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Immunohistochemical localization of inducible nitric oxide synthase on human fetal amnion in intra-amniotic infection
Immunohistochemical localization of inducible nitric oxide synthase on human fetal amnion in intra-amniotic infection Chaur-Dong Hsu, MD, MPH,a Erika Meaddough, MPH,a Li-Cheng Lu, MD,a Adina Chelouche, MD,a Ren-Ing Liang, MD,a Joshua A. Copel, MD,a and Vinita Parkash, MDb New Haven, Connecticut OBJECTIVES: Amniotic fluid levels of nitric oxide metabolites are significantly elevated in intra-amniotic infection. We hypothesized that fetal amnion is a possible site for the production of nitric oxide. Because inducible nitric oxide synthase is the key enzyme responsible for the generation of nitric oxide in patients with intra-amniotic infection, we used immunohistochemistry to localize it on human fetal amnion. STUDY DESIGN: Human fetal amnions were obtained from patients with and without intra-amniotic infection (n = 5, respectively). Intra-amniotic infection was diagnosed by positive amniotic fluid cultures and placental pathologic features. Human fetal amniotic membranes were processed into tissue blocks and embedded in paraffin. A rabbit polyclonal antibody against human inducible nitric oxide synthase was used as the primary antibody, followed by avidin-biotin immunoperoxidase localization. Normal rabbit serum was used as a negative control and ovarian carcinoma cells were used as the positive control. RESULTS: Anti–inducible nitric oxide synthase labeling of human fetal amniotic membranes in patients with intra-amniotic infection showed positive immunostaining of epithelial cells, specifically in the cytoplasm of the perinuclear area. In contrast, no anti–inducible nitric oxide synthase immunostaining on human fetal amniotic membranes could be identified in patients without intra-amniotic infection. CONCLUSIONS: Our data provide important evidence that inducible nitric oxide synthase can be induced on human fetal amnion in intra-amniotic infection. These findings strongly support our hypothesis that human fetal amnion may be a possible site for the synthesis of nitric oxide after inducible nitric oxide synthase is induced in response to infectious products in intra-amniotic infection. (Am J Obstet Gynecol 1998;179:1271-4.)
Key words: Intra-amniotic infection, inducible nitric oxide synthase, amnion, immunohistochemistry Intra-amniotic infection is a clinical or subclinical intrauterine infection associated with varied symptoms and signs1-3 and with significant perinatal morbidity and mortality.4 The diagnosis of intra-amniotic infection can be based on clinical criteria. However, clinical symptoms and signs are frequently inconsistent or subtle and many affected women are virtually symptom free.5 Direct examination of amniotic fluid obtained through amniocentesis is frequently necessary. Nitric oxide, an unstable free radical, is readily oxidized to nitrate and nitrite within a few seconds.6 Nitric oxide is formed by the action of nitric oxide synthase on arginine. During infection or inflammation, endotoxins and cytokines can induce nitric oxide synthase, which increases biologically active nitric oxide7, 8 to exert its cytotoxic and cytostatic effects. A direct relationship between
From the Departments of Obstetrics and Gynecologya and Pathology,b Yale University School of Medicine. Presented at the Eighteenth Annual Meeting of the Society of Perinatal Obstetricians, Miami, Florida, February 2-7, 1998. Reprint requests: Chaur-Dong Hsu, MD, MPH, Division of MaternalFetal Medicine, Department of Obstetrics and Gynecology, Yale University School of Medicine, New Haven, CT 06520-8063. Copyright © 1998 by Mosby, Inc. 0002-9378/98 $5.00 + 0 6/6/92297
urinary nitrite and urinary tract infection was reported by Cruickshank and Moyes9 in 1914. Because many bacterial species produce nitrate reductase, which converts nitrate to nitrite, the nitrite chemical strip has been clinically used as a rapid and simple test for the detection of urinary tract infection.10 Amniotic fluid mainly derives from fetal urine, and we have previously reported that amniotic fluid concentrations of nitric oxide metabolites were significantly higher in patients with intra-amniotic infection than in those without intra-amniotic infection.11 We then sought to define the possible endogenous source of amniotic fluid nitric oxide in intra-amniotic infection. Inducible nitric oxide synthase is the key enzyme responsible for generation of nitric oxide, and fetal amnion is proximal to amniotic fluid; we hypothesized that human fetal amnion might be a possible site for the expression of inducible nitric oxide synthase and the production of amniotic fluid nitric oxide in patients with intra-amniotic infection. Thus we localized inducible nitric oxide synthase in human fetal amnion with use of immunohistochemistry. Material and methods This investigation was carried out at Yale–New Haven Hospital with the approval of the institutional Human 1271
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Fig 1. Strong staining of human fetal amnion in intra-amniotic infection. Right arrow, Perinuclear cytoplasmic staining of amnion epithelial cells; left arrow, cytoplasmic staining of infiltrating neutrophils.
