The effect of human amnion epithelial cells on lung development and inflammation in preterm lambs exposed to antenatal inflammation

Lung inflammation and impaired alveolarization are hallmarks of bronchopulmonary dysplasia (BPD). We hypothesize that human amnion epithelial cells (hAECs) are anti-inflammatory and reduce lung injury in preterm lambs born after antenatal exposure to inflammation. Pregnant ewes received either intra-amniotic lipopolysaccharide (LPS, from E.coli 055:B5 4mg) or saline (Sal) on day 126 of gestation. Lambs were delivered by cesarean section at 128 d gestation (term ~150 d). Lambs received intravenous hAECs (LPS/hAECs: n = 7 30x10.sup.6 cells) or equivalent volumes of saline (LPS/Sal, n = 10 or Sal/Sal, n = 9) immediately after birth. Respiratory support was gradually de-escalated, aimed at early weaning from mechanical ventilation towards unassisted respiration. Lung tissue was collected 1 week after birth. Lung morphology was assessed and mRNA levels for inflammatory mediators were measured. Respiratory support required by LPS/hAEC lambs was not different to Sal/Sal or LPS/Sal lambs. Lung tissue:airspace ratio was lower in the LPS/Sal compared to Sal/Sal lambs (P<0.05), but not LPS/hAEC lambs. LPS/hAEC lambs tended to have increased septation in their lungs versus LPS/Sal (P = 0.08). Expression of inflammatory cytokines was highest in LPS/hAECs lambs. Postnatal administration of a single dose of hAECs stimulates a pulmonary immune response without changing ventilator requirements in preterm lambs born after intrauterine inflammation

Становище Заславського римо-католицького деканату в ХІХ столітті (The situation of the Zaslavsky Roman-Catholic Deanery in the 19th century)

Стаття присвячена аналізу римо-католицизму на Заславщині у ХІХ ст. Доведено, що становище Заславського римо-католицького деканату протягом ХІХ століття погіршувалося, що відповідало загальним тенденціям ситуації РКЦ в Російській імперії. (The article is devoted to the analysis of Roman Catholicism in Zaslavsk region in the 19th century. It is proved that the situation of Zaslavsky Roman Catholic deanery during the 19th century worsened, which corresponded to the general tendencies of the situation of the RCC in the Russian Empire.

Systematic review and network meta-analysis with individual participant data on cord management at preterm birth (iCOMP): study protocol

Introduction Timing of cord clamping and other cord management strategies may improve outcomes at preterm birth. However, it is unclear whether benefits apply to all preterm subgroups. Previous and current trials compare various policies, including time-based or physiology-based deferred cord clamping, and cord milking. Individual participant data (IPD) enable exploration of different strategies within subgroups. Network meta-analysis (NMA) enables comparison and ranking of all available interventions using a combination of direct and indirect comparisons. Objectives (1) To evaluate the effectiveness of cord management strategies for preterm infants on neonatal mortality and morbidity overall and for different participant characteristics using IPD meta-analysis. (2) To evaluate and rank the effect of different cord management strategies for preterm births on mortality and other key outcomes using NMA. Methods and analysis Systematic searches of Medline, Embase, clinical trial registries, and other sources for all ongoing and completed randomised controlled trials comparing cord management strategies at preterm birth (before 37 weeks’ gestation) have been completed up to 13 February 2019, but will be updated regularly to include additional trials. IPD will be sought for all trials; aggregate summary data will be included where IPD are unavailable. First, deferred clamping and cord milking will be compared with immediate clamping in pairwise IPD meta-analyses. The primary outcome will be death prior to hospital discharge. Effect differences will be explored for prespecified participant subgroups. Second, all identified cord management strategies will be compared and ranked in an IPD NMA for the primary outcome and the key secondary outcomes. Treatment effect differences by participant characteristics will be identified. Inconsistency and heterogeneity will be explored. Ethics and dissemination Ethics approval for this project has been granted by the University of Sydney Human Research Ethics Committee (2018/886). Results will be relevant to clinicians, guideline developers and policy-makers, and will be disseminated via publications, presentations and media releases

Physiological-based cord clamping in very preterm infants — Randomised controlled trial on effectiveness of stabilisation

Aim: To test whether stabilising very preterm infants while performing physiological-based cord clamping (PBCC) is at least as effective as the standard approach of time-based delayed cord clamping (DCC). Methods: A randomised contr

Effects of intra-amniotic lipopolysaccharide and maternal betamethasone on brain inflammation in fetal sheep

