Of note, a significant increase in the functional avidity of HIV-specific CD8 T cells occurred in early-treated PHI patients experiencing a virus rebound after spontaneous treatment interruption. This increase in functional avidity was associated with the accumulation of PD-1/2B4/CD160 positive cells, loss of polyfunctionality and increased TCR renewal. The increased TCR renewal may provide the mechanistic basis for the generation of high-avidity HIV-specific CD8 T cells. These results provide insights on the relationships between functional avidity, viremia, T-cell exhaustion and TCR renewal of antiviral CD8 T cell responses.The factors determining the functional avidity and its relationship with the broad heterogeneity of antiviral T cell responses remain partially understood. We investigated HIV-specific CD8 T cell responses in 85 patients with primary HIV infection (PHI) or chronic (progressive and non-progressive) infection. The functional avidity of HIV-specific CD8 T cells was not different between patients with progressive and non-progressive chronic infection. However, it was significantly lower in PHI patients at the time of diagnosis of acute infection and after control of virus replication following one year of successful antiretroviral therapy. High-avidity HIV-specific CD8 T cells expressed lower levels of CD27 and CD28 and were enriched in cells with an exhausted phenotype,  i.e. how prone T cells are to respond when they encounter low doses of antigens. In patients with chronic and established HIV infection, we observed that the sensitivity of HIV-specific CD8 T cells was not different between patients with progressive or non-progressive disease. In contrast, the sensitivity of HIV-specific CD8 T cells was significantly lower in patients with early and recent HIV infection. Furthermore, CD8 T cells of high avidity were preferentially associated with a state of functional impairment known as exhaustion. Of interest, some patients treated with antiretroviral therapy during acute infection spontaneously interrupted their treatment and experienced a rebound of virus. In these patients, the avidity of HIV-specific CD8 T cells increased and this increase was associated to stronger cell exhaustion and greater renewal of the population of antiviral CD8 T cells, thus potentially providing the mechanistic basis for the generation of high-avidity CD8 T cells. Overall, our data suggest that rapid perturbation in viremia levels drove increases in the functional avidity of HIV-specific CD8 T cells.CD8 T cells directed against virus are complex and functionally heterogeneous. One relevant component of CD8 T cells is their functional avidity which reflects their sensitivity to cognate antigens,  CD8 T cells play a critical role in antiviral immunity and a large number of studies in both human and murine models indicate that virus-specific CD8 T cells are directly involved in the control of virus replication and disease progression Functional avidity of T cells, also defined as antigen (Ag) sensitivity, is thought to be a critical component of antiviral immunity. Functional avidity reflects the ability of T cells to respond to a low Ag dose and is determined by the threshold of Ag responsiveness. There is a general consensus that high functional avidity CD8 T-cell responses are of higher efficacy against cancers However, the factors determining the level of T-cell functional avidity and its relationship with the phenotypic and functional heterogeneity of T-cell responses are only partially understood i.e. oligoclonal) Functional avidity is based on the ability of T cells to respond following stimulation with a cognate Ag and it is well established that responding CD8 T cells are clonally heterogeneous (HIV-specific CD8 T cells in primary HIV infection (PHI) are temporally associated with the initial control of viremia In the present study, we have performed a comprehensive cross-sectional characterization of HIV-specific CD8 T-cell responses in patients with PHI or chronic (progressive and non-progressive) HIV infection in both steady-state conditions as well as following virus rebound. The primary observations of the present study indicate that a) the functional avidity of HIV-specific CD8 T cells is not different between patients with progressive and non-progressive chronic infection, b) the functional avidity of HIV-specific CD8 T cells is significantly lower in PHI patients as compared to patients with chronic infections, c) increased functional avidity is associated with T-cell exhaustion and lack of expression of markers of co-stimulation, and d) great increase in functional avidity is observed after virus rebound following spontaneous interruption of antiretroviral therapy and is associated with increased TCR renewal.i.e. prior to seroconversion and incomplete western blot; hereafter referred to as PHI), 39 patients with progressive chronic infection  and 9 patients with non-progressive chronic infection (i.e. LTNP)  was determined  37 patients with very early stage of acute infection (e. LTNP) . We firse. LTNP) . As desctermined .P<0.0001; i.e. epitopes recognized by patients from distinct cohorts) were analyzed (KRWIILGLNK (i.e. KK10) epitope has been previously reported as a protective epitope The results of this analysis indicated that the functional avidity of HIV-specific CD8 T cells was lower in PHI-B as compared to CP-B or LTNP patients  in PHI-T1Y as compared to CP-T1Y patients  higher in HIV-specific CD8 T cells from PHI-B patients as compared to CP-B or LTNP  or LTNP (P = 0.003) patients  or PD-1−2B4+CD160+  as compared to PHI-B .We then performed a phenotypic characterization of HIV-specific CD8 T-cell responses and monitored CD27 and CD28 expression to assess co-stimulation and PD-1, 2B4 and CD160 expression to assess T-cell activation and exhaustion. For these analyses, only HIV-specific CD8 T cells detectable using cognate peptide-MHC class I multimers  were takpatients  and 3B. to PHI-B  and 3C. P<0.006;  and exprThese data suggest that HIV-specific CD8 T cells from patients with acute and chronic infection are functionally and phenotypically distinct.+CD28+ cells . Interestingly, this regression analysis indicated that about 28% of the functional avidity of HIV-specific CD8 T cells was explained by a combination of the proportion of CD27+CD28+ and of PD-1+2B4+CD160+ CD8 T cells . We did not, in contrast, observe any significant correlation between the functional avidity of HIV-specific CD8 T cells and their functional profile.Furthermore, we performed correlations and rank correlation's matrix to explore the partial associations of variables and to assess the dependency and potential hidden effect of confounding variables in pairs associations. These analyses indicated that the proportions of CD27Overall, these observations indicate that high-avidity HIV-specific CD8 T-cell responses are preferentially composed of cells lacking the expression of co-stimulatory molecules but co-expressing high levels of co-inhibitory receptors. However, the functional avidity can only be partially predicted from the expression of co-stimulatory or co-inhibitory molecules.i.e. viremia <50 HIV RNA copies/ml of plasma (in patients successfully treated by ART) and b) in conditions of rapid and major changes in viremia occurring in patients experiencing virus rebound following spontaneous treatment interruption (TI)  in conditions of viremia below the limit of detection, ion (TI) . In partion (TI) . Nine ouP<0.0001; i.e. further shifted toward IL-2 production) after 5 years of ART as compared to 1 year of ART (P<0.01) decrease in polyfunctionality of HIV-specific CD8 T-cell responses after TI and the significant (P = 0.03) increase in polyfunctionality of HIV-specific CD8 T-cell responses from patients who remained on ART  .Then we determined PD-1 (as well as 2B4 and CD160) expression in a subset of PHI-ATI and PHI-T5Y patients with known HIV-specific CD8 T-cell responses using cognate peptide-MHC class I multimers . As shownt #1017 . CumulatThese data indicate that major changes of viremia levels in TI patients caused reduction of polyfunctional HIV-specific CD8 T cells and were associated with an increased level of exhaustion.We then analyzed the effects of virus rebound following TI on the functional avidity of HIV-specific CD8 T cells.GPGHKARVL- and A*0301-RLRPGGKKK-specific CD8 T-cell responses significantly increased after TI (ATI) as compared to pre-TI (PTI)  differences in functional avidity were observed during the same period when a similar analysis was performed in HIV-specific CD8 T-cell responses from patients who did not interrupt ART  between the fold increase in functional avidity of HIV-specific CD8 T cells and the fold increase in the proportion of PD-1+2B4+CD160+ HIV-specific CD8 T cells  . Furtherrved ATI . Cumulat T cells .Of interest, we performed a comprehensive statistical modeling of the changes in functional avidity of HIV-specific CD8 T cells and used mixed-effect linear models i.e. neither in patients on ART  interaction between functional avidity and virus rebound. An immediate increase of functional avidity of HIV-specific CD8 T cells of about 1 order of magnitude (0.95 units) occurred directly after TI  of functional avidity were included. The statistical model revealed that the interaction between avidity and time was not significant in steady-state conditions, These observations indicated that the functional avidity of HIV-specific CD8 T cells is stable overtime in steady-state conditions regardless of viremia levels, but does increase after rapid increase in viremia levels associated with virus rebound.We recently demonstrated that the global CD8 TCR repertoire of virus-specific CD8 T cells was diverse and subjected to continuous renewal GPGHKARVL-specific CD8 T cells at week (W) 18, W96 and W125 (i.e. on ART) and a renewal of 82% between W96 and W125 (i.e. after TI). Cumulative analyses confirmed a significantly (P = 0.008) higher CDR3 renewal of HIV-specific CD8 T cells after virus rebound than in steady-state condition, i.e. during treatment (P = 0.036) with the extent of increase in functional avidity of HIV-specific CD8 T cells  the affinity of the TCR for the peptide-MHC (pMHC) molecule, i.e. the strength of the interaction