Characterization of RAGE, HMGB1, and S100β in Inflammation-Induced Preterm Birth and Fetal Tissue Injury
2009; Elsevier BV; Volume: 175; Issue: 3 Linguagem: Inglês
10.2353/ajpath.2009.090156
ISSN1525-2191
AutoresCatalin S. Buhimschi, Margaret A. Baumbusch, Antonette T. Dulay, Emily A. Oliver, Sarah Lee, Guomao Zhao, Vineet Bhandari, Richard A. Ehrenkranz, Carl P. Weiner, Joseph A. Madri, Irina A. Buhimschi,
Tópico(s)Injury Epidemiology and Prevention
ResumoImmune activation represents an adaptive reaction triggered by both noxious exogenous (microbes) and endogenous [high mobility group box-1 protein (HMGB1), S100 calcium binding proteins] inducers of inflammation. Cell stress or necrosis lead the release of HMGB1 and S100 proteins in the extracellular compartment where they act as damage-associated molecular pattern molecules (or alarmins) by engaging the receptor for advanced glycation end-products (RAGE). Although the biology of RAGE is dictated by the accumulation of damage-associated molecular pattern molecules at sites of tissue injury, the role of RAGE in mediating antenatal fetal injury remains unknown. First, we studied the relationships at birth between the intensity of human fetal inflammation and sRAGE (an endogenous RAGE antagonist), HMGB1, and S100β protein. We found significantly lower sRAGE in human fetuses that mounted robust inflammatory responses. HMGB1 levels correlated significantly with levels of interleukin-6 and S100β in fetal circulation. We then evaluated the levels and areas of tissue expression of RAGE, HMGB1, and S100β in specific organs of mouse fetuses on E16. Using an animal model of endotoxin-induced fetal damage and preterm birth, we determined that inflammation induces a significant change in expression of RAGE and HMGB1, but not S100β, at sites of tissue damage. Our findings indicate that RAGE and HMGB1 may be important mediators of cellular injury in fetuses delivered in the setting of inflammation-induced preterm birth. Immune activation represents an adaptive reaction triggered by both noxious exogenous (microbes) and endogenous [high mobility group box-1 protein (HMGB1), S100 calcium binding proteins] inducers of inflammation. Cell stress or necrosis lead the release of HMGB1 and S100 proteins in the extracellular compartment where they act as damage-associated molecular pattern molecules (or alarmins) by engaging the receptor for advanced glycation end-products (RAGE). Although the biology of RAGE is dictated by the accumulation of damage-associated molecular pattern molecules at sites of tissue injury, the role of RAGE in mediating antenatal fetal injury remains unknown. First, we studied the relationships at birth between the intensity of human fetal inflammation and sRAGE (an endogenous RAGE antagonist), HMGB1, and S100β protein. We found significantly lower sRAGE in human fetuses that mounted robust inflammatory responses. HMGB1 levels correlated significantly with levels of interleukin-6 and S100β in fetal circulation. We then evaluated the levels and areas of tissue expression of RAGE, HMGB1, and S100β in specific organs of mouse fetuses on E16. Using an animal model of endotoxin-induced fetal damage and preterm birth, we determined that inflammation induces a significant change in expression of RAGE and HMGB1, but not S100β, at sites of tissue damage. Our findings indicate that RAGE and HMGB1 may be important mediators of cellular injury in fetuses delivered in the setting of inflammation-induced preterm birth. Conventional wisdom holds that the primary causes of the high neonatal morbidity and mortality attendant preterm birth are complications of immature organ systems.1Behrman RE Butler AS Institute of Medicine Committee on Understanding Premature Birth and Assuring Health Outcomes Board on Health Sciences outcomes: Preterm Birth. Causes, Consequences, and Prevention. Institute of Medicine of the National Academies. The National Academies Press, Washington D.C.2007Google Scholar, 2Allen MC Neurodevelopmental outcomes of preterm infants.Curr Opin Neurol. 