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Background: Mycobacterium vaccae vaccine, a composition of Mycobacterium proteins, has been known to have bidirectional immunomodulatory functions. Recent studies have shown that M. vaccae has a th...

Background: Severe acute respiratory syndrome coronavirus 2 infection is associated with strong infectiousness and has no effective therapy. We aimed to explore the efficacy and safety of Mycobacterium vaccae nebulization in the treatment of Coronavirus Disease 2019 (COVID-19). Methods: In this randomized, double-blind, placebo-controlled clinical trial, we included 31 adult patients with moderate COVID-19 who were admitted to the Fourth People's Hospital of Nanning (Nanning, China) between January 22, 2020 and February 17, 2020. Patients were randomly divided into two groups: group A (standard care group) and group B (M. vaccae in combination with standard care group). The primary outcome was the time interval from admission to viral RNA negative conversion (oropharyngeal swabs were used in this study). Secondary outcomes included chest computed tomography (CT), mortality, length of hospital stay, complications during treatment, and so on. Patients were followed up to 4 weeks after discharge (reexamination of viral RNA, chest CT, etc.). Results: Nucleic acid test negative conversion time in group B was shorter than that in group A (2.9 days [2.7-8.7] vs. 6.8 days [3.3-13.8]; p = 0.045). No death and no conversion to severe or critical cases were observed in both groups. Two weeks after discharge, neither "relapse" nor "return to positive" cases were found. Four weeks after discharge, it was found that there was no case of " relapse " or "return to positive" in group B, and 1 patient in group A showed "return to positive", but there was no clinical manifestation and imaging progression. No adverse reactions related to M. vaccae were found during observation period. Conclusion: M. vaccae treatment might shorten the time interval from admission to viral RNA negative conversion, which might be beneficial to the prevention and treatment of COVID-19. Clinical Trial Registration: ChiCTR2000030016.

Background: Respiratory syncytial virus (RSV) infection is the most common cause of acute lower respiratory tract infection in children, leading to their death. Currently, no effective prevention and treatment methods for RSV infection are available. RSV and many other unknown viruses pose a serious threat to human health. Our previous study demonstrated that Mycobacterium vaccae nebulization can protect against allergic asthma. As RSV infection and asthma are closely related, we hypothesized that M. vaccae could protect against pulmonary RSV infection. Therefore, we evaluated the effect of M. vaccae on RSV infection in Balb/c mice. Methods: The mice were randomized into three groups: normal, RSV, and M. vaccae. One week before the RSV infection model was established, the mice in the M. vaccae group were nebulized with M. vaccae. On the fourth day after RSV infection, airway responsiveness, airway inflammation, pulmonary RSV infection, mRNA levels of pulmonary toll-like receptor (TLR) 7 and TLR8, and pulmonary NF09, acetylcholine, and epidermal growth factor regulator (EGFR) expression levels in all mice were measured. Results: The airway inflammation in the M. vaccae group was alleviated compared with that in the RSV group. In the M. vaccae group, the pulmonary mRNA level of RSV and the pulmonary expression levels of NF09, acetylcholine, and EGFR were decreased considerably, whereas the mRNA levels of TLR7 and TLR8 were increased significantly. Conclusions: One-week nebulization of M. vaccae can protect against RSV infection in Balb/c mice. The mechanism involves the regulation of neurotransmitters and expression of TLR7, TLR8, and EGFR.

Objectives: Bronchial asthma can be effectively controlled but not be cured, its etiology and pathogenesis are still unclear, and there are no effective preventive measures. The key characteristic ...

Background: Mycobacterium vaccae nebulization imparted protective effect against asthma in a mouse model. The Jagged2-γδT17 signal transduction pathway plays an important role in bronchial asthma. However, the effect of M. vaccae nebulization on the Jagged2-γδT17 signal transduction pathway in mouse models of asthma remains unclear. Methods: In total, 30 female C57 mice were randomized to normal control (group a), asthma control (group b), M. vaccae nebulization prevention,and M. vaccae nebulization treatment (group d) groups. Asthma mice models were created using ovalbumin (OVA). The Notch signaling pathway was blocked by DAPT inhibitors. Airway hyperreactivity (AHR) was measured by noninvasive lung function tests. Histopathological analyses using blue-periodic acid Schiff along with hematoxylin and eosin were performed. Immunohistochemistry, immunofluorescence, and a Western blotting assay allowed for the detection of lung protein expressions, while spleen expressions of IL-17+γδT+ cytokines were assessed with FLOW cytometry. One-way analysis of variance for within-group comparisons, the least significant difference t-test or Student-Newman-Keuls test for intergroup comparisons, and the nonparametric rank sum test for analysis of airway inflammation scores were used in the study. Results: Asthmatic mice models demonstrated downregulated Notch signaling pathway activation and decreased γδT cells and IL-17 cytokine secretion. There was also increased Jagged2 protein expression which correlated positively with γδT+IL-17+ secretion. In asthmatic mice, the expressions of Jagged2 and γδT17, along with airway inflammation and airway reactivity, were all decreased after M. vaccae exposure (pM. vaccae led to a significant decrease in Jagged2 and γδT17 expressions in asthmatic mice, indicating its utility in asthma prevention.

