Sample records for labeled anti-tcr alpha

  1. Atomic structure of an alphabeta T cell receptor (TCR) heterodimer in complex with an anti-TCR fab fragment derived from a mitogenic antibody.

    PubMed Central

    Wang, J; Lim, K; Smolyar, A; Teng, M; Liu, J; Tse, A G; Liu, J; Hussey, R E; Chishti, Y; Thomson, C T; Sweet, R M; Nathenson, S G; Chang, H C; Sacchettini, J C; Reinherz, E L

    1998-01-01

    Each T cell receptor (TCR) recognizes a peptide antigen bound to a major histocompatibility complex (MHC) molecule via a clonotypic alphabeta heterodimeric structure (Ti) non-covalently associated with the monomorphic CD3 signaling components. A crystal structure of an alphabeta TCR-anti-TCR Fab complex shows an Fab fragment derived from the H57 monoclonal antibody (mAb), interacting with the elongated FG loop of the Cbeta domain, situated beneath the Vbeta domain. This loop, along with the partially exposed ABED beta sheet of Cbeta, and glycans attached to both Cbeta and Calpha domains, forms a cavity of sufficient size to accommodate a single non-glycosylated Ig domain such as the CD3epsilon ectodomain. That this asymmetrically localized site is embedded within the rigid constant domain module has implications for the mechanism of signal transduction in both TCR and pre-TCR complexes. Furthermore, quaternary structures of TCRs vary significantly even when they bind the same MHC molecule, as manifested by a unique twisting of the V module relative to the C module. PMID:9427737

  2. A Co-Receptor Independent Transgenic Human TCR Mediates Anti-Tumor and Anti-Self Immunity in Mice

    PubMed Central

    Mehrotra, Shikhar; Al-Khami, Amir A.; Klarquist, Jared; Husain, Shahid; Naga, Osama; Eby, Jonathan M.; Murali, Anuradha K.; Lyons, Gretchen E.; Li, Mingli; Spivey, Natali D.; Norell, Håkan; Martins da Palma, Telma; Onicescu, Georgiana; Diaz-Montero, C. Marcela; Garrett-Mayer, Elizabeth; Cole, David J.; Le Poole, I. Caroline; Nishimura, Michael I.

    2013-01-01

    Recent advancements in T cell immunotherapy suggest that T cells engineered with high affinity T cell receptors (TCR) can offer better tumor regression. However, whether a high affinity TCR alone is sufficient to control tumor growth, or the T cell subset bearing the TCR is also important remains unclear. Using the human tyrosinase epitope reactive, CD8 independent, high affinity TCR isolated from MHC class-I restricted CD4+ T cells obtained from tumor infiltrating lymphocytes of a metastatic melanoma patient, we developed a novel TCR transgenic mouse with a C57BL/6 background. This HLA-A2 restricted TCR was positively selected on both CD4+ and CD8+ single-positive (SP) cells. However, when the TCR transgenic mouse was developed with an HLA-A2 background, the transgenic TCR was primarily expressed by CD3+CD4-CD8- double-negative (DN) T cells. TIL 1383I TCR transgenic CD4+, CD8+ and CD4-CD8- T cells were functional and retained the ability to control tumor growth without the need for vaccination or cytokine support in vivo. Furthermore, the HLA-A2+/human tyrosinase TCR double transgenic mice developed spontaneous hair depigmentation and had visual defects that progressed with age. Our data show that the expression of the high affinity TIL 1383I TCR alone in CD3+ T cells is sufficient to control the growth of murine and human melanoma and the presence or absence of CD4 and CD8 co-receptors had little effect on its functional capacity. PMID:22798675

  3. Characterizations of Anti-Alpha-Fetoprotein-Conjugated Magnetic Nanoparticles Associated with Alpha-Fetoprotein for Biomedical Applications.

    PubMed

    Liao, Shu-Hsien; Huang, Han-Sheng; Chieh, Jen-Jie; Su, Yu-Kai; Tong, Yuan-Fu; Huang, Kai-Wen

    2017-09-03

    In this work, we report characterizations of biofunctionalized magnetic nanoparticles (BMNPs) associated with alpha-fetoprotein (AFP) for biomedical applications. The example BMNP in this study is anti-alpha-fetoprotein (anti-AFP) conjugated onto dextran-coated Fe₃O₄ labeled as Fe₃O₄-anti-AFP, and the target is AFP. We characterize magnetic properties, such as increments of magnetization ΔM H and effective relaxation time Δτ eff in the reaction process. It is found that both ΔM H and Δτ eff are enhanced when the concentration of AFP, Ф AFP , increases. The enhancements are due to magnetic interactions among BMNPs in magnetic clusters, which contribute extra M H after the association with M H and in turn enhance τ eff . The screening of patients carrying hepatocellular carcinoma (HCC) is verified via ΔM H /M H . The proposed method can be applied to detect a wide variety of analytes. The scaling characteristics of ΔM H /M H show the potential to develop a vibrating sample magnetometer system with low field strength for clinic applications.

  4. Ikaros promotes rearrangement of TCR alpha genes in an Ikaros null thymoma cell line

    PubMed Central

    Collins, Bernard; Clambey, Eric T.; Scott-Browne, James; White, Janice; Marrack, Philippa; Hagman, James; Kappler, John W.

    2014-01-01

    Summary Ikaros is important in the development and maintenance of the lymphoid system, functioning in part by associating with chromatin-remodeling complexes. We have studied the functions of Ikaros in the transition from pre-T cell to the CD4+CD8+ thymocyte using an Ikaros null CD4−CD8− mouse thymoma cell line (JE131). We demonstrate that this cell line carries a single functional TCR β gene rearrangement and expresses a surface pre-TCR. JE131 cells also carry non-functional rearrangements on both alleles of their TCR α loci. Retroviral re-introduction of Ikaros dramatically increased the rate of transcription in the α locus and TCR Vα/Jα recombination resulting in the appearance of many new αβTCR+ cells. The process is RAG dependent, requires SWI/SNF chromatin-remodeling complexes and is coincident with the binding of Ikaros to the TCR α enhancer. Furthermore, knockdown of Mi2/NuRD complexes increased the frequency of TCR α rearrangement. Our data suggest that Ikaros controls Vα/Jα recombination in T cells by controlling access of the transcription and recombination machinery to the TCR α loci. The JE131 cell line should prove to be a very useful tool for studying the molecular details of this and other processes involved in the pre-T cell to αβTCR+ CD4+CD8+ thymocyte transition. PMID:23172374

  5. T cell receptor (TCR) structure of autologous melanoma-reactive cytotoxic T lymphocyte (CTL) clones: tumor-infiltrating lymphocytes overexpress in vivo the TCR beta chain sequence used by an HLA-A2- restricted and melanocyte-lineage-specific CTL clone

    PubMed Central

    1993-01-01

    HLA-A2+ melanomas express common melanoma-associated antigens (Ags) recognized in vitro by autologous cytotoxic T lymphocytes (CTL). However, it is not known whether tumor Ags can drive in vivo a selective accumulation/expansion of Ag-specific, tumor-infiltrating T lymphocytes (TIL). Therefore, to evaluate this possibility, 39 CTL clones isolated from several independent mixed lymphocyte tumor cultures (MLTC) of TIL and peripheral blood lymphocytes (PBL) of an HLA- A2+ melanoma patient and selected for T cell receptor (TCR)-dependent, HLA-restricted tumor lysis, were used for analysis of TCR alpha and beta chain structure by the cDNA polymerase chain reaction (PCR) technique with variable gene-specific primers followed by sequencing. Despite absence of oligoclonality in fresh TIL and PBL, as well as in T cells of day 28 MLTC (day of cloning), sequence analysis of TCR alpha and beta chains of TIL clones revealed a dominance of a major category of melanoma-specific, HLA-A2-restricted T cells expressing a V alpha 8.2/J alpha AP511/C alpha and V beta 2.1/D beta 1/J beta 1.1/C beta 1 TCR. The same TCR was also found in 2 out of 14 PBL clones. The other PBL clones employed a V alpha 2.1 gene segment associated with either V beta 13.2, 14, or w22. Clones A81 (V alpha 2.1/J alpha IGRJ alpha 04/C alpha and V beta 14/D beta 1/J beta 1.2/C beta 1) and A21 (V alpha 8.2/J alpha AP511/C alpha and V beta 2.1/D beta 1/J beta 1.1/C beta 1), representative of the two most frequent TCR of PBL and TIL, respectively, expressed different lytic patterns, but both were HLA-A2 restricted and lysed only HLA-A2+ melanomas and normal melanocytes, thus indicating recognition of two distinct HLA-A2-associated and tissue-related Ags. Finally, by the inverse PCR technique, the specific TCR beta chain (V beta 2.1/D beta 1/J beta 1.1/C beta 1) expressed by the dominant TIL clone was found to represent 19 and 18.4% of all V beta 2 sequences expressed in the fresh tumor sample and in the purified TIL

  6. Prevalence of -alpha(3.7) and alpha alpha alpha(anti3.7) alleles in sickle cell trait and beta-thalassemia patients in Mexico.

    PubMed

    Nava, María Paulina; Ibarra, Bertha; Magaña, María Teresa; de la Luz Chávez, María; Perea, F Javier

    2006-01-01

    The aim of this study was to determine the frequency of alpha-globin gene mutations in three groups of Mexican unrelated individuals. The first two groups were normal and sickle cell trait individuals from the Costa Chica region, a place with a 12.8% frequency of HbS carriers, and the third group comprised of Mexican mestizo patients with beta-thalassemia. We searched for -alpha(3.7) and -alpha(4.2) alpha(+)-thalassemia deletion alleles, as well as the alpha alpha alpha(anti3.7) triplication through long-gap PCR. The alleles -alpha(3.7) and alpha alpha alpha(anti3.7) were found in the heterozygote state only; 19% of the normal subjects had the -alpha(3.7) allele, and 2% showed the alpha alpha alpha(anti3.7) allele. In individuals with the sickle cell trait, 17% had the -alpha(3.7) deletion, and the alpha alpha alpha(anti3.7) triplication was observed in 3% of these individuals. We revealed that 16% of the subjects with beta-thalassemia showed the -alpha(3.7) deletion and 28% the alpha alpha alpha(anti3.7) triplication. The -alpha(4.2) deletion was not detected in any individual. The frequency of the -alpha(3.7) allele was roughly the same in the three groups studied; this can be explained by the fact that the three groups have common genes from Africa and the Mediterranean, where a high prevalence of alpha(+)-thalassemia has been observed. To our knowledge, the frequency of alpha alpha alpha(anti3.7) triplication observed in the Mexican beta-thalassemia patients is the highest reported. As the -alpha(3.7) and alpha alpha alpha(anti3.7) alleles are very common in our selected populations, we believe that there is a need to investigate systematically the alpha-globin gene mutations in all hemoglobinopathies in the Mexican population.

  7. The three-dimensional structure of a T-cell antigen receptor V alpha V beta heterodimer reveals a novel arrangement of the V beta domain.

    PubMed Central

    Housset, D; Mazza, G; Grégoire, C; Piras, C; Malissen, B; Fontecilla-Camps, J C

    1997-01-01

    The crystal structure of a mouse T-cell antigen receptor (TCR) Fv fragment complexed to the Fab fragment of a specific anti-clonotypic antibody has been determined to 2.6 A resolution. The polypeptide backbone of the TCR V alpha domain is very similar to those of other crystallographically determined V alphas, whereas the V beta structure is so far unique among TCR V beta domains in that it displays a switch of the c" strand from the inner to the outer beta-sheet. The beta chain variable region of this TCR antigen-binding site is characterized by a rather elongated third complementarity-determining region (CDR3beta) that packs tightly against the CDR3 loop of the alpha chain, without leaving any intervening hydrophobic pocket. Thus, the conformation of the CDR loops with the highest potential diversity distinguishes the structure of this TCR antigen-binding site from those for which crystallographic data are available. On the basis of all these results, we infer that a significant conformational change of the CDR3beta loop found in our TCR is required for binding to its cognate peptide-MHC ligand. PMID:9250664

  8. Domain-swapped T cell receptors improve the safety of TCR gene therapy

    PubMed Central

    Bethune, Michael T; Gee, Marvin H; Bunse, Mario; Lee, Mark S; Gschweng, Eric H; Pagadala, Meghana S; Zhou, Jing; Cheng, Donghui; Heath, James R; Kohn, Donald B; Kuhns, Michael S; Uckert, Wolfgang; Baltimore, David

    2016-01-01

    T cells engineered to express a tumor-specific αβ T cell receptor (TCR) mediate anti-tumor immunity. However, mispairing of the therapeutic αβ chains with endogenous αβ chains reduces therapeutic TCR surface expression and generates self-reactive TCRs. We report a general strategy to prevent TCR mispairing: swapping constant domains between the α and β chains of a therapeutic TCR. When paired, domain-swapped (ds)TCRs assemble with CD3, express on the cell surface, and mediate antigen-specific T cell responses. By contrast, dsTCR chains mispaired with endogenous chains cannot properly assemble with CD3 or signal, preventing autoimmunity. We validate this approach in cell-based assays and in a mouse model of TCR gene transfer-induced graft-versus-host disease. We also validate a related approach whereby replacement of αβ TCR domains with corresponding γδ TCR domains yields a functional TCR that does not mispair. This work enables the design of safer TCR gene therapies for cancer immunotherapy. DOI: http://dx.doi.org/10.7554/eLife.19095.001 PMID:27823582

  9. [Anti-TNF alpha in dermatology].

    PubMed

    Mahe, E; Descamps, V

    2002-12-01

    The discovery of the major role of TNF alpha in the physiopathology of certain inflammatory diseases and notably in rheumatoid arthritis and Crohn's disease has led to the development of anti-TNF alpha drugs. These new therapeutic arms issued from bio-technology have rapidly demonstrated their efficacy in the treatment of these two diseases. The anti-TNF alpha arsenal is currently dominated by etanercept, a fusion protein composed of a soluble TNF alpha receptor, and infliximab, a chimeric monoclonal antibody. However, new molecules will soon enrich this arsenal. TNF alpha is a major cytokine of inflammatory diseases of the skin. Many dermatological diseases will probably benefit from these new treatments. Two studies have already demonstrated their interest in cutaneous and articular psoriasis. Encouraging sporadic results suggest other potential indications (Behcet's disease, bullous dermatitis, neutrophilic dermatitis, toxic epidermal necrolysis, systemic vascularitis,.). These promising new treatments, although expensive, and with yet unknown long term side effects, justify rigorous assessment of their efficacy and tolerance in each indication. Here again the dermatologist has a major role to play in post-marketing pharmacovigilance.

  10. Mushroom acidic glycosphingolipid induction of cytokine secretion from murine T cells and proliferation of NK1.1 {alpha}/{beta} TCR-double positive cells in vitro

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Nozaki, Hirofumi; Itonori, Saki; Sugita, Mutsumi

    2008-08-29

    Interferon (IFN)-{gamma} and interleukin (IL)-4 regulate many types of immune responses. Here we report that acidic glycosphingolipids (AGLs) of Hypsizigus marmoreus and Pleurotus eryngii induced secretion of IFN- {gamma} and IL-4 from T cells in a CD11c-positive cell-dependent manner similar to that of {alpha}-galactosylceramide ({alpha}-GalCer) and isoglobotriaosylceramide (iGb3), although activated T cells by AGLs showed less secretion of cytokine than those activated by {alpha}-GalCer. In addition, stimulation of these mushroom AGLs induced proliferation of NK1.1 {alpha}/{beta} TCR-double positive cells in splenocytes. Administration of a mixture of {alpha}-GalCer and AGLs affected the stimulation of {alpha}-GalCer and generally induced a subtle Th1more » bias for splenocytes but induced an extreme Th2 bias for thymocytes. These results suggested that edible mushroom AGLs contribute to immunomodulation.« less

  11. Evolutionarily conserved TCR binding sites, identification of T cells in primary lymphoid tissues, and surprising trans-rearrangements in nurse shark.

    PubMed

    Criscitiello, Michael F; Ohta, Yuko; Saltis, Mark; McKinney, E Churchill; Flajnik, Martin F

    2010-06-15

    Cartilaginous fish are the oldest animals that generate RAG-based Ag receptor diversity. We have analyzed the genes and expressed transcripts of the four TCR chains for the first time in a cartilaginous fish, the nurse shark (Ginglymostoma cirratum). Northern blotting found TCR mRNA expression predominantly in lymphoid and mucosal tissues. Southern blotting suggested translocon-type loci encoding all four chains. Based on diversity of V and J segments, the expressed combinatorial diversity for gamma is similar to that of human, alpha and beta may be slightly lower, and delta diversity is the highest of any organism studied to date. Nurse shark TCRdelta have long CDR3 loops compared with the other three chains, creating binding site topologies comparable to those of mammalian TCR in basic paratope structure; additionally, nurse shark TCRdelta CDR3 are more similar to IgH CDR3 in length and heterogeneity than to other TCR chains. Most interestingly, several cDNAs were isolated that contained IgM or IgW V segments rearranged to other gene segments of TCRdelta and alpha. Finally, in situ hybridization experiments demonstrate a conservation of both alpha/beta and gamma/delta T cell localization in the thymus across 450 million years of vertebrate evolution, with gamma/delta TCR expression especially high in the subcapsular region. Collectively, these data make the first cellular identification of TCR-expressing lymphocytes in a cartilaginous fish.

  12. Analysis of the CDR3 length repertoire and the diversity of TCR alpha chain in human peripheral blood T lymphocytes.

    PubMed

    Yao, Xin Sheng; Diao, Ying; Sun, Wan Bang; Luo, Jun Min; Qin, Ming; Tang, Xian Ying

    2007-06-01

    Analysis of complementarity determining region 3 (CDR3) length of T lymphocyte receptors (TCRs) by immunoscope spectratyping technique has been used successfully to investigate the diversity of TCR in autoimmune diseases and infection diseases. In this study, we investigated the patterns of CDR3 length distribution for all 32 TCR AV gene families in human peripheral blood lymphocytes of four normal volunteers by the immunoscope spectratyping technique. It was found that PCR products exhibited an obscure band on 1.5% agarose gel electrophoresis. Each TCR AV family exhibited more than 8 bands on 6% sequencing gel electrophoresis. The CDR3 spectratyping of all TCR AV families showed a standard Gaussian distribution with different CDR3 length, and the expression frequency of CDR3 was similar among the gene families. Most of CDR3 in TCR AV family recombine in frame. However, some of the CDR3 showed out-of frame gene rearrangement. Additionally, we found that in some of TCR AV families there were 18 amino acid discrepancies between the longest CDR3 and shortest CDR3. These results may be helpful to further study the recombination mechanism of human TCR genes, the TCR CDR3 gene repertoire, and the repertoire drift in health people and disease state.

  13. Effects of anti-tumor necrosis factor-alpha and anti-intercellular adhesion molecule-1 antibodies on ischemia/reperfusion lung injury.

    PubMed

    Chiang, Chi-Huei

    2006-10-31

    Inhibition of neutrophil activation and adherence to endothelium by antibodies to tumor necrosis factor-alpha (TNF-alpha) and intercellular adhesion molecules (ICAM-1), respectively, might attenuate ischemia-reperfusion injury (I/R). I/R was conducted in an isolated rat lung model. Anti-TNF-alpha antibody and/or anti-ICAM-1 antibody were added before ischemia or after reperfusion. Hemodynamic changes, lung weight gain (LWG), capillary filtration coefficients (Kfc), and pathologic changes were assessed to evaluate the severity of I/R. The LWG, Kfc, pathological changes and lung injury score of treatment groups with anti-TNF-alpha antibody treatment, either pre-ischemia or during reperfusion, were less than those observed in control groups. Similar findings were found in group treated with anti-ICAM-1 antibody or combination therapy during reperfusion. In contrast, pre-I/R treatment with anti-ICAM-1 antibody induced severe lung edema and failure to complete the experimental procedure. No additional therapeutic effect was found in combination therapy. We conclude that TNF-alpha and ICAM-1 play important roles in I/R. Anti-TNF-alpha antibody has therapeutic and preventive effects on I/R. However, combined therapy with anti-TNF-alpha antibody and anti-ICAM-1 antibody may have no additive effect and need further investigation.

  14. Accelerated healing of skin burns by anti-Gal/alpha-gal liposomes interaction.

    PubMed

    Galili, Uri; Wigglesworth, Kim; Abdel-Motal, Ussama M

    2010-03-01

    Topical application of alpha-gal liposomes on burns results in rapid local recruitment of neutrophils and macrophages. Recruited macrophages are pivotal for healing of burns because they secrete cytokines/growth factors that induce epidermis regeneration and tissue repair. alpha-Gal liposomes have glycolipids with alpha-gal epitopes (Galalpha1-3Galbeta1-4GlcNAc-R) which bind anti-Gal, the most abundant natural antibody in humans constituting approximately 1% of immunoglobulins. Interaction of alpha-gal liposomes with anti-Gal within the fluid film formed on burns, activates complement and generates chemotactic complement cleavage peptides which effectively recruit neutrophils and macrophages. Anti-Gal IgG coating alpha-gal liposomes further binds to Fcgamma receptors on macrophages and activates them to secrete cytokines/growth factors. Efficacy of alpha-gal liposomes treatment in accelerating burn healing is demonstrated in the experimental model of alpha1,3galactosyltransferase knockout mice. These mice are the only available nonprimate mammals that can produce anti-Gal in titers similar to those in humans. Pairs of burns in mice were covered either with a spot bandage coated with 10mg alpha-gal liposomes, or with a control spot bandage coated with saline. On Day 3 post-treatment, the alpha-gal liposomes treated burns contained approximately 5-fold as many neutrophils as control burns, whereas macrophages were found only in alpha-gal liposomes treated burns. On Day 6, 50-100% of the surface area of alpha-gal liposomes treated burns were covered with regenerating epidermis (re-epithelialization), whereas almost no epidermis was found in control burns. The extensive recruitment of macrophages by anti-Gal/alpha-gal liposomes interaction was further demonstrated in vivo with polyvinyl alcohol (PVA) sponge discs containing alpha-gal liposomes, implanted subcutaneously. Since anti-Gal is abundant in all humans, it is suggested that treatment with alpha-gal liposomes

  15. Anti-inflammatory and anti-oxidative effects of alpha-lipoic acid in experimentally induced acute otitis media.

    PubMed

    Tatar, A; Korkmaz, M; Yayla, M; Gozeler, M S; Mutlu, V; Halici, Z; Uslu, H; Korkmaz, H; Selli, J

    2016-07-01

    To investigate the anti-inflammatory, anti-oxidative and tissue protective effects, as well as the potential therapeutic role, of alpha-lipoic acid in experimentally induced acute otitis media. Twenty-five guinea pigs were assigned to one of five groups: a control (non-otitis) group, and otitis-induced groups treated with saline, penicillin G, alpha-lipoic acid, or alpha-lipoic acid plus penicillin G. Tissue samples were histologically analysed, and oxidative parameters in tissue samples were measured and compared between groups. The epithelial integrity was better preserved, and histological signs of inflammation and secretory metaplasia were decreased, in all groups compared to the saline treated otitis group. In the alpha-lipoic acid plus penicillin G treated otitis group, epithelial integrity was well preserved and histological findings of inflammation were significantly decreased compared to the saline, penicillin G and alpha-lipoic acid treated otitis groups. The most favourable oxidative parameters were observed in the control group, followed by the alpha-lipoic acid plus penicillin G treated otitis group. Alpha-lipoic acid, with its antioxidant, anti-inflammatory and tissue protective properties, may decrease the clinical sequelae and morbidity associated with acute otitis media.

  16. A Jurkat 76 based triple parameter reporter system to evaluate TCR functions and adoptive T cell strategies.

    PubMed

    Rosskopf, Sandra; Leitner, Judith; Paster, Wolfgang; Morton, Laura T; Hagedoorn, Renate S; Steinberger, Peter; Heemskerk, Mirjam H M

    2018-04-03

    Adoptive T cell therapy using TCR transgenic autologous T cells has shown great potential for the treatment of tumor patients. Thorough characterization of genetically reprogrammed T cells is necessary to optimize treatment success. Here, we describe the generation of triple parameter reporter T cells based on the Jurkat 76 T cell line for the evaluation of TCR and chimeric antigen receptor functions as well as adoptive T cell strategies. This Jurkat subline is devoid of endogenous TCR alpha and TCR beta chains, thereby circumventing the problem of TCR miss-pairing and unexpected specificities. The resultant reporter cells allow simultaneous determination of the activity of the transcription factors NF-κB, NFAT and AP-1 that play key roles in T cell activation. Human TCRs directed against tumor and virus antigens were introduced and reporter responses were determined using tumor cell lines endogenously expressing the antigens of interest or via addition of antigenic peptides. Finally, we demonstrate that coexpression of adhesion molecules like CD2 and CD226 as well as CD28 chimeric receptors represents an effective strategy to augment the response of TCR-transgenic reporters to cells presenting cognate antigens.

  17. In vitro and in vivo comparison of binding of 99m-Tc-labeled anti-CEA MAb F33-104 with 99m-Tc-labeled anti-CEA MAb BW431/26.

    PubMed

    Watanabe, N; Oriuchi, N; Sugiyama, S; Kuroki, M; Matsuoka, Y; Tanada, S; Murata, H; Inoue, T; Sasaki, Y

    1999-01-01

    The purpose of this study was to assess the potential for radio-immunodetection (RAID) of murine anti-carcinoembryonic antigen (CEA) monoclonal antibody (MAb) F33-104 labeled with technetium-99m (99m-Tc) by a reduction-mediated labeling method. The binding capacity of 99m-Tc-labeled anti-CEA MAb F33-104 with CEA by means of in vitro procedures such as immunoradiometric assay and cell binding assay and the biodistribution of 99m-Tc-labeled anti-CEA MAb F33-104 in normal nude mice and nude mice bearing human colon adenocarcinoma LS180 tumor were investigated and compared with 99m-Tc-labeled anti-CEA MAb BW431/26. The in vitro binding rate of 99m-Tc-labeled anti-CEA MAb F33-104 with CEA in solution and attached to the cell membrane was significantly higher than 99m-Tc-labeled anti-CEA MAb BW431/261 (31.4 +/- 0.95% vs. 11.9 +/- 0.55% at 100 ng/mL of soluble CEA, 83.5 +/- 2.84% vs. 54.0 +/- 2.54% at 10(7) of LS 180 cells). In vivo, accumulation of 99m-Tc-labeled anti-CEA MAb F33-104 was higher at 18 h postinjection than 99m-Tc-labeled anti-CEA MAb BW431/26 (20.1 +/- 3.50% ID/g vs. 14.4 +/- 3.30% ID/g). 99m-Tc-activity in the kidneys of nude mice bearing tumor was higher at 18 h postinjection than at 3 h (12.8 +/- 2.10% ID/g vs. 8.01 +/- 2.40% ID/g of 99m-Tc-labeled anti-CEA MAb F33-104, 10.7 +/- 1.70% ID/g vs. 8.10 +/- 1.75% ID/g of 99m-Tc-labeled anti-CEA MAb BW431/26). 99m-Tc-labeled anti-CEA MAb F33-104 is a potential novel agent for RAID of recurrent colorectal cancer.

  18. Evolution and Function of the TCR Vgamma9 Chain Repertoire: It’s Good to be Public

    PubMed Central

    Pauza, C. David; Cairo, Cristiana

    2015-01-01

    Lymphocytes expressing a T cell receptor (TCR) composed of Vgamma9 and Vdelta2 chains represent a minor fraction of human thymocytes. Extrathymic selection throughout post-natal life causes the proportion of cells with a Vgamma9-JP rearrangement to increase and elevates the capacity for responding to non-peptidic phosphoantigens. Extrathymic selection is so powerful that phosphoantigen-reactive cells comprise about 1 in 40 circulating memory T cells from healthy adults and the subset can be expanded rapidly upon infection or in response to malignancy. Skewing of the gamma delta TCR repertoire is accompanied by selection for public gamma chain sequences such that many unrelated individuals overlap extensive in their circulating repertoire. This type of selection implies the presence of a monomorphic antigen-presenting molecule that is an object of current research but remains incompletely defined. While selection on a monomorphic presenting molecule may seem unusual, similar mechanisms shape the alpha beta T cell repertoire including the extreme examples of NKT or mucosal-associated invariant T cells (MAIT) and the less dramatic amplification of public Vbeta chain rearrangements driven by individual MHC molecules and associated with resistance to viral pathogens. Selecting and amplifying public T cell receptors whether alpha beta or gamma delta, are important steps in developing an anticipatory TCR repertoire. Cell clones expressing public TCR can accelerate the kinetics of response to pathogens and impact host survival. PMID:25769734

  19. Cerebrovascular events in inflammatory bowel disease patients treated with anti-tumour necrosis factor alpha agents.

    PubMed

    Karmiris, Konstantinos; Bossuyt, Peter; Sorrentino, Dario; Moreels, Tom; Scarcelli, Antonella; Legido, Jesus; Dotan, Iris; Naismith, Graham D; Jussila, Airi; Preiss, Jan C; Kruis, Wolfgang; Li, Andy C Y; Bouguen, Guillaume; Yanai, Henit; Steinwurz, Flavio; Katsanos, Konstantinos H; Subramaniam, Kavitha; Tarabar, Dino; Zaganas, Ioannis V; Ben-Horin, Shomron

    2015-05-01

    Cerebrovascular accidents [CVA] have rarely been reported in inflammatory bowel disease [IBD] patients treated with anti-tumour necrosis alpha [anti-TNF alpha] agents. Our aim here was to describe the clinical course of CVA in these patients. This was a European Crohn's and Colitis Organisation [ECCO] retrospective observational study, performed as part of the CONFER [COllaborative Network For Exceptionally Rare case reports] project. A call to all ECCO members was made to report on IBD patients afflicted with CVA during treatment with anti-TNF alpha agents. Clinical data were recorded in a standardised case report form and analysed for event association with anti-TNF alpha treatment. A total of 19 patients were identified from 16 centres: 14 had Crohn's disease, four ulcerative colitis and one IBD colitis unclassified [median age at diagnosis: 38.0 years, range: 18.6-62.5]. Patients received anti-TNF alpha for a median duration of 11.8 months [range: 0-62] at CVA onset; seven had previously been treated with at least one other anti-TNF alpha agent. Complete neurological recovery was observed in 16 patients. Anti-TNF alpha was discontinued in 16/19 patients. However, recurrent CVA or neurological deterioration was not observed in any of the 11 patients who received anti-TNF alpha after CVA [eight resumed after temporary cessation, three continued without interruption] for a median follow-up of 39.8 months [range: 5.6-98.2]. These preliminary findings do not unequivocally indicate a causal role of anti-TNF alpha in CVA complicating IBD. Resuming or continuing anti-TNF alpha in IBD patients with CVA may be feasible and safe in selected cases, but careful weighing of IBD activity versus neurological status is prudent. Copyright © 2015 European Crohn’s and Colitis Organisation (ECCO). Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  20. Off-label use of TNF-alpha inhibitors in a dermatological university department: retrospective evaluation of 118 patients.

    PubMed

    Sand, Freja Lærke; Thomsen, Simon Francis

    2015-01-01

    Tumor necrosis factor-alpha (TNF)-alpha inhibitors are licensed for patients with severe refractory psoriasis and psoriatic arthritis. However, TNF-alpha inhibitors have also been used off-label for various recalcitrant mucocutaneous diseases. This study aimed to evaluate the efficacy and safety of TNF-alpha inhibitors used for off-label dermatological indications. We retrospectively evaluated patient records of 118 patients treated off-label with TNF-alpha inhibitors in a dermatological university department. Patients presented with severe aphthous stomatitis/genital aphthous lesions (26), chronic urticaria (25), hidradenitis suppurativa (29), acne conglobata (11), dissecting cellulitis of the scalp (two), orofacial granulomatosis (four), sarcoidosis (four), granuloma annulare (two), granulomatous rosacea (one), granuloma faciale (one), subcorneal pustulosis (one), pyoderma gangrenosum (four), Sweet's syndrome (four), Well's syndrome (one), benign familial pemphigus (one), lichen planus (one), and folliculitis decalvans (one). A significant number of these patients went into remission during therapy with TNF-alpha inhibitors. A total of 11 patients (9%) experienced severe adverse effects during therapy. Off-label therapy with TNF-alpha inhibitors may be considered for selected patients with severe recalcitrant mucocutaneous diseases. The risk of severe adverse effects signals that a thorough benefit-risk assessment should be performed before initiating off-label treatment with TNF-alpha inhibitors for these conditions. © 2015 Wiley Periodicals, Inc.

  1. Labeled ALPHA4BETA2 ligands and methods therefor

    DOEpatents

    Mukherjee, Jogeshwar; Pichika, Ramaiah; Potkin, Steven; Leslie, Frances; Chattopadhyay, Sankha

    2013-02-19

    Contemplated compositions and methods are employed to bind in vitro and in vivo to an .alpha.4.beta.2 nicotinic acetylcholine receptor in a highly selective manner. Where such compounds are labeled, compositions and methods employing such compounds can be used for PET and SPECT analysis. Alternatively, and/or additionally contemplated compounds can be used as antagonists, partial agonists or agonists in the treatment of diseases or conditions associated with .alpha.4.beta..beta.2 dysfunction.

  2. Cutting edge: rescue of pre-TCR but not mature TCR signaling in mice expressing membrane-targeted SLP-76.

    PubMed

    Bezman, Natalie A; Baker, Rebecca G; Lenox, Laurie E; Jordan, Martha S; Koretzky, Gary A

    2009-05-01

    SLP-76 (Src homology 2 domain-containing leukocyte phosphoprotein of 76 kDa) organizes signaling from immunoreceptors, including the platelet collagen receptor, the pre-TCR, and the TCR, and is required for T cell development. In this study we examine a mouse in which wild-type SLP-76 is replaced with a mutant constitutively targeted to the cell membrane. Membrane-targeted SLP-76 (MTS) supports ITAM signaling in platelets and from the pre-TCR. Signaling from the mature TCR, however, is defective in MTS thymocytes, resulting in failed T cell differentiation. Defective thymic selection by MTS is not rescued by a SLP-76 mutant whose localization is restricted to the cytosol. Thus, fixed localization of SLP-76 reveals differential requirements for the subcellular localization of signaling complexes downstream of the pre-TCR vs mature TCR.

  3. Gold nanoparticle labeling with tyramide signal amplification for highly sensitive detection of alpha fetoprotein in human serum by ICP-MS.

    PubMed

    Li, Xiaoting; Chen, Beibei; He, Man; Xiao, Guangyang; Hu, Bin

    2018-01-01

    In this work, we developed an immunoassay based on tyramide signal amplification (TSA) and gold nanoparticles (Au NPs) labeling for highly sensitive detection of alpha fetoprotein (AFP) by inductively coupled plasma mass spectrometry (ICP-MS). AFP was captured by anti-AFP1 coating on the 96-well plate and labeled by anti-AFP2-horseradish peroxidase (HRP), in which the HRP can catalyze the deposition of biotinylated tyramine on the nearby protein. Then the streptavidin (SA)-Au NPs was labeled on the deposited biotinylated tyramine as the intensive signal probe for ICP-MS measurement. Under the optimal experimental conditions, the limit of detection of the developed method for AFP was 1.85pg/mL and the linear range was 0.005-2ng/mL. The relative standard deviation for seven replicate detections of 0.01ng/mL AFP was 5.2%. The proposed method was successfully applied to the detection of AFP in human serum with good recoveries. This strategy is highly sensitive and easy to operate, and can be extended to the sensitive detection of other biomolecules in human serum. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Increasing functional avidity of TCR-redirected T cells by removing defined N-glycosylation sites in the TCR constant domain

    PubMed Central

    Hauptrock, Beate; Malina, Victoria; Antunes, Edite; Voss, Ralf-Holger; Wolfl, Matthias; Strong, Roland; Theobald, Matthias; Greenberg, Philip D.

    2009-01-01

    Adoptive transfer of T lymphocytes transduced with a T cell receptor (TCR) to impart tumor reactivity has been reported as a potential strategy to redirect immune responses to target cancer cells (Schumacher, T.N. 2002. Nat. Rev. Immunol. 2:512–519). However, the affinity of most TCRs specific for shared tumor antigens that can be isolated is usually low. Thus, strategies to increase the affinity of TCRs or the functional avidity of TCR-transduced T cells might be therapeutically beneficial. Because glycosylation affects the flexibility, movement, and interactions of surface molecules, we tested if selectively removing conserved N-glycoslyation sites in the constant regions of TCR α or β chains could increase the functional avidity of T cells transduced with such modified TCRs. We observed enhanced functional avidity and improved recognition of tumor cells by T cells harboring TCR chains with reduced N-glycosylation (ΔTCR) as compared with T cells with wild-type (WT) TCR chains. T cells transduced with WT or ΔTCR chains bound tetramer equivalently at 4°C, but tetramer binding was enhanced at 37°C, predominantly as a result of reduced tetramer dissociation. This suggested a temperature-dependent mechanism such as TCR movement in the cell surface or structural changes of the TCR allowing improved multimerization. This strategy was effective with mouse and human TCRs specific for different antigens and, thus, should be readily translated to TCRs with any specificity. PMID:19171765

  5. TCR tuning of T cell subsets.

    PubMed

    Cho, Jae-Ho; Sprent, Jonathan

    2018-05-01

    After selection in the thymus, the post-thymic T cell compartments comprise heterogenous subsets of naive and memory T cells that make continuous T cell receptor (TCR) contact with self-ligands bound to major histocompatibility complex (MHC) molecules. T cell recognition of self-MHC ligands elicits covert TCR signaling and is particularly important for controlling survival of naive T cells. Such tonic TCR signaling is tightly controlled and maintains the cells in a quiescent state to avoid autoimmunity. Here, we review how naive and memory T cells are differentially tuned and wired for TCR sensitivity to self and foreign ligands. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  6. TCR repertoires of intratumoral T-cell subsets.

    PubMed

    Linnemann, Carsten; Mezzadra, Riccardo; Schumacher, Ton N M

    2014-01-01

    The infiltration of human tumors by T cells is a common phenomenon, and over the past decades, it has become increasingly clear that the nature of such intratumoral T-cell populations can predict disease course. Furthermore, intratumoral T cells have been utilized therapeutically in clinical studies of adoptive T-cell therapy. In this review, we describe how novel methods that are either based on T-cell receptor (TCR) sequencing or on cancer exome analysis allow the analysis of the tumor reactivity and antigen-specificity of the intratumoral TCR repertoire with unprecedented detail. Furthermore, we discuss studies that have started to utilize these techniques to probe the link between cancer exomes and the intratumoral TCR pool. Based on the observation that both the cancer epitope repertoire and intratumoral TCR repertoire appear highly individual, we outline strategies, such as 'autologous TCR gene therapy', that exploit the tumor-resident TCR repertoire for the development of personalized immunotherapy. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  7. Biodistributions, Myelosuppression, and Toxicities in Mice Treated with an Anti-CD45 Antibody Labeled with the alpha-Emitting Radionuclides Bismuth-213 or Astatine-211

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Nakamae, Hirohisa; Wilbur, D. Scott; Hamlin, Donald K.

    2009-03-15

    We previously investigated 213Bi-labeled anti-CD45 antibody to replace total body irradiation as conditioning for hematopoietic cell transplantation in a canine model. While this allowed sustained engraftment of marrow, limited availability and high cost of 213Bi led to a preliminary investigation in mice of 211At-labeled antibody for the same application. To gain an understanding of the differences between the two radionuclides, biodistribution and myelosuppression/toxicity studies were conducted with 213Bi- and 211At-labeled rat anti-murine CD45 antibody, 30F11, conjugates. After injecting mice with 2-50 μCi on 10 μg 30F11 conjugate or 20 μCi on 2 or 40 μg conjugate, biodistributions, myelosuppression and non-hematologicalmore » toxicities were evaluated. Biodistribution studies showed that the spleen had the highest concentration of radioactivity, ranging from167-417 % injected dose/gram (%ID/g) at 24 h after injection in the 211At studies and 45-166 %ID/g at 3 h after injection in the 213Bi studies. The higher concentrations observed for 211At-labeled 30F11 was likely due to its longer half-life which, permitted more localization of antibody to the spleen before decay. 211At was more effective at myelosuppression for the same (mCi) quantity of injected radioactivity. Injection of only 20 or 50 μCi 211At resulted in lethal myeloablation. There was severe reversible acute hepatic toxicity with 50 μCi 213Bi, but not with lower doses or any dose of 211At. No significant renal toxicity occurred with either radionuclide. The data suggested that considerably lower quantities of 211At-labeled anti-CD45 antibody than 213Bi-labeled antibody might be effective for myelosuppression.« less

  8. T-cell synapse formation depends on antigen recognition but not CD3 interaction: studies with TCR:ζ, a candidate transgene for TCR gene therapy.

    PubMed

    Roszik, János; Sebestyén, Zsolt; Govers, Coen; Guri, Yakir; Szöor, Arpád; Pályi-Krekk, Zsuzsanna; Vereb, György; Nagy, Peter; Szöllosi, János; Debets, Reno

    2011-05-01

    T-cell receptors (TCRs) can be genetically modified to improve gene-engineered T-cell responses, a strategy considered critical for the success of clinical TCR gene therapy to treat cancers. TCR:ζ, which is a heterodimer of TCRα and β chains each coupled to complete human CD3ζ, overcomes issues of mis-pairing with endogenous TCR chains, shows high surface expression and mediates antigen-specific T-cell functions in vitro. In the current study, we further characterized TCR:ζ in gene-engineered T cells and assessed whether this receptor is able to interact with surface molecules and drive correct synapse formation in Jurkat T cells. The results showed that TCR:ζ mediates the formation of synaptic areas with antigen-positive target cells, interacts closely with CD8α and MHC class I (MHCI), and co-localizes with CD28, CD45 and lipid rafts, similar to WT TCR. TCR:ζ did not closely associate with endogenous CD3ε, despite its co-presence in immune synapses, and TCR:ζ showed enhanced synaptic accumulation in T cells negative for surface-expressed TCR molecules. Notably, synaptic TCR:ζ demonstrated lowered densities when compared with TCR in dual TCR T cells, a phenomenon that was related to both extracellular and intracellular CD3ζ domains present in the TCR:ζ molecule and responsible for enlarged synapse areas. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Modulation of TCRβ surface expression during TCR revision.

    PubMed

    Simmons, Kalynn B; Wubeshet, Maramawit; Ames, Kristina T; McMahan, Catherine J; Hale, J Scott; Fink, Pamela J

    2012-01-01

    TCR revision is a tolerance mechanism by which self-reactive TCRs expressed by mature CD4(+) peripheral T cells are replaced by receptors encoded by genes generated by post-thymic DNA rearrangement. The downmodulation of surface TCR expression initiates TCR revision, and serves as a likely trigger for the induction of the recombinase machinery. We show here in a Vβ5 transgenic mouse model system that downregulation of the self-reactive transgene-encoded TCR is not maintained by transgene loss or diminished transcription or translation. The downregulation of surface TCR expression likely occurs in two stages, only one of which requires tolerogen expression. Copyright © 2011 Elsevier Inc. All rights reserved.

  10. Optimization of T-cell Reactivity by Exploiting TCR Chain Centricity for the Purpose of Safe and Effective Antitumor TCR Gene Therapy.

    PubMed

    Ochi, Toshiki; Nakatsugawa, Munehide; Chamoto, Kenji; Tanaka, Shinya; Yamashita, Yuki; Guo, Tingxi; Fujiwara, Hiroshi; Yasukawa, Masaki; Butler, Marcus O; Hirano, Naoto

    2015-09-01

    Adoptive transfer of T cells redirected by a high-affinity antitumor T-cell receptor (TCR) is a promising treatment modality for cancer patients. Safety and efficacy depend on the selection of a TCR that induces minimal toxicity and elicits sufficient antitumor reactivity. Many, if not all, TCRs possess cross-reactivity to unrelated MHC molecules in addition to reactivity to target self-MHC/peptide complexes. Some TCRs display chain centricity, in which recognition of MHC/peptide complexes is dominated by one of the TCR hemi-chains. In this study, we comprehensively studied how TCR chain centricity affects reactivity to target self-MHC/peptide complexes and alloreactivity using the TCR, clone TAK1, which is specific for human leukocyte antigen-A*24:02/Wilms tumor 1(235-243) (A24/WT1(235)) and cross-reactive with B*57:01 (B57). The TAK1β, but not the TAK1α, hemi-chain possessed chain centricity. When paired with multiple clonotypic TCRα counter-chains encoding TRAV12-2, 20, 36, or 38-2, the de novo TAK1β-containing TCRs showed enhanced, weakened, or absent reactivity to A24/WT1(235) and/or to B57. T cells reconstituted with these TCRα genes along with TAK1β possessed a very broad range (>3 log orders) of functional and structural avidities. These results suggest that TCR chain centricity can be exploited to enhance desired antitumor TCR reactivity and eliminate unwanted TCR cross-reactivity. TCR reactivity to target MHC/peptide complexes and cross-reactivity to unrelated MHC molecules are not inextricably linked and are separable at the TCR sequence level. However, it is still mandatory to carefully monitor for possible harmful toxicities caused by adoptive transfer of T cells redirected by thymically unselected TCRs. ©2015 American Association for Cancer Research.

  11. Src-like adaptor protein regulates TCR expression on thymocytes by linking the ubiquitin ligase c-Cbl to the TCR complex.

    PubMed

    Myers, Margaret D; Sosinowski, Tomasz; Dragone, Leonard L; White, Carmen; Band, Hamid; Gu, Hua; Weiss, Arthur

    2006-01-01

    The adaptor molecule SLAP and E3 ubiquitin ligase c-Cbl each regulate expression of T cell receptor (TCR)-CD3 on thymocytes. Here we provide genetic and biochemical evidence that both molecules function in the same pathway. TCR-CD3 expression was similar in the absence of SLAP and/or c-Cbl. SLAP and c-Cbl were found to interact, and their expression together downregulated CD3epsilon. This required multiple domains in SLAP and the ring finger of c-Cbl. Furthermore, expression of SLAP and c-Cbl together induced TCRzeta ubiquitination and degradation, preventing the accumulation of fully assembled recycling TCR complexes. These studies indicate that SLAP links the E3 ligase activity of c-Cbl to the TCR, allowing for stage-specific regulation of TCR expression.

  12. Immunoassay of serum polypeptide hormones by using 125I-labelled anti(-immunoglobulin G) antibodies.

    PubMed

    Beck, P; Nicholas, H

    1975-03-01

    1. A technique for indirectly labelling antibodies to polypeptide hormones, by combining them with radioactively labelled anti-(immunoglobulin G) is described. (a) 125I-labelled anti-(rabbit immunoglobulin G) and anti-(guinea-pig immunoglobulin G) antibodies with high specific radioactivity were prepared after purification of the antibodies on immunoadsorbents containing the respective antigens. (b) Rabbit immunoglobulin G antibodies to human growth hormone, porcine glucagon and guinea-pig immunoglobulin G antibodies to bovine insulin and bovine parathyroid hormone were combined with immunoadsorbents containing the respective polypeptide hormone antigen. (c) The immunoglobulin G antibodies to the polypeptide hormones were reacted with 125-I-labelled anti-(immunoglobulin G) antibodies directed against the appropriate species of immunoglobulin G,and the anti-hormone antibodies were combined with the hormone-containing immunoadsorbent. (d) 125I-labelled anti-(immunoglobulin G) antibodies and anti-hormone antibodies were simultaneously eluted from the hormone-containing immunoadsorbent by dilute HCl, pH 2.0. After elution the anti-(immunoglobulin G) antibodies and antihormone antibodies were allowed to recombine at pH 8.0 and 4 degrees C. 2. The resultant immunoglobulin G-anti-immunoglobulin G complex was used in immunoradiometric (labelled antibody) and two-site assays of the respective polypeptide hormone. 3. By using these immunoassays, concentrations down to 90pg of human growth hormone/ml, 100 pg of bovine insulin/ml, 80 pg of bovine parathyroid hormone/ml and 150 pg of glucagon/ml were readily detected. Assays of human plasma for growth hormone and insulin by these methods showed good agreement with results obtained by using a directly 125I-labelled anti-hormone antibody in an immunoradiometric assay of human growth hormone or by radioimmunoassay of human insulin. 4. The method described allows immunoradiometric or two-site assays to be performed starting with as

  13. Integrin alpha 3 beta 1 participates in the phagocytosis of extracellular matrix molecules by human breast cancer cells.

    PubMed

    Coopman, P J; Thomas, D M; Gehlsen, K R; Mueller, S C

    1996-11-01

    The mechanisms and receptors involved in phagocytosis by nonhematopoietic cells are not well understood. The involvement of the alpha 3 beta 1 integrin in phagocytosis of the extracellular matrix by human breast cancer cells was studied. The possible role of this integrin was suggested since alpha 3 and beta 1 but not alpha 2 subunits are concentrated at membrane sites where local degradation of fluorescently labeled gelatin occurs. Strikingly, anti-alpha 3 integrin monoclonal antibodies (mAbs) stimulate the phagocytosis of fluorescently labeled gelatin films, gelatin beads, and Matrigel films in a quantitative phagocytosis assay. Stimulation of the gelatin uptake by the anti-alpha 3 mAb is dose responsive, saturable, and time dependent. Antibodies against other integrin subunits have a lower stimulatory effect (anti-beta 1) or no significant effect (anti-alpha 2, -alpha 5, -alpha 6, and -alpha v) on gelatin phagocytosis. The synthetic HGD-6 human laminin peptide that binds specifically the alpha 3 beta 1 integrin, but not the scrambled HSGD-6 control peptide, also markedly stimulates gelatin uptake in a dose-responsive way. Furthermore, the stimulatory effects of the HGD-6 peptide and the anti-alpha 3 mAb are additive, suggesting that they might promote phagocytosis in different ways. Other laminin (YIGSR, IKVAV) and fibronectin (GRGDS) peptides have no effect on gelatin phagocytosis. Immunofluorescence shows that the alpha 3 and the beta 1, but not the alpha 2 integrin subunit, concentrate into patches on the cell surface after treatment with their respective mAbs. And, both gelatin and the alpha 3 beta 1 but not the alpha 2 beta 1 integrin are cointernalized and routed to acidic vesicles such as lysosomes. In conclusion, we demonstrate that human breast cancer cells locally degrade and phagocytose the extracellular matrix and show for the first time that the alpha 3 beta 1 integrin participates in this phagocytosis. We hypothesize that the anti-alpha 3

  14. Activation of human naïve Th cells increases surface expression of GD3 and induces neoexpression of GD2 that colocalize with TCR clusters.

    PubMed

    Villanueva-Cabello, Tania M; Mollicone, Rosella; Cruz-Muñoz, Mario E; López-Guerrero, Delia V; Martínez-Duncker, Iván

    2015-12-01

    CD4+ T helper lymphocytes (Th) orchestrate the immune response after their activation by antigen-presenting cells. Activation of naïve Th cells is reported to generate the reduction in surface epitopes of sialic acid (Sia) in α2,3 and α2,6 linkages. In this work, we report that in spite of this glycophenotype, anti-CD3/anti-CD28-activated purified human naïve Th cells show a significant increase in surface Sia, as assessed by metabolic labeling, compared with resting naïve Th cells, suggesting an increased flux of Sia toward Siaα2,8 glycoconjugates. To understand this increase as a result of ganglioside up-regulation, we observed that very early after activation, human naïve Th cells show an increased expression in surface GD3 and neoexpression of surface GD2 gangliosides, the latter clustering with the T cell receptor (TCR). Also, we report that in contrast to GM2/GD2 synthase null mice, lentiviral vector-mediated silencing of the GM2/GD2 synthase in activated human naïve Th cells reduced efficient TCR clustering and downstream signaling, as assessed by proliferation assays and IL-2 and IL-2R expression, pointing to an important role of this enzyme in activation of human naive Th cells. © The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  15. T-cell receptor V sub. alpha. and C sub. alpha. alleles associated with multiple sclerosis and myasthenia gravis

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Oksenberg, J.R.; Cavalli-Sforza, L.L.; Steinman, L.

    1989-02-01

    Polymorphic markers in genes encoding the {alpha} chain of the human T-cell receptor (TcR) have been detected by Southern blot analysis in Pss I digests. Polymorphic bands were observed at 6.3 and 2.0 kilobases (kb) with frequencies of 0.30 and 0.44, respectively, in the general population. Using the polymerase chain reaction (PCR) method, the authors amplified selected sequences derived from the full-length TcR {alpha} cDNA probe. These PcR products were used as specific probes to demonstrate that the 6.3-kb polymorphic fragment hybridizes to the variable (V)-region probe and the 2.0-kb fragment hybridizes to the constant (C)-region probe. Segregation of themore » polymorphic bands was analyzed in family studies. To look for associations between these markers and autoimmune diseases, the authors have studied the restriction fragment length polymorphism distribution of the Pss I markers in patients with multiple sclerosis, myasthenia gravis, and Graves disease. Significant differences in the frequency of the polymorphic V{sub {alpha}} and C{sub {alpha}} markers were identified between patients and healthy individuals.« less

  16. Differences in TCR-Vβ Repertoire and Effector Phenotype between Tumor Infiltrating Lymphocytes and Peripheral Blood Lymphocytes Increase with Age

    PubMed Central

    Wang, Teng; Shen, Han; Wu, Fenglin; Zhang, Wenfeng; Tao, Changli; Yuan, Yin; Bo, Huaben; Wang, Hui; Huang, Shulin

    2014-01-01

    Tumor infiltrating lymphocytes (TIL) reflect the host's anti-tumor immune response, and can be a valuable predictor of prognosis. However, many properties of TIL are not fully understood. In the present study, TCR-Vβ repertoires of cancer patients were primarily analyzed by flow cytometry. Abnormally expressed TCR-Vβ subfamilies were generally found in both TIL and peripheral blood lymphocytes (PBL) of each patient. Of note, increased patient age was associated with increasingly biased TCR-Vβ repertoire in TIL but not in PBL, and the dispersion degree of the differences of TCR-Vβ subfamilies between TIL and PBL correlated positively with age (P = 0.007). Utilizing immunoscope analysis, we identified the age-related reduction in TCR-Vβ diversity, but polyclonal pattern was predominant in significantly expanded TCR-Vβ subfamilies. In addition, we found that older patients possessed a decreased ratio of CD8+CD62L+ non-effector cells in TIL compared to PBL, implying age-related increase of CD8+CD62L− effector cells in TIL. The colocalization analysis of CD8 and CD3, however, suggested the suppressed activity of these effector cells in tumor microenvironment. These findings further elucidate the properties of TIL, showing an increasing difference between TIL and PBL with age, which may provide insight for the development of effective immunotherapies for cancer patients of different ages. PMID:25019226

  17. CRISPR-mediated TCR replacement generates superior anticancer transgenic T cells.

    PubMed

    Legut, Mateusz; Dolton, Garry; Mian, Afsar Ali; Ottmann, Oliver G; Sewell, Andrew K

    2018-01-18

    Adoptive transfer of T cells genetically modified to express a cancer-specific T-cell receptor (TCR) has shown significant therapeutic potential for both hematological and solid tumors. However, a major issue of transducing T cells with a transgenic TCR is the preexisting expression of TCRs in the recipient cells. These endogenous TCRs compete with the transgenic TCR for surface expression and allow mixed dimer formation. Mixed dimers, formed by mispairing between the endogenous and transgenic TCRs, may harbor autoreactive specificities. To circumvent these problems, we designed a system where the endogenous TCR-β is knocked out from the recipient cells using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 (Cas9) technology, simultaneously with transduction with a cancer-reactive receptor of choice. This TCR replacement strategy resulted in markedly increased surface expression of transgenic αβ and γδ TCRs, which in turn translated to a stronger, and more polyfunctional, response of engineered T cells to their target cancer cell lines. Additionally, the TCR-plus-CRISPR-modified T cells were up to a thousandfold more sensitive to antigen than standard TCR-transduced T cells or conventional model proxy systems used for studying TCR activity. Finally, transduction with a pan-cancer-reactive γδ TCR used in conjunction with CRISPR/Cas9 knockout of the endogenous αβ TCR resulted in more efficient redirection of CD4 + and CD8 + T cells against a panel of established blood cancers and primary, patient-derived B-cell acute lymphoblastic leukemia blasts compared with standard TCR transfer. Our results suggest that TCR transfer combined with genome editing could lead to new, improved generations of cancer immunotherapies. © 2018 by The American Society of Hematology.

  18. Human common acute lymphoblastic leukemia-derived cell lines are competent to recombine their T-cell receptor delta/alpha regions along a hierarchically ordered pathway.

    PubMed

    Hansen-Hagge, T E; Yokota, S; Reuter, H J; Schwarz, K; Bartram, C R

    1992-11-01

    Rearrangements of the T-cell receptor (TCR) delta locus are observed in the majority of human B-cell precursor acute lymphoblastic leukemias (ALL) with a striking predominance of V delta 2(D)D delta 3 recombinations in common ALL (cALL) patients. Recently, we and others showed that almost 20% of cALL cases are characterized by further recombination of V delta 2(D)D delta 3 segments to J alpha elements, thereby deleting the TCR delta locus in analogy to the delta Rec/psi J alpha pathway in differentiating alpha/beta-positive T cells. We report here that two human cALL-derived cell lines, REH and Nalm-6, are competent to recombine the TCR delta/alpha locus under standard tissue culture conditions. Analysis of different REH subclones obtained by limiting dilution of the initial culture showed a biased recombination of V delta 2D delta 3 to distinct J alpha elements. During prolonged tissue culture, a subclone acquired growth advantage and displaced parental cells as well as other subclones. Frequently, the DJ junctions of REH subclones contained extended stretches of palindromic sequences derived from modified D delta 3 coding elements. The other cell line, Nalm-6, started the TCR delta/alpha recombination with an unusual signal joint of a cryptic recombinase signal sequence (RSS) upstream of D delta 3 to the 3' RSS of D delta 3. The RSS dimer was subsequently rearranged in all investigated subclones to an identical J alpha element. Both cell lines might become valuable tools to unravel the complex regulation of TCR delta/alpha recombination pathways in malignant and normal lymphopoiesis.

  19. Differential TCR signals for T helper cell programming.

    PubMed

    Morel, Penelope A

    2018-05-02

    Upon encounter with their cognate antigen naïve CD4 T cells become activated and are induced to differentiate into several possible T helper (Th) cell subsets. This differentiation depends on a number of factors including antigen presenting cells, cytokines and costimulatory molecules. The strength of the T cell receptor (TCR) signal, related to the affinity of TCR for antigen and antigen dose, has emerged as a dominant factor in determining Th cell fate. Recent studies have revealed that TCR signals of high or low strength do not simply induce quantitatively different signals in the T cells, but rather qualitatively distinct pathways can be induced based on TCR signal strength. This review examines the recent literature in this area and highlights important new developments in our understanding of Th cell differentiation and TCR signal strength. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  20. Organization of the resting TCR in nanoscale oligomers.

    PubMed

    Schamel, Wolfgang W A; Alarcón, Balbino

    2013-01-01

    Despite the low affinity of the T-cell antigen receptor (TCR) for its peptide/major histocompatibility complex (pMHC) ligand, T cells are very sensitive to their antigens. This paradox can be resolved if we consider that the TCR may be organized into pre-existing oligomers or nanoclusters. Such structures could improve antigen recognition by increasing the functional affinity (avidity) of the TCR-pMHC interaction and by allowing cooperativity between individual TCRs. Up to approximately 20 TCRs become tightly apposed in these nanoclusters, often in a linear manner, and such structures could reflect a relatively generalized phenomenon: the non-random concentration of membrane receptors in specific areas of the plasma membrane known as protein islands. The association of TCRs into nanoclusters can explain the enhanced kinetics of the pMHC-TCR interaction in two dimensional versus three dimensional systems, but also their existence calls for a revision of the TCR triggering models based on pMHC-induced TCR clustering. Interestingly, the B-cell receptor and the FcεRI have also been shown to form nanoclusters, suggesting that the formation of pre-existing receptor oligomers could be widely used in the immune system. © 2012 John Wiley & Sons A/S. Published by Blackwell Publishing Ltd.

  1. Structure of the superantigen staphylococcal enterotoxin B in complex with TCR and peptide-MHC demonstrates absence of TCR-peptide contacts.

    PubMed

    Rödström, Karin E J; Elbing, Karin; Lindkvist-Petersson, Karin

    2014-08-15

    Superantigens are immune-stimulatory toxins produced by Staphylococcus aureus, which are able to interact with host immune receptors to induce a massive release of cytokines, causing toxic shock syndrome and possibly death. In this article, we present the x-ray structure of staphylococcal enterotoxin B (SEB) in complex with its receptors, the TCR and MHC class II, forming a ternary complex. The structure, in combination with functional analyses, clearly shows how SEB adopts a wedge-like position when binding to the β-chain of TCR, allowing for an interaction between the α-chain of TCR and MHC. Furthermore, the binding mode also circumvents contact between TCR and the peptide presented by MHC, which enables SEB to initiate a peptide-independent activation of T cells. Copyright © 2014 by The American Association of Immunologists, Inc.

  2. Application of four anti-human interferon-alpha monoclonal antibodies for immunoassay and comparative analysis of natural interferon-alpha mixtures

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Andersson, G.; Lundgren, E.; Ekre, H.P.

    Four different mouse monoclonal antibodies to human interferon-alpha (IFN-alpha) were evaluated for application in quantitative and comparative analysis of natural IFN-alpha mixtures. Binding to IFN-alpha subtypes in solution revealed individual reactivity patterns. These patterns changed if the IFN-alpha molecules were immobilized either passively to a surface or bound by another antibody. Also, substitution of a single amino acid in IFN-alpha 2 affected the binding, apparently by altering the conformation. Isoelectric focusing of three natural IFN-alpha preparations from different sources, followed by immunoblotting, resulted in individual patterns with each of the four mAbs and also demonstrated variation in the composition ofmore » the IFN-alpha preparations. None of the mAbs was subtype specific, but by combining the different mAbs, and also applying polyclonal anti-human IFN-alpha antibodies, it was possible to design sensitive sandwich ELISAs with broad or more limited IFN-alpha subtype specificity.« less

  3. Rag Deletion in Peripheral T Cells Blocks TCR Revision

    PubMed Central

    Hale, J. Scott; Ames, Kristina T.; Boursalian, Tamar E.; Fink, Pamela J.

    2010-01-01

    Mature CD4+Vβ5+ T cells that recognize a peripherally expressed endogenous superantigen are tolerized either by deletion or T cell receptor (TCR) revision. In Vβ5 transgenic mice, this latter tolerance pathway results in the appearance of CD4+Vβ5−TCRβ+ T cells, coinciding with Rag1, Rag2, and TdT expression and the accumulation of Vβ-DJβ recombination intermediates in peripheral CD4+ T cells. Because post-thymic RAG-dependent TCR rearrangement has remained controversial, we sought to definitively determine whether TCR revision is an extrathymic process that occurs in mature peripheral T cells. We now show that Rag deletion in post-positive selection T cells in Vβ5 transgenic mice blocks TCR revision in vivo, and that mature peripheral T cells sorted to remove cells bearing endogenous TCRβ chains can express newly generated TCRβ molecules in adoptive hosts. These findings unambiguously demonstrate post-thymic, RAG-dependent TCR rearrangement and define TCR revision as a tolerance pathway that targets mature peripheral CD4+ T cells. PMID:20435935

  4. The anti-apoptotic activity associated with phosphatidylinositol transfer protein alpha activates the MAPK and Akt/PKB pathway.

    PubMed

    Schenning, Martijn; Goedhart, Joachim; Gadella, Theodorus W J; Avram, Diana; Wirtz, Karel W A; Snoek, Gerry T

    2008-10-01

    The conditioned medium (CM) from mouse NIH3T3 fibroblast cells overexpressing phosphatidylinositol transfer protein alpha (PI-TPalpha; SPIalpha cells) demonstrates an increased anti-apoptotic activity compared with CM from wild type NIH3T3 (wtNIH3T3) cells. As previously shown, the anti-apoptotic activity acts by activating a G protein-coupled receptor, most probably a cannabinoid 1 (CB1)-like receptor as the activity was blocked by both pertussis toxin and rimonabant [M. Schenning, C.M. van Tiel, D. Van Manen, J.C. Stam, B.M. Gadella, K.W. Wirtz and G.T. Snoek, Phosphatidylinositol transfer protein alpha regulates growth and apoptosis of NIH3T3 cells: involvement of a cannabinoid 1-like receptor, J. Lipid Res. 45 (2004) 1555-1564]. The CB1 receptor appears to be expressed in mouse fibroblast cells, at levels in the order SPIalpha>wtNIH3T3>SPIbeta cells (i.e. wild type cells overexpressing PI-TPbeta). Upon incubation of SPIbeta cells with the PI-TPalpha-dependent anti-apoptotic factors, both the ERK/MAP kinase and the Akt/PKB pathway are activated in a CB1 receptor dependent manner as shown by Western blotting. In addition, activation of ERK2 was also shown by EYFP-ERK2 translocation to the nucleus, as visualized by confocal laser scanning microscopy. The subsequent activation of the anti-apoptotic transcription factor NF-kappaB is in line with the increased resistance towards UV-induced apoptosis. On the other hand, receptor activation by CM from SPIalpha cells was not linked to phospholipase C activation as the YFP-labelled C2-domain of protein kinase C was not translocated to the plasma membrane of SPIbeta cells as visualized by confocal laser scanning microscopy.

  5. Evaluation of GABAergic neuroactive steroid 3alpha-hydroxy-5alpha-pregnane-20-one as a neurobiological substrate for the anti-anxiety effect of ethanol in rats.

    PubMed

    Hirani, Khemraj; Sharma, Ajay N; Jain, Nishant S; Ugale, Rajesh R; Chopde, Chandrabhan T

    2005-07-01

    Acute systemic ethanol administration is known to elevate plasma and cerebral levels of neuroactive steroid 3alpha-hydroxy-5alpha-pregnane-20-one (3alpha, 5alpha-THP; allopregnanolone) to a concentration sufficient to potentiate GABA(A) receptors. We have earlier demonstrated that 3alpha, 5alpha-THP mediates the antidepressant-like effect of ethanol in Porsolt forced swim test. The aim of the present study is to explain the relationship between endogenous GABAergic neurosteroids and anxiolytic effect of ethanol in Sprague-Dawley rats. The mediation of 3alpha, 5alpha-THP in the anti-anxiety effect of ethanol was assessed by pharmacological interactions of ethanol with various endogenous neurosteroidal modulators and using simulated physiological conditions of altered neurosteroid content in elevated plus maze (EPM) test. Pretreatment of 3alpha, 5alpha-THP (0.5-2.5 mug/rat, i.c.v.) or neurosteroidogenic agents such as 3alpha, 5alpha-THP precursor progesterone (5 or 10 mg/kg, i.p.), 11-beta hydroxylase inhibitor metyrapone (50 or 100 mg/kg, i.p.) or the GABA(A) receptor agonist muscimol (25 ng/rat, i.c.v.) significantly potentiated the anti-anxiety effect of ethanol (1 g/kg, i.p.). On the other hand, the GABAergic antagonistic neurosteroid dehydroepiandrosterone sulphate (DHEAS) (1 mg/kg, i.p.), the GABA(A) receptor blocker bicuculline (1 mg/kg, i.p.), the 5alpha-reductase inhibitor finasteride (50 x 2 mg/kg, s.c.) or the mitochondrial diazepam binding inhibitory receptor antagonist PK11195 (1 mg/kg, i.p.) reduced ethanol-induced preference of time spent and number of entries into open arms. Anti-anxiety effect of ethanol was abolished in adrenalectomized (ADX) rats as compared to sham-operated control. This ADX-induced blockade was restored by prior systemic injection of progesterone, signifying the contribution of peripheral steroidogenesis in ethanol anxiolysis. Socially isolated animals known to exhibit decreased brain 3alpha, 5alpha-THP and GABA(A) receptor

  6. Specific lysine labeling by 18OH- during alkaline cleavage of the alpha-1-antitrypsin-trypsin complex.

    PubMed Central

    Cohen, A B; Gruenke, L D; Craig, J C; Geczy, D

    1977-01-01

    alpha-1-Antitrypsin is a serum protein that inhibits many proteolytic enzymes. Recently, it was suggested that the alpha-1-antitrypsin-trypsin complex is an acyl ester analogous to the acyl intermediate that forms between trypsin and its substrates. In previous work we showed that the alpha-1-antitrypsin-trypsin complex can be split at high pH, releasing a component of alpha-1-antitrypsin. This component had a new carboxyl-terminal lysine, and it had lost a peptide of about 4000 daltons. In order to determine whether the alpha-1-antitrypsin is bound to trypsin through the new carboxy-terminal lysine, as would be expected if the above hypothesis is correct, we split the complex in the presence of 18OH-. When the new carboxy-terminal lysine was cleaved with carboxypeptidase B, singly labeled, doubly labeled, and unlabeled lysine were recovered. These data support the hypothesis that the alpha-1-antitrypsin-trypsin complex is an acyl ester or a tetrahedral precursor that is transformed into the acyl ester form at high pH. If other enzymes are bound by a similar mechanism, the methods used may be useful in determining which amino acids on alpha-1-antitrypsin bind covalently to each enzyme. PMID:303770

  7. Anti-inflammatory activity of Pistacia lentiscus essential oil: involvement of IL-6 and TNF-alpha.

    PubMed

    Maxia, Andrea; Sanna, Cinzia; Frau, Maria Assunta; Piras, Alessandra; Karchuli, Manvendra Singh; Kasture, Veena

    2011-10-01

    The topical anti-inflammatory activity of essential oil of Pistacia lentiscus L. was studied using carrageenan induced rat paw edema and cotton pellet induced granuloma. The effect on serum tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) in rats inserted with cotton pellet was also investigated. On topical application, the oil exhibited a significant decrease in paw edema. The oil also inhibited cotton pellet-induced granuloma, and reduced serum TNF-alpha and IL-6. It can be concluded that the essential oil of Pistacia lentiscus reduces leukocyte migration to the damaged tissue and exhibits anti-inflammatory activity.

