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2.  Progesterone Interactions with the Cervix: Translational Implications for Term and Preterm Birth 
The uterine cervix plays a vital role in maintaining pregnancy and an equally important role in allowing parturition to occur. Progesterone, either endogenously produced or supplied exogenously, supports the function of the cervix in sustaining intrauterine pregnancy, and the withdrawal of progesterone, either through natural processes or pharmacologic intervention, leads to delivery which underscores the importance of the progesterone's biological activities manifest in normal gestation and pregnancy that ends prematurely. Research crossing many scientific disciplines has demonstrated that progesterone is a pleotropic compound that affects the cervix through cytoplasmic and membrane receptors with profound effects on cellular and molecular functions that influence inflammatory cascades and extracellular matrix, both of which have consequences for parturition. Beyond the local cell and molecular biology of progesterone, it has systemic effects of relevance to pregnancy as well. This paper examines the biology of the cervix from its gross to cellular structure and biological activities of its cell and molecular processes that may be affected by progesterone. The implications of these processes for preterm birth are explored, and direction of current research is in relation to translational medicine implications for diagnostic, prognostic, and therapeutic approaches to threatened preterm birth.
PMCID: PMC3206389  PMID: 22114461
4.  Mycoplasma, Ureaplasma, and Adverse Pregnancy Outcomes: A Fresh Look 
Recent work on the Molicutes that associate with genital tract tissues focuses on four species that may be of interest in potential maternal, fetal, and neonatal infection and in contributing to adverse pregnancy outcomes. Mycoplasma hominis and Ureaplasma urealyticum have historically been the subject of attention, but Mycoplasma genitalis which causes male urethritis in addition to colonizing the female genital tract and the division of Ureaplasma into two species, urealyticum and parvum, has also added new taxonomic clarity. The role of these genital tract inhabitants in infection during pregnancy and their ability to invade and infect placental and fetal tissue is discussed. In particular, the role of some of these organisms in prematurity may be mechanistically related to their ability to induce inflammatory cytokines, thereby triggering pathways leading to preterm labor. A review of this intensifying exploration of the mycoplasmas in relation to pregnancy yields several questions which will be important to examine in future research.
PMCID: PMC2913664  PMID: 20706675
5.  Tetracycline Effects on Candida Albicans Virulence Factors 
Object. To determine if tetracycline, previously reported to increase the probability of developing symptomatic vaginal yeast infections, has a direct effect on Candida albicans growth or induction of virulent phenotypes. Method. In vitro, clinical isolates of yeast were cultivated with sublethal concentrations of tetracycline and yeast cell counts, hyphal formation, drug efflux pump activity, biofilm production, and hemolysin production were determined by previously reported methods. Results. Tetracycline concentrations above 150 μg/mL inhibited Candida albicans, but at submicrogram/mL, a modest growth increase during the early hours of the growth curve was observed. Tetracycline did not inhibit hyphal formation at sublethal concentrations. Hypha formation appeared augmented by exposure to tetracycline in the presence of chemically defined medium and especially in the presence of human serum. Efflux pump CDR1 was upregulated and a nonsignificant trend toward increased biofilm formation was noted. Conclusion. Tetracycline appears to have a small growth enhancing effect and may influence virulence through augmentation of hypha formation, and a modest effect on drug efflux and biofilm formation, although tetracycline did not affect hemolysin. It is not clear if the magnitude of the effect is sufficient to attribute vaginitis following tetracycline treatment to direct action of tetracycline on yeast.
