Aged ; Alcohol Drinking ; blood ; Asian Continental Ancestry Group ; Biomarkers ; Dust ; Humans ; Male ; Middle Aged ; Occupational Exposure ; Silicon Dioxide ; toxicity ; Silicosis ; blood ; Smoking ; blood ; Uteroglobin ; blood

OBJECTIVETo investigate the dynamic changes in the expression of clara cell protein (CC16) and surfactant protein D (SP-D) in the lung tissues and bronchoalveolar lavage fluid (BALF) of silica-treated rats.

METHODSEighty-four Wistar rats were randomly divided into control group (n = 42) and silica group (n = 42). The silica group was subsequently divided into 3, 7, 14, 21, 28, and 60 d subgroups. The silicotic model was made by instilling silica suspension directly through the trachea into rat lungs. At 3, 7, 14, 21, 28, and 60 d after silica instillation, 8 rats in each group were sacrificed and their lung tissues and BALF were collected. The expression of SP-D and CC16 in lung tissues was detected by immunohistochemistry. Enzyme-linked immunosorbent assay (ELISA) was used to measure the levels of SP-D and CCl6 in BALF.

RESULTSThe immunohistochemical assay indicated that CCl6 and SP-D were expressed in lung cells. The ELISA found that in 7, 14, 21, 28, and 60 d silica subgroups, the content of CCl6 in rat BALF was 8.14±0.70, 7.15±0.66, 7.00±0.69, 6.34 ± 0.59, and 5.27±0.49 ng/L, respectively; CCl6 expression decreased gradually with the silica exposure time prolonged, indicating a negative correlation (ra = -0.953, P < 0.01). Compared with the control group, all silica subgroups had significantly decreased CCl6 levels (P < 0.05). The content of SP-D in BALF was 12.20 ± 1.57, 14.41 ± 0.65, and 12.18 ± 0.74 ng/L, respectively, in the 7, 14, and 21 d silica subgroups, significantly higher than that in the control group (P < 0.05).

CONCLUSIONThe dynamic changes in SP-D and CCl6 protein levels in the lung tissues and BALF of rats could be induced by silica exposure and are related to silica exposure time. With the extension of silica exposure, CCl6 levels are gradually reduced, while the SP-D levels first increase and then fall.

Animals ; Bronchoalveolar Lavage Fluid ; chemistry ; Epithelial Cells ; metabolism ; Lung ; metabolism ; Pulmonary Surfactant-Associated Protein D ; metabolism ; Rats ; Rats, Wistar ; Silicon Dioxide ; toxicity ; Uteroglobin ; metabolism

The present study investigated the effect of antiflammin-1 (AF-1) on LPS-induced IL-10 secretion from RAW264.7 cells through uteroglobin-binding protein (UGBP). Cultured RAW264.7 cells, a murine monocyte-macrophage cell line, were divided as following: control group, LPS group (1 µg/mL LPS), AF-1 group (100 μmol/L AF-1), LPS+AF-1 group (2 h of 100 μmol/L AF-1 pretreatment before LPS addition), and LPS+AF-1+anti-UGBP group (30 min of anti-UGBP antibody pretreatment before successive treatments with AF-1 and LPS). IL-10 concentration in the supernatants was detected by ELISA assay, and the level of IL-10 mRNA expression in macrophage was detected by using RT-PCR method. The results showed that AF-1 significantly increased LPS-induced IL-10 secretion in RAW264.7 cells in a dose dependent way, and up-regulated its mRNA level. Anti-UGBP antibody pretreatment attenuated the augmented effect of AF-1 on LPS-induced IL-10 secretion and gene expression. These results suggest that AF-1 promotes LPS-induced IL-10 secretion from macrophages, and this effect is mediated by UGBP.
Animals ; Cell Line ; Gene Expression ; Interleukin-10 ; metabolism ; Lipopolysaccharides ; Macrophages ; metabolism ; Mice ; Peptide Fragments ; metabolism ; RNA, Messenger ; Uteroglobin ; metabolism

