TCF7L2) and type 2 diabetes mellitus (T2DM) in Han Chinese people in Henan province, China.We aimed to replicate the association of the rs290487 (IVS3C/T) and rs7903146 (IVS3C/T) polymorphisms of transcription factor 7-like 2  and rs290487 (IVS3C/T). W performed a meta-analysis of the association of the risk alleles of rs7903146 (IVS3C/T) and rs290487 (IVS3C/T) in p  = 0.001), the recessive model  of rs290487 (IVS3C/T) and haplotype CC  in Han Chinese. Moreover, our meta-analyses supported the association of the T allele (IVS3C/T) of rs7903146  and T2DM but not the C allele of rs290487 (IVS3C/T)  in Han Chinese. We found no interactions between behavioral risk factors  and rs7903146 (IVS3C/T) and rs290487 (IVS3C/T) polymorphisms.We found that T2DM was associated with the CC genotype  and CC haplotype of rs7903146 (IVS3C/T) and rs290487 (IVS3C/T) in  Type 2 diabetes mellitus (T2DM) is a group of metabolic diseases characterized by hyperglycemia resulting from a progressive insulin secretory defect with insulin resistance TCF7L2) spans 215.9 kb on human chromosome 10q25 TCF7L2 gene have been evaluated in epidemiology studies of Chinese people and include rs7903146, rs290487, rs12255372, rs11196218, rs11196205, rs12775879, and DG10S478. The variants rs7903146 and rs290487 have shown inconsistent results in Han Chinese people. These two single-nucleotide polymorphisms (SNPs) have been most studied in Chinese people, but their association with T2DM in Han Chinese people is still controversial The mechanisms of T2DM remain unclear, but genetic susceptibility plays a crucial role in the etiology and manifestation of the disease TCF7L2 in a case-control study of a large population of Han Chinese people to confirm the association of these 2 polymorphisms in TCF7L2 and T2DM in Han Chinese people. We also performed a meta-analysis by combining these results with previous findings.Here, we aimd to investigate rs7903146 (IVS3C/T) and rs290487 (IVS3C/T) polymorphisms in All participants were of Northern Han Chinese ancestry and were recruited among local inhabitants of Henan Province from the outpatient clinics of several hospitals. T2DM was defined as fasting blood glucose ≧ 7.00 mmol/L or 2 h plasma glucose ≧ 11.0 mmol/L during an oral glucose tolerance test with diabetes clinical symptoms. We excluded patients with type 1 diabetes and other abnormal glucose tolerance and included patients with a previous diagnosis of T2DM in accordance with 2005 American Diabetes Association criteria All blood samples were incubated with disodium EDTA for measuring, glucose level and non-EDTA for measuring total cholesterol (TC), triglycerides (TGs), and high- -density lipoprotein-cholesterol (HDL-C) by use automatic biochemical analysis, Low-density lipoprotein-cholesterol (LDL-C) level was calculated by the Freidwald formula 2, 100 µmmol/L deoxynucleotide triphosphate (dNTP) and 0.5 U Taq polymerase), and the cycling conditions were 95°C for 10 min followed by 35 cycles of 95°C for 30 sec, 65°C [for rs7903146 (IVS3C/T)]/55°C [for rs290487 (IVS3C/T)] for 30 sec, and 72°C for 30 sec, and a final extension of 10 min at 72°C. PCR products were incubated for 10 hr with 3 U restriction enzyme in a 20-µL reaction volume and separated by 4% agarose gel electrophoresis. Genotyping success rates were 99.99% for rs7903146 (IVS3C/T) and rs290487 (IVS3C/T). To verify the reproducibility, we repeated 2% of samples at random as a quality controls for genotyping, and the concordance rate was 100% (see Genomic DNA was extracted from whole blood by use of a blood genome DNA extraction kit (Yaneng BIO). Genotyping involved PCR- restriction fragment length polymorphism (PCR-RFLP). Primers were designed by use of OLIGO Primer Analysis Software 7.0 . The primer sequences and conditions for RFLP are in supplementary 100% see .p values for risk of T2DM were calculated by logistic regression analysis after adjusting for sex, age, anthropometric measurements , and biochemical indexes such as TC, TG, HDL-C and LDL-C levels. The interactions between behavioral risk factors and SNPs were analyzed by adopting multiplicative interaction terms in a multivariate logistic regression model, including SNPs, risk factors, and interaction terms after adjusting for gender, age, and BMI. All tests were two sided and were considered statistically significant at P≤0.05. Haplotype analysis and linkage disequilibrium coefficients were calculated by use of Haploview 4.2 http://www.broad.mit.edu/haploview. P<0.05 was considered statistically significant. Calculation of population-attributable risk proportion (PARP) was based on the estimated ORs and genotypic frequencies of the SNP showing significant association with T2DM. Statistical analysis involved use of SPSS v17.0 for Windows . Power calculation involved use of PGA software.Categorical variables are described with number (percentage) and analyzed by chi-square test. Continuous variables are described with median (range) for non-normally data. Mann-Whitney-Wilcoxon and Kruskal-Wallis rank tests were used to assess differences between cases and controls. Hardy-Weinberg equilibrium for each SNP was separately calculated for cases and controls by use of the Fisher exact method TCF7L2’, ‘genetic polymorphism’, ‘Chinese’ and ‘T2D/T2DM’ for articles related to Chinese participants published in English and Chinese. We used the Metan module within the STATA 11.0 software for the meta-analysis of allele frequencies by combining the previous published data with our data. The strength of association of risk alleles and T2DM was measured by ORs with 95% CIs. The Z test was used to determine the statistical significance of the pooled OR. Power calculation involved use of PGA software. Calculation of Population-attributable risk proportion (PARP) was based on the estimated ORs and risk allele frequencies of the SNP showing significant association with T2DM.We searched MEDLINE via PubMed, EMBASE, Cohorane, and Chinese databases  from January 2007 to February 2012 using the keywords: ‘p<0.001).We included 1,842 T2DM patients  and 7,777 controls  (see supplementary p  = 7.038×10−11) but not rs7903146 (IVS3C/T) (p  = 0.063) were associated with T2DM .The distributions of both risk alleles in these two SNPs differed between cases and controls (see supplementary p  = 0.013) and CC versus TT genotype  and the recessive model  of rs290487 (IVS3C/T) were associated with T2DM. After adjustmet for potential confounders such as sex, age, anthropometric measurements, and metabolic measurements, the CC genotype  and the recessive model  and were associated with T2DM. The PARP was 4.05% for the CC genotype of rs290487 (IVS3C/T). No genotypes for rs7903146 (IVS3C/T) was associated with T2DM in the Han Chinese population with adjusted or unadjusted analysis.The CT genotype  and rs290487 (IVS3C/T) was 0.028 (r2 = 0.001), which indicated weak linkage disequilibrium for the SNPs. After analyzing all estimated haplotypes, haplotype CC was associated with T2DM  and rs290487 (IVS3C/T) in TCF7L2 but found no interactions after adjustment for gender, age, and BMI (p>0.05)  and T2DM, we analyzed the interactions of behavioral risk factors and SNPs in Linear regression analysis revealed no association of clinical and metabolic characteristics of controls and the distribution of studied genotypes with adjustment for age, gender and BMI  but not the C allele of rs290487 (IVS3C/T) . For the association of the T allele of rs7903146 (IVS3C/T) and T2DM, the PARP was 1.77% and the power of the meta-analysis was 100%.Our meta-analysis included 9 studies comprising 5,215 cases and 10,772 controls for rs290487 (IVS3C/T) and 15 studies comprising 9,744 cases and 15,246 controls for rs7903146 (IVS3C/T) in Han Chinese TCF7L2 and the haplotype CC in Han Chinese in Henan province in China. We found no association of clinical and metabolic characteristics and the distribution of the 2 SNPs in controls. As well, we found no interactions between behavioral risk factors and distribution of the SNPs in Han Chinese in Henan province, China.Our findings revealed an association of T2DM and the risk allele C and CC genotype of the SNP rs290487 (IVS3C/T) of p  = 0.001) and the recessive model  of rs290487 (IVS3C/T) and T2DM, and the power of our study was 100% to detect association based on the prevalence of T2DM in China is 9.7%. This founding is familiar to our previous meta-analysis findings for southern China  and studies by H. Zhu et al.  We found an association of the CC genotype  and T2DM. This founding was similar to that form almost all studies performed in China, except for Z. Wang et al Both our previous meta-analysis and present meta-analysis indicated the strong association of rs7903146 (IVS3C/T) and T2DM, with the power of the meta-analysis is 100% for the risk allele of rs7903146 (IVS3C/T). This result is inconsistent with our study findings. The reasons for this discrepancy may be because first, the meta-analysis pooled all results from China, and the Han Chinese are not a genetically homogenous group, so regional diversity may contribute to the discrepancy. Second, because of the lack of full information about some potential confounders such as gender, age, and BMI of subjects included in the meta-analysis, we could not perform a stratified or interaction analysis based on these factors. However, in our study, we attempted to adjust findings for the potential confounders.In the Bodhini. D et al Henan is the most populated province in China, but data were lacking on the association of rs7903146 (IVS3C/T) and rs290487 (IVS3C/T) polymorphisms and T2DM. Our study is the first to assess the associations with SNPs in Han Chinese in Henan province. As well, we analyzed the associations of metabolic measurements and genotypes of the 2 loci in Han Chinese population for the first time. In addition, our meta-analysis, by combining our data and published data, provides strong evidence for the association of the risk alleles of these 2 SNPs and T2DM in Han Chinese. Finally, the power of our study to detect the association of rs290487 (IVS3C/T) and T2DM in our study population was 100% by PGA.TCF7L2 have no major impact on T2DM; however, the CC genotype and the recessive model of rs290487 (IVS3C/T) and the haplotype CC of rs7903146 (IVS3C/T) and rs290487 (IVS3C/T) in TCF7L2 gene are associated with T2DM. We found no association of behavioral risk factors and distribution of rs7903146 (IVS3C/T) and rs290487 (IVS3C/T) in Han Chinese in Henan province, China. However, more representative and comprehensive studies of people with different ethnic backgrounds are needed to clarify the mechanisms and underlying genetic effects of T2DM in the Chinese population.In conclusion, our findings suggest that the genotypes of rs7903146 (IVS3C/T) in Table S1Primer sequences and restriction enzymes.(DOC)Click here for additional data file.Table S2Characteristics of study participants.(DOC)Click here for additional data file.Table S3Genotypic and allelic distributions of single nucleotide polymorphisms in TCF7L2 gene among Han Chinese in China.(DOC)Click here for additional data file.Table S4Association of single nucleotide polymorphisms in TCF7L2 gene and type 2 diabetes mellitus in Han Chinese in China.(DOC)Click here for additional data file.Table S5Associations of haplotypes of SNPs in TCF7L2 gene and T2DM.(DOC)Click here for additional data file.Table S6Interaction of behavioral factors and genotypes of single nucleotide polymorphisms (SNPs) in TCF7L2 for type 2 diabetes mellitus.(DOC)Click here for additional data file.Table S7Association of clinical and biochemical characteristics of normal glucose-tolerant participants and genotype.(DOC)Click here for additional data file.Table S8Meta-analyses of risk alleles in TCF7L2 and T2DM in Han Chinese population.(DOC)Click here for additional data file.


