GPC1 Is Associated with Poor Prognosis and Treg Infiltration in Colon Adenocarcinoma.

Glypican-1 (GPC1) is a glycosylated protein recognized as a promising biomarker for cancer. Nonetheless, there have been few systematic studies on GPC1 in colon adenocarcinoma (COAD). We conducted bioinformatic analysis based on The Cancer Genome Atlas (TCGA) and used clinical samples to verify that GPC1 is overexpressed in colon adenocarcinoma. Kaplan-Meier analysis showed that higher GPC1 expression was associated with poor overall survival (OS). The Cox regression model further showed that GPC1 expression is an independent negative prognostic factor for COAD. Gene set enrichment analysis demonstrated that multiple oncogenic signaling pathways were differentially enriched in GPC1 high- versus low-expressing COAD tumors, including DNA methylation, G2/M damage checkpoint, and telomere dysfunction. We observed a positive correlation between GPC1 expression and immune cell infiltration, such as regulatory T cells (Tregs), macrophages, and mast cells, and immunohistochemistry of 50 COAD tissues revealed that GPC1 expression was positively associated with Treg enrichment. Our results provide a promising candidate gene to predict the prognosis of COAD and new insights into tumor immunity. Further research is required to validate these results.

The therapeutic potential of exosomes derived from different cell sources in liver diseases.

Exosomes are small nanovesicles with a size of approximately 40-120 nm that are secreted from cells. They are involved in the regulation of cell homeostasis and mediate intercellular communication. In addition, they carry proteins, nucleic acids, and lipids that regulate the biological activity of receptor cells. Recent studies have shown that exosomes perform important functions in liver diseases. This review will focus on liver diseases (drug-induced liver injury, hepatic ischemia-reperfusion injury, liver fibrosis, acute liver failure, and hepatocellular carcinoma) and summarize the therapeutic potential of exosomes from different cell sources in liver disease.

Laparoscopic vs open surgery in ileostomy reversal in Crohn's disease: A retrospective study.

BACKGROUND: Although minimally invasive surgery is becoming more commonly applied for ileostomy reversal (IR), there have been relatively few studies of IR for patients with Crohn's disease (CD). It is therefore important to evaluate the potential benefits and risks of laparoscopy for patients with CD. AIM: To compare the safety, feasibility, and short-term and long-term outcomes of laparoscopic IR (LIR) vs open IR (OIR) for the treatment of CD. METHODS: The baseline characteristics, operative data, and short-term (30-d) and long-term outcomes of patients with CD who underwent LIR and OIR at our institution between January 2017 and January 2020 were retrieved from an electronic database and retrospectively reviewed. RESULTS: Of the 60 patients enrolled in this study, LIR was performed for 48 and OIR for 12. There were no statistically significant differences in baseline characteristics, operation time, intraoperative blood loss, days to flatus and soft diet, postoperative complications, hospitalization time, readmission rate within 30 d, length of hospitalization, hospitalization costs, or reoperation rate after IR between the two groups. However, patients in the LIR group more frequently required lysis of adhesions as compared to those in the OIR group (87.5% vs 41.7%, respectively, P < 0.05). Notably, following exclusion of patients who underwent enterectomy plus IR, OIR was more advantageous in terms of postoperative recovery of gastrointestinal function and hospitalization costs. CONCLUSION: The safety and feasibility of LIR for the treatment of CD are comparable to those of OIR with no increase in intraoperative or postoperative complications.

Laparoscopy for Crohn's disease: A comprehensive exploration of minimally invasive surgical techniques.

