In subjects with suppressed HIV VL, GBV-C was consistently associated with reduced activation in naïve, CM, EM, and effector CD4+ cells. GBV-C was associated with reduced CD4+ and CD8+ T cell surface expression of activation and proliferation markers, independent of HIV VL classification. GBV-C was also associated with higher proportions of naïve CD4+ and CD8+ T cells, and with lower proportions of EM CD4+ and CD8+ T cells. In conclusion, GBV-C infection was associated with reduced activation of CD4+ and CD8+ T cells in both HIV viremic and HIV RNA suppressed patients. Those with GBV-C infection demonstrated an increased proportion of naive T cells and a reduction in T cell activation and proliferation independent of HIV VL classification, including those with suppressed HIV VL on cART. Since HIV pathogenesis is thought to be accelerated by T cell activation, these results may contribute to prolonged survival among HIV infected individuals co-infected with GBV-C. Furthermore, since cART therapy does not reduce T cell activation to levels seen in HIV-uninfected people, GBV-C infection may be beneficial for HIV-related diseases in those effectively treated with anti-HIV therapy.GBV-C infection is associated with prolonged survival and with reduced T cell activation in HIV-infected subjects not receiving combination antiretroviral therapy (cART). The relationship between GBV-C and T cell activation in HIV-infected subjects was examined. HIV-infected subjects on cART with non-detectable HIV viral load (VL) or cART naïve subjects were studied. GBV-C VL and HIV VL were determined. Cell surface markers of activation (CD38 Chronic T cell activation accompanies HIV infection and contributes to HIV-related pathogenesis, and CD4+ T cell activation is required for efficient HIV replication Pegivirus genus of the Flaviviridaein vitro; however, low levels of replication have been documented in lymphocyte and other cell culture systems, and co-infection of peripheral blood mononuclear cells (PBMCs) with GBV-C and HIV in vitro results in inhibition of HIV replication in vivo, with average GBV-C VL levels of more than one million genome equivalents/mL plasma in HIV-infected hosts GB Virus C (GBV-C) is a human flavivirus tentatively assigned to the in vitroin vivoAlthough GBV-C interferes with HIV replication HIV-1-infected subjects were prospectively recruited from the University of Iowa HIV/AIDS Clinic and enrolled in these studies following provision of written informed consent. The study was approved by the University of Iowa Institutional Review Board. HIV was diagnosed in all subjects by documentation of both antibody and HIV VL measurement. Heparin anticoagulated blood was shipped overnight to Rush University  for expanded flow cytometric characterization of T cell subpopulations, activation, proliferation, and HIV co-receptor expression. Demographic information including age, gender, race, ethnicity, HIV transmission mode, CD4 count, CD4 percent, HIV RNA concentration , and hepatitis B and C serologies were obtained from the medical record. The lower limit of detection for HIV VL was 48 copies/mL.To determine GBV-C infection among HIV-seropositive patients, serum was prepared and stored at −70°C. RNA was extracted from serum and GBV-C RNA amplified and detected using a one-step real-time RT-PCR as described ++ and Mg++ and processed over Lymphocyte Separation Media . The interface containing PBMCs was collected, washed in PBS, resuspended in FACS buffer and stained for flow cytometric evaluation. Samples were acquired and a minimum of 35,000 cells were analyzed on a BD LSR2.0 flow cytometer using FACS Diva v6.1.3 software for each analysis. Fluorochrome conjugated antibodies against the following cell surface markers were used in these studies: CD3, CD4, CD8, CD38, CD45RA, HLA-DR, CCR7, Ki-67, CCR5, and CXCR4 (BD Biosciences). Activation was defined as the percentage of cells examined that expressed CD38 and HLA-DR for total CD4+ or CD8+ T cells, including individual T cell subsets: naïve (CD45RA (RA)+ and CCR7 (R7)+), central memory (RA−/R7+), effector (RA+/R7-) or effector memory (RA−/R7-). Proliferation was defined as the percentage of cells that expressed Ki-67. HIV co-receptor expression was defined as the percentage of cells expressing CCR5 or CXCR4 expression.Upon arrival at Rush University, plasma was prepared by centrifugation of the anticoagulated blood for 15 minutes at 400×g, and aliquots were stored at −80 C until used for further analyses. The remaining cells were diluted in PBS w/o CaThe percentage of CD38+/HLA-DR+, Ki67+, CCR5+, and CXCR4+ cells in total and naïve, CM, EM, and effector CD4+ and CD8+ T cells were used as outcomes in statistical analyses. Because there was a heterogeneity in the variance, a Kruskall-Wallace (KW) non-parametric test was used for each cell type to examine if there are differences between the 4 groups defined by HIV viremia  and GBV-C viremia (G−/+). To examine for a consistent pattern in the 4 groups between the 4 cell types, O’Brien’s test of the mean rank was used to look for similar patterns in several types of cells Sixty five subjects participated in the study. Four were excluded due to loss of virologic control on the date of sampling, one was excluded as he initiated cART in the month prior to testing, and one subject’s cells were delayed 24 hours during shipping, leaving 59 evaluable subjects. GBV-C viremia was detected in 41% (24) of the 59 remaining HIV-infected subjects. All twenty eight subjects on cART evaluated had nondetectable HIV VL values on cART for >6 months, and 31 subjects were cART naive. The 28 subjects on cART had been tested previously for GBV-C, with half (14) GBV-C viremic and the remainder non-viremic. Ten of 31 (32%) of the cART naïve subjects not previously tested had plasma GBV-C viremia detected. Based on clinical history, 2 cART naïve subjects were HIV infected for less than 6 months while the remaining 57 subjects were chronically infected.10) lower than those without GBV-C viremia . CD4 counts were not significantly different in subjects on cART (548 cell/mm3 in G+ vs. 520 cells/mm3 in G-); however, cART naïve subjects with GBV-C viremia had significantly more CD4 cells (599/mm3) than did those without GBV-C  (The study cohort included 27% women (16/59), 85% (50/59) Caucasian, 15% (9/59) African American, and 7% (4/59) of Hispanic ethnicity. GBV-C viremic and non-viremic subjects were not significantly different in age, race, gender, or ethnicity . Among t p<0.01) . All subConsistent with findings observed in recently HIV-infected subjects, GBV-C viremia was associated with a reduction in CD4+ and CD8+ T cell activation in subjects with HIV viremia as measured by CD38 and HLA-DR expression . When anThe proportion of naïve CD4+ T cells was significantly higher among G+ subjects compared to G- . AlthougThe association of GBV-C with reduced CD4+ T cell expansion following therapeutic IL-2 infusion in vitro downregulates the HIV entry co-receptor CCR5 expression by reducing steady state mRNA concentrations GBV-C infection of PBMCs Persistent immune activation is a critical component of HIV pathogenesis  These data are the first to demonstrate an association between GBV-C viremia and an increase in the proportion of naïve CD4+ and CD8+ T cells, and a reduction in proliferation. The reduction in proliferation is consistent with findings that CD4 cell expansion was impaired in G+ subjects following IL-2 infusion in vivoOur data are consistent with earlier reports that GBV-C influences CCR5 expression In summary, GBV-C viremia was associated with reduced activation and proliferation, an increased proportion of naïve CD4+ and CD8+ T cells, and reduced CCR5 expression on CD8+ T cells. The relationship between GBV-C and these variables was consistent in subjects in which HIV VL was suppressed by cART, and in treatment naïve groups. Since cART does not reduce activation levels to that seen in HIV-uninfected populations, GBV-C may provide clinical benefit to subjects effectively treated with cART. Understanding the mechanism(s) by which GBV-C might diminish T cell activation may provide novel approaches to address the persistent T cell activation observed in HIV-infected people.


HTLV-I is associated to Adult T-cell leukemia (ATL) characterized by genetic disorders and an altered mRNA expression. Individuals who have a coinfection between HTLV-I and Strongyolides stercoralis (SS) might have an increased risk to develop ATL. The mechanism for the synergism between these two infectious agents is unclear. In this study we evaluate the mRNA expression in Peripheral Blood Mononuclear Cells (PBMC) from three different groups, to determine mRNA profiles similarities or divergences among patients with a)ATL, b)HTLV-I-SS and c)Asymptomatic carriers (AC).mRNA expression of eighty genes involved in the DNA damage pathway (Sabiosciences-Qiagen) were evaluated among HTLV-1 positive patients: a)ATL (n=6), b)HTLV-I-SS (n=8) and c)Asymptomatic carriers(n=6). RNA was extracted from PBMCs and analyzed by using PCR superarray plates from Sabiosciences-Qiagen, one plate was used to analyze 80 genes for every patient. Sabiosciences sample sheet was used for calculations.The number of genes differentially expressed among these groups was: a) nine genes between ATL and AC, P values range 0.045 to 0.0073, b) nine genes between HTLV-I—SS and AC, P values range 0.0497 to 0.0098 and c) two genes between ATL and SS, P values 0.027 and 0.046.Two genes, the G-2 and S-phase expressed 1 gene and the Fanconi anemia, complementation group G gene were commonly increased in ATL and HTLV-I—SS compared to AC. Similar profiles of mRNA expression regarding of DNA damage pathway were observed among ATL and HTLV-I—SS groups, suggesting some shared regulatory mechanisms during infection in vivo.


It is known that the human immune proteins APOBEC3G and -F (hA3G/F) can inhibit Vif-deficient HIV by G-to-A mutation; however, the roles of these enzymes in the evolution of HIV are debated. We argue that if evolutionary pressure from hA3G/F exists there should be evidence of their imprint on the HIV genome in the form of (i) underrepresentation of hA3G/F target motifs  and overrepresentation of product motifs  and/or (ii) an increase in the ratio of nonsynonymous to synonymous (NS/S) G-to-A changes among hA3G/F target motifs and a decrease of NS/S A-to-G changes among hA3G/F product motifs. To test the first hypothesis, we studied the representation of hA3G/F target and product motifs in 1,932 complete HIV-1 genomes using Markov models. We found that the highly targeted motifs are not underrepresented and their product motifs are not overrepresented. To test the second hypothesis, we determined the NS/S G7A changes among the hA3G/F target and product motifs in 1,540 complete sets of nine HIV-1 genes. The NS/S changes did not show an increasing/decreasing trend within the target/ product motifs, but the NS/S changes within the motif AG was exceptionally low. We observed the same pattern by analyzing 740 human genes. Given that hA3G/F do not act on the human genome, this suggests a small NS/S change within AG has arisen by other mechanisms. We therefore find no evidence of an evolutionary footprint of hA3G/F. We postulate several mechanisms to explain why the HIV-1 genome does not contain the hA3G/F footprint.