Organisms exposed to oxidative stress respond by orchestrating a stress response to prevent further damage. Intracellular levels of antioxidant agents increase, and damaged components are removed by autophagy induction. The KEAP1-NRF2 signaling pathway is the main pathway responsible for cell defense against oxidative stress and for maintaining the cellular redox balance at physiological levels. Sulforaphane, an isothiocyanate derived from cruciferous vegetables, is a potent inducer of KEAP1-NRF2 signaling and antioxidant response element driven gene expression. In this study, we show that sulforaphane enhances the expression of the transcriptional coregulator SPBP. The expression curve peaks 6–8 hours post stimulation, and parallels the sulforaphane-induced expression of NRF2 and the autophagy receptor protein p62/SQSTM1. Reporter gene assays show that SPBP stimulates the expression of p62/SQSTM1 via ARE elements in the promoter region, and siRNA mediated knock down of SPBP significantly decreases the expression of p62/SQSTM1 and the formation of p62/SQSTM1 bodies in HeLa cells. Furthermore, SPBP siRNA reduces the sulforaphane induced expression of NRF2, and the expression of the autophagy marker protein LC3B. Both these proteins contain ARE-like elements in their promoter regions. Over-expressed SPBP and NRF2 acts synergistically on the p62/SQSTM1 promoter and colocalize in nuclear speckles in HeLa cells. Collectively, these results suggest that SPBP is a coactivator of NRF2, and hence may be important for securing enhanced and sustained expression of NRF2 induced genes such as proteins involved in selective autophagy. Oxidative stress causes damage to multiple cellular molecules, and is a major contributing factor in a variety of human diseases such as cancer, neurodegenerative disorders, inflammatory diseases, cardiovascular disease and ageing Autophagy is an essential cellular mechanism of adaption to external or internal stress. It includes degradation of intracellular components during starvation conditions, elimination of aggregated proteins, turnover of damaged or old organelles, and protection against invading microorganisms  is a 220 kDa multidomain nuclear protein expressed in most cells and tissues  [26], [2Here we have shown that expression of the transcriptional coregulator and chromatin binding protein SPBP is induced by sulforaphane. Sulforaphane is a potent inducer of ARE-driven gene expression via KEAP1-NRF2 signalling. The autophagic receptor and signalling scaffold protein p62 contains conserved ARE elements in its promoter region, and we found that SPBP enhances p62 expression via these elements. SPBP associated with the p62 promoter, and siRNA mediated knock down of SPBP significantly downregulated p62 expression and formation of p62 bodies in HeLa cells. Furthermore, knock down of SPBP affected expression of LC3B, and the sulforaphane induced expression of NRF2. NRF2 regulates its own expression via ARE elements in its promoter region, and an ARE-like element is also present in the LC3B promoter. Hence, SPBP acts as a transcriptional coactivator of NRF2, and may be important for enhanced and sustained cellular response to oxidative stress mediated by KEAP1-NRF2 signalling, including the NRF2 mediated induction of proteins involved in selective autophagy.5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTCCACCATGATGGACTTGGAGCTGCCG-3′ and 5′-GGGGACCACTTTGTACAAGAAAAGCTGGGTCTACCTAGTTTTTCTTAACATCTGGC -3′ and image clone IRAUp969G0565D as template, and recombined into pDONR221 (Invitrogen). The −1781/+46 p62 promoter construct, the −1781/+46 construct with a mutated ARE site at position −1300, and the NQO1 promoter constructs are described previously 5′-CGGGCTCGAGATCTCTCTGTCACTGCCGCCAGACC-3′. All mutation constructs were generated by PCR using Pfu Turbo polymerase (STRATAGENE) and verified by DNA sequencing with BigDye v3.1 (Applied Biosystems). The SPBP constructs are described previously 5′-CGCGAAGCTTCTCCTGCAGACGCGTCGGTGTTAGAGCC-3′ and 5′-CGCGCTGCAGTCTAAGCTTGAATTCTCTAGACAGTGTCCC-3′ for TpT, 5′-CGCGAAGCTTCTCCTGCAGCCTGGAGAATCCCGGTGCCG-3′ and 5′-CGCGCTGCAGTCTAAGCTTCACAGGATGTATATATCTGAC-3′ for 601, and 5′-CGCGAAGCTTCTCCTGCAGTAAAAGATGCTGAAGATC-3′ and 5′-CGCGCTGCAGTCTAAGCTTATAATACCGCGCCACATAGC′ for Control. Plasmids pTpT and p601, kind gifts from R. Kingston's group, were used as template. The PCR products were digested by HindIII, inserted into the HindIII sites of the p62 and NQO1 promoter constructs, and verified by DNA sequencing.cDNA constructs were subcloned into Gateway entry vectors and expression clones made as described (Invitrogen). All constructs were verified by DNA sequencing. Human NRF2 was amplified by PCR using primers 2 in a humidified atmosphere.HeLa (ATCC CCL2) and HEK293 (ATCC CRL-1573) cells were grown in Dulbecco's modified Eagle's medium (DMEM). U2OS-TA cells Cells were amino acid starved in Hanks Balanced Salts medium (HBSS) for 2 hours before treatment for 4 hours with the following pharmacological agents Bafilomycin A1 (0.2 µM), Sulforaphane (20 µM), or MG132 (0.2 µM).The following primary antibodies were used: Rabbit anti-SPBP antibody Western Blotting using total cell extracts were performed essentially as described previously 3HA (Invitrogen) was used to equalize the concentration of DNA in each transfection. Extracts were prepared 20 hours post transfection and analyzed essentially as described previously Subconfluent HEK293 cells in 24-well tissue culture dishes (Becton Dickinson) were transiently transfected using Metafectene Pro (Biontex) according to the manufacturer's protocol. All wells were cotransfected with 60 ng of luciferase reporter vectors, 100 ng or as indicated in the figures of SPBP expression vector (pDEST-HA-SPBP), 100 ng of CBP expression vector (pHA-CBP), 50 ng or as indicated in the figure of pDEST-HA-NRF2 and 5 ng of a β-galactosidase expression vector. pcDNA7 HeLa cells were used for each tested condition. Crosslinking with 1% formaldehyde was carried out for 10 minutes at 25°C. This was stopped by adding 0.125 M glycine for 5 minutes, 25°C. Harvested cells were resuspended in 500 µl Lysis Buffer , and incubated 10 minutes on ice before sonication 20 minutes in a Bioruptor (Diagenode). The DNA fragments were purified using a QIAquick Spin Kit (QIAGEN). One to five µl from a 30 µl DNA extraction were used for 35 cycles of PCR amplification. PCR primers used to amplify the p62 promoter and the Cathepsin D promoter were as described Chromatin Immunoprecipitation was performed mainly as described 5′-GGAGAAGAGCAGCTCACAGCCA-3′ and 5′-CCTTCAGCCCTGTGGGTCCCT-3′; Actin: 5′-TGACGGTCAGGTCATCACTATCGGCAATGA-3′ and 5′-TTGATCTTCATGGTGATAGGAGCGAGGGCA-3′; and GADPH: 5′-TGGGTGTGAACCACGAGAA-3′ and 5′-GGCATGGACTGTGGTCATGA-3′. The reactions were run for an initial step at 95°C for 10 min, followed by 40 cycles of amplification at 95°C for 15 s and 60°C for 1 min. All data were collected during the extension step, and a melting curve was obtained at the end of the PCR reaction to verify that only one product was produced. The calculations were carried out using Delta-delta CT method.Subconfluent HeLa cells in 6 well dishes were transfected with 20 nM SPBP siRNA or scrambled siRNA HeLa cells were cultured in 8-chambered cover slides (Nunc) and transiently transfected with 20 nM siRNA, or 50 ng pDestEGFP-NRF2 and 150 ng pDestCherry-SPBP using TransIT-LT1 (Mirus Bio) according to the manufacturer's protocol. Live cell images were taken one day post transfection using a Leica SP5 or a LSM510-META confocal laser scanning microscope. For quantification of p62 bodies, siRNA transfected cells were fixed in 4% paraformaldehyde 2 days post transfection. The cells were permeabilised by 0.1% Triton X-100, for 5 min at room temperature. Staining with antibodies was as described previously Subconfluent HeLa cells in 10 cm dishes (Nunc) were transiently co-transfected with either pDestEGFP-NRF2 and pDESTMyc-SPBP, or pDEST-EGFP and pDESTMyc-SPBP using Metafectene Pro (Biontex) according to the manufacturer's protocol. The cells were harvested 20 hours post transfection and co-immunoprecipitation performed using Chromotek-GFP-Trap (Allele) according to the manufacturer's protocol.n.s.)Data were processed in Excel (Microsoft Corporation) to generate bar charts and perform statistical analyses. Student's t-test was performed for each dependent variable. *: p< = 0.05 was considered statistically significant, and **: p< = 0.01, very significant. p>0.05 was considered not significant , is found to be associated with NRF2 . However, there is no clear correlation of their expression levels. To investigate whether SPBP may impact on the p62 expression levels in cells, siRNA mediated knock-down of SPBP was introduced into HeLa cells. Knock-down of SPBP using two different SPBP siRNAs SPBP and p62 is coexpressed in most human cells  and tissSPBP contains two nucleosome binding domains and displays strong affinity for nucleosomes We have established an U2OS cell line stably over-expressing EGFP-SPBP Reporter gene assays using the wild