Evaluating the utility of ZNF331 promoter methylation as a prognostic and predictive marker in stage III colon cancer: results from CALGB 89803 (Alliance).

While epigenomic alterations are common in colorectal cancers (CRC), few epigenomic biomarkers that risk-stratify patients have been identified. We thus sought to determine the potential of ZNF331 promoter hypermethylation (mZNF331) as a prognostic and predictive marker in colon cancer. We examined the association of mZNF331 with clinicopathologic features, relapse, survival, and treatment efficacy in patients with stage III colon cancer treated within a randomized adjuvant chemotherapy trial (CALGB/Alliance89803). Residual tumour tissue was available for genomic DNA extraction and methylation analysis for 385 patients. ZNF331 promoter methylation status was determined by bisulphite conversion and fluorescence-based real-time polymerase chain reaction. Kaplan-Meier estimator and Cox proportional hazard models were used to assess the prognostic and predictive role of mZNF331 in this well-annotated dataset, adjusting for clinicopathologic features and standard molecular markers. mZNF331 was observed in 267/385 (69.4%) evaluable cases. Histopathologic features were largely similar between patients with mZNF331 compared to unmethylated ZNF331 (unmZNFF31). There was no significant difference in disease-free or overall survival between patients with mZNF331 versus unmZNF331 colon cancers, even when adjusting for clinicopathologic features and molecular marker status. Similarly, there was no difference in disease-free or overall survival across treatment arms when stratified by ZNF331 methylation status. While ZNF331 promoter hypermethylation is frequently observed in CRC, our current study of a small subset of patients with stage III colon cancer suggests limited applicability as a prognostic marker. Larger studies may provide more insight and clarity into the applicability of mZNF331 as a prognostic and predictive marker.

A Rapid Self-Assembling Peptide Hydrogel for Delivery of TFF3 to Promote Gastric Mucosal Injury Repair.

Self-assembled peptide-based nanobiomaterials exhibit promising prospects for drug delivery applications owing to their commendable biocompatibility and biodegradability, facile tissue uptake and utilization, and minimal or negligible unexpected toxicity. TFF3 is an active peptide autonomously secreted by gastric mucosal cells, possessing multiple biological functions. It acts on the surface of the gastric mucosa, facilitating the repair process of gastric mucosal damage. However, when used as a drug, TFF3 faces significant challenges, including short retention time in the gastric mucosal cavity and deactivation due to degradation by stomach acid. In response to this challenge, we developed a self-assembled short peptide hydrogel, Rqdl10, designed as a delivery vehicle for TFF3. Our investigation encompasses an assessment of its properties, biocompatibility, controlled release of TFF3, and the mechanism underlying the promotion of gastric mucosal injury repair. Congo red/aniline blue staining revealed that Rqdl10 promptly self-assembled in PBS, forming hydrogels. Circular dichroism spectra indicated the presence of a stable ß-sheet secondary structure in the Rqdl10 hydrogel. Cryo-scanning electron microscopy and atomic force microscopy observations demonstrated that the Rqdl10 formed vesicle-like structures in the PBS, which were interconnected to construct a three-dimensional nanostructure. Moreover, the Rqdl10 hydrogel exhibited outstanding biocompatibility and could sustainably and slowly release TFF3. The utilization of the Rqdl10 hydrogel as a carrier for TFF3 substantially augmented its proliferative and migratory capabilities, while concurrently bolstering its anti-inflammatory and anti-apoptotic attributes following gastric mucosal injury. Our findings underscore the immense potential of the self-assembled peptide hydrogel Rqdl10 for biomedical applications, promising significant contributions to healthcare science.

Trefoil factor 3 can stimulate Th17 cell response in the development of type 2 diabetes mellitus.

This study aims to evaluate the role of trefoil factor 3 (TFF3) peptides in type 2 diabetes mellitus (T2DM) from an inflammatory perspective. The focus was on exploring how TFF3 affects the function of T cells. TFF3 overexpression model was constructed using lentivirus in Jurkat cell lines. We evaluated the impact of TFF3 on the proliferation, apoptosis, and IL-17A levels of Jurkat cells cultured in high glucose. The T2DM model was induced in TFF3 knockout (KO) mice through streptozotocin combined with high-fat diet. The measurements included glucose tolerance, insulin tolerance, inflammation markers, Th17 cell proportion, and pancreatic pathological changes. The T2DM modeling led to splenomegaly in mice, and increased expression of TFF3 in their spleens. Overexpression of TFF3 increased the proportion of IL-17+ T cells and the levels of Th17-related cytokines in Jurkat cells. There was no difference in body weight and blood glucose levels between wild-type and TFF3 KO mice. However, T2DM mice lacking the TFF3 gene showed improved glucose utilization, ameliorated pancreatic pathology, decreased inflammation levels, and reduced Th17 cell ratio. TFF3 may be involved in the chronic inflammatory immune response in T2DM. Its mechanism may be related to the regulation of the RORγt/IL-17 signaling pathway and its impact on T cell proliferation and apoptosis.