Investigational Committee. Intra-amniotic infection was diagnosed by positive amniotic fluid cultures and placental pathologic conditions (chorioamnionitis). Human fetal amniotic membranes were obtained from 5 patients with intra-amniotic infection11 and 5 without intra-amniotic infection (elective cesarean section without labor). The microorganisms from infected amniotic fluid cultures included 1 with Ureaplasma urealyticum, 2 with mixed anaerobic and aerobic bacteria, and 2 with αStreptococcus viridans. Human fetal amniotic tissues were immediately processed into tissue blocks and embedded in paraffin. Tissue sections were stained by the avidin-biotinylated-peroxidase complex method. Sections were deparaffinized and endogenous peroxidase was blocked with 0.6% hydrogen peroxide in methanol for 20 minutes. After being washed in phosphate-buffered saline solution and phosphate-buffered saline solution with 0.1% bovine serum albumin, they were incubated in 3% goat serum for 3 hours at room temperature to block nonspecific binding. Sections were blotted to remove excess serum and incubated overnight at 4°C with rabbit antiserum to human inducible nitric oxide synthase (Research and Diagnostic Antibodies) diluted 1:3000 to 1:6000 in phosphate-buffered saline solution containing 5% bovine serum albumin. Normal rabbit serum diluted 1:3000 to 1:6000 was used as the negative control. After another wash they were incubated for 30 minutes at room temperature with biotinylated goat antiserum to rabbit immunoglobulin G (Vector Laboratories) diluted 1:200 in phosphate-buffered saline solution with 5% bovine serum albumin. Sections were again washed and then incubated in freshly prepared avidin-biotinylatedperoxidase complex reagent (Vector Laboratories) for 60 minutes at room temperature. Peroxidase activity
was revealed with use of the hydrogen peroxide– diaminobenzidine method to give brown staining. Sections were then counterstained and mounted. The technician and investigators were blinded to the infection status of the patients. Fisher’s exact test was used for statistical analysis for the differences of immunohistochemical staining between 2 groups. Results Anti–inducible nitric oxide immunostaining was positively localized on human fetal amnion epithelial cells, specifically in the cytoplasm of the perinuclear area in the patient with intra-amniotic infection (Fig 1). We also found that inducible nitric oxide synthase was localized in infiltrating neutrophils. In contrast, no anti–inducible nitric oxide immunostaining on human fetal amniotic membrane could be identified in the patient without intra-amniotic infection (Fig 2). The difference in immunohistochemical staining between patients with and without intra-amniotic infection was statistically significant (P = .008). Comment We recently reported that pregnant women with intraamniotic infection had significantly higher amniotic fluid concentrations of nitric oxide metabolites.11 Additionally, we and others showed that amniotic fluid cytokines were significantly higher in patients with than in those without intra-amniotic infection.12, 13 These findings suggest a direct relationship between amniotic fluid nitric oxide and cytokines in patients with intraamniotic infection. Menon et al14 and Fortunato et al15 recently reported that interleukin-6 could be expressed in fetal amnion.14 They further showed that interleukin-6
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Fig 2. Negative staining of human fetal amnion without intra-amniotic infection (elective cesarean section without labor). Arrow, Amnion epithelial cells.
