Trypanosoma cruzi KMP-11 protein that is conserved among different parasite strains and that is presented by different HLA-A molecules from the A2 supertype. Because peptides presented by several major histocompatibility complex (MHC) supertypes are potential targets for immunotherapy, the aim of this study was to determine whether MHC molecules other than the A2 supertype present the TcTLE peptide.TcTLE is a nonamer peptide from + T cells that recognized the TcTLE peptide using tetramer (twenty) or functional (eight) assays, were determined. SSP-PCR was used to identify the A locus and the allelic variants. Flow cytometry was used to analyze the frequency of TcTLE-specific CD8+ T cells, and their functional activity  was induced by exposure to the TcTLE peptide. All patients tested had TcTLE-specific CD8+ T cells with frequencies ranging from 0.07–0.37%. Interestingly, seven of the twenty-eight patients had HLA-A homozygous alleles: A*24 (5 patients), A*23 (1 patient) and A*01 (1 patient), which belong to the A24 and A1 supertypes. In the remaining 21 patients with HLA-A heterozygous alleles, the most prominent alleles were A24 and A68. The most common allele sub-type was A*2402 (sixteen patients), which belongs to the A24 supertype, followed by A*6802 (six patients) from the A2 supertype. Additionally, the A*3002/A*3201 alleles from the A1 supertype were detected in one patient. All patients presented CD8+ T cells producing at least one cytokine after TcTLE peptide stimulation.From 36 HLA-A2-negative chagasic patients, the HLA-A genotypes of twenty-eight patients with CD8These results show that TcTLE is a promiscuous peptide that is presented by the A24 and A1 supertypes, in addition to the A2 supertype, suggesting its potential as a target for immunotherapy. Trypanosoma cruzi and that is primarily transmitted by blood-sucking triatomines [Chagas disease is a neglected tropical disease that is caused by the flagellated protozoan parasite atomines . Approxiatomines ,2. Afteratomines ,2. Unforatomines ,2.+ T cells are a critical component of the protective immune response against the intracellular parasite T. cruzi [+ T cells in animal models of T. cruzi acute infection demonstrated the role of these cells in infection control. For example, in CD8+ T cell-depleted mice, the disease develop faster, and these animals have higher parasitemia levels than wild-type infected mice [+ T cell activation occurs when cells specifically recognize short peptides (8 to 10 amino acids in length), which are usually derived from intracellular proteins and which are presented in the context of the major histocompatibility complex class I (MHC I) molecules [CD8T. cruzi . The depted mice –8. CD8+ olecules –11. Wherolecules ,13. Clasolecules . At leasolecules .The frequencies of HLA alleles vary greatly among different populations; thus, obtaining knowledge regarding these alleles is a critical step in the development of potential therapeutic or prophylactic immune strategies that require population-wide coverage. In this context, promiscuous peptides, which are defined as peptides that can be presented by different HLA molecules belonging to the same MHC  or even T. cruzi is a HLA-A*0201-restricted peptide [Our group recently reported that the TcTLE peptide (previously named K1 peptide) from the KMP-11 protein of  peptide . This pe peptide .+ T cells from these chagasic patients. Indeed, the finding that A24, A23 and A01 homozygous patients have TcTLE-specific CD8+ T cells demonstrated that several MHC supertypes present this peptide. TcTLE peptide-specific CD8+ T cells from non-HLA-A2 chagasic patients were also shown to be able to produce gamma interferon (IFN-γ), tumoral necrosis factor (TNFα), interleukin-2 (IL-2), perforin, and granzyme B and to express the proteins CD107a/b in response to TcTLE peptide stimulation.In the present study, the HLA-A2-restricted TcTLE peptide was observed to be presented by other HLA-A supertypes and able to induce functional activity in CD8T. cruzi antibody-positive according to both indirect immunofluorescence immunoassay (IFI) and enzyme-linked immunosorbent assay (ELISA) [Thirty-six chagasic patients from endemic areas of Colombia, including 13 males and 23 females with ages ranging from 19–76 years, were included in the study. All the subjects were recruited and clinically evaluated at the Hospital Universitario San Ignacio, Fundación Abood Clínica Shaio or Instituto Nacional de Salud in Bogotá, Colombia. All subjects were anti- (ELISA) . FifteenApproximately 20 mL of blood was collected from each individual by venipuncture as follows: 10 mL was collected in heparinized tubes for isolating peripheral blood mononuclear cells (PBMCs) by Ficoll-Paque PLUS density gradient centrifugation , 4 mL was collected in EDTA tubes for DNA extraction and 6 mL was collected for serological tests .th April 2011 and 17th June 2009 CIEs). All adult subjects who participated in the study read, accepted and signed the informed consent form.This work was approved by the Research and Ethics Committees of the Facultad de Ciencias from the Pontificia Universidad Javeriana . Once the alleles present in each individual were determined, medium-high-resolution typing was performed using A*01, A*03, A*11, A19, A*23, A*24 or A*68 SSP Unitray kits  according to the manufacturer’s protocols.HLA-A locus evaluation was performed using a commercial PCR kit based on sequence-specific primers (SSP-PCR) according to the manufacturer’s instructions . Genomic DNA was extracted from each patient’s peripheral blood using a GFX genomic blood DNA purification kit . PCRs were conducted in a Stomacher 3500 Thermal Cycler PTC-100 . The amplified products were resolved via 2% agarose gel electrophoresis and visualized following ethidium bromide staining. The amplified fragments were analyzed using UnimatchT. cruzi KMP-11 protein [6 PBMCs per tube were stained with 0.5 μg/mL tetramers and anti-CD3-PerCP (clone SK7) and anti-CD8-FITC (clone SK1) conjugates  for 20 minutes in the dark at room temperature. After the cells were washed with staining buffer , they were resuspended in 500 μL of 1X PBS. At least 50,000 events gated for CD3+ CD8+ T cells were acquired and analyzed using a FACSAria II flow cytometer (BD Immunocytometry Systems) and FlowJo 9.3.2 software . The gating strategy is shown in + T cells was established at 0.063% after determining the average background value of K1-specific CD8+ T cells from HD (0.027%) plus three standard deviations (0.012%). A cut-off point for HLA-A2/Flu-MP* and HLA-A2/CMV* tetramer CD8+ T cells was fixed at 0.1% [HLA-A2 PE-labeled tetramers loaded with TcTLE peptide (TLEEFSAKL), derived from  protein , and als protein ,26, and 6 PBMCs were cultured with the TcTLE peptide (10 μg/mL) for 6 hours at 37°C and 5% CO2 in the presence of CD28 (1 μg/mL) and CD49d (1 μg/mL). After the cells were incubated for one hour, brefeldin A (1 μg/mL) and monensin (1 μg/mL) (BD Pharmingen) were added to the cultures. PBMCs were labeled with Live/Dead Fixable Aqua for 20 minutes in the dark at room temperature. Then, the cells were stained with anti-CD3 Pacific Blue (clone UCHT1) and anti-CD8 APC-H7 (clone SK1) monoclonal antibodies (BD Biosciences). The cells were permeabilized with Cytofix/Cytoperm (BD Pharmingen) and stained with anti-IL-2 PerCP-Cy5.5 , anti-IFN-γ Alexa Fluor 700 , anti-TNFα APC , anti-Perforin PE  and anti-Granzyme B Alexa Fluor 647 (clone GB11) conjugates (BD Biosciences) for 30 minutes at 4°C. To evaluate potential cytotoxic activity, anti-CD107a (clone H4A3) and CD107b FITC (clone H4B4) conjugates (BD Biosciences) were added to the PBMCs before stimulation. In each experiment, non-stimulated cells were used as a negative control, and staphylococcal enterotoxin B (SEB) (1.5 μg/mL)  was used as a positive control (+ CD8+ T cells were acquired using a FACSAria II flow cytometer (BD Immunocytometry Systems) and analyzed with FlowJo 9.3.2 software . A positive cytokine response was defined by subtracting the background of cytokine production (cells cultured without antigen) and obtaining a value exceeding 0.05%; this frequency corresponds to the average frequency of CD8+ T cells producing cytokines from HDs cultured with the TcTLE peptide plus 3 standard deviations. Multifunctional analysis was performed using the Boolean gating strategy and visually represented with Pestle version 1.7 and SPICE software version 5.3  [To assess T cell function, 1 × 10 control  Tables. p < 0.05. GraphPad Prism version 6.0 for Mac OS X  statistical software was used for statistical analyses.Differences between two groups were assessed using the Mann-Whitney test. Tests were two-tailed, and differences were considered statistically significant when + T cells in the context of the HLA-A*0201 molecule during the natural course of Chagas disease [+ T cells from infected patients in the context of the HLA-A*0205, HLA-A*0222, HLA-A*0226, HLA-A*0259 and HLA-A*0287 alleles [+ T cells in a broader number of patients, twenty-eight of the thirty-six analyzed HLA-A2-negative chagasic patients were found to have CD8+ T cells that recognized the TcTLE peptide. Tetramer staining revealed that twenty of these patients had CD8+ T cells that were able to bind to the HLA-A2/K1-peptide tetramer at percentages ranging from 0.07–0.37% (median = 0.16%) . In order to evaluate the frequency of specific-CD8+ T cells of the irrelevant peptides, used as controls, the frequency of Flu-MP* and CMV* specific-CD8+ T cells was determined in the same assay conditions and with cells from the same patients. Indeed, it was found that although 11 out of 28 chagasic patients presented frequencies to both, TcTLE and Flu-MP* peptides, 8 out of 28 only presented frequencies to TcTLE peptide and one patient (KT-059) only presented frequencies to Flu-MP*. Additionally, the frequency of CD8+ T cells specific to CMV* was determined in the QX-036 and QX-039 patients. The results showed that while the QX-036 patient had frequencies of CD8+ T cells specific to TcTLE and CMV* peptides, the QX-039 patient only had frequencies of TcTLE peptide-specific CD8+ T cells. Together, these results demonstrate the specificity of the response.Previous studies of the KMP-11-derived TcTLE peptide revealed that it is efficiently processed, presented and recognized by CD8 disease ,28. In a= 0.16%) . The CD8Based on these results, the HLA-A genotype of these twenty-eight patients  was confTo determine the relationship between the HLA allele supertype and TcTLE recognition, tetramer- (twenty) or cytokine/cytotoxic-positive (eight) patients and healthy controls (twelve) were subjected to medium-high-resolution molecular typing . Seven H+ T cells from HLA-A2-negative patients with A24 or A1 supertypes, CD8+ T cells from twelve chronic chagasic patients were analyzed using six functional parameters after TcTLE peptide stimulation. Thus, the intracellular production of three cytokines  and the potential cytotoxic