Search

Background: Perinatal inflammation combined with hypoxia-ischemia (HI) exacerbates injury in the developing brain. Therapeutic hypothermia (HT) is standard care for neonatal encephalopathy; however, its benefit in inflammation-sensitized HI (IS-HI) is unknown., Methods: Twelve newborn piglets received a 2 µg/kg bolus and 1 µg/kg/h infusion over 52 h of Escherichia coli lipopolysaccharide (LPS). HI was induced 4 h after LPS bolus. After HI, piglets were randomized to HT (33.5 °C 1-25 h after HI, n = 6) or normothermia (NT, n = 6). Amplitude-integrated electroencephalogram (aEEG) was recorded and magnetic resonance spectroscopy (MRS) was acquired at 24 and 48 h. At 48 h, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)-positive brain cell death, microglial activation/proliferation, astrogliosis, and cleaved caspase-3 (CC3) were quantified. Hematology and plasma cytokines were serially measured., Results: Two HT piglets died. aEEG recovery, thalamic and white matter MRS lactate/N-acetylaspartate, and TUNEL-positive cell death were similar between groups. HT increased microglial activation in the caudate, but had no other effect on glial activation/proliferation. HT reduced CC3 overall. HT suppressed platelet count and attenuated leukocytosis. Cytokine profile was unchanged by HT., Conclusions: We did not observe protection with HT in this piglet IS-HI model based on aEEG, MRS, and immunohistochemistry. Immunosuppressive effects of HT and countering neuroinflammation by LPS may contribute to the observed lack of HT efficacy. Other immunomodulatory strategies may be more effective in IS-HI., Impact: Acute infection/inflammation is known to exacerbate perinatal brain injury and can worsen the outcomes in neonatal encephalopathy. Therapeutic HT is the current standard of care for all infants with NE, but the benefit in infants with coinfection/inflammation is unknown. In a piglet model of inflammation (LPS)-sensitized HI, we observed no evidence of neuroprotection with cooling for 24 h, based on our primary outcome measures: aEEG, MRS Lac/NAA, and histological brain cell death. Additional neuroprotective agents, with beneficial immunomodulatory effects, require exploration in IS-HI models., (© 2021. The Author(s).)

Background: Chorioamnionitis, an intrauterine infection of the placenta and fetal membranes, is a common risk factor for adverse pulmonary outcomes in premature infants including BPD, which is characterized by an arrest in alveolar development. As endogenous epithelial stem/progenitor cells are crucial for organogenesis and tissue repair, we examined whether intrauterine inflammation negatively affects these essential progenitor pools., Methods: In an ovine chorioamnionitis model, fetuses were intra-amniotically exposed to LPS, 2d or 7d (acute inflammation) before preterm delivery at 125d of gestation, or to intra-amniotic Ureaplasma parvum for 42d (chronic inflammation). Lung function, pulmonary endogenous epithelial stem/progenitor pools, and downstream functional markers were studied., Results: Lung function was improved in the 7d LPS and 42d Ureaplasma groups. However, intrauterine inflammation caused a loss of P63+ basal cells in proximal airways and reduced SOX-9 expression and TTF-1+ Club cells in distal airways. Attenuated type-2 cell numbers were associated with lower proliferation and reduced type-1 cell marker Aqp5 expression, indicative for impaired progenitor function. Chronic Ureaplasma infection only affected distal airways, whereas acute inflammation affected stem/progenitor populations throughout the lungs., Conclusions: Acute and chronic prenatal inflammation improve lung function at the expense of stem/progenitor alterations that potentially disrupt normal lung development, thereby predisposing to adverse postnatal outcomes., Impact: In this study, prenatal inflammation improved lung function at the expense of stem/progenitor alterations that potentially disrupt normal lung development, thereby predisposing to adverse postnatal outcomes. Importantly, we demonstrate that these essential alterations can already be initiated before birth. So far, stem/progenitor dysfunction has only been shown postnatally. This study indicates that clinical protocols to target the consequences of perinatal inflammatory stress for the immature lungs should be initiated as early as possible and ideally in utero. Within this context, our data suggest that interventions, which promote function or repair of endogenous stem cells in the lungs, hold great promise., (© 2021. International Pediatric Research Foundation, Inc.)

