SP-A mRNA Research Articles

Immunomodulatory molecules regulate adhesin gene expression in Staphylococcus aureus: Effect on bacterial internalization into bovine mammary epithelial cells

Staphylococcus aureus is a major pathogen of subclinical bovine mastitis that usually is chronic and recurrent, which has been related to its ability to internalize into bovine mammary epithelial cells (bMECs). Previously, we reported that short and medium fatty acids and cholecalciferol reduce S. aureus internalization into pretreated-bMECs with these molecules suggesting a role as immunomodulatory agents. Hence, we assessed the role of sodium butyrate (NaB), sodium octanoate (NaO) and cholecalciferol on S. aureus adhesin expression and its internalization into bMECs. S. aureus pre-treated 2 h with 0.5 mM or 2 mM NaB showed a reduction in internalization into bMECs (∼35% and ∼55%; respectively), which coincided with a down-regulated expression of clumping factor B (ClfB). Also, the S. aureus internalization reduction by 2 mM NaB (2 h) agreed with a down-regulated expression of sdrC. Moreover, the 2 mM NaB (24 h) pre-treatment induced bacterial internalization (∼3-fold), which was related with an up-regulation of spa, clfB and sdrC genes. Also, NaO (0.25 mM and 1 mM) only reduced S. aureus internalization when bacteria were grown 2 h with this molecule but there was no relationship with adhesin expression. In addition, cholecalciferol (50 nM) reduced bacteria internalization at similar levels (∼50%) when bacteria were grown 2 and 24 h in broth supplemented with this compound, which correlated with spa and sdrC mRNA expression down-regulated at 2 h, and fnba and clfB mRNA expression decreased at 24 h. In conclusion, our data support the fact that fatty acids and cholecalciferol regulate adhesin gene expression as well as bacteria internalization in nonprofessional phagocytic cells, which may lead to development of anti-virulence agents for control of pathogens.

Organic dust inhibits surfactant protein expression by reducing thyroid transcription factor-1 levels in human lung epithelial cells.

Exposure to organic dust is a risk factor for the development of respiratory diseases. Surfactant proteins (SP) reduce alveolar surface tension and modulate innate immune responses to control lung inflammation. Therefore, changes in SP levels could contribute to the development of organic-dust-induced respiratory diseases. Because information on the effects of organic dust on SP levels is lacking, we studied the effects of dust from a poultry farm on SP expression. We found that dust extract reduced SP-A and SP-B mRNA and protein levels in H441 human lung epithelial cells by inhibiting their promoter activities, but did not have any effect on SP-D protein levels. Dust extract also reduced SP-A and SP-C levels in primary human alveolar epithelial cells. The inhibitory effects were not due to LPS or protease activities present in dust extract or mediated via oxidative stress, but were dependent on a heat-labile factor(s). Thyroid transcription factor-1, a key transcriptional activator of SP expression, was reduced in dust-extract-treated cells, indicating that its down-regulation mediates inhibition of SP levels. Our study implies that down-regulation of SP levels by organic dust could contribute to the development of lung inflammation and respiratory diseases in humans.

Protective role of glucocorticosteroid prior to endotoxin exposure in cultured neonatal type II alveolar epithelial cells

BackgroundDexamethasone (DEX) is widely used for antenatal lung maturation and has been investigated to prevent premature lung injury by inhibiting postnatal inflammation. Its pharmacological mechanisms in the treatment of bacterial infection-induced injury of neonatal lung parenchymal cells remain to be clarified. We hypothesized that DEX pretreatment may attenuate endotoxin-induced growth suppression and regulate cytokine mRNA expression in cultured neonatal type II alveolar epithelial cells (AEC-II). MethodsAEC-II of newborn piglets were freshly isolated and cultured. After pretreatment of 0.01, 0.1, 1.0 and 10 μmol/l DEX (E0.01, E0.1, E1.0 and E10 group, respectively) for 24 h, the cells were cultured with 1 μg/ml lipopolysaccharides (LPS) for 7 days with medium replacement every 24 h. Messenger RNA expression of surfactant proteins (SPs), pro-inflammatory cytokines and multiple growth factors (GF) were determined by RT-PCR, along with the cell growth and apoptosis measurements. ResultsLPS without DEX pretreatment suppressed cell proliferation, enhanced expression of pro-inflammatory cytokine mRNA and apoptosis, which was ameliorated in all DEX-pretreated groups on day 3. On day 3 and 5, only cells pretreated by E1.0 and E10 showed a 20-fold increase in insulin-like GF-1 mRNA expression whereas the expression of other GFs was down-regulated. LPS exposure reduced the expression of SP-A, B, C and Aquaporin-5 mRNA on day 3–7. However, the expression of SP-C mRNA was increased in E1.0 on day 3, which was supported by in situ expression of pro-SP-C with immunocytochemical assay. ConclusionLPS-induced in vitro AEC-II injury was partially prevented by DEX pretreatment, with 1.0 μmol/l being the potentially optimal concentration. (253 words).

