MicroRNA let-7a inhibits the proliferation and invasion of nonsmall cell lung cancer cell line 95D by regulating K-Ras and HMGA2 gene expression.

MicroRNAs are closely linked to tumor metastasis and let-7a may play a role in inhibiting the proliferation, invasion, and metastasis of lung cancer. In vitro, we aim to observe the impact of let-7a on the proliferation and invasion of the nonsmall cell lung cancer cell line 95D by constructing a lentiviral vector that expresses let-7a. Cell proliferation assays and Transwell experiments were used to compare the proliferation and invasion of the 95D cell group with let-7a overexpressed or inhibited. Real-time polymerase chain reaction and immunoblotting analysis were used to compare the expression of K-RAS and HMGA2 at mRNA and the protein level in the above groups. The results showed the cells in the let-7a overexpressed group were significantly less proliferative and invasive than those in the let-7a inhibited group (p < 0.05). K-RAS and HMGA2 mRNA levels were significantly higher in the let-7a overexpressed group than those in the let-7a inhibited group (p < 0.05). However, the protein levels of K-RAS and HMGA2 were significantly lower in the let-7a overexpressed group than those in the let-7a inhibited group (p < 0.05). We suppose that let-7a inhibits the proliferation and invasion of the cell line 95D by regulating the translation of K-RAS and HMGA2 mRNA, not the transcription of the mRNA itself.

The variation of CD4+CD25+ regulatory T cells in the periphery blood and tumor microenvironment of non-small cell lung cancer patients and the downregulation effects induced by CpG ODN.

The aim of the study was to observe the variation of CD4(+)CD25(+) regulatory T cells in periphery blood and tumor microenvironment of non-small cell lung cancer (NSCLC) patients and the effects of CpG ODN. The proportion of CD4(+)CD25(+) regulatory T cells, Foxp3 gene expression, levels of tumor growth factor-ß (TGF-ß) and immunoreactive fibronectin-γ (IFN-γ) in the periphery blood of 30 NSCLC patients and 30 healthy volunteers were compared. These indicators were compared before and after CpG ODN treatment. Foxp3 gene expression in the tumor microenvironment of NSCLC patients was also observed. The results showed CD4(+)CD25(+) regulatory T cell proportion, Foxp3 expression and TGF-ß levels in the periphery blood of NSCLC patients were higher than those of healthy volunteers (p < 0.05), and these indicators of patients were significantly decreased after CpG ODN 2006 treatment (p < 0.05). Foxp3 expression in the metastatic lymph nodes was higher than that in the non-metastatic ones of NSCLC patients (p = 0.000). In conclusion, a rise in the proportion of CD4(+)CD25(+)Foxp3(+) regulatory T cells was demonstrated in the periphery blood and tumor microenvironments of NSCLC patients. CpG ODN 2006 downregulated the CD4(+)CD25(+)Foxp3(+) regulatory T cells proportion and TGF-ß levels in the periphery blood of these patients.

[The effect of lipopolysaccharide on gene expression of TLR4 and MD-2 in rat alveolar macrophage and its secretion of inflammation cytokines].

OBJECTIVE: To investigate the effect of lipopolysaccharide (LPS) on gene expression of Toll-Like receptor 4 (TLR4) and myeloid differentiation protein-2 (MD-2) in rat alveolar macrophage NR8383 cell and its secretion of inflammation cytokines with or without small interference RNA target MD-2 (MD2-siRNA) treatment. METHODS: NR8383 cell was cultured with F-12K medium and stimulated with LPS (0.01 approximately 10 mg/L) for 2 h or stimulated with 1 microg/ml LPS for 2-24 h. MD2-siRNA oligo was transfected into NR8383 cell by Lipofectamine 2000. The expression of TLR4 mRNA and MD-2 mRNA in cell were detected by semi-quantitative revels transcription polymerase (RT-PCR). The contents of TNF-alpha, IL-6 and IL-1beta in the cell cultured supernatant were tested by ELISA. One way ANOVA was used for difference comparison during groups and Pearson correlation was used for correlation analysis. RESULTS: (1) The mRNA expression of TLR4 and MD-2 in control NR8383 cell were 0.52+/-0.05 and 0.44+/-0.09, respectively. There was no obviously change after 0.01 mg/L LPS stimulation. The increase occurred in a dose dependent manner from 0.1 mg/L to 10 mg/L. The highest TLR4 and MD-2 mRNA expression were 0.72+/-0.06 and 0.65+/-0.10 (F=17.255 and 6.045, P<0.01). The changes of TNF-alpha, IL-6 and IL-1beta contents in cell cultured supernatant were similar with that of TLR4 and MD-2 gene expression. TNF-alpha, IL-6 and IL-1beta content were (25.8+/-3.4) ng/L, (62.4+/-4.7) ng/L and (31.6+/-1.7) ng/L in control group, while the corresponding contents were (58.9+/-5.3) ng/L, (96.5+/-3.9) ng/L and (55.4+/-5.4) ng/L after 10 mg/L LPS simulation (F=29.55, 54. 47 and 31.45, P<0. 01). (2) When stimulated with 1 microg/ml LPS, the mRNA expressions of TLR4 and MD-2 in NR8383 cell were increased from hour 2. The highest mRNA expressions of TLR4 and MD-2 occurred at hour 6, and then decreased slowly from hour 8. The mRNA expressions at hour 24 were still higher than those in cells without LPS stimulation (F=5.279 and 4.106, P<0.01). The changes of TNF-alpha, IL-6 and IL-1beta content in cell cultured supernatant were also similar with that of gene expression (F=10.64, 11.23 and 17.58, P<0. 01). The secretion peaks of TNF-alpha and IL-1beta occurred from hour 6 to hour 8, and the secretion peak of IL-6 occurred from hour 8 to hour12. (3) The mRNA expression of MD-2 was related with that of TLR4 positively (r=0.513, P<0.01). (4) The interfere efficiency of MD-2 siRNA was 67%. There was no obvious increase of TNF-alpha, Il-1beta and IL-6 in MD-2 siRNA treated group after LPS stimulation. CONCLUSION: Higher dose LPS can up-regulate the gene expression of TLR4 and MD-2 in rat alveolar macrophage NR8383 cell with a long time and promote the secretion of TNF-alpha, IL-6 and IL-1beta. MD-2 siRNA can inhibit NR8383 cell secrete TNF-alpha, IL-6 and IL-1beta induced by LPS.

