Trefoil Factor Family Research Articles

The Forms of the Lectin Tff2 Differ in the Murine Stomach and Pancreas: Indications for Different Molecular Functions.

The lectin TFF2 belongs to the trefoil factor family (TFF). This polypeptide is typically co-secreted with the mucin MUC6 from gastric mucous neck cells, antral gland cells, and duodenal Brunner glands. Here, TFF2 fulfills a protective function by forming a high-molecular-mass complex with the MUC6, physically stabilizing the mucus barrier. In pigs and mice, and slightly in humans, TFF2 is also synthesized in the pancreas. Here, we investigated the murine stomach, pancreas, and duodenum by fast protein liquid chromatography (FPLC) and proteomics and identified different forms of Tff2. In both the stomach and duodenum, the predominant form is a high-molecular-mass complex with Muc6, whereas, in the pancreas, only low-molecular-mass monomeric Tff2 was detectable. We also investigated the expression of Tff2 and other selected genes in the stomach, pancreas, and the proximal, medial, and distal duodenum (RT-PCR analysis). The absence of the Tff2/Muc6 complex in the pancreas is due to a lack of Muc6. Based on its known motogenic, anti-apoptotic, and anti-inflammatory effects, we propose a protective receptor-mediated function of monomeric Tff2 for the pancreatic ductal epithelium. This view is supported by a report that a loss of Tff2 promotes the formation of pancreatic intraductal mucinous neoplasms.

Abstract 704: MDSC-targeted TFF2-MSA suppresses tumor growth and increases survival in anti-PD-1 treated MC38 and CT26.wt murine colorectal cancer models

Abstract Aims: Myeloid-derived suppressor cells (MDSCs) in the tumor microenvironment are a potential therapeutic target in immune checkpoint cancer therapy, but MDSC-targeted therapies have yet been shown to improve survival. Trefoil factor family 2 (TFF2), a secreted anti-inflammatory peptide, can suppress MDSC expansion and activate tumor immunity in part through agonism of the CXCR4 receptor. The aim of this study is to investigate whether a novel TFF2 - albumin fusion peptide (TFF2-MSA) can improve survival in anti-PD-1 treated syngeneic colorectal cancer (CRC) mouse models. Methods: Two syngeneic colon carcinoma mouse models were developed using cell lines grafted subcutaneously into mice. MC38 CRC cells were engrafted into C57BL/6 mice while CT26.wt CRC cells were implanted into BALB/C mice We generated a recombinant fusion protein, designated TFF2-MSA, which contains murine TFF2 fused to murine serum albumin (MSA), for the purpose of increasing half-life and reducing dose frequency. Mice subsequently received either TFF2-MSA or anti-PD-1 antibody (clone 29F.1A12) or both, and tumor volume, and survival were measured. At the endpoint, flow cytometry was performed to examine treatment-induced effects on immune profiles. Results: In the MC38 model, administration of TFF2-MSA suppressed tumor growth (TGI 38%), the combination of TFF2-MSA and anti-PD-1 had an additive effect and suppressed tumor growth dramatically (TGI 74%). The combination also exhibited a survival rate of 90% after 50 days, while vehicle and single TFF2-MSA therapy were 30% and 65%, respectively. The percentage of exhausted CD8+ T cells was markedly reduced in the draining lymph node by the combination treatment, as measured by flow cytometry using antibodies against LAG3, TIM3 and PD-1. In the CT26.wt model, administration of TFF2-MSA alone exhibited little effect, but the combination of anti-PD-1 and TFF2-MSA showed a profound effect. In the CT26.wt model, administration of TFF2-MSA suppressed tumor growth (TGI 17%), anti-PD-1 alone (TGI 43%) and the combination of TFF2-MSA and anti-PD-1 (TGI 54%). Conclusion: Targeting MDSCs using TFF2-MSA fusion protein synergizes well with PD-1 blockade therapy in advanced and metastatic syngeneic mouse models of colorectal cancer. In a separate abstract, additive effects between TFF2-MSA and anti-PD-1 antibody were also demonstrated in a separate ACKP (Atp4b-Cre; Cdh1-/-; LSL-KrasG12D; Trp53-/-) gastric cancer model, suggesting combination therapy may also be applicable to gastric cancer. Citation Format: Bruce L. Daugherty. MDSC-targeted TFF2-MSA suppresses tumor growth and increases survival in anti-PD-1 treated MC38 and CT26.wt murine colorectal cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 704.

