Kristin Skogstrand

Intensive insulin therapy protects the endothelium of critically ill patients.

The vascular endothelium controls vasomotor tone and microvascular flow and regulates trafficking of nutrients and biologically active molecules. When endothelial activation is excessive, compromised microcirculation and subsequent cellular hypoxia contribute to the risk of organ failure. We hypothesized that strict blood glucose control with insulin during critical illness protects the endothelium, mediating prevention of organ failure and death. In this preplanned subanalysis of a large, randomized controlled study, intensive insulin therapy lowered circulating levels of ICAM-1 and tended to reduce E-selectin levels in patients with prolonged critical illness, which reflects reduced endothelial activation. This effect was not brought about by altered levels of endothelial stimuli, such as cytokines or VEGF, or by upregulation of eNOS. In contrast, prevention of hyperglycemia by intensive insulin therapy suppressed iNOS gene expression in postmortem liver and skeletal muscle, possibly in part via reduced NF-kappaB activation, and lowered the elevated circulating NO levels in both survivors and nonsurvivors. These effects on the endothelium statistically explained a significant part of the improved patient outcome with intensive insulin therapy. In conclusion, maintaining normoglycemia with intensive insulin therapy during critical illness protects the endothelium, likely in part via inhibition of excessive iNOS-induced NO release, and thereby contributes to prevention of organ failure and death.

Weight loss larger than 10% is needed for general improvement of levels of circulating adiponectin and markers of inflammation in obese subjects: a 3-year weight loss study

OBJECTIVE To investigate the effects of: I) short- (8 weeks), II) long-term (3 years) weight loss, and III) the degree of weight loss on circulating levels of adiponectin, high sensitive-C reactive protein (hs-CRP), and fibrinogen in obese subjects. Moreover, to evaluate the effect of the lipase inhibitor, orlistat, on these parameters. DESIGN Weight loss induced in 93 obese subjects (mean weight: 108.9+/-15.8 kg) through 8-week very-low-energy diet (VLED, 800 kcal/day) followed by randomization to orlistat or placebo together with lifestyle intervention for further 3 years. Adiponectin and hs-CRP were measured at baseline, after 8 weeks of VLED and 6, 12, and 36 months after the VLED by flowmetric xMAP technology (Luminex Multi-Analyte Profiling System, Luminex Corp., Austin, TX, USA). Fibrinogen was measured in a coagulation assay. RESULTS Weight loss after VLED treatment was 14.3+/-4.5 kg and after 3 years 7.7+/-8.7 kg. Orlistat-treated subjects regained 3.9 kg less than placebo-treated from the end of the VLED to 3 years (P=0.01). No differences were detected between the two groups regarding changes in adiponectin, hs-CRP, or fibrinogen. Accordingly, the groups were combined for further analyses. Serum adiponectin increased by 22% (P<0.05) after the VLED but returned to baseline after 3 years. Both short- and long-term weight losses needed to be in excess of 10% (approximately 12 kg) in order to increase adiponectin levels significantly. Weight loss was associated with a significant decrease in hs-CRP. Fibrinogen decreased by 12% (P<0.05) after 3 years. CONCLUSIONS In obese subjects, weight loss was associated with an increase in serum adiponectin and a decrease in hs-CRP and plasma fibrinogen. Long-term weight loss (3 years) must exceed 10% to induce a combined significant improvement in these inflammatory markers.