between the TCR and pMHC T-cell functional avidity reflects the ability of T cells to respond to various concentrations of Ag and may be assessed i.e. epitopes not necessarily corresponding to the autologous virus sequences. Since pMHC/TCR affinity is one of the parameters potentially influencing the functional avidity In the present study, we comprehensively investigated the functional avidity of HIV-specific CD8 T-cell responses in a cross-sectional study of different cohorts of HIV-infected patients. The evaluation of the functional avidity of HIV-specific CD8 T cells was based on optimal epitopes, HIV-specific CD8 T cells generated during acute infection were of lower functional avidity as compared to those from patients with chronic progressive or non-progressive infection. These differences were not biased by distinct peptide-HLA associations and remained significant after ART-induced control of virus replication. In addition, a preferential deletion of HIV-specific CD8 T-cell responses of higher avidity was not observed, as previously described in a cohort of early HIV infection i.e. HLA-B*27-, B*57- or B*5801-restricted T-cell responses, were consistently found in the lower range of functional avidity (data not shown). These observations do not support previous studies showing a relationship between higher avidity T-cell responses and better virus control Furthermore, consistently with previous studies Taken together, these observations suggest an association between higher avidity T-cell responses and chronic HIV infection.Of note, we also assessed the relationship between T-cell functional avidity and the expression of markers of exhaustion. It is important to underscore that HIV-specific CD8 T cells which had higher avidity in chronic infection expressed also higher levels of exhaustion markers. Therefore, these results further indicate that higher functional avidity does not correlate with better virus control but rather with the status of cells activation/exhaustion.i.e. CD27 and CD28) and exhaustion markers  on the levels of functional avidity in HIV-specific CD8 T cells using a regression model. The regression model indicated that the expression of the above markers only partially accounts for the establishment of the functional avidity of HIV-specific CD8 T cells, thus indicating that additional factors may contribute to determine the levels of functional avidity.We also determined the impact of the expression of costimulation  the formation of clusters comprising several TCRs and other molecules able to reinforce the immunological synapses Interestingly, we showed that TCR renewal was also significantly higher following virus rebound and associated with an increase in T-cell functional avidity. Therefore, our data indicate a potential role of TCR renewal in the modulation of the levels of functional avidity. However, our results do not distinguish between the recruitment of new clones, selective expansion of pre-existing high-avidity clones or depletion of low-avidity clones since the study was performed at the population level.Taken together, these results support the following model.HIV-specific CD8 T cells of lower functional avidity are generated during primary immune responses; then, persistence of detectable viremia drives an increase in functional avidity as supported by the major increase in functional avidity associated with the sudden increment in viremia levels; the increase in viremia levels is also associated with massive TCR renewal which, in turn, causes the generation/selection of T-cell clones with higher functional avidity.These results provide insights on the relationships between functional avidity, viremia, T-cell exhaustion and TCR renewal of antiviral CD8 T-cell responses.These studies were approved by the Institutional Review Board of the Centre Hospitalier Universitaire Vaudois and all subjects gave written informed consent.i.e. LTNP, as defined by documented HIV infection since >10 years, stable CD4 T-cell counts >500 cells/µl, and plasma viremia <500 HIV RNA copies/ml were also included. Clinical and virological characteristics of the different cohorts are detailed in Seventy-six patients with primary (PHI) or progressive chronic (CP) HIV infection were enrolled. Diagnosis of PHI included the presence of an acute clinical syndrome, a negative HIV antibody test, a positive test for HIV RNA in plasma, and ≤3 positive bands in a Western blot. All PHI patients started ART alone or ART+CsA within 72 h as described Epitope mapping was performed using a panel of 192 HPLC-purified (>80% purity) previously-described optimal epitopes The following antibodies were used in different combinations. CD8-PB, CD8-APCH7, CD3-APCH7, CD45RA-PECy5, PD-1-FITC, IFN-γ-APC, TNF-α-PECy7, and IL-2-PE were purchased from Becton Dickinson , CD4-ECD, CD3-ECD, CD28-ECD, CD27-APC from Beckman Coulter , Perforin-FITC (clone B-D48) from Diaclone , CCR7-FITC from R&D Systems , 2B4-PECy5.5 and CD160-APC from Biolegend .as per the manufacturer's instructions (BD Biosciences). In brief, 2×105 cryo-preserved blood mononuclear cells were stimulated with 1 µg of single peptide or peptide pools in triplicate conditions as described 6 cells from triplicate assays. Only cell samples with >80% viability after thawing were analyzed, and only assays with <50 spot forming unit (SFU)/106 cells for the negative control and >500 SFU/106 cells after SEB stimulation were considered valid. An ELISpot result was defined as positive if the number of SFUs was ≥55 SFU/106 cells and ≥4-fold the negative control.ELISPOT assays were performed 6) were stained for dead cells  and then stained with appropriately tittered peptide-MHC class I tetramer complexes at 4°C for 30′ in Ca2+-free media as described http://exon.niaid.nih.gov/spice/6 in the flow cytometry experiments.Cryo-preserved blood mononuclear cells (1–2×106) were stimulated for 6 h or overnight in 1 ml of complete media (RPMI (Invitrogen), 10% fetal bovine serum , 100 µg/ml penicillin, 100 units/ml streptomycin (BioConcept)) in the presence of Golgiplug , anti-CD28  and 1 µg/ml of peptide as described http://exon.niaid.nih.gov/spice/6. With regard to the criteria of positivity of the ICS, the background in the unstimulated controls never exceeded 0.03%. An ICS to be considered positive had to have >0.03% of cytokine-positive cells after subtraction of the background  and to be >5 fold higher that the background.Cryo-preserved blood mononuclear cells (1–2×10+ CCR7+ naïve and Ag-specific (multimer+) CD8 T cells were directly sorted  in RLT lysis buffer  containing 20 ng RNA carrier  and RNA extracted (Qiagen). Then, cDNA preparation and amplification were performed by using the SuperSMART PCR cDNA Synthesis Kit according to the manufacturer's instructions . Amplified cDNA was subjected to TRBV–TCR-b–chain C region (TRBC) PCR reactions as described The analysis of the CDR3 diversity and renewal was performed as described in vitro assays as described 50) was determined.Peptide stimulations were performed as described above. Functional avidity of T-cell responses was assessed by performing limiting peptide dilutions (ranging from 2 µg/ml to 1 pg/ml) in Four-digit HLA class I genotyping was performed by direct sequencing methods as described 10-transformed data using non-parametric tests. Associations among variables were performed by Spearman test. Rank correlations matrix and linear regression analysis were performed after log10 transformation of variables using R software. Bonferroni corrections for multiple analyses were applied. Regarding SPICE analyses of the flow-cytometry data, comparison of distributions was performed using a Student's t-test and a partial permutation test as described Mann-Whitney and Wilcoxon-matched paired tests were performed using GraphPad Prism version 6.00 . Analyses of the functional avidity of CD8 T-cell responses were performed on logFigure S1Magnitude and qualitative profiles of HIV-specific CD8 T cells during acute and chronic HIV infections.A. Associations between the magnitude and the functional avidity of HIV-specific CD8 T-cell responses from patients with either acute infection (PHI-B), untreated chronic progressive (CP-B) or non-progressive (LTNP) HIV infection. B. Representative flow cytometry examples of the functional profile of HIV-specific CD8 T cells from patients with acute (PHI-B-07 B*4402-AENLWVTVYY) and chronic progressive  and non-progressive  HIV infection on the basis of the expression of IFN-γ, TNF-α, IL-2 and perforin. C. Representative flow cytometry profiles show the identification of HIV-specific CD8 T cells using relevant peptide-MHC class I multimer complexes (upper panels) from patients with acute  or chronic progressive  and non-progressive (LTNP-2081 A*0201-SLYNTVATL) and CD27 and CD28 expression on HIV-specific CD8 T cells (bottom panels). D. Representative flow cytometry profiles show the identification of HIV-specific CD8 T cells using relevant peptide-MHC class I multimer complexes (left panels) from patients with acute  or chronic progressive  and non-progressive (LTNP-2081 A*0201-SLYNTVATL) HIV infection and 2B4, PD-1 and CD160 expression on HIV-specific CD8 T cells (right panels).(PPTX)Click here for additional data file.Figure S2Effect of the combination of Cyclosporin A with ART and T-cell responses. Analysis of the magnitude and of the functional avidity of HIV-specific CD8 T-cell responses in PHI patients treated for one year with either ART alone or ART + Cycosporin A (CsA).(PPTX)Click here for additional data file.Figure S3TRBV usage and CDR3 size pattern. Example of TRBV usage and CDR3 size pattern analysis of B*0702-GPGHKARVL-specific CD8 T cells in patient #1023 at week 18, 96 and 125. A. Profile of BV families obtained by PCR. B. CDR3 size profile obtained by genemapper analysis of BV families. TRB nomenclature is according to Wei et al. Immunogenetics (1994). The model used to define CDR3 diversity and renewal is based on Miconnet et al. J. Immunol. (2011).(PPTX)Click here for additional data file.Table S1Clinical and virological description of the distinct cohorts of HIV-infected patients.(PPTX)Click here for additional data file.Table S2HIV-derived peptide-MHC class I multimer complexes used in this study.(PPTX)Click here for additional data file.


