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PP062. Silibinin modulates NF-kB pathway and proinflammatory cytokines production by mononuclear cells of preeclamptic women
Abstracts / Pregnancy Hypertension: An International Journal of Women’s Cardiovascular Health 2 (2012) 240–339
further studies are ongoing to study the MMP and TIMP mRNA expression in these samples. Disclosure of interest: None declared
275
reflect the oxidative stress in this group of patients. This study provides evidence that Hsp70 determination may be utilized to assess the differentiation between early- and late-onset PE. Financial support: FAPESP 2010/09241-2
References [1] James JL, Whitley GS, Cartwright JE. Pre-eclampsia: fitting together the placental, immune and cardiovascular pieces. J. Pathol. 2010;221(4):363–78. [2] Staun-Ram E, Goldman S, Gabarin D, Shalev E. Expression and important of matrix metalloproteinase 2 and 9 in human trophoblast invasion. Reprod. Biol. Endocrinol. 2004;2:59. doi:10.1016/j.preghy.2012.04.171
PP061. The role of heat shock protein 60 and 70 in earlyand late-onset preeclampsia differentiation J.C. Peracoli 1,*, C.F. Bannwart-Castro 2, V.S.I. Giorgi 3, I.C. Weel 1, M. Romao 1, S.S. Witkin 1, V.T.M. Borges 1, M.V.C. Rudge 1, M.T.S. Peracoli 3 (1 Gynecology and Obstetrics, Botucatu, Brazil, 2 Botucatu Medical School – São Paulo State University, Botucatu, Brazil, 3 Microbiology and Immunology, Institute of Biosciences, São Paulo State University, Botucatu, Brazil) Introduction: Preeclampsia is a human pregnancy-specific syndrome characterized by the onset of hypertension and proteinuria. These manifestations may occur before the 34th week of gestation or from this period on, being denominated early-onset or late-onset preeclampsia respectively. The etiology of both disorders seems to differ qualitatively; therefore, different strategies of prevention and treatment must be studied. Objectives: The aim of the present study is to determine whether the plasma levels of heat-shock proteins Hsp60 and Hsp70 as well as specific antibodies anti-Hsp60 and anti-Hsp70 may differentiate early-onset from late-onset preeclampsia. Methods: We evaluated 175 pregnant women with PE (55 early-onset PE and 120 late-onset PE). Plasma was obtained from peripheral blood and Hsp60, Hsp70 as well as anti-Hsp60 and anti-Hsp70 antibody levels were determined by enzyme immunoassay. Uric acid levels were also determined in the plasma of patients. For statistical analyses, the Mann–Whitney U-test and the Spearman rank order correlation were applied with significance level set at 5%. Results: Hsp70 levels obtained from early-onset PE group were significantly higher than the late-onset PE women and showed positive correlation with uric acid (r = 0.4547; p = 0.0028). The Hsp60 production was similar in both groups. Our results also indicate that there was no significant difference of anti-Hsp60 and anti-Hsp70 antibody levels between women with early- and late-onset PE. However,these antibody levels were high,indicating a strong relationship with the production of HSP60 and Hsp70 protein. Conclusion: Association between levels of Hsp70 and uric acid in plasma of patients with early-onset PE seems to
Disclosure of interest: None declared doi:10.1016/j.preghy.2012.04.172
PP062. Silibinin modulates NF-kB pathway and proinflammatory cytokines production by mononuclear cells of preeclamptic women V.S.I. Giorgi 1, C.F. Bannwart-Castro 2, J.C. Peracoli 2, M.T.S. Peracoli 1,* (1 Microbiology and Immunology, Institute of Biosciences, São Paulo State University, Botucatu, Brazil, 2 Gynecology and Obstetrics, Botucatu Medical School, São Paulo State University, Botucatu, Brazil) Introduction: Pre-eclampsia (PE) is a complication of human pregnancy characterized by hypertension and proteinuria after 20 weeks of gestation. In addition to increased activation of monocytes and granulocytes, there is an elevated production of proinflammatory cytokines in pregnant women with PE. The nuclear transcription factor-kB (NF-kB) is present in the cells of the immune system and is responsible for transcription of genes related to inflammation. Whereas the PE is associated with intense inflammatory response, the use of substances modulating the activity of NF-kB factor could be useful in alleviating the inflammation present in these patients. Silibinin is the main component of silymarin, a polyphenolic extract obtained from fruits and seeds of Sylibum marianum with potent hepatoprotective, anti-inflammatory and anti-fibrotic activities. Objectives: The objective of this study was to assess whether silibinin modulates the activity of NF-kB and the production of inflammatory cytokines by mononuclear cells of patients with PE. Methods: We evaluated 34 pregnant women with PE, 20 normotensive pregnant women (NT) and 15 non-pregnant women (NP). Mononuclear cells (PBMC) were obtained from peripheral blood and cultured in the presence or absence of silibinin (50 uM) and stimulated with 1 ug/mL of lipopolysaccharide (LPS) for 18 h. The supernatant was employed for determination of tumor necrosis factor-alpha (TNF-a) and interleukin-1 (IL-1b) by enzyme immunoassay. The cells were also cultured for 30 min to perform the extraction and determination of the nuclear activity of NF-kB. Results: The results showed increased endogenous activation of NF-kB in PBMC of the PE group compared with the NT and NP groups. We also observed increased production of TNF-a and IL-1b by non-stimulated PBMC in the PE group compared with NT and NP groups. A positive correlation between NF-kB activity and endogenous production of TNF-a (r = 0.6509; p = 0.0047) or IL-1 b (r = 0.5106; p = 0.0304) was observed in the PE group. Silibinin showed an anti-inflammatory activity by inhibiting the spontaneous and LPS-stimulated NF-kB activation as well as the production of inflammatory cytokines in all the groups studied.
