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Gene and protein expression of Toll-like receptor-1, -2, -4, and -6 in maternal neutrophils and monocytes and activation of these cells in normal and complicated pregnancies by RPM-PT or PPT in the presence or absence of histological chorioamnionitis

Grant number: 11/17692-7
Support Opportunities:Regular Research Grants
Duration: April 01, 2012 - September 30, 2014
Field of knowledge:Health Sciences - Medicine - Maternal and Child Health
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

Abstract

Toll-like receptors (TLRs) are an important part of the innate immune system, and they are expressed in various human tissues. Few studies have focused on the role played by TLRs in pregnancy, and few data are available in relation to the expression of such receptors in maternal neutrophils and monocytes in pregnancies complicated by the preterm premature rupture of membranes (PPROM) and preterm labor (PTL). Objective: To gene and protein expression of TLR-1, -2, -4 and -6 in neutrophils and monocytes from peripheral maternal blood as well as to evaluate the state of activation of such cells during normal pregnancy and in pregnancies complicated by PPROM and PTL in the presence or not of histologic chorioamnionitis. Patients and Methods: A longitudinal study will be performed. It will include 15 TLR expression measurements in maternal neutrophils by gestational week from the 5th gestational week on, including the moment of delivery. The control group will consist of 50 non-pregnant women who must show similar age and parity to those in the study group. Concurrently, a cross-sectional study will be conducted, and it will include 45 pregnant women with PPROM and 45 pregnant women in PTL with an intact amniotic membrane, in the presence or absence of histologic chorioamnionitis. The control group will consist of pregnant women at term without obstetric complications, belonging to the longitudinal study. Samples of 35 mL of peripheral blood will be collected for the isolation and culture of neutrophils and monocytes, and 5 mL for total RNA extraction. At pregnancy resolution, fragments of the chorioamniotic membranes will be fixed, dehydrated in alcohol, diaphanized in xylol and then included into paraffin blocks. Sections of the 5mm will be stained by the Hematoxylin-Eosin (HE) method. It will be collected 20 ml of peripheral blood. The analysis of the expression of TLR-1, -2, -4 and -6 in neutrophils and monocytes will be performed by the real-time Polymerase-Chain Reaction (PCR) method and the detection of proteins of TLRs will be performed by flow cytometry. Cell cultures will be stimulated by specific PAMPs and after this, the dosages of cytokines IL-1, IL-6, IL-8 and TNF- alfa in culture supernatants will be performed by flow cytometry, using the Cytometric Bead Array (CBA) kit. Moreover, it will be evaluated the production of hydrogen peroxide, which will be held according to the method described by Pick and Keisari and adapted by Pick and Mizel. Statistical analysis will be performed by respecting the presuppositions determined by the results. The level of significance adopted will be of 5%, and the SigmaStat 9.0 (Jandel Corporation) will be used. (AU)

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