To prepare anti-mouse uteroglobin binding protein (mUGBP) polyclonal antibody, two polypeptides were synthesized based on the bioinformatics analysis of mUGBP, and New Zealand white rabbits were immunized separately with each peptide coupled with keyhole limpet hemocyanin (KLH). The data indicate that a 13-amino acid polypeptide (positions 221st-233rd) was able to generate anti-peptide antibodies. The titer of the antisera detected with ELISA was 1:10(8). The antisera were then purified with immuno-affinity chromatography to obtain antibodies. Western blot analysis of mUGBP expressed as a fusion protein with a green fluorescent protein (GFP) was performed on the cell lysates of COS-1 cells with the purified antisera, suggesting that the antisera specifically recognized UGBP. By immunohistochemistry and indirect immunofluorescence analysis, we examined the expression of UGBP in the lung tissues from a patient undergoing surgical lung resection for a tumor and from normal mouse lung tissue, and found for the first time that UGBP protein was widely expressed in both mouse and human lung tissue with the most abundant expression in bronchial epithelial cells. These results suggest that the antigen epitopes of mUGBP are well predicted by using bioinformatics analysis. We have obtained anti-mUGBP polyclonal antibody, which will be useful for further investigation.
Animals ; Antibodies ; chemistry ; COS Cells ; Carrier Proteins ; chemistry ; Cercopithecus aethiops ; Computational Biology ; Enzyme-Linked Immunosorbent Assay ; Hemocyanins ; Humans ; Immune Sera ; Immunohistochemistry ; Mice ; Rabbits ; Recombinant Proteins ; chemistry ; Uteroglobin

OBJECTIVETo investigate the effects of sevoflurane on cytosolic phospholipase A₂ (C-PLA₂) and clara cell secretory protein (CCSP) in lung tissues of rabbits with one-lung ventilation (OLV)-induced lung injuries.

METHODSThirty-six healthy Japanese white rabbits were randomized into sham-operated group, OLV group, and OLV plus sevoflurane group subdivided into 4 subgroups with sevoflurane concentrations of 1%, 2%, 3% and 4%. CCSP and C-PLA₂ mRNA and protein expressions in rabbit lung tissues were detected by Western blotting and real-time PCR, and the content of arachidonic acid (AA) was measured using ELISA. The severities of the lung injury were evaluated according to lung wet/dry weight (W/D) ratio and histological scores.

RESULTSIn the OLV group and OLV+ sevoflurane groups, pulmonary CCSP expressions were significantly lower, while C-PLA₂ expression, lung W/D ratios and lung histological scores were significantly higher than those in the sham-operated group (P<0.05). Compared with OLV group, the OLV+sevoflurane groups showed significantly increased expressions of CCSP and reduced C-PLA₂ expression, lung W/D ratios and histological scores (P<0.05). In the 4 OLV+sevoflurane groups, CCSP expressions underwent no significant changes as sevoflurane concentration increased, but C-PLA₂ expressions, lung W/D ratios and histological scores all decreased gradually as the concentrations of sevoflurane increased (P<0.05).

CONCLUSIONOLV can result in down-regulated CCSP expressions and up-regulated C-PLA₂ expressions in rabbit lung tissues. Sevoflurane can protect against OLV-induced acute lung injury possibly by inhibiting C-PLA₂ expression via up-regulation of CCSP expressions or through other mechanisms resulting in down-regulated expression of C-PLA₂.

Animals ; Female ; Lung ; metabolism ; pathology ; Male ; Methyl Ethers ; pharmacology ; One-Lung Ventilation ; adverse effects ; Phospholipases A2 ; metabolism ; Rabbits ; Uteroglobin ; metabolism ; Ventilator-Induced Lung Injury ; metabolism

BACKGROUNDClara cell secretory protein (CC16) is an important lung derived protective factor and may play an important role on the pathogenesis of acute lung injury (ALI) induced by endotoxemia. Growth hormone (GH) is an important anabolism hormone secreted by GH cells of the hypophysis. Previous research showed that GH would significantly exacerbate ALI induced by endotoxemia, but the mechanism is not very clear yet. Whether the effects are related to CC16 or not is undetermined.