2008; 21: 123-128Crossref PubMed Scopus (283) Google Scholar, 3Bhandari A Panitch HB Pulmonary outcomes in bronchopulmonary dysplasia.Semin Perinatol. 2006; 30: 219-226Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 4Robertson CM Watt MJ Yasui Y Changes in the prevalence of cerebral palsy for children born very prematurely within a population-based program over 30 years.JAMA. 2007; 297: 2733-2740Crossref PubMed Scopus (131) Google Scholar However, a growing body of investigation suggests that the poor outcome observed in many preterm children is not entirely dependent on their gestational age at birth.2Allen MC Neurodevelopmental outcomes of preterm infants.Curr Opin Neurol. 2008; 21: 123-128Crossref PubMed Scopus (283) Google Scholar, 5Tyson JE Parikh NA Langer J Green C Higgins RD National Institute of Child Health and Human Development Neonatal Research Network. Intensive care for extreme prematurity–moving beyond gestational age.N Engl J Med. 2008; 358: 1672-1681Crossref PubMed Scopus (742) Google Scholar, 6Gaudet LM Smith GN Cerebral palsy and chorioamnionitis: the inflammatory cytokine link.Obstet Gynecol Surv. 2001; 56: 433-436Crossref PubMed Scopus (52) Google Scholar After correcting for gestational age, several risk factors remain significantly associated with an increased risk of cerebral palsy, such as intra-amniotic infection, histological chorioamnionitis, prolonged rupture of the membranes, and hypoxemic fetal growth restriction.7Buhimschi CS Dulay AT Abdel-Razeq S Zhao G Lee S Hodgson EJ Bhandari V Buhimschi IA Fetal inflammatory response in women with proteomic biomarkers characteristic of intra-amniotic inflammation and preterm birth.BJOG. 2009; 116: 257-267Crossref PubMed Scopus (75) Google Scholar, 8Dammann O Leviton A Gappa M Dammann CE Lung and brain damage in preterm newborns, and their association with gestational age, prematurity subgroup, infection/inflammation and long term outcome.BJOG. 2005; 112: 4-9Crossref PubMed Scopus (99) Google Scholar, 9Ferriero DM Neonatal brain injury.N Engl J Med. 2004; 351: 1985-1995Crossref PubMed Scopus (951) Google Scholar Therefore, particularities of the fetal innate immune response to infection appear to cause pathology unique to the premature fetus. This includes a heightened inflammatory and oxidative stress state that acts synergistically with microbial insult to induce cell damage and multisystem organ failure.7Buhimschi CS Dulay AT Abdel-Razeq S Zhao G Lee S Hodgson EJ Bhandari V Buhimschi IA Fetal inflammatory response in women with proteomic biomarkers characteristic of intra-amniotic inflammation and preterm birth.BJOG. 2009; 116: 257-267Crossref PubMed Scopus (75) Google Scholar, 10Stoll BJ Hansen NI Higgins RD Fanaroff AA Duara S Goldberg R Laptook A Walsh M Oh W Hale E Very low birth weight preterm infants with early onset neonatal sepsis: the predominance of gram-negative infections continues in the National Institute of Child Health and Human Development Neonatal Research Network, 2002–2003.Pediatr Infect Dis J. 2005; 24: 635-639Crossref PubMed Scopus (274) Google Scholar, 11Eschenbach DA Amniotic fluid infection and cerebral palsy. Focus on the fetus.JAMA. 1997; 278: 247-248Crossref PubMed Google Scholar, 12Haynes RL Baud O Li J Kinney HC Volpe JJ Folkerth DR Oxidative and nitrative injury in periventricular leukomalacia.Brain Pathol. 