The development of cervical and vaginal adenosis as a result of diethylstilbestrol exposure in utero

Exposure to exogenous hormones during development can result in permanent health problems. In utero exposure to diethylstilbestrol (DES) is probably the most well documented case in human history. DES, an orally active synthetic estrogen, was believed to prevent adverse pregnancy outcome and thus was routinely given to selected pregnant women from the 1940s to the 1960s. It has been estimated that 5 million pregnant women worldwide were prescribed DES during this period. In the early 1970s, vaginal clear cell adenocarcinomas (CCACs) were diagnosed in daughters whose mother took DES during pregnancy (known as DES daughters). Follow-up studies demonstrated that exposure to DES in utero causes a spectrum of congenital anomalies in female reproductive tracts and CCACs. Among those, cervical and vaginal adenoses are most commonly found, which are believed to be the precursors of CCACs. Transformation related protein 63 (TRP63/p63) marks the cell fate decision of Müllerian duct epithelium (MDE) to become squamous epithelium in the cervix and vagina. DES disrupts the TRP63 expression in mice and induces adenosis lesions in the cervix and vagina. This review describes mouse models that can be used to study the development of DES-induced anomalies, focusing on cervical and vaginal adenoses, and discusses their molecular pathogenesis.     

小鼠阴道菌群紊乱模型的建立

目的建立小鼠阴道菌群紊乱模型,为调整阴道菌群紊乱的药物研发提供模型参考。方法采用雌激素化的C57BL/6小鼠经阴道以不同处理方式(链霉素50μg/只,加德纳菌50μL/只,链霉素50μg/只+抗链霉素加德纳菌50μL/只,每种处理方式各70只/组)诱导小鼠阴道菌群紊乱模型,采用细菌16S rDNA高通量测序考察造模后第1、第2、第3、第5、第7、第9、第11天内加德纳菌在阴道内的定植情况,以及阴道细菌结构和细菌生物多样性,并通过显微镜观察阴道组织的病理改变,比较3种造模方法的差异。另采用阳性药物(定君生,成分为德氏乳杆菌)对模型进行验证。结果采用链霉素联合抗链霉素加德纳菌能诱导形成典型的阴道菌群紊乱模型,造模后可见加德纳菌在阴道内的定植增多,优势菌以铜绿假单胞菌为主,且阴道细菌生物多样性增加,阴道组织可见明显的炎症浸润和上皮细胞坏死等病理改变。定君生能显著减少加德纳菌在阴道内的定植,减少阴道内的细菌生物多样性,并能显著改善阴道组织病理变化。结论采用链霉素联合抗链霉素加德纳菌诱导的小鼠阴道菌群紊乱模型方法更佳,模型的细菌学和组织病理学改变,以及对药物的反应性与临床有一定相似,具有临床应用价值。     

Mathematical model of the neonatal mouse ventricular action potential

Therapies for heart disease are based largely on our understanding of the adult myocardium. The dramatic differences in action potential (AP) shape between neonatal and adult cardiac myocytes, however, indicate that a different set of molecular interactions in neonatal myocytes necessitates different treatment for newborns. Computational modeling is useful for synthesizing data to determine how interactions between components lead to systems-level behavior, but this technique has not been used extensively to study neonatal heart cell function. We created a mathematical model of the neonatal (day 1) mouse myocyte by modifying, on the basis of experimental data, the densities and/or formulations of ion transport mechanisms in an adult cell model. The new model reproduces the characteristic AP shape of neonatal cells, with a brief plateau phase and longer duration than the adult (action potential duration at 80% repolarization = 60.1 vs. 12.6 ms). The simulation results are consistent with experimental data, including 1) decreased density and altered inactivation of transient outward K+ currents, 2) increased delayed rectifier K+ currents, 3) Ca2+ entry through T-type as well as L-type Ca2+ channels, 4) increased Ca2+ influx through Na+/Ca2+ exchange, and 5) Ca2+ transients resulting from transmembrane Ca2+ entry rather than release from the sarcoplasmic reticulum (SR). Simulations performed with the model generated novel predictions, including increased SR Ca2+ leak and elevated intracellular Na+ concentration in neonatal compared with adult myocytes. This new model can therefore be used for testing hypotheses and obtaining a better quantitative understanding of differences between neonatal and adult physiology.     

