A novel hemizygous CD40L mutation of X-linked hyper IgM syndromes and compound heterozygous DOCK8 mutations of hyper IgE syndromes in two Chinese families.

X-linked hyper-immunoglobulin M (X-HIGM) syndrome and autosomal recessive hyper-immunoglobulin E syndrome (HIES) are rare inborn errors of immunity characterized by recurrent infections due to immune system impairment. In this study, we identified a novel hemizygous CD40 ligand (CD40L) mutation and compound heterozygous dedicator of cytokinesis-8 (DOCK8) mutations in two Han Chinese families with X-HIGM and HIES, respectively. We aimed to investigate the association between their genotypes and phenotypes. Genomic DNA was extracted from peripheral blood samples obtained from the families. Whole exome sequencing and Sanger sequencing were performed to identify and verify pathogenic variants in the two families. Clinical analyses of the probands were also performed. A novel hemizygous mutation of CD40L in exon 2 (c.257delA) was identified in the first proband, resulting in the substitution of glycine with glutamic acid at codon 86 of the protein. This leads to premature termination of translation at downstream codon 9 (p.E86Gfs*9). Sanger sequencing confirmed that the variant was inherited from the mother. The second proband carried two novel compound heterozygous mutations in DOCK8: one at exon 14 (c.1546C > G) inherited from the father, and the other at intron 41 (c.5355 + 6C > T; splicing) inherited from the mother. This study enhances our understanding of the pathogenetic mutation spectrum of CD40L and DOCK8 genes, facilitating the prenatal diagnosis of X-HIGM and HIES and enabling timely treatment of patients.

A Case of Fetal Familial Hemophagocytic Lymphohistiocytosis Type 5 caused by STXBP2 Gene Mutation.

BACKGROUND: Familial hemophagocytic lymphohistiocytosis type 5 (FHL-5) is a rare hyper-inflammatory syndrome caused by mutations in STXBP2. Most cases present at 2 - 6 months of age, and FHL-5 is extremely rare in neonates. METHODS: Appropriate laboratory tests, abdominal ultrasonography and whole exome sequencing were carried out. Respiratory support, antibiotics, and transfusion of blood products were done. RESULTS: Laboratory tests revealed metabolic acidosis, thrombocytopenia, mild anemia, and low fibrinogen level. Blood culture, metagenomics, and TORCH screening were negative. Liver and spleen enlargements were confirmed by abdominal ultrasonography. Whole exome sequencing identified a homozygous mutation in STXBP2 c. 1432del G (p. V478Sfs*5). The heterozygous STXBP2 mutation was identified in the paternal grandfather, maternal grandfather, and parents. CONCLUSIONS: Here we report a case with a novel homozygous deletion in exon 16 of STXBP2, which caused the earliest reported case of FHL-5 in a neonate. Our results identify a new pathogenic variant for the early identification and clinical consultation of FHL-5.

Endothelial S1pr2 regulates post-ischemic angiogenesis via AKT/eNOS signaling pathway.

Aims: It is important to understand the mechanism that regulates post-ischemic angiogenesis and to explore a new therapeutic target for an effective improvement of revascularization in peripheral artery disease (PAD) patients. Post-ischemic angiogenesis is a highly orchestrated process, which involves vascular endothelial cells (ECs) proliferation, migration and assembly into capillaries. We found a significant reduction of S1pr2 (sphingosine 1-phosphate receptor 2) in endothelial cells after hindlimb ischemia (HLI). We thus hypothesized that EC-S1pr2 might be involved in the regulation of post-ischemic angiogenesis and blood flow recovery during peripheral arterial disease (PAD). Methods and Results: We generated both EC-specific S1pr2 loss-of-function and S1pr2 gain-of-function mice. Our study showed that EC-specific S1pr2 loss-of-function significantly enhanced post-ischemic angiogenesis and improved blood flow recovery upon femoral artery ligation, whereas the EC-specific S1pr2 gain-of-function severely hindered post-ischemic angiogenesis and reduced blood flow recovery in ischemic limbs. We next identified that S1pr2 inhibited AKT/eNOS signaling pathway, and thus inhibited EC proliferation/migration and angiogenic activity. As expected, pharmacological inhibition of S1pr2 by JTE013 improved post-ischemic angiogenesis and improved blood flow perfusion after femoral artery ligation. Moreover, we developed RGD-peptide magnetic nanoparticles packaging S1pr2-siRNA which specifically targeted ECs and achieved an efficient silencing of S1pr2 expression in ECs in vivo. This EC-targeted strategy to dampen S1pr2 significantly enhanced post-ischemic angiogenesis and boosted blood perfusion after HLI, supplying a novel therapy target for patients with peripheral arterial disease. Conclusions: This present study demonstrates that EC-expressing S1pr2 tightly controls post-ischemic angiogenesis and blood flow perfusion recovery. This research provides a novel strategy for EC-target knockdown of S1pr2 as a new therapeutic intervention for patients with peripheral artery disease.

