[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.

[Serum levels of IL-12, TGFbeta1 and IgE in children with asthma].

OBJECTIVE: This study examined the changes of serum levels of interleukin 12 ( IL-12), transforming growth factor beta 1 (TGFbeta 1) and immunoglobulin E ( IgE) in children with asthma as well as the correlation of IL-12 and TGFbeta 1 with IgE in order to investigate their roles in asthma. METHODS: Serum levels of IL-12 , TGFbeta 1 and IgE were detected using ELISA in 85 asthmatic children at the acute and the remission stages. Thirty healthy children served as control group. RESULTS: Compared with the control group, serum IL-12 and TGFbeta 1 levels were significantly lower and serum IgE levels were significantly higher in the asthmatic group through the acute to the remission stages. Serum IL-12 and TGFbeta 1 levels (40.42+/-15.26 ng/L and 65.41+/-22.38 pg/mL) significantly increased in the asthmatic group at the remission stage compared with those at the acute stage (28.42+/-10.73 ng/L and 40.25+/-11.73 pg/mL) (P<0.01), but remained lower levels than those in the control group (67.42+/-20.58 ng/L and 178.54+/-90.56 pg/mL) (P<0.01). The asthmatic patients at the remission stage showed significantly decreased serum IgE levels (145.67 +/-51.25 IU/mL) compared with those at the acute stage (280.35 +/-80.54 IU/mL) (P<0.01), but the IgE level in the remission stage was obviously higher than in the control group (53.61+/-13.32 IU/mL) (P<0.01). Serum IL-12 and TGFbeta 1 levels were negatively correlated with serum IgE level in asthmatic children. CONCLUSIONS: There might be an imbalance in serum IL-12, TGFbeta 1 and IgE levels in asthmatic children. IL-12, TGFbeta 1 and IgE may play an important role in the pathogenesis of asthma. They may be useful in the diagnosis and severity evaluation of asthma.

[Serum levels of IgG subclasses and vitamin A in children with recurrent respiratory tract infection].

OBJECTIVE: The development of recurrent respiratory tract infection (RRTI) is related to vitamin A deficiency and immune function abnormality in children. This study examined serum levels of IgG subclasses and vitamin A in children with recurrent respiratory tract infection. METHODS: Serum IgG subclasses levels (IgG1, IgG2, IgG3, IgG4) were detected using ELISA and serum vitamin A levels were detected using high performance liquid chromatography-Miller method in 80 children with RRTI (ranged from 2-10 years old). The values were compared with those from 80 aged-matched healthy children. RESULTS: Serum levels of IgG2 (1.52 +/- 0.18 g/L) and IgG4 (0.22 +/- 0.12 g/L) in children with RRTI were significantly lower than controls (IgG2: 2.23 +/- 0.08 g/L; IgG4: 0.28 +/- 0.01 g/L) (P < 0.05). Serum vitamin A levels in children with RRTI were also significantly lower than controls (1.16 +/- 0.22 micromol/L vs 1.56 +/- 0.12 micromol/L; P < 0.05). IgG2 and IgG4 deficiency (27%) was the most common in 22 RRTI children with vitamin A deficiency. CONCLUSIONS: Serum levels of IgG subclasses, IgG2 and IgG4, and vitamin A decrease in children with RRTI. There might be some relationship between the decreased IgG2 and IgG4 levels and vitamin A deficiency.

[Effects of recombinant human insulin-like growth factor-1 on the expression of Clara cell secretory protein in lung of hyperoxia-exposed newborn rats].