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Abstracts / Pregnancy Hypertension: An International Journal of Women’s Cardiovascular Health 2 (2012) 240–339
Conclusion: Patients with PE showed a greater activation of PBMC cells compared with NT women. Silibinin showed modulatory activity on the inflammatory response by downregulation of NF-kB activation as well as TNF-a and IL-10 production. Financial support: FAPESP 2010/00776-0 Disclosure of interest: None declared
doi:10.1016/j.preghy.2012.04.173
PP063. TLR-4 expression and pro-inflammatory cytokine production by peripheral blood monocytes from preeclamptic women M. Romao 1, J.C. Peracoli 1, C.F. Bannwart-Castro 1, 1 L.T.L. Medeiros , I.C. Weel 1, V.T.M. Borges 1, M.A. Golim 2, M.T.S. Peracoli 3,* (1 Gynecology and Obstetrics, Botucatu, Brazil, 2 Hemocenter Division, Botucatu Medical School, São Paulo State University, Botucatu, Brazil, 3 Microbiology and Immunology, Institute of Biosciences, São Paulo State University, Botucatu, Brazil) Introduction: Toll-like receptor (TLR)-4 and TLR-2 are involved in inflammatory response of monocytes. These cells are activated in pregnant women with preeclampsia (PE), and over-produce inflammatory cytokines. TLR4 may recognize endogenous ligands, the so-called danger signals released by damaged cells, leading to production of proinflammatory cytokines. Objectives: This study investigated TLR2 and TLR4 expression and cytokine production by monocytes from women with PE before and after stimulation with TLR ligands. Methods: Monocytes (5 105cell/mL) were obtained from 32 preeclamptic (PE) and 20 normotensive (NT) pregnant women in the last trimester of pregnancy. TLR2 and TLR4 expression on monocyte surface was determined by flow cytometry in non-stimulated cells, and after 18 h of culture with lipopysaccharide (LPS) and peptidoglycan (PG). TNF-a, IL-10 and IL-12p70 production by these cells stimulated or not with LPS or PG was evaluated by enzyme immunoassay. Results were analyzed by non-parametric tests with significance level set at 5%. Results: In the absence of stimulation, the basal TLR4 expression by monocytes detected by the median fluorescence intensity (MFI) was significantly higher in the PE group than in the NT group while no significant differences were observed between groups in relation to endogenous TLR2 expression. An increase in TLR4 MFIs was detected after monocytes from NT pregnant women were stimulated with LPS while TLR2 expression was increased after PGstimulation. No alterations in TLR expression was detected after LPS or PG-stimulation in monocytes from patients with PE. Evaluation of endogenous cytokine levels in supernatant culture of monocytes showed higher concentrations of TNFa and IL-12p70 in preeclamptic women in comparison with the NT group, whereas IL-10 values were significantly higher in NT pregnant women than in the PE group. In contrast, when monocytes were stimulated with the TLRs ligands
LPS and PG, the release of TNF-a was significantly reduced, while IL-12p70 levels were significantly higher in women with PE compared to NT group. IL-10 production was similar in both groups studied. Conclusion: The basal up-regulation of TLR4 expression associated with endogenous high TNF-a and IL-12p70 production by monocytes from preeclamptic women confirms the activated profile of these cells by the disease process. These findings provide new insights into possible roles for TLRs in the pathogenesis of systemic inflammation detected in PE. Financial support: FAPESP 2009/11924-3 and 2010/20207-0 Disclosure of interest: None declared doi:10.1016/j.preghy.2012.04.174
