Control of Bovine Brucellosis from Persistently Infected Holdings Using RB51 Vaccination with Test‐and‐Slaughter: A Comparative Case Report from a High Incidence Area in Portugal

Bovine brucellosis due to Brucella abortus infection causes significant reproductive and production losses in cattle and is a major zoonosis. Eradication of this disease has proved difficult to achieve in Portugal where it still occurs in some regions despite an ongoing national eradication programme. In 2004, the Alentejo region, a major cattle producing area, reported one of the highest levels of bovine brucellosis in the country, especially in one divisional area. In that area, bovine brucellosis was particularly problematic in a holding of ten herds, the largest extensive cattle unit in the country, which remained infected despite an extensive test‐and‐slaughter programme and depopulation of five herds. A 5‐year programme of RB51 vaccination with biannual test‐and‐slaughter was thus implemented in 2004. The apparent animal seroprevalence decreased from 19% (646/3,400) to 3% (88/2930) on the third herd‐level test and remained below 0.8% (27/3324) after the fourth test. After the tenth test, the holding had a prevalence of 0.1% (2/2332) and only one herd remained positive with a within‐herd prevalence of 1.1% (2/177). The results were compared to all other herds (n = 10) in the divisional area that were also persistently infected but were subject only to test‐and‐slaughter before being depopulated. In these herds, the strategy of test‐and‐slaughter did not reduce the prevalence, which remained significantly higher than the vaccinated group (median = 0.48% and 8.5% in vaccinated versus non‐vaccinated herds; Wilcoxon rank sum test; P < 0.01). The success of this pilot programme in continental Portugal provided a valuable case study to the official veterinary services by illustrating the value of RB51 vaccination with parallel testing and improved biosecurity as a comprehensive and sustainable strategy for bovine brucellosis control in persistently infected herds.     

Epigenetic abnormalities in cutaneous squamous cell carcinomas: frequent inactivation of the RB1/p16 and p53 pathways

BACKGROUND: Aberrant methylation of CpG islands in the promoter regions of cancer-related genes has been demonstrated in many human tumours. However, the methylation profile of these regions in cutaneous squamous cell carcinomas (SCCs) has not been well studied. OBJECTIVES: To examine epigenetic abnormalities of a wide range of cancer-related genes in SCCs. METHODS: We investigated the methylation status of 11 candidate cancer-related genes (CDH1, p16(INK4a), p14(ARF), DAPK1, MGMT, RB1, RASSF1, p15(INK4b), PTEN, PRDM2 and p53) in 20 cases of SCC by methylation-specific polymerase chain reaction, and comparatively examined the protein production of E-cadherin (CDH1), p16, RB1, p14, BMI1 and cyclin A by immunohistochemical analysis. RESULTS: The frequency of cancer-related gene methylation in SCCs was: CDH1 (95%), p16 (20%), p14 (15%), DAPK1 (15%), MGMT (15%), RB1 (5%), RASSF1 (5%), p15 (0%), PTEN (0%), PRDM2 (0%) and p53 (0%). Almost all cases with hypermethylation of CDH1, p16, RB1 and p14 showed no obvious production of each protein, suggesting that promoter hypermethylation of these genes contributes to the loss of protein production. The results of methylation analysis, in combination with the results of our previous mutation analysis of CDKN2A locus and p53, revealed that 70% of SCCs have alterations in the RB1/p16 or p53 pathway. CONCLUSIONS: Our findings indicate that the promoter hypermethylation of cancer-related genes, especially CDH1, is frequently shown in SCCs, and dysregulation of the RB1/p16 and/or p53 pathway through either genetic or epigenetic mechanisms, except for epigenetic abnormalities of p53 itself, should contribute to the carcinogenesis of SCCs.     

