Characterization of AST-001 non-clinical pharmacokinetics: A novel selective AKR1C3-activated prodrug in mice, rats, and cynomolgus monkeys.

AST-001 is a chemically synthesized inactive nitrogen mustard prodrug that is selectively cleaved to a cytotoxic aziridine (AST-2660) via aldo-keto reductase family 1 member C3 (AKR1C3). The purpose of this study was to investigate the pharmacokinetics and tissue distribution of the prodrug, AST-001, and its active metabolite, AST-2660, in mice, rats, and monkeys. After single and once daily intravenous bolus doses of 1.5, 4.5, and 13.5 mg/kg AST-001 to Sprague-Dawley rats and once daily 1 h intravenous infusions of 0.5, 1.5, and 4.5 mg/kg AST-001 to cynomolgus monkeys, AST-001 exhibited dose-dependent pharmacokinetics and reached peak plasma levels at the end of the infusion. No significant accumulation and gender differences were observed after 7 days of repeated dosing. In rats, the half-life of AST-001 was dose independent and ranged from 4.89 to 5.75 h. In cynomolgus monkeys, the half-life of AST-001 was from 1.66 to 5.56 h and increased with dose. In tissue distribution studies conducted in Sprague-Dawley rats and in liver cancer PDX models in female athymic nude mice implanted with LI6643 or LI6280 HepG2-GFP tumor fragments, AST-001 was extensively distributed to selected tissues. Following a single intravenous dose, AST-001 was not excreted primarily as the prodrug, AST-001 or the metabolite AST-2660 in the urine, feces, and bile. A comprehensive analysis of the preclinical data and inter-species allometric scaling were used to estimate the pharmacokinetic parameters of AST-001 in humans and led to the recommendation of a starting dose of 5 mg/m2 in the first-in-human dose escalation study.

Therapeutic effects and modulatory mechanism of Alpiniae oxyphyllae Fructus in chronic intermittent hypoxia induced enuresis in rats.

OBJECTIVE: The objective of this study was to explore the effect of Alpiniae oxyphyllae Fructus (AOF) on a rat model of chronic intermittent hypoxia (CIH)-induced enuresis. Findings of this study may help identify therapeutic targets in children with nocturnal enuresis (NE). METHODS: Female rats were randomly divided into a control group (saline gavage, 4 weeks of normal air), CIH group (saline gavage, 4 weeks of CIH), and AOF group (AOF gavage, 4 weeks of CIH). The variables measured in this study included water intake, urine output, bladder leak point pressure (BLPP), malondialdehyde (MDA) levels, and superoxide dismutase (SOD) activity. The expression levels of the purinergic P2X3 receptor, muscarinic M3 receptor, and ß3-adrenergic receptor (ß3-AR) in the bladder were also measured. The bladder was subjected to haematoxylin and eosin (HE) and Weigert staining, and histological changes were observed under a light microscope to evaluate the morphological changes in the bladder in each group. RESULTS: Compared with the control group, urine output was increased, and the BLPP was decreased in the CIH group, but AOF administration decreased urine output and increased BLPP. In addition, the serum MDA level increased and the SOD activity decreased in the CIH group compared with the control group. Administration of AOF decreased the MDA level and increased the SOD activity. Additionally, compared with the control group, HE and Weigert staining in the CIH group showed that the bladder detrusor muscle bundles were disordered and loose, some muscle bundles were broken, the content of collagen fibres in the gap was reduced, and the gap was significantly widened. However, following the administration of AOF, the bladder detrusor muscle bundles were neatly arranged, and the content of collagen fibres in the gap was increased. Furthermore, compared with the control group, the purinergic P2X3 receptor and muscarinic M3 receptor were expressed at higher levels, and ß3-AR was expressed at lower levels in the CIH group, but AOF administration decreased the expression of the purinergic P2X3 receptor and muscarinic M3 receptor and increased the expression of the ß3-AR. CONCLUSIONS: AOF improves enuresis by inhibiting oxidative stress and regulating the expression of the purinergic P2X3 receptor, muscarinic M3 receptor, and ß3 adrenergic receptor.

