Respiratory syncytial virus (RSV) is a high priority target for vaccine development. One concern in RSV vaccine development is that a non-live virus vaccine would predispose for enhanced disease similar to that seen with the formalin inactivated RSV (FI-RSV) vaccine. Since a mAb specific to RSV G protein can reduce pulmonary inflammation and eosinophilia seen after RSV infection of FI-RSV vaccinated mice, we hypothesized that RSV G peptides that induce antibodies with similar reactivity may limit enhanced disease after subunit or other non-live RSV vaccines. In support of this hypothesis, we show that FI-RSV vaccinated mice administered RSV G peptide vaccines had a significant reduction in enhanced disease after RSV challenge. These data support the importance of RSV G during infection to RSV disease pathogenesis and suggest that use of appropriately designed G peptide vaccines to reduce the risk of enhanced disease with non-live RSV vaccines merits further study.  As the single most important cause of lower respiratory tract infections in the infant and young child, respiratory syncytial virus (RSV) is a high priority target for vaccine development,2. UnforRecent studies of the role of antibodies blocking the activities of the RSV G CX3C chemokine motif suggest a new approach to improving the safety of an RSV vaccine. The RSV G has been shown to modify the immune response to RSV infection in mice, in particular by contributing to vaccine enhanced disease, by inducing pulmonary eosinophils, and increasing production of Th2 cytokines -19. StudThe study was performed in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Centers for Disease Control and Prevention (CDC) Institutional Animal Care and Use Committee (Protocol Number: 1771HAYMOUC). No surgeries were performed. All efforts were made to minimize animal suffering during all procedures performed. g, 4°C and resuspending the pellet at 1/25 of the original volume in SF-DMEM. The virus was centrifuged and the supernatant precipitated with aluminum hydroxide (4mg/ml) overnight at 4°C. The precipitate was pelleted at 1500 x g, 4°C for 15 minutes and resuspended in a 1:100 dilution of the original volume in SF-DMEM. FI-Vero was prepared as described above using uninfected Vero cell lysate.RSV A2 or B1 was propagated in Vero cells (ATCC CCL 881) as previously described . At ~ 80d-restricted 12-mer peptide from the L protein (aa 393-405) (accession number AAA84898) of RSV was used as a negative control immunogen (The following G protein peptides were synthesized and used for immunization: two G protein peptides from the Group A conserved region (amino acids (aa) 163-190) based on an RSV A2 strain aa sequence  and an Rmmunogen . The pep6 PFU equivalents of FI-A2 or FI-vero lysate. Groups of mice were then vaccinated with 50-75 µg of KLH-conjugated G peptides based on RSV A2 conserved regions, while a polymerase peptide was used as a negative control. Initial immunizations consisted of peptide emulsified 1:1 with TiterMax Gold (TiterMax) administered s.c while booster immunizations consisted of 50 µg peptide in sterile PBS administered i.p. at 14 and 28 days post-vaccination. Two weeks following final G peptide boost, the FI-RSV immunized mice, with or without the G peptide vaccination, were anesthetized by i.p. administration of Avertin , and intranasally (i.n.) challenged with 106 PFU of RSV virus, 106 PFU RSV A2, equivalent PFUs of UV-inactivated RSV A2, or uninfected, vero-lysate control in SF-DMEM. UV-inactivated RSV A2 was confirmed inactivated by standard plaque assay. BALB/c mice  were housed in accordance with the guidelines of the CDC Institutional Animal Care and Use Committee. Mice were i.m. vaccinated with 10After challenge, at the timepoints indicated, mice were anesthetized with Avertin and exsanguinated by severing the right brachial artery. Bronchoalveolar leukocyte (BAL) cells were harvested by lavaging the lungs with sterile Dulbecco’s PBS (D-PBS) and BAL cell numbers were counted. Lungs were removed and stored at -80°C until use. No fewer than three mice per treatment per time point were examined. Formalin-fixed lung tissues and BAL cell morphology were determined by hematoxylin and eosin staining and light microscopy analysis as previously described . BAL celThe procedure used for extracellular staining of BAL cells was modified for microculture staining as described . BrieflyTotal RNA was extracted from homogenized lung tissue using a RNA purification kit  per manufacturer’s protocol and expression of the RSV M gene was determined by real-time qRT-PCR (Stratagene). Results are presented as relative genome level in PFU equivalents/ml and were measured against a RSV RNA standard curve.Cell-free BAL supernatants were assayed for IFN-γ and IL-4 by multiplex bead based assay according to the manufacturer’s instructions (Bio-Rad) or by ELISA (eBioscience). Concentration of the cytokine was determined from a standard curve and data are presented as the mean pg/ml for individual mice for each group in duplicate. Mouse sera were screened for whole RSV and anti-peptide antibody titers using a modified indirect ELISA as previously described . Briefly5 PFU/well) and incubated for 1 hr at 37°C. After incubation, the virus-serum mixtures were added to microtiter plates containing 80% confluent monolayers of Vero cells, and incubated at 37°C for 2 hrs. After virus adsorption, the cell monolayers were overlaid with DMEM + 10% fetal bovine serum (DMEM+10%FBS), incubated at 37°C for three days, the monolayers fixed with a 80% to 20% acetone-PBS mixture, and immunostained with a pool of anti-F and anti-G protein mAbs  followed by horseradish peroxidase-conjugated goat anti-mouse IgG/IgM/IgA . Color was developed with ABTS (KPL) and OD measured at 410 nm. Each serum pool was assayed in triplicate, and the percent neutralization at each dilution was determined as the average value of ((ODvirus infection without antibody – ODvirus+serum antibody)/ODvirus infection without antibody)*100% for the three replicates.  Heat-inactivated serum from immunized mice was used to determine neutralizing antibody titers as previously described . Brieflyt test, where a P value of <0.05 was considered statistically significant. Data are expressed as the mean + standard error of the mean (SEM).Data were analyzed for statistical significance using a Student’s  In our FI-RSV model, peak inflammation occurs on day 5 post-challenge (p.c.) with live RSV  and we cThe RSV G peptide, aa 163-190 of RSV A2 strain (G-A2), used in this study contained the G CX3C motif and is relatively conserved among the RSV Group A strains . This G We have recently reported that vaccination with RSV G peptides containing the CX3C motif prevented body weight loss and pulmonary inflammation in BALB/c mice after acute RSV infection . To eval+ cells  and B1  led to a significant decrease in the number of BAL cells and pulmonary eosinophils and a marked decrease in IL-4 levels   and group B (52 a.a.) conserved region peptides should be capable of inducing antibodies against all known RSV strains and could protect from both natural and vaccine associated enhanced disease. A similarly constructed RSV G peptide vaccine that spans the central conserved region was recently shown to decrease pulmonary inflammation seen after primary RSV infection . One major concern about any RSV G based vaccine is based on studies that have shown that vaccination with intact or secreted RSV G can cause enhanced inflammation, including eosinophilia upon RSV challenge -18,32. IThe possibility that peptide vaccination might be used to modify the cellular immune response to RSV challenged after FI-RSV vaccination has not previously been considered. Induction of a Th2-type biased adaptive immune response by FI-RSV is considered a likely contributor to the enhanced disease with later infection ,34-38. GIn conclusion, our data demonstrate that an appropriately designed RSV G peptide vaccine can prevent enhanced disease under the most challenging circumstances, i.e. FI-RSV vaccination, and consequently suggest use of a RSV G peptide vaccine to improve the safety profile of other RSV vaccines merits further study. It is possible that co-vaccination with a RSV G peptide could provide the safe use of a non-live virus vaccine in the RSV naïve child. These data also support a role for the RSV G in the challenge virus in FI-RSV enhanced disease and are another approach to clarifying the pathogenesis of enhanced disease. RSV G peptide vaccines and their potential contribution to an RSV vaccine merits further study. Figure S1Decreased pulmonary cell inflammatory response in FI-A2 or FI-B1 and RSV G-CH17 or G-B1 peptide vaccinated mice after RSV challenge.Mice were i.m. vaccinated with 106 PFU equivalents of formalin inactivated (FI) RSV A2 (FI-A2) or FI-RSV B1 and then s.c. vaccinated with RSV CH17 G peptide (FI-A2+G-CH17) or RSV B1 G peptide (FI-B1+G-B1) as described in the Materials and Methods. Two weeks following the final boost with respective peptides, groups were challenged with either live RSV A2 (FI-A2+G-CH17) or RSV B1 (FI-B1+G-B1) and mean numbers of BAL cells/lung (A-B), total eosinophil counts in the BAL (C-D), and IL-4 levels (pg/ml) in the cell-free BAL supernatant (E-F) were determined. Error bars represent the SEM. Significance was calculated using a Student's t test comparing mice vaccinated with FI-RSV alone and mice vaccinated with both FI-RSV and respective RSV G peptide at indicated time points after RSV challenge. Representative data from three independent experiments are shown. * p<0.05 G-CH17 peptide represents the amino acid sequence (aa 163-190) for the RSV group A strain CH17 (FHFEVFNFVPCSICSNNPTCWDICKRIP), and G-B1 peptide (PPKKPKDDYHFEVFNFVPCSICGNNQLCKSICKTIPSNKPKKKPTIKPTNKP) represents the amino acid sequence (aa 155-206) for the RSV group B strain B18537. (TIF)Click here for additional data file.


