The association between selected mid-trimester amniotic fluid candidate proteins and spontaneous preterm delivery

Objective: The aim of this study was to explore inflammatory response and identify early potential biomarkers in mid-trimester amniotic fluid associated with subsequent spontaneous preterm delivery (PTD). Methods: A cohort study was performed at Sahlgrenska University Hospital/ 6stra, Gothenburg, Sweden, between 2008 and 2010. Amniotic fluid was collected from consecutive women undergoing mid-trimester transabdominal genetic amniocentesis at 14–19 gestational weeks. Clinical data and delivery outcome variables were obtained from medical records. The analysis included 19 women with spontaneous PTD and 118 women who delivered at term. A panel of 26 candidate proteins was analyzed using Luminex xMAP technology. Candidate protein concentrations were analyzed with ANCOVA and adjusted for plate effects. Results: The median gestational age at delivery was 35 + 3 weeks in women with spontaneous PTD and 40 + 0 weeks in women who delivered at term. Nominally significantly lower amniotic fluid levels of adiponectin (PTD: median 130,695 pg/mL (IQR 71,852–199,414) vs term: median 185,329 pg/mL (IQR (135,815–290,532)), granulocyte-macrophage colony stimulating factor (PTD: median 137 pg/mL (IQR 74–156) vs term: median 176 pg/mL (IQR 111–262)), and macrophage migration inhibitory factor (PTD: median 3025 pg/mL (IQR 1885–3891) vs term: median 3400 pg/mL (IQR 2181–5231)) were observed in the spontaneous PTD group, compared with the term delivery group, after adjusting for plate effects. No significant differences remained after Bonferroni correction for multiple comparisons. Conclusions: Our results are important in the process of determining the etiology behind spontaneous PTD but due to the non-significance after Bonferroni correction, the results should be interpreted with caution. Further analyses of larger sample size will be required to determine whether these results are cogent and to examine whether microbial invasion of the amniotic cavity or intra-amniotic inflammation occurs in asymptomatic women in the mid-trimester with subsequent spontaneous PTD

Effect of a toggle switch mutation in TM6 of the human adenosine A3 receptor on Gi protein-dependent signalling and Gi-independent receptor internalization

Background and Purpose: The highly conserved tryptophan (W6.48) in transmembrane domain 6 of GPCRs has been shown to play a central role in forming an active conformation in response to agonist binding. We set out to characterize the effect of this mutation on the efficacy of two agonists at multiple signalling pathways downstream of the adenosine A3 receptor. Experimental Approach: Residue W6.48 in the human adenosine A3 receptor fused to yellow fluorescent protein was mutated to phenylalanine and expressed in CHO-K1 cells containing a cAMP response element reporter gene. The effects on agonist-mediated receptor internalization were monitored by automated confocal microscopy and image analysis. Further experiments were carried out to investigate agonist-mediated ERK1/2 phosphorylation, inhibition of [3H]-cAMP accumulation and β-arrestin2 binding. Key Results: NECA was able to stimulate agonist-mediated internalization of the W6.48F mutant receptor, while the agonist HEMADO was inactive. Investigation of other downstream signalling pathways indicated that G-protein coupling was impaired for both agonists tested. Mutation of W6.48F therefore resulted in differential effects on agonist efficacy, and introduced signalling pathway bias for HEMADO at the adenosine A3 receptor. Conclusions and Implications: Investigation of the pharmacology of the W6.48F mutant of the adenosine A3 receptor confirms that this region is important in forming the active conformation of the receptor for stimulating a number of different signalling pathways and that mutations in this residue can lead to changes in agonist efficacy and signalling bias

