Sodium alginate hydrogelation mediated paper-based POCT sensor for visual distance reading and smartphone-assisted colorimetric dual-signal determination of L-lactate.

Herein, we present a paper-based POCT sensor based on lactate dehydrogenase-mediated alginate gelation combined with visual distance reading and smartphone-assisted colorimetric dual-signal analysis to determine the concentration of L-lactate in yogurt samples. In this research, L-lactate was transformed into pyruvate by lactate dehydrogenase. Pyruvate then triggered the gelation of a sol mixture, increasing the viscosity (ηs) of the mixture, which was shown as a decrease in the diffusion diameter on the paper-based sensor. In addition, protons from pyruvate accelerated the degradation of Rhodamine B, causing color fading of the mixture, which was analyzed using RGB analysis application software. Under optimal experimental conditions, the linear ranges of visual distance reading and smartphone-assisted colorimetric analysis were 0.1-15 µM and 0.3-15 µM and the detection limits were 0.03 µM and 0.07 µM, respectively. As a proof-of-concept application, we exploited the paper-based sensor to determine the concentration of L-lactate in yogurt samples. The results from the dual-signal paper-based sensor were consistent with the ones from HPLC analysis. In short, this study developed a simple, convenient, cost-effective, and feasible method for the quantitative detection of L-lactate in real samples.

Activation of mucosal insulin receptor exacerbates intestinal inflammation by promoting tissue resident memory T cells differentiation through EZH2.

BACKGROUND: Inflammatory Bowel Diseases (IBD), an autoimmune disease characterised by abnormal intestinal immunity, are related to vital morbidity around the world. However, therapeutic agents for IBD have not achieved desired benefit. Exploring new therapeutic targets for IBD, especially based on its abnormally intestinal immunity, could alleviate the flare-up and worsening of IBD. Tissue resident memory T cells (TRM) are core of multiple autoimmune diseases, including IBD. However, the mechanism of TRM differentiation remains to be investigated. METHODS: The alterations in mRNA and lncRNA profile of intestinal intraepithelial lymphocytes (IELs), the largest component of intestinal TRM, were analyzed in DSS-induced chronic colitis. Based on it, we examined the function of rectal insulin instillation in a dextran sodium sulfate (DSS) induced chronic colitis. Furthermore, we investigated the downstream-target of the insulin pathway-EZH2 and the crucial role of EZH2 in intestinal tissue resident memory T cell differentiation by utilizing EZH2fl/flCD4cre mice. RESULTS: Insulin receptor (INSR) expression was found to be significantly reduced. Activation of mucosal insulin pathway by rectal insulin instillation exacerbated colitis by disrupting IELs subgroups and up-regulating TNF-ɑ and IL-17 expression. Rectal insulin instillation promoted EZH2 expression and EZH2 inhibition alleviated chronic colitis. EZH2fl/flCD4cre mice restored the normal IEL subgroups and suppressed TNF-ɑ and IL-17 expression, exhibiting alleviated colitis. IELs from EZH2fl/flCD4cre mice exhibit significant changes in TRM related phenotype. CD4+TRM was significantly increased in chronic colitis and decreased in EZH2fl/flCD4cre mice. CONCLUSION: Insulin receptor of intestinal mucosal T-cells could promote intestinal TRM differentiation via EZH2. Our discoveries suggest that therapies targeting colonic INSR and EZH2 could be potential treatment for IBD based on its regulatory effects on TRM. Insulin receptor inhibitors rather than insulin should be applied during colitis-active phase. In addition, EZH2 shows to be a downstream signal of the insulin pathway and EZH2 inhibitor could alleviating intestinal inflammation. However, the critical role of EZH2 in TRM differentiation restricts the anti-tumor effects of EZH2 inhibitor in vivo.

Using immunohistochemistry to classify the molecular subtypes of Paget's disease of the breast.

