Cord blood stem cell­derived Angptl7 ameliorates the severity of bronchopulmonary dysplasia via anti­inflammatory and proangiogenic effects.

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. Angiopoietin­like 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 transcription­quantitative 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 interleukin­6 and monocyte chemoattractant protein­1 were negatively correlated with cord blood Angptl7 level; whereas, vascular endothelial growth factor­A levels were positively correlated with Angptl7 levels. In the LPS­induced 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, LPS­induced lung inflammation and macrophage infiltration were alleviated by Angptl7 treatment (P<0.05). In conclusion, the anti­inflammatory 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).

Corrigendum: Use of autologous cord blood mononuclear cells infusion for the prevention of bronchopulmonary dysplasia in extremely preterm neonates: a study protocol for a placebo-controlled randomized multicenter trial [NCT03053076].

[This corrects the article DOI: 10.3389/fped.2020.00136.].

Reduction of antibiotic use and multi-drug resistance bacteria infection in neonates after improvement of antibiotics use strategy in a level 4 neonatal intensive care unit in southern China.

The investigation on antibiotic stewardship in neonatal intensive care unit in China is scarce. This study aimed to analyze the effect of a comprehensive 2-year antibiotic stewardship in a level 4 NICU. During this baseline period from October 1st 2017 to October 1st 2019, continuation of empirical antibiotic therapy for ruled-out sepsis courses was beyond 72 h and for pneumonia was more than 7 days. Meropenem or vancomycin was used even if they were not the only bacterial sensitive antibiotics. The intervention period was from October 2nd 2019 to August 23rd 2021. Three areas for quality improvement were targeted in our center: discontinuation of antibiotic use in ruled-out sepsis within 72 h, treatment duration for culture-negative pneumonia less than 7 days, and vancomycin or meropenem was not used unless the cultured bacteria was only susceptible to them. The total antibiotic consumption decreased from 791.1 to 466.3 days of therapy per 1000 patient days from baseline to intervention period. Antibiotics were stopped within 72 h for 47.48% patients with rule-out sepsis and within 7 days for 75.70% patients with pneumonia compared with 11.56% and 37.69% during the baseline period respectively. The prevalence of multi-drug resistance bacteria decreased from 67.20 to 48.90%. The total use rate of meropenem or vancomycin decreased from 7.6 to 1.8%. Our quality improvement approach on antibiotic strategy significantly reduced antibiotic use and prevalence of multi-drug resistance bacteria in our NICU.

Cord blood antimicrobial peptide LL37 levels in preterm neonates and association with preterm complications.

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.

The Correlation of Antibacterial Peptides Concentration in Umbilical Cord Blood and Early Onset Sepsis in Preterm Infants.

Umbilical cord blood from singleton preterm infants was collected during delivery, and the concentration of LL37 was measured. C-reactive protein (CRP), white blood cell count (WBC), platelets (PLT), and mean platelet volume (MPV) were determined within 3 days after birth. The differences in LL37, CRP, WBC, PLT, and MPV levels between the two groups were compared. Pearson correlation method was used to analyze the correlation between these factors. The early individual value of each detected index for early onset sepsis was analyzed by ROC curve. The level of LL37 in umbilical cord blood of sepsis group was significantly higher than those in the control group (383.85 ± 46.71 vs. 252.37 ± 83.30 ng/ml). Meanwhile, the levels of CRP, WBC, and MPV in the sepsis group were significantly higher than those in the control group (CRP:5.73 ± 4.19 vs. 2.50 ± 2.77 mg/L; WBC: 13.47 ± 12.35 vs. 6.83 ± 3.55 × 109/L; MPV: 11.20 ± 1.11 vs. 8.90 ± 0.68 fL), the level of PLT was significantly lower than those in the control group (PLT: 161.00 ± 38.51 vs. 241.50 ± 49.85 × 109/L) (P < 0.05). Pearson correlation analysis showed that the expression of LL37 was negatively correlated with PLT level (r = -0.9347, P < 0.0001), and positively correlated with MPV level (r = 0.9463, P < 0.0001). ROC curve analysis showed that the area under curve of LL37 for diagnosis of early onset sepsis was 0.875, the prediction probability was 0.7, the sensitivity was 90.0% and the specificity was 80.0%.