type p62 promoter indicated that coexpression of SPBP and NRF2 results in a synergistic activation . To furtHere we have shown that the expression of the transcriptional coregulator SPBP is upregulated upon sulforaphane treatment of cells, and that SPBP may be involved in the protection of cells against oxidative stress since it acts as a coactivator of NRF2. Interestingly, a recent genome wide siRNA screen showed that SPBP (named TCF20 therein) was important for both Cyp1A1 and NQO1 expression upon cellular stress induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin www.genome.gov). This suggests that SPBP may enhance NRF2 induction via the ARE elements in the NRF2 promoter, and thereby strengthen both the magnitude and the duration of the sulforaphane mediated enhancement of NRF2 expression – and the magnitude and the duration of expression of proteins regulated by NRF2 such as p62. From this we propose that SPBP may be a player in the positive feedback loop established to enhance expression of NRF2 and NRF2 regulated genes in response to oxidative stress, contributing to the strength and duration of the oxidative stress response. Additionally, we found that over-expression of SPBP recruits NRF2 to specific nuclear speckles previously identified as chromatin-rich regions It is well known that the transcription factor NRF2 regulates the basal and inducible expression of a wide array of antioxidant genes via AREs in their control regions  Reporter constructs (60 ng) containing the indicated nucleosome position sequences inserted downstream of the transcription start site of p62 promoter (A) or NQO1 promoter (B) were transfected into HEK293 cells together with the indicated amounts of expression plasmids for SPBP or NRF2. The luciferase activity of the promoter constructs cotransfected with empty expression plasmid was set to 1. The data represent the mean of three independent experiments with standard deviations, each performed in triplicate.(TIF)Click here for additional data file.Figure S2SPBP impacts on NRF2, p62 and LC3B expression levels in HeLa and U2OS cells. (A) p62 seems to be degraded normally by autophagy in the U2OS cells over-expressing EGFP-SPBP. Cell extracts from U2OS cells overexpressing EGFP-SPBP or EGFP, and treated with DMSO or Bafilomycin A1 (0.2 µM for 4 hours), were separated by SDS-PAGE and blotted against the indicated antibodies. (B) Knock-down of SPBP reduces the expression levels of NRF2, p62 and LC3B in HeLa cells. Cell extracts of HeLa cells transfected with the indicated siRNAs for 48 hours, and stimulated by sulforaphane (20 µM) or DMSO for the last 8 hours, were separated by SDS-PAGE and blotted against the indicated antibodies. (C) NRF2 is not recruited to specific nuclear speckles when coexpressed with mCherry. HeLa cells were transiently transfected with expression vectors for EGFP-NRF2 and mCherry, and analysed 24 hours post transfection by live cell imaging using a confocal laser scanning fluorescence microscope. Pearson's colocalisation scatter was generated using Volocity (Perkin Elmer). (D) SPBP associates weakly with NRF2. HeLa cells were transfected with expression vectors for EGFP-NRF2 and Myc-SPBP, or EGFP and Myc-SPBP. EGFP-NRF2 was immunoprecipitated with GFP antibody 20 hours post transfection. Precipitated EGFP-NRF2 and co-precipitated SPBP were detected by western blotting using the indicated antibodies. (E) siRNA mediated knock-down of SPBP impairs sulforaphane induced NRF2 expression. HeLa cells were transfected with SPBP siRNAs or Control siRNA as indicated. Cells were treated with sulforaphane for eight hours two days post transfection. The cell extracts were subjected to western blot using the indicated antibodies. The graph shows fold reduction calculated and correlated to actin in two independent experiments with standard deviations.(TIF)Click here for additional data file.


CDK4) in lung cancer and its correlation with clinicopathologic features. Furthermore, the involvement of CDK4-mediated cell cycle progression and its molecular basis were investigated in the pathogenesis of lung cancer.The aim of the present study was to analyze the expression of Cyclin-dependent kinase 4  with ages ranging from 36 to 78 years and compared them to 23 normal lung tissues. Cases with cytoplasmic and nuclear CDK4 immunostaining score values greater than or equal to 7 were regarded as high expression while scores less than 7 were considered low expression. The correlation between the expression level of CDK4 and clinical features was analyzed. Furthermore, we used lentiviral-mediated shRNA to suppress the expression of CDK4 and investigate its function and molecular mechanism for mediating cell cycle progression.Using immunohistochemistry analysis, we analyzed CDK4 protein was significantly increased in lung cancer tissues compared to normal tissues (P < 0.001). In addition, high levels of CDK4 protein were positively correlated with the status of pathology classification (P = 0.047), lymph node metastasis (P = 0.007), and clinical stage (P = 0.004) of lung cancer patients. Patients with higher CDK4 expression had a markedly shorter overall survival time than patients with low CDK4 expression. Multivariate analysis suggested the level of CDK4 expression was an independent prognostic indicator (P < 0.001) for the survival of patients with lung cancer. Use of lentiviral-mediated shRNA to inhibit the expression of CDK4 in lung cancer cell line A549 not only inhibited cell cycle progression, but also dramatically suppressed cell proliferation, colony formation, and migration. Furthermore, suppressing CDK4 expression also significantly elevated the expression of cell cycle regulator p21The expression level of CDK4 is a potential unfavorable prognostic factor and mediates cell cycle progression by regulating the expression of p21 in lung cancerOverexpressed  Lung cancer is the world's most prevalent cancer according to the World Health Organization, with 1.2 million new cases every year. Nearly all lung cancers arise due to smoking and men are more frequently diagnosed than women. However, a rise in female smoking worldwide has started reversing the trend.In China, about 300,000 lung cancer patients 23/100,000) are diagnosed each year[00,000 arCDK4 is part of the cyclin-dependent kinase family. The protein encoded by this gene is a member of the Ser/Thr protein kinase family and is highly similar to the gene products of S. cerevisiae cdc28 and S. pombe cdc2. It is a catalytic subunit of the protein kinase complex important for G1 cell cycle progression. Transition through G1-S phases, is controlled by the regulatory subunits D-type cyclins(CDK4 and CDK6) and CDK inhibitor p16(INK4a). Marval et al. found that CDK4 has higher oncogenic activity than cyclin D1(CCND1) and it markedly enhanced malignant skin tumorigenesis in CDK4 transgenic mice[CDK4 has been showed in many tumor types, including oral squamous cell carcinoma[CDK4 is a key factor in promoting the initiation and development of tumors.Genetic abnormalities found in lung cancer typically affect two general classes of genes: oncogenes and tumor suppressors. Cancer-promoting oncogenes are typically activated in cancer cells, giving those cells new properties, such as hyperactive growth and division, protection against programmed cell death, or loss of respect for normal tissue boundaries. carcinoma, pancreacarcinoma,6, and ncarcinoma, suggestCDK4 in the pathogenesis of lung cancer, we explored the correlation of its protein expression with clinicopathologic features of lung cancer patients. We found that the expression levels of CDK4 were higher in lung cancer tumors compared to those in normal lung tissues. This increased CDK4 expression was associated with the progression and poor prognosis of lung cancer patients. Furthermore, suppressing the expression of CDK4 elevated tumor suppressor p21 expression, which may function to reduce cell proliferation and migration.In order to clarify the role of Eighty-nine (89) paraffin-embedded lung cancer and 23 normal lung samples were obtained from the First Affiliated Hospital of Guangdong Medical School, Zhanjiang City, China. In the 89 lung cancer cases, there were 59 males and 30 females with ages ranging from 36 to 78 years. The clinical follow-up time of patients ranged from 6 to 55 months. For use of these clinical materials for research purposes, prior consent from the patients and approval from the Ethics Committees of this hospital was obtained. Histological classification and clinicopathologic staging of the samples were performed according to the rules of according to the WHO histologic classification.CDK4 antibody (1:100)  for overnight at 4°C. After washing, the sections were incubated with biotin-labeled rabbit anti-goat antibody for 10 min at room temperature, and subsequently were incubated with streptavidin-conjugated horseradish peroxidase (HRP) . The peroxidase reaction was developed using 3, 3-diaminobenzidine chromogen solution in DAB buffer substrate. Sections were visualized with DAB and counterstained