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.

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.

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.

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.

[Analysis of clinical characteristics of 43 surgical patients with Crohn disease using the Montreal classification].

OBJECTIVE: To investigate the clinical features of Crohn disease according to the Montreal classification. METHODS: Clinical data of 43 surgical patients with Crohn disease (surgical group) and 125 non-surgical patients with Crohn disease (non-surgical group) were retrospectively analyzed and compared between two groups. The Montreal classification was used. RESULTS: In the surgical group, 28 patients (65.1%) were A2, 14 (32.6%) were A3 and only one was A1, which was not significantly different as compared to the non-surgery group. The proportions of L1, L2, L3, and L4 subtype in the surgical group were 41.9%, 25.6%, 30.2%, and 2.3%, respectively, which was not significantly different as compared to that in the non-surgery group. In the surgical group,B1 disease was found in 1 case (2.3%), B2 in 26 cases (60.5%), and B3 in 16 cases (37.2%), while in the non-surgical group, B1 was found in 79 cases (63.2%), B2 in 44 cases (35.2%) and B3 in 2 cases (1.6%). Differences were significant between two groups in disease behavior (P=0.001, P=0.004, P=0.001). CONCLUSIONS: Most surgical patients of Crohn disease are A2. L1 and L3 are the main lesion location. As disease behavior, B2 and B3 are the main reasons for operation.

Two stomach-originated Lactobacillus strains improve Helicobacter pylori infected murine gastritis.

AIM: To investigate the potential anti-Helicobacter pylori (H. pylori) and anti-inflammation in vivo effects of two lactobacillus strains from human stomach. METHODS: Forty H. pylori infected Balb/c mice were randomly divided into 4 groups: proton pump inhibitor and antibiotics triple treated group, Lactobacillus fermenti (L. fermenti) treated group, Lactobacillus acidophilus treated group and normal saline control group. Ten uninfected mice were also included as blank control group. The infection of H. pylori was detected by rapid urease tests, Giemsa staining and bacterial culture. The colonization of H. pylori was assessed in bacterial density score and gastric inflammation was assessed in histological score. The colonization of L. fermenti was performed by fluorescent probe. RESULTS: Histopathologic evaluation showed significant release of mucosal inflammation in gastric antrum and gastric body in lactobacillus treated groups and triple treated group. H. pylori eradication rate in both lactobacillus treated groups and triple treated group were higher than normal saline control group. Lactobacillus treated groups and triple treated group showed significant decrease of H. pylori bacterial density. CONCLUSION: Both lactobacillus strains have a significant anti-H. pylori activity; L. fermenti displays more efficient antagonistic activity in vivo against H. pylori infection.

[Expression and localization of strong epitope hAFP(542-550); on eukaryotic cytoplasmic membrane].

AIM: To construct the recombinant plasmid of pGPC3-EGFP containing human AFP(542-550) gene, EGFP gene and GPC3 gene to express fusion protein GPC3-hAFP(542-550)-EGFP and to discover its localization on cytoplasmic membrane. METHODS: GPC3 gene was obtained from total RNA of human placental tissues by RT-PCR; After the enhanced green fluorescent protein (EGFP) gene was amplified from pEGFP-N1 plasmid and the gene segment of-KOZAK-GPCN + afp(542-550)-was chemically synthesized, the recombinant plasmid pcDNA3.1(+)/GPCN+afp(542-550)-EGFP-GPCC (pGPC3-EGFP) containing three chimeric genes of strong epitope hAFP(542-550), GPI-anchored protein GPC3 and EGFP was constructed. The fusion protein (GPC3-hAFP(542-550)-EGFP) was detected on RNA and protein levels at 24 h and 48 h after pGPC3-EGFP was transfected into HepG2 (GPC3(+)AFP(+)) via lipofectamine 2000. EGFP expression was observed by fluorescent microscopy after pGPC3-EGFP was transfected into HepG2 using pEGFP-N1 plasmid transfection as a positive control. The fusion protein in both membrane proteins and soluble proteins extracted from the transfected 293 cells (GPC3(-)AFP(-)) was detected by Western blot using GPC3 monoclonal antibody as primary antibody. RESULTS: The recombinant plasmid pGPC3-EGFP was successfully constructed through restriction endonuclease digestion and sequencing; pGPC3-EGFP expression in HepG2 cells was detected not only by RT-PCR using specific primers (GPCN-F and EGFP-r) but also by Western blot using GFP polyclonal antibody and GPC3 monoclonal antibody. Green fluoresce was mainly found around pGPC3-EGFP transfected HepG2 cell periphery beside sporadic distribution in cytoplasm, but that of pEGFP-N1 transfected HepG2 cell was evenly distributed in the whole cell. Moreover, the fusion protein was not detected in soluble proteins but membrane proteins extracted from transfected 293 cells. CONCLUSION: The recombinant plasmid of pGPC3-EGFP based on protein engineering theory can express fusion protein (GPC3-hAFP(542-550)-EGFP) in eukaryotic cells. Furthermore, the fusion protein is still located on cytoplasmic membrane, which is a characteristic of GPI-anchored membrane protein, and is a new GPI-reanchored protein.

