南疆维吾尔族妇女宫颈癌与HPV感染及HLA-DRB1基因多态性的关系研究

目的 探讨人乳头状瘤病毒(human papilloma virus,HPV)感染及人类白细胞抗原(HLA)-DRB1基因多态性与南疆维吾尔族宫颈癌的关系.方法 采用导流杂交基因芯片技术及聚合酶链反应序列特异性寡核苷酸探针(polymerase chain reaction sequence-specific oligonucleotide,PCRSSO)法对200例南疆维吾尔族宫颈癌患者及200例正常妇女的宫颈组织检测HPV的21种型别(包括13种高危型:16,18,31,33,35,39,45,51,52,56,58,59,68;5种低危型:6,11,42,43,44;3种中国人群常见型:53,66,CP8304型)及HLA-DRB1的13个等位基因.结果 (1)200例官颈癌组织中HPV阳性者共177例(包括单一感染及多重感染),阳性率为88.5%;200例对照组中HPV阳性的标本为14例,阳性率为7.0%.HPV16在HPV阳性的宫颈癌中占的比例最高,其出现的频率为90.96%,其次为HPV18(5.08%)、HPV68(3.95%)、HPV45(3.39%)、HPV58(3.39%)、HPV39(1.69%)、HPV31(1.69%)、HPV56(1.13%)等.比较两组HPV总感染率及HPV16的阳性率,宫颈癌组明显高于对照组,两者差异有统计学意义(P＜0.0001).(2)官颈癌中HLA-DRB1*15阳性者HPV16的感染率高于阴性者,差异有统计学意义,说明携带HLA-DRB1*15等位基因的妇女更容易被HPV16感染.官颈癌中HLA-DRB1*12阳性者中HPV16的阳性率明显低于HLA-DRB1*12阴性者,说明HLA-DRB1*12对HPV16感染有保护作用.结论 南疆维吾尔族宫颈癌患者及正常人群中以HPV16感染为主,比例很高;其次为HPV18、HPV68,HPV68在第3位与文献报道不同,可能是南疆维吾尔族较易感染的类型.HLA-DRB1*15可能与维吾尔族妇女对官颈癌的遗传易感性及对HPV16感染易感性有关;HLA-DRB1*12为HPV16感染的保护基因.维吾尔族妇女官颈癌易感基因及保护基因的检测在宫颈癌高危人群的检测及群体遗传干预工作中有一定的指导意义.

Abstract：

Objective To investigate the relationships between the HPV infection and race susceptibility in the carcinogenesis of Uighur women with cervical cancer from the southern Xinjiang, one of the high risk region of cervical cancer in China. Methods To detect 21 subtypes of HPV and the 13 alleles of HLA from 200 cervical cancer cases and 200 normal tissues, by using flow-through hybridization and gene chip (HybriMax) method and polymerase chain reaction sequence-specific oligonucleotide method (PCRSSO). Results ( 1 )The proportion of HPV positive in cervical cancer and control group were 88.5% and 7.0% respecfively;HPV16 was the most common type in HPV positive cervical cancer patients with the rate of 90.96%, following were HPV18 (5.08%), HPV68(3.95% ),HPV45 (3.39%), HPV58 (3.39%),HPV39( 1.69% ), HPV31 ( 1.69% ), HPV56( 1.13% ). In cervical cancer and control group, the positive rate of HPV and HPV16 were significantly higher than that in control group. (2) In cervical cancer group the frequency of HLA-DRB1 * 15 in HPV positive cervical cancer cases was significantly higher than that among the HPV negative cases. While the frequency of HLA-DRB1 * 12 in HPV positive eervical caneer eases was significantly lower than that in the HPV negative e asea. Conclusion HPV16 was the most common type in both cervical cancer and control groups, the frequency of HPV16 in cervical carcinomas was very high, following HPV18 and HPV68, and HPV68 ranked third which was different from the results of other reports,this indicates that Uighur women are infected with this type more common. It appears that HLA-DRB1 * 15may be related to the susceptibility to cervical cancer and the HPV16 infection among the Uighur women,while the HLA-DRB1 * 12 the protective gene to HPV16 infection in Uighur women. The study of HLA alleles in the carcinogenesis of cervical carcinomas may play an important role in the gene intervention research of cervical cancer.     

