Lactate Dehydrogenase Isoform Activity Mapping in Patients with Intra-Amniotic Infection
ABSTRACT Five distinct lactate dehydrogenase isoenzymes have been described. We sought to illustrate the specific amniotic fluid lactate dehydrogenase isoenzyme activity profiles in women with intra-amniotic infection.
Amniotic fluid was retrieved from 82 women who were stratified in the following groups: (1) positive amniotic fluid cultures (n = 23 women; gestational age, 26 weeks [range, 21-32 weeks]); (2) negative amniotic fluid cultures (n = 22 women; gestational age, 30 weeks [range, 16-36 weeks]); (3) second trimester control (normal genetic karyotype; n = 17 women; gestational age, 18 weeks [range, 16-22 weeks]); and (4) third trimester control (fetal lung maturity testing; n = 20 women; gestational age, 36 weeks [range, 31-38 weeks]). The optical density of each isoform was determined relative to a standard with 5 known lactate dehydrogenase isoenzyme activities. Total lactate dehydrogenase activity was measured by the clinical laboratory immediately after retrieval and by a kinetic UV spectrophotometric assay at the time of the isoelectric focusing.
Infection increased total lactate dehydrogenase activity: positive amniotic fluid cultures (median, 762.4 [range, 169.3-3374.8]) vs negative amniotic fluid cultures (median, 203.7 [range, 57.8-1939.3]; U/L; P < .001]). Lactate dehydrogenase isoform profiling identified significant and specific increases in lactate dehydrogenase isoforms 3, 4 (P < .01), and 5 (P < .05) in positive amniotic fluid cultures compared to the negative amniotic fluid cultures group. A selective up-regulation in lactate dehydrogenase isoform 5 was identified at term in healthy subjects.
Intra-amniotic infection is characterized by an increase in the activities of lactate dehydrogenase isoforms 3, 4, and 5; advancing gestational age demonstrates an up-regulation of isoform 5 only.
- SourceAvailable from: Irina Buhimschi[Show abstract] [Hide abstract]
ABSTRACT: TLRs are pattern recognition transmembrane receptors that play key roles in innate immunity. A recently discovered soluble truncated form of TLR2 (sTLR2) acts as a decoy receptor, down-regulating the host inflammatory response to bacteria. To identify the presence and functional role of sTLR2 in modulating the intraamniotic inflammatory response to infection, we studied 109 amniotic fluid samples of women with normal pregnancy outcomes (n = 28) and women with (n = 39) and without (n = 42) intraamniotic infection. We sought to demonstrate a functional role of the amniotic fluid sTLR2 in modulating the TLR2 inflammatory signaling in vitro by using a villous explant system. Two sTLR2 forms were identified, and specificity was confirmed with neutralizing peptides. We showed that sTLR2 is present constitutively in amniotic fluid, its levels are gestational age dependent, and we determined that the sTLR2 quantity and functional engagement modulates the intensity of the intraamniotic inflammation elicited by Gram-positive bacteria. In vitro, we demonstrated that challenging placental villous explants with a specific TLR2 agonist (Pam3Cys) induced a significant cytokine response. Notably, preincubation of the preterm, but not near-term, amniotic fluid with Pam3Cys significantly inhibited the ability of this TLR2 agonist to elicit a cytokine reaction. Moreover, depletion of sTLR2 from preterm amniotic fluid removed its neutralizing property. Monensin significantly diminished sTLR2 immunoreactivity, indicating that sTLR2 is the result of intracellular posttranslational processing of TLR2. We conclude that sTLR2 is part of the amniotic fluid innate immune system and participates in regulating the inflammatory response to microbial pathogens.The Journal of Immunology 07/2009; 182(11):7244-53. DOI:10.4049/jimmunol.0803517 · 4.92 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Background and purpose: Premature rupture of membrane (PROM) is a common and challenging issue in obstetrics. Early diagnosis of PROM and the consequent infection has a pivotal role in proper management of patients. The detection of PROM is easier in a case with visible vaginal fluid, but is difficult when trivial amount of vaginal fluid presents. In this study, we aimed to assess the diagnostic value of LDH and creatinine in vaginal fluid for detecting PROM. Materials and methods: Pregnant women were recruited in the third trimester (28-42 weeks) when attending prenatal care unit at Kowsar Educational hospital, Ghazvin, Iran during 2008. Under sterile condition, vaginal fluid was collected using speculum. Fern test was performed and adequate vaginal fluid was aspirated from posterior fornix to measure LDH and creatinine. Results: 223 pregnant women with mean age of 25.4±5.5 (16-43) years were recruited into this study. The mean of gestational age was 37.6±3.2 weeks. Visible vaginal fluid was detected in 72 (32.3%) while Fern test was positive in 56 (25.1%). Visible vaginal fluid detected by a specialist is considered as a gold standard test. Using ROC curve analysis from SPSS, we have found that a cutoff point of >180 U/L for vaginal fluid LDH level is a reasonabl diagnostic value for PROM at sensitivity of 85%, specificity of 80%, PPV of 66.3%, NPV of 91.6% and an accuracy of 81%. The same analysis on vaginal creatinine using a cutoff point of >0.9 mg/dl showed diagnostic value with sensitivity of 72%, specificity of 35%, PPV of 34.7%, NPV of 72.6% and accuracy of 47%. Conclusion: Vaginal fluid LDH at a cutoff point of >180 U/L is clinically a useful diagnostic test for detection of PROM.Journal of Mazandaran University of Medical Sciences 07/2009; 20(76):43-53.
- [Show abstract] [Hide abstract]
ABSTRACT: Classic IL-6 signaling is conditioned by the transmembrane receptor (IL-6R) and homodimerization of gp130. During trans-signaling, IL-6 binds to soluble IL-6R (sIL-6R), enabling activation of cells expressing solely gp130. Soluble gp130 (sgp130) selectively inhibits IL-6 trans-signaling. To characterize amniotic fluid (AF) IL-6 trans-signaling molecules (IL-6, sIL-6R, sgp130) in normal gestations and pregnancies complicated by intra-amniotic inflammation (IAI), we studied 301 women during second trimester (n = 39), third trimester (n = 40), and preterm labor with intact (n = 131, 85 negative IAI and 46 positive IAI) or preterm premature rupture of membranes (PPROM; n = 91, 61 negative IAI and 30 positive IAI). ELISA, Western blotting, and real-time RT-PCR were used to investigate AF, placenta, and amniochorion for protein and mRNA expression of sIL-6R, sgp130, IL-6R, and gp130. Tissues were immunostained for IL-6R, gp130, CD15(+) (polymorphonuclear), and CD3(+) (T cell) inflammatory cells. The ability of sIL-6R and sgp130 to modulate basal and LPS-stimulated release of amniochorion matrix metalloprotease-9 was tested ex vivo. We showed that in physiologic gestations, AF sgp130 decreases toward term. AF IL-6 and sIL-6R were increased in IAI, whereas sgp130 was decreased in PPROM. Our results suggested that fetal membranes are the probable source of AF sIL-6R and sgp130. Immunohistochemistry and RT-PCR revealed increased IL-6R and decreased gp130 expression in amniochorion of women with IAI. Ex vivo, sIL-6R and LPS augmented amniochorion matrix metalloprotease-9 release, whereas sgp130 opposed this effect. We conclude that IL-6 trans-signaling molecules are physiologic constituents of the AF regulated by gestational age and inflammation. PPROM likely involves functional loss of sgp130.The Journal of Immunology 03/2011; 186(5):3226-36. DOI:10.4049/jimmunol.1003587 · 4.92 Impact Factor