Azithromycin inhibits neutrophil accumulation in airways by affecting interleukin-17 downstream signals.

BACKGROUND: Azithromycin can reduce neutrophil accumulation in neutrophilic pulmonary diseases. However, the precise mechanism behind this action remains unknown. Our experiment assessed whether azithromycin inhibits neutrophil accumulation in the airways by affecting interleukin-17 (IL-17) downstream signals. METHODS: Mice were pretreated with azithromycin before murine IL-17A (mIL-17) stimulation. After the mIL-17 stimulation, the levels of six neutrophil-mobilizing cytokines were determined by enzyme-linked immunosorbent assay (ELISA) tests in bronchoalveolar lavage (BAL) fluid; IL-6, CXC chemokine ligand-1 (CXCL-1), CXCL-5, macrophage inflammatory protein-2 (MIP-2), granulocyte colony-stimulating factor (G-CSF), and granulocyte macrophage colony-stimulating factor (GM-CSF). The number of neutrophils in BAL fluid were evaluated by cytospin preparations. RESULTS: (1) Azithromycin pretreatment significantly inhibited both the release of three neutrophil-mobilizing cytokines (MIP-2, CXCL-5 and GM-CSF) and the accumulation of neutrophils in airways caused by mIL-17 stimulation. (2) The levels of three neutrophil-mobilizing cytokines (IL-6, MIP-2 and GM-CSF) were positively correlated with the numbers of neutrophil in BAL fluid. CONCLUSIONS: Azithromycin can inhibit neutrophil accumulation in the airways by affecting IL-17 downstream signals. This finding suggests that macrolide antibiotic application might be useful in prevention of neutrophilic pulmonary diseases characterized by high levels of IL-17.

Enhanced immune response and protection efficacy of a DNA vaccine constructed by linkage of the Mycobacterium tuberculosis Ag85B-encoding gene with the BVP22-encoding gene.

Plasmid DNA vaccines have been widely explored for use in tuberculosis immunization but their immunogenicity needs improvement. In the present study, we incorporated the bovine herpesvirus 1 VP22 (BVP22)-encoding gene, which encodes a protein that demonstrates a capability for disseminating the expressed antigen to neighbouring cells, into a DNA vector in which it was fused to the Ag85B-encoding gene of Mycobacterium tuberculosis (Mtb), and investigated whether this linkage could enhance immune response and protective efficacy in C57BL/6 mice compared to plasmid DNA encoding Ag85B alone. After immunization in mice, Ag85B-specific ELISA antibodies and spleen lymphocyte proliferative responses induced by DNA co-expressing BVP22 and Ag85B were significantly higher than those obtained in mice immunized with Ag85B-encoding DNA alone, except for the number of gamma interferon secreting cells. In addition, based on histopathological examination and bacterial-load determination in lung and spleen, protection against intravenous Mtb H37Rv challenge evoked by the BVP22-Ag85B DNA immunization exceeded the response elicited by Ag85B DNA alone, which was not significantly different from that provided by Bacillus Calmette-Guérin (BCG). These results suggested that DNA vaccine consisting of BVP22 and Ag85B-encoding DNA enhanced immune response and protection against intravenous Mtb H37Rv challenge in mice, indicating that BVP22-encoding DNA might be a promising tool to enhance TB DNA vaccine efficacy.

Construction of a eukaryotic expression system for granulysin and its protective effect in mice infected with Mycobacterium tuberculosis.

A full-length cDNA of granulysin was inserted into the pcDNA3.1(-) vector to construct a eukaryotic expression plasmid for granulysin. The recombinant plasmids were injected intramuscularly into mice infected with Mycobacterium tuberculosis to evaluate the protective effect of granulysin. Granulysin significantly decreased the weight index (WI) of the spleen, reduced the numbers of viable bacteria in lung and spleen, and reduced the lesions of lung tissue in granulysin-rDNA-immunized mice compared with those of control group mice. In vitro, the serum of the recombinant-plasmid-immunized mice inhibited the viability of M. tuberculosis by the physical disruption of cell membranes. Therefore, granulysin has a therapeutic effect against M. tuberculosis.

[Impact of STAT6 signaling pathway blockade on apoptosis of human colon cancer cells].

OBJECTIVE: To investigate the relationship between the signal transducer and activator of transcription 6 (STAT6) and human colon cancer. METHODS: Four STAT6-specific recombinant plasmid vectors, pshRNA-STAT6-1, 2, 3, and 4 were constructed and transfected into the cultured human colon cancer cells of the line HT-29. Seventy-two hours later RT-PCR was used to detect the mRNA expression of STAT6 and the apoptosis-related genes Bcl-2 and Bax, flow cytometry (FCM) was used to detect the protein expression of phopho-STAT6 (pSTAT6). HT-29 cells were inoculated into a plate and transfected with pshRNA-STAT6-1 or pshRNA-STAT6-4, and HT-29 cells without transfection were used as controls. Seventy-two hours later FCM was used to observe the cell apoptosis. Another HT-29 cells were inoculated into a plate and transfected with pshRNA-STAT6-1 or pshRNA-STAT6-4, or blank liposome as controls. Seventy-two hours later. Western blotting was used to detect the protein expression of Bcl-2 and Bax genes. RESULTS: The p-STAT6 protein expression rate was 3.2% +/- 0.6% in the pshRNA-STAT6-1 group, significantly lower than that of the blank control group (18.2% +/- 0.9%, P < 0.01) with an inhibition rate of 82.4%, and was 7.9% +/- 0.4% in the pshRNA-STAT6-4 group, significantly lower than that in the blank control group too (P < 0.01) with an inhibition rate of 56.6%. And the p-STAT6 protein expression rates of the pshRNA-STAT6-2 and pshRNA-STAT6-3 groups were 16.6% +/- 0.5% and 17.1% +/- 0.7% respectively, both not significant different from that of the blank control group (both P > 0.05). The early cell apoptosis rates of the pshRNA-STAT6-1 and pshRNA-STAT6-4 groups were 13.0% and 8.8% respectively, both significantly higher than that of the blank control group (0.4%, both P < 0.05). The mRNA expression of Bcl-2 was significantly lower and the mRNA expression of Bax was significantly higher in the pshRNA-STAT6-1 and pshRNA-STAT6-4 groups than in the blank control and blank liposome groups (all P < 0.01). The protein expression patterns of Bcl-2 and Bax was consistent with that of their protein expression. CONCLUSION: STAT6 signaling pathway inhibits the apoptosis of colon cancer cells by regulation of the Bcl-2 and Bax genes.