Interleukin-24 as a Pulmonary Target Cytokine in Bronchopulmonary Dysplasia.

The proliferation of fetal alveolar type II cells (FATIICs) was impaired in bronchopulmonary dysplasia (BPD), which is modulated by hyperoxia and inflammatory response. Interleukin 24 (IL-24), a cytokine produced by certain cell types, plays an essential role in inflammation and host protection against infection. However, the ability of FATIICs to produce IL-24 remains unclear, and the role of IL-24 in BPD progression is yet to be determined. With reverse transcription quantitative polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay, the authors evaluated whether FATIICs produce IL-24 in physiological conditions. The authors quantified IL-24 expression in the lungs of newborn rat pups exposed to hyperoxia (70% oxygen) and in FATIICs isolated on embryonic day 19 that were exposed to 95% oxygen or lipopolysaccharide (LPS). The role of IL-24 in FATIICs, cell proliferation, cell apoptosis, and cell cycle were further evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and flow cytometric analysis. Also, they assessed caspase-3 and SOCS3 mRNA in IL-24 siRNA-treated cells by using RT-qPCR. During culture, IL-24 mRNA and protein levels in FATIICs gradually decreased with FATIIC differentiation. IL-24 expression increased significantly in rat lungs exposed to hyperoxia and FATIICs exposed to oxygen or LPS. Recombinant IL-24 enhanced cell proliferation by decreasing the proportion of apoptotic cells and increasing the proportion of cells in the S phase. The IL-24 siRNA-treated cells expressed more caspase-3 mRNA. Furthermore, suppressor of cytokine signaling 3 (SOCS3) mRNA was significantly decreased in rats and FATIICs exposed to oxygen, whereas it dramatically increased in FATIICs exposed to LPS. The IL-24 siRNA-treated cells expressed more SOCS3 mRNA. These studies suggest IL-24 is a pulmonary target cytokine in BPD, and may possibly regulate SOCS3 in oxidative stress and inflammation of the lung.

Vascular endothelial S1pr1 ameliorates adverse cardiac remodelling via stimulating reparative macrophage proliferation after myocardial infarction.

AIMS: Endothelial cell (EC) homoeostasis plays an important role in normal physiological cardiac functions, and its dysfunction significantly influences pathological cardiac remodelling after myocardial infarction (MI). It has been shown that the sphingosine 1-phosphate receptor 1 (S1pr1) was highly expressed in ECs and played an important role in maintaining endothelial functions. We thus hypothesized that the endothelial S1pr1 might be involved in post-MI cardiac remodelling. METHODS AND RESULTS: Our study showed that the specific loss of endothelial S1pr1 exacerbated post-MI cardiac remodelling and worsened cardiac dysfunction. We found that the loss of endothelial S1pr1 significantly reduced Ly6clow macrophage accumulation, which is critical for the resolution of inflammation and cardiac healing following MI. The reduced reparative macrophages in post-MI myocardium contributed to the detrimental effects of endothelial S1pr1 deficiency on post-MI cardiac remodelling. Further investigations showed that the loss of endothelial S1pr1-reduced Ly6clow macrophage proliferation, while the pharmacological activation of S1pr1-enhanced Ly6clow macrophage proliferation, thereby ameliorated cardiac remodelling after MI. A mechanism study showed that S1P/S1pr1 activated the ERK signalling pathway and enhanced colony-stimulating factor 1 (CSF1) expression, which promoted Ly6clow macrophage proliferation in a cell-contact manner. The blockade of CSF1 signalling reversed the enhancing effect of S1pr1 activation on Ly6clow macrophage proliferation and worsened post-MI cardiac remodelling. CONCLUSION: This study reveals that cardiac microvascular endothelium promotes reparative macrophage proliferation in injured hearts via the S1P/S1PR1/ERK/CSF1 pathway and thus ameliorates post-MI adverse cardiac remodelling.