Natural killer (NK) cells can kill tumor cells in a non-MHC-restricted manner. However, cancer cells frequently escape from the attack of NK cells by multiple ways. In this study, we investigated the effect of gefitinib on the interaction between NK cells and lung cancer cells.51Cr release assay, CD107a assay, and IFN-γ secretion assay were performed to detect the sensitivity of lung cancer cell lines A549 and H1975 to NK cells cytotoxicity in the presence of gefitinib. Human NK cells were co-cultured with A549 and H1975 cell lines in the presence of gefitinib. NKG2D ligands, ULBP1, ULBP2, MICA, and MHC-I on tumor cells, and NKG2D, NKp44 and NKp46 on NK cells were evaluated with flow cytometry. 51Cr release assay was performed when NKG2D antibody were added into the co-culture system. Expressions of stat3 and LC3 I/II on tumor cells were determined with western blot after co-cultured with NK cells. After treated with gefitinib, mannose-6-phosphate receptor (MPR) on H1975 cells was evaluated by flow cytometry. 51Cr release assay were performed when MPR antagonist were used.Gefitinib increased cytotoxicity of NK cells to human lung cancer H1975 cells with EGFR L858R + T790M mutations, while not in A549 cells with wild type EGFR. Gefitinib could block the immune escape by up-regulating the expression of NKG2D ligands ULBP1, ULBP2 or MICA on tumor cells and NKG2D on NK cells in the co-culture system. Gefitinib and NK cells up-regulated MHC-I expression in A549 while not in H1975 cells. NKG2D antibody blocked the enhanced NK cytotoxicity by gefitinib. The combination of NK cells and gefitinib could significantly down-regulate stat3 expression. Furthermore, NK cells-mediated tumor cell autophagy was observed in A549 cells while not in H1975 cells. Notably, gefitinib increased autophagy and MPR expression in H1975 cells, which improved the sensitivity to NK cell-based immunotherapy.Gefitinib greatly enhanced NK cell cytotoxicity to lung cancer cells with EGFR L858R + T790M resistance mutation. Combination of EGFR tyrokinase inhibitors and NK cells adoptive immunotherapy may represent a potentially effective strategy for patients with non-small cell lung cancer. Lung cancer is a leading cancer death worldwide. The useNK cells are key components of innate immunity and participate in immunity against virus-infected and neoplastic cells. NK cellHowever, several factors limited the efficiency of NK cells adoptive therapy. Except for its poor ability to home to tumor area, tumor microenvironment edited NK cells and changed NK cell response-26. ReceSeveral immunomodulatory approaches have been investigated to enhance anti-tumor therapy efficiency. Imatinib potentiates antitumor T cell responses through the inhibition of IDO. ImatiniHuman NSCLC cell lines A549 and H1975 were obtained from American Type Culture Collection  and maintained in RPMI 1640 media (Hyclone) supplemented with 10% FBS (GIBCO). NK cells were obtained from peripheral blood of different health donors by magnetic bead isolation using NK isolation kit (R&D systems) according to the manufacturer’s instructions. NK cell purity was >85%. All of the researches were performed in accordance with the Sichuan University’s Ethics Committees. NK cells were maintained in RPMI 1640 media supplemented with 200 U/ml IL-2 and 10 ng/ml IL-15 and 10% FBS.Primary NK cells were stained with CD56 (R&D systems) and CD3 antibodies (BD Pharmingen). Cells were acquired on a FACSCalibur flow cytometer and data were analyzed using Cell Quest software (BD Biosciences). NK cells were co-cultured with the indicated target cells in a ratio of 1:1 in 24-well plates for 24 hours, and 5 μg/ml gefitinib (Astra Zeneca) was added into co-culture system for another 24 hours. Afterward, NK cells were collected and examined for the expression of NKG2D, NKp44, and NKp46. ULBP1, ULBP2, MICA expression were evaluated on tumor cells. Intracellular IFN-γ staining was performed after fixation in 2% paraformaldehyde and permeabilization in 1% Trixton. IFN-γ-PE antibody was purchased from BD Pharmingen. Antibodies against NKG2D, NKp44, NKp46, ULBP1, ULBP2, MICA were purchased from R&D systems. For MPR expression, H1975 tumor cells were treated with gefitinib for 48 hours, and then the MPR levels on cell surface was evaluated by flowcytometry.NK cells were co-cultured with the indicated target cells in a ratio of 1:1 in the presence CD107a antibody (R&D systems) for 4 h in the presence or absence of 5 μg/ml gefitinib. Afterward, cells were washed and CD107a levels on the NK cells were then analyzed by flow cytometry.Tumor cells were harvested and lysed in radio-immunoprecipitation buffer for 30 min. Protein concentration was determined by Bradford assay. Cell lysates were resolved by SDS-PAGE, and transferred to PVDF membrane (Millipore). Membrane was blocked in 5% non-fat milk and then blots were probed with antibodies for stat3 and LC3  respectively. After incubated with horseradish peroxidase-conjugated secondary antibodies, probes were visualized by a chemiluminescent detection system. GAPDH as a loading control. Antibody against GAPDH was obtained from Cell Signaling Technology.2 51CrO4 for 1 h at 37°C. Cells were then washed three times with complete medium and incubated with effector cells at different E:T ratios in the presence or absence of 5 μg/ml gefitinib. After incubation for 4 h at 37°C, cell free supernatants were collected and counted on scintillation counter. Percentage of cytolysis was calculated by (sample release–spontaneous release)/(maximum release–spontaneous release). To block the cytotoxicity of NK cells, mannose-6-phosphate or 20 μg/mL NKG2D antibody (R&D system) were added into the 51Cr release assay system.Target cells were labeled with 1 mCi of Nap < 0.05 was considered statistically significant.ANOVA was used to identify significant group differences. 51Cr releasing assay was performed. Two gefitinib resistance NSCLC cell lines A549 (EGFR wt) and H1975 (EGFR L858R + T790M) were used. In the presence of gefitinib, A549 showed some more enhanced susceptibility to NK cells cytotxicity; however, there were no significant difference , CD134 OX40) and glucocorticoid-induced TNFR (GITR), which play important roles in immune synapses. CD137-s and glucRecently, immune system has been demonstrated to contribute substantially to the antitumor effects of small molecule inhibitors. Through the inhibition of IDO, imatinib potentiates antitumor T cell responses in gastrointestinal stromal tumor. ImatiniOne of the major reasons for the weak effect of cell based immunotherapy is thought to be immunosuppression. Tumor microenvironment, with abundant of immunosuppressive cells and molecules, can inhibit effector cells and result in insufficient antitumor effects-26. StatIn our present study, we also find that high purity NK cells increase autophagy in A549 cancer cells with wide type EGFR, while not in H1975 cells with EGFR L858R + T790M. Lymphocyte provides lytic signals to tumor cells, and they also promote autophagy in the remaining tumor cells. These processes are primarily mediated by NK cells. Cell-meOur present study suggests that gefitinib has multiple effects on the interaction between NK cells and tumor cells. Similar to imatinib, gefitinib has its own immunomodulatory property, which can enhance NK cell cytotoxicity. Gefitinib enhances NKG2D-NKG2D ligands interaction between NK cells and human lung cancer cells. Combination of gefitinib with NK cells down-regulates stat3 expression. MPR expression induced by gefitinib facilitates antitumor NK cell immunity. Therapeutic significance of our finding is that administration of gefitinib may offer a novel adjuvant strategy to enhance NK cells based immunotherapy in NSCLC with EGFR L858R + T790M resistance mutation.EGFR: Epithelial growth factor; NK cell: Natural killer cell; TKI: Tyrosine kinase inhibitors; MPR: Mannose-6-phosphate receptor; NSCLC: Non-small cell lung cancers.The authors declare that they have no competing interests.Conception and design: W-YS, H-SS, YT. Development of methodology: W-YS, H-SS, YT, WY. Acquisition of data: H-SS, YT, LD, G-FC, M-XL, Y-TH, S-JH, L-ZQ. Analysis and interpretation of data: W-YS, H-SS, YT, GX, WY. Writing, review, and/or revision of the manuscript: W-YS, H-SS, YT,GX. Administrative, technical, or material support: G-FC, M-XL, Y-TH, S-JH, L-ZQ Study supervision: W-YS, H-SS, YT. All authors read and approved the final manuscript.Sisi He and TaoYin are the co-first authors for this paper.


