Optimized lung expansion ventilation modulates ventilation-induced lung injury in preterm lambs.

INTRODUCTION: Preterm infants close to viability commonly require mechanical ventilation (MV) for respiratory distress syndrome. Despite commonly used lung-sparing ventilation techniques, rapid lung expansion during MV induces lung injury, a risk factor for bronchopulmonary dysplasia. This study investigates whether ventilation with optimized lung expansion is feasible and whether it can further minimize lung injury. Therefore, optimized lung expansion ventilation (OLEV) was compared to conventional volume targeted ventilation. METHODS: Twenty preterm lambs were surgically delivered after 132 days of gestation. Nine animals were randomized to receive OLEV for 24 h, and seven received standard MV. Four unventilated animals served as controls (NV). Lungs were sampled for histological analysis at the end of the experimental period. RESULTS: Ventilation with OLEV was feasible, resulting in a significantly higher mean ventilation pressure (0.7-1.3 mbar). Temporary differences in oxygenation between OLEV and MV did not reach clinically relevant levels. Ventilation in general tended to result in higher lung injury scores compared to NV, without differences between OLEV and MV. While pro-inflammatory tumor necrosis factor-α messenger RNA (mRNA) levels increased in both ventilation groups compared to NV, only animals in the MV group showed a higher number of CD45-positive cells in the lung. In contrast, mean (standard deviations) surfactant protein-B mRNA levels were significantly lower in OLEV, 0.63 (0.38) compared to NV 1.03 (0.32) (p = .023, one-way analysis of variance). CONCLUSION: In conclusion, a small reduction in pulmonary inflammation after 24 h of support with OLEV suggests potential to reduce preterm lung injury.

Multipotent adult progenitor cells prevent functional impairment and improve development in inflammation driven detriment of preterm ovine lungs.

Background: Perinatal inflammation increases the risk for bronchopulmonary dysplasia in preterm neonates, but the underlying pathophysiological mechanisms remain largely unknown. Given their anti-inflammatory and regenerative capacity, multipotent adult progenitor cells (MAPC) are a promising cell-based therapy to prevent and/or treat the negative pulmonary consequences of perinatal inflammation in the preterm neonate. Therefore, the pathophysiology underlying adverse preterm lung outcomes following perinatal inflammation and pulmonary benefits of MAPC treatment at the interface of prenatal inflammatory and postnatal ventilation exposures were elucidated. Methods: Instrumented ovine fetuses were exposed to intra-amniotic lipopolysaccharide (LPS 5 mg) at 125 days gestation to induce adverse systemic and peripheral organ outcomes. MAPC (10 × 106 cells) or saline were administered intravenously two days post LPS exposure. Fetuses were delivered preterm five days post MAPC treatment and either killed humanely immediately or mechanically ventilated for 72 h. Results: Antenatal LPS exposure resulted in inflammation and decreased alveolar maturation in the preterm lung. Additionally, LPS-exposed ventilated lambs showed continued pulmonary inflammation and cell junction loss accompanied by pulmonary edema, ultimately resulting in higher oxygen demand. MAPC therapy modulated lung inflammation, prevented loss of epithelial and endothelial barriers and improved lung maturation in utero. These MAPC-driven improvements remained evident postnatally, and prevented concomitant pulmonary edema and functional loss. Conclusion: In conclusion, prenatal inflammation sensitizes the underdeveloped preterm lung to subsequent postnatal inflammation, resulting in injury, disturbed development and functional impairment. MAPC therapy partially prevents these changes and is therefore a promising approach for preterm infants to prevent adverse pulmonary outcomes.

Umbilical cord-mesenchymal stem cells induce a memory phenotype in CD4+ T cells.