Activating adenosine A1 receptor accelerates PC12 cell injury via ADORA1/PKC/KATP pathway after intermittent hypoxia exposure.

Obstructive sleep apnea hypopnea syndrome (OSAHS) is associated with the neurocognitive deficits as a result of the neuronal cell injury. Previous studies have shown that adenosine A1 receptor (ADORA1) played an important role against hypoxia exposure, such as controlling the metabolic recovery in rat hippocampal slices and increasing the resistance in the combined effects of hypoxia and hypercapnia. However, little is known about whether ADORA1 takes part in the course of neuronal cell injury after intermittent hypoxia exposure which was the main pathological characteristic of OSAHS. The present study is performed to explore the underlying mechanism of neuronal cell injury which was induced by intermittent hypoxia exposure in PC12 cells. In our research, we find that the stimulation of the ADORA1 by CCPA accelerated the injury of PC12 cells as well as upregulated the expression of PKC, inwardly rectifying potassium channel 6.2(Kir6.2) and sulfonylurea receptor 1(SUR1) while inhibition of the ADORA1 by DPCPX alleviated the injury of PC12 cells as well as downregulated the expression of PKC, Kir6.2, and SUR1. Moreover, inhibition of the PKC by CHE, also mitigated the injury of PC12 cells, suppressed the Kir6.2 and SUR1 expressions induced by PKC. Taken together, our findings indicate that ADORA1 accelerated PC12 cells injury after intermittent hypoxia exposure via ADORA1/PKC/KATP signaling pathway.

Chronic intermittent hypoxia exposure induces kidney injury in growing rats.

OBJECTIVE: The objectives of this paper are to examine the effect of chronic intermittent hypoxia (CIH) on the morphological changes in the kidney of growing rats and to explore the mechanisms underlying the CIH-induced renal damage. METHODS: Forty Sprague-Dawley rats were randomly divided into two groups: 2 and 4 weeks CIH groups (2IH, 4IH), and in the control group 2 and 4 weeks air-stimulated groups (2C, 4C), with 10 rats in each group. Pathological changes of renal tissue were observed by HE staining, PAS staining, and Masson staining. Real-time PCR method was used to detect the mRNA expression of HIF-1α, CuZnSOD/ZnSOD, and MnSOD in renal tissue. RESULTS: (1) Intermittent hypoxia (IH) caused morphological damage in the kidney. Hypertrophy of epithelial cells in the kidney tubules and dilation in the glomeruli were observed under light microscope in HE and PAS stain, especially in 4IH group. Masson staining showed no significant fibrotic response in the IH groups. (2) Compared with the corresponding control groups, the levels of serum SOD were significantly lower in CIH groups, and especially in 4IH group. The mRNA expression of Cu/ZnSOD and MnSOD in CIH groups decreased significantly as compared to control groups. The mRNA levels of HIF-1α in the kidney were significantly higher in CIH groups than those in the corresponding control groups. CONCLUSION: Oxidative stress played a critical role in renal damage by up-regulating HIF-1α transcription and down-regulating Cu/ZnSOD and MnSOD transcription after chronic intermittent hypoxia exposure in growing rats.

Obesity in children with different risk factors for obstructive sleep apnea: a community-based study.