Rationale: Chorioamnionitis and antenatal glucocorticoids are common exposures for preterm infants and can affect the fetal brain, contributing to cognitive and motor deficits in preterm infants. The effects of antenatal glucocorticoids on the brain in the setting of chorioamnionitis are unknown. We hypothesized that antenatal glucocorticoids would modulate inflammation in the brain and prevent hippocampal and white matter injury after intra-amniotic lipopolysaccharide (LPS) exposure. Methods: Time-mated ewes received saline (control), an intra-amniotic injection of 10 mg LPS at 106d GA or 113d GA, maternal intra-muscular betamethasone (0.5 mg/kg maternal weight) alone at 113d GA, betamethasone at 106d GA before LPS or betamethasone at 113d GA after LPS. Animals were delivered at 120d GA (term=150d). Brain structure volumes were measured on T2-weighted MRI images. The subcortical white matter (SCWM), periventricular white matter (PVWM) and hippocampus were analyzed for microglia, astrocytes, apoptosis, proliferation, myelin and pre-synaptic vesicles. Results: LPS and/or betamethasone exposure at different time-points during gestation did not alter brain structure volumes on MRI. Betamethasone alone did not alter any of the measurements. Intra-amniotic LPS at 106d or 113d GA induced inflammation as indicated by increased microglial and astrocyte recruitment which was paralleled by increased apoptosis and hypomyelination in the SCWM and decreased synaptophysin density in the hippocampus. Betamethasone before the LPS exposure at 113d GA prevented microglial activation and the decrease in synaptophysin. Betamethasone after LPS exposure increased microglial infiltration and apoptosis. Conclusion: Intra-uterine LPS exposure for 7d or 14d before delivery induced inflammation and injury in the fetal white matter and hippocampus. Antenatal glucocorticoids aggravated the inflammatory changes in the brain caused by pre-existing intra-amniotic inflammation. Antenatal glucocorticoids prior to LPS reduced the effects of intra-uterine inflammation on the brain. The timing of glucocorticoid administration in the setting of chorioamnionitis can alter outcomes for the fetal brain

Reducing Brain Injury of Preterm Infants in the Delivery Room

Cerebrovascular injury is one of the major detrimental consequences of preterm birth. Recent studies have focused their attention on factors that contribute to the development of brain lesions immediately after birth. Among those factors, hypothermia and lower cerebral oxygen saturation during delivery room resuscitation and high tidal volumes delivered during respiratory support are associated with increased risk of severe neurologic injury. In preterm infants, knowledge about causes and prevention of brain injury must be applied before and at birth. Preventive and therapeutic approaches, including correct timing of cord clamping, monitoring of physiological changes during delivery room resuscitation using pulse oximetry, respiratory function monitoring, near infrared spectroscopy, and alpha EEG, may minimize brain injury, Furthermore, postnatal administration of caffeine or other potential novel treatments (e.g., proangiogenic therapies, antioxidants, hormones, or stem cells) might improve long-term neurodevelopmental outcomes in preterm infants

Ventilation Prior to Umbilical Cord Clamping Improves Cardiovascular Stability and Oxygenation in Preterm Lambs After Exposure to Intrauterine Inflammation

Background: Delaying umbilical cord clamping until after aeration of the lung (physiological-based cord clamping; PBCC) maintains cardiac output and oxygenation in preterm lambs at birth, however, its efficacy after intrauterine inflammation is not known. Given the high incidence of chorioamnionitis in preterm infants, we investigated whether PBCC conferred any benefits compared to immediate cord clamping (ICC) in preterm lambs exposed antenatally to 7 days of intrauterine inflammation.Methods: Ultrasound guided intraamniotic injection of 20 mg Lipopolysaccharide (from E. coli:055:B5) was administered to pregnant ewes at 0.8 gestation. Seven days later, ewes were anesthetized, preterm fetuses exteriorised via cesarean section, and instrumented for continuous measurement of pulmonary, systemic and cerebral pressures and flows, and systemic, and cerebral oxygenation. Lambs were then randomized to either PBCC, whereupon ventilation was initiated and maintained for 3 min prior to umbilical cord clamping, or ICC where the umbilical cord was cut and ventilation initiated 30 s later. Ventilation was maintained for 30 min.Results: ICC caused a rapid fall in systemic (by 25%) and cerebral (by 11%) oxygen saturation in ICC lambs, concurrent with a rapid increase in carotid arterial pressure and heart rate. The overshoot in carotid arterial pressure was sustained in ICC lambs for the first 20 min of the study. PBCC maintained cardiac output and prevented the fall in cerebral oxygen delivery at birth. PBCC lambs had lower respiratory compliance and higher respiratory requirements throughout the study.Conclusion: PBCC mitigated the adverse effects of ICC on oxygenation and cardiac output, and therefore could be more beneficial in preterm babies exposed to antenatal inflammation as it maintains cardiac output and oxygen delivery. The increased respiratory requirements require further investigation in this sub-group of preterm infants

