Safety and efficacy of endoscopic ultrasound-guided gastroenterostomy for gastric outlet obstruction in different sites: A single-center retrospective study.

OBJECTIVES: Endoscopic ultrasound-guided gastroenterostomy (EUS-GE) has recently been employed as a novel treatment for gastric outlet obstruction (GOO). The aim of this study was to evaluate the safety and efficacy of EUS-GE for GOO at different sites. METHODS: Consecutive hospitalized patients who underwent EUS-GE for GOO at the Department of Gastroenterology, Nanjing Drum Tower Hospital from March 2017 to April 2020 were recruited in this retrospective study. Patients were divided into three groups depending on the obstruction site. The primary outcomes included technical success and clinical success. The secondary outcomes were operation time, post-procedure length of stay (LOS), hospitalization cost, and complications such as peritonitis, bleeding, pneumoperitoneum, abdominal pain, and infection. RESULTS: A total of 51 patients were included. Technical success achieved in 100% patients with proximal GOO and in 88.9% with distal GOO (P = 0.176). Clinical success declined from the oral side to the anal side (P = 0.510). Operation time, hospitalization costs, and post-procedural LOS were similar among groups (P = 0.532, 0.520, and 0.144, respectively). Complications were observed in 28 (54.9%) patients. In approaching the mature phase of the endosopist, clinical success improved, while the secondary outcomes showed no statistically significant difference compared with the initial phase. CONCLUSIONS: EUS-GE may be challenging for distal GOO; however, it is safe and effective when carried out by experienced endoscopists. A complete preoperative evaluation to assess the difficulty of the procedure is necessary. Prospective studies with large sample size are needed to further validate our findings.

A nanobody targeting carcinoembryonic antigen as a promising molecular probe for non-small cell lung cancer.

Carcinoembryonic antigen (CEA) is a biomarker and therapy target for non­small cell lung cancer (NSCLC), which is the most common type of lung cancer. Nanobodies with high target specificity are promising candidates to function as anti­CEA probes. In the present study, the targeting effects of an anti­CEA nanobody obtained from phage display were investigated using technetium­99 m (99mTc) and fluorescence labeling. In vitro binding and immunofluorescent staining assays, as well as in vivo blood clearance and biodistribution assays were performed. High specificity and affinity of the nanobody for CEA­positive H460 cells was observed in vitro. The pharmacokinetics assay of the 99mTc­nanobody in Wistar rats demonstrated that the nanobody had appropriate T1/2α and T1/2ß, which were 20.2 and 143.5 min, respectively. The biodistribution assay using H460 xenograft­bearing nude mice demonstrated a high ratio of signal in tumor compared with background, which confirmed that the nanobody may be useful as a molecular probe for CEA­positive cancer, particularly in NSCLC.

Targeting assay of a fusion protein applied in enzyme prodrug therapy.

Tumor growth and metastasis are dependent on angiogenesis. The overexpression of integrin αvß3 on angiogenic vessels and on numerous malignant human tumor cells suggests that these labeled ligands of integrin are potentially suitable for molecular imaging and in targeted therapy of tumors. In previous studies, we added a ß-lactamase variant with reduced immunogenicity to the cyclic peptide RGD4C, resulting in the fusion protein RGD4CßL, which is suitable for use in targeted enzyme prodrug therapy (TEPT), a promising treatment for tumors. The targeting of the aforementioned fusion protein serves an important role in TEPT. In the present study, RGD4CßL was labeled with 125I and the targeting effect on integrin-positive tumors was evaluated. The results demonstrated that the 125I-RGD4CßL protein exhibited high levels of accumulation at the tumor site and rapid renal clearance, which revealed the potency and efficiency of RGD4CßL in TEPT.

A Fusion Protein of RGD4C and ß-Lactamase Has a Favorable Targeting Effect in Its Use in Antibody Directed Enzyme Prodrug Therapy.

Antibody directed enzyme prodrug therapy (ADEPT) utilizing ß-lactamase is a promising treatment strategy to enhance the therapeutic effect and safety of cytotoxic agents. In this method, a conjugate (antibody-ß-lactamase fusion protein) is employed to precisely activate nontoxic cephalosporin prodrugs at the tumor site. A major obstacle to the clinical translation of this method, however, is the low catalytic activity and high immunogenicity of the wild-type enzymes. To overcome this challenge, we fused a cyclic decapeptide (RGD4C) targeting to the integrin with a ß-lactamase variant with reduced immunogenicity which retains acceptable catalytic activity for prodrug hydrolysis. Here, we made a further investigation on its targeting effect and pharmacokinetic properties, the results demonstrated that the fusion protein retains a targeting effect on integrin positive cells and has acceptable pharmacokinetic characteristics, which benefits its use in ADEPT.

[Effect of tetrandine on gene expression of collagen type I, collagen type III and TGF-beta1 in scar tissue's of rabbits ear].

