The application of an alanine-substituted mutant of the C-terminal fragment of Clostridium perfringens enterotoxin as a mucosal vaccine in mice

Efficient delivery of antigen to mucosal immune tissues is an essential part of mucosal vaccination. Claudin-4 is expressed on the epithelial cells that cover the mucosal immune tissues. We previously found that claudin-4-targeting is a promising strategy for mucosal vaccination by using a claudin-4 binder, the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE). Substitution of Asn and Ser at positions 309 and 313, respectively, with alanine increased the affinity of C-CPE for claudin-4. However, application of the C-CPE mutant as a mucosal vaccine has never been tried. Here, we investigated whether the C-CPE mutant could serve as a mucosal vaccine. We used ovalbumin (OVA) as a model antigen and fused the C-CPE mutant to it. The resultant fusion protein was bound to claudin-4. When mice were immunized with the C-CPE mutant-fused OVA, OVA-specific serum IgG and nasal IgA increased relative to levels in mice immunized with a C-CPE-fused antigen. Immunization with the C-CPE mutant-fused OVA activated Th1- and Th2-type responses and led to increased anti-tumor activity against OVA-expressing thymoma cells relative to that of mice immunized with the C-CPE-fused antigen. These findings suggest that the alanine-substituted C-CPE mutant shows promise as a claudin-targeted mucosal vaccine.     

Claudins 1, 3, 4 and 5 in gastric carcinoma, loss of claudin expression associates with the diffuse subtype

In this study expression of claudins 1, 3, 4 and 5 were studied in 118 cases of gastric carcinoma and compared with proliferation, apoptosis and E-cadherin expression. Expression of all these claudins could be seen in gastric carcinoma, most prominently for claudin 4, and least expression was found for claudin 5. All claudins showed significantly more expression in gastric carcinomas of intestinal type. Their expression was significantly associated with each other. Expression of claudins 4 and 5 was associated with E-cadherin. Strong expression of claudin 5 was associated with higher cell proliferation and apoptosis. Claudin 3 expression had an association with a better prognosis of the patients, especially in the intestinal type. The results show that expression of claudins 1, 3, 4 and 5 is lower in diffuse-type gastric carcinomas. Possibly they play a role in determining the diffuse phenotype and loose cohesion of cells in diffuse type of gastric carcinoma in a similar manner as E-cadherin. The loss of their expression does not clearly associate with poorer prognosis of the patients, except for claudin 3, where strong expression was associated with a better outcome of the patients, a feature especially related to intestinal-type tumours.     

Folate-linked lipoplexes for short hairpin RNA targeting claudin-3 delivery in ovarian cancer xenografts

Ovarian cancers highly overexpress folate receptor α (FRα) and claudin3 (CLDN3), both of which are associated with tumor progression and poor prognosis of patients. Downregulation of FRα and CLDN3 in ovarian cancer may suppress tumor growth and promote benign differentiation of tumor. In this study, F-P-LP/CLDN3, a FRα targeted liposome loading with short hairpin RNA (shRNA) targeting CLDN3 was prepared and the pharmaceutical properties were characterized. Then, the antitumor effect of F-P-LP/CLDN3 was studied in an in vivo model of advanced ovarian cancer. Compared with Control, F-P-LP/CLDN3 promoted benign differentiation of tumor and achieved about 90% tumor growth inhibition. In the meantime, malignant ascites production was completely inhibited, and tumor nodule number and tumor weight were significantly reduced (p < 0.001). FRα and CLDN3 were downregulated together in tumor tissues treated by F-P-LP/CLDN3. The antitumor mechanisms were achieved by promoting tumor cell apoptosis, inhibiting tumor cell proliferation and reducing microvessel density. Finally, safety evaluation indicated that F-P-LP/CLDN3 was a safe formulation in intraperitoneally administered cancer therapy. We come to a conclusion that F-P-LP/CLDN3 is a potential targeting formulation for ovarian cancer gene therapy.     

血脑屏障紧密连接蛋白claudin与脑缺血及其分子机制

组成血脑屏障紧密连接的重要物质,跨膜蛋白Claudin,对维持血脑屏障的紧密连接的完整性及其正常的生理功能起着重要作用,当Claudin蛋白出现结构或功能上的障碍时,血脑屏障功能也会随之改变。目前研究显示,脑缺血对Claudin的分布和表达等都影响着血脑屏障的功能,进而影响着整个内环境的稳态。所以,研究Claudin蛋白的结构、基因表达、病理状态下的变化和与之有关的信号转导等都对了解血脑屏障的选择性开放机制、及药物治疗靶点中的作用都有着深远的意义。     

Preclinical characterization of a Fab-like CD3/CLDN18.2 XFab® bispecific antibody against solid tumors