  8. Bismuth 213-labeled anti-CD45 radioimmunoconjugate to condition dogs for nonmyeloablative allogeneic marrow grafts

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Sandmaier, B M.; Bethge, W A.; Wilbur, D. Scott

    To lower treatment-related mortality and toxicity of conventional marrow transplantation, a nonmyeloablative regimen using 200 cGy total-body irradiation (TBI) and mycophenolate mofetil (MMF) combined with cyclosporine (CSP) for postgrafting immunosuppression was developed. To circumvent possible toxic effects of external- beam gamma irradiation, strategies for targeted radiation therapy were investigated. We tested whether the short-lived (46 minutes) alpha-emitter Bi-213 conjugated to an anti-CD45 monoclonal antibody (mAb) could replace 200 cGy TBI and selectively target hematopoietic tissues in a canine model of nonmyeloablative DLA-identical marrow transplantation. Biodistribution studies using iodine 123-labeled anti-CD45 mAb showed uptake in blood, marrow, lymph nodes, spleen, andmore » liver. In a dose-escalation study, 7 dogs treated with the Bi-213-anti-CD45 conjugate (Bi-213 dose, 0.1-5.9 mCi/kg[3.7-218 MBq/kg]) without marrow grafts had no toxic effects other than a mild, reversible suppression of blood counts. On the basis of these studies, 3 dogs were treated with 0.5 mg/kg Bi-213-labeled anti-CD45 mAb (Bi-213 doses, 3.6, 4.6, and 8.8 mCi/kg[133, 170, and 326 MBq/kg]) given in 6 injections 3 and 2 days before grafting of marrow from DLA-identical littermates. The dogs also received MMF (10 mg/kg subcutaneously twice daily the day of transplantation until day 27 afterward) and CSP (15 mg/kg orally twice daily the day before transplantation until 35 days afterward). Therapy was well tolerated except for transient elevations in levels of transaminases in 3 dogs, followed by, in one dog, ascites. All dogs achieved prompt engraftment and stable mixed hematopoietic chimerism, with donor contributions ranging from 30% to 70% after more than 27 weeks of follow-up. These results form the basis for additional studies in animals and the design of clinical trials using Bi-213 as a nonmyeloablative conditioning regimen with minimal toxicity.« less

  9. Generation of TCR-Expressing Innate Lymphoid-like Helper Cells that Induce Cytotoxic T Cell-Mediated Anti-leukemic Cell Response.

    PubMed

    Ueda, Norihiro; Uemura, Yasushi; Zhang, Rong; Kitayama, Shuichi; Iriguchi, Shoichi; Kawai, Yohei; Yasui, Yutaka; Tatsumi, Minako; Ueda, Tatsuki; Liu, Tian-Yi; Mizoro, Yasutaka; Okada, Chihiro; Watanabe, Akira; Nakanishi, Mahito; Senju, Satoru; Nishimura, Yasuharu; Kuzushima, Kiyotaka; Kiyoi, Hitoshi; Naoe, Tomoki; Kaneko, Shin

    2018-06-05

    CD4 + T helper (Th) cell activation is essential for inducing cytotoxic T lymphocyte (CTL) responses against malignancy. We reprogrammed a Th clone specific for chronic myelogenous leukemia (CML)-derived b3a2 peptide to pluripotency and re-differentiated the cells into original TCR-expressing T-lineage cells (iPS-T cells) with gene expression patterns resembling those of group 1 innate lymphoid cells. CD4 gene transduction into iPS-T cells enhanced b3a2 peptide-specific responses via b3a2 peptide-specific TCR. iPS-T cells upregulated CD40 ligand (CD40L) expression in response to interleukin-2 and interleukin-15. In the presence of Wilms tumor 1 (WT1) peptide, antigen-specific dendritic cells (DCs) conditioned by CD4-modified CD40L high iPS-T cells stimulated WT1-specific CTL priming, which eliminated WT1 peptide-expressing CML cells in vitro and in vivo. Thus, CD4 modification of CD40L high iPS-T cells generates innate lymphoid helper-like cells inducing bcr-abl-specific TCR signaling that mediates effectiveanti-leukemic CTL responses via DC maturation, showing potential for adjuvant immunotherapy against leukemia. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

  10. Biodistribution of Yttrium-90-Labeled Anti-CD45 Antibody in a Nonhuman Primate Model

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Nemecek, Eneida; Hamlin, Donald K.; Fisher, Darrell R.

    2005-01-15

    Radioimmunotherapy may improve the outcome of hematopoietic cell transplantation for hematologic malignancies by delivering targeted radiation to hematopoietic organs while relatively sparing nontarget organs. We evaluated the organ localization of yttrium-90-labeled anti-CD45 (90Y-anti-CD45) antibody in macaques, a model that had previously predicted iodine-131-labeled anti-CD-45 (131I-anti-CD45) antibody biodistribution in humans. Experimental Design: Twelve Macaca nemestrina primates received anti-CD45 antibody labeled with 1 to 2 mCi of 90Y followed by serial blood sampling and marrow and lymph node biopsies, and necropsy. The content of 90Y per gram of tissue was determined by liquid scintillation spectrometry. Time-activity curves were constructed using average isotopemore » concentrations in each tissue at measured time points to yield the fractional residence time and estimate radiation absorbed doses for each organ per unit of administered activity. The biodistribution of 90Y-anti-CD45 antibody was then compared with that previously obtained with 131I-anti-CD45 antibody in macaques. Results: The spleen received 2,120, marrow 1,060, and lymph nodes 315 cGy/mCi of 90Y injected. The liver and lungs were the nontarget organs receiving the highest radiation absorbed doses (440 and 285 cGy/mCi, respectively). Ytrrium-90-labeled anti-CD45 antibody delivered 2.5- and 3.7-fold more radiation to marrow than to liver and lungs, respectively. The ratios previously observed with 131I-antiCD45 antibody were 2.5-and 2.2-fold more radiation to marrow than to liver and lungs, respectively. Conclusions: This study shows that 90Y-anti-CD45 antibody can deliver relatively selective radiation to hematopoietic tissues, with similar ratios of radiation delivered to target versus nontarget organs, as compared with the 131I immunoconjugate in the same animal model.« less

  11. High-throughput identification of antigen-specific TCRs by TCR gene capture.

    PubMed

    Linnemann, Carsten; Heemskerk, Bianca; Kvistborg, Pia; Kluin, Roelof J C; Bolotin, Dmitriy A; Chen, Xiaojing; Bresser, Kaspar; Nieuwland, Marja; Schotte, Remko; Michels, Samira; Gomez-Eerland, Raquel; Jahn, Lorenz; Hombrink, Pleun; Legrand, Nicolas; Shu, Chengyi Jenny; Mamedov, Ilgar Z; Velds, Arno; Blank, Christian U; Haanen, John B A G; Turchaninova, Maria A; Kerkhoven, Ron M; Spits, Hergen; Hadrup, Sine Reker; Heemskerk, Mirjam H M; Blankenstein, Thomas; Chudakov, Dmitriy M; Bendle, Gavin M; Schumacher, Ton N M

    2013-11-01

    The transfer of T cell receptor (TCR) genes into patient T cells is a promising approach for the treatment of both viral infections and cancer. Although efficient methods exist to identify antibodies for the treatment of these diseases, comparable strategies to identify TCRs have been lacking. We have developed a high-throughput DNA-based strategy to identify TCR sequences by the capture and sequencing of genomic DNA fragments encoding the TCR genes. We establish the value of this approach by assembling a large library of cancer germline tumor antigen-reactive TCRs. Furthermore, by exploiting the quantitative nature of TCR gene capture, we show the feasibility of identifying antigen-specific TCRs in oligoclonal T cell populations from either human material or TCR-humanized mice. Finally, we demonstrate the ability to identify tumor-reactive TCRs within intratumoral T cell subsets without knowledge of antigen specificities, which may be the first step toward the development of autologous TCR gene therapy to target patient-specific neoantigens in human cancer.

  12. delta 9-(16 alpha-/sup 125/I)iodo-19-nortestosterone: a gamma-emitting photoaffinity label for the progesterone receptor

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lamb, D.J.; Bullock, D.W.; Hoyte, R.M.

    1988-05-01

    We have synthesized 16 alpha-iodo-4,9-estradien-17 beta-ol-3-one (delta 9-16 alpha-iodo-19-nortestosterone (delta 9-INT)) labeled with 125I (delta 9-(16 alpha-125I)INT) to provide a new gamma-emitting photoaffinity ligand for the progesterone receptor that has many advantages over the currently available (3H)R5020. We have characterized the interaction of delta 9-(16 alpha-125I)INT with the rabbit uterine progesterone receptor and have demonstrated the usefulness of this compound for studies of receptor structure. The binding of 2 nM (3H)progesterone to receptor in rabbit uterine cytosol was specifically competed for by 19-nortestosterone, 16 alpha-iodo-19-nortestosterone, and delta 9-INT. Scatchard analysis demonstrated that delta 9-(16 alpha-125I)INT and (3H)progesterone estimated the samemore » number of binding sites in rabbit uterine cytosol, with a Kd for delta 9-(16 alpha-125I)INT of about 2.7 nM. The binding of delta 9-(16 alpha-125I)INT was inhibited by both progesterone and R5020, whereas testosterone, estradiol, and 5 alpha-dihydrotestosterone were ineffective. In cytosol, delta 9-(16 alpha-125I)INT covalently labeled the same mol wt receptor forms as (3H)R5020. Although the efficiency of cross-linking was similar for (3H)R5020 (3%) and delta 9-(16 alpha-125I)INT (4%), the radioactivity was 10-fold greater due to the higher specific activity of delta 9-(16 alpha-125I)INT and the lack of sample quench. The use of delta 9-(16 alpha-125I)INT greatly increases the sensitivity and efficiency of the photoaffinity labeling technique; it will provide a valuable tool for further studies of the progesterone receptor, allowing the detection of receptor in dilute cytosol after gel electrophoresis under denaturing conditions.« less

  13. Alpha-Melanocyte Stimulating Hormone: An Emerging Anti-Inflammatory Antimicrobial Peptide

    PubMed Central

    Singh, Madhuri; Mukhopadhyay, Kasturi

    2014-01-01

    The alpha-melanocyte stimulating hormone (α-MSH) is a neuropeptide belonging to the melanocortin family. It is well known for its anti-inflammatory and antipyretic effects and shares several characteristics with antimicrobial peptides (AMPs). There have been some recent reports about the direct antimicrobial activity of α-MSH against various microbes belonging to both fungal and bacterial pathogens. Similar to α-MSH's anti-inflammatory properties, its C-terminal residues also exhibit antimicrobial activity parallel to that of the entire peptide. This review is focused on the current findings regarding the direct antimicrobial potential and immunomodulatory mechanism of α-MSH and its C-terminal fragments, with particular emphasis on the prospects of α-MSH based peptides as a strong anti-infective agent. PMID:25140322

  14. Anti-alpha interferon immunization: safety and immunogenicity in asymptomatic HIV positive patients at high risk of disease progression.

    PubMed

    Gringeri, A; Santagostino, E; Mannucci, P M; Siracusano, L; Marinoni, A; Criscuolo, M; Carcagno, M; Fall, L S; M'Bika, J P; Bizzini, B

    1995-05-01

    A randomized, placebo-controlled trial was designed to evaluate safety and immunogenicity of an anti-cytokine vaccine in high risk HIV-positive patients. This strategy was aimed to modulate the impaired cytokine regulation in AIDS. Twelve asymptomatic patients on antiretroviral therapy for at least 1 year and with CD4 cell counts between 100-300/mm3 were randomized to receive adjuvanted formol-inactivated interferon alpha-2a (IFN alpha) and continue the current antiretroviral treatment, whatever it was, or to receive the adjuvant alone and the current antiretroviral treatment. All patients received 4 i.m. injections monthly, followed by booster injections every 3 months. Clinical status, immunology and virology were monitored. Immune response to vaccination was evaluated in term of antibody detection (ELISA) and serum anti-IFN alpha neutralizing capacity. Only local discomfort and transient fever were reported. All vaccines except one showed increased levels of anti-IFN alpha Abs and developed serum IFN alpha neutralizing capacity. Viral load did not increase in vaccinees while it remained unchanged or even increased in placebo-treated patients. None of them showed HIV-related symptoms and all had their CD4 cell counts stabilized over 18 months, whereas 2 placebo-treated patients developed full-blow AIDS. In conclusion, anti-IFN alpha vaccine was safe and immunogenic. Stable clinical and immunological status over 18 months was observed in vaccinees coupled to increased serum IFN alpha neutralizing capacity.

  15. Normalized Synergy Predicts That CD8 Co-Receptor Contribution to T Cell Receptor (TCR) and pMHC Binding Decreases As TCR Affinity Increases in Human Viral-Specific T Cells

    PubMed Central

    Williams, Chad M.; Schonnesen, Alexandra A.; Zhang, Shu-Qi; Ma, Ke-Yue; He, Chenfeng; Yamamoto, Tori; Eckhardt, S. Gail; Klebanoff, Christopher A.; Jiang, Ning

    2017-01-01

    The discovery of naturally occurring T cell receptors (TCRs) that confer specific, high-affinity recognition of pathogen and cancer-associated antigens remains a major goal in cellular immunotherapies. The contribution of the CD8 co-receptor to the interaction between the TCR and peptide-bound major histocompatibility complex (pMHC) has previously been correlated with the activation and responsiveness of CD8+ T cells. However, these studies have been limited to model systems of genetically engineered hybridoma TCRs or transgenic mouse TCRs against either a single epitope or an array of altered peptide ligands. CD8 contribution in a native human antigen-specific T cell response remains elusive. Here, using Hepatitis C Virus-specific precursor CTLs spanning a large range of TCR affinities, we discovered that the functional responsiveness of any given TCR correlated with the contribution of CD8 to TCR/pMHC binding. Furthermore, we found that CD8 contribution to TCR/pMHC binding in the two-dimensional (2D) system was more accurately reflected by normalized synergy (CD8 cooperation normalized by total TCR/pMHC bonds) rather than synergy (total CD8 cooperation) alone. While synergy showed an increasing trend with TCR affinity, normalized synergy was demonstrated to decrease with the increase of TCR affinity. Critically, normalized synergy was shown to correlate with CTL functionality and peptide sensitivity, corroborating three-dimensional (3D) analysis of CD8 contribution with respect to TCR affinity. In addition, we identified TCRs that were independent of CD8 for TCR/pMHC binding. Our results resolve the current discrepancy between 2D and 3D analysis on CD8 contribution to TCR/pMHC binding, and demonstrate that naturally occurring high-affinity TCRs are more capable of CD8-independent interactions that yield greater functional responsiveness even with CD8 blocking. Taken together, our data suggest that addition of the normalized synergy parameter to our previously

  16. Inverted repeats in the promoter as an autoregulatory sequence for TcrX in Mycobacterium tuberculosis

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Bhattacharya, Monolekha; Das, Amit Kumar, E-mail: amitk@hijli.iitkgp.ernet.in

    Highlights: Black-Right-Pointing-Pointer The regulatory sequences recognized by TcrX have been identified. Black-Right-Pointing-Pointer The regulatory region comprises of inverted repeats segregated by 30 bp region. Black-Right-Pointing-Pointer The mode of binding of TcrX with regulatory sequence is unique. Black-Right-Pointing-Pointer In silico TcrX-DNA docked model binds one of the inverted repeats. Black-Right-Pointing-Pointer Both phosphorylated and unphosphorylated TcrX binds regulatory sequence in vitro. -- Abstract: TcrY, a histidine kinase, and TcrX, a response regulator, constitute a two-component system in Mycobacterium tuberculosis. tcrX, which is expressed during iron scarcity, is instrumental in the survival of iron-dependent M. tuberculosis. However, the regulator of tcrX/Y has notmore » been fully characterized. Crosslinking studies of TcrX reveal that it can form oligomers in vitro. Electrophoretic mobility shift assays (EMSAs) show that TcrX recognizes two regions in the promoter that are comprised of inverted repeats separated by {approx}30 bp. The dimeric in silico model of TcrX predicts binding to one of these inverted repeat regions. Site-directed mutagenesis and radioactive phosphorylation indicate that D54 of TcrX is phosphorylated by H256 of TcrY. However, phosphorylated and unphosphorylated TcrX bind the regulatory sequence with equal efficiency, which was shown with an EMSA using the D54A TcrX mutant.« less

  17. Cutting Edge: Rag deletion in peripheral T cells blocks TCR revision.

    PubMed

    Hale, J Scott; Ames, Kristina T; Boursalian, Tamar E; Fink, Pamela J

    2010-06-01

    Mature CD4(+)Vbeta5(+) T cells that recognize a peripherally expressed endogenous superantigen are tolerized either by deletion or TCR revision. In Vbeta5 transgenic mice, this latter tolerance pathway results in the appearance of CD4(+)Vbeta5(-)TCRbeta(+) T cells, coinciding with Rag1, Rag2, and TdT expression and the accumulation of V(beta)-DJ(beta) recombination intermediates in peripheral CD4(+) T cells. Because postthymic RAG-dependent TCR rearrangement has remained controversial, we sought to definitively determine whether TCR revision is an extrathymic process that occurs in mature peripheral T cells. We show in this study that Rag deletion in post-positive selection T cells in Vbeta5 transgenic mice blocks TCR revision in vivo and that mature peripheral T cells sorted to remove cells bearing endogenous TCRbeta-chains can express newly generated TCRbeta molecules in adoptive hosts. These findings unambiguously demonstrate postthymic, RAG-dependent TCR rearrangement and define TCR revision as a tolerance pathway that targets mature peripheral CD4(+) T cells.

  18. Suppression of lethal autoimmunity by regulatory T cells with a single TCR specificity

    PubMed Central

    Hemmers, Saskia; Schizas, Michail; Faire, Mehlika B.; Konopacki, Catherine; Schmidt-Supprian, Marc; Germain, Ronald N.

    2017-01-01

    The regulatory T cell (T reg cell) T cell receptor (TCR) repertoire is highly diverse and skewed toward recognition of self-antigens. TCR expression by T reg cells is continuously required for maintenance of immune tolerance and for a major part of their characteristic gene expression signature; however, it remains unknown to what degree diverse TCR-mediated interactions with cognate self-antigens are required for these processes. In this study, by experimentally switching the T reg cell TCR repertoire to a single T reg cell TCR, we demonstrate that T reg cell function and gene expression can be partially uncoupled from TCR diversity. An induced switch of the T reg cell TCR repertoire to a random repertoire also preserved, albeit to a limited degree, the ability to suppress lymphadenopathy and T helper cell type 2 activation. At the same time, these perturbations of the T reg cell TCR repertoire led to marked immune cell activation, tissue inflammation, and an ultimately severe autoimmunity, indicating the importance of diversity and specificity for optimal T reg cell function. PMID:28130403

  19. The mechanism of chromosome 7 inversion in human lymphocytes expressing chimeric gamma beta TCR.

    PubMed

    Retière, C; Halary, F; Peyrat, M A; Le Deist, F; Bonneville, M; Hallet, M M

    1999-01-15

    Functional chimeric TCR chains, encoded by V gamma J gamma C beta or V gamma J beta C beta hybrid gene TCR, are expressed at the surface of a small fraction of alpha beta T lymphocytes in healthy individuals. Their frequency is dramatically increased in patients with ataxia-telangiectasia, a syndrome associated with inherited genomic instability. As the TCR gamma and beta loci are in an inverted orientation on chromosome 7, the generation of such hybrid genes requires at least an inversion event. Until now, neither the sequences involved in this genetic mechanism nor the number of recombinations leading to the formation of functional transcriptional units have been characterized. In this manuscript, we demonstrate that at least two rearrangements, involving classical recombination signal sequence and the V(D)J recombinase complex, lead to the formation of productive hybrid genes. A primary inversion 7 event between D beta and J gamma genic segments generates C gamma V beta and C beta V gamma hybrid loci. Within the C gamma V beta locus, secondary rearrangements between V gamma and J gamma or V gamma and J beta elements generate functional genes. Besides, our results suggest that secondary rearrangements were blocked in the C beta V gamma locus of normal but not ataxia-telangiectasia T lymphocytes. We also provide formal evidence that the same D beta-3' recombination signal sequence can be used in successive rearrangements with J gamma and J beta genic segments, thus showing that a signal joint has been involved in a secondary recombination event.

  20. Vedolizumab is an effective alternative in inflammatory bowel disease patients with anti-TNF-alpha therapy-induced dermatological side effects.

    PubMed

    Pijls, Philippe A R R; Gilissen, Lennard P L

    2016-11-01

    The treatment of patients with inflammatory bowel diseases has been revolutionized by the introduction of biological therapy with TNF-alpha blockers. However, TNF-alpha blockers are also associated with a wide variety of dermatological side effects, such as local skin infections, psoriasis and eczema. A new biological therapy, targeting the gut-specific adhesion molecule alpha4beta7 integrin, is the humanized monoclonal IgG1 antibody vedolizumab. Vedolizumab prevents leukocyte migration to the gastrointestinal tract, thereby reducing inflammation. This gut-specific therapy has the potential to reduce systemic side effects, including dermatological ones. We describe 3 inflammatory bowel disease patients who experience anti-TNF-alpha therapy-induced dermatological side effects, consisting of hidradenitis suppurativa, a folliculitis, scalp psoriasis and a dissecting folliculitis. In all patients, anti-TNF-alpha therapy-induced dermatological side effects diminished after switching to vedolizumab. Vedolizumab may be a viable alternative biological therapy in inflammatory bowel disease patients who experience anti-TNF-alpha therapy-induced dermatological side effects. Copyright © 2016 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.

  1. PU.1 regulates TCR expression by modulating GATA-3 activity

    PubMed Central

    Chang, Hua-Chen; Han, Ling; Jabeen, Rukhsana; Carotta, Sebastian; Nutt, Stephen L.; Kaplan, Mark H.

    2009-01-01

    The Ets transcription factor PU.1 is a master regulator for the development of multiple lineages during hematopoiesis. The expression pattern of PU.1 is dynamically regulated during early T lineage development in the thymus. We previously revealed that PU.1 delineates heterogeneity of effector Th2 populations. In this study, we further define the function of PU.1 on the Th2 phenotype using mice that specifically lack PU.1 in T cells using an lck-Cre transgene with a conditional Sfpi1 allele (Sfpi1lck-/-). While deletion of PU.1 by the lck-Cre transgene does not affect T cell development, Sfpi1lck-/- T cells have a lower activation threshold than wild type T cells. When TCR engagement is limiting, Sfpi1lck-/- T cells cultured in Th2 polarizing conditions secrete higher levels of Th2 cytokines and have greater cytokine homogeneity than wild type cells. We show that PU.1 modulates the levels of TCR expression in CD4+ T cells by regulating the DNA-binding activity of GATA-3 and limiting GATA-3 regulation of TCR gene expression. GATA-3 dependent regulation of TCR expression is also observed in Th1 and Th2 cells. In CD4+ T cells, PU.1 expression segregates into subpopulations of cells that have lower levels of surface TCR, suggesting that PU.1 contributes to the heterogeneity of TCR expression. Thus, we have identified a mechanism whereby increased GATA-3 function in the absence of the antagonizing activity of PU.1 leads to increased TCR expression, a reduced activation threshold and increased homogeneity in Th2 populations. PMID:19801513

  2. Alpha-v Integrin Targeted PET Imaging of Breast Cancer Angiogenesis and Low-Dose Metronomic Anti-Angiogenic Chemotherapy Efficacy

    DTIC Science & Technology

    2008-08-01

    AD_________________ Award Number: W81XWH-04-1-0697 TITLE: Alpha -v Integrin Targeted PET Imaging of...SUBTITLE 5a. CONTRACT NUMBER Alpha -v Integrin Targeted PET Imaging of Breast Cancer Angiogenesis and Low- Dose Metronomic Anti-Angiogenic...Evaluation of biodistribution and anti-tumor effect of a dimeric RGD peptide-paclitaxel conjugate in mice with breast cancer” was published in Eur J Nucl

  3. Alpha-tocopherol disappearance rates from plasma depend on lipid concentrations: Studies using deuterium labeled collard greens in younger and older adults

    USDA-ARS?s Scientific Manuscript database

    Little is known about alpha-tocopherol's bioavailability as a constituent of food or its dependence on a subject's age. To evaluate the alpha-tocopherol bioavailability from food, we used collard greens grown in deuterated water (2H collard greens) as a source of deuterium-labeled (2H) alpha-tocophe...

  4. Risk of Lymphoma in Patients With Inflammatory Bowel Disease Treated With Anti-Tumor Necrosis Factor Alpha Agents: A Systematic Review and Meta-analysis.

    PubMed

    Yang, Chen; Huang, Junlin; Huang, Xiaowen; Huang, Shaozhuo; Cheng, Jiaxin; Liao, Weixin; Chen, Xuewen; Wang, Xueyi; Dai, Shixue

    2018-05-12

    The association between anti-tumor necrosis factor alpha agents and the risk of lymphoma in patients with inflammatory bowel disease has already been sufficiently reported. However, the results of these studies are inconsistent. Hence, this analysis was conducted to investigate whether anti-tumor necrosis factor alpha agents can increase the risk of lymphoma in inflammatory bowel disease patients. MEDLINE, EMBASE and the Cochrane Library were searched to identify relevant studies which evaluated the risk of lymphoma in inflammatory bowel disease patients treated with anti-tumor necrosis factor alpha agents. A random-effects meta-analysis was performed to calculate the pooled incidence rate ratios as well as risk ratios. Twelve studies comprising 285811 participants were included. The result showed that there was no significantly increased risk of lymphoma between anti-tumor necrosis factor alpha agents exposed and anti-tumor necrosis factor alpha agents unexposed groups (random effects: incidence rate ratio [IRR], 1.43 95%CI, 0.91-2.25, p= 0.116; random effects: risk ratio [RR], 0.83 95%CI, 0.47-1.48, p=0.534). However, monotherapy of anti-tumor necrosis factor alpha agents (random effects: IRR=1.65, 95%CI, 1.16-2.35; p=0.006; random effects: RR=1.00, 95%CI, 0.39-2.59; p=0.996) or combination therapy (random effects: IRR=3.36, 95%CI, 2.23-5.05; p< 0.001; random effects: RR=1.90, 95%CI, 0.66-5.44; p=0.233) can significantly increase the risk of lymphoma. Exposition of anti-tumor necrosis factor alpha agents in patients with inflammatory bowel disease is not associated with a higher risk of lymphoma. Combination therapy and anti-tumor necrosis factor alpha agents monotherapy can significantly increase the risk of lymphoma in patients with inflammatory bowel disease.

  5. Endocytosis of GPI-linked membrane folate receptor-alpha

    PubMed Central

    1996-01-01

    GPI-linked membrane folate receptors (MFRs) have been implicated in the receptor-mediated uptake of reduced folate cofactors and folate-based chemotherapeutic drugs. We have studied the biosynthetic transport to and internalization of MFR isoform alpha in KB-cells. MFR-alpha was synthesized as a 32-kD protein and converted in a maturely glycosylated 36-38-kD protein 1 h after synthesis. 32-kD MFR-alpha was completely soluble in Triton X-100 at 0 degree C. In contrast, only 33% of the 36- 38-kD species could be solubilized at these conditions whereas complete solubilization was obtained in Triton X-100 at 37 degrees C or in the presence of saponin at 0 degree C. Similar solubilization characteristics were found when MFR-alpha at the plasma membrane was labeled with a crosslinkable 125I-labeled photoaffinity-analog of folic acid as a ligand. Triton X-100-insoluble membrane domains containing MFR-alpha could be separated from soluble MFR-alpha on sucrose flotation gradients. Only Triton X-100 soluble MFR-alpha was internalized from the plasma membrane. The reduced-folate-carrier, an integral membrane protein capable of translocating (anti-)folates across membranes, was completely excluded from the Triton X-100- resistant membrane domains. Internalized MFR-alpha recycled slowly to the cell surface during which it remained soluble in Triton X-100 at 0 degree C. Using immunoelectron microscopy, we found MFR-alpha along the entire endocytic pathway: in clathrin-coated buds and vesicles, and in small and large endosomal vacuoles. In conclusion, our data indicate that a large fraction, if not all, of internalizing MFR-alpha bypasses caveolae. PMID:8567728

  6. Endocytosis of GPI-linked membrane folate receptor-alpha.

    PubMed

    Rijnboutt, S; Jansen, G; Posthuma, G; Hynes, J B; Schornagel, J H; Strous, G J

    1996-01-01

    GPI-linked membrane folate receptors (MFRs) have been implicated in the receptor-mediated uptake of reduced folate cofactors and folate-based chemotherapeutic drugs. We have studied the biosynthetic transport to and internalization of MFR isoform alpha in KB-cells. MFR-alpha was synthesized as a 32-kD protein and converted in a maturely glycosylated 36-38-kD protein 1 h after synthesis. 32-kD MFR-alpha was completely soluble in Triton X-100 at 0 degree C. In contrast, only 33% of the 36-38-kD species could be solubilized at these conditions whereas complete solubilization was obtained in Triton X-100 at 37 degrees C or in the presence of saponin at 0 degree C. Similar solubilization characteristics were found when MFR-alpha at the plasma membrane was labeled with a crosslinkable 125I-labeled photoaffinity-analog of folic acid as a ligand. Triton X-100-insoluble membrane domains containing MFR-alpha could be separated from soluble MFR-alpha on sucrose flotation gradients. Only Triton X-100 soluble MFR-alpha was internalized from the plasma membrane. The reduced-folate-carrier, an integral membrane protein capable of translocating (anti-)folates across membranes, was completely excluded from the Triton X-100-resistant membrane domains. Internalized MFR-alpha recycled slowly to the cell surface during which it remained soluble in Triton X-100 at 0 degree C. Using immunoelectron microscopy, we found MFR-alpha along the entire endocytic pathway: in clathrin-coated buds and vesicles, and in small and large endosomal vacuoles. In conclusion, our data indicate that a large fraction, if not all, of internalizing MFR-alpha bypasses caveolae.

  7. Evolution of the antigen-specific CD8+ TCR repertoire across the life span: evidence for clonal homogenization of the old TCR repertoire.

    PubMed

    Rudd, Brian D; Venturi, Vanessa; Davenport, Miles P; Nikolich-Zugich, Janko

    2011-02-15

    Defects in T cell responses against pathogens and reduced diversity of TCRs have been described at both extremes of the life span. Yet, we still lack information on how Ag-specific T cell populations are maintained and/or altered from birth to old age. In this study, for the first time to our knowledge, we provide insight into Ag-specific TCR repertoire changes over the life span at the single-cell level. We have examined the TCR diversity of the primary CD8(+) T cell response to the immunodominant HSV-1 epitope HSV glycoprotein B 495-502 (HSV gB(498-505); SSIEFARL) (gB-8p) in neonatal, adult, and old C57BL/6 mice. The global distinctive features of the gB-8p-specific TCR repertoire were preserved in mice of different ages. However, both old and especially neonatal mice exhibited significant decreases in TCR diversity compared with that of adult mice. Still, although the neonatal Ag-specific repertoire comprised expectedly shorter germline-biased CDR3β lengths, the repertoire was surprisingly complex, and only a minority of responding cells lacked random nucleotide additions. Changes with aging included increased use of the already dominant TCRVβ10 family, a trend for lower content of the TCR containing the germline WG motif in the CDR3, and a remarkable sharing of one dominant clonotype between individual old mice, implying operation of selective mechanisms. Implications for the rational design of vaccines for neonates and the elderly are discussed.

  8. Syphilis in the Setting of Anti-tumor Necrosis Factor Alpha Therapy.

    PubMed

    Iglesias-Plaza, Ana; Iglesias-Sancho, Maribel; Quintana-Codina, Mónica; García-Miguel, Javier; Salleras-Redonnet, Montse

    2018-02-03

    Inhibitors of tumor necrosis factor-alpha (anti-TNF-alpha) are widely used in different medical specialties. The main adverse effect of these agents is the increased risk of infection. We report the case of a 30-year-old man with ankylosing spondylitis who had begun receiving golimumab two weeks earlier. He presented with a 10-day history of salmon-colored lesions on trunk, palms and soles. The clinical suspicion was secondary syphilis. Treponemal and nontreponemal tests confirmed the diagnosis of syphilis. Lumbar puncture was also performed, although there was no neurological involvement, to rule out neurosyphilis. Cases of syphilis in patients in treatment with TNF-alpha inhibitors are uncommon in the literature and there are no established protocols. Copyright © 2017 Elsevier España, S.L.U. and Sociedad Española de Reumatología y Colegio Mexicano de Reumatología. All rights reserved.

  9. TCR revision generates functional CD4+ T cells.

    PubMed

    Hale, J Scott; Wubeshet, Maramawit; Fink, Pamela J

    2010-12-01

    CD4(+)Vβ5(+) peripheral T cells in C57BL/6 mice respond to encounter with a peripherally expressed endogenous superantigen by undergoing either deletion or TCR revision. In this latter process, cells lose surface Vβ5 expression and undergo RAG-dependent rearrangement of endogenous TCRβ genes, driving surface expression of novel TCRs. Although postrevision CD4(+)Vβ5(-)TCRβ(+) T cells accumulate with age in Vβ5 transgenic mice and bear a diverse TCR Vβ repertoire, it is unknown whether they respond to homeostatic and antigenic stimuli and thus may benefit the host. We demonstrate in this study that postrevision cells are functional. These cells have a high rate of steady-state homeostatic proliferation in situ, and they undergo extensive MHC class II-dependent lymphopenia-induced proliferation. Importantly, postrevision cells do not proliferate in response to the tolerizing superantigen, implicating TCR revision as a mechanism of tolerance induction and demonstrating that TCR-dependent activation of postrevision cells is not driven by the transgene-encoded receptor. Postrevision cells proliferate extensively to commensal bacterial Ags and can generate I-A(b)-restricted responses to Ag by producing IFN-γ following Listeria monocytogenes challenge. These data show that rescued postrevision T cells are responsive to homeostatic signals and recognize self- and foreign peptides in the context of self-MHC and are thus useful to the host.

  10. Specific antibodies against Go isoforms reveal the early expression of the Go2 alpha subunit and appearance of Go1 alpha during neuronal differentiation.

    PubMed

    Rouot, B; Charpentier, N; Chabbert, C; Carrette, J; Zumbihl, R; Bockaert, J; Homburger, V

    1992-02-01

    We have previously identified two isoforms of Go alpha in membranes of N1E-115 neuroblastoma cells, using an antibody raised against the purified Go alpha subunit; one isoform of the Go alpha subunit (pI 5.80) is present in undifferentiated cells, whereas a more acidic isoform (pI 5.55) appears during differentiation [J. Neurochem. 54:1310-1320 (1990)]. Recently, the Go alpha gene has been shown to encode, by alternative splicing, two polypeptides, Go1 alpha and Go2 alpha, which differ only in their carboxyl-terminal part. To determine unambiguously whether the two Go alpha subunits detected in neuroblastoma cells were actually the products of different mRNAs, rabbit polyclonal antibodies were generated against synthetic peptides (amino acids 291-302) of both sequences. Specificity of the two affinity-purified antipeptide antibodies was assessed on Western blots by comparing their immunoreactivities with those of other G alpha antibodies. On a blotted mixture of purified brain guanine nucleotide-binding proteins, the anti-alpha o1 and anti-alpha o2 peptide antibodies only recognized the 39-kDa Go alpha subunit. Furthermore, the immunological recognition of brain membranes from 15-day-old mouse fetuses by antipeptide antibodies could be specifically blocked by addition of the corresponding antigen. When membrane proteins from differentiated neuroblastoma cells and mouse fetus brain were blotted after two-dimensional gel electrophoresis, the anti-alpha o1 and anti-alpha o2 peptide antibodies labeled a 39-kDa subunit focused at a pI value of 5.55 or 5.80, respectively. Study of the ontogenesis of both Go alpha subunits revealed the predominance of Go2 alpha in the frontal cortex at day 15 of gestation. Thereafter, there was a progressive decline of the Go2 alpha polypeptide to a very low level, concomitant with an increase in the Go1 alpha protein, which plateaued about 15 days after birth to a level 8 times higher than at gestational day 15. Similarly, on

  11. Ikaros promotes rearrangement of TCR α genes in an Ikaros null thymoma cell line.

    PubMed

    Collins, Bernard; Clambey, Eric T; Scott-Browne, James; White, Janice; Marrack, Philippa; Hagman, James; Kappler, John W

    2013-02-01

    Ikaros is important in the development and maintenance of the lymphoid system, functioning in part by associating with chromatin-remodeling complexes. We have studied the functions of Ikaros in the transition from pre-T cell to the CD4(+) CD8(+) thymocyte using an Ikaros null CD4(-) CD8(-) mouse thymoma cell line (JE131). We demonstrate that this cell line carries a single functional TCR β gene rearrangement and expresses a surface pre-TCR. JE131 cells also carry nonfunctional rearrangements on both alleles of their TCR α loci. Retroviral reintroduction of Ikaros dramatically increased the rate of transcription in the α locus and TCR Vα/Jα recombination resulting in the appearance of many new αβTCR(+) cells. The process is RAG dependent, requires switch/sucrose nonfermentable chromatin-remodeling complexes and is coincident with the binding of Ikaros to the TCR α enhancer. Furthermore, knockdown of Mi2/nucleosome remodeling and deacetylase complexes increased the frequency of TCR α rearrangement. Our data suggest that Ikaros controls Vα/Jα recombination in T cells by controlling access of the transcription and recombination machinery to the TCR α loci. The JE131 cell line should prove to be a very useful tool for studying the molecular details of this and other processes involved in the pre-T cell to αβTCR(+) CD4(+) CD8(+) thymocyte transition. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Spin-labelling study of interactions of ovalbumin with multilamellar liposomes and specific anti-ovalbumin antibodies.

    PubMed

    Brgles, Marija; Mirosavljević, Krunoslav; Noethig-Laslo, Vesna; Frkanec, Ruza; Tomasić, Jelka

    2007-03-10

    Ovalbumin (OVA) has been used continuously as the model antigen in numerous studies of immune reactions and antigen processing, very often encapsulated into liposomes. The purpose of this work was to study the possible interactions of spin-labelled OVA and lipids in liposomal membranes using electron spin resonance (ESR) spectroscopy. OVA was covalently spin-labelled with 4-maleimido-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO-maleimide), characterized and encapsulated into multilamellar, negatively charged liposomes. ESR spectra of this liposomal preparation gave evidence for the interaction of OVA with the lipid bilayers. Such an interaction was also evidenced by the ESR spectra of liposomal preparation containing OVA, where liposomes were spin-labelled with n-doxyl stearic acids. The spin-labelled OVA retains its property to bind specific anti-OVA antibodies, as shown by ESR spectroscopy, but also in ELISA for specific anti-OVA IgG.

  13. DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ozawa, Tatsuhiko; Horii, Masae; Kobayashi, Eiji

    Highlights: Black-Right-Pointing-Pointer A novel soluble TCR composed of TCR V and C regions with Ig Fc region is generated. Black-Right-Pointing-Pointer TCR-Fc protein immobilized by an anti-C{beta} antibody bound to a p/MHC tetramer. Black-Right-Pointing-Pointer Binding affinity of TCR-Fc was markedly increased by binding with anti-C{beta} antibody. -- Abstract: The identification and cloning of tumor antigen-specific T cell receptors (TCRs) and the production of the soluble form of the TCR (sTCR) contributed to the development of diagnostic and therapeutic tools for cancer. Recently, several groups have reported the development of technologies for the production of sTCRs. The native sTCR has a verymore » low binding affinity for the antigenic peptide/MHC (p/MHC) complex. In this study, we established a technology to produce high affinity, functional sTCRs. We generated a novel sTCR-Fc fusion protein composed of the TCR V and C regions of the TCR linked to the immunoglobulin (Ig) Fc region. A Western blot analysis revealed that the molecular weight of the fusion protein was approximately 60 kDa under reducing conditions and approximately 100-200 kDa under non-reducing conditions. ELISAs using various antibodies showed that the structure of each domain of the TCR-Fc protein was intact. The TCR-Fc protein immobilized by an anti-C{beta} antibody effectively bound to a p/MHC tetramer. An SPR analysis showed that the TCR-Fc protein had a low binding affinity (KD; 1.1 Multiplication-Sign 10{sup -5} M) to the p/MHC monomer. Interestingly, when the TCR-Fc protein was pre-incubated with an anti-C{beta} antibody, its binding affinity for p/MHC increased by 5-fold (2.2 Multiplication-Sign 10{sup -6} M). We demonstrated a novel method for constructing a functional soluble TCR using the Ig Fc region and showed that the binding affinity of the functional sTCR-Fc was markedly increased by an anti-C{beta} antibody, which is probably due to the stabilization of the V{alpha

  14. Enantioselective synthesis of syn/anti-1,3-amino alcohols via proline-catalyzed sequential alpha-aminoxylation/alpha-amination and Horner-Wadsworth-Emmons olefination of aldehydes.

    PubMed

    Jha, Vishwajeet; Kondekar, Nagendra B; Kumar, Pradeep

    2010-06-18

    A novel and general method for asymmetric synthesis of both syn/anti-1,3-amino alcohols is described. The method uses proline-catalyzed sequential alpha-aminoxylation/ alpha-amination and Horner-Wadsworth-Emmons (HWE) olefination of aldehydes as the key step. By using this method, a short synthesis of a bioactive molecule, (R)-1-((S)-1-methylpyrrolidin-2-yl)-5-phenylpentan-2-ol, is also accomplished.

  15. Bcl-2-interacting mediator of cell death influences autoantigen-driven deletion and TCR revision

    PubMed Central

    Hale, J. Scott; Nelson, Lisa T.; Simmons, Kalynn B.; Fink, Pamela J.

    2010-01-01

    Peripheral CD4+Vβ5+ T cells are tolerized to an endogenous mouse mammary tumor virus superantigen either by deletion or TCR revision. Through TCR revision, RAG reexpression mediates extrathymic TCRβ rearrangement and results in a population of post-revision CD4+Vβ5− T cells expressing revised TCRβ chains. We have hypothesized that cell death pathways regulate the selection of cells undergoing TCR revision to ensure the safety and utility of the post-revision population. Here, we investigate the role of Bim-mediated cell death in autoantigen-driven deletion and TCR revision. Bim deficiency and Bcl-2 overexpression in Vβ5 transgenic (Tg) mice both impair peripheral deletion. Vβ5 Tg Bim deficient and Bcl-2 Tg mice exhibit an elevated frequency of CD4+ T cells expressing both the transgene-encoded Vβ5 chain and a revised TCRβ chain. We now show that these dual-TCR expressing cells are TCR revision intermediates, and that the population of RAG-expressing, revising CD4+ T cells is increased in Bim deficient Vβ5 Tg mice. These findings support a role for Bim and Bcl-2 in regulating the balance of survival versus apoptosis in peripheral T cells undergoing RAG-dependent TCR rearrangements during TCR revision, thereby ensuring the utility of the post-revision repertoire. PMID:21148799

  16. Bcl-2-interacting mediator of cell death influences autoantigen-driven deletion and TCR revision.

    PubMed

    Hale, J Scott; Nelson, Lisa T; Simmons, Kalynn B; Fink, Pamela J

    2011-01-15

    Peripheral CD4(+)Vβ5(+) T cells are tolerized to an endogenous mouse mammary tumor virus superantigen either by deletion or TCR revision. Through TCR revision, RAG reexpression mediates extrathymic TCRβ rearrangement and results in a population of postrevision CD4(+)Vβ5(-) T cells expressing revised TCRβ chains. We have hypothesized that cell death pathways regulate the selection of cells undergoing TCR revision to ensure the safety and utility of the postrevision population. In this study, we investigate the role of Bcl-2-interacting mediator of cell death (Bim)-mediated cell death in autoantigen-driven deletion and TCR revision. Bim deficiency and Bcl-2 overexpression in Vβ5 transgenic (Tg) mice both impair peripheral deletion. Vβ5 Tg Bim-deficient and Bcl-2 Tg mice exhibit an elevated frequency of CD4(+) T cells expressing both the transgene-encoded Vβ5 chain and a revised TCRβ chain. We now show that these dual-TCR-expressing cells are TCR revision intermediates and that the population of RAG-expressing, revising CD4(+) T cells is increased in Bim-deficient Vβ5 Tg mice. These findings support a role for Bim and Bcl-2 in regulating the balance of survival versus apoptosis in peripheral T cells undergoing RAG-dependent TCR rearrangements during TCR revision, thereby ensuring the utility of the postrevision repertoire.

  17. Anti-inflammatory effects of compounds alpha-humulene and (-)-trans-caryophyllene isolated from the essential oil of Cordia verbenacea.

    PubMed

    Fernandes, Elizabeth S; Passos, Giselle F; Medeiros, Rodrigo; da Cunha, Fernanda M; Ferreira, Juliano; Campos, Maria M; Pianowski, Luiz F; Calixto, João B

    2007-08-27

    This study evaluated the anti-inflammatory properties of two sesquiterpenes isolated from Cordia verbenacea's essential oil, alpha-humulene and (-)-trans-caryophyllene. Our results revealed that oral treatment with both compounds displayed marked inhibitory effects in different inflammatory experimental models in mice and rats. alpha-humulene and (-)-trans-caryophyllene were effective in reducing platelet activating factor-, bradykinin- and ovoalbumin-induced mouse paw oedema, while only alpha-humulene was able to diminish the oedema formation caused by histamine injection. Also, both compounds had important inhibitory effects on the mouse and rat carrageenan-induced paw oedema. Systemic treatment with alpha-humulene largely prevented both tumor necrosis factor-alpha (TNFalpha) and interleukin-1beta (IL-1beta) generation in carrageenan-injected rats, whereas (-)-trans-caryophyllene diminished only TNFalpha release. Furthermore, both compounds reduced the production of prostaglandin E(2) (PGE(2)), as well as inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX-2) expression, induced by the intraplantar injection of carrageenan in rats. The anti-inflammatory effects of alpha-humulene and (-)-trans-caryophyllene were comparable to those observed in dexamethasone-treated animals, used as positive control drug. All these findings indicate that alpha-humulene and (-)-trans-caryophyllene, derived from the essential oil of C. verbenacea, might represent important tools for the management and/or treatment of inflammatory diseases.

  18. Monoclonal TCR-redirected tumor cell killing.

    PubMed

    Liddy, Nathaniel; Bossi, Giovanna; Adams, Katherine J; Lissina, Anna; Mahon, Tara M; Hassan, Namir J; Gavarret, Jessie; Bianchi, Frayne C; Pumphrey, Nicholas J; Ladell, Kristin; Gostick, Emma; Sewell, Andrew K; Lissin, Nikolai M; Harwood, Naomi E; Molloy, Peter E; Li, Yi; Cameron, Brian J; Sami, Malkit; Baston, Emma E; Todorov, Penio T; Paston, Samantha J; Dennis, Rebecca E; Harper, Jane V; Dunn, Steve M; Ashfield, Rebecca; Johnson, Andy; McGrath, Yvonne; Plesa, Gabriela; June, Carl H; Kalos, Michael; Price, David A; Vuidepot, Annelise; Williams, Daniel D; Sutton, Deborah H; Jakobsen, Bent K

    2012-06-01

    T cell immunity can potentially eradicate malignant cells and lead to clinical remission in a minority of patients with cancer. In the majority of these individuals, however, there is a failure of the specific T cell receptor (TCR)–mediated immune recognition and activation process. Here we describe the engineering and characterization of new reagents termed immune-mobilizing monoclonal TCRs against cancer (ImmTACs). Four such ImmTACs, each comprising a distinct tumor-associated epitope-specific monoclonal TCR with picomolar affinity fused to a humanized cluster of differentiation 3 (CD3)-specific single-chain antibody fragment (scFv), effectively redirected T cells to kill cancer cells expressing extremely low surface epitope densities. Furthermore, these reagents potently suppressed tumor growth in vivo. Thus, ImmTACs overcome immune tolerance to cancer and represent a new approach to tumor immunotherapy.

  19. Analysis of cytotoxic activity of the CD4+ T lymphocytes generated by local immunotherapy.

    PubMed Central

    Katsumoto, Y.; Monden, T.; Takeda, T.; Haba, A.; Ito, Y.; Wakasugi, E.; Wakasugi, T.; Sekimoto, M.; Kobayashi, T.; Shiozaki, H.; Shimano, T.; Monden, M.

    1996-01-01

    We previously reported that the anti-tumour effect of OK-432 is considerably enhanced by its intratumoral injection together with fibrinogen. In the present study, we generated killer T cells by culturing tumour-infiltrating lymphocytes from thyroid cancer patients who had received this local immunotherapy. Phenotypic analysis revealed that the T cells were positive for CD3+, CD4+, Leu8-, CD45RO+ and T-cell receptor (TCR)alpha beta+, as well as showing strong surface expression of HLA-DR, CD25, LFA-1 and ICAM-1. The generated CD4+ T cells secreted interferon (IFN)-gamma, tumour necrosis factor (TNF)-alpha, TNF-beta, and interleukin (IL)-6 (but not IL-4), and exhibited a high level of cytolytic activity against several tumour cell lines. The cytolytic activity of these T cells for Daudi cells was inhibited by preincubation with an anti-intercellular adhesion molecule (ICAM)-1 antibody, but not by preincubation with anti-TCR alpha beta, anti-CD2, or anti-LFA-1 antibodies. Pretreatment with anti-ICAM-1 antibody inhibited T-cell cytolytic activity, but not conjugation with target cells. In addition, incubation with immobilised anti-ICAM-1 enhanced the secretion of IFN-gamma by T cells. We conclude that ICAM-1 expressed on the effector cytotoxic CD4+ T lymphocytes delivers regulatory signals that enhance IFN-gamma secretion. PMID:8554971

  20. In vitro experimental (211)At-anti-CD33 antibody therapy of leukaemia cells overcomes cellular resistance seen in vivo against gemtuzumab ozogamicin.

    PubMed

    Petrich, Thorsten; Korkmaz, Zekiye; Krull, Doris; Frömke, Cornelia; Meyer, Geerd J; Knapp, Wolfram H

    2010-05-01

    Monoclonal anti-CD33 antibodies conjugated with toxic calicheamicin derivative (gemtuzumab ozogamicin, GO) are a novel therapy option for acute myeloid leukaemia (AML). Key prognostic factors for patients with AML are high CD33 expression on the leukaemic cells and the ability to overcome mechanisms of resistance to cytotoxic chemotherapies, including drug efflux or other mechanisms decreasing apoptosis. Alpha particle-emitting radionuclides overwhelm such anti-apoptotic mechanisms by producing numerous DNA double-stranded breaks (DSBs) accompanied by decreased DNA repair. We labelled anti-CD33 antibodies with the alpha-emitter (211)At and compared survival of leukaemic HL-60 and K-562 cells treated with the (211)At-labelled antibodies, GO or unlabelled antibodies as controls. We also measured caspase-3/7 activity, DNA fragmentation and necrosis in HL-60 cells after treatment with the different antibodies or with free (211)At. The mean labelling ratio of (211)At-labelled antibodies was 1:1,090 +/- 364 (range: 1:738-1:1,722) in comparison to 2-3:1 for GO. Tumour cell binding of (211)At-anti-CD33 was high in the presence of abundant CD33 expression and could be specifically blocked by unlabelled anti-CD33. (211)At-anti-CD33 decreased survival significantly more than did GO at comparable dilution (1:1,000). No significant differences in induction of apoptosis or necrosis or DNA DSB or in decreased survival were observed after (211)At-anti-CD33 (1:1,090) versus GO (1:1) treatment. Our results suggest that (211)At is a promising, highly cytotoxic radioimmunotherapy in CD33-positive leukaemia and kills tumour cells more efficiently than does calicheamicin-conjugated antibody. Labelling techniques leading to higher chemical yield and specific activities must be developed to increase (211)At-anti-CD33 therapeutic effects.

  1. TCR revision generates functional CD4+ T cells1

    PubMed Central

    Hale, J. Scott; Wubeshet, Maramawit; Fink, Pamela J.

    2010-01-01

    CD4+Vβ5+ peripheral T cells in B6 mice respond to encounter with a peripherally-expressed endogenous superantigen by undergoing either deletion or TCR revision. In this latter process, cells lose surface Vβ5 expression and undergo RAG-dependent rearrangement of endogenous TCRβ genes, driving surface expression of novel TCRs. While post-revision CD4+Vβ5−TCRβ+ T cells accumulate with age in Vβ5 transgenic mice and bear a diverse TCR Vβ repertoire, it is unknown whether they respond to homeostatic and antigenic stimuli, and thus may benefit the host. We now demonstrate that post-revision cells are functional. These cells have a high rate of steady-state homeostatic proliferation in situ and they undergo extensive MHC class II-dependent lymphopenia-induced proliferation. Importantly, post-revision cells do not proliferate in response to the tolerizing superantigen, implicating TCR revision as a mechanism of tolerance induction and demonstrating that TCR-dependent activation of post-revision cells is not driven by the transgene-encoded receptor. Post-revision cells proliferate extensively to commensal bacterial Ags and can generate I-Ab-restricted responses to Ag by producing IFNγ following Listeria monocytogenes challenge. These data show that rescued post-revision T cells are responsive to homeostatic signals and recognize self and foreign peptides in the context of self MHC, and are thus useful to the host. PMID:20971922

  2. A CD22-reactive TCR from the T-cell allorepertoire for the treatment of acute lymphoblastic leukemia by TCR gene transfer

    PubMed Central

    Jahn, Lorenz; Hagedoorn, Renate S.; van der Steen, Dirk M.; Hombrink, Pleun; Kester, Michel G.D.; Schoonakker, Marjolein P.; de Ridder, Daniëlle; van Veelen, Peter A.; Falkenburg, J.H. Frederik; Heemskerk, Mirjam H.M.

    2016-01-01

    CD22 is currently evaluated as a target-antigen for the treatment of B-cell malignancies using chimeric antigen receptor (CAR)-engineered T-cells or monoclonal antibodies (mAbs). CAR- and mAbs-based immunotherapies have been successfully applied targeting other antigens, however, occurrence of refractory disease to these interventions urges the identification of additional strategies. Here, we identified a TCR recognizing the CD22-derived peptide RPFPPHIQL (CD22RPF) presented in human leukocyte antigen (HLA)-B*07:02. To overcome tolerance to self-antigens such as CD22, we exploited the immunogenicity of allogeneic HLA. CD22RPF-specific T-cell clone 9D4 was isolated from a healthy HLA-B*07:02neg individual, efficiently produced cytokines upon stimulation with primary acute lymphoblastic leukemia and healthy B-cells, but did not react towards healthy hematopoietic and nonhematopoietic cell subsets, including dendritic cells (DCs) and macrophages expressing low levels of CD22. Gene transfer of TCR-9D4 installed potent CD22-specificity onto recipient CD8+ T-cells that recognized and lysed primary B-cell leukemia. TCR-transduced T-cells spared healthy CD22neg hematopoietic cell subsets but weakly lysed CD22low-expressing DCs and macrophages. CD22-specific TCR-engineered T-cells could form an additional immunotherapeutic strategy with a complementary role to CAR- and antibody-based interventions in the treatment of B-cell malignancies. However, CD22 expression on non-B-cells may limit the attractiveness of CD22 as target-antigen in cellular immunotherapy. PMID:27689397

  3. A CD22-reactive TCR from the T-cell allorepertoire for the treatment of acute lymphoblastic leukemia by TCR gene transfer.

    PubMed

    Jahn, Lorenz; Hagedoorn, Renate S; van der Steen, Dirk M; Hombrink, Pleun; Kester, Michel G D; Schoonakker, Marjolein P; de Ridder, Daniëlle; van Veelen, Peter A; Falkenburg, J H Frederik; Heemskerk, Mirjam H M

    2016-11-01

    CD22 is currently evaluated as a target-antigen for the treatment of B-cell malignancies using chimeric antigen receptor (CAR)-engineered T-cells or monoclonal antibodies (mAbs). CAR- and mAbs-based immunotherapies have been successfully applied targeting other antigens, however, occurrence of refractory disease to these interventions urges the identification of additional strategies. Here, we identified a TCR recognizing the CD22-derived peptide RPFPPHIQL (CD22RPF) presented in human leukocyte antigen (HLA)-B*07:02. To overcome tolerance to self-antigens such as CD22, we exploited the immunogenicity of allogeneic HLA. CD22RPF-specific T-cell clone 9D4 was isolated from a healthy HLA-B*07:02neg individual, efficiently produced cytokines upon stimulation with primary acute lymphoblastic leukemia and healthy B-cells, but did not react towards healthy hematopoietic and nonhematopoietic cell subsets, including dendritic cells (DCs) and macrophages expressing low levels of CD22. Gene transfer of TCR-9D4 installed potent CD22-specificity onto recipient CD8+ T-cells that recognized and lysed primary B-cell leukemia. TCR-transduced T-cells spared healthy CD22neg hematopoietic cell subsets but weakly lysed CD22low-expressing DCs and macrophages. CD22-specific TCR-engineered T-cells could form an additional immunotherapeutic strategy with a complementary role to CAR- and antibody-based interventions in the treatment of B-cell malignancies. However, CD22 expression on non-B-cells may limit the attractiveness of CD22 as target-antigen in cellular immunotherapy.

  4. Urokinase plasminogen activator mRNA is induced by IL-1alpha and TNF-alpha in in vitro acantholysis.

    PubMed

    Feliciani, Claudio; Toto, Paola; Wang, Binghe; Sauder, Daniel N; Amerio, Pierluigi; Tulli, Antonio

    2003-08-01

    The role of urokinase type plasminogen activator (uPA) has been well documented in the pathogenesis of pemphigus vulgaris (PV). Activation of plasminogen into active serine protease plasmin initiates extracellular proteolysis leading to acantholysis but the mechanisms underlying this process are not clearly understood. We have previously shown that keratinocyte derived cytokines IL-1alpha and TNF-alpha are involved in PV-induced acantholysis. In the present study we sought to examine whether keratinocyte-derived IL-1alpha and TNF-alpha are correlated with uPA induction in keratinocytes during acantholysis. Normal human keratinocytes were incubated with diluted PV serum. mRNAs for IL-1alpha, TNF-alpha and uPA were examined with RT-PCR at various time points and acantholysis was measured. IL-1alpha, TNF-alpha and uPA mRNAs were all induced in keratinocytes following PV serum stimulation; IL-1alpha/TNF-alpha mRNAs' expression was earlier than the expression of uPA mRNA. To further examine the role of IL-1alpha, TNF-alpha and uPA in acantholysis, we performed antibody blocking studies. Anti-IL-1alpha, anti-TNF-alpha and anti-uPA antibodies suppressed acantholysis by 76%, 80% and 90%, respectively. In addition, anti-IL-1alpha and anti-TNF-alpha antibodies inhibited uPA mRNA induction, whereas anti-uPA antibodies did not alter IL-1alpha/TNF-alpha mRNAs' expression. Our results confirm the role of uPA in acantholysis and suggest an involvement of IL-1alpha/TNF-alpha in uPA induction.

  5. Oscillatory Alpha-Band Suppression Mechanisms during the Rapid Attentional Shifts Required to Perform an Anti-Saccade Task

    PubMed Central

    Belyusar, Daniel; Snyder, Adam C.; Frey, Hans-Peter; Harwood, Mark R.; Wallman, Josh; Foxe, John J.

    2015-01-01

    Neuroimaging has demonstrated anatomical overlap between covert and overt attention systems, although behavioral and electrophysiological studies have suggested that the two systems do not rely on entirely identical circuits or mechanisms. In a parallel line of research, topographically-specific modulations of alpha-band power (~8-14Hz) have been consistently correlated with anticipatory states during tasks requiring covert attention shifts. These tasks, however, typically employ cue-target-interval paradigms where attentional processes are examined across relatively protracted periods of time and not at the rapid timescales implicated during overt attention tasks. The anti-saccade task, where one must first covertly attend for a peripheral target, before executing a rapid overt attention shift (i.e. a saccade) to the opposite side of space, is particularly well-suited for examining the rapid dynamics of overt attentional deployments. Here, we asked whether alpha-band oscillatory mechanisms would also be associated with these very rapid overt shifts, potentially representing a common neural mechanism across overt and covert attention systems. High-density electroencephalography in conjunction with infra-red eye-tracking was recorded while participants engaged in both pro- and anti- saccade task blocks. Alpha power, time-locked to saccade onset, showed three distinct phases of significantly lateralized topographic shifts, all occurring within a period of less than one second, closely reflecting the temporal dynamics of anti-saccade performance. Only two such phases were observed during the pro-saccade task. These data point to substantially more rapid temporal dynamics of alpha-band suppressive mechanisms than previously established, and implicate oscillatory alpha-band activity as a common mechanism across both overt and covert attentional deployments. PMID:23041338

  6. CD8+ TCR repertoire formation is guided primarily by the peptide component of the antigenic complex.

    PubMed

    Koning, Dan; Costa, Ana I; Hoof, Ilka; Miles, John J; Nanlohy, Nening M; Ladell, Kristin; Matthews, Katherine K; Venturi, Vanessa; Schellens, Ingrid M M; Borghans, Jose A M; Kesmir, Can; Price, David A; van Baarle, Debbie

    2013-02-01

    CD8(+) T cells recognize infected or dysregulated cells via the clonotypically expressed αβ TCR, which engages Ag in the form of peptide bound to MHC class I (MHC I) on the target cell surface. Previous studies have indicated that a diverse Ag-specific TCR repertoire can be beneficial to the host, yet the determinants of clonotypic diversity are poorly defined. To better understand the factors that govern TCR repertoire formation, we conducted a comprehensive clonotypic analysis of CD8(+) T cell populations directed against epitopes derived from EBV and CMV. Neither pathogen source nor the restricting MHC I molecule were linked with TCR diversity; indeed, both HLA-A and HLA-B molecules were observed to interact with an overlapping repertoire of expressed TRBV genes. Peptide specificity, however, markedly impacted TCR diversity. In addition, distinct peptides sharing HLA restriction and viral origin mobilized TCR repertoires with distinct patterns of TRBV gene usage. Notably, no relationship was observed between immunodominance and TCR diversity. These findings provide new insights into the forces that shape the Ag-specific TCR repertoire in vivo and highlight a determinative role for the peptide component of the peptide-MHC I complex on the molecular frontline of CD8(+) T cell-mediated immune surveillance.

  7. T follicular helper and T follicular regulatory cells have different TCR specificity

    PubMed Central

    Maceiras, Ana Raquel; Almeida, Silvia Cristina Paiva; Mariotti-Ferrandiz, Encarnita; Chaara, Wahiba; Jebbawi, Fadi; Six, Adrien; Hori, Shohei; Klatzmann, David; Faro, Jose; Graca, Luis

    2017-01-01

    Immunization leads to the formation of germinal centres (GCs) that contain both T follicular helper (Tfh) and T follicular regulatory (Tfr) cells. Whether T-cell receptor (TCR) specificity defines the differential functions of Tfh and Tfr cells is unclear. Here we show that antigen-specific T cells after immunization are preferentially recruited to the GC to become Tfh cells, but not Tfr cells. Tfh cells, but not Tfr cells, also proliferate efficiently on restimulation with the same immunizing antigen in vitro. Ex vivo TCR repertoire analysis shows that immunization induces oligoclonal expansion of Tfh cells. By contrast, the Tfr pool has a TCR repertoire that more closely resembles that of regulatory T (Treg) cells. Our data thus indicate that the GC Tfh and Tfr pools are generated from distinct TCR repertoires, with Tfh cells expressing antigen-responsive TCRs to promote antibody responses, and Tfr cells expressing potentially autoreactive TCRs to suppress autoimmunity. PMID:28429709

  8. Cutting Edge: TCR Revision Affects Predominantly Foxp3− Cells and Skews Them toward the Th17 Lineage1

    PubMed Central

    Zehn, Dietmar; Bevan, Michael J.; Fink, Pamela J.

    2009-01-01

    CD4+ T cells respond to peripheral endogenous superantigen stimulation by undergoing deletion or TCR revision. The latter involves RAG re-expression, TCR gene rearrangement, and expression of a novel TCR. TCR-revised T cells are functional and express a diverse TCR repertoire. Because TCR revision harbors the potential to create self-reactivity, it is important to explore whether T cells known to be self-reactive (regulatory T cells) or those involved in autoimmunity (Th17 cells) arise from TCR revision. Interestingly, we observed that Foxp3+ cells are excluded from revising their TCR and that only a small fraction of postrevision cells expresses Foxp3. In contrast, Th17 cells are 20 times more frequent among revised than among C57BL/6 CD4+ T cells, indicating that postrevision cells are biased toward the Th17 lineage. The link between Th17 differentiation and TCR revision might be highly relevant to the role of Th17 cells in promoting autoimmunity. PMID:17947636

  9. Intrathymic selection of NK1.1+α/β T cell antigen receptor (TCR)+ cells in transgenic mice bearing TCR specific for chicken ovalbumin and restricted to I-Ad

    PubMed Central

    Iwabuchi, Chikako; Iwabuchi, Kazuya; Nakagawa, Ken-ichi; Takayanagi, Toshiaki; Nishihori, Hiroki; Tone, Saori; Ogasawara, Kazumasa; Good, Robert A.; Onoé, Kazunori

    1998-01-01

    Generation and negative selection of NK1.1+α/β T cell receptor (TCR)+ thymocytes were analyzed using TCR-transgenic (B10.D2 × DO10)F1 and (C57BL/6 × DO10)F1 mice and Rag-1−/−/DO10 mice, which had been established by breeding and backcrossing between Rag-1−/− and DO10 mice. Almost all T cells from these mice were shown to bear Vα13/Vβ8.2 that is specific for chicken ovalbumin (cOVA) and restricted to I-Ad. A normal proportion of the NK1.1+ Vα13/Vβ8.2+ thymocytes was generated in these mice. However, the actual cell number of both NK1.1+ and NK1.1− thymocytes in I-Ad/d mice (positive selecting background) was larger than that in I-Ab/d mice (negative selecting background). Markedly low but significant proportions of NK1.1+ Vα13/Vβ8.2+ cells were detected in the spleens from I-Ad/d and I-Ab/d mice. It was shown that the splenic NK1.1+ T cells of the I-Ab/d mice were anergized against stimulation through TCR. When (B10.D2 × DO10)F1 and (C57BL/6 × DO10)F1 mice were given cOVA, extensive or intermediate elimination of NK1.1+α/βTCR+ thymocytes was induced in I-Ad/d or I-Ab/d mice, respectively. However, the clonal elimination was not as complete as that seen in the major NK1.1− thymocyte population. The present findings indicate that normal generation of NK1.1+α/βTCR+ thymocytes occurs in the absence of Vα14-Jα281 and that substantial negative selection operates on the NK1.1+α/βTCR+ cells. PMID:9653164

  10. Identification of a novel cyclosporin-sensitive element in the human tumor necrosis factor alpha gene promoter

    PubMed Central

    1993-01-01

    Tumor necrosis factor alpha (TNF-alpha), a cytokine with pleiotropic biological effects, is produced by a variety of cell types in response to induction by diverse stimuli. In this paper, TNF-alpha mRNA is shown to be highly induced in a murine T cell clone by stimulation with T cell receptor (TCR) ligands or by calcium ionophores alone. Induction is rapid, does not require de novo protein synthesis, and is completely blocked by the immunosuppressant cyclosporin A (CsA). We have identified a human TNF-alpha promoter element, kappa 3, which plays a key role in the calcium-mediated inducibility and CsA sensitivity of the gene. In electrophoretic mobility shift assays, an oligonucleotide containing kappa 3 forms two DNA protein complexes with proteins that are present in extracts from unstimulated T cells. These complexes appear in nuclear extracts only after T cell stimulation. Induction of the inducible nuclear complexes is rapid, independent of protein synthesis, and blocked by CsA, and thus, exactly parallels the induction of TNF-alpha mRNA by TCR ligands or by calcium ionophore. Our studies indicate that the kappa 3 binding factor resembles the preexisting component of nuclear factor of activated T cells. Thus, the TNF-alpha gene is an immediate early gene in activated T cells and provides a new model system in which to study CsA-sensitive gene induction in activated T cells. PMID:8376940

  11. Reduced TCR signaling potential impairs negative selection but does not result in autoimmune disease

    PubMed Central

    Hwang, SuJin; Song, Ki-Duk; Lesourne, Renaud; Lee, Jan; Pinkhasov, Julia; Li, LiQi; El-Khoury, Dalal

    2012-01-01

    Negative selection and regulatory T (T reg) cell development are two thymus-dependent processes necessary for the enforcement of self-tolerance, and both require high-affinity interactions between the T cell receptor (TCR) and self-ligands. However, it remains unclear if they are similarly impacted by alterations in TCR signaling potential. We generated a knock-in allele (6F) of the TCR ζ chain gene encoding a mutant protein lacking signaling capability whose expression is controlled by endogenous ζ regulatory sequences. Although negative selection was defective in 6F/6F mice, leading to the survival of autoreactive T cells, 6F/6F mice did not develop autoimmune disease. We found that 6F/6F mice generated increased numbers of thymus-derived T reg cells. We show that attenuation of TCR signaling potential selectively impacts downstream signaling responses and that this differential effect favors Foxp3 expression and T reg cell lineage commitment. These results identify a potential compensatory pathway for the enforcement of immune tolerance in response to defective negative selection caused by reduced TCR signaling capability. PMID:22945921

  12. Bile alcohol metabolism in man. Conversion of 5beta-cholestane-3alpha, 7alpha,12alpha, 25-tetrol to cholic acid.

    PubMed Central

    Salen, G; Shefer, S; Setoguchi, T; Mosbach, E H

    1975-01-01

    To study the role of C25-HYDROXY BILE ALCOHOLS AS PRECURSORS OF CHOlic acid, [G-3-H]5beta-cholestane-3alpha,7alpha12alpha,25-tetrol was administered intravenously to two subjects with cerebrotendinous xanthomatosis (CTX) and two normal individuals. One day after pulse labeling, radioactivity was present in the cholic acid isolated from the bile and feces of the subjects with CTX and the bile of the normal individuals. In the two normal subjects, the sp act decay curves of [G-3-H]-cholic acid were exponential, and no traces of [G-3-H]-5beta-cholestane-3alpha,7alpha,12alpha,25-tetrol were detected. In contrast, appreciable quantities of labeled 5beta-cholestane-3alpha,-7aopha,12alpha,25-tetrol were present in the bile and feces of the CTX subjects. The sp act vs. time curves of fecal [G-3-H]5beta-cholestane-3alpha,7alpha,12alpha,25-tetrol and [G-3-H]-cholic acid showed a precursor-product relationship. Although these results suggest that 5beta-cholestane-3alpha,7alpha,12alpha,25-tetrol may be a precursor of cholic acid in man, the possibility that C26-hydroxy intermediates represent the normal pathway can not be excluded. PMID:1141434

  13. 21 CFR 201.313 - Estradiol labeling.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... Pharmacopeia under the designation “Alpha Estradiol.” The substance should no longer be referred to in drug labeling as “Alpha Estradiol.” The Food and Drug Administration would not object to label references to the... referred to the presence of “Estradiol (formerly known as Alpha Estradiol).” ...

  14. 21 CFR 201.313 - Estradiol labeling.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... Pharmacopeia under the designation “Alpha Estradiol.” The substance should no longer be referred to in drug labeling as “Alpha Estradiol.” The Food and Drug Administration would not object to label references to the... referred to the presence of “Estradiol (formerly known as Alpha Estradiol).” ...

  15. 21 CFR 201.313 - Estradiol labeling.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... Pharmacopeia under the designation “Alpha Estradiol.” The substance should no longer be referred to in drug labeling as “Alpha Estradiol.” The Food and Drug Administration would not object to label references to the... referred to the presence of “Estradiol (formerly known as Alpha Estradiol).” ...