The N-Myc Downstream-Regulated Gene 4 (NDRG4), a prominent biomarker for colorectal cancer (CRC), is specifically expressed by enteric neurons. Considering that nerves are important members of the tumor microenvironment, we here establish different Ndrg4 knockout (Ndrg4 -/- ) CRC models and an indirect co-culture of primary enteric nervous system (ENS) cells and intestinal organoids to identify whether the ENS, via NDRG4, affects intestinal tumorigenesis. Linking immunostainings and gastrointestinal motility (GI) assays, we show that the absence of Ndrg4 does not trigger any functional or morphological GI abnormalities. However, combining in vivo, in vitro, and quantitative proteomics data, we uncover that Ndrg4 knockdown is associated with enlarged intestinal adenoma development and that organoid growth is boosted by the Ndrg4 -/- ENS cell secretome, which is enriched for Nidogen-1 (Nid1) and Fibulin-2 (Fbln2). Moreover, NID1 and FBLN2 are expressed in enteric neurons, enhance migration capacities of CRC cells, and are enriched in human CRC secretomes. Hence, we provide evidence that the ENS, via loss of Ndrg4, is involved in colorectal pathogenesis and that ENS-derived Nidogen-1 and Fibulin-2 enhance colorectal carcinogenesis., (© 2021 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

Chorioamnionitis, inflammation of fetal membranes, is an important cause of preterm birth and a risk factor for the development of adverse neonatal outcomes including sepsis and intestinal pathologies. Intestinal bile acids (BAs) accumulation and hepatic cytokine production are involved in adverse intestinal outcomes. These findings triggered us to study the liver and enterohepatic circulation (EHC) following intra-amniotic (IA) lipopolysaccharide (LPS) exposure. An ovine chorioamnionitis model was used in which circulatory cytokines and outcomes of the liver and EHC of preterm lambs were longitudinally assessed following IA administration of 10 mg LPS at 5, 12 or 24h or 2, 4, 8 or 15d before preterm birth. Hepatic inflammation was observed, characterized by increased hepatic cytokine mRNA levels (5h - 2d post IA LPS exposure) and increased erythropoietic clusters (at 8 and 15 days post IA LPS exposure). Besides, 12h after IA LPS exposure, plasma BA levels were increased, whereas gene expression levels of several hepatic BA transporters were decreased. Initial EHC alterations normalized over time. Concluding, IA LPS exposure induces significant time-dependent changes in the fetal liver and EHC. These chorioamnionitis induced changes have potential postnatal consequences and the duration of IA LPS exposure might be essential herein.

Background: Exposure to inflammation exacerbates injury in neonatal encephalopathy (NE). We hypothesized that brain biomarker mRNA, cytokine mRNA and microRNA differentiate inflammation (E. coli LPS), hypoxia (Hypoxia), and inflammation-sensitized hypoxia (LPS+Hypoxia) in an NE piglet model., Methods: Sixteen piglets were randomized: (i) LPS 2 μg/kg bolus; 1 μg/kg infusion (LPS; n = 5), (ii) Saline with hypoxia (Hypoxia; n = 6), (iii) LPS commencing 4 h pre-hypoxia (LPS+Hypoxia; n = 5). Total RNA was acquired at baseline, 4 h after LPS and 1, 3, 6, 12, 24, 48 h post-insult (animals euthanized at 48 h). Quantitative PCR was performed for cytokines (IL1A, IL6, CXCL8, IL10, TNFA) and brain biomarkers (ENO2, UCHL1, S100B, GFAP, CRP, BDNF, MAPT). MicroRNA was detected using GeneChip (Affymetrix) microarrays. Fold changes from baseline were compared between groups and correlated with cell death (TUNEL) at 48 h., Results: Within 6 h post-insult, we observed increased IL1A, CXCL8, CCL2 and ENO2 mRNA in LPS+Hypoxia and LPS compared to Hypoxia. IL10 mRNA differentiated all groups. Four microRNAs differentiated LPS+Hypoxia and Hypoxia: hsa-miR-23a, 27a, 31-5p, 193-5p. Cell death correlated with TNFA (R = 0.69; p < 0.01) at 1-3 h and ENO2 (R = -0.69; p = 0.01) at 48 h., Conclusions: mRNA and miRNA differentiated hypoxia from inflammation-sensitized hypoxia within 6 h in a piglet model. This information may inform human studies to enable triage for tailored neuroprotection in NE., Impact: Early stratification of infants with neonatal encephalopathy is key to providing tailored neuroprotection. IL1A, CXCL8, IL10, CCL2 and NSE mRNA are promising biomarkers of inflammation-sensitized hypoxia. IL10 mRNA levels differentiated all three pathological states; fold changes from baseline was the highest in LPS+Hypoxia animals, followed by LPS and Hypoxia at 6 h. miR-23, -27, -31-5p and -193-5p were significantly upregulated within 6 h of a hypoxia insult. Functional analysis highlighted the diverse roles of miRNA in cellular processes.