The claudin 18.2(CLDN18.2) antigen is highly expressed in gastric mucosa epithelial cells and frequently expressed in malignant tumors. Positive clinical outcomes have popularized claudin 18.2 as a novel cellular and antibody therapeutic. Here, we designed a bispecific antibody-ZWB67 using the XFab® platform, aimed at redirecting CD3+ effector T cells to CLDN18.2+ target cells or tissues. Physicochemical characterization, binding properties, T cell stimulatory activity, and T cell-dependent cellular cytotoxicity of ZWB67 were evaluated in dosage intervals using antigens of CD3 and target cells expressing CLDN18.2 or CD3. Then, the anti-tumor activity was assessed in humanized CD3EDG mice bearing MC-38-hCLDN18.2 tumors. Our data demonstrate that ZWB67 specifically binds to the human CD3e antigen (K_D = 1.04E^?08 M) and binds more strongly to CLDN18.2+ cells than to CD3+ cells (4.3- to 9.2-fold difference). ZWB67 showed good activity in the luciferase reporter system and exhibited dose-dependent activation, cytotoxicity of T cells, and cytokine release when co-cultured with CLDN18.2+ cells and CD3+ T cells. ZWB67 also exhibited high in vivo efficacy in the MC-38-hCLDN18.2 xenograft mouse model. In conclusion, the novel anti-CLDN18.2 × anti-CD3 bispecific antibody exhibited low affinity for anti-CD3, highly specific binding, potent cytotoxicity, and anti-tumor activity. These data provide a basis for future preclinical and clinical development of this therapeutic strategy.     

健脾通里中药芪黄煎剂对胃切除大鼠肠上皮紧密连接的影响

目的 观察健脾通里中药芪黄煎剂对大鼠胃切除创伤模型肠黏膜紧密连接蛋白的影响,并探讨其作用机制。方法 将45只大鼠随机分为假手术组、标准肠内营养组、中药组,每组15只。假手术组大鼠仅腹壁切开,未切除胃;其余两组采用胃切除手术方案建立大鼠模型。假手术组术后正常饮食饮水,标准肠内营养组术后注射肠内营养液,中药组注射肠内营养液+中药煎剂。干预7 d后取材,透射电镜下观察大鼠肠黏膜上皮紧密连接形态及其结构的连续性和完整性,并测量肠黏膜上皮细胞间紧密连接宽度;实时荧光定量聚合酶链式反应法检测紧密连接蛋白（zonula occludin-1,ZO-1）、封闭蛋白、咬合蛋白mRNA表达水平。结果 与假手术组比较,标准肠内营养组大鼠肠黏膜上皮细胞紧密连接宽度显著减小（P＜0.05）,小肠组织中ZO-1、封闭蛋白、咬合蛋白mRNA表达水平均显著下调（P＜0.05）;与标准肠内营养组比较,中药组大鼠肠黏膜上皮细胞紧密连接宽度显著增加（P＜0.05）,小肠组织中ZO-1、封闭蛋白、咬合蛋白mRNA表达水平均显著上调（P＜0.05）。结论 芪黄煎剂可以减轻胃切除术后大鼠肠黏膜上皮细胞间紧密连接的损伤,其作用机制与调节ZO-1、封闭蛋白、咬合蛋白mRNA的表达有关。     