Inflammation is a physiological state where immune cells evoke a response against detrimental insults. Finding a safe and effective treatment for inflammation associated diseases has been a challenge. In this regard, human mesenchymal stem cells (hMSC), exert immunomodulatory effects and have regenerative capacity making it a promising therapeutic option for resolution of acute and chronic inflammation. T cells play a critical role in inflammation and depending on their phenotype, they can stimulate or suppress inflammatory responses. However, the regulatory effects of hMSC on T cells and the underlying mechanisms are not fully elucidated. Most studies focused on activation, proliferation, and differentiation of T cells. Here, we further investigated memory formation and responsiveness of CD4+ T cells and their dynamics by immune-profiling and cytokine secretion analysis. Umbilical cord mesenchymal stem cells (UC-MSC) were co-cultured with either αCD3/CD28 beads, activated peripheral blood mononuclear cells (PBMC) or magnetically sorted CD4+ T cells. The mechanism of immune modulation of UC-MSC were investigated by comparing different modes of action; transwell, direct cell-cell contact, addition of UC-MSC conditioned medium or blockade of paracrine factor production by UC-MSC. We observed a differential effect of UC-MSC on CD4+ T cell activation and proliferation using PBMC or purified CD4+ T cell co-cultures. UC-MSC skewed the effector memory T cells into a central memory phenotype in both co-culture conditions. This effect on central memory formation was reversible, since UC-MSC primed central memory cells were still responsive after a second encounter with the same stimuli. The presence of both cell-cell contact and paracrine factors were necessary for the most pronounced immunomodulatory effect of UC-MSC on T cells. We found suggestive evidence for a partial role of IL-6 and TGFß in the UC-MSC derived immunomodulatory function. Collectively, our data show that UC-MSCs clearly affect T cell activation, proliferation and maturation, depending on co-culture conditions for which both cell-cell contact and paracrine factors are needed.

Hypoxia-Driven Changes in a Human Intestinal Organoid Model and the Protective Effects of Hydrolyzed Whey.

Many whey proteins, peptides and protein-derived amino acids have been suggested to improve gut health through their anti-oxidant, anti-microbial, barrier-protective and immune-modulating effects. Interestingly, although the degree of hydrolysis influences peptide composition and, thereby, biological function, this important aspect is often overlooked. In the current study, we aimed to investigate the effects of whey protein fractions with different degrees of enzymatic hydrolysis on the intestinal epithelium in health and disease with a novel 2D human intestinal organoid (HIO) monolayer model. In addition, we aimed to assess the anti-microbial activity and immune effects of the whey protein fractions. Human intestinal organoids were cultured from adult small intestines, and a model enabling apical administration of nutritional components during hypoxia-induced intestinal inflammation and normoxia (control) in crypt-like and villus-like HIO was established. Subsequently, the potential beneficial effects of whey protein isolate (WPI) and two whey protein hydrolysates with a 27.7% degree of hydrolysis (DH28) and a 50.9% degree of hydrolysis (DH51) were assessed. In addition, possible immune modulatory effects on human peripheral immune cells and anti-microbial activity on four microbial strains of the whey protein fractions were investigated. Exposure to DH28 prevented paracellular barrier loss of crypt-like HIO following hypoxia-induced intestinal inflammation with a concomitant decrease in hypoxia inducible factor 1 alpha (HIF1α) mRNA expression. WPI increased Treg numbers and Treg expression of cluster of differentiation 25 (CD25) and CD69 and reduced CD4+ T cell proliferation, whereas no anti-microbial effects were observed. The observed biological effects were differentially mediated by diverse whey protein fractions, indicating that (degree of) hydrolysis influences their biological effects. Moreover, these new insights may provide opportunities to improve immune tolerance and promote intestinal health.

Chorioamnionitis Causes Kidney Inflammation, Podocyte Damage, and Pro-fibrotic Changes in Fetal Lambs.

Background: Perinatal complications, such as prematurity and intrauterine growth restriction, are associated with increased risk of chronic kidney disease. Although often associated with reduced nephron endowment, there is also evidence of increased susceptibility for sclerotic changes and podocyte alterations. Preterm birth is frequently associated with chorioamnionitis, though studies regarding the effect of chorioamnionitis on the kidney are scarce. In this study, we aim to unravel the consequences of premature birth and/or perinatal inflammation on kidney development using an ovine model. Methods: In a preterm sheep model, chorioamnionitis was induced by intra-amniotic injection of lipopolysaccharide (LPS) at either 2, 8, or 15 days prior to delivery. Control animals received intra-amniotic injections of sterile saline. All lambs were surgically delivered at 125 days' gestation (full term is 150 days) and immediately euthanized for necropsy. Kidneys were harvested and processed for staining with myeloperoxidase (MPO), Wilms tumor-1 (WT1) and alpha-smooth muscle actine (aSMA). mRNA expression of tumor necrosis factor alpha (TNFA), Interleukin 10 (IL10), desmin (DES), Platelet derived growth factor beta (PDGFB), Platelet derived growth factor receptor beta (PDGFRB), synaptopodin (SYNPO), and transforming growth factor beta (TGFB) was measured using quantitative PCR. Results: Animals with extended (but not acute) LPS exposure had an inflammatory response in the kidney. MPO staining was significantly increased after 8 and 15 days (p = 0.003 and p = 0.008, respectively). Expression of TNFA (p = 0.016) and IL10 (p = 0.026) transcripts was increased, peaking on day 8 after LPS exposure. Glomerular aSMA and expression of TGFB was increased on day 8, suggesting pro-fibrotic mesangial activation, however, this was not confirmed with PDFGB or PDGFRB. The number of WT1 positive nuclei in the glomerulus, as well as expression of synaptopodin, decreased, indicating podocyte injury. Conclusion: We report that, in an ovine model of prematurity, LPS-induced chorioamnionitis leads to inflammation of the immature kidney. In addition, this process was associated with podocyte injury and there are markers to support pro-fibrotic changes to the glomerular mesangium. These data suggest a potential important role for antenatal inflammation in the development of preterm-associated kidney disease, which is frequent.

