[Correlation between fractional exhaled nitric oxide and airway reversibility in children with IgE-mediated asthma].

OBJECTIVE: To study the correlation between fractional exhaled nitric oxide (FeNO) and airway reversibility in children with IgE-mediated asthma. METHODS: A total of 86 children, aged 6-14 years, who were initially diagnosed with acute attack of asthma from September 2016 to August 2018 were enrolled as subjects. According to the results of serum specific IgE, they were divided into IgE mediated group with 61 children and non-IgE mediated group with 25 children. According to the results of allergen detection, the IgE mediated group was further divided into four groups with one, two, three, and four or more positive allergens. FeNO and the parameters of pulmonary ventilation function before and after dilation test were measured. Pearson correlation analysis was used to evaluate the correlation of FeNO with each parameter of pulmonary function. RESULTS: The IgE mediated group had significantly higher FeNO than the non-IgE mediated group (P<0.05). FeNO increased with the increase in the number of positive serum specific allergens (P<0.05). In the IgE mediated group, FeNO level was positively correlated with the change in forced expiratory volume in the first second (FEV1) and the improvement in percentage of predicted FEV1 after medication in bronchial dilation test (r=0.655 and 0.473 respectively, P<0.05). The FeNO level was not correlated with FEV1, percentage of predicted FEV1, peak expiratory flow (PEF), change in PEF after medication, percentage of predicted PEF (PEF%pred), and improvement in PEF%pred after medication (P>0.05). In the non-IgE mediated group, FeNO level was not correlated with the above indicators (P>0.05). CONCLUSIONS: FeNO level is associated with the degree of allergies. For children with IgE-mediated asthma, FeNO is positively correlated with airway reversibility, which has a certain value in the diagnosis of asthma, disease evaluation, and understanding of airway reversibility. For children with non-IgE-mediated asthma, FeNO cannot be used to evaluate airway reversibility. These two types of asthma should be treated differently.

TLR3 Plays Significant Roles against HBV-Associated HCC.

Toll-like receptor 3 (TLR3) is a pattern-recognizing receptor that is involved in immune signaling and plays a crucial role in survival by being able to recognize various viral components including double-stranded RNA (dsRNA). The role of TLR3 in hepatocellular carcinoma (HCC) with hepatitis B virus (HBV) infections is not well understood. To investigate the ability of TLR3 in regulating HBV replication in HCC, 80 cases of human HCC were collected and their tissue microarray was made. In HCC cells, the expression and location of TLR3, hepatitis-associated virus, and interstitial immunoreactive cells were assayed with immunohistochemical staining. The apoptosis of tumor cells was also detected by TUNEL stain. Correlations between TLR3 expression and HBV infection, interstitial immunoreactive cells, and cells apoptosis in HCC were investigated. In addition, we explored whether TLR3 agonist dsRNA can inhibit HepG2.2.15 cells secreting HBV. We found that the cytoplasmic expression of TLR3 in HCC is positively related to HBsAg infection and HCC with cirrhosis and promotes interstitial immunoreactive cells infiltration and cancer cells apoptosis. In HepG2.2.15 cells, dsRNA inhibited the secretion of HBV and induced apoptosis. These results indicate that TLR3 signaling activity may be involved in immune responses against HBV in HCC.

[Association of ORMDL3 single nucleotide polymorphisms with lysophosphatidylcholine and apolipoprotein B levels in children with asthma].

OBJECTIVE: To study the association of ORMDL3 single nucleotide polymorphisms (SNP) with lysophosphatidylcholine (LysoPC) and apolipoprotein B (apoB) levels. METHODS: A total of 300 children diagnosed with bronchial asthma between January 2010 and December 2012 were selected for the asthma group, and 298 children diagnosed with upper respiratory tract infection in the same period were selected for the control group. Serum LysoPC and apoB levels were measured using enzyme-linked immunosorbent assay. Genotype analysis was performed using the TaqMan probe. RESULTS: LysoPC and apoB levels were significantly higher in the asthma group than in the control group (P<0.01). Among children with various genotypes of ORMDL3 gene at locus rs12603332, the asthma group had significantly higher LysoPC and apoB levels than the control group (P<0.01). Among the children with asthma, those with CC genotype had significantly higher LysoPC and apoB levels than those with CT and TT genotypes (P<0.01). CONCLUSIONS: LysoPC and apoB may intervene in the pathological process of asthma. Pro-inflammatory gene ORMDL3 SNP rs12603332 may be associated with high LysoPC and apoB levels, which leads to the occurrence of childhood asthma.

