NADPH oxidase is a major source of reactive oxygen species (ROS) production in the kidney and contributes to renal damage in diabetes. We aimed to examine the role of the NADPH oxidase Nox1 and Nox4 in diabetic nephropathy (DN) using genetic deletion and pharmacological inhibition approaches Selleckchem Torin 1 in streptozotocin induced diabetic mice. Methods: Nox1−/yApoE−/− or Nox4−/−ApoE−/− and their respective wild type or ApoE−/− mice were rendered diabetic via streptozotocin injection. ApoE−/− non-diabetic and diabetic mice were treated with the specific Nox1/4 inhibitor (GKT137831). Animals were culled after 20 weeks and

kidneys were removed for assessment of structural damage, oxidative stress markers, as well as protein expressions extracellular matrix (ECM), pro-fibrotic and pro-inflammatory markers. In vitro, Nox4 was silenced in human podocytes and exposed to high glucose for gene expression analysis and ROS measurements. Results: Deletion of Nox4, but not of Nox1 resulted

in renal protection from glomerular injury as evidenced by attenuated albuminuria, preserved renal structure, reduced glomerular accumulation of ECM proteins as well as attenuated Neratinib glomerular macrophage infiltration. Administration of GKT137831 to diabetic ApoE−/− mice conferred a similar degree of renoprotection as did deletion of Nox4. In human podocytes, silencing of the Nox4 gene resulted in reduced ROS production and down-regulation of profibrotic markers that are implicated in diabetic

nephropathy. Conclusion: Collectively, selleck chemicals these results identify Nox4 is a key source of ROS responsible for kidney injury in diabetes and provide proof of principle for an innovative small molecule approach to treat and/or prevent DN. UJIKE HARUYO1, MAESHIMA YOHEI2, HINAMOTO NORIKAZU1, WATATANI HIROYUKI1, TANABE KATSUYUKI1, MASUDA KANA1, SUGIYAMA HITOSHI1, SATO YASUFUMI3, MAKINO HIROFUMI1 1Department of Medicine and Clinical Science, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan; 2Dept. of Chronic Kidney Disease and Cardiovascular Disease, Okayama Univ., Okayama, Japan; 3Dept. of Vascular Biology, Institute of Development, Aging, and Cancer, Tohoku Univ., Sendai, Japan Introduction: Diabetic nephropathy is the most common cause of end-stage renal disease, and albuminuria is a risk factor for progressive loss of renal function. Vasohibin-2 (VASH-2) belongs to the Vasohibin family and serves as a pro-angiogenic factor. We previously reported the protective role of exogenous Vasohibin-1, a homologous to VASH-2 and a negative feedback regulator of angiogenesis, in mouse models of diabetic nephropathy. To date, the biological role of VASH-2 in renal disorders is not clarified. In the present study, we aimed to evaluate the potential role of endogenous VASH-2 on the progression of diabetic nephropathy.

Furthermore, IWR-1 clinical trial it was noteworthy that only a fraction of the Ly-6G+ cells were positive for IL-17 immunostaining (Fig. 4 and 5), and the remaining Ly-6G+ but IL-17− cells could be either neutrophils under heterogeneous status, or other Ly-6G+ resident myeloid cells such as monocytes in the cornea [42]. Though IL-17 is generally involved in anti-infection responses [43], we show here that it can be detrimental to the clearance of pathogens in corneal tissue (Fig. 8). Considering that IL-17 expression is differentially regulated by different pathogens in the same cell [44], our conclusions concerning C. albicans may not be transferable to

infection of other pathogens. To address these concerns, we are currently undertaking comparative studies with other pathogens. In summary, we report that intrastromal inoculation Cabozantinib concentration of C. albicans blastospores does not cause keratitis in nude, IL-17A knockout, CD4+-depleted, neutrophil-depleted, and IL-23-/IL-17-neutralized mice. Our analysis of early events (<24 h) postinfection revealed that IL-17, mainly produced locally