was significantly released from cultured human fetal membranes in response to endotoxic lipopolysaccharide.15 Thus fetal amnion could play a certain role in the production of cytokines in pregnant women with intraamniotic infection. Similarly, we speculate that fetal amnion may also play a possible role in the secretion of nitric oxide into amniotic fluid in patients with intra-amniotic infection. At least 3 distinct forms of nitric oxide synthase exist, as demonstrated by complementary deoxyribonucleic acid cloning of various tissues and cells.16-20 The neuronal and endothelial isoforms are constitutive, Ca++-dependent, agonist-triggered enzymes. By contrast, macrophages have negligible nitric oxide synthase activity in basal conditions but can be massively induced at the transcriptional level by endotoxin or cytokines within 2 to 4 hours.21-23 The inducible nitric oxide synthase isoform is usually Ca++ dependent because calmodulin is very tightly bound to inducible nitric oxide synthase. Its induction by endotoxin or cytokines has also been demonstrated on vascular smooth muscle cells, neutrophils, hepatocytes, Kupffer cells, cardiac myocytes, neurons, and mesangial cells.19-23 The relevance of inducible nitric oxide synthase to human fetal membranes has not been clearly demonstrated. In addition, human inducible nitric oxide synthase, but not endothelial or neuronal nitric oxide synthase, is the principal isoform found in response to the infection or inflammation process.24, 25 Inducible nitric oxide synthase can be induced by endotoxin or cytokines on neutrophils19-23; therefore it was not unexpected to find inducible nitric oxide expressed on infiltrating neutrophils by immunohistochemical testing in patients with intra-amniotic infection. Additionally, we hypothesized that fetal amnion could be one of
the sources of inducible nitric oxide synthase expression and increased amniotic fluid nitric oxide in patients with intra-amniotic infection. Our data provide important evidence that inducible nitric oxide synthase can be induced on human fetal amnion in the patient with intraamniotic infection. These observations support our hypothesis that human fetal amnion can be a possible site for the synthesis of proinflammatory nitric oxide after inducible nitric oxide synthase is induced in response to bacterial products and cytokines in intraamniotic infection. There is one limitation in our study because patients with and without intra-amniotic infection may also differ by labor. Further comparison with patients in labor without chorioamnionitis is currently under investigation. We thank Drs Shannon Smith and Kristen Aversa, Ms Shih-Fen Hong, and Ms Marcia Wheeler for their assistance. REFERENCES
1. Gibbs RS, Castillo MS, Rogers PJ. Management of acute chorioamnionitis. Am J Obstet Gynecol 1980;136:709-13. 2. Hauth JC, Gilstrap LC, Hankins GDV, Connor KD. Term maternal and neonatal complications of acute chorioamnionitis. Obstet Gynecol 1985;66:59-62. 3. Yoder PR, Gibbs RS, Blanco JD, Castaneda YS, St Clair PJ. A prospective, controlled study of maternal and perinatal outcome after intra-amniotic infection at term. Am J Obstet Gynecol 1983;145:695-701. 4. Gilstrap LC III, Leveno KJ, Cox SM, Burris JS, Mashburn M, Rosenfeld CR. Intrapartum treatment of acute chorioamnionitis: impact on neonatal sepsis. Am J Obstet Gynecol 1988;159:579-83. 5. Romero R, Sirtori M, Oyarzun E, Avila C, Mazor M, Callahan R, et al. Infection and labor. V. Prevalence, microbiology, and clinical significance of intra-amniotic infection in women with preterm labor and intact membranes. Am J Obstet Gynecol 1989;161:817-24.
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6. Green LC, Ruiz de Luzuriaga K, Wagner DA, Rand W, Istfan N, Young UR, et al. Nitrate biosynthesis in man. Proc Natl Acad Sci U S A 1981;78:7764-8. 7. Raij L, Baylis C. Glomerular actions of nitric oxide. Kidney Int 1995;48:20-32. 8. Gross SS, Wolin MS. Nitric oxide: pathophysiological mechanisms. Annu Rev Physiol 1995;57:737-69. 9. Cruickshank J, Moyes JM. The presence and significance of nitrites in urine. BMJ 1914;2:712-3. 10. Griess P, Bermerkungen zu der Abhandlung der HH. Wesley and Benedikt Ueber einige Azoverbindungen. Ber Deutsch Chem Gen 1879;12:426-8. 11. Hsu CD, Aversa K, Meaddough E, Lee IS, Copel JA. Elevated amniotic fluid nitric oxide metabolites and cyclic guanosine 3´, 5´-monophosphate in pregnant women with intraamniotic infection. Am J Obstet Gynecol 1997;177:793-6. 12. Hsu CD, Meaddough E, Hong SF, Aversa K, Lu LC, Copel JA. Elevated amniotic fluid nitric oxide metabolites and interleukin-6 in intraamniotic infection. J Soc Gynecol Invest 1998;5:21-4. 13. Romero R, Avila C, Santhanam U, Seghal P. Amniotic fluid interleukin-6 in preterm labor: association with infection. J Clin Invest 1990;85:1392-400. 14. Menon R, Swan KF, Lyden TW, Rote NS, Fortunato SJ. Expression of inflammatory cytokines (interleukin-1 beta and interleukin-6) in amniochorionic membranes. Am J Obstet Gynecol 1995;172:493-500. 15. Fortunato SJ, Menon RP, Swan KF, Menon R. Inflammatory cytokine (interleukins 1, 6, and 8 and tumor necrosis factor-α) release from cultured human fetal membranes in response to endotoxic lipopolysaccharide mirrors amniotic fluid concentrations. Am J Obstet Gynecol 1996;174:1855-62. 16. MacMicking JD, Nathap C, Hom G, Chartrain N, Fletcher DS, Trumbauer M, et al. Altered responses to bacterial infection and endotoxic shock in mice lacking inducible nitric oxide synthase. Cell 1995;81:641-50.