with hematoxylin, mounted in neutral gum, and analyzed using a bright field microscope.Paraffin sections (4 μm) from samples were deparaffinized in 100% xylene and re-hydrated in descending ethanol series and water according to standard protocols. Heat-induced antigen retrieval was performed in 10 mM citrate buffer for 2 min at 100°C. Endogenous peroxidase activity and non-specific antigen were blocked with peroxidase blocking reagent containing 3% hydrogen peroxide and serum, followed by incubation with goat anti-human CDK4 in the nucleus and in the cytoplasm was independently evaluated. For cytoplasmic staining, the score was evaluated according to the sum of cytoplasm staining intensity and the percentage of positive staining areas of cells. The staining intensity was scored as previously described(0-3) [CDK4 (0-12). For statistical analysis, a final staining score of 0-6 or 7-12 was respectively considered to be low or high expression.The immunohistochemically stained tissue sections were reviewed and scored separately by two pathologists blinded to the clinical parameters. Expression of bed(0-3) ,9 and thCDK4 gene using the BLOCK-It RNAi Designer . The preparation of lentiviral vectors expressing human CDK4 short hairpin RNA (shRNA) was performed using the pLVTHM-GFP Lentiviral RNAi Expression System. Replication-incompetent lentivirus was produced by cotransfection of the pLVTHM/CDK4-shRNA expression vector and ViraPower packaging mix containing an optimized mixture of two packaging plasmids: psPAX2 and pMD2.G into 293FT cells. Lung cancer A549 cells were infected with lentiviral particles containing specific or negative control vectors and the single colony with strong GFP expression was selected to establish stable silencing cell lines. The total RNA of these cell clones was isolated, and the levels of CDK4 mRNA were measured using real-time PCR examination.We selected two sequences(CDK4 509: Sense:5' CGCGTCCCCGCATGTAGACCAGGACCTAAGTTCAAGAGACTTAGGTCCTGGTCTACATGCTTTTTGGAAAT 3' Antisense:5'CGATTTCCAAAAAGCATGTAGACCAGGACCTAAGTCTCTTGAACTTAGGTCCTGGTCTACATGCGGGGA 3') CDK4 1097 Sense:5'CGCGTCCCCGCAGCACTCTTATCTACATAATTCAAGAGATTATGTAGATAAGAGTGCTGCTTTTTGGAAAT 3'; Antisense:5'CGATTTCCAAAAAGCAGCACTCTTATCTACATAATCTCTTGAATTATGTAGATAAGAGTGCTGCGGGGA 3') for targeting the CDK4 antibody(1:500), anti-ACTB antibody(1:400) and p21(1:200). An HRP-conjugated anti-rabbit IgG antibody was used as the secondary antibody .Cells were lysed in RIPA Buffer , and protein concentration was determined using BCA assay . Total protein (30 μg) was resolved using a 10% SDS-PAGE gel and electro-transferred to polyvinylidene fluoride membranes , and blocked with 5% nonfat dry milk in Tris-buffered saline, pH 7.5. Membranes were immunoblotted overnight at 4°C with rabbit polyclonal anti-3 cells were seeded into a 96-well plate with quadruplicate repeat for each condition. After 24 h of incubation, MTT reagent was added to each well and incubated for 4 h. The formazan crystals formed by viable cells were then solubilized in DMSO and measured at 490 nm for the absorbance (A) values. Each experiment was performed in triplicate.Cell proliferation was analyzed using MTT assay . Briefly, 1 × 10About 100 cells were added to each well of a 6-well culture plate, and each cell group contained 2 wells. After 2 weeks of incubation, cells were washed twice with PBS and stained with Giemsa solution. The number of colonies containing ≥ 50 cells was counted under a microscope. The colony formation efficiency was calculated as: efficiency = (number of colonies/number of cells inoculated) × 100%. Each experiment was performed in triplicates.6 cells were harvested, rinsed with cold PBS, and fixed with 70% ice-cold ethanol for 48 h at 4°C. Fixed cells were rinsed with cold PBS followed by incubation with PBS containing 10 μg/mL propidium iodide and 0.5 mg/mL RNase A for 15 min at 37°C. The DNA content of labeled cells was acquired using FACS Caliber cytometry (BD Biosciences). Each experiment was performed in triplicates.To evaluate cell cycle distribution, cells were seeded on 10 cm-diameter plates in RPMI 1640 culture medium containing 10% NBCS. After 48 h of incubation, a total of 1 × 105 cells were seeded on a fibronectin-coated polycarbonate membrane insert in a transwell apparatus . In the lower chamber, 600 μl of RPMI 1640 with 10% NBCS was added as chemoattractant. After the cells were incubated for 12 h, the insert was washed with PBS, and cells on the top surface of the insert were removed by a cotton swab. Cells adhering to the lower surface were fixed with methanol, stained with Giemsa, and counted under a microscope in five predetermined fields (× 200). All assays were independently repeated at least three times.Cells growing in the log phase were treated with trypsin and re-suspended as single-cell solution. A total of 1 × 10CDK1, CDK2, CDK6, CCND1, p15, p16, p21, and p27 were first detected by real-time PCR in pLVTHM/CDK4-shRNA and control expression vector. Subsequently, genes with markedly differential expression were further validated by western blot. Real-time PCR and western blot were carried out as described above.Changes in expression of cell cycle regulators CDK4 expression levels and clinicopathologic characteristics. Survival analysis was performed using Kaplan-Meier method. Multivariate Cox proportional hazards method was used for analyzing the relationship between the variables and patient's survival time. One-way ANOVA was used to determine the differences between groups for all in vitro analyses. A P value of less than 0.05 was considered statistically significant.All data were analyzed for statistical significance using SPSS 13.0 software. The Mann-Whitney U test was applied to the examination of relationship between CDK4 protein in 89 archived paraffin-embedded lung cancer samples and 23 normal lung tissues using immunohistochemical staining  of lung cancer samples, CDK4 protein was highly expressed. In comparison, only 8.7%(2/23) of normal lung samples had highly expressed CDK4 protein, significantly lower than that in the lung cancer samples (P < 0.001) , or status of distant metastases (M classification) in 89 lung cases. However, we observed that the expression level of CDK4 was positively correlated with the status of pathology classification(P = 0.047) lymph node metastasis (N classification) (N0-N1 vs. N2-N3) (P = 0.007), and clinical stage (I-II vs. III-IV) (P = 0.004) in lung cancer patients . In addition, smoking, degree of tumor differentiation, T/N/M classifications and clinical stages were also significantly correlated with patients' survival . To determine whether CDK4 is an independent prognostic factor for lung cancer, we performed multivariate analysis of CDK4 expression adjusted for the same parameters. The results indicated that the level of CDK4 expression was an independent prognostic factor for lung cancer (P < 0.001)  . Further, decreased expression of CDK4 protein was confirmed by western blotting in these three clones compared to PLV-Ctr and A549 cells Figure . Real-tilsFigure . C1 and CDK4 expression on lung cancer cell growth in vivo. Using an MTT assay, we found that the parental lung cancer A549 cells had a similar growth rate as PLV-Ctr cells over a seven-day period, the growth of shRNA-CDK4 cells was significantly slower than the former two lines from day 3 (P < 0.05)  and D1 cells(24.33 ± 5.13) (P < 0.05)  Figure . Interes) Figure .CDK4 expression could significantly decrease cell migration of C1 cells(114 ± 26.75) and D1 cells(80 ± 7.31) compared to the parental cells(288.2 ± 41.78) or PLV-Ctr cells (254 ± 34.28) (P < 0.05)   Figure .CDK4 may play an important role in promoting the development of lung cancer. We further examined the effect of CDK4 on the expression of key regulators of G1-S cell cycle transition including CDK1, CDK2, CDK6, CCND1, p15, p16, p21, and p27. Real-time PCR indicated that reducing the levels of CDK4 significantly activates the expression of tumor suppressor p21 by 3.12-fold. Further, we confirmed the upregulated protein expression of p21 in CDK4-inhibited cells.Based on the increased population of G1-S arrested cells after inhibiting , and p27-21 were CDK4 may be involved in the development of lung cancer. Furthermore, we also demonstrated that CDK4 could serve as a potential independent prognostic factor for lung cancer patients. Due to the limited sample size of patients in our investigation, further studies would be needed to verify these findings and establish the role of CDK4 as a reliable clinical predictor for lung cancer outcome. Finally, our work is the first to present that CDK4 mediates cell cycle progression by regulating the expression of p21 expression in lung cancer.In summary, our results provide evidence that The authors declare that they have no competing interests.AW, DW, JG, WL, HY, YZ, XL, HW, and YZ performed this research. WF, ZL and ZY collected, analyzed, and interpreted data and wrote the manuscript. WF, ZL, and ZY supervised all the work. All authors have read and approved the final manuscript.


