Generation and phenotypic characterization of S100A9 gene knockout mice by CRISPR/Cas9-mediated gene targeting / 生理学报

S100 calcium binding protein A9 (S100A9) is involved in a variety of biological processes such as inflammation and tumor cell migration and invasion regulation. The purpose of this study was to construct S100A9 gene-edited mice by using CRISPR/Cas9 technology, thereby providing an animal model for exploring the biological functions of this gene. According to the S100A9 gene sequence, the single-stranded small guide RNA (sgRNA) targeting exons 2 and 3 was transcribed in vitro, and a mixture of Cas9 mRNA and candidate sgRNA was injected into mouse fertilized eggs by microinjection. Early embryos were obtained and transferred to surrogate mice, and F

Prognostic Role of S100A8 and S100A9 Protein Expressions in Non-small Cell Carcinoma of the Lung

BACKGROUND: S100A8 and S100A9 have been gaining recognition for modulating tumor growthand metastasis. This study aimed at evaluating the clinical significance of S100A8 and S100A9 innon-small cell lung cancer (NSCLC). METHODS: We analyzed the relationship between S100A8and S100A9 expressions, clinicopathological characteristics, and prognostic significance in tumorcells and peritumoral inflammatory cells. RESULTS: The positive staining of S100A8 in tumorcells was significantly increased in male (p < .001), smoker (p = .034), surgical method other thanlobectomy (p = .024), squamous cell carcinoma (SQCC) (p < .001) and higher TNM stage (p = .022)compared with female, non-smoker, lobectomy, adenocarcinoma (ADC), and lower stage. Theproportion of tumor cells stained for S100A8 was related to histologic type (p < .001) and patientsex (p = .027). The proportion of inflammatory cells stained for S100A8 was correlated with patientage (p = .022), whereas the proportion of inflammatory cells stained for S100A9 was correlatedwith patient sex (p < .001) and smoking history (p = .031). Moreover, positive staining in tumorcells, more than 50% of the tumor cells stained and less than 30% of the inflammatory cellsstained for S100A8 and S100A9 suggested a tendency towards increased survivability in SQCCbut towards decreased survivability in ADC. CONCLUSIONS: S100A8 and S100A9 expressions might be potential prognostic markers in patients with NSCLC.

The effect of smoking on myeloid-related protein-8 and myeloid-related protein-14

Abstract The aim of this study was to determine the myeloid-related protein-8 and myeloid-related protein-14 levels in the gingival crevicular fluid of smoker patients with generalized aggressive periodontitis (SAgP), smoker patients with chronic periodontitis (SCP), smoker patients with gingivitis (SG-smoker control), non-smoker patients with generalized aggressive periodontitis (AgP), non-smoker patients with chronic periodontitis (CP), and non-smoker patients with gingivitis (G-non-smoker control). The periodontal statuses of the patients were determined by periodontal clinical measurements and radiographical evaluations. The levels of myeloid-related protein-8 and myeloid-related protein-14 in the gingival crevicular fluid were assessed using enzyme-linked immuno sorbent assay. The myeloid-related protein-8 and myeloid-related protein-14 levels in the gingival crevicular fluid of patients with generalized aggressive periodontitis (non-smoker and smoker) were found to be statistically higher than patients with chronic periodontitis (non-smoker and smoker) and patients with gingivitis (non-smoker and smoker). Myeloid-related protein-8 and myeloid-related protein-14 levels of non-smokers were significantly higher than smokers in all types of periodontitis and gingivitis. The decreased myeloid-related protein-8 and myeloid-related protein-14 level could have prevented the haemostasis of calcium which plays a significant role in the migration of neutrophiles. Smoking affects myeloid-related protein-8 and myeloid-related protein-14 levels and may inhibit the antimicrobial efficiency against microorganisms. Due to these reasons smoker generalized aggressive periodontitis patients need to be treated in detail and their maintenance durations should be shortened.