多重HPV感染与宫颈病变的临床分析

目的探讨多重人乳头状瘤病毒（HPV）感染与宫颈病变之间的关系。方法采用核酸分子快速导流杂交基因芯片技术,对2008年7月至2010年7月在东莞市太平人民医院就诊且有病理确诊的332例女性患者（实验组）及100例正常妇女（对照组）进行HPV基因分型检测,比较不同宫颈病变类型与HPV多重感染的关系。结果 332例宫颈病变中HPV感染率为78.61%（261/332）,多重感染率为58.13%（168/289）,其中以二重感染为主,最常见的二重感染类型为HPV16、58及HPV52、58,以HPV16型感染多见;多重HPV感染比例随病变级别增加逐渐上升,由对照组的17.86%升高到宫颈鳞癌组的100%,各病变组与对照组相比差异有统计学意义（P〈0.05）。结论 HPV多重感染与宫颈病变的发生有关,可作为宫颈病变早诊早治的有效指标之一。     

新疆汉族、维吾尔族HPV16E6基因变异与子宫颈癌及宫颈病变的关系

目的探讨新疆地区汉族、维吾尔族宫颈癌及宫颈病变中HPV16型E6基因变异分布及频率,比较两个民族之间的差异。方法对140例HPV16阳性标本,设计E6基因特异性引物,通过PCR扩增HPV16E6全长基因,PCR产物直接测序,进行序列分析并与德国标准株对比,筛选突变位点。结果 123例汉族、维吾尔族妇女宫颈癌及宫颈病变标本中均存在HPV16E6变异株,其中突变率分别为:47.37%(27/57)和50%(33/66),E6突变位点主要是L83V、D25E、D64E、I73V、H78Y、D113E,其中L83V变异株在两个民族突变中占的比例均最高,汉族L83V突变频率为29.82%(17/57)显著低于维吾尔族的40.90%(27/66)(P0.05)。而汉族D25E突变频率为19.30%(11/57)显著高于维吾尔族的7.58%(5/66)(P0.05),D64E变异株在维族突变株中占的比例为6.1%,而汉族标本中未检出。结论两个民族HPV16 E6基因变异发生的位置和变异发生的频率存在差异,维吾尔族人群中HPV16E6基因D64E变异株可能与该地区维吾尔族宫颈癌高发存在一定的关系。     

子宫颈病变组织中低分子量蛋白酶体表达与HPV16感染的关系

目的观察低分子量蛋白酶体(low molecular-weight protein,LMP)在子宫颈病变组织中的mRNA和蛋白表达,探讨其与HPV16感染的关系。方法以152例新疆维吾尔族妇女正常子宫颈上皮、子宫颈上皮内瘤变(cervical intraepithelial neoplasia,CIN)和子宫颈鳞癌(cervical squamous cell carcinoma,CSCC)患者为研究对象,采用RT-PCR和免疫组化法鉴定LMP2和LMP7mRNA及蛋白表达水平;采用PCR技术检测相应标本HPV16感染情况。结果 (1)LMP2、LMP7随着子宫颈病变的加重其蛋白表达逐渐降低,且mRNA表达水平与蛋白表达趋势相一致。在CIN中LMP2、LMP7蛋白表达下调和缺失率分别为25.0%/15.6%、29.7%/23.4%;在子宫颈癌中LMP2、LMP7蛋白的表达下调和缺失率分别为17.5%/34.9%、23.8%/41.3%。临床病理参数进行分析发现LMP2、LMP7与子宫颈癌分化程度及淋巴结转移密切相关(P<0.05)。(2)PCR结果显示,HPV16的检出率随着子宫颈病变的进展而增加,在慢性子宫颈炎、CIN和子宫颈癌组织中阳性率分别为8%(2/25)、67.2%(43/64)和77.8%(49/63),且随着肿瘤恶性程度的增加其阳性表达率增加,各组间阳性表达差异有统计学意义(P<0.05)。在CIN中LMP7表达下调与HPV16感染有关(P<0.05),子宫颈癌中LMP2和LMP7表达下调与HPV16感染有关(P<0.05)。结论 LMP基因的转录表达下调或蛋白质表达缺失与维吾尔族妇女子宫颈癌病变进程密切相关,其中HPV16感染可能是重要原因之一。     

