M1 macrophage-related gene model for NSCLC immunotherapy response prediction.

Patients diagnosed with non-small cell lung cancer (NSCLC) have a limited lifespan and exhibit poor immunotherapy outcomes. M1 macrophages have been found to be essential for antitumor immunity. This study aims to develop an immunotherapy response evaluation model for NSCLC patients based on transcription. RNA sequencing profiles of 254 advanced-stage NSCLC patients treated with immunotherapy are downloaded from the POPLAR and OAK projects. Immune cell infiltration in NSCLC patients is examined, and thereafter, different coexpressed genes are identified. Next, the impact of M1 macrophage-related genes on the prognosis of NSCLC patients is investigated. Six M1 macrophage coexpressed genes, namely, NKX2-1, CD8A , SFTA3, IL2RB, IDO1, and CXCL9, exhibit a strong association with the prognosis of NSCLC and serve as effective predictors for immunotherapy response. A response model is constructed using a Cox regression model and Lasso Cox regression analysis. The M1 genes are validated in our TD-FOREKNOW NSCLC clinical trial by RT-qPCR. The response model shows excellent immunotherapy response prediction and prognosis evaluation value in advanced-stage NSCLC. This model can effectively predict advanced NSCLC prognosis and aid in identifying patients who could benefit from customized immunotherapy as well as sensitive drugs.

[Diagnosis of a case with Williams-Beuren syndrome by single nucleotide polymorphism array].

OBJECTIVE: To explore the genetic cause for a child with mental retardation, developmental delay and multi-systemic developmental disorders by analyzing the copy number variations (CNVs) and correlating the genotype with the phenotype. METHODS: Routine G-banding was performed to analyze the karyotype of the patient and her parents. In addition, single nucleotide polymorphisms array (SNP-array) was used to determine the CNVs, which was confirmed by fluorescence in situ hybridization (FISH). RESULTS: No karyotypic abnormality was detected upon chromosome analysis. However, SNP-array has identified a de novo hemizygous deletion of 1673 kb on chromosome region 7q11.23, which has been associated with Williams-Beuren syndrome. The microdeletion was confirmed by FISH testing. CONCLUSION: A child with Williams-Beuren syndrome has been diagnosed by SNP-array and FISH. The de novo 7q11.23 microdeletion probably underlies the clinical manifestation of the patient. Compared with routine karyotype analysis, SNP-array is more useful for diagnosing children with multiple congenital anomalies with unclear etiology.

Increased methylation at differentially methylated region of GNAS in infants born to gestational diabetes.

BACKGROUND: Offspring of pregnancy complicated with gestational diabetes (GDM) are at high risk for metabolic diseases. The mechanisms behind the association of intrauterine exposure to GDM and high risk of health problems in later life remain largely unknown. The aim of this study was to clarify the alteration in methylation levels at differentially methylated regions (DMRs) of GNAS and IGF2 in fetuses of GDM women and to explore the possible mechanisms linking maternal GDM with high risk of metabolic diseases in later life of GDM offspring. METHODS: Lymphocytes were isolated from umbilical cord blood of infants born to 87 women with GDM and 81 women with normal pregnancy. Genomic DNA was extracted and DNA methylation levels of GNAS and IGF2 DMRs were determined by Massarray quantitative methylation analysis. RESULTS: The methylation levels were detected in 7 CpG sites of GNAS DMRs and 6 sites of IGF2 DMRs. Methylation levels were significantly higher at sites 4, 5 and 7 of GNAS DMR in GDM compared to normal pregnancy (P = 0.007, 0.008 and 0.008, respectively). The methylation level at site 4 of GNAS was significantly correlated with the presence of GDM (P = 0.003), the methylation levels at site 5 and 7 were significantly correlated with the presence of GDM (P = 0.002 for both) and gestational age (P = 0.027 for both). There was no significant difference in any sites of IGF2 DMR (P > 0.05 for all). CONCLUSIONS: We concluded maternal GDM-induced hypermethylation at GNAS DMR and this condition may be among the mechanisms associating maternal GDM with increased risk of metabolic diseases in later life of offspring.