Association between serum levels of S100A8/S100A9 and clinical features of colorectal cancer patients / 中南大学学报(医学版)

OBJECTIVE@#To analyze the association between serum levels of S100A8/S100A9 and clinicopathological features of colorectal cancer patients.
@*METHODS@#A total of 82 patients with CRC and 14 healthy controls were enrolled for this study. The levels of S100A8 and S100A9 in serum were detected by ELISA assay. The association between S100A8/S100A9 and clinicopathological features was analyzed by student-t test and one-way ANOVA. Receiver Operating Characteristic curve was used to analyze diagnostic efficiency of serum S100A8 and S100A9 for colon rectal cancer. Logistic regression model was also established to analyze the possible risk factors for elevation of S100A8/S100A9.
@*RESULTS@#The levels of S100A8 and S100A9 were (1 403.3±593.7) and (2 890.3±994.9) pg/mL in patients with colon cancer, and (712.8±265.3) and (1 492.7±564.6) pg/mL in controls, respectively, with significant difference between the two groups (P<0.01). The similar results were found in rectal cancer patients, with a level of S100A8 and S100A9 at (1 417.7±666.5) and (3 026.7±887.6) pg/mL, respectively. Diagnostic sensitivity and specificity of S100A8 and S100A9 are better than traditional biomarkers. The levels of S100A9 in serum of CRC patients were correlated with clinical stages and distant metastasis. Serum levels of S100A9 in patients of stage III [(3 111.9±178.5) pg/mL] and stage IV [(3 831.4±278.5) pg/mL] were significantly (P<0.01) higher than that in stage I [(2 276.1±167.4) pg/mL], whereas there was significant change in S100A8 levels. Logistic regression showed the possible risk factors for the elevation of S100A9, including depth of invasion, lymphatic metastasis and degree of differentiation (P<0.05).
@*CONCLUSION@#Serum level of S100A8 and S100A9 in CRC patients were significantly increased and serum level of S100A9 was positively correlated with the malignant features of CRC.

Expression of S100A8 and A100A9 in giant cell tumor of bone and its relation with CT and MR imaging findings / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the mRNA and protein expression levels of S100A8 and S100A9 in giant cell tumor (GCT) of bone, and its relation with radiological findings and biological behavior.</p><p><b>METHODS</b>Forty three patient with GCT of bone admitted in Ruijin Hospital Shanghai Jiaotong University School of Medicine from January 2009 to June 2012 were enrolled in the study. The expression levels of S100A8 and S100A9 mRNA and protein were detected by using semiquantitative RT-PCR and Western blotting in 43 specimens of GCT and 6 specimens of normal bone marrow. The CT and MRI findings of patients were retrospectively reviewed, its relation with tissue expression of S100A8 and S100A9 was analyzed.</p><p><b>RESULTS</b>Among 43 GCT cases 40 showed positive expression of S100A8 and S100A9 mRNA and protein, and the expression levels were significantly higher than those in normal bone marrow P<0.05). The expression level of S100A8 protein was significantly different in bone GCT with different composition ratio on MRI (P<0.05).The expression level of S100A9 protein was significantly different in GCT with different degree of bone destruction on CT scan (P<0.05).</p><p><b>CONCLUSION</b>The expression of S100A8 and S100A9 mRNA and protein is up-regulated in GCT of bone. The expression of S100A8 and S100A9 is associated with the real composition ratio and the degree of bone destruction, respectively, indicating that S100A8 and S100A9 may be involved in the biological behavior of bone GCT.</p>

Urinary Nucleic Acid TSPAN13-to-S100A9 Ratio as a Diagnostic Marker in Prostate Cancer

The potential use of urinary nucleic acids as diagnostic markers in prostate cancer (PCa) was evaluated. Ninety-five urine samples and 234 prostate tissue samples from patients with PCa and benign prostatic hyperplasia (BPH) were analyzed. Micro-array analysis was used to identify candidate genes, which were verified by the two-gene expression ratio and validated in tissue mRNA and urinary nucleic acid cohorts. Real-time quantitative polymerase chain reaction (qPCR) was used to measure urinary nucleic acid levels and tissue mRNA expression. The TSPAN13-to-S100A9 ratio was selected to determine the diagnostic value of urinary nucleic acids in PCa (P = 0.037) and shown to be significantly higher in PCa than in BPH in the mRNA and nucleic acid cohort analyses (P < 0.001 and P = 0.013, respectively). Receiver operating characteristic (ROC) analysis showed that the area under the ROC curve was 0.898 and 0.676 in tissue mRNA cohort and urinary nucleic acid cohort, respectively. The TSPAN13-to-S100A9 ratio showed a strong potential as a diagnostic marker for PCa. The present results suggest that the analysis of urine supernatant can be used as a simple diagnostic method for PCa that can be adapted to the clinical setting in the future.

