Proliferation of Pasteurella pneumotropica at oestrus in the vagina of rats

Using a colony of Wistar-Imamichi rats contaminated with P. pneumotropica, the vaginal microflora was qualitatively and quantitatively investigated by swabbing. P. pneumotropica was the most dominant organism in the majority of rats examined. The population of P. pneumotropica and indigenous bacteria increased significantly higher at oestrus than in other oestrous stages. By the vaginal flushing technique changes in the population of P. pneumotropica and total bacteria, and changes in vaginal cell type and bacterial counts adhering to vaginal epithelial cells were consecutively investigated. The populations of P. pneumotropica and total bacteria were maximal at oestrus. The increase was correlated with an increase in cornified non-nucleated cells, with large numbers of adherent Gram-negative coccobacilli. The findings indicate that the vagina is a suitable site for colonization by P. pneumotropica in adult female rats, and that proliferation of P. pneumotropica may be due to increased affinity of the organism for cornified non-nucleated cells.     

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。     

Relationship of Vaginal Cytology to Alterations of the Vaginal Microflora of Rats During the Estrous Cycle

The influence of cyclic changes occurring in the vaginal tract during the estrous cycle upon the indigenous microflora of the vagina has been investigated by semiquantitative techniques in virgin female rats. By plate counts performed on material lavaged daily from the vaginal tract of several rats, it is apparent that bacterial counts are elevated in the proestrus and estrus phases of the cycle to values several orders of magnitude greater than those observed during metestrus or diestrus phases. Increases in vaginal bacterial counts were associated with the presence of cornified epithelial cells in the vagina; these cells were predominantly nonviable. Decreases in the vaginal bacterial content were related to the influx of leukocytes into the vagina after estrus. When leukocytes were present in the vaginal tract, they were 90 to 100% viable. From these observations it has been concluded that the female vaginal tract and the bacteria which colonize it represent a dynamic ecosystem which is responsive to cyclic events occurring in the estrous cycle. The changing cellular content of the vaginal tract may have relevance to the observed cyclic changes in the bacterial content of the vagina.     

Spatial relationship of the vaginal microflora to the vaginal epithelium of female rats: scanning electron microscopy.

The vaginal mucosa of ovariectomized female rats has been examined by scanning electron microscopy before and after estrogen [1,3,5(10)-estratriene-3,17beta-diol] treatment. Without estrogen stimulation vaginal colonization is minimal and the epithelium is characterized by a layer of epithelial cells covered with small microvillous-like projections. Progressive changes that were consistent with estrogenic cytoproliferative effects were seen after estrogen treatment. By post-treatment day 3 bacterial colonization was maximal and the epithelium was comprised of flat squamous cells that tended to become detached from the underlying tissue layers. Bacteria were seen in association with the intercellular borders of this tissue and occurred singly or as microcolonies. No distinct physical attachment structures were identified, although an amorphous extracellular material that may serve to attach the bacteria to the squamous epithelial cells was frequently seen.     

Evidence for Gardnerella vaginalis uptake and internalization by squamous vaginal epithelial cells: implications for the pathogenesis of bacterial vaginosis

Bacterial vaginosis (BV), a common condition seen in premenopausal women, is associated with preterm labor, pelvic inflammatory disease, and delivery of low birth weight infants. Gardnerella vaginalis is the predominant bacterial species associated with BV, although its exact role in the pathology of BV is unknown. Using immunofluorescence, confocal and transmission electron microscopy, we found that VK2 vaginal epithelial cells take up G. vaginalis after exposure to the bacteria. Confocal microscopy also indicated the presence of internalized G. vaginalis within vaginal epithelial cells obtained from a subject with BV. Using VK2 cells and (35)S labeled bacteria in an invasion assay, we found that a 1 h uptake of G. vaginalis was 21.8-fold higher than heat-killed G. vaginalis, 84-fold compared to Lactobacillus acidophilus and 6.6-fold compared to Lactobacillus crispatus. Internalization was inhibited by pre-exposure of cells to cytochalasin-D. In addition, the cytoskeletal protein vimentin was upregulated in VK2 cells exposed to G. vaginalis, but there was no change in actin cytoskeletal polymerization/rearrangements or vimentin subcellular relocalization post exposure. Cytoskeletal protein modifications could represent a potential mechanism for G. vaginalis mediated internalization by vaginal epithelial cells. Finally, understanding vaginal bacteria/host interactions will allow us to better understand the underlying mechanisms of BV pathogenesis.     