P<0.001). Additionally, the serum IL-21 levels in BP patients were higher than those of the healthy controls . The frequencies of Tfh cells and IL-21 levels were both positively correlated with anti-BP180-NC16A autoantibody titers . After effective therapy, the frequencies of Tfh cells as well as the serum IL-21 levels in BP patients decreased along with clinical improvement. Most importantly, Tfh depleted CD4+ T cells and anti-IL-21 neutralization antibody could inhibit the T cell-induced B cell activation and secretion of BP autoantibody in vitro. Those results suggest that Tfh cells play an important role in autoantibody production and are involved in the pathogenesis of BP.The pathogenesis of bullous pemphigoid (BP) is characterized by the T cell-dependent production of autoantibodies. Recent studies have indicated that follicular T helper cells (Tfh), the key modulator of B cell activation and autoantibody production, are critical in the development of several autoimmune diseases. Tfh cells perform their functions via IL-21, their hallmark cytokine. In the present study, the frequencies of Tfh cells were investigated in the peripheral blood samples of BP patients to evaluate whether Tfh cells involve in this clinical entity. Significantly higher Tfh cell counts were observed in the peripheral blood of BP patients than those in healthy controls (median: 11.25% vs. 4.95%, respectively;  Bullous pemphigoid (BP) is an autoimmune subepidermal blistering disease characterized by production of autoantibody directly responding to hemidesmosomal proteins within the dermal-epidermal junction + T helper cells Follicular T helper cells (Tfh) have recently emerged as a separate subset of CD4in vitro. Thus, these results have indicated the possible involvement of Tfh cells and IL-21 in the pathogenesis of BP.The present study aimed to determine whether Tfh cells play an important role in pathogenic autoantibody production in BP and to clarify their involvement in the pathogenesis of BP. The increased frequency of Tfh cells and level of IL-21 were detected in BP patients, which were positively correlated with high level of serum anti-BP180-NC16A autoantibody. In addition, we found that Tfh cells and IL-21 were the essential part for the secretion of anti-BP180-NC16A in T cell/B cell co-culture system Table S1. We collected serum samples from 14 BP patients  and 14 sex- and age-matched healthy controls. The ages of the patients and the healthy controls were not significantly different . Peripheral blood mononuclear cells (PBMCs) were isolated from 20 BP patients  and 20 sex- and age-matched healthy controls. The ages of the patients and the healthy controls were not significantly different . CD19+ B cells and CD4+ T cells were isolated from 6 BP patients  and 6 sex- and age-matched healthy controls. The ages of the patients and the healthy controls were not significantly different .Overall, 32 patients with active BP were involved in the study. The characteristics of the patients are shown in P<0.001)   and 14 age- and sex-matched healthy controls by ELISA. There was a significant increase in IL-21 levels in BP patients as compared with those in the healthy controls  . Then, w= 0.030) .+PD-1+ and CXCR5+ICOS+ cells among the CD4+ T lymphocytes were used to represent the circulating Tfh cells +PD-1+ cells among the CD4+ T lymphocytes in the BP patients as compared with those in the healthy controls   (We collected blood samples from 20 BP patients (no. 07–26) and 20 age- and sex-matched healthy controls into EDTA tubes and isolated the PBMCs. The percentages of Tfh cells in the peripheral blood of the BP patients and healthy controls were detected and analyzed by flow cytometry (FCM). The percentages of CXCR5P<0.001) . MoreoveP<0.001) . These r+PD-1+ cells among CD4+ T lymphocytes showed a positive correlation with the anti-BP180-NC16A autoantibody titers    . The cor P<0.01) .P = 0.012)  with symptomatic improvement and the scores were 0. The serum IL-21 levels of BP patients were measured before and after therapy using ELISA. The serum IL-21 levels after therapy were lower than those before therapy  .+PD-1+ cells among the CD4+ T lymphocytes were determined after therapy. A highly significant reduction in the percentage of CXCR5+PD-1+ cells among CD4+ T lymphocytes was observed after therapy as compared to those before therapy   were obtained at regular intervals following symptomatic improvement with effective therapy. The percentages of CXCR5= 0.003) .+ T cell have been shown to induce Ig production from peripheral blood B cell. We now extend these results to demonstrate the function of activated CD4+CXCR5+ T cells subset in helping B cell to secret pathogenic autoantibody. CD19+ B cells and CD4+ T cells, isolated from 6 BP patients (no. 27–32) and 6 sex- and age-matched healthy controls, were cultured alone or together for 7 days and then the secretion of autoantibody were detected. The co-culture system showed that CD4+ T cells of healthy control or BP patient activated by immobilized anti-CD3 induce anti-BP180-NC16A autoantibody secretion from BP peripheral blood B cells (+CXCR5+ T cells can significantly decrease the secretion of anti-BP180-NC16A autoantibody (P<0.05) (It has been previously reported that anti-CD3-activated human CD4 B cells . In cont(P<0.05) . To dete(P<0.05) . These r+CXCR5+ T cells can decreased the secretion of BP autoantibody in anti-CD3-activated human CD4+ T cells/B cells co-culture system, and so as to the blockade of IL-21 by neutralizing Ab. From the present results, we considered that Tfh cells and IL-21 play an important role in the production of pathogenic autoantibody are involved in the pathogenesis of BP.Tfh cells have received more attention in recent years because they were considered to be responsible for helping B cells during the antibody response + T helper cells, Tfh cells localize in the B cell follicles; the relevant transcription factor for these cells is Bcl-6, which distinguishes them from Th1, Th2, and Th17 cells + T cells has been observed using intravital microscopy As a separate subset of CD4+ T cells/B cells coculture system in vitro. This provided further evidence that Tfh cells were involved in the pathogenesis of BP and that they may play a role in the production of pathogenic autoantibodies.The production of high-affinity antibodies is the most important characteristic of many autoimmune diseases. Tfh cells likely provide inappropriate helper signals to self-reactive B cells in antibody-mediated autoimmune diseases +PD-1+ and CXCR5+ICOS+ cells among CD4+ T lymphocytes were found to be increased in BP patients as compared with those in the healthy controls. Therefore, to a certain extent, CD4+CXCR5+PD-1+ and CD4+CXCR5+ICOS+ cells can both be considered representative of circulating Tfh cells.The surface phenotype of Tfh cells, particularly “circulating Tfh cells,” has been controversial owing to only a partial knowledge of Tfh cells. In our study, CD4, CXCR5, PD-1, and ICOS were chosen as markers for Tfh cells based on the latest studies BP is a typical example of an autoimmune blistering skin disease that is well known for its intractability and propensity for recurrent attacks In conclusion, we assessed Tfh cell counts and IL-21 levels in order to explore whether Tfh cells play a role during the production of autoantibodies in BP patients. Our results suggest that Tfh cells participate in the pathogenesis of BP. Further studies are need to determine whether Tfh cells initiate autoantibody production in BP and to clarify the mechanisms involved.Written informed consent was obtained from all the subjects. This study was approved by the local ethics committee of Xijing Hospital, The Fourth Military Medical University, Xi’an, China.We collected sera from 14 BP patients and PBMCs from 20 BP patients of either sex at the Department of Dermatology, Xijing Hospital, including inpatients and outpatients from November 2009 to November 2011. All subjects were newly diagnosed with BP based on the typical clinical and histological presentation, direct or indirect immunofluorescence examination, and the presence of circulating autoantibodies. None of the patients had received any form of immunosuppressive therapy at the time of inclusion in the study. A total of 34 normal controls matched with patients in terms of sex and age were chosen from the Medical Examination Center of Xijing Hospital. None of the controls presented any evidence of chronic autoimmune diseases or acute infections in their blood. According to the scored standard for the disease severity from Tsuji-Abe et al. The following mouse anti-human mAbs and the corresponding isotype control Abs were used for flow cytometry: PE-conjugated CD3 ; FITC-conjugated CD4 (eBioscience); PE-Cy5-conjugated CD8 (eBioscience); PE-conjugated CD4 (eBioscience); PerCP-eFluor710-conjugated PD-1 (eBioscience); FITC-conjugated CXCR5 ; and PerCP/Cy5.5-conjugated ICOS .Serum IL-21 levels and anti-BP180-NC16A titers were measured using enzyme-linked immunosorbent assays (ELISAs)  according to the manufacturers’ instructions. Optical densities were measured at 450 nm for IL-21 and anti-BP180-NC16A.6 cells·mL−1 in RPMI 1640 containing 10% heat-inactivated FBS . The PBMCs were cultured overnight in six-well plates at 37°C and 5% CO2. The PBMCs were centrifuged for 10 min and resuspended in 100 µL of confining liquid containing 90% PBS and 10% FBS. The cells were then incubated for 50 min with mAbs at 4°C and washed twice with staining buffer. The cells were then resuspended in 400 µL of PBS and assayed using flow cytometry . Viable lymphocytes were chosen using a two-dimensional scatter plot chart composed of forward scatter (FSC) and side scatter (SSC). The percentages of CXCR5+PD-1+ and CXCR5+ICOS+ cells were determined among CD4+ T lymphocytes. The cells were then analyzed using the supporting flow cytometry software.Blood samples were collected into tubes containing EDTA, and PBMCs were isolated from whole blood using a lymphocyte-separating solution . PBMCs were resuspended at 2×10+ B cells and CD4+ T cells were isolated from PBMC of BP patients by using the Magcellect human cell isolation kit following the manufacturer’s instructions . Purity of the recovered CD4+ T and B cell subsets were >95%. A total of 1.5×105 CD4+ T cells and 1.5×105 purified CD19+ B cells of healthy control and BP patient were cultured alone or together in triplicate in 200 µl on the anti-CD3-precoated microtiter wells. The secretion of anti-BP180-NC16A Ab in supernatant were determined by anti-BP180-NC16A EILSA kit after 7 days and each experiment was repeated three times. To evaluate the function of Tfh cells in BP autoantibody secretion, CD4+CXCR5+ T cells were cleaved from the CD4+T cells/B cells co-cultures system by sorting on a FACSAria flow cytometer . In some experiments, the effect of inhibiting IL-21 were examined by adding anti-IL-21 neutralizing polyclonal Ab (1 µg/mL) in the T cells/B cells co-culture system.Peripheral CD19P<0.05 were considered statistically significant. GraphPad Prism 5 was used to complete the figures.The data were analyzed using the SPSS 12.0 statistical software. The Wilcoxon rank sum test was chosen for two independent samples, whereas the Wilcoxon signed-rank test was used for two related samples. Spearman’s rank correlation was used for correlation analyses. Differences with Table S1Patient characteristics(DOCX)Click here for additional data file.


