosis factor (TNF)-α expression was detected using reverse transcription polymerase chain reaction. Protective factors zonula occludens (ZO)-1 and ß-defensin 2 were detected by immunoblotting. The features of CCTCC M206119 strain were identified based on morphology, biochemical profile, and 16S RNA sequencing. RESULTS: DSS-colitis animals treated with CCTCC M206119 had markedly more severe disease, with greater weight loss, diarrhea, fecal bleeding, and shortened colon length. In addition, the CCTCC-M206119-treated group had comparatively higher histological scores and more neutrophil infiltration than the controls. Expression of protective factors ZO-1 and ß-defensin 2 was downregulated due to destruction of the mucosal barrier after CCTCC M206119 strain treatment. An in vitro assay demonstrated that CCTCC M206119 strain increased the nuclear translocation of nuclear factor-κB in epithelial cells. Intestinal proinflammatory or anti-inflammatory cytokine responses were evaluated. Proinflammatory colonic cytokine (IL-1ß, IL-6 and TNF-α) levels were clearly increased in CCTCC-M206119-treated animals, whereas anti-inflammatory colonic cytokine (IL-10) level was lowered compared with saline or 5-aminosalicylic-acid-treated DSS-colitis mice. Next, CCTCC M206119 strain was characterized as L. crispatus by microscopic morphology, biochemical tests and 16S rRNA gene level. CONCLUSION: Not all lactobacilli are beneficial for intestinal inflammation, and L. crispatus CCTCC M206119 strain is involved in exacerbation of intestinal inflammation in DSS-colitis mice.

[Suppurative arthritis caused by Gemella morbillorum in a patient with rheumatoid arthritis of the knee].

Rheumatoid arthritis of the knee is a common disease, but suppurative arthritis caused by Gemella morbillorum in the same joint is rare. We report a case of suppurative arthritis caused by Gemella morbillorum in a patient with rheumatoid arthritis. Because the infection symptoms was not typical, the diagnosis was delayed, and the delayed diagnosis and therapy led to a poor outcome of the patient.

Gastric and duodenum microflora analysis after long-term Helicobacter pylori infection in Mongolian Gerbils.

BACKGROUND: Long-term Helicobacter pylori infection leads to chronic gastritis, peptic ulcer, and gastric malignancies. Indigenous microflora in alimentary tract maintains a colonization barrier against pathogenic microorganisms. This study is aimed to observe the gastric and duodenum microflora alteration after H. pylori infection in Mongolian Gerbils model. MATERIALS AND METHODS: A total of 18 Mongolian gerbils were randomly divided into two groups: control group and H. pylori group that were given H. pylori NCTC J99 strain intragastrically. After 12 weeks, H. pylori colonization was identified by rapid urease tests and bacterial culture. Indigenous microorganisms in stomach and duodenum were analyzed by culture method. Histopathologic examination of gastric and duodenum mucosa was also performed. RESULTS: Three of eight gerbils had positive H. pylori colonization. After H. pylori infection, Enterococcus spp. and Staphylococcus aureus showed occurrences in stomach and duodenum. Lactobacillus spp. showed a down trend in stomach. The levels and localizations of Bifidobacterium spp., Bacteroides spp., and total aerobes were also modified. Bacteroides spp. significantly increased in H. pylori positive gerbils. No Enterobacteriaceae were detected. Positive colonization gerbils showed a higher histopathologic score of gastritis and a similar score of duodenitis. CONCLUSIONS: Long-term H. pylori colonization affected the distribution and numbers of indigenous microflora in stomach and duodenum. Successful colonization caused a more severe gastritis. Gastric microenvironment may be unfit for lactobacilli fertility after long-term H. pylori infection, while enterococci, S. aureus, bifidobacteria, and bacteroides showed their adaptations.

Lactobacillus species shift in distal esophagus of high-fat-diet-fed rats.

