The role of ursodeoxycholic acid in non-alcoholic steatohepatitis: a systematic review.

BACKGROUND: Non-alcoholic steatohepatitis (NASH) is a condition that occurs during the progression of non-alcoholic fatty liver disease. Effective therapy for NASH is still lacking. In this study, we investigated the effects of Ursodeoxycholic acid (UDCA) in the treatment of NASH. METHODS: Western and Chinese databases were searched by independent investigators using appropriate MESH headings to identify randomized, controlled Western and Chinese clinical trials, published between January 1990 and October 2012, testing the effects of UDCA in patients with NASH. Patient characteristics and trial endpoints were analyzed, with quality assessment according to widely acknowledged criteria. P < 0.05 was defined as statistically significant in all trials. RESULTS: Twelve qualified randomized clinical trials, including six from China and involving 1160 subjects, were selected. Seven of these trials assessed the effects of UDCA Monotherapy, with the other five testing combinations of UDCA with vitamin E, polyene phosphatidylcholine, silymarin, glycyrrhizin and tiopronin. The duration of therapy ranged from 3 to 24 months, with two studies using high doses of UDCA (23-35 mg/kg/d). The average quality point was 2.69, and was significantly lower in articles from China than in those from Western countries (2.2 ± 0.4 vs. 3.8 ± 1.1, respectively, p < 0.05). UDCA Monotherapy significantly improved liver function in five studies and improved steatosis and fibrosis in two studies. All five studies assessing UDCA combination therapy showed significant improvements liver function, while two studies also improved steatosis and inflammation. One study of high-dose UDCA showed significant improvements in ALT, γGT and liver fibrosis, whereas the other study showed no significant change in ALT and liver pathology. CONCLUSIONS: UDCA therapy is effective in NASH, especially when combined with other drugs. However, the low quality of these studies and the heterogeneity of their results precluded further meta-analysis. Additional carefully designed clinical trials are needed, especially in China.

The effect of oral tolerance on the roles of small intestinal intraepithelial lymphocytes in murine colitis induced by dextran sodium sulfate.

BACKGROUNDS AND AIMS: There is increasing evidence that gut-derived intraepithelial lymphocytes have potent cytolytic and immunoregulatory functions, which they use to sustain epithelial integrity. The aims of this study were to investigate the roles of small intestinal intraepithelial lymphocytes (SI-IELs) in oral tolerance and dextran sodium sulfate (DSS)-induced colitis. METHODS: SI-IELs or sorted γδ T cells from untreated, colitis, and colitis-extracted protein (CEP)-fed colitis mice were adoptively transferred to BALB/c mice; colitis was then induced with DSS. Cytokines were analyzed in sera from mice and culture supernatants. RESULTS: Transfer of SI-IELs or sorted γδ T cells from untreated and colitis mice all alleviated experimental colitis. Mice orally administered with five low doses of CEP showed less severe symptoms and histological injury. SI-IELs from CEP-fed colitis mice more significantly ameliorated colitis than those from control mice (weight, 94.1 ± 2.5% vs. 89.8 ± 2.6%, p < 0.05; disease activity index, 7.2 ± 1.2 vs. 8.7 ± 1.9, p < 0.05; histological scores, 22.1 ± 2.8 vs. 25.7 ± 2.1, p < 0.05, n = 8 per group); however, not did SI-γδ IELs from CEP-fed colitis mice. Alleviation of colitis was accompanied by an increase of TGF-ß1 secretion and no change of IFN-γ in sera and culture supernatants. The level of serum TGF-ß1 was negatively related to the severity of colitis. CONCLUSIONS: The protective effects of SI-IELs in DSS-induced colitis were partly accomplished by γδ T cells and could be mediated by TGF-ß but were not associated with IFN-γ. Oral tolerance strengthens the suppressive effects of regulatory subsets in SI-IELs.

Isolation of murine small intestinal intraepithelial γδt cells.

