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In vitro study on chorioamniotic membranes infection with bacterial species isolated from the lower genital tract and modulation of the innate immune response in the maternal-fetal interface

Grant number: 12/17234-1
Support Opportunities:Regular Research Grants
Duration: January 01, 2013 - December 31, 2015
Field of knowledge:Health Sciences - Medicine - Pathological Anatomy and Clinical Pathology
Principal Investigator:Márcia Guimarães da Silva
Grantee:Márcia Guimarães da Silva
Host Institution: Faculdade de Medicina (FMB). Universidade Estadual Paulista (UNESP). Campus de Botucatu. Botucatu , SP, Brazil
Associated researchers:Vera Lúcia Mores Rall


Preterm labor (PTL) is an important obstetric complication with multifactor etiology. Infection from the amniotic cavity, generally from the lower genital tract, is an important factor associated with its physiopathology as it early activates the inflammatory cascade in the maternal-fetal interface. Chorioamniotic membranes are mechanical barriers against the ascent of microorganisms, and they play a fundamental role in the immune system. Studies on the modulation of the innate immune response in such gestational tissues have shown important relevance to the understanding of the mechanisms involved in triggering preterm labor and birth. Objective: To determine the relations between microbiological status and innate immune response modulation in the maternal-fetal interface in vitro models of chorioamniotic membrane infection with bacterial species isolated in the lower genital tract of women with PTL. Material and Methods: This is a prospective study. Its first phase will be the isolation of bacterial species from the vaginal microbiota of 40 women with PTL in differents gestational age, including 20 pregnant women with PTL before 34 weeks and 20 after 34 weeks. Samples from the vaginal content will be separately collected on 3 sterile swabs at the first moment of the obstetric examination. The two of them will be placed in Amies transport medium for later seeding in order to isolate aerobic and anaerobic bacteria by using specific culture media and appropriate atmospheric conditions. The other swab will be used for quantitative determination of bacteria in the vaginal content by total DNA investigation and amplification of specific gene sequences of the most frequent bacteria through the real-time PCR technique by using specific primers for each species. For quantitative determination, the standard curve of each evaluated bacterial species will be performed by using positive controls formed by standard strains of determinate concentrations. The adherence and invasion capacity of the most frequent bacterial species in chorioamniotic membranes will be evaluated by appropriate in vitro assays. The isolated microorganisms will be stored at -80°C and used to stimulate the chorioamniotic membranes in the in vitro assays. For this phase of the study, biopsies of 6mm of chorioamniotic membranes from normal pregnant women submitted to an elective term caesarean section will be placed on sterile culture wells under appropriate temperature and atmospheric conditions. The heat inactivated bacterial suspensions from bacteria isolated in the initial phase of the study as well as those with greater adherence and invasion capacity will be added to the chorioamniotic membrane cultures in different concentrations, according to those found in the vaginal content of pregnant women with PTL. The supernatants resulting from the cultures will be evaluated for the detection of protein expression of Interleukin (IL) -1b, IL-6, IL-8, Tumor Necrosis Factor (TNF-a), Prostaglandin E2, ² defensins and Pentraxin 3 by the ELISA technique. Explants samples from the culture will be submitted to total RNA extraction and later gene evaluation of interest mediators by real time PCR. Other samples of the chorioamniotic membranes will be submitted to protein expression and evaluated as to the presence of Toll like receptor-2 and Toll like receptor-4, by using the Western Blot technique. Statistical analysis will be performed according to the presuppositions determined by the results, and the level of significance adopted will be of 5%. (AU)

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Scientific publications
(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)
NODA-NICOLAU, NATHALIA MAYUMI; POLETTINI, JOSSIMARA; DA SILVA, MARCIA GUIMARAES; PELTIER, MORGAN R.; MENON, RAMKUMAR. Polybacterial stimulation suggests discrete IL-6/IL-6R signaling in human fetal membranes: Potential implications on IL-6 bioactivity. JOURNAL OF REPRODUCTIVE IMMUNOLOGY, v. 126, p. 60-68, . (12/17234-1)
MARTIN, LAURA FERNANDES; MOCO, NATALIA PREARO; DE LIMA, MOISES DIOGO; POLETTINI, JOSSIMARA; MIOT, HELIO AMANTE; CORREA, CAMILA RENATA; MENON, RAMKUMAR; DA SILVA, MARCIA GUIMARAES. Histologic chorioamnionitis does not modulate the oxidative stress and antioxidant status in pregnancies complicated by spontaneous preterm delivery. BMC PREGNANCY AND CHILDBIRTH, v. 17, . (12/17234-1)
NODA-NICOLAU, NATHALIA MAYUMI; POLETTINI, JOSSIMARA; PELTIER, MORGAN R.; DA SILVA, MARCIA GUIMARAES; MENON, RAMKUMAR. Combinations and loads of bacteria affect the cytokine production by fetal membranes: An in vitro study. American Journal of Reproductive Immunology, v. 76, n. 6, p. 504-511, . (12/17234-1)

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