Nutrition support for critically ill patients supplemented with additional modular protein may promote skeletal muscle protein anabolism in addition to counteracting acute nitrogen loss. The present study was designed to investigate whether the essential amino acid (EAA) enriched high-protein enteral nutrition (EN) modulates the insulin-like growth factor-1 (IGF-1) system and activates the mammalian target of rapamycin (mTOR) anabolic signaling pathway in a trauma-hemorrhagic shock (T-HS) rat model.Male Sprague-Dawley rats  were randomly assigned to 5 groups: (1) normal control, (2) pair-fed, (3) T-HS, (4) T-HS and standard EN, and (5) T-HS and EAA enriched high-protein EN. Six animals from each group were harvested on days 2, 4, and 6 for serum, gastrocnemius, soleus, and extensor digitorum longus sample collection. T-HS significantly reduced muscle mass. Nutrition support maintained muscle mass, especially the EAA enriched high-protein EN. Meanwhile, a pronounced derangement in IGF-1-IGFBPs axis as well as impaired mTOR transduction was observed in the T-HS group. Compared with animals receiving standard EN, those receiving EAA enriched high-protein EN presented 18% higher serum free IGF-1 levels following 3 days of nutrition support and 22% higher after 5 days. These changes were consistent with the concomitant elevation in serum insulin and reduction in corticosterone levels. In addition, phosphorylations of downstream anabolic signaling effectors - including protein kinase B, mTOR, and ribosomal protein S6 kinase1 - increased significantly in rats receiving EAA enriched high-protein EN.Our findings firstly demonstrate the beneficial effect of EAA enriched high-protein EN on the metabolic modulation of skeletal muscle protein anabolism by regulating the IGF-1 system and downstream anabolic signaling transduction. Increased nitrogen loss and skeletal muscle wasting are metabolic features in patients under catabolic stress. Nearly 20% of total body protein can be lost in critically ill patients IGF-1 circulating in the peripheral vasculature bonds to specific binding proteins. Approximate 95% of circulating IGF-1 forms a ternary complex consisting with insulin-like growth factor binding protein 3 (IGFBP-3) and an acid-labile subunit (ALS) et al.Endogenous IGF-1 synthesis is quite sensitive to nutrient availability. In previous work, a 60% reduction in plasma IGF-1 concentrations as well as 8% decrease in gastrocnemius mass was observed in rats receiving a protein-free diet, while recovering to normal after standard casein diet refeeding The essential role of nutrition support for critically ill patients is to promote protein anabolism and protect the lean body mass and function. Historically, providing equivalent amounts of protein or amino acids in nutrition support attempted to counteract the dramatic nitrogen loss Therefore, the purpose of the present study was to investigate the role of long-term enteral nutrition (EN), in particular EAA enriched high-protein EN, upon the regulation of IGF-1 system function and downstream mTOR-related signaling transduction in a Sprague-Dawley (SD) rat model of trauma-hemorrhagic shock (T-HS). Serum levels of insulin and corticosterone were also measured, since both factors were implicated in the regulation of IGF-1 by their respective roles in IGFBP regulation ad libitum. All procedures were carried out in accordance with the “Guide for the Care and Use of Laboratory Animals” published by the National Institutes of Health (NIH publication 86-23 revised 1985) and following protocols approved by the Animal Care and Use Committee of Jinling Hospital.Male SD rats weighted 278.18±0.94 g  were housed in a temperature (25∼28°C) and light (12∶12-hour light-dark cycle) controlled environment for 1 week prior to the experiment. Water and standard chow diet were provided ad libitum overnight.Following the acclimatization period, rats were fasted overnight (Day-1). All surgeries were performed using sterile technique. At day 0, rats were anesthetized by intraperitoneal injection of ketamine (100 mg/kg body weight (BW)) and placed on a temperature-controlled heating pad. A midline laparotomy (5 cm) was made to insert a catheter  into the anterior wall of the stomach followed by suturing to the stomach wall and exteriorizing through the anterolateral abdominal wall. The catheter was then subcutaneously tunneled to the shoulder region, and connected through a customized rotating swivel to a 50 mL syringe pump . An additional groin incision was made. The left femoral artery was catheterized for bleeding, monitoring mean arterial pressure (MAP), and fluid resuscitation through a three-way microvalve. Rats were bled to a target MAP of 30∼35 mmHg within 15 minutes and maintained for 45 minutes. Blood pressure was recorded in 5-minute intervals. At the end of hemorrhagic shock period, rats were resuscitated by infusing their shed blood and lactated Ringer’s solution in a 1∶2 ratio. All rats were allowed food and water ad libitum throughout the study.Animals were randomized to 5 groups. At T-HS 3 groups, rats received either standard EN , EAA enriched high-protein EN , or a continue infusion of isotonic saline but on normal chow diet . One group that was pair-fed  with the T-HS/Ctr group was set to control for the influence of reduced food intake post-operatively. Rats in the normal control group  were under no treatment and fed standard chow diet . Additional nitrogen supplementation in the T-HS/EAA group was provided by leucine enriched EAA solution containing 0.849 g N/(kg BW·d) based on the ENSURE® diet. Therefore, the ratio of non-protein calorie to nitrogen decreases to 100∶1. The composition of the EAA mixture is provided in Following overnight recovery (Day 1), chow diet was removed from rats in the T-HS/SE and T-HS/EAA groups. A continuous EN infusion was started and provided for the subsequent 5 days, providing a non-protein calorie of 250 kcal/(kg BW·d). The T-HS/SE rats received ENSURE®  (64.38 g/(kg BW·d), providing 1.65 g N/(kg BW·d)) as the standard enteral formula, in which the ratio of non-protein calorie to nitrogen is 152∶1 . AdditioIn the T-HS/Ctr group, rats received saline at a constant rate of 0.3 mL/(100 g BW·h) through the enteral routine and had free access to chow diet. Food intake in PF rats was restricted to the T-HS/Ctr group. All rats were allowed free access to water.BW and the amount of EN solution infused were recorded daily. Frequency of diarrhea was also observed to evaluate the tolerance of EN in the T-HS/SE and T-HS/EAA rats.Rats (n = 6) were euthanized from each group on days 2, 4, and 6, respectively. EN was stopped 1 hour before sacrifice in the T-HS/SE and T-HS/EAA groups. After anesthesia, blood was collected by cardiac puncture. Serum was isolated by centrifuge at 4000 rpm at 4°C for 10 minutes and then kept at −80°C until further measurements.The gastrocnemius, soleus, and extensor digitorum longus (EDL) of the right leg were dissected out, weighted, wrapped, and then snap frozen in liquid nitrogen within 3 minutes. All samples were then stored at −80°C until analysis.In contrast to measuring total serum IGF-1 levels, we utilized a commercial IGF-1 ELISA kit from R&D Inc.  to determine the free serum IGF-1 concentrations, which represent the “bioavailable” portion of the circulating IGF-1 pool 3VO4, 25 mM β-glycerophosphate, 1 mM PMSF, and 1% protein inhibitor Cocktail  for 30 minutes on ice. The lysate was then centrifuged at 12,000 g at 4°C for 8 minutes, and the supernatant was collected. Total protein concentration was determined using the BCA assay . The supernatant was diluted (4∶1) in a 5× loading buffer  and then boiled at 100°C for 5 minutes.Muscle preparation and western blotting analysis were performed as previously detailed Equal amounts of total protein (75 ug) were separated by 8% or 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) determined by the size of target protein and then transferred to polyvinylidene difluoride membrane . Blots were blocked in 5% BSA for 1 hour at room temperature and incubated in specific primary antibodies overnight at 4°C. Blots were then washed in TBST  three times followed by incubation with secondary antibody for 2 hours at room temperature. The protein bands were visualized by a chemiluminescence detection system  with exposure to Kodak XAR film (Eastman Kodak). Band density was analyzed using Quantity One 4.6.2 software (BioRad). Specific total protein was re-probed after stripping the phospho-primary and secondary antibody. Phosphorylation data are described relative to the total protein expression after normalization by internal loading control.Antibodies that detected α-tublin (1∶1000), phospho-mTOR , S6K1 (1∶1000), phospho-S6K1 , and phospho-Akt  were from Cell Signaling Technology . Akt (1∶1000) and mTOR (1∶1000) antibodies were from Abcam . Anti-rabbit IgG horseradish peroxidase-conjugated secondary antibody (1∶3000) was from Kirkegaard & Perry Laboratories (KPL) Biotechnology Inc. .Serum amino acid concentrations were assayed by a Waters Aliance 2695 HPLC system  after pre-column derivatization with phenylisothicyanate (PITC) (Thermo Scientific (Pierce), Rockford, IL, USA) by our previous method p value <0.05 was considered significant.Results are expressed as mean ± SE. Repeated-measures ANOVA was used to characterize dietary intake and BW gain during the 6-day recovery period. For other purposes, statistical evaluation among groups was performed by one-way ANOVA followed by LSD post-hoc analysis, using SPSS software package . In the case of heterogeneous variances, we used Dunnet’s T3 test. A . Early EN failed to increase the incidence of diarrhea, indicating that rats in the T-HS/SE and T-HS/EAA groups tolerated the nutrition support. After T-HS, rats consumed less chow (p<0.05) for 4 days but achieved normal by day 5, when the food intake did not differ between the T-HS/Ctr and NC groups . The daily caloric intake of the T-HS/SE and T-HS/EAA groups is additionally provided in The average incidence of diarrhea was 0.44/day on the first day following T-HS, and gradually disappeared at day 3 . Early E. Early E. Rats in the T-HS/Ctr group presented a relatively slow gain of BW during the recovery period. Nutrition support was thought to be beneficial for achieving weight gain, especially the EAA enriched high-protein EN, however no significant difference between groups was observed at any time point.Animals weighed an average of 278.18±0.94 g at the beginning of the study. BW was low in all T-HS injured rats throughout the study, but did increase with time . Gastrocnemius masses in the T-HS/SE and T-HS/EAA groups were significantly higher than the T-HS/Ctr group by 2 days post-T-HS (p<0.01), and this trend continued throughout the remainder of the study. In addition, the T-HS/EAA group exhibited a significant increase in gastrocnemius mass compared with the T-HS/SE group on days 4 and 6, respectively (p<0.01). However, both EN treatments had limited improvement in EDL mass maintenance. For soleus mass, a slight but without significant decrease was observed after T-HS . EAA enriched high-protein EN caused a significant increase in soleus wet muscle mass by day 6, which was comparable with the NC group (p<0.05).T-HS induced significant reductions in gastrocnemius and EDL masses . Gastroc. Following this time point, free IGF-1 concentrations increased gradually, but most slowly in the T-HS/Ctr group, which remained statistically lower than the other EN-treated groups . In addition, lower free IGF-1 levels were measured in the T-HS/Ctr group than PF group at day 4 and it reached statistical