BACKGROUND: Along with the unceasing progress of medicine, Crohn's disease (CD), especially complex CD, is no longer a taboo for minimally invasive surgery. However, considering its special disease characteristics, more clinical trials are needed to confirm the safety and feasibility of laparoscopic surgery for CD. AIM: To investigate the safety and feasibility of laparoscopic enterectomy for CD, assess the advantages of laparoscopy over laparotomy in patients with CD, and discuss comprehensive minimally invasive surgical techniques in complex CD. METHODS: This study prospectively collected clinical data from patients with CD who underwent enterectomy from January 2017 to January 2020. It was registered in the Chinese clinical trial database with the registration number ChiCTR-INR-16009321. Patients were divided into a laparoscopy group and a traditional laparotomy group according to the surgical method. The baseline characteristics, operation time, intraoperative blood loss, temporary stoma, levels of abdominal adhesion, pathological characteristics, days to flatus and soft diet, postoperative complications, hospitalization time, readmission rate within 30 d, and hospitalization cost were compared between the two groups. RESULTS: A total of 120 eligible patients were enrolled into the pre-standardized groups, including 100 in the laparoscopy group and 20 in the laparotomy group. Compared with the laparotomy group, the postoperative hospitalization time in the laparoscopy group was shorter (9.1 ± 3.9 d vs 11.0 ± 1.6 d, P < 0.05), the days to flatus were fewer (2.8 ± 0.8 d vs 3.5 ± 0.7 d, P < 0.05), the days to soft diet were fewer (4.2 ± 2.4 d vs 6.2 ± 2.0 d, P < 0.05) and the intraoperative blood loss was less (103.3 ± 80.42 mL vs 169.5 ± 100.42 mL, P < 0.05). There were no statistically significant differences between the two groups in preoperative clinical data, operation time (149.0 ± 43.8 min vs 159.2 ± 40.0 min), stoma rate, levels of abdominal adhesion, total cost of hospitalization, incidence of postoperative complications [8.0% (8/100) vs 15.0% (3/20)], or readmission rate within 30 days [1.0% (1/100) vs 0.00 (0/20)]. CONCLUSION: Compared with laparotomy, laparoscopic enterectomy promotes the recovery of gastrointestinal function, shortens the postoperative hospitalization time, and does not increase the incidence of postoperative complications. Laparoscopic enterectomy combined with varieties of minimally invasive surgical techniques is a safe and acceptable therapeutic method for CD patients with enteric fistulas.

Minimally invasive surgery for glycogen storage disease combined with inflammatory bowel disease: A case report.

BACKGROUND: Inflammatory bowel disease (IBD) is rare in patients with glycogen storage disease (GSD). In GSD patients, a decrease in the number of neutrophils leads to prolonged intestinal infection, leading to the formation of chronic inflammation and eventually the development of IBD. Minimally invasive surgery for patients with IBD has been proven to reduce inflammatory responses and postoperative risks and ultimately promote rapid recovery. Herein we discuss minimally invasive surgery and the perioperative management in a patient with GSD and IBD. CASE SUMMARY: A 23-year-old male had GSD Ib associated with IBD-like disease for 10 years. Despite standard treatments, such as mesalazine, prednisone and adalimumab, the patient eventually developed colonic stenosis with incomplete ileus. After adequate assessment, the patient was treated with minimally invasive surgery and discharged in stable condition. CONCLUSION: Minimally invasive surgery for patients with IBD and GSD is safe, feasible and effective.

Graft-derived extracellular vesicles transported across subcapsular sinus macrophages elicit B cell alloimmunity after transplantation.

Despite the role of donor-specific antibodies (DSAs) in recognizing major histocompatibility complex (MHC) antigens and mediating transplant rejection, how and where recipient B cells in lymphoid tissues encounter donor MHC antigens remains unclear. Contrary to the dogma, we demonstrated here that migration of donor leukocytes out of skin or heart allografts is not necessary for B or T cell allosensitization in mice. We found that mouse skin and cardiac allografts and human skin grafts release cell-free donor MHC antigens via extracellular vesicles (EVs) that are captured by subcapsular sinus (SCS) macrophages in lymph nodes or analog macrophages in the spleen. Donor EVs were transported across the SCS macrophages, and donor MHC molecules on the EVs were recognized by alloreactive B cells. This triggered B cell activation and DSA production, which were both prevented by SCS macrophage depletion. These results reveal an unexpected role for graft-derived EVs and open venues to interfere with EV biogenesis, trafficking, or function to restrain priming or reactivation of alloreactive B cells.