Persistent trefoil factor 1 expression imprinted on mouse vaginal epithelium by neonatal estrogenization

Exposure of female mice to estrogenic substances during the neonatal period induces developmental defects in the reproductive tract such as estrogen-independent persistent proliferation of the vaginal epithelium, which often leads to carcinogenesis in adulthood. In this study, several estrogen-regulated genes have been identified in the neonatal mouse vagina by DNA microarray hybridization analysis. Among the genes up-regulated in the developing vagina by a high dose of estrogen, trefoil factor 1 (TFF1), a mucin-associated gastrointestinal growth factor, showed a unique expression pattern in accordance with the irreversible changes induced by neonatal estrogenization in the vagina. Vaginal expression of TFF1 mRNA was markedly increased by estrogen in neonatal mice but not in adults, and pronouncedly intensified expression of the gastrointestinal gene was observed in the vagina of neonatally estrogenized mice even at adulthood. The specific localization of TFF1 protein in the epithelium of neonatally estrogenized vagina was confirmed by immunohistochemistry. Moreover, without any obvious alteration in the expression of gel-forming mucin genes, the lumen of the neonatally estrogenized vagina became filled with periodic-acid-Schiff-stained mucinous gel, which was possibly caused by the overexpression of TFF1. Thus, estrogen acts directly on the developing vagina in the permanent induction of TFF1 gene expression, and the gene induction does not appear to be related to hypermethylation of the cis-promoter of the TFF1 gene. TFF1 may be a useful marker for developmental estrogenization syndrome of the mouse vagina. This work was supported by a Grant-in-Aid for Encouragement of Young Scientists from the Ministry of Education, Science, Sports, and Culture, Japan, and grants from the University of Tsukuba to M. M.     

Interactions between Trichomonas vaginalis and vaginal flora in a mouse model.

To study the role of vaginal flora and pH in the pathogenesis of Trichomonas vaginalis, an intravaginal mouse model of infection was established. By employing this model, the vaginal flora and pH of mice could be monitored for changes caused by the parasite. As a baseline, the endemic vaginal flora of BALB/c mice was examined first and found to consist mainly of Staphylococcus aureus and Enterococcus species (32-76%). Lactobacilli and enteric bacilli were moderate (16-32%) in their frequency of isolation, and the prevalence of both anaerobic species and coagulase-negative staphylococci was low (4-16%). Vaginal pH was recorded at 6.5 +/- 0.3. Estrogenization, which was required for a sustained T. vaginalis infection, did not significantly alter vaginal flora; however, a slight rise in the number of bacterial species isolated per mouse and a drop in vaginal pH (6.2 +/- 0.5) were observed. Trichomonas vaginalis-infected mice did not appear to show significant changes in vaginal flora although vaginal pH was slightly increased. This mouse model could have applications in both immunologic and pathogenic studies of T. vaginalis and, with further modifications, aid in the study of protist-bacterial interactions.     

Visualization of vaginal flora in cervical smears using a modified microwave silver-staining method

The advantage of studying the vaginal flora to determine the bacteria and fungi present in cervical smears (as opposed to cultivation of these micro-organisms) is that the micro-organisms can be observed in their natural habitat. However, they are only faintly stained by the conventional Papanicolaou method. Accordingly, contrast is weak and visualization poor. For this reason, we developed a modified microwave silver-staining method that can be performed retrospectively on stained smears. Bacteria and fungi stain distinctly black and can be studied in greater detail, and their inter-relationship can be visualized. Haematoxylin or Eosin counterstain allows us to visualize vaginal inhabitants in relation to epithelial cells. In the series presented here, we show that a modified microwave silver-staining method is well suited to studying the ecology of micro-organisms in smears taken from women presenting to their doctor with clinical symptoms. Using this staining method, we have shown that lactobacilli overgrowth is associated with symptoms. © 1998 Chapman & Hall     

乳杆菌属细菌对宫颈上皮内瘤变患者阴道菌群的影响

目的 分析乳杆菌属细菌对宫颈上皮内瘤变(CIN)患者阴道菌群的影响,为该类患者的治疗提供参考.方法 选择2019年1月至2020年1月我院收治的CIN患者137例,根据组织学病理结果分为CIN Ⅰ组(84例)和CIN Ⅱ、Ⅲ组(53例).选择同期100例宫颈检查正常者为对照组.比较各组对象阴道菌群数量.根据患者阴道内乳...     

Role of IL-10 in a neonatal mouse listeriosis model.