PURPOSE: To classify the molecular subtypes of Paget's disease of the breast, and then compare them with general breast cancer to get deeper understanding of this disease and offer better management of associate patients in clinical decisions. METHODS: We used immunohistochemistry to examine 42 cases of this disease by antibodies against estrogen and progesterone receptors, Ki-67, as well as human epidermal growth factor receptor 2 (HER-2). Due to damage and loss of specimens, etc., we obtained 36 pathological specimens from the 42 patients. For 30 of 36 pathological specimens (83.3%), we obtained a complete molecular subtype. Cause the other 6 pathological specimens have missing immunohistochemistry items. For patients with bilateral breast cancer, only information on the side with PDB is listed. For patients with recurrence, only information on the first onset was included. We finally compared and studied the molecular subtype of 26 samples. We calculated the relative frequencies of molecular subtypes including luminal A, luminal B, HER-2-enriched, and basal-like and compared them between PDB and general breast carcinomas in other studies. RESULTS: The luminal A and B subtype were found, respectively, in 3 (11.5%) and 6 (23.1%) of all patients, and 15 cases of HER-2-enriched subtype was detected (57.7%). In addition, 2 (7.7%) showed a basal-like subtype. CONCLUSION: The molecular subtypes of common breast cancer and PDB-associated breast cancer differ. Luminal subtypes are the most common in the former, while within our samples HER-2 positive subtype was the highest in PDB-associated breast carcinoma. With further understanding of this disease, rational therapies will be applied in different patients and cures for PDB and PDB-associated carcinoma will be achieved.

Adenosine and L-proline can possibly hinder Chinese Sacbrood virus infection in honey bees via immune modulation.

Sacbrood virus (SBV) infects larvae of honey bees, resulting in infected larvae becoming fluid-filled sacs. Our previous studies showed that the extract of herbal medicine, Radix Isatidis, could inhibit Chinese SBV (CSBV) infection in Asian honey bees (Apis cerana). Here, two compounds, adenosine and L-proline, which were previously reported to be associated with immune modulation, were identified in R. Isatidis extract and then selected for an evaluation of their antiviral effect on CSBV infection in A. cerana. Our results revealed that both adenosine and L-proline could significantly mitigate the impact of CSBV on the growth and development of infected larvae and modulate hosts' immune responses by downregulating the expression of immune genes in infected larvae. The results gained from this study suggest that adenosine and L-proline could possibly interfere CSBV infection via immune modulation to avoid exacerbations and nonspecific damage to infected larvae's own tissues.

Polycatechol-Derived Mesoporous Polydopamine Nanoparticles for Combined ROS Scavenging and Gene Interference Therapy in Inflammatory Bowel Disease.

Benefiting from the evolution of nanotechnology, the combination therapy by gene interference and reactive oxygen species (ROS) scavenging are expected, which holds great potential in inflammatory bowel disease (IBD) therapy. However, the functional integration of different therapeutic modules through interface modification of gene vectors for safe and efficient treatment is urgently needed. Herein, we present a catechol chemistry-mediated core-shell nanoplatform for ROS scavenging-mediated oxidative stress alleviation and siRNA-mediated gene interference in a dextran sulfate sodium (DSS)-induced colitis model. The nanoplatform is constructed by employing mesoporous polydopamine nanoparticles (MPDA NPs) with surface modification of amines as the porous core for TNF-α-siRNA loading (31 wt %) and exerts an antioxidant function, while PDA-induced biomineralization of the calcium phosphate (CaP) coating is used as the pH-sensitive protective shell to prevent siRNA from premature release. The CaP layer degraded under weakly acidic subcellular conditions (lysosomes); thus, the synergistic integration of catechol and cation moieties on the exposed surface of MPDA resulted in an efficient lysosomal escape. Subsequently, effective ROS scavenging caused by the electron-donating ability of MPDA and efficient knocking down (40.5%) of tumor necrosis factor-α (TNF-α) via sufficient cytosolic gene delivery resulted in a synergistic anti-inflammation therapeutic effect both in vitro and in vivo. This work establishes the first paradigm of synergistic therapy in IBD by ROS scavenging and gene interference.

Clinical Significance of Circulating Tumor Cells (CTCs) and Survivin on Predicting Prognosis in Thyroid Cancer Patients.