人脑肿瘤组织中SV40感染及其临床意义

目的 探讨猴病毒40（SV40）与人脑肿瘤发病的关系。方法 采用聚 一等奖反应（PCR）和原杂交（ISH）同时检测30例正常人脑组织和198例人及脑肿瘤组织以及SHG44和BT3 25两株人脑胶质瘤细胞系中SV40 DNA充列;并对SV40 DNA阳性肿瘤细胞采用免疫共沉淀和Western blot检测大T抗原（Tag）的表达及Tag-RB复合物的存在。结果 人脑肿瘤组织PCR SV40 DNA阳性率为48.5％（96／198）,其中胶质瘤47.2％（42／89）,脑膜瘤48.8％（21／43）,脑垂体腺瘤51. 4%(18/35),神经鞘瘤43.8％（7／16）,先天性肿瘤53.3％（8／15）,正常人脑组织PCR SV40 DNA阳性率为6.7％（2／30）。SHG44及BT325两株细胞中也分别检测出SV40的DNA序列。人脑肿瘤组织SV40 DNA阳性率显著高于正常人脑组织（P＜0.01）。经ISH,仅在96例PCR SV40 DNA阳性的及脑肿瘤组织中检出87例阳性,2例SV40 DNA阳性的正常人脑组织中1例ISH阳性,SHG44及BT325两株细胞ISH SV40 DNA均阳性。SV40 DNA定位于肿瘤细胞核,阳性细胞呈弥温或片灶状分布。96人列SV40 DNA阳性脑瘤组织Tag表达阳性75例,所有Tag表达阳性瘤组织均发现Tag与RB形成特异性复合物。结论 人脑肿瘤组织中存在SV40感染,提示SV40感染与有脑肿瘤有关;在人脑肿瘤组织中Tag广泛表达,Tag可能是SV40在有禽肿瘤发生发展中起作用的重要因素,S V40 Tag与RB形成特异性复合物Tag-RB,导致RB活性,有是SV40致人脑肿瘤发生的一个重要机理。     

miR-192负性调控人肝癌细胞株HepG2中RB1基因的表达

目的 探讨miR-192对人肝癌细胞株HepG2中RB1基因表达的调节作用.方法 运用生物信息学方法对miR-192进行靶基因预测并分析其潜在靶基因RB1;将含有miR-192结合位点的RB1 mRNA 3′端非编码区(3′UTR)片段和在miR-192结合位点进行突变的RB1 3′UTR突变片段克隆至报告基因载体pMIR-Report luciferase vector,重组质粒分别命名为pMIR-RB1和pMIR-RB1-mut;将重组质粒、Beta-gal内参质粒和microRNA共转染HepG2细胞,双荧光素酶报告基因系统检测各实验组中细胞荧光素酶的表达;SYBR Green荧光定量PCR和Western blot分别在mRNA和蛋白水平检测miR-192对内源性RB1表达的调节作用.结果 生物信息学分析筛选出89个miR-192的潜在靶基因,RB1基因是其中之一;测序验证重组质粒pMIR-RB1和pMIR-RB1-mut构建成功;过表达miR-192时,共转染pMIR-RB1质粒的细胞相对荧光素酶活性(4.80±0.36)较相应过表达miR-NC组(7.90±0.91)明显降低(P＜0.05),而共转染pMIR-Luc或pMIR-RB1-mut质粒的细胞相对荧光素酶活性[pMIR-Luc:(7.68±1.04);pMIR-RB1-mut:(7.56±0.99)]较相应过表达miR-NC组[pMIR-Luc:(7.86±0.73);pMIR-RB1-mut:(7.82±1.05)]无显著差异;上调miR-192水平显著降低HepG2细胞中内源性RB1 mRNA[(0.56±0.10)vs (1.05±0.13)]和蛋白(47% vs 100%)的表达.结论 RB1基因是miR-192的一个靶基因,在HepG2细胞中,miR-192通过直接结合RB1 mRNA 3′UTR而负性调控RB1基因表达.     

曲古抑菌素A对甲状腺鳞癌细胞中Cyclin D1、CDK4、pRB表达的影响

目的:观察曲古抑菌素A(TSA)对甲状腺鳞癌(SW579)细胞中细胞周期蛋白D1(Cyclin D1)、细胞周期蛋白依赖性激酶4(CDK4)、视网膜母细胞瘤基因(RB)的蛋白产物(pRB)表达的影响。方法:体外培养SW579细胞,以二甲基亚砜(DMSO)为溶剂对照组,另设空白对照(未作任何处理)组,观察50、100、200、400nmol·L-1TSA对SW579细胞生长抑制率的影响,及细胞中Cy-clin D1、CDK4、RB基因和蛋白的表达情况。结果:与溶剂对照组和空白对照组比较,各剂量TSA作用后SW579细胞的细胞生长抑制率明显升高(P<0.01),且呈剂量依赖性。与空白对照组比较,各剂量TSA作用后SW579细胞中Cyclin D1 mRNA表达明显降低(P<0.01),且随剂量增加表达降低,但CDK4、RB mRNA表达和溶剂对照组中这2个基因的表达均无明显变化(P>0.05);与空白对照组比较,各剂量TSA作用后细胞中Cyclin D1和pRB蛋白表达明显降低(P<0.01),且随剂量增加表达降低,但CDK4蛋白表达无明显变化(P>0.05)。结论:TSA能明显抑制SW579细胞的生长,且呈剂量依赖性;其机制可能与Cyclin D1基因和蛋白、pRB蛋白的表达有关。     