Abstract 5088: MDSC-targeted TFF2-MSA synergizes with PD-1 blockade therapy in diffuse-type gastric cancer

Abstract Aims: Recent studies revealed myeloid-derived suppressor cell (MDSC) in the tumor microenvironment as an appealing therapeutic target to sensitize intestinal-type gastric cancer (GC) to PD-1 blockade therapy. However, whether MDSC inhibition can improve survival in the more aggressive diffuse-type GC is unknown. Trefoil factor family 2 (TFF2), a secreted anti-inflammatory peptide, suppresses MDSC expansion partly via CXCR4 receptor. Here, we developed a novel MDSC-targeted peptide TFF2-MSA and investigated whether it can synergize with anti-PD1 to prolong survival in syngeneic diffuse GC mouse models. Methods: Murine serum albumin (MSA) was appended to the murine TFF2 to generate TFF2-MSA peptide with an extended serum half-life. ACKP (Atp4b-Cre; Cdh1-/-; LSL-KrasG12D; Trp53-/-) GC cells developed from a highly malignant diffuse GC mouse model were grafted subcutaneously into HDC-GFP transgenic mice in which the more immunosuppressive histidine decarboxylase (HDC)-expressing myeloid cells were traced with GFP. These recipient mice subsequently received either TFF2-MSA or anti-PD-1 antibody or both. Further, to assess treatment efficacy in inhibiting spontaneous lung metastasis, subcutaneous flank tumors were resected in ACKP cell-grafted mice once tumors reached 500 mm3, and mice then received treatment with TFF2-MSA or anti-PD-1 or the combination. At the endpoint, flow cytometry and histopathological analyses were performed to examine immune profiles and tumor metastasis. In another model, ACKP-luc cells were implanted orthotopically to stomach submucosa, and recipient mice received the same treatments started from 1 week later to assess their efficacy. Results: While either TFF2-MSA or PD-1 antibody showed little benefit as a single agent (TGI 15% and 25% respectively, p>0.05), their combination dramatically suppressed ACKP s.c. tumor growth (TGI 78%, p<0.0001) and markedly prolonged median survival (64 days vs. 32.5 days in control) of host mice in a synergistic manner. The combination therapy efficiently reduced HDC-GFP+ MDSCs (by 67% vs. control, p<0.01), and profoundly increased tumor-infiltrating cytotoxic CD8+ T cells (by 18 fold, p<0.001). Further, while standard chemotherapy (5-fluorouracil and oxaliplatin) showed limited efficacy in this model, addition of chemotherapy to the TFF2-MSA/PD-1 antibody combination further improved treatment efficacy (TGI 91%) and extended median survival to 73 days. In addition, combination of TFF2-MSA/PD-1 antibody significantly reduced 80% lung metastasis (vs. control, p<0.0001), in contrast to minimal inhibition observed with either monotherapy (p>0.05). Finally, in the orthotopic model, the combo regimen eradicated GC in 50% mice compared to 0% in either monotherapy treatment. Conclusion: Targeting MDSCs using TFF2-MSA synergizes with PD-1 blockade therapy in advanced and metastatic syngeneic mouse models of diffuse-type GC. Citation Format: Jin Qian, Sandra Ryeom, Bruce Daugherty, Seth Lederman, Timothy C. Wang. MDSC-targeted TFF2-MSA synergizes with PD-1 blockade therapy in diffuse-type gastric cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5088.

Investigation of the Relationship Between Trefoil Factor Family Peptides and Sinonasal Inflammation.

The trefoil factor family (TFF) is a relatively new family of peptides. In some studies, an association between trefoil factors and inflammatory diseases of the nasal and paranasal sinuses has been suggested. However, it is still not clear whether there is a relationship between trefoil peptides and inflammation of the respiratory tract. The aims of this study are to determine the presence of TFF1, TFF2, and TFF3 in the nasal mucosa and investigate their relationships with inflammation by using rat models of various sinonasal inflammations. Nasal tampon, lipopolysaccharide, and ovalbumin were used to generate rat models of sinonasal inflammation, i.e., rhinosinusitis and allergic rhinitis. The study was conducted on seventy rats in seven groups, each with ten rats: four groups with rhinosinusitis, two groups with allergic rhinitis, and a control group. Histological evaluation of sinonasal mucosa from all rats was performed, and Trefoil factors were investigated using immunohistochemical methods. All three TFF peptides were detected in rat nasal mucosa by histological evaluation. No significant differences in the trefoil factor scores were observed among the study groups. A significant correlation between the TFF1 and TFF3 scores and loss of cilia was identified (p < 0.05). In conclusion, no direct relationship between sinonasal inflammation and TFF scores was observed. However, a possible association between the TFF and epithelial damage or regeneration in sinonasal inflammation can be suggested based on the correlation observed between the TFF1 and TFF3 scores and scores of cilia loss.