Recently, Chen and Ma [A generalized shift-splitting preconditioner for saddle point problems, Applied Mathematics Letters, 43 (2015) 49-55] introduced a generalized shift-splitting preconditioner for saddle point problems with symmetric positive definite (1,1)-block. In this paper, I establish a parameterized shift-splitting preconditioner for solving the large sparse augmented systems of linear equations. Furthermore, the preconditioner is based on the parameterized shift-splitting of the saddle point matrix, resulting in an unconditional convergent fixed-point iteration, which has the intersection with the generalized shift-splitting preconditioner. In final, one example is provided to confirm the effectiveness.

Objective In this study, we aimed to identify prognostic immune-related genes and establish a prognostic model for laryngeal cancer based on these genes. Methods Transcriptome profiles and clinical data of patients with laryngeal cancer were downloaded from The Cancer Genome Atlas database. Integrated bioinformatics analyses were performed to identify genes associated with prognosis. Results Thirty prognostic immune-related genes for laryngeal cancer were identified. We constructed a regulatory network of prognosis comprising transcription factors and immune-related genes. Multivariate Cox regression analyses identified 15 immune-related genes in the network that were used to establish the prognostic model. The model exhibited excellent prognostic prediction ability with a high area under the curve value (0.916). The calculated risk score based on expression of the 15 immune-related genes was shown to be an independent prognostic factor for laryngeal cancer. Conclusion We identified prognostic immune-related genes and established a prognostic model for laryngeal cancer, which might help identify novel predictive biomarkers and therapeutic targets of laryngeal cancer.

The objective of this study was to investigate the effect of Mycobacterium vaccae on Jagged 1 and gamma delta T17 (γδT17) cells in asthmatic mice. An asthma mouse model was established through immunization with ovalbumin (OVA). Gamma-secretase inhibitor (DAPT) was used to block the Notch signaling pathway. M. vaccae was used to treat asthma, and related indicators were measured. Blocking Notch signaling inhibited the production of γδT17 cells and secretion of cytokine interleukin (IL)-17, which was accompanied by a decrease in Jagged1 mRNA and protein expression in the treated asthma group compared with the untreated asthma group. Similarly, treatment with M. vaccae inhibited Jagged1 expression and γδT17 cell production, which was associated with decreased airway inflammation and reactivity. The Notch signaling pathway may play a role in the pathogenesis of asthma through the induction of Jagged1 receptor. On the other hand, the inhibitory effect of M. vaccae on Jagged1 receptor in γδT17 cells could be used for the prevention and treatment of asthma.

Background Mycobacterium vaccae nebulization imparted protective effect against allergic asthma in a mouse model. The TGF-β/Smad signal transduction pathway plays an important role in allergic bronchial asthma. However, the effect of M. vaccae nebulization on the TGF-β/Smad signal transduction pathway in mouse models of allergic asthma remains unclear. This study investigated the preventive effect of M. vaccae nebulization during bronchial asthma in a mouse model and elucidate the implication of TGF-β/Smad signal transduction pathway in the process. Methods In total, 24 female Balb/c mice were randomized to normal control (group A), asthma control (group B), and M. vaccae nebulization (group C) groups. Both groups B and C were sensitized using ovalbumin for establishment of the asthmatic model; group A received phosphate-buffered solution. Prior to the establishment of asthma, Group C was nebulized with M. vaccae. Airway responsiveness was measured in all the groups, using a noninvasive lung function machine before and 24 h after establishment of the asthmatic model. The animals were then harvested, and bronchoalveolar lavage fluid (BALF) and lung tissue were collected. The total cell counts in BALF was estimated. Protein expression of TGF-β1, TβR1, Smad1, and Smad7 was detected by immunohistochemistry. The population of CD3 + γδT, IL-13 + CD3 + T, TGF-β + CD3 + T, IL-13 + CD3 + γδT, and TGF-β+ CD3+ γδT cells were detected by flow cytometry. One-way analysis of variance for within-group comparisons, the least significant difference t-test or Student–Newman–Keuls test for intergroup comparisons, and the nonparametric rank sum test for analysis of airway inflammation scores were used in the study. Results The eosinophil count; protein expression of TGF-β1, TβR1, and Smad1; and percentages of CD3 + γδT and IL-13 + CD3 + T cells were significantly lower in the M. vaccae nebulization group than in the asthma control group (P