A key determinant of cardiac pump function is the complex interplay between the heart and the arterial system, (i.e. arterio-ventricular coupling). Aging and pathological changes such as atherosclerosis alter elastic properties of the vessel wall, and therefore adversely influence hemodynamic parameters. Mice are commonly used to study cardiovascular disease, but very little has been published on comprehensive blood flow measurements in these models. We therefore sought to establish a compressed sensing (CS) accelerated, phase contrast MR technique (4D flow MRI) for measuring aortic 3D hemodynamics in the mouse.x/y/z=150cm/s, 2 averages) was used to encode blood flow in the aorta in 3D + time (i.e. 4D). To investigate whether or not CS can be used to accelerate the experiments, up to 3-fold undersampling and subsequent CS-reconstruction was performed in post-processing as reported previously : p. 1072-9). All image sets were processed using in-house software for noise reduction, eddy current correction, and anti-aliasing. 3D blood flow visualization (systolic 3D stream lines) and quantification  was performed using EnSight .The experiments were carried out on female C57Bl6 mice  using a quadrature driven birdcage resonator (id 33mm) on a 9.4T Agilent MR system. A four-point referenced, multi-frame phase contrast MR sequence .


Additionally, the T790M-5- and T790M-7-specific T cell lines displayed an MHC class I-restricted reactivity against NSCLC cell lines expressing both HLA-A2 and the T790M mutation. Interestingly, the NSCLC patients with antigen-specific T cell responses to these epitopes showed a significantly less frequency of EGFR-T790M mutation than those without them , indicating the negative correlation between the immune responses to the EGFR-T790M-derived epitopes and the presence of EGFR-T790M mutation in NSCLC patients. This finding could possibly be explained by the hypothesis that immune responses to the mutated neo-antigens derived from T790M might prevent the emergence of tumor cell variants with the T790M resistance mutation in NSCLC patients during EGFR-TKI treatment. Together, our results suggest that the identified T cell epitopes might provide a novel immunotherapeutic approach for prevention and/or treatment of EGFR-TKI resistance with the secondary EGFR T790M resistance mutation in NSCLC patients.Treatment with epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), such as gefitinib and erlotinib, has achieved high clinical response rates in patients with non–small cell lung cancers (NSCLCs). However, over time, most tumors develop acquired resistance to EGFR-TKIs, which is associated with the secondary EGFR T790M resistance mutation in about half the cases. Currently there are no effective treatment options for patients with this resistance mutation. Here we identified two novel HLA-A*0201 (A2)-restricted T cell epitopes containing the mutated methionine residue of the EGFR T790M mutation, T790M-5 (MQLMPFGCLL) and T790M-7 (LIMQLMPFGCL), as potential targets for EGFR-TKI-resistant patients. When peripheral blood cells were repeatedly stimulated  Lung cancer is one of the most aggressive malignancies, but recently significant progress has been made in the therapeutic strategy against this disease In recent years, the field of cancer immunology has moved forward dramatically due to the identification of numerous tumor-associated antigens In the current study, we identified HLA-A*0201 (A2)-restricted antigenic T cell epitopes containing the mutated methionine residue of the EGFR T790M resistance mutation. Given their strong immunogenicity for human T cells, the identified T cell epitopes could provide a novel and promising immunotherapeutic approach for prevention and/or treatment of the secondary EGFR T790M mutation in NSCLC patients treated with EGFR-TKIs.58-66  and HIV-derived epitope (SLYNTVATL), were provided by Thermo Fisher Scientific GmbH  at purities of higher than 90%. T2 cells and NSCLC cell lines, NCI-H1975 (HLA-A2− T790M+) and HCC827 (HLA-A2− T790M−), were obtained from the American Type Culture Collection . PC9 (HLA-A2+ T790M−), PC9/ZD (HLA-A2+ T790M+), 11–18 (HLA-A2+ T790M−), and YM-21 (HLA-A2+ T790M−) were obtained as described previously The peptides containing the wild-type (threonine) or mutated (methionine) residue at the position 790 of EGFR and control HLA-A2-restricted peptides, influenza M1Blood samples were obtained from 6 healthy donors and 22 NSCLC patients treated with EGFR-TKI, gefitinib or erlotinib, between March 2012 and February 2013, at Kurume University Hospital  or Sendai Kousei Hospital . This study was approved by the Institutional Review Board of Kurume University and Sendai Kousei Hospital, and conforms to the provisions of the Declaration of Helsinki. All of the patients had the EGFR gene mutations in exon 19 (delE746-A750) or exon 21 (L858R), and had received or were receiving gefitinib or erlotinib for treatment against advanced diseases at the blood sampling. For analysis of EGFR gene mutations in exon 19 (delE746-A750) or exon 21 (L858R), the peptic nucleic acid-locked nucleic acid (PNA-LNA) polymerase chain reaction (PCR) clamp method was adopted, using protocols described previously +) and T2 cells (T790M−) was used as a positive and negative control for this assay, respectively. Samples were prepared in duplicate for each experiment, and repeated twice to confirm reproducibility.The EGFR T790M mutation was examined in cell-free DNA obtained from plasma of the patients, since no biopsy specimens for DNA analysis could be obtained because of difficult accessibility of tumors during or after EGFR-TKI-treatment. Heparinized whole blood from patients was centrifuged for 10 min at 400 g. After centrifuging for 10 min at 2000 g again, plasma was transferred to a new tube and stored at –80°C until use. Cell-free DNA was isolated from 200 µl of thawed plasma and eluted in 60 µl extraction buffer by using QIAamp DNA Blood Mini kit . The EGFR T790M mutation was detected by the droplet digital PCR (ddPCR) system  according to the manufacturer’s instructions Clinical responses to EGFR-TKIs were evaluated by computed tomography (CT) according to the Response Evaluation Criteria in Solid Tumors (RECIST). Responses were confirmed at least every 4 weeks  or every 6 weeks (for stable disease). Among the 22 NSCLC patients, 8 patients were sensitive to EGFR-TKIs at the time of the blood sample collection. In contrast, 14 patients showed primary (n  =  2) or acquired (n  =  12) resistance to EGFR-TKIs at the blood sampling.http://www.cbs.dtu.dk/services/NetMHC) and BIMAS (http://www-bimas.cit.nih.gov/molbio/hla_bind) are among the best preforming systems for predicting HLA-A2 binding peptides Recent benchmarking has shown that NetMHC 3.2 (58-66 epitope (Flu-M1) was used as a positive control.The actual binding of predicted peptides to HLA-A2 molecules was evaluated by an MHC class I stabilization assay with TAP-deficient RMA-S cells stably transfected with the HLA-A*0201 gene (RMA-S/A2), according to the previously reported method with some modifications + healthy donors and 22 HLA-A2+ NSCLC patients after written informed consent. HLA-A2 positivity was confirmed by flow cytometry with anti-HLA-A2 mAb (BB7.2). Peripheral blood mononuclear cells (PBMCs) (1×105 cells per well) purified by Ficoll-Paque Plus  density centrifugation were incubated with 10 µg/ml of each peptide in 96 round well plates  in 200 µl of the culture medium containing 45% RPMI 1640, 45% AIM-V medium (Life Technologies), 10% FCS, IL-2 , and 0.1 mM MEM nonessential amino-acid solution (Life Technologies). At every 3 or 4 days, half of the culture medium was removed and replaced by new medium containing the corresponding peptide (10 µg/ml) and IL-2 (20 IU/mL). After 14 days, the cells were used for interferon (IFN)-γ ELSPOT or cytotoxicity assays.Peptide-specific T cell lines were generated according to the previously reported method with slight modifications 4 cells/well) were cultured in duplicate for 18 hours at 37°C with T2 cells (1×104 cells/well) loaded with or without the indicated doses of specific or control peptides in a 96-well ELISPOT plate  coated with anti-human IFN-γ mAb. After the plates were washed, biotin-conjugated anti-human IFN-γ mAb, streptavidin-ALP, and BCIP/NBT substrate were added to develop the spots, in accordance with the manufacturer’s instructions. The numbers of spots were then counted by an ELISPOT reader . The peptide-stimulated PBMCs were also tested for their reactivity against tumor cell lines by IFN-γ ELISPOT assay. In some cases, CD8+ T cells were isolated using a CD8 Negative Isolation Kit  from the peptide-stimulated PBMCs. To examine the HLA class I restriction, 10 µg/ml of anti-HLA class I mAb (W6/32: mouse IgG2a) was added into wells at the initiation of the culture. As an isotype control, anti-HLA-DR mAb (L243: mouse IgG2a) was used.For IFN-γ ELISPOT assay , the peptide-stimulated PBMCs  using a MicroBeta2 LumiJET microplate counter (PerkinElmer). Effector cells and 51Cr-labelled cells (2000 cells/well) were cultured in duplicate in 96 round well plates at the indicated effector/target ratios. The spontaneous release and maximal release were determined by the target cells cultured in medium without or with 1% Triton X-100 , respectively. The specific lysis was calculated as follows: specific lysis (%)  =  [(test release – spontaneous release)/] X 100. The means of duplicate samples were used for calculation.Peptide-stimulated PBMCs were also tested for cytotoxicity against tumor cell lines by a standard 6-hour The difference in proportion of the presence of EGFR-T790M mutation between NSCLC patients with and without