Progesterone modulates TNF receptors expression by Jurkat cells cultured with plasma from pregnant women with preeclampsia.

Pregnant women with preeclampsia (PE) present a shift in the immune response to an inflammatory profile. This deviation could be due to the interaction of tumor necrosis factor (TNF) with TNFR1 and TNFR2 receptors, besides the failure in modulation of inflammation regulatory mechanisms. This study evaluated the effects of progesterone on the expression of TNFR1 and TNFR2 by Jurkat cells after stimulation with plasma from PE and normotensive (NT) pregnant women. Jurkat cells were cultured with or without progesterone in a medium containing 20% (v/v) plasma from PE or NT women. The expression of TNF receptors was evaluated by flow cytometry. The concentration of soluble forms of TNF receptors and cytokines was determined in culture supernatant and plasma by ELISA. The plasma of PE women showed significantly higher concentrations of sTNFR1 and TNF and lower concentrations of sTNFR2 compared to the NT group. TNFR1 receptor expression was increased in Jurkat cells, while TNFR2 was decreased after culture with PE plasma when compared with Jurkat cells cultured with progesterone and plasma from NT women. The concentration of sTNFR1, TNF, and IL-10 in the culture supernatant of Jurkat cells was increased after culture with PE plasma, while the sTNFR2 receptor was decreased when compared to the NT group. Results demonstrate that in preeclamptic women a systemic inflammation occurs with an increase of inflammatory molecules, and progesterone may have a modulating effect on the expression of TNF receptors, shifting Jurkat cells towards an anti-inflammatory profile with greater expression of TNFR2.

Plasma eNOS Concentration in Healthy Pregnancy and in Hypertensive Disorders of Pregnancy: Evidence of Reduced Concentrations in Pre-Eclampsia from Two Independent Studies.

Hypertensive disorders of pregnancy (HDP), comprising gestational hypertension (GH) and pre-eclampsia (PE), are leading causes of maternal and perinatal morbidity and mortality. Both GH and PE are characterized by new-onset hypertension, but PE additionally includes proteinuria and/or end-organ damage. Impaired nitric oxide (NO) bioavailability may lead to endothelial dysfunction in GH and PE, and the primary source of vascular NO is endothelial NO synthase (eNOS). However, no previous study has investigated plasma eNOS concentrations in patients with GH and PE. In this study, we compared plasma eNOS concentrations in healthy pregnancies and HDP in two independent cohorts. The primary study included 417 subjects, with 43 non-pregnant (NP) and 156 healthy pregnant (HP) women and 122 patients with GH and 96 with PE. The replication study included 85 pregnant women (41 healthy and 44 pre-eclamptic). Plasma concentrations of eNOS were measured using a commercial ELISA kit provided by R&D Systems, and plasma nitrite concentrations were assessed using two ozone-based chemiluminescence assays. Correlations between plasma eNOS concentrations and plasma nitrite concentrations, as well as clinical and biochemical parameters, were evaluated by either Spearman's or Pearson's tests. In the primary study, NP women and HDP had significantly lower plasma eNOS concentrations compared to HP; concentrations were even lower in PE compared to GH. Plasma eNOS concentrations were reduced but not significant in early-onset PE, PE with severe features, preterm birth, and intrauterine growth restriction. No correlation was observed between plasma eNOS and nitrite levels. In HDP, there was a significant positive correlation between levels of eNOS and hemoglobin (r = 0.1496, p = 0.0336) as well as newborn weight (r = 0.1487, p = 0.0316). Conversely, a negative correlation between eNOS levels and proteinuria was observed (r = -0.2167, p = 0.0179). The replication study confirmed significantly reduced plasma concentrations of eNOS in PE compared to HP. Our findings provide evidence of reduced plasma eNOS concentrations in HDP; they were particularly lower in PE compared to GH and HP in two independent studies.

Modulatory effect of two regimens of magnesium sulfate on the systemic inflammatory response in pregnant women with imminent eclampsia.