[Prenatal diagnosis of fetal chromosome aneuploidy by massively parallel genomic sequencing].

OBJECTIVE: To perform non-invasive prenatal diagnosis (NIPD) of chromosome aneuploidy by detecting free DNA in maternal peripheral plasma by massively parallel genomic sequencing and determine the feasibility of detecting cell-free fetal DNA in chromosomal copy. METHODS: The plasma samples from 1 264 gravidas were collected from February 2012 to July 2013 at our center. Those pregnant women with a gestational age of 13 to 33 weeks having high risks in serological screening or aged over 35 years or abnormal fetus on ultrasonography. The peripheral venous blood samples were drawn from pregnant women and plasma DNA was extracted for preparing a sequencing library. By using Illumina HiSeq2000, high-throughput sequencing was performed. Amnocentesis and karyotypic analysis were conducted for positive cases and those with negative results were followed up. RESULTS: By massively parallel genomic sequencing, 20 of them showed positive results. By the standard of chromosomal karyotypic analysis, there were 21 positive cases of 13 trisomy and the diagnostic accordance rate was 100%. Among 18 positive cases of 6 trisomy, there were amniocentesis (n = 5, except for one dead fetus), trisomy 18 (n = 4) and normal karyotype with placenta chimera (n = 1). One positive case of 13 trisomy was in accordance with karyotypic analysis. CONCLUSION: Massively parallel sequencing for NIPD may be used as further screening for pregnant women with high risks of serological screening. And this technology is highly consistent with karyotypic analysis in terms of sensitivity and specificity.

Phenotypic comparison and the potential antitumor function of immortalized bone marrow-derived macrophages (iBMDMs).

Introduction: Macrophages are an important component of innate immunity and involved in the immune regulation of multiple diseases. The functional diversity and plasticity make macrophages to exhibit different polarization phenotypes after different stimuli. During tumor progression, the M2-like polarized tumor-associated macrophages (TAMs) promote tumor progression by assisting immune escape, facilitating tumor cell metastasis, and switching tumor angiogenesis. Our previous studies demonstrated that functional remodeling of TAMs through engineered-modifying or gene-editing provides the potential immunotherapy for tumor. However, lack of proliferation capacity and maintained immune memory of infused macrophages restricts the application of macrophage-based therapeutic strategies in the repressive tumor immune microenvironment (TIME). Although J2 retrovirus infection enabled immortalization of bone marrow-derived macrophages (iBMDMs) and facilitated the mechanisms exploration and application, little is known about the phenotypic and functional differences among multi kinds of macrophages. Methods: HE staining was used to detect the biosafety of iBMDMs, and real-time quantitative PCR, immunofluorescence staining, and ELISA were used to detect the polarization response and expression of chemokines in iBMDMs. Flow cytometry, scratch assay, real-time quantitative PCR, and crystal violet staining were used to analyze its phagocytic function, as well as its impact on tumor cell migration, proliferation, and apoptosis. Not only that, the inhibitory effect of iBMDMs on tumor growth was detected through subcutaneous tumor loading, while the tumor tissue was paraffin sectioned and flow cytometry was used to detect its impact on the tumor microenvironment. Results: In this study, we demonstrated iBMDMs exhibited the features of rapid proliferation and long-term survival. We also compared iBMDMs with RAW264.7 cell line and mouse primary BMDMs with in vitro and in vivo experiments, indicating that the iBMDMs could undergo the same polarization response as normal macrophages with no obvious cellular morphology changes after polarization. What's more, iBMDMs owned stronger phagocytosis and pro-apoptosis functions on tumor cells. In addition, M1-polarized iBMDMs could maintain the anti-tumor phenotypes and domesticated the recruited macrophages of receptor mice, which further improved the TIME and repressed tumor growth. Discussion: iBMDMs can serve as a good object for the function and mechanism study of macrophages and the optional source of macrophage immunotherapy.

Methylation levels at IGF2 and GNAS DMRs in infants born to preeclamptic pregnancies.