The overexpression of TSLP and DNA methylation in asthma were both risk factors the relationship was not clear. This study aimed to investigate the relationship between methylation status of TSLP promoter and mRNA/protein expression in asthmatic airway epithelial cells. Human bronchial epithelial cells were cultured in vitro and divided into: Control group, treated with PBS, model group, sensitized with LPS (10 μg/mL) for 12 h (37 °C, 5% CO2). Other groups were cultured with the pCMV3 plasmid (M + NC/pCMV), pGPH1 plasmid (M + NC/pGPH), DNMT1/pCMV3 plasmid (M + DNMT1/pCMV), and DNMT1/pGPH1 plasmid (M + DNMT1/pGPH) for 48 h. The expression of DNA methyltransferase 1 and TSLP were measured using real-time PCR and western blotting. Compared with the control group, TSLP mRNA (1.00 ± 0.00 vs. 2.82 ± 0.81 vs. 1, P

Ozone is a strong oxidant in air pollution that exacerbates respiratory disorders and is a major risk factor for acute asthma exacerbation. Ozone can induce reactive oxygen species (ROS) and airway neutrophilic inflammation. In addition, γδT17 cells contribute to IL-17A production upon ozone challenge, resulting in neutrophilic inflammation. It is known, however, that Nrf2 can ameliorate oxidative stress. We therefore investigated whether RTA-408, an Nrf2 activator, can attenuate airway inflammation and inhibit ROS production and whether this effect involves γδT17 cells. Balb/c mice were sensitized/challenged with ovalbumin (OVA) and followed by ozone exposure. We investigated the effect of Nrf2 activator RTA-408 on airway hyperresponsiveness, neutrophilic airway inflammation, cytokine/chemokine production, and OVA-specific IgE level in a mouse model of O3 induced asthma exacerbation. Furthermore, malondialdehyde (MDA) and glutathione (GSH) levels in lung and intracellular ROS were measured. IL-17+ γδT cell percentage by flow cytometer was determined. Nrf2 protein expression by western blot was also examined. We observed that RTA-408 attenuated ROS release during ozone-induced asthma exacerbation and suppressed neutrophil lung infiltration. RTA-408 decreased pro-inflammatory cytokine production and reduced the percentage of IL-17+ γδT cells. Thus, our results suggest that RTA-408 does attenuate airway inflammation in a murine model of ozone-induced asthma exacerbation.

We aimed to explore the imbalance between the T helper 17 γδT cells (γδT17) and the regulatory γδT cells (γδTreg) in asthmatic mice. Male Balb/c mice were randomly divided into the normal control group and the asthmatic model group. The asthmatic model group mice were intraperitoneally injected with the mixture of ovalbumin (OVA)/Al(OH)3 and then activated by exposure of the animals to OVA atomization. Airway hyperresponsiveness (AHR) was determined by a non-invasive lung function machine. Hematoxylin and eosin and Alcian blue-periodic acid Schiff staining were done for histopathological analysis. Interleukin (IL)-17 and IL-35 levels in bronchoalveolar lavage fluid were detected by ELISA. The percentage of IL-17+ γδT cells and Foxp3+ γδT cells in spleen cells suspension were detected and the transcription levels of RORγt and Foxp3 in the lung tissue were determined. Compared with the normal control, the severity of airway inflammation and AHR were higher in the asthmatic mice. Furthermore, mice in the asthmatic group displayed significant increases of IL-17+ γδT cells, expression of IL-17A, and RORγt, whereas control mice displayed marked decreases of Foxp3+ γδT cells, expression of IL-35, and transcription factor Foxp3. In addition, the mRNA expression of RORγt was positively correlated with the percentage of IL-17+γδT cells, and the mRNA level of Foxp3 was positively correlated with the percentage of Foxp3+ γδT cells. The imbalance of γδT17/γδTreg in the asthmatic mice may contribute to the pathogenesis of OVA-induced asthma.