Simultaneous application of BrdU and WST-1 measurements for detection of the proliferation and viability of airway smooth muscle cells

BACKGROUND: BrdU is a commonly used reagent in cell proliferation assays, and WST-1 measurement is widely used to detect cell viability. However, no previous study has formally reported the combination of the two assays, which may be used to detect the proliferation and viability simultaneously. In this study, we examined the effect of adding BrdU 2 h prior to the WST-1 assay and tried to test the possibility of the combined detection using rat airway smooth muscle cells. RESULTS: The WST-1 measurements obtained from the combined detection were consistent with those obtained from the separate detection, which suggested that the addition of BrdU 2 h prior to the WST-1 analysis did not affect the WST-1 results. The BrdU measurements obtained from the combined detection also demonstrated the same trend as that obtained from the separate detection, and dosages of 200, 400 and 800 ng/ml testing reagent significantly inhibited the proliferation of rat airway smooth muscle cells. CONCLUSIONS: Our study suggests that the BrdU and WST-1 measurements can be applied simultaneously without mutual interference, which may increase the efficacy and consistency of these measurements to a certain extent.

Role of inflammasome activation in development and exacerbation of asthma

Human airways contact with pathogen-associated molecular patterns and danger-associated molecular patterns present in many environments. Asthmatic's airways may be more susceptible to these patterns and lead to inflammasome activation; however, the participation of inflammasome in the development and exacerbation of asthma is not fully understood and remains controversial. Asthma is a heterogeneous group composed of different airway inflammation patterns with different underlying immune mechanisms. One mechanism is neutrophilic airway inflammation based on the axis of inflammasome activation, interleukin (IL) 1β/IL-18 production, T helper 17 activation, IL-8/IL-6 overproduction, and neutrophilic inflammation. The role of inflammasome activation has been highlighted in experimental asthma models and some evidence of inflammasome activation has been recently demonstrated in human neutrophilic asthmatic airways. In addition to caspase-1 activation, proteinase 3 and other protease from activated neutrophils directly cleave pro-IL-1β and pro-IL-18 to IL-1β and IL-18, which contribute to the phenotype of subsequent adaptive immune responses without inflammasome activation. Data suggests that neutrophilics in asthmatic airways may have an additional effect in initiating inflammasome activation and amplifying immune responses. Among the mediators from neutrophils, S100A9 seems to be one candidate mediator to explain the action of neutrophils in amplifying the airway inflammation in concert with inflammasome.

S100A8/A9 as a biomarker for synovial inflammation and joint damage in patients with rheumatoid arthritis

S100A8 and S100A9 are major leukocyte proteins, known as damage-associated molecular patterns, found at high concentrations in the synovial fluid of patients with rheumatoid arthritis (RA). A heterodimeric complex of S100A8/A9 is secreted by activated leukocytes and binds to Toll-like receptor 4, which mediates downstream signaling and promotes inflammation and autoimmunity. Serum and synovial fluid levels of S100A8/A9 are markedly higher in patients with RA than in patients with osteoarthritis or miscellaneous inflammatory arthritis. Serum levels of S100A8/A9 are significantly correlated with clinical and laboratory markers of inflammation, such as C-reactive protein, erythrocyte sedimentation rate, rheumatoid factor, and the Disease Activity Score for 28 joints. Significant correlations have also been found between S100A8/A9 and radiographic and clinical assessments of joint damage, such as hand radiographs and the Rheumatoid Arthritis Articular Damage score. In addition, among known inflammatory markers, S100A8/A9 has the strongest correlation with total sum scores of ultrasonography assessment. Furthermore, baseline levels of S100A8/A9 are independently associated with progression of joint destruction in longitudinal studies and are responsive to change during conventional and biologic treatments. These findings suggest S100A8/A9 to be a valuable diagnostic and prognostic biomarker for RA.