November 1998 Am J Obstet Gynecol
17. Jansenns S, Shimouchi A, Quertermous T, Block DB, Bloch KD. Cloning and expression of a cDNA encoding human endothelium-derived relaxing factor/nitric oxide synthase. J Biol Chem 1992;267:14519-22. 18. Lamas S, Marsden PA, Li GK, Tempst P, Michel T. Endothelial nitric oxide synthase: molecular cloning, and characterization of a distinct constitutive enzyme isoform. Proc Natl Acad Sci U S A 1992;89:6348-52. 19. Lyons C, Orloff G, Cunningham J. Molecular cloning and functional expression of an inducible nitric oxide synthase from a murine macrophage cell line. J Biol Chem 1992;267:6370-4. 20. Xie QW, Cho HJ, Calaycay J, Mumford RA, Swiderek KM, Lee TD, et al. Cloning and characterization of inducible nitric oxide synthase from mouse macrophages. Science 1992;256:225-8. 21. Stuehr DJ, Marietta MA. Mammalian nitrate biosynthesis: mouse macrophages produce nitrite and nitrate in response to Escherichia coli lipopolysaccharide. Proc Natl Acad Sci U S A 1985;82:7738-42. 22. Iyengar R, Stuehr DJ, Marlella MA. Macrophage synthesis of nitrite, nitrate, and N-nitrosamines: precursors and role of the respiratory burst. Proc Natl Acad Sci U S A 1987;84:6369-73. 23. Ding A, Nathan CF, Stuehr DJ. Release of reactive nitrogen intermediates and reactive oxygen intermediates from mouse peritoneal macrophages: comparison of activating cytokines and evidence for independent production. J Immunol 1988;141:2407-12. 24. Nakane M, Pollock JS, Klinghofer V, Basha F, Marsden PA, Hokari A, et al. Functional expression of three isoforms of human nitric oxide synthase in baculovirus-infected insect cells. Biochem Biophys Res Commun 1995;206:511-7. 25. Salvemini D, Manning PT, Zweifel BS, Seibert K, Connor J, Currie MG, et al. Dual inhibition of nitric oxide and prostaglandin production contributes to the antiinflammatory properties of nitric oxide synthase inhibitors. J Clin Invest 1995;96:301-8.