Objective and Design Our study aimed to determine the effect of mild hypothermia (MHT) on the expression of toll-like receptor 2 (TLR2) in lung tissue with acute lung injury. The animals were randomly divided into control, model and mild hypothermia groups. Methods A total of 40 rats was used in the study. Acute lung injury was induced by lipopolysaccharide and MHT was maintained at 32.5∼33.0°C using body surface ice-bag placement combined with animal thermostat system. The ratio of PaO 2 /FiO 2 was recorded. The mRNA and protein expressions of TLR2 were measured by real-time polymerase chain reaction and western blotting respectively. Moreover, enzyme linked immunosorbent assay were used for the quantification of TNF-α. Results The ratio of PaO 2 /FiO 2 was increased by MHT. TLR2 and TNF-α were increased in the rat lung 1h and 8h in the rats with acute lung injury while they were significantly decreased by MHT. Histological examination revealed that MHT alleviated the degree of inflammation. Conclusion Our study suggested that MHT might improve the lung function by inhibiting the inflammation via down-regulating the expressions of TLR2 in the acute injury lung tissues.

Objective To discover the effect of partial splenic embolization on the immune function of cirrhotic patients with hypersplenism. Methods Patients involved in the study were enrolled and divided into three groups, including control group, experimental group, and complication group. Numbers of CD3 + , CD4 + and CD8 + T cells and CD4 + CD25 + CDl27 low/− Treg cells in the peripheral blood of patients before surgery, 1 month, 6 months, 1 year, and 2 years after surgery were analyzed by fluorescence active cell sorting (FACS). Contents of immunoglobulins (IgA, IgG and IgM) were analyzed by auto immunoassay analyzer. Results In the peripheral blood of patients from experimental group, numbers of CD3 + , CD4 + and CD8 + T cells initially declined, but afterwards increased to normal level; in the peripheral blood of patients from complication group, CD3 + and CD8 + T cells showed the same trend, but the number of CD4 + T cells was below normal level at all detection times. Furthermore, CD3 + , CD4 + and CD8 + T cells in the peripheral blood of patients from complication group were initially less than those in experimental group, and afterwards were comparable between two groups. In patients from both experimental group and complication group, the number of CD4 + CD25 + CDl27 low/− Treg cells increased 1 month and 6 months after surgery, and gradually restored to normal level. CD4 + CD25 + CDl27 low/− Treg cell counts in patients from complication group were initially more than those in patients from experimental group 1 month and 6 months after surgery, but then they were comparable. Furthermore, contents of immunoglobulins (IgA, IgG and IgM) were comparable in three groups at all detection times. Conclusion Partial splenic embolization influenced the immune function of cirrhotic patients with hypersplenism in the short term but the immune function could afterwards gradually restore to normal. Our results implicated that measures that prevent infection and improve immune function were necessary in early stage after undergoing PSE in order to reduce complications.

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Asthma is a heterogeneous disease characterized by chronic airway inflammation. CD4(+) T-helper 9 (Th9) cells are closely linked to asthma, helping to regulate inflammation and immunity. Epidemiological studies showed that mycobacteria infections are negatively associated with asthma. Our previous research showed that inactivated Mycobacterium phlei nebulization alleviated the airway hyperresponsiveness and inflammation of asthma. However, the relationship between Th9 cells and mycobacteria remains unknown. Here, we evaluated the relationship between Mycobacterium vaccae nebulization and Th9 cells in asthmatic mice. Eighteen Balb/c mice were randomized into 3 groups of 6 mice each (normal control group, asthma control group, and nebulization asthma group [Neb. group]). The Neb. group was nebulized with M. vaccae one month before establishment of the asthmatic model with ovalbumin (OVA) sensitization, and the normal and asthma control groups were nebulized with phosphate-buffered saline. The hyperresponsiveness of the mouse airways was assessed using a non-invasive lung function machine. Lung airway inflammation was evaluated by hematoxylin and eosin and periodic acid-Schiff staining. Cytokine interlukin-9 (IL-9) concentration and OVA-specific IgE in the bronchoalveolar lavage fluid were measured by enzyme-linked immunosorbent assays. The percentages of γδTCR+ CD3+, IL-9+CD3+, IL-10+CD3+ lymphocytes, and IL9+γδT and IL-10+γδT cells were detected by flow cytometry. The airway inflammation and concentration of IL-9 and OVA-specific IgE were significantly reduced in the Neb. group compared to the asthma control group. The Neb. group had lower airway hyperresponsiveness, percentages of γδTCR+CD3+ and IL-9+CD3+ lymphocytes, and IL9+γδT cells, and higher percentages of IL-10+CD3+ lymphocytes and IL-10+γδT cells compared to the asthma control group. Thus, mouse bronchial asthma could be prevented by M. vaccae nebulization. The mechanism could involve M. vaccae-mediated effects on induction of IL-9 secretion and suppression of IL-10 secretion from γδT cells. γδT cells showed prominent IL-10 expression, indicating that they possibly belong to the Th9 family.