by neutrophils and/or CD4+ T cells, played a central role in the initiation of CaK. Future studies will investigate the sequential or spatial regulation of IL-17 production, neutrophil activation, and immune compartments that interact with IL-17/Th17 in the context of FK. Taking into account the previous report that an adaptive immune response is required to protect the host from secondary CaK, we propose a biphasic mechanism of CaK pathogenesis: in early phase, CD4+ T cells act coordinately with neutrophils to initiate CaK in an IL-17-dependent manner, and later give way to adaptive immunity processes. All animal experiments were carried out in accordance with the Chinese Ministry of Science and Technology Guidelines on the Humane Treatment of Laboratory Animals (vGKFCZ-2006–398) and the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic

and Vision Research. This study and all protocols concerning animals were approved by the Shandong Eye Institute enough Review Board with permit number SEIRB-2009–2009CB526506. All animal experiments were carried out in accordance with the Guidelines on the Humane Treatment of Laboratory Animals (Chinese Ministry of Science and Technology, 2006) and the Statement for the Use of Animals in Ophthalmic and Vision Research. WT C57BL/6J mice, BALB/c mice, and nude mice with a BALB/c background (H-2d) were purchased from the Academy of Military Medical Sciences (Beijing, China). IL-17A-deficient (IL-17A−/−) mice that were backcrossed to C57BL/6J mice for over ten generations [45, 46] were provided by Dr. Chen Dong (M.D. Anderson Cancer Center, Houston, TX, USA). All animals were maintained in pathogen-free facility and were 6–10 weeks old when the experiments were performed.

Family-based linkage studies that led to identification of disease-associated mutations in NLRP3, MEFV, PSTPIP1,

and NLRP7 have contributed significantly to our understanding of single gene Mendelian disorders such as the inflammasomopathies discussed herein. Candidate gene studies have also proven successful, in some instances, in identifying putative disease-causing mutations that affect the function of the inflammasome as illustrated by NLRP12 in hereditary periodic fever syndromes, NLRP1 as a risk gene for vitiligo, and the association of caspase-12 single nucleotide polymorphism (SNP) with severe sepsis. The advent in recent years of dbSNP databases, high-resolution haplotype maps of the human genome (HapMap) and SNP arrays capable of analyzing up to 1 million SNP simultaneously on a single array has permitted the Acalabrutinib research buy introduction of genome-wide association studies (GWAS) to tackle the heritability of complex diseases such as Crohn’s disease (CD). We discuss in this Viewpoint how conventional genetics and GWAS have been instrumental in enhancing our understanding of NLR (NOD-like receptor) biology. Inflammasomes are cellular alarms that assemble in response to microbial invasion and/or cellular damage and https://www.selleckchem.com/products/Adrucil(Fluorouracil).html alert the system by triggering an inflammatory response. They are scaffolded

by the NLR, which are germ-line encoded cytosolic pattern recognition receptors. NLRs induce inflammation by recruiting and activating caspase-1, which processes the pro-inflammatory cytokines IL-1β and IL-18 into their mature biologically active forms (Fig. 1). Considering the key role of IL-1β in inflammatory processes, it was not surprising that defective control of inflammasome activity caused Phenylethanolamine N-methyltransferase serious diseases. Among these, the most extensively studied are cryopyrinopathies (also known as cryopyrin-associated periodic fever syndromes [CAPS]). These encompass a continuum of disease states, including in increasing order of severity:

familial cold autoinflammatory syndrome, Muckle-Wells syndrome, and chronic infantile neurologic cutaneous articular syndrome. In 1999, two independent linkage studies mapped the CAPS susceptibility locus to human chromosome 1q, and 2 years later autosomal dominant mutations were identified in the gene encoding NLRP3 (originally denoted cryopyrin or CIAS1) 1, 2. CAPS-associated mutations (>40 reported so far) are mainly concentrated in exon 3 of the gene, which encodes the nucleotide-binding domain (NBD) of NLRP3 (3 and http://fmf.igh.cnrs.fr/infevers). The primary impact of these “gain-of-function” mutations is to disrupt an auto-inhibited state of NLRP3, thus potentiating constitutive inflammasome assembly 3. Two independent groups have recently reported the generation of knock-in mice that carry CAPS-associated mutations in NLRP34, 5.