activity, which was measured by evaluating CD107a/b expression as a marker of degranulation and intracellular perforin and granzyme B expression, were assessed.To evaluate the functional activity of TcTLE peptide-specific CD8+ T cells that produced at least one of these cytokines  showed three functions, three patients (25%) exhibited four functions, and four patients (33.3%) presented positive results for five of the six molecules tested. Cytokine-producing CD8+ T cells were not detected in HDs after antigenic stimulation  were measured after peptide stimulation. No TcTLE-specific CD8+ T cells exhibited all five effector functions  are functional.Subsequently, CD8unctions  in non-cunctions . Generalpatients . Overall+ T cells were demonstrated to be essential in controlling the spread of T. cruzi infection [+ T cells specific to T. cruzi antigens play an important role in protecting against T. cruzi infection in humans, and most of their epitopes are restricted to the HLA-A*0201 allele. Thus, some A2-restricted epitopes have been extensively studied to explore their use in vaccine or immunotherapy design [+ T cells from these HLA-A2-negative chronic chagasic patients was analyzed in terms of frequency and functionality, and its correlation with patient haplotype was assessed.Using experimental animal models, CD8nfection –8. In ady design –32. One y design , 27. In Mongkolsapaya et al. [Liu et al. [+ T cells from chagasic donors having A1, A2 and A24 supertypes was detected. This phenomenon of presentation by different MHC supertypes has also been identified in tumor-derived peptides [Several reports have described promiscuous T cell epitopes derived from infectious organisms, such as the dengue and Epstein-Barr (EBV) viruses, which are presented by more than one HLA-A allele and even by different HLA-A supertypes ,18. For u et al.  describepeptides ,33.Mycobacterium tuberculosis, peptides derived from the TB10.4 protein are presented by homozygous HLA-A*3001 and A*3002 alleles, which are recognized by peptide-specific CD8+ T cells from patients with active tuberculosis [Peptides from different pathogens presented in the context of homozygous HLA-A alleles confirmed the antigen presentation by alleles belonging to different supertypes. In rculosis . Cytomegrculosis . Similar+ T cell responses in T. cruzi-infected patients.Although HLA-B and HLA-C alleles may also bind and present the TcTLE peptide, our results indicate that the TcTLE peptide was able to be presented by the HLA-A*6802 allele from the A2 supertype, the supertype in which this peptide was originally described, and by other HLA-A alleles belonging to other supertypes, such as HLA-A*0101, A*3002, A*3201 and A*1101 of the A1 supertype) and A*2301 and A*2402 of the A24 supertype, which all generated measurable specific CD8+ T cells from fifteen of nineteen (78.9%) HLA-A2-positive chagasic patients were previously shown to recognize the TcTLE peptide with tetramer frequencies ranging from 0.09% to 0.34% [+ T cells from chronic chagasic patients recognized the TcTLE peptide independent of their HLA-A supertype, which indirectly indicates that the TcTLE peptide is presented by HLA-A alleles other than HLA-A2. Taken into account the published data [LEEFSAKL) peptide to other alleles of the HLA-A1 and HLA-A24 supertypes is possible because the leucine (L) at 2 and 9 primary anchors positions of the TcTLE peptide bound to the B and F pockets of the MHC molecules of these supertypes (ILGFVTTL) peptide, fits in the B and F pockets of the alleles of these supertypes, respectively , A*24 (11%) and A*68 (5%) in Brazil ,47; A*02Trypanosoma rangeli, a synanthropic non-human pathogenic parasite [Consequently, a high percentage of the Latin American population has HLA-A alleles belonging to the A1, A2 and A24 supertypes, which are capable of presenting the TcTLE peptide and of inducing immune responses through effector functions and cytotoxic activities. Additionally, the TcTLE sequence is conserved among different parasite strains, particularly if it is also present in the KMP-11 protein from parasite . Taken tparasite ,54, coulT. cruzi-derived peptide, which exhibits broad coverage of HLA presentation in the population, is a potential target for immunotherapy against Chagas disease.In summary, these results demonstrate that the TcTLE peptide epitope is recognized and presented by different HLA-A alleles from the A2, A24 and A1 supertypes and that it induces an immune response characterized by cytokine production and cytotoxic activity. This S1 Fig+ T cells to evaluated the frequency of TcTLE-specific CD8+ T cells and non-CD8+ T cells, and on total CD8+ T cells.Dot plots representative of the analysis of two HLA-A2-negative chagasic patients. Analysis were made on total CD3(TIF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.


The Murine Leukemia Virus (MLV) is a gammaretrovirus that hijack host components of the endosomal sorting complex required for transport (ESCRT) for budding. To determine the minimal requirements for ESCRT factors in MLV viral and viral-like particles (VLP) release, an siRNA knockdown screen of ESCRT(-associated) proteins was performed in MLV-producing human cells. We found that MLV VLPs and virions primarily engage the ESCRT-I factor Tsg101 and marginally the ESCRT-associated adaptors Nedd4-1 and Alix to enter the ESCRT pathway. Conversely, the inactivation of ESCRT-II had no impact on VLP and virion egress. By analyzing the effects of individual ESCRT-III knockdowns, VLP and virion release was profoundly inhibited in CHMP2A- and CHMP4B-knockdown cells. In contrast, neither the CHMP2B and CHMP4A isoforms nor CHMP3, CHMP5, and CHMP6 were found to be essential. In case of CHMP1, we unexpectedly observed that the CHMP1A isoform was specifically required for virus budding, but dispensable for VLP release. Hence, MLV utilizes only a subset of ESCRT factors, and viral and viral-like particles differ in ESCRT-III factor requirements. An essential step in the formation of an extracellular enveloped virus particle is the separation of virus and host cell membranes. For many viruses, this requires the recruitment of a network of proteins normally involved in analogous cellular membrane fission events, like intraluminal vesicle formation at the multivesicular body (MVB), separation of daughter cells during cytokinesis, and extracellular shedding of plasma membrane wounded portions ,3,4,5,6.nL and FPIV motifs found in different viruses [nL-type late domains found in equine infectious anemia virus (EIAV) and Sendai virus are responsible for binding Alix, an ESCRT-associated protein that links viral proteins to the CHMP4 subset of the downstream ESCRT-III complex, thereby bypassing the need of ESCRT-I and ESCRT-II [N-terminal C2 domain, rather than the PPXY-binding WW domain of Nedd4-2s functions as an autonomous Gag-targeting module, thereby stimulating HIV-1 budding [As ESCRT-mediated fission events resemble enveloped virus budding, a wide variety of viruses use ESCRT pathway functions to escape from the cell. Viruses recruit ESCRT components through the function of specific peptide motifs within their structural proteins referred to as late assembly domains ,11. The  viruses ,11. Thes viruses ,5,7,14.  viruses . The nee viruses ,15,16. A viruses ,18. The ESCRT-II ,5. PPXY-ESCRT-II ,20,21,22 budding ,24.Many viruses contain multiple late domains that can bind and recruit different early-acting ESCRT or ESCRT-associated factors. Due to this redundancy, the according entry sites into ESCRT pathway axes remain to be dissected. For example, murine leukemia viruses, prototypical gammaretroviruses, encode all three canonical late motifs in their Gag polyproteins with the PPPY domain being essential, while the PSAP and LYPAL motifs play complimentary roles ,25,26. AC-terminal fused, enhanced yellow fluorescent protein (YFP) (termed herein MLV.GagYFP) was generously provided by Mothes, et al. [® Site-Directed Mutagenesis Kit  was used. The primers Gag.FLAG.forward  and Gag.FLAG.reverse (5′-CGGTGCCTCTCCCGCAAGCTTATCGTCATCATCCTTATAGTCCCTGTCGGA-3′) were designed using the NEBaseChanger™ software . Mutagenesis was performed in the MLV.Gag background (termed herein MLV.GagFLAG/YFP) and in the MLV retroviral construct. In addition, the Env ORF encoded by pCL-Eco was tagged with a HA-epitope (YPYDVPDYA) that was inserted between aa 294 and 295 of Env. The insertion site is located within a proline-rich region connecting the N-terminal receptor binding domain and the C-terminal transmembrane domain of Env and had been shown to be permissive for insertions [TACCCATACGATGTTCCAGATTACGCTCCCAGTGGGACTGCAGTCTCCCCT-3′; mutant nucleotides are shown in boldface) and Env.HA.reverse (5′-AGGGGAGACTGCAGTCCCACTGGGAGCGTAATCTGGAACATCGTATGGGTATGGTTTGGTGACTGAAGG-3′) were used. The modified pCL-Eco construct is termed MLV.WTFLAG/HA herein. The pCL-MFG-LacZ reporter construct used for packaging in the tagged MLV retroviral pCL-Eco vector was purchased from Novus Biologicals .The expression vector encoding the MLV Gag full-length precursor protein with a , et al.  via Addg, et al.  and obta, et al. , whereupsertions . For mutThe following primary antibodies were used: mouse anti-ß-Actin and mouse anti-FLAG ; mouse anti-Alix, mouse anti-EAP20, rabbit anti-EAP30, rabbit anti-EAP45, and mouse anti-Tsg101 ; mouse anti-GFP ; mouse anti-HA ; and rabbit anti-Nedd4 . Peroxidase-labeled, secondary antibodies were obtained from Dianova , and fluorophore-labeled antibodies were purchased from Molecular Probes .5 cells per well of a 6-well plate were transfected with a final concentration of 30 nM siRNA according to the protocol of the supplier. After 48 h, cells were retransfected with 4 μg plasmid DNA using Lipofectamine™ Plus (Invitrogen), and transfected cells were harvested after additional 24 h. siRNA oligo sequences are provided in For transient expression studies, the human hepatocellular carcinoma cell line HuH-7 (obtained from the European Collection of Authenticated Cell Cultures) and the human embryonic kidney 293T cell line (obtained from Luise Florin) were used. To transfect cells with siRNAs, the Lipofectamine™ RNAiMAX transfection reagent  was utilized. Briefly, 5 × 10w/v) sucrose cushion . Pellets were suspended in 1× Laemmli buffer.To probe for protein expression, cells were lysed with 1× Laemmli buffer, and cell suspensions were boiled for 10 min prior to centrifugation. Cell extracts were subjected to SDS-PAGE and Western blotting (WB) analyses using standard procedures. To analyze the assembly and release of viral particles from transfected cells, clarified culture medium was concentrated by ultracentrifugation through a 20%  and Direct-zol MiniPrep , according to the protocols of the suppliers. The mRNA was treated with 5 U RNase-free, recombinant DNase I , and cDNA synthesis was performed by using the Transcriptor Universal cDNA Master Kit . For qRT-PCR, each reaction mixture (20 μL) contained 5 μL cDNA template, 1 μL forward primer (10 μM), 1 μL reverse primer (10 μM), 10 μL Fast Start Universal SYBR Green Probe Master (Roche Diagnostics), and 3 μL aqua bidest. Specific primer pairs were retrieved from PrimerBank , and seqFLAG/HA plus pCL-MFG-LacZ plasmid DNAs at a 1:1 DNA ratio. After 24 h, retroviral cell supernatants were harvested, filtrated (Ø 0.45 μm), and used immediately for infection. 