[An in vitro dendritic cells-induced T lymphocytes cytotoxicity study activated by thaw frozen K562 lysates antigen in chronic myeloid leukemia].

OBJECTIVE: To investigate the anti-chronic myeloid leukemia (CML) effect of cytotoxicity T lymphocytes (CTL) activated by dendritic cells (DC) pulsed with freeze thaw K562 lysates antigen. METHODS: DC were achieved by healthy donors peripheral blood monocytes which were induced by granulocyte-macrophage colony stimulating factor (GM-CSF) and IL-4. On the sixth day, these cells were collected. One part of them was used to prove that they were DC from morphology and phenotypes. The other part was pulsed with K562 lysates and the lysate-loaded DC were compared with immature DC from phenotypes. The expression of IL-12 and IFNgamma in supernatant, the mixed lymphocytes reaction and the specific cytotoxicity against leukemia cells were tested. RESULTS: Freeze thaw K562 lysate up-regulated the expression of various differentiation antigens of DC, such as CD(1a) (27.40 +/- 5.00)% vs (15.40 +/- 2.34)%, CD(80) (61.35 +/- 5.35)% vs (42.00 +/- 2.77)%, CD(83) (93.30 +/- 3.48)% vs (25.15 +/- 4.02)%, CD(86) (85.25 +/- 4.39)% vs (37.25 +/- 3.20)%, CD(40) (89.80 +/- 7.18)% vs (35.95 +/- 4.06)% and HLA-DR (49.50 +/- 5.45)% vs (17.15 +/- 3.61)%. Simultaneously, the expression of IL-12 rose after DC were loaded with tumor lysates after 24 hours (P < 0.05). The T cells from healthy donors and CML patients, proliferated by antigen loaded DC, could induce IFNgamma increase (P < 0.01). The proliferation of T cells had distinct relation with the time of antigen loaded with DC. These T cells had specific cytotoxicity against K562 and HLA partial matching CML cells. CONCLUSIONS: DC pulsed with K562 lysates can stimulate T cells, and can keep high immunocompetence with specific cytotoxicity against K562.

Construction of a eukaryotic expression system of HSP65 gene from Mycobacterium tuberculosis, and anti-HSP65 IgG produced in mice.

The purpose of this study was to express the HSP65 gene of Mycobacterium tuberculosis in eukaryotic cells and study its primary immune effect in animals. The HSP65 gene was amplified from the H37Rv strain of M. tuberculosis by PCR and then inserted into the expression plasmid pcDNA3.1(-). The recombinant plasmid pcHSP65 was transfected into HeLa cells by using the liposome transfection method and also injected into BALB/C mice to accomplish DNA immunization. The inserted gene was demonstrated to be identical to the reported HSP65 gene sequence. The transfected HeLa cells expressed HSP65 protein; Western blot showed the presence of a 65 kDa band of the inclusion body protein and immunofluorescence testing identified the protein expressed in cytoplasm. Specific IgG for the HSP65 protein could be identified in immunized mice. This study shows that recombinant eukaryotic expression plasmid pcHSP65 was constructed successfully, which lays a foundation for further study of gene therapy.

STAT5a regulates the GlcNAc-1-phosphate transferase gene transcription and expression.

The dolichyl-phosphate alpha-N-acetylglucosaminephosphotransferase 2 (Dpagt2) gene in the mouse has a housekeeping promoter, and its expression is regulated during the development and hormonally modulated lactogenesis of the mammary gland. Previous studies showed that the transcription of the mouse mammary Dpagt2 gene is stimulated by the lactogenic hormones, insulin, glucocorticoid receptor (GR), and prolactin. Transcription factors which bind to the Dpagt2 gene promoter region can influence the expression level of the Dpagt2 gene. It is supposed that the Dpagt2 gene promoter region (bases pairs -1462 to -5) maybe contain 10 putative STAT (signal transducer and activator of transcription) binding sites: TTN (5/6) AA. In order to identify the STAT factors involved in the transcription of the GPT gene, 32P labeling probes and lactating mouse nuclear extracts were prepared. Electrophoretic Mobility Shift Assays (EMSA) show that the region (bases pairs -386 to -322, where there is a STAT binding site, TTTCAAAAA) binds to STAT5a, not to glucocorticoid receptor (GR) or other STAT factors. The involvement of STAT5a in regulating the expression of the mouse Dpagt2 gene was further investigated by transient transfections of various Dpagt2 promoter/luciferase (Luc) constructs into COS 7 cells. The results showed that co-transfection of STAT5a or prolactin receptor can enhance Dpagt2 promoter activities in the promoter construct pGL-MX6 (from base pairs -386 to -5), but not in the promoter construct pGL-MX7 (from base pairs -322 to -5). This paper first reports that STAT5a is involved in the binding between -386 and -322 base pairs of the Dpagt2 gene promoter and stimulates the expression of the Dpagt2 gene transcription in the mouse lactating mammary gland.