Peste des Petits Ruminants Virus Tissue Tropism and Pathogenesis in Sheep and Goats following Experimental Infection

Peste des petits ruminants (PPR) is a viral disease which primarily affects small ruminants, causing significant economic losses for the livestock industry in developing countries. It is endemic in Saharan and sub-Saharan Africa, the Middle East and the Indian sub-continent. The primary hosts for peste des petits ruminants virus (PPRV) are goats and sheep; however recent models studying the pathology, disease progression and viremia of PPRV have focused primarily on goat models. This study evaluates the tissue tropism and pathogenesis of PPR following experimental infection of sheep and goats using a quantitative time-course study. Upon infection with a virulent strain of PPRV, both sheep and goats developed clinical signs and lesions typical of PPR, although sheep displayed milder clinical disease compared to goats. Tissue tropism of PPRV was evaluated by real-time RT-PCR and immunohistochemistry. Lymph nodes, lymphoid tissue and digestive tract organs were the predominant sites of virus replication. The results presented in this study provide models for the comparative evaluation of PPRV pathogenesis and tissue tropism in both sheep and goats. These models are suitable for the establishment of experimental parameters necessary for the evaluation of vaccines, as well as further studies into PPRV-host interactions.     

犬SLAM受体mRNA荧光定量PCR检测方法的建立和初步应用

目的建立荧光定量PCR方法,检测犬不同组织中SLAM受体mRNA的表达水平。方法以犬GAPDH为内参基因采用△△ct法,分析SLAM受体mRNA在犬体内不同组织中的表达。结果此方法有较高的重复性,变异系数在0．89％-2．35％。以SLAM受体在心脏的表达为1倍值,结果显示受体mRNA在脾脏中表达最高,为38．49倍;肺门淋巴结、肠系膜淋巴结、腹股沟淋巴结中表达次之,分别为9．13、8．58、6．24倍;膀胱中表达最低。结论成功建立了检测SLAM受体mRNA在不同组织中表达水平的荧光定量PCR检测方法。     

Cloning and expression levels of genes relating to the ovulation rate of the Yunling black goat

This study investigated the nucleotide sequences and tissue expression levels of genes relating to the ovulation rate in Yunling black goats, a famous Yunnan province, China, local breed with low fecundity. Five genes, FSHB, FSHR, BMP15, BMPR1B, and ESR2, were investigated; the complete cDNA sequences of these genes were 390, 2088, 1185, 1509, and 1585 bp, respectively, and compared with Boer goats (a more fecund breed), the sequence identities were 99%, 99%, 99%, 100%, and 99%, respectively. There were two base differences in FSHB and BMP15, four in FSHR, and three in ESR2. There were fewer follicles and oocytes in Yunling black goats than in Boer goats. Expression levels of FSHB, FSHR, and BMP15 genes in Yunling black goats were lower, and expression levels of BMPR1B and ESR2 genes were higher. Serum FSH content was lower in Yunling black goats, but serum estrogen content was higher. Protein expression levels of FSHR, BMP15, BMPR1B, and ESR2 in ovary and pituitary correlated positively with gene mRNA expression levels. In Yunling black goats, the mRNA expression levels of FSHB, FSHR, and BMP15 positively correlated with litter size, but those of BMPR1B and ESR2 correlated negatively. Together, base changes and variations in mRNA and protein expression levels of genes relating to the ovulation rate result in low fecundity in the Yunling black goat. Reduced BMP15 and FSHR levels may be related to the observed fewer oocytes and, consequently, fewer follicles.     