Different Forms of TFF3 in the Human Endocervix, including a Complex with IgG Fc Binding Protein (FCGBP), and Further Aspects of the Cervico-Vaginal Innate Immune Barrier.

TFF3 is a typical secretory poplypeptide of mucous epithelia belonging to the trefoil factor family (TFF) of lectins. In the intestine, respiratory tract, and saliva, TFF3 mainly exists as a high-molecular-mass complex with IgG Fc binding protein (FCGBP), which is indicative of a role in mucosal innate immunity. For the first time, we identified different forms of TFF3 in the endocervix, i.e., monomeric and homodimeric TFF3, as well as a high-molecular-mass TFF3-FCGBP complex; the latter also exists in a hardly soluble form. Immunohistochemistry co-localized TFF3 and FCGBP. Expression analyses of endocervical and post-menopausal vaginal specimens revealed a lack of mucin and TFF3 transcripts in the vaginal specimens. In contrast, genes encoding other typical components of the innate immune defense were expressed in both the endocervix and vagina. Of note, FCGBP is possibly fucosylated. Endocervical specimens from transgender individuals after hormonal therapy showed diminished expression, particularly of FCGBP. Furthermore, mucus swabs from the endocervix and vagina were analyzed concerning TFF3, FCGBP, and lysozyme. It was the aim of this study to illuminate several aspects of the cervico-vaginal innate immune barrier, which is clinically relevant as bacterial and viral infections are also linked to infertility, pre-term birth and cervical cancer.

A Trefoil factor 3-Lingo2 axis restrains proliferative expansion of type-1 T helper cells during GI nematode infection.

Host defense at the mucosal interface requires collaborative interactions between diverse cell lineages. Epithelial cells damaged by microbial invaders release reparative proteins such as the Trefoil factor family (TFF) peptides that functionally restore barrier integrity. However, whether TFF peptides and their receptors also serve instructive roles for immune cell function during infection is incompletely understood. Here, we demonstrate that the intestinal trefoil factor, TFF3, restrains (T cell helper) TH1 cell proliferation and promotes host-protective type 2 immunity against the gastrointestinal parasitic nematode Trichuris muris. Accordingly, T cell-specific deletion of the TFF3 receptor, leucine-rich repeat and immunoglobulin containing nogo receptor 2 (LINGO2), impairs TH2 cell commitment, allows proliferative expansion of interferon (IFN)g+ cluster of differentiation (CD)4+ TH1 cells and blocks normal worm expulsion through an IFNg-dependent mechanism. This study indicates that TFF3, in addition to its known tissue reparative functions, drives anti-helminth immunity by controlling the balance between TH1/TH2 subsets.

REGγ Mitigates Radiation-Induced Enteritis by Preserving Mucin Secretion and Sustaining Microbiome Homeostasis.

Radiation-induced enteritis, a significant concern in abdominal radiation therapy, is associated closely with gut microbiota dysbiosis. The mucus layer plays a pivotal role in preventing the translocation of commensal and pathogenic microbes. Although significant expression of REGγ in intestinal epithelial cells is well established, its role in modulating the mucus layer and gut microbiota remains unknown. The current study revealed notable changes in gut microorganisms and metabolites in irradiated mice lacking REGγ, as compared to wild-type mice. Concomitant with gut microbiota dysbiosis, REGγ deficiency facilitated the infiltration of neutrophils and macrophages, thereby exacerbating intestinal inflammation after irradiation. Furthermore, fluorescence in situ hybridization assays unveiled an augmented proximity of bacteria to intestinal epithelial cells in REGγ knockout mice after irradiation. Mechanistically, deficiency of REGγ led to diminished goblet cell populations and reduced expression of key goblet cell markers, Muc2 and Tff3, observed in both murine models, minigut organoid systems and human intestinal goblet cells, indicating the intrinsic role of REGγ within goblet cells. Interestingly, although administration of broad-spectrum antibiotics did not alter the goblet cell numbers or mucin 2 (MUC2) secretion, it effectively attenuated inflammation levels in the ileum of irradiated REGγ absent mice, bringing them down to the wild-type levels. Collectively, these findings highlight the contribution of REGγ in counteracting radiation-triggered microbial imbalances and cell-autonomous regulation of mucin secretion.

PTEN, ERG, SPINK1, and TFF3 Status and Relationship in a Prostate Cancer Cohort from Jordanian Arab Population.

