This ultimate answer was applied as DNA template in nested PCR and LAMP reaction. The primers for nested PCR and LAMP had been listed in Additional file 9. The procedure of bacterial isolation, nested PCR and LAMP have been carried out based on description of Wang et al. Detection of s100a4 and s100a6 expression in different tissues 3 pigs in H. parasuis infection group and manage group had been picked to the examination of s100a4 and s100a6 expression in different tissues. Total RNA from five porcine organs was isolated with RNAprep pure Tissue Kit Co. Ltd. Complete RNA was then quantified by NanoDrop one thousand Spectrophotometer. The high quality of your RNA was checked by formaldehyde denaturing gel elec trophoresis in 1. 2% agarose gels, which showed dis persed bands devoid of any apparent smearing patterns that will indicate degradation.
Two microgram of complete RNA was used for reverse transcrip tion polymerase chain response, making use of the TransSript Very first Strand cDNA Synthesis SuperMix selleck chemical according to the producers directions Co. Ltd. The qPCR assays were performed and ana lyzed as described above, with primers listed in Table three. Data for STRING and IPA examination Differentially expressed genes were analyzed making use of STRING, a database of identified and predicted protein interaction for DE gene encoded pro teins. The outcomes were obtained by inputting the record of gene symbol as identifier. Ppp1r13l gene was chosen for net perform exploration utilizing Ingenuity Pathway Evaluation. The data set containing gene identifier and corresponding expression value was uploaded into while in the application.
The identifier was mapped to its corresponding object in Ingenuitys Know-how Base. Network Eligible mole cules had been then overlaid onto a international molecular net function to ensure network of Network Eligible Molecules could possibly be algorithmically created based on their connectivity. Background The functional unit of the liver, the lobule, is roughly cylindrical, with branches of the selleck hepatic artery and por tal vein together with bile ducts at its periphery, and also a central vein branch inside the middle. Such a structure makes it possible for a normal distinction between upstream peripor tal and downstream perivenous hepatocytes, and con stitutes the basis for liver zonation. It really is frequently accepted that the homeostatic perform from the liver depends upon the practical complementation of people two zones. Distinctive, or even opposite, metabolic functions tend to be discovered involving periportal and perive nous hepatocytes. That heterogeneity is important for comprehending of many aspects of liver function and pathology, and it is extensively studied. On the other hand, minor consideration has become paid up to now to zonation like alterations related to human hepatocellular carci noma, which can be one of the most frequent visceral neoplasms globally.
To take out residual gen omic DNA, the RNA samples had been incubated with ten units of DNA free of charge DNAse I for thirty min at 37 C. The excellent and quantity on the purified RNA were established by measuring the absorbance at 260 nm280 nm applying a Nanodrop ND 1000 spectrophotometer. RNA integrity was more verified by electrophoresis by a 1. 5% agarose gel. Poly mRNA was isolated through the total RNA sam ples with oligo magnetic beads. The purified mRNA was fragmented from the RNA fragmenta tion kit and applied as template for first strand cDNA synthesis using random hexamer primers and reverse transcriptase. The 2nd strand cDNA was synthesized using RNase H and DNA polymerase I. The Illumina Genomic DNA Sample Prep kit was used to produce 120 bp paired end cDNA libraries by following the makers protocol.
The libraries selleck inhibitor have been loaded onto movement cell channels for sequencing over the Illumina HiSeq 2000 in strument through the Chinese National Human Genome Cen ter. A complete of 6 paired end cDNA libraries of zebrafish livers had been constructed for every with the check groups of WED immunized and mock immunized fish. Triplicate biological replicates have been per formed for every group. Raw data had been deposited inside the NCBI database under submission num ber SRA048658. two. Transcriptome examination The Illumina HiSeq 2000 method created 120 bp raw PE reads have been first processed by the FASTX Toolkit to clear away the reads with sequencing adaptors and of very low top quality. Then, the Burrows Wheeler Aligners Smith Waterman Alignment plan was made use of to align the remaining reads to the reference zebrafish mRNA in the Ensembl database.