EGFR-T790M-specific immune responses was statistically evaluated by the chi-squared test .The amino acid sequences (9 to 11-mer) were predicted to bind HLA-A2 molecule by NetMHC 3.2 and/or BIMAS servers. Eight peptides (10- or 11-mer) containing the mutated methionine residue at the position 790 of EGFR (T790M), which showed better scores by either or both of these prediction servers, were selected for further evaluation . But 9-mThe HLA-A2-binding capability of the selected peptides was confirmed by cell surface HLA class I stabilization assay with the TAP-deficient cell line RMA-S, which stably expressed the HLA-A*0201 gene (RMA-S/A2). As illustrated in + healthy donors were repeatedly stimulated with the synthetic peptides, T790M-5, T790M-7, or T790M-8. As shown in To examine the immunogenicity of the EGFR-T790M-derived HLA-A2-binding peptides, PBMCs from 6 different HLA-A2− T790M+), NCI-H1975-A2 (HLA-A2+ T790M+), and HCC827 (HLA-A2− T790M−), by IFN-γ ELISPOT assay. As shown in The T cell lines established after repeated stimulation of PBMCs with T790M-5 or T790M-7 were then examined for their reactivity against NSCLC cell lines, including NCI-H1975 (HLA-A251Cr-release assay. Both T790M-5- and T790M-7-stimulated T cells showed higher cytotoxic activity against NCI-H1975-A2 than HLA-A2-negative NCI-H1975 (+ T790M+), but not against the original PC9 (HLA-A2+ T790M−) or HCC827 (HLA-A2− T790M−) without the T790M mutation , NCI-H1975-A2 (HLA-A2+ T790M+), HCC827 (HLA-A2− T790M−), PC9 (HLA-A2+ T790M−), PC9/ZD (HLA-A2+ T790M+), 11–18 (HLA-A2+ T790M−), and YM-21 (HLA-A2+ T790M−), by IFN-γ ELISPOT assay. As shown in − cell lines (HCC827 and NCI-H1975), HLA-A2+ T790M+ cell lines (NCI-H1975-A2 and PC9/ZD), or HLA-A2+ T790M− cell lines .The WT-5-stimulated T cell line was then examined for their reactivity against NSCLC cell lines, including NCI-H1975 (HLA-A2+ NSCLC patients harboring the EGFR gene mutations in exon 19 (delE746-A750) or exon 21 (L858R), who had been sensitive (n  =  8) or resistant (n  =  14) to the EGFR-TKI treatment at the blood sampling (We further investigated the immunogenicity of the T790M-5 and T790M-7 epitopes in 22 HLA-A2sampling . Among thttp://www.pypop.org/popdata/2008/byfreq-A.php), and many of the peptides binding to HLA-A*0201 with high affinity have been shown to crossreact with other HLA-A2 supertype molecules, including HLA-A*0202, HLA-A*0203, HLA-A*0204, HLA-A*0205, HLA-A*0206, HLA-A*0207, HLA-A*6802, and HLA-A*6901 The clinical benefit of EGFR-TKIs has been demonstrated in NSCLC patients with activating EGFR mutations + NSCLC cells harboring wild-type EGFR. It may be possible that the TCR avidity of WT-5-stimulated T cells was too low to respond to the copy number of naturally processed WT-5 epitopes arrayed on HLA-A2+ NSCLC cells harboring wild-type EGFR.For inducing effective anti-tumor immune responses, tumor antigens should be sufficiently immunogenic to stimulate antigen-specific T cells with high avidity. However, the T cell repertoires available for non-mutated “self-antigens” might have relatively low TCR avidity to them, since T cell reactivity against them can occur only when T cell tolerance is incomplete. In contrast, the “neo-antigens” derived from mutated amino acid sequences could be tumor-specific, and T cell repertoires with high TCR avidity to them might be available due to the lack of central and/or peripheral T cell tolerance st and 3rd positions of wild type epitopes is replaced by the mutated methionine residue, respectively. Although the amino acid residues at the 2nd and C-terminal positions of epitopes have been known to act as the main anchoring sites in HLA-A*0201 st or 3rd positions of epitopes have also been reported to substantially affect the binding capability of peptides to HLA-A*0201 st position of some epitopes have been suggested to directly contact with TCRs and affect the interaction between the peptides and TCRs In T790M-5 and T790M-7, the threonine residue at the 1Recent analyses on T cell reactivity in cancer patients showed that a significant fraction of T cells recognized mutated neo-antigens, but not non-mutated self-antigens, in human cancers Interestingly, the current study showed that the NSCLC patients with antigen-specific T cell responses to EGFR-T790M-derived epitopes showed a significantly less frequency of EGFR-T790M mutation than those without them, indicating the negative correlation between the immune responses to the EGFR-T790M-derived epitopes and presence of EGFR-T790M mutation in NSCLC patients. This finding could possibly be explained by the hypothesis that immune responses to the mutated neo-antigens derived from T790M might prevent the emergence of tumor cell variants with the T790M resistance mutation in NSCLC patients during EGFR-TKI treatment. Alternatively, it may be possible that the presence/occurrence of EGFR-T790M inhibits immune responses to the EGFR-T790M-derived epitopes. To further clarify the causal relationship between immune responses to the T790M-derived neo-antigens and the presence/occurrence of EGFR-T790M in NSCLC patients, both antigen-specific immune responses and T790M mutation status remain to be monitored at different time points  in the same patients in future study.In the current study, T cells stimulated with T790M-5 showed cross-reactivity against the corresponding wild-type WT-5 peptide. However, it should be noted that the avidity of TCR to the WT-5 peptide in the T790M-5-stimulated T cells seemed to be around 100-times less than that to the T790M-5 peptide. Indeed, the T790M-5-stimulated T cells reacted to the PC9/ZD cells harboring the EGFR-T790M mutation, but not to the original PC9 cells expressing EGFR without this mutation. Based on this finding, the cross-reactivity of the T790M-5-stimulated T cells might not be high enough to induce autoimmune toxicity against normal tissues expressing wild-type EGFR.In summary, we identified two novel HLA-A2-restricted, T790M-derived epitopes, which were highly immunogenic for human T cells. Our findings suggested that recognition of T790M-derived neo-antigens can occur in healthy donors and cancer patients, even in the absence of immunotherapy. Although more studies would be required to investigate whether immune responses to the identified epitopes are associated with clinical benefits and can be selectively enhanced by immunotherapies, the current study could provide important information on individualized immunotherapy for NSCLC patients.


Important molecular tools to study ion channel regulation by PtdInsP2 in living cells have been developed in the past. These include fluorescent PH-domains as sensors for Förster resonance energy transfer (FRET), to monitor changes in plasma membrane. For controlled and reversible depletion of PtdInsP2, voltage-sensing phosphoinositide phosphatases (VSD) have been demonstrated as a superior tool, since they are independent of cellular signaling pathways. Combining these methods in intact cells requires multiple transfections. We used self-cleaving viral 2A-peptide sequences for adenovirus driven expression of the PH-domain of phospholipase-Cδ1 (PLCδ1) fused to ECFP and EYFP respectively and Ciona intestinalis VSP (Ci-VSP), from a single open reading frame (ORF) in adult rat cardiac myocytes.Most ion channels are regulated by phosphatidylinositol 4,5-bisphosphate P, EYFP-PH-PLCδ1, and Ci-VSP from a single tricistronic vector containing 2A-peptide sequences first was demonstrated in HEK293 cells by voltage-controlled FRET measurements and Western blotting. Adult rat cardiac myocytes expressed Ci-VSP and the two fluorescent PH-domains within 4 days after gene transfer using the vector integrated into an adenoviral construct. Activation of Ci-VSP by depolarization resulted in rapid changes in FRET ratio indicating depletion of PtdInsP2 in the plasma membrane. This was paralleled by inhibition of endogenous G protein activated K+ (GIRK) current. By comparing changes in FRET and current, a component of GIRK inhibition by adrenergic receptors unrelated to depletion of PtdInsP2 was identified.Expression and correct targeting of ECFP-PH-PLCδ1Expression of a FRET sensor pair and Ci-VSP from a single ORF provides a useful approach to study regulation of ion channels by phosphoinositides in cell lines and transfection-resistant postmitotic cells. Generally, adenoviral constructs containing self-cleaving 2A-peptide sequences are highly suited for simultaneous transfer of multiple genes in adult cardiac myocytes. VSPs specifically dephosphorylate PtdInsP3 and PtdInsP2Research on PtdInsP Ci-VSP, . These p2 in mammalian cell lines requires co-expression of the VSP, two FRET sensors plus the channel or its subunits respectively. Feasibility of this approach in a mammalian cell line has been demonstrated recently in seminal studies analyzing PtdInsP2 regulation of a voltage-activated K+-channel and L-type-Ca2+ channels by PtdInsP22-dependent regulation of endogenous channels in their native signaling environment.Taking advantage of these experimental tools for investigating regulation of ion channels by PtdInsPIn the present study we generated a tricistronic shuttle vector containing the cDNAs for Ci-VSP and two fluorescent PH-domains (ECFP-PH-PLCδ1 and EYFP-PH-PLCδ1) concatenated through viral 2A-peptide sequences to yield a single open reading frame. The 2A-sequences result in co translational dissociation of the polyprotein while allowing translation to continue 1a receptors results in inhibition GIRK channel current by PtdInsP2 depletion and a mechanism that is independent thereof.Expression and functionality of the two fluorescent PH-domains and the voltage-activated phosphatase, expressed