Continuous tracheal gas insufflation during protective mechanical ventilation in juvenile piglets with acute lung injury induced by endotoxin.

BACKGROUND: Low tidal volume mechanical ventilation is difficult to correct hypoxemia, and prolonged inhalation of pure oxygen can lead to oxygen poisoning. We suggest that continuous tracheal gas insufflation (TGI) during protective mechanical ventilation could improve cardiopulmonary function in acute lung injury. METHODS: Totally 12 healthy juvenile piglets were anesthetized and mechanically ventilated at PEEP of 2 cmH2O with a peak inspiratory pressure of 10 cmH2O. The piglets were challenged with lipopolysaccharide and randomly assigned into two groups (n=6 each group): mechanical ventilation (MV) alone and TGI with continuous airway flow 2 l/min. FIO2 was set at 0.4 to avoid oxygen toxicity and continuously monitored with an oxygen analyzer. RESULTS: Tidal volume, ventilation efficacy index and mean airway resistant pressure were significantly improved in the TGI group (P<0.01 or P<0.05). At 4 hours post ALI, pH decreased to below 7.20 in the MV group, and improved in the TGI group (P<0.01). Similarly, PaCO2 was stable and was significantly lower in the TGI group than in the MV group (P<0.01). PaO2 and PaO2/FIO2 increased also in the TGI group (P<0.05). There was no significant difference in heart rate, respiratory rate, mean artery pressure, central venous pressure, dynamic lung compliance and mean resistance of airway between the two groups. Lung histological examination showed reduced inflammation, reduced intra-alveolar and interstitial patchy hemorrhage, and homogenously expanded lungs in the TGI group. CONCLUSION: Continuous TGI during MV can significantly improve gas exchange and ventilation efficacy and may provide a better treatment for acute lung injury.

[Modulation of TLR9 on anti-tumor immune responses of peripheral blood mononuclear cells from patients with non-small-cell lung cancer].

OBJECTIVE: To assess the modulation of TLR9 on anti-tumor immune responses in peripheral blood mononuclear cells (PBMC) from patients with non-small-cell lung cancer (NSCLC). METHODS: PBMCs were isolated from 36 NSCLC patients. Lung cancer cells were isolated from these patients and further enriched. PBMCs were cultured in RPMI-1640 medium (blank control group), and medium with cytosine guanine oligodeoxynucleotide (CpG ODN, an TLR9 agonist) or control ODN for 72 h; and then flow cytometry was used to examine the expression of CD69 antigen on the surface of CD3 cells, [3H]-thymidine incorporation method was used to examine the cell proliferation, and the IFN-alpha level in the supernatant was measured. Another PBMCs were cultured in medium with interleukin (IL)-1 and then CpG ODN, control ODN, and CpG ODN + chloroquine or inhibitory ODN were added respectively for 24-48 h. Then the IFN-alpha in the supernatant was measured. Subsets were assessed by flow cytometry and the expression of TLR9-mRNA in freshly isolated PBMC was detected by RT-PCR. The production of interferon (IFN)-alpha in the PBMCs was measured by ELISA. The proliferation of the PBMCs was determined by [3H]-thymidine incorporation. The PBMCs co-cultured with CpG ODN and autologous lung tumor cells treated with mitomycin C were used as effector cells, and K562 cells and autologous tumor cells were used as target cells flow cytometry was used to detect the capacity of PBMCs to kill autologous lung tumor cells and K562 cells. Meanwhile we investigated the intracellular expression of IFN-gamma and IL-4 in CD8+ T. RESULTS: The expression level of TLR9 of the PBMCs from patients was not significantly different from that of the PBMCs from the healthy donors. The proportion of CD69 antigen expressing CD3+ T cells of the CpG ODN group was (39.5 +/- 8.9)%, significantly higher than those of the blank control group [(8.8 +/- 1.2)%, t = 40.30, P = 0.00] ands control ODN group [(10.6 +/- 1.0)%, t = 41.85, P = 0.00]. Examination with beta liquid scintillation counter showed that the cpm value of the CpG ODN group was (1.61 +/- 0.20) x 10(4), significantly higher than those of the blank control group [(0.27 +/- 0.14) x 10(4), t = 20.43, P = 0.00] and control ODN group [(0.34 +/- 0.13) x 10(4), t =20.20, P = 0.00]. Chloroquine and inhibitory ODN dose-dependently inhibited the IFN-alpha levels in the supernatant. The CD4 + T/CD8 + T of the CpG ODN group was (3.44 +/- 0.20), significantly higher than those of the control ODN group (1.73 +/- 0.27, t = 19.85, P = 0.00) and blank control group (1.69 +/- 0.13, t = 29.32, P = 0.00). The IFN-gamma positive CD8 + T cells of the CpG ODN group was (18.5 +/- 4.2)%, significantly higher than those of the control ODN group [(4.2 +/- 1.0)%, t = 24.12, P = 0.00] and blank control group [(3.1 +/- 1.2)%, t = 25.1, P = 0.00]. There was no significant differences in the proportion of IL-4 positive CD8 + T cells among different groups. When the E/T was 40:1 the killing capacity of PBMCs against the K562 cells was (19.5 +/- 1.0), significantly higher than those of the control ODN group (7.9 +/- 1.1, t = 19.9, P = 0.00) and blank control group (5.1 +/- 1.6, t = 21.9, P = 0.00), and the killing capacity of PBMCs against the autologous lung tumor cells was (29.8 +/- 2.1), significantly higher than those of the control ODN group (8.1 +/- 0.9, t = 36.9, P = 0.00) and blank control group (5.7 +/- 1.6, t = 35.7, P = 0.00). CONCLUSION: TLR9 signal takes part in the immunomodulation of PBMCs. The activation of TLR9 results in enhanced anti-tumor response in the PBMCs against autologous lung cancer cells and K562 cells.