  16. 21 CFR 201.313 - Estradiol labeling.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... Pharmacopeia under the designation “Alpha Estradiol.” The substance should no longer be referred to in drug labeling as “Alpha Estradiol.” The Food and Drug Administration would not object to label references to the... referred to the presence of “Estradiol (formerly known as Alpha Estradiol).” ...

  17. 21 CFR 201.313 - Estradiol labeling.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... Pharmacopeia under the designation “Alpha Estradiol.” The substance should no longer be referred to in drug labeling as “Alpha Estradiol.” The Food and Drug Administration would not object to label references to the... referred to the presence of “Estradiol (formerly known as Alpha Estradiol).” ...

  18. Cytokine appearance and effects of anti-tumor necrosis factor alpha antibodies in a neonatal rat model of group B streptococcal infection.

    PubMed Central

    Teti, G; Mancuso, G; Tomasello, F

    1993-01-01

    Cytokines are suspected of playing an important role in the pathophysiology of septic shock. This study was undertaken to determine whether tumor necrosis factor alpha (TNF-alpha) induces the production of other cytokines and mediates mortality in a neonatal rat model of sepsis caused by group B streptococci (GBS). We have measured TNF-alpha, interleukin-1 alpha (IL-1 alpha), interleukin-6 (IL-6), and gamma interferon (IFN-gamma) levels in neonatal rats infected with different strains (H738, 259, and 90) and doses (1 50% lethal dose [LD50] and 5 90% lethal doses [LD90]) of type III GBS. TNF-alpha and IL-6 were detected by the L929 cytotoxicity and the B9 proliferation assays, respectively, in serial plasma samples. IL-1 alpha and IFN-gamma were measured in spleen homogenates by enzyme-linked immunosorbent assay kits by using antibodies raised against the corresponding mouse cytokines. Plasma TNF-alpha levels significantly rose above baseline values within 12 h after intraperitoneal challenge with 5 LD90 of GBS strain H738, corresponding to 3 x 10(3) CFU. A mean peak TNF-alpha concentration of 232 +/- 124 U/ml was reached at 20 h. Peak IL-1 alpha and IL-6 levels of 766 +/- 404 U/g and 1,033 +/- 520 U/ml, respectively, were reached at 24 h after bacterial challenge. Maximal spleen concentrations of IFN-gamma (449 +/- 283 U/g) were measured at 36 h. Concentrations of TNF-alpha, but not other cytokines, remained significantly elevated at 72 h, a time when mortality approached 100%. Significant correlations were found between concentrations of each of the cytokines tested and the logs of CFU concentrations in the blood. In order to ascertain whether TNF-alpha influenced the production of other cytokines, rat pups received two injections of anti-murine TNF-alpha or normal rabbit serum at 2 h before and at 26 h after challenge with live GBS. Plasma TNF-alpha bioactivity was undetectable in anti-TNF-alpha-treated animals, while IL-6 and IFN-gamma, but not IL-1 alpha

  19. The effects of alpha2-adrenoceptor agents on anti-hyperalgesic effects of carbamazepine and oxcarbazepine in a rat model of inflammatory pain.

    PubMed

    Vucković, Sonja M; Tomić, Maja A; Stepanović-Petrović, Radica M; Ugresić, Nenad; Prostran, Milica S; Bosković, Bogdan

    2006-11-01

    In this study, the effects of yohimbine (alpha2-adrenoceptor antagonist) and clonidine (alpha2-adrenoceptor agonist) on anti-hyperalgesia induced by carbamazepine and oxcarbazepine in a rat model of inflammatory pain were investigated. Carbamazepine (10-40 mg/kg; i.p.) and oxcarbazepine (40-160 mg/kg; i.p.) caused a significant dose-dependent reduction of the paw inflammatory hyperalgesia induced by concanavalin A (Con A, intraplantarly) in a paw pressure test in rats. Yohimbine (1-3 mg/kg; i.p.) significantly depressed the anti-hyperalgesic effects of carbamazepine and oxcarbazepine, in a dose- and time-dependent manner. Both drug mixtures (carbamazepine-clonidine and oxcarbazepine-clonidine) administered in fixed-dose fractions of the ED50 (1/2, 1/4 and 1/8) caused significant and dose-dependent reduction of the hyperalgesia induced by Con A. Isobolographic analysis revealed a significant synergistic (supra-additive) anti-hyperalgesic effect of both combinations tested. These results indicate that anti-hyperalgesic effects of carbamazepine and oxcarbazepine are, at least partially, mediated by activation of adrenergic alpha2-receptors. In addition, synergistic interaction for anti-hyperalgesia between carbamazepine and clonidine, as well as oxcarbazepine and clonidine in a model of inflammatory hyperalgesia, was demonstrated.

  20. Energetic and flexibility properties captured by long molecular dynamics simulations of a membrane-embedded pMHCII-TCR complex.

    PubMed

    Bello, Martiniano; Correa-Basurto, José

    2016-04-01

    Although crystallographic data have provided important molecular insight into the interactions in the pMHC-TCR complex, the inherent features of this structural approach cause it to only provide a static picture of the interactions. While unbiased molecular dynamics simulations (UMDSs) have provided important information about the dynamic structural behavior of the pMHC-TCR complex, most of them have modeled the pMHC-TCR complex as soluble, when in physiological conditions, this complex is membrane bound; therefore, following this latter UMDS protocol might hamper important dynamic results. In this contribution, we performed three independent 300 ns-long UMDSs of the pMHCII-TCR complex anchored in two opposing membranes to explore the structural and energetic properties of the recognition of pMHCII by the TCR. The conformational ensemble generated through UMDSs was subjected to clustering and Cartesian principal component analyses (cPCA) to explore the dynamical behavior of the pMHCII-TCR association. Furthermore, based on the conformational population sampled through UMDSs, the effective binding free energy, per-residue free energy decomposition, and alanine scanning mutations were explored for the native pMHCII-TCR complex, as well as for 12 mutations (p1-p12MHCII-TCR) introduced in the native peptide. Clustering analyses and cPCA provide insight into the rocking motion of the TCR onto pMHCII, together with the presence of new electrostatic interactions not observed through crystallographic methods. Energetic results provide evidence of the main contributors to the pMHC-TCR complex formation as well as the key residues involved in this molecular recognition process.

  1. Autoradiographic localization of voltage-dependent sodium channels on the mouse neuromuscular junction using /sup 125/I-alpha scorpion toxin. I. Preferential labeling of glial cells on the presynaptic side

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Boudier, J.L.; Jover, E.; Cau, P.

    1988-05-01

    Alpha-scorpion toxins bind specifically to the voltage-sensitive sodium channel in excitable membranes, and binding is potential-dependent. The radioiodinated toxin II from the scorpion Androctonus australis Hector (alpha ScTx) was used to localize voltage-sensitive sodium channels on the presynaptic side of mouse neuromuscular junctions (NMJ) by autoradiography using both light and electron microscopy. Silver grain localization was analyzed by the cross-fire method. At the light-microscopic level, grain density over NMJ appeared 6-8x higher than over nonjunctional muscle membrane. The specificity of labeling was verified by competition/displacement with an excess of native alpha ScTx. Labeling was also inhibited by incubation in depolarizingmore » conditions, showing its potential-dependence. At the electron-microscopic level, analysis showed that voltage-sensitive sodium channels labeled with alpha ScTx were almost exclusively localized on membranes, as expected. Due to washout after incubation, appreciable numbers of binding sites were not found on the postsynaptic membranes. However, on the presynaptic side, alpha ScTx-labeled voltage-sensitive sodium channels were localized on the membrane of non-myelin-forming Schwann cells covering NMJ. The axonal presynaptic membrane was not labeled. These results show that voltage-sensitive sodium channels are present on glial cells in vivo, as already demonstrated in vitro. It is proposed that these glial channels could be indirectly involved in the ionic homeostasis of the axonal environment.« less

  2. Arzanol, an anti-inflammatory and anti-HIV-1 phloroglucinol alpha-Pyrone from Helichrysum italicum ssp. microphyllum.

    PubMed

    Appendino, Giovanni; Ottino, Michela; Marquez, Nieves; Bianchi, Federica; Giana, Anna; Ballero, Mauro; Sterner, Olov; Fiebich, Bernd L; Munoz, Eduardo

    2007-04-01

    An acetone extract of Helichrysum italicum ssp. microphyllum afforded the phloroglucinol alpha-pyrone arzanol (1a) as a potent NF-kappaB inhibitor. Arzanol is identical with homoarenol (2a), whose structure should be revised. The phloroglucinol-type structure of arzanol and the 1,2,4-trihydroxyphenyl-type structure of the base-induced fragmentation product of homoarenol could be reconciled in light of a retro-Fries-type fragmentation that triggers a change of the hydroxylation pattern of the aromatic moiety. On the basis of these findings, the structure of arenol, the major constituent of the clinically useful antibiotic arenarin, should be revised from 2b to 1b, solving a long-standing puzzle over its biogenetic derivation. An alpha-pyrone (micropyrone, 7), the monoterpene rac-E-omega-oleoyloxylinalol (10), four known tremetones (9a-d), and the dimeric pyrone helipyrone (8) were also obtained. Arzanol inhibited HIV-1 replication in T cells and the release of pro-inflammatory cytokines in LPS-stimulated primary monocytes, qualifying as a novel plant-derived anti-inflammatory and antiviral chemotype worth further investigation.

  3. Inactivation of chloroplast H(+)-ATPase by modification of Lys beta 359, Lys alpha 176 and Lys alpha 266.

    PubMed

    Horbach, M; Meyer, H E; Bickel-Sandkötter, S

    1991-09-01

    Treatment of isolated, latent chloroplast ATPase with pyridoxal-5-phosphate (pyridoxal-P) in presence of Mg2+ causes inhibition of dithiothreitol-activated plus heat-activated ATP hydrolysis. The amount of [3H]pyridoxal-P bound to chloroplast coupling factor 1 (CF1) was estimated to run up to 6 +/- 1 pyridoxal-P/enzyme, almost equally distributed between the alpha- and beta-subunits. Inactivation, however, is complete after binding of 1.5-2 pyridoxal-P/CF1, suggesting that two covalently modified lysines prevent the activation of the enzyme. ADP as well as ATP in presence of Mg2+ protects the enzyme against inactivation and concomittantly prevents incorporation of a part of the 3H-labeled pyridoxal-P into beta- and alpha-subunits. Phosphate prevents labeling of the alpha-subunit, but has only a minor effect on protection against inactivation. The data indicate a binding site at the interface between the alpha- and beta-subunits. Cleavage of the pyridoxal-P-labeled subunits with cyanogen bromide followed by sequence analysis of the labeled peptides led to the detection of Lys beta 359, Lys alpha 176 and Lys alpha 266, which are closely related to proposed nucleotide-binding regions of the alpha- and beta-subunits.

  4. Rocky Mountain Spotted Fever in a patient treated with anti-TNF-alpha inhibitors.

    PubMed

    Mays, Rana M; Gordon, Rachel A; Durham, K Celeste; LaPolla, Whitney J; Tyring, Stephen K

    2013-03-15

    Rocky Mountain Spotted Fever (RMSF) is a tick-bourne illness, which can be fatal if unrecognized. We discuss the case of a patient treated with an anti-TNF-alpha inhibitor for rheumatoid arthritis who later developed a generalized erythematous macular eruption accompanied by fever. The clinical findings were suggestive of RMSF, which was later confirmed with serology. Prompt treatment with doxyclycine is recommended for all patients with clinical suspicion of RMSF.

  5. TCR backscattering characterization for microwave remote sensing

    NASA Astrophysics Data System (ADS)

    Riccio, Giovanni; Gennarelli, Claudio

    2014-05-01

    A Trihedral Corner Reflector (TCR) is formed by three mutually orthogonal metal plates of various shapes and is a very important scattering structure since it exhibits a high monostatic Radar Cross Section (RCS) over a wide angular range. Moreover it is a handy passive device with low manufacturing costs and robust geometric construction, the maintenance of its efficiency is not difficult and expensive, and it can be used in all weather conditions (i.e., fog, rain, smoke, and dusty environment). These characteristics make it suitable as reference target and radar enhancement device for satellite- and ground-based microwave remote sensing techniques. For instance, TCRs have been recently employed to improve the signal-to-noise ratio of the backscattered signal in the case of urban ground deformation monitoring [1] and dynamic survey of civil infrastructures without natural corners as the Musmeci bridge in Basilicata, Italy [2]. The region of interest for the calculation of TCR's monostatic RCS is here confined to the first quadrant containing the boresight direction. The backscattering term is presented in closed form by evaluating the far-field scattering integral involving the contributions related to the direct illumination and the internal bouncing mechanisms. The Geometrical Optics (GO) laws allow one to determine the field incident on each TCR plate and the patch (integration domain) illuminated by it, thus enabling the use of a Physical Optics (PO) approximation for the corresponding surface current densities to consider for integration on each patch. Accordingly, five contributions are associated to each TCR plate: one contribution is due to the direct illumination of the whole internal surface; two contributions originate by the impinging rays that are simply reflected by the other two internal surfaces; and two contributions are related to the impinging rays that undergo two internal reflections. It is useful to note that the six contributions due to the

  6. Utility of anti-HSP 70, TNF-alpha, ESR, antinuclear antibody, and antiphospholipid antibodies in the diagnosis and treatment of sudden sensorineural hearing loss.

    PubMed

    Süslü, Nilda; Yilmaz, Taner; Gürsel, Bülent

    2009-02-01

    To investigate the performance of various laboratory tests used for patients with sudden sensorineural hearing loss (SSNHL). Prospective clinical trial. Thirty patients who presented with SSNHL and 30 healthy people with no cochleovestibular disorders were selected as study and control groups. The laboratory panel includes the following tests: anti-HSP 70 antibody immunoassay, tumor necrosis factor-alpha (TNF-alpha), erythrocyte sedimentation rate (ESR), antinuclear antibody (ANA), and antiphospholipid antibodies. The study group was given corticosteroid therapy and separated into two groups: the corticosteroid responders and the corticosteroid nonresponders. In the follow-up, repeat audiograms were evaluated to determine the response to treatment. TNF-alpha was found at lower titers in the study group when compared with the control group in contrast to other studies. Also, anti-HSP 70 was not found in high titers in the study group. ANA and ESR were the two parameters that were significantly more positive in the study group compared with the control group. Because of the lack of association between a positive test and response to corticosteroid treatment, detection of the anti-HSP 70 antibody, TNF-alpha, ESR, and ANA, at present, do not offer clinically useful information in the treatment of SSNHL. Also, because of the lower titers of TNF-alpha documented in patients with SSNHL, we do not recommend the use of specific TNF-alpha inhibitors in SSNHL.

  7. Variability and repertoire size of T-cell receptor V alpha gene segments.

    PubMed

    Becker, D M; Pattern, P; Chien, Y; Yokota, T; Eshhar, Z; Giedlin, M; Gascoigne, N R; Goodnow, C; Wolf, R; Arai, K

    The immune system of higher organisms is composed largely of two distinct cell types, B lymphocytes and T lymphocytes, each of which is independently capable of recognizing an enormous number of distinct entities through their antigen receptors; surface immunoglobulin in the case of the former, and the T-cell receptor (TCR) in the case of the latter. In both cell types, the genes encoding the antigen receptors consist of multiple gene segments which recombine during maturation to produce many possible peptides. One striking difference between B- and T-cell recognition that has not yet been resolved by the structural data is the fact that T cells generally require a major histocompatibility determinant together with an antigen whereas, in most cases, antibodies recognize antigen alone. Recently, we and others have found that a series of TCR V beta gene sequences show conservation of many of the same residues that are conserved between heavy- and light-chain immunoglobulin V regions, and these V beta sequences are predicted to have an immunoglobulin-like secondary structure. To extend these studies, we have isolated and sequenced eight additional alpha-chain complementary cDNA clones and compared them with published sequences. Analyses of these sequences, reported here, indicate that V alpha regions have many of the characteristics of V beta gene segments but differ in that they almost always occur as cross-hybridizing gene families. We conclude that there may be very different selective pressures operating on V alpha and V beta sequences and that the V alpha repertoire may be considerably larger than that of V beta.

  8. Unique ζ-chain motifs mediate a direct TCR-actin linkage critical for immunological synapse formation and T-cell activation.

    PubMed

    Klieger, Yair; Almogi-Hazan, Osnat; Ish-Shalom, Eliran; Pato, Aviad; Pauker, Maor H; Barda-Saad, Mira; Wang, Lynn; Baniyash, Michal

    2014-01-01

    TCR-mediated activation induces receptor microclusters that evolve to a defined immune synapse (IS). Many studies showed that actin polymerization and remodeling, which create a scaffold critical to IS formation and stabilization, are TCR mediated. However, the mechanisms controlling simultaneous TCR and actin dynamic rearrangement in the IS are yet not fully understood. Herein, we identify two novel TCR ζ-chain motifs, mediating the TCR's direct interaction with actin and inducing actin bundling. While T cells expressing the ζ-chain mutated in these motifs lack cytoskeleton (actin) associated (cska)-TCRs, they express normal levels of non-cska and surface TCRs as cells expressing wild-type ζ-chain. However, such mutant cells are unable to display activation-dependent TCR clustering, IS formation, expression of CD25/CD69 activation markers, or produce/secrete cytokine, effects also seen in the corresponding APCs. We are the first to show a direct TCR-actin linkage, providing the missing gap linking between TCR-mediated Ag recognition, specific cytoskeleton orientation toward the T-cell-APC interacting pole and long-lived IS maintenance. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Intravenous Versus Subcutaneous Anti-TNF-Alpha Agents for Crohn's Disease: A Comparison of Effectiveness and Safety.

    PubMed

    Liu, Jinan; Sylwestrzak, Gosia; Ruggieri, Alexander P; DeVries, Andrea

    2015-07-01

    In recent years, there have been a number of pharmacological innovations for Crohn's disease (CD), a difficult-to-treat condition, including new treatment philosophies (e.g., top-down therapy) and new therapeutic options in terms of the agent and the route of administration. Three anti-tumor necrosis factor (anti-TNF-alpha) agents are available for use among CD patients in the United States: infliximab, an intravenous agent, and adalimumab and certolizumab pegol, 2 newer subcutaneous products. Infliximab is considered the "gold standard" because it has the longest clinical experience, and adalimumab and certolizumab pegol have each gained significant market share. To examine differences in effectiveness and safety between currently available intravenous and subcutaneous anti-TNF-alpha agents used to treat patients with CD. Data for this retrospective, administrative claims analysis were obtained from pharmacy and medical claims from major U.S. health plans geographically dispersed across 14 states during 2007-2011. Patients had at least 1 ICD-9-CM diagnosis for CD, 6 months pre-index eligibility, and initiated anti-TNF-alpha therapy on the index date. Patients in each cohort were propensity score matched on pre-index demographics, clinical characteristics, and baseline health care use. During the post-index period, age-sex adjusted incidence rate ratios (IRRs) of CD-related symptoms, infections, cancers, and hepatic-related conditions were compared using Cox (PH) models. The matched cohorts included 515 patients in each group, with an average age of 39 years. Median follow-up was 17.5 months in the intravenous cohort and 17.7 months in the subcutaneous cohort. In terms of effectiveness outcomes, age-sex adjusted IRRs for the subcutaneous group, with the intravenous cohort as a reference, were as follows: 0.61 (95% CI = 0.32-1.18, P = 0.14) for anal fissures; 0.97 (95% CI = 0.72-1.30, P = 0.85) for abscess; 1.08 (95% CI = 0.79-1.04, P = 0

  10. Arthritis is inhibited in Borrelia-primed and infected interleukin-17A-deficient mice after administration of anti-gamma-interferon, anti-tumor necrosis factor alpha and anti-interleukin-6 antibodies.

    PubMed

    Kuo, Joseph; Warner, Thomas F; Schell, Ronald F

    2017-08-31

    The role that cytokines play in the induction of Lyme arthritis is gradually being delineated. We showed previously that severe arthritis developed in a T-cell-driven murine model, even in mice lacking interleukin-17A (IL-17A) and administered anti-gamma-interferon (IFN-γ) antibody. Increased levels of tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6), two pro-inflammatory cytokines, were detected in cultures of popliteal lymph node cells obtained from these mice. We hypothesized that concomitantly administered anti-IL-6, anti-TNF-α and anti-IFN-γ antibodies would inhibit the development of arthritis in IL-17A-deficient mice. Our results showed that swelling of the hind paws and histopathological changes consistent with arthritis were significantly reduced in IL-17A-deficient mice that administered the three anti-cytokine antibodies. These results suggest that treatment with multiple anti-cytokine antibodies can abrogate the induction of Lyme arthritis in mice. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  11. Histochemistry of nerve fibres double labelled with anti-TRPV2 antibodies and sensory nerve marker AM1-43 in the dental pulp of rat molars.

    PubMed

    Nishikawa, Sumio

    2008-09-01

    AM1-43 can label sensory nerve fibres and sensory neurons. Permeation of non-selective cation channels of the nerve cell membrane is suggested to be the mechanism responsible for labelling. To identify these channels, two candidates, TRPV1 and TRPV2 were examined by immunocytochemistry in the dental pulp and trigeminal ganglion of rats injected with AM1-43. A part of AM1-43-labelled nerve fibres was also positive for anti-TRPV2 antibody but negative for anti-TRPV1 antibody in the dental pulp. In the trigeminal ganglion, a part of the neuron showed both bright AM1-43 labelling and anti-TRPV2 immunolabelling, but neurons double labelled with AM1-43 and TRPV1 were rare. These results suggest that TRPV2 channels, but not TRPV1 channels, contribute to the fluorescent labelling of AM1-43 in the dental pulp.

  12. Abundant cytomegalovirus (CMV) reactive clonotypes in the CD8(+) T cell receptor alpha repertoire following allogeneic transplantation.

    PubMed

    Link, C S; Eugster, A; Heidenreich, F; Rücker-Braun, E; Schmiedgen, M; Oelschlägel, U; Kühn, D; Dietz, S; Fuchs, Y; Dahl, A; Domingues, A M J; Klesse, C; Schmitz, M; Ehninger, G; Bornhäuser, M; Schetelig, J; Bonifacio, E

    2016-06-01

    Allogeneic stem cell transplantation is potentially curative, but associated with post-transplantation complications, including cytomegalovirus (CMV) infections. An effective immune response requires T cells recognizing CMV epitopes via their T cell receptors (TCRs). Little is known about the TCR repertoire, in particular the TCR-α repertoire and its clinical relevance in patients following stem cell transplantation. Using next-generation sequencing we examined the TCR-α repertoire of CD8(+) T cells and CMV-specific CD8(+) T cells in four patients. Additionally, we performed single-cell TCR-αβ sequencing of CMV-specific CD8(+) T cells. The TCR-α composition of human leucocyte antigen (HLA)-A*0201 CMVpp65- and CMVIE -specific T cells was oligoclonal and defined by few dominant clonotypes. Frequencies of single clonotypes reached up to 11% of all CD8(+) T cells and half of the total CD8(+) T cell repertoire was dominated by few CMV-reactive clonotypes. Some TCR-α clonotypes were shared between patients. Gene expression of the circulating CMV-specific CD8(+) T cells was consistent with chronically activated effector memory T cells. The CD8(+) T cell response to CMV reactivation resulted in an expansion of a few TCR-α clonotypes to dominate the CD8(+) repertoires. These results warrant further larger studies to define the ability of oligoclonally expanded T cell clones to achieve an effective anti-viral T cell response in this setting. © 2016 British Society for Immunology.

  13. Hepatitis B vaccination effective in children exposed to anti-TNF alpha in utero.

    PubMed

    de Lima, Alison; Kanis, Shannon L; Escher, Johanna C; van der Woude, C Janneke

    2018-05-03

    Neonates exposed to TNF alpha inhibitors in utero are born with detectable drug levels which can still be detected throughout the first year of life. Since 2011, the hepatitis B virus (HBV) vaccine is routinely administered to all newborns in the Netherlands. Adults treated with anti-TNF have been reported to respond inadequately to the HBV vaccine. The aim of this study was to compare anti-HBs levels in anti-TNF exposed children with non- exposed children following routine Dutch HBV vaccination. We performed a cross-sectional, controlled cohort study from 2014-2017 in a single, tertiary referral center. Pregnant women treated with anti-TNF for Inflammatory Bowel Disease (IBD) and their subsequent children were recruited from the IBD preconception outpatient clinic. Pregnant women not treated with anti-TNF for IBD and their subsequent children were eligible as controls. Adherence to the Dutch National Vaccination Programme was mandatory for participation in this study. A venous blood sample was obtained one month after final HBV vaccination. Anti-HBs levels were measured by ELISA. Anti-HBs levels at 12 months did not differ between the anti-TNF exposed (n=15) and the control group (n=12) (>1000 IU/L vs >1000 IU/L, p=0.59). All children were successfully immunised against HBV, defined as anti-HBs>10 IU/L. Median anti-TNF levels determined in cord blood at birth were 9.0 µg/mL (IQR: 3.0-15.0 µg/mL) for IFX and 0.4. µg/mL (IQR: 0.3-0.6 µg/mL) for ADA. There were no differences in general birth and health outcomes. Children born with detectable anti-TNF levels can be effectively vaccinated against HBV.

  14. Antigen sensitivity of CD22-specific chimeric TCR is modulated by target epitope distance from the cell membrane.

    PubMed

    James, Scott E; Greenberg, Philip D; Jensen, Michael C; Lin, Yukang; Wang, Jinjuan; Till, Brian G; Raubitschek, Andrew A; Forman, Stephen J; Press, Oliver W

    2008-05-15

    We have targeted CD22 as a novel tumor-associated Ag for recognition by human CTL genetically modified to express chimeric TCR (cTCR) recognizing this surface molecule. CD22-specific cTCR targeting different epitopes of the CD22 molecule promoted efficient lysis of target cells expressing high levels of CD22 with a maximum lytic potential that appeared to decrease as the distance of the target epitope from the target cell membrane increased. Targeting membrane-distal CD22 epitopes with cTCR(+) CTL revealed defects in both degranulation and lytic granule targeting. CD22-specific cTCR(+) CTL exhibited lower levels of maximum lysis and lower Ag sensitivity than CTL targeting CD20, which has a shorter extracellular domain than CD22. This diminished sensitivity was not a result of reduced avidity of Ag engagement, but instead reflected weaker signaling per triggered cTCR molecule when targeting membrane-distal epitopes of CD22. Both of these parameters were restored by targeting a ligand expressing the same epitope, but constructed as a truncated CD22 molecule to approximate the length of a TCR:peptide-MHC complex. The reduced sensitivity of CD22-specific cTCR(+) CTL for Ag-induced triggering of effector functions has potential therapeutic applications, because such cells selectively lysed B cell lymphoma lines expressing high levels of CD22, but demonstrated minimal activity against autologous normal B cells, which express lower levels of CD22. Thus, our results demonstrate that cTCR signal strength, and consequently Ag sensitivity, can be modulated by differential choice of target epitopes with respect to distance from the cell membrane, allowing discrimination between targets with disparate Ag density.

  15. Myocardial and vascular adrenergic alterations in a rat model of endotoxin shock: reversal by an anti-tumor necrosis factor-alpha monoclonal antibody.

    PubMed

    Boillot, A; Massol, J; Maupoil, V; Grelier, R; Bernard, B; Capellier, G; Berthelot, A; Barale, F

    1997-03-01

    a) To investigate responsiveness to exogenous catecholamines in rat endotoxin shock by studying both myocardial and vascular functional parameters, and to determine the relationship of these parameters with other relevant biological parameters of the adrenergic pathway, such as myocardial beta-adrenergic receptors and cyclic adenosine monophosphate (cAMP); b) to investigate the role of tumor necrosis factor (TNF)-alpha via prophylactic anti-TNF-alpha monoclonal antibody administration. Experimental, comparative hospital. Laboratory in a university hospital. Male Sprague-Dawley rats, weighing 280 to 340 g. Intravenous injection of Escherichia coli endotoxin (5 mg/100 g) in the first group; injection of the same dose of endotoxin preceded by 2 mg/100 g of anti-TNF-alpha monoclonal antibody in the second group; injection of saline in the third (control) group. TNF-alpha concentration was measured before and during the first 3 hrs in all three groups. Myocardial and vascular functional parameters were obtained, respectively, from Langendorff perfused hearts and isolated aortic rings. Adrenergic biochemical parameters (catecholamines, density and affinity of beta-receptors, and isoproterenol-stimulated myocardial cAMP) were determined 3 hrs after injections in the three groups. After endotoxin injection, serum TNF-alpha concentrations peaked at 60 mins (2496 +/- 412 pg/mL) and returned slowly to control values at 3 hrs; serum TNF-alpha concentrations remained under the limit of detection in the other two groups. When compared with the control group, plasma concentrations of epinephrine and norepinephrine were significantly (p < .05) increased. Baseline values for differential left ventricular pressure and coronary flow were significantly (p < .001, p < .01, respectively) reduced in the endotoxin group; heart rate remained unchanged. In the endotoxin and control groups, isoproterenol induced a similar increase in differential left ventricular pressure and in heart rate

  16. Differential cytokine modulation and T cell activation by two distinct classes of thalidomide analogues that are potent inhibitors of TNF-alpha.

    PubMed

    Corral, L G; Haslett, P A; Muller, G W; Chen, R; Wong, L M; Ocampo, C J; Patterson, R T; Stirling, D I; Kaplan, G

    1999-07-01

    TNF-alpha mediates both protective and detrimental manifestations of the host immune response. Our previous work has shown thalidomide to be a relatively selective inhibitor of TNF-alpha production in vivo and in vitro. Additionally, we have recently reported that thalidomide exerts a costimulatory effect on T cell responses. To develop thalidomide analogues with increased anti-TNF-alpha activity and reduced or absent toxicities, novel TNF-alpha inhibitors were designed and synthesized. When a selected group of these compounds was examined for their immunomodulatory activities, different patterns of cytokine modulation were revealed. The tested compounds segregated into two distinct classes: one class of compounds, shown to be potent phosphodiesterase 4 inhibitors, inhibited TNF-alpha production, increased IL-10 production by LPS-induced PBMC, and had little effect on T cell activation; the other class of compounds, similar to thalidomide, were not phosphodiesterase 4 inhibitors and markedly stimulated T cell proliferation and IL-2 and IFN-gamma production. These compounds inhibited TNF-alpha, IL-1beta, and IL-6 and greatly increased IL-10 production by LPS-induced PBMC. Similar to thalidomide, the effect of these agents on IL-12 production was dichotomous; IL-12 was inhibited when PBMC were stimulated with LPS but increased when cells were stimulated by cross-linking the TCR. The latter effect was associated with increased T cell CD40 ligand expression. The distinct immunomodulatory activities of these classes of thalidomide analogues may potentially allow them to be used in the clinic for the treatment of different immunopathological disorders.

  17. Inhibition of Gαs/cAMP Signaling Decreases TCR-Stimulated IL-2 transcription in CD4(+) T Helper Cells.

    PubMed

    Hynes, Thomas R; Yost, Evan A; Yost, Stacy M; Hartle, Cassandra M; Ott, Braden J; Berlot, Catherine H

    2015-07-06

    The role of cAMP in regulating T cell activation and function has been controversial. cAMP is generally known as an immunosuppressant, but it is also required for generating optimal immune responses. As the effect of cAMP is likely to depend on its cellular context, the current study investigated whether the mechanism of activation of Gαs and adenylyl cyclase influences their effect on T cell receptor (TCR)-stimulated interleukin-2 (IL-2) mRNA levels. The effect of blocking Gs-coupled receptor (GsPCR)-mediated Gs activation on TCR-stimulated IL-2 mRNA levels in CD4(+) T cells was compared with that of knocking down Gαs expression or inhibiting adenylyl cyclase activity. The effect of knocking down Gαs expression on TCR-stimulated cAMP accumulation was compared with that of blocking GsPCR signaling. ZM-241385, an antagonist to the Gs-coupled A2A adenosine receptor (A2AR), enhanced TCR-stimulated IL-2 mRNA levels in primary human CD4(+) T helper cells and in Jurkat T cells. A dominant negative Gαs construct, GαsDN3, also enhanced TCR-stimulated IL-2 mRNA levels. Similar to GsPCR antagonists, GαsDN3 blocked GsPCR-dependent activation of both Gαs and Gβγ. In contrast, Gαs siRNA and 2',5'-dideoxyadenosine (ddA), an adenylyl cyclase inhibitor, decreased TCR-stimulated IL-2 mRNA levels. Gαs siRNA, but not GαsDN3, decreased TCR-stimulated cAMP synthesis. Potentiation of IL-2 mRNA levels by ZM-241385 required at least two days of TCR stimulation, and addition of ddA after three days of TCR stimulation enhanced IL-2 mRNA levels. GsPCRs play an inhibitory role in the regulation of TCR-stimulated IL-2 mRNA levels whereas Gαs and cAMP can play a stimulatory one. Additionally, TCR-dependent activation of Gαs does not appear to involve GsPCRs. These results suggest that the context of Gαs/cAMP activation and the stage of T cell activation and differentiation determine the effect on TCR-stimulated IL-2 mRNA levels.

  18. Alpha-v Integrin Targeted PET Imaging of Breast Cancer Angiogenesis and Low-Dose Metronomic Anti-Angiogenic Chemotherapy Efficacy

    DTIC Science & Technology

    2006-08-01

    64Cu -Labeled Abegrin™, a Humanized Monoclonal Antibody against Integrin αvβ3 Cancer Res. 2006;66(19):9673-81. 11. Hsu AR, Hou LC, Veeravagu A...FL, March, 2006. 5. Wu Y, Cai W, Zhang X, Chen K, Cao Q, Tice D, Chen X. In Vitro and In Vivo Characterization of 64Cu -Labeled Vitaxin, a...53rd SNM Annual meeting, San Diego, CA, June 2006 9. Cai W, Wu Y, Cao Q, Chen K, Zhang X, Tice D, Chen X 64Cu -Labeled Humanized Anti

  19. Gallium-68-labeled anti-HER2 single-chain Fv fragment: development and in vivo monitoring of HER2 expression.

    PubMed

    Ueda, Masashi; Hisada, Hayato; Temma, Takashi; Shimizu, Yoichi; Kimura, Hiroyuki; Ono, Masahiro; Nakamoto, Yuji; Togashi, Kaori; Saji, Hideo

    2015-02-01

    We aimed to develop a gallium-68 (Ga-68)-labeled single-chain variable fragment (scFv) targeting the human epidermal growth factor receptor 2 (HER2) to rapidly and noninvasively evaluate the status of HER2 expression. Anti-HER2 scFv was labeled with Ga-68 by using deferoxamine (Df) as a bifunctional chelate. Biodistribution of [(68)Ga]Df-anti-HER2 scFv was examined with tumor-bearing mice and positron emission tomography (PET) imaging was performed. The changes in HER2 expression after anti-HER2 therapy were monitored by PET imaging. [(68)Ga]Df-anti-HER2 scFv was obtained with high radiochemical yield after only a 5-min reaction at room temperature. The probe showed high accumulation in HER2-positive xenografts and the intratumoral distribution of radioactivity coincided with HER2-positive regions. Furthermore, [(68)Ga]Df-anti-HER2 scFv helped visualize HER2-positive xenografts and monitor the changes in HER2 expression after anti-HER2 therapy. [(68)Ga]Df-anti-HER2 scFv could be a promising probe to evaluate HER2 status by in vivo PET imaging, unless trastuzumab is prescribed as part of the therapy.

  20. Isolation of human hexosaminidase. cap alpha. cDNA and expression of. cap alpha. chains in E. coli

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wiktorowicz, J.E.; Whitman, J.M.

    1986-05-01

    Pooled antisera against homogeneous, glutaraldehyde cross-linked hexosaminidase (hex) A was adsorbed with E. coli lysate insolubilized on Sepharose 4B. Aliquots of a human liver lambdagtll cDNA library (50,000-100,000 pfu) were plated on E. coli Y1090. Expression of cloned cDNA, after sufficient plaque growth at 42/sup 0/, was accomplished by induction with isopropylthiogalactoside soaked nitrocellulose filters. Identification of hex cDNA clones was performed by incubation of the filters with purified antisera. Protein A labelled with I-125 was used to develop the reactive plaques. Positive plaques, identified by autoradiography, were picked, replated at a lower density, and rescreened. This was repeated severalmore » more times until all plaques yielded positive signals. Identification of the clones as containing ..cap alpha.. or ..beta.. cDNA was accomplished by replating the purified phage and rescreening the plaques with anti-hex B antiserum preadsorbed with E. coli lysate. According to this protocol several hex ..cap alpha.. clones have been identified. While these clones generate ..beta..-galactosidase: hex ..cap alpha.. fusion proteins, these findings suggest that in the future it may be possible to obtain large quantities of unmodified hex ..cap alpha.. and ..beta.. polypeptides from E. coli for the study of the structural and enzymatic properties of these polypeptides and for diagnostic purposes in the GM2 gangliosidoses.« less

  1. Characterization of Alpha-Toxin hla Gene Variants, Alpha-Toxin Expression Levels, and Levels of Antibody to Alpha-Toxin in Hemodialysis and Postsurgical Patients with Staphylococcus aureus Bacteremia

    PubMed Central

    Wu, Yuling; Tabor, David E.; Mok, Hoyin; Sellman, Bret R.; Jenkins, Amy; Yu, Li; Jafri, Hasan S.; Rude, Thomas H.; Ruffin, Felicia; Schell, Wiley A.; Park, Lawrence P.; Yan, Qin; Thaden, Joshua T.; Messina, Julia A.; Esser, Mark T.

    2014-01-01

    Alpha-toxin is a major Staphylococcus aureus virulence factor. This study evaluated potential relationships between in vitro alpha-toxin expression of S. aureus bloodstream isolates, anti-alpha-toxin antibody in serum of patients with S. aureus bacteremia (SAB), and clinical outcomes in 100 hemodialysis and 100 postsurgical SAB patients. Isolates underwent spa typing and hla sequencing. Serum anti-alpha-toxin IgG and neutralizing antibody levels were measured by using an enzyme-linked immunosorbent assay and a red blood cell (RBC)-based hemolysis neutralization assay. Neutralization of alpha-toxin by an anti-alpha-toxin monoclonal antibody (MAb MEDI4893) was tested in an RBC-based lysis assay. Most isolates encoded hla (197/200; 98.5%) and expressed alpha-toxin (173/200; 86.5%). In vitro alpha-toxin levels were inversely associated with survival (cure, 2.19 μg/ml, versus failure, 1.09 μg/ml; P < 0.01). Both neutralizing (hemodialysis, 1.26 IU/ml, versus postsurgical, 0.95; P < 0.05) and IgG (hemodialysis, 1.94 IU/ml, versus postsurgical, 1.27; P < 0.05) antibody levels were higher in the hemodialysis population. Antibody levels were also significantly higher in patients infected with alpha-toxin-expressing S. aureus isolates (P < 0.05). Levels of both neutralizing antibodies and IgG were similar among patients who were cured and those not cured (failures). Sequence analysis of hla revealed 12 distinct hla genotypes, and all genotypic variants were susceptible to a neutralizing monoclonal antibody in clinical development (MEDI4893). These data demonstrate that alpha-toxin is highly conserved in clinical S. aureus isolates. Higher in vitro alpha-toxin levels were associated with a positive clinical outcome. Although patients infected with alpha-toxin-producing S. aureus exhibited higher anti-alpha-toxin antibody levels, these levels were not associated with a better clinical outcome in this study. PMID:25392350

  2. Regional survey of tuberculosis risk assessment in rheumatology outpatients commencing anti-TNF-alpha treatment in relation to British Thoracic Society guidelines.

    PubMed

    John, H; Buckley, C; Koh, L; Obrenovic, K; Erb, N; Rowe, I F

    2009-06-01

    The aim of this study was to analyse tuberculosis (TB) risk assessment for rheumatology patients commencing anti-tumour necrosis factor-alpha (anti-TNF-alpha) therapy using the British Thoracic Society (BTS) guidelines. Data were obtained retrospectively on 856 outpatients regionally receiving anti-TNF-alpha. Prior to commencing treatment, patients had the following assessments documented: respiratory examination, 47.4%; chest X-ray, 84.5%; TB history, 92.9%; and advice about TB risk, 45.8%. Of the 856 patients, 94.3% were on immunosuppressives but 27% had a tuberculin test; 12.6% had > or =1 high-risk factors for TB. In total, 3.4% were referred to a TB specialist and of these, 24.1% had no risk factors for TB. Of patients with > or =1 risk factor, 76.9% were not referred. Only 4/28 patients at high risk for TB due to ethnicity or birthplace received chemoprophylaxis. Marked inter-unit variation was demonstrated and it was evident that patients require improved screening for TB. Greater awareness is necessary of patients with risk factors, particularly ethnicity, to facilitate more appropriate targeting of chemoprophylaxis. Multi-centre audit is a valuable clinical governance tool.

  3. High-throughput sequencing of TCR repertoires in multiple sclerosis reveals intrathecal enrichment of EBV-reactive CD8+ T cells.

    PubMed

    Lossius, Andreas; Johansen, Jorunn N; Vartdal, Frode; Robins, Harlan; Jūratė Šaltytė, Benth; Holmøy, Trygve; Olweus, Johanna

    2014-11-01

    Epstein-Barr virus (EBV) has long been suggested as a pathogen in multiple sclerosis (MS). Here, we used high-throughput sequencing to determine the diversity, compartmentalization, persistence, and EBV-reactivity of the T-cell receptor (TCR) repertoires in MS. TCR-β genes were sequenced in paired samples of cerebrospinal fluid (CSF) and blood from patients with MS and controls with other inflammatory neurological diseases. The TCR repertoires were highly diverse in both compartments and patient groups. Expanded T-cell clones, represented by TCR-β sequences >0.1%, were of different identity in CSF and blood of MS patients, and persisted for more than a year. Reference TCR-β libraries generated from peripheral blood T cells reactive against autologous EBV-transformed B cells were highly enriched for public EBV-specific sequences and were used to quantify EBV-reactive TCR-β sequences in CSF. TCR-β sequences of EBV-reactive CD8+ T cells, including several public EBV-specific sequences, were intrathecally enriched in MS patients only, whereas those of EBV-reactive CD4+ T cells were also enriched in CSF of controls. These data provide evidence for a clonally diverse, yet compartmentalized and persistent, intrathecal T-cell response in MS. The presented strategy links TCR sequence to intrathecal T-cell specificity, demonstrating enrichment of EBV-reactive CD8+ T cells in MS. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Increased Peptide Contacts Govern High Affinity Binding of a Modified TCR Whilst Maintaining a Native pMHC Docking Mode

    PubMed Central

    Cole, David K.; Sami, Malkit; Scott, Daniel R.; Rizkallah, Pierre J.; Borbulevych, Oleg Y.; Todorov, Penio T.; Moysey, Ruth K.; Jakobsen, Bent K.; Boulter, Jonathan M.; Baker, Brian M.; Yi Li

    2013-01-01

    Natural T cell receptors (TCRs) generally bind to their cognate pMHC molecules with weak affinity and fast kinetics, limiting their use as therapeutic agents. Using phage display, we have engineered a high affinity version of the A6 wild-type TCR (A6wt), specific for the human leukocyte antigen (HLA-A∗0201) complexed with human T cell lymphotropic virus type 111–19 peptide (A2-Tax). Mutations in just 4 residues in the CDR3β loop region of the A6wt TCR were selected that improved binding to A2-Tax by nearly 1000-fold. Biophysical measurements of this mutant TCR (A6c134) demonstrated that the enhanced binding was derived through favorable enthalpy and a slower off-rate. The structure of the free A6c134 TCR and the A6c134/A2-Tax complex revealed a native binding mode, similar to the A6wt/A2-Tax complex. However, concordant with the more favorable binding enthalpy, the A6c134 TCR made increased contacts with the Tax peptide compared with the A6wt/A2-Tax complex, demonstrating a peptide-focused mechanism for the enhanced affinity that directly involved the mutated residues in the A6c134 TCR CDR3β loop. This peptide-focused enhanced TCR binding may represent an important approach for developing antigen specific high affinity TCR reagents for use in T cell based therapies. PMID:23805144

  5. The CD3-Zeta Chimeric Antigen Receptor Overcomes TCR Hypo-Responsiveness of Human Terminal Late-Stage T Cells

    PubMed Central

    Awerkiew, Sabine; Schmidt, Annette; Hombach, Andreas A.; Pfister, Herbert; Abken, Hinrich

    2012-01-01

    Adoptive therapy of malignant diseases with tumor-specific cytotoxic T cells showed remarkable efficacy in recent trials. Repetitive T cell receptor (TCR) engagement of target antigen, however, inevitably ends up in hypo-responsive cells with terminally differentiated KLRG-1+ CD57+ CD7− phenotype limiting their therapeutic efficacy. We here revealed that hypo-responsiveness of CMV-specific late-stage CD8+ T cells is due to reduced TCR synapse formation compared to younger cells. Membrane anchoring of TCR components contributes to T cell hypo-responsiveness since dislocation of galectin-3 from the synapse by swainsonine restored both TCR synapse formation and T cell response. Transgenic expression of a CD3-zeta signaling chimeric antigen receptor (CAR) recovered hypo-responsive T cells to full effector functions indicating that the defect is restricted to TCR membrane components while synapse formation of the transgenic CAR was not blocked. CAR engineered late-stage T cells released cytokines and mediated redirected cytotoxicity as efficiently as younger effector T cells. Our data provide a rationale for TCR independent, CAR mediated activation in the adoptive cell therapy to avoid hypo-responsiveness of late-stage T cells upon repetitive antigen encounter. PMID:22292024

  6. Efficacy of anti-TNF alpha in severe and/or refractory Behçet's disease: Multicenter study of 124 patients.

    PubMed

    Vallet, H; Riviere, S; Sanna, A; Deroux, A; Moulis, G; Addimanda, O; Salvarani, C; Lambert, M; Bielefeld, P; Seve, P; Sibilia, J; Pasquali, Jl; Fraison, Jb; Marie, I; Perard, L; Bouillet, L; Cohen, F; Sene, D; Schoindre, Y; Lidove, O; Le Hoang, P; Hachulla, E; Fain, O; Mariette, X; Papo, T; Wechsler, B; Bodaghi, B; Rigon, M Resche; Cacoub, P; Saadoun, D

    2015-08-01

    To report the efficacy and safety of anti-TNF agents in patients with severe and/or refractory manifestations of Behçet's disease (BD). We performed a multicenter study of main characteristics and outcomes of anti-TNF alpha treatments [mainly infliximab (62%), and adalimumab (30%)] in 124 BD patients [48% of men; median age of 33.5 (28-40) years]. Overall response (i.e. complete and partial) rate was 90.4%. Clinical responses were observed in 96.3%, 88%, 70%, 77.8%, 92.3% and 66.7% of patients with severe and/or refractory ocular, mucocutaneous, joint, gastro-intestinal manifestations, central nervous system manifestations and cardiovascular manifestations, respectively. No significant difference was found with respect to the efficacy of anti-TNF used as monotherapy or in association with an immunosuppressive agent. The incidence of BD flares/patient/year was significantly lower during anti-TNF treatment (0.2 ± 0.5 vs 1.7 ± 2.4 before the use of anti-TNF, p < 0.0001). The prednisone dose was significantly reduced at 6 and 12 months (p < 0.0001). In multivariate analysis, retinal vasculitis was negatively associated with complete response to anti-TNF (OR = 0.33 [0.12-0.89]; p = 0.03). The efficacy and relapse free survival were similar regardless of the type of anti-TNF agent used. After a median follow-up of 21 [7-36] months, side effects were reported in 28% of patients, including infections (16.3%) and hypersensitivity reactions (4.1%). Serious adverse events were reported in 13% of cases. Anti-TNF alpha therapy is efficient in all severe and refractory BD manifestations. Efficacy appears to be similar regardless of the anti-TNF agent used (infliximab or adalimumab). Copyright © 2015 Elsevier Ltd. All rights reserved.

  7. Enforcement of γδ-lineage commitment by the pre-T-cell receptor in precursors with weak γδ-TCR signals.

    PubMed

    Zarin, Payam; Wong, Gladys W; Mohtashami, Mahmood; Wiest, David L; Zúñiga-Pflücker, Juan Carlos

    2014-04-15

    Developing thymocytes bifurcate from a bipotent precursor into αβ- or γδ-lineage T cells. Considering this common origin and the fact that the T-cell receptor (TCR) β-, γ-, and δ-chains simultaneously rearrange at the double negative (DN) stage of development, the possibility exists that a given DN cell can express and transmit signals through both the pre-TCR and γδ-TCR. Here, we tested this scenario by defining the differentiation outcomes and criteria for lineage choice when both TCR-β and γδ-TCR are simultaneously expressed in Rag2(-/-) DN cells via retroviral transduction. Our results showed that Rag2(-/-) DN cells expressing both TCRs developed along the γδ-lineage, down-regulated CD24 expression, and up-regulated CD73 expression, showed a γδ-biased gene-expression profile, and produced IFN-γ in response to stimulation. However, in the absence of Inhibitor of DNA-binding 3 expression and strong γδ-TCR ligand, γδ-expressing cells showed a lower propensity to differentiate along the γδ-lineage. Importantly, differentiation along the γδ-lineage was restored by pre-TCR coexpression, which induced greater down-regulation of CD24, higher levels of CD73, Nr4a2, and Rgs1, and recovery of functional competence to produce IFN-γ. These results confirm a requirement for a strong γδ-TCR ligand engagement to promote maturation along the γδ T-cell lineage, whereas additional signals from the pre-TCR can serve to enforce a γδ-lineage choice in the case of weaker γδ-TCR signals. Taken together, these findings further cement the view that the cumulative signal strength sensed by developing DN cells serves to dictate its lineage choice.

  8. PLAC1-specific TCR-engineered T cells mediate antigen-specific antitumor effects in breast cancer

    PubMed Central

    Li, Qiongshu; Liu, Muyun; Wu, Man; Zhou, Xin; Wang, Shaobin; Hu, Yuan; Wang, Youfu; He, Yixin; Zeng, Xiaoping; Chen, Junhui; Liu, Qubo; Xiao, Dong; Hu, Xiang; Liu, Weibin

    2018-01-01

    Placenta-specific 1 (PLAC1), a novel cancer-testis antigen (CTA), is expressed in a number of different human malignancies. It is frequently produced in breast cancer, serving a function in tumorigenesis. Adoptive immunotherapy using T cell receptor (TCR)-engineered T cells against CTA mediates objective tumor regression; however, to the best of our knowledge, targeting PLAC1 using engineered T cells has not yet been attempted. In the present study, the cDNAs encoding TCRα- and β-chains specific for human leukocyte antigen (HLA)-A*0201-restricted PLAC1 were cloned from a cytotoxic T-lymphocyte, generated by in vitro by the stimulation of CD8+ T cells using autologous HLA-A2+ dendritic cells loaded with a PLAC1-specific peptide (p28-36, VLCSIDWFM). The TCRα/β-chains were linked by a 2A peptide linker (TCRα-Thosea asigna virus-TCRβ), and the constructs were cloned into the lentiviral vector, followed by transduction into human cytotoxic (CD8+) T cells. The efficiency of transduction was up to 25.16%, as detected by PLAC1 multimers. TCR-transduced CD8+ T cells, co-cultured with human non-metastatic breast cancer MCF-7 cells (PLAC1+, HLA-A2+) and triple-negative breast cancer MDAMB-231 cells (PLAC1+, HLA-A2+), produced interferon γ and tumor necrosis factor α, suggesting TCR activation. Furthermore, the PLAC1 TCR-transduced CD8+ T cells efficiently and specifically identified and annihilated the HLA-A2+/PLAC1+ breast cancer cell lines in a lactate dehydrogenase activity assay. Western blot analysis demonstrated that TCR transduction stimulated the production of mitogen-activated protein kinase signaling molecules, extracellular signal-regulated kinases 1/2 and nuclear factor-κB, through phosphoinositide 3-kinase γ-mediated phosphorylation of protein kinase B in CD8+ T cells. Xenograft mouse assays revealed that PLAC1 TCR-transduced CD8+T cells significantly delayed the tumor progression in mice-bearing breast cancer compared with normal saline or negative

  9. Selection of Fecal Enterococci Exhibiting tcrB-Mediated Copper Resistance in Pigs Fed Diets Supplemented with Copper † ▿

    PubMed Central

    Amachawadi, R. G.; Shelton, N. W.; Shi, X.; Vinasco, J.; Dritz, S. S.; Tokach, M. D.; Nelssen, J. L.; Scott, H. M.; Nagaraja, T. G.

    2011-01-01

    Copper, as copper sulfate, is increasingly used as an alternative to in-feed antibiotics for growth promotion in weaned piglets. Acquired copper resistance, conferred by a plasmid-borne, transferable copper resistance (tcrB) gene, has been reported in Enterococcus faecium and E. faecalis. A longitudinal field study was undertaken to determine the relationship between copper supplementation and the prevalence of tcrB-positive enterococci in piglets. The study was done with weaned piglets, housed in 10 pens with 6 piglets per pen, fed diets supplemented with a normal (16.5 ppm; control) or an elevated (125 ppm) level of copper. Fecal samples were randomly collected from three piglets per pen on days 0, 14, 28, and 42 and plated on M-Enterococcus agar, and three enterococcal isolates were obtained from each sample. The overall prevalence of tcrB-positive enterococci was 21.1% (38/180) in piglets fed elevated copper and 2.8% (5/180) in the control. Among the 43 tcrB-positive isolates, 35 were E. faecium and 8 were E. faecalis. The mean MICs of copper for tcrB-negative and tcrB-positive enterococci were 6.2 and 22.2 mM, respectively. The restriction digestion of the genomic DNA of E. faecium or E. faecalis with S1 nuclease yielded a band of ∼194-kbp size to which both tcrB and the erm(B) gene probes hybridized. A conjugation assay demonstrated cotransfer of tcrB and erm(B) genes between E. faecium and E. faecalis strains. The higher prevalence of tcrB-positive enterococci in piglets fed elevated copper compared to that in piglets fed normal copper suggests that supplementation of copper in swine diets selected for resistance. PMID:21705534

  10. 21 CFR 660.28 - Labeling.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Blood Grouping Reagent § 660.28 Labeling. In... white, except that all or a portion of the final container label of the following Blood Grouping... panel. Blood grouping reagent Color of label paper Anti-A Blue. Anti-B Yellow. Slide and rapid tube test...

  11. 21 CFR 660.28 - Labeling.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Blood Grouping Reagent § 660.28 Labeling. In... white, except that all or a portion of the final container label of the following Blood Grouping... panel. Blood grouping reagent Color of label paper Anti-A Blue. Anti-B Yellow. Slide and rapid tube test...

  12. 21 CFR 660.28 - Labeling.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Blood Grouping Reagent § 660.28 Labeling. In... white, except that all or a portion of the final container label of the following Blood Grouping... panel. Blood grouping reagent Color of label paper Anti-A Blue. Anti-B Yellow. Slide and rapid tube test...

  13. 21 CFR 660.28 - Labeling.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Blood Grouping Reagent § 660.28 Labeling. In... white, except that all or a portion of the final container label of the following Blood Grouping... panel. Blood grouping reagent Color of label paper Anti-A Blue. Anti-B Yellow. Slide and rapid tube test...

  14. Fluorescent humanized anti-CEA antibody specifically labels metastatic pancreatic cancer in a patient-derived orthotopic xenograft (PDOX) mouse model

    NASA Astrophysics Data System (ADS)

    Lwin, Thinzar M.; Miyake, Kentaro; Murakami, Takashi; DeLong, Jonathan C.; Yazaki, Paul J.; Shivley, John E.; Clary, Bryan; Hoffman, Robert M.; Bouvet, Michael

    2018-03-01

    Specific tumor targeting can result in selective labeling of cancer in vivo for surgical navigation. In the present study, we show that the use of an anti-CEA antibody conjugated to the near-infrared (NIR) fluorescent dye, IRDye800CW, can selectively target and label pancreatic cancer and its metastases in a clinically relevant patient derived xenograft mouse model.

  15. Advanced generation anti-prostate specific membrane antigen designer T cells for prostate cancer immunotherapy.

    PubMed

    Ma, Qiangzhong; Gomes, Erica M; Lo, Agnes Shuk-Yee; Junghans, Richard P

    2014-02-01

    Adoptive immunotherapy by infusion of designer T cells (dTc) engineered with chimeric antigen receptors (CARs) for tumoricidal activity represents a potentially highly specific modality for the treatment of cancer. In this study, 2nd generation (gen) anti-prostate specific membrane antigen (PSMA) dTc were developed for improving the efficacy of previously developed 1st gen dTc for prostate cancer immunotherapy. The 1st gen dTc are modified with chimeric immunoglobulin-T cell receptor (IgTCR) while the 2nd gen dTc are engineered with an immunoglobulin-CD28-T cell receptor (IgCD28TCR), which incorporates a CD28 costimulatory signal for optimal T cell activation. A 2nd gen anti-PSMA IgCD28TCR CAR was constructed by inserting the CD28 signal domain into the 1st gen CAR. 1st and 2nd gen anti-PSMA dTc were created by transducing human T cells with anti-PSMA CARs and their antitumor efficacy was compared for specific activation on PSMA-expressing tumor contact, cytotoxicity against PSMA-expressing tumor cells in vitro, and suppression of tumor growth in an animal model. The 2nd gen dTc can be optimally activated to secrete larger amounts of cytokines such as IL2 and IFNγ than 1st gen and to proliferate more vigorously on PSMA-expressing tumor contact. More importantly, the 2nd gen dTc preserve the PSMA-specific cytotoxicity in vitro and suppress tumor growth in animal models with significant higher potency. Our results demonstrate that 2nd gen anti-PSMA designer T cells exhibit superior antitumor functions versus 1st gen, providing a rationale for advancing this improved agent toward clinical application in prostate cancer immunotherapy. © 2013 Wiley Periodicals, Inc.

  16. Definition of APC presentation of phosphoantigen (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate to Vgamma2Vdelta 2 TCR.

    PubMed

    Wei, Huiyong; Huang, Dan; Lai, Xiaomin; Chen, Meiling; Zhong, Weihua; Wang, Richard; Chen, Zheng W

    2008-10-01

    Although microbial (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) can activate primate Vgamma2Vdelta2 T cells, molecular mechanisms by which HMBPP interacts with Vgamma2Vdelta2 T cells remain poorly characterized. Here, we developed soluble, tetrameric Vgamma2Vdelta2 TCR of rhesus macaques to define HMBPP/APC interaction with Vgamma2Vdelta2 TCR. While exogenous HMBPP was associated with APC membrane in an appreciable affinity, the membrane-associated HMBPP readily bound to the Vgamma2Vdelta2 TCR tetramer. The Vgamma2Vdelta2 TCR tetramer was shown to bind stably to HMBPP presented on membrane by various APC cell lines from humans and nonhuman primates but not those from mouse, rat, or pig. The Vgamma2Vdelta2 TCR tetramer also bound to the membrane-associated HMBPP on primary monocytes, B cells and T cells. Consistently, endogenous phosphoantigen produced in Mycobacterium-infected dendritic cells was transported and presented on membrane, and bound stably to the Vgamma2Vdelta2 TCR tetramer. The capability of APC to present HMBPP for recognition by Vgamma2Vdelta2 TCR was diminished after protease treatment of APC. Thus, our studies elucidated an affinity HMBPP-APC association conferring stable binding to the Vgamma2Vdelta2 TCR tetramer and the protease-sensitive nature of phosphoantigen presentation. The findings defined APC presentation of phosphoantigen HMBPP to Vgamma2Vdelta2 TCR.

  17. CD25 and CD69 induction by α4β1 outside-in signalling requires TCR early signalling complex proteins

    PubMed Central

    Cimo, Ann-Marie; Ahmed, Zamal; McIntyre, Bradley W.; Lewis, Dorothy E.; Ladbury, John E.

    2013-01-01

    Distinct signalling pathways producing diverse cellular outcomes can utilize similar subsets of proteins. For example, proteins from the TCR (T-cell receptor) ESC (early signalling complex) are also involved in interferon-α receptor signalling. Defining the mechanism for how these proteins function within a given pathway is important in understanding the integration and communication of signalling networks with one another. We investigated the contributions of the TCR ESC proteins Lck (lymphocyte-specific kinase), ZAP-70 (ζ-chain-associated protein of 70 kDa), Vav1, SLP-76 [SH2 (Src homology 2)-domain-containing leukocyte protein of 76 kDa] and LAT (linker for activation of T-cells) to integrin outside-in signalling in human T-cells. Lck, ZAP-70, SLP-76, Vav1 and LAT were activated by α4β1 outside-in signalling, but in a manner different from TCR signalling. TCR stimulation recruits ESC proteins to activate the mitogen-activated protein kinase ERK (extracellular-signal-regulated kinase). α4β1 outside-in-mediated ERK activation did not require TCR ESC proteins. However, α4β1 outside-in signalling induced CD25 and co-stimulated CD69 and this was dependent on TCR ESC proteins. TCR and α4β1 outside-in signalling are integrated through the common use of TCR ESC proteins; however, these proteins display functionally distinct roles in these pathways. These novel insights into the cross-talk between integrin outside-in and TCR signalling pathways are highly relevant to the development of therapeutic strategies to overcome disease associated with T-cell deregulation. PMID:23758320

  18. Rab11-FIP3 Regulation of Lck Endosomal Traffic Controls TCR Signal Transduction.

    PubMed

    Bouchet, Jérôme; Del Río-Iñiguez, Iratxe; Vázquez-Chávez, Elena; Lasserre, Rémi; Agüera-González, Sonia; Cuche, Céline; McCaffrey, Mary W; Di Bartolo, Vincenzo; Alcover, Andrés

    2017-04-01

    The role of endosomes in receptor signal transduction is a long-standing question, which remains largely unanswered. The T cell Ag receptor and various components of its proximal signaling machinery are associated with distinct endosomal compartments, but how endosomal traffic affects T cell signaling remains ill-defined. In this article, we demonstrate in human T cells that the subcellular localization and function of the protein tyrosine kinase Lck depends on the Rab11 effector FIP3 (Rab11 family interacting protein-3). FIP3 overexpression or silencing and its ability to interact with Rab11 modify Lck subcellular localization and its delivery to the immunological synapse. Importantly, FIP3-dependent Lck localization controls early TCR signaling events, such as tyrosine phosphorylation of TCRζ, ZAP70, and LAT and intracellular calcium concentration, as well as IL-2 gene expression. Interestingly, FIP3 controls both steady-state and poststimulation phosphotyrosine and calcium levels. Finally, our findings indicate that FIP3 modulates TCR-CD3 cell surface expression via the regulation of steady-state Lck-mediated TCRζ phosphorylation, which in turn controls TCRζ protein levels. This may influence long-term T cell activation in response to TCR-CD3 stimulation. Therefore, our data underscore the importance of finely regulated endosomal traffic in TCR signal transduction and T cell activation leading to IL-2 production. Copyright © 2017 by The American Association of Immunologists, Inc.

  19. Human alpha-enolase from endothelial cells as a target antigen of anti-endothelial cell antibody in Behçet's disease.

    PubMed

    Lee, Kwang Hoon; Chung, Hae-Shin; Kim, Hyoung Sup; Oh, Sang-Ho; Ha, Moon-Kyung; Baik, Ja-Hyun; Lee, Sungnack; Bang, Dongsik

    2003-07-01

    To identify and recombine a protein of the human dermal microvascular endothelial cell (HDMEC) that specifically reacts with anti-endothelial cell antibody (AECA) in the serum of patients with Behçet's disease (BD), and to evaluate the usefulness of this protein in BD. The proteomics technique, with 2-dimensional gel electrophoresis and matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry, was used to identify and recombine HDMEC antigen. Western blotting and enzyme-linked immunosorbent assay (ELISA) of recombinant protein isolated by gene cloning were performed on serum from healthy controls, patients with BD, and patients with other rheumatic diseases (rheumatoid arthritis, systemic lupus erythematosus, and Wegener's granulomatosis). Eighteen of 40 BD patients had serum IgM antibody to HDMEC antigen. The purified protein that reacted with AECA in BD patient sera was found to be alpha-enolase by 2-dimensional gel electrophoresis followed by immunoblotting and MALDI-TOF mass spectrometry. Recombinant alpha-enolase protein was isolated and refined by gene cloning. On Western blots, AECA-positive IgM from the sera of patients with active BD reacted strongly with recombinant human alpha-enolase. BD patient sera positive for anti-alpha-enolase did not react with human gamma-enolase. On dot-blotting, reactivity to human alpha-enolase was detected only in the IgM-positive group. Fifteen of the 18 AECA-positive sera that were positive for the HDMEC antigen showed reactivity to recombinant alpha-enolase IgM antibody by ELISA. The alpha-enolase protein is the target protein of serum AECA in BD patients. This is the first report of the presence of IgM antibodies to alpha-enolase in endothelial cells from the serum of BD patients. Although further studies relating this protein to the pathogenesis of BD will be necessary, alpha-enolase and its antibody may prove useful in the development of new diagnostic and treatment modalities in BD.

  20. Melibiose permease and alpha-galactosidase of Escherichia coli: Identification by selective labeling using a T7 RNA polymerase/promoter expression system

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Pourcher, T.; Bassilana, M.; Sarkar, H.K.

    1990-01-23

    Identification and selective labeling of the melibiose permease and alpha-galactosidase in Escherichia coli, which are encoded by the melB and melA genes, respectively, have been accomplished by selectively labeling the two gene products with a T7 RNA polymerase expression system. Following generation of a novel EcoRI restriction site in the intergenic sequence between the two genes of the mel operon by oligonucleotide-directed, site-specific mutagenesis, melA and melB were separately inserted into plasmid pT7-6 of the T7 expression system. Expression of melB was markedly enhanced by placing a strong, synthetic ribosome binding site at an optimal distance upstream from the initiationmore » codon of melB. Expression of cloned gene products was characterized functionally and by performing autoradiographic analysis on total cell, inner membrane, and cytoplasmic proteins from cells pulse labeled with (35S)methionine in the presence of rifampicin and resolved by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The results first confirm that alpha-galactosidase is a cytoplasmic protein with an Mr of 50K; in contrast, the membrane-bound melibiose permease is identified as a protein with an apparent Mr of 39K, a value significantly higher than that of 30K previously suggested.« less

  1. In-depth Characterization of a TCR-specific Tracer for Sensitive Detection of Tumor-directed Transgenic T Cells by Immuno-PET.

    PubMed

    Yusufi, Nahid; Mall, Sabine; Bianchi, Henrique de Oliveira; Steiger, Katja; Reder, Sybille; Klar, Richard; Audehm, Stefan; Mustafa, Mona; Nekolla, Stephan; Peschel, Christian; Schwaiger, Markus; Krackhardt, Angela M; D'Alessandria, Calogero

    2017-01-01

    A number of different technologies have been developed to monitor in vivo the distribution of gene-modified T cells used in immunotherapy. Nevertheless, in-depth characterization of novel approaches with respect to sensitivity and clinical applicability are so far missing. We have previously described a novel method to track engineered human T cells in tumors using 89 Zr-Df-aTCRmu-F(ab') 2 targeting the murinized part of the TCR beta domain (TCRmu) of a transgenic TCR. Here, we performed an in-depth in vitro characterization of the tracer in terms of antigen affinity, immunoreactivity, influence on T-cell functionality and stability in vitro and in vivo . Of particular interest, we have developed diverse experimental settings to quantify TCR-transgenic T cells in vivo . Local application of 89 Zr-Df-aTCRmu-F(ab') 2 -labeled T cells in a spot-assay revealed signal detection down to approximately 1.8x10 4 cells. In a more clinically relevant model, NSG mice were intravenously injected with different numbers of transgenic T cells, followed by injection of the 89 Zr-Df-aTCRmu-F(ab') 2 tracer, PET/CT imaging and subsequent ex vivo T-cell quantification in the tumor. Using this setting, we defined a comparable detection limit of 1.0x10 4 T cells. PET signals correlated well to total numbers of transgenic T cells detected ex vivo independently of the engraftment rates observed in different individual experiments. Thus, these findings confirm the high sensitivity of our novel PET/CT T-cell tracking method and provide critical information about the quantity of transgenic T cells in the tumor environment suggesting our technology being highly suitable for further clinical translation.

  2. Anti-CD45 radioimmunotherapy using 211At with bone marrow transplantation prolongs survival in a disseminated murine leukemia model

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Orozco, Johnnie J.; Back, Tom; Kenoyer, Aimee L.

    2013-05-15

    Anti-CD45 Radioimmunotherapy using an Alpha-Emitting Radionuclide 211At Combined with Bone Marrow Transplantation Prolongs Survival in a Disseminated Murine Leukemia Model ABSTRACT Despite aggressive chemotherapy combined with hematopoietic cell transplant (HCT), many patients with acute myeloid leukemia (AML) relapse. Radioimmunotherapy (RIT) using antibodies (Ab) labeled primarily with beta-emitting radionuclides has been explored to reduce relapse.