The investigation of effect of alpha lipoic acid against damage on neonatal rat lung to maternal tobacco smoke exposure.

This study was carried out to determine the changes in the lungs of the rat pups exposed to tobacco smoke during pregnancy period and to investigate the protective effects of alpha lipoic acid, which is administered during pregnancy, on these changes. Spraque-Dawley female rats were divided into four groups: control, tobacco smoke (TS), tobacco smoke + alpha lipoic acid (TS + ALA) and alpha lipoic acid (ALA). The rats in control group were untreated. Rats were exposed to TS twice a day for one hour starting from eight weeks before mating and during pregnancy. 20 mg / kg of ALA was administered to rats. On 7th and 21st days 7 of the pups from each group were decapitated. Histological, morphometric, biochemical and quantitative real-time RT-PCR analyzes were performed. Histopathological and biochemical changes were observed in TS group. While a significant decrease was observed both in SP-A and VEGF immunoreactivities and mRNA levels, caspase-3 immunoreactivity and TUNEL positive cells were increased in TS group. It is suggested that prenatal TS exposure leads to morphological and histopathological changes on lung development by causing oxidative damage in lungs of neonatal rats and the maternal use of ALA can provide a limited protective effect on the neonatal lung development against this oxidative stress originating from TS. Although pregnant women are increasingly aware on health risks of smoking, environmental tobacco smoke exposure is still a widespread problem. For this reason, it is thought that this damage can be partially reduced by some antioxidant supplements in pregnancy.

Interleukin-6 increases the expression of key proteins associated with steroidogenesis in human NCI-H295R adrenocortical cells

Mechanisms of interleukin-6 (IL-6)-induced cortisol release (CR) were investigated by exposing H295R cells to IL-6 and determining mRNA/protein expression (PCR/western blots) for steroidogenic enzymes (SE), steroidogenic acute regulatory protein (StAR), steroidogenic factor-1 (SF-1) (enhances SE/StAR expression), activator protein 1 (AP-1) (regulates SE/StAR expression) and adrenal hypoplasia congenita-like protein (DAX-1) (inhibits SE/StAR expression). Promoter activity of StAR (SPA) was measured by a luciferase-coupled promoter. Cortisol release was increased by 10ng/mL IL-6 (24h P<0.01). Proteins/mRNAs (StAR, cholesterol side chain cleavage enzyme, SF-1, AP-1) and SPA were increased by IL-6 (60min 1–50ng/mL IL-6; 5ng/mL IL-6 30–120min P<0.05). Four other SE proteins/mRNAs were also increased by 10ng/mL IL-6 (60min P<0.01). Protein/mRNA for DAX-1 was decreased by IL-6 (60min 1–50ng/mL IL-6; 5ng/mL IL-6 30–120min P<0.01). Phosphorylation of Janus kinase (JAK) and signal transducer and activator of transcription (STAT) was increased by IL-6 (JAK2 60min 1–50ng/mL IL-6; 10ng/mL IL-6 5–60min P<0.05; STAT1 and STAT3 60min 10ng/mL IL-6 P<0.01). Inhibition of JAK/STAT with AG490 (10μM) or piceatannol (50μM) blocked (P<0.01 10ng/mL IL-6vs. IL-6 plus AG490 or piceatannol) IL-6-induced increases in SPA and StAR mRNA. In summary, IL-6-induced CR may be facilitated by increased StAR and SE mediated by increased SF-1 and AP-1, decreased DAX-1, and increased phosphorylation of JAK/STAT.