BACKGROUND: Clinical significance of circulating tumor cell (CTC) count, mesenchymal CTCs (MCTCs), and survivin in patients with thyroid cancer remains unclear. We evaluated the relationship between the expression of different CTC subtypes or survivin and the prognosis in patients with thyroid cancer. Patients and Methods. This study enrolled 164 patients with thyroid cancer who were diagnosed from January 2013 to September 2020 in our hospital. Among these patients, there were 73 cases with papillary thyroid cancer (PTC), 60 cases with follicular thyroid cancer (FTC), 12 medullary thyroid cancers (MTC), 10 poorly differentiated thyroid cancers (PDTC), 9 anaplastic thyroid cancers, and 10 control patients with nonmalignant thyroid nodules based on their histopathological characteristics. Only 5 milliliters (mL) of peripheral blood from the patients with thyroid cancer and control was used to detect the CTC cell number via CanPatrol capture technique before treatments. We also isolated mononuclear cells (MNC) from the peripheral blood and performed quantity reverse transcriptase polymerase chain reaction (qPCR) for survivin gene expression among these patients. RESULTS: The overall positive rates of CTC at diagnosis were 56.1%. The relapse and metastasis rates in PTC and FTC patients with more than 6 CTCs and positive MCTCs were significantly higher than those in the patients with 6 or less than 6 CTCs and MCTCs. It was also found that these patients with >6 CTCs and MCTCs had shorter progression-free survival (PFS). Additionally, the survivin level of the patients with thyroid cancer was strongly relative to differentiation grades of thyroid cancers. CONCLUSIONS: The detection of more than six of total CTCs and positive MCTCs in the patients with differentiated thyroid cancer is an excellent biomarker for predicting the prognosis of patients. Survivin also is a good biomarker for thyroid cancer differentiation.

Optimized extraction based on the terpenoids of Heterotrigona itama propolis and their antioxidative and anti-inflammatory activities.

Terpenoids are the main components of stingless bee propolis, their biological activities have not been fully quantified and investigated. In this study, the single-factor design and response surface methodology were applied to optimize the terpenoids extraction process in Heterotrigona itama (also called H. itama) propolis. Furthermore, the in vitro antioxidant and anti-inflammatory activities of terpenoid-rich extract were evaluated. The results showed with 95% ethanol/5% water, a material-liquid extraction yield of 1:30 g/ml, and extraction 72 hr for three extractions, the highest terpenoids content in H. itama propolis of 46.44 ± 0.07%. The H. itama propolis terpenoid-rich extract showed relative low antioxidant effect but inhibited inflammatory response by decreasing the inflammatory mediators iNOS, IL-1ß, IL-10, and increasing the antioxidant mediators HO-1. This study provided experimental parameters for the terpenoids optimal extraction from H. itama propolis and revealed their strong anti-inflammatory activity. PRACTICAL APPLICATIONS: H. itama stingless propolis is a newly founded stingless bee propolis type recently. H. itama stingless propolis has abundant terpenoids, however, scant information were provided regarding its extraction optimization. In this study, we investigated and obtained the optimal extract parameters of terpenoids in H. itama propolis，and demonstrated their strong anti-inflammatory activities; however, their antioxidant activities are relatively low. This study provided theoretical basis for the usage of H. itama stingless propolis in industry.

Axitinib overcomes multiple imatinib resistant cKIT mutations including the gatekeeper mutation T670I in gastrointestinal stromal tumors.

BACKGROUND: cKIT kinase overexpression and gain-of-function mutations are the critical pathogenesis of gastrointestinal stromal tumors (GISTs). Although the multiple kinase inhibitors such as imatinib, sunitinib, and regorafenib have been approved for GISTs, the acquisition of polyclonal secondary resistance mutations in KIT is still a limitation for GIST treatment. Here we explored the KIT inhibitory activity of axitinib in preclinical models and describe initial characterization of its activity in GIST patient-derived primary cells. METHODS: The activities of axitinib against mutant KIT were evaluated using protein-based assay and a panel of engineered and GIST-derived cell lines. The binding modes of axitinib-KIT/KIT mutants were analyzed. Four primary cells derived from GIST patients were also used to assess the drug response of axitinib. RESULTS: Axitinib exhibited potent activities against a variety of cKIT associated primary and secondary mutations. It displayed better activity against cKIT wild-type, cKIT V559D/A/G, and L576P primary gain-of-function mutations than imatinib, sunitinib, and regorafenib. In addition, it could inhibit imatinib resistant cKIT T670I and V654A mutants in vitro and in vivo GIST preclinical models. CONCLUSION: Our results provide the basis for extending the application of axitinib to GISTs patients who are unresponsive or intolerant to the current therapies.

