Modulation of porcine β-defensins 1 and 2 upon individual and combined Fusarium toxin exposure in a swine jejunal epithelial cell line

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Lactobacillus rhamnosus GG modulates intestinal barrier function and inflammation in BALB/C mice following dietary exposure to deoxynivalenol and zearalenone through changes in gut

Oral Presentation 2Conference Theme: From Experience to PerspectivesDeoxynivalenol (DON) and zearalenone are mycotoxins produced by Fusarium species, which naturally co-occur in foods and feeds. The gastrointestinal tract represents the first barrier met by exogenous food/feed compounds. The purpose of the present study was to investigate the ability of Lactobacillus rhamnosus GG (LGG) to improve intestinal barrier functions and ameliorate inflammation in Balb/c mice (6 weeks old) fed diets containing mycotoxin mixtures (i.e. DON and ZEA) through modulation of intestinal bacterial …published_or_final_versio

Modulation of mucin mRNA (MUC5AC and MUC5B) expression and protein production and secretion in Caco-2/HT29-MTX co-cultures following exposure to individual and combined Fusarium mycotoxins.

Intestinal epithelial cells (IECs) are a critical component of the innate local immune response. In order to reduce the risk of pathogen infection or xenobiotic intoxication, different host defense mechanisms have been evolved. Evidence has shown that upon ingestion of food or feed contaminated with toxins (e.g., mycotoxins), IECs respond by regulating mucin secretions, which act as a physical barrier inhibiting bacterial attachment and subsequent infection-related processes. However, the effect of Fusarium mycotoxins on mucin production remains unclear. Consequently, the aim of this study was to evaluate individual and interactive effects of four common Fusarium mycotoxins, deoxynivalenol, nivalenol, zearalenone, and fumonisins B1 on mRNA expression and secretion of mucins, MUC5AC, and MUC5B, as well as total mucin-like glycoprotein secretion, using Caco-2 (absorptive-type) and HT29-MTX (secretive-type) cells and their co-cultures (initial seeding ratios Caco-2/HT29-MTX: 90/10 and 70/30). Our results showed that individual and mixtures of mycotoxins significantly modulated MUC5AC and MUC5B mRNA and protein, and total mucin-like glycoprotein secretion as measured by quantitative polymerase chain reaction, enzyme-linked immunosorbent assay, and enzyme-linked lectin assay, respectively. Additive effects were not always observed for mixtures. Also, the present study showed that in co-cultures, lower MUC5AC and MUC5B mRNA, protein and total mucin production occurred following exposure, which might suggest higher intestinal permeability and susceptibility to toxin exposure. This study demonstrates the importance of selecting an appropriate cell model for the in vitro investigation of Fusarium mycotoxin effects either alone or in combinations on the immunological defense mechanisms of IECs, and will contribute to improved toxin risk assessments.link_to_OA_fulltex

The cell cycle effects of docosahexaenoic acid on human metastatic hepatocellular carcinoma proliferation

Given the reported side effects associated with chemotherapy and surgical resection, dietary intervention with ω-3 polyunsaturated fatty acids (PUFAs) has been postulated to be an alterative way to prevent liver cancer progression and metastasis. We studied the effects of an ω-3 PUFA, docahexaenoic acid (DHA) on COX-2 expression and the cell cycle control machinery that co-ordinately regulate the HCC cells growth. Our data showed that DHA (0-200 μM) retarded proliferation of the human metastatic HCC cell line MHCC97L dose-dependently. In addition, inhibition of cyclin A/Cdk2 interfered with S-phase progression further in agreement with the result of bivariate flow cytometric analysis which indicated that DNA synthesis time (T s) was significantly prolonged by DHA in MHCC97L. The N-myc oncogene, the heat shock proteins Hsp27 and glucose-related protein 78 (GRP78) as well as the antioxidant enzymes superoxide dismutase may play significant roles in the cell cycle control and reduced-proliferation of MHCC97L by DHA. Our data indicated that it is imperative to develop therapeutic strategy with ω-3 PUFA that simultaneously targets COX-2 and other cell cycle regulators in hepatocarcinogenesis. This study provides novel mechanistic insights into the modulation of DHA on human hepatocarcinoma.link_to_OA_fulltex