difference by day 6 (p<0.01), indicating that the depressed IGF-1 expression was sustained 6 days following T-HS after eliminating the interference of reduced food intake. Furthermore, free IGF-1 levels were 18% higher in the T-HS/EAA group than T-HS/SE group following 3 days of nutrition support (p<0.05). And a greater magnitude was observed by day 6 .Serum free IGF-1 concentrations in the PF, T-HS/Ctr, T-HS/SE, and T-HS/EAA groups were significantly decreased by day 2 . In the T-HS/Ctr group, IGFBP-3 levels remained lower than the PF group on days 4 and 6, respectively (p<0.01). Following enteral feeding, there was a significant elevation in IGFBP-3 levels compared with the T-HS/Ctr group (p<0.01). Moreover, EAA enriched high-protein EN-fed rats exhibited a 170% and 140% increase in IGFBP-3 levels compared with animals receiving standard EN on days 4 and 6, respectively (p<0.01).The pattern of serum IGFBP-3 concentrations between groups was similar to that observed in free IGF-1 . T-HS increased IGFBP-1 concentrations in the T-HS/Ctr group by 46% compared with the PF group at day 2 (p<0.01), and this trend was likely to continue throughout the study period. Although both groups of enterally fed rats exhibited significant decreases in IGFBP-1 levels compared with rats in the T-HS/Ctr group at each time point (p<0.01), the reduction was greater in the T-HS/EAA group (p<0.01).Serum IGFBP-1 levels in the NC group were markedly lower than the other groups at each time point, with the exception of day 6 when IGFBP-1 levels in the T-HS/EAA group decreased to levels comparable with the NC group p<0.05, at day 2; p<0.01, at day 4 or 6; p<0.01). Moreover, a greater magnitude of feeding-induced hyperinsulinemia was observed in the T-HS/EAA group than T-HS/SE group on days 4 and 6, respectively . Regression analysis of insulin and IGF-1 further indicated a significantly positive liner relationship .Insulin levels in the T-HS/Ctr group were significantly decreased compared with the PF group at all the time points . Meanwhile, corticosterone levels in the T-HS/EAA group appeared lower than T-HS/SE group on days 4 and 6, but without statistical difference.Serum corticosterone concentrations in rats subjected to T-HS significantly increased by day 2 , coinciding with the fact that free IGF-1 levels peaked at day 6. Meanwhile, the phosphorylation of mTOR at Ser2448 in the T-HS/Ctr group decreased to 43.3% and 45.7% compared with the NC and PF groups, respectively . As the important downstream effector of mTOR signaling, Thr389-phosphorylation of S6K1 was also measured. S6K1 phosphorylations in the PF and T-HS/Ctr groups were similar, but markedly lower than the NC group . Furthermore, phospho-S6K1 levels in the T-HS/EAA group were also higher than the T-HS/SE group (p<0.05).To further assess the signaling transduction associated with muscle protein synthesis, phosphorylations of several key proteins involved in mTOR signaling pathway were determined in gastrocnemius at day 6. After T-HS, phosphorylation of Akt at Ser473 in the T-HS/Ctr group was greatly suppressed , and the leucine and valine concentrations were even higher than that observed in the T-HS/SE group (p<0.01). Similarly, blood levels of other EAA were increased after EN treatment, but with greater in the T-HS/EAA group .There was no significant decrease in branched chain amino acid (BCAA) concentrations in the T-HS/Ctr group compared with the NC or PF group at day 6, with the exception of valine , which was in line with the previous work The composition of EAA supplementation used in this study contained a large proportion of leucine. Recently, leucine is shown to be capable of stimulating insulin secretion et al.However, Ma Endogenous glucocorticoids also mediate IGFBP-1 production. Previous work demonstrated that pretreating animals with glucocorticoid receptor antagonist RU486 partially prevented burn-induced increase in IGFBP-1 and further contributed to the increase in plasma IGF-1 levels Previous studies using a similar EAA formula or leucine alone to investigate the stimulatory effect upon skeletal muscle protein synthesis did not demonstrate changes in serum IGF-1 et al.We also detected that EAA enriched high-protein EN significantly enhanced the activation of mTOR signaling pathway; phosphorylations of mTOR and its downstream effector S6K1 in rats receiving EAA enriched high-protein formula increased nearly two-fold compared with those receiving standard EN. Recently, a large body of evidence has established that amino acid, especially leucine, can be considered as a signaling molecule to stimulate protein translation initiation through an Akt-independent pathway. Acute leucine ingestion can effectively increase protein synthesis and promote the mTOR-related signaling transduction in both normal and stress conditions et al.et al.The PI3K-Akt pathway, upstream of mTOR, can be phosphorylated by ligands including IGF-1 and insulin and that in turn stimulates the mTOR phosphorylation to induce the muscle protein translation initiation. Previous studies demonstrated the rapid development of insulin resistance in skeletal muscle following T-HS, with the earliest insulin signaling defect occurring at 60 minutes In our study, three later time points following T-HS were selected. Activated Akt was observed at day 6 in both enterally fed groups, but with greater in rats receiving EAA enriched high-protein EN, coinciding with the emergence of significant elevation in both insulin and IGF-1 levels. Previous studies demonstrated the protein retention effect of insulin was likely to be exerted by reducing the protein catabolism after trauma or surgery, because no correlation between changes in muscle protein synthesis and insulin sensitivity was found A limitation to the current study is that it is not yet clear whether the increased serum free IGF-1 levels following EAA-enriched high-protein EN are due to the overall increase in IGF-1 production or reduced IGF-1 degradation. As mentioned previously, IGFBP-3 has been shown to be regulated by proteolysis. Increasing proteolytic cleavage of IGFBP-3 causes a decreased affinity for IGF-1, which is responsible for the decrease in circulating IGF-1 levels during stress In summary, our findings firstly demonstrate the effect of EAA enriched high-protein EN upon the metabolic regulation of skeletal muscle protein anabolism by regulating the IGF-1 system and downstream anabolic signaling transduction. We demonstrated that early nutrition support favored the recovery of serum free IGF-1 levels following T-HS, and the EAA enriched high-protein EN treatment appeared more effective. It was beneficial for the muscle mass maintenance in T-HS injured rats. Moreover, long-term treatment with EAA enriched high-protein EN effectively activated the mTOR related translation initiation factors, indicating the stimulation of muscle protein synthesis. And this response might be based on cooperation between IGF-1- and nutrient- mediated signal transduction pathways.


Interleukin (IL)-21 promotes differentiation of Th17 cells, long-term maintenance of functional CD8+ T cells, and differentiation of memory B cells and antibody-secreting plasma cells. We hypothesized that administration of IL-21 will improve mucosal function in the context of pathogenic HIV/SIV infections. To test this hypothesis, we infected 12 RMs with SIVmac239 and at day 14 post-infection treated six of them with rhesus rIL-21-IgFc. IL-21-treatment was safe and did not increase plasma viral load or systemic immune activation. Compared to untreated animals, IL-21-treated RMs showed (i) higher expression of perforin and granzyme B in total and SIV-specific CD8+ T cells and (ii) higher levels of intestinal Th17 cells. Remarkably, increased levels of Th17 cells were associated with reduced levels of intestinal T cell proliferation, microbial translocation and systemic activation/inflammation in the chronic infection. In conclusion, IL-21-treatment in SIV-infected RMs improved mucosal immune function through enhanced preservation of Th17 cells. Further preclinical studies of IL-21 may be warranted to test its potential use during chronic infection in conjunction with antiretroviral therapy.In pathogenic HIV and SIV infections of humans and rhesus macaques (RMs), preferential depletion of CD4 + Th17 cells occurs during the early stage of pathogenic HIV/SIV infections and correlates with loss of mucosal integrity, microbial translocation, immune activation and disease progression. As such, therapeutic intervention aimed at preserving intestinal Th17 cells may be of critical importance. IL-21 plays an important role in promoting the differentiation and survival of Th17 cells, as well as in stimulating CD8+ T cell cytolytic function. Here, we treated SIV-infected rhesus macaques with IL-21-IgFc in the early stage of infection. Consistent with the main functions of IL-21, we found that IL-21 treated animals had higher expression of perforin and granzyme B in total and SIV-specific CD8+ T cells and higher frequencies of intestinal Th17 cells as compared to untreated controls. Remarkably, the increased proportions of Th17 cells during early infection was associated with significantly lower levels of intestinal T cell proliferation, microbial translocation and systemic activation/inflammation during chronic infection. Thus, our results suggest that IL-21 treatment in SIV-infected RMs is effective in preserving intestinal Th17 cells and results in an improvement of mucosal immune function. Further preclinical studies may be warranted to test IL-21 during chronic infection and in conjunction with antiretroviral therapy.In the gastrointestinal tract, preferential depletion of CD4 Our results indicate that in vivo administration of IL-21 during acute SIV infection beneficially impacts mucosal immune function by elevating levels of intestinal Th17 cells compared to controls during the treatment period without undesirable effects on viral load. Remarkably, higher levels of intestinal Th17 cells were associated with reduced levels of intestinal immune activation, microbial translocation and systemic inflammation in the chronic phase of infection.To test this hypothesis, in this study we infected twelve RMs with SIV239 (day 0). Starting from day 14 post-infection (p.i.), six animals were treated with five weekly doses of 50 µg/kg IL-21 (s.c.), which represents a dosage similar or lower to those tested in phase I and II clinical trials in humans  Twelve RMs were infected intravenously (i.v.) with 300 TCID50 SIVmac239 both groups of RMs experienced a rapid, exponential increase in virus replication that peaked before initiation of IL-21 treatment (at approximately day 14 p.i.) in four out of six IL-21-treated and three out of six control monkeys. Peak viremia occurred on d21 p.i. in two IL-21-treated animals (RMq8 and RFj7) and in three controls . In the IL-21 treatment group, two animals (RIp10 and RMq8) showed lower post-peak viremia, while a third animal (RRq7), showed viral load remarkably higher than the other RMs already at day 14 p.i., i.e., before IL-21 treatment. This animal did not seroconvert (data not shown) and died at day 75 p.i., thus resembling the phenotype of a rapid progressor We first examined the effects of IL-21 on the kinetics of SIV plasma viremia. As shown in + and CD8+ T cells in the sampled anatomical sites. The kinetics of the percentages .We next assessed the effects of IL-21 on the levels of T cells by determining the relative frequencies  and absolute numbers (in PB) of CD4centages  and absocentages  of circureatment , althougreatment . Similar+ and CD8+ T cells in