Discovery of 2-(4-Chloro-3-(trifluoromethyl)phenyl)-N-(4-((6,7-dimethoxyquinolin-4-yl)oxy)phenyl)acetamide (CHMFL-KIT-64) as a Novel Orally Available Potent Inhibitor against Broad-Spectrum Mutants of c-KIT Kinase for Gastrointestinal Stromal Tumors.

Starting from our previously developed c-KIT kinase inhibitor CHMFL-KIT-8140, through a type II kinase inhibitor binding element hybrid design approach, we discovered a novel c-KIT kinase inhibitor compound 18 (CHMFL-KIT-64), which is potent against c-KIT wt and a broad spectrum of drug-resistant mutants with improved bioavailability. 18 exhibits single-digit nM potency against c-KIT kinase and c-KIT T670I mutants in the biochemical assay and displays great potencies against most of the gain-of-function mutations in the juxtamembrane domain, drug-resistant mutations in the ATP binding pocket (except V654A), and activation loops (except D816V). In addition, 18 exhibits a good in vivo pharmacokinetic (PK) profile in different species including mice, rats, and dogs. It also displays good in vivo antitumor efficacy in the c-KIT T670I, D820G, and Y823D mutant-mediated mice models as well as in the c-KIT wt patient primary cells which are known to be imatinib-resistant. The potent activity against a broad spectrum of clinically important c-KIT mutants combining the good in vivo PK/pharmacodynamic properties of 18 indicates that it might be a new potential therapeutic candidate for gastrointestinal stromal tumors.

Aryl hydrocarbon receptor activation modulates γδ intestinal intraepithelial lymphocytes and protects against ischemia/reperfusion injury in the murine small intestine.

The pathogenesis of intestinal ischemia/reperfusion (I/R) is associated with dysregulation of the intestinal immune system. The aryl hydrocarbon receptor (AhR), a receptor expressed in gamma­delta (γδ) intraepithelial lymphocytes (IELs), is thought to regulate inflammation in the bowel. γδIELs are a key immunologic compartment with a capacity to modulate immune responses. In the present study, the function of the AhR in γδIELs in a mouse model of intestinal I/R injury was investigated to determine whether the AhR attenuates intestinal injury induced by intestinal I/R. Mice were assigned to three groups: sham, I/R and I/R+6­formylindolo(3,2­b)carbazole (FICZ). The sham group received no ischemia treatment, whereas the I/R and I/R+FICZ groups underwent upper mesenteric vessel ischemia for 30 min. The I/R group was injected intraperitoneally with 0.3 ml saline and the I/R+FICZ group was administered 1 µg of FICZ before a subsequent 6 h reperfusion. Then, the mice were sacrificed and the entire small intestinal tissues were collected for histologic examination. The phenotype and apoptosis of γδIELs and activation of CD4+ and CD8+ IELs were examined using flow cytometry. The cytokine mRNA and anti­apoptosis gene expression in IELs were measured by qPCR. FICZ increased the γδIEL population and anti­apoptosis genes in the γδIELs. FICZ reduced the percentage of activated CD4+ and CD8+ subpopulations and the expression of pro­inflammatory mediator genes in IELs. FICZ inhibited inflammation in the gastrointestinal tract of mice with I/R injury. These results suggest that the AhR plays an important role in protecting the small intestine from I/R and increasing the γδIEL population by decreasing apoptosis of γδIELs.

AhR activation protects intestinal epithelial barrier function through regulation of Par-6.

