Resumo: Analisar o efeito do raloxifeno sobre o epitélio vaginal de mulheres pós-menopausa. Métodos: Estudaram-se prospectivamente entre novembro de 2004 a fevereiro de 2006, 80 mulheres na pós-menopausa. Quarenta pacientes receberam 6Omg/dia de raloxifeno (GR) e 40 mulheres compuseram o grupo não tratado (grupo controle, GC), pareado por idade e tempo de menopausa. O grupo tratado foi composto por pacientes com osteoporose de coluna lombar e/ou colo do fêmur. Foram excluídos aquelas com sinais e/ou sintomas de infecção do trato genital inferior e usuárias de terapia hormonal (TH) até seis meses prévios ao estudo. Os esfregaços vaginais foram coletados em dois momentos: inicial (MO) e após seis meses de seguimento (Ml). Para avaliação do epitélio vaginal foi utilizado o valor de maturação, com a contagem de células superficias, intermediárias e parabasais. Os esfregaços foram analisados por único citopatologista, sem conhecimento dos dados das pacientes. Para análise estatística empregou- se o teste t de Student, teste Wilcoxon Mann-Witney e o teste Qui-Quadrado. Resultados: Na comparação estatística inicial os grupos foram homogêneos. Comparando os momentos inicial e final, não foram observadas diferenças estatisticamente sígnífícativas nos valores medianos de maturação do epitélio vaginal e na porcentagem de células superficiais, intermediárias e parabasais entre os grupos. Não foi constatada correlação linear significativa entre o valor de maturação e a idade, o tempo de menopausa, o uso ou não de TH prévia, tabagismo e o índice de massa corpórea, em ambos os grupos. Conclusão: O tratamento com raloxifeno por seis meses não alterou o valor de maturação do epitélio vaginal em mulheres na pós-menopausa. / Abstract: To analyze the effect of raloxifene on the vaginal epithelium of postmenopausal women. Methods: Eighty postmenopausal women were studied prospectively between November of 2004 and February of 2006. Forty patients received 6omglday of raloxifene (GR), and 40 women comprised the non-treated group (control group, CG), paired by age and time of menopause. The treated group was composed of patients with osteoporosis of the lumbar column and / or femur. Those with signs and / or symptoms of infection of the inferior genital tract and users of hormonal therapies (HT) up to six months prior to the study were excluded. Vaginal smears were collected at two moments: initial (MO) and after six months of follow-up (Ml). To evaluate the vaginal epithelium, the maturation value was determined, along with counts of superficial, intermediate and parabasal cells. Smears were analyzed by only one cytopathologist, without knowledge of patient data. For statistical analysis Student's t test, Wilcoxon Mann Witney test and Chi-Squared test were employed. Results: In the initial statistical comparison the groups were homogeneous. Comparing the initial and final moments, no statistically significant differences were observed in median values of vaginal epithelial maturation or in percentage of superficial, intermediate and parabasal cells between the groups. There was no significant linear correlation between value of vaginal epithelial maturation and age, time of menopause, use or not of previous HT, smoking or body mass index, in both groups. Conclusion: Treatment with raloxifene for six months did not alter the maturation value of vaginal epithelium in postmenopausal women. / Orientador: Jorge Nahás Neto / Coorientador: Eliana Aguiar Petri Nahás / Banca: Paulo Traiman / Banca: Lúcia Simões Costa-Paiva / Mestre

The development of antibody-based drugs is continuing to expand at a rapid pace, especially for use at mucosal surfaces to prevent or treat infectious diseases and other conditions. A better understanding of how the Fc region of antibodies interacts with Fc-binding proteins at mucosal sites can inform an optimal design for antibody-based drugs. The Human Contraception Antibody (HCA) is a human IgG1 monoclonal antibody currently under development as a topical vaginal contraceptive. HCA binds to sperm via its Fab domains and causes rapid agglutination with other sperm in close proximity resulting in near complete immobilization of sperm over a wide area. In order to determine whether HCA participates in Fc-mediated functions in the female reproductive tract (FRT), we assessed the activity of HCA and engineered variants in three assays of Fc-mediated functions: complement-dependent cytotoxicity (CDC), antibody-dependent cellular phagocytosis (ADCP), and mucus trapping. The physiological relevance of CDC was confirmed by characterizing complement levels and activity in cervical mucus. Finally, we described the activity of a novel Fc receptor expressed by vaginal epithelium. With complement, HCA significantly reduced sperm motility and increased the number of lysed sperm via CDC. Additionally, human cervical mucus was found to have sufficient levels of complement to induce the classical complement cascade. HCA-opsonized sperm associated with macrophages and were phagocytosed via ADCP. HCA also trapped sperm in ovulatory human cervical mucus, significantly reducing their progression. Variants of HCA with mutated or obstructed Fc domains had decreased abilities to perform these Fc functions, while multivalent IgM-like and IgA variants of HCA were very effective in both sperm agglutination and Fc assays. We also investigated the novel expression of Fc alpha RI (CD89) by human vaginal epithelium and provide evidence that this Fc receptor may transport IgA through the mucosa. Basal application of IgA resulted in IgA in apical supernatants which was significantly reduced following treatment with a CD89 blocker. In summary, these studies provide an improved understanding of the possible Fc functions of HCA and other antibodies in the human FRT, including interactions with complement, cervical mucus, and Fc receptors. Determining which interactions can occur in vivo and which are desired for a specific indication can inform the design of mucosally applied antibody-based drugs like HCA, a much-needed novel contraceptive antibody.

