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Made available in DSpace on 2022-05-01T09:47:28Z (GMT). No. of bitstreams: 0 Previous issue date: 2022-01-01 This study aimed to evaluate the immunomodulatory effect of vitamin D (VD) on the NLRP1 and NLRP3 inflammasomes in placental explants from preeclamptic (PE) and normotensive (NT) pregnant women. Placental explants from eight PE and eight NT pregnant women were cultured with or without hydrogen peroxide (H2O2), VD or H2O2 + VD. Gene and protein expression of NLRP1, NLRP3, HMGB1, caspase-1, IL-1β, TNF-α and IL-18 were determined by qPCR and Western blotting/ELISA. Compared to NT pregnant women, the endogenous gene expression of NLRP1, NLRP3, HMGB1, IL-1β, TNF-α and IL-18 was significantly higher in explants from PE and became decreased after VD treatment. Similarly, VD decreased the protein expression of NLRP1, NLRP3, caspase-1, HMGB1, IL-1β, TNF-α and IL-18 in PE. Placental explants from NT cultured with H2O2 showed increased gene and protein expression of NLRP1, NLRP3, caspase-1, IL-1β, TNF-α and HMGB1, while H2O2 was also able to increase TNF-α and caspase-1 gene expression in PE. Treatment with H2O2 + VD decreased gene/protein expression of NLRP1, NLRP3, caspase-1, HMGB1, IL-1β, TNF-α and IL-18 in PE and NT explants with H2O2. NLRP1 and NLRP3 are upregulated in the PE. VD may play an immunomodulatory role in the placental inflammation and downregulates oxidative stress induced in vitro by H2O2. Botucatu Medical School Sao Paulo State University (Unesp) Institute of Biosciences Sao Paulo State University (Unesp) Botucatu Medical School Sao Paulo State University (Unesp) Institute of Biosciences Sao Paulo State University (Unesp)

Made available in DSpace on 2020-12-12T02:07:46Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-01-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Preeclampsia (PE) is a pregnancy-specific disorder that affects 3-8% expecting mothers worldwide being one of the main causes of maternal and fetal morbidity and mortality. The search for altered circulating molecules in PE is an important target to better understand the pathophysiology of this disease. Therefore, we evaluated Sirtuin-1 (SIRT1) concentration in plasma from healthy pregnant (HP) women, gestational hypertensive women (GH), and preeclampsia women (PE) via enzyme-linked immunosorbent assay (ELISA). We also measured intracellular SIRT1 in HUVECs incubated with plasma from PE patients compared to HP and GH via Western Blot Assay. Statistical differences were considered when p

The formation of S-nitrosothiols (SNOs) occurs with the reaction of nitric oxide (NO) and free thiol groups in proteins. This process, called S-nitrosylation, allows NO to interfere with or even modulate a variety of cellular functions, culminating with the modification of protein trafficking, redox state, and cell cycle. Furthermore, NO plays a role in modulating a wide range of functions in endothelial cells specifically, including inflammation, apoptosis, permeability, migration, and cell growth. As such, NO acts as a mediator in several physiological processes. The interaction between endothelial nitric oxide synthase (eNOS) and proteins that are to be targeted for S-nitrosylation is a key determinant of the specificity of NO signaling. Deficits in the bioavailability of NO have been associated with pregnancy-related disorders, such as preeclampsia (PE). The study of S-nitrosylation in PE, as well as the identification of targeted proteins, may contribute to a better understanding of its pathophysiology and the development of drugs for the treatment of PE patients. In this review, we aimed to present the mechanism of S-nitrosylation, the regulatory pathways, and some proteins by which S-nitrosylation can modulate NO availability with a potential impact on PE.

Made available in DSpace on 2022-04-29T08:38:14Z (GMT). No. of bitstreams: 0 Previous issue date: 2022-01-01 ASCRS Research Foundation This study evaluated the in vitro modulatory effect of vitamin D (VD) on T cells, by determining the expression of STATs and the transcription factors of each CD4+ T cell subsets. Twenty women with preeclampsia (PE) and 20 normotensive pregnant women were studied. Peripheral blood mononuclear cells were cultured with or without VD to analyse the STATs and transcription factors by flow cytometry, and cytokines production by ELISA. The plasma levels of VD were lower in the PE group. Treatment of cells with VD decreased STAT1/STAT4/T-bet, STAT3/RORγt, and increased STAT6/GATA-3 and STAT5/FoxP3 in preeclamptic women. Treatment with VD also decreased the levels of inflammatory cytokines and increased IL-10 and TGF-β. This hormone exerts immunomodulatory effects on the STAT signalling pathway, shifting the inflammatory profiles, Th1/Th17 cells to Th2/Treg profiles, and it can be suggested as a promising strategy to regulate the systemic inflammatory response in PE. Department of Gynecology and Obstetrics Botucatu Medical School Sao Paulo State University-UNESP Department of Chemistry and Biological Sciences Institute of Biosciences Sao Paulo State University-UNESP Department of Health Science Oeste Paulista University-UNOESTE Department of Gynecology and Obstetrics Botucatu Medical School Sao Paulo State University-UNESP Department of Chemistry and Biological Sciences Institute of Biosciences Sao Paulo State University-UNESP

Made available in DSpace on 2022-05-01T11:54:05Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-01-01 This study evaluated the impact of vitamin D on Human Umbilical Vein Endothelial Cells (HUVEC) and inflammation in placental explants from women with preeclampsia (PE). HUVEC and explants from 10 late-onset PE (LOPE), 10 early-onset (EOPE), and 10 normotensive (NT) pregnant women were cultured with/without tumor necrosis factor (TNF-α) and VD. Interleukin-1β (IL-1β), 18 (IL-18), TNF-α, and TNF-related apoptosis-inducing ligand (TRAIL) were detected by ELISA. High mobility group box 1 (HMGB1) was determined by qPCR/Western blotting, and cell death by flow cytometry. Statistical significance was accepted at p

