ALTERED EX VIVO MONOKINE PRODUCTION RELATED TO HEALTHY ADULTS.
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1 HUMAN NEONATES DISPLAY ALTERED EX VIVO MONOKINE PRODUCTION RELATED TO HEALTHY ADULTS. Paulo RZ Antas, PhD Lab. de Imunologia Clínica / IOC / FIOCRUZ
2 Contributors Jessica R. Lima 1 Thaíze Q. C. Pedro 1 Eliana A. Santiago 1 Carlos G. G. Ponte 1 Fernanda C. Silva 2 Luis A. V. Melca 2 1 Clinical Immunology Laboratory, Oswaldo Cruz Institute-Fiocruz, Rio de Janeiro 2 Gaffree Guinle State University Hospital of Rio de Janeiro Supported by: FAPERJ fellowships; CNPq-PQ-2 fellowship and Fiocruz.
3 Contributors Jessica R. Lima 1 Thaíze Q. C. Pedro 1 Eliana A. Santiago 1 Carlos G. G. Ponte 1 Fernanda C. Silva 2 Luis A. V. Melca 2 1 Clinical Immunology Laboratory, Oswaldo Cruz Institute-Fiocruz, Rio de Janeiro 2 Gaffree Guinle State University Hospital of Rio de Janeiro Supported by: FAPERJ fellowships; CNPq-PQ-2 fellowship and Fiocruz.
4 Disclosure None of the authors have a commercial association that poses a conflict of interest in relation to this program/presentation.
5 Background
6 Background
7 Background There is a high global burden of Inf. Dis. in the very young. Immunity is not static; it changes with age, with many distinctive features in early life. Newborns and young infants have distinct immune ontogeny and responses to microbes. Dowling & Levy. Trend in Immunology (7), Newborns exhibit increased susceptibility to infectious agents; Generalized hypofunction of inflammatory and immune mechanisms, related to the natural dampening of the Th-1 associated immune response, increasing the risk of infection in this exposed population. The neonatal immune system is constantly maturing, but, there are virtually no comparative studies concerning ex vivo broaden analysis addressing the role of monokines in the newborn vulnerable population. Kraft et al. Immunology ,
8 Background Dowling & Levy. Trend in Immunology (7),
9 Background Dowling & Levy. Trend in Immunology (7),
10 Background There is a high global burden of Inf. Dis. in the very young. Immunity is not static; it changes with age, with many distinctive features in early life. Newborns and young infants have distinct immune ontogeny and responses to microbes. Dowling & Levy. Trend in Immunology (7), Newborns exhibit increased susceptibility to infectious agents; Generalized hypofunction of inflammatory and immune mechanisms, related to the natural dampening of the Th-1 associated immune response, increasing the risk of infection in this exposed population. The neonatal immune system is constantly maturing, but, there are virtually no comparative studies concerning ex vivo broaden analysis addressing the role of monokines in the newborn vulnerable population. Kraft et al. Immunology ,
11 Background
12 Background
13 Background
14 Background
15 Rational To reveal critical aspects of ex vivo monokine and lymphokine profiles related to both innate and adaptive immunity in a community based open-label cross-sectional population study of a Brazilian sample. The study was undertaken to compare newborn (UV) and adult (HD) plasma samples using multiplex array and ELISA approaches, and we set out to investigate whether the quantitative detection of circulating biomarkers differs between these groups.
16 Rational To reveal critical aspects of ex vivo monokine and lymphokine profiles related to both innate and adaptive immunity in a community based open-label cross-sectional population study of a Brazilian sample. The study was undertaken to compare newborn (UV) and adult (HD) plasma samples using multiplex array and ELISA approaches, and we set out to investigate whether the quantitative detection of circulating biomarkers differs between these groups.
17 Cohorts Exclusion criteria: HIV-seronegative status, a negative history of malignant, degenerative, or transmitted diseases, diabetes mellitus, and use of corticosteroids or other immunosuppressive agents at the time of the study entry. Subjects identities were omitted. The study was approved by the Institutional Review Board of the State University Hospital (#060/2009 & #089/2011).
18 Cohorts Exclusion criteria: HIV-seronegative status, a negative history of malignant, degenerative, or transmitted diseases, diabetes mellitus, and use of corticosteroids or other immunosuppressive agents at the time of the study entry. Subjects identities were omitted. The study was approved by the Institutional Review Board of the State University Hospital (#060/2009 & #089/2011).