Targeting toll-like receptor 9 with CpG oligodeoxynucleotides enhances anti-tumor responses of peripheral blood mononuclear cells from human lung cancer patients.

CpG-oligonucleotides (CpG-ODN), which induce signaling through Toll-like receptor 9 (TLR9), are currently under investigation as adjuvants in therapy against infections and cancer. However, whether the CpG-ODN alone could enhance the anti-tumor immunity and the underlying mechanisms remains unclear. Here, we investigated that stimulation of peripheral blood mononuclear cells (PBMCs) from human lung cancer patients with CpG-ODN induced proliferation responses of the PBMCs, accompanied by the elevated cytokine secretion, including IFN-alpha, IL-12 and TNF-alpha. In addition, after treatment with CpG-ODN, the cytotoxic activity of the PBMCs and the production of IFN-gamma in CD8(+) T cells were dramatically enhanced. Furthermore, we found that adoptive transfer of CpG-ODN treated PBMCs significantly inhibited the tumor progression in nude mice, which were challenged with the autologuous tumor cells from human lung cancer patients. Finally, we demonstrated that the inhibitory CpG ODN or chloroquine could dramatically abrogate the enhanced anti-tumor responses of the CpG ODN treated PBMCs. Our findings suggest that the CpG-ODN is promising as a preventive and therapeutic anti-tumor measure against pulmonary carcinoma.

[Determination and clinical implication of leukotriene B(4) and tumor necrosis factor-alpha in condensate of exhaled breath of chronic obstructive pulmonary disease patients].

OBJECTIVE: To study the changes and clinical implication of leukotriene B(4) (LTB(4)) and tumor necrosis factor-alpha (TNF-alpha) in exhaled breath condensate (EBC) of chronic obstructive pulmonary disease (COPD) patients. METHODS: EBC of 20 patients in acute episode of COPD (AECOPD), 20 patients in period of remission of COPD, and 20 persons who were having regular check-up (healthy control group) were enrolled. The concentrations of LTB(4) and TNF-alpha in EBC were assayed. Forced expiratory volume in one second (FEV1) and peak expiratory flow (PEF) of COPD patients were observed at the same time, pH, oxygenation index (PaO(2)/FiO(2)), partial pressure of oxygen in arterial blood (PaO(2)) and leukocyte count were also determined. RESULTS: (1)The concentrations of LTB(4) in EBC of AECOPD (35.43+/-14.19)ng/L and remission of COPD(24.39+/-13.75)ng/L, were significantly higher than that of the healthy control group (16.75+/-7.44)ng/L, and the concentration of LTB(4) in EBC during remission of COPD was significantly lower than that of AECOPD (all P<0.05). (2)The concentration of TNF-alpha in EBC of AECOPD (9.35+/-8.66) ng/L was significantly higher than that of remission of COPD (4.42+/-4.11)ng/L and healthy control group (4.45+/-3.92) ng/L, and the differences had statistical significance (both P<0.05). The concentration of TNF-alpha in EBC showed no significant difference between patients in remission of COPD and healthy control group. (3)The concentration of LTB(4) in EBC had negative correlation with FEV1 of AECOPD patients. Regression equation was y=or-0.51 x+0.22, r=-0.481 (P<0.05). CONCLUSION: The concentrations of LTB4 and TNF-alpha in EBC of AECOPD patients are raised when oxidation stress is reinforced, and its level reflects the severity and prognosis of COPD.

[A study on the mechanisms of mycobacterial clearance induced by CpG-oligodeoxynucleotides in mice].