Effect of oestrogen on Pasteurella pneumotropica in rat vagina

The effects of ovarian hormones on the vaginal population of Pasteurella pneumotropica in rats were investigated. Specified-pathogen-free adult female Wistar-Imamichi rats with a 4 day oestrous cycle were inoculated with P. pneumotropica in the vagina. Cyclic changes in the vaginal population of P. pneumotropica were not observed in ovariectomized rats and the bacterial population was at a similar level to that at normal dioestrus. Administration of oestrogen to ovariectomized rats caused an increase in the numbers of P. pneumotropica and total bacteria in the vagina nearly equal to that at oestrus in intact rats. The numbers of these organisms in the vagina of ovariectomized rats treated with progesterone did not change and were similar to those of control ovariectomized rats treated with sesame oil. Vaginal smears of ovariectomized rats treated with oestrogen were characterized by abundant cornified non-nucleated epithelial cells with many adherent Gram-negative coccobacilli and were similar to smears from intact rats at oestrus. These findings suggest that the proliferation of P. pneumotropica at oestrus in rat vagina may be primarily due to the environment provided by the degeneration of vaginal epithelial cells promoted by oestrogen secretion from the ovaries.     

Differential expression of p63 isoforms in female reproductive organs

p63 is the identity switch for uterine/vaginal epithelial cell fate, and disruption of p63 expression by diethylstilbestrol (DES) induces cervical/vaginal adenosis in mice. In this article, we report the expression patterns of p63 isoforms (TA, DeltaN, alpha, beta and gamma) in mice, focusing on the reproductive tract. We also present the reproductive tract phenotype of female p63-/- mice. Finally, to better evaluate the potential role of p63 in human development of DES-induced cervical/vaginal adenosis, we describe the ontogeny of p63 in human female fetuses. In adult mice, the DeltaN isoforms of p63 were expressed only in squamous/basal/myoepithelial cells of epithelial tissues, while TA isoforms of p63 were highly expressed in germ cells of the ovary and testis. In fetal mice, the DeltaN and alpha forms of p63 were expressed in the cloacal and urogenital sinus epithelia. In the female p63-/- mice, the sinus vagina developed, but p63-/- sinus vaginal epithelium failed to undergo squamous differentiation confirming an essential role of p63 in squamous epithelial differentiation. Although TAp63 was highly expressed in developing primordial germ cells/oocytes, p63-/- ovaries and oocytes developed normally. The ontogeny of p63 in female reproductive organs was essentially identical in mouse and human. In the human fetus at the susceptible stage for DES-induced cervical/vaginal adenosis, most cervical/vaginal epithelial cells were columnar and negative for p63. Therefore, inhibition of p63 expression by DES should change the cell fate of human Müllerian duct epithelial cells and cause cervical/vaginal adenosis as previously demonstrated in mouse.     

Ultrastructure of the in situ adherence of Mobiluncus to vaginal epithelial cells

From patients with bacterial vaginosis motile, anaerobic, comma-shaped bacteria can be isolated, which have recently been placed into the new genus Mobiluncus. In this study, electron microscopy was used to examine the in situ adherence of these motile curved rods to detached epithelial cells (comma cells) in vaginal fluid from two patients with bacterial vaginosis. Thin sections showed that the curved rods attached both directly to the epithelial cell surface and at various distances from it. It is concluded that after initial attachment these motile bacteria can grow at the epithelial cell surface in sessile microcolonies. Ruthenium red staining demonstrated a coating of precipitated glycocalyx material both on the surface of the curved rods and on their flagella. This may indicate that in situ the adherent curved rods were enclosed in a very hydrated matrix of exopolysaccharides. Conspicuous was the ability of the curved rods to attach to the epithelial cell surface via their cell tips. However, in situ no specialized bacteria cell surface structures were seen that might explain this polar attachment. Electron microscopy of pure cultures demonstrated that both Mobiluncus curtisii subsp. curtisii and Mobiluncus mulieris can produce a glycocalyx in vitro.     