Untersuchungen zum Einfluss von thrombozytären Wachstumsfaktoren auf den zellvermittelten Abbau eines nanopartikulären Knochenersatzstoffes auf Hydroxylapatitbasis : eine experimentelle Studie am Miniaturschwein

Ziel der vorliegenden tierexperimentellen Studie am Miniaturschwein war es, den Einfluss von plättchenreichem Plasma (PRP) auf den zellvermittelten Abbau eines nanopartikulären Hydroxylapatits (HA) in der Frühphase der Knochendefektheilung zu untersuchen. Hierzu wurden 26 männliche Miniaturschweine der Rasse Mini-Lewe in drei Versuchsgruppen eingeteilt und jeweils ein standardisierter Knochendefekt in der Intercondylarregion des rechten Femurs angelegt. Die Defekte wurden entweder mit dem Knochenersatzstoff (Gruppe I/PRP-,n = 11) oder dem Knochenersatzstoff kombiniert mit PRP (Gruppe II/PRP+, n = 11) befüllt. In einer Kontrollgruppe (n = 4) blieben die Defekte unbefüllt. Während der Implantationsoperation wurden bei sechs Tieren jeweils 250 ml Vollblut entnommen, aus dem anschließend durch fraktionierte Zentrifugation plättchenreiches Plasma gewonnen wurde. Die enthaltenen Thrombozyten wurden durch den Zusatz von Thrombin und Kalziumglukonat zur Degranulation angeregt, wodurch die enthaltenen Wachstumsfaktoren aus den alpha-Granula freigesetzt wurden. Zu diesen Wachstumsfaktoren gehören Platelet Derived Growth Factor AB und BB (PDGF AB, BB), Transforming Growth Factor ß 1 (TGF beta1), Vascular Endothelial Growth Factor (VEGF) und basic Fibroblast Growth Factor (bFGF). Die Konzentration der genannten Wachstumsfaktoren wurde mit Hilfe der ELISA-Technik bestimmt. Sie lagen zwischen Faktor 1,6 für TGF-beta1 und Faktor 24,4 für bFGF. 20 Tage post operationem fand die Explantation der operierten distalen Femura statt. Zur lichtmikroskopischen Untersuchung fanden die Knochen-Implantat-Proben Eingang in unterschiedliche Techniken der Einbettung (Paraffin-, Kunststoffeinbettungen), Präparation (Paraffinschnitte, Kunststoffschnitte und Schliffpräparationen), Färbung (Toluidinblau, Haematoxylin-Eosin, Safranin) und Histochemie (Enzym-, Immunhistochemie). Darüber hinaus wurden transmissionselektronenmikroskopische und computergestützte histomorphometrische Untersuchungen durchgeführt. Wie die Ergebnisse der Licht- und Transmissionselektronenmikroskopie aufgezeigt haben, erfolgt in den mit Knochenersatzmaterial behandelten Versuchsgruppen, unabhängig von der PRP-Applikation, die HA-Degradation hydrolytisch und Makrophagen-vermittelt. Die Makrophagen-Population wird durch Riesenzellen vom Langhans-Typ repräsentiert. Diese polarisierten Polykaryen adhärieren über ihre apikale Membrandomäne an den Implantatoberflächen. Das subplasmalemmale Zytoplasma ist immunhistochemisch durch Vimentin-Kondensationen gekennzeichnet. Nicht-adhärente, frei im Granulationsgewebe lokalisierte Polykaryen zeigen dagegen ein homogenes Vimentin-Verteilungsmuster im Zytoplasma. Der zelluläre Abbau des HA erfolgt mittels Phagozytose, indem die Polykaryen den "Fremdkörper" mit pseudopodienartigen Zytoplasmaausläufern umschließen und in ihr Zytoplasma inkorporieren. Diese Art der Degradation wird durch den post implantationem stattfindenden Zerfall des Knochenersatzmaterials in zahlreiche kleine Partikel unterstützt. Die hieraus resultierende Vergrößerung der Implantatoberfläche bietet einer Vielzahl von Zellen die Möglichkeit zur Haftung. Die festgestellten Expressionsmuster des CD44- Membranglykoproteins verweisen auf dessen funktionelle Rolle im Rahmen der Fusion mononukleärer Makrophagen zu multinukleären Riesenzellen. Die darüber hinaus beobachtete Umverteilung von CD44 von der apikalen zur basalen Membrandomäne bei Implantatassoziierten Polykaryen ist als transientes Geschehen im Zuge der Adhäsion zu interpretieren. Der hohe Aktivitätsstatus der adhärenten Polykaryen ist immunhistochemisch durch eine intensive Kathepsin K-Expression gekennzeichnet. Die vergleichende histomorphometrische Auswertung der mit HA aufgefüllten Defekte dokumentiert eine Verdopplung der Anzahl von Polykaryen in der Gruppe "Knochenersatzstoff mit PRP". Ein auf Basis der Messergebnisse durchgeführter Wilcoxon-Rangsummentest verweist auf den hochsignifikanten Einfluss (p < 0,01) des Faktors PRP auf die Ausdehnung Tartrat-resistenter saurer Phosphatase-positiver Areale in den Präparaten. Diese Effekte können sowohl auf den im PRP angereicherten Wachstumsfaktoren als auch auf dem homologen Charakter der PRP-Zubereitung