The study of intestinal intraepithelial γδ lymphocytes has been hindered by the difficulty of isolating a population of pure cells representative of their in vivo phenotype and function. Here, we describe a procedure for the purification of small intestinal intraepithelial γδ T lymphocytes in BALB/c mice, including extraction and purification of intraepithelial lymphocytes (IELs) followed by separation of γδ T cells from IELs by magnetic activated cell sorting (MACS). The small intestinal IEL isolation protocol reported here is a less time-consuming with high purity and high yields. One-step Percoll purification of IELs using a 40%-70% discontinuous Percoll gradient is less laborious yet sufficient to get exact profiles during phenotypic analysis of cell subsets. IELs obtained after two-step Percoll centrifugation using 30% Percoll, followed by a 40%-70% discontinuous Percoll gradient, are fit to purify γδ T cells by MACS. Small intestinal IELs produce much more IFN-γ and TGF-ß1 than sorted γδ T cells upon stimulation with plate-bound anti-CD3ε mAb. γδ T cells secret moderate amounts of TGF-ß1 and minimal amounts of IFN-γ. Purification of γδ T cells is helpful in investigating the phenotype and function of intestinal mucosal γδ T cells.

The protective effect of oral colitis-derived proteins in a murine model of inflammatory bowel disease is associated with an increase in gammadelta T cells in large intestinal mucosa.

BACKGROUNDS AND AIMS: Oral tolerance has previously been shown effective in preventing several immune-mediated disorders in animal models. The aims of this study were to investigate the effect of oral colitis-extracted proteins (CEP) on dextran sulfate sodium (DSS)-induced colitis in BALB/c mice and to explore the relative role of the intestinal mucosal gammadelta T cells. METHODS: The effect of five low oral doses of CEP on colitis was evaluated by clinical manifestation and histological lesions. Serum cytokines were measured by enzyme-linked immunosorbent assay. The percentages of the intestinal mucosal gammadelta T cells were evaluated by flow cytometry. RESULTS: CEP-fed colitis mice showed less severe symptoms and histological injury than bovine serum albumin (BSA)-fed control mice. Tolerized mice developed an increase in TGF-beta1 and no change in IFN-gamma serum levels. Increases in TCRgammadelta(+) T cells and CD8alpha(+)TCRgammadelta(+) T cells in small intestinal mucosal lymphocytes and no quantitative change in large intestinal mucosal lymphocytes were demonstrated in colitis mice compared to untreated mice. The proportions of TCRgammadelta(+) T cells and CD8alpha(+)TCRgammadelta(+) T cells in large intestinal mucosal lymphocytes from CEP-fed colitis mice were significantly higher compared to BSA-fed controls. The disease activity index negatively correlated with the percentages of large intestinal mucosal gammadelta T cells. Furthermore, mucosal repair in repair-period mice was also accompanied by increases in TCRgammadelta(+) T cells and CD8alpha(+)TCRgammadelta(+) T cells in large intestinal mucosal lymphocytes. CONCLUSION: Improvement of DSS-induced colitis that resulted from oral administration of colitis-extracted proteins is associated with an increase in gammadelta T cells in large intestinal mucosa.

Direct signaling of TL1A-DR3 on fibroblasts induces intestinal fibrosis in vivo.

Tumor necrosis factor-like cytokine 1A (TL1A, TNFSF15) is implicated in inflammatory bowel disease, modulating the location and severity of inflammation and fibrosis. TL1A expression is increased in inflamed mucosa and associated with fibrostenosing Crohn's disease. Tl1a-overexpression in mice causes spontaneous ileitis, and exacerbates induced proximal colitis and fibrosis. Intestinal fibroblasts express Death-receptor 3 (DR3; the only know receptor for TL1A) and stimulation with TL1A induces activation in vitro. However, the contribution of direct TL1A-DR3 activation on fibroblasts to fibrosis in vivo remains unknown. TL1A overexpressing naïve T cells were transferred into Rag-/- , Rag-/- mice lacking DR3 in all cell types (Rag-/-Dr3-/-), or Rag-/- mice lacking DR3 only on fibroblasts (Rag-/-Dr3∆Col1a2) to induce colitis and fibrosis, assessed by clinical disease activity index, intestinal inflammation, and collagen deposition. Rag-/- mice developed overt colitis with intestinal fibrostenosis. In contrast, Rag-/-Dr3-/- demonstrated decreased inflammation and fibrosis. Despite similar clinical disease and inflammation as Rag-/-, Rag-/-Dr3∆Col1a2 exhibited reduced intestinal fibrosis and attenuated fibroblast activation and migration. RNA-Sequencing of TL1A-stimulated fibroblasts identified Rho signal transduction as a major pathway activated by TL1A and inhibition of this pathway modulated TL1A-mediated fibroblast functions. Thus, direct TL1A signaling on fibroblasts promotes intestinal fibrosis in vivo. These results provide novel insight into profibrotic pathways mediated by TL1A paralleling its pro-inflammatory effects.