[Epidemiological survey of asthma among children aged 0-14 years in 2010 in urban Zhongshan, China].

OBJECTIVE: To investigate the prevalence, current treatment, and clinical characteristics of asthma, as well as the risk factors for this disease, among children aged 0-14 years in 2010 in urban Zhongshan, China. METHODS: A total of 10 336 children aged 0-14 years were selected from urban Zhongshan by cluster random sampling. The Third National Childhood Asthma Epidemiological Questionnaire 2010 was used to analyze the prevalence, current treatment, and clinical characteristics of childhood asthma, as well as the risk factors for this disease. RESULTS: Asthma was diagnosed in 179 cases (1.73%). The prevalence of asthma in male children was significantly higher than that in female children (2.25% vs 1.16%; P<0.01). Of the 179 patients, severe attacks were common in 104 cases (58.1%), 110 cases (61.5%) had slow onset, 102 cases (57.0%) had gradually relieved conditions, 61 cases (34.1%) suffered from asthma during seasonal transition, and 150 cases (83.8%) developed asthma due to respiratory tract infection. Among all asthmatic children, 71.5% had been treated with inhaled corticosteroids, and 71.5% had been treated with bronchodilator. The multivariate logistic regression analysis showed that a history of penicillin allergy, a family history of allergy, food allergy, eczema, allergic rhinitis, cesarean delivery, family mould, and perinatal passive smoking were independent risk factors for childhood asthma. CONCLUSIONS: The prevalence of childhood asthma in urban Zhongshan is on a high level, and is associated with gender. The treatment of asthma has been standardized, but still needs further improvement. The onset of asthma attack is influenced by various factors.

The expression of beclin-1, an autophagic gene, in hepatocellular carcinoma associated with clinical pathological and prognostic significance.

BACKGROUND: A role for autophagy, a conserved cellular response to stress, has recently been demonstrated in human cancers. Aberrant expression of Beclin-1, an important autophagic gene, has been reported in various human cancers. In the present study, we investigated the significance and relationship between Beclin-1 expression and cell proliferation, apoptosis, microvessel density (MVD) and clinical pathological changes or prognosis in human hepatocellular carcinoma (HCC). METHODS: A total of 103 primary HCC patients were involved in the study. Expression of Beclin-1, PCNA, NET-1, Bcl-2, Bax, Survivin in cancer cells and CD34 in stromal microvessels were evaluated immunohistochemically in tissue microarrays comprising 103 cases of HCC and 57 matched adjacent nontumor liver tissues. Correlations between clinicopathological characteristics and survival of HCC patients were explored. RESULTS: The positive rate of Beclin-1 was significantly lower in HCC tissues than adjacent tissues (72.8 vs. 89.5%, χ2 = 6.085, P = 0.015). In HCC, Beclin-1 expression was negatively correlated with cirrhosis background (r = -0.216, P = 0.029), Edmondson grade (r = -0.249, P = 0.011), vascular invasion (r = -0.246, P = 0.012), PCNA (r = -0.242, P = 0.014), NET-1 (r = -0.245, P = 0.013), anti-apoptosis protein Bcl-2 (r = -0.245, P = 0.013) and MVD (r = -0.292, P = 0.003), and positively correlated with pro-apoptosis protein Bax (r = 0.242, P = 0.014).Significant differences in the 5-year survival rates were seen among patients with Beclin-1 strong positive (++) (59.1%, 13/22), moderate positive (+) (28.3%, 15/53) and weak negative expression (-) (14.6%, 7/28) (P = 0.043). Significant differences were detected between Beclin-1 (++) and either Beclin-1 (+) (P = 0.036) or Beclin-1 (-) groups (P = 0.008), but no significant difference between Beclin-1 (+) and Beclin-1 (-) groups (P = 0.281) was observed.Survival rates were positively related to high Beclin-1 co-expressed with low PCNA, NET-1, or Bcl-2, lower MVD, and high Bax. Univariate and multivariate Cox regression analysis revealed that Beclin-1 expression was an independent indicator for overall survival in HCC patients (P < 0.05). CONCLUSIONS: The pathogenesis and progression of HCC are associated with reduced autophagy. The expression of Beclin-1 and Bax in HCC tissues may provide a synergistic effect towards inhibiting HCC proliferation, infiltration, metastasis and angiogenesis. Beclin-1 expression may be a valuable prognostic marker of HCC.