Gracilariopsis Persica (Rhodophyta) is one of the most abundant algae, introduced newly from the Indian Ocean. In this study, the main sterols of the algae have been isolated and identified. Separation and purification of the compounds was carried out on silica gel and sephadex LH20 column chromatography (CC) and high performance liquid chromatography (HPLC) to obtain five pure compounds 1-5. Structural elucidation of the compounds was based on the data obtained from H-NMR, 13C-NMR, DEPT and Mass spectroscopy. The separated compounds from Gp. Persica were identified as 22-dehydrocholesterol (1), cholesterol (2), stigmasterol (3), β-sitosterol (4) and fucosterol (5) based on the spectral data compared to those reported in literatures. Most of these sterols are noteworthy for their effectiveness in decreasing the plasma cholesterol, glucose and inflammation. The results of Brine Shrimp Cytotoxicity Assay indicated that the ethyl acetate extract of Gp. Persica showed a high cytotoxic effect against A. salina nauplii (LC50 = 4 μg/mL). The methanol extract was no effective but the aqueous methanol extract was moderately effective (LC50 = 40 μg/mL) compared to berberine hydrochloride as a positive control (LC50 = 26 μg/mL). Gracilariopsis persica (Rhodophyceae), introduced newly from the Indian Ocean, belongs to the Gracilariaceae family as a difficult group over decades  spectrometer with tetramethylsilane as an internal standard and chemical shifts are given in δ (ppm). EI-MS data were recorded on Agilent Technology (HP) instrument with 5973 Network Mass Selective Detector (MS model). Silica gel 60F254 pre-coated plates (Merck) were used for TLC. The spots were detected through spraying anisaldehyde-H2SO4 reagent followed by heating (120 ºC for 5 min).Algae materialGracilariopsis persica sp. Nov. This is a tetrasporangial algae from Iran, Persian Gulf, Bandarabbas . A voucher specimen (64-27R) has been deposited at the Herbarium of Persian Gulf Biotechnology Research Center (Gheshm Island).Red algae was collected from the northern areas of Persian Gulf in July (2008) and identified as Extractions of the marine algaeMarine algae were dried carefully and cut into small pieces. Dried powder of algae (1126 g) was extracted with ethyl acetate, methanol and aqueous methanol (50%), respectively, via percolation (72 h each time) at room temperature. Then, the solvents evaporated under reduced pressure and were dried under the vacuum in order to give dried powder of the ethyl acetate (2 g), methanol (54 g) and aqueous methanol (5 g) extracts.Isolation of the sterols3 : AcOEt , MeOH and aqueous methanol (8%), consequently, to obtain 6 fractions (A-F). The fraction B (2.3 g) was subjected to silica gel CC, with Hexane : AcOEt (9 : 1) to give three fractions (B1-B3). The fraction, B2 (3.5 mg), was the mixture of compounds 1 and 2 (3.5 mg). The fraction C (259 g) was subjected to silica gel CC, with Hexane : AcOEt (9 : 1) to give two fractions (C1 and C2). The fraction, C1 (46 mg), was the pure compound 1 (46 mg). The fraction E (2.5 g) was subjected to sephadex LH20 with MeOH : CHCl3 (7 : 3) to gain four sub fractions (E1-E4). Fraction E1 (80 mg) was submitted to the HPLC (Knauer). HPLC conditions are as follows: Vertex column C18 (250 × 20 mm I.D.), Kenauer. Gradient elution: 0.00 min 50% MeOH, 15.00 min 100% MeOH until 50.00 min. Flow-rate: 3 mL min-1. The injection volume and the detector were 2 mL and PDA (UV-spectra were collected across the range of 200-900 nm). The wave length was 210 nm for detecting the compounds. Nine fractions (E11-E19) were obtained using HPLC, of which, the fractions E12 and E16 were the pure compounds 3  and 4 , respectively. Fraction F (1.2 g) was submitted to sephadex LH20 with MeOH to obtain seven sub fractions (F1-F7). The fraction F4 (3 mg), was the pure compound 5.The methanol extract (50 g) was submitted to the silica gel column chromatography (CC) with CHClBrine shrimp cytotoxicity bioassay (BSC)Artemia salina) eggs were purchased from the Shilat Center (Tehran). The eggs were hatched in a flask containing 300 mL of artificial seawater. The flask was well aerated with the aid of an air pump and kept in a water bath at 29-30 ºC. A bright light was left on the nauplii hatched within 48 h. The extracts  were dissolved in normal saline. Different concentrations were obtained through serial dilution. Solution of each concentration (500 μL) was transferred into 24-well plates through a pipette and aerated seawater having 10-20 nauplii (500 μL) was added. A check count was performed and the number alive were noted after 24 h. The mortality end point of the bioassay was determined as the absence of controlled forward motion during 30 sec of observation. The controls used were seawater and berberine hydrochloride (LC50 = 26 μg/mL). The lethality percentage was determined and LC50 was calculated based on the Probit Analysis with 95% of confidence interval  contain primarily cholesterol -reductase in human Caco-2 and HL-60 cell lines (β-sitosterol is reported to reduce the symptoms of Benin Prostatic Hyperplasia (BPH) and also found as an anti-inflammatory agent . The MeOH extract of Gp. Persica showed no activity but the aqueous methanol extract was less effective (LC50 = 40 μg/mL) compared to berberine hydrochloride as a positive control (LC50 = 26 μg/mL). Previously, we reported the toxicity of ethyl acetate extracts of two red marine algae, Gracilaria salicornia and Hypnea flagelliformis (. The brine shrimp lethality of the ethyl acetate extract of Gp. Persica seems to be due to the presence of stigmasterol and β-sitosterol which were interestingly reported to be toxic against the brine shrimps (The results of Brine Shrimp Cytotoxicity Assay indicated that the ethyl acetate extract of liformis . The bri shrimps .Gp. Persica, which has been abundantly cultured for the animal feeding and other industrial purposes, is rich of sterols. Among the main identified sterols, fucosterol, stigmasterol and β-sitosterol are noteworthy for their effectiveness in decreasing the plasma cholesterol, glucose and inflammation.In conclusion, the results of this study show that the red marine algae


Sc) in the brain. The most complicated entity in the current classification system is MV2, since it shows wide phenotypic variations, i.e., MV2 cortical form (MV2C), MV2 with kuru plaques (MV2K), or a mixed form (MV2K + C). To resolve their complicated pathogenesis, we performed a comprehensive analysis of the three MV2 subgroups based on histopathological, molecular, and transmission properties.Sporadic Creutzfeldt-Jakob disease is classified according to the genotype at polymorphic codon 129 (M or V) of the prion protein (PrP) gene and the type (1 or 2) of abnormal isoform of PrP  and type 2 PrPSc with the 129V genotype (V2 PrPSc) that originated from V2 PrPSc, whereas MV2C + K may also contain type 2 PrPSc with the 129M genotype and cortical pathology (M2C PrPSc) that lacks infectivity to the PrP-humanized mice in addition to Mi and V2 PrPSc.In histopathological and molecular analyses, MV2C showed close similarity to MM2 cortical form (MM2C) and could be easily discriminated from the other MV2 subgroups. By contrast, MV2K and MV2K + C showed the same molecular type and the same transmission type, and the sole difference between MV2K and MV2K + C was the presence of cortical pathology characteristic of MV2C/MM2C. The remarkable molecular feature of MV2K or MV2K + C was a mixture of type 2 PrPSc origin(s).Taken together, the present study suggests that the phenotypic heterogeneity of MV2 stems from their different PrP Sc), which is converted from the normal cellular isoform (PrPC) [Sc in the brain are major determinants of the clinicopathological phenotypes of sporadic CJD (sCJD) [Sc are distinguishable according to the size of the proteinase K (PK)-resistant core of PrPSc , reflecting differences in the PK-cleavage site  [Sc typing, and histotyping, has been widely used compared with an alternative classification system proposed by others [Creutzfeldt-Jakob disease (CJD) is a lethal transmissible neurodegenerative disease caused by an abnormal isoform of prion protein (PrPm (PrPC) . There iD (sCJD) -5. Type ctively) . Accordictively) . This PaSc in certain MV2 cases shows atypical features, i.e., wide, heterogeneous fragments that migrate at approximately 20 to 19 kDa and are sometimes visible as doublets [Sc has been reported in MV2K and MV2K + C but not in MV2C. The molecular mechanisms of the atypical type 2 PrPSc formation remain elusive. In addition, the atypical type 2 PrPSc has not been recapitulated in an experimental transmission of MV2K to PrP-humanized mice carrying the 129M/V genotype [Among the seven sCJD subgroups, the most complicated entity is MV2, which accounts for 11% of total sCJD . Since Mdoublets ,11,12. Tgenotype .To characterize the MV2 subgroups in detail and to resolve their complicated pathogenesis, we performed a comprehensive analysis of the MV2 subgroups based on histopathological, molecular and transmission properties.Sc type were confirmed by PrP immunohistochemistry and western blot analysis [CJD cases included in this study were patients with clinically, genetically and histopathologically proven sCJD. Brain tissues were obtained at autopsy from CJD patients after receiving informed consent for research use. The diagnosis of sCJD, histotype, and PrPanalysis ,15. The analysis . Accordianalysis ,5, theseBrain homogenates (10%) were prepared as described . IntraceFormalin-fixed brain tissues were treated with formic acid (99% for human tissues or 60% for mouse tissues) for 1 hour to inactivate the infectivity, and embedded in paraffin. Tissue sections were pretreated by hydrolytic autoclaving before PrP immunohistochemistry . The antSc was extracted from human or mouse brains with collagenase treatment as described [Sc, samples were digested with PNGaseF (New England Biolabs) as reported [Sc- and type 2 PrPSc-specific polyclonal antibodies (designated as Tohoku 1 (T1) and Tohoku 2 (T2), respectively [PrPescribed . For degreported . Proteinreported . Type 1 ectively ) and theSc bands are expressed as mean ± SEM.Incubation times and the signal intensities of PrPHistotyping and molecular typing of MV2.i.e., a mixture of the MV2C/MM2C phenotype and MV2K phenotype. PrP plaques were absent in the brain of MV2C even in the granular cell layer of the cerebellum. Instead, MV2C and MM2C showed focal amorphous patchy PrP deposition in the molecular layer of the cerebellum as reported [At first, we performed histopathological analysis of the three subgroups of MV2  recognizes epitopes between residues 82 and 96 that are retained in type 1 PrPSc but not in type 2 PrPSc after PK-digestion. In addition, the epitopes for the T1 antibody are also retained in the intermediate type PrPSc showing intermediate electrophoretic mobility between types 1 and 2 PrPSc, which is observed in a part of dura mater-graft associated CJD [Sc-specific antibody Tohoku 2 (T2) specifically detects the N-terminal PK-cleavage site of type 2 PrPSc (at residue 97) [Sc from MM1, these heterogeneous bands were different from the co-occurrence of types 1 (21 kDa) and 2 (19 kDa) PrPSc. In contrast, MV2C and MM2C had only type 2 PrPSc located at 19 kDa. In the western blot analysis using the PrPSc type-specific antibodies, MV2K and MV2K + C had large amounts of T1-reactive PrPSc in addition to T2-reactive PrPSc  unglycosylated band in the western blot using the 3F4 antibody, since conventional western blot analysis using antibodies that react with all PrPSc types cannot detect a coexisting minority component [Sc in the cerebellum of MV2K or MV2K + C consisted of ladder-like multiple fragments, which were quite different from the T2-reactive PrPSc in the cerebrum. Thus, MV2K and MV2K + C showed similar molecular properties in the western blot analysis using the PrPSc type-specific antibodies. By contrast, MV2C and MM2C had predominant T2-reactive PrPSc with only trace amounts of T1-reactive PrPSc as reported [Sc with trace amounts of T2-reactive PrPSc as reported [Sc type-specific antibodies.Next we performed western blot analysis of PrPated CJD . Type 2 idue 97) . In the omponent . In addireported ,27. MM1 reported . Taken tSc infection regardless of the PrP genotype, these knock-in mice have an advantage over transgenic mice for evaluating the susceptibility among the genotypes [Sc in the brains from MV2K- or MV2K + C-inoculated mice using the 3F4 antibody revealed that Ki-Hu129M/M produced the 20 kDa PrPSc, Ki-Hu129M/V produced the atypical type 2 PrPSc, i.e., a mixture of the 20 kDa PrPSc and type 2 PrPSc, and Ki-Hu129V/V produced type 2 PrPSc  express human PrP with either the 129M/M, M/V, or V/V genotype at the same level. Since the expression level of PrP affects the susceptibility to PrPenotypes . After iSc with the 129V genotype (denoted as V2 PrPSc) and might contain V2 PrPSc and the intermediate type PrPSc with the 129M genotype (denoted as Mi PrPSc), since we previously found that PrPC with the 129M genotype in Ki-Hu129M/M converted into Mi PrPSc after challenge with VV2 brain material containing V2 PrPSc. These Mi PrPSc showed striking similarity to the 20 kDa PrPSc in MV2K or MV2K + C [Sc with the 129M genotype and cortical pathology (denoted as M2C PrPSc) in addition to Mi and V2 PrPSc, since MV2C showed close similarity to MM2C that contains M2C PrPSc, as described above.To resolve the complicated pathogenesis of MV2, we hypothesized that MV2K might have originated from type 2 PrPMV2K + C ,29,31. MSc into Ki-Hu129M/V, as an experimental model of MV2K. The VV2-inoculated Ki-Hu129M/V developed disease after a long incubation period , whereas VV2-inoculated Ki-Hu129V/V produced type 2 PrPSc with the 129V genotype (V2 PrPSc) as reported [Sc generated both Mi and V2 PrPSc in animals with the 129M/V genotype.To test this hypothesis, we first performed intracerebral inoculation of a VV2 brain material containing V2 PrPd Figure a, b. As s Figure c-e. VV2-reported ,29. ThesSc into the PrP-humanized mice to gain insight into the reason why MV2K + C  and MV2K (containing Mi and V2 PrPSc) could not be distinguished by transmission typing. As expected, none of the MM2C-inoculated mice had developed disease by the end of their life span. An accumulation of PrPSc was absent in the brains of the MM2C-inoculated mice  and V2 PrPSc in the brain. These results suggest that MV2K contains Mi and V2 PrPSc that originated from V2 PrPSc. In addition, we also inoculated MM2C brain material containing M2C PrPSc into the PrP-humanized mice to gain insight into the reason why MV2K and MV2K + C could not be distinguished by transmission typing. These transmission experiments revealed that M2C PrPSc lacked infectivity to the PrP-humanized mice. Therefore, the present data lead us to surmise that MV2K + C may also contain M2C PrPSc lacking infectivity to the PrP-humanized mice in addition to Mi and V2 PrPSc. Further study will be needed to verify that MV2C and MM2C are identical in transmission typing as well as histotyping and molecular typing. To grasp the whole picture of the phenotypic variability and PrPSc strain diversity in the MV2 subgroups, a transmission study of MV2C is in progress using the PrP-humanized mice.We performed a comprehensive analysis of the MV2 subgroups, Sc in the MV2 subgroups. This heterogeneous PrPSc consists of a mixture of the intermediate type PrPSc and type 2 PrPSc, denoted as types i + 2. Differentiation of the intermediate type PrPSc and type 2 PrPSc is difficult with conventional western blot analysis and requires strict conditions for PK-digestion and high resolution gel electrophoresis systems, e.g., 10-20% gradient Tris-glycine long gels or Bis-tris long gels  [Sc is a component of kuru plaques and is directly associated with the neuropathological phenotype. The amounts of Mi PrPSc might vary among brain regions, as shown in the western blot analysis of the MVi + 2 brain in the present study. Unfortunately, we could not test this possibility in detail because additional brain materials were not available. However, in the histopathological analysis, the abundance of kuru plaques varied among brain regions in the MVi + 2 or MVi + 2C patient  ,32, sugg MVi + 2 . We analreported -27, the  Mi PrPSc.Sc strains in heterozygotes of the PrP genotypes can result in a long incubation period due to interference among coexisting heterologous PrPSc, designated as heterozygous inhibition [Sc-inoculated Ki-Hu129M/V produced Mi and V2 PrPSc and showed longer incubation times compared with those of Ki-Hu129M/M despite its expression of PrPC with the 129V genotype, which is an optimal substrate for V2 PrPSc. Therefore, the long clinical course of MVi + 2 or MVi + 2C [Sc.The generation of multiple PrPhibition ,35. In tSc, MVi + 2 showed the same transmission properties as those of VV2 containing V2 PrPSc alone, as reported [Sc was originated from V2 PrPSc and showed the same transmission properties as those of the parental V2 PrPSc[Sc also showed the same transmission properties as those of VV2, since M2C PrPSc lacked infectivity to the PrP-humanized mice and did not affect the transmission properties of the coexisting Mi and V2 PrPSc. Thus, the transmission type does not reflect all existing PrPSc strains if their origins are identical or if they lack infectivity to experimental animals. Nevertheless, transmission typing will remain indispensable for the risk assessment of PrPSc infection among the PrP genotypes.Despite the coexistence of Mi and V2 PrPreported . This re V2 PrPSc. MeanwhiSc lacked infectivity to the PrP-humanized mice despite the fact that they could propagate in the MM2C/MV2C patient brain. Although there was no evidence of successful transmission in the present study, very low infectivity of M2C PrPSc was reported in a transmission study using other PrP-humanized knock-in mouse lines [Sc. Therefore, M2C PrPSc may replicate less efficiently and take a longer time to propagate compared with the other PrPSc strains.It remains unclear why M2C PrPse lines . The disse lines , suggestSc and/or Mi PrPSc to individuals with the 129M/M genotype. We reported previously that transmission of VV2 to animals with the 129M/M genotype caused p-dCJD like phenotype, i.e., widespread PrP plaques and an accumulation of Mi PrPSc, and that the transmission properties of p-dCJD were identical to those of VV2 [Sc and/or Mi PrPSc from sCJD patients with VV2, MVi + 2, or MVi + 2C. Indeed, the incidence rate of p-dCJD among total dura mater graft-associated CJD is 32% [Sc in individuals with the 129M/M genotype has never been observed in sCJD patients, it might be a characteristic fingerprint of the infection of V2 PrPSc and/or Mi PrPSc to the 129M/M individuals.Dura mater graft-associated CJD with kuru plaques (p-dCJD) might be caused by infection of V2 PrPe of VV2 ,36. In tD is 32% , which iD is 32% . Since MSc origin(s).The present study resolves the complicated pathogenesis of MV2. The phenotypic heterogeneity of MV2 stems from their different PrPThe authors declare that they have no competing interests.AK, YI, MY, MY and TK performed histopathological analysis. AK and TK performed western blot analysis and drafted the manuscript. YI, HO, MY and MY clinically examined patients and collected tissue samples. YM and SM carried out transmission experiments. All authors read and approved the final manuscript.Histotyping of the MV2 subgroups. (a-d) Histopathological features of MV2K (a), MV2K + C (b), MV2C (c), or MM2C (d). Hematoxylin and eosin stain and imunohistochemistry for PrP. Higher magnification of kuru plaques (arrows) is shown in the insets. Scale bar: 100 μm. (e-h) Regional distribution of PrP plaques in MV2K (e), MV2K + C (f), MV2C (g), or MM2C (h). Data are presented as mean ± SEM. FC, frontal cortex; TC, temporal cortex; OC, occipital cortex; BG, basal ganglia (putamen); TH, thalamus ; MB, midbrain; PO, pons; MO, medulla; CE, cerebellum (granular cell layer).Click here for filen, number of mice positive for PrP accumulation in immunohistochemical analysis; n0, number of inoculated mice. G, grey matter; W, white matter. Scale bar: 100 μm.Immunohistochemical analysis of PrP in the brains of MM2C-inoculated PrP-humanized mice. Click here for file