This study investigated the association between obesity and obstructive sleep apnea (OSA) in preschool and school-age children. Parents of obese and randomly chosen normal weight children completed a questionnaire on sleep-related symptoms, demography, family, and medical history. All subjects were invited to undergo polysomnography (PSG). OSA cases were defined as obstructive apnea hypopnea index (OAHI) ≥1. A total of 5930 children were studied with 9.5% obese (11.9% boys/6.1% girls), 205/2680 preschool and 360/3250 school children. There were 1030 children (535 obese/495 normal weight) who underwent PSG. OSA was higher in obese children and obese school children had higher OAHI, arousal index, and shorter total sleep time. However, there was no positive correlation between OSA and body mass index (BMI). The main risk factors for OSA in preschool children were adenotonsillar hypertrophy and recurrent respiratory tract infection. The main cause for OSA in school children was a history of parental snoring and obesity. Mallampati scores and sleep-related symptoms were found to be associated with OSA in both preschool and school children. CONCLUSION: We demonstrated differential risk factors for OSA in obese children, which suggest that a different mechanism may be involved in OSA development in preschool and school-age children. WHAT IS KNOWN: Various risk factors have been reported in obese children with OSA owing to the different age and different study design. Obese children have a higher prevalence and severity of obstructive sleep apnea (OSA). OSA risk factors in obese children are affected by different ages and study designs. WHAT IS NEW: A differential prevalence and risk factors for obese preschool and school-age children with OSA has been demonstrated.

Lipoxin A4 activates alveolar epithelial sodium channel gamma via the microRNA-21/PTEN/AKT pathway in lipopolysaccharide-induced inflammatory lung injury.

Lipoxin A4 (LXA4), as an endogenously produced lipid mediator, promotes the resolution of inflammation. Previously, we demonstrated that LXA4 stimulated alveolar fluid clearance through alveolar epithelial sodium channel gamma (ENaC-γ). In this study, we sought to investigate the mechanisms of LXA4 in modulation of ENaC-γ in lipopolysaccharide (LPS)-induced inflammatory lung injury. miR-21 was upregulated during an LPS challenge and downregulated by LXA4 administration in vivo and in vitro. Serum miR-21 concentration was also elevated in acute respiratory distress syndrome patients as compared with healthy volunteers. LPS increased miR-21 expression by activation of activator protein 1 (AP-1). In A549 cells, miR-21 upregulated phosphorylation of AKT activation via inhibition of phosphatase and tensin homolog (PTEN), and therefore reduced the expression of ENaC-γ. In contrast, LXA4 reversed LPS-inhibited ENaC-γ expression through inhibition of AP-1 and activation of PTEN. In addition, an miR-21 inhibitor mimicked the effects of LXA4; overexpression of miR-21 abolished the protective effects of LXA4. Finally, both AKT and ERK inhibitors (LY294002 and UO126) blocked effects of LPS on the depression of ENaC-γ. However, LXA4 only inhibited LPS-induced phosphorylation of AKT. In summary, LXA4 activates ENaC-γ in part via the miR-21/PTEN/AKT signaling pathway.

The novel HLA-B*48:01:13 allele, identified by Sanger dideoxy nucleotide sequencing in a Chinese individual.

HLA-B*48:01:13 differs from HLA-B*48:01:01:01 by one nucleotide in exon 5.

The prevalence and associated risk factors of sleep disorder-related symptoms in pregnant women in China.

PURPOSE: The sleep disorder in pregnant women remains unfamiliar to perinatal care providers, resulting in lack of appropriate care. This study was designed to investigate the prevalence of sleep disorder-related symptoms in pregnant women and to identify the associated risk factors. METHODS: Married pregnant women were enrolled from their first trimester and followed up until delivery. Nonpregnant married healthy women were selected as controls. A survey questionnaire was administered to each of them. RESULTS: We successfully performed a survey to 1,993 pregnant women and 598 nonpregnant women. The overall prevalence of sleep disorder-related symptoms in pregnant women was significantly higher than the controls (56.1 vs. 29.9 %, P < 0.05). There was higher prevalence of snoring (30.2 %), observed sleep apnea (1.1 %), mouth breathing (23.7 %), nocturnal arousal (46.5 %), insomnia (35.1 %), and daytime sleepiness (52.6 %) in pregnant women. There were no significant differences of the prevalence of bruxism (7.0 vs. 6.7 %), sleep talking (8.1 vs. 7.2 %), and sleep walking (0.4 vs. 0.2 %) between the two groups (P > 0.05). Nocturnal sleep time (8.0 ± 1.3 h) was less in the third trimester compared with the nonpregnant women (8.2 ± 1.1 h) (P < 0.05). Smoking (OR = 3.39), drinking (OR = 2.40), allergic rhinitis/asthma (OR = 1.71), an obvious difference in neck circumference (OR = 1.11), and waistline (OR = 1.07) changes between the first and third trimesters were the risk factors for sleep disorder-related problems. CONCLUSIONS: There is a high prevalence of sleep disorder-related symptoms in pregnant women. Our data may provide a baseline for prevention and treatment of sleep disturbances in pregnant women.

