[Analysis of PROC gene variant in a Chinese pedigree affected with hereditary protein C deficiency].

OBJECTIVE: To explore the molecular pathogenesis of a Chinese pedigree affected with inherited protein C (PC) deficiency. METHODS: The protein C activity (PC:A) and protein C antigen (PC:Ag) of the proband and his family members were determined by a chromogenic substrate method and enzyme-linked immunosorbent assay, respectively. The proband was subjected to whole exome sequencing. Candidate variants were verified by Sanger sequencing of other members of the pedigree. RESULTS: The PC:A and PC:Ag of proband were reduced to 15% and 11%, respectively. The above parameters of his parents and elder sister were also decreased to approximately 50% of reference values. Next generation sequencing has revealed that the proband has harbored a heterozygous c.572_574delAGA (p.Glu191_Lys192delinsGlu) variant in exon 7 and a missense c.752C>T (p.Ala251Val) variant in exon 8 of the PROC gene. His father was heterozygous for the c.572_574delAGA variant, while his mother and elder sister were heterozygous for the c.752C>T variant. According to the American College of Medical Genetics and Genomics Standards and Guidelines, the c.572_574delAGA (p.Glu191_Lys192 delinsGlu) variant was predicted to be likely pathogenic (PS1+PM4+PP3). c.752 C>T (p.Ala251Val) variant was also likely pathogenic (PS1+PM1+PP3). CONCLUSION: The deletional variant of c.572_574delAGA (p.Glu191_Lys192delinsGlu) in exon 7 and missense variant c.752C>T (p.Ala251Val) in exon 8 of the PROC gene probably underlay the inherited protein C (PC) deficiency in this pedigree. Above finding has enriched the spectrum of PROC gene variants and provided a basis for genetic counseling for this pedigree.

Th17/Treg dysregulation in allergic asthmatic children is associated with elevated notch expression.

BACKGROUND: Notch signaling pathway is critically involved in the differentiation of T helper (Th) cells, key players in the pathogenesis of allergic diseases. OBJECTIVE: The study is to explore whether Th17/Treg dysregulation in children with allergic asthma (AA) is associated with alteration of Notch expression. METHODS: Thirty-five patients with AA and thirty-five healthy control children were selected. Flow cytometry was used to detect Th17 and Treg cells. Quantitative real-time polymerase chain reaction (QRT-PCR) was used to measure the expression of Notch1 mRNA. The correlations among Notch1 mRNA expression, the percentage of Th17 cells, and Th17/Treg ratio were calculated. RESULTS: Th17 and Treg cells were significantly increased and decreased, respectively, in children with AA than in healthy control (p < 0.001). mRNA level of Notch1 was elevated in children with AA comparing to healthy controls (p < 0.001). The mRNA expression of Notch1 was positively correlated with the percentage of Th17 cells (r = 0.775, p < 0.001) and Th17/Treg ratio (r = 0.698, p < 0.001). CONCLUSION: Children with AA showed dysregulation of Th17/Treg cells in peripheral blood. Such change is accompanied with overexpression of Notch1, indicating Th17/Treg dysregulation in children with AA is associated with elevated Notch expression.

γ-Secretase Inhibitor Alleviates Acute Airway Inflammation of Allergic Asthma in Mice by Downregulating Th17 Cell Differentiation.

T helper 17 (Th17) cells play an important role in the pathogenesis of allergic asthma. Th17 cell differentiation requires Notch signaling. γ-Secretase inhibitor (GSI) blocks Notch signaling; thus, it may be considered as a potential treatment for allergic asthma. The aim of this study was to evaluate the effect of GSI on Th17 cell differentiation in a mouse model of allergic asthma. OVA was used to induce mouse asthma model in the presence and absence of GSI. GSI ameliorated the development of OVA-induced asthma, including suppressing airway inflammation responses and reducing the severity of clinical signs. GSI also significantly suppressed Th17-cell responses in spleen and reduced IL-17 levels in serum. These findings suggest that GSI directly regulates Th17 responses through a Notch signaling-dependent pathway in mouse model of allergic asthma, supporting the notion that GSI is a potential therapeutic agent for the treatment of allergic asthma.

Adipose mesenchymal stem cells-derived exosomes attenuated hyperoxia-induced lung injury in neonatal rats via inhibiting the NF-κB signaling pathway.