  3. Preparation of goat and rabbit anti-camel immunoglobulin G whole molecule labeled with horseradish peroxidase

    PubMed Central

    Abdel-Rahman, Eman Hussein; El-Jakee, Jakeen Kamal; Hatem, Mahmoud Essam; Ata, Nagwa Sayed; Fouad, Ehab Ali

    2017-01-01

    Aim: As the labeled anti-camel immunoglobulins (Igs) with enzymes for enzyme-linked immunosorbent assay (ELISA) are unavailable in the Egyptian market, the present investigation was directed for developing local labeled anti-camel IgG with horseradish peroxidase (HRP) to save hard curacy. Materials and Methods: For purification of camel IgG whole molecule, camel sera was preliminary precipitated with 50% saturated ammonium sulfate and dialyzed against 15 mM phosphate-buffered saline pH 7.2 then concentrated. This preparation was further purified by protein A sepharose affinity column chromatography. The purity of the eluted camel IgG was tested by sodium dodecyl sulfate polyacrylamide gel electrophoresi. Anti-camel IgG was prepared by immunization of goats and rabbits separately, with purified camel IgG. The anti-camel IgG was purified by protein A sepharose affinity column chromatography. Whole molecule anti-camel IgG was conjugated with HRP using glutraldehyde based assay. Sensitivity and specificity of prepared conjugated secondary antibodies were detected using positive and negative camel serum samples reacted with different antigens in ELISA, respectively. The potency of prepared conjugated antibodies was evaluated compared with protein A HRP. The stability of the conjugate at −20°C during 1 year was assessed by ELISA. Results: The electrophoretic profile of camel IgG showed four bands of molecular weight 63, 52, 40 and 33 kDa. The recorded sensitivity and specificity of the product are 100%. Its potency is also 100% compared to 58-75% of commercial protein A HRP. The conjugates are stable for 1 year at −20°C as proved by ELISA. Conclusion: Collectively, this study introduces goat and rabbit anti-camel IgG whole molecules with simple, inexpensive method, with 100% sensitivity, 100% specificity and stability up to 1 year at −20°C. The important facet of the current study is saving hard curacy. Future investigations are necessary for preparation of Ig

  4. Interpreting T-Cell Cross-reactivity through Structure: Implications for TCR-Based Cancer Immunotherapy.

    PubMed

    Antunes, Dinler A; Rigo, Maurício M; Freitas, Martiela V; Mendes, Marcus F A; Sinigaglia, Marialva; Lizée, Gregory; Kavraki, Lydia E; Selin, Liisa K; Cornberg, Markus; Vieira, Gustavo F

    2017-01-01

    Immunotherapy has become one of the most promising avenues for cancer treatment, making use of the patient's own immune system to eliminate cancer cells. Clinical trials with T-cell-based immunotherapies have shown dramatic tumor regressions, being effective in multiple cancer types and for many different patients. Unfortunately, this progress was tempered by reports of serious (even fatal) side effects. Such therapies rely on the use of cytotoxic T-cell lymphocytes, an essential part of the adaptive immune system. Cytotoxic T-cells are regularly involved in surveillance and are capable of both eliminating diseased cells and generating protective immunological memory. The specificity of a given T-cell is determined through the structural interaction between the T-cell receptor (TCR) and a peptide-loaded major histocompatibility complex (MHC); i.e., an intracellular peptide-ligand displayed at the cell surface by an MHC molecule. However, a given TCR can recognize different peptide-MHC (pMHC) complexes, which can sometimes trigger an unwanted response that is referred to as T-cell cross-reactivity. This has become a major safety issue in TCR-based immunotherapies, following reports of melanoma-specific T-cells causing cytotoxic damage to healthy tissues (e.g., heart and nervous system). T-cell cross-reactivity has been extensively studied in the context of viral immunology and tissue transplantation. Growing evidence suggests that it is largely driven by structural similarities of seemingly unrelated pMHC complexes. Here, we review recent reports about the existence of pMHC "hot-spots" for cross-reactivity and propose the existence of a TCR interaction profile (i.e., a refinement of a more general TCR footprint in which some amino acid residues are more important than others in triggering T-cell cross-reactivity). We also make use of available structural data and pMHC models to interpret previously reported cross-reactivity patterns among virus-derived peptides. Our

  5. Interpreting T-Cell Cross-reactivity through Structure: Implications for TCR-Based Cancer Immunotherapy

    PubMed Central

    Antunes, Dinler A.; Rigo, Maurício M.; Freitas, Martiela V.; Mendes, Marcus F. A.; Sinigaglia, Marialva; Lizée, Gregory; Kavraki, Lydia E.; Selin, Liisa K.; Cornberg, Markus; Vieira, Gustavo F.

    2017-01-01

    Immunotherapy has become one of the most promising avenues for cancer treatment, making use of the patient’s own immune system to eliminate cancer cells. Clinical trials with T-cell-based immunotherapies have shown dramatic tumor regressions, being effective in multiple cancer types and for many different patients. Unfortunately, this progress was tempered by reports of serious (even fatal) side effects. Such therapies rely on the use of cytotoxic T-cell lymphocytes, an essential part of the adaptive immune system. Cytotoxic T-cells are regularly involved in surveillance and are capable of both eliminating diseased cells and generating protective immunological memory. The specificity of a given T-cell is determined through the structural interaction between the T-cell receptor (TCR) and a peptide-loaded major histocompatibility complex (MHC); i.e., an intracellular peptide–ligand displayed at the cell surface by an MHC molecule. However, a given TCR can recognize different peptide–MHC (pMHC) complexes, which can sometimes trigger an unwanted response that is referred to as T-cell cross-reactivity. This has become a major safety issue in TCR-based immunotherapies, following reports of melanoma-specific T-cells causing cytotoxic damage to healthy tissues (e.g., heart and nervous system). T-cell cross-reactivity has been extensively studied in the context of viral immunology and tissue transplantation. Growing evidence suggests that it is largely driven by structural similarities of seemingly unrelated pMHC complexes. Here, we review recent reports about the existence of pMHC “hot-spots” for cross-reactivity and propose the existence of a TCR interaction profile (i.e., a refinement of a more general TCR footprint in which some amino acid residues are more important than others in triggering T-cell cross-reactivity). We also make use of available structural data and pMHC models to interpret previously reported cross-reactivity patterns among virus

  6. Purification of human alpha uterine protein.

    PubMed

    Sutcliffe, R G; Bolton, A E; Sharp, F; Nicholson, L V; MacKinnon, R

    1980-03-01

    Human alpha uterine protein (AUP) has been prepared from extracts of decudua by antibody affinity chromatography, DEAE Sepharose chromatography and by filtration through Sephadex G-150. This procedure yielded a protein fraction containing AUP, which was labelled with 125I by chloramine T. When analysed by SDS gel electrophoresis this radioiodinated protein fraction was found to contain predominantly a single species of protein which was precipitated by antibodies against AUP in antibody-antigen crossed electrophoresis. Rabbit anti-AUP precipitated 55-65% of the tracer in a double-antibody system. Sephadex G150 gel filtration of AUP obtained before and after affinity chromatography provided a molecular weight estimate of 50000. Since SDS gel electrophoresis revealed a polypeptide molecular weight of 23000-25000, it is suggested that AUP is a dimer.

  7. Butter feeding enhances TNF-alpha production from macrophages and lymphocyte adherence in murine small intestinal microvessels.

    PubMed

    Fujiyama, Yoichi; Hokari, Ryota; Miura, Soichiro; Watanabe, Chikako; Komoto, Shunsuke; Oyama, Tokushige; Kurihara, Chie; Nagata, Hiroshi; Hibi, Toshifumi

    2007-11-01

    Dietary fat is known to modulate immune functions. Intake of an animal fat-rich diet has been linked to increased risk of inflammation; however, little is known about how animal fat ingestion directly affects intestinal immune function. The objective of this study was to assess the effect of butter feeding on lymphocyte migration in intestinal mucosa and the changes in adhesion molecules and cytokines involved in this effect. T-lymphocytes isolated from the spleen were fluorescence-labeled and injected into recipient mice. Butter was administered into the duodenum, and villus microvessels of the small intestinal mucosa were observed under an intravital microscope. mRNA expression of adhesion molecules and cytokines in the intestinal mucosa were determined by quantitative PCR. The effect of butter feeding on tumor necrosis factor (TNF)-alpha mRNA expression of intestinal macrophages was also determined. Intraluminal butter administration significantly increased lymphocyte adherence to intestinal microvessels accompanied by increases in expression levels of adhesion molecules ICAM-1, MAdCAM-1 and VCAM-1. This accumulation was significantly attenuated by anti-MAdCAM-1 and anti-ICAM-1 antibodies. Butter administration significantly increased TNF-alpha in the lamina proprial macrophages but not interleukin-6. Anti-TNF-alpha treatment attenuated the enhanced expression of adhesion molecules induced by butter administration. T-lymphocyte adherence to microvessels of the small intestinal mucosa was significantly enhanced after butter ingestion. This enhancement is due to increase in expression levels of adhesion molecules of the intestinal mucosa, which is mediated by TNF-alpha from macrophages in the intestinal lamina propria.

  8. Photoluminescence of patterned CdSe quantum dot for anti-counterfeiting label on paper

    NASA Astrophysics Data System (ADS)

    Isnaeni, Yulianto, Nursidik; Suliyanti, Maria Margaretha

    2016-03-01

    We successfully developed a method utilizing colloidal CdSe nanocrystalline quantum dot for anti-counterfeiting label on a piece of glossy paper. We deposited numbers and lines patterns of toluene soluble CdSe quantum dot using rubber stamper on a glossy paper. The width of line pattern was about 1-2 mm with 1-2 mm separation between lines. It required less than one minute for deposited CdSe quantum dot on glossy paper to dry and become invisible by naked eyes. However, patterned quantum dot become visible using long-pass filter glasses upon excitation of UV lamp or blue laser. We characterized photoluminescence of line patterns of quantum dot, and we found that emission boundaries of line patterns were clearly observed. The error of line size and shape were mainly due to defect of the original stamper. The emission peak wavelength of CdSe quantum dot was 629 nm. The emission spectrum of deposited quantum dot has full width at half maximum (FWHM) of 30-40 nm. The spectra similarity between deposited quantum dot and the original quantum dot in solution proved that our stamping method can be simply applied on glossy paper without changing basic optical property of the quantum dot. Further development of this technique is potential for anti-counterfeiting label on very important documents or objects.

  9. Photoluminescence of patterned CdSe quantum dot for anti-counterfeiting label on paper

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Isnaeni,, E-mail: isnaeni@lipi.go.id; Yulianto, Nursidik; Suliyanti, Maria Margaretha

    We successfully developed a method utilizing colloidal CdSe nanocrystalline quantum dot for anti-counterfeiting label on a piece of glossy paper. We deposited numbers and lines patterns of toluene soluble CdSe quantum dot using rubber stamper on a glossy paper. The width of line pattern was about 1-2 mm with 1-2 mm separation between lines. It required less than one minute for deposited CdSe quantum dot on glossy paper to dry and become invisible by naked eyes. However, patterned quantum dot become visible using long-pass filter glasses upon excitation of UV lamp or blue laser. We characterized photoluminescence of line patterns of quantummore » dot, and we found that emission boundaries of line patterns were clearly observed. The error of line size and shape were mainly due to defect of the original stamper. The emission peak wavelength of CdSe quantum dot was 629 nm. The emission spectrum of deposited quantum dot has full width at half maximum (FWHM) of 30-40 nm. The spectra similarity between deposited quantum dot and the original quantum dot in solution proved that our stamping method can be simply applied on glossy paper without changing basic optical property of the quantum dot. Further development of this technique is potential for anti-counterfeiting label on very important documents or objects.« less

  10. Complimentary mechanisms of dual checkpoint blockade expand unique T-cell repertoires and activate adaptive anti-tumor immunity in triple-negative breast tumors

    PubMed Central

    Wei, Junping; Yang, Xiao Yi; Lei, Gangjun; Wang, Tao; Liu, Cong-Xiao; Morse, Michael A.; Gouin, Kenneth; Knott, Simon R. V.; Hartman, Zachary C.

    2018-01-01

    ABSTRACT Triple-negative breast cancer (TNBC) is an aggressive and molecularly diverse breast cancer subtype typified by the presence of p53 mutations (∼80%), elevated immune gene signatures and neoantigen expression, as well as the presence of tumor infiltrating lymphocytes (TILs). As these factors are hypothesized to be strong immunologic prerequisites for the use of immune checkpoint blockade (ICB) antibodies, multiple clinical trials testing single ICBs have advanced to Phase III, with early indications of heterogeneous response rates of <20% to anti-PD1 and anti-PDL1 ICB. While promising, these modest response rates highlight the need for mechanistic studies to understand how different ICBs function, how their combination impacts functionality and efficacy, as well as what immunologic parameters predict efficacy to different ICBs regimens in TNBC. To address these issues, we tested anti-PD1 and anti-CTLA4 in multiple models of TNBC and found that their combination profoundly enhanced the efficacy of either treatment alone. We demonstrate that this efficacy is due to anti-CTLA4-driven expansion of an individually unique T-cell receptor (TCR) repertoire whose functionality is enhanced by both intratumoral Treg suppression and anti-PD1 blockade of tumor expressed PDL1. Notably, the individuality of the TCR repertoire was observed regardless of whether the tumor cells expressed a nonself antigen (ovalbumin) or if tumor-specific transgenic T-cells were transferred prior to sequencing. However, responsiveness was strongly correlated with systemic measures of tumor-specific T-cell and B-cell responses, which along with systemic assessment of TCR expansion, may serve as the most useful predictors for clinical responsiveness in future clinical trials of TNBC utilizing anti-PD1/anti-CTLA4 ICB. PMID:29721371

  11. Complimentary mechanisms of dual checkpoint blockade expand unique T-cell repertoires and activate adaptive anti-tumor immunity in triple-negative breast tumors.

    PubMed

    Crosby, Erika J; Wei, Junping; Yang, Xiao Yi; Lei, Gangjun; Wang, Tao; Liu, Cong-Xiao; Agarwal, Pankaj; Korman, Alan J; Morse, Michael A; Gouin, Kenneth; Knott, Simon R V; Lyerly, H Kim; Hartman, Zachary C

    2018-01-01

    Triple-negative breast cancer (TNBC) is an aggressive and molecularly diverse breast cancer subtype typified by the presence of p53 mutations (∼80%), elevated immune gene signatures and neoantigen expression, as well as the presence of tumor infiltrating lymphocytes (TILs). As these factors are hypothesized to be strong immunologic prerequisites for the use of immune checkpoint blockade (ICB) antibodies, multiple clinical trials testing single ICBs have advanced to Phase III, with early indications of heterogeneous response rates of <20% to anti-PD1 and anti-PDL1 ICB. While promising, these modest response rates highlight the need for mechanistic studies to understand how different ICBs function, how their combination impacts functionality and efficacy, as well as what immunologic parameters predict efficacy to different ICBs regimens in TNBC. To address these issues, we tested anti-PD1 and anti-CTLA4 in multiple models of TNBC and found that their combination profoundly enhanced the efficacy of either treatment alone. We demonstrate that this efficacy is due to anti-CTLA4-driven expansion of an individually unique T-cell receptor (TCR) repertoire whose functionality is enhanced by both intratumoral Treg suppression and anti-PD1 blockade of tumor expressed PDL1. Notably, the individuality of the TCR repertoire was observed regardless of whether the tumor cells expressed a nonself antigen (ovalbumin) or if tumor-specific transgenic T-cells were transferred prior to sequencing. However, responsiveness was strongly correlated with systemic measures of tumor-specific T-cell and B-cell responses, which along with systemic assessment of TCR expansion, may serve as the most useful predictors for clinical responsiveness in future clinical trials of TNBC utilizing anti-PD1/anti-CTLA4 ICB.

  12. Nanozeolite bioconjugates labeled with 223Ra for targeted alpha therapy.

    PubMed

    Piotrowska, Agata; Męczyńska-Wielgosz, Sylwia; Majkowska-Pilip, Agnieszka; Koźmiński, Przemysław; Wójciuk, Grzegorz; Cędrowska, Edyta; Bruchertseifer, Frank; Morgenstern, Alfred; Kruszewski, Marcin; Bilewicz, Aleksander

    2017-04-01

    Alpha particle emitting isotopes are of considerable interest for radionuclide therapy because of their high cytotoxicity and short path length. Among the many α emitters, 223 Ra exhibits very attractive nuclear properties for application in radionuclide therapy. The decay of this radioisotope and its daughters is accompanied by the emission of four α-particles, releasing 27.9MeV of cumulative energy. Unfortunately the lack of an appropriate bifunctional ligand for radium has so far been a main obstacle for the application of 223 Ra in receptor targeted therapy. In our studies we investigated the use of nanozeolite-Substance P bioconjugates as vehicles for 223 Ra radionuclides for targeted α therapy. The sodium form of an A-type of nanozeolite (NaA) was synthesized using the template method. Next, the nanozeolite particles were conjugated to the Substance P (5-11) peptide fragment, which targets NK-1 receptors on glioma cells. The obtained bioconjugate was characterized by transmission emission spectroscopy, thermogravimetric analysis and dynamic light scattering analysis. The NaA-silane-PEG-SP(5-11) bioconjugates were labeled with 223 Ra by exchange of the Na + cation and the stability, receptor affinity and cytotoxicity of the obtained radiobioconjugates were tested. The 223 Ra-labeled nanozeolite bioconjugate almost quantitatively retains 223 Ra in vitro after 6days, while the retention of decay products varies from 90 to 95%. The synthesized 223 RaA-silane-PEG-SP(5-11) showed high receptor affinity toward NK-1 receptor expressing glioma cells and exhibited a high cytotoxic effect in vitro. Substance P functionalized nanozeolite-A represents a viable solution for the use of the 223 Ra in vivo generator as a therapeutic construct for targeting glioma cells. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. The N terminus of SKAP55 enables T cell adhesion to TCR and integrin ligands via distinct mechanisms

    PubMed Central

    Ophir, Michael J.; Liu, Beiyun C.

    2013-01-01

    The T cell receptor (TCR) triggers the assembly of “SLP-76 microclusters,” which mediate signals required for T cell activation. In addition to regulating integrin activation, we show that Src kinase–associated phosphoprotein of 55 kD (SKAP55) is required for microcluster persistence and movement, junctional stabilization, and integrin-independent adhesion via the TCR. These functions require the dimerization of SKAP55 and its interaction with the adaptor adhesion and degranulation-promoting adaptor protein (ADAP). A “tandem dimer” containing two ADAP-binding SKAP55 Src homology 3 (SH3) domains stabilized SLP-76 microclusters and promoted T cell adhesion via the TCR, but could not support adhesion to integrin ligands. Finally, the SKAP55 dimerization motif (DM) enabled the coimmunoprecipitation of the Rap1-dependent integrin regulator Rap1-GTP–interacting adaptor molecule (RIAM), the recruitment of talin into TCR-induced adhesive junctions, and “inside-out” signaling to β1 integrins. Our data indicate that SKAP55 dimers stabilize SLP-76 microclusters, couple SLP-76 to the force-generating systems responsible for microcluster movement, and enable adhesion via the TCR by mechanisms independent of RIAM, talin, and β1 integrins. PMID:24368808

  14. Nanostructured vanadium oxide thin film with high TCR at room temperature for microbolometer

    NASA Astrophysics Data System (ADS)

    Wang, Bin; Lai, Jianjun; Li, Hui; Hu, Haoming; Chen, Sihai

    2013-03-01

    In order to obtain high quality of thermal sensitive material, VOx thin film of high temperature coefficient of resistance (TCR) of 6.5%/K at room temperature has been deposited by reactive ion beam sputtering and post annealing method. AFM and XRD measurements indicate that the VOx thin film with nanostructured crystalline is composed of VO2 and V2O3. The nanostructured VOx microbolometer has been designed and fabricated. The measurement of the film system with TiN absorbing layer indicates that it has about 92% infrared absorption in the range of 8-14 μm. The performance of this bolometer, comparing with that of bolometer with common VOx, has a better result. At 20 Hz frequency and 10 μA bias current, the bolometer with high TCR has reached detectivity of 1.0 × 109 cm Hz1/2/W. It also indicates that this nanostructured VOx thin film has not only a higher TCR but also a lower noise than common VOx thin film without annealing.

  15. Different TCR-induced T lymphocyte responses are potentiated by stiffness with variable sensitivity

    PubMed Central

    Saitakis, Michael; Dogniaux, Stéphanie; Goudot, Christel; Bufi, Nathalie; Asnacios, Sophie; Maurin, Mathieu; Randriamampita, Clotilde; Asnacios, Atef; Hivroz, Claire

    2017-01-01

    T cells are mechanosensitive but the effect of stiffness on their functions is still debated. We characterize herein how human primary CD4+ T cell functions are affected by stiffness within the physiological Young’s modulus range of 0.5 kPa to 100 kPa. Stiffness modulates T lymphocyte migration and morphological changes induced by TCR/CD3 triggering. Stiffness also increases TCR-induced immune system, metabolism and cell-cycle-related genes. Yet, upon TCR/CD3 stimulation, while cytokine production increases within a wide range of stiffness, from hundreds of Pa to hundreds of kPa, T cell metabolic properties and cell cycle progression are only increased by the highest stiffness tested (100 kPa). Finally, mechanical properties of adherent antigen-presenting cells modulate cytokine production by T cells. Together, these results reveal that T cells discriminate between the wide range of stiffness values found in the body and adapt their responses accordingly. DOI: http://dx.doi.org/10.7554/eLife.23190.001 PMID:28594327

  16. Melanoma targeting with alpha-melanocyte stimulating hormone analogs labeled with fac-[99mTc(CO)3]+: effect of cyclization on tumor-seeking properties.

    PubMed

    Raposinho, Paula D; Xavier, Catarina; Correia, João D G; Falcão, Soraia; Gomes, Paula; Santos, Isabel

    2008-03-01

    Early detection of primary melanoma tumors is essential because there is no effective treatment for metastatic melanoma. Several linear and cyclic radiolabeled alpha-melanocyte stimulating hormone (alpha-MSH) analogs have been proposed to target the melanocortin type 1 receptor (MC1R) overexpressed in melanoma. The compact structure of a rhenium-cyclized alpha-MSH analog (Re-CCMSH) significantly enhanced its in vivo tumor uptake and retention. Melanotan II (MT-II), a cyclic lactam analog of alpha-MSH (Ac-Nle-cyclo[Asp-His-DPhe-Arg-Trp-Lys]-NH2]), is a very potent and stable agonist peptide largely used in the characterization of melanocortin receptors. Taking advantage of the superior biological features associated with the MT-II cyclic peptide, we assessed the effect of lactam-based cyclization on the tumor-seeking properties of alpha-MSH analogs by comparing the pharmacokinetics profile of the 99mTc-labeled cyclic peptide betaAla-Nle-cyclo[Asp-His-D-Phe-Arg-Trp-Lys]-NH2 with that of the linear analog betaAla-Nle-Asp-His-DPhe-Arg-Trp-Lys-NH2 in melanoma-bearing mice. We have synthesized and coupled the linear and cyclic peptides to a bifunctional chelator containing a pyrazolyl-diamine backbone (pz) through the amino group of betaAla, and the resulting pz-peptide conjugates were reacted with the fac-[99mTc(CO)3]+ moiety. The 99mTc(CO)3-labeled conjugates were obtained in high yield, high specific activity, and high radiochemical purity. The cyclic 99mTc(CO)3-labeled conjugate presents a remarkable internalization (87.1% of receptor-bound tracer and 50.5% of total applied activity, after 6 h at 37 degrees C) and cellular retention (only 24.7% released from the cells after 5 h) in murine melanoma B16F1 cells. A significant tumor uptake and retention was obtained in melanoma-bearing C57BL6 mice for the cyclic radioconjugate [9.26 +/- 0.83 and 11.31 +/- 1.83% ID/g at 1 and 4 h after injection, respectively]. The linear 99mTc(CO)3-pz-peptide presented lower values for

  17. Meta-Analysis of Coefficient Alpha

    ERIC Educational Resources Information Center

    Rodriguez, Michael C.; Maeda, Yukiko

    2006-01-01

    The meta-analysis of coefficient alpha across many studies is becoming more common in psychology by a methodology labeled reliability generalization. Existing reliability generalization studies have not used the sampling distribution of coefficient alpha for precision weighting and other common meta-analytic procedures. A framework is provided for…

  18. Alpha-SNAP functions in insulin exocytosis from mature, but not immature secretory granules in pancreatic beta cells.

    PubMed

    Nakamichi, Y; Nagamatsu, S

    1999-06-24

    To explore alpha-SNAP function in insulin exocytosis from either immature or mature secretory granules in pancreatic beta cells, we studied the effects of overexpression of adenovirus-mediated wild-type alpha-SNAP and C-terminally deleted alpha-SNAP mutant (1-285) on newly synthesized proinsulin and insulin release by rat islets and MIN6 cells. Rat islets overexpressing alpha-SNAP and mutant alpha-SNAP were pulse-chased. Exocytosis from immature and mature insulin secretory granules was measured as fractional (%) labeled-proinsulin release immediately after the pulse-labeling and percentage labeled-insulin release after a 3-h chase period, respectively. There was no difference in percentage labeled-proinsulin release between the control and alpha-SNAP or mutant alpha-SNAP-overexpressed islets. Although percentage labeled-insulin release after a 3-h chase period was significantly increased in alpha-SNAP-overexpressed islets, it was decreased in mutant alpha-SNAP-overexpressed islets. Thus, the results demonstrated that alpha-SNAP overexpression in rat islets primarily increased exocytosis from mature, but not immature insulin secretory granules. On the other hand, in MIN6 cells, alpha-SNAP overexpression scarcely affected glucose-stimulated insulin release; therefore, we examined the effect of mutant alpha-SNAP overexpression as the dominant-negative inhibitor on the newly synthesized proinsulin/insulin release using the same protocol as in the rat islet experiments. alpha-SNAP mutant (1-285) overexpression in MIN6 cells decreased the percentage labeled insulin release from mature secretory granules, but not percentage labeled proinsulin release from immature secretory granules. Thus, our data demonstrate that alpha-SNAP functions mainly in the mature insulin secretory granules in pancreatic beta cells. Copyright 1999 Academic Press.

  19. 111In-labeled lactam bridge-cyclized alpha-melanocyte stimulating hormone peptide analogues for melanoma imaging.

    PubMed

    Miao, Yubin; Gallazzi, Fabio; Guo, Haixun; Quinn, Thomas P

    2008-02-01

    The purpose of this study was to examine the influence of the lactam bridge cyclization on melanoma targeting and biodistribution properties of the radiolabeled conjugates. Two novel lactam bridge-cyclized alpha-MSH peptide analogues, DOTA-CycMSH (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-c[Lys-Nle-Glu-His-DPhe-Arg-Trp-Gly-Arg-Pro-Val-Asp]) and DOTA-GlyGlu-CycMSH (DOTA-Gly-Glu-c[Lys-Nle-Glu-His-DPhe-Arg-Trp-Gly-Arg-Pro-Val-Asp]), were synthesized and radiolabeled with (111)In. The internalization and efflux of (111)In-labeled CycMSH peptides were examined in B16/F1 melanoma cells. The melanoma targeting properties, pharmacokinetics, and SPECT/CT imaging of (111)In-labeled CycMSH peptides were determined in B16/F1 melanoma-bearing C57 mice. Both (111)In-DOTA-CycMSH and (111)In-DOTA-GlyGlu-CycMSH exhibited fast internalization and extended retention in B16/F1 cells. The tumor uptake values of (111)In-DOTA-CycMSH and (111)In-DOTA-GlyGlu-CycMSH were 9.53+/-1.41% injected dose/gram (% ID/g) and 10.40+/-1.40% ID/g at 2 h postinjection, respectively. Flank melanoma tumors were clearly visualized with (111)In-DOTA-CycMSH and (111)In-DOTA-GlyGlu-CycMSH by SPECT/CT images at 2 h postinjection. Whole-body clearance of the peptides was fast, with greater than 90% of the radioactivities cleared through urinary system by 2 h postinjection. There was low radioactivity (<0.8% ID/g) accumulated in blood and normal organs except kidneys at all time points investigated. Introduction of a negatively charged linker (-Gly-Glu-) into the peptide sequence decreased the renal uptake by 44% without affecting the tumor uptake at 4 h postinjection. High receptor-mediated melanoma uptakes coupled with fast whole-body clearance in B16/F1 melanoma-bearing C57 mice demonstrated the feasibility of using (111)In-labeled lactam bridge-cyclized alpha-MSH peptide analogues as a novel class of imaging probes for receptor-targeting melanoma imaging.

  20. Methanol extract of Xanthium strumarium L. possesses anti-inflammatory and anti-nociceptive activities.

    PubMed

    Kim, In-Tae; Park, Young-Mi; Won, Jong-Heon; Jung, Hyun-Ju; Park, Hee-Juhn; Choi, Jong-Won; Lee, Kyung-Tae

    2005-01-01

    As an attempt to identify bioactive natural products with anti-inflammatory activity, we evaluated the effects of the methanol extract of the semen of Xanthium strumarium L. (MEXS) on lipopolysaccharide (LPS)-induced nitric oxide (NO), prostaglandin E2 (PGE2) and tumor necrosis factor-alpha (TNF-alpha) production in RAW 264.7 cells. Our data indicate that MEXS is a potent inhibitor of NO, PGE2 and TNF-alpha production. Consistent with these findings, the expression levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein and iNOS, COX-2 and TNF-alpha mRNA were down-regulated in a concentration-dependent manner. Furthermore, MEXS inhibited nuclear factor kappa B (NF-kappaB) DNA binding activity and the translocation of NF-kappaB to the nucleus by blocking the degradation of inhibitor of kappa B-alpha (IkappaB-alpha). We further evaluated the anti-inflammatory and anti-nociceptive activities of MEXS in vivo. MEXS (100, 200 mg/kg/d, p.o.) reduced acute paw edema induced by carrageenin in rats, and showed analgesic activities in an acetic acid-induced abdominal constriction test and a hot plate test in mice. Thus, our study suggests that the inhibitions of iNOS, COX-2 expression, and TNF-alpha release by the methanol extract of the semen of Xanthium strumarium L. are achieved by blocking NF-kappaB activation, and that this is also responsible for its anti-inflammatory effects.

  1. Mapping alpha-helical induced folding within the intrinsically disordered C-terminal domain of the measles virus nucleoprotein by site-directed spin-labeling EPR spectroscopy.

    PubMed

    Belle, Valérie; Rouger, Sabrina; Costanzo, Stéphanie; Liquière, Elodie; Strancar, Janez; Guigliarelli, Bruno; Fournel, André; Longhi, Sonia

    2008-12-01

    Using site-directed spin-labeling EPR spectroscopy, we mapped the region of the intrinsically disordered C-terminal domain of measles virus nucleoprotein (N(TAIL)) that undergoes induced folding. In addition to four spin-labeled N(TAIL) variants (S407C, S488C, L496C, and V517C) (Morin et al. (2006), J Phys Chem 110: 20596-20608), 10 new single-site cysteine variants were designed, purified from E. coli, and spin-labeled. These 14 spin-labeled variants enabled us to map in detail the gain of rigidity of N(TAIL) in the presence of either the secondary structure stabilizer 2,2,2-trifluoroethanol or the C-terminal domain X (XD) of the viral phosphoprotein. Different regions of N(TAIL) were shown to contribute to a different extent to the binding to XD, while the mobility of the spin labels grafted at positions 407 and 460 was unaffected upon addition of XD; that of the spin labels grafted within the 488-502 and the 505-522 regions was severely and moderately reduced, respectively. Furthermore, EPR experiments in the presence of 30% sucrose allowed us to precisely map to residues 488-502, the N(TAIL) region undergoing alpha-helical folding. The mobility of the 488-502 region was found to be restrained even in the absence of the partner, a behavior that could be accounted for by the existence of a transiently populated folded state. Finally, we show that the restrained motion of the 505-522 region upon binding to XD is due to the alpha-helical transition occurring within the 488-502 region and not to a direct interaction with XD.

  2. Differential regulation of peripheral CD4+ T cell tolerance induced by deletion and TCR revision.

    PubMed

    Ali, Mohamed; Weinreich, Michael; Balcaitis, Stephanie; Cooper, Cristine J; Fink, Pamela J

    2003-12-01

    In Vbeta5 transgenic mice, mature Vbeta5(+)CD4(+) T cells are tolerized upon recognition of a self Ag, encoded by a defective endogenous retrovirus, whose expression is confined to the lymphoid periphery. Cells are driven by the tolerogen to enter one of two tolerance pathways, deletion or TCR revision. CD4(+) T cells entering the former pathway are rendered anergic and then eliminated. In contrast, TCR revision drives gene rearrangement at the endogenous TCR beta locus and results in the appearance of Vbeta5(-), endogenous Vbeta(+), CD4(+) T cells that are both self-tolerant and functional. An analysis of the molecules that influence each of these pathways was conducted to understand better the nature of the interactions that control tolerance induction in the lymphoid periphery. These studies reveal that deletion is efficient in reconstituted radiation chimeras and is B cell, CD28, inducible costimulatory molecule, Fas, CD4, and CD8 independent. In contrast, TCR revision is radiosensitive, B cell, CD28, and inducible costimulatory molecule dependent, Fas and CD4 influenced, and CD8 independent. Our data demonstrate the differential regulation of these two divergent tolerance pathways, despite the fact that they are both driven by the same tolerogen and restricted to mature CD4(+) T cells.

  3. Extended follow-up of anti-HBe-positive patients with chronic hepatitis B retreated with ribavirin and interferon-alpha.

    PubMed

    Carreño, V; Rico, M A; Pardo, M; Quiroga, J A

    2001-11-01

    In a pilot study of combination therapy with ribavirin and IFN alpha conducted in anti-HBe-positive individuals with chronic hepatitis B, 21% of patients achieved a sustained ALT normalization and clearance of hepatitis B virus (HBV) DNA as measured by PCR. The present work has assessed whether these sustained responses are lasting long-term. In addition, IFN gamma levels were tested serially in serum as a measure of the immune system activation during treatment. By extending the post-treatment follow-up period 2 years the occurrence of delayed HBV DNA relapses was observed. A low serum level of IFN gamma was detected during and after treatment. IFN gamma demonstrated a multiphasic time-course: the amount of IFN gamma increased in parallel with reductions in HBV DNA but also with ALT flare-ups. In conclusion, the extended follow-up study of anti-HBe-positive patients after combined treatment with ribavirin and IFN alpha has shown that sustained responses are lasting in 17% patients but also that a late onset HBV DNA relapse may occur.

  4. Drug-laden 3D biodegradable label using QR code for anti-counterfeiting of drugs.

    PubMed

    Fei, Jie; Liu, Ran

    2016-06-01

    Wiping out counterfeit drugs is a great task for public health care around the world. The boost of these drugs makes treatment to become potentially harmful or even lethal. In this paper, biodegradable drug-laden QR code label for anti-counterfeiting of drugs is proposed that can provide the non-fluorescence recognition and high capacity. It is fabricated by the laser cutting to achieve the roughness over different surface which causes the difference in the gray levels on the translucent material the QR code pattern, and the micro mold process to obtain the drug-laden biodegradable label. We screened biomaterials presenting the relevant conditions and further requirements of the package. The drug-laden microlabel is on the surface of the troches or the bottom of the capsule and can be read by a simple smartphone QR code reader application. Labeling the pill directly and decoding the information successfully means more convenient and simple operation with non-fluorescence and high capacity in contrast to the traditional methods. Copyright © 2016 Elsevier B.V. All rights reserved.

  5. Ferrocenemonocarboxylic-HRP@Pt nanoparticles labeled RCA for multiple amplification of electro-immunosensing.

    PubMed

    Su, Huilan; Yuan, Ruo; Chai, Yaqin; Mao, Li; Zhuo, Ying

    2011-07-15

    A multiple amplification immunoassay was proposed to detect alpha-fetoprotein (AFP), which was based on ferrocenemonocarboxylic-HRP conjugated on Pt nanoparticles as labels for rolling circle amplification (RCA). Firstly, the capture antibody (anti-AFP) was immobilized on glass carbon electrode (GCE) deposited nano-sized gold particles. After a typical immuno-sandwich protocol, primary DNA was immobilized by labeling secondary antibody, which acted as a precursor to initiate RCA. The products of RCA provide large amount of sites to link detection DNAs, which were labeled by signal probes (ferrocenemonocarboxylic) and horseradish peroxidase (HRP). Moreover, the enzymatic amplification signals could be produced by the catalysis of HRP and Pt nanoparticles with the addition of H₂O₂. These lead to multiple amplification signals monitoring by electrochemical instrument and further resulted in high sensitivity of the immunoassay with the detection limit of 1.7 pg/mL. Copyright © 2011 Elsevier B.V. All rights reserved.

  6. Src-like adaptor protein down-regulates T cell receptor (TCR)-CD3 expression by targeting TCRzeta for degradation.

    PubMed

    Myers, Margaret D; Dragone, Leonard L; Weiss, Arthur

    2005-07-18

    Src-like adaptor protein (SLAP) down-regulates expression of the T cell receptor (TCR)-CD3 complex during a specific stage of thymocyte development when the TCR repertoire is selected. Consequently, SLAP-/- thymocytes display alterations in thymocyte development. Here, we have studied the mechanism of SLAP function. We demonstrate that SLAP-deficient thymocytes have increased TCRzeta chain expression as a result of a defect in TCRzeta degradation. Failure to degrade TCRzeta leads to an increased pool of fully assembled TCR-CD3 complexes that are capable of recycling back to the cell surface. We also provide evidence that SLAP functions in a pathway that requires the phosphorylated TCRzeta chain and the Src family kinase Lck, but not ZAP-70 (zeta-associated protein of 70 kD). These studies reveal a unique mechanism by which SLAP contributes to the regulation of TCR expression during a distinct stage of thymocyte development.

  7. MHC drives TCR repertoire shaping, but not maturation, in recent thymic emigrants.

    PubMed

    Houston, Evan G; Fink, Pamela J

    2009-12-01

    After developing in the thymus, recent thymic emigrants (RTEs) enter the lymphoid periphery and undergo a maturation process as they transition into the mature naive (MN) T cell compartment. This maturation presumably shapes RTEs into a pool of T cells best fit to function robustly in the periphery without causing autoimmunity; however, the mechanism and consequences of this maturation process remain unknown. Using a transgenic mouse system that specifically labels RTEs, we tested the influence of MHC molecules, key drivers of intrathymic T cell selection and naive peripheral T cell homeostasis, in shaping the RTE pool in the lymphoid periphery. We found that the TCRs expressed by RTEs are skewed to longer CDR3 regions compared with those of MN T cells, suggesting that MHC does streamline the TCR repertoire of T cells as they transition from the RTE to the MN T cell stage. This conclusion is borne out in studies in which the representation of individual TCRs was followed as a function of time since thymic egress. Surprisingly, we found that MHC is dispensable for the phenotypic and functional maturation of RTEs.

  8. TCR Signal Strength Alters T–DC Activation and Interaction Times and Directs the Outcome of Differentiation

    PubMed Central

    van Panhuys, Nicholas

    2016-01-01

    The ability of CD4+ T cells to differentiate into effector subsets underpins their ability to shape the immune response and mediate host protection. During T cell receptor-induced activation of CD4+ T cells, both the quality and quantity of specific activatory peptide/MHC ligands have been shown to control the polarization of naive CD4+ T cells in addition to co-stimulatory and cytokine-based signals. Recently, advances in two-­photon microscopy and tetramer-based cell tracking methods have allowed investigators to greatly extend the study of the role of TCR signaling in effector differentiation under in vivo conditions. In this review, we consider data from recent in vivo studies analyzing the role of TCR signal strength in controlling the outcome of CD4+ T cell differentiation and discuss the role of TCR in controlling the critical nature of CD4+ T cell interactions with dendritic cells during activation. We further propose a model whereby TCR signal strength controls the temporal aspects of T–DC interactions and the implications for this in mediating the downstream signaling events, which influence the transcriptional and epigenetic regulation of effector differentiation. PMID:26834747

  9. T-cell receptor BV gene usage in colorectal carcinoma patients immunised with recombinant Ep-CAM protein or anti-idiotypic antibody.

    PubMed

    Mosolits, Szilvia; Markovic, Katja; Fagerberg, Jan; Frödin, Jan-Erik; Rezvany, Mohammad-Reza; Kiaii, Shahryar; Mellstedt, Håkan; Jeddi-Tehrani, Mahmood

    2005-06-01

    The tumour-associated antigen, Ep-CAM, is over-expressed in colorectal carcinoma (CRC). In the present study, a recombinant Ep-CAM protein or a human anti-idiotypic antibody (anti-Id) mimicking Ep-CAM, either alone or in combination, was used for vaccination of CRC patients (n=9). GM-CSF was given as an adjuvant cytokine. A cellular immune response was assessed by measuring anti-Ep-CAM lymphoproliferation, IFN-gamma production (ELISPOT) and by analysing the TCR BV gene usage within the CD4+ and CD8+ T-cell subsets followed by CDR3 fragment analysis. A proliferative and/or IFN-gamma T-cell response was induced against the Ep-CAM protein in eight out of nine patients, and against Ep-CAM-derived peptides in nine out of nine patients. Analysis of the TCR BV gene usage showed a significantly higher usage of BV12 family in CD4+ T cells of patients both before and after immunisation than in those of healthy control donors (p<0.05). In the CD8+ T-cell subset, a significant (p<0.05) increase in the BV19 usage was noted in patients after immunisation. In individual patients, a number of TCR BV gene families in both CD4+ and CD8+ T cells were over-expressed mainly in post-immunisation samples. Analysis of the CDR3 length polymorphism revealed a higher degree of clonality in post-immunisation samples than in pre-immunisation samples. In vitro stimulation with Ep-CAM protein confirmed the expansion of anti-Ep-CAM T-cell clones. The results indicate that immunisation with the Ep-CAM protein and/or anti-Id entails the induction of an anti-Ep-CAM T-cell response in CRC patients, and suggest that BV19+ CD8+ T cells might be involved in a vaccine-induced immune response.

  10. Characterization of the translation products of the major mRNA species from rabbit lactating mammary glands and construction of bacterial recombinants containing casein and alpha-lactalbumin complementary DNA.

    PubMed Central

    Suard, Y M; Tosi, M; Kraehenbuhl, J P

    1982-01-01

    Total cytoplasmic polyadenylated RNA from lactating rabbit mammary glands was analysed on methylmercury hydroxide-agarose gels. The size of the most abundant mRNA species ranged between 0.5 and 5.0 kb (kilobases), with major bands at 0.55, 0.84, 0.92, 1.18 and 2.4 kb and discrete minor bands of 1.5, 1.7, 3.0 and 3.9 kb. Translation in vitro of total mRNA with [3H]leucine or [35S]methionine as precursor yielded four major bands with apparent Mr values of 16 000, 25 000, 26 000 and 29 000. The four protein bands were identified by immunoprecipitation by using specific antisera as alpha-lactalbumin and x-, kappa- and alpha-caseins, respectively. Labelling with (35S]cysteine followed by immunoprecipitation with anti-transferrin or anti-alpha-lactalbumin sera allowed the identification of two whey proteins. Translated transferrin was resolved as an 80 000-dalton band and alpha-lactalbumin appeared as a 16 000-dalton protein. A library of recombinant plasmids containing cDNA (complementary DNA) sequences representing cytoplasmic polyadenylated RNA was used to isolate clones for the major rabbit caseins and alpha-lactalbumin. A preliminary characterization of these cDNA clones was achieved by colony hybridization with enriched RNA fractions as probes. Positive clones were identified by use of hybrid-promoted translation in vitro and immunoprecipitation of the translation products. The corresponding mRNA species were further identified by hybridizing RNA blots with radioactively labelled cDNA clones. We present the restriction map of alpha-casein and kappa-casein cDNA clones. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. PMID:6123313

  11. SDF-1 signaling via the CXCR4-TCR heterodimer requires PLC-β3 and PLC-γ1 for distinct cellular responses 1

    PubMed Central

    Kremer, Kimberly N.; Clift, Ian C.; Miamen, Alexander G.; Bamidele, Adebowale O.; Qian, Nan-Xin; Humphreys, Troy D.; Hedin, Karen E.

    2011-01-01

    The CXCR4 chemokine receptor is a G protein-coupled receptor (GPCR) that signals in T lymphocytes by forming a heterodimer with the T cell antigen receptor (TCR). CXCR4 and TCR functions are consequently highly cross-regulated, affecting T cell immune activation, cytokine secretion, and T cell migration. The CXCR4-TCR heterodimer stimulates T cell migration and activation of the ERK MAP kinase and downstream AP-1-dependent cytokine transcription in response to SDF-1, the sole chemokine ligand of CXCR4. These responses require Gi-type G proteins as well as TCR ITAM domains and the ZAP-70 tyrosine kinase, thus indicating that the CXCR4-TCR heterodimer signals to integrate GPCR-associated and TCR-associated signaling molecules in response to SDF-1. Yet, the phospholipase C (PLC) isozymes responsible for coupling the CXCR4-TCR heterodimer to distinct downstream cellular responses are incompletely characterized. Here, we demonstrate that PLC activity is required for SDF-1 to induce ERK activation, migration, and CXCR4 endocytosis in human T cells. SDF-1 signaling via the CXCR4-TCR heterodimer uses PLC-β3 to activate the Ras-ERK pathway and increase intracellular Ca2+ concentrations, while PLC-γ1 is dispensable for these outcomes. In contrast, PLC-γ1, but not PLC-β3, is required for SDF-1-mediated migration, via a mechanism independent of LAT. These results increase understanding of the signaling mechanisms employed by the CXCR4-TCR heterodimer, characterize new roles for PLC-β3 and PLC-γ1 in T cells, and suggest that multiple PLCs may also be activated downstream of other chemokine receptors in order to distinctly regulate migration versus other signaling functions. PMID:21705626

  12. Direct molecular mimicry enables off-target cardiovascular toxicity by an enhanced affinity TCR designed for cancer immunotherapy.

    PubMed

    Raman, Marine C C; Rizkallah, Pierre J; Simmons, Ruth; Donnellan, Zoe; Dukes, Joseph; Bossi, Giovanna; Le Provost, Gabrielle S; Todorov, Penio; Baston, Emma; Hickman, Emma; Mahon, Tara; Hassan, Namir; Vuidepot, Annelise; Sami, Malkit; Cole, David K; Jakobsen, Bent K

    2016-01-13

    Natural T-cell responses generally lack the potency to eradicate cancer. Enhanced affinity T-cell receptors (TCRs) provide an ideal approach to target cancer cells, with emerging clinical data showing significant promise. Nevertheless, the risk of off target reactivity remains a key concern, as exemplified in a recent clinical report describing fatal cardiac toxicity, following administration of MAGE-A3 specific TCR-engineered T-cells, mediated through cross-reactivity with an unrelated epitope from the Titin protein presented on cardiac tissue. Here, we investigated the structural mechanism enabling TCR cross-recognition of MAGE-A3 and Titin, and applied the resulting data to rationally design mutants with improved antigen discrimination, providing a proof-of-concept strategy for altering the fine specificity of a TCR towards an intended target antigen. This study represents the first example of direct molecular mimicry leading to clinically relevant fatal toxicity, mediated by a modified enhanced affinity TCR designed for cancer immunotherapy. Furthermore, these data demonstrate that self-antigens that are expressed at high levels on healthy tissue should be treated with extreme caution when designing immuno-therapeutics.

  13. Human antibodies to bovine alpha-globulin.

    PubMed

    Foucard, T; Bennich, H; Johansson, S G; Lundkvist, U

    1975-01-01

    Antibodies to bovine gamma-globulin (anti-BGG antibodies) were detectable by a radio-immunoassay in 70% of healthy blood donors but, generally, the titres were low. Significantly increased concentrations of anti-BGG antibodies were found in patients lacking IgA but not in patients with allergic disorders. The anti-BGG antibodies were shown to give rise to falsely high IgE values in the radio-immunosorbent test for IgE determination (RIST) when a sheep anti-IgE antiserum was used. Furthermore, falsely positive results can sometimes be caused by such antibodies in the determination of cow-dander- or cow's-milk-specific IgE by the radio-allergosorbent test (RAST). When a rabbit anti-IgE antiserum was used instead of the sheep anti-IgE, normal IgE levels and negative RAST results were obtained. The difference is explained by the higher degree of cross-reactivity between the anti-BGG antibodies and sheep alpha-globulin than between anti-BGG antibodies and rabbit alpha-globulin.

  14. TCR signaling by conventional CD4+ T cells is required for optimal maintenance of peripheral regulatory T cell numbers.

    PubMed

    Leichner, Theresa M; Satake, Atsushi; Kambayashi, Taku

    2016-06-01

    To maintain immune tolerance, regulatory T cell (Treg) numbers must be closely indexed to the number of conventional T cells (Tconvs) so that an adequate Treg:Tconv ratio can be maintained. Two factors important in this process are the cytokine interleukin-2 (IL-2) and T cell receptor (TCR) stimulation by major histocompatibility complex class II (MHC-II). Here, we report that in addition to TCR stimulation of Tregs themselves, the maintenance of Tregs also requires TCR signaling by Tconvs. We found that Tconvs produce IL-2 in response to self-peptide-MHC-II complexes and that Tconvs possessing more highly self-reactive TCRs express more IL-2 at baseline. Furthermore, selective disruption of TCR signaling in Tconvs led to a trend toward decreased expression of IL-2 and attenuated their ability to maintain Treg numbers. These data suggest that in order to maintain an adequate Treg:Tconv ratio, Tregs are continuously indexed to self-peptide-MHC-II-induced TCR signaling of Tconvs. These results have implications in attempts to modulate immune tolerance, as Treg numbers adjust to the self-reactivity, and ultimately IL-2 production by the T cells around them.

  15. Propensity of a single-walled carbon nanotube-peptide to mimic a KK10 peptide in an HLA-TCR complex

    NASA Astrophysics Data System (ADS)

    Feng, Mei; Bell, David R.; Zhou, Ruhong

    2017-12-01

    The application of nanotechnology to improve disease diagnosis, treatment, monitoring, and prevention is the goal of nanomedicine. We report here a theoretical study of a functionalized single-walled carbon nanotube (CNT) mimic binding to a human leukocyte antigen-T cell receptor (HLA-TCR) immune complex as a first attempt of a potential nanomedicine for human immunodeficiency virus (HIV) vaccine development. The carbon nanotube was coated with three arginine residues to imitate the HIV type 1 immunodominant viral peptide KK10 (gag 263-272: KRWIILGLNK), named CNT-peptide hereafter. Through molecular dynamics simulations, we explore the CNT-peptide and KK10 binding to an important HLA-TCR complex. Our results suggest that the CNT-peptide and KK10 bind comparably to the HLA-TCR complex, but the CNT-peptide forms stronger interactions with the TCR. Desorption simulations highlight the innate flexibility of KK10 over the CNT-peptide, resulting in a slightly higher desorption energy required for KK10 over the CNT-peptide. Our findings indicate that the designed CNT-peptide mimic has favorable propensity to activate TCR pathways and should be further explored to understand therapeutic potential.

  16. Regulation of TCR Signaling to NF-kB

    DTIC Science & Technology

    2013-03-20

    applications and benefits to basic research are discussed in detail in Chapter 4.      16   CHAPTER 2: Selective autophagy of the...To confirm the specificity of the chemical inhibitors of autophagy, we employed a genetic approach. Specifically, we in vitro differentiated Th2...MG132 treatment. These data provide strong genetic evidence that TCR-mediated degradation of endogenous Bcl10 is controlled by autophagy, with a

  17. In vitro and in vivo expression of interleukin-1alpha and tumor necrosis factor-alpha mRNA in pemphigus vulgaris: interleukin-1alpha and tumor necrosis factor-alpha are involved in acantholysis.

    PubMed

    Feliciani, C; Toto, P; Amerio, P; Pour, S M; Coscione, G; Shivji, G; Wang, B; Sauder, D N

    2000-01-01

    Keratinocyte-derived cytokines have been implicated in the pathogenesis of a number of skin diseases. In this study we examined the possible role of keratinocyte-derived cytokines in the development of acantholysis in pemphigus vulgaris. Nineteen patients with pemphigus vulgaris, demonstrating the characteristic clinical, pathologic, and immunopathologic findings were studied. In situ immunolabeling demonstrated the presence of two cytokines interleukin-1alpha and tumor necrosis factor-alpha, in lesional and perilesional areas. Results were confirmed by reverse transcriptase-polymerase chain reaction, demonstrating overexpression of both cytokines in vivo. To study the role of these cytokines in the pathogenesis of pemphigus vulgaris both in vitro and in vivo studies were performed. The results of the in vitro study demonstrated that pemphigus vulgaris IgG induced interleukin-1alpha and tumor necrosis factor-alpha mRNA in the skin. The potential pathogenic role of these mediators was demonstrated by a blocking study using antibodies against human interleukin-1alpha and tumor necrosis factor-alpha in keratinocytes cultures. A combination of anti-interleukin-1alpha and anti-tumor necrosis factor-alpha antibodies inhibited in vitro pemphigus vulgaris IgG induced acantholysis. To confirm the role of interleukin-1 and tumor necrosis factor-alpha in pemphigus, we utilized passive transfer studies using interleukin-1 deficient mice (ICE-/-, interleukin-1beta-/-) and tumor necrosis factor-alpha receptor deficient mice (TNFR1R2-/-). Both groups demonstrated a decreased susceptibility to the passive transfer of pemphigus. Our data support the role of cytokines interleukin-1 and tumor necrosis factor-alpha in the pathogenesis of pemphigus vulgaris.

  18. Synthetic. cap alpha. subunit peptide 125-147 of human nicotinic acetylcholine receptor induces antibodies to native receptor

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    McCormick, D.J.; Griesmann, G.E.; Huang, Z.

    1986-03-05

    A synthetic peptide corresponding to residues 125-147 of the Torpedo acetylcholine receptor (AChR) ..cap alpha.. subunit proved to be a major antigenic region of the AChR. Rats inoculated with 50 ..mu..g of peptide (T ..cap alpha.. 125-147) developed T cell immunity and antibodies to native AChR and signs of experimental autoimmune myasthenia gravis. They report the synthesis and preliminary testing of a disulfide-looped peptide comprising residues 125-147 of the human AChR ..cap alpha.. subunit. Peptide H ..cap alpha.. 125-147 differs from T ..cap alpha.. 125-147 at residues 139 (Glu for Gln) and 143 (Ser for Thr). In immunoprecipitation assays, antibodiesmore » to Torpedo AChR bound /sup 125/I-labelled H..cap alpha.. 125-147 antibody bound H..cap alpha.. 125-147, but monoclonal antibodies to an immunodominant region of native AChR bound neither H..cap alpha.. 125-147 nor T ..cap alpha.. 125-147. Rats immunized with H ..cap alpha.. 125-147 produced anti-mammalian muscle AChR antibodies that induced modulation of AChRs from cultured human myotubes. Thus, region 125-147 of the human AChR ..cap alpha.. subunit is extracellular in muscle, and is both antigenic and immunogenic. It remains to be determined whether or not autoantibodies to this region may in part cause the weakness or myasthenia gravis in man.« less

  19. Uneven colonization of the lymphoid periphery by T cells that undergo early TCR{alpha} rearrangements.

    PubMed

    Hendricks, Deborah W; Fink, Pamela J

    2009-04-01

    A sparse population of thymocytes undergoes TCRalpha gene rearrangement early in development, before the double-positive stage. The potential of these cells to contribute to the peripheral T cell pool is unknown. To examine the peripheral T cell compartment expressing a repertoire biased to early TCR gene rearrangements, we developed a mouse model in which TCRalpha rearrangements are restricted to the double-negative stage of thymocyte development. These mice carry floxed RAG2 alleles and a Cre transgene driven by the CD4 promoter. As expected, conventional T cell development is compromised in such Cre(+) RAG2(fl/fl) mice, and the TCRalphabeta(+) T cells that develop are limited in their TCRalpha repertoire, preferentially using early rearranging Valpha genes. In the gut, the Thy-1(+)TCRalphabeta(+) intraepithelial lymphocyte (IEL) compartment is surprisingly intact, whereas the Thy-1(-)TCRalphabeta(+) subset is almost completely absent. Thus, T cells expressing a TCRalpha repertoire that is the product of early gene rearrangements can preferentially populate distinct IEL compartments. Despite this capacity, Cre(+) RAG2(fl/fl) T cell progenitors cannot compete with wild-type T cell progenitors in mixed bone marrow chimeras, suggesting that in normal mice, there is only a small contribution to the peripheral T cell pool by cells that have undergone early TCRalpha rearrangements. In the absence of wild-type competitors, aggressive homeostatic proliferation in the IEL compartment can promote a relatively normal Thy-1(+) TCRalphabeta(+) T cell pool from the limited population derived from Cre(+) RAG2(fl/fl) progenitors.

  20. Durable donor engraftment after radioimmunotherapy using α-emitter astatine-211–labeled anti-CD45 antibody for conditioning in allogeneic hematopoietic cell transplantation

    PubMed Central

    Chen, Yun; Kornblit, Brian; Hamlin, Donald K.; Sale, George E.; Santos, Erlinda B.; Wilbur, D. Scott; Storer, Barry E.; Storb, Rainer

    2012-01-01

    To reduce toxicity associated with external γ-beam radiation, we investigated radioimmunotherapy with an anti-CD45 mAb labeled with the α-emitter, astatine-211 (211At), as a conditioning regimen in dog leukocyte antigen-identical hematopoietic cell transplantation (HCT). Dose-finding studies in 6 dogs treated with 100 to 618 μCi/kg 211At-labeled anti-CD45 mAb (0.5 mg/kg) without HCT rescue demonstrated dose-dependent myelosuppression with subsequent autologous recovery, and transient liver toxicity in dogs treated with 211At doses less than or equal to 405 μCi/kg. Higher doses of 211At induced clinical liver failure. Subsequently, 8 dogs were conditioned with 155 to 625 μCi/kg 211At-labeled anti-CD45 mAb (0.5 mg/kg) before HCT with dog leukocyte antigen-identical bone marrow followed by a short course of cyclosporine and mycophenolate mofetil immunosuppression. Neutropenia (1-146 cells/μL), lymphopenia (0-270 cells/μL), and thrombocytopenia (1500-6560 platelets/μL) with prompt recovery was observed. Seven dogs had long-term donor mononuclear cell chimerism (19%-58%), whereas 1 dog treated with the lowest 211At dose (155 μCi/kg) had low donor mononuclear cell chimerism (5%). At the end of follow-up (18-53 weeks), only transient liver toxicity and no renal toxicity had been observed. In conclusion, conditioning with 211At-labeled anti-CD45 mAb is safe and efficacious and provides a platform for future clinical trials of nonmyeloablative transplantation with radioimmunotherapy-based conditioning. PMID:22134165

  1. An optimized single chain TCR scaffold relying on the assembly with the native CD3-complex prevents residual mispairing with endogenous TCRs in human T-cells

    PubMed Central

    Knies, Diana; Klobuch, Sebastian; Xue, Shao-An; Birtel, Matthias; Echchannaoui, Hakim; Yildiz, Oezlem; Omokoko, Tana; Guillaume, Philippe; Romero, Pedro; Stauss, Hans; Sahin, Ugur; Herr, Wolfgang; Theobald, Matthias; Thomas, Simone; Voss, Ralf-Holger

    2016-01-01

    Immunotherapy of cancer envisions the adoptive transfer of T-cells genetically engineered with tumor-specific heterodimeric α/β T-cell receptors (TCRα/β). However, potential mispairing of introduced TCRα/β-chains with endogenous β/α-ones may evoke unpredictable autoimmune reactivities. A novel single chain (sc)TCR format relies on the fusion of the Vα-Linker-Vβ-fragment to the TCR Cβ-domain and coexpression of the TCR Cα-domain capable of recruiting the natural CD3-complex for full and hence, native T-cell signaling. Here, we tested whether such a gp100(280-288)- or p53(264-272) tumor antigen-specific scTCR is still prone to mispairing with TCRα. In a human Jurkat-76 T-cell line lacking endogenous TCRs, surface expression and function of a scTCR could be reconstituted by any cointroduced TCRα-chain indicating mispairing to take place on a molecular basis. In contrast, transduction into human TCRα/β-positive T-cells revealed that mispairing is largely reduced. Competition experiments in Jurkat-76 confirmed the preference of dcTCR to selfpair and to spare scTCR. This also allowed for the generation of dc/scTCR-modified cytomegalovirus/tumor antigen-bispecific T-cells to augment T-cell activation in CMV-infected tumor patients. Residual mispairing was prevented by strenghtening the Vα-Li-Vβ-fragment through the design of a novel disulfide bond between a Vα- and a linker-resident residue close to Vβ. Multimer-stainings, and cytotoxicity-, IFNγ-secretion-, and CFSE-proliferation-assays, the latter towards dendritic cells endogenously processing RNA-electroporated gp100 antigen proved the absence of hybrid scTCR/TCRα-formation without impairing avidity of scTCR/Cα in T-cells. Moreover, a fragile cytomegalovirus pp65(495-503)-specific scTCR modified this way acquired enhanced cytotoxicity. Thus, optimized scTCR/Cα inhibits residual TCR mispairing to accomplish safe adoptive immunotherapy for bulk endogenous TCRα/β-positive T-cells. PMID:27028870

  2. Staphylococcus Alpha-Toxin Action on the Rabbit Iris: Toxic Effects and Their Inhibition.

    PubMed

    Arana, Angela M; Bierdeman, Michael A; Balzli, Charles L; Tang, Aihua; Caballero, Armando R; Patel, Rupesh; O'Callaghan, Richard J

    2015-01-01

    Staphylococcus aureus infection of the anterior chamber can occur after cataract surgery, causing inflammation and extensive damage to the iris. Alpha-toxin, the most potent S. aureus corneal toxin, was tested as a possible mediator of damage to the iris, and alpha-toxin anti-serum and a chemical toxin inhibitor were tested as potential pathology-reducing agents. The hemolytic activity of alpha-toxin and its inhibition by a chemical inhibitor or anti-serum were quantified in vitro. Purified alpha-toxin, heat-inactivated toxin, or alpha-toxin plus normal serum, alpha-toxin anti-serum, or the chemical inhibitor, methyl-β-cyclodextrin-cholesterol (CD-cholesterol), was injected into the rabbit anterior chamber. Pathological changes were photographed, quantified by slit-lamp examination (SLE) scoring, and further documented by histopathological analysis. At five hours post-injection, eyes injected with alpha-toxin or heat-inactivated toxin had a mean SLE score of 7.3 ± 0.59 or 0.84 ± 0.19, respectively. Active toxin caused moderate to severe iris edema, severe erosion of the iris, and mild to moderate fibrin accumulation in the anterior chamber. Alpha-toxin plus anti-serum or CD-cholesterol, in contrast to alpha-toxin alone, caused less iris edema and epithelium sloughing as well as significantly lower SLE scores than eyes receiving alpha-toxin alone (p ≤ 0.019). Alpha-toxin caused extensive iris damage and inflammation, and either anti-alpha-toxin anti-serum or CD-cholesterol was able to significantly reduce toxin-mediated damage and inflammation.

  3. [Alpha-melanocyte-stimulating hormone. From bench to bedside].

    PubMed

    Böhm, M; Luger, T A

    2010-06-01

    Alpha-melanocyte-stimulating hormone (alpha-MSH) is a tridecapeptide that is produced by the skin itself from the precursor proopiomelanocortin. It crucially mediates ultraviolet light-induced tanning after binding to melanocortin-1 receptors (MC-1R) expressed on the surface of epidermal melanocytes. The potent pigment-inducing and also cytoprotective actions of alpha-MSH are the rationale for the performance of first phase II clinical trials with Nle4-D-Phe7-alpha-MSH (NDP-alpha-MSH), a subcutaneously administered synthetic and superpotent alpha-MSH analogue, in patients with photodermatoses such as erythropoietic protoporphyria. Since alpha-MSH has shown promising anti-inflammatory and antifibrotic properties in numerous preclinical studies, it will be most interesting to evaluate these effects in further clinical pilot studies with NDP-alpha-MSH. In addition to alpha-MSH analogues, truncated tripeptides such as KDPT which do not bind to MC-1R but have sustained anti-inflammatory properties are currently emerging as another novel therapeutic strategy in dermatology.

  4. Direct molecular mimicry enables off-target cardiovascular toxicity by an enhanced affinity TCR designed for cancer immunotherapy

    PubMed Central

    Raman, Marine C C; Rizkallah, Pierre J; Simmons, Ruth; Donnellan, Zoe; Dukes, Joseph; Bossi, Giovanna; Le Provost, Gabrielle S; Todorov, Penio; Baston, Emma; Hickman, Emma; Mahon, Tara; Hassan, Namir; Vuidepot, Annelise; Sami, Malkit; Cole, David K; Jakobsen, Bent K.

    2016-01-01

    Natural T-cell responses generally lack the potency to eradicate cancer. Enhanced affinity T-cell receptors (TCRs) provide an ideal approach to target cancer cells, with emerging clinical data showing significant promise. Nevertheless, the risk of off target reactivity remains a key concern, as exemplified in a recent clinical report describing fatal cardiac toxicity, following administration of MAGE-A3 specific TCR-engineered T-cells, mediated through cross-reactivity with an unrelated epitope from the Titin protein presented on cardiac tissue. Here, we investigated the structural mechanism enabling TCR cross-recognition of MAGE-A3 and Titin, and applied the resulting data to rationally design mutants with improved antigen discrimination, providing a proof-of-concept strategy for altering the fine specificity of a TCR towards an intended target antigen. This study represents the first example of direct molecular mimicry leading to clinically relevant fatal toxicity, mediated by a modified enhanced affinity TCR designed for cancer immunotherapy. Furthermore, these data demonstrate that self-antigens that are expressed at high levels on healthy tissue should be treated with extreme caution when designing immuno-therapeutics. PMID:26758806

  5. SLAP deficiency increases TCR avidity leading to altered repertoire and negative selection of cognate antigen-specific CD8+ T cells

    PubMed Central

    Friend, Samantha F.; Peterson, Lisa K.; Kedl, Ross M.; Dragone, Leonard L.

    2014-01-01

    How T cell receptor (TCR) avidity influences CD8+ T cell development and repertoire selection is not yet fully understood. To fill this gap, we utilized Src-like adaptor protein (SLAP)-deficient mice as a tool to increase TCR avidity on double positive (DP) thymocytes. We generated SLAP−/− mice with the transgenic MHC class I-restricted TCR (OT-1) and SLAP−/− Vβ5 mice, expressing only the β-chain of the TCR OT-1 transgene, to examine the effects of increased TCR surface levels on CD8+ T cell development and repertoire selection. In comparing SLAP−/− OT-1 and Vβ5 mice with wild-type controls, we performed compositional analysis and assessed thymocyte signaling by measuring CD5 levels. In addition, we performed tetramer and compositional staining to measure affinity for the cognate antigen, ovalbumin (OVA) peptide, presented by MHC. Furthermore, we quantified differences in α-chain repertoire in SLAP−/− Vβ5 mice. We have found that SLAP−/− OT-1 mice have fewer CD8+ thymocytes but have increased CD5 expression. SLAP−/− OT-1 mice have fewer DP thymocytes expressing Vα2, signifying increased endogenous α-chain rearrangement, and more non-OVA-specific CD8+ splenocytes upon tetramer staining. Our data demonstrate that SLAP−/− Vβ5 mice also have fewer OVA-specific cells and increased Vα2 usage in the peripheral Vβ5 CD8+ T cells that were non-OVA-specific, demonstrating differences in α-chain repertoire. These studies provide direct evidence that increased TCR avidity in DP thymocytes enhances CD8+ T cell negative selection deleting thymocytes with specificity for cognate antigen, an antigen the mature T cells may never encounter. Collectively, these studies provide new insights into how TCR avidity during CD8+ T cell development influences repertoire selection. PMID:22956467

  6. SLAP deficiency increases TCR avidity leading to altered repertoire and negative selection of cognate antigen-specific CD8+ T cells.

    PubMed

    Friend, Samantha F; Peterson, Lisa K; Kedl, Ross M; Dragone, Leonard L

    2013-03-01

    How T cell receptor (TCR) avidity influences CD8(+) T cell development and repertoire selection is not yet fully understood. To fill this gap, we utilized Src-like adaptor protein (SLAP)-deficient mice as a tool to increase TCR avidity on double positive (DP) thymocytes. We generated SLAP(-/-) mice with the transgenic MHC class I-restricted TCR (OT-1) and SLAP(-/-) Vβ5 mice, expressing only the β-chain of the TCR OT-1 transgene, to examine the effects of increased TCR surface levels on CD8(+) T cell development and repertoire selection. In comparing SLAP(-/-) OT-1 and Vβ5 mice with wild-type controls, we performed compositional analysis and assessed thymocyte signaling by measuring CD5 levels. In addition, we performed tetramer and compositional staining to measure affinity for the cognate antigen, ovalbumin (OVA) peptide, presented by MHC. Furthermore, we quantified differences in α-chain repertoire in SLAP(-/-) Vβ5 mice. We have found that SLAP(-/-) OT-1 mice have fewer CD8(+) thymocytes but have increased CD5 expression. SLAP(-/-) OT-1 mice have fewer DP thymocytes expressing Vα2, signifying increased endogenous α-chain rearrangement, and more non-OVA-specific CD8(+) splenocytes upon tetramer staining. Our data demonstrate that SLAP(-/-) Vβ5 mice also have fewer OVA-specific cells and increased Vα2 usage in the peripheral Vβ5 CD8(+) T cells that were non-OVA-specific, demonstrating differences in α-chain repertoire. These studies provide direct evidence that increased TCR avidity in DP thymocytes enhances CD8(+) T cell negative selection deleting thymocytes with specificity for cognate antigen, an antigen the mature T cells may never encounter. Collectively, these studies provide new insights into how TCR avidity during CD8(+) T cell development influences repertoire selection.

  7. KIAA1530 Protein Is Recruited by Cockayne Syndrome Complementation Group Protein A (CSA) to Participate in Transcription-coupled Repair (TCR)

    PubMed Central

    Fei, Jia; Chen, Junjie

    2012-01-01

    Transcription-coupled repair (TCR) is the major pathway involved in the removal of UV-induced photolesions from the transcribed strand of active genes. Two Cockayne syndrome (CS) complementation group proteins, CSA and CSB, are important for TCR repair. The molecular mechanisms by which CS proteins regulate TCR remain elusive. Here, we report the characterization of KIAA1530, an evolutionarily conserved protein that participates in this pathway through its interaction with CSA and the TFIIH complex. We found that UV irradiation led to the recruitment of KIAA1530 onto chromatin in a CSA-dependent manner. Cells lacking KIAA1530 were highly sensitive to UV irradiation and displayed deficiency in TCR. In addition, KIAA1530 depletion abrogated stability of the CSB protein following UV irradiation. More excitingly, we found that a unique CSA mutant (W361C), which was previously identified in a patient with UVsS syndrome, showed defective KIAA1530 binding and resulted in a failure of recruiting KIAA1530 and stabilizing CSB after UV treatment. Together, our data not only reveal that KIAA1530 is an important player in TCR but also lead to a better understanding of the molecular mechanism underlying UVsS syndrome. PMID:22902626

  8. Occurrence of the Transferable Copper Resistance Gene tcrB among Fecal Enterococci of U.S. Feedlot Cattle Fed Copper-Supplemented Diets

    PubMed Central

    Amachawadi, R. G.; Alvarado, C. A.; Mainini, T. R.; Vinasco, J.; Drouillard, J. S.; Nagaraja, T. G.