The novel HLA-A*02:07:23 allele, identified by Sanger dideoxy nucleotide sequencing in a Chinese individual.

HLA-A*02:07:23 differs from HLA-A*02:07:01:01 by one nucleotide in exon 2.

The novel HLA-B*51:381 allele, identified in a Chinese individual through Sanger dideoxy nucleotide sequencing.

HLA-B*51:381 differs from HLA-B*51:01:01:01 by one nucleotide in exon 5.

The novel HLA-B*40:536N allele, identified by Sanger dideoxy nucleotide sequencing in a Chinese individual.

HLA-B*40:536N differs from HLA-B*40:03:01:01 by one nucleotide in exon 3.

[Effect of endoplasmic reticulum stress in brain injury following chronic intermittent hypoxia in weanling rat].

OBJECTIVE: To explore the role of endoplasmic reticulum stress in brain injury following chronic intermittent hypoxia (CIH) in weanling rats. METHODS: A total of 48 male healthy Sprague-Dawley rats (3-4-week-old, 80-100 g) were randomly divided into 4 groups: 2-week-CIH (2IH) group, 4-week-CIH (4IH) group, 2-week-control (2C) group and 4-week-control (4C) group. The morphologic changes were observed by hematoxylin-eosin (HE) staining and cell apoptosis detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay. Then hippocampus and prefrontal cortices were collected for transcription and expression analysis of glucose regulated protein 78 (GRP78) by reverse transcription (RT)-PCR and Western blotting respectively. And the expressions of Caspase-12 mRNA and Caspase-12 protein in prefrontal cortex were analyzed by RT-PCR and immunohistochemistry. RESULTS: The neuronal apoptosis in hippocampus and prefrontal cortices in CIH exposed groups were more pronounced than those of the control groups (all P < 0.01), especially in the 4IH group (hippocampus: 8.78% ± 0.71% vs 3.26% ± 0.45%, cortices: 6.02% ± 0.32% vs 2.91% ± 0.29%). The expression levels of GRP78 mRNA (hippocampus: 0.424 ± 0.033 vs 0.326 ± 0.013 and 0.444 ± 0.028 vs 0.310 ± 0.015, cortices: 0.514 ± 0.038 vs 0.430 ± 0.017 and 0.524 ± 0.038 vs 0.439 ± 0.033) and GRP78 protein in hippocampus and prefrontal cortices (hippocampus: 0.221 ± 0.032 vs 0.178 ± 0.014 and 0.241 ± 0.019 vs 0.170 ± 0.013, cortices: 0.307 ± 0.012 vs 0.226 ± 0.022 and 0.311 ± 0.023 vs 0.225 ± 0.025), and the expression levels of Caspase-12 mRNA (0.396 ± 0.004 vs 0.323 ± 0.014, 0.417 ± 0.011 vs 0.313 ± 0.011) and Caspase-12 protein (0.334 ± 0.035 vs 0.197 ± 0.023, 0.368 ± 0.079 vs 0.215 ± 0.024) in prefrontal cortex in the IH groups all were more than those in the 2C and 4C groups (all P < 0.05). CONCLUSIONS: Chronic intermittent hypoxia can up-regulate the GRP78 transcription and expression in brain regions associated with learning and memory. This may induce the endoplasmic reticulum stress and activate the Caspase-12 mediated apoptosis signaling pathway. In the end, neuronal apoptosis occurs. All these factors may play an important role in the impairment of learning memory during the exposure of growing rats to chronic intermittent hypoxia.