Evaluating postoperative anal fistula prognosis by diffusion-weighted MRI.

PURPOSE: The purpose of this study was to explore whether preoperative diffusion-weighted magnetic resonance imaging (DW-MRI) can be used to evaluate the prognosis of anal fistula and identify the influence factors of postoperative recurrence. METHODS: This is a retrospective study of 117 patients with anal fistula who have undergone preoperative DW-MRI and surgery. All patients were followed up by telephone or reexamination within 2 years after surgery. Of the 117 patients, 35 were excluded due to loss of follow-up and only 82 were included in this study. MRI fistula imaging-related data were analyzed, and fistula severity was scored using criteria of both local extension of fistulas and active inflammation for a total maximum score of 22. The apparent diffusion coefficient (ADC) value of the fistula in patients with anal fistula during preoperative MRI examination was measured. According to whether anal fistula patients are accompanied by perianal abscess, they are divided into two groups, namely anal fistula group and anal fistula with abscess group. Based on whether patients with anal fistula recur after surgery, they were further divided into recurrent group and non-recurrent group. RESULTS: 82 patients with anal fistula were included in this analysis, 23 of them recurred and 59 were cured. Among patients with perianal abscess, the mean ADC value of the recurrent group was (1.19 ±â€¯0.21)×10-3 mm2/s, which is significantly lower than that of the non-recurrent group (1.36 ±â€¯0.19)×10-3 mm2/s. There were significant statistical differences in ADC values between the two groups (p = 0.03). Among patients with anal fistulas without abscesses, 15 patients recurred after surgery, with a mean ADC value of (1.45 ±â€¯0.27) ×10-3 mm2/s, and 33 patients didn't occur, with a mean ADC value of (1.44 ±â€¯0.31)×10-3 mm2/s. The ADC value of preoperative fistula in patients was negative significant correlation with MRI findings score (r= -0.332, P = 0.002). Risk factors for the recurrence after anal fistula surgery include the time interval between MRI and operation, multiple fistula tracks. Fatigue, excessive intake of spicy or greasy food and diarrhea may also be external risk factors for postoperative recurrence of patients with anal fistula. CONCLUSIONS: DW-MRI has important application value for the prognosis evaluation of anal fistula. Complex type of anal fistula and improper lifestyle are the main risk factors affecting the recurrence after anal fistula surgery.

Endothelial S1pr1 regulates pressure overload-induced cardiac remodelling through AKT-eNOS pathway.

Cardiac vascular microenvironment is crucial for cardiac remodelling during the process of heart failure. Sphingosine 1-phosphate (S1P) tightly regulates vascular homeostasis via its receptor, S1pr1. We therefore hypothesize that endothelial S1pr1 might be involved in pathological cardiac remodelling. In this study, heart failure was induced by transverse aortic constriction (TAC) operation. S1pr1 expression is significantly increased in microvascular endothelial cells (ECs) of post-TAC hearts. Endothelial-specific deletion of S1pr1 significantly aggravated cardiac dysfunction and deteriorated cardiac hypertrophy and fibrosis in myocardium. In vitro experiments demonstrated that S1P/S1pr1 praxis activated AKT/eNOS signalling pathway, leading to more production of nitric oxide (NO), which is an essential cardiac protective factor. Inhibition of AKT/eNOS pathway reversed the inhibitory effect of EC-S1pr1-overexpression on angiotensin II (AngII)-induced cardiomyocyte (CM) hypertrophy, as well as on TGF-ß-mediated cardiac fibroblast proliferation and transformation towards myofibroblasts. Finally, pharmacological activation of S1pr1 ameliorated TAC-induced cardiac hypertrophy and fibrosis, leading to an improvement in cardiac function. Together, our results suggest that EC-S1pr1 might prevent the development of pressure overload-induced heart failure via AKT/eNOS pathway, and thus pharmacological activation of S1pr1 or EC-targeting S1pr1-AKT-eNOS pathway could provide a future novel therapy to improve cardiac function during heart failure development.

[Protective effect of interleukin 22 against hyperoxia-induced lung injury in neonatal rats].