24 h before infection, murine NIH 3T3 target cells were plated on cover slips at a density of 0.4 × 104 cells per well of a 12-well plate and cultivated in DMEM supplemented with 10% fetal calf serum and 5 μg/mL Ciprofloxacin. The HuH-7–derived supernatants were titrated onto NIH 3T3 cells in the presence of 8 μg/mL polybrene. Two days post-infection, MLV infectivity was assayed by staining of cells for ß-galactosidase activity using the ß-Gal Staining Kit (Life Technologies) according to the supplied protocol. Cells were examined under a microscope by counting the number of blue cells and the total number of cells in eight random fields of view.For the infection assay, HuH-7 cells were transfected with siRNAs for 48 h and retransfected with MLV.WTFor immunostaining, siRNA- and DNA-transfected HuH-7 cells were treated with 1 μg/mL cycloheximide for 6 h to inhibit protein synthesis. Thereafter, cells were fixed and permeabilized with ice-cold methanol containing 2 mM EGTA. Cells were blocked in PBS containing 1% bovine serum albumin, incubated with the primary antibodies for 1 h at 37 °C, rinsed with PBS, and then incubated with AlexaFluor-conjugated secondary antibodies for 1 h at 37 °C. DNA was stained with Hoechst 33342 (Sigma-Aldrich). Images were acquired using a Zeiss Axiovert 200 M microscope equipped with a Plan-Apochromat 100×  and a Zeiss Axiocam digital camera (Zeiss). Axiovision software v4.7.1 (Zeiss) was used for merging pictures.C-terminally fused YFP was used (The MLV Gag polyprotein contains three putative late domains located in the matrix (MA) domain, at the junction between MA and the p12 region, and in p12 ,11,26 F. Previouwas used .YFP in an ESCRT/MVB-dependent manner. Cells were treated with control siRNA or specific siRNAs directed against Tsg101, Alix, or Nedd4-1, prior to transfection with MLV.GagYFP. For Tsg101, a single siRNA duplex was used, while Alix and Nedd4-1 were silenced with siRNA pools consisting of four different duplexes. Cell lysates were analyzed for the depletion efficacy of Tsg101, Alix, or Nedd4-1 by specific immunoblotting. All siRNAs effectively reduced the expression of their targets as compared to control siRNA-treated cells  was used. Except for cells in which Alix was knocked down, the intracellular level of MLV.GagYFP was largely unaffected , CHMP3, and CHMP2 (A or B) are recruited in this order on membranes and are thought to polymerize into a helical architecture to constrict and abscise membrane buds. The remaining ESCRT-III  exerts regulatory functions, like mediating recruitment of the Vps4 ATPase. All ESCRT-III proteins are required for MVB biogenesis and during cell division . Functioed cells . To inveP export . These rFLAG/HA construct and cell extracts and supernatants were analyzed by tag-specific WB. The knockdowns of CHMP2B, CHMP3, CHMP4A, CHMP4B, CHMP5, and CHMP6 did not affect the overall degree of synthesis and stability of Env and Gag proteins within the cells as compared to siControl-treated cells  CHMP1B-specific transcripts as compared to control cells.By using a similar approach, we studied the role of ESCRT-III in MLV virion production. In difference, siRNA-treated cells were transfected with the MLV.WTed cells A. By congYFP see , the egrgYFP see A. Similaturation A. The moTo more closely explore the crucial roles of CHMP1A, CHMP2A, and CHMP4B in MLV virus production, we inspected the degree of Gag polyprotein processing. Lysates and supernatants of ESCRT-III-depleted, MLV-producing cells (see above) were solely analyzed by FLAG-specific WB. In control cells, Gag predominantly appeared as a 65 kDa species, corresponding to the full-length, uncleaved polyprotein, together with two species of 55 and 25 kDa B. Upon mversus VLP morphogenesis, it was also important to exclude the possibility that the FLAG-tag introduced into the p12 domain of Gag somehow might contribute to the CHMP1A dependency. Therefore, we constructed an YFP-tagged MLV.Gag construct carrying an analogous FLAG epitope insertion (MLV.GagFLAG/YFP) and analyzed its behavior in siControl- and siCHMP1A-depleted cells. By probing cell lysates and supernatants by FLAG- and GFP-specific WB, we found that the CHMP1A knockdown did not impair the synthesis and release of double-tagged MLV VLPs  class I via the MVB. Unlike conventional MVB sorting, this pathway neither needs ESCRT-II nor CHMP6 but operates with the histidine domain phosphotyrosine phosphatase, a Bro1 domain-containing protein related to Alix. Similar to MLV budding, MHC class I sorting only requires a subset of ESCRT-III factors, like CHMP2A, CHMP4B, and CHMP3, whereas CHMP2B, CHMP4A, and CHMP4C are not involved . Hence, One unexpected finding of this work was the observation that CHMP1A is a dependency factor of MLV virion, but not VLP egress. In general, full particles and VLPs of one retrovirus are thought to exploit identical ESCRT factors for budding. To account for the differential functions of the two CHMP1 isoforms in MLV release, isoform-specific modifications may be involved, as CHMP1A had been shown to be a target for phosphorylation and sumoylation, and contains a putative ubiquitin-interacting motif ,56,57. MIn summary, our experiments reveal that MLV subviral and viral particle budding depends on a surprisingly small subset of ESCRT proteins, including TSG101, CHMP2A, and CHMP4B. MLV virions and VLPs share the minimal requirements for these ESCRT factors, but differ in their unequal use of CHMP1A. Albeit the fundamental role of CHMP1A played in MLV virus release remains to be elucidated, our results provide first evidence that CHMP1A may function in virus maturation and trafficking processes.


EGFR mutational status. TAE226 was more effective against cells with mutant EGFR, including the T790M mutant, than against cells with wild-type one. TAE226 preferentially inhibited phospho-EGFR and its downstream signaling mediators in the cells with mutant EGFR than in those with wild-type one. Phosphorylation of FAK and IGF-IR was not inhibited at the concentration at which the proliferation of EGFR-mutant cells was inhibited. Results of the in vitro binding assay indicated significant differences in the affinity for TAE226 between the wild-type and L858R (or delE746_A750) mutant, and the reduced affinity of ATP to the L858R (or delE746_A750) mutant resulted in good responsiveness of the L858R (or delE746_A750) mutant cells to TAE226. Of interest, the L858R/T790M or delE746_A750/T790M mutant enhanced the binding affinity for TAE226 compared with the L858R or delE746_A750 mutant, resulting in the effectiveness of TAE226 against T790M mutant cells despite the T790M mutation restoring the ATP affinity for the mutant EGFR close to that for the wild-type. TAE226 also showed higher affinity of about 15-fold for the L858R/T790M mutant than for the wild-type one by kinetic interaction analysis. The anti-tumor effect against EGFR-mutant tumors including T790M mutation was confirmed in mouse models without any significant toxicity. In summary, we showed that TAE226 inhibited the activation of mutant EGFR and exhibited anti-proliferative activity against NSCLCs carrying EGFR mutations, including T790M mutation.TAE226, a bis-anilino pyrimidine compound, has been developed as an inhibitor of focal adhesion kinase (FAK) and insulin-like growth factor-I receptor (IGF-IR). In this study, we investigated the effect of TAE226 on non-small-cell lung cancer (NSCLC), especially focusing on the  Lung cancer is the leading cause of cancer death worldwide . Lung caEGFR mutations, small deletions at exon 19 and L858R mutation at exon 21. Besides these EGFR-TKI-sensitive mutations, T790M mutation at exon 20 is known as EGFR-TKI-resistant mutation   supplemented with Complete, Mini  to extract protein. After the protein concentration was measured, equal amounts of total protein (30 μg) were separated by SDS-PAGE and transferred to PVDF membranes. The proteins on membranes were incubated overnight at 4°C with the primary antibodies. The following secondary antibodies were used: goat antirabbit or antimouse IgG-conjugated horseradish peroxidase (HRP) . To detect specific signals, the membranes were incubated by ECL plus Western Blotting Detection Reagents .Cells were cultured in 60 mm dishes overnight, and then, treated with DMSO or each concentration of TAE226 and gefitinib. Next, they were washed out with cold PBS and lysed in 1 × cell lysis buffer , which had about 20 g body weight. TAE226 was diluted by methylcellulose in the concentration of 0 mg/kg (vehicle), 30 mg/kg, 60 mg/kg and 90 mg/kg. After the tumor volume has reached 200–400 mm3, TAE226 was orally administered to each nude mouse once a day for serial 14 days. Every three days, body weight of the mice and the major axis and minor axis of each tumor were measured and calculated tumor volume using the following formula; (major axis) × (minor axis)2 × 1/2 [In this experiment, we used BALB/c-nu/nu female nude mice at 4–6 weeks of age that were purchased from Charles River Laboratories Japan, Inc . They were maintained under specific-pathogen-free conditions in accordance with the guidelines of the Animal Care and Use Committee at Okayama University. PC-9 and RPC-9 cell lines (4.0×10)2 × 1/2 , 38. Fort test was used to compare data between two groups. Data were represented as mean ± standard deviation (SD). P < 0.05 was considered as being statistically significant.Student’s 50 value obtained from independent assays are shown in EGFR mutations and two NSCLC cell lines (RPC-9 and NCI-H1975) with both TKI-sensitive and TKI-resistant EGFR mutations showed similar IC50 values, ranging from 0.086 to 0.31 μM. In contrast, the IC50 values for EGFR wild-type NSCLC cell lines (n = 10: ranged from 0.28 to 6.2 μM) were higher than those of EGFR-mutant NSCLC cell lines although two BRAF mutant cell lines and one EML4-ALK translocated cell line showed intermediate level of IC50 values . These results suggested that TAE226 was more effective in EGFR-mutant cell lines, regardless of the presence of the TKI-resistant EGFR T790M mutation, than in EGFR wild-type cell lines, especially which did not have BRAF or EML4-ALK alterations.We evaluated the anti-tumor effect of TAE226 using an MTS assay in 15 NSCLC cell lines, one breast cancer cell line and one gastric cancer cell line . Number EGFR mutation were sensitive to gefitinib , while other NSCLC cell lines harboring wild-type EGFR (n = 9) or TKI-resistant EGFR mutation (n = 2) were resistant (the IC50 were ranged from 5.3 to 32.6 μM). These data were consistent with those of previous