AIM: To analyze the microbiota shift in the distal esophagus of Sprague-Dawley rats fed a high-fat diet. METHODS: Twenty Sprague-Dawley rats were divided into high-fat diet and normal control groups of 10 rats each. The composition of microbiota in the mucosa from the distal esophagus was analyzed based on selective culture. A variety of Lactobacillus species were identified by molecular biological techniques. Bacterial DNA from Lactobacillus colonies was extracted, and 16S rDNA was amplified by PCR using bacterial universal primers. The amplified 16S rDNA products were separated by denaturing gradient gel electrophoresis (DGGE). Every single band was purified from the gel and sent to be sequenced. RESULTS: Based on mucosal bacterial culturing in the distal esophagus, Staphylococcus aureus was absent, and total anaerobes and Lactobacillus species were decreased significantly in the high-fat diet group compared with the normal control group (P < 0.01). Detailed DGGE analysis on the composition of Lactobacillus species in the distal esophagus revealed that Lactobacillus crispatus, Lactobacillus gasseri (L. gasseri) and Lactobacillus reuteri (L. reuteri) comprised the Lactobacillus species in the high-fat diet group, while the composition of Lactobacillus species in the normal control group consisted of L. gasseri, Lactobacillus jensenii and L. reuteri. CONCLUSION: High-fat diet led to a mucosal microflora shift in the distal esophagus in rats, especially the composition of Lactobacillus species.

Effects of two Lactobacillus strains on lipid metabolism and intestinal microflora in rats fed a high-cholesterol diet.

BACKGROUND: The hypocholesterolemic effects of lactic acid bacteria (LAB) have now become an area of great interest and controversy for many scientists. In this study, we evaluated the effects of Lactobacillus plantarum 9-41-A and Lactobacillus fermentum M1-16 on body weight, lipid metabolism and intestinal microflora of rats fed a high-cholesterol diet. METHODS: Forty rats were assigned to four groups and fed either a normal or a high-cholesterol diet. The LAB-treated groups received the high-cholesterol diet supplemented with Lactobacillus plantarum 9-41-A or Lactobacillus fermentum M1-16. The rats were sacrificed after a 6-week feeding period. Body weights, visceral organ and fat pad weights, serum and liver cholesterol and lipid levels, and fecal cholesterol and bile acid concentrations were measured. Liver lipid deposition and adipocyte size were evaluated histologically. RESULTS: Compared with rats fed a high-cholesterol diet but without LAB supplementation, serum total cholesterol, low-density lipoprotein cholesterol and triglycerides levels were significantly decreased in LAB-treated rats (p < 0.05), with no significant change in high-density lipoprotein cholesterol levels. Hepatic cholesterol and triglyceride levels and liver lipid deposition were significantly decreased in the LAB-treated groups (p < 0.05). Accordingly, both fecal cholesterol and bile acids levels were significantly increased after LAB administration (p < 0.05). Intestinal Lactobacillus and Bifidobacterium colonies were increased while Escherichia coli colonies were decreased in the LAB-treated groups. Fecal water content was higher in the LAB-treated groups. Compared with rats fed a high-cholesterol diet, administration of Lactobacillus plantarum 9-41-A resulted in decreases in the body weight gain, liver and fat pad weight, and adipocytes size (p < 0.05). CONCLUSIONS: This study suggests that LAB supplementation has hypocholesterolemic effects in rats fed a high-cholesterol diet. The ability to lower serum cholesterol varies among LAB strains. Our strains might be able to improve the intestinal microbial balance and potentially improve intestinal transit time. Although the mechanism is largely unknown, L. plantarum 9-41-A may play a role in fat metabolism.

GPC3 fused to an alpha epitope of HBsAg acts as an immune target against hepatocellular carcinoma associated with hepatitis B virus.

BACKGROUND: The incidence of hepatocellular carcinoma (HCC) in China is closely related to the population infected with hepatitis B virus (HBV). HCC cells with HBV secrete soluble HBsAg into blood but do not express it on the cell membrane. This study aimed to construct and investigate a new glycosyl-phosphatidylinositol (GPI)-anchored protein (GPC3+alpha+EGFP) as a DNA vaccine against HCC associated with HBV. METHODS: A recombinant plasmid (pcDNA3.1(+)/GPC3+ alpha+EGFP) was constructed and verified by restriction endonuclease digestion and sequencing. pcDNA3.1(+)/GPC3+alpha+EGFP was transfected into HepG2 cells (experimental group) using lipofectamine 2000. pEGFP-N1-transfected HepG2 cells were used as a negative control, and non-transfected HepG2 cells served as a blank control. HepG2 cells that steadily expressed the fusion protein GPC3+alpha+EGFP were screened by G418, propagated, and co-cultured with lymphocytes from healthy donors. Cell proliferation was measured by the classic sulforhodamine B assay. Apoptosis was assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), and Fas gene transcription was determined by quantitative fluorescent PCR. RESULTS: The pcDNA3.1(+)/GPC3+alpha+EGFP plasmid was successfully constructed. In the experimental group, green fluorescence was observed at the cell periphery and in the cytoplasm, whereas in the negative control group, fluorescence was evenly distributed throughout the cell. Proliferation of the experimental group significantly decreased after 72 hours compared to the negative and blank control groups. Furthermore, the number of apoptotic cells was statistically different among the three groups as determined by a contingency table Chi-square test; the experimental group had the highest incidence of apoptosis. Fas gene transcription in the experimental group was higher than in the two control groups, and an increasing trend with time in the experimental group was observed. CONCLUSION: A chimeric, membrane-anchored protein, GPC3+alpha+EGFP, localized to the membrane of HepG2 cells and inhibited proliferation and accelerated apoptosis through a Fas-FasL pathway after co-cultivation with lymphocytes.