Objective To study the protective effect of interleukin-22 (IL-22) against the hyperoxia-induced lung injury in neonatal SD rats. Methods The neonatal SD rats were randomized into control group, hyperoxia group and IL-22 (10 ng/g) treatment group. Each group was randomly divided into three subgroups of 1, 3, 7 days (n=9). Body mass in every group was detected; lung pathological changes were observed by HE staining; tumor necrosis factor α (TNF-α) in lung tissues was tested by quantitative real-time PCR; and IL-1ß in plasma was measured by ELISA. Results After exposure to hyperoxia for 1 day, the body mass in the three groups showed no significant difference, and the structure of lung tissues were normal. After exposure to hyperoxia for 3 or 7 days, the body mass in the hyperoxia group was significantly lower, IL-1ß in plasma was significantly enhanced, and the structure of lung tissues were destroyed, which aggravated with the time of hyperoxia exposure. TNF-α mRNA level increased obviously in the hyperoxia group. However, when treated with IL-22, TNF-α mRNA was significantly down-regulated in the treatment group. Conclusion IL-22 may play an protective role in hyperoxia-induced lung injury.

[The changes of Wnt7b/ß-catenin signaling pathway molecules in the differentiation of fetal alveolar epithelial type II cells].

OBJECTIVE: To investigate the changes of Wnt7b/ß-catenin signaling pathway molecules in the differentiation of fetal rat alveolar epithelial type II cells (fAEC2). METHODS: Fetal rat lung tissues were extracted from pregnant SD rats (19 days), and then fAEC2 were isolated and purified. After fAEC2 were cultured for 48, 72, 96 hours, the morphological changes of fAEC2 and the expression of ß-catenin were respectively observed by inverted microscope and immunofluorescence. The mRNA expressions of Wnt7b, cyclin D1, pulmonary surfactant C (SP-C) and aquaporin 5 (AQP5) were detected by real-time quantitative PCR. The protein expressions of cyclin D1, nucleus ß-catenin, SP-C and AQP5 were measured by Western blotting. RESULTS: ß-catenin was expressed in cell membrane when fAEC2 were cultured for 48 hours; the expression of ß-catenin decreased in cell membrane while enhanced in cytoplasm and nucleus at 72 hours; whole-cell ß-catenin expression was lowered at 96 hours. The expressions of Wnt7b and cyclin D1 mRNAs were significantly raised at 72 hours and reduced obviously at 96 hours compared with 48 hours. SP-C mRNA expression level went down gradually with the extended culture time, and AQP5 mRNA level went up gradually. The protein expressions of cyclin D1 and nucleus ß-catenin were significantly enhanced at 72 hours and weakened at 96 hours compared with 48 hours. SP-C protein expression was down-regulated with the prolonged culture time, and AQP5 protein expression was up-regulated. CONCLUSION: Wnt7b/ß-catenin signaling pathway may play an important role in fAEC2 transdifferentiation.

Effect of keratinocyte growth factor on growth and transdifferentiation of primary alveolar epithelial type II cells.

BACKGROUND/AIM: To investigate the effects of keratinocyte growth factor (KGF) on the growth and transdifferentiation of primary alveolar epithelial type II cells (AECIIs). MATERIALS AND METHODS: The number of primary AECIIs, their viability, and cell cycle and apoptosis were studied under KGF treatment using a hemocytometer, trypan blue exclusion, and flow cytometry, respectively. Positive expressions of surfactant protein C (SP-C), aquaporin 5 (AQP5), and thyroid transcription factor 1(TTF-1) were examined with indirect immunofluorescence; mRNA levels of SP-C, AQP5, and TTF-1 were determined by polymerase chain reaction. RESULTS: In response to KGF treatment, cell numbers were significantly increased, more cells were blocked in the S phase, and fewer cells were apoptotic or necrotic on days 2 and 4, but there was little effect on cell viability. In addition, KGF treatment resulted in higher levels of SP-C on days 2 and 8 while lowering them on day 4, higher levels of AQP5 on day 4 while lowering them on day 8, and higher levels of TTF-1 on days 2, 6, and 8. CONCLUSION: KGF treatment promoted proliferation of AECIIs, inhibited cell apoptosis, promoted transdifferentiation of AECIIs, and induced alveolar epithelial type I cells to revert to AECIIs.

Retinoic acid promotes primary fetal alveolar epithelial type II cell proliferation and differentiation to alveolar epithelial type I cells.