Mast Cell Tryptase Promotes Inflammatory Bowel Disease-Induced Intestinal Fibrosis.

BACKGROUND: Intestinal fibrosis is the final pathological outcome of chronic intestinal inflammation without specific therapeutic drugs, which leads to ileus and surgical intervention. Intestinal fibrosis is characterized by excessive deposition of extracellular matrix (ECM). The role of mast cells (MCs), which are members of the sentinel immune cell population, is unknown in intestinal fibrosis. METHODS: In this study, we analyzed changes in MCs, tryptase proteins, and ECM components in human fibrotic and control patient intestines. We constructed dextran sodium sulfate-induced intestinal fibrosis models using wild-type mice, MC-reconstituted mice, and MC-deficient mice to explore the role of MCs and tryptase in intestinal fibrosis. The roles and mechanisms of MCs and tryptase on fibroblasts were evaluated using human MCs (HMC-1 and LAD-2), commercial tryptase proteins, human colon fibroblasts (CCD-18Co fibroblasts), the tryptase inhibitor APC366, and the protease-activated receptor-2 (PAR-2) antagonist ENMD-1068. RESULTS: Regardless of whether the colon was a human colon or a mouse colon, the fibrotic intestinal tissue had increased MC infiltration and a higher expression of ECM proteins or genes than that of the control group. The dextran sodium sulfate-induced intestinal fibrosis in MC-deficient mice was alleviated compared with that in wild-type mice. After MC reconstruction in MC-deficient mice, the alleviating effect disappeared. Tryptase, as a content stored in MC granules, was released into fibrotic intestinal tissues in the form of degranulation, resulting in an increased expression of tryptase. Compared with the control group, the tryptase inhibition group (the APC366 group) had reduced intestinal fibrosis. The CCD-18Co fibroblasts, when cocultured with MCs or treated with tryptase proteins, were activated to differentiate into myofibroblasts and secrete more ECM proteins (such as collagen and fibronectin). The underlying mechanism of fibroblast activation by tryptase was the activation of the PAR-2/Akt/mTOR pathway. CONCLUSIONS: We found that MC tryptase promotes inflammatory bowel disease-induced intestinal fibrosis. The underlying mechanism is that tryptase promotes the differentiation of fibroblasts into fibrotic-phenotype myofibroblasts by activating the PAR-2/Akt/ mTOR pathway of fibroblasts.

iNOS/NO is required for IRF1 activation in response to liver ischemia-reperfusion in mice.

BACKGROUND: Ischemia and reperfusion (I/R) induces cytokines, and up-regulates inducible nitric oxide synthase (iNOS), interferon regulatory factor-1(IRF1) and p53 up-regulated modulator of apoptosis (PUMA), which contribute to cell death and tissue injury. However, the mechanisms that I/R induces IRF1-PUMA through iNOS/NO is still unknown. METHODS: Ischemia was induced by occluding structures in the portal triad (hepatic artery, portal vein, and bile duct) to the left and median liver lobes for 60 min, and reperfusion was initiated by removal of the clamp. Induction of iNOS, IRF1 and PUMA in response to I/R were analyzed. I/R induced IRF1 and PUMA expression were compared between iNOS wild-type and iNOS knockout (KO) mice. Human iNOS gene transfected-cells were used to determine iNOS/NO signals targeting IRF1. To test whether HDAC2 was involved in the mediation of iNOS/NO-induced IRF1 transcriptional activities and its target gene (PUMA and p21) expression, NO donors were used in vitro and in vivo. RESULTS: IRF1 nuclear translocation and PUMA transcription elevation were markedly induced following I/R in the liver of iNOS wild-type mice compared with that in knock-out mice. Furthermore, I/R induced hepatic HDAC2 expression and activation, and decreased H3AcK9 expression in iNOS wild-type mice, but not in the knock-out mice. Mechanistically, over-expression of human iNOS gene increased IRF1 transcriptional activity and PUMA expression, while iNOS inhibitor L-NIL reversed these effects. Cytokine-induced PUMA through IRF1 was p53 dependent. IRF1 and p53 synergistically up-regulated PUMA expression. iNOS/NO-induced HDAC2 mediated histone H3 deacetylation and promoted IRF1 transcriptional activity. Moreover, treating the cells with romidepsin, an HDAC1/2 inhibitor decreased NO-induced IRF1 and PUMA expression. CONCLUSIONS: This study demonstrates a novel mechanism that iNOS/NO is required for IRF1/PUMA signaling through a positive-feedback loop between iNOS and IRF1, in which HDAC2-mediated histone modification is involved to up-regulate IRF1 in response to I/R in mice.