A novel microsatellite within intron 8 in caspase10 gene in Chinese of Han nationality.

OBJECTIVE: To investigate polymorphisms in the coding exons and their splicing areas of caspase10 gene (CASP10) in Chinese of Han nationality. METHODS: Polymerase chain reaction (PCR), denaturing high-performance liquid chromatography (DHPLC), direct sequencing and clonal sequencing were used to analyze the exon 9 and its flanking sequences. RESULTS: In 70 blood samples of Chinese subjects researched the known single nucleotide polymorphisms (SNPs) in the exon 9 of CASP10 gene published in dbSNP of NCBI were not identified. We found a short sequence with more than ten continuous and repeated T nucleotides within the intron 8 near the exon 9 and the length of repeated sequence nucleotides T was different in samples. Clonal sequencing analysis indicated that it was a microsatellite of single nucleotide-repeated sequence. The recurrence and specificity of same result was further confirmed by PCR with high fidelity polymerase and sequencing. Furthermore all of 70 blood samples detected by DHPLC showed heterozygous profiles. We named it as IVS8-13(T)n temperately. CONCLUSION: Our research suggested that the site be a microsatellite of single nucleotide-repeated sequence. Meanwhile, our data further showed that it was quite different from that by querying SNP database of non-Chinese population in NCBI, in the same region a SNP of type of insertion deletion existed. It could be inferred that the difference between the populations might be associated with the genetic characteristics of ethnics. The significance of the microsatellite in CASP10 gene in Chinese of Han nationality remains to be elucidated.

[Haplotypes of four single nucleotide polymorphisms in caspase-8, -10 genes in Han nationality of Zhejiang province in China].

OBJECTIVE: To investigate single nucleotide polymorphisms (SNPs) and the distribution of their haplotypes in caspase-8, -10 genes in Zhejiang Han nationality in China. METHODS: PCR, denaturing high-performance liquid chromatography (DHPLC) and DNA sequencing were used to detect the SNPs in the 2nd-5th exons of caspase-10 gene, the 8th-10th exons of caspase-8 and their flanking sequences. Expectation Maximization (EM) algorithm was used for haplotype frequencies analysis and pairwise linkage disequilibrium (LD) test. RESULTS: (1) Two SNPs, A2823G and A12799G, were identified in caspase-10 gene, located in exon 2 and exon 5 respectively. A12799G was newly found with low informativeness. Three SNPs were identified in caspase-8 gene; A43466G, G51484A and G52951A were located in exon 8, exon 9 and intron 9, respectively. They do not change the primary structure of the encoded protein. (2) Linkage equilibrium was observed between A2823G in caspase-10 gene and the three sites in caspase-8 gene. A43466G and G52951A, and G51484A and G52951A in caspase-8 gene were also in linkage equilibrium. Their coefficients of disequilibrium were near 0. Whereas strong linkage disequilibrium was observed between A43466G and G51484A, because its coefficient of disequilibrium was near 1. (3) A total of 11 haplotypes were estimated within A2823G in caspase-10 gene and three sites in caspase-8 gene. A-2823/A-43466/G-51484/G-52951 was the main haplotype with a frequency of 0.3811. A-2823/A-43466/G-51484/A-52951 was the second haplotype with a frequency of 0.2536. The polymorphism information content of their haplotypes was 0.7106. CONCLUSION: The SNPs of caspase-8, -10 genes in Han Chinese of Zhejiang could be parsed into at least three different haplotype blocks. The polymorphism information content can be improved by using haplotype analysis of several SNPs.

[BCL-2/IgH translocation in peripheral blood cells of healthy Chinese individuals of Han nationality located in Zhejiang area].

OBJECTIVE: To investigate the relationship between the frequency of BCL-2/IgH rearrangement in peripheral blood cells of healthy Chinese individuals of Han nationality located in Zhejiang area and the low incidence of follicular lymphoma (FL). METHODS: Nested-PCR and direct DNA sequencing were used to detect the Bcl-2/IgH rearrangement in peripheral blood cells of 196 healthy individuals. DNA sequences involved were then searched and aligned in NCBI database to confine the broken points in major breakpoint region and the IgH segments involved. RESULTS: First, in this sample the frequency of BCL-2/IgH translocation in Chinese individuals of Han nationality located in Zhejiang area is 9.66%, being much lower than that in North America and Europe countries. Second, the breakpoints tend to fall into 3 clusters: 3055, 3116 and 3165 bp. Usage of J6 segment is most common. Third, There are different subclones of BCL-2/IgH rearrangements in the same individual. CONCLUSION: The low frequency of BCL-2/IgH translocation in healthy Chinese individuals of Han nationality located in Zhejiang area may be one of the reasons for the difference in the incidence of FL between China and Western countries.