The Par complex (Par-6/Par-3/aPKC) plays a key role in the maintenance of the intestinal barrier function through the regulation of epithelial junction formation. The aryl hydrocarbon receptor (AhR) has been shown to be an important regulator for intestinal homeostasis. In this study, we investigated the role of the AhR activation on the regulation of Par complex. AhR activation by 6-formylindolo (3,2-b) carbazole (FICZ) represses the abnormal expression of the Par complex in a mouse model of dextran sulphate sodium (DSS)-induced colitis. In T84 cells, overexpression of Par-6 causes intestinal barrier dysfunction. Lipopolysaccharide (LPS)-induced intestinal epithelial barrier dysfunction and increase in Par-6 expression was prevented by AhR activation. However, FICZ did not alter the expression of Par-3 or aPKC. Furthermore, AhR activation alleviated LPS-induced increase of Par-6 through repressing the expression of activating protein-2γ (Ap-2γ). These results reveal the protective effects of AhR activation on LPS induced disruption of intestinal epithelial barrier function through suppressing the expression of Par-6 expression. Our findings provide novel insights into the protective role of AhR in intestinal barrier function.

KGF inhibits hypoxia-induced intestinal epithelial cell apoptosis by upregulating AKT/ERK pathway-dependent E-cadherin expression.

OBJECTIVE: Intestinal ischemia-reperfusion (I/R) causes direct cellular damage, and the potential injury to the mucosal structure and barrier function. Keratinocyte growth factor (KGF) is highly expressed in gastrointestinal tract and exerts beneficial effects for intestinal epithelial growth and maintenance. E-cadherin plays an important role in intestinal epithelium renewal. However, the regulatory role of KGF on E-cadherin levels and I/R-induced apoptosis remain to be explored. The present study aimed to identify the effect of KGF on E-cadherin expression and I/R-induced intestinal epithelial cell apoptosis. METHODS: Caco2 cells were treated with KGF (100 ng/ml) for 0, 4, 8, 12, and 24 h under hypoxia or normoxia. An E-cadherin-knockdown model was successfully established by treatment with E-cadherin RNAi. Western blotting and immunofluorescence labeling were performed to assess E-cadherin expression. Levels of PI3K|[sol]|Akt/mitogen-activated protein kinases (MAPKs), phosphoinositide 3-kinase (PI3K|[sol]|Akt)/PI3K|[sol]|Akt pathway-related proteins, and apoptosis-related proteins were also detected by western blot. Finally, a rat model of acute intestinal I/R was established and treated with KGF. Hematoxylin-eosin (HE), terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL), and immunofluorescence staining were performed to detect morphological changes in intestinal mucosal epithelium and Caco2 cell apoptosis. RESULTS: KGF enhanced E-cadherin expression in differentiated intestinal epithelial cells under hypoxia via AKT/extracellular-regulated kinase (ERK) pathway regulation. In vitro, E-cadherin downregulation aggravates hypoxia-induced intestinal epithelial cell apoptosis. In the rat model, KGF increased E-cadherin expression, which was associated with the reduced apoptosis. CONCLUSIONS: KGF exerts protective effects on intestinal epithelial cells under hypoxia by elevating E-cadherin levels or activating AKT/ERK signaling.

6-Formylindolo(3,2-b)carbazole induced aryl hydrocarbon receptor activation prevents intestinal barrier dysfunction through regulation of claudin-2 expression.

6-Formylindolo(3,2-b)carbazole (FICZ), a high-affinity aryl hydrocarbon receptor (AhR) ligand, plays a protective role in inflammatory bowel disease (IBD) through activation of AhR. Interleukin-6 (IL-6) induced intestinal epithelial barrier dysfunction is involved in the pathological process of IBD. In this study, we investigated the protective effects of FICZ on IL-6 induced intestinal epithelial barrier injury. Our data show that AhR activation by FICZ ameliorated colonic inflammation, decreased IL-6 and claudin-2 expression, and maintained intestinal barrier function in a mouse model of dextran sulphate sodium (DSS)-induced colitis. In Caco-2 and T84 intestinal epithelial cells, FICZ also prevented the increase of intestinal epithelial permeability and claudin-2 expression induced by IL-6. Depletion of AhR expression by small interfering (si)RNA reversed FICZ induced decrease of claudin-2. Furthermore, IL-6 induced upregulation of claudin-2 was required for increased caudal-related homeobox 2 (CDX-2) and hepatocyte-nuclear factor (HNF)-1α. However, FICZ repressed the increase of CDX-2 and HNF-1α expression induced by IL-6. These results reveal the protective effects of FICZ on IL-6 induced disruption of intestinal epithelial barrier function through suppressing the expression of claudin-2. In addition, CDX-2 and HNF-1α are involved in the regulation of claudin-2 after IL-6 and FICZ treatment. Therefore compounds related to AhR ligands may be potential pharmaceutical agents to treat IBD.