Association of Surfactant Protein with Expression of Hoxa5 and Hoxb5 in Rabbit Fetal Lung

Hox genes regulate organ formation and identity of the embryo, and expressed in specific temporo-spatial patterns in the developing embryo. We compared the expression levels of the Hoxa5, Hoxb5, surfactant protein (SP)-A, and SP-B genes in immature and mature rabbit fetal lung tissues, and to uncover roles for Hoxa5, Hoxb5, SP-A, and SP-B. Cesarean sections were performed after rabbits were divided into two groups of 30-31 days of gestation (term group, n = 24) and 26-27 days of gestation (preterm group, n = 24). mRNA levels of Hoxa5, Hoxb5, SP-A, and SP-B were compared by quantitative reverse transcriptase polymerase chain reaction, and protein expression of Hoxa5 and Hoxb5 was compared by western blot analysis. Fetal lung tissue histology was observed by hematoxylin and eosin (H&E) staining. The relative expression ratios of SP-A and SP-B mRNA in the term to preterm groups were 2.45:1 and 2.94:1, respectively. Hoxb5 mRNA and protein levels decreased in the term group, with a relative expression ratio of 0.48:1 and 0.50:1, however, Hoxa5 mRNA and protein levels increased in the term group with a relative expression ration of 2.99:1 and 2.33:1, respectively, for the term to preterm groups. Moreover, a significant positive correlation was found between the expression of Hoxa5 and SP-A, SP-B in the term group. Hoxa5 gene may be essential for the expression of SP-A and SP-B in term rabbits. The Hoxb5 gene may be an important factor for lung maturation in preterm rabbits.

The ceramide inhibitor fumonisin B1 mitigates the pulmonary effects of low-dose diesel exhaust inhalation in mice

Recent studies have suggested that inhalation of diesel exhaust (DE), a major source of air pollution, results in pulmonary alterations; however, the effects of DE at low concentrations are poorly understood. Therefore, this study was conducted to elucidate the pulmonary effects of low-level exposure to DE and the potential role of a ceramide de novo biosynthesis inhibitor, fumonisin B1 (FB1) to ameliorate the DE-toxicity. Male C57BL/6J mice underwent 1- or 7-day experiments (4 equal groups/experiment) and were assigned to the control, DE (0.1mg/m3), FB1 (6.75mg/kg body weight SC at days 0, 3 and 6) or DE+FB1 groups. DE and/or FB1 treatment had no effect on the expression of Nos2, a biomarker of oxidative stress. Ceramide production in the bronchial epithelial cells and Sphk1 mRNA expression were induced in the lung after the 7-day DE exposure and were partially suppressed by the FB1 treatment. Additionally, the effects of DE on SP-A and SP-D mRNA expression were also suppressed by the FB1 treatment. These results suggest that ceramide and Sphk1 may be sensitive biomarkers for low-level DE-induced pulmonary effects. Collectively, ceramide likely contributes to the DE-induced early stage of airway inflammation, which is considered a potential pulmonary target during low-level DE exposure.

Effects of Bufei Yishen Granules Combined with Acupoint Sticking Therapy on Pulmonary Surfactant Proteins in Chronic Obstructive Pulmonary Disease Rats.

Our previous studies have demonstrated the beneficial effects of Bufei Yishen granules combined with acupoint sticking therapy (the integrated therapy) in chronic obstructive pulmonary disease (COPD), but the underlying mechanism remains unclear. Dysfunction of pulmonary surfactant proteins (SPs, including SP-A, SP-B, SP-C, and SP-D) may be included in pathophysiology of COPD. This study aimed to explore the mechanism of the integrated therapy on SPs. COPD rat models were established. The treatment groups received Bufei Yishen granules or acupoint sticking or their combination. Using aminophylline as a positive control drug. The levels of SPs in serum, BALF, and lung were measured. The results showed that the integrated therapy markedly reduced the levels of SPs in serum and increased these indicators in the lung. The integrated therapy was better than aminophylline in reducing the levels of SPs and was better than Bufei Yishen granules in reducing SP-A, SP-C, and SP-D in serum. The integrated therapy was better than aminophylline and Bufei Yishen granules in increasing SP-A, SP-B, and SP-D mRNA in the lung. SP-A and SP-D in BALF were positively correlated with PEF and EF50. The levels of SPs are associated with airway limitation. The beneficial effects of the integrated therapy may be involved in regulating pulmonary surfactant proteins.