METHODSOne hundred and twelve male Sprague-Dawley rats were randomly divided into an ALI group and a GH group. The rats in the two groups were subdivided into seven subgroups, according to injection with lipopolysaccharides (LPS) or not, then according to different intervals of time after LPS injection; 0 hour (pre-injection of LPS, acted as control group), 0.5 hour, 1 hour, 2 hours, 4 hours, 6 hours and 24 hours for subgroups. Pulmonary alveolar septa area density (PASAD) and ploymorphonuclear cells (PMN) in the lungs were analyzed morphometrically. The levels of tumor necrosis factor (TNF) and interleukin 6 (IL-6) were determined by radioimmunoassay. To analyze the expression and activation of nuclear factor kappa B (NF-κB), the numbers of NF-κB positive cells in lungs were counted after immunofluorescence staining, and the levels of NF-κB inhibitory protein-α (IκB-α) in lung homogenates of rats were detected by Western blotting. The expression levels of CC16 mRNA in lungs of the rats with ALI were determined by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). The levels of CC16 protein in lung homogenates were detected by Western blotting.

RESULTSHalf an hour after LPS injury both the PASAD and PMN numbers in lungs of the rats with ALI began to increase significantly and peaked at 6-hour post-injury. They then began to recover and reached normal levels at 24-hour post-injury. Both the PASAD and PMN numbers in the GH group increased more significantly than those in the ALI group. The levels of TNF in lungs of the rats with ALI homogenates increased significantly 0.5-hour post-injury, peaked at 1-hour and maintained a high level until 6 hours then gradually recovered. The content of TNF in the GH group lung homogenates increased more significantly than in the ALI group post-injury. The contents of IL-6 in rat lung homogenates began to increase significantly at 1-hour post-injury, peaked at 4 hours then gradually returned to normal levels by 6 hours post-injury. The levels of IL-6 in the lung homogenates of the GH group were higher than in the ALI group at different time intervals post-injury. The number of NF-κB positive cells increased dramatically at 0.5-hour post-injury, and the fluorescence intensity was enhanced. Both peaked at 4-hour post-injury. The number of NF-κB positive cells and the enhanced intensity of fluorescence began to decrease from 6-hour post-injury, but the number of NF-κB cells at 24 hours post-injury was still higher than in the control group. The number of NF-κB cells in lungs in the GH group was significantly higher than in the LPS group at the different time intervals post-injury. The IκB-α expression in lungs of the rats with ALI homogenates decreased dramatically 0.5-hour post-injury, reaching a nadir at 4-hour post-injury and then began to recover. The levels of IκB-α in GH group were significantly lower than those in ALI group. Both the levels of CC16 mRNA and protein in lungs of the rats with ALI began to decrease significantly 0.5-hour post-injury, reached a nadir at 6 hours, and then began to recover. Both the expression of CC16 mRNA and CC16 protein in the GH group were significantly lower than those in the ALI group at the different time intervals post-injury. Correlation analysis indicates that CC16 correlates significantly with all the indices mentioned above.

CONCLUSIONSDown-regulation of CC16 expression plays a critical role in the pathogenesis of acute lung injury induced by endotoxemia. The application of GH can exacerbate the lung injury induced by endotoxemia through down-regulating the expression of CC16.

Acute Lung Injury ; genetics ; metabolism ; Animals ; Bronchoalveolar Lavage Fluid ; Growth Hormone ; pharmacology ; Interleukin-6 ; metabolism ; Lung ; drug effects ; metabolism ; Male ; NF-kappa B ; metabolism ; Rats ; Rats, Sprague-Dawley ; Tumor Necrosis Factor-alpha ; metabolism ; Uteroglobin ; genetics

OBJECTIVETo study the expression of serum Clara cell secretory protein 10 (CC10) and total IgE concentration in wheezing children under 5 years old.

METHODSFifty-nine children with recurrent wheezing under 5 years old were classified into two groups: wheezing group 1 with atopic high risks (n=33) and wheezing group 2 without atopic high risks (n=26). Twenty-three children without infectious diseases served as a control group. Serum levels of CC10 and IgE were measured using a solid-phase sandwich ELISA.

RESULTSThe serum levels of CC10 in wheezing group 1 (3.95 ± 1.26 ng/mL) and wheezing group 2 (5.41 ± 1.64 ng/mL) were significantly lower than those in the control group (8.72 ± 2.23 ng/mL; P<0.01). The wheezing group 1 showed more decreased serum levels of CC10 compared with wheezing group 2 (P<0.05). The serum IgE levels in wheezing group 1 were significantly higher than those in wheezing group 2 and the control group (P<0.05). There were no significant differences in serum IgE levels between the wheezing group 2 and control group. There was a negative correlation between serum levels of CC10 and IgE in wheezing group 1 (r=-0.912, P < 0.01).