reports [We also examined the anti-tumor effect of gefitinib against 14 NSCLC cell lines . Three N reports , 28.We investigated the effect of TAE226 on FAK, IGF-IR, and EGFR-related proteins using 6 cell lines . The phosphorylation of FAK (Thy397) and IGF-IR (Tyr1131), which are targets of TAE226, was not significantly inhibited up to a concentration of 5 μM in all 6 cell lines tested . For FAKEGFR-mutant cell lines , regardless of the presence of TKI-resistant mutations. In addition, the phosphorylations of AKT and MAPK, which are downstream molecules of EGFR, were also inhibited in these EGFR-mutant cell lines. In contrast, the phosphorylations of EGFR, AKT, and MAPK were not inhibited in cell lines with wild-type EGFR (NCI-H1299 and NCI-H1819)  . These dEGFR-mutant cell lines, PC-9, HCC827, NCI-H3255, HCC4006, NCI-H1975, NCI-H820 and RPC-9; EGFR wild-type cell lines, NCI-H1819, NCI-H1299 and A549). No significant difference in the expression level of each of the proteins was observed among the cell lines   (EGFR-mutant cells by suppressing mutant EGFR protein.We examined the anti-proliferative effect of TAE226 on two HEK-293T cell lines stably transfected with wild-type EGFR or mutant EGFR (L858R) that we had previously established . The HEKctively) . TAE226 ctively) . TAE226 ctively) . These rin vitro binding assay of the variant EGFR kinases , no difference in the amounts of EGFR kinases that bound to the bead-modified ATP was noted under the control condition . Exogeno50 and Kd values for TAE226 and gefitinib against wild-type EGFR and L858R/T790M EGFR mutant were shown in 50 and Kd values for TAE226 were 0.326 μM and 0.163 μM in wild-type EGFR, and 0.0193 μM and 0.00966 μM in L858R/T790M EGFR mutant, respectively. Regarding gefitinib, those were 0.0244 μM and 0.0122 μM in wild-type EGFR, and 0.927 μM and 0.463 μM in L858R/T790M EGFR mutant, respectively. Gefitinib showed a higher affinity of about 40-fold for wild-type EGFR than for L858R/T790M EGFR mutant. On the contrary, TAE226 showed higher affinity of about 15-fold for L858R/T790M EGFR mutant than for wild-type EGFR. The values of kon and koff could not be calculated in TAE226 for the both type of EGFR and in gefitinib for L858R/T790M EGFR mutant because of the short residence time (< 1.4 min).ICin vitro data, we examined the anti-tumor effect of TAE226 on mouse xenograft models. After the subcutaneous tumor volume of the inoculated PC-9 and RPC-9 cell lines in nude mice had reached a volume of 200–400 mm3, we treated the nude mice with the oral administration of TAE226  or methylcellulose as vehicle for 14 days. The tumor volume and body weight of both xenografts were measured on the day before the oral administration of TAE226 and every three days thereafter. Obvious side effects, including the loss of body weight, did not occur in the vehicle-treated group or in the groups treated with 30mg/kg or 60mg/kg of TAE226, while a loss of body weight was observed in mice treated with 90 mg/kg of TAE226. The tumor volume in both xenografts significantly decreased over time and in a dose-dependent manner, compared with the vehicle-treated mice , HER2 (0.95), FGFR-1 (0.75), c-Met (0.16) and ALK (0.15), which are often activated in NSCLC, are less than that for EGFR (1.7)  mutant cells are similar to those for EGFR L858R (or delE746_A750) mutant cells, providing the contrasting results compared to the IC50 values of gefitinib for them. Our in vitro binding assay also indicates that the double mutant reveals higher binding affinity to TAE226 than that of the L858R (or delE746_A750) mutant. It is well known and documented that increased hydrophobic interactions between a protein and a ligand lead to an enhanced binding affinity of the ligand, occasionally by several orders of magnitude [50 of the RPC-9 cells was higher than the IC50 of the PC-9 cells for TAE226. However, the difference of IC50 between PC-9 and RPC-9 for TAE226 is slight compared to that for gefitinib. As for in vivo study, the obtained results suggest the safety of TAE226 doses suitable for the treatment of EGFR-mutant cells, regardless of the presence of EGFR T790M mutation in vivo, opening the possibility for clinical application. In addition to TAE226, related bis-anilino pyrimidine compounds have also been developed [Regarding the mechanism whereby the T790M mutation is thought to influence small molecule TKIs such as gefitinib and erlotinib, the T790M mutation has been thought to cause resistance by sterically blocking the binding of TKIs , 42. In agnitude . Kineticeveloped . FurtherEGFR-mutant NSCLC regardless of the presence of T790M mutation. Our results showed that the EGFR L858R/T790M (or delE746_A750/T790M) mutant retains the binding affinity to TAE226 comparable to that of the L858R (or delE746_A750) mutant, suggesting that TAE226, or its relatives, is promising to overcome acquired TKI resistance mediated by EGFR T790M mutation.In conclusion, we found that TAE226 inhibited the activation of EGFR-mutant kinase and exerted anti-tumor activity on S1 FigEGFR mutational status.Serum-starved NSCLC cell lines were treated with TAE226 for two hours and stimulated with IGF-I recombinant for 15 minutes. The phosphorylations of IGF-IR and AKT were completely inhibited in all cell lines regardless of (TIF)Click here for additional data file.S2 FigEGFR mutational status.There were no significant differences in the expression levels of p-FAK, FAK, p-IGF-IR and IGF-IR in 10 NSCLC cell lines regardless of (TIF)Click here for additional data file.S3 FigThe mutant EGFR-transfected HEK-293T was more sensitive to gefitinib treatment than wild-type EGFR-transfected HEK-293T. TAE226 significantly inhibited phosphorylations of EGFR and AKT in mutant EGFR-trasnsfected HEK-293T compared with wild-type EGFR-transfected HEK-293T.(TIF)Click here for additional data file.S4 Fig(A) Density of EGFR protein bands between the supernatant fraction and the precipitated fraction at 0.1 μM of TAE226 in (TIF)Click here for additional data file.S1 MethodDetailed methods are described.(DOCX)Click here for additional data file.S1 TableThis table is updated data from ref. 20.(DOCX)Click here for additional data file.S2 Table50 and SD were calculated using the value of each IC50.Mean IC(DOCX)Click here for additional data file.


Epithelial-to-Mesenchymal Transition (EMT) induced by glucose in human peritoneal mesothelial cells (HPMCs) is a major cause of peritoneal membrane (PM) fibrosis and dysfunction.To investigate serum response factor (SRF) impacts on EMT-derived fibrosis in PM, we isolated HPMCs from the effluents of patients with end-stage renal disease (ESRD) to analyze alterations during peritoneal dialysis (PD) and observe the response of PM to SRF in a rat model.Our results demonstrated the activation and translocation of SRF into the nuclei of HPMCs under extensive periods of PD. Accordingly, HPMCs lost their epithelial morphology with a decrease in E-cadherin expression and an increase in α-smooth muscle actin (α-SMA) expression, implying a transition in phenotype. PD with 4.25% glucose solution significantly induced SRF up-regulation and increased peritoneal thickness. In immortal HPMCs, high glucose  stimulated SRF overexpression in transformed fibroblastic HPMCs. SRF-siRNA preserved HPMC morphology, while transfection of SRF plasmid into HPMCs caused the opposite effects. Evidence from electrophoretic mobility shift, chromatin immunoprecipitation and reporter assays further supported that SRF transcriptionally regulated Snail, a potent inducer of EMT, by directly binding to its promoter.Our data suggested that activation of SRF/Snail pathway might contribute to the progressive PM fibrosis during PD. Peritoneal dialysis (PD) is currently used as a chronic, life-sustaining treatment by approximately 197,000 end-stage renal disease (ESRD) patients, or 11% of the global dialysis population Serum response factor (SRF), a member of a conserved DNA-binding protein family, is a master switch for the expression of contractile and cytoskeletal genes in virtually all cells across diverse species The high glucose (HG)-induced EMT of HPMCs acts as a key process in peritoneal membrane fibrosis and dysfunction. Mediated by factors including E-cadherin, α-SMA and Snail, epithelial cells may lose their epithelial characteristics and gain mesenchymal cell properties in response to certain stimuli The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki and was approved by the ethics committee of Xijing Hospital. Written informed consent was obtained from each patient. The Ethics Committee for Animal Experiments of the Fourth Military Medical University approved all animal work (Permit number: 20120023) and the experimental protocols strictly complied with the institutional guidelines and the criteria outlined in the “Guide for Care and Use of Laboratory Animals”. And all efforts were made to minimize the number of animals used and their suffering, in accordance with the ethical guidelines for animal research. All surgery was performed under sodium pentobarbital anesthesia.Human mesothelial cells obtained from the effluents of patients undergoing CAPD in our PD center in the Department of Nephrology, Xijing Hospital were collected and cultured using a previously described The immortal human HPMC line , which is an untransformed HPMC line that yields data similar to the results obtained using primary cells, was cultured in Earle's M199 medium. EMT was induced in immortal HPMCs with HG (0.5 ng/mL) , as described previously A chronic infusion model of non-uremic animal PD was used, as described previously Total protein extracts from cultured cells were loaded on SDS-PAGE gels, and Western blotting was performed according to a standard protocol. The antibodies used were as follows: anti-p-SRF  diluted 1∶500; anti-SRF diluted 1∶80; anti-E-cadherin, anti-α-SMA, and anti-Snail diluted 1∶50 (Santa Cruz Biotechnology); and anti-β-actin (Sigma) diluted 1∶4000. The antibody staining was performed as described previously Cultured cells were grown on 0.8 cm×0.8 cm slides, rinsed with ice-cold phosphate-buffered saline and fixed with 4% paraformaldehyde. Immunofluorescence staining was performed on paraffin-embedded sections or cultured cells, as described previously The siRNA plasmids that recognize human SRF (sc-36563) were purchased from Santa Cruz Biotechnology for the transient transfections. The full-length human SRF plasmids were a gift from Dr. Eric Olson . The transfections were performed using Lipofectamine 2000 , according to the manufacturer's instructions. Real-time PCR or Western blot analysis was used to assess the transfection efficiency.5′-CGTCTGTCTCCCTCACTGGACC-3′, Snail (SRE1) ChIPR1, 5′-CTCTCGGCGGCTTGAAATGC-3′, Snail (SRE2) ChIP F2, 5′-GCCCGGGCTCTCACCGCCAC-3′, and Snail (SRE2) ChIP R2, 5′-GCAGCAGCGCCGCCAACTCC-3′. The values were normalized to the input and expressed as a fold increase relative to the IgG control.ChIP was performed as