Ultrastructural study of the neovagina following the utilization of human amniotic membrane for treatment of congenital absence of the vagina

We present an ultrastructural study of the utilization of human amniotic membrane in the treatment of congenital absence of the vagina in 10 patients. All patients were surgically treated with application of an amniotic membrane graft using the modified McIndoe and Bannister technique. Sixty days after surgery, samples of the vaginal neo-epithelium were collected for transmission electron microscopy analysis. The ultrastructural findings consisted of a lining of mature squamous epithelium indicating the occurrence of metaplasia of the amniotic epithelium into the vaginal epithelium. The cells were arranged in layers as in the normal vaginal epithelium, i.e., superficial, intermediate and deep layers. There were desmosomes and cytoplasmic intermediate cytokeratin filaments, as well as some remnant features of the previous amniotic epithelium. These findings suggest that human amniotic membrane is able to complete metaplasia into squamous cells but the mechanism of this cellular transformation is unknown

Developmental and behavioral effects of postnatal amitraz exposure in rats

The effects of postnatal amitraz exposure on physical and behavioral parameters were studied in Wistar rats, whose lactating dams received the pesticide (10 mg/kg) orally on days 1, 4, 7, 10, 13, 16 and 19 of lactation; control dams received distilled water (1 ml/kg) on the same days. A total of 18 different litters (9 of them control and 9 experimental) born after a 21-day gestation were used. The results showed that the median effective time (ET50) for fur development, eye opening, testis descent and onset of the startle response were increased in rats postnatally exposed to amitraz (2.7, 15.1, 21.6 and 15.3 days, respectively) compared to those of the control pups (1.8, 14.0, 19.9 and 12.9 days, respectively). The ages of incisor eruption, total unfolding of the external ears, vaginal and ear opening and the time taken to perform the grasping hindlimb reflex were not affected by amitraz exposure. Pups from dams treated with amitraz during lactation took more time (in seconds) to perform the surface righting reflex on postnatal days (PND) 3 (25.0 ± 2.0), 4 (12.3 ± 1.2) and 5 (8.7 ± 0.9) in relation to controls (10.6 ± 1.2; 4.5 ± 0.6 and 3.4 ± 0.4, respectively); the climbing response was not changed by amitraz. Postnatal amitraz exposure increased spontaneous motor activity of male and female pups in the open-field on PND 16 (140 ± 11) and 17 (124 ± 12), and 16 (104 ± 9), 17 (137 ± 9) and 18 (106 ± 8), respectively. Data on spontaneous motor activity of the control male and female pups were 59 ± 11 and 69 ± 10 for days 16 and 17 and 49 ± 9, 48 ± 7 and 56 ± 7 for days 16, 17 and 18, respectively. Some qualitative differences were also observed in spontaneous motor behavior; thus, raising the head, shoulder and pelvis matured one or two days later in the amitraz-treated offspring. Postnatal amitraz exposure did not change locomotion and rearing frequencies or immobility time in the open-field on PND 30, 60 and 90. The present findings indicate that postnatal exposure to amitraz caused transient developmental and behavioral changes in the exposed offspring and suggest that further investigation of the potential health risk of amitraz exposure to developing human and animal offsprings may be warranted.

Neonatal handling induces anovulatory estrous cycles in rats

Since previous work has shown that stimulation early in life decreases sexual receptiveness as measured by the female lordosis quotient, we suggested that neonatal handling could affect the function of the hypothalamus-pituitary-gonadal axis. The effects of neonatal handling on the estrous cycle and ovulation were analyzed in adult rats. Two groups of animals were studied: intact (no manipulation, N = 10) and handled (N = 11). Pups were either handled daily for 1 min during the first 10 days of life or left undisturbed. At the age of 90 days, a vaginal smear was collected daily at 9:00 a.m. and analyzed for 29 days; at 9:00 a.m. on the day of estrus, animals were anesthetized with thiopental (40 mg/kg, ip), the ovaries were removed and the oviduct was dissected and squashed between 2 glass slides. The number of oocytes of both oviductal ampullae was counted under the microscope. The average numbers for each phase of the cycle (diestrus I, diestrus II, proestrus and estrus) during the period analyzed were compared between the two groups. There were no significant differences between intact and handled females during any of the phases. However, the number of handled females that showed anovulatory cycles (8 out of 11) was significantly higher than in the intact group (none out of 10). Neonatal stimulation may affect not only the hypothalamus-pituitary-adrenal axis, as previously demonstrated, but also the hypothalamus-pituitary-gonadal axis in female rats.