RBs. This part of the study is important since mucosal CD4+ T cells are severely depleted during the acute phase of HIV and SIV infections, and the severity of this depletion correlates with disease progression + T cells in the RB (+ T cells increased between wk6 and wk23 p.i. (7.90±3.00 vs. 13.89±6.51) in IL-21-treated RMs, but decreased (8.35±0.97 vs. 5.20±1.58) during the same time period in control animals , with this difference being statistically significant .The difference in the levels of intestinal CD4nfection . This an+ T cells.Together with those of + T cells in LCMV-infected mice + T cells in vitro+) CD8+ T cells, as well as their maturation subsets. Perforin and GrB analysis was performed at wks-2 (pre-infection), 2 (pre-IL-21 administration), 4 and 6 (during IL-21 treatment), 10 and 23 p.i. in PBMC; at wks-2, 2, 4, 6 and 23 in RB and at wks-2, 2, 6 and 23 in LN. As shown in + T cells in PBMC (P = 0.0038), LN (P = 0.033) and RB (P = 0.001) at wk6 p.i., i.e., after the final IL-21 dose  and at wks4 and 6 in RB (P = 0.022 and 0.0044), despite very similar levels before starting treatment  and effector memory (CD28−CD95+CCR7−) CD8+ T cell subsets of PBMC, LN and RB (data not shown). We then investigated the ability of IL-21 to induce perforin and GrB in virus specific (SIV gag tetramer+) CD8+ T cells in the six Mamu-A*01 animals (three in each group) included in the study. IL-21 did not change the frequencies of CD8+tet+ cells, which increased similarly in both groups following infection (data not shown). However, similar to the observations in total CD8+ T cells, IL-21 significantly increased the percentages of SIV-specific CD8+perforin+ T cells at wk4 p.i. in RB (P = 0.015), at wk6 in PBMC (P = 0.03), LN (P = 0.041), and RB (P = 0.028), and at wk10 in PBMC (P = 0.006) compared to control animals (+perforin+ T cells in PBMC (P = 0.0134), LN (P = 0.036) and RB (P = 0.042) significantly higher at wk6 than wk2 p.i., thus confirming the ability of IL-21 to specifically induce perforin production in virus specific CD8+T cells from different anatomical sites  at wk2 p.i. (−CD20+CD21hiCD27+IgD−) when compared to controls , 4 (during the IL-21 treatment) and 23 p.i.  infiltration in rectal biopsy tissues of IL-21-treated and control animals at wk6 post-infection . PMN migTogether, these findings indicate that administration of IL-21 during early SIV-infection of RMs significantly improved the preservation of intestinal Th17 cells and the integrity of the mucosal barrier.+ T cells are strictly associated + or CD8+ T cells expressing the activation markers HLA-DR and CD69 or the proliferation marker Ki-67 in the RBs of IL-21-treated and control RMs. While no significant differences were found at any experimental time point in the levels of CD4+ or CD8+ T cells expressing HLA-DR or CD69 (data not shown), the percentages of CD4+Ki-67+ T cells were significantly lower in IL-21-treated as compared to control RMs at wk23 p.i.  and the plasma levels of LPS  at wk23 p.i.  and the plasma levels of LPS  and sCD14   . Of noteCollectively, these data indicate a strong association between IL-21-dependent preservation of intestinal Th17 cells and limited levels of intestinal T cell proliferation and microbial translocation in IL-21-treated SIV-infected RMs.in vivo administration of IL-21, a cytokine that possesses numerous unique immunological properties, is beneficial in the context of pathogenic lentiviral infections. To test this hypothesis, we used the nonhuman primate model of SIV infection in rhesus macaques (RMs) and treated the animals with IL-21 during the acute phase of infection . The rationale for this study was based on several findings. First, a large body of evidence has shown that IL-21 is a key factor in regulating central processes that are defective and/or compromised in HIV/SIV infected humans and RMs. These processes include differentiation and homeostatic expansion of Th17 cells + T cells +IL-21+ and CD8+IL-21+ T cells are present in the rare subset of HIV-infected individuals who are able to spontaneously control HIV replication without treatment + T cells and those of Th17 cells, with a significant portion of the variability in Th17 cells being dictated by variability in CD4+IL-21+ T cells + T cells and NK cells in vitro in chronically HIV-infected individuals in vivo in a pilot study where IL-21 was administered to late-stage disease SIV-infected RMs + T cells and NK cells and the expression of ICOS and chemokine receptors  in both CD4+ and CD8+ T cells. According to these studies, the immunologic mechanisms of the antitumor action of IL-21 involve augmented NK cell and CD8+ T cell cytotoxicity and increased migratory properties of these cells to sites of inflammation In this study, we tested the hypothesis that exogenous + T cells in blood, LN, and rectal mucosa; (iii) IL-21 treatment increased the frequencies of memory B cells; (iv) IL-21 increased the levels of intestinal Th17 cells and the integrity of the mucosal barrier in the early infection; and (v) preservation of intestinal Th17 cells was associated with increased preservation of intestinal CD4+ T cells, reduced proliferation of intestinal CD4+ and CD8+ T cells, limited microbial translocation and systemic inflammation in the chronic phase of infection. The key findings of the current study are the following: (i) IL-21 treatment was safe and its administration during the acute phase of infection did not increase plasma viral load, systemic T cell activation or levels of the T cell inhibitory molecule PD-1; (ii) IL-21 significantly increased the expression of Perforin and GrB in total and virus specific CD8+ T cell subset crucial for mucosal immunity + T cells preferentially involves Th17 cells +IFN-γ+ and CD4+IL-2+ T cells were not affected by the treatment. When this study was started, IL-22 antibodies cross-reactive for RMs were not commercially available, and therefore we could not determine if the Th17 cells induced by IL-21 treatment were able to produce IL-22 in addition to IL-17. However, we could measure the plasma levels of IL-22 and found a five-fold increase (p = 0.056) in the plasma levels of IL-22 at wk6 in IL-21-treated animals compared to controls. To the best of our knowledge, this is the first study showing an intervention able to selectively preserve Th17 cells in vivo in SIV-infected RMs. Unfortunately, the effects of IL-21 on Th17 cells were temporary, as four months post-treatment the depletion of Th17 cells was comparable in IL-21-treated RMs and control animals. Overall these data underscore the need for a more prolonged treatment with IL-21 in vivo and for further studies aimed at optimizing the regimen for IL-21 administrations.As mentioned above, IL-21 has been implicated as a key factor regulating the differentiation and maintenance of Th17 cells, a CD4+ T cells + and CD8+ T cells; (ii) microbial translocation, as assessed by plasma levels of LPS and sCD14; and (iii) inflammation, as assessed by quantification of several plasma markers. In addition, the lower levels of neutrophil infiltration, commonly used as surrogate for the gut barrier integrity Depletion of intestinal Th17 cells contributes to the mucosal immune dysfunction and associated immune activation in pathogenic HIV/SIV infections with a reported association between the levels of Th17 cells and those of proliferating CD4+ T cells + T cells for up to one month after IL-21 treatment. Of note, the increase in perforin and GrB expression was confirmed also for SIV-specific CD8+ T cells, and in total and virus-specific CD8+ T cells of various anatomical locations. In our examination of B cells, we found that IL-21 had an impact on the levels of the different B cell maturation subsets, with treated animals showing higher frequencies of CD20+CD21hiCD27+ memory B cells and CD20+CD21hiCD27+IgD− switch memory B cells as compared to control animals up to one month after the last IL-21 administration. Our current data on the in vivo effects of IL-21 administration on the cytotoxic potential of T cells and expansion of memory B cells is consistent with the findings that we previously reported during chronic SIV infection in RMs + T cells, (iv) cytotoxic potential of T cells and expansion of memory B cells determined in various anatomical sites, including rectal mucosa.IL-21 may regulate two other immunological functions that are compromised in pathogenic HIV/SIV infections, i.e., the cytolytic potential of CD8+ T cell functions, viral loads were not significantly different between IL-21 treated and control RMs. Reduction of viral load may be very difficult to achieve with any single cytokines in particular during the acute phase of infection. It is also possible that IL-21 had diverse, multiple effects on viral load that counterbalance each other, thus resulting in a lack of variation in viral load. This finding led us to conclude that better preservation of intestinal Th17 cells, rather than changes in CD8+ T cells and B cells, is the main mechanism by which IL-21 impacts mucosal immune functions. Consistent with this interpretation, and despite the limited number of samples, higher levels of intestinal Th17 cells at wk6 were associated with lower levels of intestinal T cell proliferation and microbial translocation at wk23 p.i.While IL-21 treatment had a beneficial impact on CD8in situ quantitation of Th17 cells as numbers/unit of tissue by immunohistochemistry was not possible in the relatively small amount of tissues that can be collected during a longitudinal study in RMs. Furthermore, a mechanistic investigation on how IL-21 increases levels of Th17 cells is beyond the scope of the current body of work. Indeed, additional studies are needed to determine which stage of the Th17 cell differentiation pathway is mainly affected by IL-21, and why the treatment was more effective for intestinal Th17 cells than blood or LN-resident Th17 cells. In particular, these studies should investigate several non-mutually exclusive mechanisms, including (i) IL-21-mediated increase in the homing of blood and LN-resident Th17 cells to mucosal sites; (ii) IL-21-mediated increase in the levels of Th17 cell precursors; (iii) IL-21-mediated differentiation of Th17 cell precursors in fully mature Th17 cells; (iv) IL-21-mediated increase in the proliferation and/or survival of fully mature Th17 cells; and (v) IL-21-mediated decrease in HIV/SIV susceptibility by Th17 cells. Finally, it will be important to expand our investigation in an experimental setting that is more directly relevant to HIV infection in humans, i.e. IL-21 treatment performed in the chronic phase of SIV infection and in association with ART.A precise and extensive In conclusion, IL-21 treatment in SIV-infected RMs increases the levels of intestinal Th17 cells, reduces virus-associated mucosal immune dysfunction and chronic immune activation, and limits microbial translocation and systemic inflammation. These findings provide rationale for further preclinical studies aimed at exploring IL-21 as a potential immune-based intervention to be used in the context of ART during the chronic phase of infection.All animal experimentations were conducted following guidelines established by the Animal Welfare Act and the NIH for housing and care of laboratory animals and performed in accordance with Institutional regulations after review and approval by the Institutional Animal Care and Usage Committees  at the Yerkes National Primate Research Center (YNPRC). All efforts were made to minimize suffering.Mamu-B*08 and B*17 negative, while six of them were Mamu-A*01 positive . The 12 RM were randomized in two groups  of six