previously described 5′-CGTGCTGGGCGCTCCGTAAACAATCGATCGATCGAGGAACGGGTGCTCTTGGCTAGC-3′, mutSnailR1: 5′-GCTAGCCAAGAGCACCCGTTCCTCGATCGATCGATTGTTTACGGAGCGCCCAGCACG-3′, mutSnailF2: 5′-GCGCGGAGGTGACAAAGGGGCGATCGATCGATCGCCCCGCCCCTCCCACCCCCCACCA-3′, and mutSnailR2: 5′-TGGTGGGGGGTGGGAGGGGCGGGGCGATCGATCGATCGCCCCTTTGTCACCTCCGCGC-3′.The snail promoter region from −1 to −1000 was cloned into the pGL3 luciferase vector (Promega) . The primers used to create the mutant reporters  are as follows: mutSnailF1: For the transient transfection assays, HG-treated HPMCs or control cells were seeded 48 h before transfection. Then, pGL3-basic was transfected with the PRLsv40 plasmid into cells in the presence of Lipofectamine. The luciferase activity in cell lysates collected 48 h after transfection was measured as relative light units with the Fluoroscan Ascent FL  using the Dual-Luciferase Assay System (Promega). The relative luciferase activity was defined as the ratio of the firefly luciferase activity/mean Renilla luciferase activity for each construct relative to the pGL3 control vector (Promega) Nuclear proteins were isolated using the Nuclear Protein Isolation kit (Panomics). The binding reaction for the electrophoretic mobility shift assay (EMSA) contained 10 mmol/L HEPES (pH 7.5), 80 mmol/L KCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 6% glycerol, 0.5 µg of polydeoxyinosinic-deoxycytidylic acid, 0.5 µg of sonicated salmon sperm DNA, 2 pmol of a double-stranded oligonucleotide of AR/GR HRE conjugated to IR fluorophore IRDye 700 (LI-COR), and 10 µg of the nuclear extract. To verify the specificity of the AR (GR) protein-DNA complexes, a binding reaction was performed using increasing concentrations of non-conjugated HRE (cold probe) and alternating with a mutated HRE conjugated to IRDye 700. A DNA-binding reaction was performed at 16°C for 30 min in a final volume of 20 µL. The DNA-protein complexes were analyzed by Tris-borate EDTA electrophoresis on 6% polyacrylamide gels containing 0.5% glycerol. The DNA-protein complexes were visualized using an Odyssey IR scanner (LI-COR) with a 680-nm scanner channel, and linear graphs were constructed with the LI-COR Odyssey software for quantitative image analysis (LI-COR) 5′-CCTCAAGATCATCAGCAAT-3′ and R: 5′-CCATCCACAGTCTTCTGGGT-3′ ; SRF, F: 5′-AAACTGCAGCCCATGATCACC-3′ and R: 5′-CTTCAAAGCCAGTGGCACTCA-3′; CDH1, F: 5′-TTCCCTCGACACCCGATTC-3′ and R: 5′-TAGGTGGAGTCCCAGGCGTA-3′; α-SMA, F: 5′-AAGATGACCCAGATCATGTT-3′ and R: 5′-TCAT- AGATGGGGACATTGT-3′; and Snail, F: 5′-ACCCCAATCGGAAGCCTAACT-3′ and R: 5′-GGTCGTAGGGCTGCTGGAA-3′. Experiments were performed in duplicate. After amplification, the PCR products were confirmed by melting curve analysis and gel electrophoresis.Total RNA was extracted using the RNeasy kit , and cDNA was obtained from 500 ng of total RNA using an Omniscript RT kit (Qiagen). Quantitative PCR was performed using a LightCycler , SYBR Green kit (Roche Diagnostics GmbH) and the following specific primer sets: GAPDH, F: 2 test. Significant differences were determined using an analysis of variance followed by a post-hoc comparisons with Fisher's protected least-significant-difference test. The data were significant different if P<0.05.The bands from Western blotting or real-time PCR were quantified using Quantity One software . The numerical data are presented as the mean ± SE using SPSS 12.0 software . Differences between the means were assessed using Student's t-test, an ANOVA or the χMorphological features of human peritoneal mesothelial cells were markedly variable, ranging from a cobblestone-like appearance to mixed cell populations and fibroblast-like cells . Of the To examine the effect of SRF inhibition in PD, we performed PD in rats with HG-PDF  with or without CCG-1423 for 6 weeks. By histopathological examination, the thickness of the submesothelial compact zone in the HG-PDF group (139.9±5.5 µm) was significantly greater than that in the control group (9.7±0.5 µm), significantly less in the SRF-inhibitor group . So a loss of the mesothelial cell monolayer with a thicker PM was found in the parietal peritoneum from the untreated PD group compared to the saline control 1, a2, whEMT markers and fibronectin (FN) analysis by immunohistochemistry indicated that HG-PDF downregulated E-cadherin and upregulated α-SMA levels, which were inhibited by the administration of CCG-1423, suggesting EMT was tempered . A correTo pursue direct evidence of SRF on HG-induced EMT, we manipulated SRF gene expression in cultured immortal HPMCs. When the control HPMCs were incubated with HG medium (60 mmol/L) for 72 h, the cells lost intercellular junctions, scattered, and transformed into a spindled fibroblastic shape . ConcordSmall interfering SRF RNA (SRF-siRNA) in HPMCs prevented all of the above HG-induced alterations 2, e, f. To explore the downstream events of SRF-mediated EMT, we assessed target gene sequences for SRF. We found that all possible SRF target genes have one or more highly conserved CArG elements, leading to select Snail as a candidate of the EMT marker in the promoter. Indeed, Snail expression was augmented in HG-induced HPMCs or HPMCs with SRF overexpression both at the protein and mRNA levels, while levels were diminished by siRNA . These dIn addition, immunohistochemical studies also revealed pronounced Snail staining in the submesothelial layer of the abdominal walls of the PDF rats, which could be attenuated by treatment with the SRF inhibitor CCG-1423 . Snail tIn hunting for the molecular basis of the regulation of Snail by SRF, we performed chromatin immunoprecipitation (ChIP) to examine the binding of SRF to CArG elements in Snail in HPMCs. Two serum response elements, SRE1 and SRE2, in the promoter regions of the Snail gene were predicted as candidate binding sites of SRF. Fifty-fold enrichment was set as a threshold for positive SRF binding. Our results showed positive SRF binding at SRE2 in HPMCs. Moreover, the binding signal greatly increased in the HG-induced HPMCs, in agreement with their higher SRF expression . ElectroUp-regulated Snail by SRF was further confirmed by luciferase activity assays. In the control HPMCs, transfected full-length SRE (SRE1+SRE2) plasmids could stimulate their luciferase activity from the basal level either in normal medium (P = 0.030) or high glucose medium (P = 0.007) compared to control vectors. Mutation of SRE1 moderately affected their response but without statistical significance (P = 0.061). However, mutation of SRE2 alone or a double mutation of both SRE1+SRE2 eliminated the luciferase activity response to HG  1, c2. ThOur results demonstrate that HPMCs from ex vivo effluents or from PD rats in vivo undergo EMT during PD. We determined that SRF promotes EMT-mediated fibrosis and acts as a critical transcription factor for HPMCs. Moreover, infection with SRF-siRNA vectors demonstrated the HG-induced upregulation of E-cadherin and downregulation of α-SMA during EMT in HPMCs in vitro. Furthermore, we predicted and demonstrated that Snail is regulated by SRF, which can directly promote Snail transcription via SRE2. Thus, a novel SRF/Snail pathway promotes EMT-mediated peritoneal fibrosis and acts as a critical transcription factor during HPMC EMT.Increasing evidence shows that EMT, which was originally thought to be limited to tumor development and metastasis, occurs in other diseases, including kidney, liver and lung fibrosis We also found that the inhibition of SRF signaling could prevent HPMCs from acquiring a spindle-like phenotype, reverse EMT, downregulate α-SMA and upregulate E-cadherin. To our knowledge, this report is the first to demonstrate a link between the translocation and activation of SRF in HPMCs and HG-induced EMT. In addition, SRF phosphorylation, which promotes SRF transcriptional activity, may have a role in EMT.Snail is a zinc finger transcription factor expressed in cells and is the core EMT regulator that plays essential roles in fibrosis Moreover, there is still some limitation needed to show in our study. CCG-1423, a specific inhibitor of Rho pathway-mediated signaling and activation of SRF transcription, selectively inhibits DNA synthesis, proliferation and invasion of Rho-overexpressing cell lines. However, HG solutions could directly induce Rho kinases. Although a nuclear translocation of SRF in PD patients and a preservation of the peritoneal membrane in animals undergoing PD with HG solutions and receiving the inhibitor were shown in our study, any blockade of Rho kinases would reduce EMT and fibrosis, possibly irrespective of its specific SRF-dependent effects. So we added supplement data to demonstrate that Rho kinase inhibitor CCG-1423 has similar effects with SRF siRNA in vitro . It showIn all, this study is the first extensive characterization of a signaling pathway that controls EMT in a non-tumor primary cell culture model and provides basic knowledge and clinical relevance. Our results from rats experiment reveal that, CCG-1423 could weaken the EMT happening of peritoneal mesothelial cells when stimulated by glucose contained in PD fluid, and protect peritoneal member function. These results could form the rationale for developing drugs that can counteract the progressive deterioration of the PM that occurs in CAPD patients. However, lots of work is needed to explore deeply molecular mechanism and to identify the safety and detail usage of drugs.Figure S1The expression of SRF, E-cadherin and α-SMA were tested by Western blot after transfection by siRNA or SRF up-regulated plasmid in HPMCs which were stimulated by HG for 96 h.(TIF)Click here for additional data file.Figure S2CCG-1423 is stable in high glucose in vitro and in vivo. (a) For in vivo study, we injected high glucose PDF after 2 hours of DMSO with CCG-1423 injection. The other progress is the same as before. The results by immunohistochemistry with PBS show that CCG-1423 still has a strong inhibition for peritoneal membrane proliferation even in high glucose solution. Magnification is 200×. (b) We demonstrated that Rho kinase inhibitor CCG-1423 has similar effects with SRF siRNA in vitro. Treated HPMCs with CCG-1423 after high glucose treatment (G1), before high glucose treatment (G2), as well as simultaneously treatment with high glucose (G3), CCG-1423 has a similar effect on SRF inhibition, and this result could be repeated with SRF siRNA (G4). Total incubation time of CCG-1423 or SRF-siRNA for each group was 72 hours.(TIF)Click here for additional data file.Figure S3Inhibition of SRF by CCG-1423 ameliorates PD-induced PM fibrosis in vivo. (a) Masson staining and expression of FN showed PD-induced PM fibrosis in Rats in vivo by immunohistochemistry. Magnification is 200×. (b) Westren blot staining of FN in HPMCs stimulated by HG reveals that PD fluid exposure induces the EMT and fibrosis process.(TIF)Click here for additional data file.Table S1Characteristics of CAPD patients with ESRD in our study.(DOC)Click here for additional data file.