2005; 15: 225-233Crossref PubMed Scopus (153) Google Scholar The host’s response to microbial pathogens involves a series of carefully orchestrated mechanisms that include the newly described damage-associated molecular pattern molecules (DAMPs).13Medzhitov R Origin and physiological roles of inflammation.Nature. 2008; 454: 428-435Crossref PubMed Scopus (3951) Google Scholar, 14Lotze MT Zeh HJ Rubartelli A Sparvero LJ Amoscato AA Washburn NR Devera ME Liang X Tör M Billiar T The grateful dead: damage-associated molecular pattern molecules and reduction/oxidation regulate immunity.Immunol Rev. 2007; 220: 60-81Crossref PubMed Scopus (493) Google Scholar DAMPs, also known as “alarmins,”15Bianchi ME DAMPs PAM: Ps and alarmins: all we need to know about danger.J Leukoc Biol. 2007; 8: 1-5Google Scholar are a pleiotropic group of intracellular proteins that include among others the high-mobility group box-1 (HMGB1 or amphoterin) and S100β proteins.13Medzhitov R Origin and physiological roles of inflammation.Nature. 2008; 454: 428-435Crossref PubMed Scopus (3951) Google Scholar, 16Yang H Tracey KJ High mobility group box 1 (HMGB1).Crit Care Med. 2005; 33: S472-S474Crossref PubMed Scopus (39) Google Scholar When released into the extracellular compartment in excess as a result of cell activation or injury, DAMPs become “danger signals” that specifically activate the receptor of advanced glycation end-products (RAGE).14Lotze MT Zeh HJ Rubartelli A Sparvero LJ Amoscato AA Washburn NR Devera ME Liang X Tör M Billiar T The grateful dead: damage-associated molecular pattern molecules and reduction/oxidation regulate immunity.Immunol Rev. 2007; 220: 60-81Crossref PubMed Scopus (493) Google Scholar, 17Rauvala H Rouhiainen A RAGE as a receptor of HMGB1 (Amphoterin): roles in health and disease.Curr Mol Med. 2007; 7: 725-734Crossref PubMed Scopus (87) Google Scholar RAGE is a transmembrane receptor,18Neeper M Schmidt AM Brett J Yan SD Wang F Pan YC Elliston K Stern D Shaw A Cloning and expression of a cell surface receptor for advanced glycosylation end products of proteins.J Biol Chem. 1992; 267: 14998-15004Abstract Full Text PDF PubMed Google Scholar a member of the immunoglobulin superfamily, and functions as a chief receptor for products of nonenzymatic glycoxidation (advanced glycation end-products, AGEs), HMGB1, and S100β proteins.14Lotze MT Zeh HJ Rubartelli A Sparvero LJ Amoscato AA Washburn NR Devera ME Liang X Tör M Billiar T The grateful dead: damage-associated molecular pattern molecules and reduction/oxidation regulate immunity.Immunol Rev. 2007; 220: 60-81Crossref PubMed Scopus (493) Google Scholar In adult humans and animals, RAGE has been shown to be expressed on the cellular surface of cortical neurons and numerous endothelial, smooth muscle, inflammatory, and vascular cells positioned in vital organs such as the brain, lung, heart, liver, and bowel.19Brett J Schmidt AM Yan SD Zou YS Weidman E Pinsky D Nowygrod R Neeper M Przysiecki C Shaw A Survey of the distribution of a newly characterized receptor for advanced glycation end products in tissues.Am J Pathol. 1993; 143: 1699-1712PubMed Google Scholar, 20Zen K Chen CX Chen YT Wilton R Liu Y Receptor for advanced glycation endproducts mediates neutrophil migration across intestinal epithelium.J Immunol. 2007; 178: 2483-2490PubMed Google Scholar, 21Fehrenbach H Kasper M Tschernig T Shearman MS Schuh D Müller M Receptor for advanced glycation endproducts (RAGE) exhibits highly differential cellular and subcellular localisation in rat and human lung.Cell Mol Biol (Noisy-le-grand). 1998; 44: 1147-1157PubMed Google Scholar, 22Butscheid M Hauptvogel P Fritz P Klotz U Alscher DM Hepatic expression of galectin-3 and receptor for advanced glycation end products in patients with liver disease.J Clin Pathol. 