The transcription degree of each gene was deduced by deter mining PH-797804 the complete quantity of reads mapped to every single gene utilizing Picard equipment. Dif ferentially expressed genes have been recognized by the DESeq bundle in R software program, applying two fold change 1 or 1 and p value 0. 05as the threshold. Immediately after data normalization by the p value and FDR calculation, the resulting expression intensity values had been analyzed through the MA plot based mostly technique, as described by Wang et al. Functional examination of differentially expressed genes The Database for Annotation, Visualization and Inte grated Discovery was used to investi gate functional enrichment for above and below expressed genes by more than two fold during the WED immunized group relative to your mock immunized group.
Gene practical enrichment was performed working with the default parameters in DAVID to acquire an adjusted p value 0. 05 for your check gene group versus the zebra fish gene ontology annotation set. The fold enrichment minimize off suggested for DAVID practical annotation is one. five. Additionally, the appreciably up regulated genes in the differentially expressed genes dataset had been further analyzed by investigating the corresponding GO biological processes.
Cyclin related genes strongly enriched at 04 dpp in the cucum ber fruit transcriptome, such as putative homologs of CDKB1.2, CDKB2.2, CYCB12. CYCD3.1, CYCD3.3, CYCD5.1, were among those associated with the mitosis and post mitosis phases in the Arabidopsis interactome. Elevated expression of several of these genes was also observed during fruit set in pollinated vs. unpolli nated apple and cucumber flowers. In contrast, the homolog of CDKA.1, which was uni formly represented in the young cucumber fruit transcrip tome, was associated with cyclin complexes throughout the Arabidopsis cell cycle. The categories of plastid and chloroplast also were sig nificantly enriched in the 04 dpp group, then declined with age. This is consistent with the decrease in chloro phyll observed after 4dpp.
chlorophyll content per gram fresh weight peaked at 4 dpp, and then decreased until 12 dpp. The assembled contigs included 91 tran selleck chemical scripts whose homologs in Arabidopsis had annotations including one or more of the following terms chlorophyll, chloroplast, photosystem, or thylakoid. Overall patterns of transcript abundance for these genes paralleled chlorophyll content in the develop ing fruit. K means cluster analysis allowed for further identifica tion of transcripts showing progressive patterns of repre sentation with fruit age. The chloroplast and other photosynthesis related genes described above, actin related proteins, extensins, expansins, cellulose synthases, pectinase modifying enzymes, aquaporins, vacu olar HATPases, and phloem filament and lectin proteins, were among those strongly represented, as has been observed for other rapidly growing fleshy fruits such as to mato, apple, grape, and watermelon.
The major latex protein related genes also exhibited peak levels at 8 dpp, including two extremely highly transcribed genes that together accounted for more than 17,000 reads. Putative homologs of vacuolar ATP synthase kinase inhibitor p38 inhibitors subunits B, D, H and P2 showed coordinate transcript abundance, with comparable levels increasing steadily until 8 dpp, and then gradually declining Two very highly represented homologs of the vacuolar aquaporin gene, gamma tip tonoplast intrinsic protein, also peaked at 4 8dpp. All of the cucurbit specific phloem proteins listed in Table 1 and the four putative homologs of the Arabidop sis phloem protein A2 family members observed in the data set peaked somewhat later, at 816 dpp with minimal transcript levels at 0 and 4 dpp.
Cucurbits are characterized by a unique and functionally divergent network of extrafascicular phloem external to the vascular bundles. The highly expressed pro teinaceous phloem filaments, comprised of the cucurbit specific PP1 proteins, and the more widely distributed PP2 phloem lectin proteins, were found to be pri marily associated with the extrafasicular phloem.
From the mid 1980s, phospholipase A,two enzymes were identified to be really expressed from the synovial fluid of RA sufferers. PLA2 kinds a group of enzymes that meta bolise phosphoglycerides to release lipid mediators this kind of as lysophospholipids and arachidonic acid. These metabolites might be converted in to the professional inflammatory platelet activating factor and eicosanoids, respectively. Instead of cytosolic PLA2 enzymes which have physiological functions inside essentially all cells, secre tory PLA2 enzymes are acknowledged for being energetic dur ing inflammation, and as a result happen to be an appealing target for anti inflammatory drug growth. sPLA2 enzymes also have agonistic action at the M form recep tor, by way of which they’ll promote irritation by means of degranulation of mast cells, cytokine release or secretion of elastase, an activator on the complement cascade extrinsic pathway.
sPLA2 enzyme concentrations are already found to be elevated inside the synovial fluid of sufferers with RA. Correlations have also been identified concerning serum ranges of sPLA2 and clinical markers of disorder such since the variety of energetic and effused joints, erythrocyte sedimentation fee, Lansbury index, elevated platelet count, and very low mTOR kinase assay hemoglobin in RA individuals. Arthritic joints have also been shown to have higher expression of sPLA2 group IIa within the synovial lining, even though sPLA2 IIa expression in nutritious joints is vir tually absent. On top of that, intra articular injections of human recombinant sPLA2 brought on acute inflammatory arthritic like symptoms in rats and rabbits, though transgenic mice in excess of expressing human sPLA2 didn’t spontaneously develop arthritis.