Loss of enteric neuronal Ndrg4 promotes colorectal cancer via increased release of Nid1 and Fbln2.

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.

Sequential Exposure to Antenatal Microbial Triggers Attenuates Alveolar Growth and Pulmonary Vascular Development and Impacts Pulmonary Epithelial Stem/Progenitor Cells.

Perinatal inflammatory stress is strongly associated with adverse pulmonary outcomes after preterm birth. Antenatal infections are an essential perinatal stress factor and contribute to preterm delivery, induction of lung inflammation and injury, pre-disposing preterm infants to bronchopulmonary dysplasia. Considering the polymicrobial nature of antenatal infection, which was reported to result in diverse effects and outcomes in preterm lungs, the aim was to examine the consequences of sequential inflammatory stimuli on endogenous epithelial stem/progenitor cells and vascular maturation, which are crucial drivers of lung development. Therefore, a translational ovine model of antenatal infection/inflammation with consecutive exposures to chronic and acute stimuli was used. Ovine fetuses were exposed intra-amniotically to Ureaplasma parvum 42 days (chronic stimulus) and/or to lipopolysaccharide 2 or 7 days (acute stimulus) prior to preterm delivery at 125 days of gestation. Pulmonary inflammation, endogenous epithelial stem cell populations, vascular modulators and morphology were investigated in preterm lungs. Pre-exposure to UP attenuated neutrophil infiltration in 7d LPS-exposed lungs and prevented reduction of SOX-9 expression and increased SP-B expression, which could indicate protective responses induced by re-exposure. Sequential exposures did not markedly impact stem/progenitors of the proximal airways (P63+ basal cells) compared to single exposure to LPS. In contrast, the alveolar size was increased solely in the UP+7d LPS group. In line, the most pronounced reduction of AEC2 and proliferating cells (Ki67+) was detected in these sequentially UP + 7d LPS-exposed lambs. A similar sensitization effect of UP pre-exposure was reflected by the vessel density and expression of vascular markers VEGFR-2 and Ang-1 that were significantly reduced after UP exposure prior to 2d LPS, when compared to UP and LPS exposure alone. Strikingly, while morphological changes of alveoli and vessels were seen after sequential microbial exposure, improved lung function was observed in UP, 7d LPS, and UP+7d LPS-exposed lambs. In conclusion, although sequential exposures did not markedly further impact epithelial stem/progenitor cell populations, re-exposure to an inflammatory stimulus resulted in disturbed alveolarization and abnormal pulmonary vascular development. Whether these negative effects on lung development can be rescued by the potentially protective responses observed, should be examined at later time points.

Serial blood cytokine and chemokine mRNA and microRNA over 48 h are insult specific in a piglet model of inflammation-sensitized hypoxia-ischaemia.

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.

Chorioamnionitis induces changes in ovine pulmonary endogenous epithelial stem/progenitor cells in utero.

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.

Electrical stimulation promotes the angiogenic potential of adipose-derived stem cells.

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.

Pulmonary vascular changes in extremely preterm sheep after intra-amniotic exposure to Ureaplasma parvum and lipopolysaccharide.