子宫内膜癌组织KIF20A、LAPTM4B-35表达与临床病理特征及预后的关系研究

摘要 目的：探讨子宫内膜癌组织驱动蛋白家族成员20A(KIF20A)、溶酶体相关4次跨膜蛋白B-35(LAPTM4B-35)表达与临床病理特征及预后的关系。方法：选择2012年4月至2015年8月期间于我院行手术治疗的80例子宫内膜癌患者作为研究对象。检测子宫内膜癌组织以及距离肿瘤边缘2 cm以上癌旁组织中KIF20A、LAPTM4B-35 mRNA表达水平。分析子宫内膜癌组织中KIF20A、LAPTM4B-35 mRNA表达与临床病理特征的关系。分析不同KIF20A、LAPTM4B-35 mRNA表达患者5年总生存率的差异。分析子宫内膜癌患者预后的影响因素。结果：与癌旁组织相比,子宫内膜癌组织中KIF20A、LAPTM4B-35 mRNA表达水平明显升高,差异有统计学意义（P<0.05）。有淋巴结转移以及FIGO分期Ⅲ期患者的癌组织KIF20A、LAPTM4B-35 mRNA表达水平明显高于无淋巴结转移以及FIGO分期I～II期患者的癌组织,差异有统计学意义（P<0.05）。KIF20A低表达组患者5年总生存率明显高于KIF20A高表达组;LAPTM4B-35低表达组患者5年总生存率明显高于LAPTM4B-35高表达组患者,差异有统计学意义（P<0.05）。Cox回归分析结果显示：FIGO分期Ⅲ期、有淋巴结转移、KIF20A mRNA高表达和LAPTM4B-35 mRNA高表达是子宫内膜癌患者预后的影响因素（P<0.05）。结论：在子宫内膜癌组织中KIF20A、LAPTM4B-35 mRNA表达水平升高,有淋巴结转移、FIGO分期较高患者癌组织KIF20A、LAPTM4B-35 mRNA表达水平上调。KIF20A 、LAPTM4B-35高表达患者5年总生存率下降。     

骨桥蛋白在胰腺癌中的表达及其临床意义

目的:研究骨桥蛋白OPN在胰腺癌中的表达水平及其与临床病理特征的关系。方法:采用RT-PCR和免疫组织化学方法分别检测50例胰腺癌手术切除标本和20例癌旁正常胰腺组织中OPNmRNA及其蛋白水平的表达。结果:胰腺癌组织中OPNmRNA高表达率为90%(45/50)明显高于其在癌旁正常胰腺组织中的表达15%(3/20),差异有统计学意义(P<0.01);OPN蛋白的阳性率为86.0%(43/50),明显高于癌旁正常胰腺组织中的表达30%(6/20),差异有统计学意义(P<0.01);OPN在胰腺癌组织中表达与其淋巴结转移明显相关(P<0.05)。结论:OPN在胰腺癌中的过表达对胰腺癌的生长、浸润及转移起重要作用。     

Expression of FHL1 in gastric cancer tissue and its correlation with the invasion and metastasis of gastric cancer

This study was performed to analyze the expression of four and a half LIM domains 1 (FHL1) in gastric carcinoma tissue and its correlation with the clinicopathological characteristics of gastric cancer. In addition, the role of FHL1 in the invasion and metastasis of gastric cancer cells was investigated to provide an experimental basis for future treatments of gastric cancer. FHL1 mRNA and protein expression in gastric carcinoma and the adjacent normal gastric mucosa tissue were determined using RT-PCR and western blots. Correlations of FHL1 expression with the incidence, progression, and clinicopathological characteristics of gastric cancer were analyzed. Changes in the invasion and metastatic potential of MKN45 human gastric cancer cells were observed after the transient transfection with an eukaryotic expression vector containing full-length FHL1. Expression of FHL1 mRNA in gastric carcinoma tissue was significantly lower than that in the adjacent normal tissue (P < 0.05). FHL1 expression in gastric carcinoma tissue from patients who were positive for lymph node metastasis was significantly lower than those in patients who were negative for lymph node metastasis (P < 0.05). Lower FHL1 expression was correlated with lower degrees of differentiation, higher TNM stages, and greater invasive potential of the gastric cancer (P < 0.05). The FHL1 mRNA and protein expression patterns were similar in gastric cancer. FHL1 protein expression in gastric carcinoma tissue was significantly lower than that in the surrounding normal tissue (P < 0.05). FHL1 protein expression was significantly lower in gastric carcinoma tissue from patients who were positive for lymph node metastasis than that detected in patients with no lymph node metastasis (P < 0.05). Lower FHL1 protein expression was correlated with lower degrees of differentiation, higher TNM stages, and greater invasive potential in gastric cancer (P < 0.05). However, the expression of FHL1 was independent of the patient's gender, age, and tumor size (P > 0.05). Overexpression of FHL1 in the MKN45 human gastric cancer cell line using an eukaryotic expression vector resulted in a significant reduction in the invasiveness and metastatic ability of these cells as determined using the Transwell chamber invasion assay (P < 0.05). The decrease in or loss of FHL1 expression may be related to the incidence, progression, invasiveness, and metastatic potential of gastric cancer.     