animals based on age , weight (7.85±2.19 vs. 7.58±1.08) and Mamu-A*01 status (three in each group). All 12 animals were infected intravenously (i.v.) with 300 TCID50 SIVmac239 (day 0). Starting from day 14 p.i., the six animals of group 1 were treated with five weekly doses (subcutaneous) of 50 µg/kg rhesus rIL-21-IgFc (IL-21). The six RMs of group 2 remained untreated and used as controls . Perforin and Granzyme B staining in total T cells was performed as described previously + T cells on an LSRII cytometer driven by the FACS DiVa software. Analysis of the acquired data was performed using FlowJo software (TreeStar).Fourteen-parameter flow cytometric analysis was performed on whole blood, PBMC, LN and RB derived cells according to standard procedures using a panel of monoclonal antibodies that we and others have shown to be cross-reactive with RM in vitro stimulation with PMA & Ionomycin. PBMC and RB derived cells, isolated as described above, were resuspended to 1×106 cells/ml in complete RPMI 1640 medium. Cells were then incubated for 4 h at 37°C in medium containing PMA, A23187, and Golgi Stop. Following incubation, the cells were washed and stained with surface markers for 30 min in the dark at 4°C followed by fixation and permeabilization. After permeabilization, cells were washed and stained intracellularly with the antibody for the cytokines of interest for 1 h in the dark at 4°C. Following staining, cells were washed, fixed in PBS containing 1% paraformaldehyde, and acquired on an LSRII cytometer.Levels of Th17 cells were determined as the percentage of CD4+ T cells that produce IL-17 following Escherichia coli endotoxin standard provided with the assay, after background subtraction, and expressed in pg/mL. Plasma levels of sCD14 were quantified by using Human sCD14 Immunoassay kit . Ten microliters of plasma was diluted 400 fold by adding 3990 µl of calibrator diluent and assayed in duplicate as per manufacturers recommendations. LPS levels were measured in plasma (with EDTA as anticoagulant) by the limulus amebocyte lysate (LAL) chromogenic endpoint assay , as previously described 2O. Heat induced epitope retrieval (HIER) was performed by heating sections in 10 mM Citrate (pH 6.0) in a pressure cooker set at 122°C for 30 s. After HIER, slides were rinsed in ddH2O and then loaded on an IntelliPATH autostainer  and stained with optimal conditions determined empirically that consisted of a blocking step using blocking buffer (TBS with 0.05% Tween-20 and 0.5% casein) for 10 min and an endogenous peroxidase block using 1.5% (v/v) H2O2 in TBS (pH 7.4) for 10 min. Rabbit polyclonal anti-myeloperoxidase  was diluted in blocking buffer and incubated for 1 h at room temperature. Tissue sections were washed, and detected using the Rabbit Polink-1 HRP staining system  according to manufacturer's recommendations. Sections were developed with Impact DAB (Vector Laboratories). All slides were washed in ddH2O, counterstained with hematoxylin, mounted in Permount (Fisher Scientific), and scanned at high magnification (×200) using the ScanScope CS System (Aperio Technologies) yielding high-resolution data from the entire tissue section. Representative regions of interest  were identified and high-resolution images extracted from these whole-tissue scans. The percent area of the lamina propria that stained for myeloperoxidase+ neutrophils were quantified using Photoshop CS5 and Fovea tools.Immunohistochemistry was performed using a biotin-free polymer approach  on 5-µm tissue sections mounted on glass slides, which were dewaxed and rehydrated with double-distilled HSerum samples from IL-21-treated and control RMs were analyzed for their reactivity to viral antigens in a concanavalin A (ConA) ELISA + T cells; CD4+Ki-67+ T cells; Th17 cells; CD8+Ki-67+; LPS; sCD14, etc) were performed with a means model with SAS Proc Mixed (version 9) providing separate estimates of the means by weeks post-infection and treatment group. A compound-symmetic variance-covariance form in repeated measurements was assumed for each outcome and robust estimates of the standard errors of parameters were used to perform statistical tests and construct 95% confidence intervals Repeated-measures analyses for each outcome  and as fold change relative to the percentages of CD4+ T cells at wk2 (B). Values are shown for individual IL-21-treated (depicted in orange) or control (depicted in black) RMs. Shaded area represents time of IL-21 treatment.Effects of IL-21 on intestinal T cell levels in SIV-infected RMs. Longitudinal assessment of intestinal CD4(TIFF)Click here for additional data file.Figure S2+ T cells. Longitudinal assessment of the mean frequencies of (A) perforin and (B) GrB in gated live CD3+CD4+ T cells from PBMC (left panels), LN (middle panels) and RB (right panels). IL-21-treated animals are depicted in orange, controls in black. Shaded area represents time of IL-21 treatment. Averaged data are presented as mean ± SEM.Effects of IL-21 administration on perforin and GrB production by CD4(TIFF)Click here for additional data file.Figure S3A) Mean frequencies of memory B cells (CD3-CD20+CD21hiCD27+) and (B) swich memory B cells (CD3−CD20+CD21hiCD27+IgD−) in control and IL-21-treated animals. (C) Longitudinal assessment of plasma levels of anti-SIV antibodies in the two groups of animals. IL-21-treated RMs are depicted in orange, controls in black. Shaded area represents time of IL-21 treatment. Averaged data are presented as mean ± SEM.Effects of IL-21 administration on the frequency of B cell subsets and on anti-SIV antibodies. Circulating B cell subsets were analyzed longitudinally by flow cytometry. ((TIFF)Click here for additional data file.Figure S4+ T cells expressing IL-17, IFN-γ and IL-2 in SIV-infected RMs. Longitudinal assessment of the percentages of circulating (A–C) or intestinal (D–F) CD4+ T cells that express IL-17 , IFN-γ  or IL-2  in IL-21-treated (orange) and control (black) RMs. Shaded area represents time of IL-21 treatment. Averaged data are presented as mean ± SEM.Effects of IL-21 on the frequency of blood and intestinal CD4(TIFF)Click here for additional data file.Figure S5Effects of IL-21 on plasma levels of IL-22 in SIV-infected RMs. Plasma levels of IL-22 (pg/ml) were determined in IL-21-treated (orange) and control (black) RMs. Data are shown as fold change variation at wk6 (end of treatment) and wk23 (end of study) as compared to wk2 (pre-treatment) p.i. Averaged data are presented as mean ± SEM.(TIFF)Click here for additional data file.Figure S6A, B) Longitudinal assessment of intestinal (A) CD4+Ki-67+ and (B) CD8+Ki-67+ T cells in IL-21-treated and control RMs.  Longitudinal assessment of plasma levels of (C) LPS and (D) sCD14 in IL-21-treated and control RMs. Values are shown for individual IL-21-treated (depicted in orange) or control (depicted in black) RMs. Shaded area represents time of IL-21 treatment.Effects of IL-21 on intestinal T cell proliferation and microbial translocation in SIV-infected RMs. ((TIFF)Click here for additional data file.Figure S7A) CD4+Ki-67+, (B) CD8+Ki-67+, (C) CD4+PD-1+, and (D) CD8+PD-1+ T cells in IL-21-treated (orange) and control (black) RMs. Shaded area represents time of IL-21 treatment. Averaged data are presented as mean ± SEM.Effects of IL-21 on systemic T cell activation and proliferation in SIV-infected RMs. Longitudinal assessment of the percentage of circulating ((TIFF)Click here for additional data file.Table S1Modifications induced by IL-21 treatment on several immunological parameters. Summary of the parameters that were significantly different between IL-21-treated and control animals in at least one experimental time point. NA: not analyzed; NS: Not significant.(DOCX)Click here for additional data file.


Carica papaya) is a trioecious plant species that has male, female and hermaphrodite flowers on different plants. The primitive sex chromosomes genetically determine the sex of the papaya. Although draft sequences of the papaya genome are already available, the genes for sex determination have not been identified, likely due to the complicated structure of its sex-chromosome sequences. To identify the candidate genes for sex determination, we conducted a transcriptome analysis of flower samples from male, female and hermaphrodite plants using high-throughput SuperSAGE for digital gene expression analysis. Among the short sequence tags obtained from the transcripts, 312 unique tags were specifically mapped to the primitive sex chromosome (X or Yh) sequences. An annotation analysis revealed that retroelements are the most abundant sequences observed in the genes corresponding to these tags. The majority of tags on the sex chromosomes were located on the X chromosome, and only 30 tags were commonly mapped to both the X and Yh chromosome, implying a loss of many genes on the Yh chromosome. Nevertheless, candidate Yh chromosome-specific female determination genes, including a MADS-box gene, were identified. Information on these sex chromosome-specific expressed genes will help elucidating sex determination in the papaya.Papaya ( Carica papaya) is a trioecious species with male, female and hermaphrodite flowers on separate plants. It is primarily cultivated in tropical and subtropical areas. Generally, the pear-shaped fruits from hermaphrodite flowers are commercially preferred, although the female flowers can also produce fruits if pollinated. Currently, there are several critical problems in the breeding and cultivation of hermaphrodite plants. First, genetically fixing hermaphrodite characteristics is impossible due to the segregation of sex type, even when self-fertilized (see below). Thus, efforts to optimize hermaphrodite selection among the segregates are essential in the field. Second, under high summer temperatures, the flowers have been observed to change from hermaphrodite to male because of ovary abortion and abnormal carpelloid or pentandoria flowers Papaya  at a single locus, Sex1m, and therefore their genotype is mm. Males are heterozygous for m and a dominant female-suppressing allele, M, and therefore their genotype is Mm. Hermaphrodites possess an independent allele, hM, and their genotype is h mM. Individuals homozygous for the dominant alleles (MM and h MhM) and the hM M heterozygote are lethal. However, one study has suggested that a complex of genes rather than a single gene determines the sex of papaya h chromosome controls hermaphrodite sex determination. Both the X and Yh chromosomes contain an approximately 8–9 Mb MSY with low gene density and a highly repetitive sequence h chromosomes, respectively.Using the results from an analysis of the segregation of sex types after cross- or self-pollination, a genetic model of papaya sex determination was hypothesized based on three alleles  chromosome were lost due to the accumulation of mutations. A comparison of the partial genomic sequences of the Y and Yh chromosomes revealed high sequence similarity Silene spp. Sex chromosomes have been identified in 48 plant species In papaya, several genes were identified in the MSY regions on both the Y and X chromosomes, but they did not show differential expression among the sex types 5′-CATG) in the cDNA using the EcoP15I restriction enzyme as described in the Methods section. The tag extraction and PCR amplification were performed according to the original protocol for the Ht-SuperSAGE analysis using the Illumina Genome Analyzer IIx To identify candidate sex-determination genes in papaya, we conducted a large-scale transcriptome analysis of flowers from the three sex types using the Ht-SuperSAGE method. RNA was extracted from flowers at two developmental