Surfactant Components and Tracheal Aspirate Inflammatory Markers in Preterm Infants with Respiratory Distress Syndrome.

In 93 preterm infants ≤32 weeks of gestational age and 12 control infants, epithelial lining fluid disaturated-phosphatidylcholine, surfactant protein A and B, albumin, and myeloperoxidase activity were assessed after intubation and before exogenous surfactant administration. We found that disaturated-phosphatidylcholine, surfactant protein B, and myeloperoxidase were significantly higher in preterms with chorioamnionitis.

Histologic chorioamnionitis does not modulate the oxidative stress and antioxidant status in pregnancies complicated by spontaneous preterm delivery.

BACKGROUND: Infection induced-inflammation and other risk factors for spontaneous preterm birth (PTB) and preterm premature rupture of membranes (pPROM) may cause a redox imbalance, increasing the release of free radicals and consuming antioxidant defenses. Oxidative stress, in turn, can initiate intracellular signaling cascades that increase the production of pro-inflammatory mediators. The objective of this study was to evaluate the oxidative damage to proteins and antioxidant capacity profiles in amniochorion membranes from preterm birth (PTB) and preterm premature rupture of membranes (pPROM) and to determine the role of histologic chorioamnionitis in this scenario. METHODS: We included 27 pregnant women with PTB, 27 pPROM and 30 at term. Protein oxidative damage was assayed by 3-nitrotyrosine (3-NT) and carbonyl levels, using enzyme-linked immunosorbent assay (ELISA) and modified dinitrophenylhydrazine assay (DNPH), respectively. Total antioxidant capacity (TAC) was measured by ELISA. RESULTS: Protein oxidative damage determined by carbonyl levels was lower in PTB group than pPROM and term groups (p < 0.001). PTB group presented higher TAC compared with pPROM and term groups (p = 0.002). Histologic chorioamnionitis did not change either protein oxidative damage or TAC regardless of gestational outcome. CONCLUSION: These results corroborates previous reports that pPROM and term birth exhibit similarities in oxidative stress- induced senescence and histologic chorioamnionitis does not modulate oxidative stress or antioxidant status.

An animal model for chorioamnionitis at term.

OBJECTIVE: The purpose of this study was to develop an animal model for intrapartum inflammation at term to investigate the interactions between maternal and fetal inflammatory responses and adverse neurologic outcome. STUDY DESIGN: Lipopolysaccharide (160, 320, or 640 µg/kg) was administered intraperitoneally to day 20 term-pregnant Sprague Dawley rat dams 2, 4, and 6 hours before sample collection. Maternal outcomes included dam core temperature and plasma interleukin 6 (IL-6). Fetal outcomes included plasma IL-6, brain IL-6 messenger RNA expression, and brain IL-6 protein expression. Primary cortical cell cultures were prepared to examine neuronal morphologic condition. Neurite counts were obtained with the use of automated Sholl analysis. RESULTS: Maternal plasma IL-6 levels peaked 2 hours after lipopolysaccharide stimulus and rapidly resolved, except for an observed low level persistence at 6 hours with 640 µg/kg. Fetal plasma and placental IL-6 expression also peaked rapidly but only persisted in placental samples. Fetal brain IL-6 RNA and protein expression was significantly higher than control litters at 6 hours after the exposure to both 320 µg/kg (P ≤ .05) and 640 µg/kg (P ≤ .01). Cortical cells from fetuses that were exposed for 6 hours to maternal systemic inflammation showed reduced neurite number and neurite length (P < .001) with increasing lipopolysaccharide dose. CONCLUSION: Our results demonstrate that fetal brain injury follows isolated systemic maternal inflammation and that fetal brain inflammation lags after maternal stimulus, which creates a potential 4-hour clinical window for therapeutic intervention.

Escherichia coli-induced temporal and differential secretion of heat-shock protein 70 and interleukin-1ß by human fetal membranes in a two-compartment culture system.