Objective. Programmed cell death 1 (PD-1) induces negative signals to T cells during interaction with its ligands and is therefore a candidate gene in the development of autoimmune diseases such as rheumatoid arthritis (RA). Herein, we investigate the association of PDCD-1 polymorphisms with the risk of RA among Chinese patients and healthy controls. Methods. Using the PCR-direct sequencing analysis, 4 PDCD-1 SNPs  were genotyped in 320 RA patients and 309 matched healthy controls. Expression of PD-1 was determined in peripheral blood lymphocytes by flow cytometry and quantitative real-time reverse transcriptase polymerase chain reaction. Results. We observed that the GG genotype of rs36084323 was associated with a increased risk for developing RA . Patients carrying G/G genotype displayed an increased mRNA level of PD-1 (P = 0.04) compared with A/A genotype and healthy controls. Meanwhile, patients homozygous for rs36084323 had induced basal PD-1 expression on activated CD4+ T cells. Conclusion. The PDCD-1 polymorphism rs36084323 was significantly associated with RA risk in Han Chinese population. This SNP, which effectively influenced the expression of PD-1, may be a biomarker of early diagnosis of RA and a suitable indicator of utilizing PD-1 inhibitor for treatment of RA. Rheumatoid arthritis (RA) is a common chronic inflammatory autoimmune disease characterized by significant disability and early mortality, which affects ~1% of adult population worldwide . It is g PDCD-1 gene polymorphism and susceptibility to autoimmune diseases, such as systemic lupus erythematosus (SLE) [Programmed death-1  is a novel costimulatory member of B7/CD28 family, which is inducibly expressed on CD4+ T cells, CD8+ T cells, natural killer T cells, B cells, and activated monocytes . PD-1 reus (SLE) , 11, rheus (SLE) , 13, mulus (SLE) , and diaus (SLE) , 16. PDCD-1 functional SNPs, PDCD-1 −606A/G  in promoter, PDCD-1 +7146A/G  in intron 4, and PDCD-1 +7625G/A  and PDCD-1 +7786G/C  in exon 5 as prospective candidates for individuals susceptibility to RA in the Han Chinese population. Therefore, in the present study, we sought to determine the association between functional genetic variations in the PDCD-1 gene and RA risk in a Chinese population in mainland.There is mounting evidence that PD-1 is linked to human autoimmunity. In view of the pivotal role of PD-1/PD-L pathway in autoimmn immunology, it is worth considering ofThis study was approved by the Ethics Committee of Soochow University and all subjects gave informed consent for the genetic analyses. A total of 320 unrelated Chinese RA patients were recruited from the Outpatient Departments of Rheumatology in the First and the Third Affiliated Hospital of Soochow University. They were composed of 72 men and 248 women, whose mean age was 55.3 years SD = ±12.6). Individual patients with RA were diagnosed according to the diagnosis criteria established by the American College of Rheumatology and the disease severity of individual patients was evaluated using the disease activity score 28 (DAS28) [2.6. Indi PDCD-1 sequence (GeneBank accession number AF363458). The PDCD-1 −606A/G (rs36084323) in promoter, PDCD-1 +7146A/G (rs11568821) in intron 4, and PDCD-1 +7625G/A (rs2227982) and PDCD-1 +7786G/C (rs2227981) in exon 5 polymorphisms were determined by direct sequencing in an Applied Biosystems sequencer .Peripheral venous blood samples of 2 mL were drawn from each individual by standard venepuncture and stored at −20°C. Genomic DNA was isolated from peripheral blood leucocytes by standard procedures. The reference sequence is the humanμg) was used for cDNA synthesis with oligodT primers  and superscript II reverse transcriptase . PCR was performed using PCR Master  with the following primers: for PD-1 mRNA, 5′-CTCAGGGTGACAGAGAGAAG-3′ (forward) and 5′-GACACCAACCACCAGGGTTT-3′ (reverse) and for GAPDH, 5′-GTGAAGGTCGGAGTCAACG-3′ (forward) and 5′-TGAGGTCAATGAAGGGG-TC-3′ (reverse). Fold changes were normalised based on GAPDH expression, and each assay was conducted in a 96-well ABI 7900HT real-time PCR system . This procedure was performed in triplicate.Total RNA from PBMC of 30 RA patients and 24 healthy controls were extracted using Trizol reagent  according to the manufacturer's protocol. Total RNA (1 6) were incubated in wash buffer  with appropriate amounts of mAb on ice for 30 minutes. Cells were washed and were immediately analyzed on an EPICS XL-MCL flow cytometer (Beckman Coulter).All antibodies were mouse anti-human monoclonal antibodies (mAb). PE-Cy5-conjugated anti-CD4 (OKT4) were from Beckman Coulter . PE-conjugated anti-PD-1 (MIH4), fluorescein isothiocyanate (FITC)-conjugated anti-CD25 (M-A251), anti-CD69 (FN50), and anti-HLA-DR (G46-6) were from BD PharMingen . PE- or PE-Cy5-conjugated IgG1 (679.1Mc7) (Beckman Coulter), PE-conjugated IgG2a , and FITC-conjugated IgG1 (P3) (eBioscience) were used as IgG isotype controls. PBMCs  [D′ and r2 were calculated to evaluate the magnitude of LD. Haplotype frequencies were estimated using the EM algorithm coded into the haplo.stats package (http://mayoresearch.mayo.edu/mayo/research/biostat/schaid.cfm). The association analysis of haplotypes was similar to that of genotypes with logistic regression, and results were shown as OR and 95% CI. The most frequent haplotype was automatically selected as the reference category and rare haplotypes were pooled together in a group. The log-additive inheritance model was assumed by default. The significance level of all these tests was 0.05. The nonparametric Mann-Whitney U test was used for comparisons among groups with small or unequal sample sizes. Results were expressed as the mean ± SEM, and 2-tailed P values less than 0.05 were considered significant.The deviation from Hardy-Weinberg equilibrium (HWE) was examined in controls by the  NPstats) . Based oP values for Hardy-Weinberg proportions of the SNPs are shown in P < 0.05). When logistic regression was used for association analysis after modeling the SNPs' effects as additive, dominant, or recessive, rs36084323 showed significant difference in codominant , recessive , and log-additive  models  models . The log PDCD-1  was calculated for the cases and controls in the Han Chinese. We found strong LD (D′ > 0.75) between some pairs of markers in the PDCD-1 gene including rs36084323/rs2227982 (D′ = 0.7729) and rs2227982/rs2227981 (D′ = 0.7841).Because rs11568821 was nonpolymorphic in our population, it was excluded from the haplotype construction and LD analysis. Pairwise LD between the SNPs of theP = 0.00015); ACT and ACC haplotypes were less frequently observed in cases than in controls , indicating that these two haplotypes act as protective phenotype in RA. The association analysis of the haplotypes was adjusted by sex too.The association analysis of the haplotypes with RA was similar to that of genotypes by logistic regression . We foun PDCD-1 polymorphisms were associated with RA, and the GG genotype and G allele of rs36084323 that associated with increased PD-1 mRNA expression might be involved in RA development in Han Chinese population.To evaluate the association between rs36084323 polymorphism and RA, we examined whether or not the rs36084323 polymorphism was associated with an altered PD-1 mRNA expression. Significant difference was observed in the relative PD-1 mRNA expression level between patients with GG and AA genotypes Figures . We concP = 0.0014), CD4+CD69+T cells , and CD4+HLA-DR +T cells  compared with healthy controls  in promoter, PDCD-1 +7146A/G (rs11568821) in intron 4, and PDCD-1 +7625G/A (rs2227982) and PDCD-1 +7786G/C (rs2227981) in exon 5) of PDCD-1 gene and identified the association between the four polymorphisms and the risk of RA in Southeastern Chinese population.Greater than 30 single nucleotide polymorphisms (SNPs) have currently been identified in the humanulations . Underst PDCD-1 intron 4 was described as an enhancer-like structure containing binding sites for several transcription factors [P7146 G/A (PD-1.3) SNP, a regulatory SNP located in intron 4, showed to be involved in susceptibility to RA in Swedish, European American, and Mexican families and in sporadic cases. However, we found that this nucleotide is not polymorphic in our Chinese people, and this was also verified among other Chinese populations [A region of factors . P7146 Gulations , 23, 24. PDCD-1 gene, rs36084323 may also affect the transcription and activation of PDCD-1 gene, thereby influencing the development of RA. These promoter polymorphisms have been considered risk factors for RA. One group observed that the frequency of SNP PD-1.1 (rs36084323) A allele is a risk allele in Italian RA patients [As it is known, mutations in the promoter region may influence the engagement between sequence motifs and transcription factors and further disrupt the activation of gene and the initiation of transcription . Accordipatients . Anotherpatients . Previoupatients . We obsepatients . Hence, PD-1 is thought to be important for the “fine tuning” of lymphocyte activation at the level of synovial tissue, considering the wide pattern of expression of one of its ligands, PDL-1, in activated endothelial and epithelial cells , 28–30. In conclusion, the present study provides strong evidence that rs36084323 functional polymorphisms may contribute to the risk of RA. This SNP, which effectively influenced the expression of PD-1, may be a biomarker of early diagnosis of RA and a suitable indicator of utilizing PD-1 inhibitor for treatment of RA. However, our results were obtained from a sample-sized sample, and therefore this is a preliminary conclusion. Validation by a larger prospective study from a more diverse ethnic population is needed to confirm these findings.