OBJECTIVE: To investigate the possible mechanisms of mycobacterial clearance induced by CpG-oligodeoxynucleotides (CpG-ODN) in mice. METHODS: Eight-week-old female BALB/c mice were treated with intraperitoneal CpG-ODN (30 microg), while the control mice with normal saline. After 2 weeks, the control mice and the CpG-treated mice were infected with Mycobacterium tuberculosis (1 x 10(6) colony-forming units, H(37)Rv strain) through the tail vein. At 3 weeks, 4 weeks and 6 weeks after mycobacterial infection, the lung and the spleen tissues were examined for histopathological changes. Real time-PCR was performed to measure the messenger RNA (mRNA) of interleukin (IL)-12, IL-18, interferon (IFN)-gamma, IL-4, IL-10 and inducible nitric oxide synthase (iNOS) in the tissues. The homogenates of lungs and spleens were cultured, and the colonies were counted after a 4 weeks incubation period at 37 degrees C. RESULTS: At 3 weeks and 4 weeks of mycobacterial infection, CpG-ODN-pretreated mice showed less mycobacterial burden in the lungs and the spleens than that in the control mice [(0 +/- 0) x 10(6) CFU/g vs (32 +/- 11) x 10(6) CFU/g, (0 +/- 0) x 10(6) CFU/g vs (10 +/- 4) x 10(6) CFU/g; (26 +/- 4) x 10(6) CFU/g vs (56 +/- 8) x 10(6) CFU/g, (4 +/- 3) x 10(6) CFU/g vs (27 +/- 8) x 10(6) CFU/g]. At 4 weeks of mycobacterial infection, CpG-ODN-pretreated mice displayed increased levels of IL-18 mRNA, IFN-gamma mRNA, iNOS mRNA [(3.6 +/- 0.5, 1.6 +/- 1.1, 0.32 +/- 0.14) vs (0.20 +/- 0.10, 23.17 +/- 4.72, 16.18 +/- 5.09)], and decreased level of IL-12p40 mRNA (5.66 +/- 0.64 vs 14.54 +/- 1.89), but there was no difference in the levels of IL-4 mRNA and IL-10 mRNA in the lungs between CpG-ODN-pretreated mice and the control mice [(0.30 +/- 0.09 vs 0.26 +/- 0.05), (0.28 +/- 0.05 vs 0.29 +/- 0.08)]. CpG-ODN-pretreated mice displayed increased levels of IL-18 mRNA, IFN-gamma mRNA, iNOS mRNA [(5.54 +/- 1.29 vs 0.79 +/- 0.36), (0.52 +/- 0.07 vs 0.21 +/- 0.06), (9.07 +/- 1.81 vs 5.97 +/- 1.44)], and decreased levels of IL-12p40 mRNA, IL-4 mRNA and IL-10 mRNA [(2.10 +/- 0.27 vs 5.07 +/- 0.39), (0.23 +/- 0.10 vs 1.21 +/- 0.26), (0.10 +/- 0.04 vs 0.57 +/- 0.13)] in the spleens as compared with the control mice. In CpG-ODN-pretreated mice, expression level of IFN-gamma mRNA in the lungs at 6 weeks post-infection was higher than that at 4 weeks post-infection (0.95 +/- 0.27 vs 0.32 +/- 0.14). CONCLUSION: The activation of Toll-like receptor-9 (TLR-9) with CpG-ODN could enhance murine mycobacterial clearance in vivo. TLR-9-induced anti-mycobacterial activity involves increased expression of IL-18, IFN-gamma, and iNOS but decreased expression of IL-4 and IL-10.

[A study on the recurrence of ischemic stroke in nonvalvular atrial fibrillation].

OBJECTIVE: To study the recurrence of ischemic stroke in patient with nonvalvular atrial fibrillation (NVAF) and to determine the recurrence related factors of ischemic stroke in patients with NVAF. METHODS: The clinical data of 386 NVAF patients with ischemic stroke treated in our hospital from January 1992 to February 2002 were included in this study. The basic clinical information of each patient was recorded. Analysis of recurrence and recurrence related factors of ischemic stroke in patients with NVAF was conducted. RESULTS: Overall the 10-year recurrence rate of ischemic stroke in patients with NVAF was 34%. Hypertension, diabetes mellitus, transient ischemic attack (TIA), hyperlipemia and mural thrombus in left atrium were significantly correlated with the recurrence rate by univariate Cox proportional hazards regression analysis; but aspirin therapy, warfarin therapy and hypertension control were protective factors for the recurrence of ischemic stroke in patients with NVAF. Multivariate Cox proportional hazards regression analysis revealed that hypertension, TIA and mural thrombus in left atrium were independent recurrence related factors for ischemic stroke in patients with NVAF; but aspirin therapy and warfarin therapy were protective factors for the recurrence. CONCLUSIONS: Hypertension, TIA and mural thrombus in left atrium were independent recurrence related factors for ischemic stroke in patient with NVAF; but aspirin therapy and warfarin therapy were protective factors for the recurrence.

[Development and application of the software for analysis of arterial blood gases in treatment suggestions and training].

OBJECTIVE: Suggestions and training in developing a computer software for analysis of arterial blood gases in treatment to be used in clinic and teaching. METHODS: The software was edited by means of Delphi 7.0 and closely combined with Windows operating system. The knowledge base was built with Microsoft Access and HTML format files. The treatment suggestions and knowledge base of the software were built according to the correlated diagnosis and treatment guidelines of Chinese Medical Association, Practical Medicine (edition 12) edited by CHEN Hao-zhu, Medicine (edition 6) edited by YE Ren-gao, Modern Clinical Blood Gases Analysis edited by QIAN Gui-sheng, Concise Clinical Blood Gas Analysis edited by LUO Yan-jie, and the experience of the experts of our hospital. RESULTS: (1)The model of treatment suggestions consisted of the treatment objective and the treatment suggestions,and its contents included simple acid-base disturbance, mixed acid-base disturbance, respiratory failure,metabolic disturbances of sodium potassium and chloride. The calculation formulae included calculation formulae for the correction of acid-base disturbance and electrolyte disturbance. (2)Arterial blood gas analysis training software contained knowledge database of arterial blood gas analysis, clinical opinions of arterial blood gas analysis, exam database of arterial blood gas analysis, knowledge of arterial blood gas analysis graph, calculation formulas, correlated books and websites,assistance and so on. CONCLUSION: The software could be used as a reference by clinical doctors in clinic, it could also be used for teaching and training. It is scientific, state-of-the art, and valuable in clinical practice.