Trefoil factor family proteins as potential diagnostic markers for mucinous invasive ovarian carcinoma.

Ovarian cancer (OC) is the leading cause of gynecological cancer-related death. Of the main OC histologic subtypes, invasive mucinous carcinomas (MC) account for only 3% of OC cases and are frequently associated with favorable prognosis. Nevertheless, MCs differ greatly from the other OC histotypes in clinical, pathological, and biological behavior. However, the origin and molecular pathogenesis of MC are not yet fully understood. Therefore, identification of novel diagnostic markers could potentially facilitate early diagnosis of OC, particularly the MC histotype, thereby leading to the development of histotype-specific treatment regimens and improved survival rates. In the present study, Trefoil factor gene family members (TFF1, TFF2 and TFF3) were identified as MC histotype-specific biomarkers using RNA sequencing (RNA-seq) data for 95 stage I-II OCs. The diagnostic value of TFF1, TFF2 and TFF3 was then evaluated by immunohistochemistry on 206 stage I-II OCs stratified by histotype (high-grade serous carcinoma [HGSC], endometrioid carcinoma [EC], clear cell carcinoma [CCC], and MC). We showed significantly elevated intracytoplasmic protein expression levels for TFF1, TFF2 and TFF3 in MC samples, thereby revealing an association between expression of Trefoil factor gene family members and the MC histotype. Taken together, these findings suggest that the TFF proteins may play a pivotal role in tumor initiation and progression for the MC histotype. Taken together, these findings suggest that the TFF proteins may play a pivotal role in tumor initiation and progression for the MC histotype. Moreover, these novel histotype-specific diagnostic biomarkers may not only improve patient stratification of early-stage ovarian carcinomas but may also be candidates for the development of molecular targeted therapies.

Serum trefoil factor family 3 as a biomarker for the detection of colorectal cancer.

229 Background: Colorectal cancer is one of the most frequent causes of cancer-related deaths worldwide. Thus, there is a need for timely diagnosis and effective treatment. Our aim in the present study was to detect the serum level of trefoil factor family 3 (TFF3) and evaluate the diagnostic accuracy of TFF3 in patients with colorectal cancer. Methods: We prospectively collected serum samples from 72 subjects (36 patients with colorectal cancer and 36 healthy controls) in the derivation cohort. The validation cohort prospectively included 70 subjects (35 patients with colorectal cancer and 35 healthy controls). The levels of serum TFF3 and carcinoembryonic antigen (CEA) were examined using enzyme-linked immunosorbent assay. Results: The serum levels of TFF3 in patients with colorectal cancer was significantly higher compared with that in the healthy controls (9.34±12.2 ng/mL vs. 3.60±1.58 ng/mL, P = 0.008) in the derivation cohort. Using a cutoff of 3.85 ng/mL in the derivation cohort, the sensitivity, specificity, and area under the curve of serum TFF3 for the detection of advanced colorectal cancer were 0.972, 0.611, and 0.875, respectively. The sensitivity of serum TFF3 for the detection of colorectal cancer was superior (97.2%) to that of CEA (36.1%) in the derivation cohort. In the validation cohort, the sensitivity, specificity, and area under the curve of serum TFF3 for the detection of colorectal cancer were 0.771, 0.486, and 0.751, respectively. Similarly, the sensitivity of serum TFF3 for the detection of colorectal cancer was also superior (77.1%) to that of CEA (22.9%) in the validation cohort. Conclusions: Serum TFF3 is a potential biomarker for the detection of colorectal cancer and is more effective compared with CEA.

The Milk Active Ingredient, 2'-Fucosyllactose, Inhibits Inflammation and Promotes MUC2 Secretion in LS174T Goblet Cells In Vitro.

