Novel mTOR Inhibitor Enhances the Sensitivity of Hepatocellular Carcinoma Cells to Molecular Targeting Agents.

BACKGROUND: Although molecular-targeted agents are still the first choice for advanced hepatocellular carcinoma (HCC) treatment, the therapeutic efficacy of these agents is not satisfactory. Recently, the mammalian target of rapamycin (mTOR) is considered to be a promising molecular target that can enhance the sensitivity of HCC cells to antitumor therapy. However, the reported mTOR inhibitors have some shortcomings, and novel mTOR inhibitors need to be developed to enhance the antitumor effect of molecularly targeted agents on advanced HCC. METHODS: In this study, five small-molecular compounds that could serve as potential mTOR-specific inhibitors were identified by virtual screening. The activity of tert-butyl (4-(9-(2-(1,3-dioxolan-2-yl)ethyl)-6-morpholino-9H-purin-2-yl)phenyl)carbamate (compound 4) was measured by enzyme test and Western blot, and its antitumor effect on HCC was examined in nude mice subcutaneous tumor model. RESULTS: The results showed that 4 is the most effective one in inhibiting the activation of mTOR kinase (mTOR IC50 = 17.52±3.67 nmol/L) among the five lead compounds. Further research in this study indicated that treatment with 4 enhanced the sensitivity of HCC cells to the molecular-targeted agents, such as sorafenib, regorafenib, lenvatinib, anlotinib, and apatinib. In addition, this research indicated that mTOR was correlated with the poor prognosis in patients with advanced HCC who received sorafenib. CONCLUSION: Our study identified a new type of small-molecular inhibitors of mTOR and confirmed their ability to enhance the antitumor effect of molecular-targeted agents on advanced HCC.

Virtual Screening and Optimization of Novel mTOR Inhibitors for Radiosensitization of Hepatocellular Carcinoma.

BACKGROUND: Radiotherapy has an ameliorative effect on a wide variety of tumors, but hepatocellular carcinoma (HCC) is insensitive to this treatment. Overactivated mammalian target of rapamycin (mTOR) plays an important part in the resistance of HCC to radiotherapy; thus, mTOR inhibitors have potential as novel radiosensitizers to enhance the efficacy of radiotherapy for HCC. METHODS: A lead compound was found based on pharmacophore modeling and molecular docking, and optimized according to the differences between the ATP-binding pockets of mTOR and PI3K. The radiosensitizing effect of the optimized compound (2a) was confirmed by colony formation assays and DNA double-strand break assays in vitro. The discovery and preclinical characteristics of this compound are described. RESULTS: The key amino acid residues in mTOR were identified, and a precise virtual screening model was constructed. Compound 2a, with a 4,7-dihydro-[1,2,4]triazolo[1,5-a]pyrimidine scaffold, exhibited promising potency against mTOR (mTOR IC50=7.1 nmol/L (nM)) with 126-fold selectivity over PI3Kα. Moreover, 2a significantly enhanced the sensitivity of HCC to radiotherapy in vitro in a dose-dependent manner. CONCLUSION: A new class of selective mTOR inhibitors was developed and their radiosensitization effects were confirmed. This study also provides a basis for developing mTOR-specific inhibitors for use as radiosensitizers for HCC radiotherapy.

The activities of wortmannilactones against helminth electron transport chain enzymes, structure-activity relationships, and the effect on Trichinella spiralis infected mice.

Biotransformation of wortmannilactone F (3) using the marine-derived fungus DL1103 generated wortmannilactone M (1), a novel analog of wortmannilactone, which was a reduction product of 3 at the C-3 carbonyl group. The in vitro inhibitory activities of 10 wortmannilactones, including 1, against electron transport enzymes indicated that all the wortmannilactones were selective inhibitors of NADH-fumarate reductase and NADH-rhodoquinone reductase. The structure-activity relationship analysis showed that the relative configuration of C1" and C5", the positions of double bonds, the oxygen atoms in the dihydropyran moiety, and the keto-carbonyl group in the oxabicyclo-[2.2.1]-heptane moiety were important to the inhibitory activity of wortmannilactones. In vivo studies indicated that 3 significantly decreased the number and size of adult worms in Trichinella spiralis-infected mice in a dose-dependent manner. Notable changes in the cuticle and microvilli of T. spiralis were also observed. Our data provided useful information in the research and development of polyketides with dihydropyran and oxabicyclo [2.2.1] heptane moieties as antihelminthics.

