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Perinatal exposure of the neonatal lung to inflammation leads to decreased lung angiogenesis and the development of bronchopulmonary dysplasia (BPD). Notably, autologous cord blood mononuclear cells (ACBMNCs) can substantially prevent severe BPD and decrease the inflammatory response in surviving very preterm neonates. Angiopoietin‑like protein 7 (Angptl7) is one of the main paracrine cytokines in cord blood stem cells, and is capable of stimulating human hematopoietic stem and progenitor cell expansion. The present study compared Angptl7 levels between the ACBMNCs infusion and control groups (cohort 1). Subsequently, the association between cord blood Angptl7 levels and BPD incidence in a cohort of very preterm neonates was assessed (cohort 2). The hypothesis was further verified in a lipopolysaccharide (LPS)‑induced lung injury mouse model. The mRNA expression levels and protein concentrations of inflammatory cytokines in the lung tissue and mouse serum were measured using reverse transcription‑quantitative PCR and ELISA, respectively. The number and diameter of lung vessels and macrophage infiltration were assessed using immunofluorescence staining. Compared with in the control group, Angptl7 levels were significantly higher in the ACBMNCs infusion group in cohort 1. In cohort 2, the cord blood Angptl7 levels were significantly lower in infants who later developed BPD. Multiple linear regression analysis showed that higher Angptl7 level was an independent protective factor for BPD. The concentrations of interleukin‑6 and monocyte chemoattractant protein‑1 were negatively correlated with cord blood Angptl7 level; whereas, vascular endothelial growth factor‑A levels were positively correlated with Angptl7 levels. In the LPS‑induced lung injury mouse model, the LPS group presented with a significant loss of pulmonary vessels and smaller vessel diameters, which were ameliorated in the Angptl7 treatment group. Furthermore, LPS‑induced lung inflammation and macrophage infiltration were alleviated by Angptl7 treatment (P<0.05). In conclusion, the anti‑inflammatory and proangiogenic effects of Angptl7 derived from cord blood stem cells may ameliorate BPD severity. The trial for cohort 1 was registered at ClinicalTrials.gov (trial registration no. NCT02999373; date registered, December 21, 2016).

Background: Cathelicidin/LL-37 plays a significant role in the human immune defense reaction. Preterm human immature organs being exposed to inflammation-induced injury was the critical denominator leading to the common preterm associated complications. Previous study showed LL37 concentration in preterm neonates was lower in tracheal aspirates and breast milk as compared to term infants. An adults study showed decreased LL-37 levels was a risk factor for patients in developing severe chronic obstructive pulmonary disease (COPD). However, little is known about the regulation of human cord blood LL37 in preterm neonates and the association with preterm complications. This study was designed to investigate the concentration of LL37 in cord blood of preterm infants and correlation with preterm complications., Methods: Singleton infants born in June 2017 to August 2021 in the study hospital were enrolled. Maternal and neonatal clinical characteristics were collected. LL37 levels, pro-inflammatory factor interleukin-6 (IL-6) and tumor necrosis factor-a (TNF-a) in cord blood and LL37 levels in serum 48-72 hours after birth were measured by enzyme-linked immunosorbent assay. The serum level of LL37 in preterm and term neonates were compared, the perinatal factors possibly affecting the LL37 levels were investigated and the relationship between LL37 level and preterm outcomes were analyzed., Results: Cord blood LL37 levels in preterm infants were lower than that in term neonates. Cord blood LL37 level was positively correlated with gestational age in preterm. Prenatal steroid administration in preterm neonates decreased cord blood LL37 level. LL37 level was obviously lower in patients with bronchopulmonary dysplasia (BPD). Multiple line regression analysis showed higher LL37 level in cord blood was an independent protective factor for BPD. The concentration of pro-inflammatory factor IL-6 was negatively correlated with LL37., Conclusion: Cord blood LL37 levels increased during gestation and decreased after perinatal steroid usage. Very preterm infants who displayed higher cord blood LL37 level had reduced risk of developing BPD. Regulation of pro-inflammatory cytokine IL-6 may be associated with the protective effect of LL37 on BPD., (© 2022. The Author(s).)