Therapeutic effects of four strains of probiotics on experimental colitis in mice.

AIM: To investigate the therapeutic effects of four strains of probiotics (E. feacalis, L. acidophilus, C. butyricum and B. adolescentis) on dextran sulphate sodium (DSS)-induced experimental colitis in Balb/c mice. METHODS: Eighty Balb/c mice were randomly divided into 8 groups. Weight-loss, fecal character, fecal occult blood and hematochezia were recorded daily. Disease activity index (DAI) scores were also evaluated everyday. Length of colon was measured and histological scores were evaluated on the 13th day. Myeloperoxidase (MPO) activity was detected. Interleukin-1 (IL-1) and IL-4 expression was detected by ELISA and RT-PCR. RESULTS: The four strains of probiotics relieved the inflammatory condition of DSS-induced experimental colitis in mice. Weight loss was slowed down in all probiotics-treated mice. Even weight gain was observed by the end of probiotics treatment. The DAI and histological scores of probiotics-treated mice were lower than those of mice in the control group (1.9 +/- 0.2 vs 8.6 +/- 0.4, P < 0.05 for E. faecalis). The length of colon of probiotics-treated mice was longer than that of mice in the control group (10.3 +/- 0.34 vs 8.65 +/- 0.77, P < 0.05 for E. faecalis). The four strains of probiotics decreased the MP activity and the IL-1 expression, but increased the IL-4 expression. E. faecalis had a better effect on DSS-induced experimental colitis in mice than the other three strains. CONCLUSION: The four strains of probiotics have beneficial effects on experimental colitis in mice. E. faecalis has a better effect on DSS-induced experimental colitis in mice than the other three strains. Supplement of probiotics provides a new therapy for UC.

[Herceptin plus adjuvant chemotherapy for the prognosis of patients with human epithelial growth factor receptor 2 positive early-stage breast cancer: a meta-analysis].

OBJECTIVE: To evaluate the effect of herceptin(trastuzumab) plus adjuvant chemotherapy on the prognosis of patients with human epithelial growth factor receptor 2 (HER2) positive early-stage breast cancer by Meta-analysis. METHODS: Search all of randomized clinical trials (RCTs) on herceptin plus adjuvant chemotherapy for HER2 positive early-stage breast cancer in MEDLINE, EMBase, Cochrane library, Clinical Trails, ASCO Conference data, CHKD, Wanfang Database, VIP information, scholar.google.com and SIGLE. A Meta-analysis was carried out by collecting information based on the inclusion and exclusion criteria from all papers available. RESULTS: The Meta-analysis included 4 trials. A total of 9116 patients were included in the analysis(4555 in the study group and 4561 in the control group). There were statistical differences between the study group(herceptin plus adjuvant chemotherapy) and the control group(adjuvant chemotherapy) in the disease-free survival rate [relative risk(RR)=1.08, 95% CI, 1.06-1.09, P<0.001], the overall survival rate(RR=1.01, 95% CI, 1.01-1.02, P=0.0003), the distant recurrence rate(RR=0.49, 95% CI, 0.42-0.57, P<0.001), and the cardiac events rate (RR=3.93,95% CI, 1.03-15.06, P=0.05). CONCLUSION: Herceptin plus adjuvant chemotherapy can improve the disease-free survival rate and the overall survival rate, decrease distant recurrence rate of patients with HER2 positive early-stage breast cancer, but may cause heart toxicity, especially when combined with anthracycline (doxorubicin).

Targeting specificity and pharmacokinetics of asialoorosomucoid, a specific ligand for asialglycoprotein receptor on hepatocyte.