Cervical and intra-amniotic markers of preterm birth and infection

Abstract Background: Preterm delivery (PTD; < 37 gestational weeks), is one of the greatest unsolved obstetrical problems worldwide. As much as 80% of the perinatal mortality and 50% of the long-term neurological handicaps are associated with PTD. Spontaneous preterm birth (SPTD), i.e. preterm labor (PTL) or preterm prelabor rupture of membranes (PPROM) is responsible for 55% of PTD. Clinical and experimental evidence suggest that maternal infection and/or inflammation are centre stages in SPTD and the major risk factors for fetal injury. Several cytokines and chemokines play a central role in SPTD. However, in most cases the precise mechanistic pathway leading to SPTD remains unknown and good markers of prediction and therapies are few. Aim: To investigate if cervical and intra-amniotic proteins on their own and/or in combination with each other and/or with clinical characteristics could predict SPTD and intra-uterine infection/inflammation in women with singleton pregnancies in PTL. In particular the purpose was to investigate the predictive value of cervical markers (proteins or sonography) collected less invasively compared with amniotic fluid proteins collected via amniocentesis. Material and methods: A cohort of 134 women in PTL and 30 with PPROM with singleton pregnancies and gestational age less than 34 weeks were studied. Amniotic fluid (AF) was retrieved transabdominally from 107 patients in PTL and in 30 patients with PPROM. Cervical fluid (CF) was sampled from the external cervical os in all PTL women, but from none of the PPROM cases. Transvaginal sonography (TVS) assessing cervical length (CL) was performed in all patients. Polymerase chain reaction analyses for Ureaplasma urealyticum and Mycoplasma hominis and culture for aerobic and anaerobic bacteria were performed. Interleukin (IL)-6, IL-8, IL-18, monocyte chemotactic protein (MCP)-1, MCP-2, and MCP-3 were analyzed with enzyme-linked immunosorbent assay. The multiplex sandwich immunoassay, flowmetric Luminex xMAP (multiple analyte profiling) technology analyzed 27 specific proteins, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-17, IL-18, sIL-6rα, IFN-γ, TNF-α, TNF-β, MCP-1, TGF-β, MIP-1α, MIP-1β, MMP-9, TREM-1, BDNF, GM-CSF, NT-4, NT-3, sTNF RI, MIF and RANTES. Histological examinations of the placentas were performed in 42 cases in PTL and in 30 with PPROM. Maternal, antenatal and intrapartal variables were retrieved from medical records Results: Non-lacto-bacillus dominated flora was detected in CF in 25% (22/89) and 17% had microbial invasion of the amniotic cavity (MIAC) and 45% had intra-amniotic inflammation. High levels of IL-6 and IL-8 were associated with PTD ≤ 7 days from assay and ≤ 34 weeks of gestation. Cervical length assessed by TVS predicted intra-amniotic inflammation as well as PTD. Intra-amniotic levels of IL-6, IL-8, IL-18, MCP-1 and MCP-3 were all significantly higher in PTL cases with histological chorioamnionitis (HCA) whereas such relationship was not found in the PPROM group. Cervical IL-6 and IL-8 in PTL were associated HCA and an IL-8 value of 10.0 ng/mL was a strong predictor of HCA with sensitivity 100%, specificity 67%, positive predictive value 63%, and negative predicted value 100%. Several of the proteins analyzed in both AF and CF, by the xMAP technology, were associated with PTD ≤ 7 days from assay and with MIAC. Novel findings were that amniotic IL-17 and TREM1 and cervical IL-17, sIL-6r, BDNF, NT4, NT3, IL-4, IL-5, and RANTES were significantly higher in the women delivering within 7 days of assay. We found that cervical IL-17, sIL-6r, NT3, TNF-β, IL-4, and TREM1 were significantly associated with MIAC which has not previously been reported. A multivariate model combining amniotic macrophage inflammatory protein (MIP)-1β with cervical interferon (INF)-γ and MCP-1 predicted SPTD ≤ 7 days likelihood ratio (LR) 5.6 and area under the ROC-curve (AUC) 0.91 and a non-invasive multivariate model based on CL, cervical INF-γ, IL-6 and MCP-1 predicted SPTD ≤ 7 days with LR 4.7 and AUC 0.91. The best multivariate model predicting MIAC based on cervical IL-17 and MCP-1 had LR 6.0 and AUC 0.87. Conclusions: In the present studies, we have identified inflammatory markers in both cervical and amniotic fluid that together with cervical length as measured by transvaginal sonography can predict spontaneous preterm delivery, intraamniotic infection and/or inflammation and histological chorioamnionitis. It seems as the non-invasive route of sampling analytes can be used instead of the more commonly used invasive method of amniocentesis. Key words: Spontaneous preterm delivery, preterm labor, preterm prelabor rupture of membranes, intra-amniotic infection/inflammation, inflammatory proteins, histological chorioamnionitis, cervical and amniotic marker