Examination of epithelial tissue cytokine response to natural peste des petits ruminants virus (PPRV) infection in sheep and goats by immunohistochemistry

In this study, we aimed to evaluate expression of IL-4, IL-10, TNF-α, IFN-γ and iNOS in lingual, buccal mucosa and lung epithelial tissue using immunoperoxidase technique and to compare with the tissues of control animals. The tissues used in the study were collected from 17 PPRV-affected and 5 healthy sheep and goats. In PPRV positive animals, the lungs, lingual and buccal mucosa had significantly higher iNOS, IFN-γ and TNF-α expressions compared to control group animals. There was no significant difference between PPRV positive and control groups for IL-4 and IL-10 expressions of epithelial tissues. In conclusion, the epithelial tissues infected by PPRV showed significant iNOS, IFN-γ and TNF-α expressions and they might play an important role in the initiation and regulation of cytokine response, as they take place in the first host barrier to be in contact with PPRV. It is suggested that the more epithelial damage produced by PPRV the more cytokine response may result in the infected epithelial cells. The first demonstration of iNOS expression and epithelial cytokine response to PPRV in natural cases is important because it may contribute to an early initiation of systemic immunity against PPRV infection, in addition to direct elimination of the virus during the initial epithelial phase of the infection.     

膀胱癌组织CALD1 mRNA表达水平与临床病理特征和预后的关系分析

摘要 目的：探究膀胱癌组织钙调蛋白结合蛋白（CALD1）信使RNA（mRNA）表达水平与临床病理特征和预后的关系。方法：选取2015年7月-2017年7月于我院收治并确诊的111例膀胱癌患者为研究对象,收集癌组织及癌旁正常组织,采用逆转录聚合酶链式反应（RT-PCR）法检测CALD1 mRNA在膀胱癌组织与癌旁正常组织中的表达差异,分析CALD1 mRNA相对表达量与临床病理特征的关系。对患者进行5年随访,分析CALD1 mRNA相对表达量与预后的关系。结果：CALD1 mRNA在膀胱癌组织的相对表达量显著高于癌旁正常组织（P<0.05）;CALD1mRNA相对表达量与膀胱癌患者组织分化程度、TNM分期、淋巴结转移相关（P<0.05）;111例患者出院后随访5年,4例失访,107例患者获得随访,CALD1低表达组和高表达组的5年总生存率分别为72.34%（34/47）、50.00%（30/60）（P＜0.05）,CALD1低表达组5年总生存率高于高表达组;Cox比例风险回归模型分析显示,组织低分化程度、TNM高分期、淋巴结转移、CALD1mRNA高表达是影响膀胱癌患者预后的危险因素（P<0.05）。结论：CALD1在膀胱癌组织中呈高表达,CALD1mRNA相对表达量与患者组织分化程度、TNM分期、淋巴结转移及预后相关。     

NEFL mRNA expression level is a prognostic factor for early-stage breast cancer patients

