In vitro cleavage assay using the recombinant human Dicer protein also showed that synthetic 7SL RNA was processed by Dicer into fragments of different lengths. Further functional analysis suggested that 7SL RNA-derived small RNAs do not function like miRNAs, neither do they regulate the expression of 7SL RNA. In conclusion, the current study demonstrated that Dicer can process 7SL RNA, however, the biological significance remains to be elucidated.It has been reported that decreased Dicer expression leads to Alu RNAs accumulation in human retinal pigmented epithelium cells, and Dicer may process the endogenous SINE/B1 RNAs  into small interfering RNAs (siRNAs). In this study, we aimed to address whether Dicer can process Alu RNAs and their common ancestor, 7SL RNA. Using Solexa sequencing technology, we showed that Alu-derived small RNAs accounted for 0.6% of the total cellular small RNAs in HepG2.2.15 cells, and the abundance decreased when Dicer was knocked down. However, Alu-derived small RNAs showed different characteristics from miRNAs and siRNAs, the classic Dicer-processed products. Interestingly, we found that small RNAs derived from 7SL RNA accounted for 3.1% of the total cellular small RNAs in the control cells, and the abundance dropped about 3.4 folds in Dicer knockdown cells. Dicer-dependent biogenesis of 7SL RNA-derived small RNAs was validated by northern blotting.  During the past 65 million years, Alu elements have evolved to more than one million copies in the primate genomes. This makes them the most successful transposable elements in terms of copy number in vitroAlu elements can be transcribed by two independent polymerases in two different ways. “Free Alu RNAs” are transcribed by Pol III from their own promoters, while “embedded Alu RNAs” are transcribed by RNA polymerase II (Pol II) as part of protein- or non-protein-coding RNAs 7SL RNA, a component of the signal recognition particle (SRP), is the common ancestor of Alu RNAs. Alu elements probably originate from duplication of the 7SL RNA gene In this study, we addressed whether there are endogenous Alu-derived siRNAs in HepG2.2.15 cells, and whether 7SL RNA is processed by Dicer. Using Solexa sequencing technology, we demonstrated that although the abundance of Alu-derived small RNAs was reduced in Dicer knockdown cells, their characteristics were totally different from those of classic siRNAs. Interestingly, we found that 7SL RNA can be processed by Dicer, and the small RNAs derived from 7SL RNA were characterized.To address whether Alu RNAs are processed by Dicer, we sequenced small RNAs extracted from Dicer knockdown and the control HepG2.2.15 cells using Solexa technology. A total of 14,289,326 genome-matching sequence reads were identified from the control cells, and 14,623,845 from Dicer knockdown cells. The majority (48%) of small RNAs in the control cells were derived from annotated miRNA loci, and as expected, the fraction of miRNA was diminished to 32% in Dicer knockdown cells .Alu-derived small RNAs were identified in our sequencing datasets using BLAST and SOAP2 software 7SL RNA, the evolutionary precursor of Alu, is a highly structured RNA containing partial double-stranded regions in vitro cleavage assay using the recombinant human Dicer protein. As shown in To validate Dicer-dependent biogenesis of 7SL RNA-derived small RNAs, we performed northern blotting analysis. As shown in Small RNAs derived from longer non-coding RNAs, such as tRNAs or snoRNAs, have been shown to function like miRNAs MiRNA is incorporated into the RNA-Induced Silencing Complex (RISC) with Argonaute proteins to regulate gene expression Growing evidence has suggested that small RNAs are implicated in epigenetic regulation of gene expression in vitro . Dicer knockdown was performed as described previously Human embryonic kidney cells (HEK293T from ATCC) and HepG2.2.15 cells . Membranes were incubated with primary antibody, followed by horseradish peroxidase-linked secondary antibody and detected with ECL plus reagents (Millipore). The primary antibodies include: anti-Dicer , anti-Ago2  and anti-β-actin .http://transpogene.tau.ac.il/) and the human 7SL RNA gene sequence (NR_002715.1), respectively.Small RNAs ranging from 16 to 30 nt were gel-purified, and those with 5′-phosphate and 3′-hydroxyl group were ligated with the 3′- and 5′-adaptors. The ligation products were then gel-purified, reversely transcribed, and PCR-amplified. The PCR fragments were then sequenced using Illumina GAII platform. Low quality sequencing reads were discarded, and the adaptor sequences were removed before analysis of small RNA sequences. The sequences were mapped to the reference human genome (hg19) using SOAP2 program with at most two mismatches. For annotation, small RNA sequences were aligned to miRBase15.0, Rfam 9.1 and GeneBank using BLAST and SOAP2 software. Alu or 7SL RNA-derived small RNAs were identified by mapping the small RNA sequences to the human Alu sequences extracted from the TranspoGene database , and small RNAs were extracted using the E.Z.N.A.® miRNA Isolation Kit .32P]ATP labeled oligonucleotides complementary to 7SL sRNA5cd. U6 was used as loading control.To detect the full-length 7SL RNA, 10 µg total RNAs were separated by electrophoresis in an 8% polyacrylamide gel and transferred to a nylon membrane. The membrane was hybridized with [γ-32P]ATP labeled oligonucleotides complementary to 7SL sRNA5cd. U6 was used as loading control.To detect small RNAs derived from 7SL RNA, 30 µg small RNAs were separated by electrophoresis in a 15% polyacrylamide gel and transferred to a nylon membrane. The membrane was hybridized with , immunoprecipitated with an anti-Ago2 antibody  and then bound to Protein-G Sepharose  beads. The beads were washed extensively with Ago2 IP washing buffer  before elution with 0.1 M glycine (pH2.3). Ago2-bound RNAs were then isolated using Trizol LS reagent (Invitrogen). To detect 7SL sRNA5cd, the eluted RNA was poly A tailed using a Poly (A) Polymerase Tailing Kit , reversely transcribed using RT primer (ATTCTAGAGGCCGAGGCGGCCGACATGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN) and PCR amplified using the following primers: GAGTTCTGGGCTGTAGTGCGCTA and ATTCTAGAGGCCGAGGCGGCC, which only detected 7SL sRNA5cd but not the full-length RNA . MiR-17 was detected using the miR-17 RT-PCR kit from RiboBio (Guangzhou).Immunoprecipitation of Ago2 binding RNA was carried out as previously described Figure S1Quantification of small RNA northern blotting. 7SL sRNA5cd was represented by the 23 nt band.(TIF)Click here for additional data file.Figure S2Dicer cannot cleave the control RNA. 12% polyacrylamide gel electrophoresis of LacZ RNA digested by the recombinant human Dicer protein.(TIF)Click here for additional data file.Figure S3Knockdown of Ago2 in HepG2.2.15 and HEK293T cells. (A&C) representative western blot of Ago2, β-actin was used as loading control. (B&D) The relative levels of Ago2 mRNA determined by real-time RT-PCR. (A&B) Hep2.2.15 cells, (C&D) HEK293T cells.(TIF)Click here for additional data file.Figure S47SL sRNA5cd is not associated with Ago2 protein. (A) RT-PCR can specifically detect 7SL sRNA5cd but not full-length 7SL RNA. (B) RNA immnuoprecipitation experiment indicated that 7SL sRNA5cd was not associated with Ago2 protein, as a positive control, miR-17 was co-immunoprecipitated with Ago2 protein.(TIF)Click here for additional data file.Table S1Alu-derived small RNAs.(XLS)Click here for additional data file.Table S27SL RNA-derived small RNAs.(XLS)Click here for additional data file.