Objective Preeclampsia (PE) is a pregnancy-specific syndrome characterized by abnormal levels of cytokines and angiogenic factors, playing a role in the disease development. The present study evaluated whether immunological markers are associated with the gestational age and with the disease severity in preeclamptic women. Methods Ninety-five women who developed PE were stratified for gestational age as preterm PE (< 37 weeks) and term PE (≥ 37 weeks of gestation) and compared for disease severity as well as plasma concentration of angiogenic factors and cytokines. The concentrations of placental growth factor (PlGF), vascular endothelial growth factor (VEGF), Fms-like soluble tyrosine kinase (sFlt-1) and soluble endoglin (sEng), as well as the cytokines, tumor necrosis factor-α (TNF-α) and interleukin 10 (IL-10), were determined by enzyme-linked immunosorbent assay (ELISA). Results The comparison between preeclamptic groups showed a higher percentage of severe cases in preterm PE (82.1%) than in term PE (35.9%). Similarly, the concentrations of TNF-α, sFlt-1, and sEng, as well as TNF-α/IL-10 and sFlt-1/PlGF ratios were significantly higher in the preterm PE group. In contrast, concentrations of PlGF, VEGF, and IL-10 were significantly lower in women with preterm PE. Negative correlations between TNF-α and IL-10 (r = 0.5232) and between PlGF and sFlt1 (r = 0.4158) were detected in the preterm PE. Conclusion In pregnant women with preterm PE, there is an imbalance between immunological markers, with the predominance of anti-angiogenic factors and TNF-α, associated with adverse maternal clinical outcomes. Resumo Objetivo A pré-eclâmpsia (PE) é uma síndrome específica da gravidez caracterizada por níveis anormais de citocinas e fatores angiogênicos, que desempenham um papel no desenvolvimento da doença. Este estudo avaliou se os marcadores imunológicos estão associados à idade gestacional e à gravidade da doença em mulheres com pré-eclâmpsia. Métodos Noventa e cinco mulheres que desenvolveram PE foram estratificadas pela idade gestacional em PE pré-termo (< 37 semanas) e PE a termo (≥ 37 semanas de gestação) e comparadas quanto à gravidade da doença, bem como à concentração plasmática de fatores angiogênicos e citocinas. As concentrações de fator de crescimento placentário (PlGF), fator de crescimento endotelial vascular (VEGF), tirosina quinase solúvel semelhante a Fms (sFlt-1) e endoglina solúvel (sEng), bem como as citocinas, fator de necrose tumoral alfa (TNF- α) e interleucina 10 (IL-10), foram determinados porensaio de imunoabsorção enzimática (ELISA, na sigla em inglês). Resultados A comparação entre os grupos com pré-eclâmpsia mostrou maior porcentagem de casos graves em PE pré-termo (82,1%) do que em PE a termo (35,9%). Da mesma forma, as concentrações de TNF-α, sFlt-1 e sEng, bem como as razões TNF-α/IL-10 e sFlt-1/PlGF foram significativamente maiores no grupo de PE pré-termo. Em contraste, as concentrações de PlGF, VEGF e IL-10 foram significativamente menores em mulheres com PE pré-termo. Correlações negativas entre TNF-α e IL-10 (r = 0.5232) e entre PlGF e sFlt1 (r = 0.4158) foram detectadas no grupo de PE pré-termo. Conclusão Em gestantes com PE pré-termo, ocorre um desequilíbrio entre os marcadores imunológicos, com predomínio de fatores antiangiogênicos e TNF-α, associados a desfechos clínicos maternos adversos.

Made available in DSpace on 2022-04-29T08:39:57Z (GMT). No. of bitstreams: 0 Previous issue date: 2022-01-01 Preeclampsia (PE) is a multisystemic disorder characterized by abnormal placentation. Autophagy is a lysosomal degradation pathway that removes protein aggregates and damaged organelles, and it seems to be essential for cell survival during stress, hypoxia, and for implantation and development of the placenta. p62/SQSTM1 is an autophagy marker that not only binds proteins destined for elimination but is also constitutively degraded by this mechanism. Considering that the placenta plays an important role in the pathogenesis of PE, the present study aimed to evaluate the gene and protein expression of p62/SQSTM1 in placentas from pregnant women with PE. Placental tissues from 20 women with PE classified into three groups according to gestational age, 27–31 weeks (n = 8); 32–36 weeks (n = 6); 37–39 weeks (n = 6), and 20 normotensives (NT) pregnant women were collected and employed for p62/SQSTM1 expression by quantitative polymerase chain reaction (qPCR), immunohistochemistry and enzyme-linked immunosorbent assay (ELISA) techniques. p62/SQSTM1 mRNA levels were significantly lower, while protein expression was significantly higher in the placenta of pregnant women with PE than in NT pregnant women, and these results remained similar after separating the groups by gestational age. In conclusion, the results suggest that there is a reduction of autophagic activity in pregnant women with PE. Studies involving cross-talk between autophagy, inflammasomes, nuclear transcription factor (NF-κB) activation pathways, and aggregation of protein in the placenta from women with PE might help to better understand the pathogenesis of this important obstetric pathology. Department of Gynecology and Obstetrics Botucatu Medical School Sao Paulo State University Department of Chemistry and Biological Sciences Institute of Biosciences Sao Paulo State University Department of Gynecology and Obstetrics Botucatu Medical School Sao Paulo State University Department of Chemistry and Biological Sciences Institute of Biosciences Sao Paulo State University

This study evaluated the in vitro modulatory effect of vitamin D (VD) on T cells, by determining the expression of STATs and the transcription factors of each CD4