BACKGROUND: Chorioamnionitis can induce pulmonary inflammation and promote bronchopulmonary dysplasia development, distinguished by alveolar simplification and impaired vascular growth. Chorioamnionitis is more common during the extremely preterm canalicular lung stage (crucial for vascular development); and increases the risk for subsequent sepsis. We hypothesized that single/combined exposure to chronic and/or acute inflammation induces pulmonary inflammatory responses and vascular changes. METHODS: Ovine fetuses were intra-amniotically exposed to chronic Ureaplasma parvum (UP) at 24 days (d) before extreme preterm delivery at 94d (term 147d) and/or to lipopolysaccharide (LPS) 7 or 2d before delivery. Pulmonary inflammation, vascular remodeling and angiogenic factors were assessed. RESULTS: LPS exposure increased CD3-positive and myeloperoxidase-positive cells. Combined UP-LPS exposure increased pulmonary inflammation compared with 2d LPS or UP groups. The UP+2d LPS group had an increased adventitial fibrosis score when compared with UP-treated animals. A reduced wall-to-lumen ratio was found in the 7d LPS animals when compared to the 2d LPS-treated animals. Exposure to UP+2d LPS reduced VEGF and VEGFR-2 levels compared with 2d LPS-treated animals. Angiopoietin-1 (Ang1) and tunica interna endothelial cell kinase 2 (Tie-2) levels were decreased after UP+7d LPS as well as after 7d LPS, but not with UP alone. CONCLUSION: Chronic UP and subsequent LPS exposure increased pulmonary inflammation and decreased expression of angiogenic growth factors and receptors when compared to single hit-exposed animals.

Mesenchymal Stromal Cell-Derived Extracellular Vesicles Protect the Fetal Brain After Hypoxia-Ischemia.

UNLABELLED: Preterm neonates are susceptible to perinatal hypoxic-ischemic brain injury, for which no treatment is available. In a preclinical animal model of hypoxic-ischemic brain injury in ovine fetuses, we have demonstrated the neuroprotective potential of systemically administered mesenchymal stromal cells (MSCs). The mechanism of MSC treatment is unclear but suggested to be paracrine, through secretion of extracellular vesicles (EVs). Therefore, we investigated in this study the protective effects of mesenchymal stromal cell-derived extracellular vesicles (MSC-EVs) in a preclinical model of preterm hypoxic-ischemic brain injury. Ovine fetuses were subjected to global hypoxia-ischemia by transient umbilical cord occlusion, followed by in utero intravenous administration of MSC-EVs. The therapeutic effects of MSC-EV administration were assessed by analysis of electrophysiological parameters and histology of the brain. Systemic administration of MSC-EVs improved brain function by reducing the total number and duration of seizures, and by preserving baroreceptor reflex sensitivity. These functional protections were accompanied by a tendency to prevent hypomyelination. Cerebral inflammation remained unaffected by the MSC-EV treatment. Our data demonstrate that MSC-EV treatment might provide a novel strategy to reduce the neurological sequelae following hypoxic-ischemic injury of the preterm brain. Our study results suggest that a cell-free preparation comprising neuroprotective MSC-EVs could substitute MSCs in the treatment of preterm neonates with hypoxic-ischemic brain injury, thereby circumventing the potential risks of systemic administration of living cells. SIGNIFICANCE: Bone marrow-derived mesenchymal stromal cells (MSCs) show promise in treating hypoxic-ischemic injury of the preterm brain. Study results suggest administration of extracellular vesicles, rather than intact MSCs, is sufficient to exert therapeutic effects and avoids potential concerns associated with administration of living cells. The therapeutic efficacy of systemically administered mesenchymal stromal cell-derived extracellular vesicles (MSC-EVs) on hypoxia-ischemia-induced injury was assessed in the preterm ovine brain. Impaired function and structural injury of the fetal brain was improved following global hypoxia-ischemia. A cell-free preparation of MSC-EVs could substitute for the cellular counterpart in the treatment of preterm neonates with hypoxic-ischemic brain injury. This may open new clinical applications for "off-the-shelf" interventions with MSC-EVs.

Can the preterm lung recover from perinatal stress?

After birth, adequate lung function is necessary for the successful adaptation of a preterm baby. Both prenatal and postnatal insults and therapeutic interventions have an immediate effect on lung function and gas exchange but also interfere with fetal and neonatal lung development. Prenatal insults like chorioamnionitis and prenatal interventions like maternal glucocorticosteroids interact but might also determine the preterm baby's lung response to postnatal interventions ("second hit") like supplementation of oxygen and drug therapy. We review current experimental and clinical findings on the influence of different perinatal factors on preterm lung development and discuss how well-established interventions in neonatal care might be adapted to attenuate postnatal lung injury.