2007; 60: 415-418Crossref PubMed Scopus (37) Google Scholar Binding of DAMPs to the RAGE extracellular domain results in sustained activation of nuclear factor (NF)-κB and recruitment of inflammatory cells (CD68- and Cd11c-positive mononuclear phagocyte), which in turn amplify the process of tissue damage.14Lotze MT Zeh HJ Rubartelli A Sparvero LJ Amoscato AA Washburn NR Devera ME Liang X Tör M Billiar T The grateful dead: damage-associated molecular pattern molecules and reduction/oxidation regulate immunity.Immunol Rev. 2007; 220: 60-81Crossref PubMed Scopus (493) Google Scholar That RAGE and HMGB1 play a fundamental role in inflammation and oxidative stress-induced tissue injury is demonstrated by experiments in animal models where administration of quercetin (flavonoid with potent antioxidant properties and HMGB1 inhibitor)23Tang D Kang R Xiao W Zhang H Lotze MT Wang H Xiao X Quercetin prevents lipopolysaccharide-induced HMGB1 release and proinflammatory function.Am J Respir Cell Mol Biol. 2009; https://doi.org/10.1165/rcmb.2008.0119OCCrossref Google Scholar or soluble RAGE (sRAGE, an extracellular truncated form of RAGE that acts as a decoy receptor) or antibodies or peptides targeted against RAGE or HMGB1 attenuate the lethal effects of endotoxin, acetaminophen and ischemia-reperfusion.24Yang H Ochani M Li J Tanovic M Harris HE Susarla SM Ulloa L Wang H DiRaimo R Czura CJ Wang H Roth J Warren HS Fink MP Fenton MJ Andersson U Tracey KJ Reversing established sepsis with antagonists of endogenous high-mobility group box 1.Proc Natl Acad Sci USA. 2004; 101: 296-301Crossref PubMed Scopus (976) Google Scholar, 25Zhang H Tasaka S Shiraishi Y Fukunaga K Yamada W Seki H Ogawa Y Miyamoto K Nakano Y Hasegawa N Miyasho T Maruyama I Ishizaka A Role of soluble receptor for advanced glycation end products on endotoxin-induced lung injury.Am J Respir Crit Care Med. 2008; 178: 356-362Crossref PubMed Scopus (114) Google Scholar, 26Lutterloh EC Opal SM Pittman DD Keith Jr, JC Tan XY Clancy BM Palmer H Milarski K Sun Y Palardy JE Parejo NA Kessimian N Inhibition of the RAGE products increases survival in experimental models of severe sepsis and systemic infection.Crit Care. 2007; 11: R122Crossref PubMed Scopus (134) Google Scholar, 27Ekong U Zeng S Dun H Feirt N Guo J Ippagunta N Guarrera JV Lu Y Weinberg A Qu W Ramasamy R Schmidt AM Emond JC Blockade of the receptor for advanced glycation end products attenuates acetaminophen-induced hepatotoxicity in mice.J Gastroenterol Hepatol. 2006; 21: 682-688Crossref PubMed Scopus (60) Google Scholar, 28Zeng S Feirt N Goldstein M Guarrera J Ippagunta N Ekong U Dun H Lu Y Qu W Schmidt AM Emond JC Blockade of receptor for advanced glycation end product (RAGE) attenuates ischemia and reperfusion injury to the liver in mice.Hepatology. 2004; 39: 422-432Crossref PubMed Scopus (165) Google Scholar, 29Stern D Yan SD Yan SF Schmidt AM Receptor for advanced glycation endproducts: a multiligand receptor magnifying cell stress in diverse pathologic settings.Adv Drug Deliv Rev. 2002; 54: 1615-1625Crossref PubMed Scopus (245) Google Scholar, 30Muhammad S Barakat W Stoyanov S Murikinati S Yang H Tracey KJ Bendszus M Rossetti G Nawroth PP Bierhaus A Schwaninger M The HMGB1 receptor RAGE mediates ischemic brain damage.J Neurosci. 2008; 28: 12023-12031Crossref PubMed Scopus (333) Google Scholar Recently, we demonstrated that the S100A12-RAGE axis is actively engaged in modulating the intensity of the human intra-amniotic inflammatory response to infection.31Buhimschi IA Zhao G Pettker CM Bahtiyar MO Magloire LK Thung S Fairchild T Buhimschi CS The receptor for advanced glycation end products (RAGE) system in women with intra-amniotic infection and inflammation.Am J Obstet Gynecol. 