BACKGROUND: Offspring of pregnancy complicated with preeclampsia are at high risk for hypertension, stroke and possibly obesity. The mechanisms behind the association of intrauterine exposure to preeclampsia and high risk of health problems in the later life remain largely unknown. The aims of the current investigation were to determine the changes in DNA methylation at IGF2 and GNAS DMR in offspring of preeclamptic pregnancy and to explore the possible mechanisms underlying the association between maternal preeclampsia and high risk for health problems in the later life of their offspring. RESULTS: Umbilical cord blood was taken from infants born to women of preeclampsia (n=56), gestational hypertension (n=23) and normal pregnancy (n=81). DNA methylation levels of IGF2 and GNAS DMR were determined by Massarray quantitative methylation analysis. Methylation levels at IGF2 DMR were significantly lower in preeclampsia than normal pregnancy. The average methylation level at IGF2 DMR was significantly correlated with preeclampsia even after birth weight, maternal age, gestational age at delivery and fetal gender were adjusted. The difference in methylation level was not significantly different between mild and severe preeclampsia. The methylation level at GNAS DMR was not significantly correlated with birth weight, maternal age, gestational age at delivery, fetal gender, preeclampsia or gestational hypertension. CONCLUSIONS: We concluded preeclampsia induced a decrease in methylation level at IGF 2 DMR, and this might be among the mechanisms behind the association between intrauterine exposure to preeclampsia and high risk for metabolic diseases in the later life of the infants.

[First trimester and second-trimester integrated screening for Down's syndrome].

OBJECTIVE: To evaluate the effect of first and second-trimester integrated screening so as to provide an efficient screening protocol for Down's syndrome. METHODS: Using the dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA), the freeßhCG (beta human chorionic gonadotropin), PAPP-A (pregnancy associated plasma protein-A) and NT (nuchal translucency) value of type B ultrasound were assayed in the pregnancy serum during the first trimester (11-13W(+6) d) and free ßhCG and AFP (alpha fetoprotein) during the second trimester(15-20W(+6) d). By the risk calculation software, the risks during both trimesters and their integrated risk were calculated for each patient respectively. Amniocentesis and venepuncture were employed for diagnosing the high-risk patients (> 1/270). Electronic network follow-up was carried out after delivery. RESULTS: In a total of 4237 pregnant women, 98 were found to carry a high risk during the first trimester, 241 during the second trimester and 101 during the integrated screening respectively. And 2, 3 and 4 cases were diagnosed with Down's symptom at a detection rate of 50%, 75% and 100% and a detection efficiency of 1:50, 1:80 and 1:25 respectively. CONCLUSION: Integrated screening is superior to either the first or second-trimester screening. With a lower false positive rate and a higher detection rate, it reduces the chance of invasive puncture. Advanced type B ultrasonic technology is needed to improve the first-trimester diagnostic efficiency and to develop a better integrated screening protocol.

Influence of epidural dexamethasone on maternal temperature and serum cytokine concentration after labor epidural analgesia.

OBJECTIVE: To evaluate the effects of epidural dexamethasone on maternal temperature and serum cytokine levels after labor epidural analgesia. METHODS: Sixty healthy term nulliparas in spontaneous labor were randomized to receive epidural analgesia alone using bupivacaine 0.125% and fentanyl 1 µg/mL (group I) or epidural analgesia combined with dexamethasone 0.2mg/mL (group II) (n=30 per group). Maternal tympanic temperature was measured before epidural analgesia and hourly thereafter until delivery. Maternal and cord venous blood were sampled for analysis of interleukin-6 (IL-6), tumor necrosis factor-α, and interleukin-10 levels. RESULTS: There was no difference in the incidence of intrapartum fever (38 °C or more) between the 2 groups (3/30 versus 1/30, P=0.612). The mean maternal temperature increased with time in group I, with the elevation reaching statistical significance at 4 hours post analgesia and at delivery compared with baseline (P=0.012 and P=0.043, respectively). A similar trend was observed with maternal serum IL-6 levels in group I. In group II, maternal temperature and IL-6 levels did not differ from baseline at any time point during labor. CONCLUSION: Epidural dexamethasone alleviates maternal temperature elevation after epidural analgesia. This effect can be attributed to the decrease in IL-6 levels.