Community-acquired meticillin-resistant Staphylococcus aureus strain USA300 resists staphylococcal protein A modulation by antibiotics and antimicrobial peptides

Community-acquired meticillin-resistant Staphylococcus aureus (CA-MRSA) causes severe diseases through virulence factors such as staphylococcal protein A (SpA), which favours immune evasion. We have previously shown that antimicrobial peptides (AMPs) and antibiotics decrease SpA expression in CA-MRSA strains. Here we examined the effects of antibiotics and AMPs, alone and in combination, on SpA expression in various CA-MRSA strains. Six S. aureus isolates corresponding to the major worldwide CA-MRSA clones (ST8-USA300, ST80 and ST30) were selected. Strains were cultured to exponential growth phase and were subsequently incubated with antibiotics (tigecycline, linezolid, clindamycin and vancomycin) at 0.25× MIC or with AMPs [human neutrophil peptide (HNP)-1–3] at the LD50, alone and in combination. After 6h, cultures were assessed for spa mRNA by RT-PCR, whilst SpA protein was measured by specific ELISA after 18h. When used alone, antibiotics (clindamycin, linezolid and tigecycline) or HNPs significantly reduced both SpA production and mRNA levels in ST30 and ST80 strains. When used in combination, HNPs and clindamycin, linezolid or tigecycline synergistically reduced SpA production (6–100-fold) and spa mRNA levels (4–20-fold) in ST80 and ST30 strains. In contrast, for USA300 strains, among all antibiotics, clindamycin alone reduced SpA production (3.5-fold), whereas with combined treatments including HNPs, only a slight reduction in SpA production (1.7–2.2-fold) was observed. In conclusion, antibiotics and AMPs do not modulate SpA expression in USA300, unlike in other CA-MRSA clones. This observation suggests that the virulence and successful spread of USA300 strains is associated with a specific regulatory network.

DPP4 inhibitors increase differentially the expression of surfactant proteins in Fischer 344 rats.

Intact surface active agent (surfactant) composed of surfactant-associated proteins (SPs) and lipids is necessary for respiration and prevents alveoli from collapsing. CD26, a transmembrane glycoprotein exerting dipeptidyl peptidase activity (DPP4), highly expressed in lung parenchyma, is involved in inflammatory processes. A pharmacological inhibition of DPP4 influenced not only the inflammation but also elevated the SPs. Thus, DPP4 inhibitors may be a novel drug for treatment of diseases with surfactant deficiency. Therefore, we tested firstly the hypothesis that DPP4 inhibitors increase the expression of SPs in healthy rats. SP mRNA and protein expression were determined different times after nebulization of aerosolized DPP4 inhibitors [L-isoleucine-thiazolidide (L-Ile-Thia), L-valine-pyrrolidide (L-Val-Pyrr)], budesonide, saline or stereoisomers. Compared with negative controls (1) L-Ile-Thia as well as budesonide led to a significant higher and L-Val-Pyrr had the tendency to a significant higher expression of SP-A mRNA 6 h after nebulization, (2) the expression of SP-D mRNA increased significantly 6 h after nebulization with L-Ile-Thia and 3 and 6 h after nebulization with Val-pyrr, (3) SP-B mRNA levels showed significantly higher values 3 and 6 h after nebulization with L-Val-Pyrr, (4) protein levels of SP-A, SP-B and SP-C were elevated significantly 6 h after nebulization with L-Val-Pyrr as well as with budesonide, and (5) phospholipids were also increased in response to DPP4 inhibition; the minimal surface tension was comparable. DPP4 inhibition influence differently the expression of surfactant proteins in healthy rats and may be suitable to elevate surfactant synthesis in different diseases accompanied with surfactant deficiencies.

In vitro differentiation of rat amniotic fluid-derived mesenchymal stem cells into type II alveolar epithelial cells

To explore the in vitro differentiation of rat amniotic fluid-derived mesenchymal stem cells (AF-MSCs) into type II alveolar epithelial cells (AECII). Flow cytometry was used to analyze the phenotypes of AF-MSCs from 10 pregnant Sprague-Dawley rats. And the Oct-4 mRNA expression level was detected by quantitative real-time polymerase chain reaction (qRT-PCR). Rat embryonic stem cell was used as a positive control. According to different culturing methods, AF-MSCs were randomly divided into 5 groups of A (control group), B, C, D and E. After in vitro differentiation, SPA, SPB, SPC, SPD and TTF1 mRNA expressions were detected by qRT-PCR, SPA and SPC protein expressions measured by immunofluorescence and lamellar bodies observed by transmission electron microscopy. AF-MSCs could grow spirally in L-DMEM medium containing 20% fetal bovine serum and 4 µg/L basic fibroblast growth factor. The expressions of such surface antigens of AF-MSCs (third passage) as CD29 (99.1 ± 7.9)%, CD44 (99.2 ± 7.4)%, CD73 (75.6 ± 5.2)%, CD90 (98.9 ± 8.1)%, CD105 (92.9 ± 7.3)% and CD166 (89.3 ± 6.7)% were positive while CD34 and CD45 were negative. And the expression of Oct-4 mRNA (relative quantity: 0.690 ± 0.059) was significantly lower than rat embryonic stem cells (relative quantity: 1.000 ± 0.002) positive control group (P < 0.01). After in vitro differentiation, the expressions of SPA, SPB, SPC, SPD and TTF1 mRNA and SPA and SPC protein were negative in group A and positive in group B. The expressions of SPA, SPB, SPC, SPD and TTF1 mRNA (relative quantity: 0.426 ± 0.043, 0.368 ± 0.028, 0.492 ± 0.058, 0.327 ± 0.024 and 0.183 ± 0.018) and SPA and SPC protein in group B were significantly higher than other groups (all P < 0.01). Lamellar bodies could be found in the differentiated cells of group B. Rat AF-MSCs from amniotic fluid may differentiated into AECII like cells in vitro.