Key words: Intra-amniotic infection, inducible nitric oxide synthase, amnion, immunohistochemistry Intra-amniotic infection is a clinical or subclinical intrauterine infection associated with varied symptoms and signs1-3 and with significant perinatal morbidity and mortality.4 The diagnosis of intra-amniotic infection can be based on clinical criteria. However, clinical symptoms and signs are frequently inconsistent or subtle and many affected women are virtually symptom free.5 Direct examination of amniotic fluid obtained through amniocentesis is frequently necessary. Nitric oxide, an unstable free radical, is readily oxidized to nitrate and nitrite within a few seconds.6 Nitric oxide is formed by the action of nitric oxide synthase on arginine. During infection or inflammation, endotoxins and cytokines can induce nitric oxide synthase, which increases biologically active nitric oxide7, 8 to exert its cytotoxic and cytostatic effects. A direct relationship between
From the Departments of Obstetrics and Gynecologya and Pathology,b Yale University School of Medicine. Presented at the Eighteenth Annual Meeting of the Society of Perinatal Obstetricians, Miami, Florida, February 2-7, 1998. Reprint requests: Chaur-Dong Hsu, MD, MPH, Division of MaternalFetal Medicine, Department of Obstetrics and Gynecology, Yale University School of Medicine, New Haven, CT 06520-8063. Copyright © 1998 by Mosby, Inc. 0002-9378/98 $5.00 + 0 6/6/92297
urinary nitrite and urinary tract infection was reported by Cruickshank and Moyes9 in 1914. Because many bacterial species produce nitrate reductase, which converts nitrate to nitrite, the nitrite chemical strip has been clinically used as a rapid and simple test for the detection of urinary tract infection.10 Amniotic fluid mainly derives from fetal urine, and we have previously reported that amniotic fluid concentrations of nitric oxide metabolites were significantly higher in patients with intra-amniotic infection than in those without intra-amniotic infection.11 We then sought to define the possible endogenous source of amniotic fluid nitric oxide in intra-amniotic infection. Inducible nitric oxide synthase is the key enzyme responsible for generation of nitric oxide, and fetal amnion is proximal to amniotic fluid; we hypothesized that human fetal amnion might be a possible site for the expression of inducible nitric oxide synthase and the production of amniotic fluid nitric oxide in patients with intra-amniotic infection. Thus we localized inducible nitric oxide synthase in human fetal amnion with use of immunohistochemistry. Material and methods This investigation was carried out at Yale–New Haven Hospital with the approval of the institutional Human 1271
1272 Hsu et al
November 1998 Am J Obstet Gynecol
Fig 1. Strong staining of human fetal amnion in intra-amniotic infection. Right arrow, Perinuclear cytoplasmic staining of amnion epithelial cells; left arrow, cytoplasmic staining of infiltrating neutrophils.
Investigational Committee. Intra-amniotic infection was diagnosed by positive amniotic fluid cultures and placental pathologic conditions (chorioamnionitis). Human fetal amniotic membranes were obtained from 5 patients with intra-amniotic infection11 and 5 without intra-amniotic infection (elective cesarean section without labor). The microorganisms from infected amniotic fluid cultures included 1 with Ureaplasma urealyticum, 2 with mixed anaerobic and aerobic bacteria, and 2 with αStreptococcus viridans. Human fetal amniotic tissues were immediately processed into tissue blocks and embedded in paraffin. Tissue sections were stained by the avidin-biotinylated-peroxidase complex method. Sections were deparaffinized and endogenous peroxidase was blocked with 0.6% hydrogen peroxide in methanol for 20 minutes. After being washed in phosphate-buffered saline solution and phosphate-buffered saline solution with 0.1% bovine serum albumin, they were incubated in 3% goat serum for 3 hours at room temperature to block nonspecific binding. Sections were blotted to remove excess serum and incubated overnight at 4°C with rabbit antiserum to human inducible nitric oxide synthase (Research and Diagnostic Antibodies) diluted 1:3000 to 1:6000 in phosphate-buffered saline solution containing 5% bovine serum albumin. Normal rabbit serum diluted 1:3000 to 1:6000 was used as the negative control. After another wash they were incubated for 30 minutes at room temperature with biotinylated goat antiserum to rabbit immunoglobulin G (Vector Laboratories) diluted 1:200 in phosphate-buffered saline solution with 5% bovine serum albumin. Sections were again washed and then incubated in freshly prepared avidin-biotinylatedperoxidase complex reagent (Vector Laboratories) for 60 minutes at room temperature. Peroxidase activity
was revealed with use of the hydrogen peroxide– diaminobenzidine method to give brown staining. Sections were then counterstained and mounted. The technician and investigators were blinded to the infection status of the patients. Fisher’s exact test was used for statistical analysis for the differences of immunohistochemical staining between 2 groups. Results Anti–inducible nitric oxide immunostaining was positively localized on human fetal amnion epithelial cells, specifically in the cytoplasm of the perinuclear area in the patient with intra-amniotic infection (Fig 1). We also found that inducible nitric oxide synthase was localized in infiltrating neutrophils. In contrast, no anti–inducible nitric oxide immunostaining on human fetal amniotic membrane could be identified in the patient without intra-amniotic infection (Fig 2). The difference in immunohistochemical staining between patients with and without intra-amniotic infection was statistically significant (P = .008). Comment We recently reported that pregnant women with intraamniotic infection had significantly higher amniotic fluid concentrations of nitric oxide metabolites.11 Additionally, we and others showed that amniotic fluid cytokines were significantly higher in patients with than in those without intra-amniotic infection.12, 13 These findings suggest a direct relationship between amniotic fluid nitric oxide and cytokines in patients with intraamniotic infection. Menon et al14 and Fortunato et al15 recently reported that interleukin-6 could be expressed in fetal amnion.14 They further showed that interleukin-6
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Volume 179, Number 5 Am J Obstet Gynecol
Fig 2. Negative staining of human fetal amnion without intra-amniotic infection (elective cesarean section without labor). Arrow, Amnion epithelial cells.