OBJECTIVE: This study compared the modulatory effect of two intravenous magnesium sulfate (MgSO4) regimens on the systemic inflammatory response in pregnant women diagnosed with imminent eclampsia. STUDY DESIGN: In a single-blind cross-sectional study, 33 women were allocated according to the Zuspan (n = 16) and Sibai (n = 17) MgSO4 regimens, and treated for 24 h. Blood samples were collected pre-administration of the loading dose, at 24 h of the maintenance dose of MgSO4, and at 48 h, when patients were without treatment. Plasma was used to determine interleukin (IL)-1 beta (IL-1ß), IL-6, IL-10, tumor necrosis factor-alpha (TNF-α), heat shock protein (Hsp70), and heme oxygenase-1 (HO-1) by ELISA. RESULTS: The treatment with the Zuspan's regimen didn't change plasma concentrations of TNF-α, IL-10, and Hsp70 in the three-time points studied. However, it decreased IL-1ß at 24 h and 48 h and IL-6 at 48 h, and increased HO-1 concentration at 48 h. On the other hand, compared to the pre-treatment period, Sibai's regimen induced a significant decrease in TNF-α, IL-1ß, IL-6, and Hsp70, while increased HO-1 levels both at 24 h and 48 h and, IL-10 concentration at 48 h. CONCLUSIONS: Sibai's regimen determined an early and efficient immunoregulatory effect on systemic inflammatory response in preeclampsia, suggesting that the maintenance dose of two grams of MgSO4 was better than one gram in the treatment of imminent eclampsia.

Interactions between TLR2, TLR4, and mannose receptors with gp43 from Paracoccidioides brasiliensis induce cytokine production by human monocytes.

The glycoprotein gp43 is an immunodominant antigen secreted by Paracoccidioides brasiliensis, the agent of paracoccidioidomycosis. The present study evaluated whether gp43 can interact with toll-like (TLR2, TLR4) and mannose (MR) receptors on the surface of human monocytes, and how that affects their expression and cytokine production. Monocytes were incubated with or without monoclonal antibodies anti-TLR2, anti-TLR4, or anti-MR, individually or in combination, prior to the addition of gp43. The gp43 binding to monocyte surface, as well as expression of TLR2, TLR4, and MRs were analyzed by flow cytometry, while production of TNF-α and IL-10 was monitored by ELISA. The results suggested that gp43 binds to TLR2, TLR4, and MR receptors, with TLR2 and MR having the strongest effect. All three receptors influenced the production of IL-10, while TNF-α production was associated with expression of TLR4 and MR. The modulatory effect of gp43 was demonstrated by high levels of TLR4 expression associated with increased production of TNF-α after 4 h of culture. Alternatively, high levels of TLR2 expression, and elevated production of IL-10, were detected after 18 h. We showed that interaction between gp43 and monocytes may affect the innate immune response by modulating the expression of the pattern recognition receptors TLR2, TLR4 and MR, as well as production of pro- and anti-inflammatory cytokines.

Antiphospholipid Antibodies Inhibit Trophoblast Toll-Like Receptor and Inflammasome Negative Regulators.