Researchers from Eli Lilly performed a phase I clinical trial using an inhibitor of sPLA2 group IIa given intravenously selleckchem to individuals with lively RA, which offered significant improvement in swollen and tender joints after three days. Following this, a bigger scale Phase II trial was conducted to assess the oral efficacy of LY333013, a methyl ester prodrug of LY315920. The outcomes from this trial indicated that while there were vital dose response relevant enhancements after one week of treatment method, there was no considerable result following 4 and eight weeks of treatment. Poten tial explanations for this failure include the lack of suffi cient inhibitor concentration during the synovial fluid to inhibit nearby joint sPLA2, and that all patients have been currently getting disease modifying anti arthritic drug therapy through the entire trial.
Thus, there is nevertheless a have to have to establish no matter if there may be a patho genic position of sPLA2 enzymes in RA. We have now previously reported that a synthetic tiny molecule inhibitor of group IIa sPLA2 4S penta noic acid is orally active and has therapeutic efficacy in rat models of intestinal ischemia reperfusion damage and inflammatory bowel illness.
Inside the SCID HuRAg model, our information showed remarkable reductions during the quantity of inflam matory cells and the degree of cartilage erosion inside the sdAbA1 treated group compared using the manage group. Additionally, sdAbA1 treatment method prevented cartilage de struction much more efficiently than the drug infliximab. It’s been well established that proinflammatory cytokines and MMPs are concerned inside the pathogenesis of RA. CypA may possibly stimulate macrophages to degrade joint cartilage via MMP 9 expression and market irritation by means of proinflammatory cytokine manufacturing. In this study, immunohistochemical staining revealed that the MMP 9 expression within the grafts was re markably decrease in the sdAbA1 treated group compared with the management group.
The capability of sdAbA1 to inhibit MMP 9 production might explain why sdAbA1 exhibited stronger anti erosion effects compared to the anti TNF mono clonal antibody. Moreover, the serum levels of IL 6 and IL 8 within the SCID HuRAg model were considerably lowered following sdAbA1 treatment. These findings indicate selleckchem that sdAbA1 exerts anti inflammatory and anti joint damage results on RA by inhibiting MMP 9 expres sion and secretion of IL six and IL 8. Despite the fact that its largely unclear how CypA is involved in chemotaxis and MMP 9 regulation, one particular vital mechan ism takes place via the interaction of CypA with 1 of its receptors, EMMPRIN. A study re ported that fibroblast like synoviocytes from CIA mice secreted CypA and enhanced CD147 expression in mac rophages. In RA patients, upregulated expression of CD147 was also found on circulating and synovial monocytesmacrophages, and higher levels of CypA have been also detected inside the synovial fluid.
These re ports recommend that an interaction from the synovial joints involving extracellular CypA and CD147 expressed by macrophages may perhaps be a mechanism involved within the devel opment of RA. Interestingly, we observed that sdAb1 could block the CypACD147 interactions by carrying out aggressive ELISA. To even further clarify the mechanism in the therapeutic results of sdAbA1 on RA, CypA was additional Diosgenin into monocytesmacrophages from RA sufferers in vitro to mimic an in vivo surroundings of rheumatic joints. Our benefits showed that cell migration and MMP 9 secretion of RA sufferers monocytesmacro phages were remarkably inhibited by sdAbA1. Also, western blot benefits demonstrated that sdAbA1 was in a position to reverse the NF ?B action induced by CypA by means of the ERK pathway, as a result leading to downregulation of MMP 9.
In order to discover the romance in between NF ?B activation and MMP 9 production, the two NF ?B inhibitors TPCK and PDTC had been utilised. This approach showed the ranges of MMP 9 diminished substantially by including NF ?B inhibitors. Other research also sug gested that CD147 mediate the results of extracellular CypA through inducing the activation of NF ?B, which was constant with our benefits.