Structure and function of intercellular junctions in human cervical and vaginal mucosal epithelia

The mucosal epithelium is a major portal for microbial invasion. Mucosal barrier integrity is maintained by the physical interactions of intercellular junctional molecules on opposing epithelial cells. The epithelial mucosa in the female reproductive tract provides the first line of defense against sexually transmitted pathogenic bacteria and viruses, but little is known concerning the structure and molecular composition of epithelial junctions at this site. In the present study, the distribution of tight, adherens, and desmosomal junctions were imaged in the human endocervix (columnar epithelium) and ectocervix (stratified squamous epithelium) by electron microscopy, and permeability was assessed by tracking the penetration of fluorescent immunoglobulin G (IgG). To further define the molecular structure of the intercellular junctions, select junctional molecules were localized in the endocervical, ectocervical, and vaginal epithelium by fluorescent immunohistology. The columnar epithelial cells of the endocervix were joined by tight junctions that excluded apically applied fluorescent IgG. In contrast, the most apical layers of the ectocervical stratified squamous epithelium did not contain classical cell-cell adhesions and were permeable to IgG. The suprabasal and basal epithelial layers in ectocervical and vaginal tissue contained the most robust adhesions; molecules characteristic of exclusionary junctions were detected three to four cellular layers below the luminal surface and extended to the basement membrane. These data indicate that the uppermost epithelial layers of the ectocervix and vagina constitute a unique microenvironment; their lack of tight junctions and permeability to large-molecular-weight immunological mediators suggest that this region is an important battlefront in host defense against microbial pathogens.     

Serine-rich repeat proteins and pili promote Streptococcus agalactiae colonization of the vaginal tract

Streptococcus agalactiae (group B streptococcus [GBS]) is a Gram-positive bacterium found in the female rectovaginal tract and is capable of producing severe disease in susceptible hosts, including newborns and pregnant women. The vaginal tract is considered a major reservoir for GBS, and maternal vaginal colonization poses a significant risk to the newborn; however, little is known about the specific bacterial factors that promote GBS colonization and persistence in the female reproductive tract. We have developed in vitro models of GBS interaction with the human female cervicovaginal tract using human vaginal and cervical epithelial cell lines. Analysis of isogenic mutant GBS strains deficient in cell surface organelles such as pili and serine-rich repeat (Srr) proteins shows that these factors contribute to host cell attachment. As Srr proteins are heavily glycosylated, we confirmed that carbohydrate moieties contribute to the effective interaction of Srr-1 with vaginal epithelial cells. Antibody inhibition assays identified keratin 4 as a possible host receptor for Srr-1. Our findings were further substantiated in an in vivo mouse model of GBS vaginal colonization, where mice inoculated with an Srr-1-deficient mutant exhibited decreased GBS vaginal persistence compared to those inoculated with the wild-type (WT) parental strain. Furthermore, competition experiments in mice showed that WT GBS exhibited a significant survival advantage over the ΔpilA or Δsrr-1 mutant in the vaginal tract. Our results suggest that these GBS surface proteins contribute to vaginal colonization and may offer new insights into the mechanisms of vaginal niche establishment.     

Effect of estradiol on the membrane fluidity of the rat vaginal epithelial cells

Changes in the cell surface of vaginal epithelial cells were studied by scanning microscopy and fluorescence spectroscopy. Microvilli which are prominent features of the vaginal epithelial cells in proestrus and diestrus are replaced by sheet-like structures in the estrus phase. Surface morphology of vaginal epithelial cells of estradiol primed rat resembles the vaginal cells from estrus phase rats whereas vaginal cells from control rats resembles the diestrus phase. Measurement of the fluidity of the membranes indicated that the vaginal epithelial cell membrane of estrus rats is more fluid compared to proestrus and diestrus. Similarly, estradiol primed immature rat vaginal epithelial cell membrane was observed to be more fluid than the corresponding control.     