OBJECTIVE: Women with antiphospholipid antibodies (aPL) are at risk for pregnancy complications associated with poor placentation and placental inflammation. Although these antibodies are heterogeneous, some anti-ß2 -glycoprotein I (anti-ß2 GPI) antibodies can activate Toll-like receptor 4 (TLR-4) and NLRP3 in human first-trimester trophoblasts. The objective of this study was to determine the role of negative regulators of TLR and inflammasome function in aPL-induced trophoblast inflammation. METHODS: Human trophoblasts were not treated or were treated with anti-ß2 GPI aPL or control IgG in the presence or absence of the common TAM (TYRO3, AXL, and Mer tyrosine kinase [MERTK]) receptor ligand growth arrest-specific protein 6 (GAS6) or the autophagy-inducer rapamycin. The expression and function of the TAM receptor pathway and autophagy were measured by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), Western blotting, and enzyme-linked immunosorbent assay (ELISA). Antiphospholipid antibody-induced trophoblast inflammation was measured by qRT-PCR, activity assays, and ELISA. RESULTS: Anti-ß2 GPI aPL inhibited trophoblast TAM receptor function by reducing cellular expression of the receptor tyrosine kinases AXL and MERTK and the ligand GAS6. The addition of GAS6 blocked the effects of aPL on the TLR-4-mediated interleukin-8 (IL-8) response. However, the NLRP3 inflammasome-mediated IL-1ß response was not affected by GAS6, suggesting that another regulatory pathway was involved. Indeed, anti-ß2 GPI aPL inhibited basal trophoblast autophagy, and reversing this with rapamycin inhibited aPL-induced inflammasome function and IL-1ß secretion. CONCLUSION: Basal TAM receptor function and autophagy may serve to inhibit trophoblast TLR and inflammasome function, respectively. Impairment of TAM receptor signaling and autophagy by anti-ß2 GPI aPL may allow subsequent TLR and inflammasome activity, leading to a robust inflammatory response.

Increased expression of NLRP3 inflammasome in placentas from pregnant women with severe preeclampsia.

Preeclampsia is a pregnancy disorder characterized by imbalance between pro- and anti-inflammatory cytokines associated with high plasma levels of uric acid and Interleukin-1 beta (IL-1ß). The inflammasome is a protein complex that mediates innate immune responses via caspase-1 activation promoting secretion of IL-1ß and IL-18 in their active forms, and also release of the high-mobility group box 1 protein (HMGB1). As the placenta seems to play an important role in the pathogenesis of PE, the present study investigated the expression of genes and proteins related to the inflammasome in placentas from pregnant women with severe preeclampsia. Placental tissue was collected from 20 normotensive pregnant women and 20 preeclamptic women, and inflammasome components, NLRP3 (NOD-like receptor family, pyrin domain-containing protein 3), caspase-1, IL-1ß and IL-18, as well as tumor necrosis factor-alpha (TNF-α) and HMGB1 were evaluated by immunohistochemistry, enzyme-linked immunosorbent assay (ELISA) and also quantified by reverse transcription-qPCR (RT-qPCR). Compared with normotensive pregnant women, placenta from women with PE showed a significant increase in NLRP3, caspase-1, IL-1ß, TNF-α and HMGB1 mRNA. Immunohistochemical staining of NLRP3, caspase-1, IL-1ß and TNF-α in placental villi, as well as the levels of caspase-1, IL-1ß, TNF-α and HMGB1 in placental homogenate were significantly higher in the preeclamptic group than in the normotensive group. However, mRNA expression of IL-18 and its protein concentrations were lower in placentas from preeclamptic women. The results suggest that placentas from pregnant women with preeclampsia show higher expression of NLRP3 inflammasome, which may be involved in the exaggerated inflammatory state in preeclampsia.

Association between Placental Lesions, Cytokines and Angiogenic Factors in Pregnant Women with Preeclampsia.

Preeclampsia (PE) is considered the leading cause of maternal and perinatal morbidity and mortality. The placenta seems to play an essential role in this disease, probably due to factors involved in its formation and development. The present study aimed to investigate the association between placental lesions, cytokines and angiogenic factors in pregnant women with preeclampsia (PE). We evaluated 20 normotensive pregnant women, 40 with early-onset PE and 80 with late-onset PE. Placental samples were analyzed for histopathology, immunohistochemistry and determination of granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-10 (IL-10), transforming growth factor-beta 1 (TGF-ß1), tumor necrosis factor-alpha (TNF-α), placental growth factor (PlGF), vascular endothelial growth factor (VEGF), fms-like tyrosine-kinase-1 (Flt-1) and endoglin (Eng) levels. Higher percentages of increased syncytial knots and increased perivillous fibrin deposits, and greater levels of TNF-α, TGF-ß1and Flt-1 were detected in placentas from early-onset PE. Levels of IL-10, VEGF and PlGF were decreased in PE versus normotensive placentas. Both the TNF-α/IL-10 and sFlt-1/PlGF ratios were higher in placental homogenate of early-onset PE than late-onset PE and control groups. The more severe lesions and the imbalance between TNF-α/IL-10 and PlGF/sFlt-1 in placentas from early-onset PE allows differentiation of early and late-onset PE and suggests higher placental impairment in early-onset PE.