327, p<0.05, MS: r2=0.446, p<0.05). Of interest, the Treg-mediated inhibition of Tconv proliferation also positively correlated with IL-7Rα-MFIs on Tconv (HC: r2=0.175, p<0.05 MS: r2=0.587, p<0.01; Fig. 3), suggesting that IL-7Rα expression by affecting

frequencies of circulating RTE-Treg also interferes with Treg function. Proliferative responses of stimulated and unstimulated Tconv were comparable in samples obtained from MS patients and healthy donors. We measured sIL-7Rα in plasma specimens obtained from MS patients (n=20, 12 with active disease, 8 in remission) and age- and sex-matched control inidividuals (n=17) using an in-house ELISA protocol and IL-7 levels with a conventional ELISA Kit as described in the Materials and methods section. We found an inverse correlation between IL-7 plasma levels and IL-7Rα-MFIs on total Tconv in patients with MS (IL-7: HC: r2=0.142, p=0.103; MS: r2=0.252, p=0.027;

Fig. 4B). Concentrations of both IL-7 and sIL-7Rα were AG-014699 manufacturer elevated in 20 patient-derived samples as compared to 17 HC-derived PF-02341066 solubility dmso samples, which was statistically significant for IL-7 only (IL-7 [pg/mL]: HC 5.1±1.5, MS 11.2±5.9, p=0.050; sIL-7Rα [ng/mL]: HC 107.5±40.6, MS 145.0±53.7, p=0.161; Fig. 4A). Enhanced IL-7 and sIL-7Rα plasma levels were detectable in both patients with active and inactive disease. TSLP and TSLPR-expressing MDCs were previously shown to be critically involved in thymic Treg development 13. Therefore, we analyzed surface expression levels of TSLPR on circulating MDCs in blood samples obtained from MS patients (n=12, 8

with active disease, 4 in remission) and age- and sex-matched normal donors (n=11). TSLPR-MFIs were significantly lower on patient-derived MDCs (HC 96.0±15.9, MS 59.6±17.4; p<0.01; Fig. 5) and did not differ between RRMS patients with active or stable Isoconazole disease. In addition, expression levels of IL-7Rα and TSLPR strongly correlated in both study cohorts (MS: r2=0.57, p<0.05; HC: r2=0.61, p<0.05; not depicted). It was previously shown, that 10–30% of peripheral T cells and up to 99% of human Treg express two distinct TCR-Vα chains 21. Here, in both study cohorts (HC: n=33, MS: n=56) approximately a quarter of total Tconv harbored TCRs with dual specificity (HC: 31.9±14.0%, MS: 29.6±18.2%, p=0.47). In contrast, 85.6±17.1% of control-derived, but only 55.8±31.2% of patient-derived Treg expressed two TCR-Vα chains (p<0.01; Fig. 6A). Overall, there was a strong correlation of IL-7Rα-MFIs of Tconv and TSLPR-MFIs on MDCs with the amount of dual TCR specific Treg in both patients and control donors (IL-7Rα: HC: r2=0.247, p=0.011; MS: r2=0.355, p=0.008, Fig. 6B; TSLPR: HC: r2=0.214, p=0.031; MS: r2=0.333, p=0.016; not depicted). Screening for rs6897932-SNP 15–18 associated with MS, type 1 diabetes and chronic inflammatory arthropathies 19 was performed by SNAP-shot PCR.