from the single ORF is first verified in a standard mammalian cell line (HEK293). The molecular tool was then adapted for transfection resistant primary cells by construction of an adenovirus 2 and Gβγ. Their expression moderately slows replenishment of PtdInsP2 after Ci-VSP-induced depletion but neither affects activation of GIRK channels by Gβγ nor PtdInsP2 abundance required for GIRK channel function. We believe that our approach represents a versatile and powerful laboratory tool, facilitating investigations of regulation of membrane proteins by PtdInsP2 but also of control and homoeostasis of levels of PtdInsP2.PH-domains of PLCδ1 bind PtdInsP3 receptor. As shown in a representative time-resolved recording of single cell ECFP/EYFP fluorescence  are expressed, correctly processed and targeted to the plasma membrane, by transfecting HEK293 cells. As illustrated in 2 but PtdInsP3, whose abundance in the plasma membrane is very low in the absence of an activator of PI3-kinase We next studied if the three proteins encoded by the tricistronic construct P2, were induced by depolarizing voltage steps. Both, the amplitude and the on-kinetics of the FRET-response were voltage-dependent and, in line with previous studies, saturated at membrane potentials positive to +60 mV 2 via phosphatidylinositol-4-phosphate 5-kinase, was independent of the preceding depolarization . Both the kinetics of depletion at saturating voltage and the recovery showed little variation in different cells (E). In conclusion, these data demonstrate expression and correct processing of the three proteins encoded by the single ORF in a standard mammalian cell line.The positive detection of the ECFP/EYFP-fused PH-domains, which are downstream of the Ci-VSP-T2A sequence, predicts that Ci-VSP was expressed, C-terminally fused with 20 residues from the T2A-peptide 2-tool (lane 5), or the analogous vector containing the sequence for Btk-PH (lane 6). For completeness, lane 7 demonstrates cleavage of Ci-VSP2A and EYFP-PH and detection of a genuine EYFP-fused membrane protein . An unidentified XFP-positive band sometimes appeared at MW of about 60 KD , which is unrelated to any of the fluorescent constructs. According to the manufacturer's information, this is an artefact produced by the antibody itself.Positive detection of voltage-dependent changes of FRET ratio does not exclude that a fraction of the proteins escapes the self-cleaving mechanism. The relative amount of uncleaved products was estimated by Western blots using a polyclonal GFP antibody. As illustrated in 2-tool). After infection with the virus, adult rat atrial and ventricular myocytes showed membrane-delimited fluorescence within ≥48 h P2 represents a key finding in this field In order to make this molecular tool applicable to postmitotic transfection-resistant cells, the tricistronic pShuttle-CMV vector was used for recombination with the pAdEasy-1 backbone plasmid to generate a recombinant adenovirus (Ad-PIPin ≥48 h . In West2 receptor undergoes desensitization by various mechanisms K (−50 mV in the present conditions). Moreover, a holding potential negative to −40 mV at the least, is important for preventing basal activation of Ci-VSP 2-tool (B). The depolarization resulted in an instantaneous outward current that rapidly declined also in native or mock-infected myocytes due to the block by intracellular polyamines and Mg2+ underlying inward rectification 2 depletion on outward GIRK current by voltage-activation of Ci-VSP. Upon repolarization the baseline current level is reached quasi instantaneously in native or mock-infected cells, due to the rapid kinetics of the unblock. The effect of Ci-VSP-induced depletion of PtdInsP2 can be identified after repolarization as a reduction of inward current, which recovers with a half time of about 11 s in this cell. The changes in fluorescence of ECFP and EYFP recorded simultaneously with membrane current have been traced in panel C. The resulting change in FRET ratio, superimposed and scaled on the current trace (D), perfectly matches the recovery of current, which was a consistent finding (E), suggesting that PtdInsP2-binding affinities of the PH-domains and Kir3-subunits are similar. As a rule, a depolarization to +60 mV of 4 s in duration was saturating in terms of both, inhibition of current and FRET signal P22-binding of the phosphorylated channel has been suggested 2-depletion by Ci-VSP and activation of α1 receptor activation by phenylephrine  were compared. Analogous to 2 depletion. Exposure to Phe resulted in a stronger reduction of current as compared to saturating activation of Ci-VSP, whereas the change in FRET was substantially smaller. As summarized in q /PLC pathway does not directly reflect depletion of PtdInsP2.Inhibition of GIRK current in atrial myocytes by stimulation of endogenous G2 with a KD in the order of 1 µM. Their expression inevitably causes a reduction in the effective membrane concentration of the phosphoinositide. Depending on the expression level, this might exert a tonic inhibitory effect on PtdInsP2-sensitive proteins and, by competition with PIP-kinase(s), could affect kinetics of replenishment of PtdInsP2βγ2-tool (B). GIRK current in these experiments was activated by 2 µM ACh with GTP instead of GTP-γ-S in the pipette solution to maintain normal G protein cycling. Step depolarizations to +60 mV of 4 s were used to activate Ci-VSP, resulting in comparable inhibition of current by 45% and 40% respectively in these individual cells. The decrease in current by activation of Ci-VSP using this voltage step was not different with and without co-expression of the PH-domains P2 in the inner leaflet of the plasma membrane reduced by expression of the PH-domain or Ci-VSP.Fluorescent PH-PLCδ1 binds PtdInsP-domains , suggest-domains , i.e. inSP alone  suggesti22 has been localized to the pore-forming protein complex, but other mechanisms, such as interaction of PtdInsP2 with a regulatory β-subunit have been identified 2-sensitive channels and transporters are activated or kept in functional state by PtdInsP2; however, inhibition of several ion channel species by PtdInsP2 has been described. Interestingly this includes native cardiac IKs constituted by KCNQ1/KCNE1P2 levels with simultaneous measurement of channel function in their native signaling environment are desirable.Since the first publication demonstrating sensitivity of an ion channel to PtdInsPudy e.g. . For inv2 endogenous or expressed GPCRs that activate PLCs have been used in various studies in the past. This has furthered our knowledge on regulation of ion channels-inward rectifiers and KCNQ channels in particular–by this ubiquitous pathway P2 sensors 2 without activation of signaling pathways. Two laboratories introduced an elegant approach for selectively depleting PtdInsP2 by rapamycin-inducible membrane recruitment of an inositol-phosphatase 2 using this technique is rapid and selective but lacks reversibility. The remarkable potential of voltage-activated phosphatases, which can be rapidly and reversibly controlled by membrane potential on a sub-second time-scale in single cells, has been demonstrated in a number of recent studies using Xenopus oocytes or mammalian cell lines For manipulation of PtdInsP2-sensitive fluorescent sensor proteins and a voltage-activated phosphatase from a single ORF in terminally differentiated cardiac myocytes. The 2A-peptide approach has been used in the past e.g. for reconstituting protein complexes such as the T-cell CD3 complex 2 binding proteins, such as ion channel complexes. This “buffering” effect could affect basal PtdInsP2 levels as well as rates of recovery subsequent to depletion. Using endogenous GIRK current as sensor we can demonstrate that expression of the PH-domains moderately slows recovery of current subsequent to CI-VSP-induced depletion of PtdInsP2, presumably reflecting a buffering effect. Current density of ACh-activated GIRK current was unaffected by expression of PH-domains. This might suggest that the reduction of the abundance of PtdInsP2 by expression of the PH-domains is compensated by an increase in expression of the PtdInsP2-generating kinases, as has been suggested in ref. 46. Since GIRK channels are sensitive detectors of Gβγ, this also excludes significant sequestration of the βγ-complexes involved in channel activation.Standard approaches to achieve co-expresssion of proteins are transfection with several conventional or viral vectors, containing IRES sequences or multiple promoters. These strategies have been used successfully in many studies. However, the number of proteins that can be reliably expressed is limited and difficult to control. In the present work we used a molecular strategy to express two PtdInsP2. This approach facilitates investigating the PtdInsP2-dependence of ion channels with cell-type specific composition of pore-forming and ancillary subunits in their native signaling environments.Our study shows the utility of using multicistronic vectors for simultaneous expression of the present state-of-the art molecular tools for manipulating and measuring the levels of plasma membrane-associated PtdInsPDario, which have a voltage-range of activation better suited for investigating voltage-dependent ion channels in vitro or in vivo studies.The modular nature of the tricistronic vector allows for easy replacement of cDNAs encoding for different sensor constructs see  but also2 binding PH domain of phosfpholipase Cδ1  and the PtdInsP3 binding PH domain of Bruton's tyrosine kinase  were amplified by RT-PCR from rat brain total RNA with the following primer pairs:The cDNAs of the PtdInsPPLCδ1-5′-GGATCCATGGACTCGGGTAGGGACTT-3′ andTTAGAATTCATCAGCCTTTCGCAAGCA-3′5′-GCGGCCGC;Btk -5′-GGATCCATGGCTGCTGTGATACTGGA-3′ andTTAGAATTCTTTTAAGCTTCCATTCCTGTTCT-3′5′-GCGGCCGCThe underlined triplets serve as a variable stop codon. Recombinant fluorescent probes were generated by inserting the cDNA of the PH domains in frame behind ECFP or EYFP in pcDNA 3 (+) (Invitrogen) using a BamHI restriction site as linker.Thosea asigna (T2A) and porcine Teschovirus-1 (P2A), and appropriate restriction sites for recombination were attached to the cDNAs of Ci-VSP  without stop codon and ECFP-PH-PLCδ1 in a second amplification reaction by PCR using the following primer pairs:A multicistronic 2A peptide based vector for expression of Ci-VSP, EYFP-PH-PLCδ1, and ECFP-PH-PLCδ1 was obtained by linking the individual cDNAs with viral 2A oligopeptide sequences as outlined in Ci-VSP-5′-AAGCTTAGATCTGATATCGCCACCATGGAGGGATTCGACGGTT-3′ andTGGGCCGGGATTTTCCTCCACGTCCCCGCATGTTAGAAG5′-AAGCTTAAGACTTCCCCTGCCCTCGCCGGAGCCTCCGGAAATGTCTTCAGCATCTGAAAACGTAAG-3′;ECFP-PH-GGCAGCGGCGCCACAAACTTCTCTCTGCTAAA5′-GAATTCACCGGTGCAAGCAGGTGATGTTGAAGAAAACCCCGGGCCTTGGCGCGCCATGGTGAGCAAGGGCGAGGA-3′ and5′-GCGGCCGCGTTAACTTACGTACGATCAGCCTTTCGCAAGCAGG-3′.2-tool for convenience) Using analogous procedures, a vector was constructed, in which ECFP-PH-PLCδ1 was exchanged for ECFP-PH-Btk (Brutońs tyrosine kinase) to generate pShuttle-Ci-VSPT2A-EYFP-PH-PLCδ1P2A-ECFP-PH-Btk. For expression of Ci-VSP without PH-domains the cDNA was subcloned in pAdtrack-CMV, which contains a GFP sequence to generate pAd-Ci-VSP.(The underlined sequences encode for T2A and P2A). All PCR products were cloned into pCR2.1 Topo TA Cloning vector (Invitrogen) and verified by sequencing. The extended cDNAs of ECFP-PH-PLCδ1, and EYFP-PH-PLCδ1 were subcloned in series in pShuttle-CMV to generate either pShuttle-EYFP-PH-PLCδ1P2A-ECFP-PH-PLCδ1 or in combination with Ci-VSP2A . Production and purification of the recombinant viruses were performed as described in detail previously 3 receptor (M3-R) was amplified by RT-PCR from rat brain total RNA with the following primer pair:The cDNA of a muscarinic M5′–TTGGTGTGTTCTTCCTTGGAC-3′5′-CAGGTAGCTTCTCTCCCGTG-3′3-R-cDNA with stop codon was cloned into the XhoI/KpnI site of pECFP-N1.The M2. For transfection 2.5 µl of diluted polyethyleneimine  were mixed with serum free DMEM and plasmid DNA in a total volume of 200 µl and incubated for 30 min at room temperature. 800 µl serum free DMEM were added to this mixture. Cells were incubated in this solution containing 1 µg of Vector DNA for 3 h. Measurements were performed 48 h to 96 h after transfection.HEK293 cells (ATCC) were cultured on glass coverslips in DMEM supplemented with 2 mM glutamine, 10% fetal bovine serum, 0.1 mg/ml streptomycin and 100 units/ml penicillin in an atmosphere of 95% air, 5% CO3 per dish  in serum-free M199. Cells were infected about 24 h after plating. As a rule, viral titres were adjusted to yield about 50% fluorescent cells on day 3 after infection. Measurements were performed 3–5 d after infection.Wistar Kyoto rats of either sex were killed following protocols approved by the animal welfare officer of the Ruhr-University Bochum in accordance with the guidelines of the European Community (86/609/EEC). All efforts were made to minimize animal suffering. The method of enzymatic isolation of myocytes and serum-free culture conditions have been described in detail elsewhere 3 cells) infected with Ad-PIP2-tool were generated.Non-confluent HEK293 cells grown on 35 mm culture dishes were transfected and lysates were collected after 48 h. Samples were run in 12,5% SDS PAGE Gels and transferred to PVDF membranes (Millipore). Membranes were probed with anti GFP rabbit polyclonal antibody . The secondary antibody was anti rabbit HRP . Membranes were developped using an ECL detection kit  and analyzed in a Fuji LAS-4000 Chemiluminescent imager. Similarly, blots from protein extracts of cultured atrial myocytes : K+ aspartate 110, KCl 20, NaCl 10, MgCl2 1.0, MgATP 2.0, EGTA 2.0, GTP 0.02, and HEPES/KOH 10.0, pH 7.4. Where indicated, GTP was replaced by GTP-γ-S (100 µM). The difference in K+-concentrations between intra- and extracellular solutions resulted in a Nernst potential of −50 mV.For FRET and concurrent FRET and whole cell current measurements an extracellular solution of the following composition was used (mmol/L): NaCl 120, KCl 20, CaClStandard chemicals were from Merck. EGTA, HEPES, MgATP, Acetylcholine, GTP, GTP-γ-S were from Sigma.EYFP/FECFP) were corrected for direct excitation of EYFP  and bleed-through (74% of FECFP). Wide field fluorescence images were taken using a Zeiss AxioCam MRm camera and AxioVision software.FRET measurements were performed using an Axiovert 200 inverted microscope (Carl Zeiss) with a x100 oil immersion objective, a dual-emission photometric system, and a polychrome V lightsource (TILL Photonics). Illumination time was ≤50 ms at a frequency of 2–20 Hz. Excitation wavelength was set to 436±10 nm (beam splitter dichroic long pass 460 nm) and emission of single cells was recorded at 535±15 nm and 480±20 nm (beam splitter dichroic long pass 505 nm). Signals were measured by amplified photodiodes (TILL Photonics), digitized , and stored on a personal computer using Clampex 9.0 software. FRET ratios Click here for additional data file.Figure S2Stable activation of GIRK-current in atrial myocyte by GTP-γ-S in the pipette filling solution. A: Recording of membrane current as described in detail in “2. The rapid vertical deflections represent changes in membrane current due to voltage ramps from −120 mV to +60 mV within 500 ms applied at 0.1 s−1. A current-voltage relation from the ramp labeled “a”, corrected by subtraction of Ba2+-insensitive leak current is plotted in B.(TIF)Click here for additional data file.


Feline leukemia virus (FeLV)-945, a member of the FeLV-A subgroup, was previously isolated from a cohort of naturally infected cats. An unusual multicentric lymphoma of non-T-cell origin was observed in natural and experimental infection with FeLV-945. Previous studies implicated the FeLV-945 surface glycoprotein (SU) as a determinant of disease outcome by an as yet unknown mechanism. The present studies demonstrate that FeLV-945 SU confers distinctive properties of binding to the cell surface receptor.Virions bearing the FeLV-945 Env protein were observed to bind the cell surface receptor with significantly increased efficiency, as was soluble FeLV-945 SU protein, as compared to the corresponding virions or soluble protein from a prototype FeLV-A isolate. SU proteins cloned from other cohort isolates exhibited increased binding efficiency comparable to or greater than FeLV-945 SU. Mutational analysis implicated a domain containing variable region B (VRB) to be the major determinant of increased receptor binding, and identified a single residue, valine 186, to be responsible for the effect.in vivo, or by facilitating entry into a novel target cell with a low receptor density.The FeLV-945 SU protein binds its cell surface receptor, feTHTR1, with significantly greater efficiency than does that of prototype FeLV-A (FeLV-A/61E) when present on the surface of virus particles or in soluble form, demonstrating a 2-fold difference in the relative dissociation constant. The results implicate a single residue, valine 186, as the major determinant of increased binding affinity. Computational modeling suggests a molecular mechanism by which residue 186 interacts with the receptor-binding domain through residue glutamine 110 to effect increased binding affinity. Through its increased receptor binding affinity, FeLV-945 SU might function in pathogenesis by increasing the rate of virus entry and spread  Feline leukemia virus (FeLV) is a naturally occurring gammaretrovirus that infects domestic cats. The outcome of FeLV infection is variable, including malignant, proliferative and degenerative diseases of lymphoid, myeloid and erythroid origin. Determinants of disease outcome are not well understood, but likely involve both viral and host factors. FeLV, like other natural retroviruses, does not occur as a single genomic species but as a closely related, genetically complex family. Sequence variation among natural isolates occurs most commonly in the viral long terminal repeat (LTR) and in the surface-exposed envelope glycoprotein (SU) ,2. An unFeLV-945 differs in sequence from a prototype member of FeLV subgroup A, FeLV-A/61E, in the LTR and in the SU gene ,6,8,9. IAs the receptor-binding protein of the virus, natural variation in SU is associated with significant functional impact on receptor utilization, thereby influencing cell tropism, rate of spread, and disease outcome ,2,10-14.In the present study, unique properties of FeLV-945 SU were characterized that may play a role in its ability to direct disease outcome. Target cell receptor binding was compared between the FeLV-945 and FeLV-A/61E SU proteins. FeLV-945 SU was shown to exhibit an increased efficiency of receptor binding as compared to FeLV-A/61E using a variety of experimental conditions, both when presented in virus particles and in soluble form. The SU proteins of other isolates from the cohort were also found to exhibit an increase in receptor binding efficiency that was comparable to or greater than that observed with FeLV-945 SU. Mutational analyses implicated a region containing the VRB domain of FeLV-945 SU as the major determinant of the distinctive receptor-binding phenotype, and identified a single amino acid residue as primarily responsible for the effect.d; Figure Flow cytometric binding assays were first performed to assess the relative strength of receptor binding by virus particles bearing the Env protein of FeLV-945 or of prototype