L locus resistance (R) proteins are nucleotide binding (NB-ARC) leucine-rich repeat (LRR) proteins from flax (Linum usitatissimum) that provide race-specific resistance to the causal agent of flax rust disease, Melampsora lini. L5 and L6 are two alleles of the L locus that directly recognize variants of the fungal effector AvrL567. In this study, we have investigated the molecular details of this recognition by site-directed mutagenesis of AvrL567 and construction of chimeric L proteins. Single, double and triple mutations of polymorphic residues in a variety of AvrL567 variants showed additive effects on recognition strength, suggesting that multiple contact points are involved in recognition. Domain-swap experiments between L5 and L6 show that specificity differences are determined by their corresponding LRR regions. Most positively selected amino acid sites occur in the N- and C-terminal LRR units, and polymorphisms in the first seven and last four LRR units contribute to recognition specificity of L5 and L6 respectively. This further confirms that multiple, additive contact points occur between AvrL567 variants and either L5 or L6. However, we also observed that recognition of AvrL567 is affected by co-operative polymorphisms between both adjacent and distant domains of the R protein, including the TIR, ARC and LRR domains, implying that these residues are involved in intramolecular interactions to optimize detection of the pathogen and defense signal activation. We suggest a model where Avr ligand interaction directly competes with intramolecular interactions to cause activation of the R protein. Melampsora lini is the causal agent of flax rust disease. Flax produces immune-receptor proteins that recognize fungal effector proteins, and subsequently signal the activation of plant defense responses. Here we report the molecular details of interactions between L-locus immune-receptors and AvrL567-locus effectors, as well as the engineering of an enhanced flax immune-receptor. In order to investigate the role of AvrL567 amino acid residues hypothesized to mediate interactions with L-locus immune receptors, we generated a series of site-direct mutations in AvrL567 proteins. Conversely, to investigate the role of regions hypothesized to mediate interactions with AvrL567 effectors, we generated a series of chimeric L-locus immune-receptors that contain swaps between, and within protein domains. Interactions between modified immune-receptors and effector proteins were evaluated using the yeast-two-hybrid system and transient expression in planta. Our results revealed that interactions between L-locus immune receptors and AvrL567-locus effector proteins involve multiple surfaces, and that intramolecular interactions between, and within, domains of L-locus immune-receptors plays a crucial role in these interactions. Finally, the generation of an enhanced immune-receptor is an important proof-of-concept demonstrating the utility of protein engineering in generating novel disease resistance in agricultural crops.The biotrophic fungus  The plant immune system is based upon the ability to accurately perceive and appropriately respond to potential threats. In general, plants use membrane-spanning proteins with extracellular receptor domains to recognize common features of plant pathogens  and intracellular receptors to detect pathogen effectors transferred into plant cells during infection APAF-1, R protein, CED-4) domain, and a winged helical ARC2 domain Plant NB-LRR disease resistance proteins belong to the STAND  clade of AAA+ (ATPase associated with diverse cellular activities) proteins, and are similar to the Nod-like receptor (NLR) family of proteins that act as intracellular surveillance molecules in animal innate immunity The C-terminal LRR domain of R proteins generally mediates signal perception Linum usitatissimum) confer resistance to the flax rust fungus Melampsora lini through recognition of effector proteins delivered into the host cell during infection L resistance locus consists of a single gene encoding 13 allelic protein variants  that recognise different matching avirulence proteins TIR-NB-LRR resistance proteins in flax  in transient expression assays whereas the other 5 variants  are virulence alleles as they do not induce an HR in plantain plantaThe L5, L6 and L7 proteins recognise allelic variants of the et al. Wang Agrobacterium-mediated transient expression in planta - to measure R gene-dependent cell death  were associated with specificity differences ll death .et al.With one exception (see below), single amino acid changes at positions 56, 90 and 96 in AvrL567-A did not alter recognition by L5 or L6 in planta) by the single S96R substitution, but required the triple T50I/D56N/S96R substitution for full recognition. Interestingly, in the context of AvrL567-C, a K residue at position 56 was not compatible with L6 recognition, although it does not prevent recognition in the AvrL567-A or -D contexts  and the AvrL567-A mutant R96S (interacts with L5 but not L6)  and 3. T1193L5), abolished the interaction with AvrL567-D, but not -A  allowed weak interaction with AvrL567-D  did not  acquired the capacity to interact with AvrL567-A-R96S , which contain the critical 594-to-793 L5 residues along with the L5 TIR-NB-ARC domains did not interact with AvrL567-A-R96S , including the TIR and NB regions, did not increase the interaction with AvrL567-D, but a further swap including the L6 ARC1 (L6493L5556L6) restored strong interaction with AvrL567-D. This suggested that one or more of the six amino acid polymorphisms between 447 and 484 (five in ARC1 and one in ARC2) were responsible for the reduced interaction. Consistent with this, inclusion of the ARC1 and ARC2 regions of L6 along with the L5 TIR-NB (L5431L6) also restored recognition of AvrL567-D, again implicating this region in the reduced interaction. Chimeric proteins representing the inverse swaps and including the L5 LRR domain  did not interact with AvrL567-D but retained interaction with AvrL567-A  than L6 (21), L6493L5 (20), L5 (19), L6592L51193L6 (14), or L6592L5 (13)  .493L51193L6 chimera for its ability to trigger an AvrL567-dependent cell death in planta  maintained interaction with many AvrL567 variants, including -A and -J  showed an L6-like recognition repertoire  interfere with recognition in the context of the L6 LRR. However, the TIR domain is not required for L6-AvrL567 interaction, and these two residues are exposed on the surface of the TIR domain structure in a region implicated in negative regulation of L6 through intramolecular interactions Previous experiments in other systems have also demonstrated intramolecular interactions between R protein domains that are important for function. The CC, NB-ARC and LRR domains of potato Rx can interact and functionally complement each other when expressed as separate polypeptides et al.et al.To visualize structurally the L protein regions involved in AvrL567 recognition, we prepared a homology model of the L6 NB-ARC domain with the program Modeller 493L51193L6) was shown to function to induce an HR in tobacco, recapitulating the expanded recognition observed in Y2H assays. This correspondence between Avr:R interaction in yeast and HR induction in planta, now observed across a large number of L-AvrL567 pairwise combinations  according to the manufacturer's instructions. Chimeric L5–L6 proteins were constructed using native and introduced restriction sites, and/or by PCR-based fusion of overlapping sequences as described in 493L51193L6 cDNAs, were cloned into the binary vector pTNotTReg between the cauliflower mosaic virus 35S promoter and ocs terminator sequences. Agrobacterium tumefaciens (GV3101-pMP90) cells containing these constructs were grown for 36 h at 28°C in LB media supplemented with appropriate antibiotic selections. Cells were pelleted, resuspended in infiltration medium , adjusted to OD600 nm = 1 and incubated for 2 h at room temperature. Resuspended cells were infiltrated with a 1-mL needleless syringe into the leaves of near isogenic lines of flax plants containing L5, L6 (cv. Bison) or L6L11RV (cv. Ward), or into leaves of 3-week-old tobacco plants (W38). Transgenic tobacco expressing AvrL567-A was described by Dodds et al.DNA constructs encoding AvrL567 proteins lacking the signal peptide, or full-length L5, L6 or L6M7−lnLM8), with the χ2 distribution (d.f. = 2). For model M8, the empirical Bayes The non-synonymous/synonymous rate ratio parameter ω was estimated using the program CODEML Figure S1Mutational analysis of AvrL567-D interactions with L5 and L6. Upper Panels: β-galactosidase activity of yeast strain SFY526 expressing the GAL4 DNA-binding domain fused to the L5 and L6 proteins along with the corresponding GAL4 activation domain fused to AvrL567-D variants. Bottom Panels: growth of yeast strain HF7c expressing the same protein fusion constructs on selective –W,L,H plates after 4 days.(TIF)Click here for additional data file.Figure S2Immunoblot detection of fusion proteins expressed in yeast. Protein extracts from yeast strains HF7c expressing A. AvrL567::GAL4-AD, and B. chimeric L5–L6::GAL4-AD fusion proteins were separated by SDS-PAGE, blotted onto nitrocellulose membranes and detected by anti-hemagglutinin mAbs. Positions and sizes of proteins molecular mass standards are indicated.(TIF)Click here for additional data file.Figure S3Amino acid alignment of polymorphic residues of L5, L6 and all L5–L6 chimeras used in this study. Residues from L5 are coloured blue, residues from L6 are coloured red. Amino acid position numbers are indicated above the alignment and within the LRR domain residues occurring in the β-turn and β-strand regions are numbered by the repeat that they occur in (1–26). Black plus sign indicates a residue under positive selection (p>0.95), red plus sign indicates a residue under positive selection (p>0.99). i indicates a residue involved in protein-protein interactions according to the in silico docking models of L5 and AvrL567-A (TIF)Click here for additional data file.Figure S4Selected primers (black) and restriction sites (blue) used in the construction of chimeric L5–L6 proteins. See (TIF)Click here for additional data file.Figure S5592L5 and L6592L6 proteins along with the corresponding GAL4 activation domain fused to AvrL567-A or -D on selective –W,L,H plates after 4 days.Growth of yeast strain HF7c expressing GAL4 DNA-binding domain fused to the L5 L5592L5 and L L6592L6 are identical to L5 and L6, respectively, except for a W593R substitution resulting from an engineered AvrII restriction site introduced to facilitate the generation of LRR domain swaps.(TIF)Click here for additional data file.Figure S6592L51193L6 and L6493L51193L6.Recognition repertoire of L6 Growth of yeast strain HF7c expressing GAL4 DNA-binding domain fused to the L6592L51193L6 and L6493L51193L6 proteins along with the corresponding GAL4 activation domain fused to AvrL567 proteins on selective –W,L,H plates after 4 days.(TIF)Click here for additional data file.Figure S7Polymorphic amino acid residues found in the LRR domain of chimeric L5–L6 and L6–L11  resistance proteins. Residues from L5 are shaded blue, those from L6 are shaded red, and those from L11 are shaded green. LRR subunits with polymorphic residues in the β-strand/β-strand structure (xxLxLxx motif) are marked with black bars and are numbered below. All residues are listed below the corresponding L5 residues.(TIF)Click here for additional data file.Table S1Log-likelihoods (lnL), Log-likelihoods ratio test (LRT), and estimates of the model parameters  for that class) for the models M7 and M8 in CODEML.(TIF)Click here for additional data file.Text S1Supplementary experimental procedures.(DOC)Click here for additional data file.