Seasonal variation in circulating group 2 innate lymphoid cells in mugwort-allergic asthmatics during and outside pollen season.

BACKGROUND: Group 2 innate lymphoid cells (ILC2s) are a newly identified cell population with the potent capability to produce Th2-type cytokines in a non-antigen specific manner. Previous study demonstrated that enhanced circulating ILC2s in cat-allergic patient after experimental allergen challenge, whereas the effects of natural allergen exposure on peripheral ILC2s are still unclear. We therefore examined the variations in circulating ILC2s among asthmatic patients sensitized to different allergens in- and outside- pollen season. METHODS: 10 patients sensitized to mugwort, 10 patients sensitized to house dust mites (HDM) and 12 healthy controls were recruited into this study. Blood samples were collected from the patients in- and outside- pollens season, 2-3 months apart. ILC2s (Lin-CD127+ CRTH2+) were enumerated by flow cytometry, as well as intracellular IL-5 and IL-13 expression. The levels of IL-5 and IL-13 in supernatants of Lineage- and Lineage+ cells stimulated with IL-25 and/or IL-33 in the presence of IL-2 were measured using a Milliplex human cytokine array kit. RESULTS: An obvious seasonal increases in percentages of total and IL-13+ ILC2s were observed in patients with mugwort sensitization during natural pollen exposure, however, the percentages of peripheral ILC2s in HDM-allergic patients were not affected significantly. A positive correlation between FeNO and IL-13+ILC2s was found in patients sensitized to mugwort. A mixture of IL-33 and IL-25 induced a significant production of IL-13 and IL-5 from Lineage- cells of both mugwort-allergic and HDM-allergic asthmatics. Stimulation with IL-33 alone induced a significantly greater quantity of IL-13 by Lineage-cells from mugwort-allergic asthmatic compared with that from HDM-allergic asthmatics, whereas IL-25 induced a significantly greater amount of IL-5 by the Lineage-cells from mugwort-allergic asthmatic compared with that from HDM-allergic asthmatics. CONCLUSION: Within pollen season the frequencies and function profiles of circulating ILC2s among asthmatic children are altered dynamically, which may be closely related to the sensitized type of allergens.

[Distribution characteristics of serum specific IgE for inhaled allergens in children with different airway allergic diseases].

OBJECTIVE: To investigate the distribution characteristics of serum specific IgE (sIgE) for inhaled allergens in children with different airway allergic diseases. METHODS: Fluorescent enzyme-linked immunosorbent assay on the UniCAP250 system was performed to measure serum sIgE for 9 common inhaled allergens in 256 children aged 3-14 years with different airway allergic diseases. According to the clinical diagnosis, these children were divided into rhinitis group (37 children with allergic rhinitis), asthma group (82 children with bronchial asthma), and rhinitis-asthma group (137 children with allergic rhinitis complicated by bronchial asthma). The three groups were compared in terms of the detection rates of 9 inhaled allergens, sensitization level, and number of allergens. RESULTS: The detection rate of serum sIgE for inhaled allergens was 57.3% (47/82) in the asthma group, 86.5% (32/37) in the rhinitis group, and 82.5% (113/137) in the rhinitis-asthma group (P<0.05). The most common allergen in the asthma, rhinitis, and the rhinitis-asthma groups was mould fungi (32.9%, 54.1%, and 48.9% respectively), followed by dust mites (30.5%, 45.9%, and 46.0% respectively), pollen (26.8%, 35.1%, and 32.8% respectively), pets (12.2%, 27.0%, and 18.2% respectively), and cockroach (9.8%, 5.4%, and 5.8% respectively). The rhinitis group and the rhinitis-asthma group had a significantly higher detection rate of mould fungi (mx2) than the asthma group (P<0.0166). There were no significant differences in the sensitization level of 9 allergens and number of allergens between the three groups. CONCLUSIONS: In children with either bronchial asthma, allergic rhinitis, or bronchial asthma complicated by allergic rhinitis, the three most common inhaled allergens are mould fungi, dust mites, and pollens. Compared with bronchial asthma, allergic rhinitis may be more closely associated with sensitization by mould fungi. The three common airway allergic diseases have similar distribution characteristics of inhaled allergens.