The aim of the present study was to examine the effects of interleukin-5 (IL-5) inhalation on changes in the activity and number of circulating eosinophils, as well as concentrations of serum total IgE, in allergic asthmatics. A randomized double-blind, placebo-controlled study design was employed in which each subject acted as his or her own control. Eight nonsmoking patients with allergic asthma were administered recombinant human IL-5 by nebulization. Total white blood cell counts and differentials, as well as concentrations of ECP and total IgE in serum, were determined before and at 2, 24, and 48 h after inhalation. Our results demonstrated that eosinophil numbers increased from baseline (3.6 +/- 1.1 x 10(5)/ml) to 6.3 +/- 1.2 x 10(5)/ml (P0.01) at 24 h and to 5.7 +/- 0.9 x 10(5)/ml (P0.01) at 48 h after IL-5 inhalation in asthmatics. Accompanying this significantly increased blood eosinophilia were significantly elevated serum ECP levels. Compared with baseline (6.3 +/- 1.1 ng/ml), ECP levels increased with time following IL-5 inhalation, reaching 17.6 +/- 2.8 ng/ml (P0.01) at 24 h and remaining elevated at 48 h (18.1 +/- 2.9 ng/ml, P0.01). IL-5 inhalation had no significant effect on levels of serum total IgE, however. These findings provide direct evidence that nebulized IL-5 not only induces a significant blood eosinophilia but also results in the activation of circulating eosinophils. Our data further support the importance of IL-5 in the pathogenesis of bronchial asthma in humans.

To investigate diagnostic value of creatinine clearance rate (CCr) based on serum cystatin C (SCys C) in acute kidney injury (AKI), and whether it could predict the need for renal replacement therapy (RRT).The patients enrolled with the length of intensive care unit (ICU) stay over 3 days were collected from August 2010 to May 2011. According to the diagnosis of AKI during the ICU stay, patients were divided into the AKI group (n=21) and non-AKI group (n=30). After patients were admitted, the level of SCys C and creatinine (SCr) were measured so as to count CCr based on SCys C (SCys C-CCr) or on SCr (SCr-CCr) respectively, meanwhile urine volume and acute physiology and chronic health evaluation II (APACHE II) score were monitored. The value of CCr counted by SCys C and SCr on predict AKI and the correlations between RRT were compared.SCr-CCr and SCys C-CCr in AKI group both were significantly lower than non-AKI group all the way through on admission, and 2 days and 1 day before AKI diagnosed and the day AKI diagnosed. The level of SCys C-CCr on 2 days prior to AKI diagnosed was significantly lower than the day admitted (70.6±8.4 ml×min(-1)×1.73 m(-2) vs. 114.8±15.8 ml×min(-1)×1.73 m(-2), P0.01), whereas the level of SCr-CCr were not significantly changed (76.4±19.3 ml×min(-1)×1.73 m(-2) vs. 78.7±22.1 ml×min(-1)×1.73 m(-2), P0.05). Receptor operative curve (ROC) analysis indicated that SCys C-CCr could predict AKI earlier than SCr-CCr, as the area under curve (AUC) of SCys C-CCr and SCr-CCr on 2 days prior to AKI diagnosed were 0.859 and 0.664, respectively, and the sensitivity were 90.5% and 47.6%, the specificity were 76.2% and 81.0%. In AKI group 6 patients were treated with RRT, the AKI patients receiving RRT had significantly higher APACHE II score on admission (29.6±4.5 vs. 17.0±5.6, P0.05) and less urine volume within 24 hours (740±465 ml vs. 1780±1230 ml, P0.05) than patients not received RRT, however, SCys C-CCr has no significant difference between the sub-group (50.4±11.2 ml×min(-1)×1.73 m(-2) vs. 53.0±8.4 ml×min(-1)×1.73 m(-2), P0.05). SCys C-CCr did not predict the need of RRT on the day to diagnose AKI (AUC=0.65).The sensitivity of SCys C-CCr were high, but its specificity not. The SCys C-CCr may be helpful for excluding diagnose of AKI in high risk patients. However, it could not predict the need for renal replacement therapy on the day AKI diagnosed.

To investigate the role of three K+ channels-delayed rectifier K+ channel (Kv), large-conductance Ca(2+)-activated K+ channel (BK(Ca)) and ATP-sensitive K+ channel (K(ATP)) in the regulation of the resting and contracting tone of rat bronchial smooth muscle (BSM).By measuring the isometric tone of bronchial strips in vitro, the regulating effects of the three K+ channels on the tone of rat BSM were observed.(1) Kv blocker 4-aminopyridine (4-AP) caused concentration dependent contraction in resting bronchial strips, but BK(Ca) blocker tetraethylammonium (TEA) and K(ATP) blocker glibenclamide (Glib) had no such effects. (2) The bronchial epithelium had no effects on the contraction induced by 4-AP, but nifedipine, a Ca2+ channel blocker, significantly suppressed it. (3) Before or after treatment with 0.1 mmol/L histamine or 50 mmol/L KCl, administration of TEA (1 or 5 mmol/L) or 0.1 mmol/L 4-AP could significantly increase the contraction induced by histamine and KCl in bronchial strips. But Glib (10 micromol/L) had no effect on it.Not BK(Ca) and K(ATP) but Kv participated in the regulation of the resting tone in rat BSM. The closure of BK(Ca) or Kv increased the contracting tone induced by histamine or KCl in rat BSM in vitro, but K(ATP) had no such effect on it.