Neuroinflammation and structural injury of the fetal ovine brain following intra-amniotic Candida albicans exposure.

BACKGROUND: Intra-amniotic Candida albicans (C. Albicans) infection is associated with preterm birth and high morbidity and mortality rates. Survivors are prone to adverse neurodevelopmental outcomes. The mechanisms leading to these adverse neonatal brain outcomes remain largely unknown. To better understand the mechanisms underlying C. albicans-induced fetal brain injury, we studied immunological responses and structural changes of the fetal brain in a well-established translational ovine model of intra-amniotic C. albicans infection. In addition, we tested whether these potential adverse outcomes of the fetal brain were improved in utero by antifungal treatment with fluconazole. METHODS: Pregnant ewes received an intra-amniotic injection of 10(7) colony-forming units C. albicans or saline (controls) at 3 or 5 days before preterm delivery at 0.8 of gestation (term ~ 150 days). Fetal intra-amniotic/intra-peritoneal injections of fluconazole or saline (controls) were administered 2 days after C. albicans exposure. Post mortem analyses for fungal burden, peripheral immune activation, neuroinflammation, and white matter/neuronal injury were performed to determine the effects of intra-amniotic C. albicans and fluconazole treatment. RESULTS: Intra-amniotic exposure to C. albicans caused a severe systemic inflammatory response, illustrated by a robust increase of plasma interleukin-6 concentrations. Cerebrospinal fluid cultures were positive for C. albicans in the majority of the 3-day C. albicans-exposed animals whereas no positive cultures were present in the 5-day C. albicans-exposed and fluconazole-treated animals. Although C. albicans was not detected in the brain parenchyma, a neuroinflammatory response in the hippocampus and white matter was seen which was characterized by increased microglial and astrocyte activation. These neuroinflammatory changes were accompanied by structural white matter injury. Intra-amniotic fluconazole reduced fetal mortality but did not attenuate neuroinflammation and white matter injury. CONCLUSIONS: Intra-amniotic C. albicans exposure provoked acute systemic and neuroinflammatory responses with concomitant white matter injury. Fluconazole treatment prevented systemic inflammation without attenuating cerebral inflammation and injury.

Selective IL-1α exposure to the fetal gut, lung, and chorioamnion/skin causes intestinal inflammatory and developmental changes in fetal sheep.

Chorioamnionitis, caused by intra-amniotic exposure to bacteria and their toxic components, is associated with fetal gut inflammation and mucosal injury. In a translational ovine model, we have shown that these adverse intestinal outcomes to chorioamnionitis were the combined result of local gut and pulmonary-driven systemic immune responses. Chorioamnionitis-induced gut inflammation and injury was largely prevented by inhibiting interleukin-1 (IL-1) signaling. Therefore, we investigated whether local (gut-derived) IL-1α signaling or systemic IL-1α-driven immune responses (lung or chorioamnion/skin-derived) were sufficient for intestinal inflammation and mucosal injury in the course of chorioamnionitis. Fetal surgery was performed in sheep to isolate the lung, gastrointestinal tract, and chorioamnion/skin, and IL-1α or saline was given into the trachea, stomach, or amniotic cavity 1 or 6 days before preterm delivery. Selective IL-1α exposure to the lung, gut, or chorioamnion/skin increased the CD3+ cell numbers in the fetal gut. Direct IL-1α exposure to the gut impaired intestinal zonula occludens protein-1 expression, induced villus atrophy, changed the expression pattern of intestinal fatty acid-binding protein along the villus, and increased the CD68, IL-1, and TNF-α mRNA levels in the fetal ileum. With lung or chorioamnion/skin exposure to IL-1α, intestinal inflammation was associated with increased numbers of blood leukocytes without induction of intestinal injury or immaturity. We concluded that local IL-1α signaling was required for intestinal inflammation, disturbed gut maturation, and mucosal injury in the context of chorioamnionitis.

Multipotent adult progenitor cells for hypoxic-ischemic injury in the preterm brain.