Allergy march of Chinese children with infantile allergic symptoms: a prospective multi-center study.

BACKGROUND: Allergy march refers to progression of allergic diseases from infantile food allergy to the development of asthma and allergic rhinitis (AR). Evidence come mostly from studies in European countries. This study aimed to investigate allergy march in Chinese children with infantile food protein allergy (FPA) with a special focus on the effect of different formula interventions. METHODS: From 2008 to 2010, 153 infants diagnosed with FPA were recruited in five tertiary hospitals across China. They were randomly treated with amino-acid-based formula or soy-protein-based formula for a period of 3 months. Long-term follow-up was performed when they reached early school age, using questionnaires, physical examinations, and serum-specific immunoglobulin E. RESULTS: The overall follow-up rate was 73.20%. In patients who reached their early school years, the prevalence of physician-diagnosed AR and asthma were 43.75% and 23.21%, respectively. Only 40% of the subjects remained positive for food sensitizations upon follow-up. Twenty-six subjects receiving aeroallergen screening tests in infancy all proved negative, but upon follow-up, 65.57% were sensitized to aeroallergens (P=0.005). No significant difference between the effects of amino-acid-based formula and soy-protein-based formula on children's allergy march was observed. CONCLUSIONS: A high proportion (47.32%) of Chinese infants with early allergic symptoms developed respiratory allergies by their early school years. Most food-sensitized infants outgrew their condition several years later, but then aeroallergen sensitization often occurred. Amino-acid-based formula showed no advantages over soy protein-based formula with respect to arresting the allergy march.

TRAIP is involved in chromosome alignment and SAC regulation in mouse oocyte meiosis.

Recent whole-exome sequencing (WES) studies demonstrated that TRAIP is associated with primordial dwarfism. Although TRAIP was partially studied in mitosis, its function in oocyte meiosis remained unknown. In this study, we investigated the roles of TRAIP during mouse oocyte meiosis. TRAIP was stably expressed during oocytes meiosis and co-localized with CREST at the centromere region. Knockdown of TRAIP led to DNA damage, as revealed by the appearance of γH2AX. Although oocytes meiotic maturation was not affected, the proportions of misaligned chromosomes and aneuploidy were elevated after TRAIP knockdown, suggesting TRAIP is required for stable kinetochore-microtubule (K-MT) attachment. TRAIP knockdown decreased the accumulation of Mad2 on centromeres, potentially explaining why oocyte maturation was not affected following formation of DNA lesions. Securin, a protein which was prevent from precocious degradation by Mad2, was down-regulated after TRAIP knockdown. Inhibition of TRAIP by microinjection of antibody into pro-metaphase I (pro-MI) stage oocytes resulted in precocious first polar body (PB1) extrusion, and live-cell imaging clearly revealed misaligned chromosomes after TRAIP knockdown. Taken together, these data indicate that TRAIP plays important roles in oocyte meiosis regulation.

CD147 stimulates hepatoma cells escaping from immune surveillance of T cells by interaction with Cyclophilin A.

T cells play an important role in tumor immune surveillance. CD147 is a member of immunoglobulin superfamily present on the surface of many tumor cells and mediates malignant cell behaviors. Cyclophilin A (CypA) is an intracellular protein promoting inflammation when released from cells. CypA is a natural ligand for CD147. In this study, CD147 specific short hairpin RNAs (shRNA) were transfected into murine hepatocellular carcinoma Hepa1-6 cells to assess the effects of CD147 on hepatoma cells escaping from immune surveillance of T cells. We found extracellular CypA stimulated cell proliferation through CD147 by activating ERK1/2 signaling pathway. Downregulation of CD147 expression on Hepa1-6 cells significantly suppressed tumor progression in vivo, and decreased cell viability when co-cultured with T cells in vitro. Importantly, knockdown of CD147 on Hepa1-6 cells resulted in significantly increased T cells chemotaxis induced by CypA both in vivo and in vitro. These findings provide novel mechanisms how tumor cells escaping from immune surveillance of T cells. We provide a potential therapy for hepatocellular carcinoma by targeting CD147 or CD147-CypA interactions.