Expression of cytokines and chemokines in cervical and amniotic fluid: Relationship to histological chorioamnionitis

Objective. To correlate cervical and amniotic fluid cytokines and macrophage-related chemokines to the development of histological chorioamnionitis (HCA) in patients with preterm labor (PTL) and preterm prelabor rupture of the membranes (PPROM). Study design. Cervical and amniotic fluid interleukin (IL)-6, IL-8, IL-18, monocyte chemotactic protein (MCP)-1, MCP-2, and MCP-3 from pregnant women (at </=34 weeks of gestation) in PTL (N = 42) were analyzed and related to the subsequent occurrence of HCA or inflammatory signs in the placenta. For the patients with PPROM (N = 30) only amniotic fluid proteins were analyzed. Results. Intra-amniotic levels of IL-6, IL-8, IL-18, MCP-1, and MCP-3 were significantly higher in PTL cases with HCA compared to non-HCA controls, whereas no such relationship was obtained in the PPROM group. Cervical IL-8 and IL-6 (but not IL-18, MCP-1, MCP-2, and MCP-3) in PTL patients was associated with HCA, and at a cut-off level of 10.0 ng/mL cervical IL-8 was a strong predictor of HCA in the PTL cases (sensitivity 100%, specificity 67%, positive predictive value 63%, negative predictive value 100%). The cytokine and chemokine levels in the group with inflammatory signs were generally higher than in controls but lower compared to the concentrations in the HCA group. Conclusions. The amniotic levels of IL-6, IL-8, IL-18, and the CC-chemokines MCP-1 and MCP-3 in PTL patients all predicted HCA, whereas only IL-8 was a clinically useful marker of HCA in the cervical fluid. In addition there is indication that the levels of inflammatory proteins are related to the degree of inflammatory infiltration in placental tissue samples