Neurofilament, light polypeptide (NEFL) was demonstrated to be ectopically expressed in breast cancer tissues and decreased in lymph node metastases compared to the paired primary breast cancers in our previous study. Moreover, in several studies, NEFL was regarded as a tumor suppressor gene, and its loss of heterozygosity (LOH) was related to carcinogenesis and metastasis in several types of cancer. To explore the role of NEFL in the progression of breast cancer and to evaluate its clinical significance, we detected the NEFL mRNA level in normal breast tissues, primary breast cancer samples and lymph node metastases, and then analyzed the association between the NEFL expression level and several clinicopathological parameters and disease-free survival (DFS). NEFL mRNA was found to be expressed in 92.3% of breast malignancies and down-regulated in lymph node metastases compared to the paired primary tumors. NEFL mRNA level was lower in primary breast cancers with positive lymph nodes than in cancers with negative lymph nodes. Moreover, a low expression level of NEFL mRNA indicated a poor five-year DFS for early-stage breast cancer patients. Thus, NEFL mRNA is ectopically expressed in breast malignancies and could be a potential prognostic factor for early-stage breast cancer patients.     

Early Events following Experimental Infection with Peste-Des-Petits Ruminants Virus Suggest Immune Cell Targeting

Peste-des-petits ruminants virus (PPRV) is a viral pathogen that causes a devastating plague of small ruminants. PPRV is an economically significant disease that continues to be a major obstacle to the development of sustainable agriculture across the developing world. The current understanding of PPRV pathogenesis has been heavily assumed from the closely related rinderpest virus (RPV) and other morbillivirus infections alongside data derived from field outbreaks. There have been few studies reported that have focused on the pathogenesis of PPRV and very little is known about the processes underlying the early stages of infection. In the present study, 15 goats were challenged by the intranasal route with a virulent PPRV isolate, Côte d’Ivoire ’89 (CI/89) and sacrificed at strategically defined time-points post infection to enable pre- and post-mortem sampling. This approach enabled precise monitoring of the progress and distribution of virus throughout the infection from the time of challenge, through peak viraemia and into a period of convalescence. Observations were then related to findings of previous field studies and experimental models of PPRV to develop a clinical scoring system for PPRV. Importantly, histopathological investigations demonstrated that the initial site for virus replication is not within the epithelial cells of the respiratory mucosa, as has been previously reported, but is within the tonsillar tissue and lymph nodes draining the site of inoculation. We propose that virus is taken up by immune cells within the respiratory mucosa which then transport virus to lymphoid tissues where primary virus replication occurs, and from where virus enters circulation. Based on these findings we propose a novel clinical scoring methodology for PPRV pathogenesis and suggest a fundamental shift away from the conventional model of PPRV pathogenesis.     

Prognostic significance of Beclin 1 in intrahepatic cholangiocellular carcinoma

Autophagy enables cells to recycle long-lived proteins or damaged organelles. Beclin 1 plays important roles in autophagy, differentiation, apoptosis and the development and progression of cancer, but the expression of Beclin 1 and its possible role in primary intrahepatic cholangiocarcinoma (ICC) has not been reported yet. This study aimed to investigate Beclin 1 expression and its prognostic significance in ICC. First, we assessed the expression levels of Becn1 by real-time PCR in 50 ICC samples and found Becn1 mRNA expression was markedly increased in 78% (39 of 50) samples compared with normal bile duct epithelium. Beclin 1 protein expression in 108 tumor specimens from patients diagnosed with ICC was examined by immunohistochemistry and the correlation between Beclin 1 expression and clinicopathological factors were investigated. Immunopositivity for Beclin 1 was found in 72.2% (78 of 108) samples and low Beclin 1 expression was significantly associated with lymph node metastasis. The correlation between Beclin 1 expression and metastasis was validated in 46 ICC samples with lymph node metastasis. In survival analysis, low Beclin 1 expression was associated with worse overall survival (OS; p = 0.025) and disease-free survival (DFS; p = 0.027). In multivariate analysis, Beclin 1 expression, intrahepatic metastasis, lymph node metastasis and tumor size were found to be independent prognostic factors of OS. Thus, our results suggested the expression of Beclin 1 was correlated with progression and metastasis of ICC and it might serve as a novel prognostic marker for patients with ICC.     