To explore the role of gammadeltaT cells in the pathogenesis of allergic bronchial asthma.Forty patients with Der.p and Der f allergy and partly-controlled asthma were involved in a random study. Besides the fact that all patients were treated with same basic treatment. Patients in group A were nebulized with 5 mL normal saline while group B with specific allergen twice a week for six months. The percentage of gammadeltaT cells in peripheral blood and induced sputum and the percentage of IL-4(+) and IFN-gamma(+) gammadeltaT cells were detected by flow cytometry(FCM) before and after treatment.The percentage of gammadeltaT cells in group B decreased after treatment, and the percentage of IFN-gamma(+) gammadeltaT cells in both peripheral blood and induced sputum increased significantly in group B than that in group A.gammadeltaT cells, which may participate in the pathogenesis of bronchial asthma, may contribute to the prevention and treatment of bronchial asthma by desensitization of allergen nebulization.

To investigate the regulatory role of nitric oxide (NO)-nuclear factor kappaB (NF-kappaB) signal pathway in cytokine expressions, cell proliferation and apoptosis of T lymphocytes from asthmatic patients.We observed the expressions and secretions of IL-4, IL-5 and IFNgamma from T lymphocytes and measured the proliferation and apoptosis of these cells by using in situ hybridization, electrophoresis mobility shift assay (EMSA), immunofluorescence, flow cytometry, two antibodies sandwich enzyme linked immuno-sorbent assay and MTT, and by administering NO donor sodium nitroprusside (SNP) and NF-kappaB inhibitor pyrrolidine dithio-carbamic acid (PTDC).(1) The mRNA expression and protein secretion of IL-4 and IL-5 by, as well as the proliferation rate of T lymphocytes from asthmatic patients were augmented, while the mRNA expression and protein secretion of IFNgamma by, as well as the apoptosis rate of T lymphocytes from asthmatic patients were decrease as compared with the values of the control groups (P0.05). (2) Low dose of SNP (10 micromol/L) up-regulated the expression of these three cytokines, promoted the proliferation rate and suppressed the apoptosis rate of T lymphocytes. However, middle or high dose of SNP (100 micromol/L, 1 mmol/L, 10 mmol/L) dose-dependently down-regulated cytokine expressions and cell proliferation rate and promoted apoptosis rate. (3) PDTC inhibited the higher expressions of IL-5 and IFNgamma induced by 10 micromol/L SNP and strengthened the inhibitory effect of 1 mmol/L SNP on the expressions of IL-5 and IFNgamma. At the same time, PDTC weakened the promoting effect of 10 micromol/L SNP and strengthened the inhibitory effect of 1 mmol/L SNP on the proliferation of T lymphocytes. (4) The percentage of NF-kappaB-activated cells and NF-kappaB activity were significantly increased in T lymphocytes treated with low dose of SNP (P0.05); whereas these parameters were decreased in cells treated with middle or high dose of SNP (P0.05).The hyper-expression of inflammatory cytokines and the increased proliferation and decreased apoptosis of T lymphocytes from asthmatic patients are related to the abnormal over-activation of NF-kappaB. NO has bi-phasic effects on the expressions of IL-5 and IFNgamma and on the proliferation and apoptosis of T lymphocytes from asthmatic patients via its bi-phasic regulations on the activity of NF-kappaB.

To explore the inhibitory mechanism of phosphodiesterase(PDE) inhibitor on expression of IL-8 mRNA in peripheral blood mononuclear cells (PBMCs) from chronic obstructive pulmonary disease(COPD) patients.PBMCs isolated from 20 COPD patients and 15 healthy subjects were co-cultured with non-selective inhibitor theophylline or PDE type IV inhibitor Rolipram. The expression of IL-8 mRNA was assayed by RT-PCR. The expression of NF-kappaB was determined by immunocytochemical staining. The content of I-kappaB protein was detected by Western blot.The expression of IL-8 mRNA was elevated and the percentage of NF-kappaB nucleus positive cells was higher in COPD patients than in normal controls(P0.01), while the expression of I-kappaB was lower in COPD patients (P0.01). Theophylline of 1 mmol/L caused a decrease in the percentage of NF-kappaB nucleus positive cells (P0.05). The expression of IL-8 mRNA and I-kappaB were not affected by theophylline at the dose of 100 micromol/L or 1 mmol/L. Rolipram inhibited the expression of IL-8 mRNA and the activation of NF-kappaB(P0.05).The results indicated that IL-8 mRNA may play a pathogenic role in the development of COPD and that selective PDE type IV inhibitor Rolipram inhibit the expression of IL-8 mRNA via NF-kappaB.