Background and Objectives: Prognostic biomarkers in prostate cancer (PCa) include PTEN, ERG, SPINK1, and TFF3. Their relationships and patterns of expression in PCa in developing countries, including Jordan, have not yet been investigated. Materials and Methods: A tissue microarray (TMA) of PCa patients was taken from paraffin-embedded tissue blocks for 130 patients. PTEN, ERG, SPINK1, and TFF3 expression profiles were examined using immunohistochemistry (IHC) and correlated with each other and other clinicopathological factors. Results: PTEN loss of any degree was observed in 42.9% of PCa cases. ERG and TFF3 were expressed in 59.3% and 46.5% of PCa cases, respectively. SPINK1 expression was observed in 6 out of 104 PCa cases (5.4%). Among all PCa cases (n = 104), 3.8% (n = 4) showed SPINK1+/ERG+ phenotype, 1.9% (n = 2) showed SPINK1+/ERG- phenotype, 56.7% (n = 59) showed SPINK1-/ERG+ phenotype, and 37.5% showed SPINK1-/ERG- phenotype (n = 39). Among ERG positive cases (n = 63), 6.3% were SPINK1 positive. Among SPINK1 positive cases (n = 6), 66.7% were ERG positive. SPINK1 expression was predominantly observed in a subgroup of cancers that expressed TFF3 (6/6). Additionally, a statistically significant loss of PTEN expression was observed from Gleason Score 6 (GS6) (Grade Group 1 (GG1)) to GS9-10 (GG5); (p-value 0.019). Conclusions: This is the first study to look at the status of the PTEN, ERG, SPINK1, and TFF3 genes in a Jordanian Arab population. Loss of PTEN has been linked to more aggressive prostate cancer with high GSs/GGs. SPINK1 expression was predominantly observed in a subgroup of cancers that expressed TFF3. Our results call for screening these biomarkers for grading and molecular subtyping of the disease.

Intestinal Trefoil Factor 3: a new biological factor mediating gut-kidney crosstalk in diabetic kidney disease.

PURPOSE: To investigate the effect of TFF3 in the pathogenesis of Diabetic Kidney Disease (DKD), and explore the dynamic changes of TFF3 expression pattern in renal injury process. METHODS: DKD animal model was established by streptozotocin (STZ) (40 mg/kg/d, ip, for 5 days, consecutively) combined with the high fat diet (HFD) for 12 weeks. While animals were sacrificed at different time stages in DKD process (4 weeks, 8 weeks and 12 weeks, respectively). RESULTS: STZ combined with high-fat diet induced weight gain, increased blood glucose and decreased glucose tolerance in DKD mice. Compared to the control group, the DKD group exhibits extracellular matrix (ECM) accumulation and the renal injury was aggravated in a time-dependent manner. The TFF3 expression level was decreased in kidney, and increased in colon tissue. CONCLUSION: TFF3 is not only expressed in colon, but also expressed in renal medulla and cortex. TFF3 might be play a pivotal role in renal mucosal repair by gut-kidney crosstalk, and protect renal from high glucose microenvironment damage.

Delayed recanalization reduced neuronal apoptosis and neurological deficits by enhancing liver-derived trefoil factor 3-mediated neuroprotection via LINGO2/EGFR/Src signaling pathway after middle cerebral artery occlusion in rats.

Delayed recanalization at days or weeks beyond the therapeutic window was shown to improve functional outcomes in acute ischemic stroke (AIS) patients. However, the underlying mechanisms remain unclear. Previous preclinical study reported that trefoil factor 3 (TFF3) was secreted by liver after cerebral ischemia and acted a distant neuroprotective factor. Here, we investigated the liver-derived TFF3-mediated neuroprotective mechanism enhanced by delayed recanalization after AIS. A total of 327 male Sprague-Dawley rats and the model of middle cerebral artery occlusion (MCAO) with permanent occlusion (pMCAO) or with delayed recanalization at 3 d post-occlusion (rMCAO) were used. Partial hepatectomy was performed within 5 min after MCAO. Leucine-rich repeat and immunoglobulin-like domain-containing nogo receptor-interacting protein 2 (LINGO2) siRNA was administered intracerebroventricularly at 48 h after MCAO. Recombinant rat TFF3 (rr-TFF3, 30 µg/Kg) or recombinant rat epidermal growth factor (rr-EGF, 100 µg/Kg) was administered intranasally at 1 h after recanalization, and EGFR inhibitor Gefitinib (75 mg/Kg) was administered intranasally at 30 min before recanalization. The evaluation of outcomes included neurobehavior, ELISA, western blot and immunofluorescence staining. TFF3 in hepatocytes and serum were upregulated in a similar time-dependent manner after MCAO. Compared to pMCAO, delayed recanalization increased brain TFF3 levels and attenuated brain damage with the reduction in neuronal apoptosis, infarct volume and neurological deficits. Partial hepatectomy reduced TFF3 levels in serum and ipsilateral brain hemisphere, and abolished the benefits of delayed recanalization on neuronal apoptosis and neurobehavioral deficits in rMCAO rats. Intranasal rrTFF3 treatment reversed the changes associated with partial hepatectomy. Delayed recanalization after MCAO increased the co-immunoprecipitation of TFF3 and LINGO2, as well as expressions of p-EGFR, p-Src and Bcl-2 in the brain. LINGO2 siRNA knockdown or EGFR inhibitor reversed the effects of delayed recanalization on apoptosis and brain expressions of LINGO2, p-EGFR, p-Src and Bcl-2 in rMCAO rats. EGFR activator abolished the deleterious effects of LINGO2 siRNA. In conclusion, our investigation demonstrated for the first time that delayed recanalization may enhance the entry of liver-derived TFF3 into ischemic brain upon restoring blood flow after MCAO, which attenuated neuronal apoptosis and neurological deficits at least in part via activating LINGO2/EGFR/Src pathway.