视网膜母细胞瘤患者RB1基因突变筛选

目的：了解视网膜母细胞瘤患RBl基因的突变特点。方法：应用PCR-SSCP技术筛奄RB患白细胞基因组DNA，测序分析确定突变。结果：在RB患10例中，确定双眼RB患1例第8外显子37位核苷酸A-T突变，引起12位谷氨酸变为天门冬酰氨酸(G12A)。其母亲同样为第8外显子36位核苷酸A-T突变，引起12位谷氨酸变为天门冬酰氨酸(G12A)。结论：本组双眼RB患1例为遗传性，其RB1基因突变为微小突变，其母亲为突变基因携带，本研究为遗传咨询提供一些有价值的资料。     

IL-12RB1基因突变致孟德尔遗传易感分枝杆菌病2例临床特点及基因分析

目的　研究白细胞介素12受体B1基因（IL-12RB1）突变所致孟德尔遗传易感分枝杆菌病的基因资料及临床特点,提高对该病的认识。方法　检测2016—2018年中国医学科学院北京协和医院就诊的2例播散性卡介苗感染患儿基因并分析结果,同时总结患儿的临床资料。结果　2例患儿分别为11月龄和13月龄男性儿童,均于出生后接种卡介苗,接种后3个月出现同侧腋下淋巴结肿大,病原学检查提示抗酸杆菌生长。均否认结核病接触史。基因检测分析结果显示2例患儿均为IL-12RB1复合杂合基因突变,分别为c.1561C＞T,p.R521X;c.632G＞C,p.R211P;c.339-340 del CT,p.L113Lfs*15和c.1791+2T＞G。其中c.339-340 del CT,p.L113Lfs*15未见报道,是新突变。结论　对于接种卡介苗后出现感染性播散的患儿,应进行原发性免疫缺陷基因检测,相关基因突变的识别,可为早期治疗及遗传咨询提供依据。     

The significance of HOXB7 and IL17RB serum levels in prognosis of hormonally dependent breast cancer: A pilot study

PurposeImproved prognostication of a patient's outcome could allow for personalized treatment decisions in breast cancer. Homeobox B7 (HOXB7) and interleukin 17 receptor B (IL17RB) are proteins reportedly involved in the development of hormonal therapy resistance. Their prognostic value was previously investigated in tumor tissue but recent mass spectrometric detection of HOXB7 and IL17RB proteins in serum has prompted us to perform the first prognostic evaluation of their serum levels.Patients and methodsThe study included 81 premenopausal breast cancer patients that received adjuvant hormonal therapy. The median follow-up period was 61 months. HOXB7 and IL17RB serum protein levels were measured by quantitative sandwich ELISA and prognostically evaluated by Cox proportional hazards regression analysis.ResultsHOXB7 protein was detected in 96.3% and IL17RB in 33.3% of serum samples. Higher levels of serum HOXB7 significantly associated with favorable disease outcome by prognosticating distant (by HR ​= ​0.04; P ​= ​0.001) and local recurrence (by HR ​= ​0.03, P ​= ​0.001). The recurrence rates in the HOXB7^high and HOXB7^low subgroups of patients (cut-off 81.5 ​pg/mL) were 0% and 17%, respectively. Serum IL17RB levels did not significantly associate with either local or distant events. The multivariate analysis highlighted estrogen receptor, histological grade, nodal status and HOXB7 as independent prognostic parameters.ConclusionsOur findings validate the previous mass-spectrometry data by showing that HOXB7 and IL17RB cellular proteins are detectable in serum by a standard ELISA assay. Furthermore, we show that HOXB7 serum levels are the relevant prognosticator of response to hormonal therapy.     

Alterations of DNA damage response pathway: Biomarker and therapeutic strategy for cancer immunotherapy

Genomic instability remains an enabling feature of cancer and promotes malignant transformation. Alterations of DNA damage response (DDR) pathways allow genomic instability, generate neoantigens, upregulate the expression of programmed death ligand 1 (PD-L1) and interact with signaling such as cyclic GMP–AMP synthase-stimulator of interferon genes (cGAS–STING) signaling. Here, we review the basic knowledge of DDR pathways, mechanisms of genomic instability induced by DDR alterations, impacts of DDR alterations on immune system, and the potential applications of DDR alterations as biomarkers and therapeutic targets in cancer immunotherapy.