CONCLUSIONSSerum CC10 levels decrease remarkably in wheezing children, and more significant decrease is noted in patients with atopic high risks. Serum CC10 levels are negatively correlated to serum IgE levels in patients with atopic high risks.

Child, Preschool ; Female ; Humans ; Immunoglobulin E ; blood ; Infant ; Male ; Respiratory Sounds ; immunology ; Uteroglobin ; blood

OBJECTIVEto evaluate the application of GeneSearch(TM) breast lymph node assay in intraoperative detection of metastases in sentinel lymph node (SLN) from breast cancer patients.

METHODSa total of 225 SLN from 88 patients was prospectively studied. Each SLN was cut into 2 mm slabs which were examined by intraoperative imprint cytology (IIC) first, followed by GeneSearch assay and post-operative serial sectioning. GeneSearch used real-time fluorescence quantitative RT-PCR technology to detect the expression of CK19 and mammaglobin in SLN. The results of GeneSearch assay were correlated with those of IIC and post-operative serial sectioning.

RESULTSamongst the 88 cases studied, 225 SLNs were found, and obvious metastatic carcinoma cells were identified in 27 SLNs and micrometastasis in 9 SLNs. One hundred and eight-nine SLNs were considered as "negative" (with "isolated tumor cells" present in 5 SLNs). The turn-around time of intraoperative GeneSearch assay ranged from 35 to 45 minutes (mean = 40 minutes). The concordance rate between GeneSearch assay and post-operative serial sectioning was 95.6% (215/225), with a sensitivity of 86.1% (31/36), compared with 94.7% (213/225) and 72.2% (26/36) respectively for IIC. The size of metastatic foci correlated with the Ct value of CK19 and mammaglobin (P < 0.01).

CONCLUSIONSGeneSearch assay for intraoperative detection of metastase in SLN has a satisfactory performance and demonstrates a relatively higher sensitivity than IIC. The potential clinical application still requires further evaluation of larger number of cases.

Adult ; Aged ; Breast Neoplasms ; metabolism ; pathology ; surgery ; Breast Neoplasms, Male ; metabolism ; pathology ; surgery ; Carcinoma, Ductal, Breast ; metabolism ; pathology ; surgery ; Carcinoma, Intraductal, Noninfiltrating ; metabolism ; pathology ; surgery ; Female ; Humans ; Intraoperative Period ; Keratin-19 ; metabolism ; Lymph Nodes ; metabolism ; pathology ; surgery ; Lymphatic Metastasis ; Male ; Mammaglobin A ; Mastectomy ; methods ; Middle Aged ; Neoplasm Proteins ; metabolism ; Sensitivity and Specificity ; Sentinel Lymph Node Biopsy ; methods ; Uteroglobin ; metabolism

OBJECTIVETo observe the expression of Clara cell secretory protein(CCSP) in the Kunming mouse model of n-hexane long-term inhalation, and to discuss the functions of Clara cell in injury lung induced by n-hexane.

METHODS24 healthy mice were randomly divided into 4 groups: one control group and three n-hexane groups (4 w, 8 w and 12 w), 6 each group. Primary concentration of n-hexane was 17.6 g/m3, 8 hours per day, 6 d per week. After inhalation, n-hexane concentration of blood from celiac artery was detected. The lungs were embedded with paraffin and HE staining in the routine. The ratio of Clara cells with CCSP reaction in bronchiole and the number of macrophage cells with lysozyme reaction were determined by immuno-histochemistry.

RESULTSIn the poisoning groups, the average n-hexane concentration of blood was significantly higher than that of the control group (P < 0.01). There were apparent pathologic damages in lungs of the poisoning mice. In poisoning 4 w, 8 w and 12 w groups, the ratio of Clara cells was significantly decreased [(73.33 +/- 4.21)%, (60.98 +/- 4.94)%, (34.04 +/- 2.33)% in terminal bronchiole, and (75.44 +/- 7.91)%, (58.54 +/- 4.86)%, (33.35 +/- 2.67)% in respiratory bronchiole] as compared with the control mice [(80.26 +/- 6.43)% and (81.74 +/- 7.75)%, P < 0.05 or P < 0.01], meanwhile the numbers of macrophage cells were gradually increased [(21.39 +/- 7.41), (28.54 +/- 10.73), (48.97 +/- 19.55) per microscopic field at 200x] in poisoning mice than those in control mice [(7.84 +/- 3.12) per microscopic field at 200x, P < 0.05 or P < 0.01].