PP064. M1 Monocyte subpopulation is associated with pro-inflammatory cytokine production in pregnant women with preeclampsia L.T.L. Medeiros 1, J.C. Peracoli 1, M. Romao 1, C.F. BannwartCastro 1, M.A. Golim 2, V.T.M. Borges 1, M.T.S. Peracoli 3,* (1 Gynecology and Obstetrics, Botucatu, Brazil, 2 Hemocenter Division, Botucatu Medical School, Sao Paulo State University, Botucatu, Brazil, 3 Microbiology and Immunology, Institute of Biosciences, Sao Paulo State University, Botucatu, Brazil) Introduction: Monocytes from peripheral blood of pregnant women with preeclampsia are endogenously activated and secrete high levels of free radicals and inflammatory cytokines. Objectives: This study aimed at evaluating whether the inflammatory state of monocytes observed in preeclampsia is associated with the polarization of monocyte to M1 profile in peripheral blood, correlating the expression of surface receptors CD64, TLR2, TLR4, and CD163 and CD206 with cytokine production. Methods: We studied 90 pregnant women, 30 normotensive and 60 with preeclampsia, matched for gestational age. Peripheral blood monocytes obtained from normotensive pregnant or preeclamptic pregnant women were cultured for 18h, and the expression of surface receptors on M1 inflammatory monocyte subpopulation (TLR2, TLR4 and CD64) and M2 suppressor monocyte subpopulation (CD163 and CD206) were evaluated by flow cytometry, using specific monoclonal antibodies, labeled with fluorochromes. The values were expressed as the mean fluorescence intensity. Moreover, the production of proinflammatory cytokines associated with M1 profile (TNF-a, IL-12p70 and IL-23) and the anti-inflammatory cytokine associated with M2 profile (IL-10) were evaluated in the monocyte supernatant of culture by enzyme immunoassay. Results were analyzed using nonparametric tests with significance level set at 5%. Results: The expression of CD4 and TLR4 on monocyte surface, from women with preeclampsia was significantly higher, while the expression of CD163 and CD206 was significantly
further studies are ongoing to study the MMP and TIMP mRNA expression in these samples. Disclosure of interest: None declared
275
reflect the oxidative stress in this group of patients. This study provides evidence that Hsp70 determination may be utilized to assess the differentiation between early- and late-onset PE. Financial support: FAPESP 2010/09241-2
References [1] James JL, Whitley GS, Cartwright JE. Pre-eclampsia: fitting together the placental, immune and cardiovascular pieces. J. Pathol. 2010;221(4):363–78. [2] Staun-Ram E, Goldman S, Gabarin D, Shalev E. Expression and important of matrix metalloproteinase 2 and 9 in human trophoblast invasion. Reprod. Biol. Endocrinol. 2004;2:59. doi:10.1016/j.preghy.2012.04.171
PP061. The role of heat shock protein 60 and 70 in earlyand late-onset preeclampsia differentiation J.C. Peracoli 1,*, C.F. Bannwart-Castro 2, V.S.I. Giorgi 3, I.C. Weel 1, M. Romao 1, S.S. Witkin 1, V.T.M. Borges 1, M.V.C. Rudge 1, M.T.S. Peracoli 3 (1 Gynecology and Obstetrics, Botucatu, Brazil, 2 Botucatu Medical School – São Paulo State University, Botucatu, Brazil, 3 Microbiology and Immunology, Institute of Biosciences, São Paulo State University, Botucatu, Brazil) Introduction: Preeclampsia is a human pregnancy-specific syndrome characterized by the onset of hypertension and proteinuria. These manifestations may occur before the 34th week of gestation or from this period on, being denominated early-onset or late-onset preeclampsia respectively. The etiology of both disorders seems to differ qualitatively; therefore, different strategies of prevention and treatment must be studied. Objectives: The aim of the present study is to determine whether the plasma levels of heat-shock proteins Hsp60 and Hsp70 as well as specific antibodies anti-Hsp60 and anti-Hsp70 may differentiate early-onset from late-onset