Keratins 8 and 18 (K8/K18) are intermediate filament proteins that protect the liver from various forms of injury. Exonic K8/K18 variants associate with adverse outcome in acute liver failure and with liver fibrosis progression in patients with chronic hepatitis C infection or primary biliary cirrhosis. Given the association of K8/K18 variants with end-stage liver disease and progression in several chronic liver disorders, we studied the importance of keratin variants in patients with hemochromatosis.The entire K8/K18 exonic regions were analyzed in 162 hemochromatosis patients carrying homozygous C282Y HFE (hemochromatosis gene) mutations. 234 liver-healthy subjects were used as controls. Exonic regions were PCR-amplified and analyzed using denaturing high-performance liquid chromatography and DNA sequencing. Previously-generated transgenic mice overexpressing K8 G62C were studied for their susceptibility to iron overload. Susceptibility to iron toxicity of primary hepatocytes that express K8 wild-type and G62C was also assessed.We identified amino-acid-altering keratin heterozygous variants in 10 of 162 hemochromatosis patients (6.2%) and non-coding heterozygous variants in 6 additional patients (3.7%). Two novel K8 variants (Q169E/R275W) were found. K8 R341H was the most common amino-acid altering variant (4 patients), and exclusively associated with an intronic KRT8 IVS7+10delC deletion. Intronic, but not amino-acid-altering variants associated with the development of liver fibrosis. In mice, or ex vivo, the K8 G62C variant did not affect iron-accumulation in response to iron-rich diet or the extent of iron-induced hepatocellular injury.In patients with hemochromatosis, intronic but not exonic K8/K18 variants associate with liver fibrosis development. KRT genes, K proteins) represent the IFs of epithelial cells and are further subdivided into subtypes II (including K1–K8 and K71–K80) and I (K9–K28), which are both required to form obligate heteropolymers Intermediate filaments (IFs), together with microtubules and the actin-containing microfilaments constitute the three major components of eukaryotic cytoskeleton The pathogenesis of keratin variants was supported by the transgenic mice overexpressing K8 G62C variant, which are susceptible to Fas-induced apoptosis and microcystin-induced liver injury HFE gene, which is as frequent as 1∶200 in Caucasians of northern European descent HFE mutations is highly variable and does not necessarily lead to a clinically-manifest iron overload or even development of end-stage liver disease Hereditary hemochromatosis (HHC) comprises genetic disorders leading to iron overload HFE mutation and 116 age-, sex- and ethnic background matched controls. In that group, 9 patients in the HHC cohort carried the K8 R341H variant but none in the controls In a study that examined candidate gene variants that may associate with the presence of HHC, the K8 R341H heterozygous variant was identified in 3 of 13 iron overload subjects but not in 5 controls The study protocol was reviewed and approved by the local ethics committees of the participating centers Austria  and Germany  between 1991–2005, and all recruited patients gave informed and written consent.All animal experiments were conducted with the approval of UCUCA under animal protocol number 10081  for animal welfare.HFE C282Y mutation HFE mutation of the analyzed subjects was confirmed by standard genetic testing To determine the impact of keratin variants on liver disease development in HHC, we analyzed a previously described cohort of 162 homozygous carriers of the KRT8/KRT18 variants was assigned based on the mRNA sequences NM 002273.2 and NM 000224.2, respectively, while genomic sequences M34482.1 and AF179904.1 were employed for assignment of non-coding variants (www.ncbi.nlm.nih.gov). New genetic variants were reported in GenBank .Genomic DNA was isolated from peripheral whole blood using an established DNA isolation kit . For hemochromatosis patients, all 15 K8 and K18 exons and their adjacent exon-intron boundaries were amplified using previously described primers and a mixture of T Taq and Optimase Polymerases  ad libitum with 3% carbonyl iron -containing diet  for one month. Age-matched male mice kept on a standard diet were used as controls. Mice were euthanized by CO2 inhalation and blood was collected by intracardiac aspiration to measure serum alanine aminotransferase (ALT) levels. Livers were removed and cut into pieces for: (i) fixation in 10% formaldehyde for histological staining, (ii) snap freezing to measure hepatic iron concentration (HIC), or submerging into RNAlater stabilization reagent (Qiagen) for RNA analysis. Histological images were taken by Olympus BX40 with spot insight QE camera. The mRNA was transcribed into cDNA using oligo-dT primers and Superscript II reverse transcriptase . Quantitative real-time polymerase chain reaction (RT-PCR) was carried out with SYBR green Master Mix  and performed in duplicate using specific mouse hepcidin (encoded by Hamp gene), collagen1 (Col1a1), smooth muscle α-actin (SMA) and transforming growth factor-β (TGF-β) primers and L7 ribosomal protein as an internal control  overexpressing human wild-type K8 (K8 WT) or the K8 G62C variant  control . All ani3+ of 2∶1 To test whether the presence of keratin variants influences hepatocellular iron toxicity, we isolated primary hepatocytes from K8 WT and G62C males as described before The unpaired two-tailed t or chi-square tests were used, and P values less than 0.05 were considered statistically significant.HFE C282Y mutation are displayed in p<0.0001). Similarly, all assessed parameters of iron metabolism were markedly higher in males compared to females . Only a minority of patients had significant alcohol consumption.The clinical characteristics of the previously described cohort of 162 subjects carrying homozygous  females  (ferritiKRT8 505 C→G resulting in a K8 Q169E substitution within the rod domain (subdomain 1B), was found in two patients. The other novel variant, KRT8 823 C→T that codes for a K8 R275W substitution in the L2 linker sequence of KRT8, was found in one patient. The novel variants were detected in non-cirrhotic patients, and the involved amino acid residues are remarkably conserved among species and among some type II keratins  was detected that had not been described before. The frequency of amino-acid-altering variants was similar in cirrhotic and non-cirrhotic patients (6.6% vs. 6.1%). Among the described keratin variants, only K8 G62C and R341H were previously clearly implicated in liver disease development KRT8 variants were significantly overrepresented in cirrhotic patients (p = 0.02). However, neither intronic nor exonic keratin variants associated with iron accumulation in hemochromatosis patients as suggested by similar levels for serum iron, transferrin, transferrin saturation, ferritin and HIC and HII indices  and comparable levels of fibrosis-associated genes collagen 1 (Col1a1), smooth muscle α-actin (SMA) and transforming growth factor-β (TGF-β)  . In addi (TGF-β) .To test whether the presence of K8 G62C variant affects hepatocellular iron toxicity, primary hepatocytes from K8 WT and G62C mice were treated with FeNTA. FeNTA exposure led to significant increase in the release of ALT and LDH into the cell culture supernatant; which, however, did not differ significantly between the genotypes . SimilarIn the study herein, we first analyzed HHC samples for the presence of K8/K18 variants. Amino-acid-altering keratin variants were detected in 10 of 162 HHC patients (variant frequency 6.2%) and a similar frequency of keratin variants was seen in the control group (8.1%). In contrast, substantially higher keratin variant frequencies (>10%) were previously described in patients with end-stage liver disease or acute liver failure, which is likely due to the enrichment of keratin variants in patients with severe liver disease http://www.interfil.org/), further studies are needed to address the potential pathogenic role of this variant.Among single point mutations, we identified four patients with K8 R341H variant (variant frequency 2.5%), which is comparable with the frequency seen in liver-healthy Caucasians While the amino-acid altering K8/K18 variants exhibited similar frequencies in HHC patients with and without liver cirrhosis, all patients harboring the K8 G62C variant had liver cirrhosis. This mutation might therefore be of particular relevance in HHC. This hypothesis is further supported by the fact that this variant introduces a cysteine into the K8 backbone, which normally lacks cysteines, and leads to keratin crosslinking and impairment of keratin filament reorganization upon oxidative stress In summary, the combination of genetic and experimental data suggests that in contrast to hepatitis C, primary biliary cirrhosis and acute liver failure Unlike the exonic variants, the intronic variants were shown to be overrepresented in HHC patients with liver cirrhosis. This finding represents the first such liver disease association since intronic K8/K18 variants did not associate with liver fibrosis development in patients with chronic hepatitis C infection Figure S1Conservation of novel K8 variants among species and type II keratins. The sequences surrounding the amino acids K8 Q169  and K8 R275W  are displayed for selected species  and type II keratins . The sequences illustrate the single nucleotide variations leading to the highlighted amino acid change . Note that K8 Q169 is highly conserved among species and among type II keratins, while K8 R275 amino acid is highly conserved among species, and is also conserved from a charge perspective among type II keratins. The figures were assembled using the following reference sequences: K8: NM 002273.3 (Homo sapiens), NM 19937.1 (Rattus norvegicus), NM 031170.2 (Mus musculus), BC 044116.1 (Xenopus laevis) and NM 200080.2 (Danio rerio); Type II Keratins: NM 006121.3 (KRT1), NM 000423.2 (KRT2a), NM 015848.4 (KRT2p), NM 057088.2 (KRT3), NM 002272.3 (KRT4), NM 000424.3 (KRT5), NM 005554.3 (KRT6A), NM 005555.3 (KRT6B), NM 005556.3 (KRT7).(TIF)Click here for additional data file.Figure S2mRNA expression of fibrosis-related genes and hepcidin does not differ among K8 WT and G62C fed iron-rich diet. WT mRNA levels are set at 1.0 and G62C mRNA levels of the indicated transcripts are displayed relative to WT. mRNA was isolated from 4 independent livers per genotype.(TIF)Click here for additional data file.Table S1The clinical characteristics of the previously described cohort of 162 subjects carrying homozygous HFE C282Y mutation (cirrhotic patients vs non-cirrhotic patients).(DOC)Click here for additional data file.Table S2Sequence of primers used for quantitative real time-PCR and length of the amplified products.(DOC)Click here for additional data file.