食蟹猴阴道涂片的动态观察

目的　为食蟹猴实施人工授精和适时配种提供一种有效的测定排卵期的方法。方法　选择 4～ 6岁性成熟的雌性食蟹猴 30只 ,对其阴道涂片进行动态观察。结果与结论　食蟹猴阴道涂片中主要有角化上皮细胞、中层基底细胞、白细胞和细胞碎片等。分析结果说明 ,白细胞数各时期差异均具显著性 ;角化上皮细胞在月经前期和月经期没有差异 ,但其他各期差异具显著性 ;月经期和月经后期中层基底细胞数目变化差异不显著 ,但其他各期差异具显著性 ;月经前期和月经期的细胞碎片数目相似 ,但其他各期差异具显著性。各时期细胞成份变化规律是 ,角化上皮细胞从月经期到排卵期呈逐渐增加至最高 ,然后逐渐减少 ;而白细胞、中层基底细胞和细胞碎片则恰好相反。     

Commensal bacteria modulate innate immune responses of vaginal epithelial cell multilayer cultures

The human vaginal microbiome plays a critical but poorly defined role in reproductive health. Vaginal microbiome alterations are associated with increased susceptibility to sexually-transmitted infections (STI) possibly due to related changes in innate defense responses from epithelial cells. Study of the impact of commensal bacteria on the vaginal mucosal surface has been hindered by current vaginal epithelial cell (VEC) culture systems that lack an appropriate interface between the apical surface of stratified squamous epithelium and the air-filled vaginal lumen. Therefore we developed a reproducible multilayer VEC culture system with an apical (luminal) air-interface that supported colonization with selected commensal bacteria. Multilayer VEC developed tight-junctions and other hallmarks of the vaginal mucosa including predictable proinflammatory cytokine secretion following TLR stimulation. Colonization of multilayers by common vaginal commensals including Lactobacillus crispatus, L. jensenii, and L. rhamnosus led to intimate associations with the VEC exclusively on the apical surface. Vaginal commensals did not trigger cytokine secretion but Staphylococcus epidermidis, a skin commensal, was inflammatory. Lactobacilli reduced cytokine secretion in an isolate-specific fashion following TLR stimulation. This tempering of inflammation offers a potential explanation for increased susceptibility to STI in the absence of common commensals and has implications for testing of potential STI preventatives.     

Ultrastructure of the nonhuman primate vaginal mucosa: epithelial changes during the menstrual cycle and pregnancy

Ultrastructural changes in the vaginal epithelium of the rhesus monkey during the menstrual cycle and pregnancy were studied by scanning and transmission electron microscopy. During the menstrual cycle, the epithelium was keratinized but varied in thickness. Cells of the basal and parabasal layers were polyhedral in shape but as they differentiated they accumulated glycogen and filaments. Cells in the intermediate layers had keratohyaline and membrane-coating granules. Cells in the superficial layers had a thickened cell envelope, abundant keratin filaments, electron-dense intercellular material, and focal tight junctions. The epithelial surface had numerous microridges and numerous adherent bacteria; bacteria were rare on desquamating cells. The epithelium remained keratinized for about the first month of gestation, then underwent "mucification." The cells contained abundant granules and Golgi apparatus. Concomitant with this transformation, bacteria were no longer adherent to the epithelial surface and the surface cells had microvilli instead of microridges. The epithelial changes during pregnancy were roughly associated with the changing pattern of steroid hormone secretion during gestation.     