32 and Jain et CH5424802 al. 33, who showed that inactivation of Myc overexpression in Myc-transgenic mice for short periods of

time allowed further cellular differentiation of previously malignant T cells, acute myeloid leukemia cells, and osteogenic sarcoma tumors, thereby inducing a loss of transformation of these cells. Hence, upon reactivation of Myc overexpression, the differentiated, previously transformed cells were resistant to further malignant proliferation and survival. Since Myc is known to be involved in the generation of mouse plasmacytomas 34 and other mature neoplasms of mice 35 and humans (such as Burkitt’s Lymphoma), it should be able to contribute to the transformation of at least some of the compartments of mature B cells. Our findings that Myc together with Pim1 does not induce long-term proliferation of mature B cells ex vivo or in vivo suggests that different combinations of proto-oncogenes might be active in B-lineage cells at different stages of their development. If the cell cycle promoting activity of Myc also functions in mature B cells, then the inhibition of apoptosis this website supplied by another cooperating oncogene with activity in mature B cells

may be required for transformation to uncontrolled proliferation. Interesting partner candidates for Myc in mature B-cell stages are Bcl2 36, as well as BclXL 37 and Bcl6. The latter two together have recently been shown to induce proliferation of human SPTBN5 germinal center B cells ex vivo in the presence of CD40 signaling and interleukin

IL21 38. Our Pim1- and Myc-transduced inducible cell lines should be useful tools to search for additional genes with cooperating functions in the transformation of normal pre-B cells, and they also enable to screen for cooperating oncogenes active on their ways to fully malignant stages in mature B cells, memory cells and plasma cells. For the retroviral vector expressing the reverse transactivator rtTA-M2, the plasmid pSuperRetroPuro (OligoEngine, Seattle WA, USA) was cut with XhoI and ClaI (all from New England Biolabs, Ipswich MA, USA) to remove all unnecessary elements, and a linker element containing an MCS was inserted instead (=pSR-L). The phosphoglycerate kinase promoter (pgk, from pSuperRetroPuro), the rtTA gene preceded by the Kozak sequence CACC(ATG), an IRES sequence taken from pIRES (Clontech, Mountain View CA, USA), and the HistidinolR gene (from the pSV2-HIS vector) were inserted at different restriction sites in the linker (Supporting Information Fig. 1B). For the doxycycline-inducible expression vectors driving expression of Pim1, Myc, and EGFP, the puromycin resistance gene (pSuperRetroPuro) or the hygromycin resistance gene (pLHCX, Clontech) under the control of the pgk promoter were cloned into the vector pSR-L.

An amount of 0·1 g of the faecal specimens from each of the groups at 0 day (day before C. parvum infection) and daily for 13 days following infection was weighed and purified through discontinuous sucrose gradients (13). Then, 10 μL of the purified faecal matter was taken to make a smear on glass slide. The slides were air dried,

stained with modified acid-fast Fulvestrant price staining method and examined for Cryptosporidium oocysts with microscope. Cryptosporidium parvum was counted in the entire smear using a 20× objective by a blinded observer. The results were expressed as number of C. parvum/g of faeces. Results of serological assays, production of cytokines and faecal oocyst shedding after C. parvum challenge were compared using analysis of variance (anova) and t-test using the spss software. A P-value of <0·05 was considered different. To study the capacity of multivalent peptides in stimulating immune responses and protecting host from C. parvum infection, first we generated the monovalent peptide fragment rCp23 and divalent

peptide rCp15–23 through recombinant DNA techniques. To identify Selleck Compound Library these cloned genes, the plasmid constructs were sequenced and analysed by BLAST searching. As in Figure 2a, the sequences we obtained are identical to Cp23 and Cp15 genes of C. parvum reported previously (15). To determine further whether the cloned genes can generate expected peptide, immunoblotting was performed using the sera from rabbits experimentally