    2013-01-01

    Copper, an essential micronutrient, is supplemented in the diet at elevated levels to reduce morbidity and mortality and to promote growth in feedlot cattle. Gut bacteria exposed to copper can acquire resistance, which among enterococci is conferred by a transferable copper resistance gene (tcrB) borne on a plasmid. The present study was undertaken to investigate whether the feeding of copper at levels sufficient to promote growth increases the prevalence of the tcrB gene among the fecal enterococci of feedlot cattle. The study was performed with 261 crossbred yearling heifers housed in 24 pens, with pens assigned randomly to a 2×2 factorial arrangement of treatments consisting of dietary copper and a commercial linseed meal-based energy protein supplement. A total of 22 isolates, each identified as Enterococcus faecium, were positive for tcrB with an overall prevalence of 3.8% (22/576). The prevalence was higher among the cattle fed diets supplemented with copper (6.9%) compared to normal copper levels (0.7%). The tcrB-positive isolates always contained both erm(B) and tet(M) genes. Median copper MICs for tcrB-positive and tcrB-negative enterococci were 22 and 4 mM, respectively. The transferability of the tcrB gene was demonstrated via a filter-mating assay. Multilocus variable number tandem repeat analysis revealed a genetically diverse population of enterococci. The finding of a strong association between the copper resistance gene and other antibiotic (tetracycline and tylosin) resistance determinants is significant because enterococci remain potential pathogens and have the propensity to transfer resistance genes to other bacteria in the gut. PMID:23666328

  9. Expression of GDNF and GFR alpha 1 in mouse taste bud cells.

    PubMed

    Takeda, Masako; Suzuki, Yuko; Obara, Nobuko; Uchida, Nobuhiko; Kawakoshi, Kentaro

    2004-11-01

    GDNF (glial cell line-derived neurotrophic factor) affects the survival and maintenance of central and peripheral neurons. Using an immunocytochemical method, we examined whether the taste bud cells in the circumvallate papillae of normal mice expressed GDNF and its GFR alpha 1 receptor. Using double immunostaining for either of them and NCAM, PGP 9.5, or alpha-gustducin, we additionally sought to determine what type of taste bud cells expressed GDNF or GFR alpha 1, because NCAM is reported to be expressed in type-III cells, PGP 9.5, in type-III and some type-II cells, and alpha-gustducin, in some type-II cells. Normal taste bud cells expressed both GDNF and GFR alpha 1. The percentage of GDNF-immunoreactive cells among all taste bud cells was 31.63%, and that of GFR alpha 1-immunoreactive cells, 83.21%. Confocal laser scanning microscopic observations after double immunostaining showed that almost none of the GDNF-immunoreactive cells in the taste buds were reactive with anti-NCAM or anti-PGP 9.5 antibody, but could be stained with anti-alpha-gustducin antibody. On the other hand, almost all anti-PGP 9.5- or anti-alpha-gustducin-immunoreactive cells were positive for GFR alpha 1. Thus, GDNF-immunoreactive cells did not include type-III cells, but type-II cells, which are alpha-gustducin-immunoreactive; on the other hand, GFR alpha 1-immunoreactive cells included type-II and -III cells, and perhaps type-I cells. We conclude that GDNF in the type-II cells may exert trophic actions on type-I, -II, and -III taste bud cells by binding to their GFR alpha 1 receptors.

  10. How many TCR clonotypes does a body maintain?

    PubMed Central

    Lythe, Grant; Callard, Robin E.; Hoare, Rollo L.; Molina-París, Carmen

    2016-01-01

    We consider the lifetime of a T cell clonotype, the set of T cells with the same T cell receptor, from its thymic origin to its extinction in a multiclonal repertoire. Using published estimates of total cell numbers and thymic production rates, we calculate the mean number of cells per TCR clonotype, and the total number of clonotypes, in mice and humans. When there is little peripheral division, as in a mouse, the number of cells per clonotype is small and governed by the number of cells with identical TCR that exit the thymus. In humans, peripheral division is important and a clonotype may survive for decades, during which it expands to comprise many cells. We therefore devise and analyse a computational model of homeostasis of a multiclonal population. Each T cell in the model competes for self pMHC stimuli, cells of any one clonotype only recognising a small fraction of the many subsets of stimuli. A constant mean total number of cells is maintained by a balance between cell division and death, and a stable number of clonotypes by a balance between thymic production of new clonotypes and extinction of existing ones. The number of distinct clonotypes in a human body may be smaller than the total number of naive T cells by only one order of magnitude. PMID:26546971

  11. BCL11B enhances TCR/CD28-triggered NF-kappaB activation through up-regulation of Cot kinase gene expression in T-lymphocytes.

    PubMed

    Cismasiu, Valeriu B; Duque, Javier; Paskaleva, Elena; Califano, Danielle; Ghanta, Sailaja; Young, Howard A; Avram, Dorina

    2009-01-15

    BCL11B is a transcriptional regulator with an important role in T-cell development and leukaemogenesis. We demonstrated recently that BCL11B controls expression from the IL (interleukin)-2 promoter through direct binding to the US1 (upstream site 1). In the present study, we provide evidence that BCL11B also participates in the activation of IL-2 gene expression by enhancing NF-kappaB (nuclear factor kappaB) activity in the context of TCR (T-cell receptor)/CD28-triggered T-cell activation. Enhanced NF-kappaB activation is not a consequence of BCL11B binding to the NF-kappaB response elements or association with the NF-kappaB-DNA complexes, but rather the result of higher translocation of NF-kappaB to the nucleus caused by enhanced degradation of IkappaB (inhibitor of NF-kappaB). The enhanced IkappaB degradation in cells with increased levels of BCL11B was specific for T-cells activated through the TCR, but not for cells activated through TNFalpha (tumour necrosis factor alpha) or UV light, and was caused by increased activity of IkappaB kinase, as indicated by its increase in phosphorylation. As BCL11B is a transcription factor, we investigated whether the expression of genes upstream of IkappaB kinase in the TCR/CD28 signalling pathway was affected by increased BCL11B expression, and found that Cot (cancer Osaka thyroid oncogene) kinase mRNA levels were elevated. Cot kinase is known to promote enhanced IkappaB kinase activity, which results in the phosphorylation and degradation of IkappaB and activation of NF-kappaB. The implied involvement of Cot kinase in BCL11B-mediated NF-kappaB activation in response to TCR activation is supported by the fact that a Cot kinase dominant-negative mutant or Cot kinase siRNA (small interfering RNA) knockdown blocked BCL11B-mediated NF-kappaB activation. In support of our observations, in the present study we report that BCL11B enhances the expression of several other NF-kappaB target genes, in addition to IL-2. In addition, we

  12. Label-free immunosensor based on Pd nanoplates for amperometric immunoassay of alpha-fetoprotein.

    PubMed

    Wang, Huan; Li, He; Zhang, Yihe; Wei, Qin; Ma, Hongmin; Wu, Dan; Li, Yan; Zhang, Yong; Du, Bin

    2014-03-15

    In this paper, Pd nanoplates were used as a kind of electrode materials for fabrication of an electrochemical immunosensor, which was applied for detection of cancer biomarker alpha-fetoprotein (AFP). Thanks to the unique structure and properties of Pd nanoplates, the antibody of AFP (Ab) was effectively immobilized onto the surface of the Pd nanoplates modified glassy carbon electrode (GCE). Moreover, the good electrochemical properties of Pd nanoplates greatly improved the electronic transmission rate and enhanced the electrochemical signal, which led to an increase of the detection sensitivity. Based on the specific antibody-antigen interaction, a label-free immunosensor based on Pd nanoplates was developed for sensing of AFP. The current method allows us to detect AFP over a wide concentration range from 0.01 to 75.0 ng/mL with a detection limit of 4 pg/mL. The proposed immunosensor has been used to determine AFP in human serum with satisfactory results. © 2013 Elsevier B.V. All rights reserved.

  13. A HYPOTHESIS ACCOUNTING FOR THE PARADOXICAL EXPRESSION OF THE D GENE SEGMENT IN THE BCR AND THE TCR

    PubMed Central

    Cohn, Melvin

    2009-01-01

    The D gene segment expressed in both the TCR and BCR has a challenging behavior that begs interpretation. It is incorporated in three reading frames in the rearranged transcription unit but is expressed in antigen-selected cells in a preferred frame. Why was it so important to waste 2/3 of newborn cells? The hypothesis is presented that the D region is framework playing a role in both the TCR and the BCR by determining whether a signal is transmitted to the cell upon interaction with a cognate ligand. This assumption operates in determining haplotype exclusion for the BCR and in regulating the signaling orientation for the TCR. Relevant data as well as a definitive experiment challenging the validity of this hypothesis, are discussed. PMID:18546143

  14. Fluorescently labeled chimeric anti-CEA antibody improves detection and resection of human colon cancer in a patient-derived orthotopic xenograft (PDOX) nude mouse model.

    PubMed

    Metildi, Cristina A; Kaushal, Sharmeela; Luiken, George A; Talamini, Mark A; Hoffman, Robert M; Bouvet, Michael

    2014-04-01

    The aim of this study was to evaluate a new fluorescently labeled chimeric anti-CEA antibody for improved detection and resection of colon cancer. Frozen tumor and normal human tissue samples were stained with chimeric and mouse antibody-fluorophore conjugates for comparison. Mice with patient-derived orthotopic xenografts (PDOX) of colon cancer underwent fluorescence-guided surgery (FGS) or bright-light surgery (BLS) 24 hr after tail vein injection of fluorophore-conjugated chimeric anti-CEA antibody. Resection completeness was assessed using postoperative images. Mice were followed for 6 months for recurrence. The fluorophore conjugation efficiency (dye/mole ratio) improved from 3-4 to >5.5 with the chimeric CEA antibody compared to mouse anti-CEA antibody. CEA-expressing tumors labeled with chimeric CEA antibody provided a brighter fluorescence signal on frozen human tumor tissues (P = 0.046) and demonstrated consistently lower fluorescence signals in normal human tissues compared to mouse antibody. Chimeric CEA antibody accurately labeled PDOX colon cancer in nude mice, enabling improved detection of tumor margins for more effective FGS. The R0 resection rate increased from 86% to 96% with FGS compared to BLS. Improved conjugating efficiency and labeling with chimeric fluorophore-conjugated antibody resulted in better detection and resection of human colon cancer in an orthotopic mouse model. © 2013 Wiley Periodicals, Inc.

  15. Promyelocytic leukemia zinc finger turns on the effector T cell program without requirement for agonist TCR signaling.

    PubMed

    Savage, Adam K; Constantinides, Michael G; Bendelac, Albert

    2011-05-15

    Thymocytes expressing the NKT cell semi-invariant αβ TCR are thought to undergo agonist interactions with CD1d ligands prior to expressing promyelocytic leukemia zinc finger (PLZF), a broad complex, tramtrack, bric-a-brac, poxvirus, and zinc finger transcription factor that directs acquisition of the effector program of these innate-like T cells. Whether PLZF can mediate this effector conversion independently of agonist signaling has not been investigated. We demonstrated that transgenic (Tg) expression of PLZF under the CD4 promoter induced the innate effector program in two different MHC class II-restricted TCR-Tg Rag1(-/-) models examined. In CD4 thymocytes expressing a fixed Tg TCR β-chain, the associated TCRα sequences in wild-type and PLZF-Tg mice overlapped extensively, further demonstrating that PLZF could induce the effector program in most CD4 T cells that would normally be selected as naive cells. In contrast, PLZF altered the negative selection of thymocytes expressing TCR β-chains reactive against several retroviral superantigens. Thus, PLZF is remarkable in that it is a transcription factor capable of inducing an effector program in the absence of T cell agonist interactions or cell division. Its expression may also enhance the survival of agonist-signaled thymocytes.

  16. Horseradish peroxidase functionalized gold nanorods as a label for sensitive electrochemical detection of alpha-fetoprotein antigen.

    PubMed

    Guo, Jinjin; Han, Xiaowei; Wang, Junchun; Zhao, Junqing; Guo, Zilin; Zhang, Yuzhong

    2015-12-15

    In this study, a novel tracer, horseradish peroxidase (HRP) functionalized gold nanorods (Au NRs) nanocomposites (HRP-Au NRs), was designed to label the signal antibodies for sensitive electrochemical measurement of alpha-fetoprotein (AFP). The preparation of HRP-Au NRs nanocomposites and the labeling of secondary antibody (Ab2) were performed by one-pot assembly of HRP and Ab2 on the surface of Au NRs. The immunosensor was fabricated by assembling carbon nanotubes (CNTs), Au NRs, and capture antibodies (Ab1) on the glassy carbon electrode. In the presence of AFP antigen, the labels were captured on the surface of the Au NRs/CNTs via specific recognition of antigen-antibody, resulting in the signal intensity being clearly increased. Differential pulse voltammetry (DPV) was employed to record the response signal of the immunosensor in phosphate-buffered saline (PBS) containing hydrogen peroxide (H2O2) and 3,3',5,5'-tetramethylbenzidine (TMB). Under optimal conditions, the signal intensity was linearly related to the concentration of AFP in the range of 0.1-100 ng ml(-1), and the limit of detection was 30 pg ml(-1) (at signal/noise [S/N] = 3). Furthermore, the immunoassay method was evaluated using human serum samples, and the recovery obtained was within 99.0 and 102.7%, indicating that the immunosensor has potential clinical applications. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. Anti-inflammatory and anti-bacterial properties of tetramethylhexadecenyl succinyl cysteine (TSC): a skin-protecting cosmetic functional ingredient.

    PubMed

    Fernandéz, J R; Rouzard, K; Voronkov, M; Huber, K L; Stock, J B; Stock, M; Gordon, J S; Pérez, E

    2015-02-01

    The skin is the first line of defence against exposure to microbial, physical, environmental and chemical insults. In mobilizing a protective response, several different cell types located in our skin release and respond to pro-inflammatory cytokines ensuring skin homeostasis and health. However, chronic activation of this response eventually causes damage resulting in premature ageing. Diosodium tetramethylhexadecenyl succinyl cysteine (TSC or SIG1273), an isoprenylcysteine small molecule, down modulates these inflammatory signalling pathways in various cell types (keratinocytes, peripheral blood mononuclear cells (PBMCs) and endothelial cells) and possesses anti-bacterial properties. Thus, TSC represents a novel cosmetic functional ingredient that provides a broad spectrum of benefits for the skin. To assess the anti-inflammatory properties of TSC in several cutaneous cell types and further investigate its anti-microbial activity. Cultured normal human epidermal keratinocytes were exposed to chemical irritant phorbol 12-myrisate 13-acetate (TPA) or ultraviolet-B light (UVB) to induce pro-inflammatory cytokine (IL-6, IL-8 and TNF-α) production. T-cell receptor (TCR) activation of PBMCs and nickel (Ni(2+) ) treatments of human dermal microvascular endothelial cells (HDMECs) were performed resulting in IL-4, IL-6, IL-8 and IL-17 production. Streptococcus pyogenes were cultured to determine minimal inhibitory concentration values. In vitro studies demonstrate TSC blocks TPA and UVB-induced cytokine production in cultured keratinocytes. Similarly, TSC inhibits overproduction of IL-4 and IL-17 in T-cell receptor (TCR)-activated PBMCs as well as nickel induction of IL-6 and IL-8 in HDMECs. Lastly, TSC demonstrated anti-microbial properties, inhibiting cell growth of S. pyogenes. Tetramethylhexadecenyl succinyl cysteine represents a novel cosmetic functional ingredient that provides a dual modulating benefit of skin protection to individuals by reducing inflammation in

  18. 21 CFR 660.55 - Labeling.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... identify antibody specificities other than anti-IgG and anti-C3d but the reactivity of the Anti-Human... FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) BIOLOGICS ADDITIONAL STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Anti-Human Globulin § 660.55 Labeling. In addition...

  19. 21 CFR 660.55 - Labeling.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... identify antibody specificities other than anti-IgG and anti-C3d but the reactivity of the Anti-Human... AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) BIOLOGICS ADDITIONAL STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Anti-Human Globulin § 660.55 Labeling. In addition...

  20. 21 CFR 660.55 - Labeling.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... identify antibody specificities other than anti-IgG and anti-C3d but the reactivity of the Anti-Human... FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) BIOLOGICS ADDITIONAL STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Anti-Human Globulin § 660.55 Labeling. In addition...

  1. 21 CFR 660.55 - Labeling.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... identify antibody specificities other than anti-IgG and anti-C3d but the reactivity of the Anti-Human... FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) BIOLOGICS ADDITIONAL STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Anti-Human Globulin § 660.55 Labeling. In addition...

  2. Shrink-induced graphene sensor for alpha-fetoprotein detection with low-cost self-assembly and label-free assay

    NASA Astrophysics Data System (ADS)

    Sando, Shota; Zhang, Bo; Cui, Tianhong

    2017-12-01

    Combination of shrink induced nano-composites technique and layer-by-layer (LbL) self-assembled graphene challenges controlling surface morphology. Adjusting shrink temperature achieves tunability on graphene surface morphology on shape memory polymers, and it promises to be an alternative in fields of high-surface-area conductors and molecular detection. In this study, self-assembled graphene on a shrink polymer substrate exhibits nanowrinkles after heating. Induced nanowrinkles on graphene with different shrink temperature shows distinct surface roughness and wettability. As a result, it becomes more hydrophilic with higher shrink temperatures. The tunable wettability promises to be utilized in, for example, microfluidic devices. The graphene on shrink polymer also exhibits capability of being used in sensing applications for pH and alpha-fetoprotein (AFP) detection with advantages of label free and low cost, due to self-assembly technique, easy functionalization, and antigen-antibody reaction on graphene surface. The detection limit of AFP detection is down to 1 pg/mL, and therefore the sensor also has a significant potential for biosensing as it relies on low-cost self-assembly and label-free assay.

  3. Nuclear pore complex-mediated modulation of TCR signaling is required for naïve CD4+ T cell homeostasis.

    PubMed

    Borlido, Joana; Sakuma, Stephen; Raices, Marcela; Carrette, Florent; Tinoco, Roberto; Bradley, Linda M; D'Angelo, Maximiliano A

    2018-06-01

    Nuclear pore complexes (NPCs) are channels connecting the nucleus with the cytoplasm. We report that loss of the tissue-specific NPC component Nup210 causes a severe deficit of naïve CD4 + T cells. Nup210-deficient CD4 + T lymphocytes develop normally but fail to survive in the periphery. The decreased survival results from both an impaired ability to transmit tonic T cell receptor (TCR) signals and increased levels of Fas, which sensitize Nup210 -/- naïve CD4 + T cells to Fas-mediated cell death. Mechanistically, Nup210 regulates these processes by modulating the expression of Cav2 (encoding Caveolin-2) and Jun at the nuclear periphery. Whereas the TCR-dependent and CD4 + T cell-specific upregulation of Cav2 is critical for proximal TCR signaling, cJun expression is required for STAT3-dependent repression of Fas. Our results uncover an unexpected role for Nup210 as a cell-intrinsic regulator of TCR signaling and T cell homeostasis and expose NPCs as key players in the adaptive immune system.

  4. Imaging of alpha(v)beta(3) expression by a bifunctional chimeric RGD peptide not cross-reacting with alpha(v)beta(5).

    PubMed

    Zannetti, Antonella; Del Vecchio, Silvana; Iommelli, Francesca; Del Gatto, Annarita; De Luca, Stefania; Zaccaro, Laura; Papaccioli, Angela; Sommella, Jvana; Panico, Mariarosaria; Speranza, Antonio; Grieco, Paolo; Novellino, Ettore; Saviano, Michele; Pedone, Carlo; Salvatore, Marco

    2009-08-15

    To test whether a novel bifunctional chimeric peptide comprising a cyclic Arg-Gly-Asp pentapeptide covalently bound to an echistatin domain can discriminate alpha(v)beta(3) from alpha(v)beta(5) integrin, thus allowing the in vivo selective visualization of alpha(v)beta(3) expression by single-photon and positron emission tomography (PET) imaging. The chimeric peptide was preliminarily tested for inhibition of alpha(v)beta(3)-dependent cell adhesion and competition of 125I-echistatin binding to membrane of stably transfected K562 cells expressing alpha(v)beta(3) (Kalpha(v)beta(3)) or alpha(v)beta(5) (Kalpha(v)beta(5)) integrin. The chimeric peptide was then conjugated with diethylenetriaminepentaacetic acid and labeled with 111In for single-photon imaging, whereas a one-step procedure was used for labeling the full-length peptide and a truncated derivative, lacking the last five C-terminal amino acids, with 18F for PET imaging. Nude mice bearing tumors from Kalpha(v)beta(3), Kalpha(v)beta(5), U87MG human glioblastoma, and A431 human epidermoid cells were subjected to single-photon and PET imaging. Adhesion and competitive binding assays showed that the novel chimeric peptide selectively binds to alpha(v)beta(3) integrin and does not cross-react with alpha(v)beta(5). In agreement with in vitro findings, single-photon and PET imaging studies showed that the radiolabeled chimeric peptide selectively localizes in tumor xenografts expressing alphavbeta3 and fails to accumulate in those expressing alpha(v)beta(5) integrin. When 18F-labeled truncated derivative was used for PET imaging, alphavbeta3- and alpha(v)beta(5)-expressing tumors were visualized, indicating that the five C-terminal amino acids are required to differentially bind the two integrins. Our findings indicate that the novel chimeric Arg-Gly-Asp peptide, having no cross-reaction with alphavbeta5 integrin, allows highly selective alphavbeta3 expression imaging and monitoring.

  5. Active tuberculosis patients have high levels of IgA anti-alpha-crystallin and isocitrate lyase proteins.

    PubMed

    Talavera-Paulín, M; García-Morales, L; Ruíz-Sánchez, B P; Caamal-Ley, Á D; Hernández-Solis, A; Ramírez-Casanova, E; Cicero-Sabido, R; Espitia, C; Helguera-Repetto, C; González-Y-Merchand, J A; Flores-Mejía, R; Estrada-Parra, S; Estrada-García, I; Chacón-Salinas, R; Wong-Baeza, I; Serafín-López, J

    2016-12-01

    Mexico City, Mexico. To identify proteins synthetised by Mycobacterium tuberculosis in hypoxic culture, which resemble more closely a granuloma environment than aerobic culture, and to determine if they are recognised by antibodies from patients with active pulmonary tuberculosis (PTB). Soluble extracts from M. tuberculosis H37Rv cultured under aerobic or hypoxic conditions were analysed using two-dimensional polyacrylamide gel electrophoresis, and proteins over-expressed under hypoxia were identified by mass spectrometry. The presence of immunoglobulin (Ig) G, IgA and IgM antibodies against these proteins was determined in the serum of 42 patients with active PTB and 42 healthy controls. We selected three M. tuberculosis H37Rv proteins (alpha-crystallin protein [Acr, Rv2031c], universal stress protein Rv2623 and isocitrate lyase [ICL, RV0467]) that were over-expressed under hypoxia. Titres of anti-Acr and anti-ICL IgA antibodies were higher in patients than in healthy controls, with an area under the receiver operating characteristic curve of 0.71 for anti-ICL IgA antibodies. ICL could be used in combination with other M. tuberculosis antigens to improve the sensitivity and specificity of current serological TB diagnostic methods.

  6. Anti-TNF-alpha therapy does not modulate leptin in patients with severe rheumatoid arthritis.

    PubMed

    Gonzalez-Gay, M A; Garcia-Unzueta, M T; Berja, A; Gonzalez-Juanatey, C; Miranda-Filloy, J A; Vazquez-Rodriguez, T R; de Matias, J M; Martin, J; Dessein, P H; Llorca, J

    2009-01-01

    The adipocytokine leptin regulates weight centrally and participates in the regulation of the immune and inflammatory responses. Chronic systemic inflammation is of major importance in the development of atherosclerosis in rheumatoid arthritis (RA). In the present study we investigated whether inflammation, obesity or both of these characteristics are potential determinants of circulating leptin concentrations in a group of RA patients on periodical treatment with the TNF-alpha-blocker-infliximab due to severe disease. We also assessed whether the infusion of infliximab may alter circulating leptin concentrations in patients with severe RA. We investigated 33 patients with RA on periodical treatment with infliximab. Serum leptin levels were determined immediately prior to and after infliximab infusion. There was a positive correlation between body mass index of RA patients and baseline serum level of leptin (rho=0.665, p<0.001). Apart from a significant correlation with VCAM-1 (rho=0.349, p=0.04), no significant correlations between baseline leptin levels and the age at the time of the study or at the onset of the disease, disease duration, ESR and CRP levels, DAS28, lipids, insulin sensitivity, adhesion molecules, resistin, adiponectin, ghrelin or the cumulative prednisone dose at the time of the study were found. Leptin levels did not change upon infliximab infusion (p=0.48). In RA patients on TNF-alpha blocker treatment, circulating leptin levels are unrelated to disease activity but constitute a manifestation of adiposity. The beneficial effect of anti-TNF-alpha therapy on cardiovascular mortality in RA does not seem to be mediated by reduction in serum levels of leptin.

  7. Unexpected T cell regulatory activity of anti-histone H1 autoantibody: Its mode of action in regulatory T cell-dependent and -independent manners

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Takaoka, Yuki; Kawamoto, Seiji, E-mail: skawa@hiroshima-u.ac.jp; Katayama, Akiko

    2013-02-08

    Highlights: ► Anti-histone H1 autoantibody (anti-H1) acts on T cells to inhibit their activation. ► Anti-H1 suppresses T cell activation in Treg cell-dependent and -independent manners. ► Suboptimal dose of anti-H1 enhances suppressor function of Treg cells. ► High dose of anti-H1 directly inhibits T cell receptor signaling. -- Abstract: Induction of anti-nuclear antibodies against DNA or histones is a hallmark of autoimmune disorders, but their actual contribution to disease predisposition remains to be clarified. We have previously reported that autoantibodies against histone H1 work as a critical graft survival factor in a rat model of tolerogeneic liver transplantation. Heremore » we show that an immunosuppressive anti-histone H1 monoclonal antibody (anti-H1 mAb) acts directly on T cells to inhibit their activation in response to T cell receptor (TCR) ligation. Intriguingly, the T cell activation inhibitory activity of anti-H1 mAb under suboptimal dosages required regulatory T (Treg) cells, while high dose stimulation with anti-H1 mAb triggered a Treg cell-independent, direct negative regulation of T cell activation upon TCR cross-linking. In the Treg cell-dependent mode of immunosuppressive action, anti-H1 mAb did not induce the expansion of CD4{sup +}Foxp3{sup +} Treg cells, but rather potentiated their regulatory capacity. These results reveal a previously unappreciated T cell regulatory role of anti-H1 autoantibody, whose overproduction is generally thought to be pathogenic in the autoimmune settings.« less

  8. Mechanism and function of Vav1 localisation in TCR signalling.

    PubMed

    Ksionda, Olga; Saveliev, Alexander; Köchl, Robert; Rapley, Jonathan; Faroudi, Mustapha; Smith-Garvin, Jennifer E; Wülfing, Christoph; Rittinger, Katrin; Carter, Tom; Tybulewicz, Victor L J

    2012-11-15

    The antigen-specific binding of T cells to antigen presenting cells results in recruitment of signalling proteins to microclusters at the cell-cell interface known as the immunological synapse (IS). The Vav1 guanine nucleotide exchange factor plays a critical role in T cell antigen receptor (TCR) signalling, leading to the activation of multiple pathways. We now show that it is recruited to microclusters and to the IS in primary CD4(+) and CD8(+) T cells. Furthermore, we show that this recruitment depends on the SH2 and C-terminal SH3 (SH3(B)) domains of Vav1, and on phosphotyrosines 112 and 128 of the SLP76 adaptor protein. Biophysical measurements show that Vav1 binds directly to these residues on SLP76 and that efficient binding depends on the SH2 and SH3(B) domains of Vav1. Finally, we show that the same two domains are critical for the phosphorylation of Vav1 and its signalling function in TCR-induced calcium flux. We propose that Vav1 is recruited to the IS by binding to SLP76 and that this interaction is critical for the transduction of signals leading to calcium flux.

  9. Conferring indirect allospecificity on CD4+CD25+ Tregs by TCR gene transfer favors transplantation tolerance in mice

    PubMed Central

    Tsang, Julia Yuen-Shan; Tanriver, Yakup; Jiang, Shuiping; Xue, Shao-An; Ratnasothy, Kulachelvy; Chen, Daxin; Stauss, Hans J.; Bucy, R. Pat; Lombardi, Giovanna; Lechler, Robert

    2008-01-01

    T cell responses to MHC-mismatched transplants can be mediated via direct recognition of allogeneic MHC molecules on the cells of the transplant or via recognition of allogeneic peptides presented on the surface of recipient APCs in recipient MHC molecules — a process known as indirect recognition. As CD4+CD25+ Tregs play an important role in regulating alloresponses, we investigated whether mouse Tregs specific for allogeneic MHC molecules could be generated in vitro and could promote transplantation tolerance in immunocompetent recipient mice. Tregs able to directly recognize allogeneic MHC class II molecules (dTregs) were obtained by stimulating CD4+CD25+ cells from C57BL/6 mice (H-2b) with allogeneic DCs from BALB/c mice (H-2d). To generate Tregs that indirectly recognized allogeneic MHC class II molecules, dTregs were retrovirally transduced with TCR genes conferring specificity for H-2Kd presented by H-2Ab MHC class II molecules. The dual direct and indirect allospecificity of the TCR-transduced Tregs was confirmed in vitro. In mice, TCR-transduced Tregs, but not dTregs, induced long-term survival of partially MHC-mismatched heart grafts when combined with short-term adjunctive immunosuppression. Further, although dTregs were only slightly less effective than TCR-transduced Tregs at inducing long-term survival of fully MHC-mismatched heart grafts, histologic analysis of long-surviving hearts demonstrated marked superiority of the TCR-transduced Tregs. Thus, Tregs specific for allogeneic MHC class II molecules are effective in promoting transplantation tolerance in mice, which suggests that such cells have clinical potential. PMID:18846251

  10. Radionuclide therapy using ¹³¹I-labeled anti-epidermal growth factor receptor-targeted nanoparticles suppresses cancer cell growth caused by EGFR overexpression.

    PubMed

    Li, Wei; Liu, Zhongyun; Li, Chengxia; Li, Ning; Fang, Lei; Chang, Jin; Tan, Jian

    2016-03-01

    Anti-epidermal growth factor receptor (EGFR)-targeted nanoparticles can be used to deliver a therapeutic and imaging agent to EGFR-overexpressing tumor cells. (131)I-labeled anti-EGFR nanoparticles derived from cetuximab were used as a tumor-targeting vehicle in radionuclide therapy. This paper describes the construction of the anti-EGFR nanoparticle EGFR-BSA-PCL. This nanoparticle was characterized for EGFR-targeted binding and cellular uptake in EGFR-overexpressing cancer cells by using flow cytometry and confocal microscopy. Anti-EGFR and non-targeted nanoparticles were labeled with (131)I using the chloramine-T method. Analyses of cytotoxicity and targeted cell killing with (131)I were performed using the MTT assay. The time-dependent cellular uptake of (131)I-labeled anti-EGFR nanoparticles proved the slow-release effects of nanoparticles. A radioiodine therapy study was also performed in mice. The EGFR-targeted nanoparticle EGFR-BSA-PCL and the non-targeted nanoparticle BSA-PCL were constructed; the effective diameters were approximately 100 nm. The results from flow cytometry and confocal microscopy revealed significant uptake of EGFR-BSA-PCL in EGFR-overexpressing tumor cells. Compared with EGFR-BSA-PCL, BSA-PCL could also bind to cells, but tumor cell retention was minimal and weak. In MTT assays, the EGFR-targeted radioactive nanoparticle (131)I-EGFR-BSA-PCL showed greater cytotoxicity and targeted cell killing than the non-targeted nanoparticle (131)I-BSA-PCL. The radioiodine uptake of both (131)I-labeled nanoparticles, (131)I-EGFR-BSA-PCL and (131)I-BSA-PCL, was rapid and reached maximal levels 4 h after incubation, but the (131)I uptake of (131)I-EGFR-BSA-PCL was higher than that of (131)I-BSA-PCL. On day 15, the average tumor volumes of the (131)I-EGFR-BSA-PCL and (131)I-BSA-PCL groups showed a slow growth relationship compared with that of the control group. The EGFR-targeted nanoparticle EGFR-BSA-PCL demonstrated superior cellular binding and uptake

  11. Inhibitory effect of alpha-mangostin on Candida biofilms.

    PubMed

    Kaomongkolgit, Ruchadaporn; Jamdee, Kusuma

    2017-04-01

    The objective of this study was to determine the inhibitory effect of alpha-mangostin on Candida biofilms. Candida species including Candida albicans, Candida krusei, Candida tropicalis, and Candida glabrata were tested. Candida biofilms were formed in flat-bottomed 96-well microtiter plates. The metabolic activity of cells within biofilms was quantified using the XTT assay. The results demonstrated that alpha-mangostin showed a significant anti-biofilm effect on both developing biofilms and preformed biofilms of Candida species. It may be concluded that alpha-mangostin could be an anti-biofilm agent against Candida species. Further in vivo investigations are needed to uncover the therapeutic values of this medicinal plant.

  12. A novel anti-alpha-fetoprotein single-chain variable fragment displays anti-tumor effects in HepG2 cells as a single agent or in combination with paclitaxel.

    PubMed

    Ji, Xiaonan; Shen, Yanli; Sun, Hao; Gao, Xiangdong

    2016-08-01

    Human hepatocellular carcinoma (HCC) has a high rate of tumor recurrence and metastasis, resulting in shortened survival time. The function of alpha-fetoprotein (AFP) as a regulatory factor in the growth of HCC cells has been well defined. The aim of this study was to investigate the use of a novel AFP-specific single-chain variable fragment that blocked AFP and inhibited HCC cell growth. The results indicated that the anti-AFP single-chain variable fragment (scFv) induced growth inhibition of AFP-expressing HCC cell lines in vitro through induction of G1 cell cycle arrest and apoptosis. The mechanism of apoptosis probably involved with blocking AFP internalization and regulation of the PTEN/PI3K/Akt signaling network. Moreover, the anti-AFP-scFv also effectively sensitized the HepG2 cells to paclitaxel (PTX) at a lower concentration. The combination effect of PTX and anti-AFP-scFv displayed a synergistic effect on HepG2 cells both in vitro and in vivo. Our results demonstrated that targeting AFP by specific antibodies has potential immunotherapeutic efficacy in human HCC.

  13. NIR and MR imaging supported hydrogel based delivery system for anti-TNF alpha probiotic therapy of IBD

    NASA Astrophysics Data System (ADS)

    Janjic, Jelena M.; Berlec, Ales; Bagia, Christina; Liu, Lu S.; Jeric, Irenej; Gach, Michael; Janjic, Bratislav M.; Strukelj, Borut

    2016-03-01

    Current treatment of inflammatory bowel disease (IBD) is largely symptomatic and consists of anti-inflammatory agents, immune-suppressives or antibiotics, whereby local luminal action is preferred to minimize systemic side-effects. Recently, anti-TNFα therapy has shown considerable success and is now being routinely used. Here we present a novel approach of using perfluorocarbon (PFC) nanoemulsion containing hydrogels (nanoemulgels) as imaging supported delivery systems for anti-TNF alpha probiotic delivery in IBD. To further facilitate image-guided therapy a food-grade lactic acid bacterium Lactococcus lactis capable of TNFα-binding was engineered to incorporate infrared fluorescent protein (IRFP). This modified bacteria was then incorporated into novel PFC nanoemulgels. The nanoemulgels presented here are designed to deliver locally anti-TNFα probiotic in the lower colon and rectum and provide dual imaging signature of gel delivery (MRI) across the rectum and lower colon and bacteria release (NIR). NIR imaging data in vitro demonstrates high IRFP expressing and TNFα-binding bacteria loading in the hydrogel and complete release in 3 hours. Stability tests indicate that gels remain stable for at least 14 days showing no significant change in droplet size, zeta potential and pH. Flow cytometry analyses demonstrate the NIRF expressing bacteria L. lactis binds TNFα in vitro upon release from the gels. Magnetic resonance and near-infrared imaging in vitro demonstrates homogeneity of hydrogels and the imaging capacity of the overall formulation.

  14. Mechanism and function of Vav1 localisation in TCR signalling

    PubMed Central

    Ksionda, Olga; Saveliev, Alexander; Köchl, Robert; Rapley, Jonathan; Faroudi, Mustapha; Smith-Garvin, Jennifer E.; Wülfing, Christoph; Rittinger, Katrin; Carter, Tom; Tybulewicz, Victor L. J.

    2012-01-01

    Summary The antigen-specific binding of T cells to antigen presenting cells results in recruitment of signalling proteins to microclusters at the cell-cell interface known as the immunological synapse (IS). The Vav1 guanine nucleotide exchange factor plays a critical role in T cell antigen receptor (TCR) signalling, leading to the activation of multiple pathways. We now show that it is recruited to microclusters and to the IS in primary CD4+ and CD8+ T cells. Furthermore, we show that this recruitment depends on the SH2 and C-terminal SH3 (SH3B) domains of Vav1, and on phosphotyrosines 112 and 128 of the SLP76 adaptor protein. Biophysical measurements show that Vav1 binds directly to these residues on SLP76 and that efficient binding depends on the SH2 and SH3B domains of Vav1. Finally, we show that the same two domains are critical for the phosphorylation of Vav1 and its signalling function in TCR-induced calcium flux. We propose that Vav1 is recruited to the IS by binding to SLP76 and that this interaction is critical for the transduction of signals leading to calcium flux. PMID:22956543

  15. Innate signals overcome acquired TCR signaling pathway regulation and govern the fate of human CD161(hi) CD8α⁺ semi-invariant T cells.

    PubMed

    Turtle, Cameron J; Delrow, Jeff; Joslyn, Rochelle C; Swanson, Hillary M; Basom, Ryan; Tabellini, Laura; Delaney, Colleen; Heimfeld, Shelly; Hansen, John A; Riddell, Stanley R

    2011-09-08

    Type 17 programmed CD161(hi)CD8α(+) T cells contribute to mucosal immunity to bacteria and yeast. In early life, microbial colonization induces proliferation of CD161(hi) cells that is dependent on their expression of a semi-invariant Vα7.2(+) TCR. Although prevalent in adults, CD161(hi)CD8α(+) cells exhibit weak proliferative and cytokine responses to TCR ligation. The mechanisms responsible for the dichotomous response of neonatal and adult CD161(hi) cells, and the signals that enable their effector function, have not been established. We describe acquired regulation of TCR signaling in adult memory CD161(hi)CD8α(+) T cells that is absent in cord CD161(hi) cells and adult CD161(lo) cells. Regulated TCR signaling in CD161(hi) cells was due to profound alterations in TCR signaling pathway gene expression and could be overcome by costimulation through CD28 or innate cytokine receptors, which dictated the fate of their progeny. Costimulation with IL-1β during TCR ligation markedly increased proinflammatory IL-17 production, while IL-12-induced Tc1-like function and restored the response to TCR ligation without costimulation. CD161(hi) cells from umbilical cord blood and granulocyte colony stimulating factor-mobilized leukaphereses differed in frequency and function, suggesting future evaluation of the contribution of CD161(hi) cells in hematopoietic stem cell grafts to transplant outcomes is warranted.

  16. Proline substitution independently enhances H-2D(b) complex stabilization and TCR recognition of melanoma-associated peptides.

    PubMed

    Uchtenhagen, Hannes; Abualrous, Esam T; Stahl, Evi; Allerbring, Eva B; Sluijter, Marjolein; Zacharias, Martin; Sandalova, Tatyana; van Hall, Thorbald; Springer, Sebastian; Nygren, Per-Åke; Achour, Adnane

    2013-11-01

    The immunogenicity of H-2D(b) (D(b)) restricted epitopes can be significantly increased by substituting peptide position 3 to a proline (p3P). The p3P modification enhances MHC stability without altering the conformation of the modified epitope allowing for T-cell cross-reactivity with the native peptide. The present study reveals how specific interactions between p3P and the highly conserved MHC heavy chain residue Y159 increase the stability of D(b) in complex with an optimized version of the melanoma-associated epitope gp10025-33 . Furthermore, the p3P modification directly increased the affinity of the D(b)/gp10025-33 -specific T-cell receptor (TCR) pMel. Surprisingly, the enhanced TCR binding was independent from the observed increased stability of the optimized D(b)/gp10025-33 complex and from the interactions formed between p3P and Y159, indicating a direct effect of the p3P modification on TCR recognition. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Anti-neuronal anti-bodies in patients with early psychosis.

    PubMed

    Mantere, O; Saarela, M; Kieseppä, T; Raij, T; Mäntylä, T; Lindgren, M; Rikandi, E; Stoecker, W; Teegen, B; Suvisaari, J

    2018-02-01

    It may be challenging to distinguish autoimmune encephalitis associated with anti-neuronal autoantibodies from primary psychiatric disorders. Here, serum was drawn from patients with a first-episode psychosis (n=70) or a clinical high-risk for psychosis (n=6) and controls (n=34). We investigated the serum prevalence of 24 anti-neuronal autoantibodies: IgG antibodies for anti-N-methyl-d-aspartate-type glutamate receptor (anti-NMDAR), glutamate and γ-aminobutyric acid alpha and beta receptors (GABA-a, GABA-b), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPA), glycine receptor (GlyR), metabotropic glutamate receptor 1 and 5 (mGluR1, mGluR5), anti-Tr/Delta/notch-like epidermal growth factor-related receptor (DNER), contactin-associated protein-like 2 (CASPR2), myelin oligodendrocyte glycoprotein (MOG), glutamic acid decarboxylase-65 (GAD65), collapsin response mediator protein 5/crossveinless-2 (CV2), aquaporin-4 (AQP4), anti-dipeptidyl-peptidase-like protein-6 (DPPX), type 1 anti-neuronal nuclear antibody (ANNA-1, Hu), Ri, Yo, IgLON5, Ma2, zinc finger protein 4 (ZIC4), Rho GTPase-activating protein 26, amphiphysin, and recoverin, as well as IgA and IgM for dopamine-2-receptor (DRD2). Anti-NMDA IgG antibodies were positive with serum titer 1:320 in one patient with a clinical high risk for psychosis. He did not receive a diagnosis of encephalitis after comprehensive neurological evaluation. All other antineuronal autoantibodies were negative and there were no additional findings with immunohistochemistry of brain issues. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. An open-label pilot trial of alpha-lipoic acid for weight loss in patients with schizophrenia without diabetes.

    PubMed

    Ratliff, Joseph C; Palmese, Laura B; Reutenauer, Erin L; Tek, Cenk

    2015-01-01

    A possible mechanism of antipsychotic-induced weight gain is activation of hypothalamic monophosphate-dependent kinase (AMPK) mediated by histamine 1 receptors. Alpha-lipoic acid (ALA), a potent antioxidant, counteracts this effect and may be helpful in reducing weight for patients taking antipsychotics. The objective of this open-label study was to assess the efficacy of ALA (1,200 mg) on twelve non-diabetic schizophrenia patients over ten weeks. Participants lost significant weight during the intervention (-2.2 kg±2.5 kg). ALA was well tolerated and was particularly effective for individuals taking strongly antihistaminic antipsychotics (-2.9 kg±2.6 kg vs. -0.5 kg±1.0 kg). NCT01355952.

  19. Targeting of HPV-16+ Epithelial Cancer Cells by TCR Gene Engineered T Cells Directed against E6.

    PubMed

    Draper, Lindsey M; Kwong, Mei Li M; Gros, Alena; Stevanović, Sanja; Tran, Eric; Kerkar, Sid; Raffeld, Mark; Rosenberg, Steven A; Hinrichs, Christian S

    2015-10-01

    The E6 and E7 oncoproteins of HPV-associated epithelial cancers are in principle ideal immunotherapeutic targets, but evidence that T cells specific for these antigens can recognize and kill HPV(+) tumor cells is limited. We sought to determine whether TCR gene engineered T cells directed against an HPV oncoprotein can successfully target HPV(+) tumor cells. T-cell responses against the HPV-16 oncoproteins were investigated in a patient with an ongoing 22-month disease-free interval after her second resection of distant metastatic anal cancer. T cells genetically engineered to express an oncoprotein-specific TCR from this patient's tumor-infiltrating T cells were tested for specific reactivity against HPV(+) epithelial tumor cells. We identified, from an excised metastatic anal cancer tumor, T cells that recognized an HLA-A*02:01-restricted epitope of HPV-16 E6. The frequency of the dominant T-cell clonotype from these cells was approximately 400-fold greater in the patient's tumor than in her peripheral blood. T cells genetically engineered to express the TCR from this clonotype displayed high avidity for an HLA-A*02:01-restricted epitope of HPV-16, and they showed specific recognition and killing of HPV-16(+) cervical, and head and neck cancer cell lines. These findings demonstrate that HPV-16(+) tumors can be targeted by E6-specific TCR gene engineered T cells, and they provide the foundation for a novel cellular therapy directed against HPV-16(+) malignancies, including cervical, oropharyngeal, anal, vulvar, vaginal, and penile cancers. ©2015 American Association for Cancer Research.

  20. Simultaneous quantification of GABAergic 3alpha,5alpha/3alpha,5beta neuroactive steroids in human and rat serum.

    PubMed

    Porcu, Patrizia; O'Buckley, Todd K; Alward, Sarah E; Marx, Christine E; Shampine, Lawrence J; Girdler, Susan S; Morrow, A Leslie

    2009-01-01

    The 3alpha,5alpha- and 3alpha,5beta-reduced derivatives of progesterone, deoxycorticosterone, dehydroepiandrosterone and testosterone enhance GABAergic neurotransmission and produce inhibitory neurobehavioral and anti-inflammatory effects. Despite substantial information on the progesterone derivative (3alpha,5alpha)-3-hydroxypregnan-20-one (3alpha,5alpha-THP, allopregnanolone), the physiological significance of the other endogenous GABAergic neuroactive steroids has remained elusive. Here, we describe the validation of a method using gas chromatography-mass spectrometry to simultaneously identify serum levels of the eight 3alpha,5alpha- and 3alpha,5beta-reduced derivatives of progesterone, deoxycorticosterone, dehydroepiandrosterone and testosterone. The method shows specificity, sensitivity and enhanced throughput compared to other methods already available for neuroactive steroid quantification. Administration of pregnenolone to rats and progesterone to women produced selective effects on the 3alpha,5alpha- and 3alpha,5beta-reduced neuroactive steroids, indicating differential regulation of their biosynthetic pathways. Pregnenolone administration increased serum levels of 3alpha,5alpha-THP (+1488%, p<0.001), (3alpha,5alpha)-3,21-dihydroxypregnan-20-one (3alpha,5alpha-THDOC, +205%, p<0.01), (3alpha,5alpha)-3-hydroxyandrostan-17-one (3alpha,5alpha-A, +216%, p<0.001), (3alpha,5alpha,17beta)-androstane-3,17-diol (3alpha,5alpha-A-diol, +190%, p<0.01). (3alpha,5beta)-3-hydroxypregnan-20-one (3alpha,5beta-THP) and (3alpha,5beta)-3-hydroxyandrostan-17-one (3alpha,5beta-A) were not altered, while (3alpha,5beta)-3,21-dihydroxypregnan-20-one (3alpha,5beta-THDOC) and (3alpha,5beta,17beta)-androstane-3,17-diol (3alpha,5beta-A-diol) were increased from undetectable levels to 271+/-100 and 2.4+/-0.9 pg+/-SEM, respectively (5/8 rats). Progesterone administration increased serum levels of 3alpha,5alpha-THP (+1806%, p<0.0001), 3alpha,5beta-THP (+575%, p<0.001), 3alpha,5alpha

  1. Regulation by interferon alpha of immunoglobulin isotype selection and lymphokine production in mice

    PubMed Central

    1991-01-01

    Antigens and infectious agents that stimulate interferon alpha(IFN- alpha) production in mice induce antibody responses that are predominantly of the immunoglobulin (Ig)G2a isotype and contain little or no IgE. This suggested the possibility that IFN-alpha might have a role in directing Ig isotype selection. Consistent with this possibility, we have found that injection of mice with recombinant mouse IFN-alpha suppresses IgE secretion, enhances IgG2a secretion, and has no independent effect on IgG1 secretion in mice stimulated with a foreign anti-IgD antibody. Injection of mice with polyinosinic acid.polycytidylic acid (poly I.C), an inducer of macrophage IFN-alpha production, also suppresses the anti-IgD antibody-induced IgE response and stimulates the IgG2a response; these effects are blocked by a sheep antibody that neutralizes mouse IFN-alpha/beta. Both recombinant IFN- alpha and poly I.C have maximum IgE suppressive and IgG2a stimulatory effects when injected early in the anti-IgD antibody-induced immune response. Addition of IFN-alpha to mouse B cells cultured with lipopolysaccharide (LPS) + interleukin 4 (IL-4) suppresses both IgG1 and IgE production, but much less potently than IFN-gamma. IFN-alpha suppresses anti-IgD antibody-induced increases in the level of splenic IL-4 mRNA, but enhances the anti-IgD antibody-induced increase in the splenic level of IFN-gamma mRNA. These results are consistent with the effect of IFN-alpha on Ig isotype expression in mice, as IL-4 stimulates IgE and suppresses IgG2a secretion while IFN-gamma exerts opposite effects. These observations suggest that antigen presenting cells, by secreting IFN-alpha early in the course of an immune response, can influence the nature of that response both through direct effects on B cells and by influencing the differentiation of T cells. PMID:1940796

  2. Probing the active site of alpha-class rat liver glutathione S-transferases using affinity labeling by monobromobimane.

    PubMed Central

    Hu, L.; Borleske, B. L.; Colman, R. F.

    1997-01-01

    Monobromobimane (mBBr) is a substrate of both mu- and alpha-class rat liver glutathione S-transferases, with Km values of 0.63 microM and 4.9 microM for the mu-class isozymes 3-3 and 4-4, respectively, and 26 microM for the alpha-class isozymes 1-1 and 2-2. In the absence of substrate glutathione, mBBr acts as an affinity label of the 1-1 as well as mu-class isozymes, but not of the alpha-class 2-2 isozyme. Incubation of rat liver isozyme 1-1 with mBBr at pH 7.5 and 25 degrees C results in a time-dependent inactivation of the enzyme but at a slower (threefold) rate than for reactions with the mu-class isozyme 3-3 and 4-4. The rate of inactivation of 1-1 isozyme by mBBr is not decreased but, rather, is slightly enhanced by S-methyl glutathione. In contrast, 17 beta-estradiol-3,17-disulfate (500 microM) gives a 12.5-fold decrease in the observed rate constant of inactivation by 4 mM mBBr. When incubated for 60 min with 4 mM mBBr, the 1-1 isozyme loses 60% of its activity and incorporates 1.7 mol reagent/mol subunit. Peptide analysis after thermolysin digestion indicates that mBBr modification is equally distributed between two cysteine residues at positions 17 and 111. Modification at these two sites is reduced equally in the presence of the added protectant, 17 beta-estradiol-3,17-disulfate, suggesting that Cys 17 and Cys 111 reside within or near the enzyme's steroid binding sites. In contrast to the 1-1 isozyme, the other alpha-class isozyme (2-2) is not inactivated by mBBr at concentrations as high as 15 mM. The different reaction kinetics and modification sites by mBBr suggest that distinct binding site structures are responsible for the characteristic substrate specificities of glutathione S-transferase isozymes. PMID:9007975

  3. Probing the active site of alpha-class rat liver glutathione S-transferases using affinity labeling by monobromobimane.

    PubMed

    Hu, L; Borleske, B L; Colman, R F

    1997-01-01

    Monobromobimane (mBBr) is a substrate of both mu- and alpha-class rat liver glutathione S-transferases, with Km values of 0.63 microM and 4.9 microM for the mu-class isozymes 3-3 and 4-4, respectively, and 26 microM for the alpha-class isozymes 1-1 and 2-2. In the absence of substrate glutathione, mBBr acts as an affinity label of the 1-1 as well as mu-class isozymes, but not of the alpha-class 2-2 isozyme. Incubation of rat liver isozyme 1-1 with mBBr at pH 7.5 and 25 degrees C results in a time-dependent inactivation of the enzyme but at a slower (threefold) rate than for reactions with the mu-class isozyme 3-3 and 4-4. The rate of inactivation of 1-1 isozyme by mBBr is not decreased but, rather, is slightly enhanced by S-methyl glutathione. In contrast, 17 beta-estradiol-3,17-disulfate (500 microM) gives a 12.5-fold decrease in the observed rate constant of inactivation by 4 mM mBBr. When incubated for 60 min with 4 mM mBBr, the 1-1 isozyme loses 60% of its activity and incorporates 1.7 mol reagent/mol subunit. Peptide analysis after thermolysin digestion indicates that mBBr modification is equally distributed between two cysteine residues at positions 17 and 111. Modification at these two sites is reduced equally in the presence of the added protectant, 17 beta-estradiol-3,17-disulfate, suggesting that Cys 17 and Cys 111 reside within or near the enzyme's steroid binding sites. In contrast to the 1-1 isozyme, the other alpha-class isozyme (2-2) is not inactivated by mBBr at concentrations as high as 15 mM. The different reaction kinetics and modification sites by mBBr suggest that distinct binding site structures are responsible for the characteristic substrate specificities of glutathione S-transferase isozymes.

  4. TCR-engineered, customized, antitumor T cells for cancer immunotherapy: advantages and limitations.

    PubMed

    Chhabra, Arvind

    2011-01-05

    The clinical outcome of the traditional adoptive cancer immunotherapy approaches involving the administration of donor-derived immune effectors, expanded ex vivo, has not met expectations. This could be attributed, in part, to the lack of sufficient high-avidity antitumor T-cell precursors in most cancer patients, poor immunogenicity of cancer cells, and the technological limitations to generate a sufficiently large number of tumor antigen-specific T cells. In addition, the host immune regulatory mechanisms and immune homeostasis mechanisms, such as activation-induced cell death (AICD), could further limit the clinical efficacy of the adoptively administered antitumor T cells. Since generation of a sufficiently large number of potent antitumor immune effectors for adoptive administration is critical for the clinical success of this approach, recent advances towards generating customized donor-specific antitumor-effector T cells by engrafting human peripheral blood-derived T cells with a tumor-associated antigen-specific transgenic T-cell receptor (TCR) are quite interesting. This manuscript provides a brief overview of the TCR engineering-based cancer immunotherapy approach, its advantages, and the current limitations.

  5. Guinea pig hepatocyte alpha 1A-adrenoceptors: characterization, signal transduction and regulation.

    PubMed

    García-Sáinz, J A; Romero-Avila, T; Olivares-Reyes, J A; Macías-Silva, M

    1992-11-02

    Activation of guinea pig hepatocyte alpha 1-adrenoceptors increases phosphatidylinositol (PI) labeling, [3H]inositol phosphate production and phosphorylase activity. These adrenergic actions were not altered by pretreatment with chlorethylclonidine but were blocked by 5-methyl urapidil and prazosin (the former being 3- to 10-fold more potent than the latter), indicating that alpha 1A-adrenoceptors were involved. When the cells were incubated in buffer without calcium and containing EGTA, the alpha 1A-adrenergic stimulation of PI labeling was diminished but not abolished and that of phosphorylase was not affected. The alpha 1A-adrenergic effects were insensitive to pertussis toxin treatment. Phorbol myristate acetate inhibited the alpha 1A-adrenergic actions, although at relatively large concentrations, and also those of other agents such as angiotensin II and NaF. Our data clearly indicate that guinea pig hepatocytes express alpha 1A-adrenoceptors whose activation stimulates phosphoinositide turnover, via a pertussis toxin-insensitive process; the alpha 1A-adrenergic effects were at least partially independent of extracellular calcium.

  6. Vasodilating effect of norethisterone and its 5 alpha metabolites: a novel nongenomic action.

    PubMed

    Perusquía, Mercedes; Villalón, Carlos M; Navarrete, Erika; García, Gustavo A; Pérez-Palacios, Gregorio; Lemus, Ana E

    2003-08-15

    Estrogens are generally administered in hormone replacement therapy in combination with synthetic progestins. Studies of cardiovascular risk factors in postmenopausal women have shown a variety of responses according to the molecular structure of the progestin used in hormone replacement therapy schemes. The present study sets out to determine the vasoactive effects of norethisterone and its 5alpha-dihydro (5alpha-norethisterone) and -tetrahydro (3alpha,5alpha-norethisterone and 3beta,5alpha-norethisterone) metabolites in isolated precontracted rat thoracic aorta. The addition of norethisterone and 3alpha,5alpha-norethisterone in rat aorta exhibited a potent, concentration-response inhibition of noradrenaline-induced contraction, while 5alpha- and 3beta,5alpha-norethisterone had very little, if any, vasorelaxing effect. Relaxation to norethisterone and 3alpha,5alpha-norethisterone had very rapid time-courses and it was neither affected by the absence of endothelium nor by the inhibitor of nitric oxide synthase, Nomega-nitro-L-arginine methyl ester (L-NAME). The addition of specific anti-androgen, anti-progestin and anti-estrogen compounds and protein synthesis inhibitors did not preclude the vasorelaxing effect of norethisterone and its 3alpha,5alpha-reduced metabolite. The results strongly suggest that these effects are not mediated by nuclear sex steroid hormone receptors. The overall data document a novel nongenomic endothelium-independent vasorelaxing action of a 19-nor synthetic progestin and one of its A-ring-reduced derivatives.

  7. The complex and specific pMHC interactions with diverse HIV-1 TCR clonotypes reveal a structural basis for alterations in CTL function

    PubMed Central

    Xia, Zhen; Chen, Huabiao; Kang, Seung-gu; Huynh, Tien; Fang, Justin W.; Lamothe, Pedro A.; Walker, Bruce D.; Zhou, Ruhong

    2014-01-01

    Immune control of viral infections is modulated by diverse T cell receptor (TCR) clonotypes engaging peptide-MHC class I complexes on infected cells, but the relationship between TCR structure and antiviral function is unclear. Here we apply in silico molecular modeling with in vivo mutagenesis studies to investigate TCR-pMHC interactions from multiple CTL clonotypes specific for a well-defined HIV-1 epitope. Our molecular dynamics simulations of viral peptide-HLA-TCR complexes, based on two independent co-crystal structure templates, reveal that effective and ineffective clonotypes bind to the terminal portions of the peptide-MHC through similar salt bridges, but their hydrophobic side-chain packings can be very different, which accounts for the major part of the differences among these clonotypes. Non-specific hydrogen bonding to viral peptide also accommodates greater epitope variants. Furthermore, free energy perturbation calculations for point mutations on the viral peptide KK10 show excellent agreement with in vivo mutagenesis assays, with new predictions confirmed by additional experiments. These findings indicate a direct structural basis for heterogeneous CTL antiviral function. PMID:24522437

  8. The complex and specific pMHC interactions with diverse HIV-1 TCR clonotypes reveal a structural basis for alterations in CTL function

    NASA Astrophysics Data System (ADS)

    Xia, Zhen; Chen, Huabiao; Kang, Seung-Gu; Huynh, Tien; Fang, Justin W.; Lamothe, Pedro A.; Walker, Bruce D.; Zhou, Ruhong

    2014-02-01

    Immune control of viral infections is modulated by diverse T cell receptor (TCR) clonotypes engaging peptide-MHC class I complexes on infected cells, but the relationship between TCR structure and antiviral function is unclear. Here we apply in silico molecular modeling with in vivo mutagenesis studies to investigate TCR-pMHC interactions from multiple CTL clonotypes specific for a well-defined HIV-1 epitope. Our molecular dynamics simulations of viral peptide-HLA-TCR complexes, based on two independent co-crystal structure templates, reveal that effective and ineffective clonotypes bind to the terminal portions of the peptide-MHC through similar salt bridges, but their hydrophobic side-chain packings can be very different, which accounts for the major part of the differences among these clonotypes. Non-specific hydrogen bonding to viral peptide also accommodates greater epitope variants. Furthermore, free energy perturbation calculations for point mutations on the viral peptide KK10 show excellent agreement with in vivo mutagenesis assays, with new predictions confirmed by additional experiments. These findings indicate a direct structural basis for heterogeneous CTL antiviral function.

  9. Human and Murine Clonal CD8+ T Cell Expansions Arise during Tuberculosis Because of TCR Selection

    PubMed Central

    Nunes-Alves, Cláudio; Booty, Matthew G.; Carpenter, Stephen M.; Rothchild, Alissa C.; Martin, Constance J.; Desjardins, Danielle; Steblenko, Katherine; Kløverpris, Henrik N.; Madansein, Rajhmun; Ramsuran, Duran; Leslie, Alasdair; Correia-Neves, Margarida; Behar, Samuel M.

    2015-01-01

    The immune system can recognize virtually any antigen, yet T cell responses against several pathogens, including Mycobacterium tuberculosis, are restricted to a limited number of immunodominant epitopes. The host factors that affect immunodominance are incompletely understood. Whether immunodominant epitopes elicit protective CD8+ T cell responses or instead act as decoys to subvert immunity and allow pathogens to establish chronic infection is unknown. Here we show that anatomically distinct human granulomas contain clonally expanded CD8+ T cells with overlapping T cell receptor (TCR) repertoires. Similarly, the murine CD8+ T cell response against M. tuberculosis is dominated by TB10.44-11-specific T cells with extreme TCRβ bias. Using a retrogenic model of TB10.44-11-specific CD8+ T cells, we show that TCR dominance can arise because of competition between clonotypes driven by differences in affinity. Finally, we demonstrate that TB10.4-specific CD8+ T cells mediate protection against tuberculosis, which requires interferon-γ production and TAP1-dependent antigen presentation in vivo. Our study of how immunodominance, biased TCR repertoires, and protection are inter-related, provides a new way to measure the quality of T cell immunity, which if applied to vaccine evaluation, could enhance our understanding of how to elicit protective T cell immunity. PMID:25945999

  10. Human and Murine Clonal CD8+ T Cell Expansions Arise during Tuberculosis Because of TCR Selection.

    PubMed

    Nunes-Alves, Cláudio; Booty, Matthew G; Carpenter, Stephen M; Rothchild, Alissa C; Martin, Constance J; Desjardins, Danielle; Steblenko, Katherine; Kløverpris, Henrik N; Madansein, Rajhmun; Ramsuran, Duran; Leslie, Alasdair; Correia-Neves, Margarida; Behar, Samuel M

    2015-05-01

    The immune system can recognize virtually any antigen, yet T cell responses against several pathogens, including Mycobacterium tuberculosis, are restricted to a limited number of immunodominant epitopes. The host factors that affect immunodominance are incompletely understood. Whether immunodominant epitopes elicit protective CD8+ T cell responses or instead act as decoys to subvert immunity and allow pathogens to establish chronic infection is unknown. Here we show that anatomically distinct human granulomas contain clonally expanded CD8+ T cells with overlapping T cell receptor (TCR) repertoires. Similarly, the murine CD8+ T cell response against M. tuberculosis is dominated by TB10.44-11-specific T cells with extreme TCRβ bias. Using a retro genic model of TB10.44-11-specific CD8+ Tcells, we show that TCR dominance can arise because of competition between clonotypes driven by differences in affinity. Finally, we demonstrate that TB10.4-specific CD8+ T cells mediate protection against tuberculosis, which requires interferon-γ production and TAP1-dependent antigen presentation in vivo. Our study of how immunodominance, biased TCR repertoires, and protection are inter-related, provides a new way to measure the quality of T cell immunity, which if applied to vaccine evaluation, could enhance our understanding of how to elicit protective T cell immunity.

  11. TCR Signal Strength Regulates Akt Substrate Specificity To Induce Alternate Murine Th and T Regulatory Cell Differentiation Programs.

    PubMed

    Hawse, William F; Boggess, William C; Morel, Penelope A

    2017-07-15

    The Akt/mTOR pathway is a key driver of murine CD4 + T cell differentiation, and induction of regulatory T (Treg) cells results from low TCR signal strength and low Akt/mTOR signaling. However, strong TCR signals induce high Akt activity that promotes Th cell induction. Yet, it is unclear how Akt controls alternate T cell fate decisions. We find that the strength of the TCR signal results in differential Akt enzymatic activity. Surprisingly, the Akt substrate networks associated with T cell fate decisions are qualitatively different. Proteomic profiling of Akt signaling networks during Treg versus Th induction demonstrates that Akt differentially regulates RNA processing and splicing factors to drive T cell differentiation. Interestingly, heterogeneous nuclear ribonucleoprotein (hnRNP) L or hnRNP A1 are Akt substrates during Treg induction and have known roles in regulating the stability and splicing of key mRNAs that code for proteins in the canonical TCR signaling pathway, including CD3ζ and CD45. Functionally, inhibition of Akt enzymatic activity results in the dysregulation of splicing during T cell differentiation, and knockdown of hnRNP L or hnRNP A1 results in the lower induction of Treg cells. Together, this work suggests that a switch in substrate specificity coupled to the phosphorylation status of Akt may lead to alternative cell fates and demonstrates that proteins involved with alternative splicing are important factors in T cell fate decisions. Copyright © 2017 by The American Association of Immunologists, Inc.

  12. Alpha4 containing nicotinic receptors are positioned to mediate postsynaptic effects on serotonin neurons in the rat dorsal raphe nucleus

    PubMed Central

    Commons, Kathryn G.

    2008-01-01

    Nicotinic acetylcholine receptors containing the alpha4 and beta2 subunits constitute the most abundant high-affinity binding site of nicotine in the brain and are critical for the addictive qualities of nicotine. Serotonin neurotransmission is thought to be an important contributor to nicotine addiction. Therefore in this study it was examined how alpha4-containing receptors are positioned to modulate the function of serotonin neurons using ultrastructural analysis of immunolabeling for the alpha4 receptor subunit in the dorsal raphe nucleus (DR), a primary source of forebrain serotonin in the rat. Of 150 profiles labeled for the alpha4 subunit, 140 or 93% consisted of either soma or dendrites, these were often small-caliber (distal) dendrites <1.5 um in diameter (63/150 or 42%). The majority (107/150 or 71%) of profiles containing labeling for alpha4 were dually labeled for the synthetic enzyme for serotonin, tryptophan hydroxylase (TPH). Within dendrites immunogold labeling for alpha4 was present on the plasma membrane or near postsynaptic densities. However, labeling for alpha4 was commonly localized to the cytoplasmic compartment often associated with smooth endoplasmic reticulum, plausibly representing receptors in transit to or from the plasma membrane. Previous studies have suggested that nicotine presynaptically regulates activity onto serotonin neurons, however alpha4 immunolabeling was detected in only 10 axons in the DR or 7% of profiles sampled. This finding suggest that alpha4 containing receptors are minor contributors to presynaptic regulation of synaptic activity onto serotonin neurons, but rather alpha4 containing receptors are positioned to influence serotonin neurons directly at postsynaptic sites. PMID:18403129

  13. TCR-pMHC encounter differentially regulates transcriptomes of tissue-resident CD8 T cells.

    PubMed

    Yoshizawa, Akihiro; Bi, Kevin; Keskin, Derin B; Zhang, Guanglan; Reinhold, Bruce; Reinherz, Ellis L

    2018-01-01

    To investigate the role of TCR-pMHC interaction in regulating lung CD8 tissue-resident T cell (T R ) differentiation, polyclonal responses were compared against NP 366-374 /D b and PA 224-233 /D b , two immunodominant epitopes that arise during influenza A infection in mice. Memory niches distinct from iBALTs develop within the lamina propria, supporting CD103 + and CD103 - CD8 T R generation and intraepithelial translocation. Gene set enrichment analysis (GSEA) and weighted gene co-expression network analysis (WGCNA) identify dominant TCR, adherens junction, RIG-I-like and NOD-like pattern recognition receptor as well as TGF-β signaling pathways and memory signatures among PA 224-233 /D b T cells consistent with T resident memory (T RM ) status. In contrast, NP 366-374 /D b T cells exhibit enrichment of effector signatures, upregulating pro-inflammatory mediators even among T RM . While NP 366-374 /D b T cells manifest transcripts linked to canonical exhaustion pathways, PA 224-233 /D b T cells exploit P2rx7 purinoreceptor attenuation. The NP 366-374 /D b CD103 + subset expresses the antimicrobial lactotransferrin whereas PA 224-233 /D b CD103 + utilizes pore-forming mpeg-1, with <22% of genes correspondingly upregulated in CD103 + (or CD103 - ) subsets of both specificities. Thus, TCR-pMHC interactions among T R and antigen presenting cells in a tissue milieu strongly impact CD8 T cell biology. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Recall of anti-tobacco advertising and information, warning labels and news stories in a national sample of Aboriginal and Torres Strait Islander smokers.

    PubMed

    Nicholson, Anna K; Borland, Ron; Sarin, Jasmine; Wallace, Sharon; van der Sterren, Anke E; Stevens, Matthew; Thomas, David P

    2015-06-01

    To describe recall of anti-tobacco advertising (mainstream and targeted), pack warning labels, and news stories among a national sample of Aboriginal and Torres Strait Islander smokers, and to assess the association of these messages with attitudes that support quitting, including wanting to quit. A quota sampling design was used to recruit participants from communities served by 34 Aboriginal community-controlled health services and one community in the Torres Strait. We surveyed 1643 Aboriginal and Torres Strait Islander smokers from April 2012 to October 2013. Frequency of recall of advertising and information, warning labels and news stories; recall of targeted and local advertising; attitudes about smoking and wanting to quit. More smokers recalled often noticing warning labels in the past month (65%) than recalled advertising and information (45%) or news stories (24%) in the past 6 months. When prompted, most (82%) recalled seeing a television advertisement. Just under half (48%) recalled advertising that featured an Aboriginal or Torres Strait Islander person or artwork (targeted advertising), and 16% recalled targeted advertising from their community (local advertising). Frequent recall of warning labels, news stories and advertising was associated with worry about health and wanting to quit, but only frequent advertising recall was associated with believing that society disapproves of smoking. The magnitude of association with relevant attitudes and wanting to quit increased for targeted and local advertising. Strategies to tackle Aboriginal and Torres Strait Islander smoking should sustain high levels of exposure to anti-tobacco advertising, news stories and warning labels. More targeted and local information may be particularly effective to influence relevant beliefs and subsequently increase quitting.

  15. Nucleotide Excision Repair and Transcription-coupled DNA Repair Abrogate the Impact of DNA Damage on Transcription*

    PubMed Central

    Nadkarni, Aditi; Burns, John A.; Gandolfi, Alberto; Chowdhury, Moinuddin A.; Cartularo, Laura; Berens, Christian; Geacintov, Nicholas E.; Scicchitano, David A.

    2016-01-01

    DNA adducts derived from carcinogenic polycyclic aromatic hydrocarbons like benzo[a]pyrene (B[a]P) and benzo[c]phenanthrene (B[c]Ph) impede replication and transcription, resulting in aberrant cell division and gene expression. Global nucleotide excision repair (NER) and transcription-coupled DNA repair (TCR) are among the DNA repair pathways that evolved to maintain genome integrity by removing DNA damage. The interplay between global NER and TCR in repairing the polycyclic aromatic hydrocarbon-derived DNA adducts (+)-trans-anti-B[a]P-N6-dA, which is subject to NER and blocks transcription in vitro, and (+)-trans-anti-B[c]Ph-N6-dA, which is a poor substrate for NER but also blocks transcription in vitro, was tested. The results show that both adducts inhibit transcription in human cells that lack both NER and TCR. The (+)-trans-anti-B[a]P-N6-dA lesion exhibited no detectable effect on transcription in cells proficient in NER but lacking TCR, indicating that NER can remove the lesion in the absence of TCR, which is consistent with in vitro data. In primary human cells lacking NER, (+)-trans-anti-B[a]P-N6-dA exhibited a deleterious effect on transcription that was less severe than in cells lacking both pathways, suggesting that TCR can repair the adduct but not as effectively as global NER. In contrast, (+)-trans-anti-B[c]Ph-N6-dA dramatically reduces transcript production in cells proficient in global NER but lacking TCR, indicating that TCR is necessary for the removal of this adduct, which is consistent with in vitro data showing that it is a poor substrate for NER. Hence, both global NER and TCR enhance the recovery of gene expression following DNA damage, and TCR plays an important role in removing DNA damage that is refractory to NER. PMID:26559971

  16. Alpha-beta T cells provide protection against lethal encephalitis in the murine model of VEEV infection

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Paessler, Slobodan; Yun, Nadezhda E.; Judy, Barbara M.