女性HPV感染与宫颈鳞状上皮内病变

目的 研究女性HPV感染与其宫颈鳞状上皮内病变关系,为临床预防宫颈癌发生发展提供基础数据.方法 537例HPV感染阳性患者样本来源于解放军第210医院妇科门诊,分析患者感染HPV基因亚型与数量及患者年龄数据,研究其与患者宫颈鳞状上皮内病变统计学关系.结果 单一高危亚型HPV感染患者宫颈鳞状上皮内病变发病率高于单一低危亚型HPV感染患者,差别有统计学意义(P＜0.05).单一高危亚型HPV感染患者4种具体宫颈鳞状上皮内病变发病率均高于低危亚型HPV感染患者,其中ASC-H及LSIL发病率差别有统计学意义(P＜0.05).不同类型多重HPV感染患者宫颈鳞状上皮内病变发病率差别未见统计学意义(P＞0.05).多重HPV感染患者宫颈鳞状上皮内病变发病率高于单一HPV感染患者,差别有统计学意义(P＜0.05).多重HPV感染患者ASC-US发病率高于单一HPV感染患者,差别有统计学意义(P＜0.05).HPV感染患者宫颈鳞状上皮内病变发病率在41 ～ 50年龄组最高,65.04％(80/123),与其它各年龄组比较差别均有统计学意义(P＜0.05).结论 HPV感染基因亚型种类、数量及感染者年龄均与宫颈鳞状上皮内病变发生有一定关联,其中高危亚型HPV感染、多重HPV感染与中年妇女三种因素,与宫颈鳞状上皮内病变发生发展关系密切.     

斑点分子杂交检测HPV感染与宫颈癌的关系

采用ＤＮＡ－ＤＮＡ分子杂交技术，对经病理组织学确诊的慢性宫颈炎，宫颈癌和正常宫颈的宫颈活检组织中ＨＰＶ６，ＨＰＶ１１，ＨＰＶ１６，ＨＰＶ１８型ＤＮＡ进行同源序列检测，结果表明ＨＰＶ６，ＨＰＶ１１，ＨＰＶ１６，ＨＰＶ１８型ＤＮＡ的检出率在正常宫颈均为０；在慢性宫颈炎分别为１６．０９％，１２．６４％，１１．４９％，３．４５％；在宫颈癌组分别为３．９６％，１．９８％，４６．５３％，７．９２％。在宫颈癌组     

女性下生殖道HPV感染和HPV相关的宫颈肿瘤

子宫颈病变是女性最常见的疾患之一.在女性癌瘤中,宫颈癌的发病率仅次于乳腺癌.人乳头状瘤病毒(HPV)感染在宫颈肿瘤的发病机制中起着重要的作用,许多学者关注HPV疫苗的预防和治疗.在女性生殖道HPV传播及继发性感染是局部性的,因此,针对这种局部性传播疫苗的有效性最好能用局部免疫的参数来评价.     

高危亚型HPV33和HPV52感染与宫颈病变的关系

目的 探讨人乳头瘤病毒(HPV)高危亚型HPV33和HPV52感染与宫颈病变的关系。方法 选取316例于我院进行病理组织活检联合HPVDNA检测的病例,对其进行HPVDNA提取、PCR扩增、导流杂交,其中对194例HPV33和HPV52亚型阳性患者资料进行分析。结果 194例HPV33和HPV52阳性患者中,宫颈良性病变225例,宫颈上皮内瘤变(CIN)为114例和宫颈恶性肿瘤为55例,利用统计学χ~2检验发现在HPV33和HPV52阳性患者中,CIN和宫颈恶性肿瘤的阳性率明显高于宫颈良性病变的阳性率(p<0.05)。结论 HPV33和HPV52阳性患者患宫颈病变的几率增大,高危型HPV33和HPV52是引起宫颈恶性病变的致病基因型。     

300例妇科宫颈病变患者石蜡切片HPV基因分型结果分析

探讨女性生殖道感染人乳头瘤病毒(HPV)和不同宫颈病变的关系及HPV检测在宫颈癌防治方面的应用价值.对阴道镜活检病理诊断的300例不同的宫颈病变患者进行HPV-DNA检测(同时检测5种低危型和18种高危型HPV亚型).结果显示,慢性宫颈炎组HPV阳性87例,阴性48例,阳性率64.5%;子宫颈上皮内瘤变(CIN)组(C...     