Human Umbilical Cord Mesenchymal Stem Cells Prevent Bacterial Biofilm Formation.

Biofilm formation is easily found in patients suffered from ventilator-associated pneumonia (VAP) in neonatal intensive care unit (NICU) and makes the VAP infections not only harder to be treated but easier to relapse. In order to find some novel ways to inhibit biofilm formation, this study describe a previously unrecognized role for the human umbilical cord mesenchymal stem cells (hUCMSCs). In addition to multiple differentiation, hUCMSCs have the ability to prevent the biofilms formation in vitro by secreting antibacterial peptides (LL-37 and hBD-2). This occurred while P. aeruginosa PA27853 and hUCMSCs were cocultured, and the filtrated medium, which was the supernatant containing antibacterial peptides (5.9 ng/ml of LL-37, 1.77 ng/ml of hBD-2), and inhibited the growth of the bacterial biofilm on the surface of tracheal tube (2.5#, for preterm infant). Using microarrays, we were able to demonstrate that the antibacterial peptides from hUCMSC affected biofilm formation by downregulating the gene-encoded polysaccharide biosynthesis protein. In addition, in order to find out the most suitable concentration of hUCMSCs, P. aeruginosa was cocultured with eight-level concentrations of hUCMSCs, and we found that the concentration of LL-37 was positively correlated with the concentration of hUCMSCs. Meanwhile, the concentration of LL-37 became stable while the hUCMSC concentration reaches higher than 5 × 106 cells/ml. But the concentration of hBD-2 had no significant correlation with hUCMSCs. The collection of these stem cells is not only limited by ethics but also reduces host rejection. This makes it possible to use autologous hUCMSCs to treat neonatal VAP.

The Correlation Between Bronchopulmonary Dysplasia and Platelet Metabolism in Preterm Infants.

Background: Platelets play an important role in the formation of pulmonary blood vessels, and thrombocytopenia is common in patients with pulmonary diseases. However, a few studies have reported on the role of platelets in bronchopulmonary dysplasia. Objective: The objective of the study was to explore the relationship between platelet metabolism and bronchopulmonary dysplasia in premature infants. Methods: A prospective case-control study was performed in a cohort of premature infants (born with a gestational age <32 weeks and a birth weight <1,500 g) from June 1, 2017 to June 1, 2018. Subjects were stratified into two groups according to the diagnostic of bronchopulmonary dysplasia: with bronchopulmonary dysplasia (BPD group) and without bronchopulmonary dysplasia (control group). Platelet count, circulating megakaryocyte count (MK), platelet-activating markers (CD62P and CD63), and thrombopoietin (TPO) were recorded and compared in two groups 28 days after birth; then serial thrombopoietin levels and concomitant platelet counts were measured in infants with BPD. Results: A total of 252 premature infants were included in this study. Forty-eight premature infants developed BPD, 48 premature infants without BPD in the control group who were matched against the study infants for gestational age, birth weight, and admission diagnosis at the age of postnatal day 28. Compared with the controls, infants with BPD had significantly lower peripheral platelet count [BPD vs. controls: 180.3 (24.2) × 109/L vs. 345.6 (28.5) × 109/L, p = 0.001]. Circulating MK count in the BPD group was significantly more abundant than that in the control group [BPD vs. controls: 30.7 (4.5)/ml vs. 13.3 (2.6)/ml, p = 0.025]. The level of CD62p, CD63, and TPO in BPD group was significantly higher than the control group [29.7 (3.1%) vs. 14.5 (2.5%), 15.4 (2.0%) vs. 5.8 (1.7%), 301.4 (25.9) pg/ml vs. 120.4 (14.2) pg/ml, all p < 0.05]. Furthermore, the concentration of TPO was negatively correlated with platelet count in BPD group with thrombocytopenia. Conclusions: Our findings suggest that platelet metabolism is involved in the development of BPD in preterm infants. The possible mechanism might be through increased platelet activation and promoted TPO production by feedback.

Mesenchymal Stem Cells: Potential Therapeutic Prospect of Paracrine Pathways in Neonatal Infection.

