Snare-assisted endoscopic resection of gastric subepithelial tumors originating from the muscularis propria layer: a multicenter study.

BACKGROUND AND AIM: Although successful endoscopic resection of gastric subepithelial tumors (SETs) originating from the muscularis propria (MP) layer has been frequently reported, it requires a relatively complicated technique and has a high perforation rate. In this retrospective study, we evaluated the efficacy and safety of the snare-assisted endoscopic resection (SAER) method which is performed using a snare and insulated-tip (IT) knife via a single-channel endoscope to reduce the perforation rate. METHODS: In this study, fifty-six patients with gastric SETs originating from the MP layer treated by the SAER method at three institutions between July 2017 and December 2017 were reviewed. The procedure involved multiple steps as shown in Fig. 2. Data were obtained on demographics, SET features, histopathological diagnoses, procedure time, en bloc resection rate, R0 resection (negative margins) status, and adverse events. RESULTS: Endoscopic resection was successfully performed in all patients. The median overall procedure time was 43.5 min (range 26-106 min). The mean size of resected specimens was 19.73 mm (range 10-33 mm). The overall rate of en bloc resection was 96.4% (54/56). In addition, the perforation rate was 7.1% (4/56), and defects in the stomach wall were very small and easily closed using metallic clips. No postprocedural bleeding occurred in any case. CONCLUSIONS: The SAER method is an effective, safe, less costly technique for the removal of some gastric SETs originating from the MP layer with an appropriate size.

3-L split-dose is superior to 2-L polyethylene glycol in bowel cleansing in Chinese population: a multicenter randomized, controlled trial.

Large volume (4 L) of polyethylene glycol (PEG) solution would ensure a better quality of bowel cleansing but might be poorly tolerated. Due to the smaller body size, lower body weight, and different diet habits, the large volume of 4-L PEG might be poorly tolerated by the Chinese population. In view of this, a balance should be made between the volume and effectiveness. This study aimed to compare the effectiveness, compliance, and safety between 3-L split-dose and 2-L PEG in Chinese population. Consecutive patients scheduled for colonoscopy were recruited from 5 tertiary medical centers in South China between April and July, 2014. Patients were prospectively randomized into 2 groups: 3-L split-dose PEG (3L-group) and 2 L PEG (2L-group). The primary endpoint was bowel cleansing and was defined according to Ottawa Bowel Preparation Scale (OBPS). The safety and compliance were also evaluated. A total of 318 patients were included in the analysis. The mean total OBPS score was significantly higher in 2L-group than in 3L-group (4.4 ±â€Š2.7 vs 2.9 ±â€Š2.4, P < 0.001). Both the intention-to-treat and per-protocol analysis found that rates of successful and excellent bowel preparation were much higher in 3L-group (89.9% and 78.0%) than 2L-group (79.2% and 48.4%), respectively (P < 0.001). The average cecum intubation time was significantly shorter in 3L-group (8.2 ±â€Š3.7 min) than in 2L-group (10.3 ±â€Š4.2 min) (P = 0.04). Adenoma detection rate in right colon was slightly higher in 3L-group than in 2L-group (17.6% vs 12.6%, P = 0.21). The safety and compliance including the taste, smell, and dosage of PEG were similar between 2 groups (P > 0.05). 3-L split-dose PEG is superior to 2-L PEG by better bowel cleansing, improved safety and compliance, shorter cecum intubation time, and potentially higher adenoma detection rate in rightward colon in Chinese population.

miR-449a and CDK6 in gastric carcinoma.