Objective(s): Our objective was to evaluate the effects of a triple antioxidant combination  on the cholesterol and glutathione levels, and the fatty acid composition of liver and muscle tissues in diabetic rats. Materials and Methods: Forty-three Wistar rats were randomly divided into five groups. The first group was used as a control. The second, third and fourth groups received STZ (45 mg/kg) in citrate buffer. The fourth and fifth groups were injected with intraperitoneal (IP) 50 mg/kg DL-AT and 50 mg /kg DL-LA four times per week and received water-soluble vitamin C (50 mg/kg) in their drinking water for a period of six weeks.Results: Liver cholesterol levels in the AT+AA+LA group were lower than the control (P<0.05). Glutathione level was lower in D-2 (P<0.05) and were higher in D+AT+AA+LA and AT+AA+LA groups than the control groups (P≤ 0.05). The muscle cholesterol levels in the D-1 and D+AT+AA+LA groups were higher than the control group (P≤ 0.05). The levels of oleic acid were higher in the D-1 group and lower in the D-2 group (P<0.001). The arachidonic acid level in the D-1 and D-2 groups were lower (P<0.05), and higher in the D+AT+AA+LA group. Conclusion: Our results revealed that glutathione levels and the Stearoyl CoA Desaturase enzyme products in liver tissues of diabetic and non-diabetic rats were increased by triple antioxidant mixture. Diabetes mellitus is a common degenerative disease as one of the leading global causes of morbidity and mortality . Increasde novo synthesis of fatty acid (FA), myocytes rely on the supply of FA from extracellular sources to cover their need of these substrates. Fatty acids are supplied to cardiac and skeletal muscle either bound to plasma albumin (non-esterified fatty acids) or in the form of triacylglycerols in the core of circulating lipoproteins  in the diet of subjects belonging to the high-risk group for the development of insulin resistance . The aimAnimals, diets, experimental designad libitum access to rat chow and water. Animals were randomly divided into five groups. The first group was used as a control (n = 6), the second group D-1 (n = 10), the third group D-2 (n = 10), the fourth group D+AT+AA+LA (n = 10), the fifth group AT+AA+LA (n =7). Rats in D-1, D-2, and D+AT+AA+LA groups were made diabetic via a single intraperitoneal injection (IP) of 45 mg/kg STZ in citrate buffer (pH = 4.5). Control group were injected IP with the buffer alone. Two days following administration of STZ, the tail vein blood glucose level was measured in all the animals. Those with blood glucose level of 250 mg/dl and above were considered diabetics. Following formation of diabetic, rats in D-1 and D+AT+AA+LA groups received daily subcutaneous injections of 8 IU/kg insulin; rats in the D-2 group were not injected. Rats in the D+AT+AA+LA group were injected intraperitoneal (IP) 50 mg / kg DL-AT and 50 mg/kg DL-LA four times per week and received water-soluble vitamin C (50 mg/kg) in their drinking water. These treatments were continued for six weeks, after which each rat was anesthetized with ether, liver and muscle tissue samples were collected and stored at –85OC prior to biochemical analyses.A total 43 Wistar rats  were used. The animals were housed in cages in an air-conditioned room with a 12 hour light/dark cycle where they had Laboratory analyseset al  using the method of Folch et al  as descret al . Liver aet al . Aliquotmethanol  and the Cholesterol analysisClass VP software. The results were expressed as µmol/g tissue weight.For the cholesterol analysis, fully automatic HPLC  had been used. A Supelcosil LC 18TMDB column  was used as the HPLC column. The mobile phase used was acetonitrile – isopropyl alcohol (70:30 v/v) at a flow rate of 1 ml/ min . DetectiDetermination of GSH in Liver and muscle tissues3PO4 was used as the mobile phase with a flow rate of 1 ml/min  was used and 50 mM NaClO4 0.1 % H1 ml/min . Detecti1 ml/min , samplesFatty acid methyl ester analysisOC, 5OC/min and the final temperature was held for 15 min. Injector and FID temperatures were 240 and 280OC, respectively. Individual methyl esters were identified by comparison with authentic external standard mixtures analyzed under the same conditions. Class GC 10 software version 2.01 was used to process the data. The results were expressed as percent tissue weight.Fatty acids in lipid extracts were converted to methyl esters by using 2% sulfuric acid (v/v) in methanol . Fatty aStatistical analysisThe experimental results were reported as mean ± SE. Statistical analysis was performed using SPSS. Analysis of variance (ANOVA) and Least Significant Difference (LSD) test were used to compare the experimental groups with the controls. Total lipid, cholesterol and reduced glutathione (GSH) levelsP>0.05), however, the cholesterol levels in D-1, D-2, and AT+AA+LA were lower than the control group . While the GSH level did not differ between the control and D-1 groups, its level decreased in the D-2 group (P<0.05). However, the GSH level increased in D+AT+AA+LA and AT+AA+LA groups . The effects of triple antioxidant combination on the cholesterol and GSH levels of liver are shown in P<0.05). However, the cholesterol level decreased in the D-2 group (P<0.01). Although GSH level did not differ between the control, D-1 and D+AT+AA+LA groups, its level decreased in the D-2 group, but its level in the AT+AA+LA group was higher than the other groups , but stearic acid level decreased in the same groups (P<0.001). While the liver palmitoleic acid level  did not differ between control, D-1 and D-2 groups, its level increased in the D+AT+AA+LA group (P<0.01). Although liver oleic acid level was high  in the D+AT+AA+LA and AT+AA+LA groups (P<0.01), its level decreased in the D-2 group (P<0.05). Liver linoleic acid level  was high in the D-1, D- 2 and D+ AT+AA+LA groups (P<0.01), however arachidonic acid  level was low in the D-2 and D+ D+ AT+AA+LA groups (P<0.001). Docosahexaenic acid  level increased in the D-2 and AT+AA+LA groups , although there was no differences between control, D-1 and D+AT+AA+LA groups (P>0.05)  .P<0.01, P<0.001). Muscle oleic acid levels increased in the D-1 group, but its level decreased in the D-2 group (P<0.001). While the linoleic acid levels of muscle was high in the D-1 and D-2 groups (P<0.01), muscle arachidonic acid level was low in the same groups (P<0.05). However, muscle arachidonic acid level increased in the D+AT+AA+LA and AT+AA+LA groups (P<0.05). While DHA level in muscle decreased in the D-1 group, its level increased in the D-2 and AT+AA+LA groups (P<0.05) ((P<0.05) . reductase enzyme (HMG-CoA), which is a key enzyme in cholesterol biosynthesis. Therefore, cholesterol synthesis increases and facilitates the storage of fuels and macromolecules in liver, muscle and fat (Experimental and clinical studies revealed that free radicals are formed disproportionately in diabetes by glucose oxidation, non-enzymatic glycation of proteins , 27. Abn and fat . In the  and fat . AT supp and fat . In addi and fat , 32. et al (de novo synthesis of GSH in many cell types. It has been revealed to be potent antioxidants of the LA, to regenerate through redox cycling other antioxidants like AA and α -tocopherol, and to raise intracellular GSH levels (et al (et al (et al (et al (et al (The results of the present study revealed that the level of GSH in D-2 group was lower than the control group. However, GSH level was high in the treated triple antioxidant group. GSH is the most prevalent low molecular weight antioxidant within cells and it protects cellular constituents from oxidative damage by reacting directly with oxidants or by acting as the substrate for glutathione peroxidase to scavenge peroxides . GSH alset al  suggesteH levels , 38. CliH levels , 39. Yils (et al  found thl (et al  reportedl (et al  suggestel (et al  found thl (et al  suggestel (et al .de novo synthesis by the activity of cytoplasmic FA synthase (in vivo Δ9-desaturase activity. The significant increase in the proportions of 16:1, n-7 and 18:1, n-9 may reflect higher activity of Δ9 desaturase in the cell. An upsurge in the Δ9 desaturase activity has been reported to be related to hyperinsulinemia. It has been found to induce an over-expression of Δ9 desaturase by the effect of insulin in animals (In the FA composition of liver tissue, although palmitic acid levels in D-1 and D-2 and D+AT+AA+LA groups were higher than the control group, stearic acid levels were low in the same groups. In mammalian cells, palmitic and stearic acids are major products of synthase . The pre animals -47. The  animals . et al (Lipid peroxidation is a significant aspect of oxidative damage and oxidative modification of phospholipid polyunsaturated fatty acid residues in membrane structure . In the et al  revealedet al , 50. It et al (9 desaturase activity and consequently enhanced monounsaturated acids in all tissues of experimental diabetes. In the present results, treatment of triple antioxidants to diabetic groups increased the level of monounsaturated fatty acids. This increase might be due to the effects of AT, AA, LA and insulin administration. Skeletal muscle is a major site of insulin action, and insulin sensitivity may be related to the fatty acid composition of muscle lipids. Mohan et al  reportedet al  suggesteAntioxidant therapy alone had no effect on the measured parameters in either the diabetic or control animals. However, combined insulin and antioxidant therapies normalized blood pressure, plasma MDA and urinary protein in the diabetic animals . InsulinIn conclusion, it was observed that GSH levels in liver and muscle tissue of diabetic and non-diabetic rats were increased by triple antioxidant mixture; in particular, the administration of LA increased the level of GSH in non-diabetic rats. In addition, the administration of triple antioxidant mixture and insulin were increased the level of products the SCD in liver tissue.