PMCID: PMC2408679  PMID: 18528520
6.  Adherence and Blocking of Candida Albicans to Cultured Vaginal Epithelial Cells: Treatments to Decrease Adherence 
Background. Pathogenesis of mucosal microorganisms depends on adherence to the tissues they colonize and infect. For Candida albicans, cell surface hydrophobicity may play a significant role in tissue binding ability. Methods. A continuous cell line of vaginal epithelial cells (VEC) was grown in keratinocyte serum-free medium (KSFM) with supplements and harvested by trypsinization. VEC were combined with yeast cells to evaluate adherence and inhibition of adherence. In this experimental setup, yeast stained with fluorescein isothiocyanate were allowed to attach to VEC and the resulting fluorescent VEC were detected by flow cytometry. Results. VEC were cultured and examined daily after plating and showed morphology similar to basal epithelial cells. Culture media supplemented with estradiol showed increased VEC proliferation initially (first 24 h) but cell morphology was not altered. Fluorescinated Candida cells bound effectively to the cultured VEC. Using fresh cells exposed to various preparations of K-Y, we showed that all formulations of the product reduced Candida binding to VEC by 25% to 50%. While VEC were generally harvested for use in experiments when they were near confluent growth, we allowed some cultures to grow beyond that point and discovered that cells allowed to become overgrown or stressed appeared to bind yeast cells more effectively. Conclusion. Flow cytometry is a useful method for evaluating binding of stained yeast cells to cultured VEC and has demonstrated that commercially available products have the ability to interfere with the process of yeast adherence to epithelial cells.
PMCID: PMC1581476  PMID: 17485817
7.  Surface modifying substances that reduce apparent yeast cell hydrophobicity. 
OBJECTIVE: To determine whether several topical compounds and other chemical entities are able to diminish the surface hydrophobicity of yeast cells. METHOD: Hydrophobicity of yeast cells was determined by binding styrene microspheres to the surface of untreated yeast or yeast pre-incubated with various substances with potential for cell surface modification. The degree of microsphere adherence to yeast cells was measured by flow cytometry. RESULTS: A significant reduction in cell surface hydrophobicity was observed when yeast was incubated in protein-containing media. Other compounds that effectively reduced microsphere binding were various formulations of K-Y and heparin. Divalent cations (Ca+ + , Mg+ + , Zn+ + , Cu + + ) were also potent inhibitors of microsphere adherence. It was possible to remove substances contributing to microsphere binding by chemical extraction of the yeast. Yeast having reduced microsphere binding activity also showed diminished binding of concanavalin A. CONCLUSIONS: Several commercially available compounds were able to block binding of styrene microspheres to yeast. Some of the binding activity appeared to be attributable to mannose-containing surface components. These findings have implications for formulating therapeutic products that might block yeast binding to tissues.
PMCID: PMC1784570  PMID: 16126503
8.  Evaluation of relative yeast cell surface hydrophobicity measured by flow cytometry. 
OBJECTIVE: To develop an efficient method for evaluating cell surface hydrophobicity and to apply the method to demonstrate the effects of fungal growth conditions on cell surface properties. METHODS: Yeast isolates were suspended in phosphate-buffered saline and mixed with deep blue-dyed polystyrene microspheres. Flow cytometry was used to detect the degree of microsphere binding to yeast cells. Different strains of yeast were compared for intrinsic microsphere binding activity and changes in growth conditions were invoked to modify the relative surface hydrophobicity. RESULTS: Commercially available blue-dyed polystyrene microspheres showed strong fluorescence in the FL3 channel, whereas yeast cells did not show appreciable FL3 fluorescence. Microspheres and yeast were generally distinguishable on the basis of size revealed by forward light scatter. This method showed a wide variation in intrinsic cell surface hydrophobicity among Candida albicans strains. Likewise, variation in hydrophobicity of non-albicans yeast species was observed. Growth on solid media, incubation at 25 degrees C, or 250 mg/dl glucose concentration increased hydrophobicity compared with growth in liquid media, incubation at 37 degrees C, or 50 mg/dl glucose, respectively. Growth in 1 x 10(-9) M estradiol had no appreciable effect on hydrophobicity. CONCLUSIONS: Stained latex microspheres fluoresced in the FL3 channel of the flow cytometer and bound to yeast cells to an extent related to the surface hydrophobicity of the yeast. Binding detected by flow cytometry showed that clinical yeast isolates varied in intrinsic binding capacity and this binding ability was altered by different growth conditions. The implications for virulence regulation among yeast isolates are discussed.
PMCID: PMC1784552  PMID: 16040327
9.  Relationship of Fungal Vaginitis Therapy to Prior Antibiotic Exposure 
Objective: To address the putative association of antibiotic use and subsequent yeast vaginitis in a population of non-pregnant women.