stages in the male (from TM1), female and hermaphrodite (from Sunrise Solo) plants as described in the Methods section, and these flower samples were designated as P1 to P6, respectively , Table 1After converting the raw sequence data to the FASTA format, the reads containing the exact index sequences and the NlaIII site were selected. In total, 9,273,744 SuperSAGE tag-encoding sequences were selected and classified into six groups according to the index sequences that corresponded to each sample. Subsequently, the 26-bp tags were extracted from the NlaIII sites, and their counts were calculated to obtain an expression (tag) profile for each sample. The total tag counts and numbers of the unique tags in each sample are shown in h and X chromosomes from GenBank (h). These perfectly matched tags were designated as SC-tags  (By comparing the tag profiles among the six samples (P1–P6), many differentially expressed tags were identified . These t GenBank  and desiomosomes . Other t30 tags) . To eval30 tags) . This ra30 tags) , showingTo identify their corresponding genes, these SC-tags were subjected to BLAST searches against the sequences of predicted genes from the draft genome sequence h) or in both females and hermaphrodites. Only a few tags were expressed uniquely in males or hermaphrodites, but no female-specific expressed tags were observed in this analysis.From the 312 sex-chromosome-specific SC-tags, we selected 47 tags  that werh chromosome (15 tags), 12 tags showed expression in the male flowers carrying the Y chromosome, which was potentially due to the sequence similarity between the Y and Yh chromosomes. Approximately 1 kb of the genomic sequences upstream of these Yh -specific tags, except for the retroelement tags , was subjected to a BLASTN search against the BAC clone sequences of the X chromosome (h (Y) chromosome-specific loci, as determined using in silico analysis.Within the sex-dependent SC-tags uniquely mapped to the Yromosome  to identh-specific tags, Cp2671 and Cp12204, to validate their presence or absence on the X chromosome. The annotation analysis revealed that the gene corresponding to Cp2671 encoded the MADS-box protein and the gene corresponding to Cp12204 encoded monodehydroascorbate reductase (MDAR). RT-PCR analysis of the Cp2671 gene demonstrated its specific expression in male and hermaphrodite flower samples , which were good candidate genes for elucidating the mechanism of sex determination in papaya.We employed the previously established Ht-SuperSAGE analysis h chromosome was previously shown to have significant sequence similarity to the Y chromosome h chromosome-specific tags (30 tags) than X chromosome-specific tags (252 tags); a limited number of tags (30 tags) were commonly mapped to both the X and Yh chromosomes. Although directly associating the SC-tags with predicted genes on the sex chromosomes remained difficult, the gene prediction and transcriptome data indicated the loss of functional genes on the Yh chromosome.According to the model of plant sex-chromosome evolution h) chromosome is predicted to possess a dominant mutant allele for the female-sterility gene and a wild-type allele for the male-sterility gene h-specific tags that were absent from the X chromosome. These genes might involve the wild-type allele for the male-sterility  locus because this allele was absent from the X chromosome, leading to male sterility in the absence of the Y or Yh chromosome. Indeed, the experimental results definitively demonstrated that the MADS-box gene, which corresponded to the Cp2671 tag, was Y and Yh chromosome-specific. In Silene latifolia, the presence of the Y chromosome determines sex through the regulation of floral organ development SISUP), which is responsible for female organ development SLM2). The MADS-box gene, which corresponds to Cp2671, encodes a protein with 85% similarity to the Short Vegetative Phase (SVP) protein in ArabidopsisFT) Regardless of the degeneration and gene loss, the Y , and the expression of these genes is compensated through the inactivation of one of the X chromosomes in female mammals h chromosomes. To determine its role in sex determination, further functional analysis of this gene, including genetic transformation in papaya, is necessary. Furthermore, the other genes for sex determination might be present in the results obtained from the transcriptome analysis performed in this study; therefore, the structural and functional analysis of those genes will be required to better understand sex determination in papaya.In the present study, we analyzed the transcriptome of genes on the papaya sex chromosomes and identified the MADS-box gene for Cp2671 as a candidate for sex determination because of its presence only on the Y and YThe plant materials used in this study were obtained from the Hawaiian papaya cultivar “Sunrise solo”, and the breeding material TM1 was derived from a cross between cv. “Wonder frea” and Okinawan land race IG4. The female and hermaphrodite flower buds were harvested from the Sunrise solo cultivar, and the male flower buds were obtained from TM1. The flowers in the early (no longer than 7 mm) and late (approximately 20 mm in length) developmental stages were harvested from one-year-old plants of the three sex types grown in a greenhouse. To avoid a high-temperature-induced sex change, the samples were collected in March and April (spring season). Each developmental stage of the flower sample from each sex type was designated as P1 to P6 . Twenty 5′-TTCCTCATTCTCTCAAGCAGAAGACGGCATACGAAATGATACGGCGACCACCGACAGGTCTAACGATGTACGCAGCAGCATG-3′ and 5′-CTGCTGCGTACATCGTTAGACCTGTCGGTGGTCGCCGTATCATTTCGTATGCCGTCTTCTGCTTGAGAGAATGAGGAA-amino-3′) were synthesized and annealed. For the adapter-1-SLD, two oligonucleotides  primer (5′-bio-CTGATCTAGAGGTACCGGATCCCAGCAGTTTTTTTTTTTTTTTTT-3′). Purified cDNA was digested with NlaIII. The resulting fragments were bound to streptavidin-coated beads (Dynabeads streptavidin M-270), and the non-biotinylated cDNA fragments were removed by washing. Adapter-2SLD was ligated to the cDNA fragments on the beads and digested with EcoP15I after washing. EcoP15I-digested and released fragments (adapter-2SLD-tags) were ligated to adapter-1SLD with defined index sequences for sample identification.5′- CCACTACGCCTCCGCTTTCCTCTC -3′) and P2 (5′- CTGCCCCGGGTTCCTCATTCTCTCAAGCAGAAGA -3′) primers. The PCR program consisted of denaturation at 98°C for 1 min followed by 12 cycles of 98°C for 30 sec and 60°C for 30 sec. Eight tubes from this PCR amplification (15 µl each) were pooled, and the concentrated PCR products were purified using the MinElute reaction purification kit (Qiagen) and separated on an 8% non-denaturing polyacrylamide gel. After staining with SYBR green (Takara Bio), the 160-bp band was excised from the gel, and the DNA was eluted from the gel pieces. The PCR product from each sample was analyzed on an Agilent Bioanalyzer 2100. Equal quantities of the PCR products from all samples were mixed, and the mixture was analyzed using the Applied Biosystems SOLiD3.The tags located between the two adapters were amplified using PCR with PhusionHigh polymerase and the P1 . The BAC clone sequences of the sex chromosomes . These cDNAs were used as templates for the RT-PCR analysis (Genomic DNA was extracted from the mature leaf of each papaya plant using the DNeasy Plant Mini Kit (Qiagen). For the RT-PCR analysis, two pools from each flower sample (P1–P6) were harvested from independent plants and subjected to total RNA extraction. Single-stranded cDNA was synthesized from the total RNA by reverse transcription with the adapter-dT primer (5′-GCTGTCAACGATACGCTACGTAACGGCATGACAGTG(T)analysis .5′-GAGATGCACCATTCTAGTTCC-3′), Cp2671-F2 (5′-CAGATCGGGCACTTTAATGTTG-3′), Cp2671-F3 (5′-TGGCAAACTCTGTACTTGCA-3′), Cp2671-F3 (5′-TGGCAAACTCTGTACTTGCA-3), Cp2671-R2 (5′-GGAGCTCCAAGGATTAAGCA-3′), Cp2671-R4 (5′-GCAGGGAATACAATTGATGG-3′) and Cp2671-R5 (5′-TCGGAGAATGTAGCGTCTGA -3′). For the amplification of genes corresponding to Cp3177 (MDAR gene), the primers Cp3177-LPF1 (5′-GTGTGAAATGATGGAAGCCGTGCACGAGCTGCAGATGAAT-3′), Cp3177-LPF2 (5′-CGTGTTCCAAGAACTAACTCAATTCCTAAGAAAATATGCGGAG-3′), Cp3177-F1 (5′-GAGTGATGATGTAACAAGTTAG-3′) and Cp3177-R2 (5′-CATGGTGTTCCCTGAAGCAC-3′) were employed. As a control, the amplification of an actin gene was performed using the primers Cp47F (5′-GTCCTAGACACACAGGTGTCATGG-3′) and Cp47R (5′-GACTTGTCCATCAGGCAACTCGTA-3′). For the RT-PCR shown in The primer sequences for the amplification of the genes corresponding to the Cp2671 tag (MADS-box protein) are listed as follows: Cp2671-F1  Partial fragment of the gene for Cp11249 (encoding putative zinc finger protein) was amplified from male (P1), female (P3) and hermaphrodite (P5) cDNA. In the lane “m”, a 100 bp ladder size marker was loaded. Template cDNA was prepared as described in the Method. The Cp11249F (5′- CATATATGGATTGGGGAAAC-3′) and Cp11249RV (5′- ACCTGGAGCTGTATGGTAAGATT -3′) primers were used for amplification. B) RT-PCR experiments with primers for the partial Cp11249 gene in two additional replicated flower samples . In the lane “m”, a lambda HindIII digested size marker was loaded. C) An amplified fragment from each sample  was applied to the sequencing analysis, and aligned. The Cp11249 tag sequence is shown above the alignments, and the tag regions in the cDNA sequences are surrounded by a square. The SNP in P1  was underlined.(PPT)Click here for additional data file.Figure S2RT-PCR analysis of three genes in replicated flower RNA samples. RT-PCR experiments with primers for the MADS-box protein gene (for Cp26719) as shown in (PPT)Click here for additional data file.Table S1List of analyzed SuperSAGE tags from papaya flowers*. * Count for individual tag from all six samples (P1 to P6) were combined and indicated as “SUM” in the table, and only the tags, showing more than 10 counts of “SUM”, were listed in this table.(XLS)Click here for additional data file.Table S2BAC clones of the papaya sex chromosomes used for mapping of SuperSAGE tags. * These clones were regarded as BAC clones on the X chromosome.(XLS)Click here for additional data file.Table S3SuperSAGE tags specifically mapped on the sex chromosome (SC-tags). * Predicted gene or EST, which showed perfect match to 26-bp tag sequence. ** Predicted gene, which was found within 2 kbp upstream of the tag sequence. *** Deduced proteins were determined by BLASTX searching of genomic sequences upstream of the SuperSAGE tags as described in method.(XLS)Click here for additional data file.Table S4SuperSAGE tags mapped on the sex chromosome and other chromosomes simultaneously. * Deduced proteins were determined by BLASTX searching of genomic sequences upstream of the SuperSAGE tags as described in method.(XLS)Click here for additional data file.Table S5SuperSAGE tags mapped on the sex chromosome and organelle genome sequences simultaneously. * Deduced proteins were determined by BLASTX searching of genomic sequences upstream of the SuperSAGE tags as described in method.(XLS)Click here for additional data file.Table S6Expression patterns of SC-tags among male, female and hermaphrodite. *Sex types are designated as M for male, F for female and H for hermaphrodite. **SC-tags were mapped on both X and Yh chromosome.(XLS)Click here for additional data file.Table S7Transcription activity of genes mapped on the X chromosome among papaya flowers from different sex types.(XLS)Click here for additional data file.Table S8List of the sex dependent SC-tags. * Deduced proteins were determined by BLASTX searching of genomic sequences upstream of the SuperSAGE tags as described in method.(XLSX)Click here for additional data file.