O-glycosylation and phosphorylation operate the functional expression of cellular proteins, this switching on and off the protein expression is difficult to monitor in vivo. By using neural network based prediction methods, we propose that alternate O-β-GlcNAc modification and phosphorylation on Ser 204 control the binding of IGFBP-6 with IGF-II. This information may be used for developing new therapies by regulating IGFBP-6 assembly with IGF-II to minimize the risk of viral associated hepatocellular carcinoma. We can conclude that during HCV/HBV infection, O-β-GlcNAc of IGFBP-6 at Ser 204 diminish their binding with IGF-II, increase IGF-II cellular expression and promote cancer progression which can lead to hepatocellular carcinoma. Furthermore, this site can be used for developing new therapies to control the IGF-II actions during viral infection to minimize the risk of hepatocellular carcinoma.Hepatocellular carcinoma is mainly associated with viral hepatitis B and C. Activation of cell growth stimulator IGF-II gene is observed in tumor formation especially in viral associated hepatocellular carcinoma. Elevated IGF-II levels are indicator of increased risk for cholangiocellular and hepatocellular carcinomas through over saturation of IGF-II binding capacities with IGF receptors leading to cellular dedifferentiation. In HCV, core protein is believed to trans-activate host IGF-II receptor through PKC pathway and the inhibition of tumor cell growth can be achieved by blocking IGF-II pathway either at transcriptional level or increasing its binding with IGFBPs (Insulin like growth factor proteins) at C-terminal, so that it is not available in free form. IGFBP-6 is a specific inhibitor of IGF-II actions. Affinity of IGFBPs with IGFs is controlled by post-translational modifications. Phosphorylation of IGFBPs inhibits IGFs action on target cells while O-glycosylation prevents binding of IGFBP-6 to glycosaminoglycans and cell membranes and resulting in a 10-fold higher affinity for IGF-II.  Among the highly malignant human tumors, hepatocellular carcinoma is the most common. It is the fifth most prominent tumor in the world and is the third most widespread cause of cancer-related death . HCC proIGF-I binds with high affinity to IGF-I receptor whereas IGF-II binds with IGF-II receptor more effectively . IGF-II IGFBP-6 an important member of IGFBPs family is a relatively specific inhibitor of IGF-II actions. IGFBP has the highest affinity among IGFBPs to bind with IGF-II along with 20 to 100 fold preference of binding to IGF-II rather than IGF-I. Non proliferative state is usually related with IGFBP-6 expression. IGFBP-6 expression is generally stimulated by different agents like retinoic acid . It consO-glycosylation prevents binding of IGFBP-6 to glycosaminoglycans and cell membranes and resulting in a 10-fold higher affinity for IGF-II. O-glycosylation also delays and resists the proteolysis of IGFBP by chymotrypsin and trypsin than non-glycosylated IGFBP-6 [Cell systems in which IGFBP-6 has been shown to inhibit IGF-II induced effects such as proliferation, differentiation, cell adhesion, and colony formation include osteoblasts, keratinocytes, myoblasts, and colon cancer cells . Post-tr IGFBP-6 ,20.β-GlcNAc sites, and their possible interplay in IGFBP-6 leading to change affinity with IGF-II and modify the functions of IGF-II, which have been predicted and analyze using different prediction methods available. On the basis of potential phosphorylation and glycosylation interplay in conserved residues of IGFBP-6, the possible roles played by these post-translational modifications in regulating the functions of IGFBP-6 are analyzed.It has been documented but not extensively studied that high levels of IGF-II in hepatocellular carcinoma can be control by parental imprinting or changes in methylation pattern in IGF-II genes . In thisHomo sapiens was retrieved from the SWISS-PROT sequence database [sapiens , Mus musculus , Ovis aries , Rattus norvegicus , Sus sacrofa  and Bos taurus . All the six sequences were multiple aligned using ClustalW [The sequence data used to predict potential phosphorylation and glycosylation sites of IGFBP-6 of database . The entdatabase . The seaClustalW . ClustalThe IGFBP-6 sequence used in this study was  "MTPHRLLPPLLLLLALLLAASPGGALARCPGCGQGVQAGCPGGCVEEEDGGSPAEGCAEAEGCLRREGQECGVYTPNCAPGLQCHPPKDDEAPLRALLLGRGRCLPARAPAVAEENPKESKPQAGTARPQDVNRRDQQRNPGTSTTPSQPNSAGVQDTEMGPCRRHLDSVLQQLQTEVYRGAQTLYVPNCDHRGFYRKRQCRSSQGQRRGPCWCVDRMGKSLPGSPDGNGSSSCPTGSSG".http://www.cbs.dtu.dk/services/NetPhos/) server [http://www.ist.temple.edu/disphos/) server [Phosphorylation potential for human IGFBP-6 was predicted by using NetPhos 2.0  server . This is) server  was alsohttp://cbs.dtu.dk/services/NetPhosK) [http://kinasephos.mbc.nctu.edu.tw/case2.html) server [Kinase specific phosphorylation sites in human IGFBP-6 were predicted by NetPhosK 1.0 server (etPhosK) . The Net) server  was alsohttp://phospho.elm.eu.org) was used [For the purpose of evaluating experimentally verified phosphorylation sites on human IGFBP-6, Phospho.ELM database . This program can predict the potential phosphorylation sites as well and hence predicting the Yin-Yang sites with highly uneven threshold that is adjusted in accordance with amino acid surface accessibility [http://ogpet.utep.edu/OGPET/) [O-glycosylation at Ser and Thr residues.sibility -37. We a/OGPET/)  for predhttp://zhanglab.ccmb.med.umich.edu/I-TASSER/) [http://jmol.sourceforge.net/) [http://www.pymol.org/export) [http://www.cbs.dtu.dk/services/NetSurfP/) was used [http://scansite.mit.edu/motifscan_seq.phtml) to check surface ability of IGFBP-6 to solvents and for PTMs [As there was no template model of IGFBP-6 available in protein data bank , we desiTASSER/) . Data inge.net/)  and PYmo/export)  programswas used . We alsofor PTMs .Above described prediction methods are artificial neural network-based and have been extensively used in predicting the potentials of proteins for post-translational modifications and in biological sequence analysis . These mTo determine conserved and conserved substituted Ser and Thr residues within each subtype, human IGFBP-6 protein FASTA sequence was aligned with other mammals Figure . It is chttp://phospho.elm.eu.org) and UniprotKB (http://www.uniprot.org). Although all IGFBP sub forms are subjected to be phosphorylated, however, there is no experimentally verified phosphorylation site.Data for experimentally confirmed S/T/Y residues was searched from Phospho.ELM  and Thr (126 and 176). Cdc2 can phosphorylate Ser  and Thr (126 and 176). Similarly Cdk5 can phosphorylate Ser (225) and Thr . CK II can phosphorylate Ser 52, PKA phosphorylate serine 120, DNAPK and ATM both can phosphorylate serine 144 and 204. EGFR can phosphorylate Tyr 179 and 196 while GSK3 can phosphorylate Ser 221 and 225 respectively.The other neural network based program Kinasephos was also used to assess the possible kinases on IGFBP-6. It also predicts many kinases that may be involved in IGFBP-6 protein phosphorylation as given in Table O-GlcNAc modification is known to be dynamic and analogous to phosphorylation. The prediction results obtained from YinOYang 1.2 for O-β-GlcNAc showed that there are many high potential O-β-GlcNAc sites for O-linked modifications. The human IGFBP-6 has 12 potential sites for O-β-GlcNAc modifications at Ser 144, 148, 152, 204, 231, 232, 233, 238 and 239 and at Thr 143, 146 and 236, with five Yin-Yang sites marked by asterisk . Besides these, there were many other Ser and Thr residues that were predicted to be non-glycosylated, but the phosphorylation potential predicted was either much higher than the threshold value or very close to it. These are also conserved residues in mammals. Such residues appear to be as false-negative sites. These may act as possible Yin Yang sites other than those which have been predicted by the YinOYang 1.2 method. According to our results these sites may be Ser 120 and 169; and Thr 75 and 126 Table .Many studies have tried to find out the role of host factors in causing hepataocellular carcinoma linked to HBV and HCV. One of the factors mediating the HCV and HBV associated hepatocellular carcinoma is IGFs. HBV stimulate the expression of IGF-IR and modulate the transcription of IGFII using P3 and P4 promoter . WhereasO-glycosylation of IGFBP-6 enhances its resistance to proteolysis by chymotrypsin and trypsin [Post-translational modifications in proteins play very important role in regulation of their functions and induce conformational changes that allow the protein to interact with other proteins ,48. Bind trypsin . The IGF trypsin .c and it is also a Yin Yang site [O-β-GlcNAc modification can occur on these Ser and Thr residues where kinases are involved in phosphorylation as it is well known that kinases and OGT can compete for same site modification [O-glycosylation is known and experimentally verified post-translational modification in IGFBP-6 [O-linked glycosylation  found that the IGF-II binding site is located on C-terminal of IGFBP-6 from Leu 174, Gly 206 . Regulatang site . Our Net004 foundfication -37,52. T IGFBP-6 . YinOYanO-β-GlcNAc modification are Ser-144, 148, 152 and 204; and Thr 146. NetPhos 2.0 and Disphos prediction results showed that there are many Ser and Thr residues which are not predicted as Yin Yang but have high potential for phosphorylation, same as; YinOYang 1.2 and OGPET also predicted such type of residues to have high potential for O-β-GlcNAc modification  sites , Waqar Ahmad (M Phil Chemistry) and Gull S (MSc Biochemistry) are Research Officer; Shabbiri K is lecturer while Nazar S, Nazar N and Fouzia K are BS (Hons) student at GC University, Lahore. Kausar H, Sawar MT, Shahid I and Asad S are PhD scholars. Sajida Hassan (PhD Molecular Biology) is Principal Investigator at CEMB, University of the Punjab, Lahore


P = 0.025), the proliferation marker Ki-67 (P = 0.001), and the recurrence rate of TNBC patients (P<0.001). In TNBC patients with Aur-A high expression, the risk of distant recurrence peaked at the first 3 years and declined rapidly thereafter, whereas patients with Aur-A low expression showed a relatively constant risk of recurrence during the entire follow-up period. Univariate and multivariate analysis showed that overexpression of Aur-A predicted poor overall survival (P = 0.002) and progression-free survival (P = 0.012) in TNBC. Furthermore, overexpression of Aur-A, associated with high Ki-67, predicted an inferior prognosis compared with low expression of both Aur-A and Ki-67. Importantly, we further found that Aur-A was overexpressed in TNBC cells, and inhibition of this kinase inhibited cell proliferation and prevented cell migration in TNBC. Our findings demonstrated that Aur-A was a potential therapeutic target for TNBC and inhibition of Aur-A kinase was a promising regimen for TNBC cancer therapy.Triple negative breast cancer (TNBC) acquires an unfavorable prognosis, emerging as a major challenge for the treatment of breast cancer. In the present study, 122 TNBC patients were subjected to analysis of Aurora-A (Aur-A) expression and survival prognosis. We found that Aur-A high expression was positively associated with initial clinical stage ( Breast cancer is one of the leading causes of cancer-related death among adult females in the world Aur-A , as a member of mitotic serine/threonine Aurora kinase family, is essential in accurate timing of mitosis and maintenance of bipolar spindles In breast cancer, Aur-A expression was found to be elevated at protein and mRNA levels in the tumor specimens The purpose of this study was to detect the protein expression of Aur-A in TNBC patients and analyze