2007; 196: 181.e1-181.e13Abstract Full Text Full Text PDF Scopus (69) Google Scholar, 32Buhimschi IA Christner R Buhimschi CS Proteomic biomarker analysis of amniotic fluid for identification of intra-amniotic inflammation.BJOG. 2005; 112: 173-181Crossref PubMed Scopus (175) Google Scholar We attributed a key role to the presence and activity of amniotic fluid (AF) sRAGE.31Buhimschi IA Zhao G Pettker CM Bahtiyar MO Magloire LK Thung S Fairchild T Buhimschi CS The receptor for advanced glycation end products (RAGE) system in women with intra-amniotic infection and inflammation.Am J Obstet Gynecol. 2007; 196: 181.e1-181.e13Abstract Full Text Full Text PDF Scopus (69) Google Scholar In this study we sought to evaluate the role of RAGE, HMGB1, and S100β proteins as mediators of fetal organ injury in the context of infection and/or inflammation. Specifically, we have begun by assessing whether the intensity of the human maternal and fetal inflammation impacts on the fetal systemic levels of sRAGE (as marker of the RAGE system activation),33Bopp C Hofer S Weitz J Bierhaus A Nawroth PP Martin E Büchler MW Weigand MA sRAGE is elevated in septic patients and associated with patients outcome.J Surg Res. 2008; 147: 79-83Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar HMGB1, or S100β levels at birth. Given that sRAGE acts as a decoy for RAGE we anticipated that in the setting of a robust fetal inflammatory response the circulatory levels of sRAGE are low. We thought that this may be related to inhibition or dysfunction of the mechanisms responsible for synthesis of this decoy receptor in the setting of overwhelming cytokemia. Alternatively, low levels of total sRAGE may be related to successful removal/detoxification of AGEs known to be generated in high amounts in the context of increased metabolic and oxidative stress, such as that associated with fetal prematurity and infection.34Azpurua H Dulay AT Buhimschi IA Bahtiyar MO Funai E Abdel-Razeq SS Luo G Bhandari V Copel JA Buhimschi CS Fetal artery impedance as assessed by Doppler ultrasound in pregnancies complicated by intra-amniotic inflammation and preterm birth.Am J Obstet Gynecol. 2009; 200: 203.e1-203.e11Abstract Full Text Full Text PDF Scopus (15) Google Scholar, 35Buhimschi IA Buhimschi CS Weiner CP Protective effect of N-acetylcysteine against fetal death and preterm labor induced by maternal inflammation.Am J Obstet Gynecol. 2003; 188: 203-208Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar To elucidate whether RAGE, HMGB1, and S100β are participants in the mechanisms underlying infection/inflammation induced fetal cell/organ damage, we turned to an animal model of preterm birth induced by maternal administration of endotoxin (lipopolysaccharide, LPS).35Buhimschi IA Buhimschi CS Weiner CP Protective effect of N-acetylcysteine against fetal death and preterm labor induced by maternal inflammation.Am J Obstet Gynecol. 2003; 188: 203-208Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar First, we evaluated the level of RAGE, HMGB1, and S100β tissue expression and regional distribution in specific organs of mouse fetuses at developmental stage E16. Furthermore, we aimed to determine whether altered expression of RAGE, HMGB1, or S100β co-exists with tissue inflammation and cellular damage in vital organs such as fetal brain and liver. In a prospective study design, we enrolled 121 consecutive preterm singletons born to mothers who had a clinically indicated amniocentesis to rule out infection. All women were recruited at Yale New Haven Hospital (YNHH) following their admission to Labor and Birth or to the High Risk antepartum units. This study extended from May 2004 to October 2007. The Human Investigation Committee of Yale University approved the study protocol. Amniocentesis was indicated independent of our research protocol. Amniotic fluid was collected by ultrasound-guided amniocentesis and each woman was