This study was undertaken to test the hypothesis that altered IL-10 production plays a role in the increased susceptibility of neonates to listeriosis. Plasma IL-10 levels were measured in neonatal and adult mice at various times after infection with Listeria monocytogenes. Relative to adults, neonatal mice had markedly increased IL-10 levels early in the course of infection with Listeria using a 90% lethal dose. Higher neonatal IL-10 responses were also observed after injecting adults and pups with equal doses of killed organisms. Splenic macrophages from neonates produced higher IL-10 levels than those of adults after in vitro stimulation with killed bacteria, confirming in vivo observations. Moreover, IL-10 blockade had differential effects in neonates and adults infected with live Listeria. In adult mice, anti-IL-10 Abs decreased bacterial burden early in the course of infection, but were no longer effective at 6 days or later after challenge. In the pups, however, the same treatment had beneficial effects both early and late during infection and resulted in increased survival. Collectively, our data suggest that an overproduction of IL-10 by macrophages may at least partially explain the increased susceptibility of neonates to listeriosis, and provide further evidence that cytokine production is different in adults and neonates.     

A knock-out mouse model for methylmalonic aciduria resulting in neonatal lethality

Methylmalonic aciduria is a human autosomal recessive disorder of organic acid metabolism resulting from a functional defect in the activity of the enzyme methylmalonyl-CoA mutase. Based upon the homology of the human mutase locus with the mouse locus, we have chosen to disrupt the mouse mutase locus within the critical CoA binding domain using gene-targeting techniques to create a mouse model of methylmalonic aciduria. The phenotype of homozygous knock-out mice (mut-/-) is one of early neonatal lethality. Mice appear phenotypically normal at birth and are indistinguishable from littermates. By 15 h of age, they develop reduced movement and suckle less. This is followed by the development of abnormal breathing, and all of the mice with a null phenotype die by 24 h of age. Urinary levels of methylmalonic and methylcitric acids are grossly increased. Measurement of acylcarnitines in blood shows elevation of propionylcarnitine with no change in the levels of acetylcarnitine and free carnitine. Incorporation of [14C]propionate in primary fibroblast cultures from mut-/- mice is reduced to approximately 6% of normal level, whereas there is no detectable synthesis of mut mRNA in the liver. This is the first mouse model that recapitulates the key phenotypic features of mut0 methylmalonic aciduria.     

宫颈癌癌前病变及宫颈癌患者阴道微生态失调相关因素

目的 探讨宫颈癌癌前病变及宫颈癌患者阴道微生态失调相关因素。 方法 选择2016年7月至2018年12月我院收治的200例宫颈癌和癌前病变患者为研究对象,其中宫颈癌患者100例(宫颈癌组),癌前病变患者100例(癌前组),另选50例健康女性为对照组。观察各组对象阴道微生态指标(菌群密集度、菌群多样性、pH和H2O2)水平、HPV感染情况及乳杆菌分布情况。分析患者阴道微生态变化与HPV感染的关系。 结果 癌前组和宫颈癌组阴道菌群密集度Ⅰ-Ⅳ级的患者分别占33.00%和42.00%,显著高于对照组的10.00%(χ2=15.762 9,P=0.000 1);菌群多样性Ⅰ-Ⅳ级的患者分别占35.00%和41.00%,同样高于对照组的6.00%(χ2=19.783 1,P4.5的患者分别占53.00%和56.00%,高于对照组的12.00%(χ2=29.267 3,P2O2阴性患者占比分别为63.00%和70.00%,显著高于对照组的18.00%(χ2=39.343 7,P2=63.624 2,P2=8.742 1,P结论 宫颈癌前病变和宫颈癌的发生与患者阴道微生态失调、HPV感染、乳杆菌减少密切相关;同时癌前病变的发展与阴道微生态失调具有相关性。     

Inter-laboratory comparison of the CD-1 neonatal mouse logistic dose-response model for Cryptosporidium parvum oocysts

cryptosporidium parvum oocyst viability can be determined by vital dyes, in vitro excystation, and cell culture; however, neonatal mouse infectivity assays are the reference method. Unfortunately, there have been few efforts to standardize methods for infectivity assays thus casting a veil of uncertainty over the significance and comparability of results. In order to address this issue, two laboratories proficient in measuring oocyst infectivity conducted independent dose titration studies with neonatal CD-1 mice using standardized protocols and a well-characterized isolate of Cryptosporidium parvum. The resulting independent logistic dose-response models derived by regression analysis were compared with each other and with a published model. The comparisons showed these dose-response functions to be reproducible under standardized conditions. It is important to standardize mouse strain, age of mice at inoculation and necropsy, oocyst isolate, and age of oocysts. However, other factors, including methods used to detect infectivity and to count oocyst doses, appear less critical. Adopting a standardized assay for oocyst infectivity will provide both a basis for comparing data from various oocyst disinfection studies and a suitable platform for evaluating new or existing in vitro viability surrogates such as excystation, vital dyes or cell culture.     