its clinicopathological/prognostic significances in this tumor subtype. Our results showed that high expression of Aur-A in TNBC was positively associated with malignant pathological features and predicted adverse overall survival (OS) and progression-free survival (PFS). Moreover, we found that inhibition of Aur-A could inhibit cell proliferation and reduce cell migration in TNBC cells, emerging as a potential targeted treatment for TNBC.Between January 1997 and October 2003, a total of 628 female breast cancer patients who received postoperative adjuvant chemotherapy (Cyclophosphamide/Adriamycin/5-Fluorouracil (CAF) or Cyclophosphamide/Methotrexate/5-Fluorouracil (CMF)) at Cancer Center of Sun Yat-sen University  were included in the present study. Among the 628 breast cancer patients, 149 patients were classified as TNBC. Staging was performed according to American Joint Committee on Cancer guidelines 2 incubator.Human breast epithelial cell lines  were obtained from the American Type Culture Collection (ATCC). MDA-MB-231, MCF-7, SKBR3 and MDA-MB-435 were routinely maintained in high-glucose DMEM . BT549, ZR75-1, MDA-MB-468 and BT474 were routinely maintained in RPMI1640 . All media were supplemented with 10% fetal bovine serum , penicillin , and streptomycin . All cells were cultured at 37°C in humidified 5% COThe tissue microarrays (TMAs) were constructed as a method described previously The immunohistochemistry (IHC) assay was performed as previously described The staining intensity and extent of Aur-A was graded as described previously, and the merged overall score >5 was regarded as high staining MTT  assay was used to assess the growth of MDA-MB-231, MCF-7, MDA-MB-468, and MDA-MB-435 cells. The cells under indicating treatments were plated in 96-well plates and culture for 24 h. Cell survival was assessed as described previously Transwell assay was performed as described previously Western blot analysis was performed as described previously The siRNA oligonucleotides targeting to Aur-A  and control (sense 5′UUCUCCGAACGUGUCACGU TT3′ anti-sense 5′ACGUGACACGUUCGGAGAATT3′) were transfected into tumor cells  for 48 h using Lipofectamine 2000  according to manufacturer’s instructions. Downregulation of the proteins were demonstrated by Western blot.All TNBC patients (n = 122) had a follow-up record for over 8 years. After the completion of therapy, patients were observed at 3 month intervals during the first 3 years and at 6 month intervals thereafter. The latest follow-up was updated in September 2011. OS was defined as the time from the date of definitive surgery to the date of last follow-up or death; PFS was defined as the time from the date of definitive surgery to the date of last follow-up or disease relapse 2χ test and student t test were used to make statistical comparisons between groups. The effect of predictive variables was evaluated by Cox univariate and multivariate regression models. The relationships between Aur-A expression and OS, PFS were determined by Kaplan-Meier analysis. The log-rank tests were performed to value the difference in survival probabilities between patient subsets. All P values quoted were two-sided and P<0.05 was considered statistically significant.Statistical analysis was performed using SPSS V. 17.0 . The Clinical features of the 122 TNBC patients, including age, family history, pathological characteristics, lymph node status, initial clinical stage, tumor stage, Ki-67, adjuvant radiotherapy, adjuvant chemotherapy, and recurrence, were summarized in P = 0.025, P = 0.001, P<0.001, Our data showed that Aur-A high expression was positively correlated with initial clinical stage  As the most aggressive subtype of breast cancer, TNBC occurs in approximately 20∼25% of all patients with breast cancer, associating with an unfavorable prognosis Recently, the proliferation index Ki-67 is used to further classification of TNBC with different response and prognosis As the underlying mechanism of Aur-A involving in cancer progression and prognosis, it remains complicated and varies in various types of human cancers. Previously, we found that Aur-A promoted epithelial–mesenchymal transition and invasion in nasopharyngeal carcinoma (NPC) mediated by mitogen-activated protein kinase (MAPK) phosphorylation In our previous studies, VX-680, a selective Aurora kinase inhibitor, was demonstrated to effectively suppress cell growth and migration and potently sensitized cancer cells to therapy, leading to significant cell death in LSCC In conclusion, the findings reported here provide several new insights to the understanding of TNBC as following: (a) Aur-A is useful to identify TNBC patients that are at high risk of early recurrence and progression. (b) Overexpression of Aur-A, associated with high expression of Ki-67, predicts a shorter survival of TNBC, serving as a novel independent prognostic biological marker for TNBC. (c) Aur-A is demonstrated as a potential therapeutic target for TNBC and inhibition of Aur-A kinase is a promising regimen for cancer therapy in TNBC patients with Aur-A high expression.Figure S1Whole picture of TMA with Aur-A staining.(TIF)Click here for additional data file.Figure S2Immunohistochemistry analysis of Aur-A and Ki-67 expression in TNBC tissues. (A1) Ki-67 low expression and (A2) Aur-A high expression was shown in a TNBC patient sample (100×). (B1) Ki-67 low expression and (B2) Aur-A low expression was shown in a TNBC patient sample (100×). (C1) Ki-67 high expression and (C2) Aur-A high expression was shown in a TNBC patient sample (100×). (D1) Ki-67 high expression and (D2) Aur-A low expression was shown in another sample (100×). (A1 ’), (A2 ’), (B1 ’), (B2 ’), (C1 ’), (C2 ’), (D1 ’), (D2 ’) demonstrated the higher magnification (200×) from the area of the box in (A1), (A2), (B1), (B2), (C1), (C2), (D1), (D2) respectively.(TIF)Click here for additional data file.Figure S3Inhibition of Aur-A kinase inhibited TNBC cell proliferation and reduced cell migration. (A) TNBC cell MDA-MB-468 and non-TNBC cell MDA-MB-435 were incubated with indicated doses of VX-680 , or DMSO for 24 h; Cells were harvested, and subjected to Western blot analysis for the indicated proteins. (B) MDA-MB-468 and MDA-MB-435 cells were exposed to different concentrations of VX-680  or DMSO for 24 h. Cell survival rates were measured by MTT assay, *P<0.05; **P<0.01; ***P<0.001. (C) and (D) MDA-MB-468 cells were seeded for transwell migration assay in the presence of DMSO or increasing doses of VX-680. After incubation for 24 h, migration rates were quantified by counting the migrated cells in five random fields. Original magnification, 100×. Data summarized three independent experiments. Columns, average cell number; bars, SD. (E) TNBC cell MDA-MB-468 and non-TNBC cell MDA-MB-435 were treated with targeted RNAis or control for 48 h, then cells were harvested, and subjected to Western blot analysis for the indicated proteins. (F) MDA-MB-231 and MCF-7 cells were treated with different RNAis for 48 h. Cell survival rates were then measured by MTT assay. (G) and (H) MDA-MB-468 cells were treated with different RNAis for 24 h, then seeded for transwell migration assay for 24 h, migration rates were quantified by counting the migrated cells in five random fields. Original magnification, 100×.(TIF)Click here for additional data file.


Systemic lupus erythematosus (SLE) is an autoimmune disorder characterized by production of autoantibodies and immune complex deposition in various organs. Aberrations in the T lymphocyte compartment and dysregulated cytokine production are key features of SLE pathogenesis and disease progression. Recently, the role of the interleukin (IL)-17/IL-23 axis in the pathogenesis of SLE has been reported. IL-23 and IL-23R are essential for expansion of pathogenic IL-17-producing T lymphocytes and have been shown to be important in the pathogenesis of lupus in animal models.+ and CD8+ T lymphocytes in peripheral blood mononuclear cells (PBMCs) of SLE patients and control subjects were examined by flow cytometry. Twenty-nine SLE patients and 10 control subjects were recruited in this study. Patients were divided into active and inactive groups based on the SLE disease activity index (SLEDAI). As another disease control population, five psoriatic patients were recruited in this study.In this study, the expression of IL-23R and IL-17 in CD4+ CD4+ and IL-23R+ CD8+ T cell subsets were significantly higher in freshly isolated PBMCs from both groups of SLE patients compared to control subjects . In addition, this difference was maintained after ex vivo stimulation with plate-bound anti-CD3/CD28 antibodies . When the fold increase in IL-17+ T cells after ex vivo stimulation for three days was compared between patients and controls, SLE patients exhibited significantly higher increases in CD4+ IL-17+ and CD8+ IL-17+ T cells, suggesting that PBMCs from SLE patients promoted the expansion of IL-17-producing T cells upon stimulation more vigorously than control PBMCs. These trends were not observed in psoriasis patients. The correlations between IL-23R+ T cells and IL-17+ T cells and IL-23R+ CD8+ T cells and SLEDAI scores in patients were also found to be statistically significant.Percentages of both IL23R+ T cell subsets in this autoimmune disease.The results of our study confirmed the relevance of the IL-23/IL-17 axis in the pathogenesis of SLE and further highlighted the importance of IL-23R Systemic lupus erythematosus (SLE) is an autoimmune disorder that affects multiple organs and is characterized by production of autoantibodies and immune complex deposition in various organs, leading to inflammation and tissue destruction. T lymphocytes and their cytokines play essential roles in the immunopathogenesis of the disease . Studies+ T cells (Th17), CD8+ T cells, CD3+ CD4- CD8- T cells, γδ T cells, NK cells and neutrophils [IL-23p19- and IL-12p40-deficient mice showed distinct phenotypes in terms of susceptibility to autoimmune diseases helped to establish Th17 as a unique helper T cell subset distinct from Th1 and Th2 cells [IL-23 is a heterodimeric cytokine produced predominantly by activated antigen presenting cells, such as macrophages and dendritic cells. The cytokine is composed of a unique p19 subunit and a p40 subunit that is shared with the Th1 signature cytokine, IL-12 [h2 cells ,6. The mcells Th1, CD8+ T The IL-23/IL-17 axis, therefore, is one of the main cytokine axes driving the pathogenesis of various autoimmune diseases, a role that had been previously attributed to Th1 cells. The receptor for IL-23 is composed of IL-12Rβ1, a common subunit shared with IL-12R, and a unique IL-23R . IL-23R - CD8- double negative T cells was uniquely expanded and identified as a source of IL-17 in PBMCs and kidney biopsies from patients with SLE [IL-23p19 and IL-12p40 were found in psoriatic skin lesions, and increased in circulating Th1, Th17 and Th22 were found in psoriasis [IL23R gene were reported to affect psoriatic phenotypes, which strongly link this disease with IL-23/Th17 axis [Since the identification of the Th17 lineage and its associated cytokines, multiple reports have documented the involvement of the IL-23/IL-17 axis in animal models of SLE and in SLE patients . Sera ILwith SLE . The reswith SLE . Increassoriasis . In addih17 axis .