TCT联合HPV检测与多基因甲基化检测在宫颈病变诊断中应用价值分析

目的 分析液基薄层细胞学检测(TCT)联合人乳头瘤病毒(HPV)检测与多基因甲基化检测在宫颈病变诊断中的应用价值.方法 将2018年6月至2020年5月于成都医学院第二附属医院核工业四一六医院妇科就诊的298例宫颈病变高风险患者作为研究对象,均进行TCT联合HPV检测、多基因甲基化检测和阴道镜下宫颈病理活检,以病理结果...     

新疆维吾尔族患者HPV感染型别临床分析

目的分析新疆维吾尔族妇女子宫颈HPV感染类型及其分布的规律。方法采用导流杂交基因分型技术对318名妇科维吾尔族就诊患者进行HPV基因分型检测。结果 HPV-DNA检测阳性者占21.70%（69/318）。低危型感染12例（20.69%）,以HPV 6和HPV 1l为主;高危型感染46例（79.31%）,以HPV 16、HPV58和HPV68感染为主。多重感染11例,感染率为3.46%。从年龄分布来看,≤25岁的患者占31.48%,HPV感染率最高,其次为50~55岁年龄段的患者占25.71%。结论 HPV分布存在地域和种族差异,加强对新疆各年龄人群,多种型别HPV的筛查,有助于预防宫颈癌的发生及了解HPV感染的转归,并为新疆地区宫颈癌干预提供理论基础。     

DAPK1, MGMT and RARB promoter methylation as biomarkers for high-grade cervical lesions

Gene promoter methylation may be used a potential biomarker for detecting solid tumor including cervical cancer. Here, we used methylation sensitive-high resolution melting (MS-HRM) analysis to detecting promoter methylation ratios of DAPK1, MGMT and RARB gene in patients with different cervical disease grade. The detection of gene promoter methylation was conducted in two hundred fifty patients’ samples including normal cytology (n=48), cervical intraepithelial neoplasia grade 1 (CIN1, n=54), cervical intraepithelial neoplasia grade 2 (CIN2, n=47), cervical intraepithelial neoplasia grade 3 (CIN3, n=56) and cervical squamous cell carcinomas (SCS, n=45). We found there were a significant positive correlation between the promoter methylation status of DAPK1 and cervical disease grade (P=0.022). In addition, the methylated promoters of DAPK1 combined with MGMT, MGMT combined with RARB, DAPK1 combined with RARB were positive correlated with cervical disease grade (P < 0.05). All three genes promoters methylated were positive correlated with cervical disease grade (P < 0.001). Receiver operating characteristic (ROC) curves was conducted to evaluate whether the three genes methylation could be used to be a potential marker for diagnosing high grade cervical disease (HSIL and SCC). The cutoff values for the methylation rates of all these genes were 0-5%. Regrettably, only the methylation of MGMT combined with DAPK1 gave 43.4% sensitivity and 68.6% specificity. The current results indicated that MS-HRM-based testing for DNA methylations of MGMT plus DAPK1 genes holds some promise for high grade cervical disease screening.     

Expression of Bcl-2 and Survivin in uterine cervical carcinogenesis and correlation between the expression and infection of HPV_(16/18)

Carcinomaofthecervixisthesecondleading causeofdeathamongwomenworldwide.Each year,anestimated500000casesarenewlydiag nosed[1].Manystudiesshowedinfectionofhu manpapillomavirus(HPV)wasoneofthemost importantetiologicfactorsforcervicalcarcinoma[24].Morethan95%ofallcervicalcarcinomas havebeenfoundtobeassociatedwithHPV(ma inlytypes16and18)[5].Thestudiesinmolec ularoncologyrecentlyshowedthatitresultedin occurrenceanddevelopmentofcervicalcarcinomasthatcarcinogenicfactorsmadeproto oncogene activatean…     

Association between high risk human papillomavirus infection and co-infection with Candida spp. and Trichomonas vaginalis in women with cervical premalignant and malignant lesions