Effects of histologic chorioamnionitis induced by lipopolysaccharide intra-amniotic injection on fetal lung maturation and development in preterm fetal rats

Objective With various doses of lipopolysaccharide (LPS) intra-amniotic injection in pregnant rats to establish histologic chorioamnionitis (HCA) model,and to determine the effects of HCA on fetal lung maturation and development in preterm fetal rats.Methods Thirty pregnant Sprague-Dawley rats were randomly assigned to four groups.On 19 days of gestation,rats in three model groups (n=8) were given intra-amniotic injection of LPS 2,8,10 μg,respectively,and those in the only one control group (n=6)was given normal saline (NS) 40 μl.Fetal rats were taken out 48 hours after LPS injection.Placenta,fetal membrane,fetal lung and umbilical cord were collected for pathological examination.mRNAs of surfactant protein (SP)-A,B and C and keratinocyte growth factor (KGF) in lung were determined by quantitative real-time polymerase chain preaction.KGF protein expression in lung was measured by immunohistochemistry.Morphologic observation of lung was performed on postnatal day 3.R× C table Chi-square test,KruskalWallis test and analysis of variance were used as statistical methods.Results (1) Fetal rat mortality in control,LPS2,LPS8 and LPS10 group was 0.0％ (0/55),22.5％ (18/80),51.4％ (36/70) and 87.5％(35/40),respectively,which increased with increasing dose of LPS (x2=46.183,P＜0.005).(2) HCA,fetal lung inflammation and umbilical vasculitis were induced in LPS groups,and the severity was dosedependent (fetal lung inflammation:Hc=39.84,HCA:Hc=41.13,umbilical vasculitis:Hc=41.52,all P＜0.01).(3) The expression of SP-A,SP-B,SP-C and KGF mRNAs in the four groups (control,LPS2,8,10) had statistical differences (SP-A mRNA:0.99±0.28,2.48±0.34,1.09±0.31 and 0.09±0.09,F=78.051; SP-B mRNA:0.99±0.34,2.23±0.53,1.49±0.51 and 0.14±0.06,F=28.327; SP-C mRNA:1.20±0.39,2.00±0.20,1.04±0.37 and 0.12±0.16,F=39.546; KGF mRNA:0.97±0.19,2.18±0.61,0.93±0.09 and 0.21±0.11,F=37.544; all P＜0.01).All mRNA expressions in LPS2 group were higher than those in the control group and those in LPS10 group was lower (all P＜0.05).(4) The expression of KGF protein in LPS2 group was significantly higher than that in other groups (0.60±0.20 vs 0.28±0.12,0.37±0.22,0.24±0.12,F=17.280,all P＜0.01).(5) Alveolarization was significantly inhibited in LPS8 group on postnatal day 3,and less maturity of pulmonary tissue was observed.Conclusions Various doses of LPS intra-amniotic injection in rats could induce HCA,fetal lung inflammation and umbilical vasculitis with different degrees.Histological inflammation would be worse with increasing LPS dosage.Moderate inflammation could promote lung maturation without inducing bronchopulmonary dysplasia,and KGF may play a role in this process. Key words: Chorioamnionitis; Infant, premature; Lung; Fetal organ maturity; Fibroblast growth factor7; Disease models, animal; Lipopolysaccharides

The Expression of Surfactant Proteins A and D in the Intestines and Pancreas of Murine Fetuses