beruhen. Die beobachteten Polykaryen – sogenannte "Fremdkörperriesenzellen" – sind auch immer Indikatoren einer stattfindenden Entzündungsreaktion. Die histomorphometrisch dargestellte, deutlich verstärkte Fremdkörperreaktion in Gruppe II/PRP+ kann auf die PRP-Applikation zurückgeführt werden. Im weiteren Heilungsverlauf kann dies zu einer Verzögerung der knöchernen Konsolidierung der Defekte führen.Aim of the current experimental study in Minipigs was to examine the effects of homologous platelet-rich plasma (PRP) on the cell-mediated degradation of a nanoparticulate hydroxyapatite (HA) during the early phase of bone defect healing. Twenty-six male "Lewe" minipigs were divided into three groups. Standardized bone defects were created in the intercondylar region of the right femur of each pig and were filled with HA (Group I/PRP-, n = 11) or HA + PRP (Group II/PRP+, n = 11). The defects of the control group (n = 4) were left empty. During the implantation procedure blood was drawn from six minipigs (250 ml each). PRP was isolated from these blood samples after several centrifugation steps. After the addition of thrombin and calcium gluconate growth factors were released from the alpha-granules of the thrombocytes which were enriched within the PRP. Some of these growth factors are Platelet Derived Growth Factor AB and BB (PDGF AB, BB), Transforming Growth Factor ß 1 (TGF beta1), Vascular Endothelial Growth Factor (VEGF) and basic Fibroblast Growth Factor (bFGF). The level of enrichment of these growth factors was controlled by the ELISA technique. Growth factor enrichment within the PRP ranged from 1.6 fold (TGF-beta1) to 24.4 fold (bFGF). After 20 days the treated distal femura were explanted. For light microscopical examination different tissue embedding methods (paraffine, plastic, resin), sectioning techniques (paraffine sections, plastic and resin sections, sawing and grinding sections), staining procedures (toluidine blue, hematoxylin eosin, safranin) and histochemical methods (enzyme- and immunohistochemistry) were performed. Additionally transmission electron microscopy and computer-assisted histomorphometry were used. The results of light microscopy and transmission electron microscopy showed that regardless of the addition of PRP, the HA is degraded by hydrolysis and macrophages. The population of macrophages consists of Langhans-type giant cells. The adhesion of the polarized polykaryons at the surfaces of the implant is mediated by the apical domain of the plasmamembranes. Vimentin condensations of the cytoplasm are attached to the apical plasmalemma. In contrast, non-adherent polykaryons of the granulation tissue reveal a homogeneous Vimentin distribution pattern within their cytoplasma. As shown ultrastructurally, the implant is degraded by means of phagocytosis. The implant particles are encircled by pseudopodia of the polykaryons and become incorporated into the cytoplasma. The degradation process is supported by disintegration of the bone substitute into numerous small particles after implantation. This disintegration causes enlargement of the implant surface and increases the probability of phagocyte adhesion. The pattern of CD44 expression points towards a functional role of the molecule during fusion of mononucleated macrophages into multinucleated giant cells. Implant-associated polykaryons show CD44 immunoreactivity only along the basal domains of the cytomembrane. This pattern can be interpreted as a temporal event during adhesion. Adherent polykaryons are further characterized by strong cathepsin K expression. The histomorphometric examination demonstrates twice as much foreign body giant cells in "Group II/PRP+" as in "Group I/PRP-". Based on these results, a Wilcoxon-signed-rank test was performed and a highly significant effect (p < 0.01) of PRP on the expansion of tartrate resistent acid phophatase (TRAP)-positive areas within bone defects could be demonstrated. This effect could be a result of the substution of PRP or of its homologous character. The polykaryons descibed in this work - so-called Foreign Body Giant Cells - are also indicators of inflammation. The enhanced cellular reaction observed in Group II/PRP+ must be interpreted as a strong foreign body reaction, triggered by the addition of PRP. It cannot be excluded that the strong inflammation reaction will lead to delayed bone formation in the course of healing