[The effects of CpG-oligodeoxynucleotides on airway remodeling in chronic asthmatic mice].

OBJECTIVE: To investigate if CpG-oligodeoxynucleotides (CpG-ODN) intervention has inhibitory effects on the development of airway remodeling in an ovalbumin (OVA)-sensitized mouse model of chronic asthma. METHODS: Forty female C57BL/6 mice were randomly divided into four groups (n = 10): (1) Group A (chronic asthma model): mice were sensitized by intraperitoneal injection of OVA (10 microg) precipitated with aluminium hydroxide (100 microg) on days 1 and 14. From day 21, the mice were challenged by nebulized 2.5% OVA solution (30 min/d, three times a week for 8 weeks). (2) Group B (CpG-ODN intervention group): mice were sensitized and challenged as above, and were given 60 microg CpG-ODN by intraperitoneal injection for once every two weeks. (3) Group C (GpG-ODN control): Mice were given GpC-ODN instead of CpG motifs, other treatments same as Group B. (4) Group D (saline control): mice were sensitized and challenged by saline. All mice were killed 24 h after the final OVA challenge. Blood was obtained for eosinophil counts and measurement of serum IgE by enzyme-linked immunoabsorbent assay (ELISA). Bronchoalveolar lavage fluid (BALF) was collected for total and differential counts. The concentration of interleukin-13 (IL-13) and transforming growth factor-beta1 (TGF-beta(1)) in BALF was measured by ELISA. The left lung was isolated for pathological examination. Lung sections were stained with hematoxylin and eosin (HE), and Masson's trichrome. Other sections were prepared for immunohistochemistry using monoclonal antibodies against alpha-smooth muscle actin (alpha-SMA) and TGF-beta(1). RESULTS: The eosinophil count [(89 +/- 10) x 10(4)/ml], serum IgE [(279 +/- 53) ng/ml], BALF eosinophils [(6.30 +/- 1.30) x 10(5)/ml] and the concentrations of BALF IL-13 [(4 015 +/- 361) pg/ml] and TGF-beta(1) [(356 +/- 64) pg/ml] in the OVA-sensitized mice (Group A) showed significant difference as compared with those in the NS control group (Group D, t values are 24.0, 15.7, 14.7, 18.4, 12.0 and 18.9 respectively, all P < 0.01). In Group A, the percentages of positive staining area in Masson's trichrome, alpha-SMA staining and TGF-beta(1) staining were (29.7 +/- 4.2)%, (45 +/- 7)% and (34 +/- 4)% respectively. These percentages were significantly different from those in the NS control group (Group D, t values are 18.0, 15.6 and 17.9 respectively, all P < 0.01). In mice treated with CpG-ODN (Group B), the percentages of positive staining area in Masson's trichrome, alpha-SMA staining and TGF-beta(1) staining were (13.8 +/- 3.2)%, (24.7 +/- 3.1)%, (18 +/- 4)% respectively, which were significantly different from those in Group A (t values are 9.5, 8.9 and 9.8 respectively, all P < 0.05). CONCLUSIONS: This study demonstrated that CpG-ODN could prevent Th2 responses, eosinophilic inflammation and the development of airway remodeling. Its inhibitory effect on airway remodeling might, in part, be due to inhibition of the expression of cytokines such as TGF-beta(1) and IL-13.

[Therapeutic effects and mechanisms of CpG oligodeoxynucleotides in mice infected with Mycobacterium tuberculosis].