OBJECTIVE: The development of neonatology and the availability of pulmonary surfactant have been helpful in effective reduction of the mortality of very low birth weight infants at the expense of an increasing number of survivors with bronchopulmonary dysplasia (BPD) caused by lung immaturity. BPD is a common syndrome in newborns, especially in preterm infants, when treated with hyperoxia and mechanical ventilation. Unfortunately, there have been no effective measure for the prevention and treatment of BPD. The purpose of this study was to investigate the influence of recombinant human insulin-like growth factor-1 (rh-IGF-1) on cell apoptosis and Clara cell secretory protein (CCSP) expression during the lung injury induced by hyperoxia, so as to assess its effect on the inflammatory lung injury and its developmental repair. METHODS: Eighty full term neonatal Wistar rats under the same condition were divided randomly into four groups on the second day after birth. Group I was air control, group II was exposed to hyperoxia, group III air + rh-IGF-1, and group IV was treated with hyperoxia + rh-IGF-1. The pups in the control group were kept in room air, while pups in hyperoxia group were kept in a Plexiglas chamber and exposed to over 85% oxygen. Pups in group III were under the same raising condition except for exposure to room air and treated with intraperitoneal injection of rh-IGF-1 (1 microg/Kg) everyday from the third day. Pups in group IV were treated with intraperitoneal injection of rh-IGF-1 (1 microg/Kg) everyday from the third day of exposure to hyperoxia. Lung tissue sections of the neonatal rats were stained with hematoxylin and eosin (HE) after 7 d of hyperoxia exposure, expression of CCSP was examined by immunohistochemical method, and apoptotic cell index of lung tissue was calculated by using TUNEL method. RESULTS: It was observed from immunohistochemical examination that positive staining of CCSP was distributed mainly in distal and respiratory bronchioles. The percentage of Clara cells in distal and respiratory bronchioles epithelium decreased in hyperoxia group (32.17 +/- 3.19)% compared to that in air control group (68.32 +/- 2.04)%, P < 0.01. Statistically significant differences were found in intensity of positiveness of Clara cells between hyperoxia (29.45 +/- 5.56) and air control group (42.37 +/- 3.24), P < 0.01. TUNEL assay showed that most apoptotic cells were alveolar and bronchial epithelial cells. The apoptotic index increased significantly in the hyperoxia group (55.77 +/- 6.09)% compared to the air control group (16.41 +/- 4.01)%, (P < 0.01). The positive rate (52.98 +/- 2.68)% of Clara cells and the expression (41.22 +/- 6.36) of CCSP in hyperoxia + rh-IGF-1 group increased significantly when compared with hyperoxia group, and the differences between these two group were also statistically significant (P < 0.01). The apoptotic index increased significantly in the hyperoxia + rh-IGF-1 group (27.98 +/- 3.09)% compared to the hyperoxia group (P < 0.01). CONCLUSIONS: Hyperoxia exposure can promote the pneumocyte apoptosis and inhibit the expression of CCSP. Rh-IGF-1 can remove the block of the formation of lung alveoli, increase the secretion of CCSP, mitigate inflammatory responses in airway and alleviate lung injury via pneumocyte apoptosis. Therefore, the results of this study provide a theoretic and experimental evidence for clinical application of rh-IGF-1 in prevention and treatment of BPD.

[Regulatory effects of inhaled steroids on expression of matrix metalloproteinase-9 and its inhibitor in asthmatic rats].

OBJECTIVE: To investigate the role of matrix metalloproteinase-9 (MMP-9) and its tissue inhibitor (TIMP-1) in the pathogenesis of bronchial asthma and assess the effect of steroid treatment on MMP-9 and TIMP-1 levels. Matrix metalloproteinases are a family of zinc and calcium-dependent endopeptidases. Many MMPs such as MMP-1, MMP-2, MMP-3 are associated with asthma, in which MMP-9 is the key factor in asthma. Tissue inhibitor-1 of metalloproteinases is a specific inhibitor of MMP-9; the MMP-9 and TIMP-1 imbalance could lead to airway inflammation and remodeling in lung disease such as asthma. METHODS: Forty Wistar rats were divided into 4 groups randomly: control, asthma model 7 days (7-day group), asthma model 21 days (21-day group) and steroid treatment groups. Asthma model of rats were established by ovalbumin (OVA) sensitization and challenge with mist inhalation. The expression of MMP-9 and TIMP-1 in lung tissues was detected by immunocytochemistry, RT-PCR and Western blotting. RESULTS: (1) By observing the changes of action, tracing respiratory curves, detecting level of serum IgE level and observing the lung tissues sections, the authors demonstrated that the rat asthmatic models were successfully established. (2) The lung tissue sections of the asthma groups stained with hematoxiline and eosin (HE) showed many inflammatory cell infiltrations around the bronchioli and accompanying arterioles, hyperplasia of caliciform cells, broken bronchial mucous membrane and thickening of submucosal layer. The hyperplasia of airway smooth muscle and basement membrane were more significant in asthma model 21-day group than that in 7-day group. These changes were improved after treatment. (3) The expression of MMP-9 in rat's lung tissues: the expression was 2.71 +/- 0.37 in 7-day group, 1.76 +/- 0.27 in 21-day group, 0.88 +/- 0.18 in the treatment group and 0.52 +/- 0.10 in the control group (F = 151.52, P < 0.01). The expression of TIMP-1 in rat's lung tissues was 1.13 +/- 0.19 in the 7-day group, 1.55 +/- 0.24 in 21-day group, 0.77 +/- 0.15 in the treatment group and 0.47 +/- 0.08 in the control group (F = 69.46, P < 0.01). (4) The results of immunocytochemistry and protein expression were consistent with those of RT-PCR. CONCLUSION: The protein and mRNA expression level of MMP-9 and TIMP-1 was high in asthmatic rat's lung tissues. Down-regulation of the expression of MMP-9 and TIMP-1 by steroids may be one of the mechanisms by which airway inflammation and remodeling are inhibited in asthma.