Expression of myeloid-related protein complex in association with circulating endothelial cells in children with acute Kawasaki disease / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To investigate the expression of myeloid-related protein complex (MRP-8/14) in children with acute Kawasaki Disease (KD).</p><p><b>METHODS</b>A total of 41 children with acute KD and 40 age- and sex-matched control children with upper respiratory tract infection were recruited. Serum levels of MRP-8/MRP-14 complex were measured by ELISA, messenger ribonucleic acid (mRNA) abundance of MRP-8 and MRP-14 in circulating granulocytes and monocytes was determined by RT-PCR, and the number of circulating endothelial cells was determined by flow cytometry.</p><p><b>RESULTS</b>When the analysis was stratified according to the presence or absence of coronary artery ectasia in the KD patient group, serum levels of MRP-8/MRP-14 complex, MRP-8 and MRP-14 mRNA abundance in granulocytes, and the number of circulating endothelial cells were all significantly higher in KD patients with coronary artery ectasia than in KD patients without coronary artery ectasia (P<0.05). Serum levels of MRP-8/MRP-14 complex were positively correlated with the number of endothelial cells in the circulation (r=0.69, P<0.05).</p><p><b>CONCLUSIONS</b>Serum levels of MRP-8/MRP-14 complex are elevated in a positive association with the number of circulating endothelial cells in KD children with coronary artery ectasia, suggesting a causative role in the development of coronary artery lesions.</p>

Expression of galectin-7 and S100A9 and development of cervical squamous carcinoma / 中南大学学报(医学版)

OBJECTIVE@#To observe the correlation between the expression of galectin-7 and S100A9 with the development of cervical squamous carcinoma.@*METHODS@#Immunohistochemical SP staining was used to detect the expression of galectin-7 and S100A9 in 243 patients with cervical intraepithelial neoplasia (CIN) or cervical squamous carcinoma. The association of clinical data with galectin-7 and S100A9 expression was examined.@*RESULTS@#The expression of galectin-7 and S100A9 in CIN and cervical squamous carcinoma was significantly different (P0.05). Expression of galectin-7 was associated with the survival rate of patients with cervical squamous carcinoma (P<0.05). Univariate analysis of Cox proportional hazards regression model revealed that the FIGO stage, lymph nodes metastasis, and the expression of galectin-7 were relevant to the 5 year survival rate of patients with cervical squamous carcinoma, which was confirmed by multiple analysis of Cox proportional hazards regression model.@*CONCLUSION@#Expression of galectin-7 and S100A9 is related with cervical the tumorigenesis of carcinoma, clinical stage, and lymph nodes of cervical squamous carcinoma. Galectin-7 is probably associated with the prognosis. The long-term survival of patients with cervical carcinoma may be associated with FIGO stage, lymph node metastasis, and the expression of galectin-7.