OBJECTIVE: To observe the effect of tetrandine on gene expression of collagen type I, collagen type III, transformation growth factor-beta1 and to investigate the inhibitory effect of tetrandine on the scar tissue hyperplasia in rabbits' ears. METHODS: After the scar model was formed on the rabbits' ears, the rabbits were divided into 4 groups to receive intro-lesion injection with saline, or prednisolone (Pre) or tetrandrine in low concentration (L-Tet, 1.0 mg/ml) or tetrandrine in high concentration (H-Tet, 7.5 mg/ml). The morphological changes of scar tissue were observed. The changes of fibroblasts quantity and collagen expression were observed with HE and Masson staining. Immunohistochemical study was used to observe the expression level of collagen type I and collagen type III and TGF-beta1. Collagen type I and collagen type III and TGF-beta1, and signal factor Smad 3 mRNA were detected with RT-PCR. RESULTS: (1) 24 days after injury, all the wounds healed completely with formation of red, tough and hypertrophic scar. HE and Masson staining showed significant increase of fibroblasts and collagen density with irregularly arrangement. (2) Compared with that in saline group, the scar in other groups became softer, lighter and thinner, especially in H-Tet group. (3) HE and Masson staining shows the scar in Tet and Pre groups contained less fibroblasts and lower collagen dentsity with comparatively regular arrangement than that in saline group (P < 0.01), especially in H-Tet group. (4) According to the immunohistochemical study, the expression of collage type I and III and TGF-beta was positive in all the groups, but the positive rate and the ratio of collagen density I to III decreased in the order of saline, L-Tet, H-Tet and Pre groups (P < 0.01). (5) PT-PCR detection results showed that the amplification bands brightness of collagen type I and III and TGF-beta1 and signal molecular Smad 3 mRNA in scar tissue were obviously different. Compared with that in saline group, the expression of collagen type I and III and TGF-beta1 and Smad 3 mRNA decreased in Tet and Pre groups (P < 0.01). H-Tet group showed the most obvious reduce in the expression of type I collagen and TGF-beta1 and Smad 3 mRNA. Conclusions Tetrandine can significantly suppress the expression of collagen type I and collagen type III and TGF-beta1 on hypertrophic scar of rabbit ears, and reduce signal factor Smad 3 mRNA' s expression. It may be one of the important mechanism for its inhibitory effect on scar hyperplasia.

[Prokaryotic expression, purification and biological activity analysis of recombinant ß-Lactamase protein].

AIM: To prepare RGD4CßL fusion protein using prokaryotic expression system and evaluate the biological activity of the RGD4CßL. METHODS: RGD4CßL gene was cloned into pColdII to contruct ß-Lactamase prokaryotic expression vector. After transformation, the recombinant vector was induced to express recombinant protein RGD4CßL by IPTG in E.coli BL(DE3). The recombinant protein was purified by Ni-NTA resin under denaturing condition and then dialyzed to renature. The tumor cell targeting ability of the recombinant protein was analyzed by flow cytometric analysis. RESULTS: After cleavage and purification, ß-Lactamase moiety showed the expected size of 42 000 on Tricine-SDS-PAGE, and was further confirmed by Western blotting. Based on flow cytometric analysis, the purified protein specially targeted breast cancer cell line MCF-7. CONCLUSION: This research successfully estiblished a method for prokaryotic expression and purification of ß-lactamase. These results suggest the potential use of the protein as an agent for ADEPT.

(6R,7R)-3-Hydroxymethyl-7-(2-phenyl-acetamido)-3-cephem-4-carboxylic acid lactone.

In the title compound {systematic name: N-[(4R,5R)-3,11-dioxo-10-oxa-6-thia-2-aza-tricyclo-[6.3.0.0(2,5)]undec-1(8)-en-4-yl]-2-phenyl-acetamide}, C(16)H(14)N(2)O(4)S, the four- and five-membered rings adopt planar conformations (with r.m.s. deviations of 0.0349 and 0.0108 Šrespectively) while the six-membered ring adopts a half-chair, or envelope-like, conformation with the S atom in the flap position. In the crystal, mol-ecules are linked by N-H⋯O hydrogen bonds.

Construction, expression and functional characterization of the ß-lactamase with αv integrin ligands.

Antibody-directed enzyme prodrug therapy (ADEPT) delivers chemotherapeutic agents at high concentration to tumor tissues while minimizing systemic drug exposure. ß-Lactamases are particularly useful enzymes for ADEPT systems due to their unique substrate specificity, which allows the activation of a variety of lactam-based prodrugs with minimal interference from mammalian enzymes. This study used integrin α(v)ß(3) as a target for tumor-specific delivery of ß-Lactamase. ß-Lactamase was fused with ACDCRGDCFCG peptide (RGD4C) by recombinant DNA technology. Likewise, this study cloned a fused cDNA and successfully expressed active recombinant protein in E. coli purified with Ni-NTA resin. After purification, ß-Lactamase moiety showed the expected size of 42 kDa on Tricine-SDS-PAGE, and was further confirmed by Western blotting. Based on flow cytometric analysis, the purified protein was found to be active for specificity in breast cancer cell line, MCF-7, which supports the utility of the protein as an agent for ADEPT.