BACKGROUND: Preterm infants are at risk for hypoxic-ischemic encephalopathy. No therapy exists to treat this brain injury and subsequent long-term sequelae. We have previously shown in a well-established pre-clinical model of global hypoxia-ischemia (HI) that mesenchymal stem cells are a promising candidate for the treatment of hypoxic-ischemic brain injury. In the current study, we investigated the neuroprotective capacity of multipotent adult progenitor cells (MAPC®), which are adherent bone marrow-derived cells of an earlier developmental stage than mesenchymal stem cells and exhibiting more potent anti-inflammatory and regenerative properties. METHODS: Instrumented preterm sheep fetuses were subjected to global hypoxia-ischemia by 25 min of umbilical cord occlusion at a gestational age of 106 (term ~147) days. During a 7-day reperfusion period, vital parameters (e.g., blood pressure and heart rate; baroreceptor reflex) and (amplitude-integrated) electroencephalogram were recorded. At the end of the experiment, the preterm brain was studied by histology. RESULTS: Systemic administration of MAPC therapy reduced the number and duration of seizures and prevented decrease in baroreflex sensitivity after global HI. In addition, MAPC cells prevented HI-induced microglial proliferation in the preterm brain. These anti-inflammatory effects were associated with MAPC-induced prevention of hypomyelination after global HI. Besides attenuation of the cerebral inflammatory response, our findings showed that MAPC cells modulated the peripheral splenic inflammatory response, which has been implicated in the etiology of hypoxic-ischemic injury in the preterm brain. CONCLUSIONS: In a pre-clinical animal model MAPC cell therapy improved the functional and structural outcome of the preterm brain after global HI. Future studies should establish the mechanism and long-term therapeutic effects of neuroprotection established by MAPC cells in the developing preterm brain exposed to HI. Our study may form the basis for future clinical trials, which will evaluate whether MAPC therapy is capable of reducing neurological sequelae in preterm infants with hypoxic-ischemic encephalopathy.

Chorioamnionitis-induced fetal gut injury is mediated by direct gut exposure of inflammatory mediators or by lung inflammation.

Intra-amniotic exposure to proinflammatory agonists causes chorioamnionitis and fetal gut inflammation. Fetal gut inflammation is associated with mucosal injury and impaired gut development. We tested whether this detrimental inflammatory response of the fetal gut results from a direct local (gut derived) or an indirect inflammatory response mediated by the chorioamnion/skin or lung, since these organs are also in direct contact with the amniotic fluid. The gastrointestinal tract was isolated from the respiratory tract and the amnion/skin epithelia by fetal surgery in time-mated ewes. Lipopolysaccharide (LPS) or saline (controls) was selectively infused in the gastrointestinal tract, trachea, or amniotic compartment at 2 or 6 days before preterm delivery at 124 days gestation (term 150 days). Gastrointestinal and intratracheal LPS exposure caused distinct inflammatory responses in the fetal gut. Inflammatory responses could be distinguished by the influx of leukocytes (MPO(+), CD3(+), and FoxP3(+) cells), tumor necrosis factor-α, and interferon-γ expression and differential upregulation of mRNA levels for Toll-like receptor 1, 2, 4, and 6. Fetal gut inflammation after direct intestinal LPS exposure resulted in severe loss of the tight junctional protein zonula occludens protein 1 (ZO-1) and increased mitosis of intestinal epithelial cells. Inflammation of the fetal gut after selective LPS instillation in the lungs caused only mild disruption of ZO-1, loss in epithelial cell integrity, and impaired epithelial differentiation. LPS exposure of the amnion/skin epithelia did not result in gut inflammation or morphological, structural, and functional changes. Our results indicate that the detrimental consequences of chorioamnionitis on fetal gut development are the combined result of local gut and lung-mediated inflammatory responses.

Systemic G-CSF attenuates cerebral inflammation and hypomyelination but does not reduce seizure burden in preterm sheep exposed to global hypoxia-ischemia.