Preeclampsia: from epidemiological observations to molecular mechanisms

Preeclampsia is the main cause of maternal mortality and is associated with a five-fold increase in perinatal mortality in developing countries. In spite of this, the etiology of preeclampsia is unknown. The present article analyzes the contradictory results of the use of calcium supplementation in the prevention of preeclampsia, and tries to give an explanation of these results. The proposal of an integrative model to explain the clinical manifestations of preeclampsia is discussed. In this proposal we suggest that preeclampsia is caused by nutritional, environmental and genetic factors that lead to the creation of an imbalance between the free radicals nitric oxide, superoxide and peroxynitrate in the vascular endothelium. The adequate interpretation of this model would allow us to understand that the best way of preventing preeclampsia is the establishment of an adequate prenatal control system involving adequate antioxidant vitamin and mineral supplementation, adequate diagnosis and early treatment of asymptomatic urinary and vaginal infections. The role of infection in the genesis of preeclampsia needs to be studied in depth because it may involve a fundamental change in the prevention and treatment of preeclampsia.

Effects of melatonin on the ovarian response to pinealectomy or continuous light in female rats: similarity with polycystic ovary syndrome

The current study was conducted to investigate the relationship between melatonin and chronic anovulation. Adult (3-4 months old) female Wistar rats were submitted to pinealectomy: group I: pinealectomized ovariectomized melatonin-treated (N = 10); group II: pinealectomized ovariectomized placebo-treated (N = 12); group III: pinealectomized light-treated placebo-treated(N = 10) or maintained under continuous light; group IV: maintained under continuous light, ovariectomized melatonin-treated (N = 22); group V: maintained under continuous light, ovariectomized placebo-treated (N = 10); group VI: maintained under continuous light placebo-treated (N = 10). In order to assess ovarian modifications, unilateral ovariectomy was performed during the fourth month in groups I, II, IV, V and the other ovary was removed after 8 months. Ovariectomy was performed in groups III and VI only after eight months. Melatonin (200 µg/100 g body weight) dissolved in 0.02 ml absolute ethanol was injected intramuscularly daily during the last 4 months into groups I and IV. The other groups were treated with placebo (NaCl). The ovarian cysts were analyzed and their area, perimeter and maximum diameter, as well as the thickness of the ovarian capsule were measured. Daily colpocytological smears were performed throughout the study. Persistent estrous condition and ovarian cysts were observed in all groups. In pinealectomized rats the ovarian and vaginal alterations disappeared at the end of the study and in rats maintained under continuous light the vaginal and ovarian polycystic aspect was reversed only in those treated with melatonin. We conclude that melatonin may act on the ovarian response reverting chronic anovulation induced by pinealectomy or continuous light.

Quantification of fecal estradiol and progesterone metabolites in Syrian hamsters (Mesocricetus auratus)

Alternative methods to the utilization of laboratory animal blood and its by-products are particularly attractive, especially regarding hamsters due to their small size and difficulties in obtaining serial blood samples. Steroid hormone metabolite quantification in feces, widely used in studies of free-ranging or intractable animals, is a non-invasive, non-stressor, economical, and animal saving technique which allows longitudinal studies by permitting frequent sampling of the same individual. The present study was undertaken to determine the suitability of this method for laboratory animals. Estradiol and progesterone metabolites were quantified by radioimmunoassay in feces of intact, sexually mature female Syrian hamsters during the estrous cycle (control) and in feces of superovulated females. Metabolites were extracted by fecal dilution in ethanol and quantified by solid phase radioimmunoassay. Median estrogen and progesterone concentrations were 9.703 and 180.74 ng/g feces in the control group, respectively. Peaks of estrogen (22.44 ± 4.54 ng/g feces) and progesterone (655.95 ± 129.93 ng/g feces) mean fecal concentrations respectively occurred 12 h before and immediately after ovulation, which is easily detected in this species by observation of a characteristic vaginal postovulatory discharge. Median estrogen and progesterone concentrations (28.159 and 586.57 ng/g feces, respectively) were significantly higher in superovulated animal feces (P < 0.0001). The present study demonstrated that it is possible to monitor ovarian activity in Syrian hamsters non-invasively by measuring fecal estradiol and progesterone metabolites. This technique appears to be a quite encouraging method for the development of new endocrinologic studies on laboratory animals.