[A comparision of the effects of subcutaneous and sublingual immunotherapy on immunological responses in children with asthma].

OBJECTIVE: To compare the difference in the effects of subcutaneous immunotherapy (SCIT) and sublingual immunotherapy (SLIT) on immunological responses in children with asthma. METHODS: A total of 86 children with asthma caused by dust mites were enrolled and divided into a SLIT group (n=29), a SCIT group (n=13), a group receiving complete SCIT course (complete SCIT group; n=14), and a group receiving conventional medication (control group, n=30). Peripheral blood mononuclear cells were isolated and stimulated with house dust mite extract for 48 hours in vitro, and the percentage of regulatory T cells (Treg%) in CD4+ T cells was measured by flow cytometry. Analysis of variance with repeated measures was applied to compare the changes in humoral immunological indices and therapeutic effects in the SCIT and SLIT groups before treatment and after 6 and 12 months of treatment. RESULTS: Before antigenic stimulation, Treg% in CD4(+) T cells in the SCIT group was significantly higher than that in the SLIT and control groups; after antigenic stimulation was given, Treg% in the four groups decreased significantly. After 6 and 12 months of immunotherapy, the SCIT group had significant changes in serum sIgE and sIgG4 levels, while the SLIT group only showed a significant change in serum sIgE level. CONCLUSIONS: Temporal difference exists in different immunotherapies to cause immunological responses in children with asthma, and immunological responses induced by SCIT may occur earlier.

[Related factors for asthmatic children's responses to long-term treatment].

OBJECTIVE: To study the changes in pulmonary function and fractional exhaled nitric oxide in exhaled breath (FeNO) in asthmatic children who have different responses to regular treatment. METHODS: A total of 52 asthmatic children who had a good compliance with regular stepped control treatment were selected as subjects. They were followed up every three months to evaluate the asthma control level, pulmonary ventilation function, and FeNO for 9 months. Besides, medications for asthma control were recorded. RESULTS: At three follow-up points (months 3, 6, and 9), the percentage of asthmatic children who used the first or the second level of control treatment in the stable group (with stable response to the treatment) was significantly higher than in the unstable group (with unstable response to the treatment) (P<0.05), while the percentage of asthmatic children who used the third level of control treatment in the stable group was significantly lower than in the unstable group (P<0.05). At the three follow-up points, the stable group had a significantly higher ratio of forced expiratory volume in 1 second to forced vital capacity (FEV1/FVC) than the unstable group (P<0.05); at the 3-month and 9-month follow-up points, the stable group had a significantly higher percentage of predicted maximum mid-expiratory flow (MMEF%) than the unstable group (P<0.05); at the initial evaluation and 3-month follow-up point, the stable group had a significantly higher FeNO than the unstable group (P<0.05). CONCLUSIONS: Continuously monitoring FEV1/FVC, MMEF% and FeNO is useful in the early evaluation of the responses to treatment in children with asthma.

[Quality control for maximal expiratory flow-volume curve as a pulmonary function test in school-age children].

OBJECTIVE: To assess the quality control for the maximal expiratory flow-volume (MEFV) curve in school-age children. METHODS: Eight hundred and sixty-two children who had two or more MEFV manoeuvres were classified into ≥6-year-old (n=379), ≥8-year-old (n=210), ≥10-year-old (n=64), and 12-17-year-old groups (n=109). The parameters of quality control and concordance with quality control criteria for MEFV were compared between the two groups. In addition, patients who were diagnosed with asthma were classified into two groups, one with normal pulmonary function (n=155) and the other with abnormal pulmonary function (n=62), based on the results of spirometry. Differences in the parameters of quality control for spirometry were compared between the two groups. RESULTS: Eight hundred and sixty-two children underwent 2 367 MEFV manoeuvres, 97.8% of which met the start of test criterion for backward extrapolated volume (VBE) of less than 0.15 L, with the highest concordance in the ≥6-year-old group and the lowest concordance in the 12-17-year-old group. Three hundred and eighty-one children (44.2%) met the end of test criterion for forced expiratory time (FET) and the concordance in children over 10 years of age was lower than that in children under 10 years of age (P<0.05). Differences in two best forced expiratory volume in first second (FEV1) and forced vital capacity (FVC) manoeuvres were within 150 mL in 91.9% and 84.8%, respectively, of the children. The parameters of quality control for spirometry were better for asthmatic children with abnormal pulmonary function compared with asthmatic children with normal pulmonary function (P<0.05). CONCLUSIONS: Concordance with the start of test criteria and the manoeuvre repeatability criteria is high, whereas the concordance with the end of test criteria is low. It is suggested that the concordance with the FET criteria should be improved.

