AIM: To investigate the role of potassium channels in the regulation of intracellular free calcium concentration (_i) of pulmonary artery smooth muscle cells (PASMCs) in rats. METHODS: The fluorescence Ca 2+ indicator Fura-2/AM was used to observe _i of rat PASMCs in normal and chronic hypoxic condition. The influences of potassium channels on PASMCs proliferation were assessed by MTT assay. RESULTS: 1. In normoxic condition,_i was (156.91?8.60) nmol/L,and in hypoxic condition,_i was (294.01?16.81) nmol/L. 2. In normoxic condition,the voltage-dependent K +-channel antagonist 4-aminopyridine (4AP),but not the Ca 2+ -activated K +-channel antagonist tetraethylammonium (TEA) and the ATP-sensitive K +-channel antagonist glibenclamide (Glib) increased _i. 3. In hypoxic condition,4AP and TEA caused the rise in _i [(422.41?24.28) nmol/L,(380.84?11.02) nmol/L,respectively],but Glib had no effect on _i. 4. MTT assay showed that 4AP increased the value of absorbing light degree ( A value) in normoxic and hypoxic condition (0.582?0.062,0.873?0.043,respectively, P

AIM: To investigate the effect of chronic hypoxia on the expression of voltage-gated potassium channels ? subunits in pulmonary tissue. METHODS: Twelve Wistar rats matched with age and body weight were randomly divided into control and chronic hypoxic groups. The expression of mRNA and protein of Kv1.5, Kv2.1, Kv9.3 in pulmonary tissues were detected with reverse transcription-PCR (RT-PCR) and Western Blot. RESULTS: The expression of Kv1.5, Kv2.1 and Kv9.3 gene were found in pulmonary tissue of rats exposed either to normoxia or chronic hypoxia. Chronic hypoxia decreased mRNA expression of Kv channel subunit (Kv1.5, Kv2.1, Kv9.3) in pulmonary tissue from 0.473?0.022, 0.912?0.084, and 0.319?0.019 to 0.256?0.008, 0.406?0.031 and 0.114?0.009, respectively (P

AIM: To elucidate the effect of ET-1 on the expression of voltage-gated K~+ channel ? subunits Kv2.1, Kv9.3 in pulmonary artery smooth muscle cells in rats. METHODS: The mRNA expression of Kv2.1, Kv9.3 in the 2nd, 3rd, 4th subculture of intrapulmonary artery smooth muscle cells isolated from Wistar rats, which exposed to either normoxia or chronic hypoxia, were detected with reverse transcription-PCR (RT-PCR). At the same time, PASMCs were treated with BQ123, an ET_A receptor antagonist. RESULTS: The expression of Kv2.1 and Kv9.3 gene were found in the subculture PASMCs of rats exposed either to normoxia or chronic hypoxia. Chronic hypoxia decreased mRNA expressions of Kv channel subunit Kv2.1, Kv9.3 in PASMCs from 0.827?0.126 and 0.388?0.026 to 0.378?0.015 and 0.184?0.009, respectively (P

AIM: To study the effect of BQ123 on voltage-gated K+ current in pulmonary artery smooth muscle cells(PASMCs) from chronic hypoxic rats.METHODS: Twelve age and body weight matched Wistar rats were randomly divided into control and chronic hypoxic group.Single PASMCs were obtained with acute enzyme(collagnaseⅠ plus papain) dispersing method.Using the whole cell patch-clamp technique in freshly isolated PASMCs from normorxic and hypoxic rats,the effects of ET-1 and BQ123,a selective ETA receptor antagonist,on voltage-gated K+ current were recorded.RESULTS:(1) ET-1(10-8 mol?L-1) caused inhibition of K+ current in PASMCs from normoxic and hypoxic rats.The effect of ET-1 on K+ current in PASMCs from hypoxic rats was greater than that from normoxic rats [+50 mV,percent inhibition were(71.04?6.58)% and(60.21?5.32)%,respectively,P0.05,n=5),nor ETA receptor blockade had change of ET-1 mediated IKV inhibition.(3) In chronic hypoxic PASMCs,BQ123 significantly reduced the effect of ET-1 mediated IKV inhibition,from(28.49?6.69) pA/pF to(74.19?9.74) pA/pF at +50 mV(P

Objective To evaluate the medical indexes for student pilots from different areas , to discover the major different indexes between different areas ,and to establish the space distribution model of military pilots .Methods A cross-section survey was conducted among student pilots , and 290 student pilots sampled as respondents were interviewed with questionnaires and subjected to a physical examination , involving distant vision , heart function , and pulmonary function , before a database was established , cleaned and analyzed by EpiData 3.02, SAS 9.13 with double checking .Results There was no difference between the medical indexes of student pilots from 7 areas, but the psychological selection performance record and the entrance examination record were different .Student pilots from area E had the highest psychological selection performance record while those from area D had the highest entrance examination record .Conclusion Student pilots have area difference ,so we should pay close attention to their birth place during recruitment of student pilots .