In several mice inflammatory models, human milk oligosaccharides (HMOs) were shown to protect the intestinal barrier by promoting mucin secretion and suppressing inflammation. However, the functions of the individual HMOs in enhancing mucin expression in vivo have not been compared, and the related mechanisms are not yet to be clarified. In this study, we investigated the modulatory effects of 2′-fucosyllactose (2′-FL), 3′-sialyllactose (3′-SL), galacto-oligosaccharide (GOS) and lactose (Lac) on goblet cells’ functions in vitro. The appropriate dosage of the four chemicals was assessed in LS174T cells using the CCK-8 method. Then they were supplemented into a homeostasis and inflammatory environment to further investigate their effects under different conditions. Mucin secretion-related genes, including mucin 2 (MUC2), trefoil factor family 3 (TFF3), resistin-like β (RETNLB), carbohydrate sulfotransferase 5 (CHST5) and galactose-3-O-sulfotransferase 2 (GAL3ST2), in LS174T cells were detected using quantitative RT-qPCR. The results showed that 2′-FL (2.5 mg/mL, 72 h) was unable to increase MUC2 secretion in a steady-state condition. Comparatively, it exhibited a greater ability to improve mucin secretion under an inflammatory condition compared with GOS, demonstrated by a significant increase in TFF3 and CHST5 mRNA expression levels (p > 0.05). However, 3′-SL and Lac exhibited no effects on mucin secretion. To further investigate the underlying mechanism via which 2′-FL enhanced goblet cells’ secretion function, the NOD-like receptor family pyrin domain containing 6 (NLRP6) gene, which is closely related to MUC2 secretion, was silenced using the siRNA method. After silencing the NLRP6 gene, the mRNA expression levels of MUC2, TFF3 and CHST5 in the (2′-FL + tumor necrosis factor α (TNF-α) + NLRP6 siRNA) group were significantly decreased compared with the (2′-FL + TNF-α) group (p > 0.05), indicating that NLRP6 was essential for MUC2 expression in goblet cells. We further found that 2′-FL could significantly decrease toll-like receptor 4 (TLR4, p < 0.05), myeloid differential protein-88 (MyD88, p < 0.05) and nuclear factor kappa-B (NF-κB, p < 0.05) levels in LS174T inflammatory cells, even when the NLRP6 was silenced. Altogether, these results indicated that in goblet cells, 2′-FL exerts its function via multiple processes, i.e., by promoting mucin secretion through NLRP6 and suppressing inflammation by inhibiting the TLR4/MyD88/NF-κB pathway.

Different Molecular Forms of TFF3 in the Human Respiratory Tract: Heterodimerization with IgG Fc Binding Protein (FCGBP) and Proteolytic Cleavage in Bronchial Secretions

The polypeptide TFF3 belongs to the trefoil factor family (TFF) of lectins. TFF3 is typically secreted from mucous epithelia together with mucins. Both intestinal and salivary TFF3 mainly exist as disulfide-linked heterodimers with IgG Fc binding protein (FCGBP). Here, we investigated bronchial tissue specimens, bronchial secretions, and bronchoalveolar lavage (BAL) fluid from patients with a chronic obstructive pulmonary disease (COPD) background by fast protein liquid chromatography and proteomics. For the first time, we identified different molecular forms of TFF3 in the lung. The high-molecular mass form represents TFF3-FCGBP oligomers, whereas the low-molecular mass forms are homodimeric and monomeric TFF3 with possibly anti-apoptotic activities. In addition, disulfide-linked TFF3 heterodimers with an Mr of about 60k and 30k were detected in both bronchial secretions and BAL fluid. In these liquids, TFF3 is partly N-terminally truncated probably by neutrophil elastase cleavage. TFF3-FCGBP is likely involved in the mucosal innate immune defense against microbial infections. We discuss a hypothetical model how TFF3 might control FCGBP oligomerization. Furthermore, we did not find indications for interactions of TFF3-FCGBP with DMBT1gp340 or the mucin MUC5AC, glycoproteins involved in mucosal innate immunity. Surprisingly, bronchial MUC5AC appeared to be degraded when compared with gastric MUC5AC.

Trefoil Factor 3 Protein and Sepsis

Sepsis is one of the most common causes of death in hospitalized patients. Disruption of intestinal barrier homeostasis is one of its main hallmarks. Trefoil factor family proteins are known for their role in protecting and repairing the intestinal mucosa. It has been repeatedly shown that the TFF3 protein is involved in maintaining the intestinal barrier. For that reason, it has been studied in the search for objective measures to predict the onset or outcome of sepsis. Several studies have been performed on rodent sepsis models and on sepsis patients, both children and adults. From the limited research available to date, it appears that TFF3 is involved in the pathogenesis of sepsis, but the exact mechanism is not yet clear. Its potential as a sepsis biomarker has so far been low, but more extensive studies on its role in predicting disease severity and outcome, as well as organ dysfunction, may lead to finding specific patient groups or sepsis stages for which it would be suitable.

Effect of electroacupuncture on the repair of stress ulcer injury in neurocritical patients: A randomized clinical trial.