Staufen基因在结直肠癌组织中的表达

为研究甲基化差异相关基因Staufen在结直肠癌不同组织中的表达情况,采用RT-PCR和免疫组织化学等方法在结直肠癌病人中检测Staufen基因在腺癌、癌旁粘膜和相应远端切缘正常组织中的表达。研究发现,在mRNA水平上,远端切缘正常组织中Staufen基因的表达水平显著高于癌旁粘膜和腺癌（P<0.05）;从正常组织、癌旁粘膜到腺癌,Staufen基因的表达有降低的趋势;未发现性别、年龄、肿瘤部位（结肠/直肠）、分化程度、淋巴结转移等临床病理因素与Staufen基因的表达有关（P均>0.05）。正常组织与腺癌组织的Staufen蛋白的表达水平显著高于癌旁粘膜（P<0.05）,而正常组织与腺癌组织之间未发现有统计学差别（P>0.05）。结果表明,Staufen基因在癌旁粘膜和肿瘤中有低表达的趋势,该基因可能在结直肠癌的发生、发展中起作用。     

Expression of GLP-1R protein and its clinical role in intrahepatic cholangiocarcinoma tissues

The study investigates the expression and clinical role of GLP-1R in intrahepatic cholangiocarcinoma (ICC) tissues. ICC tissue, tissue around tumour and normal liver tissue samples from 176 ICC patients were investigated for GLP-1R expression by immunohistochemistry and western blots. Expression levels were correlated to clinical variables and to the postoperative outcome. High GLP-1R expression levels were detected in tumor tissue samples. Kaplan–Meier method was used for survival analysis of patients follow-up data. Results showed that median survival time of patients with high GLP-1R positive expression in ICC tissue were 22 months. Median survival time of patients with low GLP-1R positive expression in ICC tissue were 19.8 months. There wasn’t statistical difference (p = 0.332) between two groups. Immunohistochemistry semi-quantitative analysis showed that tissue differentiation is not prognostic risk factors. In patients with GLP-1R positive expression in ICC tissue, lymph node metastasis was important prognostic factors (p = 0.001). Although statistical analysis showed that GLP-1R can not be judged as a risk prognostic factors, GLP-1 might become a new target for therapy of ICC.     

Signaling lymphocytic activation molecule is expressed on CD40 ligand-activated dendritic cells and directly augments production of inflammatory cytokines.

Dendritic cells (DC) comprise a key part of the innate immune system that, upon activation, profoundly influences the nature of the adaptive T cell response. In this study, we present evidence that signaling lymphocytic activation molecule (SLAM), a molecule first identified in activated T and B cells, is strongly up-regulated in DC activated through CD40, as well as in response to inflammatory stimuli, including polyinosinic polycytidylic acid and LPS. mRNA encoding both membrane-bound and soluble secreted isoforms of SLAM was detected in CD40 ligand-activated DC, comprising two of the four known SLAM isoforms. Expression of membrane-bound SLAM protein peaked at 12 h poststimulation with CD40 ligand, gradually returning to baseline levels after 6 days. SLAM up-regulation appears to be a direct result of the induction of DC maturation, as inflammatory cytokines released during this process do not affect SLAM expression. Functionally, engagement of SLAM enhances DC production of IL-12 and IL-8, while having no effect on production of IL-10. Because SLAM is involved in the activation of T cells, the expression of SLAM on DC may provide a bidirectional signaling mechanism in which interacting DC and T cells are simultaneously and synergistically activated to mount proinflammatory Th1 responses.     