OBJECTIVEThe aim of this study was to establish a standard protocol for detection of EGFR mutations in cytologic specimens.

METHODS287 cytologic samples were collected from the patients who were suspected of having lung cancer at six hospitals in Beijing. A detection protocol for EGFR mutations was designed. Two comparative experiments were carried out for the coincidence in EGFR mutation rates between direct sequencing (Seq) and amplification refractory mutation system (ARMS) methods, and between 40 matched cytologic samples with formaldehyde-fixed paraffin embedded (FFPE) cytologic blocks and cytospin slides.

RESULTSTumor cells were found in 236 out of 287 cases (82.2%, 236/287) . Among them, there were 31 cases (13.1%, 31/236) of low tumor cell content samples and 205 cases (86.9%, 205/236) of high tumor cell content samples. 180 cases in the high tumor cell content samples (87.8%, 180/205) were diagnosed to be consistent with NSCLC. 25 out of 194 cases were ruled out or indefinite to be diagnosed as NSCLC by immunohistochemistry. By direct sequencing, the mutation rate of EGFR was 27.8% (50/180) in NSCLC samples and 28.2% (50/177) in adenocarcinoma samples (high tumor content samples) . By ARMS, the mutation rate of EGFR was 45.6% (82/180) in NSCLC samples and 46.3% (82/177) in adenocarcinoma samples (high tumor content samples). The EGFR mutation rate in low tumor content samples was 38.7% (12/31) , there was no significant difference in EGFR mutation rates between the groups of low tumor cell content samples and high tumor cell content samples (P = 0.12). The concordance rate of EGFR mutation rates was 100% between scraping tumor cells from slides samples and from FFEP blocks in the 40 matched samples. Forty-eight out of 180 definitive NSCLC patients received Gefitinib therapy. The FPS was 12 months in the gefitinib-treated ARMS⁺ group and 2 months in the ARMS⁻ group (P < 0.001), and the OS was 19 months in the gefitinib-treated ARMS⁺ group and 7 months in the ARMS⁻ group (P = 0.003), but no significant differences were found in the efficacy (PFS and OS) of Gefitinib between Seq⁺ and Seq⁻ groups (P = 0.227, P = 0.510, respectively), and Seq⁺/ARMS⁺ and Seq⁻/ARMS⁺ groups (P = 0.354, P = 0.334, respectively).

CONCLUSIONSThe detection protocol for EGFR mutations in cytological specimens introduced in this study is tested to be reliable and feasible. Pathological evaluation and immunohistochemistry are important in the detection procedure of EGFR mutations in cytologic specimens. High sensitivity methods should be selected for detection of EGFR mutations in cytologic samples.

Adenocarcinoma ; metabolism ; Carcinoma, Non-Small-Cell Lung ; metabolism ; Humans ; Lung Neoplasms ; diagnosis ; epidemiology ; metabolism ; Mutation ; Mutation Rate ; Polymerase Chain Reaction ; Receptor, Epidermal Growth Factor ; genetics ; metabolism

OBJECTIVEThe aim of this study was to establish a standardized protocol for detection of ALK protein expression and gene fusion in cytologic specimens.

METHODSLung adenocarcinoma cytologic specimens were collected from seven hospitals in Beijing city. A detection protocol for ALK protein expression and gene fusion was designed according to the results of comparative experiment. Ventana immunohistochemical (IHC) ALK(D5F3) detecting ALK protein expression was performed in 203 prepared formalin-fixed paraffin-embedded (FFPE) cell blocks. ALK gene fusion in 98 EGFR gene wild type cytologic specimens and in 4 bronchoalveolar lavage fluid (BL) samples was detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR). ALK gene fusion in the Ventana IHC ALK (D5F3) positive samples was further tested by fluorescence in situ hybridization (FISH). Six patients with ALK IHC-positive result were followed up to analyze the responses of crizotinib therapy. Comparative experiments: (1) Comparison of the results of 4% neutral buffered formalin fixed for different time (24 h, 48 h, 72 h) on the Ventana IHC ALK (D5F3) staining was conducted in two cases of IHC ALK positive FFPE cell blocks; (2) Comparing qRT-PCR results for ALK fusion in samples from FFPE cell blocks and cytospin prepared slides in 10 cases of lung adenocarcinoma cytologic specimens.