OBJECTIVE: To investigate the protective effects and mechanisms of CpG oligodeoxynucleotides (CpG ODN) in mice infected with Mycobacterium tuberculosis. METHODS: Ninety-six female BALB/c mice were randomized into 4 groups, namely CpG ODN treatment (A group), control ODN treatment (B group), infection control (C group) and healthy control (D group) (n = 24 each). A group and B group were treated once with CpG ODN or control ODN (30 microg/mouse) intraperitoneally 2 weeks before infection with Mycobacterium tuberculosis. 1 x 10(6) bacili in a volume of 300 microl saline was injected into the tail vein of mice from A group, B group and C group. Twelve mice from each group were sacrificed at 3 weeks postinfection. Pathologic changes in lung tissues were observed. The expression of Toll-like receptor 9 (TLR9) mRNA and cytokine mRNA was detected by RT-PCR. The numbers of viable bacteria in lung and spleen were counted. The rest 12 mice from each group were monitored for 60 days to observe the mortality. RESULTS: CpG ODN was shown to increase the survival time of mice infected with Mycobacterium tuberculosis. The body weights of mice from A group [(20.37 +/- 1.12) g] were higher than those of B group [(17.50 +/- 0.62) g] and C group [(17.15 +/- 0.97) g, P < 0.01]. The lung weights of mice from A group [(0.25 +/- 0.02) g] were similar to those of B group [(0.27 +/- 0.34) g, P > 0.05], but less than those of C group [(0.28 +/- 0.26) g, P < 0.01]. The spleens of mice from A group [(0.63 +/- 0.37) g] were larger than those of B group [(0.39 +/- 0.05) g] and C group [(0.38 +/- 0.02) g, P < 0.01]. The inflammation in lung tissue of mice from A group was less than that of B group and C group. There was no mycobacterial outgrowth in lungs and spleens of mice from A group. The expression of TLR9 mRNA in lungs and spleens of mice from A group (0.61 +/- 0.29 and 0.72 +/- 0.48) was similar to that in B group (0.58 +/- 0.35 and 0.64 +/- 0.28) and C group (0.60 +/- 0.32 and 0.65 +/- 0.31, P > 0.05), but higher than that in D group (0.11 +/- 0.08 and 0.26 +/- 0.22, P < 0.01). CpG ODN did not affect the expression of TLR9 mRNA. The expression of IFN-gamma mRNA in lungs and spleens of mice from A group (0.44 +/- 0.07 and 0.76 +/- 0.09) was higher than that in B group (0.19 +/- 0.05 and 0.22 +/- 0.05) and C group (0.16 +/- 0.04 and 0.18 +/- 0.08, P < 0.01). The expression of IL-6 mRNA in lungs and spleens of mice from A group (1.56 +/- 0.29 and 8.21 +/- 0.82) was higher than that in B group (0.86 +/- 0.55 and 0.16 +/- 0.09) and C group (0.78 +/- 0.21 and 0.06 +/- 0.04, P < 0.01). The expression of IL-4 mRNA in lungs and spleens of mice from A group (0.18 +/- 0.05 and 0.06 +/- 0.02) was lower than that in B group (0.31 +/- 0.06 and 1.22 +/- 0.01) and C group (0.35 +/- 0.04 and 1.31 +/- 0.31, P < 0.01). The expression of IL-10 mRNA in spleens of mice from A group (0.05 +/- 0.02) was lower than that in B group (0.57 +/- 0.09) and C group (0.65 +/- 0.15, P < 0.01). CONCLUSION: CpG ODN could increase the immunity of mice against tuberculosis through up-regulation of Th1 immunity and down-regulation of Th2 immunity.

[The combined effect of isoniazid and rifampicin on the activities of CYP4501A2 and CYP4503A4 in primary hepatocytes from healthy human adults].

OBJECTIVE: To study the combined effect of isoniazid and rifampicin on the activities of CYP1A2 and 3A4 in primary hepatocytes from healthy human adults. METHODS: The primary hepatocytes were isolated from adult healthy human livers, and cultured for 3 days. Then the cells were divided into 15 groups including two negative control groups (culture media alone) and 13 drug intervention groups, to which the following drugs were added: isoniazid (25 micromol/L, 50 micromol/L), rifampicin (12.5 micromol/L, 25 micromol/L) or both of them with different concentrations (CYP 1A2: rifampicin 12.5 micromol/L + isoniazid 50 micromol/L, rifampicin 25 micromol/L + isoniazid 50 micromol/L; CYP3A4: rifampicin 12.5 micromol/L + isoniazid 50 micromol/L, rifampicin 25 micromol/L + isoniazid 25 micromol/L, rifampicin 25 micromol/L + isoniazid 50 micromol/L) respectively. All the concentrations were consistent with the range of maximum clinical blood concentrations. After culture for 2 days, substrates (phenacetin for CYP1A2 , testosterone for CYP3A4) were added, and then the peak area (unit: mAU. min) of their metabolites was measured with high performance liquid chromatography (HPLC) to assess the activities of CYP450 1A2 and 3A4. RESULTS: (1) The activity of CYP1A2 in isoniazid groups with concentrations of 25 micromol/L and 50 micromol/L was (3.33 +/- 0.65), (3.03 +/- 0.38) mAU.min respectively, significantly different compared with that in the negative control group [(5.23 +/- 0.31) mAU.min, P < 0.01]. The activity of CYP450 1A2 in rifampicin groups with a concentration of 12.5 micromol/L was (6.07 +/- 0.55) mAU.min, which had significant difference compared with that in the negative control group (P < 0.05). There was no statistical difference of CYP1A2 activity between rifampicin with 25 micromol/L [(4.93 +/- 0.57) mAU.min] and the negative control group (P > 0.05). The activity of CYP1A2 of groups with two kinds of different concentrations of isoniazid and rifampicin combined groups was (3.27 +/- 0.96), (3.97 +/- 0.25) mAU.min respectively, which had significant difference compared with that in the negative control group (P < 0.05). (2) The activity of CYP3A4 in isoniazid groups with concentrations of 25 micromol/L and 50 micromol/L was (5.40 +/- 1.35), (2.63 +/- 0.06) mAU.min respectively, which had significant difference compared with that in the negative control group [(12.53 +/- 0.51) mAU.min, P < 0.01]. The activity of CYP3A4 in rifampicin groups with concentrations of 12.5 micromol/L and 25 micromol/L was (165.17 +/- 11.47), (120.20 +/- 15.73) mAU.min respectively, which had significant difference compared with that in the negative control group (P < 0.01). The activity of CYP3A4 in the three isoniazid and rifampicin combined groups with three kinds of different concentrations was (118.37 +/- 8.90), (77.53 +/- 6.91), (68.73 +/- 4.72) mAU.min respectively, which had significant difference compared with that in the negative control group (P < 0.01), but they were lower than those in rifampicin groups with corresponding concentrations (P < 0.05). CONCLUSIONS: Isoniazid and rifampicin in the range of maximum clinical blood concentration have no significant inducing or inhibiting effect on the activity of CYP1A2 of healthy adult human primary hepatocytes. Isoniazid in the range of maximum clinical blood concentration can inhibit the activity of CYP3A4, while rifampicin can induce the activity of CYP3A4; the combined effect of isoniazid and rifampicin being the induction of CYP3A4 activity, but the inducing effect was less than that of rifampicin alone with the same concentration.