To explore the method of selective culture of gamma delta T cells in peripheral blood and bronchoalveolar lavage fluid(BALF) for obtaining pure gamma delta T cell subset.A novel attack-panning method for selectively culturing gamma delta T cells was set up. The peripheral blood mononuclear cells (PBMCs) and BALF were isolated by Ficoll-hypaque density gradient centrifugation (n=10). alpha beta T cells in PBMCs were depleted by complement-dependent-cytotoxicity(CDC) after the monocytes/macrophages were removed by adherence. The gamma delta T cells in PBMCs were cultivated selectively using anti-TCR gamma delta monoclonal antibody and IL-2. The proliferation of gamma delta T cells were observed by plotting growth curve. The purity of gamma delta T cells were detected by flow cytometry and immunohistochemistry.Stimulated by anti-TCR gamma delta mAb and IL-2, gamma delta T cells of peripheral blood and BALF could proliferate for a few days. The purity of gamma delta T cells obtained by Attack-panning method was 81%-99%.The attack-panning method can get pure gamma delta T cells from peripheral blood and BALF.

To explore the mode of immune responses of gammadeltaT cells in asthma and the role of gammadeltaT cell subsets in the pathogenesis of asthma.Wistar rats were sensitized and challenged with ovalbumin to establish an asthmatic model (n = 10, for each group). "Attack-panning" method was used to culture selective gammadeltaT cells. The percentage of gammadelta T cells among cultured cells was determined by flow cytometry. The levels of interleukin (IL)-4 and interferon-gamma (IFN-gamma) were measured by ELISA. The expression of IL-4 mRNA and IFN-gamma mRNA was determined by in situ hybridization.In the asthmatic group, IL-4 and IFN-gamma were detectable at the same time in cultured supernatant of gammadeltaT cells in peripheral blood mononuclear cells (PBMC) or broncho-alveolar fluid (BALF), and the level of IL-4 [PBMC: (40.5 +/- 3.7) ng/L, BALF: (49.6 +/- 3.1) ng/L] increased as compared with the control group [PBMC: (26.1 +/- 2.1) ng/L, BALF: (23.6 +/- 1.7) ng/L; all P0.01], while the level of IFN-gamma [PBMC: (17.6 +/- 2.5) ng/L, BALF: (28.5 +/- 3.6) ng/L] decreased as compared with the control group [PBMC: (24.3 +/- 1.7) ng/L, BALF: (38.4 +/- 2.8) ng/L; all P0.01]. In the asthmatic group, the percentage of IL-4 mRNA positive-staining cells [PBMC: (76.2 +/- 7.2)%, BALF: (85.7 +/- 8.4)%] among the gammadeltaT cells significantly increased as compared with the control group [PBMC: (20.6 +/- 5.3)%, BALF: (25.0 +/- 6.8)%; all P0.01], while the percentage of IFN-gammamRNA positive-staining cells [PBMC: (30.9 +/- 6.7)%, BALF: (41.5 +/- 3.6)%] among the gammadeltaT cells significantly decrease as compared with the control group [PBMC: (60.1 +/- 4.2)%, BALF: (53.8 +/- 5.1)%; all P0.01].gammadeltaT cells can secrete both IL-4 and IFN-gamma at the same time, or there are two subsets of gammadeltaT cells which can secrete either IL-4 or IFN-gamma in PBMC or BALF in asthma. Th1/Th2 immune response mode is present in gammadeltaT cells of asthma and Th2-cytokine profile predominates. gammadeltaT cells is involved in the pathogenesis of asthma.