Vaginal lactic acid bacteria in the mare: evaluation of the probiotic potential of native Lactobacillus spp. and Enterococcus spp. strains

Lactic acid bacteria (LAB) are important members of the human vaginal microbiota and their presence is considered beneficial. However, little is known about native vaginal bacteria in other animal species such as the horse. The aim of this work was to quantify the vaginal lactic acid bacteria and lactobacilli of mares and to establish if selected equine vaginal lactic acid bacteria, particularly Lactobacillus and Enterococcus spp. strains, could exhibit potential as probiotics. The vaginal lactic acid bacteria and lactobacilli of 26 mares were quantified by plate counts. Five strains (three Lactobacillus spp. and two Enterococcus spp.) were characterised and adhesion to vaginal epithelial cells, antimicrobial activity and ability to form biofilms were evaluated. Lactic acid bacteria were recovered from the 26 samples and lactobacilli counts were detected in 18 out of 26 mares (69%). Probiotic properties tested in this study varied among the isolates and showed promising features for their use as equine probiotics.     

Lactobacillus acidophilus contributes to a healthy environment for vaginal epithelial cells

Lactobacillus species in the female genital tract are thought to act as a barrier to infection. Several studies have demonstrated that lactobacilli can adhere to vaginal epithelial cells. However, little is known about how the adherence of lactobacilli to vaginal epithelial cells affects the acidity, cell viability, or proliferation of the lactobacilli themselves or those of vaginal epithelial cells. Lactobacillus acidophilus was co-cultured with immortalized human vaginal epithelial cells (MS74 cell line), and the growth of L. acidophilus and the acidity of the culture medium were measured. MS74 cell density and viability were also assessed by counting cell numbers and observing the cell attachment state. L. acidophilus showed exponential growth for the first 6 hr until 9 hr, and the pH was maintained close to 4.0-5.0 at 24 hr after culture, consistent with previous studies. The growth curve of L. acidophilus or the pH values were relatively unaffected by co-culture with MS74 cells, confirming that L. acidophilus maintains a low pH in the presence of MS74 cells. This co-culture model could therefore potentially be used to mimic vaginal conditions for future in vitro studies. On the other hand, MS74 cells co-cultured with L. acidophilus more firmly attached to the culture plate, and a higher number of cells were present compared to cells cultured in the absence of L. acidophilus. These results indicate that L. acidophilus increases MS74 cell proliferation and viability, suggesting that lactobacilli may contribute to the healthy environment for vaginal epithelial cells.     

Effect of Two Probiotic Strains of Lactobacillus on In Vitro Adherence of Listeria monocytogenes,Streptococcus agalactiae,and Staphylococcus aureus to Vaginal Epithelial Cells

The lactobacilli probiotics maintain a normal vaginal biota and prevent disease recurrence. This microorganisms form a pellicle on the vaginal epithelium that acts as a biologic barrier against colonization by pathogenic bacteria. In this paper were realized assays of exclusion, competition, and displacement. For these test, vaginal epithelial cells, two strains of lactobacilli and pathogenic bacteria (Staphylococcus aureus, Streptococcus agalactiae and Listeria monocytogenes) were used. The lactobacilli strains showed a great capacity of adherence, with a mean of 83.5 ± 26.67 Lactobacillus fermentum cells and 56.2 ± 20.87 Lactobacillus rhamnosus cells per vaginal epithelial cells. L. fermentum and L. rhamnosus were able to reduce the adherence of S. aureus, S. agalactiae and L. monocytogenes in a significant level in this assay (P < 0.01). The lactobacilli used in this study protect the vaginal epithelium through a series of barriers and interference mechanisms. The aim of present study was to assess the ability of vaginal Lactobacillus strains, selected for their probiotic properties, to block the adherence of pathogenic microorganisms in vitro by displacement, competition, and exclusion mechanisms.     

Complete genome sequence of Lactococcus lactis subsp. lactis CV56, a probiotic strain isolated from the vaginas of healthy women

Lactic acid bacteria that exist in the urinogenital system play an important role in maintaining the health of the host. Here, we report the finished and annotated genome of a Lactococcus strain that was isolated from the vaginas of healthy women and shows probiotic properties, including nisin A production and adhesion to vaginal epithelial cells.