The insulin -23Hph and IGF2 Apa polymorphisms were genotyped in Romanian patients with T1DM (n = 204), T2DM (n = 215) or obesity (n = 200) and normoponderal healthy subjects (n = 750). The genotypes of both polymorphisms were distributed in concordance with Hardy-Weinberg equilibrium in all groups. The -23Hph AA genotype increased the risk for T1DM , especially in patients without macroalbuminuria . No other significant association between the alleles or genotypes of insulin -23Hph and IGF2 Apa and diabetes or obesity was identified. The Insulin -23HphI and IGF2 Apa are other polymorphisms that have recently received attention  and predisposition to some pathologic traits (Insulin and IGF2 (11p15.5) are candidate genes for complex traits, including type I diabetes mellitus (T1DM) , type IIttention , becauseIn this study we tested the possible association between two polymorphisms from the insulin – IGF2 region and T1DM, T2DM and obesity.2. T2DM patients were included if they had had more than two fasting glycemia values above 140 mg/dL after the age of 30 years old, a BMI of 18.5-30 kg/m2 and required no treatment with insulin. Obese patients were selected based on BMI values between 30-40 kg/m2 and glycemia in a fasting state ≤ 110 mg/dL. The presence or absence of hypertension (blood pressure above 130/85 mm Hg or treatment with antihypertensive medication), smoking  and drinking  habits were recorded for all participants.A total of 1369 unrelated Romanian Caucasians , older than 18 years, was enrolled in the study. Patients with T1DM (n = 204), T2DM (n = 215) or obesity (n = 200) diagnosed for at least two years were selected from the N Paulescu Institute . T1DM patients were selected if they were younger than 20 years at the onset of the disease, required daily insulin treatment and had a BMI between 18.5-25 kg/mThe control subjects comprised a total of 750 individuals  selected from visitors attending two medical clinics from Bucharest for routine checking. They were matched for age, sex, ethnicity and birth place with patients and were equally distributed into Hc1, Hc2 and Hc3 groups. All normal individuals had no relatives with DM, obesity or other genetic syndromes.Informed consent was obtained from all subjects before enrolment. The design of the study was in accordance with internationally agreed ethical standards  and approved by the N Paulescu hospital ethical committee., rs680) was based on a RFLP method. The INSF 5'tccaggacaggctgcatcag3', INSR 5'agcaatgggcggttggctca3' and IGF2F 5'cttggactttgagtcaaattgg3', IGF2R 5'cctcctttggtcttactggg3' primer pairs (Sigma Genosys) were used for PCR (Corbett Research thermocycler). Amplicons (5 μL) were electrophoresed on agarose gel (2%) after digestion with 6 U of HphI or ApaI endonucleases (New England BioLabs). Two researchers who did not know the clinical data of the subjects independently scored all genotypes. Ten percent of randomly selected samples were re-genotyped as a quality control. No errors in genotyping were observed.Fasting plasma glucose, serum total cholesterol, serum triglycerides and HbA1C levels were measured using standard laboratory protocols. Genomic DNA was isolated using a commercial kit  from venous blood (1 mL) collected into tubes containing EDTA. Genotyping for insulin -23Hph  and IGF2 Apa .After checking the normality of distribution for continuous variables by means of the Shapiro-Wilk W test, comparisons of these parameters between groups were performed through Mann-Whitney U non-parametric tests. Categorical variables between groups were compared through chi-squared tests, that were also used for verifying genotype departures from Hardy-Weinberg equilibrium within each group and for comparing genotype frequencies between affected and control subjects. Odds ratio (ORs) and their corresponding 95% confidence intervals were calculated for estimating disease risks corresponding to genotypes AA from -23Hph and IGF2ApaThe main characteristics of the patients and controls are presented in 2 = 0.84, DF = 4, p = 0.93) and IGF2 Apa  genotypes are similar in the control lots, Hc1, Hc2 and Hc3. We observed a different distribution of -23Hph genotypes in T1DM and Hc1 lots. The -23Hph AA genotype is significantly associated with T1DM . This genotype remains also more frequent in T1DM than in controls (89.66% vs. 50.26%) if patients with macroalbuminuria and their matched controls were excluded from the analysis. In this case, the association between AA genotype and T1DM increases . The disease risk remains non-significant if the T2DM patients with macroalbuminuria and their matched controls were excluded from analysis.The distribution of the -23Hph and IGF2 Apa genotypes were in Hardy-Weinberg equilibrium in all groups . The Inse.g. age of disease onset, sex).We did not observe any other significant associations between investigated markers and clinical and biochemical data (e.g. OR > 2.0) between normal and clinical phenotypes using relatively small sample sizes (n = 200)  . The subThe variants of Insulin -23Hph (-23Hph A: 73.53%) and IGF2 Apa (IGF2 A: 77.3%) polymorphisms in our healthy controls are similar to those reported for Caucasians .AA = OR: 3.22, 95% CI: 2.09-4.98, p < 0.0001). This result is in agreement with previous studies based on Caucasian cohorts (We found a strong association between insulin -23Hph polymorphism and T1DM (ORvs. 50.26%) and the disease risk  increases when patients with macroalbuminuria and their matched controls were excluded from analyses. We did not identify a similar effect in patients with T2DM. The -23Hph A allele is in almost complete linkage disequilibrium with the INS VNTR class I. It has been previously reported that the prevalence of proteinuria in insulin-requiring diabetic patients is significantly higher in homozygotes for INS VNTR class I variants than in carriers of other genotypes , but not all populations . In our study the results were non-significant even when the gender was taken into account (p > 0.05).We did not observe a significant association between -23Hph genotypes and obesity in middle aged patients (p > 0.05). This result is in agreement with other studies, in which IDDM2 markers were also found to not change the risk for obesity in middle-aged subjects  and non-genetic  factors.A predisposition for diabetes and obesity may be triggered by genetic and environmental determinants of growth patterns from prenatal to childhood life . We cann


TNF-related apoptosis-inducing ligand  has been shown to promote intestinal cell differentiation. Nuclear factor of activated T cells (NFAT) participates in the regulation of a variety of cellular processes, including differentiation. Here, we examined the role of NFAT in the regulation of TRAIL in human intestinal cells. Treatment with a combination of phorbol 12-myristate 13-acetate (PMA) plus the calcium ionophore A23187 (Io) increased NFAT activation and TRAIL expression; pretreatment with the calcineurin inhibitor cyclosporine A (CsA), an antagonist of NFAT signaling, diminished NFAT activation and TRAIL induction. In addition, knockdown of NFATc1, NFATc2, NFATc3, and NFATc4 blocked PMA/Io increased TRAIL protein expression. Expression of NFATc1 activated TRAIL promoter activity and increased TRAIL mRNA and protein expression. Deletion of NFAT binding sites from the TRAIL promoter did not significantly abrogate NFATc1-increased TRAIL promoter activity, suggesting an indirect regulation of TRAIL expression by NFAT activation. Knockdown of NFATc1 increased Sp1 transcription factor binding to the TRAIL promoter and, importantly, inhibition of Sp1, by chemical inhibition or RNA interference, increased TRAIL expression. These studies identify a novel mechanism for TRAIL regulation by which activation of NFATc1 increases TRAIL expression through negative regulation of Sp1 binding to the TRAIL promoter. The mammalian intestinal mucosa undergoes a process of continual renewal, characterized by active proliferation of stem cells localized near the base of the crypts, progression of these cells up the crypt-villus axis with cessation of proliferation and subsequent differentiation into one of the four primary cell types Waf1 and p27Kip1Tumor necrosis factor-related apoptosis-inducing ligand , a novel member of the TNF family, was identified based on homology to the extracellular domains of TNF and FasL (CD95L) kip1 expression and decreases Akt phosphorylation and that NFAT activation is required for sodium butyrate mediated-intestinal cell differentiation The nuclear factor of activated T cells (NFATc) proteins are a family of transcription factors whose activation is controlled by calcineurin, a calcium-dependent phosphatase. Four distinct genes encoding closely related NFATc proteins (NFATc1–4) Previously, we cloned the human TRAIL promoter and identified a number of putative transcription factor binding sites including NFAT and Sp1 sequences 2SO); this induction was attenuated by pretreatment with CsA. To test whether PMA/Io activates NFAT, we performed EMSAs with nuclear extracts obtained from HT29 cells treated for 60 min with PMA/Io using a commercial oligonucleotide with consensus binding sites for NFAT as the probe. As shown in Previously, we have shown that inhibition of PI3-kinase, which augments enterocyte-like differentiation of HT29 and Caco-2 human colon cancer cells b. The specificity of this binding was confirmed by competition studies using cold wild type and mutant competitor probes. Addition of anti-NFATc1 antibody to the mixture abolished bands b and c. Band b was also abolished when anti-NFATc2 antibody was added to the mixture and a supershifted band (band d) was observed. When anti-NFATc4 antibody was added to the mixture, band a was abolished; there was no effect on the binding complex with addition of anti-NFATc3 antibody. These results demonstrate that NFATc1, NFATc2, and NFATc4 isoforms are present in HT29 cells. Treatment with PMA/Io increased band b, suggesting a role of NFATc1 or NFATc2 in PMA/Io induction of TRAIL expression in HT29 cells.Four classical isoforms of NFAT have been identified To confirm the contribution of each isoform to TRAIL expression, individual NFAT isoforms were silenced by transfection of HT29 cells with the relevant siRNA. As shown in To further determine the role of NFAT in TRAIL regulation, HT29 cells were transfected with a plasmid encoding NFATc1, or the control pDF30 vector; TRAIL protein expression was assayed by Western blotting. Overexpression of NFATc1 by transfection with the pSH-NFATc1 vector increased TRAIL protein expression compared with the control pDF30 vector . NFATc1 To determine whether NFAT induction of TRAIL expression was regulated at the level of promoter activity, Caco-2 human colon cancer cells