Stress ulcer (SU) is one of the main causes of prolonged hospital stay, poor prognosis, and increased mortality in critically ill patients. This study aimed to investigate the effect of electroacupuncture (EA) on SU in patients with severe neurological diseases and explore its possible mechanisms. Taking patients with SU in adult neurocritical care as the research object, they were randomly divided into the EA group and the control group. Through the perioperative EA intervention, the following indicators were documented: main observation indicator (the effective rate of SU treatment), secondary observation indicators (gastric juice pH, gastric juice occult blood test, and stool occult blood test), related mechanisms [repair factors trefoil factor family 2 (TFF2), vascular endothelial growth factor (VEGF), and heat shock protein 70 (HSP70)], complications during hospitalization, and intensive care unit (ICU) stay time. Compared with the control treatment, EA increased the effective rate of SU treatment (85.4% for the EA group, 57.5% for the control group, risk difference: 27.9% (95% CI: 8.3%-45.1%); P < 0.01). EA increased the success rate of gastric juice pH treatment on days 1, 2, and 3 (P < 0.01 for day 1, P < 0.05 for days 2 and 3). EA lowered the positive rate of gastric occult blood test on days 1 and 3 (all P-values < 0.05) and the positive rate of fecal occult blood test on day 3 (P < 0.05). EA also reduced the ICU stay time (P < 0.01) and total hospitalization time (P < 0.05). Compared with day 0, all serum repair factors (VEGF, HSP70, and TFF2) of both groups significantly increased on days 1, 3, and 5 (all P-values < 0.01). Compared with the control group, VEGF in the EA group was increased on days 3 and 5 (all P-values < 0.01); HSP70 was increased on days 1, 3, and 5 (P < 0.05 for day 1, P < 0.01 for days 3 and 5); and TFF2 was increased on days 1, 3, and 5 (all P-values < 0.01). Electroacupuncture promoted the repair of SU damage in severe neurological disease, and its effect was related to enhancing the expression of gastric mucosal repair factors. [https://www.chictr.org.cn/showprojen.aspx?proj=127012], identifier [ChiCTR2100046701].

Effects of dietary Bopu powder supplementation on intestinal development and microbiota in broiler chickens.

This study aimed to investigate the effect of dietary supplementation with Bopu powder on intestinal development and bacterial community composition in broiler chickens. A total of 486 1-day-old arbor acres broilers were fed a basal diet (CON group), a basal diet supplemented with 50 mg/kg aureomycin (AB group), or a basal diet supplemented with 40 mg/kg Bopu powder (BP group). The results showed that the BP group had significantly lower serum tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, and diamine oxidase concentrations and had significantly higher serum IL-10 concentrations than CON group (p < 0.05). Groups AB and BP had a significantly higher weight per unit length of the small intestine and villus height than the CON group (p < 0.05), and BP group had a significantly higher ratio of villus height to crypt depth than groups CON and AB (p < 0.05). Compared to the CON group, dietary Bopu powder or aureomycin supplementation significantly increased transforming growth factor-α concentration and mRNA expressions of zonula occludens-1 (ZO-1) and occludin, and decreased intestinal mucosal concentrations of TNF-α, IL-6, IL-10, caspase-3, and caspase-8 and mRNA expressions of nuclear factor-kappa-B and Bax/Bcl-2 ratio in the intestinal mucosa (p < 0.05). Meanwhile, BP group had significantly higher ZO-1, secretory immunoglobulin A, interferon-γ concentrations, and mRNA expressions of glucose transporter type-2 and sirtuin-1, and significantly lower IL-1β concentration than groups CON and AB in intestinal mucosa (p < 0.05). Dietary Bopu powder supplementation significantly increased the concentration of trefoil factor family member and mRNA expressions of superoxide dismutase-1 and bcl-2 associated X, and significantly reduced casepase-9 concentration and myeloid differentiation primary response-88 expression in the intestinal mucosa of broiler chickens relative to CON group (p < 0.05). Moreover, results of high-throughput sequencing showed that broilers in the BP group had microbial community structure distinct from that in CON group, and the addition of Bopu powder increased the abundances of Faecalibacterium and Colidextribacter (p < 0.05). Therefore, our study suggests a synergic response of intestinal development and microbiota to the Bopu powder, and provides a theoretical basis as a potential substitute for antibiotics.

Effect of live Eimeria vaccination or salinomycin on growth and immune status in broiler chickens receiving in-feed inclusion of gelatin and vitamin E