Human papillomavirus infection and its association with cervical dysplasia in Ecuadorian women attending a private cancer screening clinic

Women living in Latin American countries bear a disproportionate burden of cervical cancer, a condition caused by infection with the human papillomavirus (HPV). We performed a study in Santa Elena, Guayas (currently Santa Elena Province), Ecuador, to determine how often HPV could be detected in women attending a private cancer screening clinic. Participants underwent a Pap test, and vaginal and cervical swabs were performed for HPV testing by the polymerase chain reaction (PCR). Each participant completed a verbally administered survey. The mean age of 302 participants was 37.7 years (range 18 to 78 years). The majority of cervical and vaginal specimens contained sufficient DNA to perform PCR. Overall, 24.2% of the participants had either a cervical or vaginal swab that tested positive for HPV. In general, there was a good correlation between the HPV types detected in the cervical and vaginal swabs from the participants, but vaginal swabs were more likely to contain HPV DNA than were cervical swabs. The high-risk HPV types 16, 52, 58, and 59 and the low-risk HPV types 62, 71, 72, and 83 were the most frequently detected HPV types. The number of lifetime sexual partners was positively associated with detection of any HPV type, detection of oncogenic HPV, and abnormal Pap smears. Further studies are needed to determine if these results are representative of all Ecuadorian women and to determine if cervical cancers in Ecuadorian women are caused by the same HPV types found in the swab specimens obtained in this study.

Identification and in vitro production of Lactobacillus antagonists from women with or without bacterial vaginosis

Lactobacilli isolated from the vaginal tract of women with and without bacterial vaginosis (BV) were identified and characterized for the production of antagonists. Bacterial samples were isolated from healthy women (N = 16), from patients with clinical complaints but without BV (N = 30), and from patients with BV (N = 32). Identification was performed using amplified ribosomal DNA restriction analysis. Production of antagonistic compounds was evaluated by the double-layer diffusion technique using Gram-positive (N = 9) and Gram-negative bacteria (N = 6) as well as yeast (N = 5) as indicator strains. Of a total of 147 isolates, 133 were identified as pertaining to the genus Lactobacillus. Lactobacillus crispatus was the species most frequently recovered, followed by L. johnsonii and L. jensenii. Statistical analysis showed that L. crispatus was more frequent in individuals without BV (P < 0.05). A higher production of antagonists was noted in L. crispatus isolates from healthy women (P < 0.05). More acidic local pH and higher H2O2 production by isolated lactobacilli from healthy women suggest these mechanisms as the possible cause of this antagonism. In conclusion, a significant correlation was detected between the presence and antagonistic properties of certain species of Lactobacillus and the clinical status of the patients.

Maternal serum progesterone, estradiol and estriol levels in successful dinoprostone-induced labor

Hormone-mediated quiescence involves the maintenance of a decreased inflammatory responsiveness. However, no study has investigated whether labor induction with prostanoids is associated with changes in the levels of maternal serum hormones. The objective of this study was to determine whether labor induction with dinoprostone is associated with changes in maternal serum progesterone, estradiol, and estriol levels. Blood samples were obtained from 81 pregnant women at term. Sixteen patients had vaginal birth after spontaneous labor, 12 required cesarean section after spontaneous labor and 16 underwent elective cesarean. Thirty-seven patients had labor induction with dinoprostone. Eligible patients received a vaginal insert of dinoprostone (10 mg) and were followed until delivery. Serum progesterone (P4), estradiol (E2) and estriol (E3) levels and changes in P4/E2, P4/E3 and E3/E2 ratios were monitored from admission to immediately before birth, and the association of these measures with the resulting clinical classification outcome (route of delivery and induction responsiveness) was assessed. Progesterone levels decreased from admission to birth in patients who underwent successful labor induction with dinoprostone [vaginal and cesarean birth after induced labor: 23% (P < 0.001) and 18% (P < 0.025) decrease, respectively], but not in those whose induction failed (6.4% decrease, P > 0.05). Estriol and estradiol levels, P4/E2, P4/E3 and E3/E2 ratios did not differ between groups. Successful dinoprostone-induced labor was associated with reduced maternal progesterone levels from induction to birth. While a causal relationship between progesterone decrease and effective dinoprostone-induced labor cannot be established, it is tempting to propose that dinoprostone may contribute to progesterone withdrawal and favor labor induction in humans.