Preeclampsia (PE) is characterized by abnormal activation of the immune system. The intense systemic inflammatory reaction, could be related to the presence of molecules released after cell stress or death, that are capable of inducing inflammation and are known as damage-associated molecular patterns (DAMP). This study evaluated the profile of T cells through the analysis of transcription factors and the cytokines produced after culture with or without DAMPs: heat shock protein 70 (Hsp70), hyaluronan (HA) and monosodium urate (MSU). Twenty pregnant women with PE, 20 normotensive (NT) pregnant women and 20 non-pregnant (NP) women were studied. The results showed polarization toward Th1/Th17 and a decrease in Th2/Treg profiles in preeclamptic women associated with elevated levels of TNF, IFN-γ, and IL-17A and diminished levels of TGF-β1 and IL-10 when compared to the normotensive group. In addition, preeclamptic women had a higher percentage of cells co-expressing T-bet/GATA-3 and T-bet/RORγt and fewer T-bet/FoxP3 cells when compared to normotensive group. MSU induced an increase in IFN-γ and IL-22 in all studied groups. MSU, HA, and Hsp70 induced significant higher production of TNF in the PE and NP groups. The PE group showed elevated levels of TGF-β1 after incubation with MSU, HA, and Hsp70, whereas HA and Hsp70 decreased TGF-β1 production in NT group. The results suggest that these alarmins may play a role in the activation of innate and adaptive immune systems by skewing CD4 + T cells and increasing the release of inflammatory cytokines, thereby contributing to the pathogenesis of this important syndrome.

The coronavirus disease 2019 (COVID‐19), which had spread to the world from Wuhan (China) in late December, was announced as a pandemic by the World Health Organization in March 2020. In addition to acute respiratory syndrome symptoms, this virus also affects nonrespiratory organs, according to existing data. ACE2 and TMPRSS2, which play a critical role in the entrance of SARS‐COV‐2 into the cell, are coexpressed in placental development stages, so the placenta also carries a risk for this virus. Many studies have shown that this virus causes some histopathological changes in the placenta. The vertical transmission of the virus is not yet clear, but available data have shown that the indirect effects of the virus can be seen on the fetus. This article focuses on revealing the effects of the virus on the placenta in all aspects., Highlights •The placenta also shows antiviral activity due to placental growth factors, cytokines, and chemokines, so the use of placental extracts as therapeutic agents in the treatment of COVID‐19 needs authority regulations and clinical guidelines.

Made available in DSpace on 2021-06-25T15:06:18Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-01-01. Added 1 bitstream(s) on 2021-07-15T14:36:46Z : No. of bitstreams: 1 S0100-879X2021000800604.pdf: 632798 bytes, checksum: 7a3373bbfe0a871d577a001fd4fb9313 (MD5) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Swedish Research Council Swedish Heart and Lung Foundation NovoNordisk The study evaluated the effect of the supernatant of placental explants from preeclamptic (PE) and normotensive (NT) pregnant women after tissue treatment with or without vitamin D (VD) on oxidative stress and nitric oxide (NO) bioavailability in human umbilical vein endothelial cells (HUVEC). Placental explants were prepared from eight NT and eight PE women, and supernatants were obtained after incubation with or without hydrogen peroxide (H2O2) and/or VD. HUVEC were cultured for 24 h with supernatants, and the following parameters were analyzed in HUVEC cultures: NO, nitrate (NO3-), and nitrite (NO2-) levels, lipid peroxidation, and intracellular reactive oxygen species (ROS). Results showed that the production of NO3-, NO2-, malondialdehyde (MDA), and ROS were significantly higher in HUVEC treated with explant supernatant from PE compared to NT pregnant women, while the supernatant of PE explants treated with VD led to a decrease in these parameters. A significantly high production of NO was detected in HUVEC cultured with control supernatant of NT group, and in cultures treated with supernatant of PE explants treated with VD. Taken together, these results demonstrated that cultures of placental explants from PE women with VD treatment generated a supernatant that decreased oxidative stress and increased the bioavailability of NO in endothelial cells. Univ Estadual Paulista, Fac Med Botucatu, Dept Ginecol & Obstet, Botucatu, SP, Brazil Karolinska Inst, Dept Physiol & Pharmacol, Stockholm, Sweden Univ Estadual Paulista, Inst Biociencias, Dept Biofis & Farmacol, Botucatu, SP, Brazil Univ Estadual Paulista, Fac Med Botucatu, Dept Ginecol & Obstet, Botucatu, SP, Brazil Univ Estadual Paulista, Inst Biociencias, Dept Biofis & Farmacol, Botucatu, SP, Brazil FAPESP: 2016/23452-2 FAPESP: 2019/00234-8 Swedish Research Council: 2016-01381 Swedish Research Council: 2020-01645 Swedish Heart and Lung Foundation: 20170124 Swedish Heart and Lung Foundation: 20180568 NovoNordisk: 0055026

Made available in DSpace on 2021-06-25T10:52:06Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-04-01 This study evaluated the in vitro modulatory effect of progesterone (PG) and vitamin D (VD) on NLRP1/NLRP3 inflammasomes and TLR4/NF-κB pathway in monocytes from pregnant women with preeclampsia (PE). Monocytes from 20 preeclamptic and 20 normotensive (NT) pregnant women, and THP-1 cells were cultured with/without hyaluronan (HA), PG, or VD to determine gene and protein expression of TLR4 receptor, phosphorylated NF-κB, IκBα, TLR4, MYD88, NF-κB, NLRP1, NLRP3, caspase-1, IL-1β, IL-18, TNF-α, and IL-10. Higher endogenous activation of inflammatory genes and higher protein expression of TLR4 and NF-κB was detected in monocytes of PE group and decreased after PG or VD treatment. Monocyte from PE stimulated with HA increased while treatment with PG or VD decreased the expression of genes and proteins related to the inflammasomes. THP-1 cells showed a similar immune response profile as monocytes from PE. These results demonstrate that PG and VD play an immunomodulatory role in monocyte activation. Department of Gynaecology and Obstetrics Botucatu Medical School Sao Paulo State University (UNESP) Department Health Science Oeste Paulista University (UNOESTE) Department of Chemistry and Biological Sciences Institute of Biosciences Sao Paulo State University (UNESP), Botucatu Department of Gynaecology and Obstetrics Botucatu Medical School Sao Paulo State University (UNESP) Department of Chemistry and Biological Sciences Institute of Biosciences Sao Paulo State University (UNESP), Botucatu