Infectious and inflammatory mechanisms in preterm birth and cerebral palsy

Background: International studies of women in preterm labor (PTL) and preterm pre-labor rupture of the membranes (pPROM) have shown a significant association between microbial invasion of the amniotic cavity (MIAC), some cytokines and chemokines and preterm birth (PTB). These studies have been performed in countries with higher incidence of PTB than that in Sweden. Cerebral palsy (CP) has also been shown to be associated with infectious and inflammatory mechanisms in international epidemiological studies. Our aim was to examine the role of inflammatory mechanisms in PTB and CP in a setting with a low incidence of PTB and perinatal infections.Material and Methods: Amniotic fluid (AF) was retrieved transabdominally from 61 patients in PTL and 47 patients with pPROM, before 34 weeks of gestation in both groups. Forty-five women at term (= 37 weeks) were included. These women were scheduled for elective cesarean section after uncomplicated pregnancies. Cervical fluid was obtained from the external cervical os in all patients in PTL and in all term patients. Polymerase chain reaction analyses for Ureaplasma urealyticum and Mycoplasma hominis and culture for aerobic and anaerobic bacteria were performed. Interleukin (IL)-6, IL-8, IL-18 and monocyte chemotactic protein (MCP)-1 were analyzed with enzyme-linked immunosorbent assay.In order to examine inflammatory mechanisms in CP, a population-based series of 148 preterm infants with spastic CP, born 1983-90, were included and matched with a control group (n=296). Subgroup analyses of patients with spastic diplegia and hemiplegia and those born at <32 and =32 weeks were performed. Maternal, antenatal and intrapartal variables were retrieved from obstetric records. Results: MIAC was detected in 16% of women in PTL and 25 % of women with pPROM. Patients in PTL with MIAC had significantly elevated levels of IL-6, IL-8 and IL-18. The levels of IL-6, IL-8 and MCP-1 were elevated in MIAC cases in women with pPROM. There was also a significant association between elevated levels of IL-6, IL-8, IL-18 and MCP-1 and short amniocentesis-delivery interval (= 7 days) and PTB (< 34 weeks) in women in PTL, whereas this association was less evident in women with pPROM. A receiver-operator-characteristic curve was used to identify the best cut-off levels of IL-6 and IL-8 in AF for delivery within 7 days. This value was used to define an inflammatory response. The inflammatory response rate was 46 % in the PTL group and 51% in the pPROM group. Elevated IL-18 and MCP-1 were related to an inflammatory response in the women in PTL; MCP-1 was also related to an inflammatory response in women with pPROM. There were higher levels of IL-18 and MCP-1 in the cervical fluid of women in PTL, compared with non-laboring women at term. There were elevated levels of MCP-1 in the cervical fluid of women in PTL who gave birth within 7 days or before 34 weeks of gestation, who had MIAC or had intra-amniotic inflammation.In the case-control study of CP, clinical chorioamnionitis/pyelonephritis, long interval between rupture of membranes and birth and admission-delivery interval <4 hours just significantly increased the risk of CP. Apgar scores of <7 at 5 and 10 minutes were strongly associated with an increased CP risk. Abruptio placentae and pathological non-stress test (reason for delivery) were significant risk factors for CP only in the moderately preterm and hemiplegic groups, whereas fever prior to delivery was a significant risk factor in the very preterm and spastic diplegic groups. Conclusion: The occurrence of intra-amniotic microbial invasion and inflammation in this population of Swedish women in PTL and pPROM was similar to that reported in data from populations with a higher incidence of PTB. In addition, our data support an association between antenatal infection/inflammation and CP.Background: International studies of women in preterm labor (PTL) and preterm pre-labor rupture of the membranes (pPROM) have shown a significant association between microbial invasion of the amniotic cavity (MIAC), some cytokines and chemokines and preterm birth (PTB). These studies have been performed in countries with higher incidence of PTB than that in Sweden. Cerebral palsy (CP) has also been shown to be associated with infectious and inflammatory mechanisms in international epidemiological studies. Our aim was to examine the role of inflammatory mechanisms in PTB and CP in a setting with a low incidence of PTB and perinatal infections. Material and Methods: Amniotic fluid (AF) was retrieved transabdominally from 61 patients in PTL and 47 patients with pPROM, before 34 weeks of gestation in both groups. Forty-five women at term (≥ 37 weeks) were included. These women were scheduled for elective cesarean section after uncomplicated pregnancies. Cervical fluid was obtained from the external cervical os in all patients in PTL and in all term patients. Polymerase chain reaction analyses for Ureaplasma urealyticum and Mycoplasma hominis and culture for aerobic and anaerobic bacteria were performed. Interleukin (IL)- 6, IL-8, IL-18 and monocyte chemotactic protein (MCP)-1 were analyzed with enzyme-linked immunosorbent assay. In order to examine inflammatory mechanisms in CP, a population-based series of 148 preterm infants with spastic CP, born 1983-90, were included and matched with a control group (n=296). Subgroup analyses of patients with spastic diplegia and hemiplegia and those born at <32 and ≥32 weeks were performed. Maternal, antenatal and intrapartal variables were retrieved from obstetric records. Results: MIAC was detected in 16% of women in PTL and 25 % of women with pPROM. Patients in PTL with MIAC had significantly elevated levels of IL-6, IL-8 and IL-18. The levels of IL-6, IL-8 and MCP-1 were elevated in MIAC cases in women with pPROM. There was also a significant association between elevated levels of IL-6, IL-8, IL-18 and MCP-1 and short amniocentesis-delivery interval (≤ 7 days) and PTB (< 34 weeks) in women in PTL, whereas this association was less evident in women with pPROM. A receiver-operator-characteristic curve was used to identify the best cut-off levels of IL-6 and IL-8 in AF for delivery within 7 days. This value was used to define an inflammatory response. The inflammatory response rate was 46 % in the PTL group and 51% in the pPROM group. Elevated IL-18 and MCP-1 were related to an inflammatory response in the women in PTL; MCP-1 was also related to an inflammatory response in women with pPROM. There were higher levels of IL- 18 and MCP-1 in the cervical fluid of women in PTL, compared with non-laboring women at term. There were elevated levels of MCP-1 in the cervical fluid of women in PTL who gave birth within 7 days or before 34 weeks of gestation, who had MIAC or had intra-amniotic inflammation. In the case-control study of CP, clinical chorioamnionitis/pyelonephritis, long interval between rupture of membranes and birth and admission-delivery interval <4 hours just significantly increased the risk of CP. Apgar scores of <7 at 5 and 10 minutes were strongly associated with an increased CP risk. Abruptio placentae and pathological non-stress test (reason for delivery) were significant risk factors for CP only in the moderately preterm and hemiplegic groups, whereas fever prior to delivery was a significant risk factor in the very preterm and spastic diplegic groups. Conclusion: The occurrence of intra-amniotic microbial invasion and inflammation in this population of Swedish women in PTL and pPROM was similar to that reported in data from populations with a higher incidence of PTB. In addition, our data support an association between antenatal infection/inflammation and CP. Keywords: preterm birth, preterm labor, preterm prelabor rupture of membranes, intra-uterine infection, intra-amniotic inflammation, interleukin-6, interleukin-8, interleukin-18, monocyte chemotactic protein-1, cerebral palsy, antenatal risk factors, chorioamnionitis