Expression of Tight Junction Proteins and Cadherin 17 in the Small Intestine of Young Goats Offered a Reduced N and/or Ca Diet

Diets fed to ruminants should contain nitrogen (N) as low as possible to reduce feed costs and environmental pollution. Though possessing effective N-recycling mechanisms to maintain the N supply for rumen microbial protein synthesis and hence protein supply for the host, an N reduction caused substantial changes in calcium (Ca) and phosphate homeostasis in young goats including decreased intestinal transepithelial Ca absorption as reported for monogastric species. In contrast to the transcellular component of transepithelial Ca transport, the paracellular route has not been investigated in young goats. Therefore, the aim of the present study was to characterise the effects of dietary N and/or Ca reduction on paracellular transport mechanisms in young goats. Electrophysiological properties of intestinal epithelia were investigated by Ussing chamber experiments. The expression of tight junction (TJ) and adherens junction (AJ) proteins in intestinal epithelia were examined on mRNA level by qPCR and on protein level by western blot analysis. Dietary N reduction led to a segment specific increase in tissue conductances in the proximal jejunum which might be linked to concomitantly decreased expression of cadherin 17 mRNA. Expression of occludin (OCLN) and zonula occludens protein 1 was increased in mid jejunal epithelia of N reduced fed goats on mRNA and partly on protein level. Reduced dietary Ca supply resulted in a segment specific increase in claudin 2 and claudin 12 expression and decreased the expression of OCLN which might have been mediated at least in part by calcitriol. These data show that dietary N as well as Ca reduction affected expression of TJ and AJ proteins in a segment specific manner in young goats and may thus be involved in modulation of paracellular Ca permeability.     

Prognostic significance of Beclin 1 in intrahepatic cholangiocellular carcinoma

Autophagy enables cells to recycle long-lived proteins or damaged organelles. Beclin 1 plays important roles in autophagy, differentiation, apoptosis and the development and progression of cancer, but the expression of Beclin 1 and its possible role in primary intrahepatic cholangiocarcinoma (ICC) has not been reported yet. This study aimed to investigate Beclin 1 expression and its prognostic significance in ICC. First, we assessed the expression levels of Becn1 by real-time PCR in 50 ICC samples and found Becn1 mRNA expression was markedly increased in 78% (39 of 50) samples compared with normal bile duct epithelium. Beclin 1 protein expression in 108 tumor specimens from patients diagnosed with ICC was examined by immunohistochemistry and the correlation between Beclin 1 expression and clinicopathological factors were investigated. Immunopositivity for Beclin 1 was found in 72.2% (78 of 108) samples and low Beclin 1 expression was significantly associated with lymph node metastasis. The correlation between Beclin 1 expression and metastasis was validated in 46 ICC samples with lymph node metastasis. In survival analysis, low Beclin 1 expression was associated with worse overall survival (OS; p = 0.025) and disease-free survival (DFS; p = 0.027). In multivariate analysis, Beclin 1 expression, intrahepatic metastasis, lymph node metastasis and tumor size were found to be independent prognostic factors of OS. Thus, our results suggested the expression of Beclin 1 was correlated with progression and metastasis of ICC and it might serve as a novel prognostic marker for patients with ICC.     

TEM1mRNA 在非小细胞肺癌中的表达及临床意义*

目的:揭示TEM1其与非小细胞肺癌侵袭和转移的可能关系,为靶向治疗提供理想的药物作用靶点。方法:实时荧光定量PCR方法检测56例非小细胞肺癌肿瘤组织及癌旁组织中TEM1 mRNA表达水平,分析其在不同组中的表达差异。结果:TEM1在56例非小细胞肺癌组织中都有表达。TEM1表达水平在肿瘤组织中比癌旁组织表达高,并且其表达水平与淋巴结转移及肿瘤分期密切相关(P<0.05),但与患者的病理类型,年龄及性别无关(P>0.05)。结论:TEM1表达水平与非小细胞肺癌分期密切相关,表明其可能是一个参与非小细胞肺癌侵袭及转移有价值的分子标记物。TEM1可能成为潜在的基因治疗靶点。