Increased pulse pressure (PP) is a strong predictor of cardiovascular mortality and it has been associated with an increased risk of developing diabetes and its microvascular complications. However, the mechanism underlying the association between PP and abnormalities of glucose metabolism is unclear.To study how PP obtained by 24h ambulatory blood pressure monitoring (ABPM) is related to fasting and 2-h plasma glucose in subjects with different degrees of glucose tolerance.In a cross-sectional study, 128 subjects  were submitted to a 75-g OGTT (measurement of glucose and insulin) and divided according to glucose tolerance status . 24-h ABPM was performed. Mean 24h PP was calculated as the difference between mean systolic and diastolic blood pressure (BP), obtained through 24-h ABPM. Fasting C-peptide and A1c were collected. Body size (BMI) and central obesity (waist circumference) were assessed. Insulin sensitivity index (ISI Stumvoll), insulin resistance (HOMA-IR) and β-cell function  were estimated. A two-sided P value < 0.05 was considered significant.By ABPM, 24-hour PP progressively increased from NGT to DM . 24-hour PP was positively related to age , waist circumference , BMI , A1C , 2-h glucose level , C-peptide , HOMA-IR  and it was inversely related to ISI Stumvoll  and to insulinogenic index . While adjusting for age and waist circumference, pulse pressure was independently associated with 2 hour plasma glucose  and A1c .According to our data, pulse pressure obtained by 24-h ABPM increases with decreasing of glucose tolerance. It is also an independent predictor of higher 2 hour plasma glucose and A1c levels.


We show that cells without both cis- and trans-ligands can mediate Notch-dependent developmental events during Drosophila oogenesis, indicating ligand-independent Notch activity occurs when the receptor is free of cis- and trans-ligands. Furthermore, cis-ligands can reduce Notch activity in endogenous and genetically induced situations of elevated trans-ligand-independent Notch signaling. We conclude that cis-expressed ligands exert their repressive effect on Notch signaling in cases of trans-ligand-independent activation, and propose a new function of cis-inhibition which buffers cells against accidental Notch activity.The Notch pathway is integrated into numerous developmental processes and therefore is fine-tuned on many levels, including receptor production, endocytosis, and degradation. Notch is further characterized by a twofold relationship with its Delta-Serrate (DSL) ligands, as ligands from opposing cells  in an adjacent cell. However, if the Serrate or Delta ligands are present on the surface of the same cell as Notch they turn the receptor off, rather than activate it. Notch can also work without being activated by Serrate or Delta, but whether the ligands can inhibit this ‘ligand-independent’ Notch activation if they are on the surface of the same cell as the Notch receptor was unknown.Palmer et al. study Notch signaling in the fruit fly equivalent of the ovary, in cells that are naturally deficient in Serrate and from which Delta was artificially removed. The Notch protein was activated when these ligands were not present. Furthermore, the developmental processes that are activated by Notch were able to proceed as normal when triggered by ligand-independent Notch signaling. In total, Palmer et al. investigated three different types of fruit fly cell, and found that ligand-independent Notch signaling can occur in all of them.Reintroducing Delta to the same cell as Notch turns the receptor off, suggesting that ligands on the surface of the same cell as the receptor can inhibit ligand-independent Notch activity. Many genetic diseases and cancers have been linked to Notch being activated when it should not be; therefore, understanding how Notch is controlled could help guide the development of new treatments for these conditions.DOI:http://dx.doi.org/10.7554/eLife.04415.002 ICD— into the nucleus to act as a transcriptional co-activator  in an adjacent cell, which leads to γ-secretase-dependent cleavage of Notch, and translocation of the intracellular domain—Nctivator . Notch mctivator . DSL ligreceptor . Howevercis and in trans, as Dl is the sole signaling source and the signal sending and receiving cells can be easily distinguished (Hindsight (Hnt), downregulation of Cut, and the polyploidization of the follicle cells , the follicle cells failed to downregulate Cut expression, which persisted past stage 7, indicative of a failure to activate Notch  show correct Hnt and Cut expression from stage 7  are comparable to wild-type cells before entry to endocycle (cis- and trans-Dl through knockdown of Dl by RNA interference (RNAi) simultaneously in the germline and soma confirmed this finding  system (Dl-/Dl- clones driving expression of NotchRNAi show a significantly higher proportion (p < 0.0001) of late Cut-expressing cells than the Dl-/Dl- clones alone, indicating that Notch is still required for the mitotic-to-endocycle switch (Su(H)47, in RNAi-induced Dl-/Dl- clones also show late Cut expression (p < 0.0001)  system . The MARe switch . Likewis 0.0001)  in compacontrols . A Notchcontrols  was also stage 6 , suggests-ligand . This NRs-ligand , perhapss-ligand . As repoDl expression and a very low level of Ser expression, which had no effect on Notch signaling  , and our observation that Notch accumulates in Dl-/Dl- clones   . Notch ativation . This istivation   . These rcis-inhibition can also block ligand-independent Notch activity induced in aberrant genetic backgrounds. The Notch target, Wingless (Wg) is normally expressed along the dorsoventral boundary of the wing disc  homozygous mutant (lgdd7) larvae display overgrown imaginal discs and ubiquitous ligand-independent Notch activation in the wing pouch region, as shown by upregulation of Wg , an E3 ubiquitin ligase that stimulates Notch monoubiquitination and promotes its trafficking to the lysosomal limiting membrane, has also been shown to induce ligand-independent Notch activation specifically in the ventral wing pouch region (patched (ptc)-GAL4 to drive expression of UAS-Dx with either UAS-Dl or UAS-Serdel3, whose ectopic expression leads to a reduction of Wg staining along the dorsoventral boundary  in the Notch activation compared with transfecting pMT-NFL alone  reduced the level of Notch activation (lgd or shrub (a component of the ESCRT-III complex). Lgd dsRNA induced an increase in Notch activation by a factor of 1.73 compared with GFP dsRNA-treated cells (p = 0.00286) (shrub dsRNA caused a 3.93-fold increase (p < 0.0001) in Notch activation in S2 cells   . LikewisS2 cells  (ThompsoS2 cells . Express 0.0257) .cis-acting ligands might block endogenous raised levels of ligand-independent Notch activation, in addition to the raised levels induced by genetic defects, we examined the effect of increased ligand expression in crystal cells in the larval lymph gland, which have recently been shown to have ligand-independent Notch activation (Lozenge (Lz)-GAL4, a crystal cell lineage-specific driver  (UAS-SerWT led to a significant decrease of the Notch reporter E(spl):mβ-CD2expression in mature crystal cells  . Furtheral cells . Reducedl cells,  was unafl cells, . These ocis-expressed DSL ligands in numerous tissues. Cis-inhibition can also attenuate DSL-ligand independent Notch activity both in endogenous and genetically induced situations. Mechanistically, this could be explained if DSL ligands sequestered Notch at the membrane, made Notch more sensitive to degradation, or increased the stability of the heterodimer as it travels through the endosomal pathway. As we and others , hs-FLP122; act-GAL4 UAS-GFP;FRT82B Gal80, UAS-NotchRNAi (VDRC #1112—no expression in germline cells), UAS-DeltaRNAi (BDSC #34322—able to express in germline cells); hsFLP GFPstau; act > y+ > GAL4, UAS-GFP, hs-flp122; Gal80 FRT40A; tubGAL4 UASGFP, Su(H)47FRT40A (Myc (a gift from Marc Muskavitch), tsg101111019 from Kyoto stock center, UAS-SerWT (BDSC #5815), UAS-Serdel3−tom (a gift from Robert J Fleming) (d740A (BDSC #25087), dppGAL4 (BDSC #7007), lz-GAL4 UAS-GFP (BDSC #6314). To create FRT82B, DlRevF10 germline/follicle cell clones by the FLP/FRT or MARCM methods  outliers, which were omitted from further analyses.The following fly stocks were used for Drosophila crosses. hs-flpT82B RFP , FRT82B   , UAS-Del methods  , mouse anti-NECD , mouse anti-Wingless , mouse anti-Dl , rabbit anti-βGal , or rabbit anti-GFP  primary antibodies. Tissues were mounted on slides after PBT washes and secondary antibody incubation. 4',6-Diamidino-2-phenylindole (DAPI) was used to stain nuclei. Samples were then analyzed with a Zeiss 510 or Leica SP2 confocal microscope and after analysis with the Image J software. Nuclear volume quantification was done with the Volumest plug-in for ImageJ.Ovaries, imaginal discs, or lymph glands were dissected in phosphate-buffered saline (PBS), fixed in 10% formaldehyde, washed three times in PBS + Triton-X (PBT), and then blocked for at least 1 hr in PBT with goat serum. Tissues were then either stained overnight with mouse anti-Cut , mouse anti-Hindsight , mouse anti-N6 cells per milliliter were seeded into either 24-well plates or 96-well plates for experiments with or without dsRNA treatment, respectively. Transfections were carried out with Qiagen Effectene  transfection reagent according to the manufacturer's instruction. Plasmids used for transfection were pMT-NotchFL (a gift from Renjie Jiao), pMT-GAL4 (DGRC #1042), pUASt-Serdel3 (a gift from Robert J Fleming), pMT-Deltex (a gift from Spyros Artavanis-Tsakonas), NRE-firefly luciferase (a gift from Sarah Bray), or Renilla luciferase (a gift from Sarah Bray). Aliquots (75 ng for 24-well plates or 50 ng for 96-well plates) of each non-luciferase plasmid were added and, where applicable, 10 ng of each luciferase plasmid. DNA concentration between transfections was kept constant with an empty vector. For experiments without dsRNA treatment, CuSO4 was added to a concentration of 500 µM 24 hr after transfection, and cells were assayed 24 hr later. dsRNA was transcribed in vitro using the RiboMAX large-scale RNA production system-T7 kit . The following primers were used to amplify genomic DNA taken from a single male fly from the NRE-GFP stock:S2 cells were grown under standard conditions and passaged once every three days in serum-free Gibco media  supplemented with antibiotics. In preparation for transfection 10Forward: GAATTAATACGACTCACTATAGGGCAGGGGACGAATTTCATTTGReverse: GAATTAATACGACTCACTATAGGGAAAACCCTGCGCTTTCTTCTForward: GAATTAATACGACTCACTATAGGGAATCGCCAACAATCAAGTCCReverse: GAATTAATACGACTCACTATAGGGCGTGCAGCACTACTTTCCAAForward: GAATTAATACGACTCACTATAGGGAGATGCCTCTGAGGAACCCGTCCAGReverse: GAATTAATACGACTCACTATAGGGAGAGTGTGGGTTCTGGGGCAGCAGTForward: GAATTAATACGACTCACTATAGGGACTTTTATGCAGGGACGTGGReverse: GAATTAATACGACTCACTATAGGGTCCCTCGCTTCGAACTAAAAForward: GAATTAATACGACTCACTATAGGGTCTCACCAACCAACCAATCAReverse: GAATTAATACGACTCACTATAGGGCACAATATAGAGCGCGACGAForward: GAATTAATACGACTCACTATAGGGAGCTGGACGGCGACGTAAACReverse: GAATTAATACGACTCACTATAGGGATGGGGGTGTTCTGCTGGTAG4 was added to a concentration of 500 µM later that day. Cells were incubatedfor five days, with an additional treatment of dsRNA on the fourth day.Cells were treated with dsRNA at a concentration of 50 nM, and then transfected shortly after. CuSOt test was used to test for statistical significance.Cells were transfected with plasmids of interest together with an NRE-driving firefly luciferase expression and a constitutively activated Renilla luciferase to control for transfection efficiency. Luciferase measures were inspected with the Dual-Luciferase Assay Kit (Promega) in 96-well luminometer plates. Each transfection was performed in duplicate and repeated several times. Student's  review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent , a Reviewing editor, and 3 reviewers.Thank you for sending your work entitled “Cis-interactions between Notch and its ligands block ligand-independent Notch activity” for consideration at The Reviewing editor and the reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.In general, all reviewers agreed that this is an imaginative paper that addresses an important issue. We would like to see this paper published if the authors address the issues raised by the reviewers.eLife, it is essential that it is copyedited and simplified by the authors and perhaps in consultation with a non-Drosophila colleague. Also, many of the figures need to be improved.1) Repeatedly, in post review discussions, it came up that the paper was written in a confusing form in many places. To make this paper more understandable by the broad readership of 2) In the Abstract, the authors state that the paper is about the ligand-independent N activity in the ovary; however, the suggested mechanism of DSL-independent N activation  is shown only in the wing disk. If this mechanism is to be extrapolated to explain how ligand-independent N signaling acts in the ovarian soma, the authors should at least show that “endosomal pathway mutant” follicle cells also have precocious N activation.3) Mosaic analyses in follicle cells provide evidence that these cells without both cis and trans ligand sources are still able to activate Notch through what is presumed to be a non-canonical pathway. It is not shown what the receptor does in these cells even though the consequences of its activation are well documented. I would have liked to see where the Notch receptor is under those conditions and presume that an immunocytological following of the Notch receptor could be informative. The conclusion that “Therefore, ligands expressed in the same cell as the Notch receptor have an endogenous role in buffering against DSL-independent Notch auto-activation” is too general and too strong and while this is possible it is certainly not conclusive. There are other roles that cis inhibition serves or indeed can serve. 1) Repeatedly, in post review discussions, it came up that the paper was written in a confusing form in many places. To make this paper more understandable by the broad readership of eLife, it is essential that it is copyedited and simplified by the authors and perhaps in consultation with a non-Drosophila colleague. Also, many of the figures need to be improved.Drosophila biologists, we have gone through the manuscript with non-specialists who have helped us to locate areas which may not be clear, and have attempted to clarify these points. For example, we added a figure to show the schematic drawing of oogenesis, which shows where the follicle cells and germline cells are located. We also include a WT wing disc stained with Wg for comparison and label regions of interest on this disc  which we refer to in the text.We have made substantial changes in the revision to clarify points that may have been a source of confusion before, especially for 2) In the Abstract, the authors state that the paper is about the ligand-independent N activity in the ovary; however, the suggested mechanism of DSL-independent N activation  is shown only in the wing disk. If this mechanism is to be extrapolated to explain how ligand-independent N signaling acts in the ovarian soma, the authors should at least show that “endosomal pathway mutant” follicle cells also have precocious N activation.tsg101 mutant follicle cells.Several earlier studies have reported that different trafficking mutants such as Su(Dx) , tsg101 , and lgd  show precocious Notch activation in the follicle cells. We have added a sentence that makes these past results of others more explicit, and also added an example figure showing early Notch activity in 3) Mosaic analyses in follicle cells provide evidence that these cells without both cis and trans ligand sources are still able to activate Notch through what is presumed to be a non-canonical pathway. It is not shown what the receptor does in these cells even though the consequences of its activation are well documented. I would have liked to see where the Notch receptor is under those conditions and presume that an immunocytological following of the Notch receptor could be informative. The conclusion that “Therefore, ligands expressed in the same cell as the Notch receptor have an endogenous role in buffering against DSL-independent Notch auto-activation” is too general and too strong and while this is possible it is certainly not conclusive. There are other roles that cis inhibition serves or indeed can serve.Dl-/Dl- clones, and noted that we did observe Notch accumulation, consistent with other cases of ligand-independent Notch activation. From the comments, we are not sure whether “an immunocytological following” also refer to a pulse-chase experiment (i.e. pulse with NECD antibody then chase for varying amounts of time before fixation and secondary antibody wash), this technique, although it works in the imaginal disc, has not been successful in the follicle cells, which was noted in Yan et al, 2009, Dev Cell 17. We agree with the reviewer that the conclusion we made on cis-inhibition might be too general and too strong. We therefore have reworded this to the following: “Therefore, we propose that a role of cis-ligands could be to keep the Notch pathway in a state of readiness by buffering against unintentional stochastic Notch activity resulting from normal processing through the endosomes.”We have included figures of Notch distribution in different stages of oogenesis in


IL-17A −197A/G and IL-17F +7488C/T polymorphisms in T1DM and CP and determined their associations with IL-17 production and occurrence of periopathogens. Totally 154 controls, 125 T1DM, and 244 CP patients were genotyped using 5′ nuclease TaqMan® assays. Bacterial colonization was investigated by a DNA-microarray kit. Production of IL-17 after in vitro stimulation of mononuclear cells by mitogens and bacteria was examined by the Luminex system. Although no differences in the allele/genotype frequencies between patients with CP and T1DM + CP were found, the IL-17A −197 A allele increased the risk of T1DM (P < 0.05). Levels of HbA1c were significantly elevated in carriers of the A allele in T1DM patients (P < 0.05). Production of IL-17 by mononuclear cells of CP patients  was associated with IL-17A A allele (P < 0.05). IL-17A polymorphism increased the number of Tannerella forsythia and Treponema denticola in patients with CP and T1DM + CP, respectively (P < 0.05). IL-17A gene variability may influence control of T1DM and the “red complex” bacteria occurrence in patients with CP and T1DM + CP. Our findings demonstrated the functional relevance of the IL-17A polymorphism with higher IL-17 secretion in individuals with A allele.Interleukin-17 contributes to the pathogenesis of type 1 diabetes mellitus (T1DM) and chronic periodontitis (CP). We analyzed A complex interplay between genetic and environmental factors participates in developing of T1DM and manifestation of its systemic and oral complications. Periodontitis has been identified as the sixth complication of diabetes; the other five complications are retinopathy (DR), nephropathy (DN), neuropathy (DPN), macrovascular disease, and poor wound healing  in subgingival sulci/pockets was investigated in a subgroup of controls (N = 51), CP patients (N = 182), and T1DM patients with CP (N = 38) before subgingival scaling. This test determined the individual pathogens semiquantitatively as follows: (−) undetected, which corresponds to the number of bacteria less than 103, (+) slightly positive corresponding to the number of bacteria 103 to 104, (++) positive corresponding to the number of bacteria 104 to 105, and (+++) strongly positive, with the number of bacteria higher than 105.DNA-microarray analyses of oral pathogens based on a periodontal pathogen detection kit  were previously used and described . BacteriCultivation of periodontopathic bacteria and immunological examination were performed in the laboratory of the Institute of Clinical and Experimental Dental Medicine, General University Hospital and First Faculty of Medicine, Charles University, Prague, Czech Republic.A.a., T.f., P.g., and P.i.) and mitogens or Heat Shock Protein (HSP) 70 were described in detail in our previous article [IL-17 levels were measured in a subgroup of 15 healthy controls and 30 patients with CP. IL-17 levels were determined in mononuclear cells isolated from 20 mL of heparinized blood using the Luminex multiplex method . The isolation, cultivation, and stimulation of cells by selected bacteria ( article .