Differential Patterns of 27 Cord Blood Immune Biomarkers Across Gestational Age

OBJECTIVES. Inflammation has been associated with preterm delivery and adverse neonatal outcomes such as cerebral palsy and chronic lung disease. However, no study to date has simultaneously examined a wide range of inflammatory mediators and their relationship to gestational age. We sought to describe the distribution of immune biomarkers in cord blood across gestational age and to investigate the association between biomarker level patterns and preterm birth. PATIENTS AND METHODS. As part of a large-scale molecular epidemiological study of preterm birth conducted at Boston Medical Center, this study analyzed both clinical and biomarker data from 927 births. Twenty-seven biomarkers were simultaneously quantified by immunoassay. The associations between the quartiles of 27 biomarkers and 3 gestational groups (≤32, 33–36, and ≥37 weeks) were analyzed. Biomarkers found to be significant were further analyzed for dose-response correlation with preterm birth by logistic regression, adjusted for pertinent demographic and clinical factors. RESULTS. The 27 biomarkers could be classified into 1 of 3 groups: (1) biomarkers increased in preterm birth (interleukin [IL]-2, IL-4, IL-5, IL-8, IL-10, monocyte chemoattractant protein 1, macrophage inflammatory protein [MIP]-1α, MIP-1β, soluble IL-6 receptor α, tumor necrosis factor α, soluble tumor necrosis factor receptor I, and TREM-1 [triggering receptor expressed on myeloid cells 1]); (2) biomarkers decreased in preterm birth (brain-derived neurotrophic factor, IL-1β, IL-18, matrix metalloproteinase 9, and neurotrophin 3); and (3) biomarkers not associated with preterm birth (IL-6, IL-12, IL-17, granulocyte/macrophage colony-stimulating factor, interferon γ, macrophage migration inhibitory factor, neurotrophin 4, RANTES [regulated on activation, normal T-cell expressed and secreted], transforming growth factor β, and tumor necrosis factor β). CONCLUSIONS. Biomarkers have different directions of association with prematurity; for significant biomarkers, the strength of association increases with biomarker concentration. Our results provide important information that could be used to guide additional studies aimed at determining mechanisms that contribute to preterm birth.

Perinatal Systemic Inflammatory Response Syndrome and Retinopathy of Prematurity

Fetal and neonatal inflammation is associated with several morbidities of prematurity. Its relationship to retinopathy of prematurity (ROP) has not been investigated. Our objective was to determine the relationship between cytokine levels and ROP in the first 3 postnatal wks. Data for this study were derived from the NICHD Cytokine Study. Dried blood spots (DBS) were obtained from infants <1000 g on days 0–1, 3 ± 1, 7 ± 2, 14 ± 3, and 21 ± 3. Infants were classified into three groups—no, mild, and severe ROP. Multiplex Luminex assay was used to quantify 20 cytokines. Temporal profiles of cytokines were evaluated using mixed-effects models after controlling for covariates. Of 1074 infants enrolled, 890 were examined for ROP and 877 included in the analysis. ROP was associated with several clinical characteristics on unadjusted analyses. Eight cytokines remained significantly different across ROP groups in adjusted analyses. IL-6 and IL-17 showed significant effects in early time periods (D0–3); TGF-β, brain-derived neurotrophic factor (BDNF), and regulated on activation, normal T cell expressed and secreted (RANTES) in later time periods (D7–21) and IL-18, C-reactive protein (CRP), and neurotrophin-4 (NT-4) in both early and later time periods. We conclude that perinatal inflammation may be involved in the pathogenesis of ROP.

Effects of blood sample handling procedures on measurable inflammatory markers in plasma, serum and dried blood spot samples

The interests in monitoring inflammation by immunoassay determination of blood inflammatory markers call for information on the stability of these markers in relation to the handling of blood samples. The increasing use of stored biobank samples for such ventures that may have been collected and stored for other purposes, justifies the study hereof. Blood samples were stored for 0, 4, 24, and 48 h at 4 degrees C, room temperature (RT), and at 35 degrees C, respectively, before they were separated into serum or plasma and frozen. Dried blood spot samples (DBSS) were stored for 0, 1, 2, 3, 7, and 30 days at the same temperatures. 27 inflammatory markers in serum and plasma and 25 markers in DBSS were measured by a previously validated multiplex sandwich immunoassay using Luminex xMAP technology. The measurable concentrations of several cytokines in serum and plasma were significantly increased when blood samples were stored for a period of time before the centrifugation, for certain cytokines more than 1000 fold compared to serum and plasma isolated and frozen immediately after venepuncture. The concentrations in serum generally increased more than in plasma. The measurable concentrations of inflammatory markers also changed in DBSS stored under various conditions compared to controls frozen immediately after preparation, but to a much lesser degree than in plasma or serum. The study demonstrates that trustworthy measurement of several inflammatory markers relies on handling of whole blood samples at low temperatures and rapid isolation of plasma and serum. Effects of different handling procedures for all markers studied are given. DBSS proved to be a robust and convenient way to handle samples for immunoassay analysis of inflammatory markers in whole blood.