This experiment determined if 2% of gelatin, to improve the levels of proline and glycine in the diet, and 70 mg/kg of vitamin E supplementation would relieve the impaired performance of male Cobb broilers vaccinated for coccidiosis. Half of the chicks were vaccinated via water (live oocysts), while the other half received medication (salinomycin) in the feed until 35 d of age. The effects of coccidiosis vaccine on performance and mRNA levels of genes involved in mucin synthesis, cytokines, trefoil family factor-2 (TFF2), and metabolic processes (CD36) in the jejunum of broilers were measured. Vaccination negatively affected performance in the first 21 d; however, the inclusion of gelatin and vitamin E reduced this negative response. Additionally, supplementation with these nutrients led to an improvement in broilers receiving the coccidiostat (P < 0.05). From 21 to 35 d, birds treated with gelatin and coccidiosis vaccine experienced better body weight gain than birds without gelatin and vitamin E (P < 0.05). Vaccinated chickens had decreased body weight and decreased anti-inflammatory cytokine expression. Furthermore, they had increased inflammatory cytokine expression, mucin 2 expression, and TFF2 compared to salinomycin-fed broilers (P < 0.05). Transcripts for IL-1B, IFN-y, MUC2, TFF2 were decreased while mRNAs for IL-4 and IL-10 increased in salinomycin-fed broilers compared to vaccinated broilers (P < 0.05). In conclusion, broilers vaccinated against coccidiosis increase their pro-inflammatory immune status and mucin expression compared to broilers receiving salinomycin. These events may contribute to lower performance in vaccinated broiler chicks. Moreover, vitamin E and gelatin can minimize the vaccine's negative immune effects and promote better performance.

Supplementation with paraformic acid in the diet improved intestinal development through modulating intestinal inflammation and microbiota in broiler chickens.

The aim of this study was to explore the effects of supplementing paraformic acid (PFA) to the diet of broiler chickens on intestinal development, inflammation, and microbiota. A total of 378 healthy 1-day-old Arbor Acres broilers with similar birth weight were used in this study, and randomly assigned into two treatment groups. The broiler chickens were received a basal diet or a basal diet supplemented with 1,000 mg/kg PFA. Results showed that PFA supplementation increased (P < 0.05) small intestinal villus height and villus height/crypt depth ratio, elevated intestinal mucosal factors (mucin 2, trefoil factor family, and zonula occludens-1) concentrations, and upregulated mNRA expression of y + L amino acid transporter 1. Moreover, PFA supplementation decreased (P < 0.05) the concentrations of inflammatory cytokines (tumor necrosis factor-alpha, interleukin-1beta, interleukin-6, and interleukin-10), activities of caspase-3 and caspase-8, and mNRA expressions of Toll-like Receptor 4, nuclear factor-kappa B, Bax, and Bax/Bcl-2 ratio in small intestinal mucosa. Dietary PFA supplementation also increased (P < 0.05) alpha diversity of cecal microbiota and relative abundance of Alistipes. The present study demonstrated that supplementation of 1,000 mg/kg PFA showed beneficial effects in improving intestinal development, which might be attributed to the suppression of intestinal inflammation and change of gut microbiota composition in broiler chickens. These findings will aid in our knowledge of the mechanisms through which dietary PFA modulates gut development, as well as support the use of PFA in poultry industry.

Downregulation of the Tumor Suppressor TFF1 Is Required during Induction of Colon Cancer Progression by L1.

Simple SummaryAberrant activation of Wnt/β-catenin signaling and the subsequent induction of downstream target genes is a hallmark of colorectal cancer (CRC) development. Previously, we found that overexpression of the immunoglobulin-like cell adhesion receptor L1CAM (L1), a target of the Wnt/β-catenin pathway, confers enhanced proliferation, motility, tumorigenesis, and liver metastasis in CRC cells. Transcriptomic and proteomic analyses revealed changes in both pro-tumorigenic and potential tumor-suppressor genes in L1-overexpressing CRC cells. We wished to identify such tumor suppressor/s, and found that trefoil family factor 1 (TFF1) was involved in L1-mediated CRC progression. TFF1 overexpression suppressed the growth, motility and tumorigenesis of L1-expressing CRC cells by inhibiting the NF-κB pathway. In human CRC tissue, TFF1-positive staining was evident in goblet cells of the normal mucosa, while in CRC tissue, TFF1 expression was lost in >50% of the tumor samples. Our results support a tumor-suppressor role of TFF1 in human CRC, and we suggest that TFF1 could be used for CRC detection and as a novel therapeutic target in L1-mediated CRC.The immunoglobulin family cell adhesion receptor L1 is induced in CRC cells at the invasive front of the tumor tissue, and confers enhanced proliferation, motility, tumorigenesis, and liver metastasis. To identify putative tumor suppressors whose expression is downregulated in L1-expressing CRC cells, we blocked the L1–ezrin–NF-κB signaling pathway and searched for genes induced under these conditions. We found that TFF1, a protein involved in protecting the mucus epithelial layer of the colon, is downregulated in L1-expressing cells and displays characteristics of a tumor suppressor. Overexpression of TFF1 in L1-transfected human CRC cells blocks the pro-tumorigenic and metastatic properties conferred by L1 by suppressing NF-κB signaling. Immunohistochemical analyses revealed that human CRC tissue samples often lose the expression of TFF1, while the normal mucosa displays TFF1 in goblet cells. Identifying TFF1 as a tumor suppressor in CRC cells could provide a novel marker for L1-mediated CRC development and a potential target for therapy.