preeclampsia. Methods: We evaluated 175 pregnant women with PE (55 early-onset PE and 120 late-onset PE). Plasma was obtained from peripheral blood and Hsp60, Hsp70 as well as anti-Hsp60 and anti-Hsp70 antibody levels were determined by enzyme immunoassay. Uric acid levels were also determined in the plasma of patients. For statistical analyses, the Mann–Whitney U-test and the Spearman rank order correlation were applied with significance level set at 5%. Results: Hsp70 levels obtained from early-onset PE group were significantly higher than the late-onset PE women and showed positive correlation with uric acid (r = 0.4547; p = 0.0028). The Hsp60 production was similar in both groups. Our results also indicate that there was no significant difference of anti-Hsp60 and anti-Hsp70 antibody levels between women with early- and late-onset PE. However,these antibody levels were high,indicating a strong relationship with the production of HSP60 and Hsp70 protein. Conclusion: Association between levels of Hsp70 and uric acid in plasma of patients with early-onset PE seems to
Disclosure of interest: None declared doi:10.1016/j.preghy.2012.04.172
PP062. Silibinin modulates NF-kB pathway and proinflammatory cytokines production by mononuclear cells of preeclamptic women V.S.I. Giorgi 1, C.F. Bannwart-Castro 2, J.C. Peracoli 2, M.T.S. Peracoli 1,* (1 Microbiology and Immunology, Institute of Biosciences, São Paulo State University, Botucatu, Brazil, 2 Gynecology and Obstetrics, Botucatu Medical School, São Paulo State University, Botucatu, Brazil) Introduction: Pre-eclampsia (PE) is a complication of human pregnancy characterized by hypertension and proteinuria after 20 weeks of gestation. In addition to increased activation of monocytes and granulocytes, there is an elevated production of proinflammatory cytokines in pregnant women with PE. The nuclear transcription factor-kB (NF-kB) is present in the cells of the immune system and is responsible for transcription of genes related to inflammation. Whereas the PE is associated with intense inflammatory response, the use of substances modulating the activity of NF-kB factor could be useful in alleviating the inflammation present in these patients. Silibinin is the main component of silymarin, a polyphenolic extract obtained from fruits and seeds of Sylibum marianum with potent hepatoprotective, anti-inflammatory and anti-fibrotic activities. Objectives: The objective of this study was to assess whether silibinin modulates the activity of NF-kB and the production of inflammatory cytokines by mononuclear cells of patients with PE. Methods: We evaluated 34 pregnant women with PE, 20 normotensive pregnant women (NT) and 15 non-pregnant women (NP). Mononuclear cells (PBMC) were obtained from peripheral blood and cultured in the presence or absence of silibinin (50 uM) and stimulated with 1 ug/mL of lipopolysaccharide (LPS) for 18 h. The supernatant was employed for determination of tumor necrosis factor-alpha (TNF-a) and interleukin-1 (IL-1b) by enzyme immunoassay. The cells were also cultured for 30 min to perform the extraction and determination of the nuclear activity of NF-kB. Results: The results showed increased endogenous activation of NF-kB in PBMC of the PE group compared with the NT and NP groups. We also observed increased production of TNF-a and IL-1b by non-stimulated PBMC in the PE group compared with NT and NP groups. A positive correlation between NF-kB activity and endogenous production of TNF-a (r = 0.6509; p = 0.0047) or IL-1 b (r = 0.5106; p = 0.0304) was observed in the PE group. Silibinin showed an anti-inflammatory activity by inhibiting the spontaneous and LPS-stimulated NF-kB activation as well as the production of inflammatory cytokines in all the groups studied.