氧化苦参碱通过下调miR-155-5p对胃癌细胞的增殖、凋亡、迁移、侵袭作用

目的 探讨氧化苦参碱通过调节miR-155-5p表达对胃癌细胞的增殖、凋亡、迁移、侵袭中的作用和其潜在机制。方法 使用TRIzol法提取组织标本和细胞系的总RNA,检测胃癌组织与癌旁组织中的miR-155-5p表达情况。采用qRT-PCR评估miR-155-5p在人胃癌细胞系（SGC7901、MGC803）与正常胃黏膜上皮（GES-1、AGS）细胞中的表达情况。将MGC803分为空白组、转染错义序列siRNA组、miR-155-5p模拟物组、miR-155-5p抑制剂组、氧化苦参碱（100 μmol/L）组、氧化苦参碱＋miR-155-5p模拟物组。采用MTT法观察不同干预下胃癌细胞的生长抑制作用,用细胞集落形成试验检测不同干预下胃癌细胞的增殖情况。用流式细胞技术分析不同干预对胃癌细胞凋亡的影响。Transwell法检测不同干预后胃癌细胞的迁移、侵袭情况。蛋白质印迹法检测MGC803细胞中Claudin 1、c-Myc、cyclin D1、Bcl-2、Caspase-3蛋白的相对表达量。结果 与癌旁组织和胃黏膜细胞（GES-1、AGS）相比,在胃癌组织和胃癌细胞系（SGC7901、MGC803）中miR-155-5p相对表达量升高（P＜0.001）。而氧化苦参碱通过调节miR-155-5p表达抑制胃癌细胞生长。MTT实验表明,miR-155-5p模拟物的转染后MGC803细胞活力显著增加,而氧化苦参碱可显著抑制胃癌细胞、转染和miR-155-5p模拟物的细胞活力（P＜0.001）。与空白组、错义序列siRNA组相比,miR-155-5p模拟物转染后MGC803细胞活力显著增加,细胞集落生成增加,而氧化苦参碱可显著抑制胃癌细胞的活力和转染miR-155-5p模拟物的细胞活力,及细胞集落生成数（P＜0.001）。miR-155-5p-模拟物组的细胞迁移和侵袭细胞数显著增加、凋亡比例降低,而氧化苦参碱组和miR-155-5p-抑制剂组细胞迁移和侵袭细胞数量降低、凋亡比例升高（P＜0.001）。miR-155-5p模拟组的Claudin 1、c-Myc、Cyclin D1、Bcl-2相对表达量增加,而Caspase-3相对表达量降低（P＜0.01、0.001）。而miR-155-5p抑制剂组、氧化苦参碱组和氧化苦参碱＋miR-155-5p抑制剂组的Claudin 1、c-Myc、Cyclin D1、Bcl-2表达较miR-155-5p模拟组显著下降,Caspase-3表达增加（P＜0.001）。结论 miR-155-5p可能是胃癌的治疗靶点,氧化苦参碱可通过miR-155-5p的调节作用发挥抗肿瘤作用。     

Claudin 14/15 play important roles in early wallerian degeneration after rat sciatic nerve injury

PurposeWallerian degeneration (WD) is an antegrade degenerative process distal to peripheral nerve injury. Numerous genes are differentially regulated in response to the process. However, the underlying mechanism is unclear, especially the early response. We aimed at investigating the effects of sciatic nerve injury on WD via CLDN 14/15 interactions in vivo and in vitro.MethodsUsing the methods of molecular biology and bioinformatics analysis, we investigated the molecular mechanism by which claudin 14/15 participate in WD. Our previous study showed that claudins 14 and 15 trigger the early signal flow and pathway in damaged sciatic nerves. Here, we report the effects of the interaction between claudin 14 and claudin 15 on nerve degeneration and regeneration during early WD.ResultsIt was found that claudin 14/15 were upregulated in the sciatic nerve in WD. Claudin 14/15 promoted Schwann cell proliferation, migration and anti-apoptosis in vitro. PKCα, NT3, NF2, and bFGF were significantly upregulated in transfected Schwann cells. Moreover, the expression levels of the β-catenin, p-AKT/AKT, p-c-jun/c-jun, and p-ERK/ERK signaling pathways were also significantly altered.ConclusionClaudin 14/15 affect Schwann cell proliferation, migration, and anti-apoptosis via the β-catenin, p-AKT/AKT, p-c-jun/c-jun, and p-ERK/ERK pathways in vitro and in vivo. The results of this study may help elucidate the molecular mechanisms of the tight junction signaling pathway underlying peripheral nerve degeneration.     

Claudin18 associated with corticosteroid-induced expression of surfactant proteins in pulmonary epithelial cells

Objective: The present study was undertaken to test the hypothesis that if Cldn18 is associated with lung maturation and play a key role in the corticosteroid-induced expression of surfactant proteins.

Methods: Mouse embryonic lung tissue at different developmental stages (E14.5, E16.5 and E18.5) was collected to detect the temporal-spatial expression of Cldn18 by Western blot and immunofluorescence staining. Dexamethasone?+?cAMP?+?isobutylmethylxanthine (DCI), a commonly used agent for inducing maturation of alveolar epithelial cell in vitro, was used to mimic the roles of corticosteroid. Mouse alveolar epithelial cell line MLE12 treated with or without DCI were harvested, expression of Cldn18 and surfactant proteins were assessed by real-time RT-PCR. For Si-RNA experiments, MLE-12 was transfected with control siRNA or Cldn18 siRNA for 24?h before DCI treatment. Expression of Cldn18 and surfactant proteins was assessed by real-time RT-PCR.

Results: Expression of Cldn18 was detected in peripheral lung epithelial cells, and increased expression was detected from E14.5 to E18.5 during lung development. Expression of Cldn18 and surfactants (SPA, SPB and SPC) were significantly induced in MLE12 after DCI treatment. Silencing of Cldn18 effectively suppressed the DCI-induced expression of SPB and SPC but not SPA.

Conclusion: Our results demonstrated that expression of Cldn18 is associated with peripheral lung epithelial cell maturation and play a key role in the corticosteroid-induced expression of surfactant proteins.