Application of the CellDetect® staining technique in diagnosis of human cervical cancer.

BACKGROUND: CellDetect® staining technique is a newly invented technique for cancer diagnosis. It easily distinguishes between normal and neoplastic cells including pre-cancer and squamous cell carcinoma (SCC) cells, based on staining color and morphology. In this study, application of CellDetect® staining technique was assessed in diagnosis of human cervical cancer as compared with hematoxylin and eosin (H&E) staining in conventional slides and Thinprep cytologic test (TCT) smears. METHODS: The conventional slides and TCT smears of 600 patients were stained and observed while comparing with H&E staining to assess sensitivity and specificity of CellDetect® staining technique in diagnosis of cervical cancer. Conventional smear slides (440 cases) were fixed in 95% ethanol or with CYTOFIX® Spray. TCT smears (160 cases) were processed based on manual. The paraffin sections from cervical intraepithelium neoplasia (CIN) 2-3 and SCC cases were prepared by biopsy. RESULTS: CellDetect® staining exhibited well cell morphology, simultaneously, showed dual color discrimination, the stain targeted cytoplasm in normal cells in green and dysplastic cells or neoplastic cells in purple/red. Both cervical cell smears or both fixation methods in conventional slides did not affect CellDetect® staining diagnosis, especially in tissue biopsies CellDetect® staining exhibited well epithelium layers to benefit the diagnosis of CIN grade. The sensitivity and specificity of CellDetect® staining technology in diagnosing CIN and SCC were 94.34% and 88.73%, respectively. CONCLUSIONS: CellDetect® staining technique provided a dual color discrimination and morphological analysis. It has the potential to become one of the most effective methods for cervical screening and early diagnosis.

Study of RNA Interference Targeting NET-1 Combination with Sorafenib for Hepatocellular Carcinoma Therapy In Vitro and In Vivo.

The aim of this study is to explore the inhibitory effects of RNA interference (RNAi) targeting NET-1 or combined with sorafenib on HCC in vitro and in vivo and the possible underlying mechanisms. The expressions of NET-1 mRNA and protein were detected by RT-QPCR and western blot. The ability of proliferation was determined by CCK-8 assay. Apoptosis was examined by flow cytometry (FCM). Abilities of migration and invasion were measured by scratch-wound assay and transwell assay. MHCC97H cells with stable transfection of NET-1shRNA were injected subcutaneously to prepare nude mice model of HCC and Caspase-3, Caspase-8, and Caspase-9 mRNAs of tumor tissues in different groups were examined. NET-1 mRNA and protein were reduced sharply in MHCC97H cells transfected with NET-1shRNA. The abilities of proliferation and migration were inhibited and apoptosis was promoted in either NET-1shRNA or sorafenib as compared with untreated cells in vitro and in vivo (P < 0.05). The mRNA levels of caspase-3, caspase-8, and caspase-9 of tumor tissues were reduced in different treatment groups compared with untreated group, particularly in combination group. (P < 0.05). The combination NET-1shRNA with sorafenib dramatically enhanced the effects of sorafenib antitumor ,which may involve in blocking ras signaling pathway and stimulating apoptotic pathways simultaneously.

A synthetic dsRNA, as a TLR3 pathwaysynergist, combined with sorafenib suppresses HCC in vitro and in vivo.