followed prospectively until delivery. Eligible women had a gestational age at delivery ≥23.1 weeks, preterm labor contractions refractory to tocolysis, preterm premature rupture of membranes (PPROM), or advanced cervical dilatation (≥3 cm). Exclusion criteria were the presence of anhydramnios, human immunodeficiency or hepatitis viral infections, congenital anomalies, abnormal karyotype, or any known maternal medical conditions. Gestational age was determined based on last menstrual period confirmed by an ultrasound examination before 20 weeks.36Hadlock FP Deter RL Harrist RB Park SK Computer assisted analysis of fetal age in the third trimester using multiple fetal growth parameters.J Clin Ultrasound. 1983; 11: 313-316Crossref PubMed Scopus (65) Google Scholar Preterm labor was defined as the presence of regular uterine contractions and documented cervical effacement and/or dilatation in patients <37 weeks of gestation. PPROM was confirmed by vaginal AF “pooling,” “nitrazine,” “ferning,” or an amniocentesis-dye positive test. Corticosteroids and antibiotics were recommended as clinically indicated.37ACOG Committee on Practice Bulletins-Obstetrics ACOG Practice Bulletin No. 80: premature rupture of membranes. Clinical management guidelines for obstetrician-gynecologists.Obstet Gynecol. 2007; 109: 1007-1019Crossref PubMed Scopus (287) Google Scholar The neonatology resuscitation team was present at the time of delivery for all neonates. Following retrieval under sterile conditions, amniotic fluid was analyzed by the YNHH clinical and microbiological laboratories for glucose concentration, lactate dehydrogenase activity, white blood cell count, Gram stain, and standard culturing methods for aerobic and anaerobic bacteria, including Ureaplasma and Mycoplasma species. These results were available to the clinical team for management of the case. An amniotic fluid glucose cut-off of ≤15 mg/dL, an lactate dehydrogenase level ≥419 U/L, a positive Gram stain and/or culture result were considered suggestive of intra-amniotic infection. The results of the microbiological tests were available for case management and were reported as final 5 days after culturing. The remaining amniotic fluid was transported to the research laboratory, spun at 3000 × g at 4°C for 20 minutes, aliquoted into polypropylene cryotubes, and stored at −80°C until analysis. To confirm or exclude the presence of intra-amniotic inflammation, an amniotic fluid proteomic fingerprint (mass restricted [MR] score) was generated using surface enhanced laser desorbtion ionization-time of flight mass spectrometry. The method for generation of the MR score has been previously described.32Buhimschi IA Christner R Buhimschi CS Proteomic biomarker analysis of amniotic fluid for identification of intra-amniotic inflammation.BJOG. 2005; 112: 173-181Crossref PubMed Scopus (175) Google Scholar Briefly, the MR score is comprised of four proteomic biomarkers: defensin-2, defensin-1, S100A12 (calgranulin C), and S100A8 (calgranulin A). The MR score ranges from 0 to 4, depending on the presence or absence of each of the 4 protein biomarkers. A value of 1 was assigned if a biomarker peak was present and 0 if absent. Based on our previous results, we stratified the study population based on the “severity” of inflammation (MR 0: “no” inflammation; MR 1–2: “minimal” inflammation; MR 3–4: “severe” inflammation).38Buhimschi CS Bhandari V Hamar BD Bahtiyar MO Zhao G Sfakianaki AK Pettker CM Magloire L Funai E Norwitz ER Paidas M Copel JA Weiner CP Lockwood CJ Buhimschi IA Proteomic profiling of the amniotic fluid to detect inflammation, infection, and neonatal sepsis.PLoS Med. 2007; 4: e18Crossref PubMed Scopus (142) Google Scholar Scorings of the amniotic fluid surface enhanced laser desorbtion