Blockade of lysophosphatidic acid receptors LPAR1/3 ameliorates lung fibrosis induced by irradiation.

Lung fibrosis is a common and serious complication of radiation therapy for lung cancer, for which there are no efficient treatments. Emerging evidence indicates that lysophosphatidic acid (LPA) and its receptors (LPARs) are involved in the pathogenesis of fibrosis. Here, we reported that thoracic radiation with 16Gy in mice induced development of radiation lung fibrosis (RLF) accompanied by obvious increases in LPA release and LPAR1 and LPAR3 (LPAR1/3) transcripts. RLF was significantly alleviated in mice treated with the dual LPAR1/3 antagonist, VPC12249. VPC12249 administration effectively prolonged animal survival, restored lung structure, inhibited fibroblast accumulation and reduced collagen deposition. Moreover, profibrotic cytokines in radiation-challenged lungs obviously decreased following administration of VPC12249, including transforming growth factor ß1 (TGFß1) and connective tissue growth factor (CTGF). In vitro, LPA induced both fibroblast proliferation and CTGF expression in a dose-dependent manner, and both were suppressed by blockade of LPAR1/3. The pro-proliferative activity of LPA on fibroblasts was inhibited by siRNA directed against CTGF. Together, our data suggest that the LPA-LPAR1/3 signaling system is involved in the development of RLF through promoting fibroblast proliferation in a CTGF-dependent manner. The LPA-LPAR1/3-CTGF pathway may be a potential target for RLF therapy.

Ability of low-molecular-weight heparin to alleviate proteinuria by inhibiting respiratory syncytial virus infection.

AIM: Low-molecular-weight heparin (LMWH) is a negatively charged glycoprotein and has a very similar structure to that of cell surface heparin sulfate (HS). Thus, LMWH, an analog of HS, may inhibit positively charged respiratory syncytial virus (RSV) infection through cooperative electrostatic association. METHODS: In this study, rats were respectively treated with 400 IU/kg LMWH before, during or after being inoculated with 6 x 10(6) plaque-forming unit (PFU) RSV. RSV and normal control groups were respectively inoculated by RSV and virus-free Dulbecco's modified Eagle's medium (DMEM). HeLa cells in vitro were pretreated with LMWH, elastase (ELA), heparinase (HpaIII) and protamine before being inoculated with 6 x 10(1) PFU RSV. RSV infectivity was determined by in situ hybridization and plaque assay. RESULTS: After inoculation, the urinary protein excretion and serum parameters in LMWH-treated rats were significantly lower than those in the RSV group. No abnormalities of glomerular structure were observed in LMWH-treated groups whereas swelling and slight hypercellularity in minority glomeruli and foot process effacement were observed in the RSV group. RSV RNA of LMWH-treated rats had weaker expression than that of the RSV group. In vitro, RSV infection in RSV + LMWH, HpaIII + ELAI, protamine + ELAI, ELAI, HpaIII and protamine treatment cells were significantly lower than that of the RSV control, and that in RSV + LMWH was the least. There were no significant differences in RSV infection between ELAI + LMWH and RSV control. CONCLUSION: Our study confirmed that there is a correlation between RSV and proteinuria in rats. LMWH can alleviate proteinuria in rats through inhibiting RSV from binding with HS which plays an important role in the onset of RSV infection.

[The construction and expression of eukaryotic expression plasmid of Legionella pneumophila immunogenic protein].

OBJECTIVE: To construct the recombinant plasmid of Legionella pneumophila immunogenic protein gene (ip) and detect the immunoprotein expression in NIH3T3 cells. METHODS: The immunogenic protein gene was amplified from DNA of Legionella pneumophila by polymerase chain reaction (PCR), then inserted into pcDNA3. 1 (+) vector. The recombinant plasmid was named as pcDNA3. 1-ip and analyzed with restriction-endonuclease Hind III and BamH I digestion, PCR and DNA sequencing techniques. The NIH3T3 cell was transfected by recombinant plasmid pcDNA3. 1-ip with lipofection strategy. The transient and stable expression products of immunogenic protein gene were detected by immunofluorescence and Western blot. RESULTS: We have successfully constructed the recombinant plasmid of Legionella pneumophila immunogenic protein gene. It was found that there was dark green fluorescence on the cell membrane and inside the cell. Our results showed that NIH3T3 cell was transfected by pcDNA3. 1-ip successfully. The rabbit serum antibody of Legionella pneumophila detected the NIH3T3 cell transfected stably by pcDNA3. 1-ip to express the immunogenic protein with the relative molecular mass 29 X 1093). CONCLUSION: We have successfully expressed immunogenic protein of Legionella pneumophila. The expressed product showed the good immunogenicity and immunoreactivity.