Infection is the leading cause of admission and mortality in neonatal intensive care units. Immature immune function and antibiotic resistance make the treatment more difficult. However, there is no effective prevention for it. Recently, more and more researches are focusing on stem cell therapy, especially mesenchymal stem cells (MSCs); their potential paracrine effect confer MSCs with a major advantage to treat the immune and inflammatory disorders associated with neonatal infection. In this review, we summarize the basal properties and preclinical evidence of MSCs and explore the potential mechanisms of paracrine factors of MSCs for neonatal infection.

Safety and feasibility of umbilical cord blood collection from preterm neonates after delayed cord clamping for the use of improving preterm complications.

BACKGROUND: Umbilical cord blood (UCB) is a new and convenient source of stem cells reported to be safe and effective in preventing and treating preterm complications. The initial processing step for this therapy involves cord blood collection and isolation of the mononuclear cell (MNC) layer. However, there is limited information regarding the feasibility and safety of cord blood collection in preterm infants, and whether cord blood cell quality and quantity are adequate for treating complications in preterm infants. UCB units from preterm infants are currently discarded due to safety concerns regarding collection and owing to the harvesting of inadequate volumes for banking. This study aimed to investigate the feasibility and safety of UCB collection following delayed cord clamping (DCC) for preventing and treating complications in preterm infants. METHODS AND MATERIALS: Singleton preterm infants below 35 weeks gestation were assigned to two cohorts: cord blood collection and non-cord blood collection groups. Mortality and preterm complications in the two groups were compared to evaluate the safety of cord blood collection in preterm infants. The characteristics of the cord blood cells in preterm infants were investigated by comparing the cord blood parameters before and after processing with those of term infants born during the same period. RESULTS: There were 90 preterm infants and 120 term neonates enrolled in this study. Compared to those of the term group, the preterm neonates had significantly less cord blood volume and fewer cell numbers. Nevertheless, the MNC number in the preterm group was 1.92±1.35×108 per kg, which fulfilled the previously reported targeted cell dose (5×107 cells/kg) suitable for application to improve preterm complications. There was no significant difference regarding complications in the preterm neonates with or without cord blood collection. CONCLUSIONS: The collection of UCB after DCC in preterm infants is feasible and safe. The cell numbers and quality fulfill the criteria for use in improving preterm complications. Cord blood MNCs from preterm neonates should be reconsidered as an ideal source for use in stem cell therapy for preterm complications.

Placental pathology and neonatal outcomes in pre-eclampsia with gestational diabetes mellitus.

OBJECTIVE: To investigate histopathological placental lesions and adverse neonatal outcomes by Pre-eclampsia (PE) with Gestational Diabetes Mellitus (GDM). METHODS: This was a retrospective cohort study of pregnancies with PE delivered between 1 January 2012 to 1 January 2014. Pregnant women with PE were recruited, and divided into PE with GDM (PE + GDM) group (n = 278) and PE without GDM (PE - GDM) group (n = 586). We compared the placental pathology and neonatal outcomes between the two groups. RESULTS: The (PE + GDM) group was significantly associated with high placenta weight (534.8 ± 124.1 vs 519.3 ± 132.3 g, p = .011), the large diameter of the placenta (17.8 ± 2.2 vs 16.2 ± 2.7 cm, p = .016) than (PE - GDM) group. The incidence of chorioamnionitis in (PE + GDM) group was significantly higher than (PE - GDM) group [48.9% (136/278) vs 41.5% (243/586), p = .028], whereas there were no significant differences in umbilical cord length and infarction between the two groups. The (PE + GDM) group had a higher rate of prematurity [44.9% (125/278) vs 39.9% (234/586), p = .042] than (PE - GDM) group, in (PE + GDM) group the incidence of LGA [15.1% (42/278) vs 1.0% (6/586), p = .034], RDS [18.7% (52/278) vs 9.2% (54/586), p = .011] and hyperbilirubinemia [10.7% (30/278) vs 1.0% (6/586), p = .038] were higher than (PE - GDM) group. CONCLUSIONS: GDM increased the offspring's complication in pregnancy with PE, the potential mechanism might be that GDM increased the placenta inflammation.

Cord Blood Levels of Angiopoietin-Like 7 (ANGPTL7) in Preterm Infants.