OBJECTIVE: Bronchopulmonary dysplasia (BPD) is the most common chronic morbidity in extremely preterm infants. Mesenchymal stem cells-derived exosomes (MSC-Exos) therapies have shown prospects in animal models of BPD. Our study aimed to evaluate the effect of adipose mesenchymal stem cells-derived exosomes (AMSC-Exos) on BPD and the role of the NF-κB signaling pathway in this process. METHODS: The AMSCs were extracted and AMSC-Exos were isolated by ultracentrifugation method. Newborn rats were exposed to hyperoxia (90% O2) continuously for 7 days to establish a BPD model. The rats were treated with AMSC-Exos by intratracheal administration on postnatal day 4 (P4). Pulmonary morphology, pulmonary vasculature, inflammatory factors, and NF-κB were assessed. Hyperoxia-induced primary type II alveolar epithelial cells (AECIIs) and AMSC-Exos treatment with or without a pan-NF-κB inhibitor (PDTC) were established to explore the potential mechanism. RESULTS: Hyperoxia-exposed rats showed alveolar simplification with decreased radial alveolar count and increased mean linear intercept, low CD31, and vascular endothelial growth factor expression, reduced microvessel density, increased the expression of TNF-α, IL-1ß, and IL-6 and decreased the expression of IL-10, and induced NF-κB phosphorylation. AMSC-Exos protected the neonatal lung from the hyperoxia-induced arrest of alveolar and vascular development, alleviated inflammation, and inhibited NF-κB phosphorylation. Hyperoxia decreased viability, increased apoptosis, enhanced inflammation, and induced NF-κB phosphorylation of AECIIs but improved by AMSC-Exos, PDTC, or AMSC-Exos+PDTC. The effect of AMSC-Exos+PDTC in AECIIs was the same as AMSC-Exos, but more notable than PDTC alone. CONCLUSION: AMSC-Exos attenuated the hyperoxia-induced lung injury in neonatal rats by inhibiting the NF-κB signaling pathway partly.

[Coordinated regulation of PI3K and Notch signal pathways in the activation and proliferation of CD4(+)T lymphocytes in asthma mice].

OBJECTIVE: To explore whether the signal pathways of phosphoinositide 3-kinase (PI3K) and Notch can realize coordinated regulation on the activation and proliferation of CD4(+)T lymphocytes. METHODS: Male BALB/c mice were randomly divided into control and asthma groups. Then the murine model of asthma was established by the method of ovalbumin (OVA) challenge. The CD4(+)T lymphocytes were isolated by magnetic activated cell sorter (MACS) and then activated with phytohaemagglutinin (PHA) (10 µg/ml) and IL-2 (1000 U/ml) for 6 h. Those cells were then divided into Group A: without any treatment; Group B: treatment with PI3K inhibitor (LY294002); Group C: treatment with Notch inhibitor (gamma-secretase inhibitor, DAPT); Group D: treatment with PI3K inhibitor and Notch inhibitor. The protein and transcription levels of Cyclin A, Cyclin D1 and P27(kip1) of CD4(+)T lymphocytes were assessed by flow cytometry and reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: The results of flow cytometry showed that the purity of MACS-isolated CD4(+)T lymphocytes was 90.0% ± 5.2% and the survival rate 94.8% ± 3.2%. The protein (28.0% ± 3.5%, 14.9% ± 3.4%) and mRNA levels (0.55 ± 0.16, 1.38 ± 0.42) of Cyclin A and Cyclin D1 in CD4(+)T lymphocytes of asthma group were significantly higher than those of the control group (13.4% ± 3.5%, 7.7% ± 1.8% and 0.32 ± 0.10, 0.92 ± 0.37) (P = 0.002, 0.036 and P = 0.007, 0.042). The protein and mRNA levels (23.3% ± 3.9% and 0.16 ± 0.03) of P27(kip1) of asthma group were significantly lower than those of control group (37.5% ± 5.8% and 0.32 ± 0.03, P = 0.006 and P = 0.000). The protein and mRNA levels of Cyclin D1 in groups A, B, C and D-treated CD4(+)T lymphocytes were 12.2% ± 3.7%, 7.3% ± 3.0%, 8.1% ± 2.3%, 4.2% ± 1.7% and 1.71 ± 0.44, 1.07 ± 0.31, 1.21 ± 0.32 and 0.62 ± 0.20 respectively; groups B, C and D decreased markedly compared with group A (all P < 0.01) while group D decreased significantly compared with groups B and C (all P < 0.05). The protein levels of P27(kip1) in groups A, B, C and D were 22.9% ± 3.0%, 31.6% ± 5.3%, 28.4% ± 5.6% and 44.6% ± 2.8% respectively; group B was significantly higher than that of group A (P = 0.016) while group D was significantly higher than those of groups A, B and C (P = 0.003, 0.004, 0.000). Meanwhile P27(kip1) mRNA levels in each group were 0.16 ± 0.07, 0.36 ± 0.09, 0.63 ± 0.08 and 0.99 ± 0.21 respectively; groups B, C and D were much higher than that of group A (P = 0.016, 0.000, 0.000) while group D was significantly higher than those of groups B and C (P = 0.000, 0.023). The protein and mRNA levels of CylinA showed no statistical significance among different experimental groups (all P > 0.05). CONCLUSION: The signal pathways of PI3K and Notch may coordinately up-regulate the expression of positive regulatory factor cylinD1 and down-regulation the expression of negative regulatory factor P27(kip1) of CD4(+)T lymphocytes.