Hypoxic-ischemic encephalopathy (HIE) is common in preterm infants, but currently no curative therapy is available. Cell-based therapy has a great potential in the treatment of hypoxic-ischemic preterm brain injury. Granulocyte-colony stimulating factor (G-CSF) is known to mobilize endogenous hematopoietic stem cells (HSC) and promotes proliferation of endogenous neural stem cells. On these grounds, we hypothesized that systemic G-CSF would be neuroprotective in a large translational animal model of hypoxic-ischemic injury in the preterm brain. Global hypoxia-ischemia (HI) was induced by transient umbilical cord occlusion in instrumented preterm sheep. G-CSF treatment (100µg/kg intravenously, during five consecutive days) was started one day before the global HI insult to ascertain mobilization of endogenous stem cells within the acute phase after global HI. Mobilization of HSC and neutrophils was studied by flow cytometry. Brain sections were stained for microglia (IBA-1), myelin basic protein (MBP) and myeloperoxidase (MPO) to study microglial proliferation, white matter injury and neutrophil invasion respectively. Electrographic seizure activity was analyzed using amplitude-integrated electroencephalogram (aEEG). G-CSF effectively mobilized CD34-positive HSC in the preterm sheep. In addition, G-CSF caused marked mobilization of neutrophils, but did not influence enhanced invasion of neutrophils into the preterm brain after global HI. Microglial proliferation and hypomyelination following global HI were reduced as a result of G-CSF treatment. G-CSF did not cause a reduction of the electrographic seizure activity after global HI. In conclusion, G-CSF induced mobilization of endogenous stem cells which was associated with modulation of the cerebral inflammatory response and reduced white matter injury in an ovine model of preterm brain injury after global HI. G-CSF treatment did not improve neuronal function as shown by seizure analysis. Our study shows that G-CSF treatment has neuroprotective potential following hypoxic-ischemic injury in the preterm brain.

Mesenchymal stem cells induce T-cell tolerance and protect the preterm brain after global hypoxia-ischemia.

Hypoxic-ischemic encephalopathy (HIE) in preterm infants is a severe disease for which no curative treatment is available. Cerebral inflammation and invasion of activated peripheral immune cells have been shown to play a pivotal role in the etiology of white matter injury, which is the clinical hallmark of HIE in preterm infants. The objective of this study was to assess the neuroprotective and anti-inflammatory effects of intravenously delivered mesenchymal stem cells (MSC) in an ovine model of HIE. In this translational animal model, global hypoxia-ischemia (HI) was induced in instrumented preterm sheep by transient umbilical cord occlusion, which closely mimics the clinical insult. Intravenous administration of 2 x 10(6) MSC/kg reduced microglial proliferation, diminished loss of oligodendrocytes and reduced demyelination, as determined by histology and Diffusion Tensor Imaging (DTI), in the preterm brain after global HI. These anti-inflammatory and neuroprotective effects of MSC were paralleled by reduced electrographic seizure activity in the ischemic preterm brain. Furthermore, we showed that MSC induced persistent peripheral T-cell tolerance in vivo and reduced invasion of T-cells into the preterm brain following global HI. These findings show in a preclinical animal model that intravenously administered MSC reduced cerebral inflammation, protected against white matter injury and established functional improvement in the preterm brain following global HI. Moreover, we provide evidence that induction of T-cell tolerance by MSC might play an important role in the neuroprotective effects of MSC in HIE. This is the first study to describe a marked neuroprotective effect of MSC in a translational animal model of HIE.

Intraamniotic lipopolysaccharide exposure changes cell populations and structure of the ovine fetal thymus.

RATIONALE: Chorioamnionitis induces preterm delivery and acute involution of the fetal thymus which is associated with postnatal inflammatory disorders. We studied the immune response, cell composition, and architecture of the fetal thymus following intraamniotic lipopolysaccharide (LPS) exposure. METHODS: Time-mated ewes received an intraamniotic injection of LPS 5, 12, or 24 hours or 2, 4, 8, or 15 days before delivery at 125 days gestational age (term = 150 days). RESULTS: The LPS exposure resulted in decreased blood lymphocytes within 5 hours and decreased thymic corticomedullary ratio within 24 hours. Thymic interleukin 6 (IL6) and IL17 messenger RNA (mRNA) increased 5-fold 24 hours post-LPS exposure. Increased toll-like receptor 4 (TLR4) mRNA and nuclear factor κB positive cells at 24 hours after LPS delivery demonstrated acute thymic activation. Both TLR4 and IL1 mRNA increased by 5-fold and the number of Foxp3-positive cells (Foxp3+ cells) decreased 15 days after exposure. CONCLUSION: Intraamniotic LPS exposure caused a proinflammatory response, involution, and a persistent depletion of thymic Foxp3+ cells indicating disturbance of the fetal immune homeostasis.