DMOG ameliorates IFN-γ-induced intestinal barrier dysfunction by suppressing PHD2-dependent HIF-1α degradation.

Hypoxia-inducible factor 1α (HIF-1α) has been well established as a protective factor for intestinal barrier function in intestinal epithelial cells. Recently, a study found that increased HIF-1α-induced intestinal barrier dysfunction. We proposed that lymphocyte-derived interferon-gamma (IFN-γ) might be responsible for the intestinal barrier dysfunction caused by increased HIF-1α. HT-29 cell monolayers were grown in the presence or absence of IFN-γ under hypoxia. Then, the transepithelial electrical resistance was measured, and HIF-1α-modulated intestinal barrier protective factors were quantified by polymerase chain reaction (PCR). PCR, western blotting, and chromatin immunoprecipitation of HIF-1α were performed. Dimethyloxalyglycine (DMOG), an inhibitor of prolyl-hydroxylases (PHDs) that stabilizes HIF-1α during normoxia, and RNA interference of PHDs were also used to identify the signal pathway between IFN-γ and HIF-1α. We demonstrated that IFN-γ caused barrier dysfunction in hypoxic HT-29 cell monolayers via suppressing HIF-1α and HIF-1α-modulated intestinal barrier protective factors. We found that IFN-γ decreased HIF-1α protein expression instead of affecting HIF-1α transcription or transcriptional activity. Study also showed that DMOG reversed the IFN-γ-induced decrease in HIF-1α protein expression. Further, we found that PHD2 is the major regulator of IFN-γ-induced HIF-1α degradation by PHD inhibition and RNA interference. We conclude that IFN-γ caused barrier dysfunction by promoting PHD-, especially PHD2-, dependent HIF-1α degradation, and DMOG or PHD2 inhibition reversed this HIF-1α suppression and ameliorated barrier dysfunction. Combined with other studies demonstrating HIF-1α activation in lymphocytes promotes IFN-γ secretion, these findings suggest a mechanism by which increased HIF-1α-induced intestinal barrier dysfunction.

Up-regulation of intestinal epithelial cell derived IL-7 expression by keratinocyte growth factor through STAT1/IRF-1, IRF-2 pathway.

BACKGROUND: Epithelial cells(EC)-derived interleukin-7 (IL-7) plays a crucial role in control of development and homeostasis of neighboring intraepithelial lymphocytes (IEL), and keratinocyte growth factor (KGF) exerts protective effects on intestinal epithelial cells and up-regulates EC-derived IL-7 expression through KGFR pathway. This study was to further investigate the molecular mechanism involved in the regulation of IL-7 expression by KGF in the intestine. METHODS: Intestinal epithelial cells (LoVo cells) and adult C57BL/6J mice were treated with KGF. Epithelial cell proliferation was studied by flow cytometry for BrdU-incorporation and by immunohistochemistry for PCNA staining. Western blot was used to detect the changes of expression of P-Tyr-STAT1, STAT1, and IL-7 by inhibiting STAT1. Alterations of nuclear extracts and total proteins of IRF-1, IRF-2 and IL-7 following IRF-1 and IRF-2 RNA interference with KGF treatment were also measured with western blot. Moreover, IL-7 mRNA expressions were also detected by Real-time PCR and IL-7 protein level in culture supernatants was measured by enzyme linked immunosorbent assay(ELISA). RESULTS: KGF administration significantly increased LoVo cell proliferation and also increased intestinal wet weight, villus height, crypt depth and crypt cell proliferation in mice. KGF treatment led to increased levels of P-Tyr-STAT1, RAPA and AG490 both blocked P-Tyr-STAT1 and IL-7 expression in LoVo cells. IRF-1 and IRF-2 expression in vivo and in vitro were also up-regulated by KGF, and IL-7 expression was decreased after IRF-1 and IRF-2 expression was silenced by interfering RNA, respectively. CONCLUSION: KGF could up-regulate IL-7 expression through the STAT1/IRF-1, IRF-2 signaling pathway, which is a new insight in potential effects of KGF on the intestinal mucosal immune system.