Did prostate size affect the complication and outcome of plasmakinetic enucleation of the prostate?

OBJECTIVE: To evaluate surgical complications and outcomes based on prostate size in patients with benign prostatic hyperplasia (BPH) treated with plasmakinetic enucleation of the prostate (PKEP). METHODS: A retrospective review was conducted of PKEP performed between July 2008 and January 2013. According to the prostate size on preoperative transrectal ultrasonography measurement, patients were divided into three groups: group 1: <40 ml, group 2: 40-80 ml and group 3: >80 ml. Baseline, perioperative and postoperative data were obtained. RESULTS: There were significant differences among the three groups regarding the mean operative time (p < 0.001) and the mean resected tissue weight (p < 0.001). But enucleation efficiency (p < 0.001) in gm tissue per minute increased significantly as prostate size increased. Mean hemoglobin decrease (p > 0.05), mean postoperative irrigation time (p > 0.05), mean catheter time (p > 0.05) and mean hospital stay (p > 0.05) did not differ significantly among three groups. The three groups had a similar and significant postoperative improvement in International Prostate Symptom Score, quality of life, maximum uroflow rate and post-void residual urine volume independent of prostate size (p < 0.001), but no significant difference was found among three groups at the 12-month follow-up (p > 0.05). Perioperative and postoperative complications did not depend on prostate size (p > 0.05). CONCLUSIONS: Although patients with a larger BPH required significantly longer operation time in PKEP, prostate size did not affect perioperative and postoperative complications or micturition improvement.

Plasmakinetic enucleation of the prostate versus transvesical open prostatectomy for benign prostatic hyperplasia >80 mL: 12-month follow-up results of a randomized clinical trial.

OBJECTIVE: To prospectively evaluate perioperative results and 12-month follow-up after plasmakinetic enucleation of the prostate (PKEP) and transvesical open prostatectomy (OP) for benign prostatic hyperplasia (BPH) >80 mL. METHODS: A total of 83 patients with a prostate >80 mL were randomized to either PKEP or OP. Perioperative and postoperative outcome data were obtained during a 12-month follow-up. RESULTS: No statistical differences were observed in the preoperative data. Both groups resulted in a similar and significant postoperative improvement in International Prostate Symptom Score (IPSS), quality of life (QOL), maximum uroflow rate (Qmax), postvoid residual (PVR) urine volume and prostate specific antigen (PSA), but no significant difference was found between the groups at the 12-month follow-up. Compared to OP, operation time (111.2 ± 27.1 minutes vs 109.6 ± 28.2 minutes, P = .708) were not significantly different between the groups, but blood loss was significantly less (10.2 ± 4.5 g/l vs 15.1 ± 4.3 g/l, P <.001), and bladder irrigation (2.4 ± 1.0 days vs 4.3 ± 1.1 days, P <.001), catheterization time (3.3 ± 1.1 days vs 6.2 ± 1.3 days, P <.001), and hospital stay (5.4 ± 1.2 days vs 9.3 ± 1.1 days, P <.001) were significantly shorter in the PKEP group. Effects on erectile function were similar in both groups, but adverse events were less frequent in the PKEP group. CONCLUSION: PKEP can be performed safely and is an equally effective procedure for treatment of large BPH with OP, with minimal complications and faster postoperative recovery. The PKEP helps to reduce the morbidity associated with OP and may become the attractive alternative to OP for patients with large BPH.