FeLV-A/61E. For this purpose, equivalent infectious titer of particles bearing either Env protein were allowed to bind to feline 3201 T-lymphoid cells, after which binding was detected using monoclonal antibody C11D8 directed against FeLV SU. The results demonstrated that virus particles bearing FeLV-945 SU bind to the cell surface receptor significantly more efficiently than do particles bearing the FeLV-A/61E SU  within FeLV-945 SU responsible for the increased binding affinity, we first considered VRA since that domain has been previously identified as the major determinant of receptor interaction in murine and feline gammaretroviral SU proteins -21. We bHaving determined that the divergent residues within consensus VRA do not determine receptor-binding affinity, a more comprehensive region surrounding VRA was then examined through the use of substitution mutants. Segments of the FeLV-A/61E SU gene were replaced with corresponding segments of FeLV-945 SU so that the resultant proteins would be substituted of either a VRA domain-containing region or both VRA- and PRR-containing regions . Comparative binding assays using equivalent mass amounts of SU proteins demonstrated the binding phenotype of N147S SU to be indistinguishable from FeLV-A/61E Table . A recipComputational modeling of the putative SU receptor-binding surface demonstrates a change in structure associated with the isoleucine-to-valine change at position 186, and reveals a mechanism that may account for the influence of that change on the binding affinity of FeLV-945 SU Figure , the relFeLV-945 was isolated from a natural cohort of infected cats in which the predominant disease presentation was a distinctive multicentric lymphoma of non-T-cell origin -5. FeLV-in vivo, or by facilitating entry into a novel target cell with a low receptor density. The analysis further demonstrated a 2-fold difference in dissociation constant  are localized within consensus VRB. Mutational analysis, however, demonstrated that these residues could not confer the enhanced binding phenotype of FeLV-945 VRB. Mutational analysis of four other residues  that neighbor consensus VRB and are shared among other cohort isolates similarly demonstrated that these do not influence relative binding affinity. Indeed, only when the isoleucine-to-valine change at position 186 was incorporated into the mutants was the binding phenotype affected Table . By contd relative to prototype FeLV-A SU. Computational modeling and mutational analyses implicate a region containing the VRB domain of FeLV-945 SU as the major determinant of increased binding affinity, although a region containing the VRA domain appears to play a role. The results implicate a single residue adjacent to consensus VRB, valine 186, as the major determinant of increased binding affinity. Computational modeling suggests a molecular mechanism by which residue 186 interacts with residue Q110 to effect increased binding affinity.The SU protein of FeLV-945, a unique natural isolate of feline leukemia virus, directs the induction of non-T-cell multicentric lymphoma through an as yet unknown mechanism. FeLV-945 SU is shown here to bind its cell surface receptor, feTHTR1, with significantly greater efficiency than does prototype FeLV-A SU when present on the surface of virus particles or in soluble form. The increased binding activity demonstrates a 2-fold difference in KMus dunni tail fibroblasts engineered to express the FeLV-A receptor, feTHTR1, and were maintained in DMEM with 10% FBS and 0.6 mg/ml geneticin . An empty vector control, designated pCS2/Ctrl, was generated by religation of BamHI-digested CS2-FeLV-A-61E-SU-HA. 61E/945-VRA and 61E/945-VRA/VRB were generated by replacing a HindIII-to-HindIII fragment or an NdeI-to-Bsu36I fragment of CS2-FeLV-A-61E-SU-HA, respectively, with the corresponding fragment of CS2-FeLV-945-SU-HA. 61E/945-VRA/PRR was generated by replacing a Bsu36I-to-NcoI fragment of 61E/945-VRA with the corresponding fragment of CS2-FeLV-945-SU-HA. 945/61E-VRA/VRB was generated by replacing a NdeI-to-Bsu36I fragment of CS2-FeLV-945-SU-HA with the corresponding fragment of CS2-FeLV-61E-SU-HA. 61E/945-VRB was generated by replacing a HindIII-to-HindIII fragment of 61E/945-VRA/VRB with the corresponding fragment of CS2-FeLV-61E-SU-HA. A reciprocal mutant, 945/61E-VRB, was made by replacing a HindIII-to-HindIII fragment of 945/61E-VRA/VRB with the corresponding fragment of CS2-FeLV-945-SU-HA. SU genes bearing point mutations were generated by site-directed mutagenesis  using CS2-FeLV-61E-SU-HA as template and using the indicated primers where the introduced mutation in each case is underlined: 1) the mutant designated 61E/945-5 using primer 5'-ACTAGTGTTGGATCCTAACAACGTTCGGCATGGAGCTAGGTATAGCAGTAGCAAATATGGATGTAAAACTACAGATAG-3', 2) the mutant designated VRB3aa using primer 5'-GAGGGAGTAATCAGGACAATAGCTGCACAGGAAAATGCAACCCCC-3', 3) the mutant designated N147S using primer 5'-GGGAGTAGTCAGGACAATAGCTGTGAGGG-3', 4) the mutant designated K128N/S130T using primer 5'-GCAACCCCCTAGTCTTACAGTTCACCCAGAAGGGAAGACAAGCCTCTTGG-3', 5) the mutant designated I156V/K164R using primer 5'-GGAGAAGCTTGGTGGAATCCCACCTCCTCATGG-3', and 6) the mutant designated I186V using primer 5'-GGATATGACCCTGTCGCTTTATTCACGGTGTCCCGGCAGG-3'. The mutant designated V186I was generated using CS2-FeLV-945-SU-HA as template and using primer 5'-GGATATGACCCTATCGCCTTATTCACGGTATCCCGGCAGG-3'. The mutants designated K128N/S130T/I186V and I156V/K164R/I186V were generated using primer 5'-GGATATGACCCTGTCGCTTTATTCACGGTGTCCCGGCAGG-3' and using K128N/S130T or I156V/K164R as template, respectively.CS2-FeLV-A-61E-SU-HA , a gift 293T/17 cells were transfected with SU expression plasmids using Lipofectamine LTX reagent  as per the manufacturer's instructions. Approximately 48 hours after transfection, SU-containing cell supernatants were collected and filtered to remove cellular debris. Cell-free supernatants were mixed with Laemmli Buffer  and 2.5% beta-mercaptoethanol, boiled for 5 minutes, and electrophoresed under reducing conditions in NuPAGE Novex Bis-Tris Mini Gels . Proteins were transferred to nitrocellulose and western blot analysis was performed to detect SU proteins using either the primary antibody C11D8  or mouse anti-HA  and an infrared dye-conjugated secondary antibody . SU protein preparations were thereby quantified using the Odyssey Infrared Imaging System (Li-Cor). Equivalent mass amounts of each SU protein were then visualized by western blot analysis using chemiluminescent detection  to confirm the validity of the quantitation.6 TCID50 of either 61E/945L or 61E/945SL were incubated with 5 × 105 3201 cells in the presence of 8 μg/ml hexadimethrine bromide  for one hour at 37°C. Cells were washed in standard azide buffer  and incubated with C11D8 monoclonal antibody for 1.5 hours at 4°C to detect FeLV SU. Cells were then washed again in SAB and incubated with a goat anti-mouse Alexa 488-conjugated secondary antibody  for 30 minutes at 4°C. Virus-bound cells were analyzed on a Becton Dickson FACSCalibur flow cytometer with BD Biosciences CELLQuest Pro Software. In some cases, binding assays were performed with pseudotyped particles where 5 × 105 FFU of pseudotyped particles bearing envelope proteins of FeLV-945, FeLV-922, FeLV-1049, FeLV-1306, or FeLV-1046A [4 3201 cells and processed as described above. Binding assays were also performed using equivalent mass amounts of soluble SU proteins quantified as described above. Soluble SU proteins were incubated with 5 × 105 target cells for one hour at 37°C. Cells were washed in phosphate buffered saline containing 2% horse serum and incubated with either C11D8 antibody or mouse anti-HA antibody  for one hour at 4°C. Cells were then washed as before, incubated with goat anti-mouse Alexa 488-conjugated secondary antibody for 45 minutes at 4°C, and analyzed by flow cytometry. To determine the relative dissociation constants (Kd), binding assays were performed on quadruplicate samples at increasing concentrations of SU protein from FeLV-A/61E or from FeLV-945. The relative Kd was then calculated from the geometric mean fluorescence intensities obtained in three replicate assays at each concentration by nonlinear regression analysis using saturation binding kinetics equations in GraphPad Prism5.0  with an assumption of one site-specific binding. The relative Kd values were used to calculate receptor occupancy using the following equation:Analysis of virus binding to target cells was performed using cell-free supernatants that contained viral particles which had been titered by quantifying the 50% tissue culture infectious dose (TCID50). This was accomplished by using limiting dilution assays for infectivity on FEA cells as described previously . For binBased on the previously reported crystal structure of the receptor binding domain of FeLV subgroup B , the corStatistical analysis of the data from replicate binding assays was performed using one-way ANOVA and Bonferroni post test.The authors declare that they have no competing interests.LSL conceived of the study, participated in study design and implementation, data analysis and interpretation and, together with LLB, drafted the manuscript. LLB developed SU exchange mutants and point mutants (except 61E/945-5), expressed and quantified soluble SU proteins, performed SU binding assays, and together with LSL, played the primary role in experimental design and interpretation. CC and PLR developed assay systems, quantified virus particles and performed virus binding assays. LMA performed the computational modeling, developed the SU point mutant 61E/945-5 and performed the saturation binding analysis. All authors read and approved the manuscript.