OBJECTIVE: ANGPTL7 is a member of the angiogenin-like protein family. Compared to other members, ANGPTL7 is the least known. Recent studies have explored the relationship between ANGPTL7 and multiple pathological processes and diseases. However, there is no research about ANGPTL7 in neonates. This study was designed to investigate the concentration of ANGPTL7 in cord blood of preterm infants. METHOD: Singleton infants born in November 2017 to June 2019 in the study hospital were enrolled in the study. Maternal and neonatal clinical data were collected. ANGPTL7 levels in cord blood and serum on the third day after birth were measured by an enzyme-linked immunosorbent assay. RESULT: A total of 182 infants were enrolled in this study. Patients were categorized into two groups by gestational age (102 preterm, 80 full-term). ANGPTL7 levels in preterm infants were significantly higher than that in full-term babies (t = 15.4, P < 0.001). In multiple line regression analysis, ANGPTL7 levels independently correlated with gestational age (ß = -0.556, P < 0.001). There is also no correlation between preterm outcomes and ANGPTL7 levels. Cord blood levels of ANGPTL7 were significantly higher than those in serum on the third day after birth (t = 13.88, P < 0.001). CONCLUSION: Cord blood ANGPTL7 levels are higher in preterm infants than full-term babies. The levels are independently influenced by gestational ages and attenuated significantly after birth. The underlying mechanism needs to be further studied.

Pathological staging of chorioamnionitis contributes to complications in preterm infants.

OBJECTIVE: To investigate the effect of pathological staging of chorioamnionitis (CA) on complications in preterm infants; METHODS: A single-center, retrospective study was conducted to choose singleton preterm infants (gestational age < 37 weeks) from the Department of Obstetrics and Gynecology in our hospital from December 2016 to December 2017. The basic data and placental pathological results were retrospectively collected. According to the placental pathological results of whether inflammation infiltrating amnion, CA 0/I phase was classified into non-amnionitis group, CA II/III phase was classified into amnionitis group, the incidence of common complications in preterm infants was compared. Further, logistic regression was used to analyze the effects of amnionitis on complications after being adjusted to gestational age, birth weight and thrombocytopenia. RESULTS: A total of 221 preterm infants were enrolled, including 186 cases in non-amnionitis group and 35 cases in amnionitis group. The gestational age of amnionitis group (32.00 ± 2.71 weeks) was significantly lower than non-amnionitis group (34.14 ± 2.06 weeks), birth weight (1.93 ± 0.64 kg) was significantly lower than that of non-amnionitis group (2.26 ± 0.58 kg), and the hospital stay in amnionitis group was significantly longer (25.71 ± 19.23 days), all of the difference above was statistically significant(P < 0.05). The incidence of intraventricular hemorrhage (IVH) in amnionitis group (37.14%) was significantly higher than that in non-amnionitis group (13.98%) (P = 0.002), and the risk of IVH was significantly increased by amnionitis (OR = 3.636, 95%CI: 1.632-8.102); after correction of gestational age, birth weight and thrombocytopenia, the risk of IVH was still significantly increased (OR = 2.471, P = 0.046, 95% CI: 1.015-6.015). And the late-onset IVH was more common (P = 0.009). CONCLUSION: Amnionitis leads to a significant reduction in gestational age and birth weight in preterm infants, and it is an independent risk factor for IVH.

Use of Autologous Cord Blood Mononuclear Cells Infusion for the Prevention of Bronchopulmonary Dysplasia in Extremely Preterm Neonates: A Study Protocol for a Placebo-Controlled Randomized Multicenter Trial [NCT03053076].