Identification of Immune Cell Landscape and Construction of a Novel Diagnostic Nomogram for Crohn's Disease.

Crohn's disease (CD) has an increasing incidence and prevalence worldwide. The etiology of CD remains unclear and there is no gold standard for diagnosis. The dysregulated immune response and different infiltration status of immune cells are critical for CD pathogenesis; therefore, it is important to provide an overview of immune-cell alterations in CD and explore a novel method for auxiliary diagnosis. Here we analyzed microarray datasets from Gene Expression Omnibus (GEO), and an extended version of Cell-type Identification By Estimating Relative Subsets Of RNA Transcripts (CIBERSORTx) was utilized to estimate the fraction of 22 types of immune cells. Differentially expressed genes (DEGs) and a protein-protein interaction (PPI) network were identified, and we performed gene set enrichment analysis (GSEA) and gene set variation analysis (GSVA) to identify differentially regulated pathways in CD. Least absolute shrinkage and selection operator (LASSO) regression was conducted to filter features, and a diagnostic nomogram based on logistic regression was built and validated in an independent validation cohort. In the derivation cohort, we found a proportion of 17 immune-cell types to be significantly altered between CD and healthy controls and a total of 150 DEGs were identified, which were mostly related to the immune response. Among the 15 hub genes based on the PPI network, C-X-C chemokine ligand 8 (CXCL8) and interleukin-1B (IL-1B) showed the highest degree of interaction. Additionally, GSEA and GSVA identified five significantly enriched pathways, among which the nucleotide-binding oligomerization domain (NOD)-like receptor signaling pathway was critical in the CD development. Furthermore, six variables comprising of CXCL8, IL-1B, M1 macrophages, regulatory T cells, CD8+ T cells, and plasma cells were identified by LASSO regression and incorporated into a logistic regression model. The nomogram displayed a good prediction, with a 0.915 area under the receiver operating curve (AUC) and a C-index of 0.915 [95% confidence interval (CI): 0.875-0.955]. Similar results were found in the validation cohort, with an AUC of 0.884 and a 0.884 C-index (95% CI: 0.843-0.924). These results provide novel in silico insight into cellular and molecular characteristics of CD and potential biomarkers for diagnosis and targeted therapy.

Interferon regulatory factor 1 priming of tumour-derived exosomes enhances the antitumour immune response.

BACKGROUND: Tumour-derived exosomes (TEXs) have a potential for application in cancer vaccines. Whether TEXs after induction by interferon regulatory factor 1 (IRF-1) are capable of enhancing the antitumour response remains to be determined. METHODS: Exosomes released by tumour cells infected with IRF-1-expressing adenovirus (IRF-1-Exo) or treated with interferon-γ (IFN-Exo) were isolated via ultracentrifugation. The IRF-1 target proteins IL-15Rα and MHC class I (MHC-I) were analysed by western blot. Exosomes along with CpG adjuvant were injected into tumour models to assess the antitumour effects. Tumours were harvested for immunofluorescence staining. Splenocytes from tumour-bearing mice were co-cultured with tumour cells. The IFNγ-positive and granzyme B-positive CD8α+ splenocyte cells were quantified by flow cytometry. RESULTS: The IRF-1-Exo or IFN-Exo displayed increased IL-15Rα and MHC-I expression. Injection of IRF-1-Exo or IFN-Exo combined with CpG had improved antitumour effects in mice. This effect may be a result of increased infiltration of tumours by CD4+ and CD8α+ T cells. Antibody-mediated depletion of CD4+ or CD8+ T cells abrogated the antitumour effects. Splenocytes isolated from CpG+IRF-1-Exo-injected Hepa 1-6 tumour mice had increased IFNγ-positive and granzyme B-positive CD8+ cells after co-culturing with Hepa 1-6 cells as compared with MC38 cells. CONCLUSIONS: The IRF-1 priming of TEXs enhances antitumour immune response.