Molecular adjuvant interleukin-33 enhances the antifertility effect of Lagurus lagurus zona pellucida 3 DNA vaccine administered by the mucosal route

It has been shown that cytokines can act as molecular adjuvant to enhance the immune response induced by DNA vaccines, but it is unknown whether interleukin 33 (IL-33) can enhance the immunocontraceptive effect induced by DNA vaccines. In the present study, we explored the effects of murine IL-33 on infertility induced by Lagurus lagurus zona pellucida 3 (Lzp3) contraceptive DNA vaccine administered by the mucosal route. Plasmid pcD-Lzp3 and plasmid pcD-mIL-33 were encapsulated with chitosan to generate the nanoparticle chi-(pcD-Lzp3+pcD-mIL-33) as the DNA vaccine. Sixty female ICR mice, divided into 5 groups (n=12/group), were intranasally immunized on days 0, 14, 28, and 42. After intranasal immunization, the anti-LZP3-specific IgG in serum and IgA in vaginal secretions and feces were determined by ELISA. The results showed that chi-(pcD-Lzp3+pcD-mIL-33) co-immunization induced the highest levels of serum IgG, secreted mucosal IgA, and T cell proliferation. Importantly, mice co-immunized with chi-(pcD-Lzp3+pcD-mIL-33) had the lowest birth rate and mean litter size, which correlated with high levels of antibodies. Ovaries from infertile female mice co-immunized with chi-(pcD-Lzp3+pcD-mIL-33) showed abnormal development of ovarian follicles, indicated by atretic follicles and loss of oocytes. Our results demonstrated that intranasal delivery of the molecular adjuvant mIL-33 with chi-pcD-Lzp3significantly increased infertility by enhancing both systemic and mucosal immune responses. Therefore, chi-(pcD-Lzp3+pcD-mIL-33) co-immunization could be a strategy for controlling the population of wild animal pests.

EXTRAÇÃO DOS ALCALÓIDES: CAFEÍNA E TRIGONELINA DOS GRÃOS DE CAFÉ COM C SUPERCRÍTICO

A descafeinação do café canephora é vantajosa tanto para a obtenção da cafeína quanto para valorização desta espécie. A cafeína, subproduto da descafeinação, atua como estimulante e diurético no organismo e é usado pelas indústrias de bebidas de refrigerante e farmacêutica. Existem patentes industriais aplicando CO2 supercrítico na extração da cafeína do café. Porém pouco é divulgado sobre a extração da trigonelina (alcalóide importante na formação da vitamina‘ niacina’), que atua no sistema nervoso central, na secreção da bile e no intestino. O objetivo deste trabalho foi levantar subsídios experimentais e teóricos para a extração de alcalóides dos grãos de café canephora, variedade robusta, usando CO2 supercrítico. Os dados foram obtidos num aparelho de extração a altas pressões onde as variáveis termodinâmicas são independentemente controladas. Os alcalóides foram analisados por Cromatografia Liquida de Alta Eficiência (CLAE). As solubilidades da cafeína pura e a obtida dos grãos de café no CO2 supercrítico foram determinadas a 313, 323 e 343 K, de 9,5 a 23,5 MPa. Os resultados revelam a existência de um comportamento retrógrado para a solubilidade da cafeína pura e a obtida dos grãos de café; também mostram a seletividade do CO2 supercrítico pela cafeína quando comparada a trigonelina. A modelagem termodinâmica do equilíbrio sólido-fluido usando uma equação de estado de quarta ordem correlacionou satisfatoriamente as solubilidades da cafeína pura no solvente supercrítico.

EXTRAÇÃO DE OLEORESINA DE CÚRCUMA (Curcuma longa L) COM CO2 SUPERCRÍTICO

Dentre as fontes de corantes naturais mais utilizadas na indústria de alimentos, encontra-se a cúrcuma (Curcuma longa L), um rizoma do qual podem ser obtidas substâncias como a curcumina, demetoxicurcumina e bis-demetoxicurcumina. Estes pigmentos possuem coloração amarela e capacidade de substituir corantes artificiais. Com a finalidade de verificar a influência do pré-tratamento de secagem na extração, foram realizados experimentos de extração de oleoresina de cúrcuma com CO2 supercrítico, na unidade de extração do Laboratório de Engenharia Química da Universidade Federal do Pará, submetendo-se a matéria-prima a uma secagem nas temperaturas de 70 e 105oC. As extrações foram feitas a pressões de 200, 250 e 300 bar, e na temperatura de 45oC. Os resultados estão apresentados em tabelas e gráficos, em termos de rendimentos totais e teor de curcumina presente na oleoresina. A secagem a 70oC favoreceu a extração de oleoresina em termos de tempo de extração, e contribuiu para a manutenção de curcumina na matéria-prima.