The Role of the Multiple Banded Antigen of Ureaplasma parvum in Intra-Amniotic Infection: Major Virulence Factor or Decoy?

The multiple banded antigen (MBA) is a predicted virulence factor of Ureaplasma species. Antigenic variation of the MBA is a potential mechanism by which ureaplasmas avoid immune recognition and cause chronic infections of the upper genital tract of pregnant women. We tested whether the MBA is involved in the pathogenesis of intra-amniotic infection and chorioamnionitis by injecting virulent or avirulent-derived ureaplasma clones (expressing single MBA variants) into the amniotic fluid of pregnant sheep. At 55 days of gestation pregnant ewes (n = 20) received intra-amniotic injections of virulent-derived or avirulent-derived U. parvum serovar 6 strains (2×104 CFU), or 10B medium (n = 5). Amniotic fluid was collected every two weeks post-infection and fetal tissues were collected at the time of surgical delivery of the fetus (140 days of gestation). Whilst chronic colonisation was established in the amniotic fluid of animals infected with avirulent-derived and virulent-derived ureaplasmas, the severity of chorioamnionitis and fetal inflammation was not different between these groups (p>0.05). MBA size variants (32–170 kDa) were generated in vivo in amniotic fluid samples from both the avirulent and virulent groups, whereas in vitro antibody selection experiments led to the emergence of MBA-negative escape variants in both strains. Anti-ureaplasma IgG antibodies were detected in the maternal serum of animals from the avirulent (40%) and virulent (55%) groups, and these antibodies correlated with increased IL-1β, IL-6 and IL-8 expression in chorioamnion tissue (p<0.05). We demonstrate that ureaplasmas are capable of MBA phase variation in vitro; however, ureaplasmas undergo MBA size variation in vivo, to potentially prevent eradication by the immune response. Size variation of the MBA did not correlate with the severity of chorioamnionitis. Nonetheless, the correlation between a maternal humoral response and the expression of chorioamnion cytokines is a novel finding. This host response may be important in the pathogenesis of inflammation-mediated adverse pregnancy outcomes

IL-1α Mediated Chorioamnionitis Induces Depletion of FoxP3+ Cells and Ileal Inflammation in the Ovine Fetal Gut

Endotoxin induced chorioamnionitis increases IL-1 and provokes an inflammatory response in the fetal ileum that interferes with intestinal maturation. In the present study, we tested in an ovine chorioamnionitis model whether IL-1 is a major cytokine driving the inflammatory response in the fetal ileum.Sheep bearing singleton fetuses received a single intraamniotic injection of recombinant ovine IL-1α at 7, 3 or 1 d before caesarian delivery at 125 days gestational age (term = 150 days).3 and 7 d after IL-1α administration, intestinal mRNA levels for IL-4, IL-10, IFN-γ and TNF-α were strongly elevated. Numbers of CD3+ and CD4+ T-lymphocytes and myeloidperoxidase+ cells were increased whereas FoxP3+ T-cells were detected at low frequency. This increased proinflammatory state was associated with ileal mucosal barrier loss as demonstrated by decreased levels of the intestinal fatty acid binding protein and disruption of the tight junctional protein ZO-1.Intraamniotic IL-1α causes an acute detrimental inflammatory response in the ileum, suggesting that induction of IL-1 is a critical element in the pathophysiological effects of endotoxin induced chorioamnionitis. A disturbed balance between T-effector and FoxP3+ cells may contribute to this process