Interferon regulatory factor 1-Rab27a regulated extracellular vesicles promote liver ischemia/reperfusion injury.

The role and regulators of extracellular vesicle (EV) secretion in hepatic ischemia/reperfusion (IR) injury have not been defined. Rab27a is a guanosine triphosphatase known to control EV release. Interferon regulatory factor 1 (IRF-1) is a transcription factor that plays an important role in liver IR and regulates certain guanosine triphosphatases. However, the relationships among IRF-1, Rab27a, and EV secretion are largely unknown. Here, we show induction of IRF-1 and Rab27a both in vitro in hypoxic hepatocytes and in vivo in warm IR and orthotopic liver transplantation livers. Interferon γ stimulation, IRF-1 transduction, or IR promoted Rab27a expression and EV secretion. Meanwhile, silencing of IRF-1 decreased Rab27a expression and EV secretion. Rab27a silencing decreased EV secretion and liver IR injury. Ten putative IRF-1 binding motifs in the 1,692-bp Rab27a promoter region were identified. Chromatin immunoprecipitation and electrophoretic mobility shift assay verified five functional IRF-1 binding motifs, which were confirmed by a Rab27a promoter luciferase assay. IR-induced EVs contained higher oxidized phospholipids (OxPL). OxPLs on the EV surface activated neutrophils through the toll-like receptor 4 pathway. OxPL-neutralizing E06 antibody blocked the effect of EVs and decreased liver IR injury. CONCLUSION: These findings provide a novel mechanism by which IRF-1 regulates Rab27a transcription and EV secretion, leading to OxPL activation of neutrophils and subsequent hepatic IR injury. (Hepatology 2018;67:1056-1070).

Upregulation of heme oxygenase-1 in Kupffer cells blocks mast cell degranulation and inhibits dendritic cell migration in vitro.

Kupffer cells (KCs) influence liver allografts by interacting with other non­parenchymal cells. However, the exact mechanism remains unclear. Upregulation of heme oxygenase-1 (HO-1) in KCs upon interaction with mast cells (MCs), and the effects on dendritic cell (DC) function, were investigated in the present study. KCs, MCs and DCs were prepared from 8­10­week­old C57BL/6 mice. KCs were pretreated with PBS, dimethyl sulfoxide, hemin (50 µM; HO­1 inducer), and zinc protoporphyrin (50 µM; HO­1 inhibitor) for 8 h. Reverse transcription­polymerase chain reaction and western blotting was performed to determine HO­1 mRNA and protein levels in KCs, respectively. C­C motif chemokine receptor 7 (CCR7) surface molecules were measured using flow cytometry, and prostaglandin E2 (PGE2), C­C motif chemokine ligand (CCL) 19 and CCL21 were measured by ELISA. The Transwell model was used to investigate the migration of DCs. Pretreatment of KCs with hemin induced HO­1 transcription and protein expression, and interacted with and stabilized MC membranes. When co­cultured with MCs, the expression of CCR7 on DCs was reduced, and PGE2, CCL19 and CCL21 were similarly decreased. DC migration was also impaired. Upregulation of HO­1 in KCs blocked MC degranulation and reduced DC migration.

The role of mast cells in ischemia and reperfusion injury.