Tff3 Deficiency Differentially Affects the Morphology of Male and Female Intestines in a Long-Term High-Fat-Diet-Fed Mouse Model.

Trefoil factor family protein 3 (Tff3) protects the gastrointestinal mucosa and has a complex mode of action in different tissues. Here, we aimed to determine the effect of Tff3 deficiency on intestinal tissues in a long-term high-fat-diet (HFD)-fed model. A novel congenic strain without additional metabolically relevant mutations (Tff3-/-/C57Bl6NCrl strain, male and female) was used. Wild type (Wt) and Tff3-deficient mice of both sexes were fed a HFD for 36 weeks. Long-term feeding of a HFD induces different effects on the intestinal structure of Tff3-deficient male and female mice. For the first time, we found sex-specific differences in duodenal morphology. HFD feeding reduced microvilli height in Tff3-deficient females compared to that in Wt females, suggesting a possible effect on microvillar actin filament dynamics. These changes could not be attributed to genes involved in ER and oxidative stress, apoptosis, or inflammation. Tff3-deficient males exhibited a reduced cecal crypt depth compared to that of Wt males, but this was not the case in females. Microbiome-related short-chain fatty acid content was not affected by Tff3 deficiency in HFD-fed male or female mice. Sex-related differences due to Tff3 deficiency imply the need to consider both sexes in future studies on the role of Tff in intestinal function.

Small molecule inhibition of TFF3 overcomes tamoxifen resistance and enhances taxane efficacy in ER+ mammary carcinoma.

Even though tamoxifen has significantly improved the survival of estrogen receptor positive (ER+) mammary carcinoma (MC) patients, the development of drug resistance with consequent disease recurrence has limited its therapeutic efficacy. Trefoil factor-3 (TFF3) has been previously reported to mediate anti-estrogen resistance in ER+MC. Herein, the efficacy of a small molecule inhibitor of TFF3 (AMPC) in enhancing sensitivity and mitigating acquired resistance to tamoxifen in ER+MC cells was investigated. AMPC induced apoptosis of tamoxifen-sensitive and resistant ER+MC cells and significantly reduced cell survival in 2D and 3D culture in vitro. In addition, AMPC reduced cancer stem cell (CSC)-like behavior in ER+MC cells in a BCL2-dependent manner. Synergistic effects of AMPC and tamoxifen were demonstrated in ER+MC cells and AMPC was observed to improve tamoxifen efficacy in tamoxifen-sensitive cells and to re-sensitize cells to tamoxifen in tamoxifen-resistant ER+MC in vitro and in vivo. Additionally, tamoxifen-resistant ER+MC cells were concomitantly resistant to anthracycline, platinum and fluoropyrimidine drugs, but not to Taxanes. Taxane treatment of tamoxifen-sensitive and resistant ER+MC cells increased TFF3 expression indicating a combination vulnerability for tamoxifen-resistant ER+MC cells. Taxanes increased CSC-like behavior of tamoxifen-sensitive and resistant ER+MC cells which was reduced by AMPC treatment. Taxanes synergized with AMPC to promote apoptosis and reduce CSC-like behavior in vitro and in vivo. Hence, AMPC restored the sensitivity of tamoxifen and enhanced the efficacy of Taxanes in tamoxifen-resistant ER+MC. In conclusion, pharmacological inhibition of TFF3 may serve as an effective combinatorial therapeutic strategy for the treatment of tamoxifen-resistant ER+MC.

[Effect of TFF3 on tight junction protein in eosinophilic chronic sinusitis and its related mechanism].