infected with C. parvum. The through bands appeared at 27 and 46 kDa position indicated that the sizes of the peptides were the same as estimated molecular weights (Figure 2b). To examine the antigen specificity of the proteins, rCp15–23, rCp23 or crude extract of C. parvum was used to coat 96-well plate and reacted with sera from rabbits experimentally infected with C. parvum oocysts. We found that all of the three antigens had higher specific immune reaction with the sera and the immune response using rCp15–23 generated stronger reaction than that in either rCp23 or crude extract group (Figure 3). Immunization of BALB/c mice three times with 10 μg of the proteins at 2-week intervals resulted in the generation of specific antibody responses against rCp23 protein, crude extract of C. parvum and rCp15–23 fusion protein (Figure 4). The concentrations of IgG remained at low levels until days 14 after the first vaccination, whereas the second dose of vaccine rapidly and significantly boosted the responses with the titre at 1 : 22 810 for rCp23 and 1 : 81 280 for rCp15–23. A peak concentration was observed after third boost (with the titre of 1 : 51 200 for rCp23 and 1 : 102 400 for rCp15–23). The vaccination of the mice with both the recombinant Cp15–23 fusion protein and rCp23 induced stronger antibody response than crude extract (P < 0·05).

We compared changes in fluorescence ratios when a triggering dose of 1 ng DNP-HSA was added to non-desensitized cells, to desensitized cells and to cells that had not been sensitized with anti-DNP IgE. DNP-desensitized cells showed 90% inhibition of calcium mobilization (see Fig. 2B), indicating that calcium-dependent

events are impaired during desensitization. Because calcium mobilization is key to arachidonic acid metabolization and generation of prostaglandins and leukotrienes, we studied arachidonic acid products. Thirty minutes after 1 ng DNP-HSA challenge, cell supernatant was analyzed by reverse-phase high-performance liquid chromatography (RP-HPLC); buy Erlotinib cysteinyl leukotriene C4 (LTC4), leucotriene B4 (LTB4), and 12(S)-hydroxyheptadeca-5Z, 8E, 10E-trienoic acid (12-HHT) were identified with retention times of 21.4, 23.7 and 24.4 min, respectively, with prostaglandin B2 (PGB2) as an internal standard. In contrast, LTB4, LTC4 and 12-HHT were not detected in rapidly desensitized cell supernatants or in cells treated with 1 ng HSA (see Fig. 2C). This result indicates a lack of arachidonic acid metabolization

with desensitization. Other proinflammatory mediators are released from mast cells upon activation, such as TNF-α and IL-6 cytokines. Pre-formed TNF-α is released upon IgE stimulation in the early-phase response, while secretion of de novo synthesized TNF-α and IL-6 production occurs several hours post-stimulation, in the late-phase dipyridamole response. Because early-phase activation events may influence late-phase responses, and because desensitization may affect early and late-phase responses differently, see more we studied TNF-α, a product of mast cell responses in both phases, and IL-6, a cytokine not typically stored but produced in the late phase. Pre-formed TNF-α released with 1 ng DNP-HSA challenge was 490 pg±15%, while in rapid-desensitized cells the release was 185 pg±23%, a significant 62% reduction (see Fig. 2D, white bars). During the late-phase response, 4 h after activation or desensitization,

the release of newly generated TNF-α from DNP-activated cells was 978 pg±23%, while rapid-desensitized cells released 272 pg±22%, a significant 72% reduction (see Fig. 2D, black bars). The production of IL-6 assessed 4 h after activation or desensitization (see Fig. 2E) was 14362 pg±42% and 3665 pg±35%, respectively, showing a 75% reduction. Those results indicate that desensitization impaired early- and late-phase mast cell responses. It has been reported that STAT6 plays a pivotal role in antigen/IgE/FcεRI-mediated cytokine release from mast cells and that STAT6 phosphorylation occurs not only through the JAK-STAT pathway after IL-4 receptor activation but also after antigen crosslinking of FcεRI/IgE 18. Since our previous studies showed that STAT6-null BMMCs from BALB/c and C57BL/6 mice could not be desensitized 16, we explored how rapid desensitization targeted STAT6.