The vagina is one organ of the body which has not been studied exhaustively. Moreover, most of the studies found in the contemporary literature have been performed on women affected by a variety of genital diseases. In the present study the vaginal epithelium was examined with a histological method, morphometry, whereby cyclical changes related to hormonal variation during the menstrual cycle were demonstrated. Determination of the quantity of estrogen receptors in the vaginal epithelium on two occasions during the menstrual cycle revealed a significantly greater number in the follicular than in the luteal phase. The results of these studies indicate the presence of a menstrual variation in the vaginal epithelium comparable to that in the endometrium. Phenoxymethylpenicillin (pcV) was used as a marker substance to study the dynamics of the transport mechanisms into the vagina. PcV was found to accumulate in the vaginal fluid and high concentrations persisted for a long period of time. In hysterectomized women, the appearance of pcV in the vaginal fluid followed the same pattern. Consequently, the substance is transported through the vaginal wall and need not enter with the secretions from the internal genitalia. The greatest concentration of pcV was in the distal portion of the vagina, possibly due to the specific internal circulation of fluid within the vagina. Bacterial vaginosis as an example of an ‘ecological disease’ has been studied with regard to the formation of endotoxin, a constituent of the cell wall of Gram- negative bacteria. Large amounts of endotoxin were found and the clinical implication of this finding has been pointed out. Furthermore, the influence of pcV on the vaginal microbial flora of healthy women has been investigated. A change from a situation with predominance of lactobacilli to the appearance of Gram-negative rods was observed. In one of the women the lactobacilli disappeared completely and were replaced by E. coliand high levels of endotoxin in the vaginal fluid were found. This study demonstrates the complexity of the ecological balance of the vaginal microbial flora and illustrates the difficulty of defining a ‘normal’ vaginal condition. Is there any unquestionable state of ‘normality’ even in a healthy woman free from symptoms of genital disease? / <p>S. 1-22: sammanfattning, s. 25-64: 6 uppsatser</p> / digitalisering@umu

Made available in DSpace on 2014-06-11T19:26:18Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-12-07Bitstream added on 2014-06-13T20:54:35Z : No. of bitstreams: 1 delmanto_a_me_botfm_prot.pdf: 1594635 bytes, checksum: 59b8850497a3a71e0f5ae2ec36c4ccb0 (MD5) / Fundação para o Desenvolvimento Médico e Hospitalar (Famesp) / Analisar o efeito do raloxifeno sobre o epitélio vaginal de mulheres pós-menopausa. Métodos: Estudaram-se prospectivamente entre novembro de 2004 a fevereiro de 2006, 80 mulheres na pós-menopausa. Quarenta pacientes receberam 6Omg/dia de raloxifeno (GR) e 40 mulheres compuseram o grupo não tratado (grupo controle, GC), pareado por idade e tempo de menopausa. O grupo tratado foi composto por pacientes com osteoporose de coluna lombar e/ou colo do fêmur. Foram excluídos aquelas com sinais e/ou sintomas de infecção do trato genital inferior e usuárias de terapia hormonal (TH) até seis meses prévios ao estudo. Os esfregaços vaginais foram coletados em dois momentos: inicial (MO) e após seis meses de seguimento (Ml). Para avaliação do epitélio vaginal foi utilizado o valor de maturação, com a contagem de células superficias, intermediárias e parabasais. Os esfregaços foram analisados por único citopatologista, sem conhecimento dos dados das pacientes. Para análise estatística empregou- se o teste t de Student, teste Wilcoxon Mann-Witney e o teste Qui-Quadrado. Resultados: Na comparação estatística inicial os grupos foram homogêneos. Comparando os momentos inicial e final, não foram observadas diferenças estatisticamente sígnífícativas nos valores medianos de maturação do epitélio vaginal e na porcentagem de células superficiais, intermediárias e parabasais entre os grupos. Não foi constatada correlação linear significativa entre o valor de maturação e a idade, o tempo de menopausa, o uso ou não de TH prévia, tabagismo e o índice de massa corpórea, em ambos os grupos. Conclusão: O tratamento com raloxifeno por seis meses não alterou o valor de maturação do epitélio vaginal em mulheres na pós-menopausa. / To analyze the effect of raloxifene on the vaginal epithelium of postmenopausal women. Methods: Eighty postmenopausal women were studied prospectively between November of 2004 and February of 2006. Forty patients received 6omglday of raloxifene (GR), and 40 women comprised the non-treated group (control group, CG), paired by age and time of menopause. The treated group was composed of patients with osteoporosis of the lumbar column and / or femur. Those with signs and / or symptoms of infection of the inferior genital tract and users of hormonal therapies (HT) up to six months prior to the study were excluded. Vaginal smears were collected at two moments: initial (MO) and after six months of follow-up (Ml). To evaluate the vaginal epithelium, the maturation value was determined, along with counts of superficial, intermediate and parabasal cells. Smears were analyzed by only one cytopathologist, without knowledge of patient data. For statistical analysis Student's t test, Wilcoxon Mann Witney test and Chi-Squared test were employed. Results: In the initial statistical comparison the groups were homogeneous. Comparing the initial and final moments, no statistically significant differences were observed in median values of vaginal epithelial maturation or in percentage of superficial, intermediate and parabasal cells between the groups. There was no significant linear correlation between value of vaginal epithelial maturation and age, time of menopause, use or not of previous HT, smoking or body mass index, in both groups. Conclusion: Treatment with raloxifene for six months did not alter the maturation value of vaginal epithelium in postmenopausal women.

The vagina is the port of entry for sexually transmitted diseases in women. Its epithelium constitutes the luminal border, thus comprising an important defence barrier. The objective of this work was to investigate the mechanisms of importance in the immune defence of the vaginal epithelium of healthy, fertile women, and possible menstrual cycle changes. Effects of hormonal contraceptive usage on oestrogen receptor (ER) and progesterone receptor (PR) expression were studied. The contribution of epithelial cell to the immune defence was estimated by assaying their expression of antimicrobial defensins and the epithelial thickness. Vaginal biopsies and serum samples were collected during the follicular and luteal phases in regularly menstruating women (controls) and in users of combined oral contraceptives (COCs), levonorgestrel implants (LNGs), or depot-medroxyprogesterone acetate injections (DMPAs). Fifteen healthy women (aged 20–34 years) were enrolled in each group. Morphometry was performed on vaginal tissue stained with haematoxylin/eosin and by immunohistochemistry using monoclonal antibodies against immune cell markers, PR, and ER. Expression of mRNA for human α-defensins HD-5 and HD-6, and human β-defensins (HBD) 1 to 4 were determined by real-time qRT-PCR and in situ hybridization. In controls, the epithelium was 261 ± 16 μm thick and harboured 241 ± 35 leukocytes (CD45+) per mm2. T lymphocytes (CD3+) dominated. Both αβ T cells and γδ T cells were present with an approximate 4-fold dominance of αβ T cells. Cytotoxic T cells (CD8+) were more frequent than T helper cells (CD4:CD8 ratio: 0.7 ± 0.1). Macrophages (CD68+) constituted the second-largest population, followed by Langerhans cells (CD1a+). B cells, natural killer cells, monocytes and granulocytes were generally absent. No differences were found between the follicular and luteal phase. All four β-defensins analysed for were detected in vaginal epithelium and most samples expressed at least two. HBD-2 and HBD-3 were most frequent. HBD-3 and HBD-4 expressing cells were localized in the parabasal and intermediate cell layers. α-defensins were not detected. The epithelium was significantly thicker (333 ± 9 μm) in COC, LNG, and DMPA users than in controls, and commonly showed hyperplasia. In DMPA and LNG users the frequency of intraepithelial leukocytes (CD45+) was increased, explained by increased frequencies of both αβ and γδ T cells. In DMPA users there was also a selective increase in CD8+ T cells. PR expression was significantly reduced in DMPA users compared with controls, COC and LNG users. COC and particularly DMPA users often had undetectable levels of serum E2. In conclusion, both adaptive immunity, i.e. intraepithelial T cells, and innate defence mechanisms, i.e. intraepithelial macrophages and β-defensins, are believed to contribute to the immune defence in the human female lower genital tract. These parameters did not change during the menstrual cycle but hormonal contraceptive usage, especially DMPA, affected the quality of the epithelium. The use of DMPA and LNG was correlated with the accumulation of T cells within the epithelium. The effects of these changes on the risk of contracting infections are yet to be determined.

