Objective:To analyze the changes of intestinal microflora and to predict the metabolic function of intestinal microflora in severe burn patients at early stage by 16S ribosomal RNA ( rRNA) high-throughput sequencing. Methods:In this prospective observational study, 48 patients with severe burns who met the inclusion criteria were admitted to Department of Burns and Plastic Surgery of Affiliated Hospital of Jiangsu University from January 2018 to December 2019 were included in burn group, and 40 healthy volunteers who met the inclusion criteria and underwent physical examination at the Physical Examination Center of Affiliated Hospital of Jiangsu University in the same period were included in healthy group. Fecal samples were collected from patients in burn group in about 1 week after admission and from volunteers in healthy group on the day of physical examination. The 16S rRNA V4 gene sequencing was performed in the feces of patients in burn group and volunteers in healthy group to analyze the relative abundance of various bacteria. The operational classification unit (OTU) was divided by Mothur software to analyze the dominant bacteria. The OTU number, Chao1 index, Ace index, and Shannon index of fecal microflora were analyzed by QIIME1.9.0 software. The principal component analysis for relative abundance of fecal microflora was performed by Canoco Software 5.0. The metabolic function of fecal microflora was predicted by Kyoto Encyclopedia of Genes and Genomes. Data were statistically analyzed with independent sample t test, and Mann-Whitney U test, and Bonferroni correction. Results:The relative abundance of Bacteroides, Enterococcus, Acinetobacter, Macrococcus, and Staphylococcus in feces of patients in burn group was significantly higher than that of volunteers in healthy group ( Z=-5.20, -2.37, -5.17, -4.41, -6.03, P<0.05 or P<0.01), and the relative abundance of unclassified-Helicobacillae, Prevotella, Cecobacteria, unclassified-Rumencocci, Pseudobutyrivibrio, Brautia, and unclassified-Digiestive Streptococcaceae ( Z=-8.03, -3.21, -7.63, -5.88, -8.05, -8.05, -6.77, P<0.01) and other 12 species of bacteria in the feces of volunteers in healthy group was significantly higher than that of patients in burn group. The diversity of fecal microflora of volunteers in healthy group was better than that of patients in burn group, the main dominant microflora of volunteers in healthy group were Bacteroides, unclassified- Helicobacillae, Prevotella, unclassified- Enterobacteriaceae, Brautia, Parabacteroides, Escherichia coli, etc., and the main dominant microflora of patients in burn group were Bacteroides, Prevotella, unclassified-Enterobacteriaceae, and Parabacteroides. The OTU number, Ace index, Chao1 index, and Shannon index of fecal microflora of patients in burn group were 149±47, 199±45, 190±45, 2.0±0.9, which were significantly lower than 266±57, 323±51, 318±51, 3.8±0.5 of volunteers in healthy group ( t=10.325, 11.972, 12.224, 11.662, P<0.01). The relative abundance of fecal microflora of patients in burn group and volunteers in healthy group was clearly divided into two groups by principal component 1, and the contribution rate of principal component 1 was 32.50%, P<0.01. The fecal microflora of volunteers in healthy group were more concentrated on principal component 2, the fecal microflora of patients in burn group were dispersed in principal component 2, and the contribution rate of principal component 2 was 13.44%, P>0.05. The metabolic levels of alanine-aspartate-glutamate, arginine- proline, cysteine-methionine, glycine-serine-threonine, phenylalanine, tryptophan, and tyrosine in amino acid, tricarboxylic acid cycle, glucose and mannose, galactolipin, glycolysis/gluconiogenesis, starch and sucrose in carbohydrate of fecal microflora of patients in burn group were significantly lower than those of volunteers in healthy group ( Z=-4.75, -4.54, -4.75, -4.62, -3.71, -3.28, -4.19, -3.82, -4.72, -4.35, -4.75, -4.71, P<0.01). The levels of lipoic acid metabolism and coenzyme Q synthesis of fecal microflora of patients in burn group were significantly higher than those of volunteers in healthy group ( Z=-6.07, -4.51, P<0.01). The metabolic level of arachidonic acid of fecal microflora of patients in burn group was similar to that of volunteers in healthy group ( P>0.05). Conclusions:There are significant differences in intestinal microflora between severe burn patients at the early stage and healthy people, and the species and diversity of microflora are decreased, and the nutrient metabolism level is decreased in burn patients by 16S rRNA high-throughput sequencing.