BACKGROUND: Recent studies have demonstrated that synthetic dsRNAs may produce therapeutic effects in a target-independent manner through stimulation of the toll-like receptor-3 (TLR3)/interferon pathway; as a result, angiogenesis and proliferation of tumor cells are inhibited. Thus, this pathway may become a potential target of dsRNA in tumor suppression. In this study, we evaluated the role of synthetic dsRNA as a TLR3 synergist and by combining with sorafenib in anti-hepatocellular carcinoma (HCC) in vitro and in vivo. METHODS: Four dsRNAs were designed and synthesized. One of them that was capable of activating TLR3 most effectively in human HCC cell line (HepG2.2.15) was selected as a TLR3 synergist (called BM-06). Subsequently, the expression of proteins relating to TLR3 signaling pathway, such as NF-κB, caspase 8 survivin, bcl-2 and PCNA affected by BM-06, sorafenib alone or in combination, was compared. The migration, proliferation and apoptosis of HepG2.2.15 cells were evaluated in presence of BM-06, sorafenib alone or in combination of both. The similar treatments were also applied in an SD rat primary HCC model. RESULTS: qRT-PCR data showed that the expression of TLR3 and NF-κB in HepG2.2.15 cells was enhanced. BM-06 was selected as a TLR3 synergist capable of activating the TLR3/interferon pathway most effective among 4 synthetic dsRNAs. The migration and proliferation were significantly inhibited in treated HepG2.2.15 cells with BM-06 or Sorafenib alone as compared with PBS-sham control (P<0.01). However, the role of combination BM-06 with Sorafenib was the most prominent. Tumor cell apoptotic rate was increased by BM-06 or combination when compared to PBS or poly(I:C) (P<0.05). Similarly, in orthotopic HCC SD rats, the effect of the combination was superior to either agent alone on the inhibition of tumor growth and induction of HCC cell apoptosis (P<0.05). CONCLUSIONS: dsRNA alone was capable of inhibiting the proliferation of HepG2.2.15 cells and tumor growth of orthotopic HCC SD rats, but the effect of combination of dsRNA with sorafenib was more prominent. These findings implicate the potential role of combined use of a dsRNA, a TLR3 synergist, and sorafenib in inhibition of HCC.

Inhibition of hepatocellular carcinoma growth and angiogenesis by dual silencing of NET-1 and VEGF.

Simultaneous silencing of multiple up-regulated genes is an attractive and viable strategy to treat many incurable diseases including cancer. Herein we used dual gene targeted siRNA (DGT siRNA) conjugate composed of NET-1 and VEGF siRNA sequences in the same backbone could inhibit growth and angiogenesis HCC. DGT siRNA showed a further down regulation on VEGF mRNA and protein levels compared with NET-1 siRNA or VEGF siRNA, but not on NET-1 expression. It also exhibited greater suppression on proliferation and trigger of apoptosis in HepG2 cells than NET-1 siRNA or VEGF siRNA; this could be explained by the significant down regulation of cyclin D1 and Bcl-2. A lower level of ANG2 mRNA and protein was detected in HUVEC cultured with supernatant of HepG2 cells treated with DGT siRNA than that of VEGF siRNA or NET-1 siRNA, resulting in much more inhibited angiogenesis of HUVEC. Tumor growth was inhibited and microvessel density dropped in the xenograft tumor models compared to the untreated controls. NET-1 and VEGF silencing play a key role in inhibiting hepatocellular cell proliferation, promoting apoptosis, and reducing angiogenesis. Simultaneous silencing of NET-1 and VEGF using DGT siRNA construct may provide an advantageous alternative in development of therapeutics for Hepatocellular carcinoma.

[Spectrum of pathogens for community-acquired pneumonia in children].