RESULTSAmong the specimens examined using the standardized protocol recommended by this study, 229 cases of cytologic specimens met the diagnostic criteria of lung adenocarcinoma. Among them, 207 cases obtained ALK gene test results (by at least one method), with an ALK test ratio of 90.4% (207/229). FFPE cell blocks were successfully prepared in 203 cases, Ventana IHC ALK (D5F3) were successfully performed in all the 203 FFPE cell blocks (100%), and the ALK protein positive detection rate was 10.3% (21/203). ALK fusion was tested in 98 FFPE cytologic samples of EGFR wild types by qRT-PCR, and 96 out of 98 (97.96%) cytologic samples were successfully performed.18 out of 19 IHC ALK-positive cases were verified to be of ALK fusion status by qRT-PCR. The concordance rate was 94.7% (Kappa=0.967, P<0.001) between Ventana IHC ALK (D5F3) and qRT-PCR, and the sensitivity of the Ventana IHC ALK (D5F3) assay compared with qRT-PCR was 100% and the specificity was 98.7%. FISH assay was used to verify the positive cases detected by Ventana IHC ALK (D5F3) staining. Two cases of low tumor cell content FFPE samples obtained indefinite results by FISH test. The six patients with positive ALK protein expression received crizotinib therapy, and 5 paitents got treated effectively. For two ALK IHC positive cases, which were 4% neutral buffered formalin fixed for 72 h, the result of Ventana IHC ALK (D5F3) staining became weakened obviously and uneven. In 10 cases of samples, total RNA was extracted from FFPE cytologic sections and cytospin prepared slides, and the results of qRT-PCR test and ALK gene fusion showed good concordance.

CONCLUSIONSThe standardized protocol recommended in this study expands the detection types and quantity of cytologic specimens for ALK protein expression and gene fusion and increased the detection rate. Ventana IHC ALK (D5F3) is a reliable method for detecting ALK protein expression in FFPE cell blocks. The pathologic quality control procedure prior to Ventana IHC ALK (D5F3) is crucial for the accuracy of testing the ALK gene status. When FFPE cell blocks could not be prepared or prepared unsuccessfully from the cytologic specimens, qRT-PCR may be an alternative option for the detection of ALK gene fusion.

Adenocarcinoma ; drug therapy ; enzymology ; genetics ; pathology ; Alkaline Phosphatase ; genetics ; metabolism ; Gene Fusion ; Genes, erbB-1 ; Humans ; Immunohistochemistry ; In Situ Hybridization, Fluorescence ; Lung Neoplasms ; drug therapy ; enzymology ; genetics ; pathology ; Protein Kinase Inhibitors ; therapeutic use ; Proteomics ; Pyrazoles ; therapeutic use ; Pyridines ; therapeutic use ; Sensitivity and Specificity

Objective: To investigate the characteristics and clinical significance of the immunomicroenvironment typing based on the expression of programmed death-ligand 1 (PD-L1) and the infiltration of CD8+ T cells in the stroma in patients with non-small cell lung cancer (NSCLC). Methods: Paraffin tissue specimens and relevant clinicopathological data of 74 NSCLC patients admitted to our hospital from January 2016 to July 2018 were collected.All patients received EGFR gene test, and none received radiotherapy, chemotherapy or targeted therapy. Immunohistochemistry was used to detect the expression of PD-L1 in tissues and the infiltration of CD8+T cells in interstitium, and the relationship between PD-L1, CD8+T cells, and the immune microenvironment typing based on both, and the pathological parameters and the survival of patients was analyzed. Results: PD-L1 expression in the primary tumor of NSCLC patients showed statistical differences in gender, pathological type, smoking history, EFGR gene mutation status ( P <0.05). The infiltration of CD8+ T lymphocytes in tumor microenvironment showed statistically significant differences in different TNM stage and lymph node metastasis ( P <0.05), PD-L1 expression was significantly correlated with EGFR mutation ( P =0.000), while CD8+T lymphocyte infiltration was not correlated with EGFR mutation ( P =0.605). The immunomicroenvironment of EGFR wild-type patients was mainly (CD8+ PD-L1+) (type I), and the mutants were mainly (CD8-PD-L1-) (type II) and (CD8+PD-L1-) (type IV). The distribution of immune microenvironmental typing in each group with different EGFR mutation, smoking history and pathological differentiation degree was significantly different ( P <0.05) and significantly correlated with EGFR mutation ( P <0.05). Follow-up showed that the patients with disease free survival, recurrence and metastasis and death were the most in type I, type II and type I, respectively. Conclusions: In this study, the distribution of tumor immunomicroenvironmental typing in NSCLC patients was mainly the highest in type I and the lowest in type Ⅲ, which was related to EGFR mutation, smoking history and pathological differentiation. Patients with EGFR mutations were mainly of type Ⅱand type Ⅳ, and were associated with low expression of PD-L1. ··