Effects of four Bifidobacteria on obesity in high-fat diet induced rats.

AIM: To compare the effects of four Bifidobacteria strains (Bifidobacteria L66-5, L75-4, M13-4 and FS31-12, originated from normal human intestines) on weight gain, lipid metabolism, glucose metabolism in an obese murine model induced by high-fat diet. METHODS: Forty-eight Sprague-Dawley rats were randomly divided into six groups. Control group received standard chow, model group received high-fat diet, and intervention groups received high-fat diet added with different Bifidobacteria strains isolated from healthy volunteers' fresh feces. All rats were executed at the 6th weekend. Body weight (BW), obese indexes, oral glucose tolerance test, serum and liver lipid and serum insulin (INS) were tested. Liver lipid deposition was classified pathologically. RESULTS: Compared with the model group, B. M13-4 improved BW gains (264.27 +/- 26.91 vs 212.55 +/- 18.54, P = 0.001) while B. L66-5 induced a decrease in BW (188.47 +/- 11.96 vs 212.55 +/- 18.54, P = 0.043). The rest two strains had no significant change in BW. All the four strains can reduce serum and liver triglyceride and significantly alleviate the lipid deposition in liver. All strains showed a trend of lowing serum and liver total cholesterol while B. L66-5 and B. FS31-12 did so more significantly. In addition, all the four strains showed no significant differences in serum INS and glucose level. CONCLUSION: The response of energy metabolism to administration of Bifidobacteria is strain dependent. Different strains of Bifidobacteria might drive different directions of fat distribution.

[Inhibition of the growth of hepatocarcinoma xenograft in Balb/c mice induced by dendritic cells immunized with AFP cDNA fragement].

OBJECTIVE: To investigate the antitumor immune response induced by dendritic cells vaccine coding AFPcDNA fragment with signal peptide (AFP(1)) and without signal peptide (AFP(2)), and to determine the inhibiting effect of the vaccine on the growth of hepatocarcinoma xenograft in Balb/c mice. METHODS: pcDNA3.1/AFP(1) and pcDNA3.1/AFP(2) were transfected into dendritic cells (DCs) by calcium phosphate nanoparticles and became DCs vaccine. Mouse spleen lymphocytes were stimulated by AFP(1)/DC and AFP(2)/DC. A Balb/c mouse model bearing mouse HCC xenograft was established on the day 14 after transplantation. Forty mice were divided equally into AFP(2)/DC group, AFP(1)/DC group and plasmid control group. The treated mice received DCs vaccine and the same amount of control plasmid. RESULTS: AFP(2)/DC stimulated T lymphocytel proliferation in vitro and improved CTL activity. The effects were better than AFP(1)/DC. The tumor-bearing mice injected intralesionally with AFP(1)/DC and AFP(2)/DC at a dose of 0.5 ml per mouse showed inhibition of tumor growth and prolongation of survival time. The tumor inhibition rate of the AFP(2)/DC group was 79.2% and the AFP(1)/DC group was 39.7% at 2 weeks after treatment. The tumor volume of AFP(2)/DC group was (726.7 +/- 298.2) mm(3), significantly smaller than the (1486.2 +/- 457.2) mm(3) of the AFP(1)/DC group and (2137.2 +/- 547.2) mm(3) of the plasmid control group (P < 0.05). The mean survival time of mice in the AFP(2)/DC group [(58.5 +/- 4.2) d] and AFP(1)/DC group [(45.2 +/- 4.8) d] were significantly longer than that of plasmid control group [(30.6 +/- 6.2) d, P < 0.05]. Bax-positive cell percentage was increased in the xenografts of AFP(2)/DC-treatment group compare with that of plasmid control group. CONCLUSION: AFP(2)/DC and AFP(1)/DC vaccines show evident inhibiting effect on the growth of H22 xenograft in Balb/c mice through inducing efficient and specific immune response against the hepatocarcinoma cells.