Interlaboratory Standardization of the Sandwich Enzyme-Linked Immunosorbent Assay Designed for MATS, a Rapid, Reproducible Method for Estimating the Strain Coverage of Investigational Vaccines.

International audienceThe meningococcal antigen typing system (MATS) sandwich enzyme-linked immunosorbent assay (ELISA) was designed to measure the immunologic cross-reactivity and quantity of antigens in target strains of a pathogen. It was first used to measure the factor H-binding protein (fHbp), neisserial adhesin A (NadA), and neisserial heparin-binding antigen (NHBA) content of serogroup B meningococcal (MenB) isolates relative to a reference strain, or “relative potency” (RP). With the PorA genotype, the RPs were then used to assess strain coverage by 4CMenB, a multicomponent MenB vaccine. In preliminary studies, MATS accurately predicted killing in the serum bactericidal assay using human complement, an accepted correlate of protection for meningococcal vaccines. A study across seven laboratories assessed the reproducibility of RPs for fHbp, NadA, and NHBA and established qualification parameters for new laboratories. RPs were determined in replicate for 17 MenB reference strains at laboratories A to G. The reproducibility of RPs among laboratories and against consensus values across laboratories was evaluated using a mixed-model analysis of variance. Interlaboratory agreement was very good; the Pearson correlation coefficients, coefficients of accuracy, and concordance correlation coefficients exceeded 99%. The summary measures of reproducibility, expressed as between-laboratory coefficients of variation, were 7.85% (fHbp), 16.51% (NadA), and 12.60% (NHBA). The overall within-laboratory measures of variation adjusted for strain and laboratory were 19.8% (fHbp), 28.8% (NHBA), and 38.3% (NadA). The MATS ELISA was successfully transferred to six laboratories, and a further laboratory was successfully qualified