19 Cohorts Exclusion criteria: HIV-seronegative status, a negative history of malignant, degenerative, or transmitted diseases, diabetes mellitus, and use of corticosteroids or other immunosuppressive agents at the time of the study entry. Subjects identities were omitted. The study was approved by the Institutional Review Board of the State University Hospital (#060/2009 & #089/2011).
20 Methods HD=28 UV=28 Plasma - Fresh venous (HD) or cord (UV) blood. - 2 vials of plasma kept at -70 C. - Extensive evaluations of pro- and anti-inflammatory pathway cytokines (biomarkers) by: -Protein multiarray system (Bio-Rad, Hercules, CA, USA) to quantify human IL-2, IL-4, IL-5, IL-10, IL-12, IL- 13, GM-CSF, TNFα and IFNγ. -ELISA (DuoSet R&D, Minneapolis, MN, USA) to quantify human IL-1α, IL-18, IL-23, IL-27, IL-33 and TGF-β1 in parallel.
21 Methods HD=28 UV=28 Plasma - Fresh venous (HD) or cord (UV) blood. - 2 vials of plasma kept at -70 C. - Extensive evaluations of pro- and anti-inflammatory pathway cytokines (biomarkers) by: -Protein multiarray system (Bio-Rad, Hercules, CA, USA) to quantify human IL-2, IL-4, IL-5, IL-10, IL-12, IL- 13, GM-CSF, TNFα and IFNγ. -ELISA (DuoSet R&D, Minneapolis, MN, USA) to quantify human IL-1α, IL-18, IL-23, IL-27, IL-33 and TGF-β1 in parallel.
22 Methods HD=28 UV=28 Plasma - Fresh venous (HD) or cord (UV) blood. - 2 vials of plasma kept at -70 C. - Extensive evaluations of pro- and anti-inflammatory pathway cytokines (biomarkers) by: -Protein multiarray system (Bio-Rad, Hercules, CA, USA) to quantify human IL-2, IL-4, IL-5, IL-10, IL-12, IL- 13, GM-CSF, TNFα and IFNγ. -ELISA (DuoSet R&D, Minneapolis, MN, USA) to quantify human IL-1α, IL-18, IL-23, IL-27, IL-33 and TGF-β1 in parallel.
23 Table 1: Characteristics of the neonate population. Neonatal growth parameters UV Gestational age (weeks) 39.2 ± 0.07 a Birth weight (kg) 0.04 ± 0.02 Birth length (cm) 51.1 ± 0.2 Mode of delivery Induced vaginal Vaginal UV ND 2 (13%) b Elective cesarean 12 (80%) Emergency cesarean NA 1 (7%) c 13 (46%) d a Mean ± SEM; b Duration: 5.3h; c Duration: 4h; d IRB restrictions.
24 Table 2: Ex vivo human cytokine levels (pg/ml) determined in thawed healthy donor adult plasma (HD=28) and umbilical cord blood samples (UV=28) using commercially available protein multiarray system and enzyme linked immunosorbent assay (ELISA). Cytokines UV HD IL-1a 0.07 ± 0.01 a 0.06 ± 0.01 IL ± ± 2.1 IL ± ± 7.3 IL ± ± 3.6 IL ± ± 9.5 IL ± ± 1.5 IL ± ± 0.9 IL ± ± 0.1 IFNg 69.8 ± ± 12.0 TNFa 46.2 ± ± 9.1 GM-CSF 22.3 ± ± 4.0 IL ± 0.5 b 12.3 ± 3.4 a Mean ± SEM; b p < , when compared to HD group and based on statistical significance using the Mann-Whitney U test.
25 Table 2: Ex vivo human cytokine levels (pg/ml) determined in thawed healthy donor adult plasma (HD=28) and umbilical cord blood samples (UV=28) using commercially available protein multiarray system and enzyme linked immunosorbent assay (ELISA). Cytokines UV HD IL-1a 0.07 ± 0.01 a 0.06 ± 0.01 IL ± ± 2.1 IL ± ± 7.3 IL ± ± 3.6 IL ± ± 9.5 IL ± ± 1.5 IL ± ± 0.9 IL ± ± 0.1 IFNg 69.8 ± ± 12.0 TNFa 46.2 ± ± 9.1 GM-CSF 22.3 ± ± 4.0 IL ± 0.5 b 12.3 ± 3.4 a Mean ± SEM; b p < , when compared to HD group and based on statistical significance using the Mann-Whitney U test.