Bacillus subtilis programs the differentiation of intestinal secretory lineages to inhibit Salmonella infection.

The role of intestinal microbiota on fate determination of intestinal epithelial cells has not been extensively examined. In this study, we explore the effect of Bacillus subtilis on programmed intestinal epithelial differentiation. We find that B.subtilis stimulates the differentiation of intestinal secretory cells. Moreover, B.subtilis inhibits the Notch pathway to reduce the expression of hairy and enhancer of split 1, thereby shifting intestinal stem cell differentiation toward a secretory cell fate. Moreover, we demonstrate that the programming effect of B.subtilis on intestinal differentiation is Toll-like receptor 2 pathway dependent. B.subtilis is associated with increased numbers of Paneth and goblet cells in the intestine. This results in the production of antimicrobial peptides to protect the intestinal mucosal barrier against Salmonella typhimurium. This study demonstrates that B.subtilis contributes to the differentiation of secretory cells by affecting Notch pathway signaling to maintain the intestinal barrier.

Trefoil factor 1 inhibits the development of esophageal adenocarcinoma from Barrett's epithelium

AbstractTrefoil factor family 1 (TFF1) is one of three members of the trefoil factor family that are abundantly expressed in the gastrointestinal mucosal epithelium. Recent studies have shown that TFF1 acts as a tumor suppressor in gastric, pancreatic and hepatocellular carcinogenesis; however, little is known about its function in esophageal carcinogenesis, especially in esophageal adenocarcinoma (EAC). Barrett's epithelium is the metaplastic columnar epithelium of the esophagus and a known premalignant lesion of EAC. To investigate the role of TFF1 in EAC development, a mouse model of Barrett's epithelium was employed, and human specimens of EAC were assessed by immunohistochemistry (IHC) and methylation-specific PCR. Wild-type (WT) mice underwent gastrojejunostomy on the forestomach, resulting in the development of Barrett's epithelium-like (BE-like) epithelium adjacent to the anastomotic site. BE-like epithelium in these mice expressed TFF1, indicating the association of TFF1 with esophageal adenocarcinoma. TFF1-knockout (TFF1KO) mice underwent the same procedure as well, revealing that a deficiency in TFF1 resulted in the development of adenocarcinoma in the anastomotic site, presumably from BE-like epithelium. IHC of human samples revealed strong TFF1 expression in Barrett's epithelium, which was lost in some EACs, confirming the association between TFF1 and EAC development. Aberrant DNA hypermethylation in TFF1 promoter lesions was detected in TFF1-negative human EAC samples, further confirming not only the role of TFF1 in EAC but also the underlying mechanisms of TFF1 regulation. In addition, IHC revealed the nuclear translocation of β-catenin in human and mouse EAC, suggesting that activation of the Wnt/β-catenin pathway was induced by the loss of TFF1. In conclusion, these results indicate that TFF1 functions as a tumor suppressor to inhibit the development of esophageal carcinogenesis from Barrett's epithelium.

Role of the mucin-like glycoprotein FCGBP in mucosal immunity and cancer.

IgGFc-binding protein (FCGBP) is a mucin first detected in the intestinal epithelium. It plays an important role in innate mucosal epithelial defense, tumor metastasis, and tumor immunity. FCGBP forms disulfide-linked heterodimers with mucin-2 and members of the trefoil factor family. These formed complexes inhibit bacterial attachment to mucosal surfaces, affect the motility of pathogens, and support their clearance. Altered FCGBP expression levels may be important in the pathologic processes of Crohn’s disease and ulcerative colitis. FCGBP is also involved in regulating the infiltration of immune cells into tumor microenvironments. Thus, the molecule is a valuable marker of tumor prognosis. This review summarizes the functional relevance and role of FCGBP in immune responses and disease development, and highlights the potential role in diagnosis and predicting tumor prognosis.