ionization-time of flight tracings were performed by an investigator without knowledge of the maternal outcome, the results of the placental histological examination, or umbilical cord HMGB1, S100β protein, and interleukin (IL)-6 levels. The results of the MR score were not used for patient clinical management. Within 10 minutes of delivery, cord blood samples (umbilical artery and vein) were collected in pre-heparinized 1-ml syringes, which were then capped and transported to the laboratory. The acid-base status was determined with the ABL 800 FLEX blood gas analyzer (Radiometer Medical A/S, Denmark). Umbilical cord blood was also obtained by aseptic puncture of the clamped umbilical vein. Following collection, the cord blood was immediately centrifuged at 1000 × g at 4°C for 15 minutes. Serum was aliquoted in sterile polypropylene tubes and stored at −80°C until IL-6, HMGB1, and S100β protein levels were examined. Placental tissues were available for all 121 neonates included in this analysis. H&E-stained sections of the amniochorionic membranes and umbilical cord were read by a perinatal pathologist unaware of the results of the MR score, fetal outcome, or umbilical cord blood analyses. Each section was examined systematically for the presence or absence of inflammation, and funisitis was diagnosed when neutrophils infiltrated the umbilical vessel walls or Wharton’s jelly. Three histological stages of chorioamnionitis were complemented by the histological grading system devised by Salafia et al, which includes four grades of inflammation of the amnion, chorion-decidua, and umbilical cord.39Naeye RL Disorders of the placenta and decidua.in: Naeye RL Disorder of the Placenta, Fetus and Neonate: Diagnosis and Clinical Significance. Mosby, St. Louis1992: 118Google Scholar, 40Salafia CM Weigl C Silberman L The prevalence and distribution of acute placental inflammation in uncomplicated term pregnancies.Obstet Gynecol. 1989; 73: 383-389PubMed Google Scholar Histopathological evidence of maternal and/or fetal immunoresponse was assessed as previously described by Ghidini and collaborators,41Ghidini A Salafia CM Kirn V Doria V Spong CY Biophysical profile in predicting acute ascending infection in preterm rupture of membranes before 32 weeks.Obstet Gynecol. 2000; 96: 201-206Crossref PubMed Scopus (27) Google Scholar and scored as either absent, mild (grades 1–2), or severe (grades 3–4). Severe inflammation in the amnion was considered a marker of activation of the maternal immune system in response to ascending infection, whereas severe inflammation of umbilical cord and/or chorionic vessels was considered to indicate a response of the fetal immune system.41Ghidini A Salafia CM Kirn V Doria V Spong CY Biophysical profile in predicting acute ascending infection in preterm rupture of membranes before 32 weeks.Obstet Gynecol. 2000; 96: 201-206Crossref PubMed Scopus (27) Google Scholar Following delivery, all 121 neonates were admitted to the YNHH Newborn Special Care Unit were per institutional clinical guidelines; all had blood specimens and cultures obtained within 2 hours from the time of birth. Blood cultures were performed in the clinical microbiology laboratory for aerobic bacteria, anaerobic bacteria, and Mycoplasma species. Cerebrospinal and urine cultures were performed when clinically indicated. Antibiotic treatment was begun in all neonates after blood cultures were obtained. Early-onset neonatal sepsis (EONS) was defined as the presence of confirmed or suspected sepsis at ≤72 hours after birth. Confirmed sepsis represented the presence of a positive blood or any other body fluid microbial culture. A diagnosis of EONS was based on clinical symptoms corroborated with hematological laboratory results.42Bhandari V Wang C Rinder C Rinder H Hematologic profile of sepsis in neonates: neutrophil CD64 as a diagnostic marker.Pediatrics. 