OBJECTIVE: To investigate the spectrum of pathogens for community-acquired pneumonia (CAP) in children, and to provide a basis for the diagnosis and treatment of CAP. METHODS: Respiratory secretions and venous blood samples were collected from 1560 children with CAP aged from one month to 9 years within 2 hours after admission, for detection of multiple pathogens. Respiratory virus antigens in nasopharyngeal swab specimens were detected by immunofluorescence. Sputum was used for bacterial culture. Levels of Mycoplasma pneumoniae (MP)-IgM and Chlamydia pneumoniae (CP)-IgM in venous blood were measured by enzyme-linked immunosorbent assay. RESULTS: A total of 579 strains of bacteria were isolated from all respiratory secretions, including 213 (36.8%) Gram-positive strains and 366 (63.2%) Gram-negative strains. The five most common strains were Haemophilus influenzae (7.50%), Streptococcus pneumoniae (6.73%), Staphylococcus aureus (6.35%), Moraxella catarrhalis (5.19%), and Escherichia coli (3.46%), wherein the beta-lactamase-producing strains accounted for 3.3% of all strains. The non-bacterial pathogens mainly included respiratory syncytial virus (12.88%), MP (7.88%), and CP (8.91%). Mixed infection of pathogens was serious, and the mixed infection of respiratory syncytial virus with Haemophilus influenzae infections were the most common. For most pathogens, the infection rate was higher in children aged under one year than in those aged over one year. CONCLUSIONS: Haemophilus influenzae, respiratory syncytial virus, MP and CP are the main pathogens for children with CAP. For most pathogens, the infection rate is higher in children aged under one year than in those aged over one year. Mixed infection rate of pathogens is high.

[Study of genetic susceptibility in 198 children with asthma].

OBJECTIVE: To analyze the frequency distribution of single nucleotide polymorphisms (SNPs) of four asthma-related gene loci (ACE I/D; ADRB2 Arg16Gly; TNF-α G-308A; MS4A2 Glu237Gly) in 198 asthmatic children, and to investigate its association with genetic susceptibility to childhood asthma and some clinical phenotypes of asthma. METHODS: Polymerase chain reaction product electrophoresis identification and real-time quantitative PCR detecting system were used to determine the frequency distributions of the SNPs of the four asthma-related gene loci in 198 asthmatic children and 110 healthy controls. The serum total IgE (TIgE) levels and blood eosinophil proportion (%EOS) of the asthmatic children were measured. Different genotypes at the four asthma-related gene loci were compared in terms of TIgE and %EOS. RESULTS: The genotype DD of angiotensin-converting enzyme (ACE) had a significantly higher frequency in the asthmatic children than in the healthy controls (χ2= 30.667, P<0.01), and the frequency of D allele was also significantly higher in the asthmatic children than in the healthy controls (χ2=7.151, P<0.01). No correlation was found between the polymorphism of each gene locus and serum TIgE level and %EOS (P>0.05). CONCLUSIONS: Genotype DD of ACE is related to genetic susceptibility to childhood asthma and may be the risk factor for childhood asthma.

[Comparison study on knee osteoarthritis in rabbits induced by different concentrations of papain].

OBJECTIVE: To compare the knee osteoarthritis (OA) models in rabbits by different concentrations of papain and provide data for exploring pathogenesis and treatments of this disease. METHODS: Sixty New Zealand white rabbits were randomly divided into four groups of 15 each and given injections into the right knee on days 1, 3 and 5 including intra-articular injections of 2%, 5% or 10% (w/v) papain and 0.03 mol/L L-cysteine at the dose of 0.1 ml/kg (experimental groups). The 0.9% NaCl (w/v) with a dose of 0.1 ml/kg were injected intra-articularly into the right knees of rabbits in the control group. The rabbits were sacrificed at 2, 4, 6 weeks respectively after the initiation of papain injection and these OA models were evaluated through recording the width of knee joint, performing the morphological observation and histological evaluation of articular cartilage and synovium. RESULTS: The degenerative changes were demonstrated in knee joints of rabbit in all experimental groups, such as thinner articular cartilage, fibrillation and destroyed cartilage matrix, and inflammation, proliferation, and degeneration of the synovial tissue. All these changes were much worse with increased concentration and prolonged observation time. CONCLUSION: Different severity of OA are established through intra-articular injections of 2%, 5% or 10% papain and 0.03 mol/L L-cysteine at the dose of 0.1 ml/kg. These models are of the characters of short period and a good reproducibility.