Antimicrobial peptides (AMPs) are small proteins produced by epithelial surfaces, which have broad-spectrum antimicrobial and immunomodulatory activities. In the lung, skin and alimentary tract AMPs are known to be important in infectious and inflammatory conditions. Far less is known regarding the role of AMPs within the female reproductive tract, but as infection and inflammation are causes of preterm labour, AMPs may have a key function in maintain and protecting pregnancy. The major groups of human AMPs include the human beta defensins (HBDs), two antileukoproteinases (secretory leukocyte protease inhibitor (SLPI) and Trappin-2/Elafin), and the human cathelicidin hCAP18/LL-37, with several studies identifying their presence at sites throughout the reproductive tract. The cervix in pregnancy is positioned between the upper genital tract containing the developing fetus and the lower tract where infections usually arise. I hypothesise that AMPs are fundamental to mucosal immune defence of the cervix in pregnancy, preventing ascending infection and excessive inflammation that can cause preterm labour. This thesis focused on the human cathelicidin hCAP18/LL-37 and its role within the cervix and vagina. The aims of this thesis were to; investigate the inflammatory effects of LL-37 from cervical and vaginal derived epithelial cells and determine the pathways and receptors in which LL-37 may elicit its effects and how production may be regulated; investigate the role of CRAMP in a mouse model of preterm birth; and determine the production of AMPs by the pregnant cervix whilst investigating the relationship between AMP concentrations in cervicovaginal secretions and preterm labour. The inflammatory effect of LL-37 was investigated using cell lines derived from endocervical, ectocervical and vaginal epithelium. The study of these cell lines suggests divergent responses of cervical and vaginal epithelial cells. LL-37 mediated induction of IL-8 and IL-6 production from endocervical epithelial cells was observed in a dose-dependent and time-dependent manner, whilst ectocervical and vaginal cells also respond to treatment with LL-37 through IL-8 and IL-6 production. To determine a possible mechanism of action of LL-37 on IL-8 and IL-6 in the three cell lines, inhibitors against MAPK cascades, ERK, p38 MAPK and JNK, and known LL-37 receptors were investigated. In endocervical cells LL-37 mediated IL-8 occurs via activation of unidentified GPCRs, whilst in ectocervical cells this effect on IL‐8 and IL-6 is via the activation of ERK and p38 MAPK cascades. The mechanism by which LL-37 induces IL-8 secretion in vaginal epithelial cells remains unknown. Expression of LL-37 was shown to be mediated by vitamin D3 in vitro in cervical and vaginal epithelial cells. However when this relationship was investigated in vivo, using matched serum and cervicovaginal secretions from woman at early pregnancy, no correlation was observed between circulating vitamin D and cervicovaginal or circulating hCAP18/LL-37. However, the majority of women in this study reported with insufficient levels of vitamin D, which may effect the relationship observed with hCAP18/LL-37. Using a mouse model of LPS-induced preterm labour, to mimic the presence of intrauterine infection bacterial infection, I aimed to characterise the role of CRAMP, the mouse orthologue of hCAP18/LL-37, in the lower inflammatory and immune response that results in preterm labour. Wild type C57Bl/6J mice receiving an intrauterine injection of LPS deliver prematurely, within 24 hours of injection. However mice deficient in CRAMP (Camp -/-) receiving an intrauterine injection of LPS deliver significantly later and have a non-significant increase in pup survival compared to wild type C57Bl/6J mice. Cervical tissue collected post partum showed no difference in inflammatory markers between wild type C57Bl/6J and Camp -/- mice, however there was increased expression of the neutrophil chemoattractant marker, Cxcl5, and the neutrophil marker, Ngp in Camp -/- mice. In the lower genital tract, levels of antimicrobial peptides were determined in samples of cervicovaginal secretions collected from pregnant women. AMPs, hCAP18/LL-37, HBD-2 and SLPI were found in cervicovaginal secretions, and levels of hCAP18/LL-37 were increased in women with the common vaginal infection bacterial vaginosis. However no relationship was identified between the concentration of AMPs and preterm birth in this study. This work has shown that the lower genital tract, where infections that are associated with preterm labour originate, expresses the human cathelicidin hCAP18/LL-37. It may play an important role in modulating the immune response to invading infection associated with preterm labour. Further investigation of these responses may increase understanding of the physiology and pathophysiology of labour, and lead to strategies for the prevention of premature delivery.