2008; 121: 129-134Crossref PubMed Scopus (134) Google Scholar, 43Rodwell RL Taylor KM Tudehope DI Gray PH Hematologic scoring system in early diagnosis of sepsis in neutropenic newborns.Pediatr Infect Dis J. 1993; 12: 372-376Crossref PubMed Scopus (81) Google Scholar Sepsis was suspected in the presence of ≥2 hematological criteria in the absence of a positive blood culture.44Smulian JC Bhandari V Campbell WA Rodis JF Vintzileos AM Value of umbilical artery and vein levels of interleukin-6 and soluble intracellular adhesion molecule-1 as predictors of neonatal hematologic indices and suspected early sepsis.J Matern Fetal Med. 1997; 6: 254-259Crossref PubMed Google Scholar EONS was dichotomized into present (when sepsis was either confirmed or suspected) or absent.38Buhimschi CS Bhandari V Hamar BD Bahtiyar MO Zhao G Sfakianaki AK Pettker CM Magloire L Funai E Norwitz ER Paidas M Copel JA Weiner CP Lockwood CJ Buhimschi IA Proteomic profiling of the amniotic fluid to detect inflammation, infection, and neonatal sepsis.PLoS Med. 2007; 4: e18Crossref PubMed Scopus (142) Google Scholar Sepsis categorization and neonatal hematological indices were assessed by investigators who analyzed all of the data, unaware of the results of the umbilical cord analyte levels, proteomic profiling of the amniotic fluid, or histological evaluation of the placenta. IL-6 (Pierce-Endogen, Rockford, IL), sRAGE (R&D Systems, Minneapolis, MN), HMGB1 (IBL International, Hamburg, Germany), and S100β (BioVendor, Candler, NC) immunoassays were performed in duplicate according to manufacturers’ instructions by investigators unaware of the clinical presentation and umbilical cord blood sample origin. The minimal detectable concentration for IL-6, HMGB1, S100β, and sRAGE proteins was 1 pg/ml, 1 ng/ml, 20 pg/ml, or 4.12 pg/ml, respectively. The inter- and intra-assay coefficients of variation were <10% for all analytes. Several of the cord blood samples were previously used in studies aimed at exploring the relationships between fetal inflammation (IL-6) and the proteomic biomarkers of the MR score.7Buhimschi CS Dulay AT Abdel-Razeq S Zhao G Lee S Hodgson EJ Bhandari V Buhimschi IA Fetal inflammatory response in women with proteomic biomarkers characteristic of intra-amniotic inflammation and preterm birth.BJOG. 2009; 116: 257-267Crossref PubMed Scopus (75) Google Scholar The animal model of preterm birth and fetal injury has previously been reported.35Buhimschi IA Buhimschi CS Weiner CP Protective effect of N-acetylcysteine against fetal death and preterm labor induced by maternal inflammation.Am J Obstet Gynecol. 2003; 188: 203-208Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar The University of Maryland, Institutional Animal Review Board (Baltimore, MD) approved the animal experimentation. Briefly, C57BL/6 mice were purchased from Harlan Sprague Dawley (Madison, WI), maintained on a 12:12 hours light cycle and given free access to food and water. On E15.5 (E0 = sperm plug observed), ten pregnant mice were assigned to receive either one 10 μg of i.p. injection of LPS (from E. Coli 0111:B4, Calbiochem, La Jolla,) diluted in 0.2 ml endotoxin-free saline (LPS, n = 5) or saline alone (control: CRL, n = 5). To evaluate the effect of maternal inflammation on fetal survivability, we sacrificed all pregnant animals by cervical dislocation at a specific time following LPS or saline injection. The uterus was quickly opened and the fetuses assessed for viability. A viable fetus was denoted by presence of a detectable motor response to mechanical stimulation. We found that 58% of fetuses were already dead when animals were sacrificed on E16
Referência(s)