INTRODUCTION: Ischemia and reperfusion (IR) injury is a challenging clinical problem that is triggered by ischemia in an organ followed by subsequent restoration of the blood supply. The effects of mast cell (MC) in IR injury are not totally clear. MATERIALS AND METHODS: We review the body of literature on the role of MCs in IR injury based on an unrestricted Pubmed search for the descriptors "mast cell", "ischemia" and "reperfusion injury", as well as discuss implications for treatment and future directions. RESULTS: Shortly after IR, chemicals released by MC can trigger vasoactive substance formation, tissue leakage, upregulation of adhesive molecules followed by leukocyte recruitment and infiltration, and pronecrotic pathway activation, among other physiologic changes. In the long term, MCs may influence tissue remodeling and repair as well as blood restoration after IR. Consistent with these findings, methods and drugs that target MCs have been shown to attenuate IR injury. CONCLUSION: It has been demonstrated that MCs play a role in IR injury, but the mechanisms are complex and need to be further studied.

Inhibiting mast cell degranulation by HO-1 affects dendritic cell maturation in vitro.

OBJECTIVE AND DESIGN: Mast cell (MC) degranulation can break peripheral immune tolerance. However, its mechanism remains unclear. Our goal was to study the stabilization of MC membranes by heme oxygenase-1 (HO-1) in order to influence dendritic cell (DC) function. MATERIAL: Mast cells and dendritic cells were prepared from 8-week-old to 10-week-old C57BL/6 mice; spleen mononuclear cells (SMCs) were prepared from 8-week-old to 10-week-old C57BL/6 and Balb/c mice. TREATMENT: Mast cells were pretreated with PBS, DMSO, Hemin (50 µl/ml), and Znpp (50 µl/ml) for 8 h. METHOD: Real-time PCR and western-blot tested the HO-1 of MC mRNA and protein. The co-stimulatory molecules of DCs (CD80, CD86, CD40) were measured by flow cytometry, and levels of TNF-α, IL-6, and IFN-γ were measured by ELISA. We set up a one-way mixed lymphocyte reaction (MLR) model to test the proliferation of SMCs after MC/DC interaction. *P < 0.05 (t test) was taken as the level of statistical significance. RESULT: MCs pretreated with hemin induced HO-1 mRNA and protein expression, then interacted with DCs; expression of the co-stimulatory molecules was attenuated. The TNF-α, IL-6, and IFN-γ levels in the co-culture system were decreased. These DCs couldn't stimulate the proliferation of SMCs. CONCLUSION: Inhibiting MC degranulation by HO-1 restrained DC maturation and attenuated the proliferation of SMCs.

Mast cell degranulation promotes ischemia-reperfusion injury in rat liver.

BACKGROUND: Mast cells (MCs) play a role in ischemia-reperfusion (I/R) injury in many organs. However, a recent study found that MCs are not involved in I/R injury in isolated rat livers that were perfused only for 1 h. The purpose of this study is to reevaluate the role of MCs in hepatic I/R injury in rat. MATERIALS AND METHODS: A warm hepatic I/R injury model of 1 h ischemia followed by 24 h of reperfusion was used. MC modulation was induced via cromolyn injection or a method called MC depletion using compound 48/80. The effects of MC modulation were evaluated by toluidine blue staining and assessment of mast cell tryptase in sera. The role of MCs in I/R injury was evaluated by hematoxylin and eosin staining graded by Suzuki criteria, alanine aminotransferase and aspartate aminotransferase levels in sera, and malondialdehyde levels in liver homogenates. RESULTS: First, MC degranulation peaked after 2 h of reperfusion and liver damage peaked after approximately 6 h of reperfusion. Second, a method called MC depletion previously used in the skin with repeated injections of compound 48/80 worked similarly in the hepatic setting. Third, stabilization of MCs with cromolyn or depletion of MCs with compound 48/80 each decreased hepatic I/R injury. The most noticeable effects of cromolyn and compound 48/80 treatment were observed after approximately 6 h of reperfusion. CONCLUSIONS: MC degranulation promotes hepatic I/R injury in rats.