Methods: Three hundred and sixteen women who received medical care in rural family medicine clinics enrolled in this study. Participants were pre-menopausal and non-pregnant and were followed until they used a course of antifungal therapy for vaginitis, became pregnant or moved from the catchment area. At entry subjects were free of vaginitis symptoms and had taken no antibiotics for 30 days. Patients were followed by repeated review of clinic records, hospital records and telephone or personal interviews. Data collection included documentation of episodes of antifungal treatment for vulvovaginal candidiasis and confirmed antibiotic treatment or credible history of antibiotic use prior to the use of antifungal therapy. Physician-reported uses of antibiotic and antifungal as well as patient-reported uses of these were recorded.
Results: There were four reported cases of antifungal therapy following within a month of antibiotic use, in contrast to 484 antibiotic uses not followed by antifungal use. If time of observation was extended to 6 months from antibiotic use, there were 13 uses of antifungal therapy after antibiotics and 475 uses of antibiotics not followed by antifungal therapy.
Conclusion: Our results cast doubt on the association of antibiotics as a putative cause of yeast vulvovaginitis.
PMCID: PMC1852283  PMID: 15022876
10.  Differences in Innate Immunologic Response to Group B Streptococcus Between Colonized and Noncolonized Women 
Objective: To evaluate the functional capacity of granulocytes and monocytes from pregnant and nonpregnant women in relation to group B streptococcus (GBS) colonization status.
Methods: Engulfment of fluorescent GBS by peripheral blood phagocytes from GBS-colonized and noncolonized women was measured by flow cytometry. Intracellular superoxiode generated in response to GBS challenge to monocytes and granulocytes enriched from peripheral blood of these women was also measured by flow cytometry, and extracellular superoxide was determined by colorimetric assay.
Results: Monocytes and granulocytes from pregnant, GBS-colonized women engulfed significantly greater numbers of GBS than phagocytes from pregnant, noncolonized women. No difference in intracellular superoxide production was detected between any of the groups of women; however, monocytes from pregnant, colonized women released significantly more superoxide into the extracellular milieu than did granulocytes from the same women. No differences in extracellular release of superoxide were observed among noncolonized women whether they were pregnant or not.
Conclusions: Monocytes from pregnant, colonized women engulf more GBS and release more of the superoxide into the extracellular environment, where it is unlikely to be an effective defense mechanism against intracellular bacteria. This suggests that components of the innate immune system that should serve in a protective role may function suboptimally, thereby contributing to the colonization process by GBS.
PMCID: PMC1784651  PMID: 11516060
11.  Candidiasis During Pregnancy May Result From Isogenic Commensal Strains 
Objective: Our laboratory previously demonstrated that asymptomatic vaginal colonization during pregnancy is a factor predisposing patients to subsequent symptomatic vulvovaginal candidiasis. It is unknown whether symptoms result from strain replacement or a change in host relationship to the original colonizing strain. This study was undertaken to determine whether Candida albicans isolates from asymptomatic women could be responsible for subsequent symptomatic vaginitis.
Methods: We retained isolates of C. albicans from women followed longitudinally through pregnancy, and identified six pairs of cultures from women who were colonized without symptoms and who later became symptomatic (average time 14 weeks). We used a random amplification of polymorphic DNA (RAPD) analysis to determine whether isolates from our study patients were genetically similar or dissimilar.
Results: Analysis of these pairs of yeast strains by RAPD revealed that five of the six women had symptoms apparently due to the same yeast strain that was found initially as a commensal strain. To increase the power of these observations, we also performed RAPD analysis on six randomly selected yeast strains from other women in this study who had not become symptomatic to determine whether any of these unrelated strains matched strains from those women who became symptomatic.
Conclusion: Symptomatic yeast vaginitis is usually due to strains of C. albicans already carried in the lower genital tract, underscoring the need to understand regulation of growth and virulence of the organism in vivo.
PMCID: PMC1784641  PMID: 11495556
12.  Antifungal Effect of Hydrogen Peroxide on Catalase-Producing Strains of Candida spp. 
Objective: Clinical isolates of Candida were tested for the presence of catalase and susceptibility to hydrogen peroxide.
Methods: MIC was tested by broth dilution technique and catalase was determined by a spectrophotometric procedure.