Uninterrupted transmission of highly pathogenic avian influenza virus (HPAIV) H5N1 of clade 2.2.1 in Egypt since 2006 resulted in establishment of two main genetic clusters. The 2.2.1/C group where all recent human and majority of backyard origin viruses clustered together, meanwhile the majority of viruses derived from vaccinated poultry in commercial farms grouped in 2.2.1.1 clade.2 3.2± 4.2). The hemagglutinin (HA) and neuraminidase (NA) genes of the isolated virus were closely related to viruses in 2.2.1/C group isolated from poultry in live bird market (LBM) and backyards or from infected people. Conspicuous mutations in the HA and NA genes including a deletion within the receptor binding domain in the HA globular head region were observed.In the present investigation, an HPAIV H5N1 was isolated from twenty weeks old layers chickens that were vaccinated with a homologous H5N1 vaccine at 1, 7 and 16 weeks old. At twenty weeks of age, birds showed cyanosis of comb and wattle, decrease in egg production and up to 27% mortality. Examined serum samples showed low antibody titer in HI test (LogDespite repeated vaccination of layer chickens using a homologous H5N1 vaccine, infection with HPAIV H5N1 resulted in significant morbidity and mortality. In endemic countries like Egypt, rigorous control measures including enforcement of biosecurity, culling of infected birds and constant update of vaccine virus strains are highly required to prevent circulation of HPAIV H5N1 between backyard birds, commercial poultry, LBM and humans. A devastating highly pathogenic avian influenza virus (HPAIV) of H5N1 subtype first emerged in poultry and further transmitted to human in Hong Kong in 1997. To dateIn Egypt, H5N1 isolated from humans and a large group of viruses isolated from backyard ducks and chickens clustered in a distinct genetic group designated “2.2.1/C”, meanwhiBackyard birds and commercial poultry are kept in very close contact with humans due to integration of both farms and houses in the same buildings. Employees in commercial farms usually maintain their own household birds. Furthermore, selling of remaining feed, utensils and equipment from commercial farms to the rural family poultry often occurs in Egypt. Backyard chickens, ducks and geese are mostly reared together and roam freely in the vicinity of the house in close contact with human, particularly children. More than 70% of the Egyptian poultry production from commercial or backyard sectors is marketed through LBM. All H5NHere we describe the isolation and molecular characterization of an HPAI H5N1 virus isolated from twenty weeks old chicken layer flock, which was vaccinated three times with a commercial inactivated H5N1 vaccine.A commercial chicken farm with 23,699 Hisex-brown layers kept in cages; chickens were vaccinated three times at weeks 1, 7, and 16 with a commercial inactivated H5N1 vaccine seeded by A/Goose/Guangdong/1/1996/H5N1 . Vaccination of birds was conducted by the owner as part of usual practice. Ten tracheal swabs and ten cloacal swabs were collected randomly at day 7 after onset of clinical signs. At the same time, ten serum samples were collected for further laboratory investigation. Ten swabs were pooled according to Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. No expe50/0.1mL) according to Reed and Muench isolated from geese in live bird market (LBM) in the same region and referred to as LBM/Gs. (2) A/duck/Egypt/0918-NLQP/2009 [GenBank: GU002674 and HQ908452] isolated from backyard birds referred to as BY/Dk. (3) A/Egypt/N02039/2009 [GenBank: CY041918 and CY041919] was detected from 32 months old boy few days before the incidence of the current outbreak in commercial-layer flock from the same village  and an increase of the number of misshaped eggs . The course of the disease took more than 21 days from onset of clinical illness until depopulation of the flock.50/0.1mL. Serum samples collected at 7 days after the onset of clinical signs had mean HI titer of 3.2 log2 (standard deviation ± 4.3 log2) using homologous A/Goose/Guangdong/1/1996 H5N1 antigen, supplied by the vaccine-producing company .After inoculation of embryonated chicken eggs with tracheal and cloacal swabs media, all embryos died within 48 hours post inoculation. Hemagglutination activity in the harvested allantoin fluid was determined. The isolated virus was confirmed to be an H5N1 virus by partial amplifications of the M, HA and NA genes of avian influenza viruses using specific real-time reverse transcription– polymerase chain reaction (RT-qPCR). The median egg infectious dose was 7.2 EIDThe full coding H5 and N1 gene sequences generated in this study were submitted to GenBank under accession numbers GU366078 and HQ908461 for the HA and NA genes, respectively. A/chicken/Egypt/0987-NLQP/2009(H5N1) isolated in this study had 99.76% and 99.65% HA and 99.85% and 99.78% NA nucleotides and amino acids identity, respectively with an H5N1 virus isolated from LBM from the same village as shown in Table The proteolytic cleavage site of the HA had polybasic amino acid motif “ERRRKKR/GLF”, typical for HPAIV of clade 2.2. A deletion within the receptor binding site at position 129S (H5 numbering) and additional four amino acid substitutions namely; D43N, S120N, I151T and S320G of viral H5 protein in comparison to H5N1 virus introduced into Egypt in 2006 (Index/2006) were found. All mutations located in the HA1 on the globular head domain  in comparison to GsGd/96. Furthermore, in comparison to H5N1 virus introduced into Egypt in 2006, A/chicken/Egypt/0987-NLQP/2009 had five amino acid substitutions in the NA protein namely; A46D, N200D, L224M, S339V, and S450G and 14 synonymous mutations in the NA gene. There was no molecular marker of neuraminidase inhibitor resistance. The NA protein had three predicted potential glycosylation sites; residues 68, 126 and 215. On the contrary, GsGd/96 virus had additional four potential glycosylation sites that located in the deleted stalk region of A/chicken/Egypt/0987-NLQP/2009.Twenty weeks old layer chickens flock vaccinated three times with inactivated H5N1 vaccine suffered from respiratory distress and some birds had signs commonly seen in poultry infected with HPAIV. HPAIV H5N1 was isolated from tracheal and cloacal swabs media collected after onset of clinical signs during regular national surveillance. Full H5 and N1 coding gene sequences showed a close genetic relationship between the obtained virus and viruses of LBM, backyards and human origins. Due to insufficiency of epidemiological data, the source of this infection through either LBM, backyard birds or from humans cannot be deduced.2 indicated sufficient protection of vaccinated chickens against HPAIV H5N1 infection. In this study, anti-H5 antibody titer was low with variable individual titers (3.2 ± 4.3 log2) after three successive vaccinations of Hisex brown-layers. Recent experimental studies showed that specific-pathogen-free (SPF) chickens vaccinated with this inactivated H5N1 vaccine evoked high HI titer (7.0 ± 0.8 log2) and were protected against challenge with an Egyptian H5N1 virus belonged to the 2.2.1/C group[Despite repeated vaccination using a homologous H5N1 vaccine, the chickens exhibited observable distress and mortality. This suggests updated poultry vaccine may be necessary in endemic areas like Egypt. Tian et al., found th/C group. Therefo/C group could beThe high level of genetic identity of A/chicken/Egypt/0987-NLQP/2009 to viruses in LBM markets and backyard birds suggests a direct transmission link which is not uncommon scenario in Egypt. On the other hand, close genetic relationship between A/chicken/Egypt/0987-NLQP/2009 and H5N1 virus isolated from 32 months old boy from the same village could be explained by existence of a common source of infection; most probably backyard birds and/or LBM-37. It i. Deletion of RBD 129S found in A/chicken/Egypt/0987-NLQP/2009 . It has It is well known that N-linked glycosylation of the HA of influenza A viruses can affect receptor binding preferences or mask antigenic regions. A/chickIn conclusion, circulation of HPAIV H5N1 in vaccinated birds continues to devastate the poultry industry in Egypt. Birds in backyards and LBM remain the main potential source of H5N1 infection to both commercial poultry and humans in Egypt. Targeted surveillance to elucidate the spread of HPAIV H5N1 among commercial poultry workers and/or householders should be considered.AIV: Avian influenza virus; D: Aspartic acid; EID: Egg-infective dose; HA: Hemagglutinin; HI: Hemagglutinin inhibition; HPAI: Highly pathogenic avian influenza; I: Isoleucine; G: Glycine; LBM: Live birds markets; N: Asparagine; NLQP: National laboratory for quality control on poultry production; OIE: World organization of animal health; RBD: Receptor binding domain; RT-qPCR: Real-time reverse transcription polymerase chain reaction; SPF: Specific pathogen free; S: Serine; T: Threonine.The authors declare that they have no competing interests.EFE carried out sample collection and examination and helped to draft the manuscript. AA carried out the sequence of the isolated virus. WHK participated in virus isolation and/or serological examination. EMA did sequence and phylogenetic analyses and helped to draft the manuscript. MMA and HMH conceived and coordinated the study and helped to draft the manuscript. All authors read and approved the final manuscript.