    2007-10-25

    We evaluated the safety and immunogenicity of a chimeric alphavirus vaccine candidate in mice with selective immunodeficiencies. This vaccine candidate was highly attenuated in mice with deficiencies in the B and T cell compartments, as well as in mice with deficient gamma-interferon responsiveness. However, the level of protection varied among the strains tested. Wild type mice were protected against lethal VEEV challenge. In contrast, alpha/beta ({alpha}{beta}) TCR-deficient mice developed lethal encephalitis following VEEV challenge, while mice deficient in gamma/delta ({gamma}{delta}) T cells were protected. Surprisingly, the vaccine potency was diminished by 50% in animals lacking interferon-gamma receptor alpha chain (R1)-chainmore » and a minority of vaccinated immunoglobulin heavy chain-deficient ({mu}MT) mice survived challenge, which suggests that neutralizing antibody may not be absolutely required for protection. Prolonged replication of encephalitic VEEV in the brain of pre-immunized mice is not lethal and adoptive transfer experiments indicate that CD3{sup +} T cells are required for protection.« less

  17. 18O stable isotope labeling, quantitative model experiments, and molecular dynamics simulation studies on the trans-specific degradation of the bitter tasting iso-alpha-acids of beer.

    PubMed

    Intelmann, Daniel; Demmer, Oliver; Desmer, Nina; Hofmann, Thomas

    2009-11-25

    The typical bitterness of fresh beer is well-known to decrease in intensity and to change in quality with increasing age. This phenomenon was recently shown to be caused by the conversion of bitter tasting trans-iso-alpha-acids into lingering and harsh bitter tasting tri- and tetracyclic degradation products such as tricyclocohumol, tricyclocohumene, isotricyclocohumene, tetracyclocohumol, and epitetracyclocohumol. Interestingly, the formation of these compounds was shown to be trans-specific and the corresponding cis-iso-alpha-acids were found to be comparatively stable. Application of 18O stable isotope labeling as well as quantitative model studies combined with LC-MS/MS experiments, followed by computer-based molecular dynamics simulations revealed for the first time a conclusive mechanism explaining the stereospecific transformation of trans-iso-alpha-acids into the tri- and tetracyclic degradation products. This transformation was proposed to be induced by a proton-catalyzed carbon/carbon bond formation between the carbonyl atom C(1') of the isohexenoyl moiety and the alkene carbon C(2'') of the isoprenyl moiety of the trans-iso-alpha-acids.

  18. Thiols in the alphaIIbbeta3 integrin are necessary for platelet aggregation.

    PubMed

    Manickam, Nagaraj; Sun, Xiuhua; Hakala, Kevin W; Weintraub, Susan T; Essex, David W

    2008-07-01

    Sulfhydryl groups of platelet surface proteins are important in platelet aggregation. While p-chloromercuribenzene sulphonate (pCMBS) has been used in most studies on platelet surface thiols, the specific thiol-proteins that pCMBS reacts with to inhibit aggregation have not been well defined. Since the thiol-containing P2Y(12) ADP receptor is involved in most types of platelet aggregation, we used the ADP scavenger apyrase and the P2Y(12) receptor antagonist 2-MeSAMP to examine thiol-dependent reactions in the absence of contributions from this receptor. We provide evidence for a non-P2Y(12) thiol-dependent reaction near the final alphaIIbbeta3-dependent events of aggregation. We then used 3-(N-maleimidylpropionyl)biocytin (MPB) and pCMBS to study thiols in alphaIIbbeta3. As previously reported, disruption of the receptor was required to obtain labelling of thiols with MPB. Specificity of labelling for thiols in the alphaIIb and beta3 subunits was confirmed by identification of the purified proteins by mass spectrometry and by inhibition of labelling with 5,5'-dithiobis-(2-nitrobenzoic acid). In contrast to MPB, pCMBS preferentially reacted with thiols in alphaIIbbeta3 and blocked aggregation under physiological conditions. Similarly, pCMBS preferentially inhibited signalling-independent activation of alphaIIbbeta3 by Mn(2+). Our results suggest that the thiols in alphaIIbbeta3 that are blocked by pCMBS are important in the activation of this integrin.

  19. HIV-1 Nef limits communication between linker of activated T cells and SLP-76 to reduce formation of SLP-76-signaling microclusters following TCR stimulation.

    PubMed

    Abraham, Libin; Bankhead, Peter; Pan, Xiaoyu; Engel, Ulrike; Fackler, Oliver T

    2012-08-15

    Signal initiation by engagement of the TCR triggers actin rearrangements, receptor clustering, and dynamic organization of signaling complexes to elicit and sustain downstream signaling. Nef, a pathogenicity factor of HIV, disrupts early TCR signaling in target T cells. To define the mechanism underlying this Nef-mediated signal disruption, we employed quantitative single-cell microscopy following surface-mediated TCR stimulation that allows for dynamic visualization of distinct signaling complexes as microclusters (MCs). Despite marked inhibition of actin remodeling and cell spreading, the induction of MCs containing TCR-CD3 or ZAP70 was not affected significantly by Nef. However, Nef potently inhibited the subsequent formation of MCs positive for the signaling adaptor Src homology-2 domain-containing leukocyte protein of 76 kDa (SLP-76) to reduce MC density in Nef-expressing and HIV-1-infected T cells. Further analyses suggested that Nef prevents formation of SLP-76 MCs at the level of the upstream adaptor protein, linker of activated T cells (LAT), that couples ZAP70 to SLP-76. Nef did not disrupt pre-existing MCs positive for LAT. However, the presence of the viral protein prevented de novo recruitment of active LAT into MCs due to retargeting of LAT to an intracellular compartment. These modulations in MC formation and composition depended on Nef's ability to simultaneously disrupt both actin remodeling and subcellular localization of TCR-proximal machinery. Nef thus employs a dual mechanism to disturb early TCR signaling by limiting the communication between LAT and SLP-76 and preventing the dynamic formation of SLP-76-signaling MCs.

  20. Characterization of salivary alpha-amylase binding to Streptococcus sanguis

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Scannapieco, F.A.; Bergey, E.J.; Reddy, M.S.

    1989-09-01

    The purpose of this study was to identify the major salivary components which interact with oral bacteria and to determine the mechanism(s) responsible for their binding to the bacterial surface. Strains of Streptococcus sanguis, Streptococcus mitis, Streptococcus mutans, and Actinomyces viscosus were incubated for 2 h in freshly collected human submandibular-sublingual saliva (HSMSL) or parotid saliva (HPS), and bound salivary components were eluted with 2% sodium dodecyl sulfate. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western transfer, alpha-amylase was the prominent salivary component eluted from S. sanguis. Studies with {sup 125}I-labeled HSMSL or {sup 125}I-labeled HPS also demonstrated a componentmore » with an electrophoretic mobility identical to that of alpha-amylase which bound to S. sanguis. Purified alpha-amylase from human parotid saliva was radiolabeled and found to bind to strains of S. sanguis genotypes 1 and 3 and S. mitis genotype 2, but not to strains of other species of oral bacteria. Binding of ({sup 125}I)alpha-amylase to streptococci was saturable, calcium independent, and inhibitable by excess unlabeled alpha-amylases from a variety of sources, but not by secretory immunoglobulin A and the proline-rich glycoprotein from HPS. Reduced and alkylated alpha-amylase lost enzymatic and bacterial binding activities. Binding was inhibited by incubation with maltotriose, maltooligosaccharides, limit dextrins, and starch.« less

  1. Platypus TCRμ provides insight into the origins and evolution of a uniquely mammalian TCR locus.

    PubMed

    Wang, Xinxin; Parra, Zuly E; Miller, Robert D

    2011-11-15

    TCRμ is an unconventional TCR that was first discovered in marsupials and appears to be absent from placental mammals and nonmammals. In this study, we show that TCRμ is also present in the duckbill platypus, an egg-laying monotreme, consistent with TCRμ being ancient and present in the last common ancestor of all extant mammals. As in marsupials, platypus TCRμ is expressed in a form containing double V domains. These V domains more closely resemble Ab V than that of conventional TCR. Platypus TCRμ differs from its marsupial homolog by requiring two rounds of somatic DNA recombination to assemble both V exons and has a genomic organization resembling the likely ancestral form of the receptor genes. These results demonstrate that the ancestors of placental mammals would have had TCRμ but it has been lost from this lineage.

  2. TCR gamma chain diversity in the spleen of the duckbill platypus (Ornithorhynchus anatinus).

    PubMed

    Parra, Zuly E; Arnold, Tamara; Nowak, Melissa A; Hellman, Lars; Miller, Robert D

    2006-01-01

    TCR gamma (TRG) chain diversity in splenic gammadelta T cells was determined for an egg-laying mammal (or monotreme), the duckbill platypus. Three distinct V subgroups were found in the expressed TRG chains and these three subgroups are members of a clade not found so far in eutherian mammals or birds. Each subgroup contains approximately five V gene segments, and their overall divergence is much less than is found in eutherians and birds, consistent with their recent evolution from an ancestral V gene segment. The platypus TRG locus also contains three C region genes and many of the residues involved in TCR function, such as interactions with CD3, were conserved in the monotreme C regions. All non-eutherian mammals (monotremes and marsupials) lacked the second cysteine residue necessary to form the intradomain disulfide bond in the C region, a loss apparently due to independent mutations in marsupials and monotremes. Monotreme TRGC regions also had among the most variation in the length of the connecting peptide region described for any species due to repeated motifs.

  3. Tritiated-nicotine- and /sup 125/I-alpha-bungarotoxin-labeled nicotinic receptors in the interpeduncular nucleus of rats. II. Effects of habenular destruction

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Clarke, P.B.; Hamill, G.S.; Nadi, N.S.

    1986-09-15

    The cholinergic innervation of the interpeduncular nucleus (IPN) is wholly extrinsic and is greatly attenuated by bilateral habenular destruction. We describe changes in the labeling of putative nicotinic receptors within this nucleus at 3, 5, or 11 days after bilateral habenular lesions. Adjacent tissue sections of the rat IPN were utilized for /sup 3/H-nicotine and /sup 125/I-alpha-bungarotoxin (/sup 125/I-BTX) receptor autoradiography. Compared to sham-operated controls, habenular destruction significantly reduced autoradiographic /sup 3/H-nicotine labeling in rostral (-25%), intermediate (-13%), and lateral subnuclei (-36%). Labeling in the central subnucleus was unchanged. Loss of labeling was maximal at the shortest survival time (3more » days) and did not change thereafter. In order to establish whether this loss was due to a reduction in the number or the affinity of /sup 3/H-nicotine-binding sites, a membrane assay was performed on microdissected IPN tissue from rats that had received surgery 3 days previously. Bilateral habenular lesions produced a 35% reduction of high-affinity /sup 3/H-nicotine-binding sites, with no change in binding affinity. Bilateral habenular lesions reduced /sup 125/I-BTX labeling in the intermediate subnuclei, and a slight increase occurred in the rostral subnucleus. In the lateral subnuclei, /sup 125/I-BTX labeling was significantly reduced (27%) at 3 days but not at later survival times. In view of the known synaptic morphology of the habenulointerpeduncular tract, it is concluded that a subpopulation of /sup 3/H-nicotine binding sites within the IPN is located on afferent axons and/or terminals. This subpopulation, located within rostral, intermediate, and lateral subnuclei, may correspond to presynaptic nicotinic cholinergic receptors. Sites that bind /sup 125/I-BTX may include a presynaptic subpopulation located in the lateral and possibly the intermediate subnuclei.« less

  4. 64Cu-labeled alpha-melanocyte-stimulating hormone analog for microPET imaging of melanocortin 1 receptor expression.

    PubMed

    Cheng, Zhen; Xiong, Zhengming; Subbarayan, Murugesan; Chen, Xiaoyuan; Gambhir, Sanjiv Sam

    2007-01-01

    The alpha-melanocyte-stimulating hormone (alpha-MSH) receptor (melanocortin type 1 receptor, or MC1R) plays an important role in the development and growth of melanoma cells. It was found that MC1R was overexpressed on most murine and human melanoma, making it a promising molecular target for melanoma imaging and therapy. Radiolabeled alpha-MSH peptide and its analogs that can specifically bind with MC1R have been extensively explored for developing novel agents for melanoma detection and radionuclide therapy. The goal of this study was to evaluate a 64Cu-labeled alpha-MSH analog, Ac-Nle-Asp-His-D-Phe-Arg-Trp-Gly-Lys(DOTA)-NH2 (DOTA-NAPamide), as a potential molecular probe for microPET imaging of melanoma and MC1R expression in melanoma xenografted mouse models. 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) conjugated NAPamide was synthesized and radiolabeled with 64Cu (t1/2=12 h) in NH4OAc (0.1 M; pH 5.5) buffered solution for 60 min at 50 degrees C. Cell culture studies reveal rapid and high uptake and internalization of 64Cu-DOTA-NAPamide in B16F10 cells. Over 90% of receptor-bound tracer is internalized at 3 h incubation. A cellular retention study demonstrates that the receptor-bound 64Cu-DOTA-NAPamide is slowly released from the B16F10 cells into the medium; 66% of the radioactivity is still associated with the cells even after 3 h incubation. The biodistribution of 64Cu-DOTA-NAPamide was then investigated in C57BL/6 mice bearing subcutaneous murine B16F10 melanoma tumors with high capacity of MC1R and Fox Chase Scid mice bearing human A375M melanoma with a relatively low number of MC1R receptors. Tumor uptake values of 64Cu-DOTA-NAPamide are found to be 4.63 +/- 0.45% and 2.49 +/- 0.31% ID/g in B16F10 and A375M xenografted melanoma at 2 h postinjection (pi), respectively. The B16F10 tumor uptake at 2 h pi is further inhibited to 2.29 +/- 0.24% ID/g, while A375M tumor uptake at 2 h pi remains 2.20 +/- 0.41% ID/g with a coinjection of excess

  5. Enhanced clinical-scale manufacturing of TCR transduced T-cells using closed culture system modules.

    PubMed

    Jin, Jianjian; Gkitsas, Nikolaos; Fellowes, Vicki S; Ren, Jiaqiang; Feldman, Steven A; Hinrichs, Christian S; Stroncek, David F; Highfill, Steven L

    2018-01-24

    Genetic engineering of T-cells to express specific T cell receptors (TCR) has emerged as a novel strategy to treat various malignancies. More widespread utilization of these types of therapies has been somewhat constrained by the lack of closed culture processes capable of expanding sufficient numbers of T-cells for clinical application. Here, we evaluate a process for robust clinical grade manufacturing of TCR gene engineered T-cells. TCRs that target human papillomavirus E6 and E7 were independently tested. A 21 day process was divided into a transduction phase (7 days) and a rapid expansion phase (14 days). This process was evaluated using two healthy donor samples and four samples obtained from patients with epithelial cancers. The process resulted in ~ 2000-fold increase in viable nucleated cells and high transduction efficiencies (64-92%). At the end of culture, functional assays demonstrated that these cells were potent and specific in their ability to kill tumor cells bearing target and secrete large quantities of interferon and tumor necrosis factor. Both phases of culture were contained within closed or semi-closed modules, which include automated density gradient separation and cell culture bags for the first phase and closed GREX culture devices and wash/concentrate systems for the second phase. Large-scale manufacturing using modular systems and semi-automated devices resulted in highly functional clinical-grade TCR transduced T-cells. This process is now in use in actively accruing clinical trials and the NIH Clinical Center and can be utilized at other cell therapy manufacturing sites that wish to scale-up and optimize their processing using closed systems.

  6. Overexpression of cellular repressor of E1A-stimulated genes inhibits TNF-{alpha}-induced apoptosis via NF-{kappa}B in mesenchymal stem cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Peng, Cheng-Fei; Cardiovascular Research Institute and Department of Cardiology, Shenyang Northern Hospital, Shenyang; Han, Ya-Ling, E-mail: hanyaling53@gmail.com

    2011-03-25

    Research highlights: {yields} CREG protected MSCs from tumor necrosis factor-{alpha} (TNF-{alpha}) induced apoptosis. {yields} CREG inhibits the phosphorylation of I{kappa}B{alpha} and prevents the activation of NF-{kappa}B. {yields} CREG inhibits NF-{kappa}B nuclear translocation and pro-apoptosis protein transcription. {yields} CREG anti-apoptotic effect involves inhibition of the death receptor pathway. {yields} p53 is downregulated by CREG via NF-{kappa}B pathway under TNF-{alpha} stimulation. -- Abstract: Bone marrow-derived mesenchymal stem cells (MSCs) show great potential for therapeutic repair after myocardial infarction. However, poor viability of transplanted MSCs in the ischemic heart has limited their use. Cellular repressor of E1A-stimulated genes (CREG) has been identified asmore » a potent inhibitor of apoptosis. This study therefore aimed to determine if rat bone marrow MSCs transfected with CREG-were able to effectively resist apoptosis induced by inflammatory mediators, and to demonstrate the mechanism of CREG action. Apoptosis was determined by flow cytometric and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling assays. The pathways mediating these apoptotic effects were investigated by Western blotting. Overexpression of CREG markedly protected MSCs from tumor necrosis factor-{alpha} (TNF-{alpha}) induced apoptosis by 50% after 10 h, through inhibition of the death-receptor-mediated apoptotic pathway, leading to attenuation of caspase-8 and caspase-3. Moreover, CREG resisted the serine phosphorylation of I{kappa}B{alpha} and prevented the nuclear translocation of the transcription factor nuclear factor-{kappa}B (NF-{kappa}B) under TNF-{alpha} stimulation. Treatment of cells with the NF-{kappa}B inhibitor pyrrolidine dithiocarbamate (PDTC) significantly increased the transcription of pro-apoptosis proteins (p53 and Fas) by NF-{kappa}B, and attenuated the anti-apoptotic effects of CREG on MSCs. The results of this

  7. SLAT promotes TCR-mediated, Rap1-dependent LFA-1 activation and adhesion through interaction of its PH domain with Rap1

    PubMed Central

    Côte, Marjorie; Fos, Camille; Canonigo-Balancio, Ann J.; Ley, Klaus; Bécart, Stéphane; Altman, Amnon

    2015-01-01

    ABSTRACT SLAT (also known as DEF6) promotes T cell activation and differentiation by regulating NFAT-Ca2+ signaling. However, its role in TCR-mediated inside-out signaling, which induces integrin activation and T cell adhesion, a central process in T cell immunity and inflammation, has not been explored. Here, we show that SLAT is crucial for TCR-induced adhesion to ICAM-1 and affinity maturation of LFA-1 in CD4+ T cells. Mechanistic studies revealed that SLAT interacts, through its PH domain, with a key component of inside-out signaling, namely the active form of the small GTPase Rap1 (which has two isoforms, Rap1A and Rap1B). This interaction has been further shown to facilitate the interdependent recruitment of Rap1 and SLAT to the T cell immunological synapse upon TCR engagement. Furthermore, a SLAT mutant lacking its PH domain drastically inhibited LFA-1 activation and CD4+ T cell adhesion. Finally, we established that a constitutively active form of Rap1, which is present at the plasma membrane, rescues the defective LFA-1 activation and ICAM-1 adhesion in SLAT-deficient (Def6−/−) T cells. These findings ascribe a new function to SLAT, and identify Rap1 as a target of SLAT function in TCR-mediated inside-out signaling. PMID:26483383

  8. SLAT promotes TCR-mediated, Rap1-dependent LFA-1 activation and adhesion through interaction of its PH domain with Rap1.

    PubMed

    Côte, Marjorie; Fos, Camille; Canonigo-Balancio, Ann J; Ley, Klaus; Bécart, Stéphane; Altman, Amnon

    2015-12-01

    SLAT (also known as DEF6) promotes T cell activation and differentiation by regulating NFAT-Ca(2+) signaling. However, its role in TCR-mediated inside-out signaling, which induces integrin activation and T cell adhesion, a central process in T cell immunity and inflammation, has not been explored. Here, we show that SLAT is crucial for TCR-induced adhesion to ICAM-1 and affinity maturation of LFA-1 in CD4(+) T cells. Mechanistic studies revealed that SLAT interacts, through its PH domain, with a key component of inside-out signaling, namely the active form of the small GTPase Rap1 (which has two isoforms, Rap1A and Rap1B). This interaction has been further shown to facilitate the interdependent recruitment of Rap1 and SLAT to the T cell immunological synapse upon TCR engagement. Furthermore, a SLAT mutant lacking its PH domain drastically inhibited LFA-1 activation and CD4(+) T cell adhesion. Finally, we established that a constitutively active form of Rap1, which is present at the plasma membrane, rescues the defective LFA-1 activation and ICAM-1 adhesion in SLAT-deficient (Def6(-/-)) T cells. These findings ascribe a new function to SLAT, and identify Rap1 as a target of SLAT function in TCR-mediated inside-out signaling. © 2015. Published by The Company of Biologists Ltd.

  9. The activation threshold of CD4+ T cells is defined by TCR/peptide-MHC class II interactions in the thymic medulla.

    PubMed

    Stephen, Tom Li; Tikhonova, Anastasia; Riberdy, Janice M; Laufer, Terri M

    2009-11-01

    Immature thymocytes that are positively selected based upon their response to self-peptide-MHC complexes develop into mature T cells that are not overtly reactive to those same complexes. Developmental tuning is the active process through which TCR-associated signaling pathways of single-positive thymocytes are attenuated to respond appropriately to the peptide-MHC molecules that will be encountered in the periphery. In this study, we explore the mechanisms that regulate the tuning of CD4(+) single-positive T cells to MHC class II encountered in the thymic medulla. Experiments with murine BM chimeras demonstrate that tuning can be mediated by MHC class II expressed by either thymic medullary epithelial cells or thymic dendritic cells. Tuning does not require the engagement of CD4 by MHC class II on stromal cells. Rather, it is mediated by interactions between MHC class II and the TCR. To understand the molecular changes that distinguish immature hyperactive T cells from tuned mature CD4(+) T cells, we compared their responses to TCR stimulation. The altered response of mature CD4 single-positive thymocytes is characterized by the inhibition of ERK activation by low-affinity self-ligands and increased expression of the inhibitory tyrosine phosphatase SHP-1. Thus, persistent TCR engagement by peptide-MHC class II on thymic medullary stroma inhibits reactivity to self-Ags and prevents autoreactivity in the mature repertoire.

  10. Off-Label Uses of Anti-TNF Therapy in Three Frequent Disorders: Behçet's Disease, Sarcoidosis, and Noninfectious Uveitis

    PubMed Central

    Sánchez-Cano, Daniel; Callejas-Rubio, José Luis; Ruiz-Villaverde, Ricardo; Ríos-Fernández, Raquel; Ortego-Centeno, Norberto

    2013-01-01

    Tumoral necrosis factor α plays a central role in both the inflammatory response and that of the immune system. Thus, its blockade with the so-called anti-TNF agents (infliximab, etanercept, adalimumab, certolizumab pegol, and golimumab) has turned into the most important tool in the management of a variety of disorders, such as rheumatoid arthritis, spondyloarthropatties, inflammatory bowel disease, and psoriasis. Nonetheless, theoretically, some other autoimmune disorders may benefit from these agents. Our aim is to review these off-label uses of anti-TNF blockers in three common conditions: Behçet's disease, sarcoidosis, and noninfectious uveitis. Due to the insufficient number of adequate clinical trials and consequently to their lower prevalence compared to other immune disorders, this review is mainly based on case reports and case series. PMID:23983404

  11. Screening alpha-glucosidase and alpha-amylase inhibitors from natural compounds by molecular docking in silico.

    PubMed

    Jhong, Chien-Hung; Riyaphan, Jirawat; Lin, Shih-Hung; Chia, Yi-Chen; Weng, Ching-Feng

    2015-01-01

    The alpha-glucosidase inhibitor is a common oral anti-diabetic drug used for controlling carbohydrates normally converted into simple sugars and absorbed by the intestines. However, some adverse clinical effects have been observed. The present study seeks an alternative drug that can regulate the hyperglycemia by down-regulating alpha-glucosidase and alpha-amylase activity by molecular docking approach to screen the hyperglycemia antagonist against alpha-glucosidase and alpha-amylase activities from the 47 natural compounds. The docking data showed that Curcumin, 16-hydroxy-cleroda-3,13-dine-16,15-olide (16-H), Docosanol, Tetracosanol, Antroquinonol, Berberine, Catechin, Quercetin, Actinodaphnine, and Rutin from 47 natural compounds had binding ability towards alpha-amylase and alpha-glucosidase as well. Curcumin had a better biding ability of alpha-amylase than the other natural compounds. Analyzed alpha-glucosidase activity reveals natural compound inhibitors (below 0.5 mM) are Curcumin, Actinodaphnine, 16-H, Quercetin, Berberine, and Catechin when compared to the commercial drug Acarbose (3 mM). A natural compound with alpha-amylase inhibitors (below 0.5 mM) includes Curcumin, Berberine, Docosanol, 16-H, Actinodaphnine/Tetracosanol, Catechin, and Quercetin when compared to Acarbose (1 mM). When taken together, the implication is that molecular docking is a fast and effective way to screen alpha-glucosidase and alpha-amylase inhibitors as lead compounds of natural sources isolated from medicinal plants. © 2015 International Union of Biochemistry and Molecular Biology.

  12. Functional coupling of rat myometrial alpha 1-adrenergic receptors to Gh alpha/tissue transglutaminase 2 during pregnancy.

    PubMed

    Dupuis, Morgan; Lévy, Arlette; Mhaouty-Kodja, Sakina

    2004-04-30

    Gh alpha protein, which exhibits both transglutaminase and GTPase activities, represents a new class of GTP-binding proteins. In the present study, we characterized Gh alpha in rat uterine smooth muscle (myometrium) and followed its expression during pregnancy by reverse transcription-PCR and Western blot. We also measured transglutaminase and GTP binding functions and used a smooth muscle cell line to evaluate the role of Gh alpha in cell proliferation. The results show that pregnancy is associated with an up-regulation of Gh alpha expression at both the mRNA and protein level. Gh alpha induced during pregnancy is preferentially localized to the plasma membrane. This was found associated with an increased ability of plasma membrane preparations to catalyze Ca(2+)-dependent incorporation of [(3)H]putrescine into casein in vitro. In the cytosol, significant changes in the level of immunodetected Gh alpha and transglutaminase activity were seen only at term. Activation of alpha1-adrenergic receptors (alpha1-AR) enhanced photoaffinity labeling of plasma membrane Gh alpha. Moreover, the level of alpha1-AR-coupled Gh alpha increased progressively with pregnancy, which parallels the active period of myometrial cell proliferation. Overexpression of wild type Gh alpha in smooth muscle cell line DDT1-MF2 increased alpha1-AR-induced [(3)H]thymidine incorporation. A similar response was obtained in cells expressing the transglutaminase inactive mutant (C277S) of Gh alpha. Together, these findings underscore the role of Gh alpha as signal transducer of alpha1-AR-induced smooth muscle cell proliferation. In this context, pregnant rat myometrium provides an interesting physiological model to study the mechanisms underlying the regulation of the GTPase function of Gh alpha

  13. 64Cu antibody-targeting of the T-cell receptor and subsequent internalization enables in vivo tracking of lymphocytes by PET

    PubMed Central

    Griessinger, Christoph M.; Maurer, Andreas; Kesenheimer, Christian; Kehlbach, Rainer; Reischl, Gerald; Ehrlichmann, Walter; Bukala, Daniel; Harant, Maren; Cay, Funda; Brück, Jürgen; Nordin, Renate; Kohlhofer, Ursula; Rammensee, Hans-Georg; Quintanilla-Martinez, Leticia; Schaller, Martin; Röcken, Martin; Pichler, Bernd J.; Kneilling, Manfred

    2015-01-01

    T cells are key players in inflammation, autoimmune diseases, and immunotherapy. Thus, holistic and noninvasive in vivo characterizations of the temporal distribution and homing dynamics of lymphocytes in mammals are of special interest. Herein, we show that PET-based T-cell labeling facilitates quantitative, highly sensitive, and holistic monitoring of T-cell homing patterns in vivo. We developed a new T-cell receptor (TCR)-specific labeling approach for the intracellular labeling of mouse T cells. We found that continuous TCR plasma membrane turnover and the endocytosis of the specific 64Cu-monoclonal antibody (mAb)–TCR complex enables a stable labeling of T cells. The TCR–mAb complex was internalized within 24 h, whereas antigen recognition was not impaired. Harmful effects of the label on the viability, DNA-damage and apoptosis-necrosis induction, could be minimized while yielding a high contrast in in vivo PET images. We were able to follow and quantify the specific homing of systemically applied 64Cu-labeled chicken ovalbumin (cOVA)-TCR transgenic T cells into the pulmonary and perithymic lymph nodes (LNs) of mice with cOVA-induced airway delayed-type hypersensitivity reaction (DTHR) but not into pulmonary and perithymic LNs of naïve control mice or mice diseased from turkey or pheasant OVA-induced DTHR. Our protocol provides consequent advancements in the detection of small accumulations of immune cells in single LNs and specific homing to the sites of inflammation by PET using the internalization of TCR-specific mAbs as a specific label of T cells. Thus, our labeling approach is applicable to other cells with constant membrane receptor turnover. PMID:25587131

  14. Therapeutic Efficacy of Alpha-RIT Using a (213)Bi-Anti-hCD138 Antibody in a Mouse Model of Ovarian Peritoneal Carcinomatosis.

    PubMed

    Derrien, Aurélie; Gouard, Sébastien; Maurel, Catherine; Gaugler, Marie-Hélène; Bruchertseifer, Frank; Morgenstern, Alfred; Faivre-Chauvet, Alain; Classe, Jean-Marc; Chérel, Michel

    2015-01-01

    Ovarian peritoneal carcinomatosis is a pathology for which effective cures are currently lacking. New research protocols seek to eradicate residual micrometastases following cytoreductive surgery by using hyperthermic intraperitoneal chemotherapy (HIPEC) or radioimmunotherapy (RIT). This study aims to first develop alpha-RIT using an anti-CD138 mAb radiolabeled with an alpha-emitter, bismuth-213 ((213)Bi-B-B4) and HIPEC in a nude mouse model and second to compare and combine these techniques. A murine model of postoperative ovarian peritoneal carcinomatosis was established. A pilot group of six mice received an intraperitoneal injection of luciferase-tagged SHIN-3 cells and bioluminescence was measured every day. Cytoreductive surgery was performed at day 14 (n = 4) and 29 (n = 2). Because the residual bioluminescence signal measured after surgery was equivalent to that obtained 3 days after the graft, HIPEC or alpha-RIT treatments were applied 3 days after the graft. Ten mice were treated by HIPEC with cisplatine (37.5 mg/mL), 11 with 7.4 MBq of (213)Bi-B-B4, seven with 11.1 MBq of (213)Bi-B-B4, and 10 mice were treated with the combined therapy (HIPEC + 7.4 MBq of (213)Bi-B-B4). Eleven mice received no treatment. Bioluminescence imaging and survival were assessed. Alpha-RIT 7.4 MBq and 11.1 MBq significantly improved survival (p = 0.0303 and p = 0.0070, respectively), whereas HIPEC and HIPEC + alpha-RIT treatments did not significantly ameliorate survival as compared to the control group. Survival was significantly increased by alpha-RIT treatment in mice with peritoneal carcinomatosis of ovarian origin; however, HIPEC alone or in combination with alpha-RIT had no significant effect.

  15. A structural model of anti-anti-[sigma] inhibition by a two-component receiver domain: the PhyR stress response regulator

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Herrou, Julien; Foreman, Robert; Fiebig, Aretha

    2012-05-09

    PhyR is a hybrid stress regulator conserved in {alpha}-proteobacteria that contains an N-terminal {sigma}-like (SL) domain and a C-terminal receiver domain. Phosphorylation of the receiver domain is known to promote binding of the SL domain to an anti-{sigma} factor. PhyR thus functions as an anti-anti-{sigma} factor in its phosphorylated state. We present genetic evidence that Caulobacter crescentus PhyR is a phosphorylation-dependent stress regulator that functions in the same pathway as {sigma}{sup T} and its anti-{sigma} factor, NepR. Additionally, we report the X-ray crystal structure of PhyR at 1.25 {angstrom} resolution, which provides insight into the mechanism of anti-anti-{sigma} regulation. Directmore » intramolecular contact between the PhyR receiver and SL domains spans regions {sigma}{sub 2} and {sigma}{sub 4}, likely serving to stabilize the SL domain in a closed conformation. The molecular surface of the receiver domain contacting the SL domain is the structural equivalent of {alpha}4-{beta}5-{alpha}5, which is known to undergo dynamic conformational change upon phosphorylation in a diverse range of receiver proteins. We propose a structural model of PhyR regulation in which receiver phosphorylation destabilizes the intramolecular interaction between SL and receiver domains, thereby permitting regions {sigma}{sub 2} and {sigma}{sub 4} in the SL domain to open about a flexible connector loop and bind anti-{sigma} factor.« less

  16. Platypus TCRμ provides insight into the origins and evolution of a uniquely mammalian TCR locus1

    PubMed Central

    Wang, Xinxin; Parra, Zuly E.; Miller, Robert D.

    2011-01-01

    TCRμ is an unconventional TCR that was first discovered in marsupials and appears to be absent from placental mammals and non-mammals. Here we show that TCRμ is also present in the duckbill platypus, an egg-laying monotreme, consistent with TCRμ being ancient and present in the last common ancestor of all extant mammals. As in marsupials, platypus TCRμ is expressed in a form containing double V domains. These V domains more closely resemble antibody V than that of conventional TCR. Platypus TCRμ differs from its marsupial homologue by requiring two rounds of somatic DNA recombination to assemble both V exons and has a genomic organization resembling the likely ancestral form of the receptor genes. These results demonstrate that the ancestors of placental mammals would have had TCRμ but it has been lost from this lineage. PMID:21976776

  17. Nucleotide Excision Repair and Transcription-coupled DNA Repair Abrogate the Impact of DNA Damage on Transcription.

    PubMed

    Nadkarni, Aditi; Burns, John A; Gandolfi, Alberto; Chowdhury, Moinuddin A; Cartularo, Laura; Berens, Christian; Geacintov, Nicholas E; Scicchitano, David A

    2016-01-08

    DNA adducts derived from carcinogenic polycyclic aromatic hydrocarbons like benzo[a]pyrene (B[a]P) and benzo[c]phenanthrene (B[c]Ph) impede replication and transcription, resulting in aberrant cell division and gene expression. Global nucleotide excision repair (NER) and transcription-coupled DNA repair (TCR) are among the DNA repair pathways that evolved to maintain genome integrity by removing DNA damage. The interplay between global NER and TCR in repairing the polycyclic aromatic hydrocarbon-derived DNA adducts (+)-trans-anti-B[a]P-N(6)-dA, which is subject to NER and blocks transcription in vitro, and (+)-trans-anti-B[c]Ph-N(6)-dA, which is a poor substrate for NER but also blocks transcription in vitro, was tested. The results show that both adducts inhibit transcription in human cells that lack both NER and TCR. The (+)-trans-anti-B[a]P-N(6)-dA lesion exhibited no detectable effect on transcription in cells proficient in NER but lacking TCR, indicating that NER can remove the lesion in the absence of TCR, which is consistent with in vitro data. In primary human cells lacking NER, (+)-trans-anti-B[a]P-N(6)-dA exhibited a deleterious effect on transcription that was less severe than in cells lacking both pathways, suggesting that TCR can repair the adduct but not as effectively as global NER. In contrast, (+)-trans-anti-B[c]Ph-N(6)-dA dramatically reduces transcript production in cells proficient in global NER but lacking TCR, indicating that TCR is necessary for the removal of this adduct, which is consistent with in vitro data showing that it is a poor substrate for NER. Hence, both global NER and TCR enhance the recovery of gene expression following DNA damage, and TCR plays an important role in removing DNA damage that is refractory to NER. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. Immunoreactive transforming growth factor alpha is commonly present in colorectal neoplasia.

    PubMed Central

    Tanaka, S.; Imanishi, K.; Yoshihara, M.; Haruma, K.; Sumii, K.; Kajiyama, G.; Akamatsu, S.

    1991-01-01

    Surgical specimens from 19 patients with invasive colorectal cancers and 12 specimens of normal mucosa from the same patients were examined immunohistochemically for the production of the immunoreactive (IR-) transforming growth factor (TGF)-alpha and IR-epidermal growth factor (EGF) with an anti-TGF-alpha monoclonal antibody (MAb) OAL-MTG01 and anti-EGF MAb KEM-10. Immunoreactive TGF-alpha was detected in 16 (84.2%) of 19 colorectal cancers. In contrast, there was no IR-TGF-alpha in the gland cells of normal mucosa. Immunoreactive EGF was detected in 7 (36.8%) of 19 colorectal cancers and 1 (8.3%) of 12 cases of normal mucosa. The production of both IR-TGF-alpha and IR-EGF in colorectal cancer did not differ by histologic type and Dukes' stage. Immunoreactive TGF-alpha was detected at significantly higher incidence than IR-EGF in colorectal cancer. These results indicate that IR-TGF-alpha should prove valuable as a possible tumor marker in colorectal cancers, and it may be very useful in understanding the biology of colorectal cancer. Images Figure 2 Figure 3 Figure 4 Figure 5 PMID:1853928

  19. Immune selection of tumor cells in TCR β-chain transgenic mice.

    PubMed

    Silaeva, Yulia Yu; Grinenko, Tatyana S; Vagida, Murad S; Kalinina, Anastasia A; Khromykh, Ludmila M; Kazansky, Dmitry B

    2014-10-01

    The concept of immunological surveillance implies that immunogenic variants of tumor cells arising in the organism can be recognized by the immune system. Tumor progression is provided by somatic evolution of tumor cells under the pressure of the immune system. The loss of MHC Class I molecules on the surface of tumor cells is one of the most known outcomes of immune selection. This study developed a model of immune selection based on the immune response of TCR 1d1 single β-chain transgenic B10.D2(R101) (K(d)I(d)D(b)) mice to allogeneic EL4 (H-2(b)) thymoma cells. In wild-type B10.D2(R101) mice, immunization with EL4 cells induced a vigorous CTL response targeted to the H-2K(b) molecule and results in full rejection of the tumor cells. In contrast, transgenic mice developed a compromised proliferative response in mixed-lymphocyte response assays and were unable to reject transplanted allogeneic EL4 cells. During the immune response to EL4 cells, CD8(+) T-lymphocytes with endogenous β-chains accumulated predominantly in the spleen of transgenic mice and only a small part of the T-lymphocytes expressing transgenic β-chains became CD8(+)CD44(+)CD62L(-) effectors. Then, instead of a full elimination of tumor cells as in wild-type mice, a reproducible prolonged equilibrium phase and subsequent escape was observed in transgenic mice that resulted in death of 90% of the mice in 40-60 days after grafting. Prolonged exposure of tumor cells to the pressure of the immune system in transgenic mice in vivo resulted in a stable loss of H-2K(b) molecules on the EL4 cell surface. Genetic manipulation of the T-lymphocyte repertoire was sufficient to reproduce the classic pattern of interactions between tumor cells and the immune system, usually observed in reliable syngeneic models of anti-tumor immunity. This newly-developed model could be used in further studies of immunoregulatory circuits common for transplantational and anti-tumor immune responses.

  20. TCR-independent CD28-mediated gene expression in peripheral blood lymphocytes from donors chronically infected with HIV-1.

    PubMed Central

    Wong, J G; Smithgall, M D; Haffar, O K

    1997-01-01

    Complete activation of peripheral blood T cells requires both T-cell receptor (TCR) stimulation and CD28 costimulation. Signalling pathways associated specifically with CD28 are not well understood, however, because ligation of CD28 in the absence of TCR stimulation does not give rise to cellular responses in normal cells. In peripheral blood lymphocytes (PBL) from donors chronically infected with human immunodeficiency virus-1 (HIV-1), CD28 can induce viral replication through an alternative pathway that does not require TCR ligation. We have exploited this observation to study CD28-mediated signal transduction using reverse transcriptase-mediated polymerase chain reaction (RT-PCR) to amplify viral RNA. Independent ligation of CD28 on donor PBL induced expression of the HIV-1 tat gene but not the interleukin-2 (IL-2) gene. Viral induction did not occur following pretreatment of cells with actinomycin D, suggesting it was mediated through transcriptional activation of the viral long terminal repeat (LTR). tat was induced in the presence of the protein kinase C inhibitor H-7, but was inhibited by cyclosporin A. Our results demonstrate that CD28 is linked directly to specific signalling pathways leading to de novo induction of genes in PBL. Images Figure 1 Figure 2 Figure 3 PMID:9135558

  1. Palladium-109 labeled anti-melanoma monoclonal antibodies

    DOEpatents

    Srivastava, S.C.; Fawwaz, R.A.; Ferrone, S.

    1984-04-30

    The invention consists of new monoclonal antibodies labelled with Palladium 109, a beta-emitting radionuclide, the method of preparing this material, and its use in the radiotherapy of melanoma. The antibodies are chelate-conjugated and demonstrate a high uptake in melanomas. (ACR)

  2. Combinations of ERK and p38 MAPK inhibitors ablate tumor necrosis factor-alpha (TNF-alpha ) mRNA induction. Evidence for selective destabilization of TNF-alpha transcripts.

    PubMed

    Rutault, K; Hazzalin, C A; Mahadevan, L C

    2001-03-02

    Tumor necrosis factor-alpha (TNF-alpha) is a potent proinflammatory cytokine whose synthesis and secretion are implicated in diverse pathologies. Hence, inhibition of TNF-alpha transcription or translation and neutralization of its protein product represent major pharmaceutical strategies to control inflammation. We have studied the role of ERK and p38 mitogen-activated protein (MAP) kinase in controlling TNF-alpha mRNA levels in differentiated THP-1 cells and in freshly purified human monocytes. We show here that it is possible to produce virtually complete inhibition of lipopolysaccharide-stimulated TNF-alpha mRNA accumulation by using a combination of ERK and p38 MAP kinase inhibitors. Furthermore, substantial inhibition is achievable using combinations of 1 microm of each inhibitor, whereas inhibitors used individually are incapable of producing complete inhibition even at high concentrations. Finally, addressing mechanisms involved, we show that inhibition of p38 MAP kinase selectively destabilizes TNF-alpha transcripts but does not affect degradation of c-jun transcripts. These results impinge on the controversy in the literature surrounding the mode of action of MAP kinase inhibitors on TNF-alpha mRNA and suggest the use of combinations of MAP kinase inhibitors as an effective anti-inflammatory strategy.

  3. Co-Introduced Functional CCR2 Potentiates In Vivo Anti-Lung Cancer Functionality Mediated by T Cells Double Gene-Modified to Express WT1-Specific T-Cell Receptor

    PubMed Central

    Asai, Hiroaki; Fujiwara, Hiroshi; An, Jun; Ochi, Toshiki; Miyazaki, Yukihiro; Nagai, Kozo; Okamoto, Sachiko; Mineno, Junichi; Kuzushima, Kiyotaka; Shiku, Hiroshi; Inoue, Hirofumi; Yasukawa, Masaki

    2013-01-01

    Background and Purpose Although gene-modification of T cells to express tumor-related antigen-specific T-cell receptor (TCR) or chimeric antigen receptor (CAR) has clinically proved promise, there still remains room to improve the clinical efficacy of re-directed T-cell based antitumor adoptive therapy. In order to achieve more objective clinical responses using ex vivo-expanded tumor-responsive T cells, the infused T cells need to show adequate localized infiltration into the tumor. Methodology/Principal Findings Human lung cancer cells variously express a tumor antigen, Wilms' Tumor gene product 1 (WT1), and an inflammatory chemokine, CCL2. However, CCR2, the relevant receptor for CCL2, is rarely expressed on activated T-lymphocytes. A HLA-A2402+ human lung cancer cell line, LK79, which expresses high amounts of both CCL2 and WT1 mRNA, was employed as a target. Normal CD8+ T cells were retrovirally gene-modified to express both CCR2 and HLA-A*2402-restricted and WT1235–243 nonapeptide-specific TCR as an effector. Anti-tumor functionality mediated by these effector cells against LK79 cells was assessed both in vitro and in vivo. Finally the impact of CCL2 on WT1 epitope-responsive TCR signaling mediated by the effector cells was studied. Introduced CCR2 was functionally validated using gene-modified Jurkat cells and human CD3+ T cells both in vitro and in vivo. Double gene-modified CD3+ T cells successfully demonstrated both CCL2-tropic tumor trafficking and cytocidal reactivity against LK79 cells in vitro and in vivo. CCL2 augmented the WT1 epitope-responsive TCR signaling shown by relevant luciferase production in double gene-modified Jurkat/MA cells to express luciferase and WT1-specific TCR, and CCL2 also dose-dependently augmented WT1 epitope-responsive IFN-γ production and CD107a expression mediated by these double gene-modifiedCD3+ T cells. Conclusion/Significance Introduction of the CCL2/CCR2 axis successfully potentiated in vivo anti-lung cancer

  4. Development of a mouse-feline chimeric antibody against feline tumor necrosis factor-alpha.

    PubMed

    Doki, Tomoyoshi; Takano, Tomomi; Hohdatsu, Tsutomu

    2016-10-01

    Feline infectious peritonitis (FIP) is a fatal inflammatory disease caused by FIP virus infection. Feline tumor necrosis factor (fTNF)-alpha is closely involved in the aggravation of FIP pathology. We previously described the preparation of neutralizing mouse anti-fTNF-alpha monoclonal antibody (mAb 2-4) and clarified its role in the clinical condition of cats with FIP using in vitro systems. However, administration of mouse mAb 2-4 to cat may lead to a production of feline anti-mouse antibodies. In the present study, we prepared a mouse-feline chimeric mAb (chimeric mAb 2-4) by fusing the variable region of mouse mAb 2-4 to the constant region of feline antibody. The chimeric mAb 2-4 was confirmed to have fTNF-alpha neutralization activity. Purified mouse mAb 2-4 and chimeric mAb 2-4 were repeatedly administered to cats, and the changes in the ability to induce feline anti-mouse antibody response were investigated. In the serum of cats treated with mouse mAb 2-4, feline anti-mouse antibody production was induced, and the fTNF-alpha neutralization effect of mouse mAb 2-4 was reduced. In contrast, in cats treated with chimeric mAb 2-4, the feline anti-mouse antibody response was decreased compared to that of mouse mAb 2-4-treated cats.

  5. Plasma binding of an alpha-blocking agent, nicergoline--affinity for serum albumin and native and modified alpha 1-acid glycoprotein.

    PubMed

    Robert, L; Migne, J; Santonja, R; Zini, R; Schmid, K; Tillement, J P

    1983-06-01

    The binding of nicergoline, an alpha-blocking drug, by human plasma proteins was studied using gel filtration, polyacrylamide gel electrophoresis, and equilibrium dialysis techniques. 3H-labeled nicergoline added to plasma was eluted together with two major protein fractions, one containing mainly serum albumin, the other glycoproteins such as alpha 1-acid glycoprotein (alpha 1-AG). Equilibrium dialysis experiments with pure human serum albumin and alpha 1-AG as well as with its chemically modified forms, desialylated, carboxymethylated, and both desialylated and carboxymethylated alpha 1-AG gave the following results: nicergoline has about a 4-fold higher affinity for alpha 1-AG than for serum albumin. There are two binding sites per molecule on serum albumin and one on alpha 1-AG. The binding parameters of alpha 1-AG were not significantly modified by desialylation or carboxymethylation. Only desialylated and carboxymethylated alpha 1-AG showed a decreased binding for nicergoline, suggesting conformational modifications induced by these combined treatments. The fact that desialylated alpha 1-AG keeps its affinity for nicergoline suggests the possibility of a selective introduction of this drug in cells possessing the Ashwell-type specific receptor for desialylated alpha 1-AG, for instance hepatocytes. Increased serum alpha 1-AG concentration induced by inflammatory reactions will also modify the distribution of bound nicergoline between serum albumin and alpha 1-AG and as a consequence its half-life and cell distribution.

  6. Alpha-beta T cells provide protection against lethal encephalitis in the murine model of VEEV infection

    PubMed Central

    Paessler, Slobodan; Yun, Nadezhda E.; Judy, Barbara M.; Dziuba, Natallia; Zacks, Michele A.; Grund, Anna H.; Frolov, Ilya; Campbell, Gerald A.; Weaver, Scott C.; Estes, D. Mark

    2007-01-01

    We evaluated the safety and immunogenicity of a chimeric alphavirus vaccine candidate in mice with selective immunodeficiencies. This vaccine candidate was highly attenuated in mice with deficiencies in the B and T cell compartments, as well as in mice with deficient gamma-interferon responsiveness. However, the level of protection varied among the strains tested. Wild type mice were protected against lethal VEEV challenge. In contrast, alpha/beta (αβ) TCR-deficient mice developed lethal encephalitis following VEEV challenge, while mice deficient in gamma/delta ( γδ) T cells were protected. Surprisingly, the vaccine potency was diminished by 50% in animals lacking interferon-gamma receptor alpha chain (R1)-chain and a minority of vaccinated immunoglobulin heavy chain-deficient (μMT) mice survived challenge, which suggests that neutralizing antibody may not be absolutely required for protection. Prolonged replication of encephalitic VEEV in the brain of pre-immunized mice is not lethal and adoptive transfer experiments indicate that CD3+ T cells are required for protection. PMID:17610927

  7. Anti-Tribbles Homolog 2 Autoantibodies in Japanese Patients with Narcolepsy

    PubMed Central

    Toyoda, Hiromi; Tanaka, Susumu; Miyagawa, Taku; Honda, Yutaka; Tokunaga, Katsushi; Honda, Makoto

    2010-01-01

    Study Objectives: Narcolepsy is a sleep disorder characterized by excessive daytime sleepiness and cataplexy. The association with human leukocyte antigen (HLA)-DQB1*0602 and T-cell receptor alpha locus suggests that autoimmunity plays a role in narcolepsy. A recent study reported an increased prevalence of autoantibodies against Tribbles homolog 2 (TRIB2) in patients with narcolepsy. To replicate this finding, we examined anti-TRIB2 autoantibodies in Japanese patients with narcolepsy. Design: We examined anti-TRIB2 autoantibodies against a full-length [35S]-labeled TRIB2 antigen in Japanese patients with narcolepsy-cataplexy (n = 88), narcolepsy without cataplexy (n = 18), and idiopathic hypersomnia with long sleep time (n = 11). The results were compared to Japanese healthy controls (n = 87). Thirty-seven healthy control subjects were positive for HLA-DRB1*1501-DQB1*0602. We also examined autoantibodies against another Tribbles homolog, TRIB3, as an experimental control. Measurements and Results: Autoantibodies against TRIB2 were found in 26.1% of patients with narcolepsy-cataplexy, a significantly higher prevalence than the 2.3% in healthy controls. We found that anti-TRIB3 autoantibodies were rare in patients with narcolepsy and showed no association with anti-TRIB2 indices. No significant correlation was found between anti-TRIB2 positivity and clinical information. Conclusions: We confirmed the higher prevalence and specificity of anti-TRIB2 autoantibodies in Japanese patients with narcolepsy-cataplexy. This suggests a subgroup within narcolepsy-cataplexy might be affected by an anti-TRIB2 autoantibody-mediated autoimmune mechanism. Citation: Toyoda H; Tanaka S; Miyagawa T; Honda Y; Tokunaga K; Honda M. Anti-Tribbles homolog 2 autoantibodies in Japanese patients with narcolepsy. SLEEP 2010;33(7):875-878. PMID:20614847

  8. Anti-nociceptive effect of thalidomide on zymosan-induced experimental articular incapacitation.

    PubMed

    Vale, Mariana L; Cunha, Fernando Q; Brito, Gerly A C; Benevides, Verônica M; Ferreira, Sérgio H; Poole, Stephen; Ribeiro, Ronaldo A

    2006-05-01

    The anti-nociceptive effect of thalidomide on zymosan-induced articular knee joint incapacitation in rats was investigated. Thalidomide (5-45 mg/kg), given 30 min before but not 2 h after the intra-articular injection of zymosan, inhibited the nociceptive response in a dose-dependent manner. Furthermore, thalidomide pretreatment significantly reduced the concentration of tumor necrosis factor-alpha (TNF-alpha, -68.4%) in the exudate of zymosan-injected joints, but not those of interleukin-1beta, interleukin-6, CINC-1 or interleukin-10. The expression of TNF-alpha, determined by immunohistochemical staining, in synovial tissues obtained from articular joints injected with zymosan was also inhibited by thalidomide pretreatment. The anti-nociceptive effect of thalidomide was not reversed by the co-administration of an opioid receptor antagonist, naloxone, suggesting that endogenous opioids do not mediate the anti-nociceptive effect of thalidomide in this model. In conclusion, the anti-nociceptive activity of thalidomide in zymosan-induced articular incapacitation is associated with the inhibition of TNF-alpha by resident synovial cells.

  9. Evaluation of a new reagent for anti-cytomegalovirus and anti-Epstein-Barr virus immunoglobulin G.

    PubMed Central

    Gutierrez, J; Maroto, M D; Piédrola, G

    1994-01-01

    The Enzygnost alpha method was tested against the complement fixation test and anti-VCA immunofluorescence to determine the respective titers of anti-cytomegalovirus and anti-Epstein-Barr virus immunoglobulin G antibodies. For cytomegalovirus, the Enzygnost results showed 97.99% agreement with the readings obtained by the alternative method, with 100% sensitivity and 93.7% specificity. For Epstein-Barr virus, Enzygnost showed 97.71% agreement, 100% sensitivity, and 91.11% specificity. PMID:7814510

  10. A structural model of anti-anti-[sigma];#963; inhibition by a two-component receiver domain: the PhyR stress response regulator

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Herrou, Julien; Foreman, Robert; Fiebig, Aretha

    2012-03-30

    PhyR is a hybrid stress regulator conserved in {alpha}-proteobacteria that contains an N-terminal {sigma}-like (SL) domain and a C-terminal receiver domain. Phosphorylation of the receiver domain is known to promote binding of the SL domain to an anti-{sigma} factor. PhyR thus functions as an anti-anti-{sigma} factor in its phosphorylated state. We present genetic evidence that Caulobacter crescentus PhyR is a phosphorylation-dependent stress regulator that functions in the same pathway as {sigma}{sup T} and its anti-{sigma} factor, NepR. Additionally, we report the X-ray crystal structure of PhyR at 1.25 {angstrom} resolution, which provides insight into the mechanism of anti-anti-{sigma} regulation. Directmore » intramolecular contact between the PhyR receiver and SL domains spans regions {sigma}{sub 2} and {sigma}{sub 4}, likely serving to stabilize the SL domain in a closed conformation. The molecular surface of the receiver domain contacting the SL domain is the structural equivalent of {alpha}4-{beta}5-{alpha}5, which is known to undergo dynamic conformational change upon phosphorylation in a diverse range of receiver proteins. We propose a structural model of PhyR regulation in which receiver phosphorylation destabilizes the intramolecular interaction between SL and receiver domains, thereby permitting regions {sigma}{sub 2} and {sigma}{sub 4} in the SL domain to open about a flexible connector loop and bind anti-{sigma} factor.« less

  11. Characterization of synthesis and storage of TGF-alpha in rat parotid acinar and intercalated duct cells.

    PubMed

    Login, G R; Yang, J; Bryan, K P; Digenis, E C; McBride, J; Elovic, A; Quissell, D O; Dvorak, A M; Wong, D T

    1997-03-01

    Although the expression and biological role of transforming growth factor-alpha (TGF-alpha) have been explored in a variety of normal cells in mammalian species, little is known about the storage of TGF-alpha in secretory cells of exocrine organs. Parotid glands from four rats were homogenized for RNA isolation followed by reverse transcription-polymerase chain reaction to determine the presence of TGF-alpha message. In situ hybridization using a hamster-specific TGF-alpha riboprobe was done on paraffin sections. Parotid gland and isolated acinar cells were processed for transmission electron microscopy (TEM) and postembedding immunogold labeled for TGF-alpha. Gold particles were counted on approximately 200 granules in 10 acinar cells and in 10 intercalated duct cells. Labeling density was calculated as the number of gold particles per square micrometer +/- SD. Statistical significance was calculated using one-way analysis of variance. Using multiple technologies, we have established that rat parotid acinar and intercalated duct cells synthesize TGF-alpha and store the precursor form of this cytokine in their secretory granules.

  12. Lipid Lowering Effect of Antioxidant Alpha-Lipoic Acid in Experimental Atherosclerosis

    PubMed Central

    Amom, Zulkhairi; Zakaria, Zaiton; Mohamed, Jamaluddin; Azlan, Azrina; Bahari, Hasnah; Taufik Hidayat Baharuldin, Mohd; Aris Moklas, Mohd; Osman, Khairul; Asmawi, Zanariyah; Kamal Nik Hassan, Mohd

    2008-01-01

    Accumulating data demonstrated that hypercholesterolemia and oxidative stress play an important role in the development of atherosclerosis. In the present study, a protective activity of alpha-lipoic acid; a metabolic antioxidant in hypercholesterolemic-induced animals was investigated. Eighteen adult male New Zealand White (NZW) rabbit were segregated into three groups labelled as group N, HCD and ALA (n = 6). Group N (normal control) was fed with normal chow, the rest (HCD and ALA) were fed with 100 g/head/day of 1% cholesterol rich diet to induce hypercholesterolemia. Four point two mg/body weight of alpha lipoic acid was concomintantly supplemented to the ALA group. Drinking water was given ad-libitum. The study was designed for 10 weeks. Blood sampling was taken from the ear lobe vein at the beginning, week 5 and week 10. Plasma was prepared for lipid profile estimation and microsomal lipid peroxidation index indicated with malondialdehyde (MDA) formation. At the end of the experiment, the animals were sacrificed and the aorta were excised for intimal lesion analysis. The plasma total cholesterol (TC) and low density lipoprotein (LDL) levels were found to be significantly low in ALA group compared to that of the HCD group (p<0.05). Similarly, low level of MDA (p<0.05) in ALA group was observed compared to that of the HCD group showing a significant reduction of lipid peroxidation activity. Histomorphometric intimal lesion analysis of the aorta showing less of atheromatous plaque formation in alpha lipoic acid supplemented group (p<0.05) compared to HCD group. These findings suggested that alpha lipoic acid posses a dual lipid lowering and anti-atherosclerotic properties indicated with low plasma TC and LDL levels and reduction of athero-lesion formation in hypercholesterolemic-induced rabbits. PMID:18818758

  13. Deficiency of a alpha-1-antitrypsin influences systemic iron homeostasis

    EPA Science Inventory

    Abstract Background: There is evidence that proteases and anti-proteases participate in the iron homeostasis of cells and living systems. We tested the postulate that alpha-1 antitrypsin (A1AT) polymorphism and the consequent deficiency of this anti-protease in humans are asso...

  14. Accelerated Loss of TCR Repertoire Diversity in Common Variable Immunodeficiency

    PubMed Central

    Wong, Gabriel K.; Millar, David; Penny, Sarah; Heather, James M.; Mistry, Punam; Buettner, Nico; Bryon, Jane; Huissoon, Aarnoud P.

    2016-01-01

    Although common variable immunodeficiency (CVID) has long been considered as a group of primary Ab deficiencies, growing experimental data now suggest a global disruption of the entire adaptive immune response in a segment of patients. Oligoclonality of the TCR repertoire was previously demonstrated; however, the manner in which it relates to other B cell and T cell findings reported in CVID remains unclear. Using a combination approach of high-throughput TCRβ sequencing and multiparametric flow cytometry, we compared the TCR repertoire diversity between various subgroups of CVID patients according to their B cell immunophenotypes. Our data suggest that the reduction in repertoire diversity is predominantly restricted to those patients with severely reduced class-switched memory B cells and an elevated level of CD21lo B cells (Freiburg 1a), and may be driven by a reduced number of naive T cells unmasking underlying memory clonality. Moreover, our data indicate that this loss in repertoire diversity progresses with advancing age far exceeding the expected physiological rate. Radiological evidence supports the loss in thymic volume, correlating with the decrease in repertoire diversity. Evidence now suggests that primary thymic failure along with other well-described B cell abnormalities play an important role in the pathophysiology in Freiburg group 1a patients. Clinically, our findings emphasize the integration of combined B and T cell testing to identify those patients at the greatest risk for infection. Future work should focus on investigating the link between thymic failure and the severe reduction in class-switched memory B cells, while gathering longitudinal laboratory data to examine the progressive nature of the disease. PMID:27481850

  15. A hormone pulse induces transient changes in the subcellular distribution and leads to a lysosomal accumulation of the estradiol receptor alpha in target tissues.

    PubMed

    Qualmann, B; Kessels, M M; Thole, H H; Sierralta, W D

    2000-06-01

    An intrauterine pulse-stimulation with estradiol induced changes in the subcellular localization of estrogen receptor alpha in porcine endometrium, as detected with F(ab') fragments of various anti-receptor antibodies covalently linked to nanogold. The low-sterically hindered immunoreagents--recognizing different epitopes within the hormone binding domain--allowed for an efficient immunolabeling of estradiol receptor alpha, detecting it both in the cytoplasm and the nucleus of nonstimulated epithelium cells. In the cytoplasm, the receptor often seemed to be associated with actin filaments and the endoplasmatic reticulum. After the stimulation with estradiol, a predominantly nuclear localization and a labeling of nucleoli was observed. Our immunoelectron microscopy study demonstrates a localization of the receptor in cytoplasmic organelles that increased after the hormone pulse. These organelles exhibited the morphological properties of lysosomes and relocated to the perinuclear area. In analogous cytoplasmic organelles, the presence of cathepsin D was detected via indirect immunogold labeling, justifying their classification as lysosomes. Quantitative examinations revealed that not only the number of lysosomes in the proximity of the nucleus but also their immunostaining for estradiol receptor alpha increased significantly after the hormone pulse. Thus, estradiol induces both the rapid shift of receptor into the nucleus, a slower perinuclear accumulation of lysosomes and an increase of lysosomal ERalpha-immunoreactivity. These results suggest a role for lysosomes in the degradation of receptor shuttling out of the nucleus. This could serve as termination of the estradiol receptor alpha-dependent activation of target cells. This hypothesis is strengthened by the fact that the receptor content in uterine tissue declined drastically few hours after the hormone pulse.

  16. Anti-Dementia Drugs, Gait Performance and Mental Imagery of Gait: A Non-Randomized Open-Label Trial.

    PubMed

    Beauchet, Olivier; Barden, John; Liu-Ambrose, Teresa; Chester, Victoria L; Annweiler, Cedric; Szturm, Tony; Grenier, Sébastien; Léonard, Guillaume; Bherer, Louis; Allali, Gilles

    2016-09-01

    Few studies have examined the effect of anti-dementia drugs (i.e., acetylcholinesterase inhibitors and N-methyl-D-aspartate receptor antagonists) on gait performance. Past studies have focused on the stride time (i.e., gait cycle duration) but not on the mental imagery of gait. To compare mental imagery of gait and spatiotemporal gait parameters in patients with dementia [i.e., Alzheimer's disease (AD) and non-AD] before and after the use of anti-dementia drugs (i.e., acetylcholinesterase inhibitors and memantine) and in controls (i.e., patients with dementia who did not take anti-dementia drugs). A total of 112 patients (mean age 82.5 ± 4.2 years, 68.8 % female) with mild-to-moderate AD and non-AD dementia were included in this non-randomized open-label trial (n = 56 in the Intervention group, and n = 56 in the Control group matched for age, sex, and stage and type of dementia) nested in a cohort study (mean follow-up 238.5 ± 79.8 days). Mental imagery of gait was assessed with the actual and imagined Timed Up and Go tests (aTUG and iTUG) and the difference between aTUG and iTUG (i.e., delta-TUG). Spatiotemporal gait parameters were measured with the GAITRite(®) system during normal walking. Participants in the Intervention group had a longer iTUG time (p < 0.001) and a lower delta-TUG value (p = 0.001) at the follow-up compared with those in the Control group. There was a significant increase in iTUG (p = 0.001) and decrease in delta-TUG (p < 0.001) from baseline to the follow-up only in the Intervention group. Multiple linear regression showed that the use of anti-dementia drugs was associated with a longer iTUG time and a lower delta-TUG value (best performance, p < 0.002). Our findings showed an improvement in mental imagery of gait with the use of anti-dementia drugs, but no changes in actual gait performance. NCT01315704.

  17. Activated PLC-γ1 is Catalytically Induced at LAT but Activated PLC-γ1 is Localized at both LAT- and TCR-Containing Complexes

    PubMed Central

    Cruz-Orcutt, Noemi; Vacaflores, Aldo; Connolly, Sean F.; Bunnell, Stephen C.; Houtman, Jon C.D.

    2014-01-01

    Phospholipase C-γ1 (PLC-γ1) is a key regulator of T cell receptor (TCR)-induced signaling. Activation of the TCR enhances PLC-γ1 enzymatic function, resulting in calcium influx and the activation of PKC family members and RasGRP. The current model is that phosphorylation of LAT tyrosine 132 facilitates the recruitment of PLC-γ1, leading to its activation and function at the LAT complex. In this study, we examined the phosphorylation kinetics of LAT and PLC-γ1 and the cellular localization of activated PLC-γ1. We observed that commencement of the phosphorylation of LAT tyrosine 132 and PLC-γ1 tyrosine 783 occurred simultaneously, supporting the current model. However, once begun, PLC-γ1 activation occurred more rapidly than LAT tyrosine 132. The association of LAT and PLC-γ1 was more transient than the interaction of LAT and Grb2 and a pool of activated PLC-γ1 translocated away from LAT to cellular structures containing the TCR. These studies demonstrate that LAT and PLC-γ1 form transient interactions that catalyze the activation of PLC-γ1, but that activated PLC-γ1 resides in both LAT and TCR clusters. Together, this work highlights that our current model is incomplete and the activation and function of PLC-γ1 in T cells is highly complex. PMID:24412752

  18. Mapping of the acetylcholine binding site of the nicotinic acetylcholine receptor: ( sup 3 H)nicotine as an agonist photoaffinity label

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Middleton, R.E.; Cohen, J.B.

    1991-07-16

    The agonist ({sup 3}H)nicotine was used as a photoaffinity label for the acetylcholine binding sties on the Torpedo nicotinic acetylcholine receptor (AChR). ({sup 3}H)Nicotine binds at equilibrium with K{sub eq} = 0.6 {mu}M to the agonist binding sites. Irradiation with 254-nm light of AChR-rich membranes equilibrated with ({sup 3}H)nicotine resulted in covalent incorporation into the {alpha}- and {gamma}-subunits, which was inhibited by agonists and competitive antagonists but not by noncompetitive antagonists. Inhibition of labeling by d-tubocurarine demonstrated that the {alpha}-subunit was labeled via both agonist sites but the {gamma}-subunit was labeled only via the site that binds d-tubocurarine with highmore » affinity. Chymotryptic digestion of the {alpha}-subunit confirmed that Try-198 was the principal amino acid labeled by ({sup 3}H)nicotine. This confirmation required a novel radiosequencing strategy employing o-phthalaldehyde ({sup 3}H)Nicotine, which is the first photoaffinity agonist used, labels primarily Tyr-198 in contrast to competitive antagonist affinity labels, which label primarily Tyr-190 and Cys-192/Cys-193.« less

  19. Peroxisome proliferator-activated receptor alpha (PPARalpha) agonists down-regulate alpha2-macroglobulin expression by a PPARalpha-dependent mechanism.

    EPA Science Inventory

    Peroxisome proliferator-activated receptor alpha (PPARα) regulates transcription of genes involved both in lipid and glucose metabolism as well as inflammation. Fibrates are PPARα ligands used to normalize lipid and glucose parameters and exert anti-inflammatory effects. Fibrates...

  20. Redefining face contour with a novel anti-aging cosmetic product: an open-label, prospective clinical study

    PubMed Central

    Garre, Aurora; Martinez-Masana, Gemma; Piquero-Casals, Jaime; Granger, Corinne

    2017-01-01

    Background Skin aging is accelerated by multiple extrinsic factors: ultraviolet radiation, smoking and pollution increase oxidative activity, damaging cellular and extracellular components such as DNA, proteins, and lipids. With age, collagen and hyaluronic acid levels decline, resulting in loss of elasticity and moisture of the skin. Over time this damage leads to characteristic signs that make the skin look older: altered facial contour, sagging skin, wrinkles, and an uneven complexion. This study evaluated the anti-aging effects of a new facial cream formulated with carnosine, Alteromonas ferment extract, crosspolymer hyaluronic acid, and a tripeptide. Methods An open-label intra-individual study to assess the anti-aging efficacy of the investigational product in 33 women aged 45 to 65 years. The product was applied twice daily for 56 days. Facial contour and skin deformation, elasticity, hydration, and complexion were measured with specialized equipment at baseline and days 28 and 56. Additionally, subjects completed questionnaires at days 28 and 56 on the perceived efficacy and cosmetic characteristics of the product. Results After 56 days of use of the investigational product, a redefining effect was observed, with a significant decrease in sagging jawline (7%). Skin was significantly more hydrated (12%), firmer (29%), and more elastic (20%) (P<0.001 for all). On complexion assessment, skin texture (a measure of skin smoothness) and spots (brown and red skin lesions) also improved significantly (12% and 6% decrease, respectively). In the subjective self-evaluation, the majority of subjects reported that the skin was visibly tightened and more elastic, flexible, and moisturized (91%, 88%, 91%, and 90%, respectively). The product was well tolerated with no adverse events reported during the study. Conclusion This new cosmetic product demonstrated anti-aging effects after 56 days of use, most notably a redefined facial contour and improved complexion. It is a

  1. Redefining face contour with a novel anti-aging cosmetic product: an open-label, prospective clinical study.

    PubMed

    Garre, Aurora; Martinez-Masana, Gemma; Piquero-Casals, Jaime; Granger, Corinne

    2017-01-01

    Skin aging is accelerated by multiple extrinsic factors: ultraviolet radiation, smoking and pollution increase oxidative activity, damaging cellular and extracellular components such as DNA, proteins, and lipids. With age, collagen and hyaluronic acid levels decline, resulting in loss of elasticity and moisture of the skin. Over time this damage leads to characteristic signs that make the skin look older: altered facial contour, sagging skin, wrinkles, and an uneven complexion. This study evaluated the anti-aging effects of a new facial cream formulated with carnosine, Alteromonas ferment extract, crosspolymer hyaluronic acid, and a tripeptide. An open-label intra-individual study to assess the anti-aging efficacy of the investigational product in 33 women aged 45 to 65 years. The product was applied twice daily for 56 days. Facial contour and skin deformation, elasticity, hydration, and complexion were measured with specialized equipment at baseline and days 28 and 56. Additionally, subjects completed questionnaires at days 28 and 56 on the perceived efficacy and cosmetic characteristics of the product. After 56 days of use of the investigational product, a redefining effect was observed, with a significant decrease in sagging jawline (7%). Skin was significantly more hydrated (12%), firmer (29%), and more elastic (20%) ( P <0.001 for all). On complexion assessment, skin texture (a measure of skin smoothness) and spots (brown and red skin lesions) also improved significantly (12% and 6% decrease, respectively). In the subjective self-evaluation, the majority of subjects reported that the skin was visibly tightened and more elastic, flexible, and moisturized (91%, 88%, 91%, and 90%, respectively). The product was well tolerated with no adverse events reported during the study. This new cosmetic product demonstrated anti-aging effects after 56 days of use, most notably a redefined facial contour and improved complexion. It is a safe and effective anti-aging product.