+ T lymphocytes in SLE patients regardless of disease severity, but this trend was not observed in psoriasis. Furthermore, we found significantly increased IL-17+ CD4+ and CD8+ T cells in SLE patients and a positive correlations between IL-23R+ and IL-17+ T cells, IL-23R+ T cells and SLEDAI scores in SLE patients.In this study, we investigated the expression profile of IL-23R and IL-17 in T lymphocytes of PBMCs from SLE patients and examined its correlation with disease severity. We found significant increases in IL-23Rn = 13) and SLE patients with inactive SLE . Normal subjects were recruited as healthy controls. Psoriatic patients were recruited from King Chulalongkorn Memorial Hospital. The demographic data of subjects recruited in this study and the treatment each patient received are summarized in Table Twenty-nine Thai SLE patients were recruited from King Chulalongkorn Memorial Hospital and Bhumibol Adulyadej Hospital . Diagnosis of SLE was established according to the Revised American College of Rheumatology (ACR) criteria, and disease activity was evaluated by the SLE disease activity index (SLEDAI) 2000 score. Active SLE disease was defined as a SLEDAI-2K score of ≥6 and inactive SLE disease was defined as a SLEDAI-2K score of <6 ,18. RecrFor cell surface staining, anti-CD4-APC and anti-CD8-PerCP antibodies and FITC conjugated-streptavidin were purchased from BD Biosciences ; biotinylated anti-IL-23 receptor antibody was obtained from R&D Systems . Staining for intracellular cytokines was performed with the anti-IL-17A-PE antibody purchased from eBioscience .6 cells/ml) were immediately stained with cell surface markers and intracellular cytokines (referred to as Day 0) or incubated (1 × 106 cells/ml/well) in 24-well tissue culture plates pre-coated with anti-CD3 and anti-CD28 antibodies (1 μg/ml) (BD Pharmingen) in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 100 U/mL of penicillin, 100 μg/ml of streptomycin and non-essential amino acids  for 72 hours (referred to as Day 3). Ex vivo stimulated PBMCs were harvested and stained with cell surface markers and intracellular cytokines using the antibodies described above, and the data were analyzed by flow cytometry.Seventeen milliliters of venous peripheral blood from patients and normal subjects was collected in ACD blood collection tubes . PBMCs were isolated by Ficoll-Hypaque Isoprep . Isolated PBMCs (1 × 10Cells treated as indicated were resuspended in staining buffer (PBS supplemented with 0.02% sodium azide and 1% BSA). The conjugated antibodies for cell surface staining were added and cells were incubated at 4°C in the dark for 30 minutes. This step was followed by washing and fixing in 4% paraformaldehyde  for 20 minutes at room temperature in the dark. Fixed cells were washed and resuspended in permeabilization buffer  for 10 minutes at room temperature in the dark. The anti-IL-17-PE antibody was added to cells and the mixture was incubated for 30 minutes at 4°C in the dark before analysis on a flow cytometer. All samples were analyzed on a four-color FACSCalibur (BD Biosciences). Data analysis was performed with Summit software 5.0 .P < 0.05 were considered statistically significant. All probabilities were two-tailed.Statistical analysis was performed using GraphPad Prism 4 software . Differences among groups were compared using the Mann-Whitney U-test. Spearman's rank correlation test was used to assess the correlation of two variables. Probability values of + IL-23R+ and CD8+ IL-23R+ T lymphocytes were analyzed after gating on lymphocytes. As shown in Figures + IL-23R+ and CD8+ IL-23R+ than control subjects (P = 0.0021 for CD4+ and P = 0.0006 for CD8+ T cells). When SLE patients were divided into active and inactive groups based on SLEDAI scores, differences between each patient group and the control subjects remained statistically significant  in T lymphocytes from SLE patients, PBMCs were isolated and immediately stained for cell surface markers  and intracellular cytokines (IL-17) (Day 0). The percentages of CD4 Figures  and 2a. ex vivo. Upon ex vivo stimulation by plate-bound anti-CD3 and anti-CD28 antibodies for three days (Day 3), the higher proportion of CD4+ IL-23R+ and CD8+ IL-23R+ in all SLE patients remained statistically significant compared to the control group . Similar to the results obtained from Day 0, no statistical difference was found between inactive and active patients in percentages of IL-23R+ cells at Day 3. This may be due to the effect of the higher dose of immunosuppressive drugs given to patients in the active group. When we compared data from SLE patients with those from psoriatic patients, psoriatic patients showed significantly lower percentages of IL-23R+ T cells than SLE patients  but no difference from controls in percentages of CD4+ IL-17+ cells . Because the difference in IL-17+ T cell subsets between patients and the control group was not found at Day 0, the higher percentages of IL-17+ T cells after ex vivo stimulation strongly implies that T cells in the PBMCs of SLE patients preferentially skew towards IL-17 production and expand more vigorously than those of the control, both in the CD4+ and CD8+ compartments. Investigation into whether all IL-17+ T cells were limited to the IL-23R+ subset revealed that IL-17+ T cells exhibited both an IL-23R+ and IL-23R- phenotype (data not shown). No differences were found between normal group and psoriatic patient group  and in total SLE cases   . In addition, when we analyzed the correlation between disease severity using SLEDAI score and percentages of IL-23R+ T cells or IL-17+ T cells in SLE patients, only the positive correlation between SLEDAI and IL-23R+CD8+ T cells on Day 3 in active SLE group was found , but it did not reached statistical significance   are one of the characteristics exhibited by SLE patients and may be useful biomarkers for detection of severity and disease stage in SLE patients.When the correlation between percentages of IL-23R) Figure . Similar) Figure . The core Figure . Further) Figure . These rEven though the protective role of the IL-23/IL-17 axis is essential for certain bacterial and fungal infections, its contribution to the immunopathology of various autoimmune diseases has been highlighted . Recent + T lymphocytes as compared to those of the control subjects. Expression of IL-23R is reported to be restricted to effector and memory T lymphocytes and possibly to Th17 lineage cells. Therefore, our data suggest that SLE patients may have high numbers of effector and memory Th17 cells in circulation. This is consistent with a previous study using a lupus animal model, which showed that IL-23R-deficiency prevented the development of lupus nephritis in lpr/lpr mice [ex vivo stimulation, the percentages of IL-23R+ T lymphocytes in SLE patients remained higher than those of the control subjects. Because IL-23R can be expressed in both effector and memory cells, it will be of interest to test whether subsets of cells we detected in this study have effector or memory phenotypes. Human Th17 cells selectively expressing IL-17 with a memory phenotype can be further identified by chemokine markers, including CCR6 and CCR4 [In this study, we found that PBMCs from SLE patients contained higher percentages of IL-23Rlpr mice . Upon exand CCR4 .+ T cells from Day 0 to Day 3 upon ex vivo PBMCs stimulation, significant differences were detected between the control group and SLE patients. The positive correlation between IL-23R+ and IL-17+ T cells in SLE patients further highlighted the importance of IL-23R+ IL-17+ T cells in this disease. These data suggest that IL-17+ T cells vigorously expanded during ex vivo stimulation and probably reflect the higher frequency of IL-23R+ T cells in freshly isolated PBMCs from SLE patients. When we analyzed data from psoriatic patients, no detectable increase in either IL-23R+ or IL-17+ T cells were found. Since psoriasis is an autoimmune disease with both genetic and immunopathogenic links with IL-23/Th17 axis, this is somewhat unexpected. However, most reports on the links between psoriasis and increases in cells or molecules of the IL-23/Th17 axis focused on psoriatic skin lesions, and the report on increase in circulating Th17 in psoriasis did not use IL-23R but other markers to identify the Th17 population [+ T cells may be a specific characteristic of SLE but not psoriasis.When IL-17 was assessed in samples of freshly isolated PBMCs, we did not detect any statistical significance between controls and patients. Similarly, we found no detectable level of IL-17 in the sera of patients in our study; when sera IL-17 was measured by ELISA, only two sera samples from SLE patients yielded detectable IL-17 (5.12 and 6.17 pg/ml), while the rest of the samples from both the SLE patients and control subjects did not reach detectable levels (data not shown). In contrast, other studies have reported the elevation of both IL-17 and IL-23 in sera from patients [pulation ,24. Ther+ or IL-17+ T cells was not found in inactive SLE groups, but the correlation were found in active SLE patients between IL-23R+CD8+ T cells only after ex vivo stimulation. Even though this correlation did not reach statistical significance, this may imply that higher percentages of IL-23R+ T lymphocytes per se may not directly promote pathogenesis, but perhaps the cytokines produced by these cells act as effector molecules in the pathogenesis of SLE. Upon ex vivo stimulation, these pathogenic groups of cells are probably selectively expanded. Therefore, as long as the immunosuppressive drugs keep the cytokine-producing effector cells in check, the disease outcome may not be as severe, but the subsets of cells ready to produce such pro-inflammatory cytokines may be present in patients even when the disease is in an inactive stage.When SLE patients recruited in this study were classified into active and inactive groups based on disease severity by SLEDAI score, the correlation between disease severity and percentages of IL-23R+ T lymphocytes with an IL-23R+ phenotype were higher in SLE patients. Whether this subset of CD8+ T cell also produces IL-17 in vivo and whether it plays a role in the pathogenesis of SLE have not been documented. Recent evidence suggests that IL-23/IL-23R drives pathogenic IL-17-producing CD8+ T cells [+ T cells positive for IL-17 were higher in SLE patients. Interestingly, expanded, double-negative αβ T cells in SLE patients have been found to produce IL-17 and infiltrate the kidneys, and this subset of cells can be derived from CD8+ T cells with an inflammatory effector phenotype [+ CD8+ T cells may become double-negative T cells upon stimulation.In addition, we also found that CD8 T cells . Fold inhenotype ,26. FromTaken together, we added another supportive piece of evidence for the role of the IL-23/IL-17 axis in SLE, suggesting that these cytokines may play a central role in the pathogenesis of the disease. The results from our study may be useful for therapeutic intervention in SLE patients.+ T lymphocytes (both CD4+ and CD8+ subsets) than the control subjects in PBMCs and psoriatic patients. Upon ex vivo stimulation, PMBCs from SLE patients preferentially expanded IL-17 producing T cells, when compared with the normal subjects. The statistically significant correlation between percentages of IL-23R+ T cells and IL-17+ T cells, and IL-17+ T cells and SLEDAI scores were found in active SLE patients. Taken together, this study suggests that the IL-23/IL-17 axis is implicated in the pathogenesis of SLE.SLE patients, regardless of disease severity, have higher percentages of IL-23RIL: interleukin; PMBCs: peripheral blood mononuclear cells; SLE: systemic lupus erythematosus; SLEDAI: SLE disease activity index.The authors declare that they have no competing interests.All authors contributed to the design of the study, and to the acquisition and interpretation of data. HP and PS carried out the analysis by flow cytometry. YA, NH and JW evaluated patients' data. TP and NH drafted the manuscript. All authors read and approved the final manuscript.