Results: All 38 strains tested were inhibited by hydrogen peroxide in concentrations ranging from 4.4 to 88 mM/l, with non-albicans isolates generally requiring higher concentrations of hydrogen peroxide for inhibition. Growth media consisting of glucose and protein diminished the antifungal effectiveness of hydrogen peroxide, as did the presence of hemoglobin, in incubation mixtures. However, hydrogen peroxide exerted greater inhibition at pH 4 than at pH 7. Although all Candida isolates tested possessed catalase, there was no apparent correlation between the catalase activity of individual isolates and the minimal antifungal concentration of hydrogen peroxide.
Conclusions: This study suggested that, despite the production of catalase by vaginal microorganisms, hydrogen peroxide may exert a regulating influence which may be further modified by the proteins found in the vaginal milieu.
PMCID: PMC2364421  PMID: 18476024
13.  Virulence Attributes of Low-Virulence Organisms 
The vast majority of infections involving female pelvic structures arise from organisms that are members of the normal flora. In addition, exogenous organisms that invade through the lower genital tract must interact with organisms that are part of the host's flora. In contrast to the concept that the normal flora is entirely innocuous, recent research has begun to identify what appear to be virulence attributes among these ordinarily low-virulence organisms. Most of our understanding of virulence has been derived from highly virulent organisms, of which Neisseria gonorrhoeae provides an example of relevance to the female genital tract. A review of the virulence factors of the gonococcus is presented to serve as an example of the variety of virulence properties associated with pathogenic bacteria. Molecular biology has begun to clarify one of the important paradigms of pathogenic bacteriology—that bacteria change their expression of virulence properties in response to their location within a host or to the stage of infection. Thus, infection involves not only the possession of virulence factors, but also the carefully controlled use of those factors. Virulence is often controlled by the coordinate expression of many virulence-associated genes in response to one environmental signal. With regard to low- virulence organisms present in the female lower genital tract, we are beginning to identify some of their virulence attributes. Examples from the work of our laboratory include the hemolysin of Gardnerella vaginalis and an immunosuppressive mycotoxin produced by Candida albicans. Demonstrating the coordinate expression (or other control mechanisms) of virulence factors in these sometimes innocuous and sometimes inimical organisms represents the next frontier in the study of normal vaginal microbiology.
PMCID: PMC2364362  PMID: 18475373
14.  Development of a High Performance Liquid Chromatographic Assay Measuring Mezlocillin in Serum or Tissue 
Objective: This study evaluated the blood and uterine tissue concentration of mezlocillin, a broadspectrum penicillin.
Methods: We adapted a liquid chromatographic method to measure mezlocillin in serum and tissue. Mezlocillin reference standard was diluted in water, chromatographed on a reversed phase C18 column eluted at 1.5 ml/min with acetonitrile and phosphate buffer (1:3 v:v), and detected spectrophotometrically at 210 nm. Mezlocillin was administered to 14 premenopausal women scheduled to undergo vaginal hysterectomy. Each patient received a 4 g IV infusion of the drug 30 to 60 min prior to surgery. During surgery, tissue was removed from the uterine cervix and blood was obtained for assay of mezlocillin content.
Results: Chromatography of the mezlocillin standard furnished a discrete peak with a retention time of 2.4 min. The sensitivity of the assay was 0.1 µg/ml with a linear response up to 100 µg/ml. The correlation coefficient for the standard curve was 0.9997. When reference standard was diluted in pooled human serum, the assay was complicated by interfering compounds. These were removed by ether extraction. The sensitivity of the assay performed in serum was 3 µg/ml. Serum samples contained from 81.2 to 358 µg of mezlocillin/ml with an average serum concentration of 207.5 µg/ml. When serum containing a known amount of mezlocillin was homogenized for a period of time similar to that required to homogenize tissue samples, a deteatable loss of drug was observed and was applied as a correction factor to the measured tisulevels. After correction, the average tissue level was 117.2 µg/ml and ranged from 27% to 98% of the serum levels.
Conclusions: The serum concentration of mezlocillin after IV infusion of 4 g was greater than that required to inhibit the majority of the most significant organisms responsible for post-hysterectomy sepsis. Although tissue levels appeared to be consistently lower than serum levels, they could be expected to provide an inhibitory effect against many of the bacterial strains that contaminate the surgical site.
PMCID: PMC2364671  PMID: 18476212

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