Mid-pregnancy circulating cytokine levels, histologic chorioamnionitis and spontaneous preterm birth

Some spontaneous preterm deliveries (PTD) are caused by occult infections of the fetal membranes (histologic chorioamnionitis [HCA]). High levels of infection-related markers, including some cytokines, sampled from maternal circulation in mid-pregnancy have been linked to PTD, but whether these specifically identify HCA has not been established. We have tested associations between 13 Th1, Th2 and Th17 cytokines and PTD with and without HCA in a prospective cohort study. The study sample included 926 Pregnancy Outcomes and Community Health Study subcohort women; women with medically indicated PTD or incomplete data excluded. A panel of cytokines was assessed using a multiplex assay in maternal plasma collected at 15-27 weeks of gestation. Severe HCA was scored by a placental pathologist blinded to clinical variables. Multivariable polytomous logistic regression was used to estimate adjusted odds ratios (OR) per 1 standard deviation (S.D.) increase in cytokine levels using a 5 level outcome variable: PTD <35 weeks with HCA, PTD <35 weeks without HCA, PTD 35-36 weeks with HCA, PTD 35-36 weeks without HCA, and term (referent). Interleukin (IL)-1beta, IL-2, IL-12, interferon-gamma, IL-4, IL-6 and transforming growth factor-beta were all significantly associated with PTD <35 weeks with HCA, with ORs of 1.6-2.3 per S.D. increase. None of these were associated with PTD <35 weeks without HCA or PTD 35-36 weeks with HCA. Although the tissues of origin of circulating cytokines are unclear, the observed elevations across many cytokines among women who later delivered <35 weeks with HCA may represent a robust immune response to infection within gestational tissues. These results suggest that women with HCA could be identified using relatively non-invasive means.

Mid-pregnancy maternal plasma levels of interleukin 2, 6, and 12, tumor necrosis factor-alpha, interferon-gamma, and granulocyte-macrophage colony-stimulating factor and spontaneous preterm delivery

Background. Few studies have investigated the relationship between inflammation and spontaneous preterm delivery (sPTD) in women before preterm labour. The authors examine whether mid‐pregnancy plasma cytokine levels are associated with sPTD, and whether associations vary by maternal age, body mass index, prior preterm delivery, or gravidity. Methods. This case‐control study was nested within the Danish National Birth Cohort, a cohort of women with 101,042 pregnancies from 1997 to 2002. Included in this study are 61 women delivering at 24–29 weeks, 278 delivering at 30–33 weeks, 334 delivering at 34–36 weeks, and 1,125 delivering at ≥37 weeks. Maternal plasma interleukin (IL)‐2, IL‐6, tumor necrosis factor (TNF)‐α, interferon (IFN)‐γ, and granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) at 25 weeks’ gestation were measured using multiplex flow cytometry. Results. For IL‐2, TNF‐α, and GM‐CSF, the proportion of women with levels >75th or >90th percentile did not differ by gestational age at delivery. IFN‐γ >90th percentile was associated with an increased risk of delivering at 30–33 weeks (crude odds ratio (cOR): 1.56; 95% confidence interval (CI): 1.07–2.30), while IFN‐γ >75th percentile and IL‐6 >75th percentile were associated with an increased risk of delivering at 34–36 weeks (cOR: 1.32; 95% CI: 1.01–1.73); estimates changed little after adjusting for confounders. There was no effect‐measure modification by maternal factors. Conclusion. Elevated mid‐pregnancy plasma IL‐2, TNF‐α, and GM‐CSF did not appear to be associated with an increased risk of sPTD, while elevated IFN‐γ and IL‐6 levels were weakly associated with moderate and late sPTD. The value of using mid‐pregnancy cytokines in predicting spontaneous preterm delivery appears limited.

Intraamniotic inflammatory response to bacteria: analysis of multiple amniotic fluid proteins in women with preterm prelabor rupture of membranes.