[Efficacy of specific sublingual immunotherapy with dermatophagoides farinae drops in the treatment of cough variant asthma in children].

OBJECTIVE: To study the efficacy and safety of specific sublingual immunotherapy with dermatophagoides farinae drops in the treatment of cough variant asthma in children. METHODS: A total of 106 children aged 4-14 years old with cough variant asthma and positive skin prick test responses to dermatophagoides farinae allergens were randomly divided into two groups: SLIT group (n=53), which received specific sublingual immunotherapy with dermatophagoides farinae drops as well as standardized treatment and conventional treatment group (n=53), which received standardized treatment alone. Improvement in cough/asthma symptom scores and the time taken for symptoms to improve were observed after treatment. Serum eosinophil (EOS) level and peak expiratory flow (PEF) were measured after treatment. The side effects were observed. RESULTS: Compared with the conventional treatment group, the SLIT group showed significant decrease in symptom scores and serum EOS level and significant increase in PEF (P<0.05). The time at which symptoms began to improve in the SLIT group was earlier than in the conventional treatment group (P<0.05). The effective rate in the SLIT group was significantly higher than in the conventional treatment group (85% vs 68%; P<0.05). Local reactions such as redness, swelling, and itching occurred in some children of the SLIT group but disappeared on the following day. CONCLUSIONS: Specific sublingual immunotherapy with dermatophagoides farinae drops is an effective and highly safe treatment for cough variant asthma in children.

The effect of NET-1 on the proliferation, migration and endocytosis of the SMMC-7721 HCC cell line.

To explore the effect of NET-1 on the proliferation, migration and endocytosis in the hepatocellular carcinoma (HCC) cell line SMMC-7721, we constructed the pU6H1-NET-1-siRNA (NET-1siRNA) and pcDNA3.1/myc-NET-1 (myc-NET-1) vectors and transfected them into SMMC-7721 cells. The expression levels of NET-1 mRNA and protein were detected using real-time quantitative RT-QPCR and western blotting. The proliferation rates of SMMC-7721 cells were determined by CCK-8 assays, flow cytometry (FCM) and immunohistochemistry staining. The migration in two or three dimensional space of SMMC-7721 cells were determined by wound-healing assay and in vitro invasion assay. The extent of endocytosis in SMMC-7721 cells was estimated by observing the amount of transferrin (Tfn) absorbed with capture ELISA assays, and Tfn endocytosis was observed under confocal immunofluorescence microscopy. The results show that: i) after transfecting NET-1 siRNA, the expression of NET-1 mRNA and protein in SMMC-7721 cells decreased significantly, the growth of cells was suppressed, which induced cell cycle arrest, the proliferation rates were dramatically reduced and the expression of Ki67 declined, and migration and endocytosis in cells were inhibited, compared with untreated cells (every P<0.01); ii) Following transfection with myc-NET-1, the expression of NET-1 mRNA and protein in SMMC-7721 cells increased, and both the proliferation of cells and the cell cycle were promoted (P<0.01, respectively). However, the abilities of cell migration and endocytosis were not affected compared with untreated cells. These data suggest that: i) the NET-1 gene may play an important role in proliferation, migration and endocytosis of cells; ii) siRNA technology may efficiently suppress the expression and function of NET-1 in HCC, suggesting that NET-1 may be a therapeutic target for HCC.

[Etiology and risk factors of infantile wheezing].

OBJECTIVE: To study the etiology and risk factors of infantile wheezing. METHODS: The clinical data of 180 infants with wheezing were retrospectively studied. The risk factors for wheezing attacks were investigated by logistic regression analysis. RESULTS: Viral infection (33.3%) was the most common cause for wheezing attacks, followed by asthma (19.4%), parental smoking and special environments (15.6%), gastroesophageal reflux disease (12.8%), premature delivery (7.8%), Mycoplasma infection (6.7%), and bronchopulmonary dysplasia (4.4%). The multivariate logistic regression analysis showed 7 factors that significantly correlated with wheezing attacks: allergic history of parents, sensitization to alimentary or inspiratory allergens, viral or Mycoplasma infection, premature delivery and special environments. CONCLUSIONS: The commonest cause of infantile wheezing is viral infection, followed by asthma. Genetic factors, individual atopic constitution and environmental factors play important roles in wheezing attacks.