INTRODUCTION: Escherichia coli is recognized as an etiological bacteria associated with chorioamnionitis and the preterm premature rupture of fetal membranes. This pathological condition induces pro-inflammatory cytokines and degradative metalloproteinases, which are considered biological markers secreted in an acute stage of infection. Heat-shock proteins (HSPs) are an important component of the innate immunity response and are found in different pathological conditions. They have not been previously measured in human fetal membranes in response to infectious conditions. We hypothesized that the choriodecidual tissue and amniotic epithelium secreted temporal and differential Hsp-60, Hsp-70, and interleukin (IL)-1ß mediated by E. coli infection. METHODS: Fetal membranes were mounted in a two-compartment culture system and infected with two passes of live E. coli at different doses (10², 104, 105, and 106 colony-forming units (CFU)/mL) and intervals of incubation (3, 6, and 24 h). The culture medium was collected, and Hsp-60, Hsp-70, and IL-1ß were assessed using the enzyme-linked immunosorbent assay (ELISA) method. RESULTS: After 3 and 6 h of infection, E. coli induced an increase in Hsp-70 secretion in the choriodecidual tissue. However, after 24 h of incubation, Hsp-70 was downregulated and we observed an increase in IL-1ß secretion. By contrast, E. coli induced a lower Hsp-60 secretion in the amnion compared to Hsp-70. DISCUSSION: Human fetal membranes responded actively to E. coli infection, with an increase in Hsp-70 during the first hours of infection. After 24 h, there was an increase in the liberation of IL-1ß.

Gene expression and protein localization of TLR-1, -2, -4 and -6 in amniochorion membranes of pregnancies complicated by histologic chorioamnionitis.

OBJECTIVES: To determine whether histologic chorioamnionitis is associated with changes in gene expression of TLR-1, -2, -4 and -6, and to describe the localization of these receptors in fetal membranes. STUDY DESIGN: A total of 135 amniochorion membranes with or without histologic chorioamnionitis from preterm or term deliveries were included. Fragments of membranes were submitted to total RNA extraction. RNA was reverse transcribed and the quantification of TLRs expression measured by real time PCR. RESULTS: All amniochorion membranes expressed TLR-1 and TLR-4, whereas 99.1% of membranes expressed TLR-2 and 77.4% expressed TLR-6. TLR-1 and TLR-2 expressions were significantly higher in membranes with histologic chorioamnionitis as compared to membranes without chorioamnionitis in preterm pregnancies (p=0.003 and p<0.001, respectively). Among the membranes of term pregnancies there were no differences in the expressions of such receptors regardless of inflammatory status. Regarding TLR-4 and TLR-6 expression, there was no difference among membranes with or without histologic chorioamnionitis, regardless gestational age at delivery. TLR-1, TLR-2, TLR-4 and TLR-6 expressions were observed in amniotic epithelial, chorionic and decidual cells. CONCLUSION: Amniochorion membranes express TLR-1, TLR-2, TLR-4 and TLR-6 and increased expression of TLR-1 and TLR-2 is related to the presence of histologic chorioamnionitis in preterm pregnancies. This study provides further evidence that amniochorion membranes act as a mechanical barrier to microorganisms and as components of the innate immune system.

Interleukin 18 messenger RNA and proIL-18 protein expression in chorioamniotic membranes from pregnant women with preterm prelabor rupture of membranes.

OBJECTIVE: To quantify the expression of IL-18 mRNA and protein in the chorioamniotic membranes of pregnant women with PPROM and correlate expression with histological chorioamnionitis. STUDY DESIGN: A case control study that included 42 pregnant women not in labor in the following groups: PPROM (n=28) and controls with intact membranes submitted to selective cesarean section at term (n=14). Expression of IL-18 mRNA in chorioamniotic membranes was determined by real-time polymerase chain reaction, and IL-18 protein expression was measured by western blot. Histopathological analyses and immunolocalization of IL-18 by immunohistochemistry were also performed. Analyses were performed using the Mann-Whitney or Fisher's exact tests and the group effect was considered significant if the adjusted p-values were <0.05 and the magnitude of change was greater than 2-fold for mRNA expression. RESULTS: IL-18 mRNA was present in 100% of samples and no difference in expression was observed between term vs. PPROM membranes (fold-change 0.12; p=0.88). In the PPROM group, no difference was observed in IL-18 mRNA regarding gestational age (fold-change 0.11; p=0.42) or the presence of histological chorioamnionitis (fold-change 0.26; p=0.15). ProIL-18 was present in all samples. IL-18 was immunolocalized to amnion, chorion and decidua cells, with intense immunohistochemical staining at the choriodecidual junction. CONCLUSION: Chorioamniotic membranes are sources of IL-18 mRNA and proIL-18, and their expression is unrelated to PPROM or histological chorioamnionitis.

An experimental mixed bacterial infection induced differential secretion of proinflammatory cytokines (IL-1ß, TNFα) and proMMP-9 in human fetal membranes.