Figure S4+ precursor ion m/z 757.11. (B) Scheme providing the interpretation of MS/MS spectrum in ‘A’ leading to the identification of molecular structure of the intermediate.ppApA is another intermediate of the DAC reaction. (A) MALDI-MS/MS spectrum of [M+H](TIF)Click here for additional data file.


Interferon-gamma induced protein 10 (IP-10) was suggested to be involved in liver injury in viral hepatitis. This study aimed to investigate the impact of the single nucleotide polymorphisms (SNP) G-201A (rs1439490) in IP-10 gene on disease progression of hepatitis B virus (HBV) infection.In vitro IP-10 promoter activity was compared between haplotype GG and AA homozygotes using luciferase reporter system in HepG2 cells. In vivo expression of IP-10 was compared between patients with -201 AA genotype and GG genotype.The -201 SNP in IP-10 promoter was genotyped from 577 patients with different illness categories and 275 health controls; In vitro IP-10 promoter-driven luciferase activity in pGL3-Enhancer-201A transfected HepG2 cells was 1.43-fold higher than that in pGL3-Enhancer-201G transfected HepG2 cells (P<0.01). In vivo IP-10 transcriptional expression of peripheral blood mononuclear cells was 1.38-fold higher in patients with -201 AA genotype than in patients with -201 GG genotype (P<0.01).The detected frequency of G-201A SNP was 17.8%, 25.3%, 26.6%, and 13.8% for patients with acute hepatitis B (AHB), patients with mild chronic hepatitis B (CHB-M), patients with severe chronic hepatitis B (CHB-S), and health controls, respectively. G-201A in promoter region of IP-10 gene was associated with liver disease progression in patients with HBV infection through up-regulating IP-10 expression. Hepatitis B virus (HBV) is non-cytopathic itself and the liver damage caused by HBV infection is mainly immune-mediated Interferon gamma (IFN-γ) was involved in both innate and adapted immune responses, and IFN-γ induces protein 10 (IP-10) is a cytokine that chemo-attracts T cells and NK cells that express CXCR3 ligands in vitro and in vivo.The mechanism affecting IP-10's binding affinity to CXCR3 is not fully understood. In previous study, 21 SNPs was recognized by sequence analysis of the 4228 bp length IP-10 gene, and none of them was in the coding region The study population consisted of 577 unrelated Chinese HBV patients who visited Beijing 302 hospital and 275 healthy controls who donated at the blood donor center in Beijing 302 Hospital. The patients were categorized into AHB (196), CHB-M (193), and CHB-S (188) sub-groups with increasing disease severity. The categorization was based on the diagnostic criteria of 2000 Xi'an Viral Hepatitis Management Scheme issued by the Chinese Society of Infectious Diseases and Parasitology, and the Chinese Society of Hepatology of the Chinese Medical Association 2 = 1, in a validation sample of 387) in previous study 5′-CATCCTAGGATCAGGCTACTGT-3′) and R (5′- AGCTGACAACTTAGATACCAAC -3′); the primers for the second-round nested PCR were F (5′-GCAGATACTGTCTCAGAACCTGGTA-3′) and R (5′- TGTCACCATCTCTCATTTTGATTGT-3′). The first-round PCR consisted of a denaturing at 94°C for 3 min and 30 cycles at 94°C for 25 s, 52°C for 25 s, 72°C for 50 s. The second-round PCR consisted of a denaturing at 94°C for 3 min and 30 cycles at 94°C for 20 s, 54°C for 20 s, 72°C for 40 s. Amplicons of 499-base pair lengths were the PCR products, which were purified by QIAquick PCR purification kit . After purification, 5 µL of the PCR products were digested within 15 U Xba I  for the genotyping of C-1596T polymorphisms. Cleaved DNA fragments were identified by ultraviolet light after electrophoresis in 2% agarose gel stained by ethidium bromide. The homozygotes with C-1596T alleles yielded restriction fragments of 174/325 base pairs.The SNPs G-201A polymorphisms in the 5′ flanking region of the IP-10 gene was the research interest. As the polymorphisms G-201A and C-1596T were in absolute LD  and R (5′-TGGCAGTTTGATTCATGGTGC-3′), the second-round PCR were F (5′- GGAGGACTGTCCAGGTAAATCACTG-3′) and R (5′-TGAGGAATGTCTCAGAAAACGTGG-3′). The conditions for the first-round and second-round PCR amplifying G-201A were the same as that of amplifying C-1596T. Sequencing was performed using an ABI 3730xl DNA Analyzers .The accuracy of genotyping information for each -201A alleles that referred from PCR-RFLP analysis was validated by sequencing of 45% samples randomly from the studied population. G-201A polymorphisms were amplified by PCR method. The primers for the first-round PCR were F (5′-GAACCCCATCGTAAATCAACCTG-3′) and R (5′-CCTTGAATGCCACTTAGAGTCAG-3′); for the second-round PCR were F (CGGGGT ACCCAAGGTCTGTCTCTATGCGTGC-3′5′- ) (introduced Kpn I as underlined) and R (CCCAAG CTTGCAGCAAATCAGAATGGCAGTTTG-3′5′-) (introduced Hind III). The conditions for the first-round and second-round PCR were the same as described above. PCR products were purified by QIAquick PCR Purification Kit (Qiagen), after purification, the products were incubated with dATP and Taq DNA polymerase for adding A, and purified by a QIAquick Gel Extraction Kit. The ligation and transformation were performed according to the manufacturer's instructions of pGEM-T Vector System II . The plasmids were extracted and digested with Kpn I and Hind III, and the IP-10 promoter DNA fragments were purified by a QIAquick Gel Extraction Kit. The fragment was ligated to pGL3-Enhancer Vector and then transiently transfected HepG2 cells . All reporter constructs and transformation was confirmed by direct sequencing after digestion with Kpn I and Hind III.Genomic DNAs from haplotype GG and AA homozygotes of G-201A polymorphisms were chosen for reporter plasmid construction. DNA fragments corresponding to IP-10 promoter region from nucleotides −875 to +27 (relative to the first nucleotide of the open reading frame of IP-10) were amplified. The primers for the first-round PCR were F (5) were divided into 6-well plates before transfection. Every well was transfected with 2 µg of one of the constructs together with 0.2 µg of pRL-TK vector (Promega), which served as the internal control for transfection efficiency, by FuGENE HD transfection reagent . After 48 hours, cells were collected and the luciferase activities were measured using the Dual-Luciferase Reporter Assay system (Promega). The measure was performed in triplicate.HepG2 cells  using the RNeasy Mini kit (Qiagen) according to the manufacturer's instructions. The IP-10 mRNA levels were determined by SYBR Green real-time PCR (Roche) with beta actin as the internal control. The primers for IP-10 were t test was used to analyze the luciferase reporter assays and the transcriptional expression of IP-10 in G-201A and G-201G genotype in vitro and in vivo. P values less than 0.05 were considered as statistical significant.Statistical analysis was performed in SPSS . Chi-squared analysis was used to calculate the difference of G-201A genotype frequencies among groups. Cochran-Armitage test was used to examine the linear trend. Student P value among four groups was 0.0037 by general chi- square test. Cochran-Armitage linear trend test  indicated an increasing trend of G-201A frequency among the groups .The percentages of -201A containing genotype at -201 were 17.8%, 25.3%, 26.6%, and 13.8% for patients with AHB, CHB-M, CHB-S, and healthy controls, respectively. The overall e groups . Paired provided . For theP<0.01)  , suggestP<0.01)  .Many methods are practically available to detect SNPs, and the present study adapted a PCR-RFLP method for easy serum samples SNP detection. Comparing with the direct sequencing method, the PCR-RFLP method is fast, cost-effective, less laboratory demanding, thus favorable for study of large population. The assay was validated by direct sequencing for 45% samples and the two assays showed a 98.7% identity with statistical consistence.in vitro and in vivo functional comparison.A single SNP may cause a disease such as Mendelian disease, while a single SNP often function in coordination with other SNPs to manifest a disease condition as seen in osteoporosis and Hirschsprung's disease Elevation of IP-10 affects host immune responses to infection by altering activity of cells that expresses CXCR3 receptors in vitro and up-regulated the transcriptional expression of IP-10 in vivo. These results suggested that SNP at -201 played a role in how humans respond to the virus and progress the persistent infection.Multiple factors may contribute to the elevation of IP-10. Decreased expression of CXCR3 receptor on plasmacytoid dendritic cells has been observed concomitant with an increased IP-10 level in plasma in patients with chronic HBV However, we did not investigate potential SNPs outside IP-10 gene that co-work with G-201A, neither did we investigate other identified SNPs in IP-10 gene due to the consideration that the most frequent SNP C-1513T was equally presented between HBV non-progressed and progressed carriers in pervious study In summary, the current study explained the contribution of the G-201A in promoter region of IP-10 gene to the disease progression of CHB. Possible mechanisms of G-201A influencing the disease progression were altering immune responses through up-regulating IP-10 expression.