One can find two leading limitations inside of this review a fairly minor size from the tested cohort, in addition to a broad variation from the time interval between the blood samples collected in advance of the onset from the condition and the RA diagnosis. These samples were not collected frequently but had been retrieved from the current financial institution of blood donors. Stratification within the studied topics diminished the numbers in every single group and manufactured extra complica tions for the detection of statistical significance. Conclusions In summary, greater amounts of survivin in patients were detected many years before the onset of RA signs and symptoms, par ticularly in individuals folks beneficial for anti CCP anti bodies. The substantial levels of survivin seem for being extra linked to your cytokines created through the Th1 and Th17 subsets.
These results recommend a position for survivin on the early presymptomatic stages of RA. Introduction Rheumatoid arthritis is a chronic inflammatory dis ease characterized by synovial inflammation and hyperpla sia, leading to progressive cartilage and bone destruction. Typical synovium selleck chemicals NSC 74859 forms a thin membrane with the edges of joints and provides lubrication and nutrients to the cartil age. In RA, this thin synovial lining layer radically increases and transforms into an inflammatory mass, often called the pannus. This tissue mass expands and attaches to and invades the adjacent cartilage and sub chondral bone, causing erosion. The main cell kind ac counting for the thickened lining layer and resultant pannus could be the activated RA synovial fibroblasts.
Likewise as mediating tissue destruction, RASFs perform a significant role in catalyzing and sustaining RA by producing inflammatory cytokines selleck this kind of as interleukin 1 beta and tumor necrosis aspect alpha, proangiogenic aspects, and matrix degrading enzymes. Of equal concern, RASFs collaborate with and help the recruitment, survival, activation, and dif ferentiation of T cells, B cells, macrophages, mast cells, osteoclasts, and endothelial cells throughout the RA synovium. As soon as activated, the aggressive phenotype of RASFs can exist independent of inflammation. This was demon strated by scientific studies performed from the significant combined im munodeficient mouse model of RA, through which implanted human RASFs degraded co implanted human cartilage within the absence of inflammatory cells, and RASFs mi grated by means of the bloodstream to implanted cartilage at a distant site, spreading RA to unaffected joints. These information clearly indicates that RASFs aren’t passive by standers, but are energetic participants in joint destruction in RA. Endothelial protein C receptor is definitely an endothelial transmembrane glycoprotein capable to bind to a organic anticoagulant, protein C, and its activated kind, APC, with related affinity.
Basal gene and protein expression levels have been analysed by qRT PCR and western blotting. ChIP analyses had been performed and demonstrated that BRCA1 regulates basal gene expression by means of a transcriptional mechanism involving c myc. Benefits We’ve previously carried out microarray primarily based expression profiling to examine variations in gene expression when BRCA1 is reconstituted in BRCA1 mutated HCC1937 breast cancer cells. We observed that p cadherin plus the cytokeratin 5 and cytokeratin 17 genes, that are strongly correlated with the basal phenotype, are differentially expressed when BRCA1 is reconstituted. Additionally, qRT PCR and ChIP evaluation of BRCA1 reconstituted cells show that BRCA1 represses the expression of those basal genes by a transcriptional mechanism.
Furthermore, abrogation of endogenous BRCA1 protein inside the T47D cell line using siRNA final results in re expression of these basal genes, suggesting inhibitor NSC319726 that BRCA1 expression levels may possibly be important in basal gene expression. We’ve also demonstrated that BRCA1 is physically connected with all the promoter regions of basal genes by way of an association with c myc. Consequently, we’ve confirmed that siRNA inhibition of c myc in T47D cells results in re expression of those genes. Conclusions Our benefits suggest that BRCA1 is involved in the transcriptional regulation of genes associated with all the basal phenotype and that BRCA1 controls basal gene expression by way of a transcriptional mechanism involving c myc. Additional work is now concentrating on defining the relationship amongst BRCA1 and basal gene expression and how this may possibly affect clinical responses to breast cancer chemotherapy.