BackgroundHuman papillomavirus (HPV) is the necessary cause of cervical cancer. Cervico-vaginal infection with pathogens like Chlamydia is a likely cofactor. The interactions between HPV, Trichomonas vaginalis (TV) and Candida spp. are less understood, though inflammation induced by these pathogens has been demonstrated to facilitate oncogenesis.ObjectiveOur study aimed to evaluate the association between Candida spp. and TV co-infection with HPV in cervical oncogenesis.Study designWomen with normal cervix who were high-risk HPV-negative (N = 104) and HPV-positive (N = 105); women with CIN 1 (N = 106) and CIN 2/CIN 3 (N = 62) were recruited from a community based cervical cancer screening program. Cervical cancer patients (N = 106) were recruited from a tertiary care oncology clinic. High-risk HPV was detected by Hybrid Capture II technique; Candida spp. and TV were detected by culturing the high vaginal swabs followed by microscopic examination in all. The disease status was established by histopathology in all the women.ResultHPV-positive women had significantly higher risk of having precursor lesions (of any grade) and cancer compared to HPV-negative women. Candida spp. or TV infection did not alter the risk of low grade or high grade lesions among HPV- positive women. HPV positive women co-infected with TV had higher risk of cervical cancer but not those co-infected with Candida spp.ConclusionThe higher risk of cancer observed in the women co-infected with HPV and TV without any enhanced risk of CIN 3 suggests secondary infection of the malignant growth by TV rather than any causal role. Co-infection with Candida spp. and/or TV infection did not increase the carcinogenic effect of HPV on cervix.     

宫颈鳞状细胞癌组织中hMSH2启动子甲基化及蛋白表达的研究

目的检测宫颈鳞癌组织中hMSH2启动子甲基化及蛋白表达，探讨hMSH2在宫颈鳞癌发生、发展中的作用。方法采用甲基化特异性PCR方法和免疫组织化学方法分别检测正常宫颈组织、宫颈鳞癌组织中hMSH2启动子甲基化状况及蛋白表达。结果正常组织中未见hMSH2启动子甲基化，其蛋白表达为阳性表达；宫颈鳞癌组织中可见启动子甲基化，蛋白表达也表现异常或缺失。结论错配修复基因的正常表达是保证基因稳定的重要因素，hMSH2启动子区域甲基化及蛋白表达缺失在宫颈鳞状细胞癌的发生、发展过程中起一定作用。     

Comparing triage algorithms using HPV DNA genotyping,HPV E7 mRNA detection and cytology in high-risk HPV DNA-positive women

BackgroundHigh-risk human papillomavirus (hrHPV) DNA positive women require triage testing to identify those with high-grade cervical intraepithelial neoplasia or cancer (≥CIN2).ObjectiveComparing three triage algorithms (1) E7 mRNA testing following HPV16/18/31/33/45/52/58 genotyping (E7 mRNA test), (2) HPV16/18 DNA genotyping and (3) cytology, for ≥CIN2 detection in hrHPV DNA-positive women.Study designhrHPV DNA-positive women aged 18–63 years visiting gynecology outpatient clinics were included in a prospective observational cohort study. From these women a cervical scrape and colposcopy-directed biopsies were obtained. Cervical scrapes were evaluated by cytology, HPV DNA genotyping by bead-based multiplex genotyping of GP5+6+-PCR-products, and presence of HPV16/18/31/33/45/52/58 E7 mRNA using nucleic acid sequence-based amplification (NASBA) in DNA positive women for respective HPV types. Sensitivities and specificities for ≥CIN2 were compared between E7 mRNA test and HPV16/18 DNA genotyping in the total group (n = 348), and E7 mRNA test and cytology in a subgroup of women referred for non-cervix-related gynecological complaints (n = 133).ResultsSensitivity for ≥CIN2 of the E7 mRNA test was slightly higher than that of HPV16/18 DNA genotyping (66.9% versus 60.9%; ratio 1.10, 95% CI: 1.0002–1.21), at similar specificity (54.8% versus 52.3%; ratio 1.05, 95% CI: 0.93–1.18). Neither sensitivity nor specificity of the E7 mRNA test differed significantly from that of cytology (sensitivity: 68.8% versus 75.0%; ratio 0.92, 95% CI: 0.72–1.17; specificity: 59.4% versus 65.3%; ratio 0.91, 95% CI: 0.75–1.10).ConclusionFor detection of ≥CIN2 in hrHPV DNA-positive women, an algorithm including E7 mRNA testing following HPV16/18/31/33/45/52/58 DNA genotyping performs similar to HPV16/18 DNA genotyping or cytology.     