were transfected with plasmids encoding NFATc1, NFATc2, NFATc3, NFATc4 or control vectors pDF30 or pcDNA3.1 together with a 1371 bp TRAIL proximal promoter construct Previously, we have reported two NFAT binding sites identified in the 1371 bp TRAIL promoter by computer sequence analysis using MatInspector V2.2 software a, b, c, respectively) or diminished binding of the protein-DNA binding complex. We did not identify NFATc4 binding to these two sites.To determine whether PMA/Io treatment affects the binding activity of these potential NFAT binding sites, EMSA was performed. Treatment with PMA/Io increased N4 and N5, but not N1, N2, and N3 binding activity , suggestTo determine the contribution of these NFAT binding sites to TRAIL promoter activation, deletional analyses were performed. Caco-2 cells were transfected with two 5′- deletion promoter constructs together with control vector pDF30 or a plasmid encoding NFATc1. As shown in To further identify the NFATc1 responsive elements in the −165 TRAIL promoter, five oligonucleotide probes were synthesized. These probes contained all of the putative transcriptional binding sites identified between −169 and −33 by computer sequence analysis . HT29 ceTo further confirm NFATc1 regulation of TRAIL expression, HT29 cells stably transfected with the shRNA targeting NFATc1 and the control shRNA (CON) were established. Complete knockdown of NFATc1 in clones 33, 36 and 35 and partial knockdown of NFATc1 in clone 34 were confirmed in HT29 cells transfected with NFATc1 shRNA by RT-PCR . The incSince both probe 4 and probe 5 contain putative Sp1 binding sites , we nextTo further confirm these findings, occupancy of the TRAIL promoter by Sp1 in cells was further analyzed using a ChIP assay. Cross-linked chromatin was prepared from HT29 cells transfected with control siRNA or siRNA targeting NFATc1. The human TRAIL promoter region containing the Sp1 binding sites was precipitated using either the anti-Sp1 antibody or IgG, and using the probe 1 (forward) and the probe 5 (reverse) shown in GGGCGG binding sequence, and effectively blocks the binding of Sp1 Since Sp1 protein directly binds to the TRAIL promoter and knockdown of NFATc1 decreased TRAIL expression and increased Sp1 binding, we next investigated whether Sp1 transcription factor regulates TRAIL expression. Mithramycin has affinity for the Previously, we demonstrated that intestinal cell differentiation is associated with the induction of TRAIL expression Although five NFAT transcription factor binding sites were identified in the TRAIL promoter, our results showed that NFATc1 increased TRAIL promoter activity not by direct binding but, alternatively, through negative regulation of Sp1 binding to the promoter. Recently, we have found that knockdown of NFATc1 decreased PTEN expression and increased the phosphorylation levels of Akt while overexpression of NFATc1 increased PTEN expression and decreased Akt phosphorylation in HT29 and Caco-2 cells Similar to our findings showing induction of TRAIL by NFAT, activation of NFAT isoforms, including NFATc1, up-regulated FasL expression Sp1 recognizes and binds to GC/GT boxes and regulates a large number of mammalian genes in normal and transformed cells Sp1 binds the Myc-associated zinc finger protein (MAZ) promoter region and represses promoter activity In summary, our current study identifies the NFAT molecular pathway as an important regulator of TRAIL. Furthermore, we demonstrate that Sp1 inhibition increases TRAIL expression. Importantly, the induction of TRAIL expression by NFATc1 is not through the direct binding of NFATc1 to the TRAIL promoter but through negative regulation of Sp1 binding. TRAIL has been shown to play an important role in intestinal cell differentiation 32P] ATP  was from Amersham Pharmacia Biotech . Immobilon-P nylon membranes for Western blots were purchased from Millipore , and x-ray film was purchased from Eastman Kodak . Total RNA was isolated using Ultraspec RNA from Biotecx Laboratories . The enhanced chemiluminescence (ECL) system for Western immunoblot analysis was from Amersham . Tissue culture media was obtained from GIBCO-BRL . All other reagents were of molecular biology grade and purchased from either Sigma or Amresco .Phorbol 12-myristate 13-acetate (PMA) was purchased from Sigma Chemical Company . A23187 calcium ionophore (Io) was from Alexis Corporation . The calcineurin inhibitor cyclosporine A (CsA) was from Calbiochem . The plasmids encoding Flag-tagged NFATc1, NFATc2, NFATc3 and empty vector (pDF30) were kindly provided by Dr. Gerald R. Crabtree . Mouse anti-TRAIL antibody used for Western blot was purchased from IMGENEX Corporation . Mouse antibodies against NFATc1, NFATc2 and NFATc4 were from Affinity BioReagents, Inc. . Rabbit anti-β-actin antibody was from Sigma. Rabbit polyclonal anti-Sp1 antibody (07-645) was from Upstate . Mouse anti-NFATc3 antibody, the NFATc, NFATc mutant, Sp1, and ETS oligonucleotides were from Santa Cruz Biotechnology . Human NFATc1, NFATc2, NFATc3, NFATc4, Sp1, and control siRNA were purchased from Dharmacon, Inc. . [γ-Human colon cancer cell lines HT29 and Caco-2  were maintained in McCoy's 5A supplemented with 10% fetal calf serum (FCS) and MEM supplemented with 15% of FCS, respectively. The cells were pretreated with CsA for 30 min and then treated with PMA/Io in the presence or absence of CsA.Total protein was extracted and resolved on a 10% polyacrylamide gel and transferred to Immobilon-P nylon membranes as we have described previously CCGGCATCGAGATAACCTCGTGCTTCTCGAGAAGCACGAGGTTATCTCGATGTTTTT (TRCN0000017336), CCGGCGTCAGTTTCTACGTCTGCAACTCGAGTTGCAGACGTAGAAACTGACGTTTTT (TRCN0000017335), CCGGCCCGCCAACGTTCCAATTATACTCGAGTATAATTGGAACGTTGGCGGGTTTTT (TRCN0000017334), CCGGCGGCAACATTAGAAAGTGATTCTCGAGAATCACTTTCTAATGTTGCCGTTTTT (TRCN0000017333). HT29 cells were infected with the control shRNA or shRNA to human NFATc1 lentivirus particles and stably expressing cells were selected with puromycin at a concentration of 2 µg/ml.MISSION shRNA Lentiviral Particles with short-hairpin RNAs (shRNAs) were purchased from Sigma. The control shRNA  contains 4 base pair mismatches within the short hairpin sequence to any known human or mouse genes. A set of four shRNAs to human NFATc1  were used; the sequences were as follows: 32P-labeled consensus oligonucleotide in a buffer containing 4% glycerol, 50 mM NaCl, 1 mM MgCl2, 0.5 mM dithiothreitol, 0.5 mM EDTA, 10 mM Tris-HCl; pH 7.5) and 0.5 µg/ml poly(dI·dC) in a final volume of 20 µl, for 15 min at room temperature. For supershift studies, 2 µl of antiserum was added to the nuclear protein for 20 min at room temperature prior to the addition of labeled probe. The complexes were fractionated on 6% native polyacrylamide gels run in 1× TBE buffer , dried, and exposed to Kodak X-AR film at −70°C. Competition binding experiments were performed by the addition of the nonradioactive oligonucleotide, in 100-fold molar excess, at the time of addition of radioactive probe.The nuclear extracts were prepared using a kit according to the manufacturer's protocol. EMSAs were performed as described previously 5′- CATGGATCCTGAGGGCAAGG-3′ and 5′-TTGGCCCCACCCACATCTATTGAACC-3′.The ChIP assay was performed using the ChIP-IT Express Enzymatic Kit (Active Motif) according to the manufacturer's protocol. PCR of the human TRAIL promoter containing the Sp1 binding sites was performed using total (input) or immunoprecipitated chromatin with the following pair of oligonucleotide primers: T values were analyzed in Microsoft Excel using the comparative CT(ΔΔCT) method as described by the manufacturer (Applied Biosystems). The amount of target (2−ΔΔCT) was obtained by normalizing to the endogenous reference (18S) and relative to a calibrator .Total RNA was isolated using the Ultraspec™ RNA reagent. Real-time RT-PCR was performed using Applied Biosystems assays-on-demand 20× assay mix of primers and Taq-Man MGB probes (FAM™ dye-labeled) for target gene TRAIL (ID Hs00234355_m1) and predeveloped 18S rRNA(VIC™ dye-labeled probe) TaqMan® assay reagent (P/N 4319413E) for endogenous control. Separate tubes (singleplex) one-step RT-PCR was performed with 80 ng of RNA for both target gene and endogenous control. The reagent was TaqMan one-step RT-PCR master mix reagent kit (P/N 4309169). The cycling parameters for one-step RT-PCR were: reverse transcription 48°C for 30 min, AmpliTaq activation 95°C for 10 min, denaturation 95°C for 15 s, and annealing/extension 60°C for 1 min (repeat 40 times). Duplicate C5′-CTTCACAGTGCTCCTGCAGT-3′ and 5′-TTAGCCAACTAAAAAGGCCCC-3′), NFATc1 (5′- GGAAGGGCGGCTTCTGCGAC-3′ and 5′- AGGCGTGCGGGCGCAGCAG-3′), NFATc2 (5′- CTGCCTCGCCACATCTACC-3′ and 5′-TGGTAGAAGGCGTGCGGCTT-3′), NFATc3 (5′-TGGATCTCAGTATCCTTTAA-3′ and 5′- TACACGAAACACAAGTCGTA-3′), NFATc4 (5′- TACAGATGTTCATCGGCAC-3′ and 5′ CGAAGCTCAATGTCTGAAT-3′).RT-PCR was performed using a Titan One-Tube RT-PCR kit  according to the manufacturer's protocol. Cycling conditions were as follows: 95°C for 30 s, 54°C for 30 s, and 68°C for 50 s for 31 cycles. Approximately 8 µl of product was run on a 0.5× Tris-borate-EDTA-1% agarose gel. Primer sets were synthesized by Sigma as follows: TRAIL  as we have described previously Due to the heterogeneous variability among groups, data in