[Correlation between the area of arterial blood gases graph and the acid-base disturbance in 92 cases with chronic obstructive pulmonary disease].

OBJECTIVE: To study the correlation between the area of arterial blood gases graph (ABGG) and the acid-base disturbance (ABD). METHODS: Using the software developed by ourselves, the results of arterial blood gases and concomitant electrolyte (K(+), Na(+), Cl(-), HCO(3)(-)) determination in 313 episodes were analyzed in 92 cases with chronic obstructive pulmonary diseases (COPD) during hospitalization, and the correlation was studied. RESULTS: (1)On admission, among 92 cases, in 9 cases the results of blood gas analysis were in the area of compensated ventilation and deranged gas exchange (area IV), in 82 cases the results were in the area of insufficient ventilation and deranged gas exchange (area V), and 1 case in the area of excessive ventilation and deranged gas exchange (area VI). There were 8 types of the ABDs, and respiratory acidosis (RAC)+metabolic alkalosis (MAL), RAC, RAC+metabolic acidosis (MAC) with increase in anion gap (AG) ranked in above order. (2)At the time of discharge, in 2 cases the distribution of ABGG was in the normal area (area I), 5 cases in the area IV, 20 cases in the area V, and 1 case in the area VI. There were 5 types of the ABDs. RAC+MAL and RAC ranked in the first two positions. (3)During hospitalization, the correlations between the area of ABGG and ABD in 313 episodes were as follows: ABD normal and MAC of AG increased in the area I; ABD normal and MAL, MAC with AG increased, RAC+MAC, respiratory alkalosis (RAL)+MAL, MAL+MAC of AG increased, RAC+MAL+MAC of AG increased and RAL+MAL+MAC of AG increased in the area IV; RAC, MAL, RAC+MAL, RAC+MAC of AG increased, MAL+MAC of AG increased, and RAC+MAL+MAC of AG increased in the area V; MAC of AG increased, RAC+MAC, RAL+MAL, MAL+MAC of AG increased, and RAL+MAL+MAC of AG increased in the area VI. No ABD occurred in the areas II and III. CONCLUSION: The correlation between ABGG and ABD can be analyzed easily and quickly and the accuracy of results is ensured by using the software.

[The expression of survivin messenger RNA in sputum and cancerous tissue in human lung cancer].

OBJECTIVE: To study the diagnostic significance of the expression of survivin mRNA in transbronchial biopy samples and sputum samples in lung cancer. METHODS: The resected lung cancer tissues and their para-carcinomatous normal tissue specimens of 41 patients with lung cancer and tissue specimens of 9 patients with benign pulmonary diseases were studied. 110 bronchial biopsy specimens from 80 patients with lung cancer and 30 patients with benign pulmonary diseases and their 160 sputum samples were also evaluated. RT-PCR was performed for the detection of survivin mRNA expression in specimens. The results were compared with their histological or cytological examinations. RESULTS: (1) The positive rate of survivin mRNA in resected lung cancer tissues (29/41; 70.7%) was higher than that in the para-carcinomatous normal tissues (7/41; 17.1%) and the benign pulmonary tissues (1/9; 11.1%, P < 0.05). There was no statistical difference (P > 0.05) in the positive rate of survivin mRNA between para-carcinomatous normal tissues and the benign pulmonary disease tissues. In the bronchial biopsy samples, the positive rate of survivin mRNA in 80 lung cancer tissues (51/80; 63.8%) was also higher than that in the benign pulmonary tissues (4/30; 13.3%, P < 0.05). There were no relationships between survivin mRNA expression and age, sex, histopathological subtype, differentiation, or lymph node metastases of the patients. The sensitivity and specificity of diagnosis for lung cancer by detecting survivin mRNA in resected cancer tissue and transbronchial biopsy tissue were 63.8% - 70.7% and 86.7% - 88.9%, respectively. (2) The sensitivity of cytological examinations combined with detecting survivin mRNA in sputum samples was higher than that of either cytological examination or survivin mRNA detection of sputum samples alone (P < 0.05). The sensitivity of the diagnosis for lung cancer increased from 47.1% (sputum cytology alone) to 80.2% (sputum survivin mRNA detection combined with sputum cytology, P < 0.05) and the negative predictive value increased from 37.9% for sputum cytology alone to 57.9% (P < 0.01) for sputum survivin mRNA detection combined with sputum cytology. The specificity did not change significantly. CONCLUSIONS: Detecting survivin mRNA expression from bronchial biopsy specimens might be used as one of the specific molecular markers for diagnosis of lung cancer, while the detection of survivin mRNA from sputum samples as a new ancillary diagnostic method for lung cancer.

[The relationship between vitamin D binding protein gene polymorphism and chronic obstructive pulmonary disease].