Cancer Research UK, London Investigation Institute, South Mimms, UK Breast Cancer Investigation 2006, GDC-0199 clinical trial 8 S5 Background Inherited mutations in BRCA2 are related with a predisposition to early onset breast cancers. The underlying basis of tumourigenesis is believed to become linked to defects in DNA double strand break repair by homologous recombination, as indicated by the spontaneous chromosomal instability phenotype of BRCA2 defective cell lines. The BRCA2 protein interacts with ssDNA along with the RAD51 recombination protein, and is proposed to recruit RAD51 for the harm internet site for the HR repair. Approaches Recombinant BRCA2 fragments that cover the complete length of BRCA2 were tested for interaction with RAD51 and for their phosphorylation employing cell cost-free extracts.
An antibody that especially recognises BRCA2 phosphorylated at serine 3291 was generated and applied to analyse the phosphorylation status of endogenous BRCA2 for the duration of the cell cycle and soon after DNA damaging treatment. A cell line that stably expresses a C terminal BRCA2 fragment was generated, to enable the analysis of RAD51 interactions and ability to market homologous recombinational repair.
1% DMSO. For generation of clonally derived cell lines, Ca1a cells had been double sorted and single cells plated straight into 96 properly dishes containing conditioned DMEMF12 media supple mented with 5% heat inactivated HS. These wells containing a single cell had been identified microscopically and expanded. Flow cytometric evaluation and sorting Anti human CD44 allophycocyanin and anti human CD24 phycoeryth rin or anti human CD24 fluorescein were employed for each evaluation and live sorting. 7 aminoactinomy cin D was used for livedead cell distinction. For flow cytometric analysis, cells were stained with a PBS answer containing 0.1% BSA and 0. 1% sodium azide for 25 min at 4 C followed by two washes with this similar buffer. For dual stain ing of CD24 and vimentin cells were stained with CD24 FITC as described above followed by fixa tion and permeabilization.
Staining was performed inside a PBS option containing 0. 1% BSA, 0. 1% sodium azide, and 0. 5% selleck inhibitor Tween 20 for 25 min at four C followed by two washes with this same buffer. Analysis was performed on either a BD Bio sciences FACSCalibur or LSR II. For dissociated xenografts, gates were established post compensation with lineageneg cells that were not exposed to anti human CD44 or anti human CD24 antibodies. For reside sorting, cells have been stained within a PBS remedy containing 1. 0% FBS, 100 unitsml penicillin streptomycin, and 1g ml Amphotericin B for 25 min at 4 C. Gates had been estab lished with unstained cells. Cell sorting was performed on a BD Biosciences FACSAria operating at Low Pressure utilizing a 100M nozzle. Cell clusters and doublets had been elec tronically gated out.
Cells had been routinely double sorted and post sort analysis usually indicated purities of 90% with minimal cell death. Flow cytometry data have been ana lyzed making use of selleckchem FlowJo v8. eight. five. In vivo tumorigenicity and processing of xenografts In vivo tumorigenicity was assessed by both frequency and latency of tumor formation inside the abdominal mammary gland fat pad of 8 wk old athymic NCr nunu mice obtained in the NCI colony. All animal experiments were performed in accord with accepted standards of humane animal care and approved by the Animal Care and Use Committee at the National Institutes of Wellness. Five days before injection of cells, the bone marrow suppressant etopo side was administered intraperitoneally. animals also received a subcutaneous estrogen pellet. Cells were suspended within a F12 Matrigel mixture and injected in to the mammary fat pad inside a 50l volume. Mice have been anesthetized by an ip injection of ketaminexylazine in 200l Hanks Bal anced Salt Solution prior to surgically exposing the gland for injection. Tumor size was measured weekly working with a caliper. Experiments had been terminated after a xenograft reached 1.0
Additionally, SL0101 substantially impairs MSP and TGF b1 induced cell migration, which can be a function connected with EMT. Impact of elevated RSK expression in MSP induced EMT like activity in cancer cells To study the impact of RSK2 on MSP induced EMT in extra detail, two human cancer cell lines L3. 6pl and HT 29 have been selected based on their variations in RSK1 and RSK2 levels and similarities in RON and TGF b receptor expression. Pancreatic cancer L3. 6pL cells expressed regular levels of RSK1 and RSK2. MSP and TGF b1 stimulation brought on elongated cell morphology, decreased E cadherin expression, and elevated vimentin expression. Combined MSP and TGF b1 therapy additional enhanced the mod ulating effect on E cadherin and vimentin expression. These final results indicated that L3.