was significantly released from cultured human fetal membranes in response to endotoxic lipopolysaccharide.15 Thus fetal amnion could play a certain role in the production of cytokines in pregnant women with intraamniotic infection. Similarly, we speculate that fetal amnion may also play a possible role in the secretion of nitric oxide into amniotic fluid in patients with intra-amniotic infection. At least 3 distinct forms of nitric oxide synthase exist, as demonstrated by complementary deoxyribonucleic acid cloning of various tissues and cells.16-20 The neuronal and endothelial isoforms are constitutive, Ca++-dependent, agonist-triggered enzymes. By contrast, macrophages have negligible nitric oxide synthase activity in basal conditions but can be massively induced at the transcriptional level by endotoxin or cytokines within 2 to 4 hours.21-23 The inducible nitric oxide synthase isoform is usually Ca++ dependent because calmodulin is very tightly bound to inducible nitric oxide synthase. Its induction by endotoxin or cytokines has also been demonstrated on vascular smooth muscle cells, neutrophils, hepatocytes, Kupffer cells, cardiac myocytes, neurons, and mesangial cells.19-23 The relevance of inducible nitric oxide synthase to human fetal membranes has not been clearly demonstrated. In addition, human inducible nitric oxide synthase, but not endothelial or neuronal nitric oxide synthase, is the principal isoform found in response to the infection or inflammation process.24, 25 Inducible nitric oxide synthase can be induced by endotoxin or cytokines on neutrophils19-23; therefore it was not unexpected to find inducible nitric oxide expressed on infiltrating neutrophils by immunohistochemical testing in patients with intra-amniotic infection. Additionally, we hypothesized that fetal amnion could be one of
the sources of inducible nitric oxide synthase expression and increased amniotic fluid nitric oxide in patients with intra-amniotic infection. Our data provide important evidence that inducible nitric oxide synthase can be induced on human fetal amnion in the patient with intraamniotic infection. These observations support our hypothesis that human fetal amnion can be a possible site for the synthesis of proinflammatory nitric oxide after inducible nitric oxide synthase is induced in response to bacterial products and cytokines in intraamniotic infection. There is one limitation in our study because patients with and without intra-amniotic infection may also differ by labor. Further comparison with patients in labor without chorioamnionitis is currently under investigation. We thank Drs Shannon Smith and Kristen Aversa, Ms Shih-Fen Hong, and Ms Marcia Wheeler for their assistance. REFERENCES
1. Gibbs RS, Castillo MS, Rogers PJ. Management of acute chorioamnionitis. Am J Obstet Gynecol 1980;136:709-13. 2. Hauth JC, Gilstrap LC, Hankins GDV, Connor KD. Term maternal and neonatal complications of acute chorioamnionitis. Obstet Gynecol 1985;66:59-62. 3. Yoder PR, Gibbs RS, Blanco JD, Castaneda YS, St Clair PJ. A prospective, controlled study of maternal and perinatal outcome after intra-amniotic infection at term. Am J Obstet Gynecol 1983;145:695-701. 4. Gilstrap LC III, Leveno KJ, Cox SM, Burris JS, Mashburn M, Rosenfeld CR. Intrapartum treatment of acute chorioamnionitis: impact on neonatal sepsis. Am J Obstet Gynecol 1988;159:579-83. 5. Romero R, Sirtori M, Oyarzun E, Avila C, Mazor M, Callahan R, et al. Infection and labor. V. Prevalence, microbiology, and clinical significance of intra-amniotic infection in women with preterm labor and intact membranes. Am J Obstet Gynecol 1989;161:817-24.