B. melitensis vaccine M5-90 is currently used for vaccination of sheep and goats in China. In the study, a periplasmic protein BP26 from M5-90 was characterized for its epitope reactivity with mouse monoclonal and sheep antibodies. A total of 29 monoclonal antibodies (mAbs) against recombinant BP26 (rBP26) were produced, which were tested for reactivity with a panel of BP26 peptides, three truncated rBP26 and native BP26 containing membrane protein extracts (NMP) of B. melitensis M5-90 in ELISA and Western-Blot. The linear, semi-conformational and conformational epitopes from native BP26 were identified. Two linear epitopes recognized by mAbs were revealed by 28 of 16mer overlapping peptides, which were accurately mapped as the core motif of amino acid residues 93DRDLQTGGI101 (position 93 to 101) or residues 104QPIYVYPD111, respectively. The reactivity of linear epitope peptides, rBP26 and NMP was tested with 137 sheep sera by ELISAs, of which the two linear epitopes had 65–70% reactivity and NMP 90% consistent with the results of a combination of two standard serological tests. The results were helpful for evaluating the reactivity of BP26 antigen in M5-90.More than 35,000 new cases of human brucellosis were reported in 2010 by the Chinese Center for Disease Control and Prevention. An attenuated  Brucellae are gram-negative intracellular bacterial pathogens of both humans and animals. More than 500,000 new cases of human brucellosis are annually reported, which may greatly be underestimated according to the World Health Organization B. melitensis infected sheep or goats B. melitensis vaccine M5-90 has been mostly used for vaccination of sheep and goats in China Brucella and has been identified as an important diagnostic antigen in brucellosis B. abortus, B. suis, B. ovis, B. canis, B. neotomae and B. melitensis, and was sensitive and specific for diagnosis of Brucella infection in animals by enzyme immunoassays (EIAs) B. melitensis M5-90 vaccine.Protein BP26 is located in the periplasma of B. melitensis M5-90 was produced by gene expression in E. coli and used as the immunogen for monoclonal antibody preparation. A panel of 16mer overlapping peptides, 9mer shortened peptides and mutated peptides derived from M5-90 BP26 were synthesized for mAb's recognition analysis and epitope mapping , 1 IgG2a (k), 8 IgG2b (k), 1 IgG3 (k) and 1 IgA (k) were identified (Recombinant periplasmic protein BP26 (rBP26) of  mapping Table 1.To classify mAb's epitope recognitions, 29 mAbs were tested for reactivity with a panel of 28 of 16mer overlapping peptides and native BP26 containing membrane protein extracts (NMP) of M5-90, respectively. Eleven mAbs reacted with three peptides P11, P12 or P13 in peptide-ELISA Table 2. and Of 11 mAbs reacted with peptides  and 2, mAbsTo localize the epitopes of BP26, three truncated proteins of rBP26-1 (aa 29–250), rBP26-2 (aa 48–131) and rBP26-3 (aa 129–250) were tested by ELISA for reacting with 16 semi-conformational and conformational mAbs. All of those mAbs reacted with the truncated rBP26-1 protein, 5 reacted with rBP26-2, 1 reacted with rBP26-3 and 1 reacted with both rBP26-2 and rBP26-3 Table 2.93DRDLQTGGI101 (position 93 to 101 within BP26). The bindings of mAbs 2A4 and 5A5 to P12 coated plates were competitively inhibited by five 9mer peptides P1202, P1203, P1204, P1205 and P1206 and P12 itself . Two linear epitope peptide conjugates (P11-KLH and P12′-KLH) were reactive to 65–70% sheep sera in peptide-ELISA consistent with the standard serological tests (P<0.001 and 0.002)   of M5-90 by ELISAs. Of 137 sheep sera, 59 (39 infected and 20 vaccinated) were positive using either Standard Tube Agglutination Test (SAT) or Rose Bengal Plate Test (RBPT). Sheep antibody response to epitope peptides, rBP26 or NMP were analyzed in line with the results obtained by combination of SAT and RBPT, and their ROC curves were plotted Figure 3Brucella melitensis vaccine strain Rev.1 is recognized worldwide as the best vaccine available against brucellosis in sheep and goats B. melitensis vaccine strain M5-90 is mostly used in China Brucella infected animals using the conventional serological tests. In addition, those attenuated vaccines still keep residual virulence for humans and may result in pregnant sheep abortion B. melitensis in sheep or B. ovis in rams The live attenuated data not shown), which also indicated that BP26 was responsible for the mixed Th cell response. The 62% (18/29) IgG1 of murine mAbs obtained in the present study, it suggested that BP26 might be associated with Th2 cytokine responses. Additionally the presence of BP26 enhanced antibody response to the chaperone protein, trigger factor (TF) Brucella infections Previous data demonstrated that BP26 provided a strong protection antigen within Rev. 1 inducing not only high IgG1 titers but also cellular response of IFN-γ, IL-4, IL-5 and IL-6 B. melitensis M5-90. Two linear epitopes were initially discovered using 28 overlapping peptides spanning the whole sequence of BP26. The core motifs of those two linear epitopes were accurately defined as amino acid sequences 93DRDLQTGGI101 and 104QPIYVYPD111 recognized by 11 of 29 mAbs, indicating that both were dominant epitopes within the BP26 antigen. Additionally the semi-conformational and conformational epitopes of native BP26 recognized by two groups of each eight mAbs were identified. All 16 mAbs were reactive to the truncated BP26-1 protein (aa 29–250) that did not contain 28 amino acid signal peptide. Four semi-conformational and one conformational epitopes were approximately localized between aa 48–131, and one semi-conformational epitope was identified between aa 129–250, which suggested that amino acids 48–131 within BP26 contained major semi-conformational and conformational epitopes. MAb 3C3 recognized both aa 48–131 and aa 129–250 of truncated rBP26 proteins, which indicated the conformational epitope was structurally formed by those two parts of BP26 protein. Other un-identified 9 mAbs might be involved in more complicated epitope recognitions, mostly conformational.Molecular epitopes of BP26 to both B cells and T cells have not been explored extensively. A region of amino acid residues position 55 to 152 within a truncated BP26 protein had been previously found reactive with two monoclonal antibodies, but the epitopes recognized had not been identified Brucella infected or M5-90 vaccinated sheep, which were 65% or 70% consistent with results of the combined SAT and RBPT. The reactivity of two linear epitopes of BP26 to mAbs could be inactivated by mutations of two critical amino acids, which suggested that molecular modification for BP26 epitopes of M5-90 could be genetically realized without altering the protein structure. Compared with rBP26, the NMP appeared more sensitive and specific in ELISA for detection of antibodies to Brucella from sheep, and had 90% agreement with the combination of SAT and RBPT.Recombinant BP26 was investigated for diagnosis of brucellosis in sheep and goats B. melitensis BP26 and finally mapped two novel linear epitopes reacting with antibodies in sheep. Taking together with T cell epitopes of BP26, the results were helpful for evaluation of BP26 antigen function within vaccine M5-90.In this study, we identified linear, semi-conformational and conformational B cell epitopes within All animal care and procedures were in accordance with national and institutional policies for animal health and well-being. Mouse experimentation and Sheep blood sample collection and field study were approved by Southern Medical University (SMU) Animal Care and Use Committee (permit numbers: NFYY-2008-043 and NFYY-2010-076). All mouse surgery was performed under anesthesia, and all efforts were made to minimize suffering of animals.B. melitensis vaccine strain M5-90 was cultured in the broth of Trypticase Soy medium (TSB) supplemented with 0.1% (w/v) yeast extract, which was used for extraction of DNA or membrane proteins. A total of 137 sheep serum samples  were collected from the farms in Brucella epidemic area of Shihezi, Xinjiang or provided by the Chinese Center for Disease Control and Prevention, Beijing, China. Sera were tested by Standard Tube Agglutination Test (SAT) and Rose Bengal Plate Test (RBPT), and positive samples were confirmed by either or both tests.Attenuated E. coli induced for 4 hours by 1 mM Isopropyl-1-thio-D-galactopyloranoside (IPTG) at 37°C. Soluble rBP26 was purified by Ni-NTA agarose  and analyzed by Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis (SDS-PAGE).By using pET-28a plasmid, recombinant entire BP26 (rBP26), truncated rBP26-1 (aa 29–250), truncated rBP26-2 (aa 48–131) and truncated rBP26-3 (aa 129–250) were expressed in A panel of 44 peptides was synthesized by a commercial company . Twenty-eight of 16mer peptides with 7mer overlap were designated as P01 to P28 spanning the 250 amino acids of the entire BP26 periplasmic protein. Twelve of 9mer peptides shortened from P11 and P12 were designated as P1101 to 06 or P1201 to 06. One of 16mer peptide was designated as P12′, as it's only the first amino acid overlapped with the C-terminal amino acid of P11. Two mutated P11 and P12′ were designated as MutP11 and MutP12′, respectively. One HCV NS3 peptide is used as a negative control. All peptides had above 90% purity.P11 and P12′ conjugates coupled with keyhole limpet hemocyanin (P11-KLH and P12′-KLH) were prepared by a commercial company .B. melitensis strain M5-90 by ReadyPrep Protein Extraction Kit (membrane II) . The cells were sonicated in Lysis buffer to fractionate and isolate membrane proteins by sodium carbonate extraction methods applied in the kit Native BP26 containing membrane proteins (NMP) were extracted from the attenuated Three 6-weeks old BALB/c female mice were housed at the Laboratory Animal Center, Southern Hospital, Southern Medical University, Guangzhou, China. Mice were inoculated with rBP26 antigens for three times at 2-week intervals. The first subcutaneous injection contained 100 µg of rBP26 in a final volume of 100 µl (100 µg/100 µl) of complete Freund adjuvant . The second subcutaneous injection was 50 µg/100 µl rBP26 antigen with incomplete Freund adjuvant . The final boost was intraperitoneally injected with 50 µg of rBP26 in 100 µl of PBS. The antibody titers to rBP26 were determined on day 3 after the final injection, and the best responding mice were sacrificed and the spleens were obtained. Pre- and post-immunization sera were collected and used as negative or positive controls for screening monoclonal antibodies (mAbs).The spleen cells of rBP26 immunized mice were fused with SP2/0 myeloma cells in a ratio of 5∶1 by polyethylene glycol 4000 . Mouse myeloma cell line SP2/0 was provided by the laboratory, which was originated from Division of Transfusion Medicine, Department of Haematology, University of Cambridge, UK Nunc Immuno microtiter plates were coated with 5 µg/ml of 16mer peptides in 0.1 M carbonate buffer (pH 9.6) and were incubated overnight at 4°C as described previously To map epitopes accurately, a panel of 9mer shorter peptides derived from P11 or P12 was synthesized for competitive binding to mAbs in peptide-ELISA. An HCV NS3 peptide was used as negative control.The rBP26 or native BP26 containing membrane protein extracts (NMP) of M5-90 were denatured and electrophoresed on SDS-PAGE, then transferred to Polyvinylidene Fluoride membranes , and finally blocked in TBST containing 5% non-fat dry milk for 2 h. Protein bound membranes were saturated with the diluted supernatants of mAbs cultures for 1 h at room temperature. The membranes were washed three times with TBST and incubated with 1∶10000 diluted goat anti-mouse IgG and IgM HRP-conjugate in TBST for 1 h. MAb bound membranes were washed and visualized by adding immunochemiluminescence reagent . An unrelated mAb to HCV rNS3 was used as negative control.PVDF membranes (8 mm×8 mm) were soaked in methanol for 1 min and then dried at room temperature. One microliter of native BP26 containing membrane protein extract (NMP) of M5-90 was dropped onto the center of each membrane and dried at room temperature. MAbs of cloned supernatant cultures were added to NMP on the membranes and the bound mAbs were detected by goat anti-mouse HRP-conjugate. The membranes were visualized by adding diaminobenzidine (DAB) substrate for color development. An unrelated mAb to HCV rNS3 was used as negative control.Brucella from sheep sera were calculated using receiver operating characteristic (ROC) curves. Optimal cutoff values were defined using the highest sum of sensitivity and specificity. For each optimal cutoff value, sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were calculated P value was obtained for comparing the differences between the areas under ROC curve (AUC) and reference (0.5) by the Kolmogorov-Smirnov Z analysis in SPSS software version 13.0.The overall sensitivity of Peptide-ELISA, rBP26-ELISA, NMP-ELISA for detection of antibodies to