[Value of fractional nitric oxide concentration in exhaled breath in assessing level of asthma control in children].

OBJECTIVE: To investigate the value of fractional nitric oxide concentration in exhaled breath (FeNO) in assessing the level of asthma control in children. METHODS: A total of 226 asthmatic children were divided into controlled asthma (n= 86), partially controlled asthma (n=63), and uncontrolled asthma groups (n=77). Ninety healthy children were enrolled as controls. FeNO was measured for both asthmatic and healthy children using the Swedish-designed NIOX system. RESULTS: The control group had an FeNO of 14±6 ppb, the controlled asthma group had an FeNO of 29±26 ppb, the partially controlled asthma group had an FeNO of 32±30 ppb, and the uncontrolled asthma group had an FeNO of 40±32 ppb. The three asthma groups showed significantly higher FeNO than the control group (P<0.05). The uncontrolled asthma group showed significantly higher FeNO than the controlled asthma group (P<0.05), but there were no significant differences in FeNO between the partially controlled and uncontrolled asthma groups and between the partially controlled and controlled asthma groups (P>0.05). CONCLUSIONS: Asthmatic children have significantly higher FeNO than healthy children, and FeNO is correlated with the level of asthma control.

[Experimental treatment using combined Fructus psoraleae and dihydroartemisinin in mouse cryptosporidiosis].

A combination of Fructus psoraleae (3.4 g/kg) and dihydroartemisinin (60 mg/(kg x d)) was used for 7 d in mice infected with Cryptosporidium parvum infection induced by oral administration of dexamethasone. The results indicated that number of Cryptosporidium parvum oocysts in feces, proportion of CD4+, CD3+ T lymphocytes in blood, serum IFN-gamma, and nitric oxide (NO) content in intestinal tissue were all higher than those of infected control group (P < 0.01). The pathogenic change in intestine of treated mice was lighter than that of infected control. It indicates that the combination of Fructus psoraleae and dihydroartemisinin is effective to mice cryptosporidiosis probably through upregulating serum IFN-gamma, proportion of CD4+ and CD3+ T cells and NO concentration in intestine.

[Immunological regulation and treatment of Brucea javanica and Fructus Psoraleae on rats with Pneumocystis carinii pneumonia].

OBJECTIVE: To study the immunological regulation and treatment of Brucea javanica and Fructus Psoraleae, traditional Chinese medicine, on rats with Pneumocystis carinii pneumonia (PCP). METHODS: Rats were injected subcutaneously by dexamethasone. When the rats got Pc infected, they were divided into two groups: rats in one group were treated with the mixture of Brucea javanica and Fructus Psoraleae and another group was used as infected control. Control with normal rats was also established. Observations were made on the number of cysts in lungs and the changes of CD4+ T cells, CD8+ T cells and TNF-alpha in the serum to demonstrate the immunological regulation and killing effect of the medicine on cysts in the infected rats. RESULTS: The body weight of the rats treated with Brucea javanica and Fructus Psoraleae increased considerably than that of immunosuppressed rats and the normal control. The damaged lung got improved and repaired, and a significant cyst reduction was shown in the treated group. The CD4+ T cells, CD8+ T cells and level of TNF-alpha in serum also increased in the treated group significantly. CONCLUSION: The mixture of Brucea javanica and fructus psoraleae plays an immunological regulation on rats with Pneumocystis carinii pneumonia and shows certain killing effect on the cysts.

[Electron microscopical observation on rats with Pneumocystis carinii pneumonia treated with Brucea javanica and Fructus Psoraleae].

Rats with Pneumocystis carinii pneumonia (PCP) were established by hypodermic injection of dexamethasone and treated by Brucea javanica combined with Fructus Psoraleae 2 ml (Brucea javanica 0.12 mg and Fructus Psoraleae 1.0 mg) per rat per day for 7 days or 14 days. The effect of cyst-killing and the pathological change were observed under transmission electron microscope. Lung damage was alleviated or repaired, and a significant reduction of cysts was shown in the treatment group. The results show that a combination of Brucea javanica and Fructus Psoraleae played a significant restraining and killing effect on cysts, and helped repair the impairment of pneumonia.