276
Abstracts / Pregnancy Hypertension: An International Journal of Women’s Cardiovascular Health 2 (2012) 240–339
Conclusion: Patients with PE showed a greater activation of PBMC cells compared with NT women. Silibinin showed modulatory activity on the inflammatory response by downregulation of NF-kB activation as well as TNF-a and IL-10 production. Financial support: FAPESP 2010/00776-0 Disclosure of interest: None declared
doi:10.1016/j.preghy.2012.04.173
PP063. TLR-4 expression and pro-inflammatory cytokine production by peripheral blood monocytes from preeclamptic women M. Romao 1, J.C. Peracoli 1, C.F. Bannwart-Castro 1, 1 L.T.L. Medeiros , I.C. Weel 1, V.T.M. Borges 1, M.A. Golim 2, M.T.S. Peracoli 3,* (1 Gynecology and Obstetrics, Botucatu, Brazil, 2 Hemocenter Division, Botucatu Medical School, São Paulo State University, Botucatu, Brazil, 3 Microbiology and Immunology, Institute of Biosciences, São Paulo State University, Botucatu, Brazil) Introduction: Toll-like receptor (TLR)-4 and TLR-2 are involved in inflammatory response of monocytes. These cells are activated in pregnant women with preeclampsia (PE), and over-produce inflammatory cytokines. TLR4 may recognize endogenous ligands, the so-called danger signals released by damaged cells, leading to production of proinflammatory cytokines. Objectives: This study investigated TLR2 and TLR4 expression and cytokine production by monocytes from women with PE before and after stimulation with TLR ligands. Methods: Monocytes (5 105cell/mL) were obtained from 32 preeclamptic (PE) and 20 normotensive (NT) pregnant women in the last trimester of pregnancy. TLR2 and TLR4 expression on monocyte surface was determined by flow cytometry in non-stimulated cells, and after 18 h of culture with lipopysaccharide (LPS) and peptidoglycan (PG). TNF-a, IL-10 and IL-12p70 production by these cells stimulated or not with LPS or PG was evaluated by enzyme immunoassay. Results were analyzed by non-parametric tests with significance level set at 5%. Results: In the absence of stimulation, the basal TLR4 expression by monocytes detected by the median fluorescence intensity (MFI) was significantly higher in the PE group than in the NT group while no significant differences were observed between groups in relation to endogenous TLR2 expression. An increase in TLR4 MFIs was detected after monocytes from NT pregnant women were stimulated with LPS while TLR2 expression was increased after PGstimulation. No alterations in TLR expression was detected after LPS or PG-stimulation in monocytes from patients with PE. Evaluation of endogenous cytokine levels in supernatant culture of monocytes showed higher concentrations of TNFa and IL-12p70 in preeclamptic women in comparison with the NT group, whereas IL-10 values were significantly higher in NT pregnant women than in the PE group. In contrast, when monocytes were stimulated with the TLRs ligands
LPS and PG, the release of TNF-a was significantly reduced, while IL-12p70 levels were significantly higher in women with PE compared to NT group. IL-10 production was similar in both groups studied. Conclusion: The basal up-regulation of TLR4 expression associated with endogenous high TNF-a and IL-12p70 production by monocytes from preeclamptic women confirms the activated profile of these cells by the disease process. These findings provide new insights into possible roles for TLRs in the pathogenesis of systemic inflammation detected in PE. Financial support: FAPESP 2009/11924-3 and 2010/20207-0 Disclosure of interest: None declared doi:10.1016/j.preghy.2012.04.174
PP064. M1 Monocyte subpopulation is associated with pro-inflammatory cytokine production in pregnant women with preeclampsia L.T.L. Medeiros 1, J.C. Peracoli 1, M. Romao 1, C.F. BannwartCastro 1, M.A. Golim 2, V.T.M. Borges 1, M.T.S. Peracoli 3,* (1 Gynecology and Obstetrics, Botucatu, Brazil, 2 Hemocenter Division, Botucatu Medical School, Sao Paulo State University, Botucatu, Brazil, 3 Microbiology and Immunology, Institute of Biosciences, Sao Paulo State University, Botucatu, Brazil) Introduction: Monocytes from peripheral blood of pregnant women with preeclampsia are endogenously activated and secrete high levels of free radicals and inflammatory cytokines. Objectives: This study aimed at evaluating whether the inflammatory state of monocytes observed in preeclampsia is associated with the polarization of monocyte to M1 profile in peripheral blood, correlating the expression of surface receptors CD64, TLR2, TLR4, and CD163 and CD206 with cytokine production. Methods: We studied 90 pregnant women, 30 normotensive and 60 with preeclampsia, matched for gestational age. Peripheral blood monocytes obtained from normotensive pregnant or preeclamptic pregnant women were cultured for 18h, and the expression of surface receptors on M1 inflammatory monocyte subpopulation (TLR2, TLR4 and CD64) and M2 suppressor monocyte subpopulation (CD163 and CD206) were evaluated by flow cytometry, using specific monoclonal antibodies, labeled with fluorochromes. The values were expressed as the mean fluorescence intensity. Moreover, the production of proinflammatory cytokines associated with M1 profile (TNF-a, IL-12p70 and IL-23) and the anti-inflammatory cytokine associated with M2 profile (IL-10) were evaluated in the monocyte supernatant of culture by enzyme immunoassay. Results were analyzed using nonparametric tests with significance level set at 5%. Results: The expression of CD4 and TLR4 on monocyte surface, from women with preeclampsia was significantly higher, while the expression of CD163 and CD206 was significantly