For immunotherapy of human papillomavirus (HPV) -16-associated cervicalcancers the E7 protein is considered a prime candidate. However it is a poor inducer ofcytotoxic T-cell response, when being used as a singular antigen in protein vaccination.Hence, in this study we focused on the utilization of a vaccine delivery system for preventionor treatment of cervical cancer.In this experimental study, we designed and evaluated anovel fusion protein comprising HPV16 E7 antigen fused to Shiga toxin B-subunit(STxB) as both an antigen vector and an adjuvant. Then we designed two preventiveand therapeutic tumor models to investigate the prevention and inhibitionof TC-1 cell growth in female C57BL/6 mice, respectively. In each model, micewere immunized with the recombinant protein of E7-STxB or E7 without anyadjuvant.We demonstrated that prophylactic immunization of E7-STxB protected miceagainst TC-1 cells. Also in the therapeutic model, E7-STxB inhibited TC-1 tumor growthinlungs. The results were significant when compared with the immunization of E7 singularly.We concluded that immunization with the E7-STxB protein without anyadjuvant could generate anti-tumor effect in mice challenged with TC-1 cells.This researchverifies the clinical applications and the future prospects of developing HPV16E7 therapeutic vaccines fused to immunoadjuvants. Cervical cancer is the second most commoncause of cancer-related deaths in women worldwide.Human papillomavirus (HPV) is the mostprevalent, accounting for more than half of cervicalcancer cases. The HPV oncogenic proteins, E6and E7, are important in the induction and maintenanceof cellular transformation and co-expressedin most HPV-containing cervical cancers -4.VacciIn order to overcome many of the disadvantagesof peptide vaccines, using of full-sizeproteins as model antigens can be considered.As the antigen cannot be introduced into theMHC class I antigen presentation pathway, theadministration of soluble proteins alone mostlymay not induce cytotoxic T lymphocytes (CTL)responses. Hence their efficiency to elicit antibodyis not also often comparable, requiring theuse of adjuvant , 10. ThuOne the other hand, Shiga toxin from Shigelladysenteriae is composed of an A subunit, whichmediates toxicity, and a B subunit (StxB), a nontoxichomopentamericprotein responsible fortoxin binding and internalization into target cellsby interacting with the glycolipid globotriaosylceramide(Gb3 or CD77)  which isE. coli BL21 (DE3). This study focuses on theutilization of a vaccine delivery system for preventionor treatment of cervical cancer.As STxB can efficiently target peptides intothe MHC class I pathway and induced peptidespecificCTL in mice, it has the potential to actas both an antigen vector and an adjuvant inenhancing antigen-specific tumor immunity. Soit is tempting to propose the use of STxB fortumor cell delivery purposes . In thisshigella dysenteriae type I  was prepared. Following themethod we previously described (E. coli DH5αand E. coli BL21 (DE3) was used for cloning andexpression experiments. Plasmid pGEM-T Vectorand pET-28a (+) were used as cloning and expressionvectors respectively.Pfu and taq DNA polymerase , enzymes NdeI, SalI and NotI, IPTG ,Vector pET-28a (+) (Novagen USA), VectorpGEM-T , and stxB gene fromescribed , 18, and2.TC-1 cells expressing HPV16-E6 and HPV16-E7 proteins were purchased from a cell bank (PasteurInstitute of Iran). The tumor cell line, TC-1,was derived from primary lung epithelial cells ofC57BL/6 mice. The cells were immortalized withthe amphotropic retrovirus vector LXSN16E6E7and subsequently transformed with the pVEJBplasmid expressing the activated human c-Ha-rasoncogene .They werSix to seven week-old female C57BL/6 mice wereobtained from Animal Lab, Shiraz University ofMedical Sciences, Iran. Given free access to foodand water, the mice were housed for one week beforethe experiment, and were maintained at standardcondition. All experiments were done in accordancewith the Animal Care and Usage Protocol of ShirazUniversity of Medical Sciences, Iran.shigella dysenteriaetype I and synthetic codon optimizedHPV16 E7 were fused as E7-STxB chimericgene (E. coli DH5α. The E7-stxB fragment wassubcloned into indigested pET28a (+) as anexpression vector and to construct pET28a(+)-E7-stxB. Separately, the amplified E7 genewas subcloned into undigested pET28a (+) vectorto construct pET28a (+)-E7. Subsequently,the pET-28a (+)-E7-stxBwas confirmed by PCRand restriction enzyme digestion.As previously described , the ampricgene . The ampE. coli BL21 (DE3) after 4 hours of inductionby isopropyl-β-d-thiogalactoside(IPTG). Inclusion body and supernatant wereanalyzed by SDS-PAGE ona 12% gel. Theseproteins were expressed as inclusion bodies.The inclusion body that contained theE7 or E7-STxB protein was then sonicatedand washed twice with 100 ml solution  and centrifuged at 8000 rpm for 20minutes at 4˚C. The resultant pellet was resuspendedin 30 ml buffer solution , sonicatedand centrifuged at 16,000 rpm for 20minutes at 4˚C. The supernatant (solubleprotein) was dialyzed three times in 1l solution at 4˚C for 1 hour. Subsequently, theprotein solution was dialyzed extensivelywith PBS at 4˚C and the final supernatant(soluble protein solution) was obtained bycentrifuging at 16,000 rpm for 20 minutesat 4˚C. Removal of contaminating LPS wasconducted using ToxinEraserTM EndotoxinRemoval Resin (GenScript USA Inc.)Expression of E7 (13 kDa) and E7-STxB(28kDa) proteins followed the procedure wepreviously described . The E7 The identity and the purity of the recombinantproteins were determined by SDS-PAGE. Concentrationsof proteins were measured by the Bradfordassay. To confirm the characteristics of the recombinantproteins, human HPV16 E7 antibody  wereused to verify all the purified proteins by westernblot analysis.5 TC-1 cells in the right flank seven days afterthe second immunization. After 90 days, tumorgrowth was monitored and tumor incidence wasalso recorded.In the preventive tumor model, 12 femaleC57BL/6 mice from both the control and the experimentgroup were immunized subcutaneouslywith 1.5 nmol of either E7 or E7-STxB protein in100 μl PBS as experiment groups and also withonly 100 μl PBS as the control group. A secondequivalent dose of the same protein was givenby intraperitoneal injection two weeks later. Themice were again injected subcutaneously with1×105TC-1 cells, and then immunized subcutaneouslywith 1.5 nmol of protein on the next day. Themodel consisted of three groups with eight micein each. The groups were divided into two experimentgroups of E7 and E7-STxB, and one controlgroup of PBS. One week later, mice were immunizedby intraperitoneal injection with the samedose of protein. Starting 7-10 days later and every3-4 days there after, the area was observed andpalpated for the presence of a tumor nodule. Fourweeks later, six in each group were randomly byCO2 inhalation. The lungs were resected andinspected for tumor growth using anatomicaldissecting microscopy with ×2 magnification.The number of tumor nodules on lungs wascounted and the weight of the body and lungswere recorded. Subsequently, the average meanof all tumor sizes were calculated and reportedin millimeters. Tumor diameters were measuredin two orthogonal dimensions using electronicdigital calipers. Tumor volumes were calculatedfrom these measurements according to:(length×width2)/2. Tumor sizes (in millimeters)were reported as the average of all measured dimensions.In therapeutic tumor model, 24 mice were injectedintravenously into thetail vein with 1×10All data expressed as means ± SD are representativeof two to four different experiments. Allthe statistical analyses were analyzed by onewayANOVA followed by Tukey’s test. In allcases, the differences showing (p<0.05) wereconsidered as significant.The E7 and E7-STxB proteins were characterizedby SDS-PAGE electrophoresis andsubsequent western blot analysis .The pur5 TC-1 cells, all the mice of thecontrol group who were immunized with only100 μl PBS developed tumors, whereas no tumorwas developed in all the mice immunized withE7-STxB protein for over 90 days. The percentageof tumor incidence for the mice immunizedwith E7 protein was not significant in comparisonwith the control group.A preventive tumor model was used to test theanti-tumor immunity of E7-STxB protein. FemaleC57BL/6 mice were immunized twice withsoluble E7-STxB or E7 protein. After being challengedwith 1×10In the therapeutic tumor model, four weeks afterthe last injection, six mice of each group weresacrificed randomly and the number of tumor noduleson lungs was counted . In figu2b)/2. It was 624.3 ± 62.6 in the PBSgroup, 182.4 ± 41.3 in the E7 group and 125.3 ± 0.8 inthe E7-STxB group. There were significant decreasesin tumors sizes and improvements in the mice treatedwith E7 (p<0.001) and E7-STxB (p<0.001) comparedto PBS only. The mean tumor volumes and tumor sizeswere not significantly different between the E7andthe E7-STxB groups (p=0.09).The volume of tumors was computed using theformula: V=, but no significantdifference between PBS group and E7 groupwas observed. Also there was significant difference betweenE7-STxB and E7 group (p<0.05).The value of Lungs weight/Body weight ×100showed that there was significant difference betweenE7-STxB and the PBS or the E7 group (p<0.001). Thedifference between the E7 group and the PBS groupshowed a p value of 0.055. These data suggested thatE7-STxB immunization could elicit stronger inhibitionof TC-1 growth on lungs compared to E7 immunization.These findings raise the possibility that STxBmay be an efficient agent in generating cell mediatedimmune responses. Therefore, the STxB protein mayincrease the therapeutic potential of E7 protein-basedvaccine against cervical cancer in women.Immunization with E7-STxB protein without anyadjuvant could produce efficient anti-tumor effectin mice challenged with TC-1 cells, compared toE7 only-based immunization. This study verifiesthe clinical applications and the future prospects ofdeveloping HPV16 E7 therapeutic vaccines fusedto immunoadjuvants.