Objective: To study the effect of trefoil factor family (TFF) 3 on the expression of tight junctions (TJs) in the nasal mucosa epithelium of eosinophilic chronic rhinosinusitis (eCRS) and its mechanism. Methods: From September to December 2020, eligible patients from the Department of Otorhinolaryngology of the First Affiliated Hospital of Nanchang University were recruited, including 11 control patients and 37 patients with chronic rhinosinusitis with nasal polyps (CRSwNP), from whom nasal mucosa and nasal polyp tissue samples were collected. Immunohistochemistry (IHC) was used to detect the localization and expression intensity of TFFs (TFF1, TFF2 and TFF3) and TJs (occudin, claudin-1 and ZO-1) in nasal mucosa. Real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) and western blot (WB) were used to detect the mRNA and protein expression. A cell model of tight junction injury in human nasal epithelial cells (HNECs) through stimulation with interleukin (IL)-13 was also established. The optimal modeling concentration and time for HNECs were determined, which were subsequently treated with TFF3 and/or a phosphoinositide 3-kinase (PI3K)-specific inhibitor (LY294002). Finally, RT-qPCR and WB were used to assess the effects of TFF3 on tight junctions and the PI3K/serine/threonine kinase (Akt) signaling pathway. Data were analyzed statistically using GraphPad Prism 7 software. Results: IHC results showed that the expression of TFF1 and TFF3 in nasal mucosa of eCRS group was significantly higher than that of control group (t=4.62, P=0.002; t=5.89, P<0.001), respectively, mainly expressed in goblet cell. The expression of occludin, claudin-1 and ZO-1 in the nasal mucosa of the eCRS group was lower than that of the control group (occludin t=3.98, P=0.019; claudin-1 t=5.15, P=0.002; ZO-1 t=5.42, P=0.001), respectively. WB results showed that the expression of TFF3 in non-eosinophilic chronic sinusitis (Non-eCRS) group and eCRS group was higher than that in the control group (t=3.62, P=0.036; t=5.93, P<0.001). The expression of occludin, claudin-1 and ZO-1 in eCRS group was lower than that in the control group (occludin t=5.14, P=0.002; claudin-1 t=6.35, P<0.001; ZO-1 t=6.64, P<0.001), respectively. The RT-qPCR results showed that compared with the control group, the levels of TFF1 and TFF3 mRNA were increased in the nasal mucosal epithelium of the Non-eCRS and eCRS groups (TFF1 t=3.98, P=0.046, t=4.89, P=0.002; TFF3 t=3.50, P=0.044, t=6.78, P<0.001). There was no statistically significant difference in TFF2 mRNA levels between the Non-eCRS and eCRS groups (t=1.34, P=0.061; t=3.37, P=0.055). Compared with the control group, Non-eCRS and eCRS groups showed a decrease in the mRNA levels of occludin, claudin-1 and ZO-1 (occludin t=4.27, P=0.011, t=5.61, P=0.007; claudin-1 t=3.62, P=0.036, t=6.80, P<0.001; ZO-1 t=3.47, P=0.047, t=7.86, P<0.001). The mRNA levels of TFF3 and TJs in eCRS nasal mucosa tissue showed a moderate positive correlation (occludin r=0.661, claudin-1 r=0.614, ZO-1 r=0.548, all P<0.001); TFF1 showed a low degree of positive correlation with the expression of occludin, claudin-1 and ZO-1 (occludin r=0.467, P=0.040; claudin-1 r=0.362, P=0.012; ZO-1 r=0.425, P=0.025). The establishment of cell models showed that compared with normal HNECs, the mRNA expression of TFF3 was most significantly increased at a concentration of 50 ng/ml stimulated by IL-13 (t=3.72, P=0.013); The mRNA expression of occludin, claudin-1 and ZO-1 decreased (occludin t=3.18, P=0.031; claudin-1 t=3.86, P=0.010; ZO-1 t=5.16, P=0.002). The expression of TFF3 mRNA increased most significantly after 15 hours of IL-13 stimulation (t=3.14, P=0.034); The mRNA expression of occludin, claudin-1 and ZO-1 decreased (occludin t=3.97, P=0.010; claudin-1 t=4.78, P=0.004; ZO-1 t=5.16, P=0.004). TJs damage model could be established by treating HNECs with 50 ng/ml IL-13 for 15 hours. Intervention experiments showed that compared with the IL-13 group, the IL-13+TFF3 group showed an increase in TJs mRNA expression (occludin t=6.10, P=0.009; claudin-1 t=5.90, P=0.013; ZO-1 t=9.44, P=0.007). Compared with the IL-13 group, the expression of TJs protein in the IL-13+TFF3 group increased (occludin t=3.23, P=0.013; claudin-1 t=9.40, P=0.017; ZO-1 t=2.23, P=0.032); The expression of TJs protein decreased in the IL-13+TFF3+LY294002 group (occludin t=4.73, claudin-1 t=8.77, ZO-1 t=3.51, all P<0.001). Compared with the IL-13+TFF3 group, the IL-3+TFF3+LY294002 group showed a decrease in PI3K and p-Akt/Akt protein expression (PI3K t=13.29, p-Akt/Akt t=10.30, all P<0.001). The increased mRNA and protein expression of occludin, claudin-1 and ZO-1 induced by TFF3 were also inhibited by LY294002. Conclusion: TFF3 can up-regulate the expression of occludin, claudin-1, and ZO-1 through PI3K/Akt pathway, and has a certain protective effect on the nasal mucosal epithelial barrier, providing a new idea for treating eCRS.