Virulence profile of ten Paracoccidioides brasiliensis isolates: association with morphologic and genetic patterns.

Ten isolates of Paracoccidioides brasiliensis were examined for differences in virulence in outbred mice intravenously inoculated with the fungus, associated with mycelial morphology, and genetic patterns measured by random amplified polymorphic DNA (RAPD). Virulence was evaluated by viable yeast cell recovery from lungs and demonstration of histopathologic lesions in different organs. The results showed that the isolates presented four virulence degrees: high virulence, intermediate, low and non-virulence. RAPD clustered the isolates studied in two main groups with 56% of genetic similarity. Strains with low virulence, Pb265 or the non-virulent, Pb192, showed glabrous/cerebriform morphology and high genetic similarity (98.7%) when compared to the other isolates studied. The same was observed with Bt79 and Bt83 that shared 96% genetic similarity, cottony colonies and high virulence. The RAPD technique could only discriminate P. brasiliensis isolates according to glabrous/cerebriform or cottony colonies, and also high from low virulence strains. Isolates with intermediate virulence such as Pb18, Pb18B6, Bt32 and Bt56 showed variability in their similarity coefficient suggesting that RAPD was able to detect genetic variability in this fungal species. Virulence profile of P. brasiliensis demonstrated that both mycelial morphologic extreme phenotypes may be associated with fungal virulence and their in vitro subculture time. Thus, RAPD technique analysis employed in association with virulence, morphologic and immunologic aspects might prove adequate to detect differences between P. brasiliensis isolates.

Production of pro- and anti-inflammatory cytokines by monocytes from patients with paracoccidioidomycosis.

Monocytes and macrophages can produce a large repertoire of cytokines and participate in the pathogenesis of granulomatous diseases. We investigated the production of pro- and anti-inflammatory cytokines by monocytes from patients with active paracoccidioidomycosis. Peripheral blood monocytes from 37 patients and 29 healthy controls were cultivated with or without 10 microg/ml of lipopolysaccharide (LPS) for 18 h at 37 degrees C, and the cytokine levels were determined in the culture supernatants by enzyme immunoassay. The results showed that the endogenous levels of tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-1beta), IL-6, IL-8, IL-10 and transforming growth factor beta detected in the supernatant of patient monocytes cultivated without stimulus were significantly higher than those produced by healthy controls. These data demonstrated that monocytes from patients with active paracoccidioidomycosis produce high levels of cytokines with both inflammatory and anti-inflammatory activities. However, patient monocytes produced significantly lower TNF-alpha and IL-6 levels in response to LPS when compared to normal subjects, suggesting an impairment in their capacity to produce these cytokines after LPS stimulation. Concentrations of IL-1beta, IL-8 and IL-10 in cultures stimulated with LPS were higher in patients than in controls. These results suggest that an imbalance in the production of pro- and anti-inflammatory cytokines might be associated with the pathogenesis of paracoccidioidomycosis.

Monocytes from pregnant women with pre-eclampsia are polarized to a M1 phenotype.