The present study aimed to identify the association between microRNA (miR/miRNA)-449a, the cyclin-dependent kinase (CDK)6 protein and gastric carcinoma, and discuss the effect of miR-449a on the expression of the CDK6 protein. Quantitative (q)PCR and western blot analysis were used to analyze the expression of the miR-449a and the CDK6 protein in gastric carcinoma and tumor-adjacent normal tissues. The real-time cell analyzer and the DAPI staining test were used to monitor the different miR-449a levels regulating the proliferation and apoptosis of the MGC-803 cell line. Immunofluorescence and western blot analyses were used to detect the expression level of the CDK6 protein in the cells of the miR-449a upregulation and downregulation groups, and a control group. A scratch test was used to study the effects of miR-449a expression on migration and invasion. It was found that the expression of miR-449a was downregulated and the expression of CDK6 protein was upregulated in gastric carcinoma tissue. The level of MGC-803 cell proliferation was decreased and the apoptosis level was increased by the upregulation of miR-449a expression, and the opposite effect was shown by the downregulation of expression. The expression of the CDK6 protein in the MGC-803 cells was downregulated by upregulating the expression of miR-449a. The distance of the scratch was shortened markedly after 12 h by downregulating the expression of miR-449a in the MGC-803 cells. The present study identified that a lower expression level of miR-449 and a higher expression level of CDK6 may contribute to the occurrence and development of gastric cancer. Furthermore, it was shown that miR-449a is able to regulate the expression of the CDK6 protein.

Activating transcription factor 4 promotes esophageal squamous cell carcinoma invasion and metastasis in mice and is associated with poor prognosis in human patients.

BACKGROUND: Activating transcription factor 4 (ATF4) is a stress response gene that is involved in homeostasis and cellular protection. However, its expression and function in esophageal squamous cell carcinoma (ESCC) remains unknown. In this study, we aimed to determine the clinicopathologic significance of ATF4 in ESCC and its potential role in ESCC invasion and metastasis. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrated that ATF4 overexpression is correlated with multiple malignant characteristics and indicates poor prognosis in ESCC patients. ATF4 expression was an independent factor that affected the overall survival of patients with ESCC after surgical resection. ATF4 promoted cell invasion and metastasis by promoting matrix metalloproteinase (MMP)-2 and MMP-7 expression, while its silencing significantly attenuated these activities both in vitro and in vivo. CONCLUSIONS/SIGNIFICANCE: We report that ATF4 is a potential biomarker for ESCC prognosis and that its dysregulation may play a key role in the regulation of invasion and metastasis in ESCC cells. The targeting of ATF4 may provide a new strategy for blocking ESCC metastasis.

Activating transcription factor 4 mediates a multidrug resistance phenotype of esophageal squamous cell carcinoma cells through transactivation of STAT3 expression.

Multidrug resistance (MDR) is a major challenge to the clinical treatment of esophageal cancer. The stress response gene activating transcription factor 4 (ATF4) is involved in homeostasis and cellular protection. However, relatively little is known about the expression and function of ATF4 in esophageal squamous cell carcinoma (ESCC) MDR. In this study, we investigate the potential role and mechanisms of ATF4 in ESCC MDR. We demonstrated that overexpression of ATF4 promotes the MDR phenotype in ESCC cells, while depletion of ATF4 in the MDR ESCC cell line induces drug re-sensitization. We also demonstrated that ATF4 transactivates STAT3 expression by directly binding to the signal transducers and activators of transcription 3 (STAT3) promoter, resulting in MDR in ESCC cells. Significantly, inhibition of STAT3 by small interfering RNA (siRNA) or a selective inhibitor (JSI-124) reintroduces therapeutic sensitivity. In addition, increased Bcl-2, survivin, and MRP1 expression levels were observed in ATF4-overexpressing cells. In conclusion, ATF4 may promote MDR in ESCC cells through the up-regulation of STAT3 expression, and thus is an attractive therapeutic target to combat therapeutic resistance in ESCC.

Undifferentiated embryonal sarcoma of the liver mistaken for hepatic abscess in an adult.