Blockade of adenosine A2A receptor alleviates cognitive dysfunction after chronic exposure to intermittent hypoxia in mice.

Obstructive sleep apnea-hypopnea syndrome (OSAHS) is widely known for its multiple systems damage, especially neurocognitive deficits in children. Since their discovery, adenosine A2A receptors (A2ARs) have been considered as key elements in signaling pathways mediating neurodegenerative diseases such as Huntington's and Alzheimer's, as well as cognitive function regulation. Herein, we investigated A2AR role in cognitive impairment induced by chronic intermittent hypoxia (CIH). Mice were exposed to CIH 7 h every day for 4 weeks, and intraperitoneally injected with A2AR agonist CGS21680 or A2AR antagonist SCH58261 half an hour before IH exposure daily. The 8-arm radial arm maze was utilized to assess spatial memory after CIH exposures.To validate findings using pharmacology, the impact of intermittent hypoxia was investigated in A2AR knockout mice. CIH-induced memory dysfunction was manifested by increased error rates in the radial arm maze test. The behavioral changes were associated with hippocampal pathology, neuronal apoptosis, and synaptic plasticity impairment. The stimulation of adenosine A2AR exacerbated memory impairment with more serious neuropathological damage, attenuated long-term potentiation (LTP), syntaxin down-regulation, and increased BDNF protein. Moreover, apoptosis-promoting protein cleaved caspase-3 was upregulated while anti-apoptotic protein Bcl-2 was downregulated. Consistent with these findings, A2AR inhibition with SCH58261 and A2AR deletion exhibited the opposite result. Overall, these findings suggest that A2AR plays a critical role in CIH-induced impairment of learning and memory by accelerating hippocampal neuronal apoptosis and reducing synaptic plasticity. Blockade of adenosine A2A receptor alleviates cognitive dysfunction after chronic exposure to intermittent hypoxia in mice.

[Association of single nucleotide polymorphisms in the promoter region of the TLR9 gene with childhood atopic asthma].

OBJECTIVE: To investigate the distribution characteristics of the single nucleotide polymorphisms (SNPs) in the promoter region of the toll-like receptor 9 gene (TLR9) in Chinese Han children from Zhejiang province, and their associations with asthma susceptibility and phenotypes. METHODS: A case-control study was conducted. A total of 312 asthmatic children aged between 1.9 and 11.6 and 339 age matched healthy controls were enrolled in this study from April 2007 to November 2008. The -1486 C/T in rs187084 and -1237 C/T in rs5743836 loci of the TLR9 gene were genotyped by direct DNA sequencing of the PCR products. Serum levels of IFN gamma, IL-12 and IL-4 were detected by enzyme linked immunosorbent assay.Serum levels of total IgE were detected by chemiluminescence, and serum levels of antigen specific IgE antibodies were detected by fluoroenzymeimmunoassay. RESULTS: (1) The -1486 C/T polymorphism was identified in both groups. The genotype frequencies of TT, TC and CC at -1486 C/T were 41.0%, 44.3%, 14.7% in the healthy controls, and 38.8%, 48.4%, 12.8% in the asthmatic children. The -1237 C/T polymorphism was not detected in the population. (2) There were no statistically significant differences in the allele and genotype frequencies at the -1486 C/T locus between the two groups (P;>0.05). (3) Serum levels of IFN gamma and IL-4 differed significantly among the three genotypes at the -1486 C/T locus in asthmatic children (P<0.01). The CC genotype had the lowest levels of serum IFN gamma and the highest levels of serum IL-4 among the three genotypes. There were no significant differences in these cytokines among the healthy controls (P>0.05). No statistical differences of serum IL-12 were found among the three genotypes in the two groups (P>0.05). (4) There were no significant differences of total IgE (log-transformed) among the three genotypes in the asthmatic children (P>0.05). CONCLUSION: The -1237 C/T polymorphism of TLR9 gene was not detected in Chinese Han children in this study. The -1486 C/T polymorphism was associated with the levels of serum IFN gamma and IL-4 in children with asthma. However, there were no correlations between the -1486C/T polymorphism and serum IL-12 levels, total IgE levels or asthmatic susceptibility.