OBJECTIVE: To testify that the asialoorosomucoid (ASOR) prepared by us has liver-targeting specificity and to investigate its pharmacokinetic characteristics. METHODS: The distribution of 125I-ASOR in vivo was determined by single photon emission computed tomography (SPECT) and immunohistochemical technique after 125I-ASOR was injected into Sprague-Dawley (S-D) rats through their caudal veins. In vitro, different doses of pEGFP-N1 plasmid were transfected into both HepG2 cells and HT1080 cells with the use of ASOR-poly-L-lysine. At 24 and 48 h after transfection, the expression of green fluorescent protein (GFP) was determined under fluorescent microscope. Pharmacokinetic parameters were calculated according to two-compartment open system model with first-order kinetics. RESULTS: SPECT images showed that 125I-ASOR was located only in liver/stomach and root of caudal vein/bladder at 10 min after injection. The 125I-ASOR radioactivities of organs taken out from S-D rats were different at different times, and about 63% of 125I-ASOR was located in the liver at 10 min after injection. At 30 min after injection a peak of radioactivity was seen in stomach. The times of these two radioactivity peaks were different. Immunohistochemical study of liver frozen sections showed that ASOR was combined mainly with hepatocyte membrane, especially in areas with rich blood flow. In vitro study showed that ASOR targeted specifically cells with asialoglycoprotein receptor (ASGr). GFP expression was detected in HepG2 cells but not in HT1080 cells. Furthermore, the more quantity of pEGFP-N1 transfected and the longer expression time, the higher GFP expression level was in HepG2 cells. The 125I-ASOR pharmacokinetics equation for liver was Ct=662216e-3.362t+8896e-2343t. 125I-ASOR was excreted from liver slowly after an initial rapid decrease. The pharmacokinetic equation for stomach was Ct=-114815e-1.7t+1148153e-15t and the half-life of 125I-ASOR in stomach was 4.62 h. CONCLUSIONS: ASOR prepared by us could be an efficient gene transfer vector, ASOR was distributed mainly in the liver and stomach and had high targeting specificity to hepatocytes or hepatic originating cells.

[Effect of Bacillus acidi lactici CL22 strain on Helicobacter Pylori infectious gastritis in Balb/c mouse model].

OBJECTIVE: To observe the therapeutic effect of Bacillus acidi lactici on Helicobacter Pylori (Hp) infectious gastritis in Balb/c mouse model so as to explore a possible non-antibiotic treatment for Hp. METHODS: To establish a Balb/c mouse model with Hp infectious gastritis through inoculation of mankind Hp,32 Balb/c mice infected by Hp were randomly divided into 4 groups:Group 1(PPI trigeminy treatment group),Group 2 (Bacillus acidi lactici CL22 treatment group),Group 3 (Bacillus acidi lactici CL24 treatment group),and Group 4 (normal saline control group). Intragastric administration was given continuously for 10 days. Another 8 normal mice were chosen as Group 5(blank control group). All mice were killed after 4 weeks since last intragastric administration. Hp was detected by rapid urease test,Giemsa dying, and bacterial culture,and histopathologic changes in the gastric mucosa of mice were determined by H-E staining. RESULTS: There were significant differences in pathohistologic scores in sinus ventriculi among the 5 groups (F = 7.932, P = 0.000). The scores in Group 1, Group 2, Group 3, and Group 5 were obviously lower than those in Group 4 (P < 0.05), but there were not significant differences among Group 1, 2, and 5 (P>0.05). The pathohistologic score in Group 3 was obviously higher than that in Group 5 (P <0.05). There were significant differences in pathohistologic scores in corpus ventriculi among the 5 groups (F = 6.241, P = 0.001). The scores in Group 1,Group 2,Group 3,and Group 5 were obviously lower than those in Group 4(P <0.05), but there were not significant differences among Group 1, 2, 3,and 5 (P>0.05). There was significant difference in Hp eradication rates in sinus ventriculi among the 5 groups (chi2 = 16.923, P=0.002). The Hp eradication rates in Group 1 and 2 were obviously lower than those in Group 4 (P <0.05), but there was not significant difference between Group 1 and Group 2, Group 3 and Group 4 (P>0.05). There also were significant differences in Hp eradication rate in corpus ventriculi among the 5 groups (chi2 = 14.295, P=0.006). Of them, Group 1 and Group 2 were higher than Group 4 (P <0.05), but there were not obviously differences between Group 1 and 2,Group 3 and 4 (P>0.05). CONCLUSION: Bacillus acidi lactici strain CL22 can effectively inhibit and eradicate Hp in Balb/c mouse model with Hp infectious gastritis in vivo. The therapeutic effect of Bacillus acidi lactici strain CL22 is equal to PPI + antibiotics and could be another choice of nonjantibiotic treatment for Hp.

Primary shunt hyperbilirubinaemia in a large four-generation family confirming autosomal dominant genetic disorder.

AIM: To describe the pattern of inheritance and confirm the diagnostic criteria of primary shunt hyperbilirubinaemia (PSH). METHODS: Forty members of a family pedigree across four generations were included in this study. All family members were interviewed and investigated by physical examination, hematology and liver function test and the pattern of inheritance was analyzed. RESULTS: Nine of the forty family members suffered primary shunt hyperbilirubinaemia. The mature erythrocytes of the propositus were irregular in shape and size. The pedigree showed transmission of the trait through four generations with equal distribution in male and female. No individual with a primary shunt hyperbilirubinaemia was born to unaffected parents. The penetrance was complete in adult. CONCLUSION: The pattern of inheritance is autosomal dominant. The abnormality of erythrocytes and decrease in white blood cell could be supplemented in the diagnosis of PSH. The PSH is a genetic disorder and could by renamed as hereditary shunt hyperbilirubinaemia.