Objective: To analyse whether intraamniotic inflammation in response to bacteria is different below and above gestational age 32 weeks in pregnancies complicated by preterm prelabor rupture of membranes (PPROM). Methods: A prospective study was performed, and 115 women with singleton pregnancies complicated by PPROM at gestational ages between 240/7 and 366/7 weeks were included in the study. Transabdominal amniocenteses were performed. Amniotic fluid was analysed using polymerase chain reactions for genital mycoplasmas and cultured for aerobic and anaerobic bacteria. The concentrations of 26 proteins in the amniotic fluid were determined simultaneously using multiplex technology. Results: Bacteria were found in the amniotic fluid of 43% (49/115) of the women. The women were stratified into two subgroups according to gestational age 32 weeks. The amniotic fluid levels of four (interleukin-6, interleukin-10, CC chemokine ligands 2, and 3) and one specific (CC chemokine ligands 2) proteins were higher in women with the presence of bacteria in the amniotic fluid below and above 32 gestational weeks, respectively. Conclusions: An intraamniotic inflammatory response to bacteria in pregnancies complicated by PPROM seems to be different below and above 32 weeks of gestation.

Prediction of spontaneous preterm delivery in women with preterm labor: analysis of multiple proteins in amniotic and cervical fluids.

OBJECTIVE: To analyze whether specific proteins in amniotic and cervical fluids, alone or in combination with risk factors, can identify women in preterm labor with intact membranes who will deliver spontaneously within 7 days of sampling. METHODS: In a cohort of 89 women in preterm labor, amniotic and cervical fluids were collected between 22 and 33 weeks of gestation. Twenty-seven proteins were analyzed simultaneously using multiplex technology. Individual levels of each protein were compared and calculations performed to find associations among different proteins, background variables, and spontaneous preterm delivery within 7 days of sampling. The area under the curve (AUC) was calculated using receiver operating characteristic curve analysis, and prediction models were created based on stepwise logistic regression. RESULTS: We found two multivariable models that predicted spontaneous preterm delivery better than any single variable. One combined multivariable prediction model was based on amniotic macrophage inflammatory protein-1&bgr;, cervical interferon-&ggr;, and monocyte chemotactic protein-1. This model predicted outcome with 91% sensitivity, 84% specificity, 78% positive predictive value, and 94% negative predictive value, with a likelihood ratio of 5.6 and AUC of 0.91. An alternative, noninvasive model based on cervical length, cervical interferon-&ggr;, interleukin-6, and monocyte chemotactic protein-1 predicted delivery within 7 days with 85% sensitivity, 82% specificity, 74% positive predictive value, and 90% negative predictive value, with a likelihood ratio of 4.7 and AUC of 0.91. CONCLUSION: A combination of proteins from amniotic fluid and cervical fluid or cervical length can help determine which women will deliver preterm. LEVEL OF EVIDENCE: II

Cytokines and Neurodevelopmental Outcomes in Extremely Low Birth Weight Infants

OBJECTIVE To determine if selected pro-inflammatory and anti-inflammatory cytokines and/or mediators of inflammation reported to be related to the development of cerebral palsy (CP) predict neurodevelopmental outcome in extremely low birth weight infants. STUDY DESIGN Infants with birth weights ≤1000 g (n = 1067) had blood samples collected at birth and on days 3 ± 1, 7 ± 1, 14 ± 3, and 21 ± 3 to examine the association between cytokines and neurodevelopmental outcomes. The analyses were focused on 5 cytokines (interleukin [IL] 1β; IL-8; tumor necrosis factor-α; regulated upon activation, normal T-cell expressed, and secreted (RANTES); and IL-2) reported to be most predictive of CP in term and late preterm infants. RESULTS IL-8 was higher on days 0-4 and subsequently in infants who developed CP compared with infants who did not develop CP in both unadjusted and adjusted analyses. Other cytokines (IL-12, IL-17, tumor necrosis factor-β, soluble IL rα, macrophage inflammatory protein 1β) were found to be altered on days 0-4 in infants who developed CP. CONCLUSIONS CP in former preterm infants may, in part, have a late perinatal and/or early neonatal inflammatory origin.