Purpose: Surfactant proteins exist in the digestive tract and may play an important role in the host defense. However, the expression of surfactant proteins in the premature digestive system remains unclear. The aim of this study was to investigate the expression of surfactant proteins in the intes-tines and pancreas of murine fetuses. Methods: Immunostaining for SP-A and SP-D was assessed in the small intestine and pancreas of ICR murine fetuses on days 15, 16, 17 and 18 of gestation (normal duration of pregnancy: 19 - 21 days). RT-PCR was performed to detect the expression of spa and spd mRNA in the small intestine and pancreas on day 16, 17 and 18 of gestation. Results: Immunoreactivity for SP-A and SP-D in the acinar cells of pancreas and intestinal mucosal surface were positive on day 16 of gestation onward. RT-PCR revealed that the expression of spa and spd mRNA was significant in the pancreas but weak in the small intestine. Conclusions: Our data revealed that surfactant proteins are present in the fetal intestines and pancreas and that a significant expression of spa and spd mRNA is detected in the fetal pancreas. Pancreas may be a possible organ involved in the synthesis and secretion of surfactant proteins into the intestinal lumen.

Effect of SP-A/B in lipoic acid on acute paraquat poisoning.

This study was undertaken to observe the concentration of SP-A/B and the pulmonary surfactant in the lung tissue of rats with acute lung injury/acute respiratory distress syndrome caused by paraquat poisoning after the treatment of metabolic antioxidant-lipoic acid and whether its influence was related to TNF-α. Sixty-six male Sprage-Dawley rats were randomly divided into three groups: normal control group (NS group), 6 rats; paraquat poisoning group (PQ group), 30 rats; and paraquat+lipoic acid treatment group (LA group), 30 rats. The rats in the PQ and LA groups were subdivided into 3-, 6-, 12-, 24-, 48-hour subgroups, with 6 rats in each group. After the rats were sacrificed, lung tissue from the same part was taken from the rats. After HE staining, histological changes were observed in the tissue under a light microscope. Lung tissue was also taken to test the levels of superoxide dismutase (SOD) and malondialdehyde (MDA). Whole blood (0.8 mL) without anticoagulant was drawn from the tail vein of rats for the determination of the TNF-α level. The total RNA of the lung tissue was collected, and the Rt-PCR method was used to measure the levels of SP-A and SP-B mRNA. HE staining showed that histopathological changes were milder in the LA group than in the PQ group. There were significant differences in MDA and SOD levels between different intervals both in intergroups and intragroups except the 3-hour subgroup (P<0.01). Likewise, the significant differences in the levels of TNF-α were also present between the three groups and between different intervals (P<0.01). The significant differences in SP-A mRNA and SP-B mRNA amplification ratio were seen between the three groups at the same intervals (P<0.01), but the differences between different intervals in the PQ group were statistically significant (P<0.05). The differences between different intervals in the LA group were statistically significant (P<0.01). Lipoic acid in acute paraquat poisoning could diminish lung tissue damage by regulating directly tumor necrosis factor and indirectly the content of pulmonary surfactant so as to reduce pulmonary edema, improve lung compliance, and finally protect lung tissues.

Pilot Study Exploring Lung Allograft Surfactant Protein A (SP-A) Expression in Association With Lung Transplant Outcome

Primary graft failure and chronic lung allograft dysfunction (CLAD) limit lung transplant long–term outcomes. Various lung diseases have been correlated with surfactant protein (SP) expression and polymorphisms. We sought to investigate the role of SP expression in lung allografts prior to implantation, in relation to posttransplant outcomes. The expression of SP-(A, B, C, D) mRNA was assayed in 42 allografts. Posttransplant assessments include pulmonary function tests, bronchoscopy, broncho-alveolar lavage fluid (BALF) and biopsies to determine allograft rejection. BALF was assayed for SP-A, SP-D in addition to cytokines IL-8, IL-12 and IL-2. The diagnosis of CLAD was evaluated 6 months after transplantation. Lung allografts with low SP-A mRNA expression prior to implantation reduced survival (Log-rank p < 0.0001). No association was noted for the other SPs. Allografts with low SP-A mRNA had greater IL-2 (p = 0.03) and IL-12 (p < 0.0001) in the BALF and a greater incidence of rejection episodes (p = 0.003). Levels of SP-A mRNA expression were associated with the SP-A2 polymorphisms (p = 0.015). Specifically, genotype 1A1A0 was associated with lower SP-A mRNA expression (p < 0.05). Lung allografts with low levels of SP-A mRNA expression are associated with reduced survival. Lung allograft SP-A mRNA expression appears to be associated with SP-A gene polymorphisms.