Analysis of differentially expressed proteins in normal cervix, cervical intraepithelial neoplasia and cervical squamous carcinoma tissues / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To explore the differentially expressed proteins in normal cervix, cervical intraepithelial neoplasia (CIN) and squamous cervical carcinoma (SCC) tissues by differential proteomics, and to provide a basis for studies on CIN molecular pathogenesis, clinical diagnosis and treatment.</p><p><b>METHODS</b>Uterine cervical tissue specimens from the patients treated between August 2008 and September 2009 in the Department of Oncology of Beijing Obstetrics and Gynecology Hospital were collected. There were samples of normal cervix (n = 9), CIN (n = 23, CIN I = 7, CIN II = 8, CIN III = 8) and SCC (n = 7). 2-D DIGE and DeCyder software were used to detect the differentially expressed protein-spots. Then MALDI-TOF/TOF MS was used to analyze the differentially expressed proteins. Collect normal cervix(n = 20), CIN (n = 60) and SCC (n = 20), immunohistochemistry (IHC) and Western blot were used to verify the differentially expressed proteins of S100A9 (S100 calcium-binding protein A9) , eEF1A1 (eukaryotic elongation factor 1-alpha-1) and PKM2 (pyruvate kinase isozymes M2) among the normal cervix, CIN and SCC tissues. Immunohistochemistry was used to detect the differentially expressed S100A9, eEF1A1 and PKM2 in the cervical tissues.</p><p><b>RESULTS</b>2D gel electrophoresis images with high resolution and good repeatability were obtained. Forty-six differentially expressed proteins (27 were up-regulated and 19 were down-regulated) were selected among the normal, CIN, and SCC, and 26 proteins were successfully identified. Immunohistochemistry showed that protein S100A9 was mainly expressed in the cytoplasm, and its positive expression rate was 20.0% in normal cervical mucosa, 70.0% in CIN, and 100.0% in squamous cell carcinoma, with a significant difference between them (P = 0.006). eEF1A1 was mainly expressed in the cell plasma. Its positive expression rate was 70.0% in normal cervix, 73.3%in CIN and 60.0% in SCC tissues, with a non-significant difference between them (P = 0.758). The protein PKM2 was mainly expressed in the cell nuclei. Its positive expression rate was 100.0% in normal cervix, 93.3% in CIN and 75.0% in SCC tissues, showing a difference close to statistical significance (P = 0.059) between them. The results of Western blot were similar with that of immunohistochemical examination.</p><p><b>CONCLUSIONS</b>There are differentially expressed proteins among normal cervix, CIN and SCC. S100A9, eEF1A1 and PKM2 may become candidate markers for early diagnosis of cervical cancer and new targets for therapy. It also provides a further basis for studies of the pathogenetic mechanism of CIN developing to cervical cancer.</p>

MRP8 promotes Th17 differentiation via upregulation of IL-6 production by fibroblast-like synoviocytes in rheumatoid arthritis

Myeloid-related protein (MRP)8/MRP14 is an endogenous Toll-like receptor 4 (TLR4) ligand and is abundant in synovial fluid (SF) of rheumatoid arthritis (RA) patients. Belonging to damage-associated molecular patterns, it amplifies proinflammatory mediators and facilitates a wide range of inflammatory and autoimmune diseases. Interleukin (IL)-17-producing T-helper (Th)17 cells have a crucial role in RA pathogenesis, and IL-6 is the key factor promoting Th17 differentiation. We investigated whether the level of MRP8/MRP14 is positively associated with IL-6 and IL-17 levels in RA SF and found that MRP8/MRP14 level had a significant correlation with IL-6 and IL-17 levels in RA SF. We also observed that MRP8-induced IL-17 production by peripheral blood mononuclear cells but MRP14 did not. Upon stimulation with MRP8, IL-6 production was enhanced by RA fibroblast-like synoviocytes (FLS) and was further elevated by coculturing RA FLS with activated CD4+ T cells. Moreover, we demonstrated that MRP8-activated IL-6 production by RA FLS promoted differentiation of Th17 cells using the coculture system consisting of CD4+ T cells and RA FLS. In addition, IL-6 blockade attenuated Th17 polarization of CD4+ T cells in the cocultures. Inhibitor studies revealed that MRP8 increased IL-6 production in RA FLS via TLR4/phosphoinositide 3-kinase/nuclear factor-kappaB and mitogen-activated protein kinase signaling pathways. Our results show that MRP8 has a crucial role in stimulating IL-6 expression by RA FLS, and subsequently promotes Th17 differentiation in RA, suggesting that neutralizing MRP8 level in RA synovium may be an effective therapeutic strategy in RA treatment.