The first digit following the KIR acronym corresponds to the number of immunoglobulin-like domains in the molecule and the ‘D’ denotes ‘domain’. The D is followed by either an ‘L’, indicating a ‘Long’ cytoplasmic tail, (these proteins have inhibitory function), or ‘S’ indicating a ‘Short’

cytoplasmic tail, (these proteins have activating function), or a ‘P’ for ‘pseudogene’. The final digit indicates the number of the gene encoding a protein with MAPK Inhibitor Library order this structure. Where two or more genes have very similar structures and have very similar sequences, they may be given the same number but distinguished by a final letter, for example, the KIR2DL5A and KIR2DL5B genes.17 KIR alleles are named in a similar fashion to alleles of the HLA system (Fig. 1). Hence, the first three digits distinguish alleles differing in exon sequences that lead to non-synonymous changes. (The HLA nomenclature is on the point of being changed to allow for the expansion in the number of alleles). The next two digits indicate alleles that differ in exon sequences leading to synonymous changes and the last two digits are used for those alleles that only differ in an intron, promoter or other non-coding region. The HLA class I molecules act as ligands for some of the KIR genes. The alleles of the HLA-C locus can be distinguished into two groups of ligands (C1 and C2) by the amino acid present at position 80 of the molecule with approximately

50% of alleles being in each group. HLA-C group 1 with asparagine at position Sirolimus in vivo 80 provides the ligand for KIR2DL2 and KIR2DL3, whereas HLA-C group 2 with lysine at position 80 provides the ligand for KIR2DL1.

Recently it has been shown that whereas KIR2DL1 has only interaction with HLA-C2 group, KIR2DL2, and to a weaker Cepharanthine extent KIR2DL3, also bind to HLA-C2 group.18KIR3DL1 has specificity for the HLA-Bw4 epitope at residues 77–83, present on some HLA-A molecules in addition to many of the HLA-B alleles as each HLA-B allele has a Bw4 or Bw6 epitope. KIR3DL2 has as its ligand HLA-A3 and HLA-A11 allele families but only when certain virally derived peptides are loaded and HLA-G is the ligand for KIR2DL4. As all individuals will carry an HLA-C allele, HLA-C may be more important in the regulation of NK cells. As many of the laboratories interested in typing for presence/absence of KIR genes were histocompatibility laboratories the tendency was to use methods familiar to the laboratory, i.e. sequence-specific primers (SSP)19–22 and sequence-specific oligonucleotide probes (SSOP).23 However, these methods are not able to determine the number of gene copies present. Allele typing is limited and has been performed in only a few laboratories. Continuous discovery of new alleles and the difficulties inherent because of similarity in sequences, even between alleles of different genes, requires constant revision of the SSP and SSOP typing systems.

Although genome-wide linkage analysis of IgAN has revealed several susceptibility loci, the causative genes have not been identified. From the point of view of genetic heterogeneity of familial IgAN, an oligo/polygenic and multiple susceptibility gene model for the disease has been proposed. Recently, exome learn more sequencing has emerged as a powerful and cost-effective strategy for dissecting the genetic basis of diseases. Methods: To identify the genetic causality of familial IgAN,

we applied exome sequencing to a family comprising four biopsy-proven IgAN patients clustered in a dominant transmission mode. The whole exomes of four affected, two unmanifested carriers, and two unaffected individuals were captured and subjected to massive parallel sequencing. Variants identified by exome sequencing were filtered on the basis of variant annotation, functional expectation, and allele frequency. The affected individuals in the family were expected to share the same causal variant. Genome-wide linkage analysis was concurrently