Cyclophilin A (CypA) is a typical member of the cyclophilin family of peptidyl-prolyl isomerases and is involved in the replication of several viruses. Previous studies indicate that CypA interacts with influenza virus M1 protein and impairs the early stage of the viral replication. To further understand the molecular mechanism by which CypA impairs influenza virus replication, a 293T cell line depleted for endogenous CypA was established. The results indicated that CypA inhibited the initiation of virus replication. In addition, the infectivity of influenza virus increased in the absence of CypA. Further studies indicated that CypA had no effect on the stages of virus genome replication or transcription and also did not impair the nuclear export of the viral mRNA. However, CypA decreased the viral protein level. Additional studies indicated that CypA enhanced the degradation of M1 through the ubiquitin/proteasome-dependent pathway. Our results suggest that CypA restricts influenza virus replication through accelerating degradation of the M1 protein. Influenza virus is an enveloped negative-sense RNA virus that causes major public health problems worldwide. The matrix protein (M1) is the most abundant protein in the viral particle and forms the bridge between the viral envelope and the core. M1 protein is a multifunctional protein in the influenza virus life cycle including uncoating, transcription, the nuclear export of vRNP, assembly and budding. Several host cell factors have been determined possibly to be required for regulation of influenza virus replication through interacting with M1 at different stages of infection Post-translational modification of proteins by ubiquitin is a key regulatory event in many cellular activities, such as signal transduction, transcription, nuclear transport, membrane protein trafficking, autophagy, and immune responses CypA is a member of the immunophilin superfamily that has peptidyl-prolyl cis-trans isomerase activity. Several lines of evidence implicate that CypA can aid protein folding due to its isomerase activity, and it is also active in cell signaling A cell line depleted of endogenous CypA would be a useful model to understand the precise functions of CypA in the influenza virus life cycle. Therefore, in the present study, a stable RNAi 293T cell line with maximally decreased CypA expression (293T/CypA−) was established as described in To better evaluate the function of CypA during viral infection, a 293T cell line depleted of CypA expression (293T/CypA−) was established as described in the To determine the impact of CypA on influenza virus replication, the one-step growth curves of influenza A/WSN/33 were examined in the 293T/CypA+ and 293T/CypA− cell lines . These cTo determine the infectivity of influenza virus in both 293T/CypA+ and 293T/CypA− cell lines, indirect immunofluorescence assays were performed to detect NP protein at 4 h post-infection (MOI = 1) in both cell lines . An analTo identify the exact stage of viral replication regulated by CypA, real-time PCR assays were performed to detect M1 mRNA, vRNA, and cRNA levels upon influenza virus infection in the 293T/CypA+ and 293T/CypA− cell lines. The results demonstrated that the M1 mRNA level was similar in both 293T/CypA+ and 293T/CypA− cells , and theAs was shown, CypA did not affect the transcription and genome replication of influenza virus. To determine if CypA has an effect on viral mRNA nuclear export, we compared the nuclear and cytoplasmic abundance of M1 and NP mRNA upon influenza virus infection at 4 h post-infection in the 293T/CypA+ and 293T/CypA− cell lines. In addition, we also rescued the expression of CypA in the 293T/CypA− cell line and then analyzed the nuclear and cytoplasmic abundance of M1 and NP mRNA upon influenza virus infection. First, we determined the CypA protein level in the 293T/CypA+, 293T/CypA− and “CypA re-expression” 293T/CypA− cell lines . Then nuAs demonstrated above, CypA had no effect on influenza virus genome transcription, replication, or on the nuclear export of viral mRNA. Thus, we investigate if CypA impairs the viral protein level upon infection. The protein levels of M1 after infection were detected at various time points in both cell lines . At 6 h To analyze whether CypA influences viral protein stability, protein degradation assays were performed. First, both 293T/CypA+ and 293T/CypA− cell lines were transiently transfected with pcDNA3-FLAG-M1 or pcDNA3-FLAG-NP, respectively. Cycloheximide (CHX) was then added 24 h post-transfection. Cells were collected at 0, 1, 2, 3, 4, 5, and 6 h post-treatment with CHX, and western blot analyses were performed for M1 or NP . The qua4Cl. Treatment with MG-132 significantly inhibited the degradation of M1 in both cell lines, whereas the NH4Cl treatment did not inhibit M1 degradation  Tax oncoprotein and promote malignant transformation As the major intracellular protein degradation pathway, the UPS also regulates many basic cellular processes. Inhibition of the UPS affects the replication of several viruses, As for HIV-1, TRIM 5α accelerates the degradation of cytosolic capsid protein normally associated with productive HIV-1 entry The Madin-Darby Canine Kidney (MDCK) cell line (ATCC CCL-34), human embryonic kidney 293T 5′GATCCCCGGGTTCCTGCTTTCACAGATTCAAGAGATCTGTGAAAGCAGGAACCCTTTTTGGAAA-3′ and 5′-AGCTTTTCCAAAAAGGGTTCCTGCTTTCACAGATCTCTTGAATCTGTGAAAGCAGGAACCCGGG-3′BglII/HindIII sites of pSRgfp/neo. A stable cell line expressing the shRNA to CypA was generated using a retroviral spin infection. Retroviral supernatant was generated from 293T cells transfected with the pSR-shCypA with the pCL-Eco packaging construct and pCL-VSVG. The 293T cells were grown to sub-confluence in a 12-well dish. Cells and viral supernatant were then centrifuged at 2,500 rpm for 2 h at 32°C with 8 µg/ml polybrene. Several clonal populations of GFP-expressing cells were obtained and measured for CypA depletion. To measure CypA depletion, a short-length (<1 kb) cDNA library was generated from total RNA using an Aurum total RNA mini kit  and an iScript cDNA Synthesis kit . CypA primers, position 1–20 and position 147–166, were used for quantitative real-time PCR. Primers targeting the CypA intron, β-actin, and GAPDH were used as controls. Primer sequences are available upon request. SYBR green supermix, the MyiQ single-color PCR detection system, and optical system software (Bio-Rad) were used for real-time PCR The pSUPER RNAi System  was used to generate a viral vector (pSR-CypA) to produce an shRNA to CypA. The oligos used for the shRNA were The 293T/CypA+ and 293T/CypA− cells, growing on glass cover slips, were infected with WSN at an MOI of 0.1. The cells were then washed with PBS three times, fixed in 4% paraformaldehyde for 30 min at room temperature, permeabilized with 0.5% triton X-100 in PBS (PBST) for 20 min, and stained with appropriate antibodies. Cell nuclei were stained with 5 µg/ml DAPI (Sigma) in PBS. Following staining, cover slips were analyzed using an LSCMFV500 confocal microscope.6 cells at a confluency of 100% in a 12-well chamber) were washed with PBS and infected with different dilutions of virus for 1 h at 37°C. The virus inoculums were removed by washing with PBS. Cell monolayers were then overlaid with agar overlay medium (DMEM supplemented with 1% low melting point agarose and 1 µg/ml TPCK-treated trypsin) and incubated at 37°C. Visible plaques were counted at 4 days post-infection, and the virus titers were determined. All data are expressed as the mean of three independent experiments.MDCK cell monolayers . At 4 h post-infection, the cells were fixed, and analyzed by indirect immunofluorescence using anti-NP antibody and FITC-conjugated anti-rabbit IgG antibody.5′-AGTAGAAACAAGG-3′), 10 pmol of vRNA-specific primer  Equal numbers of 293T/CypA+ and 293T/CypA− cells were cultivated and infected with WSN (MOI = 0.1). Total RNA was extracted at 0, 2, 4, 6, and 8 h post-infection using Trizol reagent (Invitrogen). The purified RNA samples were treated with DNase I (TaKaRa) for 30 min at 37°C. Total RNA (1.5 µg) was reverse transcribed by AMV reverse transcriptase (Promega) according to the protocol provided by the manufacturer and 10 pmol of cRNA-specific primer (primer: 5′-TCTGATCCTCTCGTCATTGCAGCAA-3′) and M1 reverse (5′-AATGACCATCGTCAACATCCACAGC-3′) 5′-GGTGGTCTCCTCTGACTTCAACA-3′) and GAPDH reverse (5′-GTTGCTGTAGCCAAATTCGTTGT-3′), as described in The analysis of the relative M1 gene expression was performed using Corbett 6200 and PCR primers: M1 forward (2) containing 0.5% Nonidet P-40, 10% glycerol, and 100 units/ml rRNasin (Promega). Nuclei were further washed with 1% Tween-40 and 0.5% sodium deoxycholate, and RNA from both cytoplasmic and nuclear fractions was purified using Trizol (Invitrogen). The purified RNA samples were treated with DNase I (TaKaRa) for 30 min at 37°C. One microgram of total RNA was used for cDNA synthesis with the oligo(dT)18 primer, followed by real-time RT-PCR quantification. Negative controls (amplifications in the absence of RNA or primers) were included in parallel to ascertain absence of contamination by template nucleic acids and the efficiency in RT inactivation. The resulting relative M1 and NP mRNA levels were normalized by the geometric mean of the housekeeping gene (GAPDH).Nuclear and cytoplasmic RNA was fractionated from cells as described in 4Cl were used at the same time as CHX, and the cells were harvested 8 h after treatment.Both 293T/CypA+ and 293T/CypA− cell lines were transfected with pcDNA3-FLAG-M1 or pcDNA3-FLAG-NP plasmids. CHX (100 µg/ml) was added to the medium 24 h post-transfection, and the cells were harvested at different time points. Western blots were probed with anti-β-actin, anti-M1, anti-NP, and anti-CypA antibodies. MG-132 and NH6-tagged proteins by Ni2+-nitrilotriacetic acid (NTA) beads. The cell pellet was lysed in buffer A  and incubated with Ni2+-NTA beads (Qiagen) for 4 h at room temperature. The beads were then washed with buffer A, buffer B , and buffer C , and bound proteins were eluted with buffer D . The eluted proteins were analyzed by western blotting using an anti-FLAG monoclonal antibody.293T/CypA− cells were transfected with expression constructs for Myc-CypA, FLAG-tagged full-length M1, and His-tagged ubiquitin. Thirty-two hours post-transfection, the proteasome inhibitor MG-132 (dissolved in DMSO) was added to the cells for 4 h at a final concentration of 10 µM. Cells from each plate were collected into two aliquots. One aliquot (10%) was used for conventional western blotting to confirm expression and degradation of the transfected proteins. The remaining cells (90%) were used for purification of the His


In approx. 50% of cases, heart failure is caused by an isolated diastolic dysfunction (DD) in the presence of a preserved systolic function but with comparably devastating outcome. Among others, echocardiography (EC) categorized DD mainly according to early (E-wave) and late (A-wave) diastolic mitral blood flow (MBF) as well as tissue-doppler imaging (TDI) showing characteristic S`-E`-A` lateral wall velocity patterns.Cardiovascular magnetic resonance (CMR) has excellent capabilities to assess blood flow and myocardial tissue motion using phase contrast (PC-CMR) imaging but has not been used to quantify DD similar to the EC approach.We introduce TDI-comparable tissue-phase-contrast imaging (TPCI) of the lateral wall and present reference values for MBF and TPCI in a normal collective.In 120 male/female healthy volunteers divided into three age groups  MBF and TPCI was measured by single-slice short axis PC-CMR  comparable to typical EC locations at the tip of mitral leaflets in diastole on a 1.5T whole body MRI system (Philips Achieva). Similar to EC, mitral E-and A-waves, lateral S`-E`-A-velocities, E/A-, E`/A`- and E/E`-ratios were calculated and compared using ANOVA statistics (p<0.05=significant).From group 1 to 3, for MBF there is a significant E-wave decrease, A-wave increase and E-A ratio decrease (p<0.05). Similarly albeit not significant for TPCI, we measured an S`-and E`-wave decrease but A`-wave increase. The E`/A` ratio decreased significantly (p=0.04) whereas E/E` remained unchanged for groups 1-3 (figure By applying PC-CMR to a normal collective we showed for the first time that TPCI is feasible and can be utilized for evaluation of DD similar to the echocardiography approach in a reasonable scanning and analysis time. The generated reference values for MBF and TPCI could be potentially utilized for future evaluations for DD. However, further studies have to be conducted comparing PC-CMR with conventional EC.