1453-P: Possible Delta- to Beta-Cell Transdifferentiation in Aged Diabetic Mutant CRY1 Transgenic Mice

Cysteine414 (zinc-binding site of mCRY1) -alanine mutant mCRY1 transgenic mice (TG) show diabetes characterized by the reduction of β-cell proliferation due to the characters of senescence-associated secretory phenotype (SASP) in β-cells as we have reported at ADA Scientific Sessions in 2015. We also showed that various structural remodeling progresses in the islets of TG age-dependently: the increase of the population ratios of α-cell to β-cell and of ∂-cell to β-cell as well as fibrillation, angiogenesis and also the generation of mucin-producing atypical intra-islet ducts. Also, we revealed that trefoil factor family 2 (TFF2) is up-regulated in the β-cell of aged TG (Okano S. et al., ADA Scientific Sessions in 2020). To explore the mechanisms of keeping of the amount of β-cells under hyperglycemia for prolonged period in TG, we further conducted detailed double immune-staining experiments in the pancreatic sections of various ages (approximately 1, 10, and 19 months) of TG. The results demonstrated that, somatostatin and insulin double-positive cells were observed exclusively in aged TG (19 months of age). In wild-type mice (WT) of all stages, the double-positive cells were not observed. No glucagon and insulin double-positive cell was observed in islets of both TG and WT of all stages. In addition, scrutinized analyses demonstrated that not only β-cells but also small portion of somatostatin-positive cells express TFF2 in islets of aged TG. Taken together, these results suggest the possible ∂-cell to β-cell transdifferentiation in aged TG, that is, ∂-cells may play some role as the source for β-cells to supply new β-cells for islets in TG. Disclosure S.Okano: None. A.Yasui: None. K.Satoh: None. S.Kanno: None. M.Igarashi: None. O.Nakajima: None. Funding Grants-in-Aid for Scientific Research (19K07498). The Joint Research Program of IDAC, Tohoku University (38).

Runx1 regulates Tff1 expression to expedite viability of retinal microvascular endothelial cells in mice with diabetic retinopathy

Diabetic retinopathy (DR) represents a major complication of diabetes, and molecular mechanisms related to vascular dysfunction, particularly endothelial dysfunction, in DR remains unclear. In the present work, we generated a DR animal model using mice and a cell model in mouse retinal microvascular endothelial cells (mRMECs) to examine the role of Trefoil factor family 1 (Tff1) in DR. Tff1 was poorly expressed in DR mice and high glucose (HG)-treated mRMECs. Overexpression of Tff1 significantly attenuated streptozotocin-induced retinal proliferation and angiogenesis in DR mice and reduced the secretion of inflammatory factors. In HG-treated mRMECs, overexpression of Tff1 remarkably reduced the proliferation and angiogenesis of mRMECs. In further experiments, we found that Tff1 was transcriptionally repressed by Runt-related transcription factor 1 (Runx1) directly, and Tff1 expression was indirectly modulated by Runx1 via the core-binding factor subunit beta (CBF-β)/nuclear factor, erythroid 2/microRNA-423-5p axis and the CBF-β/estrogen receptor 1 (ESR1) axis. Moreover, Tff1 could inhibit the activation of NF-κB signaling pathway, which in turn attenuated retinal endothelial cell proliferation and angiogenesis. It was thus proposed that Runx1/Tff1/NF-κB axis may be a potential target for the treatment strategy of DR, and further studies are needed.

TFF1 in Aqueous Humor—A Potential New Biomarker for Retinoblastoma

Simple SummaryRetinoblastoma is the most common pediatric intraocular malignancy with high cure rates in developed countries. Nevertheless, useful predictive biomarkers providing reliable evidence for therapy decisions are urgently needed to optimize therapy regimes. TFF1 is a promising candidate as it is expressed in a more advanced subtype of retinoblastoma. Additionally, TFF1 is a naturally secreted peptide. Thus, TFF1 might be detectable in the aqueous humor of RB patients’ eyes, providing the opportunity to determine its expression prior to therapy without the necessity of a tumor biopsy. We therefore investigated for the first time aqueous humor samples of retinoblastoma patients in order to test for the availably and expression status of TFF1 as well as to compare it with the original tumor and established corresponding primary cell cultures.Retinoblastoma (RB) is the most common childhood eye cancer. The expression of trefoil factor family peptide 1 (TFF1), a small secreted peptide, has been correlated with more advanced RB stages and it might be a promising new candidate as a RB biomarker. The study presented addressed the question of if TFF1 is detectable in aqueous humor (AH) of RB patients’ eyes, providing easy accessibility as a diagnostic and/or therapy accompanying predictive biomarker. The TFF1 expression status of 15 retinoblastoma AH samples was investigated by ELISA and Western blot analyses. The results were correlated with the TFF1 expression status in the tumor of origin and compared to TFF1 expression in established corresponding primary tumor cell cultures and supernatants. Nine out of fifteen AH patient samples exhibited TFF1 expression, which correlated well with TFF1 levels of the original tumor. TFF1 expression in most of the corresponding primary cell cultures reflects the levels of the original tumor, although not all TFF1-expressing tumor cells seem to secret into the AH. Together, our findings strongly suggest TFF1 as a reliable new RB biomarker.