  2. Prostaglandin E and F2 alpha receptors in human myometrium during the menstrual cycle and in pregnancy and labor

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Giannopoulos, G.; Jackson, K.; Kredentser, J.

    The binding of prostaglandins E1 and F2 alpha has been studied in the human myometrium and cervix during the menstrual cycle and in the myometrium of pregnant patients at term before and during labor. Tritium-labeled prostaglandin E1 and F2 alpha binding was saturable and reversible. Scatchard analysis of tritium-labeled prostaglandin E1 binding was linear, which suggests a single class of high-affinity binding sites with an estimated apparent equilibrium dissociation constant of 2.5 to 5.4 nmol/L and inhibitor affinities of 0.9, 273, 273, and 217 nmol/L for prostaglandins E2, A1, B1, and F2 alpha, respectively. Scatchard analysis of tritium-labeled prostaglandin F2more » alpha, binding was also linear, but the affinity of these binding sites was much lower, with an average dissociation constant of 50 nmol/L and inhibitor affinities of 1.6, 2.2, and 11.2 nmol/L for prostaglandins E1, E2, and A1, respectively. In nonpregnant patients, the concentrations and affinities of tritium-labeled prostaglandin E1 binding sites were similar in the myometrium during the proliferative and secretory phases of the menstrual cycle, but the concentration of these sites was much lower in the cervix. The concentration of the tritium-labeled prostaglandin E1 binding sites was significantly lower in the myometrium of pregnant patients at term than in the myometrium of nonpregnant patients. The concentrations and affinities of tritium-labeled prostaglandin E1 binding sites were not significantly different in the upper and lower myometrium of pregnant patients at term or in the myometrium of such patients before and during labor. The concentrations of the tritium-labeled prostaglandin F2 alpha binding sites during the menstrual cycle and in pregnancy at term were similar to those of tritium-labeled prostaglandin E1 binding sites.« less

  3. Gold nanoparticles and polyethylene glycols functionalized conducting polyaniline nanowires for ultrasensitive and low fouling immunosensing of alpha-fetoprotein.

    PubMed

    Hui, Ni; Sun, Xiaotian; Song, Zhiling; Niu, Shuyan; Luo, Xiliang

    2016-12-15

    An ultrasensitive biosensor for alpha-fetoprotein was developed based on electrochemically synthesized polyaniline (PANI) nanowires, which were functionalized with gold nanoparticles (AuNPs) and polyethylene glycols (PEG). The prepared PEG/AuNPs/PANI composite, combining the electrical conductivity of the AuNPs/PANI with the robust antifouling ability of PEG, offered an ideal substrate for the development of low fouling electrochemical biosensors. Alpha-fetoprotein (AFP), a well-known hepatocellular carcinoma biomarker, was used as a model analyte, and its antibody was immobilized on the PEG/AuNPs/PANI for the construction of the AFP immunosensor. Using the redox current of PANI as the sensing signal, in addition to the good biocompatibility of PEG/AuNPs and the anti-biofouling property of PEG, the developed immunosensor showed improved biosensing performances, such as wide linear range and ultralow detection limit (0.007pgmL(-1)). More importantly, it is label-free, reagentless and low fouling, making it capable of assaying AFP in real serum samples without suffering from significant interference or biofouling. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. A BioDesign Approach to Obtain High Yields of Biosimilars by Anti-apoptotic Cell Engineering: a Case Study to Increase the Production Yield of Anti-TNF Alpha Producing Recombinant CHO Cells.

    PubMed

    Gulce Iz, Sultan; Inevi, Muge Anil; Metiner, Pelin Saglam; Tamis, Duygu Ayyildiz; Kisbet, Nazli

    2018-01-01

    Recent developments in medical biotechnology have facilitated to enhance the production of monoclonal antibodies (mAbs) and recombinant proteins in mammalian cells. Human mAbs for clinical applications have focused on three areas, particularly cancer, immunological disorders, and infectious diseases. Tumor necrosis factor alpha (TNF-α), which has both proinflammatory and immunoregulatory functions, is an important target in biopharmaceutical industry. In this study, a humanized anti-TNF-α mAb producing stable CHO cell line which produces a biosimilar of Humira (adalimumab) was used. Adalimumab is a fully human anti-TNF mAb among the top-selling mAb products in recent years as a biosimilar. Products from mammalian cell bioprocesses are a derivative of cell viability and metabolism, which is mainly disrupted by cell death in bioreactors. Thus, different strategies are used to increase the product yield. Suppression of apoptosis, also called anti-apoptotic cell engineering, is the most remarkable strategy to enhance lifetime of cells for a longer production period. In fact, using anti-apoptotic cell engineering as a BioDesign approach was inspired by nature; nature gives prolonged life span to some cells like stem cells, tumor cells, and memory B and T cells, and researchers have been using this strategy for different purposes. In this study, as a biomimicry approach, anti-apoptotic cell engineering was used to increase the anti-TNF-α mAb production from the humanized anti-TNF-α mAb producing stable CHO cell line by Bcl-xL anti-apoptotic protein. It was shown that transient transfection of CHO cells by the Bcl-xL anti-apoptotic protein expressing plasmid prolonged the cell survival rate and protected cells from apoptosis. The transient expression of Bcl-xL using CHO cells enhanced the anti-TNF-α production. The production of anti-TNF-α in CHO cells was increased up to 215 mg/L with an increase of 160% after cells were transfected with Bcl-xL expressing plasmid

  5. Immature blood vessels in rheumatoid synovium are selectively depleted in response to anti-TNF therapy.

    PubMed

    Izquierdo, Elena; Cañete, Juan D; Celis, Raquel; Santiago, Begoña; Usategui, Alicia; Sanmartí, Raimon; Del Rey, Manuel J; Pablos, José L

    2009-12-02

    Angiogenesis is considered an important factor in the pathogenesis of Rheumatoid Arthritis (RA) where it has been proposed as a therapeutic target. In other settings, active angiogenesis is characterized by pathologic, immature vessels that lack periendothelial cells. We searched for the presence of immature vessels in RA synovium and analyzed the dynamics of synovial vasculature along the course of the disease, particularly after therapeutic response to TNF antagonists. Synovial arthroscopic biopsies from RA, osteoarthritis (OA) and normal controls were analyzed by double labeling of endothelium and pericytes/smooth muscle mural cells to identify and quantify mature/immature blood vessels. To analyze clinicopathological correlations, a cross-sectional study on 82 synovial biopsies from RA patients with variable disease duration and severity was performed. A longitudinal analysis was performed in 25 patients with active disease rebiopsied after anti-TNF-alpha therapy. We found that most RA synovial tissues contained a significant fraction of immature blood vessels lacking periendothelial coverage, whereas they were rare in OA, and inexistent in normal synovial tissues. Immature vessels were observed from the earliest phases of the disease but their presence or density was significantly increased in patients with longer disease duration, higher activity and severity, and stronger inflammatory cell infiltration. In patients that responded to anti-TNF-alpha therapy, immature vessels were selectively depleted. The mature vasculature was similarly expanded in early or late disease and unchanged by therapy. RA synovium contains a significant fraction of neoangiogenic, immature blood vessels. Progression of the disease increases the presence and density of immature but not mature vessels and only immature vessels are depleted in response to anti-TNFalpha therapy. The different dynamics of the mature and immature vascular fractions has important implications for the

  6. Effects of thalidomide, cytochrome P-450 and TNF-alpha on angiogenesis in a three-dimensional collagen gel-culture.

    PubMed

    Fujita, Keiko; Asami, Yoshiko; Murata, Eiko; Akita, Masumi; Kaneko, Katsuji

    2002-10-01

    The anti-angiogenic effects of thalidomide were examined in mouse aortae grown in a three-dimensional collagen gel-culture. In our in vitro model, (+/-)-thalidomide and (-)-thalidomide exhibited no anti-angiogenic effects. On the other hand, when the culture was treated with thalidomide plus cytochrome P-450, both types of thalidomides significantly inhibited angiogenesis. Co-administration of 100 microg/ml thalidomide plus 200 microg/ml cytochrome P-450 inhibited angiogenesis more strongly than thalidomide plus cytochrome P-450 at other concentrations (10 microg/ml + 200 microg/ml and 100 microg/ml + 20 microg/ml). To study the relation between the anti-angiogenic effect and TNF-alpha, we also evaluated the concentration of TNF-alpha in the culture medium. We found that the concentration of TNF-alpha was correlated to the strength of the anti-angiogenic effect. The inhibition of angiogenesis by thalidomide and cytochrome P-450 takes place through a suppression of TNF-alpha and involves the metabolism of the thalidomide.

  7. Epithelial V-Like Antigen Mediates Efficacy of Anti-Alpha4 Integrin Treatment in a Mouse Model of Multiple Sclerosis

    PubMed Central

    Wright, Erik; Rahgozar, Kusha; Hallworth, Nicholas; Lanker, Stefan; Carrithers, Michael D.

    2013-01-01

    Natalizumab inhibits the transmigration of activated T lymphocytes into the brain and is highly efficacious in multiple sclerosis (MS). However, from a pharmacogenomic perspective, its efficacy and safety in specific patients remain unclear. Here our goal was to analyze the effects of epithelial V-like antigen (EVA) on anti-alpha4 integrin (VLA4) efficacy in a mouse model of MS, experimental autoimmune encephalomyelitis (EAE). EVA has been previously characterized in human CD4 T lymphocytes, mouse thymic development, and choroid plexus epithelial cells. Further analysis here demonstrated expression in B lymphocytes and an increase in EVA+ lymphocytes following immunization. Following active induction of EAE using the MOG35–55 active immunization model, EVA deficient mice developed more severe EAE and white matter tissue injury as compared to wild type controls. This severe EAE phenotype did not respond to anti-VLA4 treatment. In both the control antibody and anti-VLA4 conditions, these mice demonstrated persistent CNS invasion of mature B lymphocyte (CD19+, CD21+, sIgG+), increased serum autoantibody levels, and extensive complement and IgG deposition within lesions containing CD5+IgG+ cells. Wild type mice treated with control antibody also demonstrated the presence of CD19+, CD21+, sIgG+ cells within the CNS during peak EAE disease severity and detectable serum autoantibody. In contrast, wild type mice treated with anti-VLA4 demonstrated reduced serum autoantibody levels as compared to wild type controls and EVA-knockout mice. As expected, anti-VLA4 treatment in wild type mice reduced the total numbers of all CNS mononuclear cells and markedly decreased CD4 T lymphocyte invasion. Treatment also reduced the frequency of CD19+, CD21+, sIgG+ cells in the CNS. These results suggest that anti-VLA4 treatment may reduce B lymphocyte associated autoimmunity in some individuals and that EVA expression is necessary for an optimal therapeutic response. We postulate that

  8. WC1 is a hybrid γδ TCR coreceptor and pattern recognition receptor for pathogenic bacteria.

    PubMed

    Hsu, Haoting; Chen, Chuang; Nenninger, Ariel; Holz, Lauren; Baldwin, Cynthia L; Telfer, Janice C

    2015-03-01

    WC1 proteins are uniquely expressed on γδ T cells and belong to the scavenger receptor cysteine-rich (SRCR) superfamily. While present in variable, and sometimes high, numbers in the genomes of mammals and birds, in cattle there are 13 distinct genes (WC1-1 to WC1-13). All bovine WC1 proteins can serve as coreceptors for the TCR in a tyrosine phosphorylation dependent manner, and some are required for the γδ T cell response to Leptospira. We hypothesized that individual WC1 receptors encode Ag specificity via coligation of bacteria with the γδ TCR. SRCR domain binding was directly correlated with γδ T cell response, as WC1-3 SRCR domains from Leptospira-responsive cells, but not WC1-4 SRCR domains from Leptospira-nonresponsive cells, bound to multiple serovars of two Leptospira species, L. borgpetersenii, and L. interrogans. Three to five of eleven WC1-3 SRCR domains, but none of the eleven WC1-4 SRCR domains, interacted with Leptospira spp. and Borrelia burgdorferi, but not with Escherichia coli or Staphylococcus aureus. Mutational analysis indicated that the active site for bacterial binding in one of the SRCR domains is composed of amino acids in three discontinuous regions. Recombinant WC1 SRCR domains with the ability to bind leptospires inhibited Leptospira growth. Our data suggest that WC1 gene arrays play a multifaceted role in the γδ T cell response to bacteria, including acting as hybrid pattern recognition receptors and TCR coreceptors, and they may function as antimicrobials. Copyright © 2015 by The American Association of Immunologists, Inc.

  9. TNF-alpha antagonist induced lupus on three different agents.

    PubMed

    Mudduluru, Bindu Madhavi; Shah, Shalin; Shamah, Steven; Swaminath, Arun

    2017-03-01

    Tumor necrosis factor alpha (TNF alpha) antagonists are biologic agents used in the management of inflammatory conditions such as rheumatoid arthritis, seronegative spondyloarthropathies and inflammatory bowel disease. These agents have been recently shown to cause a syndrome called anti-TNF induced lupus (ATIL), a rare condition which has similar clinical manifestations to idiopathic systemic lupus erythematosus (SLE). Given that extra-intestinal manifestations of inflammatory bowel disease include arthritis, it can be difficult to separate arthritis due to underlying disease from drug-induced arthritis. We present a case of a 28-year-old female with Crohn's disease, who developed disabling arthritis as a clinical manifestation of ATIL following treatment with three anti-TNF agents, namely infliximab, adalimumab and certolizumab.

  10. Photoaffinity labeling of the Torpedo californica nicotinic acetylcholine receptor with an aryl azide derivative of phosphatidylserine

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Blanton, M.P.; Wang, H.H.

    1990-02-06

    A photoactivatable analogue of phosphatidylserine, {sup 125}I-labeled 4-azidosalicylic acid-phosphatidylserine ({sup 125}I ASA-PS), was used to label both native acetylcholine receptor (AchR)-rich membranes from Torpedo californica and AchR membranes affinity purified from Torpedo reconstituted into asolectin vesicles. The radioiodinated arylazido group attaches directly to the phospholipid head group and thus probes for regions of the AchR structure in contact with the negatively charged head group of phosphatidylserine. All four subunits of the AchR incorporated the label, with the {alpha} subunit incorporating approximately twice as much as each of the other subunits on a per mole basis. The regions of the AchRmore » {alpha} subunit that incorporated {sup 125}I ASA-PS were mapped by Staphylococcus aureus V8 protease digestion. The majority of label incorporated into fragments representing a more complete digestion of the {alpha} subunit was localized to 11.7- and 10.1-kDa V8 cleavage fragments, both beginning at Asn-339 and of sufficient length to contain the hydrophobic region M4. An 18.7-kDa fragment beginning at Ser-173 and of sufficient length to contain the hydrophobic regions M1, M2, and M3 was also significantly labeled. In contrast, V8 cleavage fragments representing roughly a third of the amino-terminal portion of the {alpha} subunit incorporated little or no detectable amount of probe.« less

  11. Natura-Alpha Targets Forkhead Box M1 and Inhibits Androgen-Dependent and -Independent Prostate Cancer Growth and Invasion

    PubMed Central

    Li, Yirong; Ligr, Martin; McCarron, James P; Daniels, Garrett; Zhang, David; Zhao, Xin; Ye, Fei; Wang, Jinhua; Liu, Xiaomei; Osman, Iman; Mencher, Simon K; Lepor, Hebert; Wang, Long G; Lee, Peng

    2011-01-01

    Purpose The development of new effective therapeutic agents with minimal side effects for prostate cancer treatment is much needed. Indirubin, an active molecule identified in the traditional Chinese herbal medicine – Qing Dai (Indigo Naturalis), has been used to treat leukemia for decades. However, the anti-cancer properties of Natura-alpha, an indirubin derivative, are not well studied in solid tumors, particularly in prostate cancer. Experimental Design Human prostate cancer cell lines were treated with or without Natura-alpha followed by cell growth and invasion assays measured. The anti-tumor effects of Natura-alpha were examined in nude mice tumor xenograft models, as well as in a patient with advanced hormone refractory metastatic prostate cancer. Signal network proteins targeted by Natura-alpha were analyzed using Proteomic Pathway Array Analysis (PPAA) on xenografts. Results Natura-alpha inhibited the growth of both androgen-dependent (LNCaP), and androgen-independent (LNCaP-AI, PC-3, and DU145) prostate cancer cells with IC50 between 4 to 10 Μm, also inhibits invasion of androgen-independent prostate cancer cells. Its anti-tumor effects were further evident in vivo tumor reduction in androgen-dependent and -independent nude mice tumor xenograft models as well as reduced tumor volume in the patient with hormone refractory metastatic prostate cancer. PPAA revealed that anti-proliferative and anti-invasive activities of Natura-alpha on prostate cancer might primarily be through its down-regulation of Forkhead box M1 (FOXM1) protein. Forced over-expression of FOXM1 largely reversed the inhibition by Natura-alpha. Conclusion Natura-alpha could serve as a novel and effective therapeutic agent for treatment of both hormone sensitive and hormone refractory prostate cancer with minimal side effects. PMID:21606178

  12. [Anti-TNF-alpha therapy in ulcerative colitis].

    PubMed

    Lakatos, Péter László; Lakatos, László

    2008-05-18

    The most important factors that determine treatment strategy in ulcerative colitis (UC) are disease extent and severity. Orally-topically administered 5-aminosalicylates (5-ASA) remain the treatment of choice in mild-to-moderate UC. In contrast, the treatment of refractory (to steroids, azathioprine or 5-ASA) and fulminant cases is still demanding. New evidence supports a role for infliximab induction and/or maintenance therapy in these subgroup of patients leading to increased remission and decreased colectomy rates. The aim of this paper is to review the rationale for the use of TNF-alpha inhibitors in the treatment of UC.

  13. An endogenous RNA transcript antisense to CNG(alpha)1 cation channel mRNA.

    PubMed

    Cheng, Chin-Hung; Yew, David Tai-Wai; Kwan, Hiu-Yee; Zhou, Qing; Huang, Yu; Liu, Yong; Chan, Wing-Yee; Yao, Xiaoqiang

    2002-10-01

    CNG channels are cyclic nucleotide-gated Ca(2+)-permeable channels that are suggested to be involved in the activity-dependent alterations of synaptic strength that are thought to underlie information storage in the CNS. In this study, we isolated an endogenous RNA transcript antisense to CNG(alpha)1 mRNA. This transcript was capable of down-regulating the expression of sense CNG(alpha)1 in the Xenopus oocyte expression system. RT-PCR, Northern blot, and in situ hybridization analyses showed that the transcript was coexpressed with CNG(alpha)1 mRNA in many regions of human brain, notably in those regions that were involved in long-term potentiation and long-term depression, such as hippocampal CA1 and CA3, dentate gyrus, and cerebellar Purkinje layer. Comparison of expression patterns between adult and fetal cerebral cortex revealed that there were concurrent developmental changes in the expression levels of anti-CNG1 and CNG(alpha)1. Treatment of human glioma cell T98 with thyroid hormone T(3) caused a significant increase in anti-CNG1 expression and a parallel decrease in sense CNG(alpha)1 expression. These data suggest that the suppression of CNG(alpha)1 expression by anti-CNG1 may play an important role in neuronal functions, especially in synaptic plasticity and cortical development. Endogenous antisense RNA-mediated regulation may represent a new mechanism through which the activity of ion channels can be regulated in the human CNS.

  14. [Relationship between the anti-hemolysin activity and the structure of catechins and theaflavins].

    PubMed

    Ikigai, H; Toda, M; Okubo, S; Hara, Y; Shimamura, T

    1990-11-01

    We examined the corresponding isomers of catechins and theaflavins for anti-hemolysin activities against Staphylococcus aureus alpha-toxin and Vibrio cholerae O1 hemolysin. Catechins and theaflavins showed anti-hemolysin activities in a dose-dependent manner. Among the catechins tested, (-)catechin gallate, (-)epicatechin gallate and (-)epigallocatechin gallate having galloyl groups in their molecules showed more potent anti-hemolysin activities against both toxins. On the other hand, free catechins, i. e. (-)catechin, (-)gallocatechin, (-) epicatechin and (-)epigallocatechin had low anti-hemolysin activities against alpha-toxin. Although (-)catechin or (-)gallocatechin had no effect on cholera hemolysin, (-) epicatechin and (-)epigallocatechin were slightly inhibitory. Among dextrocatechins, (+) epicatechin and (+)epigallocatechin proved to be more effective than (+)catechin and (+) gallocatechin. The anti-hemolysin activities of theaflavins against alpha-toxin and cholera hemolysin were dependent on the number of the galloyl group in their structure. These results suggest that the tertiary structure of the catechin or theaflavin and the active site of hemolysin, that affects the interaction between them, plays an important role in the anti-hemolysin activity.

  15. Anti-D immunoglobulin preparations: the stability of anti-D concentrations and the error of the assay of anti-D.

    PubMed

    Hughes-Jones, N C; Hunt, V A; Maycock, W D; Wesley, E D; Vallet, L

    1978-01-01

    An analysis of the assay of 28 preparations of anti-D immunoglobulin using a radioisotope method carried out at 6-montly intervals for 2--4.5 years showed an average fall in anti-D concentration of 10.6% each year, with 99% confidence limits of 6.8--14.7%. The fall in anti-D concentration after storage at 37 degrees C for 1 month was less than 8%, the minimum change that could be detected. No significant change in physical characteristics of the immunoglobulin were detected. The error of a single estimate of anti-D by the radioisotope method (125I-labelled anti-IgG) used here was calculated to be such that the true value probably (p = 0.95) lay between 66 and 150% of the estimated value.

  16. Gallium-67-labeled lactam bridge-cyclized alpha-melanocyte stimulating hormone peptide for primary and metastatic melanoma imaging.

    PubMed

    Guo, Haixun; Yang, Jianquan; Shenoy, Nalini; Miao, Yubin

    2009-12-01

    The purpose of this study was to examine the melanoma imaging properties of a novel 67Ga-labeled lactam bridge-cyclized alpha-melanocyte stimulating hormone (alpha-MSH) peptide. A lactam bridge-cyclized alpha-MSH peptide, DOTA-GlyGlu-CycMSH {DOTA-Gly-Glu-c[Lys-Nle-Glu-His-DPhe-Arg-Trp-Gly-Arg-Pro-Val-Asp]}, was synthesized and radiolabeled with 67Ga. The melanoma targeting and pharmacokinetic properties of 67Ga-DOTA-GlyGlu-CycMSH were determined in B16/F1 flank primary melanoma-bearing and B16/F10 pulmonary metastatic melanoma-bearing C57 mice. Flank primary melanoma and pulmonary metastatic melanoma imaging were performed by small animal single photon emission computed tomography (SPECT)/CT using 67Ga-DOTA-GlyGlu-CycMSH as an imaging probe. 67Ga-DOTA-GlyGlu-CycMSH was readily prepared with greater than 95% radiolabeling yield. 67Ga-DOTA-GlyGlu-CycMSH exhibited substantial tumor uptake (12.93 +/- 1.63%ID/g at 2 h postinjection) and prolonged tumor retention (5.02 +/- 1.35%ID/g at 24 h postinjection) in B16/F1 melanoma-bearing C57 mice. The uptake values for nontarget organs were generally low (<0.30%ID/g) except for the kidneys at 2, 4, and 24 h postinjection. 67Ga-DOTA-GlyGlu-CycMSH exhibited significantly (p < 0.05) higher uptakes (1.44 +/- 0.75%ID/g at 2 h postinjection and 1.49 +/- 0.69%ID/g at 4 h postinjection) in metastatic melanoma-bearing lung than those in normal lung (0.15 +/- 0.10%ID/g and 0.17 +/- 0.11%ID/g at 2 and 4 h postinjection, respectively). Both flank primary B16/F1 melanoma and B16/F10 pulmonary melanoma metastases were clearly visualized by SPECT/CT using 67Ga-DOTA-GlyGlu-CycMSH as an imaging probe 2 h postinjection. 67Ga-DOTA-GlyGlu-CycMSH exhibited favorable melanoma targeting and imaging properties, highlighting its potential as an effective imaging probe for early detection of primary and metastatic melanoma.

  17. Absorption, tissue distribution and excretion of radiolabelled compounds in rats after administration of [14C]-L-alpha-glycerylphosphorylcholine.

    PubMed

    Abbiati, G; Fossati, T; Lachmann, G; Bergamaschi, M; Castiglioni, C

    1993-01-01

    The kinetics and metabolism of L-alpha-glycerylphosphoryl-choline (alpha-GPC) were investigated in male and female rats after i.v. (10 mg/kg) and oral doses (100-300 mg/kg). alpha-GPC was labelled with [14C]-glycerol ([14G]-GPC) or [14C]-choline ([14C]-GPC). Different kinetic and metabolic profiles were observed after i.v. and oral administration. It is assumed that alpha-GPC is hydrolyzed by phosphodiesterases in the gut mucosa. The different labelled metabolites have different kinetic properties of absorption, distribution and clearance, leading to different blood concentration-time curves of total radioactivity. Both labelled compounds gave a wide distribution of radioactivity, particularly concentrated in the liver, kidney, lung and spleen compared to blood. Brain concentrations of [14C]-GPC were comparable to ([14G]-GPC) or lower than ([14C]-GPC) total blood radioactivity. The metabolite profile in the perfused brain showed a small amount of choline and two unknown metabolites, probably the same as in blood. In addition, choline was incorporated into brain phospholipids in increasing amounts within 24 h of dosing. In all cases renal and fecal excretion of radioactivity was low and comparable for [14G]-GPC and [14C]-GPC. Mostly the administered radioactivity was exhaled as 14CO2, this degradation being faster and more pronounced for the glycerol-labelled metabolites than for the choline-labelled metabolites for both routes of administration. In all cases the results were the same for male and female rats.

  18. Epidermal growth factor system is a physiological regulator of development of the mouse fetal submandibular gland and regulates expression of the alpha6-integrin subunit.

    PubMed

    Kashimata, M; Gresik, E W

    1997-02-01

    Epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) regulate branching morphogenesis of fetal mouse submandibular gland (SMG) rudiments in vitro. The EGF system (EGF, TGF-alpha, and their shared receptor, EGFR) also regulates expression of integrins and their ligands in the extracellular matrix. We show here that inhibition of EGFR tyrosine-kinase activity by a tyrphostin retards in vitro development of SMGs. Using total RNA isolated from pooled SMGs taken from intact mouse fetuses, mRNA transcripts for EGF, TGF-alpha, and EGFR were detected by reverse transcription-polymerase chain reaction (RT-PCR), and age-dependent variations in the levels of these mRNA were quantitatively determined by nuclease protection assays. These findings suggest that the EGF system is operative in the in vivo development of this gland. alpha6-Integrin subunit was localized by immunofluorescence at the basal surface of epithelial cells. Branching morphogenesis of cultured SMG rudiments was inhibited by anti-alpha6 antibodies. Synthesis of alpha6-subunit in cultured SMGs, detected by metabolic labeling and immunoprecipitation, was increased by EGF and drastically reduced by tyrphostin. RT-PCR revealed that mRNAs for alpha6- and beta1- and beta4-integrin subunits are expressed at all ages between embryonic day 13 and postnatal day 7. These findings suggest that 1) the EGF system is a physiologic regulator of development of fetal mouse SMG, and 2) one mechanism by which it acts may be by regulating expression of integrins, which in turn control interaction of epithelial cells with the extracellular matrix.

  19. The reactivities of human erythrocyte autoantibodies anti-Pr2, anti-Gd, Fl and Sa with gangliosides in a chromatogram binding assay.

    PubMed Central

    Uemura, K; Roelcke, D; Nagai, Y; Feizi, T

    1984-01-01

    The thin layer chromatogram binding assay was used to study the reaction of several natural-monoclonal autoantibodies which recognize sialic acid-dependent antigens of human erythrocytes. Immunostaining of gangliosides derived from human and bovine erythrocytes was achieved with four autoantibodies designated anti-Pr2, anti-Gd, Sa and Fl, each of which has a different haemagglutination pattern with untreated and proteinase-treated erythrocytes and with cells of I and i antigen types. From the chromatogram binding patterns of anti-Pr2 with gangliosides of the neolacto and the ganglio series, it is deduced that this antibody reacts best with N-acetylneuraminic acid when it is alpha 2-3- or alpha 2-6-linked to a terminal Gal(beta 1-4)Glc/GlcNAc GlcNAc sequence and to a lesser extent when it is alpha 2-3-linked to a terminal Gal(beta 1-3)GalNAc sequence or to an internal galactose and when it is alpha 2-8-linked to another, internal N-acetylneuraminic acid residue. The other three antibodies differ from anti-Pr2 in their lack of reaction with glycolipids of the ganglio series. They react with the NeuAc(alpha 2-3)Gal(beta 1-4)Glc/GlcNAc sequence as found in GM3 and in glycolipids of the neolacto series, but show a preference for the latter, longer sequences. Thus all four antibodies react with sialylated oligosaccharides containing i type (linear) and I type (branched) neolacto backbones. Fl antibody differs from the other three in its stronger reaction with branched neolacto sequences in accordance with its stronger agglutination of erythrocytes of I rather than i type. The four antibodies show a specificity for N-acetyl- rather than N-glycolyl-neuraminic acid. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. PMID:6204642

  20. Label-free sensing of the binding state of MUC1 peptide and anti-MUC1 aptamer solution in fluidic chip by terahertz spectroscopy.

    PubMed

    Zhao, Xiang; Zhang, Mingkun; Wei, Dongshan; Wang, Yunxia; Yan, Shihan; Liu, Mengwan; Yang, Xiang; Yang, Ke; Cui, Hong-Liang; Fu, Weiling

    2017-10-01

    The aptamer and target molecule binding reaction has been widely applied for construction of aptasensors, most of which are labeled methods. In contrast, terahertz technology proves to be a label-free sensing tool for biomedical applications. We utilize terahertz absorption spectroscopy and molecular dynamics simulation to investigate the variation of binding-induced collective vibration of hydrogen bond network in a mixed solution of MUC1 peptide and anti-MUC1 aptamer. The results show that binding-induced alterations of hydrogen bond numbers could be sensitively reflected by the variation of terahertz absorption coefficients of the mixed solution in a customized fluidic chip. The minimal detectable concentration is determined as 1 pmol/μL, which is approximately equal to the optimal immobilized concentration of aptasensors.

  1. Digoxigenylated wheat germ agglutinin visualized with alkaline phosphatase-labeled anti-digoxigenin antibodies--a new, sensitive technique with the potential for single and double tracing of neuronal connections.

    PubMed

    Veh, R W

    1991-01-02

    For double tracing experiments, wheat germ agglutinin (WGA) molecules labeled with two different haptens are desirable. In the present report the suitability of digoxigenylated WGA (DIG-WGA) for retrograde tracing was investigated. For this purpose the new tracer was pressure injected into rat brains and the transported DIG-WGA visualized via its digoxigenyl group with an alkaline phosphatase linked anti DIG antibody in permanently stained sections of high quality. With fixatives containing 2.5% glutaraldehyde only few positive cells were found. However, at milder fixation conditions (4% paraformaldehyde, 0.05% glutaraldehyde 0.2% picric acid, 30 min) retrogradely labeled cells were detected with a sensitivity comparable to tetramethylbenzidine protocols for conventional WGA-HRP (horseradish peroxidase) tracing. Preliminary experiments suggest excellent suitability for double labeling.

  2. Systematic review with network meta-analysis: the efficacy of anti-tumour necrosis factor-alpha agents for the treatment of ulcerative colitis.

    PubMed

    Stidham, R W; Lee, T C H; Higgins, P D R; Deshpande, A R; Sussman, D A; Singal, A G; Elmunzer, B J; Saini, S D; Vijan, S; Waljee, A K

    2014-04-01

    Antibodies against tumour necrosis factor-alpha (anti-TNF) are effective therapies in the treatment of ulcerative colitis (UC), but their comparative efficacy is unknown. To perform a network meta-analysis comparing the efficacy of anti-TNF agents in UC. After screening 506 studies, reviewers extracted information on seven studies. Traditional meta-analysis (TMA) was used to compare each anti-TNF agent to placebo. Bayesian network meta-analysis (NMA) was performed to compare the effects of anti-TNF agents to placebo. In addition, sample sizes for comparative efficacy trials were calculated. Compared to placebo, TMA revealed that anti-TNF agents result in a higher likelihood of induction of remission and response (RR: 2.45, 95% CI: 1.72-3.47 and RR: 1.65, 95% CI: 1.37-1.99 respectively) as well as maintenance of remission and response (RR: 2.00, 95% CI: 1.52-2.62 and RR: 1.76, 95% CI: 1.46-2.14 respectively). Individually, infliximab, adalimumab and goliumumab resulted in a higher likelihood of induction and maintenance for both remission and response. NMA found nonsignificant trends in comparisons of the individual agents. The required sample sizes for direct head-to-head trials between infliximab and adalimumab for induction and maintenance are 174 and 204 subjects respectively. This study demonstrates that, compared to placebo, infliximab, adalimumab and golimumab are all effective for the induction and maintenance of remission in ulcerative colitis. However, network meta-analysis demonstrates that no single agent is clinically superior to the others and therefore, other factors such as cost, safety, route of administration and patient preference should dictate our choice of anti-TNF agents. A randomised comparative efficacy trial between infliximab and adalimumab in UC is of practical size and should be performed. © 2014 John Wiley & Sons Ltd.

  3. 42 CFR 414.930 - Compendia for determination of medically-accepted indications for off-label uses of drugs and...

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... indications for off-label uses of drugs and biologicals in an anti-cancer chemotherapeutic regimen. 414.930... indications for off-label uses of drugs and biologicals in an anti-cancer chemotherapeutic regimen. (a... specialty compendium, for example a compendium of anti-cancer treatment. A compendium— (i) Includes a...

  4. 42 CFR 414.930 - Compendia for determination of medically-accepted indications for off-label uses of drugs and...

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... indications for off-label uses of drugs and biologicals in an anti-cancer chemotherapeutic regimen. 414.930... indications for off-label uses of drugs and biologicals in an anti-cancer chemotherapeutic regimen. (a... specialty compendium, for example a compendium of anti-cancer treatment. A compendium— (i) Includes a...

  5. 42 CFR 414.930 - Compendia for determination of medically-accepted indications for off-label uses of drugs and...

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... indications for off-label uses of drugs and biologicals in an anti-cancer chemotherapeutic regimen. 414.930... indications for off-label uses of drugs and biologicals in an anti-cancer chemotherapeutic regimen. (a... specialty compendium, for example a compendium of anti-cancer treatment. A compendium— (i) Includes a...

  6. 42 CFR 414.930 - Compendia for determination of medically-accepted indications for off-label uses of drugs and...

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... indications for off-label uses of drugs and biologicals in an anti-cancer chemotherapeutic regimen. 414.930... indications for off-label uses of drugs and biologicals in an anti-cancer chemotherapeutic regimen. (a... specialty compendium, for example a compendium of anti-cancer treatment. A compendium— (i) Includes a...

  7. 42 CFR 414.930 - Compendia for determination of medically-accepted indications for off-label uses of drugs and...

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... indications for off-label uses of drugs and biologicals in an anti-cancer chemotherapeutic regimen. 414.930... indications for off-label uses of drugs and biologicals in an anti-cancer chemotherapeutic regimen. (a... specialty compendium, for example a compendium of anti-cancer treatment. A compendium— (i) Includes a...

  8. Lysophospholipid presentation by CD1d and recognition by a human Natural Killer T-cell receptor

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    López-Sagaseta, Jacinto; Sibener, Leah V.; Kung, Jennifer E.

    2014-10-02

    Invariant Natural Killer T (iNKT) cells use highly restricted {alpha}{beta} T cell receptors (TCRs) to probe the repertoire of lipids presented by CD1d molecules. Here, we describe our studies of lysophosphatidylcholine (LPC) presentation by human CD1d and its recognition by a native, LPC-specific iNKT TCR. Human CD1d presenting LPC adopts an altered conformation from that of CD1d presenting glycolipid antigens, with a shifted {alpha}1 helix resulting in an open A pocket. Binding of the iNKT TCR requires a 7-{angstrom} displacement of the LPC headgroup but stabilizes the CD1d-LPC complex in a closed conformation. The iNKT TCR CDR loop footprint onmore » CD1d-LPC is anchored by the conserved positioning of the CDR3{alpha} loop, whereas the remaining CDR loops are shifted, due in part to amino-acid differences in the CDR3{beta} and J{beta} segment used by this iNKT TCR. These findings provide insight into how lysophospholipids are presented by human CD1d molecules and how this complex is recognized by some, but not all, human iNKT cells.« less

  9. Differential effect of isotype on efficacy of anti-tumor necrosis factor alpha chimeric antibodies in experimental septic shock

    PubMed Central

    1994-01-01

    Immune complexes containing human gamma (g)1 or murine g2a antibodies generate secondary effector mechanisms via Fc receptor binding or complement activation, whereas those containing human g4 or murine g1 antibodies generally do not. Therefore, isotype selection of therapeutic antibodies may have important clinical consequences. In a rabbit model of human tumor necrosis factor (rhuTNF)-induced pyrexia, a murine/human chimeric g4 anti-human TNF-alpha monoclonal antibody (mAb) (cCB0011) showed a dose-dependent inhibition of pyrexia, whereas a g1 isotype variant of the same mAb gave a marked pyrexia that was seen at all doses indicative of an immune complex-mediated response. To investigate whether isotype difference could influence mAb efficacy in pathological disease states, hamster/murine chimeric g1 and g2a anti- murine TNF-alpha mAbs (TN3g1, TN3g2a) were studied in experimental shock in mice and rats. In lipopolysaccharide-induced shock in mice, treatment with TN3g1 mAb at 30 and 3 mg/kg resulted in 90% survival by 72 h (p < or = 0.004), and prolonged survival to 45 h (p < or = 0.05), respectively, compared with 100% mortality by 27 h in controls. In contrast, a g2a isotype variant of the same mAb (30 mg/kg) resulted in only 10% survival by 72 h (p < or = 0.05). In a neutropenic sepsis model in rats there was greater survival in animals receiving the g1 isotype of TN3 compared with g2a isotype variant (70 vs. 27%; p < or = 0.005) with 100% mortality in the controls. These differences were not due to the pharmacokinetic profiles of the mAbs. In models of experimental shock antibody isotype can affect outcome with inactive isotypes (human g4 and murine g1) being more efficacious than active isotypes (human g1 and murine g2a). PMID:8113678

  10. Anti-Inflammatory Effects of Cajaninstilbene Acid and Its Derivatives.

    PubMed

    Huang, Mei-Yan; Lin, Jing; Lu, Kuo; Xu, Hong-Gui; Geng, Zhi-Zhong; Sun, Ping-Hua; Chen, Wei-Min

    2016-04-13

    Cajaninstilbene acid (CSA) is one of the active components isolated from pigeon pea leaves. In this study, anti-inflammatory effects of CSA and its synthesized derivatives were fully valued with regard to their activities on the production of nitric oxide (NO) and pro-inflammatory cytokines tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) in vitro cell model, as well as their impacts on the migration of neutrophils and macrophages in fluorescent protein labeled zebrafish larvae model by live image analysis. Furthermore, the anti-inflammatory mechanism of this type of compounds was clarified by western-blot and reverse transcription-polymerase chain reaction (RT-PCR). The results showed that CSA, as well as its synthesized derivatives 5c, 5e and 5h, exhibited strong inhibition activity on the release of NO and inflammatory factor TNF-α and IL-6 in lipopolysaccharides (LPS)-stimulated murine macrophages. CSA and 5c greatly inhibited the migration of neutrophils and macrophages in injury zebrafish larvae. CSA and 5c treatment greatly inhibited the phosphorylation of proteins involved in nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) pathways. Moreover, we found that peroxisome proliferator-activated receptor gamma (PPARγ) inhibitor GW9662 could reverse partly the roles of CSA and 5c, and CSA and 5c treatment greatly resist the decrease of PPARγ mRNA and protein induced by LPS stimulation. Our results identified the promising anti-inflammatory effects of CSA and its derivatives, which may serve as valuable anti-inflammatory lead compound. Additionally, the mechanism studies demonstrated that the anti-inflammatory activity of CSA and its derivative is associated with the inhibition of NF-κB and MAPK pathways, relying partly on resisting the LPS-induced decrease of PPARγ through improving its expression.

  11. Lens proteome map and alpha-crystallin profile of the catfish Rita rita.

    PubMed

    Mohanty, Bimal Prasanna; Bhattacharjee, Soma; Das, Manas Kumar

    2011-02-01

    Crystallins are a diverse group of proteins that constitute nearly 90% of the total soluble proteins of the vertebrate eye lens and these tightly packed crystallins are responsible for transparency of the lens. These proteins have been studied in different model and non-model species for understanding the modifications they undergo with ageing that lead to cataract, a disease of protein aggregation. In the present investigation, we studied the lens crystallin profile of the tropical freshwater catfish Rita rita. Profiles of lens crystallins were analyzed and crystallin proteome maps of Rita rita were generated for the first time. alphaA-crystallins, member of the alpha-crystallin family, which are molecular chaperons and play crucial role in maintaining lens transparency were identified by 1- and 2-D immunoblot analysis with anti-alphaA-crystallin antibody. Two protein bands of 19-20 kDa were identified as alphaA-crystallins on 1-D immunoblots and these bands separated into 10 discrete spots on 2-D immunoblot. However, anti-alphaB-crystallin and antiphospho-alphaB-crystallin antibodies were not able to detect any immunoreactive bands on 1- and 2-D immunoblots, indicating alphaB-crystallin was either absent or present in extremely low concentration in Rita rita lens. Thus, Rita rita alpha-crystallins are more like that of the catfish Clarias batrachus and the mammal kangaroo in its alphaA- and alphaB-crystallin content (contain low amount from 5-9% of alphaB-crystallin) and unlike the dogfish, zebrafish, human, bovine and mouse alpha-crystallins (contain higher amount of alphaB-crystallin from 25% in mouse and bovine to 85% in dogfish). Results of the present study can be the baseline information for stimulating further investigation on Rita rita lens crystallins for comparative lens proteomics. Comparing and contrasting the alpha-crystallins of the dogfish and Rita rita may provide valuable information on the functional attributes of alphaA- and alphaB-isoforms, as

  12. Negative interference by rheumatoid factor in alpha-fetoprotein chemiluminescent microparticle immunoassay.

    PubMed

    Wang, Hui; Bi, Xiaohui; Xu, Lei; Li, Yirong

    2017-01-01

    Background Rheumatoid factor causes positive interference in multiple immunoassays. Recently, negative interference has also been found in immunoassays in the presence of rheumatoid factor. The chemiluminescent microparticle immunoassay is widely used to determine serum alpha-fetoprotein. However, it is not clear whether the presence of rheumatoid factor in the serum causes interference in the chemiluminescent microparticle immunoassay of alpha-fetoprotein. Methods Serum alpha-fetoprotein was determined using the ARCHITECT alpha-fetoprotein assay. The estimation of alpha-fetoprotein recovery was carried out in samples prepared by diluting high-concentration alpha-fetoprotein serum with rheumatoid factor-positive or rheumatoid factor-negative serum. Paramagnetic microparticles coated with hepatitis B surface antigen-anti-HBs complexes were used to remove rheumatoid factor from the serum. Results The average recovery of alpha-fetoprotein was 88.4% and 93.8% in the rheumatoid factor-positive and rheumatoid factor-negative serum samples, respectively. The recovery of alpha-fetoprotein was significantly lower in the rheumatoid factor-positive serum samples than in the rheumatoid factor-negative serum samples. In two of five rheumatoid factor-positive samples, a large difference was found (9.8%) between the average alpha-fetoprotein recoveries in the serially diluted and initial recoveries. Fourteen rheumatoid factor-positive serum samples were pretreated with hepatitis B surface antigen-anti-HBs complex-coated paramagnetic microparticles. The alpha-fetoprotein concentrations measured in the pretreated samples increased significantly. Conclusions It was concluded that the alpha-fetoprotein chemiluminescent microparticle immunoassay is susceptible to interference by rheumatoid factor, leading to significantly lower results. Eliminating the incidence of negative interference from rheumatoid factor should be an important goal for immunoassay providers. In the meantime

  13. Alpha-fetoprotein detection by using a localized surface plasmon coupled fluorescence fiber-optic biosensor

    NASA Astrophysics Data System (ADS)

    Chang, Ying-Feng; Chen, Ran-Chou; Li, Ying-Chang; Yu, Chih-Jen; Hsieh, Bao-Yu; Chou, Chien

    2007-11-01

    Alpha-fetoprotein (AFP) detection by using a localized surface plasmon coupled fluorescence (LSPCF) fiber-optic biosensor is setup and experimentally demonstrated. It is based on gold nanoparticle (GNP) and coupled with localized surface plasmon wave on the surface of GNP. In this experiment, the fluorophores are labeled on anti-AFP which are bound to protein A conjugated GNP. Thus, LSPCF is excited with high efficiency in the near field of localized surface plasmon wave. Therefore, not only the sensitivity of LSPCF biosensor is enhanced but also the specific selectivity of AFP is improved. Experimentally, the ability of real time measurement in the range of AFP concentration from 0.1ng/ml to 100ng/ml was detected. To compare with conventional methods such as enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA), the LSPCF fiber-optic biosensor performs higher or comparable detection sensitivity, respectively.

  14. Pontin is required for pre-TCR signaling at the β-selection checkpoint in T cell development.

    PubMed

    Boo, Kyungjin; Baek, Sung Hee; Lee, Ho

    2014-04-25

    Pontin is a chromatin remodeling factor that possesses both ATPase and DNA helicase activities. Based on high expression in lymphoid tissues, we examined whether Pontin has a T cell-specific function. We generated Pontin(f/f);Lck-Cre mice, in which Pontin can be conditionally deleted in T cells and then explored T cell-specific function of Pontin in vivo. Here, we show that specific abrogation of Pontin expression in T cells almost completely blocked development of αβ T cells at the β-selection checkpoint by inducing cell apoptosis indicating that Pontin is essential for early T cell development. Pontin-deficient thymocytes show a comparable expression level of T cell receptor (TCR)β chain, but have enhanced activation of p53 and Notch signaling compared to wild-type thymocytes. Intriguingly, the developmental block of αβ T cells can be partially rescued by loss of p53. Together, our data demonstrate a novel role of Pontin as a crucial regulator in pre-TCR signaling during T cell development. Copyright © 2014 Elsevier Inc. All rights reserved.

  15. T-bet Down-Modulation in Tolerized Th1 Effector CD4 Cells Confers a TCR-Distal Signaling Defect That Selectively Impairs IFN-γ Expression1

    PubMed Central

    Long, Meixiao; Slaiby, Aaron M.; Hagymasi, Adam T.; Mihalyo, Marianne A.; Lichtler, Alexander C.; Reiner, Steven L.; Adler, Adam J.

    2010-01-01

    When Th1 effector CD4 cells encounter tolerizing Ag in vivo, their capacity to express the effector cytokines IFN-γ and TNF-α is lost more rapidly than noneffector functions such as IL-2 production and proliferation. To localize the relevant intracellular signaling defects, cytokine expression was compared following restimulation with Ag vs agents that bypass TCR-proximal signaling. IFN-γ and TNF-α expression were both partially rescued when TCR-proximal signaling was bypassed, indicating that both TCR-proximal and -distal signaling defects impair the expression of these two effector cytokines. In contrast, bypassing TCR-proximal signaling fully rescued IL-2 expression. T-bet, a transcription and chromatin remodeling factor that is required to direct the differentiation of naive CD4 cells into IFN-γ -expressing Th1 effectors, was partially down-modulated in tolerized Th1 effectors. Enforcing T-bet expression during tolerization selectively rescued the ability to express IFN-γ, but not TNF-α. Conversely, expression of a dominant-negative T-bet in Th1 effectors selectively impaired the ability to express IFN-γ, but not TNF-α. Analysis of histone acetylation at the IFN-γ promoter further suggested that down-modulation of T-bet expression during Th1 effector CD4 cell tolerization does not impair IFN-γ expression potential through alterations in chromatin structure. PMID:16393991

  16. Anti-fibrin antibody binding in valvular vegetations and kidney lesions during experimental endocarditis.

    PubMed

    Yokota, M; Basi, D L; Herzberg, M C; Meyer, M W

    2001-01-01

    In Streptococcus sanguinis (sanguis) induced experimental endocarditis, we sought evidence that the development of aortic valvular vegetation depends on the availability of fibrin. Endocarditis was induced in New Zealand white rabbits by catheter placement into the left ventricle and inoculation of the bacteria. Fibrin was localized in the developing vegetation with 99mTechnetium (Tc)-labeled anti-fibrin antibody one or three days later. When rabbit anti-fibrin antibody was given intravenously on day 1, the mass of aortic valvular vegetation was significantly reduced at day 3; infusion of non-specific rabbit IgG showed no effect. The 99mTc-labeled anti-fibrin antibody also labeled kidneys that showed macroscopic subcapsular hemorrhage. To learn if the deposition of fibrin in the kidneys was a consequence of endocarditis required a comparison of farm-bred and specific pathogen-free rabbits before and after the induction of endocarditis. Before induction, the kidneys of farm-bred rabbits were labeled, but specific pathogen-free rabbits were free of labeling and signs of macroscopic hemorrhage. After 3 days of endocarditis, kidneys of 10 of 14 specific pathogen-free rabbits labeled with 99mTc-labeled anti-fibrin antibody and showed hemorrhage. Kidney lesions were suggested to be a frequent sequellae of S. sanguinis infective endocarditis. For the first time, fibrin was shown to be required for the continued development of aortic valvular vegetations.

  17. Comparative Evaluation of Anti-HER2 Affibody Molecules Labeled with 64Cu Using NOTA and NODAGA

    PubMed Central

    Yim, Cheng-Bin; Rajander, Johan; Perols, Anna; Karlström, Amelie Eriksson; Haaparanta-Solin, Merja; Grönroos, Tove J.; Solin, Olof; Orlova, Anna

    2017-01-01

    Imaging using affibody molecules enables discrimination between breast cancer metastases with high and low expression of HER2, making appropriate therapy selection possible. This study aimed to evaluate if the longer half-life of 64Cu (T1/2 = 12.7 h) would make 64Cu a superior nuclide compared to 68Ga for PET imaging of HER2 expression using affibody molecules. The synthetic ZHER2:S1 affibody molecule was conjugated with the chelators NOTA or NODAGA and labeled with 64Cu. The tumor-targeting properties of 64Cu-NOTA-ZHER2:S1 and 64Cu-NODAGA-ZHER2:S1 were evaluated and compared with the targeting properties of 68Ga-NODAGA-ZHER2:S1 in mice. Both 64Cu-NOTA-ZHER2:S1 and 64Cu-NODAGA-ZHER2:S1 demonstrated specific targeting of HER2-expressing xenografts. At 2 h after injection of 64Cu-NOTA-ZHER2:S1, 64Cu-NODAGA-ZHER2:S1, and 68Ga-NODAGA-ZHER2:S1, tumor uptakes did not differ significantly. Renal uptake of 64Cu-labeled conjugates was dramatically reduced at 6 and 24 h after injection. Notably, radioactivity uptake concomitantly increased in blood, lung, liver, spleen, and intestines, which resulted in decreased tumor-to-organ ratios compared to 2 h postinjection. Organ uptake was lower for 64Cu-NODAGA-ZHER2:S1. The most probable explanation for this biodistribution pattern was the release and redistribution of renal radiometabolites. In conclusion, monoamide derivatives of NOTA and NODAGA may be suboptimal chelators for radiocopper labeling of anti-HER2 affibody molecules and, possibly, other scaffold proteins with high renal uptake. PMID:29097939

  18. Anti-inflammatory and antioxidant effect of ginger in tuberculosis.

    PubMed

    Kulkarni, Rashmi Anant; Deshpande, Ajit Ramesh

    2016-06-01

    Tuberculosis (TB) has reemerged to become the world's leading cause of death from a single infectious agent. Inflammatory cytokines play an important role during the course of the disease and may be responsible for tissue damage by lipid peroxidation. The study was aimed to explore the anti-inflammatory and antioxidant effect of ginger in pulmonary TB patients. A total of 69 pulmonary TB patients participated in a randomized and placebo-controlled study. The intervention group received 3 g of ginger extract daily for 1 month and placebo group was supplemented with starch capsule. Participants of both groups were taking standard antitubercular treatment during the study. The concentrations of tumor necrosis factor (TNF) alpha, ferritin and malondialdehyde (MDA) in blood samples were analyzed before and after the intervention by using enzyme-linked immunosorbent assay for TNF alpha and ferritin and spectrophotometry for MDA. Ginger supplementation significantly reduced the levels of TNF alpha, ferritin and MDA in ginger supplemented group in comparison to baseline. Ginger supplementation with antitubercular treatment significantly lowered TNF alpha, ferritin and MDA concentrations in comparison to control group. Ginger was found to be effective as an anti-inflammatory and antioxidant supplement along with anti-TB therapy as it possesses strong free radical scavenging property.

  19. Extraordinary TCR in Carbon Nanotube-Polymer Composites and Device Implications in Bolometric Infrared Detection

    DTIC Science & Technology

    2015-03-24

    were made via filtration of 1 mL of 1 mg/L SWCNT solution (Nanointegris Inc.) through a 20 nm pore anodized aluminum oxide ( AAO ) membrane filter to...prepared by attaching a rectangular piece cut from the AAO membrane filter onto a glass substrate. Contacts were made with silver paste to the extremities...NUMBER(S) 14. ABSTRACT _ The development of high TCR materials, such as vanadium oxide (VOx), has enabled the introduction of bolometric infrared

  20. Photoaffinity labelling of MSH receptors on Anolis melanophores: effects of catecholamines, calcium and forskolin.

    PubMed

    Eberle, A N; Girard, J

    1985-01-01

    Photoaffinity labelling of MSH receptors on Anolis melanophores was used as a tool for studying the effects of catecholamines, calcium and forskolin on hormone-receptor interaction and receptor-adenylate cyclase coupling. Covalent attachment of photoreactive alpha-MSH to its receptor was suppressed in calcium-free buffer but was hardly influenced by catecholamines or forskolin. The longlasting signal generated by the covalent MSH-receptor complex was readily and reversibly abolished by adrenaline, noradrenaline, dopamine or clonidine or by the absence of calcium. The suppression of pigment dispersion by catecholamines was blocked by the simultaneous presence of yohimbine but not prazosin, indicating that the catecholamines antagonize the alpha-MSH signal by inhibitory action on the adenylate cyclase system through an alpha-2 receptor. Forskolin, which stimulates melanophores by direct action on the catalytic unit of the adenylate cyclase and at about the same speed as alpha-MSH, produced a slower and weaker response in the presence of noradrenaline. If MSH receptors were covalently labelled and then exposed to noradrenaline, the characteristics of the forskolin-induced response were identical to those of unlabelled cells that had not been exposed to noradrenaline. This may point to a partial restoration of receptor-adenylate cyclase coupling by forskolin. The results show that the longlasting stimulation of Anolis melanophores by photoaffinity labelling proceeds via a permanently stimulated adenylate-cyclase system whose coupling to the receptor depends on calcium and is abolished by alpha-2 receptor agonists. Calcium is also essential for hormone-receptor binding.

  1. The quantum chemical causality of pMHC-TCR biological avidity: Peptide atomic coordination data and the electronic state of agonist N termini.

    PubMed

    Antipas, Georgios S E; Germenis, Anastasios E

    2015-06-01

    The quantum state of functional avidity of the synapse formed between a peptide-Major Histocompatibility Complex (pMHC) and a T cell receptor (TCR) is a subject not previously touched upon. Here we present atomic pair correlation meta-data based on crystalized tertiary structures of the Tax (HTLV-1) peptide along with three artificially altered variants, all of which were presented by the (Class I) HLA-A201 protein in complexation with the human (CD8(+)) A6TCR. The meta-data reveal the existence of a direct relationship between pMHC-TCR functional avidity (agonist/antagonist) and peptide pair distribution function (PDF). In this context, antagonist peptides are consistently under-coordinated in respect to Tax. Moreover, Density Functional Theory (DFT) datasets in the BLYP/TZ2P level of theory resulting from relaxation of the H species on peptide tertiary structures reveal that the coordination requirement of agonist peptides is also expressed as a physical observable of the protonation state of their N termini: agonistic peptides are always found to retain a stable ammonium (NH3 (+)) terminal group while antagonist peptides are not.

  2. An overview of targeted alpha therapy with 225Actinium and 213Bismuth.

    PubMed

    Morgenstern, Alfred; Apostolidis, Christos; Kratochwil, Clemens; Sathekge, Mike; Krolicki, Leszek; Bruchertseifer, Frank

    2018-05-01

    Recent reports of the remarkable therapeutic efficacy of 225Ac-labeled PSMA-617 for therapy of metastatic castration-resistant prostate cancer have underlined the clinical potential of targeted alpha therapy. This review describes methods for the production of 225Ac and its daughter nuclide 213Bi and summarizes the current clinical experience with both alpha emitters with particular focus on recent studies of targeted alpha therapy of bladder cancer, brain tumors, neuroendocrine tumors and prostate cancer. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  3. Antioxidant status and alpha1-antiproteinase activity in subarachnoid hemorrhage patients.

    PubMed

    Marzatico, F; Gaetani, P; Tartara, F; Bertorelli, L; Feletti, F; Adinolfi, D; Tancioni, F; Rodriguez y Baena, R

    1998-01-01

    The antiproteasic activity of alpha1-antitrypsin (alpha1-AT) is reduced in cases of subarachnoid hemorrhage from ruptured intracranial aneurysm and particularly in patients currently smoking; alpha1-AT is very sensitive to oxidant agents. About 50% of physiological anti-oxidant systemic capacity is represented by Vitamin A, E and C. Plasmatic amounts of alpha1-AT, alpha1-AT Collagenase Inhibitory Capacity (CIC) and levels of vitamin A, vitamin E and vitamin C were analyzed in 39 patients, 26 women and 13 men, operated for intracranial aneurysm; 11 patients with unruptured intracranial aneurysm were considered as controls while 28 patients were included within 12 hours from subarachnoid hemorrhage (SAH). Plasmatic levels of vitamin A and vitamin E were significantly lower (p=0.038 and p=0.0158) in patients suffering SAH than in controls, while no statistically significant differences were found in mean plasmatic vitamin C levels. Level of alpha1-AT was not statistically different in controls and in patients with SAH; however, the activity of alpha1-AT, evaluated as CIC, is significantly reduced in patients with SAH (p=0.019). We have observed that systemic plasmatic levels of vitamins did not significantly differ in relation to smoking habit. Vitamin A and E represent an important defensive system against free radicals reactions. Particularly, vitamin E acts as an antioxidant by scavenging free-radicals. A reduced anti-oxidant status might be related to the higher sensibility of alpha1-AT to oxidative reactions and the activity of alpha1-AT is dependent on the antioxidant capacity of liposoluble vitamins. We can speculate that an acute systemic oxidative stress condition might influence the rupture of intracranial aneurysms.

  4. Radionuclide and Fluorescence Imaging of Clear Cell Renal Cell Carcinoma Using Dual Labeled Anti-Carbonic Anhydrase IX Antibody G250.

    PubMed

    Muselaers, Constantijn H J; Rijpkema, Mark; Bos, Desirée L; Langenhuijsen, Johan F; Oyen, Wim J G; Mulders, Peter F A; Oosterwijk, Egbert; Boerman, Otto C

    2015-08-01

    Tumor targeted optical imaging using antibodies labeled with near infrared fluorophores is a sensitive imaging modality that might be used during surgery to assure complete removal of malignant tissue. We evaluated the feasibility of dual modality imaging and image guided surgery with the dual labeled anti-carbonic anhydrase IX antibody preparation (111)In-DTPA-G250-IRDye800CW in mice with intraperitoneal clear cell renal cell carcinoma. BALB/c nu/nu mice with intraperitoneal SK-RC-52 lesions received 10 μg DTPA-G250-IRDye800CW labeled with 15 MBq (111)In or 10 μg of the dual labeled irrelevant control antibody NUH-82 (20 mice each). To evaluate when tumors could be detected, 4 mice per group were imaged weekly during 5 weeks with single photon emission computerized tomography/computerized tomography and the fluorescence imaging followed by ex vivo biodistribution studies. As early as 1 week after tumor cell inoculation single photon emission computerized tomography and fluorescence images showed clear delineation of intraperitoneal clear cell renal cell carcinoma with good concordance between single photon emission computerized tomography/computerized tomography and fluorescence images. The high and specific accumulation of the dual labeled antibody conjugate in tumors was confirmed in the biodistribution studies. Maximum tumor uptake was observed 1 week after inoculation (mean ± SD 58.5% ± 18.7% vs 5.6% ± 2.3% injected dose per gm for DTPA-G250-IRDye800CW vs NUH-82, respectively). High tumor uptake was also observed at other time points. This study demonstrates the feasibility of dual modality imaging with dual labeled antibody (111)In-DTPA-G250-IRDye800CW in a clear cell renal cell carcinoma model. Results indicate that preoperative and intraoperative detection of carbonic anhydrase IX expressing tumors, positive resection margins and metastasis might be feasible with this approach. Copyright © 2015 American Urological Association Education and Research

  5. Characterization and charge distribution of the asparagine-linked oligosaccharides on secreted mouse thyrotropin and free alpha-subunits

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Gesundheit, N.; Gyves, P.W.; DeCherney, G.S.

    1989-06-01

    Mouse hemipituitaries in vitro secrete TSH, composed of an alpha-beta heterodimer, as well as excess (free) alpha-subunits. By dual metabolic labeling with (35S)sulfate and (3H)mannose, we have characterized oligosaccharides from secreted TSH alpha, TSH beta, and free alpha-subunits released from the apoprotein by enzymatic deglycosylation. Oligosaccharides from each subunit displayed a distinct anion exchange HPLC profile due to a specific pattern of sialylation and sulfation. Six species were obtained from TSH alpha (with two glycosylation sites), including neutral oligosaccharides as well as those with one or two negative charges. For TSH beta (with one glycosylation site) at least eight oligosaccharidemore » species were noted, representing nearly every permutation of sialylation and sulfation; approximately 30% contained three or more negative charges. Analysis of (3H)mannose-labeled oligosaccharides on Concanavalin-A-agarose showed 85% binding for those from TSH alpha, 70% for free alpha, and 50% for those from TSH beta. These data demonstrate that oligosaccharides from secreted TSH beta were more sialylated and sulfated, consistent with a more complex branching pattern, than those from TSH alpha. Oligosaccharides from free alpha-subunit were more sialylated than those from TSH alpha, and the net negative charge was intermediate between those of TSH alpha and TSH beta. Although great microheterogeneity is present even at the single glycosylation site on the beta-subunit of secreted TSH, a pattern of sialylation and sulfation could be discerned.« less

  6. Site preference, magnetism and lattice vibrations of intermetallics Lu₂Fe 17–xT x (T=Cr, Mn, Ru)

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Li, Jin-Chun; Qian, Ping, E-mail: qianpinghu@sohu.com; Zhang, Zhen-Feng

    We present an atomistic study on the phase stability, site preference and lattice constants of the rare earth intermetallics Lu₂Fe 17–xT x (T=Cr, Mn, Ru). The calculated preferential occupation site of ternary element T is found to be the 4f site. The order of site preference is given as 4f, 12k, 12j and 6g for Lu₂Fe 17–xT x. The calculated lattice parameters are corresponding to the experimental results. We have calculated the magnetic moments of Lu₂Fe 17–xT x compounds. Results show that the calculated total magnetic moment of Lu₂Fe₁₇ compound is M=37.34 μ B/f.u. In addition, the total and partialmore » phonon densities of states are evaluated first for these complicated structures. - Graphical abstract: The vibrational modes are mostly excited by Fe atoms, Lu contributes to the lower frequencies modes, and the contribution of Ru atoms is the same as Fe atoms. Highlights: • There are no reports on lattice vibrations of Lu₂(Fe, T) 17–x (T=Cr, Mn, Ru) compounds. • The phase stability and site preference are evaluated first for the complex structures of Lu₂(Fe, T) 17–x (T=Cr, Mn, Ru) compounds. • The lattice inversion method to obtain the interatomic pair potential is the unique one.« less

  7. CDR3 analysis of TCR Vβ repertoire of CD8⁺ T cells from chickens infected with Eimeria maxima.

    PubMed

    Ren, Chao; Yin, Guangwen; Qin, Mei; Suo, Jingxia; Lv, Qiyao; Xie, Li; Wang, Yunzhou; Huang, Xiaoxi; Chen, Yuchen; Liu, Xianyong; Suo, Xun

    2014-08-01

    CD8(+) T cells play a major role in the immune protection of host against the reinfection of Eimeria maxima, the most immunogenic species of eimerian parasites in chickens. To explore the dominant complementarity-determining regions 3 (CDR3) of CD8(+) T cell populations induced by the infection of this parasite, sequence analysis was performed in this study for CDR3 of CD8(+) T cells from E. maxima infected chickens. After 5 days post the third or forth infection, intraepithelial lymphocytes were isolated from the jejunum of bird. CD3(+)CD8(+) T cells were sorted and subjected to total RNA isolation and cDNA preparation. PCR amplification and cloning of the loci between Vβ1 and Cβ was conducted for the subsequent sequencing of CDR3 of T cell receptor (TCR). After the forth infection, 2 birds exhibited two same frequent TCR CDR3 sequences, i.e., AKQDWGTGGYSNMI and AGRVLNIQY; while the third bird showed two different frequent TCR CDR3 sequences, AKQGARGHTPLN and AKQDIEVRGPNTPLN. No frequent CDR3 sequence was detected from uninfected birds, though AGRVLNIQY was also found in two uninfected birds. Our result preliminarily demonstrates that frequent CDR3 sequences may exist in E. maxima immunized chickens, encouraging the mining of the immunodominant CD8(+) T cells against E. maxima infection. Copyright © 2014 Elsevier Inc. All rights reserved.

  8. Photoaffinity labelling of the ATP-binding site of the epidermal growth factor-dependent protein kinase.

    PubMed

    Kudlow, J E; Leung, Y

    1984-06-15

    Epidermal growth factor (EGF), after binding to its receptor, activates a tyrosine-specific protein kinase which phosphorylates several substrates, including the EGF receptor itself. The effects of a photoaffinity analogue of ATP, 3'-O-(3-[N-(4-azido-2-nitrophenyl)amino]propionyl)adenosine 5'-triphosphate (arylazido-beta-alanyl-ATP) on the EGF-dependent protein kinase in A431 human tumour cell plasma membrane vesicles was investigated. This analogue was capable of inactivating the EGF-receptor kinase in a photodependent manner. Partial inactivation occurred at an analogue concentration of 1 microM and complete inactivation occurred at 10 microM when a 2 min light exposure was used. Arylazido-beta-alanine at 100 microM and ATP at 100 microM were incapable of inactivating the enzyme with 2 min of light exposure. The photodependent inactivation of the enzyme by the analogue could be partially blocked by 20 mM-ATP and more effectively blocked by either 20 mM-adenosine 5'-[beta gamma-imido]triphosphate or 20 mM-guanosine 5'-[beta gamma-imido]triphosphate, indicating nucleotide-binding site specificity. Arylazido-beta-alanyl-[alpha-32P]ATP was capable of labelling membrane proteins in a photodependent manner. Numerous proteins were labelled, the most prominent of which ran with an apparent Mr of 53000 on polyacrylamide-gel electrophoresis. A band of minor intensity was seen of Mr corresponding to the EGF receptor (170000). Immunoprecipitation of affinity-labelled and solubilized membranes with an anti-(EGF receptor) monoclonal antibody demonstrated that the Mr 170000 receptor protein was photoaffinity labelled by the analogue. The Mr 53000 peptide was not specifically bound by the anti-receptor antibody. The affinity labelling of the receptor was not enhanced by EGF, suggesting that EGF stimulation of the kinase activity does not result from changes in the affinity of the kinase for ATP. These studies demonstrate that arylazido-beta-alanyl-ATP interacts with the ATP

  9. Anti- and Pro-Immigrant Entrepreneurs Labeling Theory Revisited

    ERIC Educational Resources Information Center

    Wilson, Tamar Diana

    2009-01-01

    Almost forty years ago, noted immigration scholar Jorge A. Bustamante published an article in the "American Journal of Sociology" applying Howard Becker's labeling theory to the phenomenon of deviantizing and stigmatizing the undocumented. While immigration laws and some of the players involved have changed since his article was published,…

  10. [Cloning of VH and VL Gene of Human anti-IL1RAP McAb and Construction of Recombinant Chimeric Receptor].

    PubMed

    Yin, Ling-Ling; Ruan, Su-Hong; Tian, Yu; Zhao, Kai; Xu, Kai Lin

    2015-10-01

    To clone the variable region genes of human anti-IL1RAP (IL-1 receptor accessory protein) monoclonal antibodies (McAb) and to construct IL1RAP chimeric antigen receptors (CARs). The VH and VL DNA of IL1RAP single chain antibodies were amplified by RACE and overlap extension PCR from total RNA extracted from 3H6E10 and 10D8A7 hybridoma and ligated into specific IL1RAP single-chain variable fragments (scFv). CD8α transmembrane domain, CD137 intracellular domain, TCR ζ chain, human CD8α signal peptide and scFv-anti-IL1RAP were cloned into plasmid LV-lac. Recombinant lentiviruses were generated by co-transfection of recombinant plasmid LV-lac, pMD2. G, and psPAX2 helper vectors into 293FT packing cells. The VH and VL genes of 2 human anti-IL1RAP McAb were acquired. The 3H6E10 VH and VL genes consisted of 402 bp and 393 bp encoding 134 and 131 aminoacid residues, respectively; 10D8A7 VH and VL genes consisted of 423 bp and 381 bp encoding 141 and 127 amine acid residues, respectively. Recombinant expression vertors LV-3H6E10 scFv-ICD and LV-10D8A7 scFv-ICD (ICD: CD8α transmembrane domain-CD137 intracellular domain-TCR ζ chain) were constructed. The target fragments were demonstrated by sequencing analysis. Recombinant plasmids were transfected into 293FT cells and lentiviral particles were acquired. Human anti-IL1RAP recombinant receptors are constructed successfully and lay a good foundation for the construction of IL1RAP-CAR killer T cell vaccine.