Keratinocyte growth factor pretreatment prevents radiation-induced intestinal damage in a mouse model.

BACKGROUND: Radiation-induced gastrointestinal syndrome is usually severe in clinical practice. Keratinocyte growth factor (KGF) plays an important role in the intestinal mucosal growth and repair of intestinal injury. This study was to investigate the effects of KGF on radiation-induced intestinal damage, especially the barrier dysfunction, in a mouse model. METHODS: Adult C57BL/6J mice were randomized into three groups: normal control, irradiation group, and KGF-treated group. Mice in the later two groups received irradiation with a dose of 6 Gy from Co-60 source. In the KGF-treated group, KGF was intraperitoneally given once daily (5 mg/kg/day) for 5 consecutive days before irradiation. Mice were killed at 3 days after irradiation and the small bowel was collected for histology. Epithelial cell proliferation was studied by immunohistochemistry for proliferating cell nuclear antigen. Claudin-1 and ZO-1 expressions were determined by western blot assay and immunohistochemistry. Epithelial barrier function was assessed with transepithelial resistance. RESULTS: KGF significantly promoted the recovery of mucosa from radiation-induced injury demonstrated by mucosal histology, villus height, crypt depth, and crypt cell proliferation. KGF also improved the disrupted distribution of tight junction proteins and the epithelial barrier dysfunction after irradiation. CONCLUSION: KGF pretreatment could improve radiation-induced intestinal injury including the epithelial structure and function in a mouse model.

Keratinocyte growth factor improves epithelial structure and function in a mouse model of intestinal ischemia/reperfusion.

BACKGROUND: Intestinal ischemia/reperfusion (I/R) induces the desquamation of the intestinal epithelium, increases the intestinal permeability, and in patients often causes fatal conditions including sepsis and multiple organ failure. Keratinocyte growth factor (KGF) increases intestinal growth, although little is known about KGF activity on intestinal function after intestinal I/R. We hypothesized that KGF administration would improve the intestinal function in a mouse model of intestinal I/R. METHODS: Adult C57BL/6J mice were randomized to three groups: Sham, I/R group and I/R+KGF group. Mice were killed on day 5, and the small bowel was harvested for histology, wet weight, RNA and protein content analysis. Epithelial cell (EC) proliferation was detected by immunohistochemistry for PCNA, and apoptosis was determined by TUNEL staining. The expressions of Claudin-1 and ZO-1 were detected by immunohistochemistry. Epithelial barrier function was assessed with transepithelial resistance (TER). RESULTS: KGF significantly increased the intestinal wet weight, contents of intestinal protein and RNA, villus height, crypt depth and crypt cell proliferation, while KGF resulted in the decrease of epithelial apoptosis. KGF also stimulated the recovery of mucosal structures and attenuated the disrupted distribution of TJ proteins. Moreover, KGF attenuated the intestinal I/R-induced decrease in TER and maintained the intestinal barrier function. CONCLUSION: KGF administration improves the epithelial structure and barrier function in a mouse model of intestinal I/R. This suggests that KGF may have clinical applicability.

Keratinocyte growth factor up-regulates Interleukin-7 expression following intestinal ischemia/reperfusion in vitro and in vivo.

BACKGROUND: Epithelial cell (EC)-derived Interleukin-7 (IL-7) plays a crucial role in control of neighboring intestinal intraepithelial lymphocytes (IEL) development and homeostasis, and IEL derived keratinocyte growth factor (KGF) promotes intestinal epithelial growth, which was regulated by EC-derived IL-7. On this basis, we hypothesize that there is a crosstalk between IELs and ECs, and KGF could regulate the EC-derived IL-7 expression, which should be associated with the protective effects by KGF on intestinal injury. METHODS: Histological evaluation was performed in small intestine tissues of patients with intestinal obstruction and IL-7 expression was detected by immunofluorescence. Intestinal epithelial cells (LoVo) and adult C57BL/6J mice undergoing ischemia/reperfusion injury were treated with recombinant KGF. KGF, KGF receptor(KGFR) and IL-7 expressions were measured with western blot and immunofluorescence analysis. RESULTS: IL-7 expression increased in the mild ischemia while decreased in severe ischemia small intestinal tissues of patients with intestinal obstruction. KGF expression significantly decreased while IL-7 expression increased early after acute intestinal I/R administration in a mouse model. KGF treatment significantly increased the IL-7 expression both in vitro and in vivo, while when the KGFR was blocked, the findings above were absent. In addition, our results showed changes of IL-7 expression at different stages after acute intestinal I/R administration, KGF treatment significantly attenuated the decreasing of IL-7 expression caused by acute intestinal I/R. CONCLUSION: KGF could up-regulate the IL-7 expression both in vitro and in vivo through KGFR pathway, which should have associated with the protective effects of KGF in intestinal injury.