Clinicopathological significance of expression of Tspan-1, Jab1 and p27 in human hepatocellular carcinoma.

The aim of this study was to investigate the expression of Tspan-1, Jab1 and p27 in human hepatocellular carcinoma (HCC) and their clinicopathological significance. The expression of Tspan-1, Jab1 and p27 was detected in HCC tissues, the tissues around cancer (76 cases), and the normal tissues around the liver hemangiomas (10 cases). The overexpression of Tspan-1 and Jab1 was found in HCC tissues, positively correlated with clinical stage and negatively correlated with survival rate. The expression of p27 was found inversely linked to which of Tspan-1 and Jab1. In conclusion, the expression of Tspan-1, Jab1 and p27 is significantly associated with development of HCC. Overexpression of Tspan-1 and Jab1 suggests poor prognosis but overexpression of p27 may expect good prognosis for patients with HCC.

[Packaging and concentrating of high titer of retrovirus containing IL-4RA].

OBJECTIVE: To construct a recombination retroviral expression vector pLNC-IL-4RA with high efficiency transfection and carrying a screening label. METHODS: IL-4RA was inserted into retroviral vector pLNC-Laz to get recombination retroviral expression vector pLNC-IL-4RA and then transfected into packaging cell line PA317 by liposome transfection. The transfected PA317 cells were obtained and amplified by G418 pressure screening. The cell culture supernatants containing viruses were harvested and the viral titer was determined by NIH3T3 cells infection. RESULTS: The G418 resistant clones were titrated and checked for the presence of replication virus. The results showed that the highest titer of viral supernatant was 1 x 10(4) CFU/mL. Genome DNA isolated from the cell clone of the highest titer showed the function gene, IL-4RA cDNA, had integrated into the genome of host cells verified by PCR. CONCLUSIONS: The recombination retroviral vector pLNC-IL-4RA encoding IL-4RA after packaging PA317 cells have higher viral titer. This provides a basis for gene treatment of asthma.

TSPAN1 protein expression: a significant prognostic indicator for patients with colorectal adenocarcinoma.

AIM: To determine if TSPAN1 overexpression is associated with clinicopathological and prognostic factors in human colorectal adenocarcinoma. METHODS: Total RNA was extracted in 20 human adenocarcinoma tissues for TSPAN1 mRNA assay by RT-PCR. Eighty-eight specimens of human colorectal adenocarcinoma were surgically removed. TSPAN1 protein levels in cancer tissues were determined by immunohistochemistry using a polyclonal antibody against self-prepared TSPAN1. The correlation between TSPAN1 expression and the clinicopathological factors and the overall survival rate was analyzed by univariate and multivariate assay. RESULTS: TSPAN1 mRNA was detected in 90.0% (18/20) of cancerous tissues. The light density of TSPAN1 mRNA expression levels was 0.89 +/- 0.30 in adenocarcinoma by gel-image system. TSPAN1 protein expression was detected in 78.41% (69/88) and weakly expressed in 40% normal colorectal tissues. There were significant differences between colorectal adenocarcinoma and normal control epithelium (P < 0.05). TSPAN1 protein expression in colorectal cancerous tissue was significantly correlated with the histological grade, cell expression PCNA, lymph nodal metastasis and TNM staging of the disease. Patients with TSPAN1 protein overexpression had a significantly shorter survival period than that in patients with TSPAN1 protein negative or weak expression, respectively (P < 0.05). Furthermore, by multivariate analysis, TSPAN1 protein expression demonstrated an independent prognostic factor for human colorectal cancers (P < 0.05, relative risk 0.755; 95% confidence interval 0.302-1.208). CONCLUSION: The expression of TSPAN1 gene is increased in colorectal carcinoma, suggesting that TSPAN1 might serve as an independent prognostic factor for the colorectal adenocarcinoma patients.