Retinoic acid (RA) plays an important role in lung development and maturation. Many stimuli can induce alveolar epithelial cell damage which will result in the injury of lung parenchyma. The aim of this study was to observe the effect of RA on the proliferation and differentiation of primary fetal alveolar epithelial type II cells (fAECIIs). Primary fAECIIs were isolated from fetal rats at 19 d of gestation and purified by a differential centrifugation and adhesion method. The cells were randomly divided into control (dimethyl sulfoxide, DMSO) and RA groups. Cell proliferation, viability, apoptosis, cycle, and expression of target protein were examined at 24, 48, and 72 h. We found that the proliferation and viability of cells in the RA-exposed group significantly increased compared with the DMSO control group. The proportion (%) of cells in the G2 and S phases in the RA group was significantly higher than that in control group cells. The proportion (%) of both early apoptotic cells and late apoptotic cells decreased significantly in cells exposed to RA compared with cells exposed to DMSO. RA significantly enhanced the expression of aquaporin 5 (AQP5). The expression level of pulmonary surfactant C (SPC) was elevated after cells were exposed to RA for 24 and 72 h but was inhibited when cells were exposed to RA for 48 h. These results suggest that RA promotes fAECII proliferation by improving cell viability, promoting S phase entry and inhibiting apoptosis and RA promotes fAECIIs differentiation to alveolar epithelial type I cells (AECIs).

Effect of hyperoxia on the viability and proliferation of the primary type II alveolar epithelial cells.

To observe the effect of hyperoxia on the growth of type II alveolar epithelial cells (AEC II). The lungs of 19-day gestation fetal rats were primary cultured and the AEC II were purified by differential adhesion method. The cells were divided into control (normoxia) group and hyperoxia group. The cell growth, cell viability, cell apoptosis, and cell cycle were examined at 2, 4, 6, and 8 days of normoxia or hyperoxia exposure. The number of cells in hyperoxia-exposed group significantly decreased as compared to those of air control group. Number of cells in hyperoxia group was the highest at day 2 of exposure and gradually decreased with time. The viability of cells exposed to hyperoxia was substantially reduced compared with cells exposed to air. Percentage of cells in G1 phase and S phase in hyperoxia group increased gradually with increase in exposure duration and significant differences were seen at day 4 and day 6 compared with either the preceding time points and also with corresponding air-exposed cells. The percentage of both early apoptotic cells (Annexin-V(+)/PI(-)) and late apoptotic cells and necrotic cells (Annexin-V(+)/PI(+)) increased significantly in cells exposed to hyperoxia compared with cells exposed to air. Hyperoxia inhibits proliferation, viability and growth of AEC II and promotes apoptosis.

[Therapeutic effect and its mechanism of "huo xue bu qi fang" on fetal rats with intrauterine growth retardation].

OBJECTIVE: To probe into therapeutic effect and its mechanism of "Huo Xue Bu Qi Fang" (HXBQF) on fetal rats with intrauterine growth retardation (IUGR). METHOD: The model of pregnant rat with IUGR was established by passive smoking method. Forty pregnant rats with IUGR were randomly divided into intervention group (with high-, middle- and low-dose Chinese traditional medicine) and non-intervention group. In addition, 10 normal pregnant rats were taken into control group. Intervention group was adminstered with 16.2, 5.4 and 1.62 g/kg HXBQF respectively. Non-intervention group and control group were administered with 10 ml/kg saline. Fetal rats were taken out, and blood and urine samples were collected from pregnant rats on day 21 of the pregnancy. The weight, nose-hip-length and poundera index of these fetal rats were measured. Serum NO, plasma ET-1 and urine 8-epi-PGF2 alpha level from pregnant rats were determined by nitro-reductase method, radioimmunoassay (RIA) and enzyme immunoassay (EIA) respectively. RESULT: Compared with fetal rats from non-intervention group, fetal weight, distance between nose-hip, poundera index serum NO level and NO/ET-1 were increased, plasma ET-1 level and urine 8-epi-PGF2 alpha level were decreased in those from intervention group (P < 0.01). CONCLUSION: There is an enhancement of lipid peroxidation and NO/ET-1 ratio imbalance in pregnant rats with IUGR. HXBQF has good therapeutic effect on IUGR since it can inhibit lipid oxidation and regulate NO/ET-1 balance.