TFF3 as a Diagnostic Biomarker in Kidney Transplant Patients.

Intestinal trefoil factor 3 (TFF3) is a protein secreted by many cell types, and its serum and urine levels vary in patients with kidney disease. Therefore, the present study aimed to determine the diagnostic value of TFF3 in allogeneic kidney transplant patients included in the one-year follow-up. To analyze the influence of the diagnostic method used, we studied the type of biological material and the time elapsed since renal transplantation on the parameter's value. The study also aimed to investigate the relationship between TFF3 levels and creatinine and estimated glomerular filtration rate (eGFR) values in the serum and urine of the patients studied. The study used blood and urine samples from adult patients (n = 19) 24-48 h, 6 months, and 12 months after kidney transplantation. We collected one-time blood and urine from healthy subjects (n = 5) without renal disease. We applied immunoenzymatic ELISA and xMap Luminex flow fluorimetry to determine TFF3 in serum and urine. There was a significant difference in TFF3 levels in the serum of patients collected on the first one or two days after kidney transplantation compared to the control group (determined by ELISA and Luminex) and six months and one year after kidney transplantation (ELISA). We observed a correlation between creatinine concentration and urinary TFF3 concentration (ELISA and Luminex) and a negative association between eGFR and urinary (ELISA) and serum (Luminex) TFF3 concentration in patients on the first and second days after kidney transplantation. We noted significant correlations between eGFR and TFF3 levels in the serum and urine of patients determined by the two methods six months and one year after transplantation. In women, we observed that urinary TFF3 concentration increased significantly with increasing creatinine and that with increasing eGFR, urinary TFF3 concentration determined by two methods decreased significantly. In the present study, the choice of diagnostic method for the determination of TFF3 in serum and urine significantly affected the concentration of this biomarker. The values of this parameter determined by ELISA were higher than those assessed using the Luminex assay. Based on the presented results, we can conclude that TFF3 has great potential to monitor renal transplant patients. Determination of this protein in parallel with creatinine and eGFR levels in serum and urine may provide helpful diagnostic information.

Evaluation of trefoil factor 3 as a non-invasive biomarker of gastric intestinal metaplasia and gastric cancer in a high-risk population / Evaluación de Trefoil factor 3 como un biomarcador no invasivo para la detección de metaplasia intestinal y cáncer gástrico en una población de alto riesgo

Background: Adenocarcinoma is preceded by chronic atrophic gastritis, gastric intestinal metaplasia and dysplasia. Trefoil factor 3 (TFF3) is a peptide secreted by goblet cells, which is abundantly present in intestinal metaplasia. Aim: To evaluate the utility of serum TFF3 as a non-invasive biomarker for the diagnosis of intestinal metaplasia and gastric cancer. Methods: Single-center, cross-sectional study of 274 patients who consecutively underwent upper gastrointestinal endoscopy with gastric biopsies (updated Sydney system). TFF3 levels were measured in serum by a commercial ELISA kit. Patients with normal histology or chronic atrophic gastritis without intestinal metaplasia comprised the control group. In addition, 14 patients with invasive gastric cancer were included as a reference group. The association between TFF3 levels and intestinal metaplasia was assessed by logistic regression. Results: Patients with intestinal metaplasia (n=110) had a higher median TFF3 level as compared to controls (n=164), 13.1 vs. 11.9ng/mL, respectively (p=0.024). Multivariable logistic regression showed a no significant association between TFF3 levels and intestinal metaplasia (OR=1.20; 95%CI: 0.87–1.65; p-trend=0.273). The gastric cancer group had a median TFF3 level of 20.5ng/mL, and a significant association was found (OR=3.26; 95%CI: 1.29–8.27; p-trend=0.013). Conclusion: Serum levels of TFF3 do not discriminate intestinal metaplasia in this high-risk Latin American population. Nevertheless, we confirmed an association between TFF3 levels and invasive gastric cancer.(AU)