To explore the effect of nitric oxide on NF-kappaB activation in rat alveolar macrophage(AM) induced by cigarette smoke extract(CSE) and the involved mechanism.AMs were co-cultured with NO precursor L-arginine (L-Arg) or inhibitor of inducible nitric oxide synthase L-N(6)(1-iminoethyl)lysine (L-NIL) and CSE. The expression of NF-kappaB was determined by immunocytochemistrical staining, the content of I-kappaB protein was detected by Western blot, the level of NO in supernatant was assayed by Griess reaction.CSE could induce NF-kappaB activation and decrease level of I-kappaB. L-Arg augmented CSE-induced NF-kappaB activation and decreased I-kappaB content at low concentration while inhibited CSE-induced NF-kappaB activation at high concentration, but there was not any significant difference of the expression of I-kappaB. The percentage of NF-kappaB nucleus dyeing positive cells was lower and the expression of I-kappaB was higher in response to CSE plus various concentrations of L-NIL as compared with that in response to CSE alone (P0.01). The changes induced by L-NIL were concentration-dependent.NO may up-regulate and down-regulate the activation of NF-kappaB induced by CSE according to difference in NO concentration in AMs.

To investigate the role of delayed rectifier potassium channels (K(V)) in the regulation of bronchial smooth muscle tension in asthmatic rats and their effects on bronchial reactivity in vitro.By using a specific potassium channel blocker, the bronchial contraction induced by spasmogens was observed in normal and asthmatic rats with the isometric tension recording technique.(1) K(V) blocker 4-aminopyridine (4-AP) caused concentration-dependent bronchial contraction in vitro. The concentration-response curve to 4-AP in the asthmatic group showed a left displacement with bigger pD(2) (the negative logarithm of the drug concentration causing 50% of maximal effect, 2.58 +/- 0.07, n = 10, P0.001) and no change in E(max) [maximal effect, (32 +/- 5) mg/mg, P0.05], compared with pD(2) (2.12 +/- 0.04, n = 10) and E(max) [(31 +/- 6) mg/mg] obtained in the control group; (2) In the control group, pre-treatment with 0.1 mmol/L 4-AP displaced the concentration-response curves to ET-1 and histamine to the left. pD(2) (for ET-1 and histamine) before and after 4-AP treatment was 6.27 +/- 0.38, 5.59 +/- 0.27 and 6.80 +/- 0.47, 6.42 +/- 0.14 respectively (P0.01). E(max) before and after 4-AP treatment was (36 +/- 8) mg/mg, (36 +/- 8) mg/mg and (40 +/- 8) mg/mg, (39 +/- 9) mg/mg respectively (P0.05); (3) In the asthmatic group, pre-treatment with 0.1 mmol/L 4-AP had no effect on the concentration-response curve to ET-1 and histamine. There was no significant difference (P0.05) in pD(2) before (6.51 +/- 0.07, 5.86 +/- 0.14 respectively) and after (6.48 +/- 0.16, 5.96 +/- 0.08 respectively) 4-AP treatment, and in E(max) before [(61 +/- 8) mg/mg, (54 +/- 11) mg/mg respectively] and after [(65 +/- 10) mg/mg, (55 +/- 9) mg/mg respectively] 4-AP treatment.The activity of K(V) decreases in bronchial smooth muscle isolated from asthmatic rats compared with that obtained from normal ones. This change may be involved in the bronchial hyperreactivity to some spasmogens in vitro in asthmatic rats.

To investigate whether the relaxation of bronchial smooth muscle induced by isoprenaline and aminophylline is mediated by large conductance Ca2+-activated K+ channels (BK(Ca)) via cAMP-dependent mechanism.With isometric tension recording, the role of BK(Ca) in relaxations of rat bronchial strips induced by isoprenaline and aminophylline was determined. With perforated patch-clamp technique, BK(Ca) currents were observed in freshly isolated rat bronchial myocytes.Tetraethylammonium 5 mmol/L, a BK(Ca) blocker, caused a significant rightward shift in the concentration-response curves of isoprenaline and aminophylline (about 4.26-fold and 3.78-fold, respectively) in methacholine-precontracted rat bronchial strips. Isoprenaline 1 micromol/L caused a significant increase in BK(Ca) current from (94+/-15) pA/pF to (186+/-30) pA/pF (voltage steps from -60 mV to +50 mV, n=10, P0.01), which was partly abolished by Rp-cAMP 100 micromol/L, a protein kinase A inhibitor. Furthermore, current-voltage relationship(I-V) curve exhibited an upward shift, and the peak current density was significantly raised (n=10, P0.01) by ramp depolarization from -100 mV to +100 mV. Aminophylline 1 mmol/L caused a significant increase in BK(Ca) current from (90+/-10) pA/pF to (166+/-25) pA/pF (voltage steps from -60 mV to +50 mV, n=11, P0.01), which was partly abolished by Rp-cAMP 100 micromol/L. Furthermore, the I-V curve exhibited an upward shift, and the peak current density was significantly raised (n=11, P0.01) by ramp depolarization from -100 mV to +100 mV.The relaxations induced by isoprenaline and aminophylline were, at least partly, mediated by cAMP-stimulation of BK(Ca) in rat bronchial smooth muscle.