Prenatal Hypoxia Downregulates the Expression of Pulmonary Vascular Endothelial Growth Factor and Its Receptors in Fetal Mice

Background: Previous reports showed that prenatal hypoxia delays the process of lung maturation. Vascular endothelial growth factor (VEGF) and its receptors were important for lung development. However, the role of VEGF and VEGF receptors in altered fetal lung development and maturation induced by prenatal hypoxia remains unknown. Objectives: To elucidate the role of VEGF and VEGF receptors in altered fetal lung development and maturation induced by prenatal hypoxia. Methods: Lung sections of control and maternal hypoxic fetal mice were used for the determination of lung development and total RNA isolated from lung homogenates were used for determination of the expression patterns of VEGF, Flt-1, Flk-1, hypoxia-inducible factor (HIF)-1α, HIF-2α, surfactant protein (SP)-A, SP-B, SP-C, and SP-D by quantitative real-time RT-PCR. Results: Prenatal hypoxia resulted in fetal mice body weight gain impairment, delayed fetal pulmonary aeration and maturation. Pulmonary SP-A, SP-B, SP-C, and SP-D mRNA were all decreased in the prenatal hypoxia group. In addition, we demonstrated that prenatal hypoxia inhibited the developmental increase of pulmonary HIF-1α and HIF-2α expression and resulted in decreasing VEGF and its receptors (Flt-1 and Flk-1) at the mRNA expression level and VEGF protein level in fetal lungs. These inhibitory effects persisted and progressed even when the dams were returned to air. Conclusions: We suggest that prenatal hypoxia insults, at least in late gestation, influence pulmonary VEGF and VEGF receptor expression through the down-regulation of HIF pathways and impair fetal lung growth and maturation.

Effects of subinhibitory concentrations of antibiotics on virulence factor expression by community-acquired methicillin-resistant Staphylococcus aureus

To examine the effect of subinhibitory concentrations (sub-MICs) of antistaphylococcal drugs on Panton-Valentine leucocidin (PVL), α-haemolysin (Hla) and protein A (SpA) expression by community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA). Five clinical isolates representing the main worldwide CA-MRSA clones were grown with sub-MICs (1/8, 1/4 and 1/2 MIC) of five antibiotics (clindamycin, daptomycin, linezolid, tigecycline and vancomycin). After 4 and 6 h of incubation, culture pellets were used for relative quantitative RT-PCR with primers specific for pvl, hla, spa and gyrB. The PVL, Hla and SpA concentrations were measured in the supernatant (for PVL and Hla) and in the cell pellet (for SpA) using specific ELISAs. For all strains tested, clindamycin and linezolid dramatically reduced mRNA levels of PVL and SpA. Tigecycline also decreased the PVL and SpA mRNA levels of 3/5 and 4/5 strains tested, respectively, whereas daptomycin and vancomycin had no significant effect. PVL and SpA quantification confirmed the concentration-dependent inhibition of PVL and SpA production by clindamycin and, to a lesser extent, by linezolid and tigecycline. Only clindamycin decreased Hla mRNA expression, whereas linezolid, tigecycline and daptomycin showed heterogeneous strain-dependent results, and vancomycin had no significant effect. Analysis of the Hla level revealed a stronger concentration-dependent inhibition of Hla release by clindamycin than by linezolid. The effect of sub-MICs on virulence expression depended on the antibiotic and the virulence factor. Clindamycin and linezolid consistently suppressed the expression of different virulence factors by CA-MRSA, whereas tigecycline specifically suppressed PVL expression. Daptomycin and vancomycin seem to have no significant effects at these concentrations.

Lipopolysaccharide-induced expression of surfactant proteins A1 and A2 in human renal tubular epithelial cells

BackgroundSurfactant protein A (SP-A), encoded by two functional genes, SP-A1 and SP-A2, is essential for the inflammatory process and host defence in the lungs. Recent studies have demonstrated the extrapulmonary expression of SP-A. Similar to the lungs, the kidneys are organs exposed to external pathogens. The present study evaluated the expression and location of SP-A in the kidneys. The effect of lipopolysaccharide (LPS) on the expression of SP-A subtypes was also studied in renal tubular epithelial (HK-2) cells.MethodsImmunohistochemical staining was performed using polyclonal antibody against SP-A. RT-PCR was also performed using mRNA from normal human renal tissues and HK-2 cells. The expressions of the SP-A1 and SP-A2 genes were determined by PCR-based RFLP analysis, gene-specific amplification, and direct sequencing of RT-PCR products. Western blot was conducted to analyse the SP-A protein. HK-2 cells were treated with LPS at various concentrations (0, 0.1, 1, 2, 5, and 10 μg/mL) for 8 h and at 5 μg/mL at various time points (0, 2, 4, 8, 16, and 24 h). The LPS-induced expressions of SP-A1 and SP-A2 mRNA and protein were analysed by RT-PCR and Western blot.ResultsSP-A was localised in the renal tubular epithelial cells in the proximal and distal convoluted tubules. SP-A1 and SP-A2 mRNA and protein were expressed in HK-2 cells and human renal tissues, which were significantly increased in time- and dose-dependent manners after LPS treatment (P < 0.05).ConclusionsHuman renal tubular epithelial cells can express both SP-A1 and SP-A2 genes, which may play important roles in the inflammatory modulation of the kidney.