Introducción: El adenocarcinoma gástrico es precedido por la gastritis crónica atrófica, metaplasia intestinal y displasia gástrica. Trefoil factor 3 (TFF3) es un péptido secretado por las células caliciformes, que están abundantemente presentes en la metaplasia intestinal. Objetivo: Evaluar la utilidad de TFF3 sérico como biomarcador no invasivo para el diagnóstico de metaplasia intestinal y cáncer gástrico. Métodos: Estudio transversal, de 274 pacientes a los que se les realizó endoscopia digestiva alta consecutivamente con biopsias gástricas (sistema Sydney actualizado). Los niveles de TFF3 se midieron en suero mediante un kit de ELISA comercial. Los pacientes con histología normal o gastritis crónica atrófica sin metaplasia intestinal formaron el grupo control. Además, se incluyeron como grupo de referencia 14 pacientes con cáncer gástrico avanzado. La asociación entre los niveles de TFF3 y la metaplasia intestinal se evaluó mediante una regresión logística. Resultados: Los pacientes con metaplasia intestinal (n=110) presentaron una mediana de TFF3 más alta en comparación con el grupo control (n=164), 13,1 vs. 11,9ng/ml, respectivamente (p=0,024). Sin embargo, la regresión logística multivariable no mostró una asociación significativa entre los niveles de TFF3 y la metaplasia intestinal (OR=1,20; IC95%: 0,87-1,65; p-trend=0,273). El grupo de cáncer gástrico tuvo una mediana significativamente mayor de TFF3 de 20,5ng/ml (OR=3,26; IC95%: 1,29-8,27; p-trend=0,013). Conclusión: Los niveles séricos de TFF3 no permiten el diagnóstico no invasivo de metaplasia intestinal en esta población latinoamericana de alto riesgo. La asociación entre los niveles de TFF3 y el cáncer gástrico avanzado fue confirmada.(AU)

Modulation of the Intestinal Barrier Integrity and Repair by Microbiota Extracellular Vesicles through the Differential Regulation of Trefoil Factor 3 in LS174T Goblet Cells.

Trefoil factor 3 (TFF3) plays a key role in the maintenance and repair of intestinal mucosa. TFF3 expression is upregulated by the microbiota through TLR2. At the posttranscriptional level, TFF3 is downregulated by miR-7-5p. Reduced TFF3 levels have been detected in the damaged tissue of IBD patients. Here, we investigate the regulation of TFF3 expression by microbiota extracellular vesicles (EVs) in LS174T goblet cells using RT-qPCR and inhibitors of the TLR2 or PI3K pathways. To evaluate the subsequent impact on epithelial barrier function, conditioned media from control and vesicle-stimulated LS174T cells were used to treat Caco-2 monolayers. The barrier-strengthening effects were evaluated by analysing the expression and subcellular distribution of tight junction proteins, and the repairing effects were assessed using wound-healing assays. The results showed a differential regulation of TFF3 in LS174T via EVs from the probiotic EcN and the commensal ECOR12. EcN EVs activated the TFF3 production through TLR2 and downregulated miR7-5-p through PI3K. Consistently, high levels of secreted TFF3 reinforced the tight junctions and stimulated wound healing in the Caco-2 cells. ECOR12 EVs did not cause these effects. TFF3 is a potential therapeutic target in IBD. This study contributes to understanding the molecular players (microbiota EVs) connecting gut microbes to health and may help in designing better nutritional interventions based on microbiota bioactive compounds.

Salivary trefoil factor family peptide 3 (TFF3) and flow rate in persons with and without obstructive sleep apnea: A preliminary study.

OBJECTIVES: Obstructive sleep apnea (OSA) is one of the most common chronic diseases. Trefoil factor family 3 (TFF3) peptides are secreted by major and minor salivary glands and may be involved in the pathogenesis of OSA. This study aimed to evaluate salivary TFF3 and flow rate between those with and without OSA. MATERIAL AND METHODS: This was a prospective experimental study that enrolled patients with OSA and non-OSA. Total unstimulated saliva was collected, the salivary flow rate was measured, and the TFF3 level was analyzed by using a modified sandwich enzyme-linked immunosorbent assay. Baseline characteristics, TFF3 level, and salivary flow rate were compared between both groups. Factors associated with the TFF3 level and flow rate were computed by using multivariate linear regression analysis. RESULTS: Twenty-eight participants were recruited in the study: 20 patients with OSA (71.42%) and 8 non-OSA as control. The TFF3 and salivary flow rates between both groups of non-OSA versus OSA were comparable (TFF3 non-OSA 61.06 vs. OSA 96.00 ng/mg; p = .276 and flow rate non-OSA 0.40 vs. OSA 0.35 mL/min; p = .320). Factors associated with the TFF3 level were neck circumference with a negative coefficient of -16.419 (p = .042). For the salivary flow rate, only age was a significant factor with the coefficient of -0.013 (p = .044). CONCLUSIONS: TFF3 and salivary flow rate were comparable between patients with OSA and non-OSA. The factor associated with TFF3 level was neck circumference, while age was negatively associated with the salivary flow rate in patients with OSA.

Expression of trefoil factor 2 and 3 and adrenomedullin in chronic periodontitis subjects with coronary heart disease.