During the early infection stage, infected C57BL/6 mice exhibited more severe lung injury, slower recovery from lung damage, less effective viral clearance, higher levels of interlukine (IL)-6, monocyte chemotactic protein (MCP)-1, and IL-1β, and lower levels of tumor necrosis factor (TNF)-α and interferon (IFN)-γ than infected BALB/c mice. These results suggest that TNF-α and IFN-γ may help suppress viral gene expression and increase viral clearance, and that IL-6 and MCP-1 may contribute to lung injury in A/H7N9-infected individuals. In addition, lung damage and the distribution of virus antigen in tissues were similar in young and middle-aged mice. These results suggest that the more serious lung injury in middle-aged or older H7N9 cases is not mainly caused by differences in viral replication in the lung but probably by a dysregulated immune response induced by underlying comorbidities. These results indicate that the extent of dysregulation of the host immune response after H7N9 virus infection most probably determines the outcome of H7N9 virus infection.Avian influenza A/H7N9 virus infection causes pneumonia in humans with a high case fatality rate. However, virus-induced modulation of immune responses is being recognized increasingly as a factor in the pathogenesis of this disease. In this study, we compared the pathogenicity of A/H7N9 infection in BALB/c and C57BL/6 mouse models, and investigated the putative involvement of proinflammatory cytokines in lung injury and viral clearance. In both mouse strains, A/Anhui/1/2013(H7N9) infection with 10 During March 2013, a novel avian influenza A/H7N9 virus was identified in Shanghai and Anhui, China The dysregulation of proinflammatory cytokines and chemokines, or a “cytokine storm”, a severe adverse reaction created by the secretion of large amounts of proinflammatory cytokines, may aggravate lung injury observed in A/H5N1- and A/H7N9-infected patients Clinical data show that the fatality risks of patients admitted to hospital differ substantially depending on age. Increasing age is associated with greater disease severity in patients infected with seasonal influenza BALB/c and C57BL/6 mice have been used widely to study the pathogenesis of infectious diseases and exhibit different susceptibilities and immune responses to invading pathogens. For example, Otte et al. All of the procedures involving animals were approved by the Laboratory Animal Center, State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology (Permit number BIME 2013-15). The animal studies were conducted in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals. All of the procedures involving live A/H7N9 viruses were carried out in an approved biosafety level 3 facility.Specific pathogen-free young C57BL/6 and BALB/c mice  and female middle-aged BALB/c mice (10 months old) were obtained from the Laboratory Animal Center, Academy of Military Medical Sciences, Beijing, China. A/Anhui/1/2013 (H7N9) virus (Anhui/1/H7N9) was initially isolated by the National Institute for Viral Diseases Control and Prevention, Chinese Center for Diseases Control and Prevention (CDC) from a throat-swab specimen of the third case of laboratory-confirmed A/H7N9 virus 50) of 106 H7N9 virus in a volume of 20 μl. The infected mice were sacrificed at the times indicated and the lung, spleen, liver, kidney, brain, and intestine were harvested for virological and pathological analyses. Mouse sera were also collected for cytokine analysis. In addition, the young C57BL/6 and BALB/c mice were monitored daily to assess weight loss following H7N9 virus infection.The juvenile C57BL/6 and BALB/c mice and the middle-aged BALB/c mice were anesthetized via intraperitoneal injection of ketamine (80 mg/kg) and then inoculated intranasally (i.n.) with a 50% tissue culture infective dose  at 450 nm. The levels of mouse serum cytokines were determined using standard curves.Sera were collected from the infected mice at the times indicated and stored at −20°C before being analyzed. The levels of IL-1β, TNF-α, IL-6, IFN-γ, MCP-1, and keratinocyte-derived cytokine (KC) (a mouse homologue of IL-8) in the mouse sera were determined quantitatively using an enzyme-linked immunosorbent assay (ELISA) , according to the manufacturer's protocol. Briefly, 100 μl of diluted mouse serum was added to a plate precoated with a monoclonal antibody specific for individual mouse cytokines and incubated at 37°C for 90 min. After washing, biotinylated-detection antibodies specific to each cytokine were added and incubated at 37°C for 1 h. Plates were then washed and incubated for 1 h with 100 μl of HRP-conjugate streptavidin. The reactions were developed with 3,3′,5,5′-tetramethylbenzidine (TMB) substrate  at 37°C for 30 min, followed by adding 1 N H50, as described previously 10 TCID50/g of tissue The tissues of infected mice were harvested at the times indicated and homogenized in minimal essential medium (MEM) plus antibiotics to produce 10% (w/v) suspensions. The viral titers in tissues were determined as the TCIDA portion of lung tissue was excised, fixed with 10% neutral buffered formalin, dehydrated, and then embedded in paraffin before performing routine histology. Sections measuring 6 μm in thickness were stained with hematoxylin and eosin (H&E) and examined by light microscopy. The extent of lung injury was scored according to the levels of degeneration and necrosis of the bronchi and bronchiolar epithelium, infiltration of inflammatory cells, alveoli degeneration and collapse, expansion of parenchymal wall, hemorrhage, and interstitial edema Formalin-fixed, paraffin-embedded lung sections were de-paraffinized with xylene and hydrated using a graded alcohol series. The changes in the levels of HA protein of A/H7N9 virus and infiltrating neutrophils were assessed using a rabbit polyclonal antibody to A/H7N9 HA  and neutrophil marker NIMP-R14 , respectively. Antibodies were detected using a standard streptavidin-biotin detection system , according to the manufacturer's instructions.Neutrophil infiltration was assessed semi-quantitatively by examining in a blinded manner for the presence of neutrophils in 10 arbitrarily selected 40× objective fields of lung parenchyma in each lung section by light microscopy. The cumulative scores for each animal were expressed as the number of positive fields per 100 fields (%) All of the analyses were performed using Graphpad Prism version 5.01. The differences in the gross lesion areas between mouse strains were analyzed using a nonparametric Mann-Whitney test. The differences between mouse groups at the times indicated were analyzed using a two-way ANOVA and a Bonferroni post test.6 TCID50 of AH/1/H7N9 virus. Both C57BL/6 and BALB/c mice exhibited ruffled fur and hunched backs during the days following infection (data not shown). Although none of the mice died after infection with AH/1/H7N9 virus, body weight loss was clearly observed in all of the infected mice. Compared with BALB/c mice, which had an average body weight loss of 25% on day 8 postviral infection, C57BL/6 mice exhibited more severe weight loss, particularly on days 9–10 postviral infection. Moreover, infected C57BL/6 mice recovered body weight loss more slowly than infected BALB/c mice , with mean gross lesion areas of 28% and 45% in BALB/c mice and C57BL/6 mice, respectively. Gross presentations in both mice after A/H7N9 virus infection indicated more severe lung damage in C57BL/6 mice than in BALB/c mice , an indicator of cell proliferation. The number of PCNA-positive cells was similar in the two strains of mice at 0, 3, and 5 days after virus infection (data not shown). On days 7 and 14 after viral infection, however, the number of regenerative cells among bronchial epithelial cells and in the interstitial tissues of the lungs of BALB/c mice was higher than that in the lungs of C57BL/6 mice , therebyTo determine whether an association existed between the severity of lung injury and the tissue distribution of A/H7N9 virus, the distribution of the virus was followed at different time points after viral inoculation. The results demonstrated that many bronchial epithelial cells, but few pneumocytes or terminal bronchiole cells, were infected on day 1 postviral infection in either mouse . On day The dysregulation of proinflammatory cytokines and chemokines, which is sometimes referred to as a cytokine storm, is associated with lung injury and pneumonia in H7N9-infected patients Neutrophil infiltration was observed as early as day 1 and it reached a peak on day 5 after A/H7N9 virus infection. Neutrophils were present mainly in the parenchyma around bronchi on days 1 and 3, whereas they were found mainly in the necrotic areas of lungs on day 5 postviral challenge. Neutrophil infiltration disappeared at 14 days postviral infection. During the early stage of viral infection, however, more neutrophils were observed in the parenchyma around the bronchi and lung tissues of BALB/c mice than in the parenchyma of C57BL/6 mice, whereas there was no significant difference in this respect between the two mouse strains on day 14 . These rTo clarify whether the clinical severity of lung injury in elderly patients is related to virus replication, we examined the lung lesions in healthy naïve young and middle-aged BALB/c mice infected with H7N9 virus after H&E staining. The lung tissue distribution of the H7N9 virus was also followed by the HA antigen detection assay. The results showed that lung lesions were similar in middle-aged and young mice on day 5 postviral infection . The HA The novel avian influenza H7N9 virus has caused outbreaks of influenza in China, which have resulted in 139 infected cases and a high case fatality rate (32%) vs 25%, vs 28%, Previous reports show that BALB/c and C57BL/6 mice differ in their susceptibility and immune responses to infection by HPAI H5N1 and pandemic H1N1 (pH1N1) viruses It is widely believed that proinflammatory cytokine and chemokine dysregulation, referred to as a cytokine storm, contributes to lung injury and severe pneumonia during the progression of a viral infection. In the present study, we compared the levels of proinflammatory cytokines in C57BL/6 and BALB/c mice infected with A/H7N9 at different time points after viral infection. We found that the levels of proinflammatory cytokines, including IL-6, MCP-1, IL-1β, TNF-α, and IFN-γ, increased immediately after infection and peaked on day 3 postviral infection, before returning to their baseline levels about 7–10 days after infection in both mouse strains. However, A/H7N9-infected C57BL/6 mice exhibited higher levels of IL-6, MCP-1, and IL-1β, but lower levels of TNF-α and IFN-γ, than A/H7N9-infected BALB/c mice at day 3 postviral infection . These fMCP-1 plays an important role in the inflammatory and fibrotic responses to influenza A virus infection under neonatal hyperoxia in vivo antiviral effects TNF-α, which is generally considered to be a proinflammatory and proimmune cytokine, plays an important role in the early stages of host defense against influenza infection Neutrophils, which migrate readily into lung tissues in response to inflammatory stimuli, occur at higher levels in pulmonary capillaries than in systemic blood. Thus, neutrophils are considered to be the host immune system's first line of defense against infection. Tumpey et al. Epidemiological studies of human A/H7N9 virus infections show that the most severe cases occur among middle-aged and elderly patients and the age distribution differs from that of HPAI H5N1-infected cases