Overall, 1-4% of all births in the US are complicated by choriamnionitis. Choriamnionitis is a polymicrobial infection most often due to ascending genital microbes which, in over 65% of positive amniotic fluid cultures, involves two or more organisms. In this study, we determine the cytokines expression (IL-1ß, TNFα) and prometalloproteinase activation (proMMP-2 and proMMP-9) after double o single infection an in vitro model of human fetal membranes. Fetal membranes at term were mounted in the Transwell culture system and after 24 h of infection, choriodecidual, and amnion media was collected. IL-1ß and TNFα were evaluated by ELISA, whereas proMMP-9 and proMMP-2 were determined by substrate gel zymography. The choriodecidual and amnion compartments actively respond to the infectious process, which induced the secretion of IL-1ß, TNFα, and proMMP-9 after either mixed or single infection. The proMMP-2 secretion profile was the same after all experimental conditions. There was no synergy between Streptococcus agalactiae and Escherichia coli for inducing the secretion of inflammatory factors or degradative metalloproteinase. In conclusion, these two bacteria could initiate different pathways to induce chorioamnioitis.

Expression of ß defensins 1, 3 and 4 in chorioamniotic membranes of preterm pregnancies complicated by chorioamnionitis.

OBJECTIVE: To quantify the expression of human ß-defensins (HBDs) 1, 3 and 4 in chorioamniotic membranes in pregnancies complicated by prematurity associated with histologic chorioamnionitis. STUDY DESIGN: The study group included 23 fragments of chorioamniotic membranes with histologic chorioamnionitis from women with preterm premature rupture of membranes (PPROM) and preterm labor (PTL) with intact membranes, who had preterm deliveries. As a control group, 30 chorioamniotic membranes without chorioamnionitis at the same gestational age as those in the study group were included. Chorioamniotic membranes were collected for histopathological analyses, and HBD mRNA expression quantification was analyzed by real time PCR. Comparisons were performed using the Mann-Whitney or Kruskal-Wallis tests and all the women were informed and provided written consent. RESULTS: The expression of HBDs mRNA in the fetal membranes was similar in patients without histologic chorioamnionitis (HBD1: 0.7-fold, HBD3: 0.9-fold, HBD4: 0.3-fold) compared to patients with chorioamnionitis (HBD1: 1.1-fold, HBD3: 0.9-fold, HBD4: 0.4-fold; p>0.05). Regarding the gestational complications that resulted in premature delivery, PPROM or PTL, the relative quantification of HBD1, HBD3 and HBD4 showed no statistically significant differences in either the absence or presence of chorioamnionitis. Among patients with histologic chorioamnionitis, patients with PPROM (HBD1: 2.7-fold, HBD3: 0.3-fold, HBD4: 0.7-fold) presented similar mRNA expression to those with PTL (HBD1: 0.7-fold, HBD3: 1.2-fold, HBD4: 0.13-fold; p>0.05). CONCLUSIONS: Chorioamniotic membranes are sources of ß defensins 1, 3 and 4; however, considering the sample size and the methodology applied, mRNA concentrations were not related to the presence of histologic chorioamnionitis.

[Local expression of tumor necrosis factor-alpha on premature rupture of membranes]. / Expressão local do fator de necrose tumoral alfa na ruptura prematura de membranas.

PURPOSE: to compare the expression of tumor necrosis factor-alpha (TNF-alpha) in ovular membranes with premature rupture (MPR) and with opportune rupture; to verify the association between the expression of the TNF-alpha in ovular membranes and the degree of chorioamnionitis, correlating the expression of the TNF-alpha and the membranes' time of rupture. METHODS: ovular membranes from 31 parturients with MPR, with gestational ages over 34 weeks, and from parturients with opportune membranes' rupture, with gestational ages equal or over 37 weeks. Chorioamnionitis detection has been done by histopathological analysis. The evaluation of the TNF-alpha expression has been done by immune-histochemical technique, using the labile streptavidin-biotin-peroxidase (LSAB) method. RESULTS: the average rupture time was 16.6 hours. The ratio of the TNF-alpha expression in the Control and Study Groups did not show a significant difference (chi(2)=6.6; p=0.08). In the Study Group, there was no correlation between the degree of chorioamnionitis and the intensity of TNF-alpha expression (Spearman's coefficient (Rs)=0.4; p=0.02). CONCLUSIONS: there was no significant difference between the TNF-alpha expression in ovular membranes with premature or opportune rupture; in the Study Group, there was significant association between TNF-alpha expression and the degree of chorioamnionitis, and there was no association between rupture time and the intensity of TNF-alpha expression.