[The haplotypes of three single nucleotide polymorphisms in caspase-3 gene in Han nationality of Zhejiang province in China].

OBJECTIVE: To investigate the single nucleotide polymorphisms (SNPs) and the distribution of their haplotypes in caspase-3 gene in Zhejiang Han nationality in China. METHODS: Denaturing high-performance liquid chromatography (DHPLC) and DNA sequencing were used to detect the SNPs in the regulatory region and the exons 2-7 and their flanking sequences in caspase-3 gene. Expectation maximization (EM) algorithm was used for haplotype frequencies analysis and pairwise linkage disequilibrium test. RESULTS: (1) Three SNPs were identified in caspase-3 gene; the three sites C829A, A17532C and C20541T were located in 5' regulatory region, intron 4 and 3' regulatory region, respectively. (2) Strong linkage disequilibrium was found among these SNPs; site A17532C and C20541T were in complete linkage disequilibrium. (3) C-829/A-17532/C-20541 (54.3%) was the main haplotype of Zhejiang Han nationality. CONCLUSION: The above findings indicated there is strong linkage disequilibrium among the three SNPs in caspase-3 gene in Han nationality of Zhejiang province and the main haplotype of Han nationality is obviously different from that of North American.

Helicobacter pylori and Crohn's disease: a retrospective single-center study from China.

AIM: To investigate the association between Helicobacter pylori (H. pylori) infection and the prevalence of Crohn's disease (CD). METHODS: Subjects were selected from patients admitted the gastrointestinal (GI) department at The First Affiliated Hospital School of Medicine (Zhejiang University) for abdominal pain, hematochezia, diarrhea and other GI symptoms between January 2008 and September 2012. CD was diagnosed by endoscopy and biopsy. H. pylori infection was detected by a (14)C-urea breath test and culturing of the biopsy sample. Demographic, anthropometric and serologic data were collected for each patient. H. pylori infection rate was compared between CD and control groups, followed by a subgroup analysis based on extent and severity of CD. Student's t, Mann-Whiney U, and χ(2) tests were used to analyze the data. RESULTS: A total of 447 patients were analyzed, including 229 in the CD group and 248 in the control group. There were no significant differences in age, sex, and rates of hypertension or diabetes. However, the CD group showed significantly higher rates of smoking history (34.9% vs 18.1%), alcohol intake (17.4% vs 8.1%), white blood cell count (9.7 ± 2.9 × 10(9)/L vs 4.3 ± 0.9 × 10(9)/L), and C-reactive protein (36.3 ± 20.8 mg/L vs 5.5 ± 2.3 mg/L) but lower body mass index (24.5 ± 2.0 kg/m(2) vs 26.0 ± 2.2 kg/m(2)) than the control group. The H. pylori infection rate in the CD group was 27.1%, significantly lower than that of 47.9% in the control group. Furthermore, the H. pylori infection rates in patients with colonic, small intestine, ileocolonic and extensive CD were 31.1%, 28.9%, 26.8% and 25.9% respectively, all of which were significantly lower than in the control group. Finally, the H. pylori infection rates in patients with remission, moderate and severe CD were 34.3%, 30.7% and 22.0% respectively, which were also significantly lower than in the control group. CONCLUSION: Lower H. pylori infection in CD patients suggests a correlation between bacterial infection and CD, suggesting caution when considering H. pylori eradication in CD patients.

Uncoupling protein and nonalcoholic fatty liver disease.