PROBLEM: This study evaluated whether the monocyte inflammatory state in pre-eclampsia (PE) might be associated with polarization to either M1 classically or M2 alternatively activated monocyte subsets. METHOD OF STUDY: Eighty-five women with (PE) and 52 normotensive (NT) pregnant women matched for gestational age were included. Expression of surface receptors characteristic of M1, such as Toll-like receptor (TLR)2, TLR4, and CD64, or M2, such as CD163 and CD206 monocyte subsets were evaluated in peripheral blood monocytes by flow cytometry. Tumour necrosis factor-alpha (TNF-α), interleukin-(IL)-12p40, IL-12p70, and IL-10 were evaluated in the supernatant of monocyte cultures by ELISA. RESULTS: Expression of TLR4 and CD64 by monocytes from pre-eclamptic women was significantly higher, while the expression of CD163 and CD206 expression was significantly lower compared with NT pregnant women. Endogenous production of TNF-α, IL-12p40, and IL-12p70 by monocytes was increased, while synthesis of IL-10 was lower in women with PE than in NT pregnant women. CONCLUSIONS: Monocytes from women with PE are classically activated, producing higher levels of pro-inflammatory cytokines, and express surface receptors characteristic of the M1 subset. These results provide evidence that the systemic inflammatory environment in PE may differentiate and polarize these cells to the M1 phenotype.

sFlt-1/PlGF ratio as a prognostic marker of adverse outcomes in women with early-onset preeclampsia.

Soluble fms-like tyrosine kinase 1 (sFlt-1) is an anti-angiogenic factor released in higher amounts by preeclamptic placentas and it has been implicated in the endothelial dysfunction observed in the disease. In this study we evaluated if circulating sFlt-1/PlGF ratio is useful to predict adverse outcomes in women with early-onset preeclampsia. This is a cohort study of 88 preeclamptic women with singleton pregnancies at ⩽35weeks of gestation. According to definitions used, adverse outcomes occurred in 46.5% (N=43) of the patients. The median sFlt1/PlGF ratio (25th-75th centile) for all patients evaluated was of 42.26 (13.1-226.1). The median sFlt-1/PlGF ratio among women who had any adverse outcome (N=43) versus no adverse outcomes (N=45) was of 227.6 (80.3-346.1) versus 14.4 (3.35-30.0), (P<0.0001). According to our analyses a sFlt-1/PlGF ratio cut-point of ⩾85 gave a sensitivity of 74.0% and specificity of 97.0%. The positive predictive value and the negative predictive value were 96.0% and 80.0%, respectively. The median sFlt-1/PlGF ratio (25th-75th centile) for patients who delivered within <7days was 260.0 (127.7-404.7) as compared to 14.4 (3.35-34.97) for those patients who delivered within two weeks or more (P<0.0001). Our results suggest that sFlt-1/PlGF ratio is a promising marker for adverse outcomes in women with early-onset preeclampsia.

High levels of heat shock protein 70 are associated with pro-inflammatory cytokines and may differentiate early- from late-onset preeclampsia.

Preeclampsia (PE), a specific syndrome of pregnancy, can be classified into early and late onset, depending on whether clinical manifestations occur before or after 34 weeks' gestation. We determined whether plasma concentrations of Hsp60 and Hsp70 were related to circulating cytokine levels, as well as kidney and liver functions, in early- and late-onset PE. Two hundred and thirty-seven preeclamptic women (95 with early- and 142 with late-onset PE) were evaluated. Plasma levels of Hsp60, Hsp70, and their specific antibodies, tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1, IL-10, IL-12, and soluble TNF-α-receptor I (sTNFRI) concentrations, were determined by enzyme-linked immunosorbent assay (ELISA). Concentrations of Hsp70, TNF-α, IL-1ß, IL-12, and sTNFRI were significantly elevated in patients with early-onset PE compared with women with late-onset PE; IL-10 levels were significantly lower in the early-onset PE group. Concentrations of urea, uric acid, proteinuria, glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), and lactate dehydrogenase (LDH) were also significantly higher in early-onset PE. The percentage of infants with intrauterine growth restriction was also significantly higher in women with early-onset PE. There were positive correlations between Hsp70 levels and TNF-α, TNFRI, IL-1ß, IL-12, GOT, GPT, LDH, and uric acid concentrations in early-onset PE group. Thus, early-onset PE was associated with greater maternal and fetal impairment. There are differences in pathophysiology between early- and late-onset PE, highlighting by the difference in Hsp70 levels.

sFlt-1 and IP-10 in women with early-onset preeclampsia.