HPV感染和p53异常表达与宫颈鳞癌的关系

目的研究宫颈鳞癌与p53蛋白表达和HPV感染的关系,探讨宫颈鳞癌的形成机制。方法采用RT-PCR法分别检测p53基因在39例宫颈鳞癌组织和39例正常宫颈黏膜组织中的表达情况以及PCR方法检测HPV在这些组织中的感染情况。结果 HPV在宫颈鳞癌组中阳性率为48.72%(19/39),正常组织中为14.81%(4/27),p53基因表达在宫颈鳞癌组中阳性率为53.85%(21/39),正常组织中为18.52%(5/27),宫颈癌组的HPV感染和p53表达均高于正常组(P0.05)。结论 HPV感染与p53基因的异常表达与宫颈鳞癌发生密切相关,联合检测能提高准确性。     

Retinoic acid receptor beta promoter methylation and risk of cervical cancer

Cervical cancer is one of the leading causes of death in women worldwide, particularly in developing countries. Human papillomavirus has been reported as one of the key etiologic factors in cervical carcinoma. Likewise, epigenetic aberrations have ability to regulate cancer pathogenesis and progression. Recent research suggested that methylation has been detected already at precancerous stages, which methylation markers may have significant value in cervical cancer screening. The retinoic acid receptor beta (RARβ) gene, a potential tumor suppressor gene, is usually expressed in normal epithelial tissue. Methylation of CpG islands in the promoter region of the RARβ gene has been found to be associated with the development of cervical cancer. To investigate whether RARβ methylation is a potential biomarker that predicts the progression of invasive cancer, we reviewed 14 previously published articles related to RARβ methylation. The majority of them demonstrated that the frequency of RARβ promoter methylation was significantly correlated with the severity of cervical epithelium abnormalities. However, methylation of a single gene may not represent the best approach for predicting disease prognosis. Analyzing combinations of aberrant methylation of multiple genes may increase the sensitivity, and thus this approach may serve as a better tool for predicting disease prognosis.     

Methylation‐specific digital karyotyping of HPV16E6E7‐expressing human keratinocytes identifies novel methylation events in cervical carcinogenesis

Transformation of epithelial cells by high‐risk human papillomavirus (hrHPV) types can lead to anogenital carcinomas, particularly cervical cancer, and oropharyngeal cancers. This process is associated with DNA methylation alterations, often affecting tumour suppressor gene expression. This study aimed to comprehensively unravel genome‐wide DNA methylation events linked to a transforming hrHPV‐infection, which is driven by deregulated expression of the viral oncogenes E6 and E7 in dividing cells. Primary human keratinocytes transduced with HPV16E6E7 and their untransduced counterparts were subjected to methylation‐specific digital karyotyping (MSDK) to screen for genome‐wide DNA‐methylation changes at different stages of HPV‐induced transformation. Integration of the obtained methylation profiles with genome‐wide gene expression patterns of cervical carcinomas identified 34 genes with increased methylation in HPV‐transformed cells and reduced expression in cervical carcinomas. For 12 genes (CLIC3, CREB3L1, FAM19A4, LFNG, LHX1, MRC2, NKX2‐8, NPTX‐1, PHACTR3, PRDM14, SOST and TNFSF13) specific methylation in HPV‐containing cell lines was confirmed by semi‐quantitative methylation‐specific PCR. Subsequent analysis of FAM19A4, LHX1, NKX2‐8, NPTX‐1, PHACTR3 and PRDM14 in cervical tissue specimens showed increasing methylation levels for all genes with disease progression. All six genes were frequently methylated in cervical carcinomas, with highest frequencies (up to 100%) seen for FAM19A4, PHACTR3 and PRDM14. Analysis of hrHPV‐positive cervical scrapes revealed significantly increased methylation levels of the latter three genes in women with high‐grade cervical disease compared to controls. In conclusion, MSDK analysis of HPV16‐transduced keratinocytes at different stages of HPV‐induced transformation resulted in the identification of novel DNA methylation events, involving FAM19A4, LHX1, NKX2‐8, PHACTR3 and PRDM14 genes in cervical carcinogenesis. These genes may provide promising triage markers to assess the presence of (pre)cancerous cervical lesions in hrHPV‐positive women. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.