[In vitro expression of Lgeionella pneumophila pilE gene and its immunogenicity].

OBJECTIVE: To construct a recombinant plasmid containing Lgeionella pneumophila pilE gene, detect its expression in NIH3T3 cells and evaluate its immunogenicity. METHODS: PilE gene (LP) was amplified from Legionella pneumophila DNA by PCR and inserted into pcDNA3.1(+) vector to construct the recombinant plasmid pcDNA3.1-pilE, which as verified by restriction endonuclease digestion, PCR and DNA sequencing analysis. NIH3T3 cells were transfected with the recombinant plasmid with Lipofection strategy. Transient and stable pilE gene products were detected by immunofluorescence and Western blotting, respectively. To evaluate the immunogenicity of pcDNA3.1-pilE, the recombinant plasmid was used as a DNA vaccine to immunize female BALB/c mice intramuscularly and the specific antibodies, lymphocyte proliferation response, interferon (IFN)-gamma production and cytotoxic T-lymphocyte response of the immunized mice were detected and evaluated. RESULTS: The pilE gene of 429 bp in length was amplified. After transfection of NIH3T3 cells with the recombinant plasmid, strong green fluorescence was observed on the cell membrane and inside the cell. A protein with relative molecular mass of 15.7 kD was detected in the transfected cells with Western blotting, suggesting successful protein expression of pilE gene. pcDNA3.1-pilE resulted in much stronger immune response in the immunized mice than pcDNA3.1(+) (P<0.01). CONCLUSION: The recombinant plasmid containing Lgeionella pneumophila pilE gene constructed in this study is capable of expression in NIH3T3 cells, and can induce specific humoral and cellular immune responses in mice.

[The cloned ctxA gene of vibrio cholerae expresses in E. coli].

OBJECTIVE: To construct the expression vector of Vibrio cholerae ctxA gene, and realize the Vibrio cholerae ctxA gene to express in E. coli, and lay a basis for future research on the immunogenicity and immunoadjuvant. METHODS: The ctxA gene, an cholera toxin subunit gene (ctxA) of Vibrio cholerae, was obtained by polymerase chain reaction (PCR) from DNA of Vibrio cholerae, and cloned into prokaryotic expressed vector pET32a(+) with thioredoxin (Trx) gene. The recombinant plasmid (pET-ctxA) was analyzed to identify with restriction-endonuclease digestion, PCR and DNA sequencing analysis. And then the pET-ctxA was transferred into E. coli strain BL21 (DE3) for transformation. The ctxA expression of pET-ctxA was induced with isopropy-beta-D-thiogalactoside (IPTG) and the fused protein Trx-CTA was examined by SDS-PAGE and Western blot techniques. RESULTS: Restriction endonuclease digestion, PCR and DNA sequencing analyses showed that the ctxA gene of 787 bp was amplified from Vibrio cholerae DNA, and the recombinant plasmid pET-ctxA was successfully constructed, and the ctxA expression in prokaryotic cell was detected by SDS-PAGE and Western blot techniques. CONCLUSION: The ctxA gene of Vibrio cholerae, in fused protein form with Trx, got a high expression in E. coli.

The Relationship Between Atmospheric Transport and the Particle Scattering Coefficient at the Grand Canyon.

Air trajectory and particle scattering data (bsp) for the period 1984-1989 are used to determine the relationship between atmospheric transport and visual air quality at the Grand Canyon National Park. Using cluster analysis, 72-hour back-trajectories arriving four times per day were grouped into distinct transport patterns. Northwesterly and southerly/southwesterly flow dominate in the winter and summer seasons, respectively. Comparisons of bsp values accompanying different transport patterns showed a clear relationship between air flow pathway and light scattering due to small particles during the non-summer months only. An index is defined which describes the percentage of annual trajectories belonging to specific transport routes delivering predominantly clear air to the GCNP.