Perinatal exposure to di-(2-ethylhexyl) phthalate leads to restricted growth and delayed lung maturation in newborn rats.

BACKGROUND: Pregnant women and infants have significant exposures to the most commonly used plasticizer di-(2-ethylhexyl) phthalate (DEHP). OBJECTIVES: This study was designed to evaluate the effects of DEHP exposure on growth and lung maturation in rats and determine if DEHP regulation of 11beta-hydroxysteroid dehydrogenase type 1 gene (Hsd11b1) expression in the lung tissue plays a role in its effects on lung maturation. METHOD: Pregnant Sprague-Dawley rats were treated from gestational day 12 to postnatal day (PND) 21 with DEHP orally at dosages of 0, 10, 100 or 750 mg/kg/day, respectively (n=8 for each group). Two rat pups (one male and one female) from each litter were sacrificed at PND 1 and 21. Body weight was measured and the lung was processed for histology and calculation of lung interstitial tissue proportion as well as real-time PCR determination of the expressions of Hsd11b1, surfactant associated protein-A1 gene (Sftpa1) and B gene (Sftpb). RESULTS: The perinatal DEHP exposure led to a dose dependent intrauterine and postnatal growth restriction (P<0.001). High dose DEHP (750 mg/kg/day) exposure led to decreased gas-exchange space as evidenced by increased lung interstitial tissue proportion (P<0.001), but did not cause significant changes in Hsd11b1, Sftpa1 or Sftpb gene expression in the rat lung at PND 1. The DEHP-induced change in lung histology remained significant at PND 21 with improvement despite continual exposure to DEHP. CONCLUSION: Perinatal DEHP exposure leads to growth restriction and delayed lung maturation in newborn rats.

[Effect of nitric oxide derived from endothelial nitric oxide synthase on tumor angiogenesis].

BACKGROUND AND OBJECTIVE: Studies have shown that nitric oxide (NO) derived from endothelial nitric oxide synthase (eNOS) is expressed widely in tumor tissues and regulates tumor angiogenesis. However, the results are controversial. This study was to investigate the effect of NO on tumor angiogenesis and its mechanism. METHODS: C57BL/6 mice inoculated with Lewis lung cancer cells were randomly divided into three groups. Mice in the NO group were inoculated with lung cancer cells transfected with eNOS gene, mice in the L-NAME group with L-NAME, an eNOS antagonist, and mice in the control group with normal saline. Plasma concentration of NO and the number of endothelial progenitor cells (EPCs) in peripheral blood were detected . Tumor vessel density, CD133+ cells, and the expression of VEGF-VEGFR in tumor tissues were also measured. RESULTS: Four weeks after inoculation of Lewis cells, tumor volume was significantly larger in control group [ (3022 +/- 401) mm(3)] than in the L-NAME group [ (1204 +/-97) ) mm(3)] and in the eNOS group [(1824 +/- 239) mm(3)] (P<0.01). eNOS protein and NO production increased significantly in Lewis lung cancer cells transfected with eNOS gene. But the number of CD133-positive cells and vessel density in tumors were significantly lower in the eNOS group than in the control group [(48+/-19) / HPF vs. ( 103 +/- 27)/ HPF, (19+/- 7) HPF vs. (31 +/- 9) HPF, P<0.05]. The number of EPCs in peripheral blood was not statistically different between each group. The levels of NO in blood and tumor tissue significantly decreased after the treatment of L-NAME, while the tumor vessel density reduced to 12+/- 5/ HPF (P<0.01, vs. the control group; P<0.05, vs the eNOS transfected group). The number of EPCs in blood and that of CD133-positive cells in tumor tissue were significantly smaller in the L-NAME group than in the control group (P<0.05). CONCLUSION: No derived from eNOS inhibits angiogenesis and tumor growth, which may be due to its suppression on either the mobilization or homing of EPCs via VEGF binding to VEGFR.