Preeclampsia (PE) is a multifactorial disease that is characterized by inflammation. Some of the factors responsible for this inflammation are the cells of the innate and adaptive immune systems and their interactions. The use of natural products, such as silibinin (SB), can contribute to the control of this inflammation and gestational success. The present study evaluated whether the flavonoid SB has an in vitro immunomodulatory effect on the signal transducers and transcription activators (STATs) signaling pathway and transcription factors of CD4

Made available in DSpace on 2018-12-11T17:17:54Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-05-01 Pre-eclampsia (PE) is a human pregnancy syndrome with abnormal activation of the innate immune response. The study evaluated the involvement of molecular structures called damage-associated molecular patterns (DAMPs), such as hyaluronan (HA) and heat shock proteins (Hsp) on NLRP1 and NLRP3 inflammasomes activation in peripheral blood monocytes. Twenty pre-eclamptic women, 20 normotensive pregnant women (NT) and 20 non-pregnant women (NP) were studied. Enzyme immunoassay was employed for the determination of HA, Hsp70 and High mobility group Box 1 (HMGB1) in plasma, as well as for the detection of Interleukin-1β (IL-1β), IL-18 and tumor necrosis factor alpha (TNF-α) in the supernatant of monocytes cultured with or without HA and Hsp70. The inflammasomes induction was evaluated by the quantification of mRNA for NLRP1, NLRP3, caspase-1, IL-1β IL-18, HMGB1 and TNF-α by qPCR in monocyte culture. The results showed significantly higher plasma levels of HA, Hsp70 and HMGB1 in pre-eclamptic women than in NT and NP women. Monocytes from women with PE showed endogenous activation of NLRP1 and NLRP3 inflammasomes, and expressed high amounts of IL-1β IL-18, HMGB1 and TNF-α. The stimulation of monocytes with HA increased the gene expression of NLRP1, NLRP3, caspase-1, TNF-α IL-1β HMGB1 and IL-18 and the production of IL-1β in pre-eclamptic women. Monocytes cultured with Hsp70 produced elevated levels of IL-1β and TNF-α through a mechanism independent of inflammasomes activation. These results suggest the participation of these DAMPs in the systemic inflammatory response that is characteristic of PE. Department of Microbiology and Immunology Institute of Biosciences Botucatu São Paulo State University – UNESP Department of Gynaecology and Obstetrics Botucatu Medical School Botucatu São Paulo State University – UNESP Department of Microbiology and Immunology Institute of Biosciences Botucatu São Paulo State University – UNESP Department of Gynaecology and Obstetrics Botucatu Medical School Botucatu São Paulo State University – UNESP

Made available in DSpace on 2022-04-29T08:38:07Z (GMT). No. of bitstreams: 0 Previous issue date: 2022-02-01 Preeclampsia (PE) is characterized by abnormal activation of the immune system. The intense systemic inflammatory reaction, could be related to the presence of molecules released after cell stress or death, that are capable of inducing inflammation and are known as damage-associated molecular patterns (DAMP). This study evaluated the profile of T cells through the analysis of transcription factors and the cytokines produced after culture with or without DAMPs: heat shock protein 70 (Hsp70), hyaluronan (HA) and monosodium urate (MSU). Twenty pregnant women with PE, 20 normotensive (NT) pregnant women and 20 non-pregnant (NP) women were studied. The results showed polarization toward Th1/Th17 and a decrease in Th2/Treg profiles in preeclamptic women associated with elevated levels of TNF, IFN-γ, and IL-17A and diminished levels of TGF-β1 and IL-10 when compared to the normotensive group. In addition, preeclamptic women had a higher percentage of cells co-expressing T-bet/GATA-3 and T-bet/RORγt and fewer T-bet/FoxP3 cells when compared to normotensive group. MSU induced an increase in IFN-γ and IL-22 in all studied groups. MSU, HA, and Hsp70 induced significant higher production of TNF in the PE and NP groups. The PE group showed elevated levels of TGF-β1 after incubation with MSU, HA, and Hsp70, whereas HA and Hsp70 decreased TGF-β1 production in NT group. The results suggest that these alarmins may play a role in the activation of innate and adaptive immune systems by skewing CD4 + T cells and increasing the release of inflammatory cytokines, thereby contributing to the pathogenesis of this important syndrome. Department of Chemistry and Biological Sciences Institute of Biosciences Sao Paulo State University - Unesp Department de Gynecology and Obstetrics Botucatu Medical School Sao Paulo State University - Unesp Department Health Science Oeste Paulista University (UNOESTE) Department of Chemistry and Biological Sciences Institute of Biosciences Sao Paulo State University - Unesp Department de Gynecology and Obstetrics Botucatu Medical School Sao Paulo State University - Unesp

Made available in DSpace on 2022-04-29T08:36:39Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-01-01 Vitamin D (VD) is a multifunctional prohormone and low VD status in pregnancy may contribute to the risk of adverse perinatal outcomes, such as preeclampsia (PE). This molecule may modulate the polarization of T cell subsets during gestation. This study evaluated the in vitro immunomodulatory effect of VD [1,25(OH)2D3] on the gene expression of transcription factors and on cytokine production by T cell subsets. Twenty pregnant women with PE and twenty normotensive (NT) pregnant women were studied. Plasma concentration of VD, [25(OH)D3], was evaluated by chemiluminescence. PBMCs from preeclamptic and NT pregnant women were cultured in the absence or presence of VD to determine gene expression of T-bet (Th1), GATA-3 (Th2), RORγt, and RUNX1 (Th17), FoxP3 (regulatory T cell- Treg), and the receptors of VD (VDR) and IL-23 (IL-23R) by quantitative PCR. The concentration of cytokines in the PBMC supernatant culture was determined by cytometric bead array and ELISA immunoassay. The results showed that plasmatic levels of VD were significantly lower in the PE group. The treatment of PBMCs from PE pregnant women with VD induced downregulation of genes related to inflammatory profiles (Th1 and Th17), as well as an increase of the Th2 and Treg profiles. Thus, VD treatment decreased the release of IFN-γ, TNF-α, IL-17, IL-6, and IL-23 while it increased the levels of IL-10 in the PE group. VD induces an immunomodulatory effect in T cell subsets from pregnant women with PE, polarizing these cells to an anti-inflammatory and regulatory profile. Department of Gynecology and Obstetrics Botucatu Medical School Sao Paulo State University-UNESP Department of Chemistry and Biological Sciences Institute of Biosciences Sao Paulo State University-UNESP Department of Gynecology and Obstetrics Botucatu Medical School Sao Paulo State University-UNESP Department of Chemistry and Biological Sciences Institute of Biosciences Sao Paulo State University-UNESP