Undifferentiated embryonal sarcoma of the liver (UESL) predominantly occurs in children under the age of 10 years, and ~90% of cases occur in children <15 years old. Patients may complain of abdominal pain, fever or other symptoms. No significant decrease has been identified in the hepatic function or elevation of α-fetoprotein, which differentiates UESL from primary carcinomas of the liver. In the present study, a rare and misdiagnosed case of an UESL arising in a male, which was mistaken for a hepatic abscess and retrospectively re-diagnosed, is reported. This case was misdiagnosed as a hepatic abscess initially, and it was diagnosed as UESL subsequent to performing tests, including a type-B ultrasonic scan and computed tomography (CT), and evaluating pathological findings. The rapid recurrence of the tumor in this patient was identified by CT, and this is associated with the malignancy of the disease. Currently, patients with UESL have a poor prognosis as there is not a successful treatment strategy. The present study analyzes the course of diagnosis and potential treatment for the disease.

The protective effect of recombinant Lactococcus lactis oral vaccine on a Clostridium difficile-infected animal model.

BACKGROUND: Oral immunization with vaccines may be an effective strategy for prevention of Clostridium difficile infection (CDI). However, application of previously developed vaccines for preventing CDI has been limited due to various reasons. Here, we developed a recombinant Lactococcus lactis oral vaccine and evaluated its effect on a C. difficile-infected animal model established in golden hamsters in attempt to provide an alternative strategy for CDI prevention. METHODS: Recombinant L. lactis vaccine was developed using the pTRKH2 plasmid, a high-copy-number Escherichia coli-L. shuttle vector: 1) L. lactis expressing secreted proteins was constructed with recombinant pTRKH2 (secreted-protein plasmid) carrying the Usp45 signal peptide (SPUsp45), nontoxic adjuvanted tetanus toxin fragment C (TETC), and 14 of the 38 C-terminal repeats (14CDTA) of nontoxic C. difficile toxin A (TcdA); and 2) L. lactis expressing secreted and membrane proteins was constructed with recombinant pTRKH2 (membrane-anchored plasmid) carrying SPUsp45, TETC, 14CDTA, and the cell wall-anchored sequence of protein M6 (cwaM6). Then, 32 male Syrian golden hamsters were randomly divided into 4 groups (n = 8 each) for gavage of normal saline (blank control) and L. lactis carrying the empty shuttle vector, secreted-protein plasmid, and membrane-anchored plasmid, respectively. After 1-week gavage of clindamycin, the animals were administered with C. difficile spore suspension. General symptoms and intestinal pathological changes of the animals were examined by naked eye and microscopy, respectively. Protein levels of anti-TcdA IgG/IgA antibodies in intestinal tissue and fluid were analyzed by enzyme-linked immunosorbent assay (ELISA). A cell culture cytotoxicity neutralization assay was done by TcdA treatment with or without anti-TcdA serum pre-incubation or treatment. Apoptosis of intestinal epithelial cells was examined by flow cytometry (FL) assay. Expression of mucosal inflammatory cytokines in the animals was detected by polymer chain reaction (PCR) assay. RESULTS: After the C. difficile challenge, the animals of control group had severe diarrhea symptoms on day 1 and all died on day 4, indicating that the CDI animal model was established in hamster. Of the 3 immunization groups, secreted-protein and membrane-anchored plasmid groups had significantly lower mortalities, body weight decreases, and pathological scores, with higher survival rate/time than the empty plasmid group (P < 0.05). The tilter of IgG antibody directed against TcdA was significantly higher in serum and intestinal fluid of secreted-protein and membrane-anchored plasmid groups than in the empty plasmid group (P < 0.05) while the corresponding titer of IgA antibody directed against TcdA had no substantial differences (P > 0.05). The anti-TcdA serum of membrane-anchored plasmid group neutralized the cytotoxicity of 200 ng/ml TcdA with the best protective effect achieved by anti-TcdA serum pre-incubation. The incidences of TcdA-induced death and apoptosis of intestinal epithelial cells were significantly reduced by cell pre-incubation or treatment with anti-TcdA serum of membrane-anchored plasmid group (P < 0.05). MCP-1, ICAM-1, IL-6, and Gro-1 mRNA expression levels were the lowest in cecum tissue of the membrane-anchored groups compared to the other groups. CONCLUSION: Recombinant L. lactis live vaccine is effective for preventing CDI in the hamster model, thus providing an alternative for immunization of C. difficile-associated diseases.