[The pathological effects of snoring on pregnant women].

OBJECTIVE: To discuss the pathological effect of snoring on pregnant women in Wenzhou area. METHODS: The study was performed between January 2006 and February 2008, 601 women with pregnancies being in clinic or the ward were surveyed about snoring occur, measuring physiological and biochemical parameters in the 13th, 28th week of pregnancy and before delivery, recording the complication and pregnancy outcome. According to their pregnancy and snoring occur, they were divided into the first, the second and the third trimester snoring group and non-snoring group. RESULT: Compared with the non-snoring group, The BMI, abdominal perimeter, the neck circumference and systolic blood pressure in snoring group of every trimester increased significantly (P<0.05). There were no significant differences about the hip circumference of snoring group in the first trimester (P>0.05), but they increased significantly in the second and the third trimester (P<0.05). There were no significant differences about the diastolic blood pressure of snoring group in the first and the second trimester (P>0.05), but they increased significantly in the third trimester (P<0.05). There were no significant differences about the snoring group's BMI, abdominal perimeter, the neck circumference, the hip circumference and blood pressure between the groups of every trimester (P>0.05). Compared with the non-snoring group, the incidence of snoring group's gestational hypertension, premature birth and abdominal delivery increased significantly every trimester of pregnancy (P<0.05). There were no significant differences Between the snoring groups of every trimester (P>0.05). CONCLUSION: The snore makes pregnant women physiological characteristics changed, the incidence of gestational hypertension, premature delivery and abdominal delivery increased. So we should pay more attentions to them in their perinatal stage.

[Genotyping for Auxiliary Identification of Anti-C Alloantibody with Anti-f Autoantibody Mimicking Alloantibody].

OBJECTIVE: To identify the difficult antibody specificity of 1 PNH patient with blood transfusion history by genotyping. METHODS: RH typing of this patient was performed using gel card method, the antibody specificity was identified by panel cells, the RH-unrelated phenotype were excluded by genotyping method in difficult condition of serologic identification, furthmore different RH phenotype cells were used for adsorption-elution so as to re-examine the reactivity of antibodies in this patient's serum, and finally different RH phenotype cells were combined to exclude other unrelated antibodies. RESULTS: The RH phenotype presented as double population for C antigen, and positive agglutination for the other antigens. The results of RHD zygote, together with RHD and RHCE sequencing showed that the RHD genotype was homozygous RHD/RHD, and the c.122A＞G mutation did not found in RHCE gene, thus CW antigen was excluded; 48G lies in the 1st exon, the 5th exon showed nt676 G/C heterozygosity, the 2nd-4th, 6th-10th exons did not show mutations, however a new mutation was found in the 4th intron IVS4+29A＞C. It can be judged that the RH genotype was Dce/DcE, and its phenotype should be ccDEe. The anti-C alloantibody and rare anti-f autoantibody mimicking alloantibody in serum was determined by different RH phenotypic cell absorption-elution. Finally, cross-matched AB ccDEE blood units were selected for the patient and there was no adverse reactions of blood transfusion occurred. CONCLUSION: Genotyping can be auxiliarily applied to the identification of difficult antibodies in serum of a patient, thereby reducing the risk of blood transfusion.

Survival Benefit and Genetic Profile of Pemetrexed as Initial Chemotherapy in Selected Chinese Patients With Advanced Lung Adenocarcinoma.