Autologous fat transfer (AFT) is limited by post-operative volume loss due to ischemia-induced cell death in the fat graft. Previous studies have demonstrated that electrical stimulation (ES) promotes angiogenesis in a variety of tissues and cell types. In this study we investigated the effects of ES on the angiogenic potential of adipose-derived stem cells (ASC), important progenitor cells in fat grafts with proven angiogenic potential. Cultured human ASC were electrically stimulated for 72 hours after which the medium of stimulated (ES) and non-stimulated (control) ASC was analysed for angiogenesis-related proteins by protein array and ELISA. The functional effect of ES on angiogenesis was then assessed in vitro and in vivo. Nine angiogenesis-related proteins were detected in the medium of electrically (non-)stimulated ASC and were quantified by ELISA. The pro-angiogenic proteins VEGF and MCP-1 were significantly increased following ES compared to controls, while the anti-angiogenic factor Serpin E1/PAI-1 was significantly decreased. Despite increased levels of anti-angiogenic TSP-1 and TIMP-1, medium of ES-treated ASC significantly increased vessel density, total vessel network length and branching points in chorio-allantoic membrane assays. In conclusion, our proof-of-concept study showed that ES increased the angiogenic potential of ASC both in vitro and in vivo.

Co-existing infection/inflammation and birth asphyxia potentiate the risk of developing neonatal encephalopathy (NE) and adverse outcome. In a newborn piglet model we assessed the effect of E. coli lipopolysaccharide (LPS) infusion started 4 h prior to and continued for 48 h after hypoxia on brain cell death and systemic haematological changes compared to LPS and hypoxia alone. LPS sensitized hypoxia resulted in an increase in mortality and in brain cell death (TUNEL positive cells) throughout the whole brain, and in the internal capsule, periventricular white matter and sensorimotor cortex. LPS alone did not increase brain cell death at 48 h, despite evidence of neuroinflammation, including the greatest increases in microglial proliferation, reactive astrocytosis and cleavage of caspase-3. LPS exposure caused splenic hypertrophy and platelet count suppression. The combination of LPS and hypoxia resulted in the highest and most sustained systemic white cell count increase. These findings highlight the significant contribution of acute inflammation sensitization prior to an asphyxial insult on NE illness severity.

Chorioamnionitis is caused by intrauterine infection with microorganisms including Candida albicans (C.albicans). Chorioamnionitis is associated with postnatal intestinal pathologies including necrotizing enterocolitis. The underlying mechanisms by which intra-amniotic C.albicans infection adversely affects the fetal gut remain unknown. Therefore, we assessed whether intra-amniotic C.albicans infection would cause intestinal inflammation and mucosal injury in an ovine model. Additionally, we tested whether treatment with the fungistatic fluconazole ameliorated the adverse intestinal outcome of intra-amniotic C.albicans infection. Pregnant sheep received intra-amniotic injections with 10(7) colony-forming units C.albicans or saline at 3 or 5 days before preterm delivery at 122 days of gestation. Fetuses were given intra-amniotic and intra-peritoneal fluconazole treatments 2 days after intra-amniotic administration of C.albicans. Intra-amniotic C.albicans caused intestinal colonization and invasive growth within the fetal gut with mucosal injury and intestinal inflammation, characterized by increased CD3(+) lymphocytes, MPO(+) cells and elevated TNF-α and IL-17 mRNA levels. Fluconazole treatment in utero decreased intestinal C.albicans colonization, mucosal injury but failed to attenuate intestinal inflammation. Intra-amniotic C.albicans caused intestinal infection, injury and inflammation. Fluconazole treatment decreased mucosal injury but failed to ameliorate C.albicans-mediated mucosal inflammation emphasizing the need to optimize the applied antifungal therapeutic strategy.

Background: Intrauterine inflammation activates the fetal immune system and can result in organ injury and postnatal complications in preterm infants. As the spleen is an important site for peripheral immune activation, we asked how the fetal spleen would respond to intrauterine inflammation over time. We hypothesized that intraamniotic lipopolysaccharide (IA LPS) exposure induces acute and persistent changes in the splenic cytokine profile and T-cell composition that may contribute to the sustained fetal inflammatory response after chorioamnionitis., Methods: Fetal sheep were exposed to IA LPS 5, 12, and 24 h and 2, 4, 8, or 15 d before delivery at 125 d of gestational age (term = 150 d). Splenic cytokine mRNA levels and cleaved caspase-3, CD3, and Foxp3 expression were evaluated., Results: IA LPS increased interleukin (IL)1, IL4, IL5, and IL10 mRNA by twofold 24 h after injection. Interferon gamma increased by fivefold, whereas IL23 decreased 15 d post-LPS exposure. Cleaved caspase-3-positive cells increased 2 and 8 d after LPS exposure. CD3 immunoreactivity increased within 5 h with increased Foxp3-positive cells at 12 h., Conclusion: Intrauterine inflammation induced a rapid and sustained splenic immune response with persistent changes in the cytokine profile. This altered immune status may drive sustained inflammation and injury in other fetal organs.