OBJECTIVE: To review the current advances on the role of uncoupling protein (UCP) in the pathogenesis and progress of nonalcoholic fatty liver disease (NAFLD). DATA SOURCES: A comprehensive search of the PubMed literature without restriction on the publication date was carried out using keywords such as UCP and NAFLD. STUDY SELECTION: Articles containing information related to NAFLD and UCP were selected and carefully analyzed. RESULTS: The typical concepts, up-to-date findings, and existing controversies of UCP2 in NAFLD were summarized. Besides, the effect of a novel subtype of UCP (hepatocellular down regulated mitochondrial carrier protein, HDMCP) in NAFLD was also analyzed. Finally, the concept that any mitochondrial inner membrane carrier protein may have, more or less, the uncoupling ability was reinforced. CONCLUSIONS: Considering the importance of NAFLD in clinics and UCP in energy metabolism, we believe that this review may raise research enthusiasm on the effect of UCP in NAFLD and provide a novel mechanism and therapeutic target for NAFLD.

Changes of CD8⁺ T cells in dextran sulfate sodium-induced colitis mice pretreated with oral immune regulation.

BACKGROUND: It has been reported that CD8(+) regulatory cells could be induced upon oral tolerance. The purpose of this study was to investigate the changes of CD8α(+) T cells in dextran sulfate sodium (DSS)-induced colitis mice pretreated by oral immune regulation. METHODS: The effects of five low oral doses of colitis-extracted proteins (CEP) on colitis were evaluated by clinical manifestation and histological lesions. The percentages of CD8α(+) T cells gating on CD3(+) T cells were evaluated in the gut-associated lymphoid tissues (GALT) and the spleens by flow cytometry. Differences between the two groups were compared by Student's t test or Mann-Whitney U test. RESULTS: Compared to bovine serum albumin (BSA)-fed control mice, administration of CEP resulted in marked alleviation of colitis. The proportion of CD8α(+) T cells, not only in intraepithelial lymphocytes (IELs) and lamina propria lymphocytes (LPLs) of the large intestine (LI) but also in spleen from CEP-fed colitis mice, was significantly higher than that from BSA-fed colitis mice (LI-IELs: (71.5 ± 5.4)% vs. (60.1 ± 4.3)%, P < 0.01; LI-LPLs: (60.7 ± 5.2)% vs. (51.9 ± 4.7)%, P < 0.01; spleen: (24.1 ± 3.6)% vs. (20.3 ± 4.1)%, P < 0.05; n = 8). Mucosal repair in repair-period mice five days after termination of DSS treatment was also accompanied by an increase of CD8α(+) T cells in large intestinal mucosal lymphocytes (LI-IELs: (72.1 ± 3.7)% vs. (61.5 ± 4.5)%, P < 0.01; LI-LPLs: (62.1 ± 5.7)% vs. (52.7 ± 3.6)%, P < 0.01; n = 8). The proportion of CD3(+) T cells increased in Peyer's patches (PPs) and decreased in mesenteric lymph nodes (MLNs) from colitis mice compared to untreated mice, whereas the change pattern of CD3(+) T cells in PPs and MLNs from CEP-fed colitis mice was just on the contrary. CONCLUSION: Improvement of DSS-induced colitis resulted from oral immune regulation is associated with an increase in CD8α(+) T cells in spleen and large intestinal mucosa.

Effects of appendectomy and oral tolerance on dextran sulfate sodium colitis.

AIM: To evaluate the concomitant effects of appendectomy and oral tolerance on colitis. METHODS: Delayed-type hypersensitivity (DTH) was investigated at a 7-d interval after ovalbumin (OVA) administration and immunization under normal and colitis conditions in appendectomized or sham-operated mice. Pathological scores for the colon were graded after ingestion of colon-extracted protein (CEP) and induction of dextran sulfate sodium (DSS) colitis in appendectomized or sham-operated mice. Thereafter, Th1 and Th2 in Peyer's patches and spleen lymphocytes were detected in CEP-treated and bovine serum albumin (BSA)-treated control mice. RESULTS: In appendectomized mice, DTH was not inhibited at day 7 after OVA administration and at the initial phase of DSS colitis, whereas it was inhibited at day 14 and day 21. However, in sham-operated mice, it was inhibited during the whole procedure and the onset of DSS colitis. The protective role of CEP against DSS colitis was present in sham-operated mice, with predominant improvement of colonic pathological changes, while vanished in the appendectomized mice. A shift from Th1 to Th2 in Peyer's patches resulted from a decrease of Th1 cells with the ingestion of CEP. Compared with BSA in the sham-operated group, no predominant changes were observed in the appendectomized mice. CONCLUSION: Appendectomy interferes with the protective role of CEP in DSS colitis via a shift from Th2 to Th1 during oral tolerance induction.