Objective: This study investigated survival in selected Chinese patients with advanced lung adenocarcinoma who received initial chemotherapy with pemetrexed. We also explored the relationship between genetic biomarkers and pemetrexed efficacy. Methods: We retrospectively collected patients (n = 1,047) enrolled in the Chinese Patient Assistance Program from multiple centers who received pemetrexed alone or combined with platinum as initial chemotherapy and continued pemetrexed maintenance therapy for advanced lung adenocarcinoma from November 2014 to June 2017. The outcomes were duration of treatment (DOT) and overall survival (OS). Clinical features were analyzed for their influence on the treatment effect and prognosis. Next-generation sequencing (NGS) was performed to identify genetic biomarkers associated with the efficacy of pemetrexed. Results: The median DOT was 9.1 months (95% CI: 8.5-9.8), and the median OS was 26.2 months (95% CI: 24.2-28.1). OS was positively correlated with DOT (r = 0.403, P < 0.001). Multivariable analysis showed that smoking status and Eastern Cooperative Oncology Group (ECOG) performance status (PS) were independently associated with DOT; smoking status, ECOG PS, targeted therapy, and EGFR/ALK/ROS1 status were independently associated with OS. NGS in 22 patients with available samples showed genes with high mutation rates were: TP53 (54.5%), EGFR (50.0%), MYC (18.2%), and PIK3CA (13.6%). When grouped based on progression-free survival (PFS) reported in the PARAMOUNT study, the DOT > 6.9 months set was associated with PIK3CA, ALK, BRINP3, CDKN2A, CSMD3, EPHA3, KRAS, and RB1 mutations, while ERBB2 mutation was observed only in the DOT ≤ 6.9 months set. Conclusion: This study shows that initial chemotherapy with pemetrexed is an effective regimen for advanced lung adenocarcinoma in selected Chinese patients. There is no specific genetic profile predicting the benefit of pemetrexed found by NGS. Biomarkers predicting the efficacy of pemetrexed need further exploration.

[Study of CD4+ CD25+ regulatory T cells and expression of Foxp3 mRNA in bronchiolitis and glucocorticoid regulation].

OBJECTIVE: To explore the pathogenesis of respiratory syncytial viruses (RSV) bronchiolitis and its treatment with glucocorticoids. METHODS: The pediatric patients with RSV bronchiolitis were divided into an atopic group (n = 50) and a non-atopic group (n = 50) based on whether there were IgE elevation, eczema and dermatitis. Another 25 normal subjects were chosen as a control group. Divided into mild, medium and severe groups, they were finally randomly divided into hormone (dexamethasone) and non-hormone groups. The proportion of CD4+ CD25+ regulatory T cells (Treg) and the expression of Foxp3 mRNA were tested by flow cytometry and RT-PCR retrospectively. RESULTS: The proportion of CD4+ CD25+ Treg and the Foxp3 mRNA expression in the control, non-atopic, and atopic groups reached (10.5 +/- 1.6)% and 0.34 +/- 0.11, (8.8 +/- 2.2)% and 0.26 +/- 0.08, (7.6 +/- 1.8)% and 0.21 +/- 0.09, respectively. There were significant differences among these groups (all P < 0.05). The mild, medium and severe bronchiolitis groups reached (9.7 +/- 1.6)% and 0.28 +/- 0.08, (7.8 +/- 2.1)% and 0.24 +/- 0.06, (6.7 +/- 1.3)% and 0.20 +/- 0.07 respectively (all P < 0.05). The proportion of CD4+ CD25+ Treg and the expression of Foxp3 showed significantly negative correlations with severity (r = -0.62, -0.71, both P < 0.01). That is, they correlated with the severity of disease. The proportion of the CD4+ CD25+ Treg and the expression of Foxp3 mRNA of the hormone group were higher than those of the non-hormone group [(9.5 +/- 2.1)% and 0.33 +/- 0.10 vs (8.5 +/- 1.8)% and 0.27 +/- 0.12, P < 0.05 and < 0.01] respectively. CONCLUSION: CD4+ CD25+ Treg and Foxp3 mRNA are involved in the inflammation of bronchiolitis. And the levels of CD4+ CD25+ Treg and Foxp3 mRNA level is an objective indicator of the severity of RSV bronchiolitis. The effect of glucocorticoids upon RSV bronchiolitis may be in part due to the direct enhancement of production and functions of CD4+ CD25+ Treg and Foxp3 mRNA.