BACKGROUND: The current study aims to determine the expression of trefoil factor 2 (TFF2), trefoil factor 3 (TFF3), and adrenomedullin (ADM) in salivary samples of periodontitis patients with and without coronary heart disease (CHD). METHODS: A total of 75 patients were selected based on the inclusion and exclusion criteria and divided into three groups of 25 patients each: generalized periodontitis (GP) only; GP+CHD; and CHD only. Demographic, periodontal, and cardiac parameters were recorded, and unstimulated saliva samples were collected and analyzed for the expression of TFF2, TFF3, and ADM. RESULTS: Among the demographic variables, the means for age, weight, and body mass index were significantly different between the groups on statistical analysis. Plaque index, bleeding on probing, probing pocket depth, clinical attachment level, and the expression of TFF2 were highest in the GP+CHD group, and ADM was highest in the CHD group, with P values of < 0.01 as compared to the other groups. TFF2, TFF3, and ADM were also correlated with the demographic and periodontal parameters. CONCLUSIONS: The study demonstrates significantly elevated levels of TFF2 in CHD and GP patients, and a higher expression of ADM in CHD patients only, suggesting the possibility of an underlying inflammatory mechanism.

Evaluation of trefoil factor 3 as a non-invasive biomarker of gastric intestinal metaplasia and gastric cancer in a high-risk population.

BACKGROUND: Adenocarcinoma is preceded by chronic atrophic gastritis, gastric intestinal metaplasia and dysplasia. Trefoil factor 3 (TFF3) is a peptide secreted by goblet cells, which is abundantly present in intestinal metaplasia. AIM: To evaluate the utility of serum TFF3 as a non-invasive biomarker for the diagnosis of intestinal metaplasia and gastric cancer. METHODS: Single-center, cross-sectional study of 274 patients who consecutively underwent upper gastrointestinal endoscopy with gastric biopsies (updated Sydney system). TFF3 levels were measured in serum by a commercial ELISA kit. Patients with normal histology or chronic atrophic gastritis without intestinal metaplasia comprised the control group. In addition, 14 patients with invasive gastric cancer were included as a reference group. The association between TFF3 levels and intestinal metaplasia was assessed by logistic regression. RESULTS: Patients with intestinal metaplasia (n=110) had a higher median TFF3 level as compared to controls (n=164), 13.1 vs. 11.9ng/mL, respectively (p=0.024). Multivariable logistic regression showed a no significant association between TFF3 levels and intestinal metaplasia (OR=1.20; 95%CI: 0.87-1.65; p-trend=0.273). The gastric cancer group had a median TFF3 level of 20.5ng/mL, and a significant association was found (OR=3.26; 95%CI: 1.29-8.27; p-trend=0.013). CONCLUSION: Serum levels of TFF3 do not discriminate intestinal metaplasia in this high-risk Latin American population. Nevertheless, we confirmed an association between TFF3 levels and invasive gastric cancer.

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.

Intestinal Injury Biomarkers Predict Mortality in Pediatric Severe Malaria.

Severe malaria (SM) increases the risk of invasive bacterial infection, and there is evidence to suggest increased gastrointestinal permeability. Studies have shown sequestration of infected erythrocytes in intestinal microvasculature, and in vivo studies of rectal mucosa have demonstrated disruption of microvascular blood flow. However, the extent of intestinal injury in pediatric malaria is not well characterized. In this study, two serum biomarkers of intestinal injury, trefoil factor 3 (TFF3) and intestinal fatty acid binding protein (I-FABP), were analyzed in 598 children with SM and 120 healthy community children (CC), 6 months to 4 years of age. Serum was collected at enrollment and 1 month for laboratory studies, and participants were monitored for 12 months. Intestinal injury biomarkers were significantly elevated in children with SM, with 18.1% having levels of TFF3 and/or I-FABP greater than the 99th percentile of CC levels. TFF3 levels continued to be elevated at 1 month, while I-FABP levels were comparable to CC levels. Both markers predicted in-hospital mortality {odds ratio (OR) (95% confidence interval [CI]), 4.4 (2.7, 7.3) and 2.3 (1.7, 3.1)} for a natural log increase in TFF3 and I-FABP, respectively. TFF3 was also associated with postdischarge mortality (OR, 2.43 [95% CI, 1.1, 4.8]). Intestinal injury was associated with acute kidney injury (AKI), acidosis (P < 0.001 for both), and angiopoietin 2, a maker of endothelial activation. In conclusion, intestinal injury is common in pediatric severe malaria and is associated with an increased mortality. It is strongly associated with AKI, acidosis, and endothelial activation. IMPORTANCE In children with severe malaria, intestinal injury is a common complication associated with increased mortality. Intestinal injury is associated with acute kidney injury, acidosis, and endothelial activation. Interventions promoting intestinal regeneration and repair represent novel approaches to improve outcomes.