Made available in DSpace on 2019-10-06T16:38:00Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-06-15 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Preeclampsia (PE) is a pregnancy-specific syndrome characterized by a systemic inflammatory response that polarizes peripheral blood monocytes to the M1 phenotype. The classically activated M1 monocytes comprise immune effector cells with an acute inflammatory phenotype. CD163 is a scavenger receptor expressed by monocytes/macrophages that may be shed from their cell membrane after proteolytic cleavage, producing the soluble CD163 molecule (sCD163). This study evaluated CD163 expression by monocytes and sCD163 as well as pro- and anti-inflammatory cytokine concentration in the plasma of pregnant women with PE. Fifty-six women with PE and 28 normotensive pregnant women were included. Plasma levels of sCD163, interleukin-1 beta (IL-1β), IL-6, IL-10, transforming growth factor beta (TGF-β1), and tumor necrosis factor-alpha (TNF-α) were determined by ELISA, and CD163 expression by monocytes was assessed by flow cytometry. The expression of CD163 by monocytes was significantly lower in severe and mild PE than in normotensive pregnant. Plasma concentrations of IL-1β, TGF-β1, and TNF-α were higher in severe PE than in mild PE and normotensive pregnant women. Both groups of preeclamptic women showed decreased plasma levels of sCD163 and IL-10. Negative correlations between sCD163 and IL-1β (r = − 0.45; P = 0.014) and between sCD163 and TNF-α concentrations (r = − 0.54; P = 0.001) were observed in the severe PE group. The association between the pro-inflammatory cytokine profile and lower concentrations of sCD163 and IL-10 in plasma from women with severe PE suggests an impairment in the modulation of the systemic inflammatory response in this group of pregnant women with preeclampsia. Department of Microbiology and Immunology Institute of Biosciences of Botucatu São Paulo State University UNESP, Rubião Junior s/n Department of Gynecology and Obstetrics Botucatu Medical School São Paulo State University UNESP Department of Microbiology and Immunology Institute of Biosciences of Botucatu São Paulo State University UNESP, Rubião Junior s/n Department of Gynecology and Obstetrics Botucatu Medical School São Paulo State University UNESP FAPESP: 2010/20207-0 FAPESP: 2013/12260-7

Preeclampsia (PE) is a human pregnancy-specific syndrome with abnormal activation of cells from the innate immune system. The present study evaluated whether silibinin (SB) treatment of monocytes from preeclamptic women could modulate NLRP1 and NLRP3 inflammasomes as well as TLR4/NF-&kappa, B pathway activation. Peripheral blood monocytes from 20 preeclamptic and 20 normotensive (NT) pregnant women, as well as the THP-1 cell line, were cultured with or without monosodium urate (MSU) or SB. NLRP1, NLRP3, Caspase-1, TLR4, MyD88, NF-&kappa, B, IL-1&beta, IL-18, TNF-&alpha, and IL-10 gene expression by monocytes was analysed by quantitative real-time polymerase chain reaction (qPCR), while inflammatory cytokine production and p65NF-&kappa, B activity were determined by enzyme-linked immunosorbent assays (ELISAs). TLR4/MyD88/NF-&kappa, B and NLRP1/NLRP3 inflammasomes pathways in THP-1 cells were evaluated by flow cytometry and western blot respectively. Compared with NT women, monocytes from preeclamptic women showed The Ethics Committee of the Botucatu Medical School approved the study (protocol number 2.333.216)higher endogenous activation of NLRP1/NLRP3 inflammasomes and the TLR4/NF-&kappa, B pathway as well as higher gene and protein expression of IL-1&beta, IL-18 and TNF-&alpha, and lower expression of IL-10. Monocyte stimulation with MSU increased inflammation-related genes as well as NF-&kappa, B activity. In vitro, SB treatment of monocytes from preeclamptic women reduced the basal activation of these cells by decreasing NLRP1/NLRP3 inflammasomes and p65NF-&kappa, B activity. THP-1 cells exhibited a similar immunological response profile to monocytes from preeclamptic women when cultured with or without MSU or SB. These results suggest uric acid participates in the systemic inflammatory response characteristic of preeclampsia and that in vitro SB treatment can modulate the sterile inflammation established in monocytes from preeclamptic women.