Background: Despite the rapid advance of neonatal care, bronchopulmonary dysplasia (BPD) remains a significant burden for the preterm population, and there is a lack of effective intervention. Stem cell depletion because of preterm birth is regarded as one of the underlying pathological mechanisms for the arrest of alveolar and vascular development. Preclinical and small-sample clinical studies have proven the efficacy and safety of stem cells in treating and preventing lung injury. However, there are currently no randomized clinical trials (RCTs) investigating the use of autologous cord blood mononuclear cells (ACBMNC) for the prevention of BPD in premature infants. The purpose of this study is to investigate the effects of infusion of ACBMNC for the prevention of BPD in preterm neonates <28 weeks. Methods: In this prospective, randomized controlled double-blind multi-center clinical trial, 200 preterm neonates <28 weeks gestation will be randomly assigned to receive intravenous ACBMNC infusion (5 × 107 cells/kg) or placebo (normal saline) within 24 h after birth in a 1:1 ratio using a central randomization system. The primary outcome will be survival without BPD at 36 weeks of postmenstrual age or at discharge, whichever comes first. The secondary outcomes will include the mortality rate, other common preterm complication rates, respiratory support duration, length, and cost of hospitalization, and long-term outcomes after a 2-year follow-up. Conclusion: This will be the first randomized, controlled, blinded trial to evaluate the efficacy of ACBMNC infusion as a prevention therapy for BPD. The results of this trial will provide valuable clinical evidence for recommendations on the management of BPD in extremely preterm infants. Clinical Trial Registration: ClinicalTrials.gov, NCT03053076, registered 02/14/2017, retrospectively registered, https://register.clinicaltrials.gov/prs/app/action/SelectProtocol?sid=S0006WN4&selectaction=Edit&uid=U0002PLA&ts=2&cx=9y23d4 (Additional File 2).

Autologous cord blood cell infusion in preterm neonates safely reduces respiratory support duration and potentially preterm complications.

Preterm birth and its complications are the leading cause of neonatal death. The main underlying pathological mechanisms for preterm complications are disruption of the normal maturation processes within the target tissues, interrupted by premature birth. Cord blood, as a new and convenient source of stem cells, may provide new, promising options for preventing preterm complications. This prospective, nonrandomized placebo controlled study aimed at investigating the effect of autologous cord blood mononuclear cells (ACBMNC) for preventing preterm associated complications. Preterm infants less than 35 weeks gestational age were assigned to receive ACBMNC (5 × 107 cells/kg) intravenous or normal saline within 8 hours after birth. Preterm complication rates were compared between two groups to demonstrate the effect of ACBMNC infusion in reducing preterm complications. Fifteen preterm infants received ACBMNC infusion, and 16 infants were assigned to the control group. There were no significant differences when comparing mortality and preterm complication rates before discharge. However, ACBMNC infusion demonstrated significant decreases in duration of mechanical ventilation (3.2 days vs 6.41 days, P = .028) and oxygen therapy (5.33 days vs 11.31 days, P = .047). ACBMNC infusion was effective in reducing respiratory support duration in very preterm infants. Due to the limited number of patients enrolled, powered randomized controlled trials are needed to better define its efficacy.

Clcn3 deficiency ameliorates high-fat diet-induced obesity and adipose tissue macrophage inflammation in mice.

Obesity induces accumulation of adipose tissue macrophages (ATMs) and ATM-driven inflammatory responses that promote the development of glucose and lipid metabolism disorders. ClC-3 chloride channel/antiporter, encoded by the Clcn3, is critical for some basic cellular functions. Our previous work has shown significant alleviation of type 2 diabetes in Clcn3 knockout (Clcn3-/-) mice. In the present study we investigated the role of Clcn3 in high-fat diet (HFD)-induced obesity and ATM inflammation. To establish the mouse obesity model, both Clcn3-/- mice and wild-type mice were fed a HFD for 4 or 16 weeks. The metabolic parameters were assessed and the abdominal total adipose tissue was scanned using computed tomography. Their epididymal fat pad tissue and adipose tissue stromal vascular fraction (SVF) cells were isolated for analyses. We found that the HFD-fed Clcn3-/- mice displayed a significant decrease in obesity-induced body weight gain and abdominal visceral fat accumulation as well as an improvement of glucose and lipid metabolism as compared with HFD-fed wild-type mice. Furthermore, the Clcn3 deficiency significantly attenuated HFD-induced ATM accumulation, HFD-increased F4/80+ CD11c+ CD206- SVF cells as well as HFD-activated TLR-4/NF-κB signaling in epididymal fat tissue. In cultured human THP-1 macrophages, adenovirus-mediated transfer of Clcn3 specific shRNA inhibited, whereas adenovirus-mediated cDNA overexpression of Clcn3 enhanced lipopolysaccharide-induced activation of NF-κB and TLR-4. These results demonstrate a novel role for Clcn3 in HFD-induced obesity and ATM inflammation.