Detection of Surfactant Proteins A, B, C, and D in Human Nasal Mucosa and Their Regulation in Chronic Rhinosinusitis with Polyps

The nasal mucosa is characterized by a multirow high prismatic ciliated epithelium representing the first barrier of the immune defense system against microbial and other environmental pathogenic influences. A number of nonspecific defense mechanisms, including the presence of lactoferrin, peroxidases, proteases, interferons, and lysozymes in nasal secretions, act to counter inflammatory processes. The surfactant proteins (SPs) known from the lungs are important components of the innate immune system. They also influence the rheology of fluids and reduce the surface tension of gas-fluid interphases. The objective of this study was to investigate the protein expression of all four SPs. A specific aim was detection and characterization of SP-C, which had previously not been confirmed in human nasal mucosa. The expression of mRNA for SP-A, -B, -C and -D was investigated using reverse transcriptase polymerase chain reaction on samples of both healthy nasal mucosa and nasal mucosa altered by inflammatory processes (allergic rhinitis and chronic rhinosinusitis). The distribution of all four proteins was determined with monoclonal antibodies using Western blot analysis as well as immunohistochemical methods. The results show that all four SPs, including SP-C not detected before this, are nasal mucosa components. A shift was also observed in the expression behavior of the SP-A, -B, and -D in nasal mucosa with inflammatory changes. Based on these results, SPs appear to have an important function in immunologic and rheological process of the nasal mucosa and support the prospective therapeutic use of liposomal nasal sprays.

The effects of mitogen-activated protein kinases on the genes expression of the pulmonary surfactants from alveolar epithelial cells during ventilation induced lung injury in rats

Objective To investigate the effects of mitogen-activated protein kinases (MAPK) on the genes expression of the pulmonary surfactants from alveolar epithelial cells during ventilation induced lung injury in rats.Methods Anesthetized male SD rats were randomized into six groups:① Control group(C) received no mechanical ventilation.② Low-volume ventilation(LV) group received mechanical ventilation with Vt=6 ml/kg,RR=40 bmp,I∶E=1∶2,FiO2=21％,positive end-expiratory pressure =0.③ Hyperventilation(HV) group received mechanical ventilation with the same respiratory parameters as the LV group and the Vt was 40 ml/kg.The inhibitors of MAPK including the JNK's:SP600125,the ERK's:PD98059 and the p38's:SB203580 were pretreated 30 min before ventilation just like in the HV group.④ SP600125 group (SP600125 + HV group).⑤ PD98059 group (PD98059 + HV group).⑥ SB203580 group (SB203580 + HV group).After 4 h ventilation,the animals in each group were killed and the lungs were removed for detecting the expression of SP-A,B,C and RTI40 mRNA by RT-PCR.Results ① Compared with group C,the expression of RTI40 mRNA of lung tissue increased significantly in the LV group and the HV group (P＜0.05),but the expression of SP-A,SP-C mRNA decreased in the HV group,SP600125 group and SB203580 group (P＜0.05),while there was no significant difference in the expression of SP-A、C mRNA in LV groups and PD98059 group (P＜0.05).② Compared with the HV group,the SP-A,C gene expressions of lung tissue in SP600125 group,PD98059 group and SB203580 group increased significantly (P＜0.05),but RTI40 gene expressions decreased(P＜0.05).③ The SP-A,C gene expressions of lung tissue in PD98059 group increased significantly than those of SP600125 group and SB203580 group.④ The SP-B gene expression had no difference in all groups.Conclusions The results of our study show that mechanical ventilation inhibited the expressions of the SP-A and SP-C,but upregulated RTI40 gene expressions.The inhibitors of MAPK attenuated the changes of SP-A,C and RTI40 mRNA during the process of high volume ventilation,which indicates that MAPK,at least in part,may play a role in the functions of Ⅰ and Ⅱ alveolar epithelial cells in the lung injury induced by mechanical ventilation. Key words: Ventilation induced lung injury; Pulmonary surfactant; Genes expression; Mitogen-activated protein kinases