Expressão local do fator de necrose tumoral alfa na ruptura prematura de membranas / Local expression of tumor necrosis factor-alpha on premature rupture of membranes

OBJETIVO: comparar a expressão do fator de necrose tumoral alfa (TNF-α) em membranas ovulares com ruptura prematura (RPM) e com ruptura oportuna das mesmas; verificar a associação entre a expressão do TNF-α em membranas ovulares e o grau de corioamnionite das mesmas e correlacionar a expressão do TNF-α e o tempo de ruptura das membranas. MÉTODOS: foram analisadas as membranas ovulares de 31 parturientes com RPM, com idade gestacional acima de 34 semanas, e de 14 parturientes com ruptura oportuna das membranas, com idade gestacional igual ou maior de 37 semanas. A detecção da corioamnionite foi feita por meio de estudo histopatológico. A avaliação da expressão do TNF-α foi feita por meio de técnica imunoistoquímica, na qual foi empregado o método streptavidina-biotina-peroxidase (LSAB). RESULTADOS: o tempo médio de ruptura foi de 16,6 horas. A frequência da expressão de TNF-α, nos Grupos Controle e Estudo, não mostrou diferença significante (χ2=6,6; p=0,08). No Grupo Estudo, houve correlação entre o grau de corioamnionite e a intensidade da expressão de TNF-α (coeficiente de Spearman (Rs)=0,4; p=0,02). CONCLUSÕES: não houve diferença significante entre as expressões do TNF-α em membranas ovulares com ruptura prematura e com ruptura oportuna das mesmas; no Grupo Estudo, constatou-se associação significante entre a expressão do TNF-α e o grau de corioamnionite e não houve associação entre o tempo de ruptura e a intensidade da expressão do TNF-α.

PURPOSE: to compare the expression of tumor necrosis factor-alpha (TNF-α) in ovular membranes with premature rupture (MPR) and with opportune rupture; to verify the association between the expression of the TNF-α in ovular membranes and the degree of chorioamnionitis, correlating the expression of the TNF-α and the membranes' time of rupture. METHODS: ovular membranes from 31 parturients with MPR, with gestational ages over 34 weeks, and from parturients with opportune membranes' rupture, with gestational ages equal or over 37 weeks. Chorioamnionitis detection has been done by histopathological analysis. The evaluation of the TNF-α expression has been done by immune-histochemical technique, using the labile streptavidin-biotin-peroxidase (LSAB) method. RESULTS: the average rupture time was 16.6 hours. The ratio of the TNF-α expression in the Control and Study Groups did not show a significant difference (χ2=6.6; p=0.08). In the Study Group, there was no correlation between the degree of chorioamnionitis and the intensity of TNF-α expression (Spearman's coefficient (Rs)=0.4; p=0.02). CONCLUSIONS: there was no significant difference between the TNF-α expression in ovular membranes with premature or opportune rupture; in the Study Group, there was significant association between TNF-α expression and the degree of chorioamnionitis, and there was no association between rupture time and the intensity of TNF-α expression.

Inflammatory cytokine mRNA detection by real time PCR in chorioamniotic membranes from pregnant women with preterm premature rupture of membranes.

OBJECTIVE: To quantify the expression of interleukin (IL)-1beta, IL-6, IL-8 and tumor necrosis factor alpha (TNF-alpha) in chorioamniotic membranes of PPROM pregnant women with chorioamnionits. STUDY DESIGN: The study included 25 PPROM women in labor, 15 PPROM without labor, and 25 pregnant women in preterm labor (PTL). Chorioamniotic membranes were collected for histopathological analyses and cytokine mRNA expression quantification by real time PCR. Comparisons were performed using the Mann-Whitney, Kruskal-Wallis, Fisher's exact test or z test with significance set at p<0.05. The software employed was the SigmaStat version 3.1. RESULTS: During the study PPROM incidence was 4.6% and chorioamnionits was present in 75% of the samples. IL-1beta, IL-6, and IL-8 mRNA expression did not statistically differ among study groups. TNF-alpha mRNA expression was statistically higher in PTL. No difference in the mRNA concentration of the cytokines studied in the presence of chorioamnionitis was observed. CONCLUSION: Chorioamniotic membranes are sources of IL-1beta, IL-6, IL-8, and TNF-alpha and their mRNA concentrations in PPROM are not related to the presence of chorioamnionitis.

Small thymus in very low birth weight infants born to mothers with subclinical chorioamnionitis.

Chorioamnionitis, a major cause of preterm birth with significant neonatal morbidity and mortality, frequently occurs in mothers who are free of symptoms. A combined clinical, radiologic, and pathologic study of 129 very low birth weight infants indicated a significant association between a markedly decreased thymic size at birth and subclinical chorioamnionitis.