performed for the family using the high-throughput linkage analysis system SNP HiTLink. Sequence analysis of the EEA1 gene was performed in other members of the family and in 27 additional cases with IgAN. The Human Genetic Variation database was used as a reference for the exome sequence data of the Japanese population. Results: Several filtering procedures for extracting candidates with disease-causing variants were effectively used as follows. The first step involved performing variant annotation on the basis of dbSNP FDA approved Drug Library cell assay entries, 1000 Genome O-methylated flavonoid Project, and amino acid substitutions to retain novel nonsynonymous variants. The next filtering

stage was performed on the basis of allele frequency, and an interval of 30%–70% was used as the cut-off threshold. Finally, 13 variants that were shared only by the affected individuals in the family were selected as candidate genes for familial IgAN. Linkage analysis of the family revealed linkage signals at nine loci. Among the candidates, a novel missense variant F161Y in EEA1 that encodes early endosome antigen 1 (a Rab5 effector protein that facilitates the docking and tethering of incoming endocytic vesicles) was located within a linkage locus with a maximum LOD score of 1.68. Furthermore, the F161Y variant completely cosegregated in the family, and this variant is present in a highly conserved region across zebrafish to human. Sequence analysis of EEA1 revealed that among the additional 27 familial IgAN cases, six families carried three other variants (R1262W, N1072K, and E1010G) within EEA1 with reduced penetrance. The frequencies of these EEA1 variants in familial IgAN were significantly higher than those in the Human Genetic Variation database.

g. CD2 or CD28, with their ligands on APCs. During antigen-specific T-cell activation, these surface receptors, along with intracellular signaling or scaffolding proteins, organize in supramolecular

activation clusters (SMACs) and form an immunological synapse 1, 2. Functionally, this immune synapse provides a stop signal on APCs for migrating T cells 3 and is important for enhancing, directing or terminating T-cell immunity 4. Since the immune synapse has an important function in T-cell selleck activation, sustained signaling, and effector functions 4, 5, it is important to elucidate whether clinically used immunosuppressive drugs interfere with immune synapse formation or stabilization. Glucocorticoids are commonly used immunosuppressants in organ transplantation or the treatment of dermatitis, arthritis, or inflammatory bowel disease. The immunosuppressive action of glucocorticoids is thought to be mainly based on the inhibition of cytokine expression and dependent on the regulation of cytoplasmic glucocorticoid receptors (GRs). Whether glucocorticoids influence costimulatory signals required for immune synapse formation and the dynamic actin rearrangement of untransformed

human T cells was so far unexplored. It has been known for a long time that the formation and stabilization of the immune synapse requires dynamic rearrangements of the actin cytoskeleton as well as costimulation 6, 7. We have recently shown that expression of the actin-bundling protein L-plastin is crucial for actin polymerization after antigen encounter, immune synapse maturation, Selleckchem EPZ-6438 and sustained T-cell signaling 5. L-plastin is post-translationally regulated by phosphorylation on Ser5 and this phosphorylation is induced in primary human T cells via costimulation, i.e. TCR/CD3 plus CD28 or CD2 8, 9. This phosphorylation facilitates the surface transport of activation-induced receptors like CD69

8. Furthermore, it was demonstrated by others that phosphorylated L-plastin has a higher affinity toward F-actin in HEK293T cells 10. Although it is known that Y-27632 2HCl expression of L-plastin is mandatory for the maturation of the immune synapse 5, the role of L-plastin phosphorylation on Ser5 in that process remained as yet unclear. Moreover, it was unknown whether commonly used immunosuppressive drugs influence the actin regulatory functions of L-plastin required for the formation of the immune synapse upon antigen encounter. Here, we demonstrate that phosphorylation of the actin-bundling protein L-plastin is crucial for the formation of a stable immune synapse and the increased F-actin content in superantigen-stimulated untransformed human T cells. Interestingly, the immunosuppressive drug dexamethasone interferes with L-plastin phosphorylation and T-cell functions that rely on L-plastin phosphorylation, such as actin polymerization and immune synapse formation.