6pl cells show EMT like phenotypic modifications right after MSP and TGF b1 stimulation in addition to a synergistic activity involving RON and TGF bRI II signaling in induction of EMT like phenotype. HT 29 cells expressed incredibly low levels of RSK1 selleck chemicalNMS-873 and RSK2. Therapy of cells with MSP, TGF b1 or both brought on barely any morphological adjustments. Western blot evaluation also failed to observe any modifications in E cadherin and vimentin expression in MSP plus TGF b1 stimulated HT 29 cells. However, RSK2 overex pression by pRSK2 plasmid transfection resulted in cell morphological modifications following MSP stimulation. We observed comparable modifications when transfected HT 29 cells have been stimulated with TGF b1 or MSP plus TGF b1. Evaluation of E cadherin and vimentin expression in pRSK2 transfected HT 29 cells confirmed that MSP and TGF b1 stimulation caused E cadherin reduction and vimentin induction.
These final results sug gested that rising RSK2 expression renders HT 29 cells responsive to MSP and TGF b1 induced EMT like activities. Impact of RSK specific siRNA on selleckchem MSP induced cell migration To further confirm the role of RSK2, we transiently transfected L3. 6pl cells with specific siRNA to silence RSK1 or RSK2 mRNA expression. Final results in Figure 7A showed that siRNA precise to RSK1 proficiently silenced RSK1 expression but had no effect on RSK2 expression. RSK2 specific siRNA only silenced RSK2 expression but had no effect on RSK1 expression. These final results con firmed specificities of siRNA used to silence RSK1 and RSK2, respectively. Analysis of MSP and TGF b1 regu lated epithelial and mesenchymal proteins revealed that silencing RSK1 expression did not avoid MSP and TGF b1 induced reduction of E cadherin and induction of vimentin.
In contrast, knockdown of RSK2 expression restored E cadherin expression and prevented vimentin induction. We also observed these effects in cells treated with TGF b1 and MSP plus TGf b1, indicating that RSK2 was required for MSP and TGF b1 induced EMT like biochemical modifications. We additional studied the impact of siRNA mediated RSK2 knockdown on cell migration by the wound heal ing assay.
These data recommend that robust Src household kinase activity might result in the activation of inhibitory signals suppressing T cell activation. In summary, we’ve got shown that stimulation with iAbs induces unique feedback regulation than sAbs therapy. sAbs result in sturdy and rapid acti vation of Src kinases and subsequently for the phosphor ylation of inhibitory molecules, which terminate signaling. However, iAbs induce only slight improve in kinase activity and an Erk Lck good feedback loop, which might be needed to prevent rapid Lck dephosphorylation by SHP 1 or other phosphatases, and as a result bring about sustained activation. Discussion Signaling through a number of plasma membrane connected receptors leads to cell choice processes like cell proliferation, differentiation, survival, and motil ity.
Considerable proof suggests that the magnitude and also the duration of a signal ascertain the functional outcome. As tiny is recognized on the mechanisms regulat ing signaling kinetics correlating with cellular responses in T cells, we’ve analyzed TCR mediated signaling below conditions top to either T cell unresponsive ness or to proliferation. We selleck chemical employed sAbs and iAbs stimulation which induce qualitatively unique signals and T cell responses. We discovered striking differences in TCR signaling kinetics and feedback regulation. In deed, under proliferation inducing conditions, TCR mediated signaling is prolonged by a optimistic feedback loop involving Erk and Lck. Conversely, stimulation with sAbs strongly activates inhibitory molecules that probably terminate signaling.
These observations are in agreement with the model proposed by Acuto et al, that the signal amplitude and kinetics in double optimistic thymocytes de read full report pend around the sort of the applied stimulus. Here, we show that a equivalent principle could apply also to mature T cells. An essential question that demands additional investiga tions is how signals using a widespread origin in the TCR are split to activate distinctive effector molecules. Through thymocyte development, it has been proposed that a molecule or complex functioning as signal splitter senses the intensity of signals emanating from the TCR and discriminates involving negatively and positively deciding on ligands. However, such a molecule has not however been identified in immature T cells.
We propose that Lck might function as signal splitter in mature T cells directing signals emanating in the TCR toward unre sponsiveness if the signal is at higher intensity. This security mechanism could be set in mo tion in case of an inappropriate stimulation that could result in T cell hyperactivation and the improvement of autoimmunity. The concept that the molecular switch is situated in the apical a part of the cascade could represent an advantage from the method.