Background: Gut immaturity is linked with postnatal intestinal disorders. However, biomarkers to assess the intestinal developmental stage around birth are lacking. The aim of this study was to gain more insight on intestinal fatty acid-binding protein (I-FABP) as an indicator of gut maturity., Methods: Antenatal I-FABP distribution and release was investigated in extremely premature, moderately premature, and term lambs, and these findings were verified in human urinary samples. Ileal I-FABP distribution was confirmed in autopsy material within 24 h postnatally., Results: Median (range) serum I-FABP levels were lower in extremely premature lambs compared with moderately premature lambs (156 (50.0-427) vs. 385 (100-1,387) pg/ml; P = 0.02). Contrarily, median early postnatal urine I-FABP levels in human infants were higher in extremely premature compared with moderately premature and term neonates (1,219 (203-15,044) vs. 256 (50-1,453) and 328 (96-1,749) pg/ml; P = 0.008 and P = 0.04, respectively). I-FABP expression was most prominent in nonvacuolated enterocytes and increased with rising gestational age (GA) in ovine and human tissue samples. The epithelial distribution pattern changed from a phenotype displaying I-FABP-positive enterocytes merely in the crypts early in gestation into a phenotype with I-FABP expressing cells exclusively present in the villus tips at term in ovine and human tissue., Conclusion: In this ovine and human study, increasing GA is accompanied by an increase in I-FABP tissue content. Cord I-FABP levels correlate with gestation in ovine fetuses, identifying I-FABP as a marker for gut maturation. Raised postnatal urine I-FABP levels in preterm human infants may indicate intestinal injury and/or inflammation in utero.

Background: Antenatal inflammation and maternal corticosteroids induce fetal lung maturation but interfere with late lung development. Canonical Wingless-Int (Wnt) signaling directs lung development and repair. We showed that intra-amniotic (IA) lipopolysaccharide (LPS) exposure disrupted developmental signaling pathways in the preterm lamb lungs. Therefore, we hypothesized that pulmonary Wnt signaling was altered by exposure to IA LPS and/or antenatal corticosteroids., Methods: Ovine fetuses were exposed to IA LPS, maternal intramuscular betamethasone, a control saline injection, or a combination thereof at 107 and/or 114 d gestational age (term = 150 d gestational age) before delivery at 121 d gestational age., Results: IA LPS exposure decreased the lung expression of lymphoid enhancer-binding factor 1 (LEF1), a major Wnt pathway effector. WNT1, WNT4, and downstream messenger β-catenin decreased after LPS exposure. WNT7b mRNA increased fourfold 14 d post-LPS exposure. Betamethasone treatment 7 d before LPS exposure prevented the reduction in LEF1 expression, whereas betamethasone administration after LPS normalized the LPS-induced increase in Wnt7b mRNA., Conclusion: IA LPS exposure decreased canonical Wnt signaling in the developing lung. Antenatal corticosteroids before or after IA inflammation had different effects on pulmonary Wnt signaling. This study provides new insights into possible mechanisms by which prenatal inflammation affects lung development and how corticosteroid can be beneficial in this setting.

Background: Reintroduction of enteral nutrition in neonates with necrotizing enterocolitis (NEC) should take place when the gut is ready for its normal function. Too early a start of oral feeding might lead to disease relapse, whereas prolonged discontinuation of enteral nutrition is associated with impaired gut function and parenteral nutrition-related complications. This study evaluated whether noninvasive urinary measurement of intestinal fatty acid binding protein (I-FABP) at the time of refeeding can predict clinical outcome in neonates with NEC., Methods: Urinary I-FABP concentrations were measured in 21 infants with NEC just before reintroducing enteral nutrition. Poor outcome was defined as unsuccessful reintroduction of enteral feeding (EF), (re)operation for NEC, or death related to NEC after reintroduction of EF., Results: Median urinary I-FABP levels in neonates with poor outcome (n = 5) were significantly higher as compared with I-FABP levels in neonates with good outcome (n = 16) (P < 0.01). A clinically significant cutoff value of 963 pg/ml was found to discriminate between infants with poor outcome and those with good outcome (sensitivity 80%, specificity 94%)., Conclusion: Noninvasive urinary I-FABP measurement at time of refeeding differentiates neonates with poor outcome from neonates with good outcome in NEC. Urinary I-FABP measurement may therefore be helpful in the timing of EF in neonates with NEC.