Identification of local and circulating cancer stem cells in human liver cancer

Increasing evidence has revealed the importance of cancer stem cells (CSCs) in carcinogenesis. Although liver CSCs have been identified in hepatocellular carcinoma (HCC) cell lines, no data have shown the presence of these cells in human settings. The present study was designed to delineate CSCs serially from HCC cell lines, human liver cancer specimens to blood samples, using CD90 as a potential marker. The number of CD90+ cells increased with the tumorigenicity of HCC cell lines. CD45-CD90+ cells were detected in all the tumor specimens, but not in the normal, cirrhotic, and parallel nontumorous livers. In addition, CD45-CD90+ cells were detectable in 90% of blood samples from liver cancer patients, but none in normal subjects or patients with cirrhosis. A significant positive correlation between the number of CD45-CD90+ cells in the tumor tissues and the number of CD45-CD90+ cells in the blood samples was identified. CD90+ cells sorted from cell lines and CD45-CD90+ cells from the tumor tissues and blood samples of liver cancer patients generated tumor nodules in immunodeficient mice. Serial transplantation of CD90+ cells from tumor xenografts generated tumor nodules in a second and subsequently third batch of immunodeficient mice. Treatment of CD90+ CSCs with anti-human CD44 antibody induced cell apoptosis in a dose-dependent manner. Conclusion: Identification of CD45 -CD90+ CSCs in both tumor tissues and circulation suggests that CD45-CD90+ could be used as a marker for human liver cancer and as a target for the diagnosis and therapy of this malignancy. Copyright © 2007 by the American Association for the Study of Liver Diseases.link_to_subscribed_fulltex

Molecular features and functional consequence of CD44 activation by a novel recurrent IGH translocation t(11;14) (p13;q32) in mature B-cell lymphoid neoplasm

Poster Session 8 - Functional Identification of New Cancer Genes: abstract no. 258Dysregulation of an oncogene by translocation to Ig locus is a common event in the pathogenesis of most non-Hodgkin’s lymphoma. Using inverse-PCR, CD44 on 11p13 was identified as a novel translocation partner of IgH in 9 of 114 cases of gastric, non-gastric extranodal, follicular and nodal diffuse large B-cell lymphomas (DLBCLs) analyzed. IgHSμ/CD44 translocation juxtaposes the enhancer of IgHSμ to the 5’ regulatory region of CD44 in a tail-to-head orientation, leading to the removal of exon 1 of CD44. Sequencing analysis showed microhomology sequences at the junction breakpoints of all the IGHSμ/CD44 translocations, suggesting that these translocations were the results of illegitimate switch recombination facilitated by homologous sequences present on both chromosomes. By interphase-FISH using home-grown CD44 dual-color break-apart probes (consisting of BACs RP4-607I7 and RP4-683L5), breakage at the CD44 locus in each of the nine IGHSμ/CD44 translocation-positive cases was confirmed. By 5’ RACE analysis, fusion Iμ-CD44ΔEx1 hybrid transcripts (with a splicing of the Iμ exon upstream of Sμ to exon 2 of CD44), were identified in all the nine lymphomas with IGHSμ/CD44 translocations. Notably, these translocations were detected exclusively in GCB-type DLBCLs. However, CD44 mRNA was minimally or not expressed in CD10+ microdissected reactive GCB cells. The IGHSμ/CD44 translocation substitute Sμ for the CD44 promoter and remove exon 1 of CD44, resulting in over-expression of the Iμ-CD44 hybrid transcript which encodes for a new CD44 variant lacking leader peptide sequence but retaining a unique C-terminus (CD44ΔEx1). The Iμ-CD44ΔEx1 ORF would encode for the CD44ΔEx1 protein starting from the ATG at nucleotide 254 with strong Kozak sequence. The new CD44ΔEx1 variant showed some similarity to CD44v5, but it lacked the leader peptide. The IGHSμ/CD44 translocation was detected in patients with advanced-stage disease (stages III and IV). When overexpressed in vitro in the CD44-negative GCB-DLBCL cell line BJAB, the CD44ΔEx1-GFP was localized to the cytoplasm and nucleus, while CD44s-GFP (standard form) was localized to the plasma membrane. The ectopic expression of CD44ΔEx1 in BJAB cells enhanced the cell proliferation rate and its clonogenic ability. These findings indicate a possible pathogenic role of the recurrent translocation in several malignant B-cell lymphomas.link_to_OA_fulltextThe 101st Annual Meeting of the American Association for Cancer Research (AACR 2010), Washington D.C., 17-21 April 2010. In AACR Meeting Abstracts, 201

