Leukotriene receptor 1 and 2 expression in tonsilar B and T lymphocyte of children with tonsillar enlargement: comparison between allergic and non-allergic

• Grant number: 13/19500-3
• Support Opportunities: Regular Research Grants
• Start date: February 01, 2014
• End date: January 31, 2016
• Field of knowledge: Health Sciences - Medicine
• Principal Investigator: Renata Cantisani Di Francesco
• Grantee: Renata Cantisani Di Francesco
• Host Institution: Hospital das Clínicas da Faculdade de Medicina da USP (HCFMUSP). Secretaria da Saúde (São Paulo - Estado). São Paulo , SP, Brazil
• Associated researchers: Bruno Peres Paulucci ; Juliana Pereira

Abstract

Tonsil and adenoid hyperplasia (AH) is the main cause of the syndrome of obstructive sleep apnea in children and does not have fully understood etiology. It is suggested an association with allergic diseases such as rhinitis.Allergies have leukotrienes as a key modulator. The most studied receptors are CysLT1R and CysLT2R which are expressed on lymphocytes and other cells, especially in situations of allergic response. Some studies also demonstrate expression in environments of cell proliferation, such as cancer.Whereas leukotriene receptors are enrolled in mitotic processes even in the absence of allergic responses, our hypothesis is that the lymphocyte proliferation of adenotonsillar hyperplasia occurs mediated by CysLT1R and CysLT2R in patient with and without respiratory allergic diseases. We too believe that the expression is greater in allergic people.The objectives of this study are:Primary: to compare quantitatively the expression of CysLT1R and CysLT2R in the surface of tonsillar B and T lymphocytes from children allergic and non-allergic with OSAS by AH.Secondary: to compare, in each anatomical sub-region of tonsil, the expression of CysLT1R and CysLT2R on the surface of lymphocytes of children allergic and non-allergic with OSAS by HA.Will include patients with: 4- 9 years, having snoring and nocturnal apnea due to AH.Initially will be performed double prick test for most common respiratory allergens. Thus will be formed 2 groups:Allergic: prick test response concordant in at least one of the testsNon allergic: prick test with negative response in all testsWill be excluded patients with: prick test discordant, overweight or obesity, craniofacial deformities or genetic syndromes; use of corticosteroids, anti-leukotrienes or antihistamines in three months before surgery; recurrent tonsillitis.Then the patients will undergo adenotonsillectomy. A fragment of tissue removed will be examined histologically with hematoxylin and eosin for confirmation of hyperplasia, this fragment will be stored in paraffin.Flow cytometry: the remaining tissue will be mechanically dissociated in RPMI, centrifuged and the cell pellet resuspended in RPMI. Tonsillar B lymphocytes and T are defined by an antibody against human CD3 FITC and PerCP antibody against human CD19. Antibodies anti CysLT1R and CysLT2R will be used to evaluate its expression on the surface of the lymphocytes.The cells will then be labeled with fluorochrome PE-conjugated F (AB0) secondary anti-rabbit antibody. The fluorescence will be read on a FACScan cytofluorimeter.Immunofluorescence: as a secondary objective, this step will be performed if there be viable tissue. In blades of successive cuts will be performed: marking of CysLT1R and CysLT2R using antibodies against antigenic tag in one of the blades; the adjacent blade will be marked antigen CD3 (T lymphocytes), and CD20 (B lymphocytes). Then will be added fluorescent secondary antibodies. The tissue reading is done via a confocal fluorescent microscope.Statistical analysis: the expression of CysLT1R and CysLT2R will be the main outcome of this study. It will be made the next comparison between groups:Flow cytometry: percentile of each cell type lymphocytes expressing CysLT1R and CysLT2R. Immunofluorescence: degree of receptor expression in each sub-anatomical region (zone and follicular germinal center)Will use the student's t-test for analysis of the variable under study.As there are no similar studies in the literature, we can not predict the degree of difference between the groups and it is not possible to calculate prior sample. Thus, there will be an early test with 20 patients (10 in each group). From the results obtained, we will evaluate the need for inclusion of new patients. (AU)

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)

PAULUCCI, BRUNO PERES; PEREIRA, JULIANA; PICCIARELLI, PATRICIA; LEVY, DEBORA; DI FRANCESCO, RENATA CANTISANI. Expression of CysLTR1 and 2 in Maturating Lymphocytes of Hyperplasic Tonsils Compared to Peripheral Cells in Children. Inflammation, v. 39, n. 3, p. 1216-1224, JUN 2016. (13/19500-3)