Changes in innate and permissive immune responses after hbv transgenic mouse vaccination and long-term-siRNA treatment [corrected].

BACKGROUND: Currently, no licensed therapy can thoroughly eradicate hepatitis B virus (HBV) from the body, including interferon α and inhibitors of HBV reverse-transcription. Small interfering RNA (siRNA) seem to be a promising tool for treating HBV, but had no effect on the pre-existing HBV covalently closed circular DNA. Because it is very difficult to thoroughly eradicate HBV with unique siRNAs, upgrading the immune response is the best method for fighting HBV infection. Here, we aim to explore the immune response of transgenic mice to HBV vaccination after long-term treatment with siRNAs and develop a therapeutic approach that combines siRNAs with immunopotentiators. METHODOLOGY/PRINCIPAL FINDINGS: To explore the response of transgenic mice to hepatitis B vaccine, innate and acquired immunity were detected after long-term treatment with siRNAs and vaccination. Antiviral cytokines and level of anti-hepatitis B surface antigen antibody (HBsAg-Ab) were measured after three injections of hepatitis B vaccine. RESULTS: Functional analyses indicated that toll-like receptor-mediated innate immune responses were reinforced, and antiviral cytokines were significantly increased, especially in the pSilencer4.1/HBV groups. Analysis of CD80+/CD86+ dendritic cells in the mouse liver indicated that dendritic cell antigen presentation was strengthened. Furthermore, the siRNA-treated transgenic mice could produce detectable HBsAg-Ab after vaccination, especially in the CpG oligonucleotide vaccine group. CONCLUSIONS/SIGNIFICANCE: For the first time, our studies demonstrate that siRNAs with CpG HBV vaccine could strengthen the immune response and break the immune tolerance status of transgenic mice to HBV. Thus, siRNAs and HBV vaccine could provide a sharp double-edged sword against chronic HBV infection.

[PCR-based assembly of the DNA sequence coding for tetanus toxin C fragment].

OBJECTIVE: To develop a PCR-based method for gene assembly of tetanus toxin C fragment (TETC) DNA sequence from a large number of oligodeoxyribonucleotides (oligos). METHODS: To allow for its cloning and expression in Lactococcus lactis, the TETC gene sequence was designed according to the known TETC gene sequence (GenBank accession number M12739, 367-1719) and the amino acid coding in Lactococcus lactis. The sequence contained 1383 nucleotides (nt) with Sal I site added to its 5' end and Xho I and Hind III sites to its 3' end. There were 209 synonymous codon substitutions in the designed gene sequence as compared with the sequence reported in GenBank for amino acid coding in Lactococcus lactis and elimination of the restriction site of EcoR I and Kpn I. The 1380 nt of the sequence was divided into 68 oligos designated as TETC 1 to TETC 68, each containing 40 nt. A 16 nt oligos designated as TETC 69 was designed as the downstream primer. The TETC 1-24 fragment was acquired using the oligos TETC 1 to TETC 24 by PCR-based gene assembly method, and the TETC 23-46 and TETC45-68 fragments were assembled similarly. The full-length TETC gene was assembled using TETC 1 and TETC 69 as the primers when the 3 fragments were mixed. The target gene was gel-purified and digested with Sal I and Hind III, followed by ligation to the pBluescript II SK(+) and digestion with the same enzymes. The positive clones were confirmed by restriction enzyme excision and sequencing. RESULTS: Three 500-bp fragments were acquired by PCR-based gene assembly, and the full-length TETC gene was obtained from the 3 fragment mixed at a equal concentration by a second PCR-based gene assembly using TETC 1 and TETC 69 as the primers. The target gene was cloned to pBluescript II SK(+) vector, and sequence analysis of the positive clones indicated that the assembled sequence was identical to the designed coding sequence of TETC gene. CONCLUSION: PCR-based assembly of the synthesized constitutive gene fragments into the complete sequence can be an effective strategy for synthesis of long DNA sequences in vitro.