Made available in DSpace on 2021-06-25T11:05:11Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-12-01 Preeclampsia (PE) is a pregnancy-specific syndrome featuring intense activation of circulating monocytes and an imbalance between pro- and anti-inflammatory cytokines. The present study evaluated the immunomodulatory effect of silibinin (Sb) on the expression of surface markers and the nuclear transcription factor NF-κB signalling pathway of monocytes from preeclamptic women. Monocytes were cultured with or without Sb, and the mean fluorescence intensity of the surface molecules TLR4, CD64, and CD163 as well as the intracellular transcription factors IκB-α and NF-κBp65 was analysed by flow cytometry. The concentration of cytokines in the monocyte culture supernatant was determined by cytometric bead array and ELISA immunoassay. The results showed that the in vitro treatment of monocytes from preeclamptic women with Sb downregulated the endogenous activation of NF-κB and the expression of surface receptors TLR4 and CD64, and reduced the synthesis of the pro-inflammatory cytokines interleukin 1 (IL-1β), IL-6, IL-8, IL-12p70, IL-23, and tumour necrosis factor alpha (TNF-α) compared with cultures not treated with Sb. The presence of this flavonoid in monocyte cultures increased the expression of CD163 and IκBα and the release of IL-10 and transforming growth factor beta (TGF-β) in the culture supernatants, polarising these cells from the M1-like profile to the M2-like profile. The anti-inflammatory activity of Sb on the NF-κB activation pathway and induction of cell polarisation to the M2 profile was confirmed by an in vitro assay using monocytes from healthy, non-pregnant women. Treatment of monocytes from preeclamptic women with Sb polarises the cells to the M2-like phenotype, suggesting that this flavonoid has an immunomodulatory effect on the sterile inflammation characteristic of PE. Botucatu Medical School Sao Paulo State University (Unesp) Department Health Science Oeste Paulista University (UNOESTE), Jau Institute of Biosciences of Botucatu Sao Paulo State University (Unesp) Botucatu Medical School Sao Paulo State University (Unesp) Institute of Biosciences of Botucatu Sao Paulo State University (Unesp)

Made available in DSpace on 2018-12-11T17:21:58Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-10-01 Background: The placenta is a multifunctional organ that can suffer with imbalances between pro- and antioxidant molecules, contributing for inflammatory imbalance. The inflammation generated by oxidative stress may induce inflammasome activation, an essential complex for pro-inflammatory cytokine production. Objective: The aim of this study was to evaluate whether hydrogen peroxide (H2O2) mediated oxidative stress induces inflammasome activation on placental explants. Study design: Tissue cultures of placental explants obtained from normotensive pregnant women were performed in different concentrations of H2O2. Gene expressions of NLRP3, caspase-1, IL-1β TNF-α and IL-10 were evaluated by qPCR. Superoxide dismutase (SOD), catalase, Heat shock protein 70 (Hsp70), Caspase-1, TNF-α IL-1β IL-10 and human Chorionic Gonadotropin (hCG) were determined by ELISA. Results: Concentrations of catalase, Hsp70, hCG and SOD were higher in cultures with 100 and 1000 µM H2O2 compared to controls. Gene and protein expressions of TNF-α and IL-1β were elevated in cultures with 1000 μM H2O2 compared to controls. This concentration led to inflammasome activation, by increasing gene expressions of NLRP3, caspase-1 and IL-1β. In contrast, gene and protein expressions of IL-10 were reduced at 100 and 1000 μM H2O2. Protein expression of caspase-1 was higher in cultures of 100 μM H2O2 compared to controls. Treatment with Glybenclamide at 200 μM was used to prevent NLRP3 inflammasome activation. This concentration reduced protein expression of caspase-1 compared to culture with only H2O2 and control cultures. Conclusions: Our results confirm that H2O2 induces oxidative stress on placental explants and demonstrate that cell responses to this stress involve inflammasome activation. Department of Gynaecology and Obstetrics Medical School Botucatu Sao Paulo State University (Unesp) Department of Microbiology and Immunology Institute of Biosciences Botucatu Sao Paulo State University (Unesp) Department of Gynaecology and Obstetrics Medical School Botucatu Sao Paulo State University (Unesp) Department of Microbiology and Immunology Institute of Biosciences Botucatu Sao Paulo State University (Unesp)

Introduction Pregnancies complicated by preeclampsia (PE) are associated with placental oxidative stress and related to the activation of NLRP3 inflammasome, an important complex for activation of caspase-1, interleukin-1 beta (IL-1β) and high-mobility group box-1 (HMGB1). The hyperactivation of NLRP3 inflammasome in placental tissues may be involved in the huge systemic inflammatory state in PE, which could be modulate by administration of substances with anti-inflammatory properties such as vitamin D (VD). Objectives This study aimed to evaluate vitamin D immunomodulatory effect on the NLRP3 inflammassome in placental explants from normotensive (NT) and preeclamptic women cultured with hydrogen peroxide. Methods Placental explants from 7 PE and 7 NT women in the third trimester of pregnancy were obtained from women undergoing elective cesarean section. Explants were cultured with or without H2O2 and H2O2 plus VD. NLRP3, HMGB1, caspase-1 and IL-1β gene expression were analysed by qPCR. Protein expression were determined by western blotting or ELISA. Results Endogenous gene expression of NLRP3 and HMGB1 were significantly higher in PE than in NT explants. NLRP3, caspase-1, IL-1β and HMGB1 gene expression were higher in cultures treated with H2O2 compared to control cultures, while explants treated with H2O2 plus VD led to lower gene expression in NT and PE groups. High protein expression of NLRP3, caspase-1, IL-1β and HMGB1 was detected in explants cultured with H2O2 compared to controls whereas cultures treated with H2O2 plus VD decreased these protein expression compared to H2O2 stimuli. Conclusion Vitamin D was able to decrease the gene and protein expression of NLRP3 inflammasome and HMGB1 production in placental explants cultured with H2O2 in PE and NT groups. These results suggest that vitamin D may play an immunomodulatory role in the systemic inflammatory response present in preeclampsia. Financial support FAPESP 2016/18155-9 and 2016/23452-2.