[Value of the laparoscopy combined with double-balloon enteroscopy in diagnosis and treatment of intestinal diseases].

OBJECTIVE: To evaluate the value of laparoscopy combined with double-balloon enteroscopy (DBE) for the diagnosis and treatment of intestinal diseases. METHODS: Clinical data of 69 cases with suspected small bowel diseases undergoing laparoscopic and DBE for the diagnosis and treatment were retrospectively analyzed. RESULTS: The lesions were found in 48 cases by laparoscopy. DBE was required in the remaining 21 patients to identify the underlying condition. All the operations were successfully completed using the laparoscopic approach, including totally laparoscopic bowel resection (n=11), and laparoscopic-assisted bowel resection (n=58). There were no anastomotic leakage, postoperative bleeding, intestinal obstruction, or wound infection. All the patients were discharged within 7 to 9 days after surgery. Postoperative pathological examination showed vascular abnormally (n=10), gastrointestinal stromal tumor (n=20), intestinal adenocarcinoma (n=5), intestinal neurofibroma (n=2), diverticulum (n=5), intestinal mucosal ulceration (n=8), intestinal tuberculosis (n=3), postoperative pouch bleeding (n=1), intestinal polyp (n=6), Crohn's disease (n=5), Meckel diverticulum (n=2), metastatic kidney cancer (n=1), and metastatic lung cancer (n=1). Length of follow up ranged from 3 months to 4 years, during which no re-bleeding occurred, 2 patients with gastrointestinal stromal tumor died of local recurrence and liver metastasis, 1 patient with adenocarcinoma died of local recurrence involving pancreatic head, duodenum, and mesenteric vessels, 2 patients with metastatic disease died of peritoneal recurrence and liver metastasis. CONCLUSION: Laparoscopic combined with DBE has a high detection rate for small intestinal disease with accurate localization, less trauma, and quicker recovery.

Identification of CD44+CD24+ gastric cancer stem cells.

OBJECTIVE: Purification and characterization of cancer stem cells (CSCs) can lead to the identification of targets for therapeutic interventions of cancer. With regard to gastric cancer, studies have not yet defined and characterized CSCs. METHODS: The expression of the cell surface markers CD44 and CD24 was examined in gastric cell lines AGS and gastric cancer tissues from five patients with fluorescence-activated cell sorting analysis (FACS). The tumorigenic properties, self-renewal, and differentiated progeny in the two distinct cell populations CD44+CD24+ and CD44-CD24- were identified in vivo serial transplantation and in vitro culture. Real-time RT-PCR was used to assess the expression of sonic hedgehog (SHH), patched 1 (PTCH1), and GLI3 signaling molecules in CD44+CD24+ and CD44-CD24- cells. RESULTS: As few as 200 CD44+CD24+ cells injected in NOD-SCID mice were able to generate tumors in 50% of mice (6 of 12), while tumors did not form in mice until at least 10,000 CD44-CD24- cells were injected, where only one of 12 mice formed a tumor, further verifying that CD44+CD24+ gastric cancer cells have the capacity to both self-renew and produce differentiated progeny. Moreover, SHH, PTCH1, and GLI3 mRNA expression increased significantly in the CD44+CD24+ subpopulation when compared with the CD44-CD24- subpopulation. CONCLUSIONS: These studies strongly suggest that the CD44+CD24+ subpopulation of human gastric cancer cell lines, AGS, is gastric cancer stem cells.