Controlled vaginal delivery of antibodies in the mouse.

Controlled delivery of monoclonal antibodies to the mucus secretions of the vagina might provide women with passive immunoprotection against both sexually transmitted diseases and unwanted pregnancy. We have developed intravaginal devices composed of poly(ethylene-co-vinyl acetate) (EVAc) that continuously release IgG antibodies for over 30 days into buffered saline, and we have tested these devices in the vagina of mice. Polymeric devices containing either BSA (as a test reagent for proteins) or anti-hCG antibody, when inserted into the vaginas of mice, provided a continuous supply of either BSA or hCG-binding antibodies to the vaginal mucus for 30 days. Antibodies released by the devices achieved high concentration in the mucus within the lumen of the vagina, but did not significantly ascend into the uterine horns, as determined by epifluorescence microscopy of fluorescently labeled mouse IgG and by immunohistochemical localization of rabbit IgG. Our results suggest that long-term intravaginal delivery of functionally intact antibodies can be achieved with devices composed of EVAc.     

宫颈高危型HPV感染后的转归与阴道微生态的相关性分析

对宫颈高危型HPV HR-HPV感染后的转归进行阴道微生态及其危险因素分析, 以探讨宫颈高危型HPV感染后的转归与阴道微生态的相关性。

选取2019年7月至2020年9月在中山市博爱医院妇科门诊就诊的进行机会性宫颈癌筛查女性, 对其中符合研究纳入标准及排除标准的291例HR-HPV阳性者进行阴道微生态分析并随访, 在排除宫颈病变后, 6~12个月后再次进行HR-HPV检测, 若再次阳性者为持续感染组, 转阴性者为病毒清除组。分析2组的阴道微生态是否存在差异, 并进一步分析HR-HPV持续感染的危险因素。

符合研究纳入标准及排除标准的291例HR-HPV阳性者中持续感染组173例, 占59.45%, 平均年龄43.02岁±9.16岁(22~64岁); 病毒清除组118例, 占40.55%, 平均年龄40.26岁±9.00岁(24~66岁)。2组间年龄比较差异无统计学意义(F=0.041, P=0.839)。2组的阴道微生态分析表明, 病毒持续感染组的细菌性阴道病感染率、乳杆菌异常率、pH值异常率、白细胞酯酶阳性率分别为15.03%、41.62%、74.41%和22.54%, 这4项指标均大于病毒清除组, 2组间差异有统计学意义(χ²=7.088、14.369、9.585、4.487, P=0.008、＜0.001、0.002、0.034)。而2组在外阴阴道假丝酵母菌感染、滴虫性阴道炎、菌群密集度、多样性、过氧化氢酶、唾液酸苷酶、β-葡萄糖醛酸苷酶和乙酰氨基葡萄糖苷酶等指标方面的差异无统计学意义。Logistic回归分析显示乳杆菌异常、pH值异常是HR-HPV持续感染的危险因素, 其OR值分别为2.076(95%CI: 1.156~3.728)、2.121(95%CI: 1.218~3.688)(P＜0.05)。

宫颈HR-HPV感染后的转归与细菌性阴道病、白细胞酯酶阳性、乳杆菌异常及pH值异常存在一定的相关性, 尤其是乳杆菌异常、pH值异常是HR-HPV持续感染的危险因素, 需要重视。同时, 临床工作中可以考虑通过积极治疗下生殖道感染性疾病, 重建阴道微生态平衡, 以利于HR-HPV的清除, 降低宫颈病变的发生率。

     

阴道乳杆菌与宫颈癌相关性研究进展

人类阴道微生物群是由200多种细菌组成的复杂系统,其组成受到基因、民族背景、行为以及环境因素等的影响。其中乳杆菌是女性阴道内的优势菌,在健康女性的阴道内主要有L. iners、L. crispatus、L. gasseri和L. jensenii这4种乳杆菌。阴道内乳杆菌主要通过代谢阴道上皮细胞内的糖原产生乳酸,维持阴道正常的酸性环境。此外,阴道乳杆菌还可通过产生过氧化氢(H2O2)和细菌素等物质发挥抵御致病微生物入侵的作用。近年来的研究显示女性阴道HPV感染与阴道微生态失衡尤其是乳杆菌含量的减低有关。本文将围绕阴道内乳杆菌的生理作用及其与宫颈癌发病的相关性作一综述。