Pre-eclampsia (PE) is an obstetric pathology characterized by abnormal activation of the innate and adaptive immune systems dependent on the imbalance of T helper subsets. The present study aimed to evaluate the gene and protein expression of T helper type 1 (Th1)/Th2/Th17/regulatory T (Treg) cell transcription factors in peripheral blood lymphocytes from pregnant women with PE employing quantitative RT-PCR and flow cytometry techniques, as well as the cytokine profile produced by these CD4+ T-cell subsets in the plasma of pregnant women with PE, classified as early-onset PE (n = 20), late-onset PE (n = 20) and normotensive pregnant women (n = 20). Results showed a higher percentage of CD4+ T cells expressing the RORc transcription factor (Th17) and a lower percentage of cells expressing FoxP3 (Treg) in women with early-onset PE compared with late-onset PE and normotensive groups. A lower gene expression of GATA-3 transcription factor was detected in cells of women with early-onset PE compared with the late-onset PE group. Endogenous plasma levels of interleukin-6 (IL-6), IL-17 and tumour necrosis factor-α were significantly higher in the early-onset PE group than in the late-onset PE and normotensive groups, whereas IL-4 (Th2 profile) and IL-22 (Th17 profile), were not significantly different between the studied groups. The endogenous levels of transforming growth factor-β and IL-10 were significantly lower in the pre-eclamptic than in the normotensive groups of the same gestational age, with a significant difference between early- and late-onset PE. The results show that in women with PE there is an imbalance between inflammatory and anti-inflammatory profiles in CD4+ T-cell subsets, with polarization to Th17 profiles in the early-onset PE, considered as the severe form of PE.

Introduction Preeclampsia (PE) is a pregnancy specific syndrome characterized by a systemic inflammatory response. An imbalance between Th17 and regulatory T cell detected in this pathology seems to be dependent on the deficiency of regulatory factors capable of modulating this inflammation. Vitamin D generates an immune tolerogenic status through its regulatory activities on the inflammatory response. Objectives The present study aimed to evaluate the plasma levels of vitamin D, cytokines and to analyze the profile of CD4+ T cells subsets as well as the expression of vitamin D receptor (VDR) in peripheral blood mononuclear cells from pregnant women with PE. Methods Twenty women with early-onset PE, 20 women with late-onset PE and 20 normotensive (NT) pregnant women were studied. Plasma concentrations of vitamin D was evaluated by chemiluminescence and the levels of IL-10, IL-17 and TNF-a were determined by ELISA. Gene expression of VDR, transcription factors RORc (Th17) and FoxP3 (Treg) were evaluated by qPCR. Results were analyzed by non-parametric tests at 5% significance level. Results Plasma concentration of vitamin D was significantly lower in early-onset PE group compared to late-onset PE and NT groups, while the levels of TNF-α and IL-17 were significantly higher in early-onset PE group. On the other hand, IL-10 levels were significantly lower in both PE groups than in NT group. Gene expression of VDR was significantly decreased in early-onset PE group compared to NT group, while higher expression of mRNA RORc and lower expression of mRNA FoxP3 were observed in preeclamptic women. Positive correlation between vitamin D vs VDR and VDR vs FoxP3 was observed in PE (r = 0.44, p Conclusion In preeclamptic women there is a decrease in regulatory status, suggesting a deficient immunomodulatory role of vitamin D that result in higher inflammatory profile, more evident in the early-onset PE. This imbalance in maternal tolerance indicate that vitamin D may participate in the pathogenesis of the disease.

Introduction Preeclampsia (PE) is characterized by a state of abnormal activation of the innate immune system. It’s associated with hyperuricemia and elevated serum levels of inflammatory cytokines. Uric acid crystals can activate inflammasome, a multiprotein structure important for processing and release of inflammatory cytokines. The imbalance between pro- and anti-inflammatory cytokines in PE seems to be dependent on the deficiency of regulatory factors capable of modulating inflammatory response, which could be alleviated by administration of substances with anti-inflammatory and modulatory properties such as silibinin (SB). Objective The study aimed to evaluate the modulatory effect of silibinin on NLRP1 and NLRP3 inflammasomes and on NF-κB pathway activation in monocytes from preeclamptic women stimulated with monosodium urate (MSU). Methods Twenty preeclamptic women, 20 normotensive pregnant women (NT) and THP-1 cells were cultured with or without SB and MSU. Gene expression of NLRP1, NLRP3, Caspase-1, TLR4, MyD88, NF-κB, IL-1β, IL-18, TNF-α and IL-10 was performed by qPCR. Inflammatory cytokines and p65NF-κB activity were evaluated by ELISA. Results Monocytes of preeclamptic women presented higher endogenous activation of the inflammatory genes, as well as p65NF-κB basal activity and the inflammatory cytokines production than NT group. MSU stimulation increased the expression of these parameters, whereas SB treatment reduced them. Additionally, THP-1 cells had a similar immunological response profile of monocytes from preeclamptic women when cultured with or without MSU and SB. Conclusion These results suggest the MSU participation in the systemic inflammatory response, characteristic of preeclampsia, and that silibinin treatment is capable of modulating the sterile inflammation established in monocytes of preeclamptic women, demonstrating that this flavonoid plays a relevant role in the regulation of the inflammatory response in preeclampsia. Financial support FAPESP 2015/26147-3, 2016/18155-9 and 2016/22854-0

Introduction Preeclampsia is a specific pregnancy syndrome characterized by a systemic inflammatory response associated with the production of proinflammatory cytokines and with the presence of molecular structures associates with stress and cell death and called ”danger signals” or damage associated molecular patterns (DAMPs). HMGB1 is a proinflammatory cytokine and considered as DAMP, can interact with the acute phase protein haptoglobin, an endogenous HMGB1 inhibitor, forming complexes that can be removed from the circulation by interaction with CD163 receptor, present on the surface of monocytes/macrophages. Objective This study aimed to analyze the levels of haptoglobin, HMGB1 and inflammatory cytokines in the plasma of pregnant women with PE. Methods The plasma was obtained from 90 pregnant women, 30 pregnant women with severe preeclampsia (severe PE), 30 pregnant women with mild preeclampsia (mild PE) and 30 normotensive pregnant women (NT). The concentrations of haptoglobin, HMGB1 and IL-1β, IL-6, IL-10, TGF-β and TNF-α cytokines were determined by ELISA. The results were analyzed using parametric tests at 5% significance level. Results Plasma levels of haptoglobin and IL-10 were significantly lower in the severe PE group whereas the concentrations of TNF-α, IL-1β and TGF- β were higher in the severe PE group in comparison to the mild PE and NT. On the other hand, the results of HMGB1 and IL-6 only showed difference between pregnant women with PE and NT. Discussion The high endogenous concentration of HMGB1, IL-1β, IL-6, TGF-β and TNF-α in the plasma of pregnant women with PE confirms the inflammatory activation profile of this pathology, while the lower concentrations of haptoglobin and IL-10 suggest a deficiency in a mechanism capable of correcting this inflammatory process in PE.

