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Evaluation of genes expression involved in Nod-like receptor (NLR) activation in PBMCs from sequential samples of patients with sepsis secondary to real-time array PCR pneumonia

Grant number: 15/17109-0
Support type:Scholarships in Brazil - Scientific Initiation
Effective date (Start): September 01, 2015
Effective date (End): August 31, 2016
Field of knowledge:Biological Sciences - Immunology - Cellular Immunology
Principal Investigator:Reinaldo Salomão
Grantee:Felipe Barros de Francisco
Home Institution: Escola Paulista de Medicina (EPM). Universidade Federal de São Paulo (UNIFESP). Campus São Paulo. São Paulo , SP, Brazil
Associated research grant:11/20401-4 - Sepsis: integrating basic research and clinical research II, AP.TEM

Abstract

Assessment of expression of genes involved in activation of the Nod-like receptor (NLR) in peripheral blood mononuclear cells (PBMC) of sequential samples of patients with from sepsis secondary to pneumonia by to real-time PCR. Sepsis is a systemic inflammatory manifestation produced by the host in response to an infection. It can be classified in different degrees of severity: sepsis, severe sepsis or septic shock. There is a complex modulation of immune response during sepsis and hyper or hypo response may be observed. Despite the lack of comprehensive epidemiological studies in Brazil sepsis is clearly a public health problem. The Brazilian Sepsis Epidemiogical Study (BASES) and a multicenter survey developed by Sales et al showed that the incidence of sepsis is high and mortality reaches almost 50% of the cases. The initial inflammatory process of sepsis is triggered by the innate immune system. This response involves receptors such as Toll-like Receptors (TLR) that recognize pathogen-associated molecular patterns (PAMPs). The cells also have intracellular receptors, such as Nucleotide-binding oligomerization domain (NOD) or Nod-like receptor (NLR). NOD receptors are capable of signaling danger to the cells and induce several functions activating the innate immune response, activating the NF-kB and MAPK pathways, modulating the expression of MHC receptors, inhibiting inflammatory signaling and activating the oligomerization of inflammasomes. This work aims to evaluate the expression of genes involved in the NLR activation cascade in PBMC of septic patients to understand the role of these receptors in the immune response to sepsis. Septic patients will be included within 48 hours after diagnosis (day zero - D0) and after seven days of follow-up (D7). The sample size to be involved in the study should contain approximately 30 patients with sepsis secondary to pneumonia hospitalized in ICU and 15 healthy subjects for comparison. During the analysis results of patients will also be divided into two groups, survivors and non-survivors. The PBMC are separated and kept in liquid nitrogen until the time of RNA extraction using the RNeasy mini kit (Qiagen). Then the complementary DNA synthesis will be done after this. PCR array plates (Qiagen) will be customized for analysis of 39 genes of interest. After obtaining the raw data, robust analyzes with the aid of bioinformatics software will be used to understand the modulation of the expression of genes involved in the cascade of NLR, such as Genesis and Ingenuity.