CD44 expression is regulated by translocation and promoter hypermethylation in gastric lymphoma

One of the major genetic features of non-Hodgkin’s lymphomas (NHL) is the chromosomal translocation, most frequently involving the immunoglobulin heavy chain (IGH) gene at band 14q32. By employing the IgH dual color, break apart rearrangement probe (Vysis Inc.) for interphase FISH; followed by the long-distance inverse polymerase chain (LDI-PCR) strategy, we identified CD44 at chromosome 11p13 as a novel translocation partner gene of IGH in primary GL. The IGH/CD44 t(11;14)(p13;q32) was detected in 40% (4/10) mucosa-associated lymphoid tissue (MALT) lymphoma (3 positive cases are transformed MALT lymphoma cases) and 10% (4/40) diffuse large B-cell lymphoma (DLBCL) cases. This finding makes CD44 as the first cell adhesion molecule involving the IGH translocation and the third gene identified that involves the IGH translocation in MALT lymphoma. Immunohistochemistry showed that CD44 was expressed strongly in the membrane and cytoplasm of the tumor cells in 40% (4/10) MALT cases and 67.5% (27/40) DLBCL cases, including 8 IGH/CD44 translocation positive cases, while the remaining cases showed very weak or no CD44 expression. We next determined whether the hypermethylation of the promoter region of CD44 gene was responsible for the downregulation of CD44 in GL cases lacking CD44 expression. We first analyzed 10 mature B-cell lymphoma cell lines for the CD44 methylation status by methylation-specific PCR (MSP) and bisulfite genomic sequencing (BGS). Methylated alleles were detected in all the cell lines lacking CD44 expression but not in cell lines with CD44 expression. The treatment of the cell lines lacking CD44 with the demethylation agent 5-Aza-dC restored the CD44 expression. In the GL cases, methylated alleles were detected in 42% (21/50) cases and they significantly correlated with weak or no CD44 protein expression in the tumor cells. The most interesting observation was that the 3 CpG sites contained by E2F, Sp1 and EGR1 were nearly always methylated in cell lines lacking CD44 expression but not in the cell lines with CD44 expression. Similar results were also found in the GL cases. The correlation of CD44 expression and clincopathological parameters showed that strong CD44 expression was associated with poor prognosis in GL, and the cases with unmethylated CD44 were positively correlated with high stage (p=0.023). Overall, our results show that CD44 expression is regulated by different mechanisms operating simultaneously in GL, chromosomal translocation in cases with strong CD44 expression and gene silencing by promoter hypermethylation in cases lacking CD44

CD44 activation in mature B-cell malignancies by a novel recurrent IGH translocation

Using inverse polymerase chain reaction, we identified CD44, located on chromosome 11p13, as a novel translocation partner of IGH in 9 of 114 cases of gastric, nongastric extranodal, follicular, and nodal diffuse large B-cell lymphoma (DLBCL). Notably, these translocations involving IGHSμ were detected in follicular lymphomas and exclusively in germinal center B cell - like (GCB) - DLBCLs. CD44 is not expressed in reactive GC B cells. The IGHSμ/CD44 translocations substitute Sμ for the CD44 promoter and remove exon 1 of CD44, resulting in the overexpression of Iμ-CD44 hybrid mRNA transcripts activated from derivative 11 that encode a new CD44 variant lacking the leader peptide and with a unique C-terminus (CD44ΔEx1). When overexpressed in vitro in the CD44 - GCB-DLBCL cell line BJAB, CD44ΔEx1 - green fluorescent protein localized to the cytoplasm and nucleus, whereas CD44s - green fluorescent protein (standard form) localized to the plasma membrane. The ectopic expression of CD44ΔEx1 in BJAB cells enhanced their proliferation rate and clonogenic ability, indicating a possible pathogenic role of the translocation. © 2010 by The American Society of Hematology.link_to_OA_fulltex