Up-regulation of the expression of S100A8 and S100A9 in lung adenocarcinoma and its correlation with inflammation and other clinical features / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>S100A8 and S100A9 are two members of the S100 protein family characterized by the presence of two Ca2+-binding sites of the EF-hand type. Previous studies suggested that the whole S100 family displays significant functions in tumor growth, progression and invasion. This study aimed to determine the expression of the two indices of the family, S100A8 and S100A9, in lung cancer tissues and normal lung tissues and its correlation with clinical features.</p><p><b>METHODS</b>A total of 60 cases with a variety of clinical data that were diagnosed with different histological subtypes of lung cancer were investigated. Semi-quantitative reverse transcriptase-PCR (Sq-Rt-PCR) and immunohistochemical staining of cancer, adjacent and peripheral lung tissues were executed to distinguish the expression patterns of S100A8 and S100A9 and to further clarify their correlation with clinical features.</p><p><b>RESULTS</b>Immunohistochemical staining of both proteins showed a significant up-regulation in lung cancer tissue (S100A8, S100A9, P<0.0001), and PCR revealed that the levels of S100A8 and S100A9 expression were significantly higher in lung cancer tissues (S100A8 P=0.002/0.004; S100A9 P=0.022/0.026). The higher expression was found to be correlated with the clinical characteristics of adenocarcinoma, inflammation and stage IV lesion.</p><p><b>CONCLUSIONS</b>S100A8, S100A9 up-regulation was found in the lung adenocarcinoma and end stage lung cancer tissue, the correlation of which with their higher expression in inflammatory lung tissues may indicate the collaborative effect of inflammation on the progression of cancer.</p>

Correlation of myeloid related proteins [MRP8/MRP14] and disease activity in juvenile rheumatoid arthritis

To evaluate the expression of myeloid related proteins MRP8/MRP14 in the serum and synovial fluid of JRA and their correlation with local and systemic parameters of disease activity. Thirty JRA patients [Group I], and ten controls [Group II] were included in the study. The patients were subjected to thorough history taking and clinical examination. Synovial fluid aspiration was done from ten JRA patients. Laporatory investigations included CBC, ESR, RE, ASO, ANA, CRP, ALT, urine analysis and synovial fluid analysis for white blood cell count [SF-WBCs], lymphocytes%, and acute phase serum amyloid. MRP8/MRP14 was assessed with ELISA technique in serum and synovial fluid samples. Serum level of MRP8/MRP14 was elevated in Group I in comparison to up II. The serum and synovial levels of MRP8/MRP14 in Group I showed no significant inter-correlation. The MRP8/MRP14 in group I showed significant positive correlation with ESR, CRP, DAS, and A-SAA. The SF MRP8/MRP14 in group I showed positive significant correlation with SF-WBCs and A-SAA. The elevated MRP8/MRP14 in the serum and synovial fluids of patients with JRA showed a significant correlation with local and systemic disease activity parameters. So, it can be used to monitor disease activity and patient's response to treatment

Analysis of in vitro anti-leukemia effect of 5-aza-2'-deoxycitydine / 中南大学学报(医学版)

OBJECTIVE@#To investigate the effect of methylation transferase inhibitor 5-aza-2'-deoxycitydine (5-aza-2 dC) on the growth, differentiation and apoptosis of human acute myeloid leukemia(AML) cell line HL-60, and to explore the possible anti-leukemia mechanism of 5-aza-2 dC.@*METHODS@#HL-60 cells were treated by 5-aza-2 dC at various concentrations for different periods of time. The effect of 5-aza-2 dC on the growth of HL-60 cells were detected by MTT assay. The effect on the cell cycle and differentiation were detected by flow cytometry. The effect on the apoptosis were detected by Hochest33342 staining and flow cytometry. The expression of S100A8 and S100A9 was detected by reverse transcription-polymerase chain reaction (RT-PCR).@*RESULTS@#(1) 5-aza-2 dC inhibited the growth of HL-60 cells in a concentration- and time-dependent manner, and HL-60 cells were arrested at G2/M phases; (2) 5-aza-2 dC enhanced the expression of cell differentiation antigen CD11b at HL-60 cells, especially at the low drug concentration; (3) 5-aza-2 dC induced HL-60 cell apoptosis in a concentration- and time-dependent manner, especially at the high drug concentration; (4) 5-aza-2 dC increased the expression levels of S100A8 and S100A9 mRNA in HL-60 cells.@*CONCLUSION@#5-aza-2 dC can inhibit the growth of HL-60 cells accompanied with G2/M phase arrest, induce the differentiation and apoptosis of the cells, and increase the expression levels of S100A8 and S100A9 mRNA, which may be the anti-AML mechanism of 5-aza-2 dC.