Pancreatic cancer is the one of the deadliest of all malignancies. The five year survival rate for patients with this disease is 3-5%. Thus, there is a compelling need for novel therapeutic strategies to improve the clinical outcome for patients with pancreatic cancer.  Several groups have demonstrated for other types of solid tumors that early passage human tumor xenograft models can be used to define some genetic and molecular characteristics of specific human tumors. Published studies also suggest that murine tumorgraft models (early passage xenografts derived from direct implantation of primary tumor specimens) may be useful in identifying compounds with efficacy against specific tumor types.  Because pancreatic cancer is a fatal disease and few well-characterized model systems are available for translational research, we developed and characterized a panel of pancreatic tumorgraft models for biological evaluation and therapeutic drug testing.  Of the 41 primary tumor specimens implanted subcutaneously into mice, 35 produced viable tumorgraft models.  We document the fidelity of histological and morphological characteristics and of KRAS mutation status among primary (F0), F1, and F2 tumors for the twenty models that have progressed to the F3 generation.  Importantly, our procedures produced a take rate of 85%, higher than any reported in the literature. Primary tumor specimens that failed to produce tumorgrafts were those that either contained <10% tumor cells or that were obtained from significantly smaller primary tumors. In view of the fidelity of characteristics of primary tumor specimens through at least the F2 generation in mice, we propose that these tumorgraft models represent a useful tool for identifying critical characteristics of pancreatic tumors and for evaluating potential therapies.   Pancreatic cancer (PC) continues to have one of the poorest prognoses among adult solid tumors. Despite comprising only 2% of all cases of cancer in adult patients, PC is the fourth leading cause of cancer related deaths in both men and women in the United States ,2. In 20in vitro for many generations. Established cell lines virtually all accumulate genotypic or phenotypic changes that confer a survival advantage in vitro, undergo clonal expansion, and display alterations in characteristics that depend on the interaction of the tumor cells with adjacent stromal tissue in vivo. Consequently, the degree to which these cell lines and the xenografts derived therefrom reflect characteristics of the primary tumors from which they were derived is controversial ) . ConsistWe randomly selected three independent primary (F0) tumors, and analyzed the sequence of KRAS codons 12 and 13 of these tumors. We also analyzed three F1 progeny derived from each F0 tumor (three “sets” of three F1 sister tumorgrafts) and four F2 progeny of one tumor from each set of F1 tumors (three “sets” of four F4 sister tumorgrafts) 2. The dHaving demonstrated fidelity in F0, F1, and F2 tissue specimens with respect to the mutation status of codon 12 of KRAS, we next addressed fidelity of tissue morphology of the same twenty models. As shown in Histology was evaluated using criteria for clinical staging and diagnosis of PDAC, a subtype that accounts for 90% of all cases of PC. PDAC displays characteristic lesions that have at least one of three specific histological traits: pancreatic intraepithelial neoplasia (PanIN), intraductal papillary mucinous neoplasm (IPMN), or mucinous cystic neoplasia (MCN) . Among ta-h); similar analyses of the remaining twelve tumors are depicted and described in Supporting Information (j-u). Summaries of the histological and morphological characteristics (by LNC) of each of the twenty tumorgraft models that have progressed to the F3 passage are based on: degree of differentiation, atypical gland formation, cytonuclear atypia, and nuclear abnormalities . These characteristics comprise the predominant indicators of PDAC malignancy in primary patient tumors (F0) and are used to describe the degree to which tumorgraft  progeny reflected characteristics of F0 tissue. We defined the degree of differentiation based on the presence or absence of glandular formation. Tumors containing gland tissue were designated as well-differentiated or moderately differentiated. Tissue closely resembling normal pancreatic (NP) tissue is routinely designated as well-differentiated. However, none of our tumor specimens  ratio and also more nuclear pleomorphism. F1 and F2 tissues show more apparent necrotic debris (red arrow) within the glandular lumina (black arrow) than the F0 tissue from which they were derived. F0 through F2 specimens all show characteristics typical of invasive PDAC with PanIN-3 features. Tissues from all three types of specimens  are moderately differentiated, with comparable amounts of peritumoral stroma. There is marked increase in cytologic atypia (particularly increased N:C ratio) in F1 compared to F0 tissues. There is also a noticeable increase in architectural complexity, with F1 tissue glands showing more crowding (red circle) and increased cribriforming (black circle). All three generations display similar cytology and glandular morphology. F1 and F2 tissues are most similar to each other in terms of cytology. Also, compared to F0, F1 and F2 tissues of UAB-PA3 display more homogeneous nuclear features (hyperchromasia and polarity) and more homogeneous N:C ratios. F0 through F2 display PDAC with PanIN-3 characteristics. Peritumoral (or periglandular) stroma and moderate differentiation is maintained from F0 through F2 tissues; however, F0 tissue has more apparent architectural and cytologic atypia than F1 tissue. The F1 tissue acquired clear cell features and increased nuclear polarity but still maintains the micropapillations (red arrow) seen in F0 tissue. As was observed for Tumor UAB-PA3, F1 and F2 tissues of Tumor UAB-PA4 are more similar in morphology to each other than to the F0 specimen, in that they have clear cell-like features and nuclear features. F2 tissue has decreased N:C ratio compared to F1 tissue and also has fewer micropapillations. While some unique features distinguish the specimens, F0 through F2 specimens are all classified as PDAC with PanIN-3 characteristics. F0 through F2 tissues are all moderately differentiated with intact but complex gland formation. All have a moderate amount of peritumoral stroma. Cytologically, all three generations show apical cytoplasmic clearing and conserved nuclear polarity. The N:C ratio is higher in F0 and F1 tissues than in F2 tissue; but all three types of specimens represent PDAC tumors with PanIN-2 features. F0 through F2 tissues display moderate differentiation. The F2 tissue has less apparent peritumoral stroma, compared to F0 and F1 tissues. There is overall maintenance of architectural integrity between F0 and F1 tissues. Both have complex gland formation with cribriforming. Compared to F0 and F1, F2 tissues has an increased N:C ratio and loss of nuclear polarity. Although overall degree of differentiation is maintained in terms of gland formation/architecture, the cytology of F2 tissue is higher grade than F0 and F1 tissues. F0 through F2 are classified as PDAC with PanIN-3 features. F1 and F2 tissues are similar in terms of cellularity and cytology with F2 tissue showing slightly more cytologic atypia (increased loss of nuclear polarity) (black arrow indicates cell with this characteristic). There is progressive loss of peritumoral stroma from F0 to F2 tissues, but all are considered moderately differentiated PDAC. F0 through F2 tissues show moderate differentiation with slightly greater peritumoral stroma demonstrated in F0 and F2 tissues than in F1 tissue. In terms of glandular morphology, F0 and F1 tissues are very similar. Both show a wide array of architectural atypia including cribriforming (black circle). The F1 specimen shows more glands/glandular crowding (red circle) and fewer small glands. F1 and F2 tissues show slightly increased cytologic atypia as compared to F0 tissue. F0 through F2 are classified as PDAC with PanIN-3 features. F0 through F2 tissues show moderate differentiation. There is progressively less peritumoral stroma in F1 tumors compared to F0 and in F2 tumors compared to F1. F0 and F1 tissues are very similar in architecture and cytology. Both show complex glandular architecture (black circle) and increased nuclear pleomorphism. F1 tissue has increased glandular crowding, and F0 tissue has a more fibrotic/desmoplastic background, compared to F1 and F2 tissue. F0 through F2 tissues show similar architectural and cytologic morphology. F2 tissue contained less mucin and necrotic debris than F0 and F1 tissue. F0, F1 and F2 specimens are all classified as PDAC with PanIN-3 features. The photomicrograph in the In summary, morphological and histological characteristics and degree of differentiation were well preserved among F0, F1 and F2 tissue for each of the twenty models evaluated. Minor variations among the generations of a given model were noted, but all tumorgraft models (F1 and F2 