CONCLUSIONIn injury lungs after n-hexane inhalation, Clara cells are the target cells of n-hexane toxicity effect. Clara cells play an extensive protective role in lung inflammation.

Animals ; Epithelial Cells ; metabolism ; Hexanes ; toxicity ; Inhalation Exposure ; Lung Injury ; etiology ; metabolism ; Mice ; Mice, Inbred Strains ; Toxicity Tests, Chronic ; Uteroglobin ; metabolism

BACKGROUNDThe number of Clara cells and the Clara cell 16-kDa protein (CC16) levels of the lung decrease in patients with chronic obstructive pulmonary disease (COPD). N-acetylcysteine (NAC) is a powerful antioxidant and can reduce the frequency of acute exacerbations of COPD. But the exact mechanism is unclear. The present study was designed to investigate the effects of NAC on Clara cells in rats with cigarette smoke exposure.

METHODSEighteen adult male Wistar rats were randomly divided into 3 groups, 12 exposed to cigarette smoke (CS) thrice a day, 10 cigarettes for 30 minutes each time for 1 week, without (CS group) or with (CS + NAC group) oral intake of NAC 80 mg x kg(-1) x d(-1), and another 6 rats exposed to fresh air (control group). Clara cells were observed by an electron microscope. The mRNA expression of CC16 and CC16 protein in lungs were determined by reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry respectively. The glutathion (GSH) level in plasma and lung tissue were tested by fluorimetry assay.

RESULTSCompared with the controls, the pathologic score of small airways significantly increased in the CS exposed rats (20.3 +/- 14.7 vs. 53.7 +/- 11.5, P < 0.05). The Clara cell particles in cytoplasm decreased in the CS group (P < 0.05). The percentage of CC16-positive cells in bronchioles in the CS group (27.8 +/- 4.3 and 29.5 +/- 2.4 in terminal bronchioles and respiratory bronchioles, respectively) significantly decreased as compared with the control group (37.1 +/- 3.8 and 43.8 +/- 5.8 in terminal bronchioles and respiratory bronchioles, respectively) (P < 0.05). No significant difference was observed in GSH level ((181 +/- 26) nmol/L in the control group vs. (170 +/- 18) nmol/L in the CS group) between the two groups. After treatment with NAC, the pathologic score of small airways (24.1 +/- 17.5) decreased (P < 0.05). Clara cell particles in cytoplasm of Clara cells increased and GSH level in plasma ((213 +/- 40) nmol/L vs. (170 +/- 18) nmol/L in the CS group) increased too (P < 0.05), while the increase in the proportions of CC16 positive cells in bronchioles (30.1 +/- 6.4 and 34.3 +/- 6.3 in terminal bronchioles and respiratory bronchioles, respectively) did not reach the statistical significance (P > 0.05). No significant difference was found in the expression of CC16 mRNA among the three groups. Correlation analysis indicated that the percentage of CC16-positive cells in bronchioles negatively correlated with the pathologic score of small airways (r = -0.592, P < 0.05), but not with GSH level.

CONCLUSIONSOne-week CS exposure decreased the number of Clara cells and the expression of CC16 in bronchioles in rats. NAC might provide protection of the Clara cells from oxidative damage and possibly through the elevation of the synthesis and secretion of CC16. These data indicate that NAC decreases airway inflammation induced by CS via induction of CC16.

Acetylcysteine ; metabolism ; Animals ; Bronchioles ; cytology ; drug effects ; metabolism ; Fluorometry ; Glutathione ; metabolism ; Immunohistochemistry ; Male ; Microscopy, Electron, Transmission ; Random Allocation ; Rats ; Reverse Transcriptase Polymerase Chain Reaction ; Smoking ; adverse effects ; Uteroglobin ; genetics ; metabolism