EXTRAÇÃO DE LIPÍDEOS DA AMÊNDOA DE CASTANHA DE CAJU COM CO2 SUPERCRÍTICO

Nos últimos anos, vem crescendo o interesse em alimentos com baixo índice de calorias. Nos métodos tradicionais de extração de lipídeos com solventes orgânicos, devido as condições de extração, a integridade dos compostos extraídos e da matriz pode ser afetada pela decomposição térmica ou por contaminação pelo solvente. A extração com fluido supercrítico (EFSC) pode proporcionar um método alternativo para remover lipídeos, sem que haja redução significativa nas propriedades organolépticas do produto. O objetivo deste trabalho é extrair parcialmente o óleo contido na amêndoa da castanha de caju (Anacardium occidentale) com dióxido de carbono (CO2) supercrítico em condições de pressão de 100-170 bar e temperatura de 40-80 (C, para obter um produto de valor calórico reduzido que poderá ser usado como substituto para o amendoim e outras amêndoas na indústria de confeitos. Os experimentos foram realizados em um extrator de 300 ml, dispondo de sistemas de agitação magnética e variação de temperatura. Os resultados experimentais mostraram que o aumento da pressão acarretou um aumento na eficiência de extração. O efeito da temperatura foi o mesmo porém menos significativo.

NOVAS APLICAÇÕES DE SISTEMAS SFE "HOME MADE".: I. PLANTAS MEDICINAIS BRASILEIRAS

Neste trabalho, apresenta-se uma visão geral da evolução do uso da SFE (extração com fluido supercrítico) na análise de plantas medicinais brasileiras dentro das pesquisas desenvolvidas pelo Laboratório de Cromatografia do IQSC-USP. Utilizou-se como fluido extrator o CO2, puro ou modificado com solventes de polaridade baixa a alta (pentano, solventes halogenados, álcoois, água), para a extração seletiva de princípios ativos de Maytenus aquifolium ("espinheira santa") e Mikania glomerata ("guaco"), respectivamente contendo triterpenos e cumarina. Os extratos obtidos por SFE foram analisados por CGAR (cromatografia gasosa de alta resolução), CLAE-DAD (cromatografia líquida de alta eficiência com detector" photodiodearray") ou CCD (cromatografia de camada delgada). A SFE forneceu resultados similares ou melhores do que os obtidos com métodos convencionais de extração (Soxhlet, maceração, etc), indicando o potencial uso da SFE para análise e/ou produção de preparações contendo estas plantas medicinais.

NOVAS APLICAÇÕES DE SISTEMAS SFE" HOME MADE".: II. PLANTAS DA AMÉRICA DO SUL

O Laboratório de Cromatografia do IQSC-USP tem desenvolvido pesquisas em conjunto com outros grupos de pesquisa do Brasil e da América Latina, visando a futura implantação da SFE (extração com fluido supercrítico) nestes laboratórios. No presente trabalho, apresenta-se alguns dos resultados obtidos na cooperação com pesquisadores do Chile e da Venezuela, direcionados para a SFE de substâncias de potencial interesse econômico, princípios ativos ou substâncias tóxicas: triglicerídeos, ácidos graxos, flavonóides, alcalóides, etc. Foram estudadas plantas nativas do Chile e da Venezuela, algumas delas nunca estudadas por métodos fitoquímicos convencionais e pertencentes a várias famílias vegetais: Leguminosae, Chrysobalanaceae, Annonnaceae, etc. O uso da SFE em conjunto com CGAR-EM (cromatografia gasosa de alta resolução - espectrometria de massas) ou CLAE-DAD (cromatografia líquida de alta eficiência com detector" photodiodearray") permitiu a identificação de algumas substâncias presentes em baixa concentração, a partir de pequenas quantidades de material vegetal; estes dados são de grande importância para a caracterização química das espécies estudadas.