Introduction Preeclampsia (PE) is a specific pregnancy syndrome characterized by an imbalance between the inflammatory T cell (Th1 and Th17) and the anti-inflammatory (Th2 and Treg) profiles. It seems to be dependent on the deficiency of regulatory factors such as vitamin D (VD), which exerts modulatory effects on cells of the adaptive immune system, especially T cells. Objective The study aimed to evaluate the modulatory effect of VD on T cell subsets and the production of cytokines in preeclamptic women. Methods Peripheral blood mononuclear cells obtained from 12 pregnant women with PE and 12 NT were cultured with or without VD. The cytokines IL-6, IL-10, IL-17 and TNF- α were evaluated by cytometric bead array (CBA) or ELISA. The gene expression of the transcription factors T-bet (Th1), GATA-3 (Th2), ROR γ t and RUNX1 (Th17), FoxP3 (Treg) and vitamin D receptor (VDR) were determined by the qPCR. Results Endogenous mRNA levels of T-bet, ROR γ t and RUNX1 were significantly higher, whereas GATA-3 and FoxP3 mRNA levels were lower in PE group when compared to NT women. Cell treatment with VD decreased the gene expression of T-bet, ROR γ t and RUNX1 and increased that of GATA-3, FoxP3 and VDR in preeclamptic women. Regarding the endogenous cytokines production IL-17 and TNF- α were significantly higher, while IL-10 was decreased in PE when compared to NT group. VD treatment decreased the production of IL-17 and led to a tendency of decreased levels of IL-6 and TNF- α in PE group. On the other hand, IL-10 levels increased significantly after treatment with VD in both groups. Discussion VD treatment increased GATA-3, FoxP3 and IL-10, demonstrating that this molecule is able to polarize the inflammatory response present in PE to the anti-inflammatory profile. It confirms the important immunomodulatory effect of VD in this disease. Financial support FAPESP 2016/23450–0 e 2016/18155–9

Introduction Preeclampsia (PE) is a pregnancy specific syndrome, characterized by abnormal levels of cytokines and angiogenic factors, suggesting that immunological factors play a role in the disease development. Objective The objective of this study was to determine whether immunological markers are associated with the gestational age and with the disease severity at the time of the disease diagnosis. Methods Ninety-five women who developed PE were stratified for gestational age as preterm PE ( α ) and Interleukin 10 (IL-10) were determined by immunoenzymatic assay (ELISA). Results were analyzed by non-parametric test with significance set at p Results The comparison between preterm and term PE showed higher percentage of severe cases in the preterm PE (76.7%) than in the term PE (23.3%) group. Similarly the concentrations of TNF- α , TNF- α /IL-10 ratio, sFlt-1, sEng and sFlt-1/PlGF ratio were significantly higher in the preterm PE group. On the other hand, concentrations of PlGF, VEGF and IL-10 were significantly lower in the preterm PE group. Negative correlations between TNF- α and IL-10 (r = −0.5232; p Discussion Pregnant women with preterm PE showed an imbalance between immunological markers, with predominance of anti-angiogenic factors and TNF- α , associated with adverse maternal clinical outcomes.

Introduction Preeclampsia (PE) is a systemic disease characterized by multiple alterations in the maternal organism and clinically identified by hypertension and proteinuria that manifest in the second half of gestation. Studies indicate that PE has an exacerbated inflammatory response and a state of maladaptation of immunological tolerance, characterized by abnormal activation of the innate and adaptive immune system. Objectives This study characterized the Th cell subsets (Th1, Th2, Th17 and Treg) in pregnant women with PE and normotensive (NT) pregnant women. Methods Thirty-eigth pregnant women were studied, 20 women with PE and 18 NT, matched by gestational age. Mononuclear cells from peripheral blood obtained from preeclamptic and NT women were analyzed for the endogenous expression of transcription factors involved in the characterization of Th cell subsets. The expression of intracytoplasmic transcription factors T-bet (Th1), GATA-3 (Th2), ROR γ T (Th17) and FoxP3 (Treg) were evaluated by flow cytometry, using specific monoclonal antibodies labeled with fluorochromes. The gene expression of these transcription factors were determined by quantitative real-time polymerase chain reaction (RT-qPCR). Differences between groups were analyzed by non-parametric tests with significance level set at 5%. Results Flow cytometry analysis demonstrated an inflammatory profile in PE group. Th1 and Th17 subsets showed higher media fluorescence intensity (MFI) and percentage of cells expressing the transcription factor T-bet and ROR γ T respectively, compared with NT women. The anti-inflammatory Th2 profile was decreased in percentage of cells expressing GATA-3, but MFI in PE was elevated when compared to NT. No significant difference of MFI was observed in Treg profile between groups, but the percentage of lymphocytes expressing FoxP3 was increased in NT than in PE group. The gene expression of the transcription factors demonstrated greater values of T-bet and ROR γ T in women with PE when compared to the NT. Significantly lower expression of GATA-3 and FoxP3 genes were detected in preeclamptic women than in NT group. Conclusion The increase of inflammatory profiles Th1 and Th17 and the decrease of anti-inflammatory subsets Th2 and Treg both in gene expression and intracytoplasmic transcription factors in women with PE suggest that there is an imbalance in maternal tolerance in PE and confirms the activation of inflammatory profile in subpopulations of Th cells in the pathogenesis of the disease. Financial support – FAPESP 2014/25124-7 and 2012/24697-8.