generations) shown in in vivo F3 generation preserved the histological features of its tumor of origin. We next compared the growth patterns of F1 and F2 generations of each of these twenty models. To date, we have sufficient material for histologic analysis from the F3 generation of only model UAB-PA2. Notably, as shown in , S3a and S3c. The F1 tumors demonstrated a range of growth rates that differed by ~12-fold, based on an approximated slope during exponential growth . F1 tumors reached ~1,500 mm3 11-38 weeks after implantation. The growth curves of F1 tumors produced by individual F0 tumor specimens following implantation into mice are shown in , S3b and S3d), generated from data obtained from 2-4 mice per tumor model, show that while F2 tumors grew at rates similar to each other, differences were observed in the interval between implantation and measurable tumor progression (lag phase). Compare, for example, the slopes of the lines representing UAB-PA2-F2 tumor growth compared to UAB-PA16-F2 after a ~7-week lag period. F2 tumors reached ~1,500 mm3 6-25 weeks after transplantation. F2 tumor growth curves (in vivo growth characteristics of F2 tumors derived from frozen F1 specimens (UAB-PA2-F2-FV tumors) compared to fresh F1 specimens with the UAB-PA2 model. As outlined in Methods, after dissection, F1 specimens were either transplanted directly into mice (to produce UAB-PA2-F2) or stored in liquid nitrogen for a minimum of one month and then transplanted into mice to produce UAB-PA2-F2-FV tumors. H&E-stained sections of F2 tumors produced by both fresh and frozen F1 specimens showed the same degree of moderate differentiation and similar morphology: complex gland formation and increased N:C ratio with relatively intact nuclear polarity  histological fidelity in primary (F0), F1 and F2 specimens, based on H&E staining; (ii) genetic fidelity through at least two passages in immunocompromised mice, based on KRAS codon 12 mutation status; (iii) the observation that specimens derived from primary tumors at least 2.5 cm in greatest dimension have a higher take rate than smaller tumors; (iv) a higher tumor take rate in immunocompromised mice than has been reported previously -40; and Although a potential association between tumorgraft development and time between surgical resection and implantation is somewhat controversial , our datin vitro prior to implantation into the mice, and no Matrigel or collagenase was used. These factors, in addition to rapid implantation, may also have affected take rate. Notably, our take rate of a minimum of 85% allows for evaluation of all stages of PDAC tumors, without introducing biases that might result if specimens from only particular tumor subclasses could be successfully propagated. Our models encompass all stages of PDAC  were conserved in the F1 and F2 tumorgrafts derived from each tumor.Electropherograms show mutations in codon 12 of the KRAS gene in six primary PDAC tumors (F0) and in the F1 and F2 tumorgrafts derived from each tumor. Results for additional tumors are shown in Figure 1(c) and Table 3 of the main manuscript. Data for a total of 20 F0 tumors and corresponding F1 and F2 tumorgrafts are reported in this study. (TIF)Click here for additional data file.Figure S2Histological evaluation of twelve F1 and F2 tumorgrafts demonstrates morphological fidelity of these tumors with the F0 tumors from which they were derived.Histologic analyses are provided for a total of 20 F0 tumors and their corresponding F1 and F2 tumorgrafts in this study. See also Figure 3. Tumor UAB-PA8 (j): Morphologic features are highly conserved from F0 through F2. The tumor remains moderately differentiated across generations with comparable gland formation in F0 and F1. F2 tumors display decreased, but recognizable, gland formation. Cytologically, F0 and F1 tumors display similar N:C ratios, with F2 showing decreased N:C ratio. There is a decreasing amount of peritumoral stroma across generations with F0 displaying the most and F2 the least. F0 through F2 are classified as PDAC. Tumor UAB-PA13 (k): Tumor features are well conserved across generations (F0-F2). Morphologically, F0 through F2 tissues show moderate differentiation. The N:C ratio is preserved in F0 and F1, but decreased in F2. Also, the nuclear features of F2 are more dysplastic than those of F0 or F1. Peritumoral stroma appears decreased from F0 to F2. Interestingly, F1 has increased peritumoral adipose tissue compared to F0 and F2. F0 through F2 are classified as PDAC with PanIN-3 features. Tumor UAB-PA20 (l): Tumor morphology appears conserved across generations in terms of maintained gland formation and similar amounts of peritumoral stroma. Cytologically, the tumor cell nuclei remain hyperchromatic and round across generations. However, the N:C ratio decreases significantly from F0 to F1, with a slight increase in F2. This may be secondary to reactive changes in F1 (mucin depletion). F1 displays features of PanIN-3/PDAC, while F1 and F2 display features of PDAC. Tumor UAB-PA22 (m): Morphologically, the tumor remains moderately differentiated across generations. There is a progressive decrease in peritumoral stroma from F0 through F2. Cytologically, F1 appears to have decreased N:C ratio compared to F0 and F2. However, F1 appears to have more reactive epithelial changes (mucin depletion) which may explain the higher cytologic atypia. F2 displays increased N:C ratio with significant loss of nuclear polarity as well as decreased, though intact, gland formation. All generations display features of PDAC. Tumor UAB-PA26 (n): The primary tumor appears moderately to poorly differentiated while F1 and F2 generations appear moderately differentiated. The amount of peritumoral stroma appears to vary from F0 to F2. Cytologically, all exhibit very increased N:C ratio compared to cytology expected for normal glandular mucosa with moderate nuclear atypia. All exhibit features of PDAC. Tumor UAB-PA28 (o): The tumor appears moderately differentiated across all generations. There appears to be a progressive decrease in peritumoral stroma from F0 to F2. Cytologically, the tumor seems to have maintained a high N:C ratio across all generations with conserved atypical nuclear features . All generations display features of PDAC. Tumor UAB-PA29 (p): Interestingly, this tumor appears to have progressed from moderately to poorly differentiated in F0 to moderately differentiated in F1 and F2. There appears to be a decreasing amount of peritumoral stroma from F0 to F2. Cytologically, F1 and F2 tumor cells appear to have a lower N:C ratio than F0. Both F1 and F2 tumors have better gland formation than F0, and F2 has improved gland formation over F1. All have features of PDAC, with F1 showing PDAC/PanIN-3 features. Tumor UAB-PA30 (q): The tumor has similar characteristics from F0 to F2 and remains moderately differentiated across generations. There appears to be more peritumoral stroma in F0 and F1 than in F2. Cytologically, the degree of nuclear atypia appears stable from F0 to F2, while the N:C ratio appears to decrease slightly from one generation to the next. All display characteristics of PDAC. Tumor UAB-PA33 (r): The tumor appears well differentiated in F0 and moderately differentiated in F1 and F2. There appears to be a significant decrease in peritumoral stroma from F0 to F2. Cytologically both nuclear and atypia and N:C ratio increases from F0 to F2. All display characteristics of PDAC. Tumor UAB-PA34 (s): F0 has more peritumoral stroma and smaller tumor load than either F1 and F2. Though there are fewer tumor cells/glands, the F0 tumor displays characteristics of moderate differentiation, similar to F1 and F2 tumors. There is a decrease in peritumoral stroma from F1 to F2. Cytologically, F2 tumors appear to have increased nuclear atypia compared to F0 and F1. The N:C ratio appears variable across generations, with F1 having lower N:C ratio than F0 and F2. All display characteristics of PDAC. Tumor UAB-PA36 (t): The F0 generation tumor is moderately to poorly differentiated, while F1 and F2 tumors are moderately differentiated. F1 and F2 tumors have more peritumoral stroma than F0, although there seems to be a decrease between F1 and F2. The cytology of F0 is more high grade than F1 and F2 in terms of nuclear atypia and N:C ratio. All display characteristics of PDAC, with F1 displaying features of PDAC/PanIN-3. Tumor UAB-PA37 (u): The F0 generation appears moderately to poorly differentiated compared to F1 and F2, both of which appear moderately differentiated. There is slightly more peritumoral stroma in F1 and F2 than in F0. In terms of cytology, both F1 and F2 have lower grade nuclear atypia and N:C ratio than F0. All display characteristics of PDAC. (TIF)Click here for additional data file.Figure S3(a). Growth curves for six first generation (F1) tumorgrafts. See (b). Growth curves for six second generation (F2) tumorgrafts. See (c). Growth curves for six first generation (F1) tumorgrafts. See (d). Growth curves for six second generation (F2) tumorgrafts. See (TIF)Click here for additional data file.