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6. Green LC, Ruiz de Luzuriaga K, Wagner DA, Rand W, Istfan N, Young UR, et al. Nitrate biosynthesis in man. Proc Natl Acad Sci U S A 1981;78:7764-8. 7. Raij L, Baylis C. Glomerular actions of nitric oxide. Kidney Int 1995;48:20-32. 8. Gross SS, Wolin MS. Nitric oxide: pathophysiological mechanisms. Annu Rev Physiol 1995;57:737-69. 9. Cruickshank J, Moyes JM. The presence and significance of nitrites in urine. BMJ 1914;2:712-3. 10. Griess P, Bermerkungen zu der Abhandlung der HH. Wesley and Benedikt Ueber einige Azoverbindungen. Ber Deutsch Chem Gen 1879;12:426-8. 11. Hsu CD, Aversa K, Meaddough E, Lee IS, Copel JA. Elevated amniotic fluid nitric oxide metabolites and cyclic guanosine 3´, 5´-monophosphate in pregnant women with intraamniotic infection. Am J Obstet Gynecol 1997;177:793-6. 12. Hsu CD, Meaddough E, Hong SF, Aversa K, Lu LC, Copel JA. Elevated amniotic fluid nitric oxide metabolites and interleukin-6 in intraamniotic infection. J Soc Gynecol Invest 1998;5:21-4. 13. Romero R, Avila C, Santhanam U, Seghal P. Amniotic fluid interleukin-6 in preterm labor: association with infection. J Clin Invest 1990;85:1392-400. 14. Menon R, Swan KF, Lyden TW, Rote NS, Fortunato SJ. Expression of inflammatory cytokines (interleukin-1 beta and interleukin-6) in amniochorionic membranes. Am J Obstet Gynecol 1995;172:493-500. 15. Fortunato SJ, Menon RP, Swan KF, Menon R. Inflammatory cytokine (interleukins 1, 6, and 8 and tumor necrosis factor-α) release from cultured human fetal membranes in response to endotoxic lipopolysaccharide mirrors amniotic fluid concentrations. Am J Obstet Gynecol 1996;174:1855-62. 16. MacMicking JD, Nathap C, Hom G, Chartrain N, Fletcher DS, Trumbauer M, et al. Altered responses to bacterial infection and endotoxic shock in mice lacking inducible nitric oxide synthase. Cell 1995;81:641-50.
November 1998 Am J Obstet Gynecol
17. Jansenns S, Shimouchi A, Quertermous T, Block DB, Bloch KD. Cloning and expression of a cDNA encoding human endothelium-derived relaxing factor/nitric oxide synthase. J Biol Chem 1992;267:14519-22. 18. Lamas S, Marsden PA, Li GK, Tempst P, Michel T. Endothelial nitric oxide synthase: molecular cloning, and characterization of a distinct constitutive enzyme isoform. Proc Natl Acad Sci U S A 1992;89:6348-52. 19. Lyons C, Orloff G, Cunningham J. Molecular cloning and functional expression of an inducible nitric oxide synthase from a murine macrophage cell line. J Biol Chem 1992;267:6370-4. 20. Xie QW, Cho HJ, Calaycay J, Mumford RA, Swiderek KM, Lee TD, et al. Cloning and characterization of inducible nitric oxide synthase from mouse macrophages. Science 1992;256:225-8. 21. Stuehr DJ, Marietta MA. Mammalian nitrate biosynthesis: mouse macrophages produce nitrite and nitrate in response to Escherichia coli lipopolysaccharide. Proc Natl Acad Sci U S A 1985;82:7738-42. 22. Iyengar R, Stuehr DJ, Marlella MA. Macrophage synthesis of nitrite, nitrate, and N-nitrosamines: precursors and role of the respiratory burst. Proc Natl Acad Sci U S A 1987;84:6369-73. 23. Ding A, Nathan CF, Stuehr DJ. Release of reactive nitrogen intermediates and reactive oxygen intermediates from mouse peritoneal macrophages: comparison of activating cytokines and evidence for independent production. J Immunol 1988;141:2407-12. 24. Nakane M, Pollock JS, Klinghofer V, Basha F, Marsden PA, Hokari A, et al. Functional expression of three isoforms of human nitric oxide synthase in baculovirus-infected insect cells. Biochem Biophys Res Commun 1995;206:511-7. 25. Salvemini D, Manning PT, Zweifel BS, Seibert K, Connor J, Currie MG, et al. Dual inhibition of nitric oxide and prostaglandin production contributes to the antiinflammatory properties of nitric oxide synthase inhibitors. J Clin Invest 1995;96:301-8.