χ2). The association was described by odds ratios (OR) with 95% confidence intervals (95% CI). Only the values of P less than 0.05 were considered as statistically significant.Statistical analysis was performed using the statistical package Statistica v. 10 . Standard descriptive statistics were applied in the analysis: absolute and relative frequencies for categorical variables and mean with standard deviation (SD) or median with quartiles for quantitative variables. To compare independent groups, one-way analysis of variance (ANOVA) and Kruskal-Wallis (ANOVA) were performed to compare continuous variables. The allele frequencies were calculated from the observed numbers of genotypes. The differences in the allele frequencies were tested by the Fisher-exact test; Hardy-Weinberg equilibrium (HWE) and genotype frequencies were calculated by the chi-square test (P > 0.05). There were also no significant differences between the subjects with T1DM or CP and the controls relating to males/females ratio or smoking status. All diabetic patients (N = 38) who were examined at the Periodontology Department were affected by periodontitis. Duration of DM and diabetic complications  in the whole diabetic cohort versus subgroup of T1DM patients with CP were not statistically different.The demographic data of the study population are shown in P > 0.05). Considering the fact that, in the Czech population, SNP IL-17F +7488C/T  TT genotype occurred in 93.7%, we analyzed this polymorphism only in the subgroup of subjects .Both studied polymorphisms were in the HWE in the control group ( IL-17A −197A/G (rs2275913) variant are shown in P < 0.05, OR = 1.36, 95% CI = 0.96–1.92). Moreover, mean levels of HbA1c were significantly elevated in carriers of the A allele (AA and AG genotypes in comparison to GG homozygotes) in a group of T1DM patients , whereas, in CP patients, this allele was found more frequently in nonsmokers . Although a similar distribution of alleles or genotypes between the groups of control nonsmokers versus CP nonsmokers was found, frequencies of the A allele , AA genotype , and AA + AG versus GG genotypes  were more frequent in the control smokers in comparison with the smokers with CP (N = 269) and males (N = 254) showed no significant difference in the IL-17A −197A/G (rs2275913) allele or genotype frequencies (data not shown). Due to the small number of T1DM patients with confirmed CP, this group was not analyzed according to gender or smoking status.Although no significant differences in thehole set , stratif with CP . However IL-17A −197A/G (rs2275913) variant and the occurrence of seven selected periodontal bacteria in subgingival pockets were analyzed. The IL-17A −197 A allele carriers had an increased risk of T.f. occurrence in 182 CP patients  and a similar but nonsignificant trend was observed for the presence of P.i. . On the other hand, in patients with T1DM and CP carrying A allele of the IL-17A −197 polymorphism, T.d. in subgingival biofilm occurred less frequently than in subjects without this allele , from those seven selected ones (data not shown).Possible links between the IL-17A −197A/G (rs2275913) polymorphism in selected patients with CP and healthy controls. The IL-17 levels were measured after a 3-day in vitro cultivation of mononuclear cells, without or with stimulation by dental plaque bacteria, mitogens, or HSP70 in the CP patients (N = 30). Carriers of genotype with the A allele of IL-17A −197A/G (rs2275913) SNP had a higher production of IL-17 by unstimulated monocytes  and also after stimulation with P.g.  than GG homozygotes (N = 15), unstimulated IL-17 levels were 0.10 (0.08–0.26) pg/mL  without any significant relationship with IL-17A polymorphism. After pooling both groups (N = 45), production of IL-17 was significantly associated with IL-17A polymorphism in the unstimulated mononuclear cells (P < 0.05), but not after stimulation by P.g. . IL-17A −197A/G SNP had no significant effect on IL-17 production after stimulation with other periodontal bacteria, HSP70, and/or mitogens (data not shown).We analyzed IL-17 levels in relation to theozygotes . In the Inflammation is a physiological immune response triggered during infection and injury in an attempt to prevent infection and promote regeneration. However, persistent and unwarranted inflammation can result in host tissue damage . Regulat IL-17A −197A/G (rs2275913) and IL-17F +7488C/T  SNPs in a group of adults with and without T1DM and/or CP from Czech population. As the present study identified a low variability of IL-17F at position +7488C/T (similarly as other studies in other populations: http://www.snpedia.com/index.php/Rs763780), the TT genotype occurred in 93.7%, and no CC homozygote was detected, we investigated this polymorphism only in a subgroup of 190 subjects. Further, no associations of this polymorphism with aggressive periodontitis (AgP) or CP [ IL-17A −197A/G SNP was analyzed in the whole set of our 523 subjects. The minor allele frequency (MAF) of this SNP was 0.36, which is in line with the allele frequency in other European population [ IL-17A −197A/G or IL-17F +7488C/T variants with T1DM or periodontitis in European white population has been published. Although no significant differences were found in the genotype frequencies between the healthy subjects and T1DM or CP patients, the A allele was marginally associated with an increased risk of T1DM (P < 0.05). This allele displayed a higher affinity for the nuclear factor of activated T cells (NFAT), a critical transcription factor involved in the IL-17 regulation [ IL-17A −197A/G (rs2275913) produced significantly more IL-17 after in vitro T cells stimulation than those without this allele. Shao et al. [We evaluatedP) or CP –33 have pulation . To thisgulation , 44. Espgulation  reportedo et al.  describe IL-6 [ IL-18 separately [ IL-12B gene [In the studied diabetic population, significantly higher levels of HbA1c were present in T1DM carriers of the genotype with the A allele (AA + AG genotypes) versus the GG homozygotes (76.6 mmol/mol ± 16.5 mmol/mol versus 69.8 mmol/mol ± 13.9 mmol/mol). To our knowledge, no previous study has focused on this issue; however, polymorphisms in other genes, for example, IL-6  and IL-1parately  or in co12B gene , have be IL-17A −197A/G (rs2275913) allele or genotype frequencies between the healthy subjects and patients with CP, not even after stratification by sex (data not shown). No studies on this topic from European but only from Iranian and Brazilian populations have been reported. In the Iranian population, the CC genotype of another IL-17A variant (rs10484879) was associated with CP and peri-implantitis [ IL-17A −197A/G (rs2275913) gene in relation to periodontal disease with controversial results. In the study by Saraiva et al. [ IL-17A polymorphism and periodontal status  was found. However, Gürsoy et al. [ IL-17A variant in the subgroup of T1DM patients with CP were closer to the values in nondiabetic CP than T1DM patients. The different findings in Czech population may be due to differences in European versus Brazilian populations.In addition, we found no significant differences in thelantitis . Three Ba et al. , the A aa et al.  and Zacaa et al.  found thy et al.  recently IL-17A −197A/G alleles or genotypes between healthy and periodontitis nonsmokers. However, the G allele and the GG genotype were marginally significantly associated with an increased risk of periodontitis in smokers. Analysis of the allele frequencies in nonsmokers versus smokers showed borderline significant differences in both studied groups but in the opposite trend, in which 68.1% versus 56.8% for healthy controls and 64.5% versus 73.8% for patients with CP. Due to a relatively small number of subjects in the individual subgroups, number of comparisons performed, and only marginally significant differences, our results should be interpreted carefully.In contrast to findings in the Brazilian cohort where the AA genotype was identified as a risk factor for CP in nonsmoker Caucasians , our res IL-17A gene variability in relation to the presence of periodontopathic bacteria in subgingival pockets in patients with CP and T1DM + CP. In CP population, IL-17A −197 A allele carriers had an increased risk of T.f. occurrence and, in CP nonsmokers only, this allele increased risk of the occurrence of P.i., but the same A allele was protective for the presence of T.d. in subgingival biofilm in T1DM patients with CP. In connection with periodontopathic bacteria, gene variability has been investigated in a few other interleukins to this date. The specific IL-8 variants were associated with subgingival colonization with A.a. in AgP and T.f. in CP in the Czech population [ IL-8 haplotype influenced the presence of the “red complex” bacteria in gingival sulci. Several studies by Nibali et al. [ IL-6 and Fc γ receptor genes. Additionally, SNPs in the IL-1 gene cluster [ interferon γ, and IL-2 were associated with the presence of various periodontopathic bacteria, especially A.a. and the “red complex” bacteria. However, in our study, no other bacteria from those studied were associated with IL-17A −197 variant and there were no significant differences in the occurrence of these bacteria among groups.In the next step, according to the hypothesis about biological functions and regulation of IL-17 which plays a role in host defense , we assepulation . Finoti pulation  also foui et al. –56 focus cluster , interfe in vitro by blood mononuclear cells with the IL-17A −197A/G (rs2275913) gene polymorphism in the subgroup of patients with CP. In Czech periodontitis patients, IL-17A −197 AA + AG carriers had higher IL-17 levels in unstimulated mononuclear cells than GG homozygotes. Our results are in accordance with the conclusion of Espinoza et al. [ in vitro after T cells stimulation. Even greater differences in IL-17 production were measured in CP carriers with the A allele in genotype after stimulation with P.g. It is in agreement with previous findings that P.g. LPS is a mediator of IL-17 release from human periodontal ligament cells [ P.g. induced innate cell IL-17 production and promoted Th17 polarization. In a pooled group of CP and healthy subjects (N = 45), production of IL-17 was associated with the IL-17A polymorphism only in unstimulated mononuclear cells, but not after stimulation by P.g., other periodontal bacteria, HSP70, and/or mitogens. In addition, recently Azman et al. [Finally, in functional study, we associated IL-17 productiona et al. , who connt cells . Also Mont cells  proved tn et al.  demonstrA/G rs22713 gene pn et al. .P < 0.05) without multitest corrections. Therefore, risk of relatively high false discovery rate means that our results should be interpreted with caution. Secondly, levels of IL-17 in plasma or in gingival tissue were not measured and the presence of periodontal pathogens was examined only in the 271 subjects. However, this study also has several strengths. This is the first study of the IL-17 gene polymorphisms in T1DM population and in European patients with CP that was conducted in a relatively homogenous population of white Caucasians in Central Europe of the Czech origin. Secondly, the size of studied healthy subjects and CP patients is greater than in previous Iranian or Brazilian studies. Thirdly, we examined not only the polymorphisms alone but their relationship with clinical, bacterial, and biochemical parameters, which allowed a better biological assessment of the detected associations.There are many possible limitations in this study. The present study is mainly limited by relatively low numbers of subjects and especially by the fact that from the group of 125 T1DM subjects, only 38 patients were evaluated for periodontal status. The negative findings of single marker analysis in this subgroup could be a result of a lack of statistical power to detect minor differences . In addition, most of the associations found were tightly below statistical significance ( IL-17A gene variability may partially influence T1DM control and the “red complex” bacteria occurrence in patients with CP and diabetic patients with CP. Additionally, our findings confirmed the functional relevance of the IL-17A polymorphism with higher IL-17 secretion in the individuals with the A allele. However, the results of this study need to be proven in a larger independent cohort.In conclusion,