Adipose Stem Cells Derived Exosomes Alleviate Bronchopulmonary Dysplasia and Regulate Autophagy in Neonatal Rats.

BACKGROUND: Mesenchymal stem cell-derived exosomes (MSC-Exos) therapies have shown prospects in preclinical models of pathologies relevant to neonatal medicine, such as bronchopulmonary dysplasia (BPD). Adipose-derived stem cells (ADSCs) have been recognized as one of the most promising stem cell sources. Autophagy plays a key role in regulating intracellular conditions, maintaining cell growth and development, and participating in the pathogenesis of BPD. OBJECTIVES: To investigate the potential therapeutic role of ADSC-Exos on BPD and to illustrate the role of autophagy in this process. METHOD: ADSC-Exos was isolated from media conditioned of ADSCs by ultracentrifugation and characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blotting (WB). Newborn rats were exposed to hyperoxia (90% O2) to mimic BPD, treated with ADSC-Exos by intratracheal or intravenous administration on postnatal day 4 (P4) and returned to room air on P7 until P14. Treated animals and appropriate controls were harvested on P7 and P14 for assessment of pulmonary parameters. RESULTS: Hyperoxia-exposed rats were presented with pronounced alveolar simplification with decreased radial alveolar count (RAC) and increased mean linear intercept (MLI), impaired vascular development with low vascular endothelial growth factor (VEGF) and CD31 expression, and stimulated inflammation with increased expression of TNF-α, IL-1ß, and IL-6, and decreased expression of IL-10. Meanwhile, the rats with hyperoxia exposure blocked autophagic flux with lower levels of Beclin1, LC3B, LC3BII/I ratio and higher levels of p62. ADSC-Exos administration protected the neonatal lung tissues from the hyperoxia-induced arrest of alveolar and vascular development, reduced inflammation, and facilitated autophagy. Intratracheal administration was more efficacious than intravenous administration. CONCLUSION: The intratracheal administration of ADSC-Exos significantly improved alveolarization and pulmonary vascularization arrest in hyperoxia-induced BPD, which was associated with facilitating autophagy in part.

Low-Dose Vitamin D Protects Hyperoxia-Induced Bronchopulmonary Dysplasia by Inhibiting Neutrophil Extracellular Traps.

Background and Objective: As bronchopulmonary dysplasia (BPD) can lead to considerable mortality and morbidity, this disease is the focus of attention in neonatology. Vitamin D (VD), which has anti-inflammatory properties and promotes lung growth, may have a therapeutic effect on BPD. The overexpression of neutrophil extracellular traps (NETs) has been demonstrated to be involved in the pathogenesis of BPD in our previous study. This study aimed to elucidate the effect of VD on BPD and the role of NETs in this process. Methods: Newborn rats were exposed to 90% oxygen continuously for 7 days to mimic BPD, and rats under hyperoxia were injected with 1,25(OH)2D3 at different doses (0.5 ng/g, 3 ng/g). Alveolarization, pulmonary vascular development, inflammatory cytokines and NETs were assessed. Results: Hyperoxia increased mortality, decreased body weight, impaired alveolarization with a decrease in radial alveolar count (RAC) and an increase in mean linear intercept (MLI), and impaired vascular development with low vascular endothelial growth factor (VEGF) expression. Meanwhile, hyperoxia enhanced expression of the proinflammatory factors TNF-α, IL-1ß, and IL-6, and elevated NETs in lung tissues and plasma. Low-dose VD (0.5 ng/g) administration increased the survival rate, attenuated developmental retardation, improved alveolarization, and pulmonary vascular development in hyperoxia-induced BPD, and reduced the expression of proinflammatory factors and NETs. However, high-dose VD (3 ng/g) treatment did not attenuate lung injury or NETs significantly, and even led to more severe developmental retardation and a higher mortality rate. Conclusions: Low-dose VD increased the survival rate, attenuated developmental retardation, and improved alveolarization and pulmonary vascularization arrest in hyperoxia-induced BPD partially by inhibiting NETs.