OBJECTIVE: To determine the association between vitamin D binding protein (VDBP) gene and chronic obstructive pulmonary disease (COPD) susceptibility in Han Nationality in China. METHODS: This was a case-control study. Blood samples were taken from sixty-nine smokers with COPD (group A) and fifty-two smokers without COPD (Group B). DNA was extracted from the white blood cells of both groups. Genotypes of VDBP were measured with polymerase chain reaction and restriction fragment length polymorphism analysis. RESULTS: In group A the proportion of allele 1F homozygote was significantly Higher than that in group B (33.3% versus 11.5%, p = 0.005). The odds ratio for 1F-1F was 3.5 (95%confidence interval 1.9294-9.429) for COPD. Phenotype 2-2 showed a decreasing trend in group A, but the difference was not significant due to the small sample size. The frequency of allele 1F in group A was 0.558 compared with 0.404 in group B (p = 0.018). The frequency of allele 2 in group A was 0.196 compared with 0.308 in group B (p = 0.044). CONCLUSIONS: Our result suggests that allele 1F is one of the risk factors for COPD associated with smoking. 1F homozygote may increase the risk of COPD. On the other hand, allele 2 might have a protective effect on pathogenesis of COPD.

[Development and its clinical application of an acid-base balance computer software].

OBJECTIVE: To develop acid-base balance computer software for estimating acid-base disturbance rapidly and accurately. METHODS: The data module was set up, based on Henderson-Hasselbalch (H-H) and compensation formula, and the program was written with Delphi 5.0. RESULTS: The right RESULTS was 100.0 percent in 118 cases of acid-base disturbance patients by the software, while those with the right RESULTS was 89.8 percent by hands (P<0.05). In 20 cases pure acid-base disturbance, 70 cases double acid-base disturbance and 28 cases with triple acid-base disturbance, it took only (9.0+/-0.6) seconds, (8.9+/-1.3) seconds and (9.1+/-1.6)seconds respectively, with software, while which were much less than those by hands ((20.4+/-16.8) seconds, (92.4+/-45.3) seconds and (128.6+/-66.4) seconds, respectively, P<0.001). CONCLUSION: This software has a terse interface, by which the acid-base disturbance was concluded exactly and quickly.

[A retrospective study on the treatment of patients with chronic obstructive pulmonary disease by face mask mechanical ventilation].

OBJECTIVE: To assess the effect and the influencing factors in face mask mechanical ventilation (FMMV) for the treatment of COPD with respiratory failure (RF), and to compare the efficacy and efficiency of FMMV with endotracheal intubation mechanical ventilation (ETMV). METHODS: From 1991 to 2001, 4 ventilating periods were named initial, middle, late, and extending stage according to ventilators, face masks and mechanical ventilating methods. The results of FMMV in the 4 periods were compared. The results in patients with FMMV who reached the endotracheal intubation criteria were also compared with those in ETMV ones from 1988 to 1990. RESULTS: 385 patients with COPD and RF were admitted to RICU. The proportion of FMMV in all MV patients increased [48% (11/23), 79% (15/19), 85% (29/34), 91% (223/246)]; at the same time the effective rate of FMMV [46% (5/11), 67% (10/15), 83% (24/29), 89% (198/223)] and the total effective rate [71% (35/49), 71% (20/28), 81% (34/42), 88% (234/266)] also increased. 83 percent of the patients who suffered from severe disturbances of consciousness in the late and extending stage were successfully weaned from FMMV. Complications including the rates of nasal bridge erosion [27% (3/11), 13% (2/15), 7% (2/29), 2% (4/223)] and the rates of stomach distension [46% (5/11), 40% (6/15), 21% (6/29), 5% (11/223)] all decreased. The ventilation times and length of stay were significantly shorter than those in patients with ETMV. The rate of nosocomial pneumonia and the mortality of inpatients were significantly lower. CONCLUSION: The ventilating techniques, ventilators and face masks were the main factors influencing FMMV results. FMMV could be used rationally for most COPD patients with RF as a valuable alternative to ETMV.

[Clinical application of a software for the analysis of arterial blood gases graph in 231 patients with chronic obstructive pulmonary disease].

OBJECTIVE: To evaluate the clinical significance of a computer software for the analysis of arterial blood gases graph (ABGG) in chronic obstructive pulmonary disease(COPD). METHODS: The software was developed with Win98 as the operating platform and the visual software Delphi 5.0 from Borland Company, and it was used for evaluation of the changes in arterial blood gases (ABG) of 231 COPD cases. RESULTS: (1) With the software it took only (4.7+/-0.5)s to draw and analyze an ABGG of COPD patients during oxygen inhalation; the time was much shorter than manual analysis (90.2+/-4.9)s, P<0.001. (2) During acute attack, the distributions of the arterial blood gases parameters on ABGG were as follows: 55.4% of cases in the area of insufficient ventilation and deranged gas exchange (area 5), 22.9% of cases in the area of compensated ventilation and deranged gas exchange (area 4), 21.6% of cases in the area of excessive ventilation and deranged gas exchange (area 6). (3) When the COPD patient's condition improved, the location of ABG in ABGG shifted from area 5 to area 4 or area 6. The distributions of the arterial blood gases parameters on ABGG of 106 cases on admission were significantly different from those at the time of discharge. (4) With deterioration of patient's condition, it shifted to area 5. Before death, the arterial blood gases parameters were exclusively in the area 5. CONCLUSION: The computer software for ABGG shortened the time to draw and evaluate ABG during oxygen inhalation, and it could reflect the changes in patient's condition promptly.