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. ··

Objective To systematically evaluate the effect of HLA-DPB1 mismatch on the clinical outcomes of unrelated donor hematopoietic stem cell transplantation. Methods Relevant studies analyzing the effect of HLA-DPB1 mismatch upon the clinical outcomes of unrelated donor hematopoietic stem cell transplantation published from January 1995 to December 2016 were retrieved from the PubMed, Embase, Center of International Bone Marrow Transplant Registration, Cochrane Central Register of Controlled Trials, Cochrane Database of Systematic Reviews, Chinese BioMedical Literature Database and China National Knowledge Infrastructure database. The survival rate, the incidence of implantation failure, graft-versus-host disease (GVHD), recurrence rate, transplantation-related mortality rate and other adverse events were analyzed by Review Manager 5.3 software. Funnel plot was utilized to assess the publication bias. Results A total of 19 literatures consisting of 26634 patients were retrieved. Meta-analysis demonstrated that compared with the recipients in the HLA-DPB1 match group, those in the HLA-DPB1 mismatch group had lower disease-free survival rate and overall survival rate in non-T cell-depleted transplantation. In the HLA-DPB1 mismatch group, the incidence of severe GVHD and transplantation-related mortality(TRM) rate were higher. And the TRM rate of two loci mismatch was more evident than that of one locus mismatch. Conclusions HLA-DPB1 locus exerts significant effect on the survival rate and incidence of adverse events after unrelated donor hematopoietic stem cell transplantation. The status of HLA-DPB1 match should be considered when selecting the unrelated donors.

Objective To compare the newer antifungal agents micafungin and voriconazole for prophylaxis effects on the clinical outcomes.Methods We electronically searched the database of Cochrane Central Register of Controlled Trials,Pubmed,EMbase,China Biology Medicine (CBM),China National Knowledge Infrastructure(CNKI),and relevant database articles (1996.01-2014.12).Comparative studies were carried out on proved fungal infections,mortality,and adverse effects.Meta-analysis was performed by Review Manager 5.3 software.Results We found 1 564 records and 16 studies totaling 4 234patients included in analyses.Pooled comparisons of studies found that antifungal prophylaxis with the new agents did reduce the incidence of invasive fungal infections and transplant related mortality than fluconazole or itraconazole [OR =0.41 (0.21 ～ 0.80) and OR =0.40 (0.24 ～ 0.66),respectively,P ＜ 0.01].Voriconazole had higher rates of liver dysfunction,lower gastrointestinal side effects over fluconazole,and lower rates of nephrotoxic effects than amphotericin B.Voriconazole had significant decrease in adverse events requiring drug discontinuation compared to itraconazole [OR =0.43 (0.27 ～ 0.68),P ＜ 0.01].Conclusions This analysis indicated the 2 agents appear to be well tolerated with manageable side effects and beneficial in the prophylaxis of invasive fungal infection (IFI).

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 The aim of this study was to establish a standardized protocol for detection of ALK protein expression and gene fusion in cytologic specimens. Methods Lung 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. Results Among 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. Conclusions The 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.

Objective The aim of this study was to establish a standardized protocol for detection of ALK protein expression and gene fusion in cytologic specimens. Methods Lung 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. Results Among 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. Conclusions The 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.

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

OBJECTIVE:To investigate the effect of Bidouyan oral liquid(BOL) on granulocyte-macrophage colony-stimulating factor(GM-CSF) release,ultrastructure changes of eosinophil and epithelial cell proliferation in cultured tissue of nasal polyps METHODS:Nasal polyp tissue was cultured in medium containing different concentrations of BOL The growth of epithelial cells surrounding tissue block and ultrastructure changes of eosinophil in culture tissue were observed The content of GM-CSF in supernate was detected by ELISA RESULTS:In comparison with control,the neogenetic epithelial cells cultured in medium with BOL were fewer in number The contents of GM-CSF were decreased with the increase of the concentrations of BOL with significant differences(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