Intense inflammatory response and an anti-angiogenic state have been implicated in the pathogenesis of preeclampsia. Here, we investigated this hypothesis evaluating the serum concentrations of CXCL10/IP-10, sFlt-1, and PlGF in women with early-onset preeclampsia. CXCL10/IP-10 was measured by Cytometric Bead Array. sFlt-1 and PlGF were measured by automated electrochemiluminescence immunoassay. The median serum concentration of CXCL10/IP-10 was 109.5pg/mL in preeclamptic women, as compared with 52.28pg/mL in the controls (P=0.0028). The mean serum level of sFlt-1 was 13,636pg/mL in preeclamptic women, as compared with 2020pg/mL in the controls (P<0.0001). PlGF levels were significantly lower in women with preeclampsia.

Increased reactive oxygen species and tumor necrosis factor-alpha production by monocytes are associated with elevated levels of uric acid in pre-eclamptic women.

PROBLEM: To evaluate associations between hyperuricemia and increases in production of reactive oxygen species (ROS) and tumor necrosis factor alpha (TNF-α) in pre-eclamptic pregnancies. METHOD OF STUDY: This study investigated serum uric acid levels, monocyte production of TNF-α, superoxide anion (O(2)(-)) and hydrogen peroxide (H(2)O(2)), as well as superoxide dismutase (SOD) and catalase (CAT) activities in erythrocytes from 30 women with pre-eclampsia (PE) compared with 30 normotensive (NT) pregnant women in the last trimester of pregnancy. RESULTS: Serum uric acid levels (6.1 versus 2.8 mg/dL) as well as endogenous O(2)(-) (2.2 versus 1.6 nm), H(2)O(2) (1.8 versus 1.4 nm) and TNF-α (91.6 versus 40.4 pg/mL) released from monocytes were significantly higher in the pre-eclamptic group than in the NT group (P < 0.05). SOD activity in erythrocytes was also significantly elevated in the PE group (5969.2 versus 4834.7 U/g Hb). No significant difference between groups was observed in relation to CAT activity. CONCLUSIONS: Elevated serum uric acid levels are correlated with higher O(2)(-) and TNF-α production by monocytes in women with PE. This may contribute to the enhanced oxidative and inflammatory state characteristic of this disorder.

Virulence profile of ten Paracoccidioides brasiliensis isolates: Association with morphologic and genetic patterns

Dez isolados de P. brasiliensis foram avaliados em relação à patogenicidade por inoculação intravenosa em camundongos e associação com morfologia miceliana e padrão genético por amplificação genônica do DNA polimórfico (RAPD). A patogenicidade, avaliada por recuperação de fungos viáveis a partir de tecido pulmonar e por lesões histopatológicas em diferentes órgãos, mostrou que os isolados apresentaram quatro graus de virulência: alta virulência, virulência intermediária, baixa virulência e não virulência. A técnica de RAPD agrupou os isolados em dois grupos com 56% de similaridade genética. Amostras com baixa virulência Pb265 ou não virulência Pb192 apresentaram morfologia glabra/cerebriforme e alta similaridade genética (98,7%) quando comparadas com os outros isolados estudados. O mesmo foi observado com os isolados Bt79 e Bt83, que compartilharam 96% de semelhança genética, colônias cotonosas e alta virulência. Essa técnica pode discriminar apenas isolados com morfologia glabra da cotonosa e com alta e baixa virulência. Isolados com virulência intermediária como Pb18, Pb18B6, Bt32 e Bt54 mostraram variabilidade no coeficiente de similaridade, sugerindo que a técnica de RAPD permite mostrar variabilidade genética nessa espécie fúngica. O estudo do perfil de virulência das amostras de P. brasiliensis demonstrou que os dois fenótipos extremos de morfologia miceliana podem ser associados com a virulência do fungo e com o tempo de subcultivo in vitro. Assim, a análise de RAPD, utilizada em conjunto com aspectos de virulência, morfológicos e imunológicos pode ser considerada adequada para detectar diferenças entre isolados de P. brasiliensis.