High Neutrophil-to-Lymphocyte Ratio Is an Early Predictor of Bronchopulmonary Dysplasia.

Background and Objective: Bronchopulmonary dysplasia (BPD) is a common complication in preterm infants; predicting the degree of BPD at an early life stage is difficult. Inflammation is a crucial risk factor for BPD pathogenesis, and the neutrophil-to-lymphocyte ratio (NLR) is a potential systemic inflammatory biomarker. We aimed to assess the predictive value of the NLR for BPD. Methods: We carried out a retrospective, single-center, observational study of neonates with gestational ages (GAs) <32 weeks and assessed the association between the NLR and BPD. Results: The study population included 296 preterm infants with BPD (n = 144) or without BPD (n = 152). Among the infants, 75 (25.3%) had mild BPD, 37 (12.5%) had moderate BPD, and 32 (10.8%) had severe BPD. The BPD group had a higher NLR at birth and at 72 h than the non-BPD group. The NLR cutoff value at 72 h for the prediction of BPD was 3.035 (sensitivity = 0.519, specificity = 0.964), and the area under the curve (AUC) was 0.714. The NLR cutoff value at 72 h for predicting severe BPD was 3.105 (sensitivity = 0.607, specificity = 0.819), with an AUC of 0.756. At the NLR cutoff value at 72 for the prediction of BPD, the AUCs were 0.640 and 0.970 in the preterm infants with EOS and congenital pneumonia, respectively. Conclusions: The NLR is an inexpensive, accessible and convenient tool; an increase in the NLR at 72 h could be an early predictor of BPD, especially severe BPD. Additionally, the NLR at 72 h could be a predictor of BPD in preterm infants with intrauterine infections.

[Diagnosis and prognosis of spontaneous pneumomediastinum in eighteen children].

OBJECTIVE: To analyze the diagnosis, treatment and prognosis of spontaneous pneumomediastinum (SPM) in children. METHOD: A retrospective analysis of the clinical data of 18 children diagnosed with SPM in Yuying Children's Hospital Affiliated to Wenzhou Medical University from December 2007 to February 2013 was performed. Information of the sequelae and recurrence of SPM was obtained by telephone follow-up. SPM was diagnosed according to Versteegh's standard. SPM cases due to mechanical ventilation, trauma, inhaled foreign body or as a result of the underlying disease were not included. Also cases of secondary pneumothorax pneumomediastinum and neonatal mediastinal emphysema were excluded. RESULT: Fifteen of 18 cases were boys and 3 were girls, the range of age was from 9 to 17 years. Predisposing factors included sport activities, severe cough or without a known cause. Clinical manifestations included chest pain, chest tightness, dyspnea, neck pain, back pain, foreign body sensation or pain on swallowing, throat pain of swelling. Chest CT of 18 cases showed pneumomediastinum, 8 cases displayed varied degrees of air in neck, chest; 18 cases of SPM responded well to bed rest, oxygen, antitussive and anti-infection treatment. Fifteen cases received chest CT or X-ray inspection after therapy, showing that the pneumomediastinum disappeared or significantly absorbed, 3 cases improved in clinical symptom. Among 18 patients, telephone follow-up of 14 were successful and 4 cases were lost. An average follow-up time was (24 ± 17) months. None of the cases had any serious consequences, and recurrence happened in one case. CONCLUSION: Children's spontaneous pneumomediastinum is a benign disease. When a child has chest pain or chest tightness, SPM should be considered after excluding the common diseases. SPM can be diagnosed in association with clinical feature and chest CT examination. Patients respond well to conservative therapy and most of them had no severe sequelae.