Summary: Background: Bronchopulmonary dysplasia (BPD) is the primary severe complication of preterm birth. Severe BPD was associated with higher risks of mortality, more postnatal growth failure, long term respiratory and neurological developmental retardation. Inflammation plays a central role in alveolar simplification and dysregulated vascularization of BPD. There is no effective treatment to improve BPD severity in clinical practice. Our previous clinical study showed autologous cord blood mononuclear cells (ACBMNCs) infusion could reduce the respiratory support duration safely and potential improved BPD severity. Abundant preclinical studies have reported the immunomodulation effect as an important mechanism underlying the beneficial results of stem cell therapies in preventing and treating BPD. However, clinical studies assessing the immunomodulatory effect after stem cells therapy were rare. This study was to investigate the effect of ACBMNCs infusion soon after birth on prevention for severe BPD and long term outcomes in very preterm neonates. The immune cells and inflammatory biomarkers were detected to investigate the underlying immunomodulatory mechanisms. Methods: This single-center, prospective, investigator-initiated, non-randomized trial with blinded outcome assessment aimed to assess the effect of a single intravenous infusion of ACBMNCs in preventing severe BPD (moderate or severe BPD at 36 weeks of gestational age or discharge home) in surviving very preterm neonates less than 32 gestational weeks. Patients admitted to the Neonatal Intensive Care Unit (NICU) of Guangdong Women and Children Hospital from July 01, 2018 to January 1, 2020 were assigned to receiving a targeted dosage of 5 × 107 cells/kg ACBMNC or normal saline intravenously within 24 h after enrollment. Incidence of moderate or severe BPD in survivors were investigated as the primary short term outcome. Growth, respiratory and neurological development were assessed as long term outcomes at corrected age of 18–24 month-old. Immune cells and inflammatory biomarkers were detected for potential mechanism investigation. The trial was registered at ClinicalTrials.gov (NCT02999373). Findings: Six-two infants were enrolled, of which 29 were enrolled to intervention group, 33 to control group. Moderate or severe BPD in survivors significantly decreased in intervention group (adjusted p = 0.021). The number of patients needed to treat to gain one moderate or severe BPD-free survival was 5 (95% confidence interval: 3–20). Survivors in the intervention group had a significantly higher chance to be extubated than infants in the control group (adjusted p = 0.018). There was no statistical significant difference in total BPD incidence (adjusted p = 0.106) or mortality (p = 1.000). Incidence of developmental delay reduced in intervention group in long term follow-up (adjusted p = 0.047). Specific immune cells including proportion of T cells (p = 0.04) and CD4+ T cells in lymphocytes (p = 0.03), and CD4+ CD25+ forkhead box protein 3 (FoxP3)+ regulatory T cells in CD4+ T cells increased significantly after ACBMNCs intervention (p ＜ 0.001). Anti-inflammatory factor IL-10 was higher (p = 0.03), while pro-inflammatory factor such as TNF-a (p = 0.03) and C reactive protein (p ＜ 0.001) level was lower in intervention group than in control group after intervention. Interpretation: ACBMNCs could prevent moderate or severe BPD in surviving very premature neonates and might improve neurodevelopmental outcomes in long term. An immunomodulatory effect of MNCs contributed to the improvement of BPD severity. Funding: This work was supported by National Key R&D Program of China (2021YFC2701700), National Natural Science Foundation of China (82101817, 82171714, 8187060625), Guangzhou science and technology program (202102080104).

BackgroundPreterm-associated complications remain the main cause of neonatal death. Survivors face the challenges of short- and long-term complications. Among all complications, bronchopulmonary dysplasia (BPD) remains the first important cause of neonatal mortality and morbidity. Current treatment does not address this main preterm complication. Cord blood is regarded as a convenient source of stem cells. The paracrine bioactive factors of stem cells contribute to tissue repair and immune modulation. Our clinical studies and those of others have shown that cord blood cell infusion is both safe and possibly effective in the prevention and treatment of BPD. The therapeutic use of cord blood has emerged as a promising therapy. However, the genetic heterogeneity between control and intervention groups may reduce the comparability especially among small sample trials. The purpose of this study protocol is to investigate the effects of autologous cord blood mononuclear cell (ACBMNC) infusion on the prevention of BPD in very preterm monozygotic twins of less than 32 gestation weeks.MethodsIn this prospective, randomized, placebo-controlled, double-blinded multicenter clinical trial, 60 pairs of monozygotic twin preterm neonates of less than 32 weeks admitted to the Neonatal Intensive Care Unit are randomly assigned to receive intravenous ACBMNC infusion (targeted at 5 × 107 cells/kg) or placebo (normal saline) within 24 h after birth in a 1:1 ratio. The primary outcome will be survival without BPD at 36 weeks of postmenstrual age. The secondary outcomes will include the mortality rate, BPD severity, other common preterm complication rates, respiratory support duration, length and cost of hospitalization, and long-term respiratory and neurodevelopmental outcomes during a 2-year follow-up. Furthermore, we will perform single-cell RNA sequencing for cord blood cells and blood cells 3–10 days after intervention and detect whether reactive oxygen species and inflammatory cytokines are present.ConclusionThis will be the first randomized, placebo-controlled, double-blinded trial to evaluate the efficacy of ACBMNC infusion to prevent BPD in monozygotic twin premature infants and investigate the underlying protective mechanisms. The results of this trial will provide valuable clinical evidence for translational application of cord blood cell therapy in very preterm infants.Trial registration: ClinicalTrials.gov, NCT05087498, registered 10/09/2021, https://register.clinicaltrials.gov/prs/app/action/SelectProtocol?sid=S000BAD7&selectaction=Edit&uid=U0002PLA&ts=2&cx=qvyylv.