26 The ex vivo human IL-18 & IL-23 levels (ng/ml) were determined in thawed healthy donor adult plasma (HD; n=28) and umbilical cord blood samples (UV; n=28) using commercially available enzyme linked immunosorbent assay (ELISA) kits. HD UV HD UV The horizontal bars represent mean values. **p < 0.01, based on statistical significance using the Mann-Whitney U test.
27 The ex vivo human IL-18 & IL-23 levels (ng/ml) were determined in thawed healthy donor adult plasma (HD; n=28) and umbilical cord blood samples (UV; n=28) using commercially available enzyme linked immunosorbent assay (ELISA) kits. HD UV The horizontal bars represent mean values. **p < 0.01, based on statistical significance using the Mann-Whitney U test.
28 The ex vivo human IL-18 & IL-23 levels (ng/ml) were determined in thawed healthy donor adult plasma (HD; n=28) and umbilical cord blood samples (UV; n=28) using commercially available enzyme linked immunosorbent assay (ELISA) kits. HD UV The horizontal bars represent mean values. **p < 0.01, based on statistical significance using the Mann-Whitney U test.
29 Correlation analysis intra-group (UV; n=28) of IL-18 and IL-23 plasma levels (ng/ml). ρ = 0.52; p-level = 0.02 Spearman`s rank coefficient test (ρ).
30 Correlation analysis intra-group (UV; n=28) of IL-18 and IL-23 plasma levels (ng/ml). ρ = 0.52; p-level = 0.02 Spearman`s rank coefficient test (ρ).
31 The ex vivo human TGF-β1 levels (ng/ml) were determined in thawed healthy donor adult plasma (HD; n=28) and umbilical cord blood samples (UV; n=28) using an commercially available enzyme linked immunosorbent assay (ELISA) kit. HD UV The horizontal bars represent mean values. **p < 0.01, based on statistical significance using the Mann-Whitney U test.
32 The ex vivo human TGF-β1 levels (ng/ml) were determined in thawed healthy donor adult plasma (HD; n=28) and umbilical cord blood samples (UV; n=28) using an commercially available enzyme linked immunosorbent assay (ELISA) kit. HD UV The horizontal bars represent mean values. **p < 0.01, based on statistical significance using the Mann-Whitney U test.
33 Cytokine Network Maturação de Células B IL-6 IL-1 B IL-6 IL-12 Monócito IL-10 Atividade Lítica IFN-α IFN-β Mtb MHCII B7.2 B7.1 Inibição da síntese de citocinas NK Macrófago IFN-γ T TNF-α IL-12 IFN-γ Produção do granuloma IL-12 IL-18 Defesa antibacteriana Ativação da resposta Th1 Promove atividade de IFN-γ e fenótipo Th1 Atividade NK Produção de IFN-γ
34 Considerations Several factors may be implicated in those neonatal alterations, such as inherent immaturity or regulatory T cell-mediated inhibition. The apparent superior performance of the ELISA compared to the multiplex approach was an anticipated bias, due to our selective choice to quantify monokines based on own previous data. Previously, UV showed high IL-10 levels and/or decreased expression of the beta-2-microglobulin.
35 Considerations Several factors may be implicated in those neonatal alterations, such as inherent immaturity or regulatory T cell-mediated inhibition. The apparent superior performance of the ELISA compared to the multiplex approach was an anticipated bias, due to our selective choice to quantify monokines based on own previous data. Previously, UV showed high IL-10 levels and/or decreased expression of the beta-2-microglobulin.
36 Considerations Several factors may be implicated in those neonatal alterations, such as inherent immaturity or regulatory T cell-mediated inhibition. The apparent superior performance of the ELISA compared to the multiplex approach was an anticipated bias, due to our selective choice to quantify monokines based on own previous data. Previously, UV showed high IL-10 levels and/or decreased expression of the beta-2-microglobulin.
37 Conclusion Term human newborns unveil a differential monokine production patterns when compared to healthy adults, and those variations seem to be corrected during the immune system development.
38 Perspective Additional characterization of a broader cytokine panel might reveal other future candidates linked to that common underlying mechanism in order to better understand the functional capability of the neonatal immune system.
39 OBRIGADO (Thank You!) Paulo RZ Antas ( address:
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