  11. Anti-interferon-gamma antibodies in the treatment of autoimmune diseases.

    PubMed

    Skurkovich, Boris; Skurkovich, Simon

    2003-02-01

    Interferon (IFN)-gamma is an important immune regulator in normal immunity. When IFN gamma production is disturbed, various autoimmune diseases (ADs) can develop, in which we suggest that anti-IFN gamma could have a beneficial effect. Depending on the cell type in which IFN gamma synthesis is disturbed, different clinical manifestations may result. We have also proposed to remove tumor necrosis factor (TNF)-alpha, together with certain types of IFNs, to treat various ADs and AIDS, also an autoimmune condition. Anti-IFN gamma has been tested in several T-helper cell (Th1) ADs, including rheumatoid arthritis (RA), multiple sclerosis (MS), corneal transplant rejection, uveitis, Type I diabetes, schizophrenia (anti-IFN gamma and anti-TNF alpha), and various autoimmune skin diseases (alopecia areata, psoriasis vulgaris, vitiligo, pemphigus vulgaris and epidermolysis bullosa). A strong, sometimes striking, therapeutic response followed administration of anti-IFN gamma, indicating that it may be a promising therapy for Th1 ADs.

  12. Liposomal formulation of {alpha}-tocopheryl maleamide: In vitro and in vivo toxicological profile and anticancer effect against spontaneous breast carcinomas in mice

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Turanek, Jaroslav; Wang Xiufang; Knoetigova, Pavlina

    2009-06-15

    The vitamin E analogue {alpha}-tocopheryl succinate ({alpha}-TOS) is an efficient anti-cancer drug. Improved efficacy was achieved through the synthesis of {alpha}-tocopheryl maleamide ({alpha}-TAM), an esterase-resistant analogue of {alpha}-tocopheryl maleate. In vitro tests demonstrated significantly higher cytotoxicity of {alpha}-TAM towards cancer cells (MCF-7, B16F10) compared to {alpha}-TOS and other analogues prone to esterase-catalyzed hydrolysis. However, in vitro models demonstrated that {alpha}-TAM was cytotoxic to non-malignant cells (e.g. lymphocytes and bone marrow progenitors). Thus we developed lyophilized liposomal formulations of both {alpha}-TOS and {alpha}-TAM to solve the problem with cytotoxicity of free {alpha}-TAM (neurotoxicity and anaphylaxis), as well as the low solubilitymore » of both drugs. Remarkably, neither acute toxicity nor immunotoxicity implicated by in vitro tests was detected in vivo after application of liposomal {alpha}-TAM, which significantly reduced the growth of cancer cells in hollow fiber implants. Moreover, liposomal formulation of {alpha}-TAM and {alpha}-TOS each prevented the growth of tumours in transgenic FVB/N c-neu mice bearing spontaneous breast carcinomas. Liposomal formulation of {alpha}-TAM demonstrated anti-cancer activity at levels 10-fold lower than those of {alpha}-TOS. Thus, the liposomal formulation of {alpha}-TAM preserved its strong anti-cancer efficacy while eliminating the in vivo toxicity found of the free drug applied in DMSO. Liposome-based targeted delivery systems for analogues of vitamin E are of interest for further development of efficient and safe drug formulations for clinical trials.« less

  13. Binding-dependent disorder-order transition in PKI alpha: a fluorescence anisotropy study.

    PubMed

    Hauer, J A; Taylor, S S; Johnson, D A

    1999-05-25

    The conformational flexibility of peptidyl ligands may be an essential element of many peptide-macromolecular interactions. Consequently, the alpha-carbonyl backbone flexibility of the 8 kDa protein kinase inhibitor (PKI alpha) peptide of cAMP-dependent protein kinase (cAPK) free in solution and bound to cAPK was assessed by time-resolved fluorescence anisotropy. Specifically, three full-length, single-site PKI alpha mutants (V3C, S28C, and S59C) were prepared, and fluorescein iodoacetamide (FI) was selectively conjugated to the side chains of each substituted cysteine. The time-resolved anisotropy decay profiles of the labeled mutants were well fit to a model-free nonassociative biexponential equation. Free in solution, the three labeled proteins had very similar anisotropy decays arising primarily from local alpha-carbonyl backbone movements. Only a small fraction of the anisotropy decay was associated with slower, whole-body tumbling, confirming that PKI alpha is highly disordered at all three locations. Complexation of the mutants with the catalytic (C) subunit of cAPK decreased the rate of whole-body tumbling for all three mutants. The effects on the rapid decay processes, however, were dependent upon the site of conjugation. The anisotropy decay profiles of both FI-V3C- and FI-S28C-PKI alpha were associated with significantly reduced contributions from the fast decay processes, while that of FI-S59C-PKI alpha was largely unaffected by binding to the C-subunit. The results suggest that the cAPK-binding domain of PKI alpha extends from the its N-terminus to residues beyond Ser28 but does not include the segment around Ser59, which is still part of a highly flexible domain when bound to the C-subunit.

  14. Fab fragment labeled with ICG-derivative for detecting digestive tract cancer.

    PubMed

    Yano, Hiromi; Muguruma, Naoki; Ito, Susumu; Aoyagi, Eriko; Kimura, Tetsuo; Imoto, Yoshitaka; Cao, Jianxin; Inoue, Shohei; Sano, Shigeki; Nagao, Yoshimitsu; Kido, Hiroshi

    2006-09-01

    In previous studies, we generated infrared ray fluorescence-labeled monoclonal antibodies and developed an infrared ray fluorescence endoscope capable of detecting the monoclonal antibodies to establish a novel diagnostic technique for gastrointestinal cancer. Although the whole IgG molecule has commonly been used for preparation of labeled antibodies, labeled IgG displays insufficient sensitivity and specificity, probably resulting from non-specific binding of the Fc fragment to target cells or interference between fluorochromes on the identical labeled antibody, which might be caused by molecular structure. In this in vitro study, we characterized an Fc-free fluorescence-labeled Fab fragment, which was expected to yield more specific binding to target cells than the whole IgG molecule. An anti-mucin antibody and ICG-ATT, an ICG derivative, were used as the labeled antibody and labeling compound, respectively. Paraffin sections of excised gastric cancer tissues were subjected to staining. The labeled whole IgG molecule (ICG-ATT-labeled IgG) and the labeled Fab fragment (ICG-ATT-labeled Fab) were prepared according to a previous report, and the fluorescence properties, antibody activities, and features of fluorescence microscope images obtained from paraffin sections were compared. Both ICG-ATT-labeled Fab and ICG-ATT-labeled IgG were excited by a near infrared ray of 766nm, and maximum emission occurred at 804nm. Antibody activities of ICG-ATT-labeled Fab were shown to be similar to those of unlabeled anti-MUC1 antibody. The fluorescence intensity obtained from paraffin sections of excised gastric cancer tissues revealed a tendency to be greater with ICG-ATT-labeled Fab than with ICG-ATT-labeled IgG. The infrared ray fluorescence-labeled Fab fragment was likely to be more specific than the conventionally labeled antibodies. Fragmentation of antibodies is considered to contribute to improved sensitivity and specificity of labeled antibodies for detection of micro

  15. Identification of a major continuous epitope of human alpha crystallin

    NASA Technical Reports Server (NTRS)

    Takemoto, L.; Emmons, T.; Spooner, B. S. (Principal Investigator)

    1992-01-01

    Human lens proteins were digested with trypsin or V8 protease, and the resulting peptides resolved on a C18 reverse phase column. Fractions from this column were probed with polyclonal antiserum made against the whole alpha crystallin molecule. Peptides in the seropositive fraction were purified to homogeneity, then characterized by mass spectral analysis and partial Edman degradation. The tryptic and V8 digests contained only one seropositive peptide that was derived from the C-terminal region of the alpha-A molecule. To determine the exact boundaries of the epitope, various size analogues of this region were synthesized and probed with anti-alpha serum. Together, these studies demonstrate that the major continuous epitope of the alpha-A chain includes the sequence KPTSAPS, corresponding to residues 166-172 of the human alpha-A crystallin chain.

  16. The effect of low-carbohydrates calorie-restricted diet on visceral adipose tissue and metabolic status in psoriasis patients receiving TNF-alpha inhibitors: results of an open label controlled, prospective, clinical study.

    PubMed

    Campanati, A; Molinelli, E; Ganzetti, G; Giuliodori, K; Minetti, I; Taus, M; Catani, M; Martina, E; Conocchiari, L; Offidani, A

    2017-05-01

    TNF alpha inhibitors are usually associated with anthropometric changes over the time, however whether and how the visceral adipose tissue (VAT) is involved in this phenomenon, still remains unclear. Aim of the study is to evaluate if the increases in trunk fat percentage (TF%) and VAT are directly involved in anthropometric changes occurring during treatment, and whether and how a calorie restricted diet could prevent these changes. Twenty patients receiving TNF-alpha inhibitors for psoriasis was evaluated at baseline (T0) and after 24 weeks of therapy (T24), and then compared with 25 patients receiving a combined treatment based on TNF alpha inhibitors and low-carbohydrates calorie-restricted diet. TNF-alpha inhibitors do not influence the VAT expression. The combined treatment is associated with a significant decrease in body weight (kg) (p < .0001), BMI (p = .0001), WC (cm) (p < .0001), TF% (p < .0001), VAT (p < .0001), serum levels of triglycerides (mg/dL) (p = .0018) and total cholesterol (mg/dL) (p = .0005). The administration of TNF-alpha inhibitors can induce anthropometric changes after 24 weeks, but it does not cause an increase in VAT. The association between low-carbohydrates calorie-restricted diet and anti-TNF-alpha therapy seems to be able to improve the anthropometric profile of psoriasis patients.

  17. MHC drives TCR repertoire shaping, but not maturation, in recent thymic emigrants1

    PubMed Central

    Houston, Evan G.; Fink, Pamela J.

    2009-01-01

    After developing in the thymus, recent thymic emigrants (RTEs) enter the lymphoid periphery and undergo a maturation process as they transition into the mature naïve (MN) T cell compartment. This maturation presumably shapes RTEs into a pool of T cells best fit to function robustly in the periphery without causing autoimmunity; however, the mechanism and consequences of this maturation process remain unknown. Using a transgenic mouse system that specifically labels RTEs, we tested the influence of MHC molecules, key drivers of intrathymic T cell selection and naive peripheral T cell homeostasis, in shaping the RTE pool in the lymphoid periphery. We found that the TCRs expressed by RTEs are skewed to longer CDR3 regions compared to those of MN T cells, suggesting that MHC does streamline the TCR repertoire of T cells as they transition from the RTE to the MN T cell stage. This conclusion is borne out in studies in which the representation of individual TCRs was followed as a function of time since thymic egress. Surprisingly, we found that MHC is dispensable for the phenotypic and functional maturation of RTEs. This is an author-produced version of a manuscript accepted for publication in The Journal of Immunology (The JI). The American Association of Immunologists, Inc. (AAI), publisher of The JI, holds the copyright to this manuscript. This version of the manuscript has not yet been copyedited or subjected to editorial proofreading by The JI; hence, it may differ from the final version published in The JI (online and in print). AAI (The JI) is not liable for errors or omissions in this author-produced version of the manuscript or in any version derived from it by the U.S. National Institutes of Health or any other third party. The final, citable version of record can be found at www.jimmunol.org PMID:19915060

  18. Secondary anchor polymorphism in the HA-1 minor histocompatibility antigen critically affects MHC stability and TCR recognition

    PubMed Central

    Nicholls, Sarah; Piper, Karen P.; Mohammed, Fiyaz; Dafforn, Timothy R.; Tenzer, Stefan; Salim, Mahboob; Mahendra, Premini; Craddock, Charles; van Endert, Peter; Schild, Hansjörg; Cobbold, Mark; Engelhard, Victor H.; Moss, Paul A. H.; Willcox, Benjamin E.

    2009-01-01

    T cell recognition of minor histocompatibility antigens (mHags) underlies allogeneic immune responses that mediate graft-versus-host disease and the graft-versus-leukemia effect following stem cell transplantation. Many mHags derive from single amino acid polymorphisms in MHC-restricted epitopes, but our understanding of the molecular mechanisms governing mHag immunogenicity and recognition is incomplete. Here we examined antigenic presentation and T-cell recognition of HA-1, a prototypic autosomal mHag derived from single nucleotide dimorphism (HA-1H versus HA-1R) in the HMHA1 gene. The HA-1H peptide is restricted by HLA-A2 and is immunogenic in HA-1R/R into HA-1H transplants, while HA-1R has been suggested to be a “null allele” in terms of T cell reactivity. We found that proteasomal cleavage and TAP transport of the 2 peptides is similar and that both variants can bind to MHC. However, the His>Arg change substantially decreases the stability and affinity of HLA-A2 association, consistent with the reduced immunogenicity of the HA-1R variant. To understand these findings, we determined the structure of an HLA-A2-HA-1H complex to 1.3Å resolution. Whereas His-3 is accommodated comfortably in the D pocket, incorporation of the lengthy Arg-3 is predicted to require local conformational changes. Moreover, a soluble TCR generated from HA-1H-specific T-cells bound HA-1H peptide with moderate affinity but failed to bind HA-1R, indicating complete discrimination of HA-1 variants at the level of TCR/MHC interaction. Our results define the molecular mechanisms governing immunogenicity of HA-1, and highlight how single amino acid polymorphisms in mHags can critically affect both MHC association and TCR recognition. PMID:19234124

  19. Pak2 is required for actin cytoskeleton remodeling, TCR signaling, and normal thymocyte development and maturation

    PubMed Central

    Phee, Hyewon; Au-Yeung, Byron B; Pryshchep, Olga; O'Hagan, Kyle Leonard; Fairbairn, Stephanie Grace; Radu, Maria; Kosoff, Rachelle; Mollenauer, Marianne; Cheng, Debra; Chernoff, Jonathan; Weiss, Arthur

    2014-01-01

    The molecular mechanisms that govern thymocyte development and maturation are incompletely understood. The P21-activated kinase 2 (Pak2) is an effector for the Rho family GTPases Rac and Cdc42 that regulate actin cytoskeletal remodeling, but its role in the immune system remains poorly understood. In this study, we show that T-cell specific deletion of Pak2 gene in mice resulted in severe T cell lymphopenia accompanied by marked defects in development, maturation, and egress of thymocytes. Pak2 was required for pre-TCR β-selection and positive selection. Surprisingly, Pak2 deficiency in CD4 single positive thymocytes prevented functional maturation and reduced expression of S1P1 and KLF2. Mechanistically, Pak2 is required for actin cytoskeletal remodeling triggered by TCR. Failure to induce proper actin cytoskeletal remodeling impaired PLCγ1 and Erk1/2 signaling in the absence of Pak2, uncovering the critical function of Pak2 as an essential regulator that governs the actin cytoskeleton-dependent signaling to ensure normal thymocyte development and maturation. DOI: http://dx.doi.org/10.7554/eLife.02270.001 PMID:24843022

  20. Shikonins, phytocompounds from Lithospermum erythrorhizon, inhibit the transcriptional activation of human tumor necrosis factor alpha promoter in vivo.

    PubMed

    Staniforth, Vanisree; Wang, Sheng-Yang; Shyur, Lie-Fen; Yang, Ning-Sun

    2004-02-13

    Tumor necrosis factor alpha (TNF-alpha) contributes to the pathogenesis of both acute and chronic inflammatory diseases and has been a target for the development of new anti-inflammatory drugs. Shikonins, the naphthoquinone pigments present in the root tissues of Lithospermum erythrorhizon Sieb. et Zucc. (Boraginaceae), have been reported to exert anti-inflammatory effects both in vitro and in vivo. In this study, we evaluated the effects of shikonin and its derivatives on the transcriptional activation of human TNF-alpha promoter in a gene gun-transfected mouse skin system by using a luciferase reporter gene assay. The crude plant extract of L. erythrorhizon as well as derived individual compounds shikonin, isobutyryl shikonin, acetyl shikonin, dimethylacryl shikonin and isovaleryl shikonin showed significant dose-dependent inhibition of TNF-alpha promoter activation. Among the tested compounds, shikonin and isobutyryl shikonin exhibited the highest inhibition of TNF-alpha promoter activation and also showed significant suppression of transgenic human TNF-alpha mRNA expression and protein production. We demonstrated that shikonin-inhibitory response was retained in the core TNF-alpha promoter region containing the TATA box and a 48-bp downstream sequence relative to the transcription start site. Further our results indicated that shikonin suppressed the basal transcription and activator-regulated transcription of TNF-alpha by inhibiting the binding of transcription factor IID protein complex (TATA box-binding protein) to TATA box. These in vivo results suggest that shikonins inhibit the transcriptional activation of the human TNF-alpha promoter through interference with the basal transcription machinery. Thus, shikonins may have clinical potential as anti-inflammatory therapeutics.

  1. CD4+ CD25high Foxp3+ regulatory T cells downregulate human Vδ2+ T-lymphocyte function triggered by anti-CD3 or phosphoantigen

    PubMed Central

    Mahan, C Scott; Thomas, Jeremy J; Boom, W Henry; Rojas, Roxana E

    2009-01-01

    Vδ2+ T cells, the major circulating T-cell receptor-γδ-positive (TCR-γδ+) T-cell subset in healthy adults, are involved in immunity against many microbial pathogens including Mycobacterium tuberculosis. Vδ2+ T cells recognize small phosphorylated metabolites (phosphoantigens), expand in response to whole M. tuberculosis bacilli, and complement the protective functions of CD4+ T cells. CD4+ CD25high Foxp3+ T cells (Tregs) comprise 5–10% of circulating T cells and are increased in patients with active tuberculosis (TB). We investigated whether, in addition to their known role in suppressing TCR-αβ+ lymphocytes, Tregs suppress Vδ2+ T-cell function. We found that depletion of Tregs from peripheral blood mononuclear cells increased Vδ2+ T-cell expansion in response to M. tuberculosis (H37Ra) in tuberculin-skin-test-positive donors. We developed a suppression assay with fluorescence-activated cell sorting-purified Tregs and Vδ2+ T cells by coincubating the two cell types at a 1 : 1 ratio. The Tregs partially suppressed interferon-γ secretion by Vδ2+ T cells in response to anti-CD3 monoclonal antibody plus interleukin-2 (IL-2). In addition, Tregs downregulated the Vδ2+ T-cell interferon-γ responses induced by phosphoantigen (BrHPP) and IL-2. Under the latter conditions there was no TCR stimulus for Tregs and therefore IL-2 probably triggered suppressor activity. Addition of purified protein derivative (PPD) increased the suppression of Vδ2+ T cells, suggesting that PPD activated antigen-specific Tregs. Our study provides evidence that Tregs suppress both anti-CD3 and antigen-driven Vδ2+ T-cell activation. Antigen-specific Tregs may therefore contribute to the Vδ2+ T-cell functional deficiencies observed in TB. PMID:19019089

  2. V(D)J recombination and allelic exclusion of a TCR beta-chain minilocus occurs in the absence of a functional promoter.

    PubMed

    Alvarez, J D; Anderson, S J; Loh, D Y

    1995-08-01

    Transcriptional activation of rearranging Ag receptor gene segments has been hypothesized to regulate their accessibility to V(D)J recombination. We analyzed the role of a functional promoter in the rearrangement of the murine TCR beta-chain locus using two transgenic minilocus constructs. These miniloci each contain an unrearranged V beta 8.3 gene. One has a wild-type V beta 8.3 gene, but the other has a V beta 8.3 gene with a promoter mutation that was previously shown to abrogate transcription in tissue culture. FACS analysis of thymus and lymph node cells from transgenic mouse lines showed that only the lines with the wild-type V beta 8.3 gene promoter express an 8.3 TCR beta-chain. Consistent with the protein expression data, V beta 8.3 gene transcripts were found only in the transgenic lines with the wild-type promoter. Using a quantitative PCR-based assay, it was shown that both types of transgenic lines recombine the V beta 8.3 gene at similar levels. Rearrangement of the transgenes was normal with respect to thymic development and junctional reading frame. Interestingly, both types of miniloci also underwent allelic exclusion in that recombination was blocked by the expression of a rearranged TCR beta-chain transgene. We conclude that a functional V beta gene promoter is not necessary for proper V(D)J recombination to occur.

  3. Mono-PEGylation of Alpha-MMC and MAP30 from Momordica charantia L.: Production, Identification and Anti-Tumor Activity.

    PubMed

    Sun, Yun; Sun, Fenghui; Li, Jianlong; Wu, Minlu; Fan, Xiang; Meng, Yanfa; Meng, Yao

    2016-10-31

    PEGylation is a well-established and effective strategy to decrease immunogenicity, which can increase the stability and in vivo half-life time. However, the generation of multi-site modified products is inevitable due to the lysine chemistry, which will bring difficulties in subsequent research, such as purification and quantification. Site-specific modification by mPEG-succinimidyl carbonate (mPEG-SC) is a widely used method for N -terminal conjugation. In this study, we used it for site-directed modification on two ribosome-inactivating proteins (RIPs), alpha-momorcharin (α-MMC) and momordica anti-HIV protein (MAP30), from Momordica charantia L. According to the optimization of previous modification conditions, we compared Macro-Cap SP with SP-Sepharose FF chromatography for separating the final mPEGylated RIPs. Two kinds of methods both can obtain homogenous mPEGylated RIPs which were identified by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE), isoelectric focusing electrophoresis (IEF), and matrix-assisted laser desorption ionization-time of flight/time of flight (MALDI-TOF/TOF) analysis. We also used iodine staining method to detect the amount of unmodified PEG. Furthermore, the inhibition activity of both mPEGylated and non-PEGylated RIPs against human lung adenocarcinoma epithelial A549 cells was detected. All of the results suggested that the mPEGylated α-MMC/MAP30 might be potentially developed as new anti-tumor drugs.

  4. Isolation of mouse pancreatic alpha, beta, duct and acinar populations with cell surface markers.

    PubMed

    Dorrell, Craig; Grompe, Maria T; Pan, Fong Cheng; Zhong, Yongping; Canaday, Pamela S; Shultz, Leonard D; Greiner, Dale L; Wright, Chris V; Streeter, Philip R; Grompe, Markus

    2011-06-06

    Tools permitting the isolation of live pancreatic cell subsets for culture and/or molecular analysis are limited. To address this, we developed a collection of monoclonal antibodies with selective surface labeling of endocrine and exocrine pancreatic cell types. Cell type labeling specificity and cell surface reactivity were validated on mouse pancreatic sections and by gene expression analysis of cells isolated using FACS. Five antibodies which marked populations of particular interest were used to isolate and study viable populations of purified pancreatic ducts, acinar cells, and subsets of acinar cells from whole pancreatic tissue or of alpha or beta cells from isolated mouse islets. Gene expression analysis showed the presence of known endocrine markers in alpha and beta cell populations and revealed that TTR and DPPIV are primarily expressed in alpha cells whereas DGKB and GPM6A have a beta cell specific expression profile. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  5. Tumor necrosis factor-alpha antagonists improve aortic stiffness in patients with inflammatory arthropathies: a controlled study.

    PubMed

    Angel, Kristin; Provan, Sella Aarrestad; Gulseth, Hanne Løvdahl; Mowinckel, Petter; Kvien, Tore Kristian; Atar, Dan

    2010-02-01

    The chronic inflammatory state of rheumatoid arthritis and other inflammatory arthropathies, such as ankylosing spondylitis and psoriatic arthritis, contributes to the accelerated atherosclerosis associated with these conditions. This study evaluates the effect of treatment with tumor necrosis factor (TNF)-alpha antagonists on arterial stiffness in patients with inflammatory arthropathies. A total of 60 patients with rheumatoid arthritis, ankylosing spondylitis, or psoriatic arthritis and clinical indication for anti-TNF-alpha therapy were included. Thirty-five patients started with anti-TNF-alpha therapy and were compared with a nontreatment group of 25 patients. Aortic stiffness (aortic pulse wave velocity), augmentation index, and disease activity were assessed at baseline and after 3 months. Aortic pulse wave velocity (mean+/-SD) was reduced in the treatment group but not in the control group (-0.50+/-0.78 m/s versus 0.05+/-0.54 m/s, respectively; P=0.002). Concomitantly, C-reactive protein and the disease activity score were reduced in the treatment group (-9.3+/-20.2 mg/L [P<0.001] and -0.74+/-0.91 [P=0.004]). Augmentation index remained unchanged in both groups (0.1+/-7.1% versus -1.0+/-5.8%, respectively; P=0.53). In a multivariate linear regression model, only treatment with TNF-alpha antagonist and change in mean arterial pressure predicted alterations in aortic pulse wave velocity. In summary, anti-TNF-alpha therapy improved aortic stiffness in patients with inflammatory arthropathies. These findings support the idea that anti-inflammatory treatment has a favorable effect on cardiovascular risk in patients with inflammatory arthropathies.

  6. Synthesis, Characterisation, Molecular Docking, Anti-microbial and Anti-diabetic Screening of Substituted 4-indolylphenyl-6-arylpyrimidine-2-imine Derivatives.

    PubMed

    Ramya, Veerasamy; Vembu, Santhirakasu; Ariharasivakumar, Ganesan; Gopalakrishnan, Manathusamy

    2017-09-01

    The purpose of the research is to synthesise a novel series of (E)-2-(4-(1H-indol-3-yl)-6-p-substituted phenylpyrimidin-2-yl)dimethylguanidine derivatives since 3-(1H-indol-3-yl)-1-p-substituted phenylprop-2-en-1-one and evaluate their molecular docking studies, antimicrobial, and anti-diabetic activities. Among all the synthesized compounds ( 11a-g ), compound 11a exhibits excellent CDOCKER energy (-11.36 kcal/mol). The entire compounds ( 11a-g ) confirm very good antimicrobial activity towards the tested microorganisms. In the in vitro anti-diabetic studies, compounds (11a, 11c, and 11g) confirm higher alpha-amylase and alpha-glucosidase inhibition activity. In the in vivo anti-diabetic activities, the synthesized compounds (11a-g) (10 mg/kg, p.o.) investigated by the streptozotocin (60 mg/kg, ip) -nicotinamide (120 mg/kg, p.o.) - induced model in adult male albino Wistar rat and these derivatives show considerable fasting blood glucose level when compared to metformin hydrochloride a potent and well-known anti-diabetic drug as a reference. © Georg Thieme Verlag KG Stuttgart · New York.

  7. T-cell receptor variable genes and genetic susceptibility to celiac disease: an association and linkage study.

    PubMed

    Roschmann, E; Wienker, T F; Gerok, W; Volk, B A

    1993-12-01

    Genetic susceptibility of celiac disease is primarily associated with a particular combination of and HLA-DQA1/DQB1 gene; however, this does not fully account for the genetic predisposition. Therefore, the aim of this study was to examine whether T-cell receptor (TCR) genes may be susceptibility genes in celiac disease. HLA class II typing was performed by polymerase chain reaction amplification in combination with sequence-specific oligonucleotide hybridization. TCR alpha (TCRA), TCR gamma (TCRG), and TCR beta (TCRB) loci were investigated by restriction fragment length polymorphism analysis. Allelic frequencies of TCRA, TCRG, and TCRB variable genes were compared between patients with celiac disease (n = 53) and control patients (n = 67), and relative risk (RR) estimates were calculated. The RR was 1.67 for allele C1 at TCRA1, 3.35 for allele D2 at TCRA2, 1.66 for allele B2 at TCRG, and 1.35 for allele B at TCRB, showing no significant association. Additionally, linkage analysis was performed in 23 families. The logarithm of odd scores for celiac disease vs. the TCR variable genes at TCRA, TCRG, and TCRB showed no significant linkage. These data suggest that the analyzed TCR variable gene segments V alpha 1.2, V gamma 11, and V beta 8 do not play a major role in susceptibility to celiac disease.

  8. Identification of the protein responsible for pyruvate transport into rat liver and heart mitochondria by specific labelling with [3H]N-phenylmaleimide.

    PubMed

    Thomas, A P; Halestrap, A P

    1981-05-15

    1. N-Phenylmaleimide irreversibly inhibits pyruvate transport into rat heart and liver mitochondria to a much greater extent than does N-ethylmaleimide, iodoacetate or bromopyruvate. alpha-Cyanocinnamate protects the pyruvate transporter from attack by this thiol-blocking reagent. 2. In both heart and liver mitochondria alpha-cyanocinnamate diminishes labelling by [3H]N-phenylmaleimide of a membrane protein of subunit mol.wt. 15000 on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 3. Exposure of mitochondrial to unlabelled N-phenylmaleimide in the presence of alpha-cyanocinnamate, followed by removal of alpha-cyanocinnamate and exposure to [3H]N-phenylmaleimide, produced specific labelling of the same protein. 4. Both labelling and kinetic experiments with inhibitors gave values for the approximate amount of carrier present in liver and heart mitochondria of 100 and 450 pmol/mg of mitochondrial protein respectively. 5. The turnover numbers for net pyruvate transport and pyruvate exchange at 0 degrees C were 6 and 200 min-1 respectively.

  9. Anti-inflammatory effect of resveratrol on TNF-{alpha}-induced MCP-1 expression in adipocytes

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Zhu Jian; Key Laboratory of Human Functional Genomics of Jiangsu Province, School of Basic Medical Science, Jiangsu Province Diabetes Center, Nanjing Medical University, 140 Hanzhong Road, Nanjing 210029; Yong Wei

    2008-05-02

    Chronic low-grade inflammation characterized by adipose tissue macrophage accumulation and abnormal cytokine production is a key feature of obesity and type 2 diabetes. Adipose-tissue-derived monocyte chemoattractant protein (MCP)-1, induced by cytokines, has been shown to play an essential role in the early events during macrophage infiltration into adipose tissue. In this study we investigated the effects of resveratrol upon both tumor necrosis factor (TNF)-{alpha}-induced MCP-1 gene expression and its underlying signaling pathways in 3T3-L1 adipoctyes. Resveratrol was found to inhibit TNF-{alpha}-induced MCP-1 secretion and gene transcription, as well as promoter activity, which based on down-regulation of TNF-{alpha}-induced MCP-1 transcription. Nuclearmore » factor (NF)-{kappa}B was determined to play a major role in the TNF-{alpha}-induced MCP-1 expression. Further analysis showed that resveratrol inhibited DNA binding activity of the NF-{kappa}B complex and subsequently suppressed NF-{kappa}B transcriptional activity in TNF-{alpha}-stimulated cells. Finally, the inhibition of MCP-1 may represent a novel mechanism of resveratrol in preventing obesity-related pathologies.« less

  10. Tumor necrosis factor alpha mediates resistance to Trypanosoma cruzi infection in mice by inducing nitric oxide production in infected gamma interferon-activated macrophages.

    PubMed Central

    Silva, J S; Vespa, G N; Cardoso, M A; Aliberti, J C; Cunha, F Q

    1995-01-01

    Cell invasion by Trypanosoma cruzi and its intracellular replication are essential for continuation of the parasite life cycle and for production of Chagas' disease. T. cruzi is able to replicate in nucleated cells and can be killed by activated macrophages. Gamma interferon (IFN-gamma) is one of the major stimuli for the activation of macrophages and has been shown to be a key activation factor for the killing of intracellular parasites through a mechanism dependent upon nitric oxide (NO) biosynthesis. We show that although the addition of exogenous tumor necrosis factor alpha (TNF-alpha) does not potentiate the trypanocidal activity of IFN-gamma in vitro, treatment of resistant C57BI/6 mice with an anti-TNF-alpha monoclonal antibody increased parasitemia and mortality. In addition, the anti-TNF-alpha-treated animals had decreased NO production, both in vivo and in vitro, suggesting an important role for TNF-alpha in controlling infection. In order to better understand the role of TNF-alpha in the macrophage-mediating killing of parasites, cultures of T. cruzi-infected macrophages were treated with an anti-TNF-alpha monoclonal antibody. IFN-gamma-activated macrophages failed to kill intracellular parasites following treatment with 100 micrograms of anti-TNF-alpha. In these cultures, the number of parasites released at various time points after infection was significantly increased while NO production was significantly reduced. We conclude that IFN-gamma-activated macrophages produce TNF-alpha after infection by T. cruzi and suggest that this cytokine plays a role in amplifying NO production and parasite killing. PMID:7591147

  11. The future role of anti-tumour necrosis factor (TNF) products in the treatment of rheumatoid arthritis.

    PubMed

    Camussi, G; Lupia, E

    1998-05-01

    Tumour necrosis factor-alpha (TNF alpha) is a pleiotropic cytokine which is overproduced in rheumatoid joints primarily by macrophages. This cytokine has a potential pathogenic role in the establishment of rheumatoid synovitis, in the formation of pannus tissue and in the process of joint destruction, as it increases synoviocyte proliferation and triggers a cascade of secondary mediators involved in the recruitment of inflammatory cells, in neo-angiogenesis and in the process of joint destruction. These findings made TNF alpha a potential target for anticytokine therapy. Experimental studies have shown that TNF alpha blockade by monoclonal antibodies or by soluble TNF receptor reduced the extent and severity of arthritis both in collagen-induced arthritis in mice and in transgenic mice overexpressing TNF alpha, which develop a rheumatoid-like destructive arthritis. Clinical studies based on the use of anti-TNF alpha antibodies or soluble receptors have suggested a potential beneficial effect of TNF alpha-blocking therapy in inducing amelioration of inflammatory parameters in patients with long-standing active disease. In these patients anti-TNF alpha therapy induces a rapid improvement in multiple clinical assessment of disease activity, including morning stiffness, pain score, Ritchie articular index and swollen joint count. The clinical benefits are associated with an improvement in some serological parameters, such as C-reactive protein and serum amyloid-A, erythrocyte sedimentation rate, blood cytokine levels, haemoglobin, white cells and platelet counts, rheumatoid factor titre and histological features of the synovium. However, it remains to be determined whether anti-TNF alpha therapy may be useful in the long term management of rheumatoid patients and in the achievement of better outcomes of disease. Because TNF alpha production also serves a specific function in host defence against infections and tumours, the adverse effects of long term anti-TNF alpha

  12. The effect of anti-tumor necrosis factor alpha agents on the outcome in pediatric uveitis of diverse etiologies.

    PubMed

    Deitch, Iris; Amer, Radgonde; Tomkins-Netzer, Oren; Habot-Wilner, Zohar; Friling, Ronit; Neumann, Ron; Kramer, Michal

    2018-04-01

    This study aimed to report the clinical outcome of children with uveitis treated with anti-tumor necrosis factor alpha (TNF-α) agents. This included a retrospective cohort study. Children with uveitis treated with infliximab or adalimumab in 2008-2014 at five dedicated uveitis clinics were identified by database search. Their medical records were reviewed for demographic data, clinical presentation, ocular complications, and visual outcome. Systemic side effects and the steroid-sparing effect of treatment were documented. The cohort included 24 patients (43 eyes) of whom 14 received infliximab and 10 received adalimumab after failing conventional immunosuppression therapy. Mean age was 9.3 ± 4.0 years. The most common diagnosis was juvenile idiopathic arthritis-related uveitis (n = 10), followed by Behçet's disease (n = 4), sarcoidosis (n = 1), and ankylosing spondylitis (n = 1); eight had idiopathic uveitis. Ocular manifestations included panuveitis in 20 eyes (46.5%), chronic anterior uveitis in 19 (44.2%), and intermediate uveitis in 4 (9.3%). The duration of biologic treatment ranged from 6 to 72 months. During the 12 months prior to biologic treatment, while on conventional immunosuppressive therapy, mean visual acuity deteriorated from 0.22 to 0.45 logMAR, with a trend of recovery to 0.25 at 3 months after initiation of biologic treatment, remaining stable thereafter. A full corticosteroid-sparing effect was demonstrated in 16 of the 19 patients (84.2%) for whom data were available. Treatment was well tolerated. Treatment of pediatric uveitis with anti-TNF-α agents may improve outcome while providing steroid-sparing effect, when conventional immunosuppression fails. The role of anti-TNF-α agents as first-line treatment should be further investigated in controlled prospective clinical trials.

  13. Inhibition of anti-CD3 monoclonal antibody-induced T-cell proliferation and interleukin-2 secretion by physiologic combinations of dexamethasone and prostaglandin E2.

    PubMed

    Elliott, L; Brooks, W; Roszman, T

    1993-12-01

    1. The purpose of these studies was to characterize further previous observations from our laboratory indicating that physiologic concentrations of dexamethasone (DEX) and prostaglandin E2 (PGE2) added together result in synergistic inhibition of the proliferative response of T cells stimulated via the T-cell receptor CD3 signaling complex (TCR/CD3). 2. Various physiologic concentrations of DEX and PGE2 were added to T cells stimulated with immobilized anti-CD3 monoclonal antibody (mAb) and cultured at optimal and suboptimal cell densities. The results demonstrate that the proliferative response of anti-CD3 mAb-stimulated T cells cultured at a suboptimal cell density is more suppressed than that of T cells cultured at optimal concentrations. 3. The proliferative response of CD4+ T cells to immobilized anti-CD3 mAb was also determined in the presence of PGE2 and DEX. The data indicate that the CD4+ subset of T cells is more sensitive to the synergistic antiproliferative effects of DEX and PGE2 compared to whole T-cell populations. 4. Various concentrations of DEX and/or PGE2 were added to T cells stimulated with anti-CD3 mAb and the secretion of interleukin-2 (IL-2) was determined. The results demonstrate that concentrations of DEX and PGE2 which individually do not significantly suppress IL-2 synthesis act together to inhibit the synthesis of IL-2 synergistically. 5. The addition of an exogenous source of recombinant IL-2 (rIL-2) to T cells stimulated in the presence of DEX and PGE2 completely reversed the synergistic antiproliferative effect of these two compounds. This reversal was even more pronounced with T cells cultured at a suboptimal cell density. Additionally, PGE2 and DEX did not affect expression of the IL-2 receptor (IL-2R), as measured by upregulation of the alpha chain, on anti-CD3 mAb stimulated T cells. 6. Collectively these data indicate that physiologic concentrations of PGE2 and DEX, which alone have no effect on anti-CD3 mAb-induced T

  14. Label-free optical biosensing with slot-waveguides.

    PubMed

    Barrios, Carlos A; Bañuls, María José; González-Pedro, Victoria; Gylfason, Kristinn B; Sánchez, Benito; Griol, Amadeu; Maquieira, A; Sohlström, H; Holgado, M; Casquel, R

    2008-04-01

    We demonstrate label-free molecule detection by using an integrated biosensor based on a Si(3)N(4)/SiO(2) slot-waveguide microring resonator. Bovine serum albumin (BSA) and anti-BSA molecular binding events on the sensor surface are monitored through the measurement of resonant wavelength shifts with varying biomolecule concentrations. The biosensor exhibited sensitivities of 1.8 and 3.2 nm/(ng/mm(2)) for the detection of anti-BSA and BSA, respectively. The estimated detection limits are 28 and 16 pg/mm(2) for anti-BSA and BSA, respectively, limited by wavelength resolution.

  15. CACA-TOCSY with alternate 13C-12C labeling: a 13Calpha direct detection experiment for mainchain resonance assignment, dihedral angle information, and amino acid type identification.

    PubMed

    Takeuchi, Koh; Frueh, Dominique P; Sun, Zhen-Yu J; Hiller, Sebastian; Wagner, Gerhard

    2010-05-01

    We present a (13)C direct detection CACA-TOCSY experiment for samples with alternate (13)C-(12)C labeling. It provides inter-residue correlations between (13)C(alpha) resonances of residue i and adjacent C(alpha)s at positions i - 1 and i + 1. Furthermore, longer mixing times yield correlations to C(alpha) nuclei separated by more than one residue. The experiment also provides C(alpha)-to-sidechain correlations, some amino acid type identifications and estimates for psi dihedral angles. The power of the experiment derives from the alternate (13)C-(12)C labeling with [1,3-(13)C] glycerol or [2-(13)C] glycerol, which allows utilizing the small scalar (3)J(CC) couplings that are masked by strong (1)J(CC) couplings in uniformly (13)C labeled samples.

  16. Aerosol-administered alpha-tocopherol attenuates lung inflammation in rats given lipopolysaccharide intratracheally.

    PubMed

    Hybertson, Brooks M; Chung, Jin H; Fini, Mehdi A; Lee, Young M; Allard, Jenny D; Hansen, Brian N; Cho, Okyong J; Shibao, Gayle N; Repine, John E

    2005-04-01

    Intrapulmonary administration of bacterial lipopolysaccharide (LPS) induces a well-characterized lung inflammatory response involving alveolar macrophage activation, proinflammatory cytokine elaboration, and neutrophil influx. Vitamin E, a lipophilic antioxidant consisting of a family that includes tocopherols and tocotrienols, has previously been shown to have a variety of anti-inflammatory effects, raising interest in its possible uses in disease prevention or therapy. Because aerosol delivery is a specific and rapid way to administer agents to the lungs, the authors undertook to determine whether inhaled vitamin E aerosols would have an anti-inflammatory effect in the lungs. Using a rat model of acute lung inflammation caused by intratracheally administered LPS (10 microg Pseudomonas aeruginosa LPS), the authors examined the effect of aerosol-administered vitamin E, in this case alpha-tocopherol, on several indices of lung inflammation which are increased by LPS treatment. It was found that inhaled alpha-tocopherol aerosol, but not inhaled alpha-tocopherol acetate aerosol, decreased tumor necrosis factor alpha (TNFalpha) and cytokine-induced neutrophil chemoattractant-1 (CINC-1) mRNA levels in lung tissue, TNFalpha and CINC-1 immunoreactive protein levels in lung lavage, and the number of neutrophils recoverable by lung lavage from rats given LPS intratracheally. These results contribute to the increasing body of work describing immunomodulatory functions of alpha-tocopherol, and support the idea that direct aerosol administration of alpha-tocopherol may play a beneficial role in strategies to control inflammatory lung illnesses.

  17. Tannic acid induces in vitro acantholysis of keratinocytes via IL-1alpha and TNF-alpha.

    PubMed

    Feliciani, C; Ruocco, E; Zampetti, A; Toto, P; Amerio, Pa; Tulli, A; Amerio, P; Ruocco, V

    2007-01-01

    The mechanism of acantholysis in pemphigus vulgaris (PV) is an intriguing argument since several chemical mediators are implicated. We previously reported a central role for IL-1alpha and TNF- alpha, both able to regulate complement activation and plasminogen activators. Very little is known about what triggers the disease (drugs, viruses or food). In this study, we evaluate the molecular role of tannins in acantholysis. By HPLC chromatography we measured tannic acid (TA) and gallic acid (GA) in blister fluid of 4 groups of patients divided according to their dietary habits, including a regular diet, a diet rich in tannins, a diet free of tannins, and a group of pemphigus patients. Blister fluid was obtained from patients using a suction blister apparatus. We show that people with a diet rich in tannins have increased tannin metabolites (TA and GA) in the skin in respect to controls (tannin-rich diet: GA = 194.52+/-2.39 nmol/ml; TA = 348.28+/-1.4 nmol/ml versus tannin-Mediterranean diet: GA = 15.28+/-1.63 nmol/ml; TA = 22.81+/-1.68 nmol/ml). PV patients showed similar values to the Mediterranean diet population (PV patients: GA = 95.8+/-1.97 nmol/ml; TA = 199.09+/-4.15 nmol/ml versus Mediterranean diet: GA = 83.53+/-2.35 nmol/ml; TA = 195.1+/-2.50 nmol/ml). In an in vitro acantholysis system using TA and PV-IgG we show that TA 0.1 mM in NHEK culture is able to induce acantholysis. This effect was able to amplify the acantholytic action of PV-IgG in vitro. A blocking study using anti IL-1 alpha and anti TNF-alpha antibodies showed a reduction in TA-induced acantholysis. Taken together, these results suggest that a diet rich in tannins could be a trigger in genetically predisposed patients. If these data are confirmed, a complementary diet poor in tannins may be useful in patients affected by PV.

  18. A Higher Activation Threshold of Memory CD8+ T Cells Has a Fitness Cost That Is Modified by TCR Affinity during Tuberculosis.

    PubMed

    Carpenter, Stephen M; Nunes-Alves, Cláudio; Booty, Matthew G; Way, Sing Sing; Behar, Samuel M

    2016-01-01

    T cell vaccines against Mycobacterium tuberculosis (Mtb) and other pathogens are based on the principle that memory T cells rapidly generate effector responses upon challenge, leading to pathogen clearance. Despite eliciting a robust memory CD8+ T cell response to the immunodominant Mtb antigen TB10.4 (EsxH), we find the increased frequency of TB10.4-specific CD8+ T cells conferred by vaccination to be short-lived after Mtb challenge. To compare memory and naïve CD8+ T cell function during their response to Mtb, we track their expansions using TB10.4-specific retrogenic CD8+ T cells. We find that the primary (naïve) response outnumbers the secondary (memory) response during Mtb challenge, an effect moderated by increased TCR affinity. To determine whether the expansion of polyclonal memory T cells is restrained following Mtb challenge, we used TCRβ deep sequencing to track TB10.4-specific CD8+ T cells after vaccination and subsequent challenge in intact mice. Successful memory T cells, defined by their clonal expansion after Mtb challenge, express similar CDR3β sequences suggesting TCR selection by antigen. Thus, both TCR-dependent and -independent factors affect the fitness of memory CD8+ responses. The impaired expansion of the majority of memory T cell clonotypes may explain why some TB vaccines have not provided better protection.

  19. A Higher Activation Threshold of Memory CD8+ T Cells Has a Fitness Cost That Is Modified by TCR Affinity during Tuberculosis

    PubMed Central

    Carpenter, Stephen M.; Nunes-Alves, Cláudio; Booty, Matthew G.; Way, Sing Sing; Behar, Samuel M.

    2016-01-01

    T cell vaccines against Mycobacterium tuberculosis (Mtb) and other pathogens are based on the principle that memory T cells rapidly generate effector responses upon challenge, leading to pathogen clearance. Despite eliciting a robust memory CD8+ T cell response to the immunodominant Mtb antigen TB10.4 (EsxH), we find the increased frequency of TB10.4-specific CD8+ T cells conferred by vaccination to be short-lived after Mtb challenge. To compare memory and naïve CD8+ T cell function during their response to Mtb, we track their expansions using TB10.4-specific retrogenic CD8+ T cells. We find that the primary (naïve) response outnumbers the secondary (memory) response during Mtb challenge, an effect moderated by increased TCR affinity. To determine whether the expansion of polyclonal memory T cells is restrained following Mtb challenge, we used TCRβ deep sequencing to track TB10.4-specific CD8+ T cells after vaccination and subsequent challenge in intact mice. Successful memory T cells, defined by their clonal expansion after Mtb challenge, express similar CDR3β sequences suggesting TCR selection by antigen. Thus, both TCR-dependent and -independent factors affect the fitness of memory CD8+ responses. The impaired expansion of the majority of memory T cell clonotypes may explain why some TB vaccines have not provided better protection. PMID:26745507

  20. CP-25, a Novel Anti-inflammatory and Immunomodulatory Drug, Inhibits the Functions of Activated Human B Cells through Regulating BAFF and TNF-alpha Signaling and Comparative Efficacy with Biological Agents.

    PubMed

    Zhang, Feng; Shu, Jin-Ling; Li, Ying; Wu, Yu-Jing; Zhang, Xian-Zheng; Han, Le; Tang, Xiao-Yu; Wang, Chen; Wang, Qing-Tong; Chen, Jing-Yu; Chang, Yan; Wu, Hua-Xun; Zhang, Ling-Ling; Wei, Wei

    2017-01-01

    Paeoniflorin-6'- O -benzene sulfonate (code: CP-25) was the chemistry structural modifications of Paeoniflorin (Pae). CP-25 inhibited B cells proliferation stimulated by B cell activating factor belonging to the TNF family (BAFF) or Tumor necrosis factor alpha (TNF-alpha). CP-25, Rituximab and Etanercept reduced the percentage and numbers of CD19 + B cells, CD19 + CD20 + B cells, CD19 + CD27 + B cells and CD19 + CD20 + CD27 + B cells induced by BAFF or TNF-alpha. There was significant difference between CP-25 and Rituximab or CP-25 and Etanercept. CP-25 down-regulated the high expression of BAFFR, BCMA, and TACI stimulated by BAFF or TNF-alpha. The effects of Rituximab and Etanercept on BAFFR or BCMA were stronger than that of CP-25. CP-25, Rituximab and Etanercept down-regulated significantly the expression of TNFR1 and TNFR2 on B cell stimulated by BAFF or TNF-alpha. CP-25, Rituximab and Etanercept down-regulated the expression of MKK3, P-p38, P-p65, TRAF2, and p52 in B cells stimulated by BAFF and the expression of TRAF2 and P-p65 in B cells stimulated by TNF-alpha. These results suggest that CP-25 regulated moderately activated B cells function by regulating the classical and alternative NF-κB signaling pathway mediated by BAFF and TNF-alpha-TRAF2-NF-κB signaling pathway. This study suggests that CP-25 may be a promising anti-inflammatory immune and soft regulation drug.

  1. Ultrasensitive Label-free Electrochemical Immunosensor based on Multifunctionalized Graphene Nanocomposites for the Detection of Alpha Fetoprotein

    PubMed Central

    Wang, Yaoguang; Zhang, Yong; Wu, Dan; Ma, Hongmin; Pang, Xuehui; Fan, Dawei; Wei, Qin; Du, Bin

    2017-01-01

    In this work, a novel label-free electrochemical immunosensor was developed for the quantitative detection of alpha fetoprotein (AFP). Multifunctionalized graphene nanocomposites (TB-Au-Fe3O4-rGO) were applied to modify the electrode to achieve the amplification of electrochemical signal. TB-Au-Fe3O4-rGO includes the advantages of graphene, ferroferric oxide nanoparticles (Fe3O4 NPs), gold nanoparticles (Au NPs) and toluidine blue (TB). As a kind of redox probe, TB can produce the electrochemical signal. Graphene owns large specific surface area, high electrical conductivity and good adsorption property to load a large number of TB. Fe3O4 NPs have good electrocatalytic performance towards the redox of TB. Au NPs have good biocompatibility to capture the antibodies. Due to the good electrochemical performance of TB-Au-Fe3O4-rGO, the effective and sensitive detection of AFP was achieved by the designed electrochemical immunosensor. Under optimal conditions, the designed immunosensor exhibited a wide linear range from 1.0 × 10−5 ng/mL to 10.0 ng/mL with a low detection limit of 2.7 fg/mL for AFP. It also displayed good electrochemical performance including good reproducibility, selectivity and stability, which would provide potential applications in the clinical diagnosis of other tumor markers. PMID:28186128

  2. Anti-idiotypic antibodies that protect cells against the action of diphtheria toxin

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Rolf, J.M.; Gaudin, H.M.; Tirrell, S.M.

    1989-03-01

    An anti-idiotypic serum prepared against the combining site (idiotype) of specific anti-diphtheria toxoid antibodies was characterized with respect to its interaction with highly diphtheria toxin-sensitive Vero cells. Although the anti-idiotypic serum protected Vero cells against the cytotoxic action of diphtheria toxin, it did not prevent the binding of /sup 125/I-labeled diphtheria toxin to the cells but did inhibit the internalization and degradation of /sup 125/I-labeled toxin. This anti-idiotypic serum immunoprecipitated a cell-surface protein from radiolabeled Vero cells with an apparent Mr of approximately 15,000. These results are consistent with the hypothesis that the anti-idiotypic serum contains antibodies that carry anmore » internal image of an internalization site on the toxin and that a cell-surface protein involved in toxin internalization possesses a complementary site recognized by both the toxin and the anti-idiotypic antibodies.« less

  3. Copper-64-labeled anti-bcl-2 PNA-peptide conjugates selectively localize to bcl-2-positive tumors in mouse models of B-cell lymphoma.

    PubMed

    Jia, Fang; Balaji, Baghavathy S; Gallazzi, Fabio; Lewis, Michael R

    2015-11-01

    The bcl-2 gene is overexpressed in non-Hodgkin's lymphoma (NHL). We have reported micro-SPECT/CT imaging of Mec-1 human lymphoma xenografts in SCID mice, using [(111)In]DOTA-anti-bcl-2-PNA-Tyr(3)-octreotate. In order to reduce normal organ accumulation and improve imaging contrast, modified monomers with neutral hydrophilic (serine, TS) or negatively charged (aspartic acid, TD) residues were synthesized as substitutes for glycine at T(14) in the PNA sequence. The parent and modified PNA-peptide conjugates were labeled with (64)Cu and evaluated in biodistribution studies and high resolution PET/CT imaging of SCID mice bearing bcl-2-positive Mec-1 xenografts as well as bcl-2-negative Ramos xenografts. Mice were administered the (64)Cu-labeled conjugates for biodistribution and imaging studies. Biodistributions were obtained from 1 to 48 h post-injection. Mice were imaged from 1 to 48 h post-injection. The parent glycine conjugate and two modified conjugates all showed selective tumor uptake in Mec-1 xenografts. The liver uptake of the serine conjugate was significantly reduced compared to the two other PNA conjugates. Its kidney uptake was highest of the three at 47.1% ID/g at 1h and dropped to 20.6% ID/g at 24h. [Copper-64]DOTA-anti-bcl-2-TS-PNA-Tyr(3)-octreotate showed tumor uptake of 1.38% ID/g at 1h and 1.06% ID/g at 24h. The tumor-to-blood ratio was increased by factor of 2 from 1h to 24h. This compound detected Mec-1 tumors by micro-PET/CT as early as 1h post-injection and at time points out to 48 h. However, the negative control Ramos tumor could not be detected. These (64)Cu-labeled, amino acid-modified PNA conjugates showed selective tumor targeting in vivo, and tumor xenografts were detected by micro-PET/CT as early as 1h post-injection, suggesting that bcl-2 expression at the mRNA level can detected by PET in mouse models of NHL. Advances in knowledge and implications for patient care Down-regulating bcl-2, an anti-apoptotic proto-oncogene, is a mechanism

  4. Antioxidant and anti-inflammatory activities of alpha lipoic acid protect against indomethacin-induced gastric ulcer in rats.

    PubMed

    Gomaa, Asmaa M S; Abd El-Mottaleb, Nashwa A; Aamer, Hazem A

    2018-05-01

    Little is known about the role of tumor necrosis factor-alpha (TNF-α), plasminogen activator inhibitor-1 (PAI-1), and inducible nitric oxide synthase (iNOS) in the gastric ulcer and the effect of alpha lipoic acid (ALA) in their modulation. Hence, this experimental study was designed to assess the possible protective effect of ALA against indomethacin (IND)-induced gastric ulcer in rats, as well as to determine the possible underlying mechanisms with a special focus on TNF-α, PAI-1, and iNOS. Adult male rats (n = 28) were divided into four equal groups: the control group received distilled water, the vehicle group received 0.5% carboxymethylcellulose, the ulcer group received a single oral dose of IND (50 mg/kg) and the ALA-treated group received ALA (100 mg/kg) orally for 3 days before ulcer induction. Four hours after IND administration, all rats were sacrificed. The ulcer index, and gastric tissue homogenate contents of total antioxidant capacity (TAC), malondialdehyde (MDA), TNF-α, and PAI-1 were evaluated. Immunohistochemical evaluation of iNOS protein expression and histopathological examination of gastric tissue were investigated. The results revealed that ALA pretreatment significantly decreased the ulcer index, the gastric levels of MDA, TNF-α, PAI-1, and iNOS protein expression while increased the gastric levels of TAC as well as improved the histopathological appearance of gastric tissues. In conclusion, ALA ameliorated the IND-induced gastric ulceration. This could be attributed to its antioxidant and anti-inflammatory activities via suppression of TNF-α-induced elevation of both PAI-1 level and iNOS expression in the gastric tissue. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

  5. Estimating TCR using an integrated model-observation framework that accounts for spatio-temporal variability and pre-industrial radiative imbalances.

    NASA Astrophysics Data System (ADS)

    Haustein, K.; Schurer, A. P.; Venema, V.

    2016-12-01

    Apart from a few exceptions (e.g. Aldrin et al. 2012, Skeie et al. 2013) TCR estimates with EBMs are based on global data. Since these estimates don't represent the true spatial-temporal behaviour for observed temperature as well as external forcing (Marvel et al. 2015), we have developed a two-box EBM framework that accounts for these effects. In addition, external forcing from anthropogenic aerosol and GHGs tends to have different response times in comparison to volcanic stratospheric aerosols. Using PMIP3 and an extended ensemble of HadCM3 simulations (Euro500; Schurer et al. 2014) GCM simulations for the pre-industrial period, we obtain the fast and slow response time constants required in the EBM. With the most recent anthropogenic and natural forcing estimates, we test a range of TCR values against observations. The TCR/ECS ratio necessary to achieve that goal is taken from CMIP5 as observationally OHC-based estimates are notoriously unreliable. Given that observed and modelled OHC estimates are in agreement (Cheng et al. 2016), we argue that this should be the standard procedure the make inferences about ECS. Alternatively, it should be distinguished between equilibrium and effective climate sensitivity. The preliminary best estimate for TCR is 1.6K (1.1-2.2K) with an associated ECS value of 2.9K (2.0-4.0K). This is in good agreement with other D&A techniques that do use spatio-temporal patterns as well (e.g. Jones et al. 2016, Gillet et al. 2013). Correcting for natural ENSO variability and tas/tos-related inaccuracies (Richardson et al. 2016) further increases the robustness of the estimated sensitivity range. Our results also indicate that the small radiative imbalance caused by the period of very strong volcanic eruptions just before the CMIP5 historical period starts (1809-1840) has noteworthy implications for the response to later volcanic eruptions and the temperature evolution after 1850. Simply put, CMIP5-type simulations are slightly more sensitive

  6. Treatment of rheumatoid arthritis with chimeric monoclonal antibodies to tumor necrosis factor alpha.

    PubMed

    Elliott, M J; Maini, R N; Feldmann, M; Long-Fox, A; Charles, P; Katsikis, P; Brennan, F M; Walker, J; Bijl, H; Ghrayeb, J

    1993-12-01

    To evaluate the safety and efficacy of a chimeric monoclonal antibody to tumor necrosis factor alpha (TNF alpha) in the treatment of patients with rheumatoid arthritis (RA). Twenty patients with active RA were treated with 20 mg/kg of anti-TNF alpha in an open phase I/II trial lasting 8 weeks. The treatment was well tolerated, with no serious adverse events. Significant improvements were seen in the Ritchie Articular Index, which fell from a median of 28 at study entry to a median of 6 by week 6 (P < 0.001), the swollen joint count, which fell from 18 to 5 (P < 0.001) over the same period, and in the other major clinical assessments. Serum C-reactive protein levels fell from a median of 39.5 mg/liter at study entry to 8 mg/liter at week 6 (P < 0.001), and significant decreases were also seen in serum amyloid A and interleukin-6 levels. Treatment with anti-TNF alpha was safe and well tolerated and resulted in significant clinical and laboratory improvements. These preliminary results support the hypothesis that TNF alpha is an important regulator in RA, and suggest that it may be a useful new therapeutic target in this disease.

  7. Phosphorylation and desensitization of alpha1d-adrenergic receptors.

    PubMed Central

    García-Sáinz, J A; Vázquez-Cuevas, F G; Romero-Avila, M T

    2001-01-01

    In rat-1 fibroblasts stably expressing rat alpha(1d)-adrenoceptors, noradrenaline and PMA markedly decreased alpha(1d)-adrenoceptor function (noradrenaline-elicited increases in calcium in whole cells and [(35)S]guanosine 5'-[gamma-thio]triphosphate binding in membranes), suggesting homologous and heterologous desensitizations. Photoaffinity labelling, Western blotting and immunoprecipitation identified alpha(1d)-adrenoceptors as a broad band of 70-80 kDa. alpha(1d)-Adrenoceptors were phosphorylated in the basal state and noradrenaline and PMA increased it. The effect of noradrenaline was concentration-dependent (EC(50) 75 nM), rapid (maximum at 1 min) and transient. Phorbol ester-induced phosphorylation was concentration-dependent (EC(50) 25 nM), slightly slower (maximum at 5 min) and stable for at least 60 min. Inhibitors of protein kinase C decreased the effect of phorbol esters but not that of noradrenaline. Evidence of cross-talk of alpha(1d)-adrenoceptors with receptors endogenously expressed in rat-1 fibroblasts was given by the ability of endothelin, lysophosphatidic acid and bradykinin to induce alpha(1d)-adrenoceptor phosphorylation. In summary, it is shown for the first time here that alpha(1d)-adrenoceptors are phosphoproteins and that receptor phosphorylation is increased by the natural ligand, noradrenaline, by direct activation of protein kinase C and via cross-talk with other receptors endogenously expressed in rat-1 fibroblasts. Receptor phosphorylation has functional repercussions. PMID:11171057

  8. Tc-labeling of Peptidomimetic Antagonist to Selectively Target alpha(v)beta(3) Receptor-Positive Tumor: Comparison of PDA and EDDA as co-Ligands.

    PubMed

    Shin, In Soo; Maeng, Jin Soo; Jang, Beom-Su; You, Eric; Cheng, Kenneth; Li, King C P; Wood, Bradford; Carrasquillo, Jorge A; Danthi, S Narasimhan; Paik, Chang H

    2010-01-01

    OBJECTIVES: The aim of this research was to synthesize radiolabeled peptidomimetic integrin alpha(v)beta(3) antagonist with (99m)Tc for rapid targeting of integrin alpha(v)beta(3) receptors in tumor to produce a high tumor to background ratio. METHODS: The amino terminus of 4-[2-(3,4,5,6-tetra-hydropyrimidin-2-ylamino)-ethyloxy]benzoyl-2-(S)-[N-(3-amino-neopenta-1-carbamyl)]-aminoethylsulfonyl-amino-beta-alanine hydrochloride (IAC) was conjugated with N-hydroxysuccinimide ester of HYNIC and labeled with (99m)Tc using tricine with either 1,5-pyridinedicarboxylic acid (PDA) or ethylenediamine-N,N'-diacetic acid (EDDA) as the co-ligand. The products, (99m)Tc EDDA(2)/HYNIC-IAC (P1) and (99m)Tc PDA (tricin)/HYNIC-IAC (P2) were subjected to in vitro serum stability, receptor-binding, biodistribution and imaging studies. RESULTS: P1 and P2 were synthesized with an overall yield of >80%. P1 was slightly more stable than P2 when incubated in serum at 37 degrees C for 18 hrs (84 vs 77% intact). The In vitro receptor-binding of P1 was higher than that of P2 (78.02 +/- 13.48 vs 51.05 +/- 14.05%) when incubated with alpha(v)beta(3) at a molar excess (0.8 muM). This receptor binding was completely blocked by a molar excess of an unlabeled peptidomimetic antagonist. Their differences shown in serum stability and the receptor-binding appeared to be related to their biological behaviors in tumor uptake and retention; the 1 h tumor uptakes of P1 and P2 were 3.17+/-0.52 and 2.13+/-0.17 % ID/g, respectively. P1 was retained in the tumor longer than P2. P1 was excreted primarily through the renal system whereas P2 complex was excreted equally via both renal and hepatobiliary systems. Thus, P1 was retained in the whole-body with 27.25 +/- 3.67% ID at 4 h whereas 54.04 +/- 3.57% ID of P2 remained in the whole-body at 4 h. This higher whole-body retention of P2 appeared to be resulted from a higher amount of radioactivity retained in liver and intestine. These findings were supported by

  9. Crossing symmetry in alpha space

    NASA Astrophysics Data System (ADS)

    Hogervorst, Matthijs; van Rees, Balt C.

    2017-11-01

    We initiate the study of the conformal bootstrap using Sturm-Liouville theory, specializing to four-point functions in one-dimensional CFTs. We do so by decomposing conformal correlators using a basis of eigenfunctions of the Casimir which are labeled by a complex number α. This leads to a systematic method for computing conformal block decompositions. Analyzing bootstrap equations in alpha space turns crossing symmetry into an eigenvalue problem for an integral operator K. The operator K is closely related to the Wilson transform, and some of its eigenfunctions can be found in closed form.

  10. T-cell receptor accessory and co-receptor molecules in channel catfish

    USDA-ARS?s Scientific Manuscript database

    T cell receptor (TCR) associated invariant chains CD3gamma/delta,epsilon, and zeta as well as TCR co-receptors CD8alpha and CD8beta were isolated from the channel catfish, Ictalurus punctatus, at both the gene and cDNA levels. All of catfish CD3 sequences encode for proteins that resemble their resp...

  11. Combination alpha-interferon and lamivudine therapy for alpha-interferon-resistant chronic hepatitis B infection: results of a pilot study.

    PubMed

    Mutimer, D; Naoumov, N; Honkoop, P; Marinos, G; Ahmed, M; de Man, R; McPhillips, P; Johnson, M; Williams, R; Elias, E; Schalm, S

    1998-06-01

    Alpha-interferon achieves seroconversion in about one third of naive patients. Attempts to achieve seroconversion in patients who have previously failed alpha-interferon have proved disappointing. Combination chemotherapy (alpha-interferon with a nucleoside analogue) might provide a treatment alternative for these patients. We have undertaken a phase 2 study in 20 patients who had previously failed at least one course of alpha-interferon. The study was designed to assess the safety, tolerability and efficacy of the combination. All patients were treated for 16 weeks with alpha-interferon in combination with 12 or 16 weeks of Lamivudine (3'TC). Patients were followed for 16 weeks post-treatment. Pharmacokinetic studies were performed to identify/exclude significant pharmacokinetic drug interaction. The combination was well tolerated, and side-effects of the combination were indistinguishable from the recognised side-effects of alpha-interferon. Pharmacokinetic studies performed on days 1 and 29 did not show any significant interaction. All patients achieved HBV DNA clearance during treatment, but 19 relapsed at the end of treatment. HBeAg/anti-HBe seroconversion was observed for four patients, but was sustained for a single patient (who also had sustained DNA clearance). Combination therapy with alpha-interferon and lamivudine given for 16 weeks appears safe and is well tolerated. However, for this group of patients who had previously failed interferon monotherapy, the efficacy of combination interferon/lamivudine therapy appears disappointing, and other treatment strategies should be investigated.

  12. 6-Gingerol inhibits ROS and iNOS through the suppression of PKC-{alpha} and NF-{kappa}B pathways in lipopolysaccharide-stimulated mouse macrophages

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lee, Tzung-Yan, E-mail: joyamen@mail.cgu.edu.tw; Lee, Ko-Chen; Center for Traditional Chinese Medicine, Chang Gung Memorial Hospital, Taoyuan, Taiwan

    2009-04-24

    Inflammation is involved in numerous diseases, including chronic inflammatory diseases and the development of cancer. Many plants possess a variety of biological activities, including antifungal, antibacterial and anti-inflammatory activities. However, our understanding of the anti-inflammatory effects of 6-gingerol is very limited. We used lipopolysaccharide (LPS)-stimulated macrophages as a model of inflammation to investigate the anti-inflammatory effects of 6-gingerol, which contains phenolic structure. We found that 6-gingerol exhibited an anti-inflammatory effect. 6-Gingerol could decrease inducible nitric oxide synthase and TNF-{alpha} expression through suppression of I-{kappa}B{alpha} phosphorylation, NF-{kappa}B nuclear activation and PKC-{alpha} translocation, which in turn inhibits Ca{sup 2+} mobilization and disruptionmore » of mitochondrial membrane potential in LPS-stimulated macrophages. Here, we demonstrate that 6-gingerol acts as an anti-inflammatory agent by blocking NF-{kappa}B and PKC signaling, and may be developed as a useful agent for the chemoprevention of cancer or inflammatory diseases.« less

  13. The opposite role of two UBA-UBX containing proteins, p47 and SAKS1 in the degradation of a single ERAD substrate, α-TCR.

    PubMed

    Park, Eun Sil; Yoo, Yung Joon; Elangovan, Muthukumar

    2017-01-01

    The UBA-UBX domain-containing proteins can interact with ubiquitinated substrates and p97 during endoplasmic reticulum-associated degradation (ERAD). Here, we found that the expressions of all UBA-UBX genes p47, SAKS1, UBXD8, FAF1, and UBXD7 were elevated upon ER stress, albeit with different levels. Of which p47, SAKS1, and UBXD8 are 'immediate' respondents whereas FAF1 and UBXD7 were 'late' respondents to ER stress. Interestingly, the expression of specific UBA-UBX genes were altered in cells stably expressing three different ERAD substrates such as α-TCR, α1-antitrypsin, and δCD3. We first found that p47 and UBXD8 expression levels were increased in α-TCR and α1-antitrypsin stable cell lines, respectively, whereas SAKS1 expression level was reduced in all the three ERAD substrates tested. Of note, we also found p47 promotes, whereas SASK1 delays the degradation of a single ERAD substrate, α-TCR. Additionally, we found that SAKS1 selectively inhibits the degradation of ERAD substrates without affecting cytosolic proteasomal substrates. Taken together, our results identified that UBA-UBX proteins possess substrate selectivity and opposite role of two different UBA-UBX proteins in the degradation of a single ERAD substrate.

  14. Preliminary in vivo efficacy studies of a recombinant rhesus anti-alpha(4)beta(7) monoclonal antibody.

    PubMed

    Pereira, L E; Onlamoon, N; Wang, X; Wang, R; Li, J; Reimann, K A; Villinger, F; Pattanapanyasat, K; Mori, K; Ansari, A A

    2009-01-01

    Recent findings established that primary targets of HIV/SIV are lymphoid cells within the gastrointestinal (GI) tract. Focus has therefore shifted to T-cells expressing alpha(4)beta(7) integrin which facilitates trafficking to the GI tract via binding to MAdCAM-1. Approaches to better understand the role of alpha(4)beta(7)+ T-cells in HIV/SIV pathogenesis include their depletion or blockade of their synthesis, binding and/or homing capabilities in vivo. Such studies can ideally be conducted in rhesus macaques (RM), the non-human primate model of AIDS. Characterization of alpha(4)beta(7) expression on cell lineages in RM blood and GI tissues reveal low densities of expression by NK cells, B-cells, naïve and TEM (effector memory) T-cells. High densities were observed on TCM (central memory) T-cells. Intravenous administration of a single 50mg/kg dose of recombinant rhesus alpha(4)beta(7) antibody resulted in significant initial decline of alpha(4)beta(7)+ lymphocytes and sustained coating of the alpha(4)beta(7) receptor in both the periphery and GI tissues.

  15. Alpha-1 antitrypsin reduces ovariectomy-induced bone loss in mice

    USDA-ARS?s Scientific Manuscript database

    Alpha-1antitrypsin (AAT) is a multifunctional protein with proteinase inhibitor and anti-inflammatory activities. Recent studies showed that AAT has therapeutic effect for diseases associated with inflammation, such as type 1 diabetes and arthritis. Proinflammatory cytokines are primary mediators of...

  16. IgG1 antimycobacterial antibodies can reverse the inhibitory effect of pentoxifylline on tumour necrosis factor alpha (TNF-alpha) secreted by mycobacterial antigen-stimulated adherent cells.

    PubMed

    Thakurdas, S M; Hasan, Z; Hussain, R

    2004-05-01

    Chronic inflammation associated with cachexia, weight loss, fever and arthralgia is the hallmark of advanced mycobacterial diseases. These symptoms are attributed to the chronic stimulation of tumour necrosis factor (TNF)-alpha. Mycobacterial components directly stimulate adherent cells to secrete TNF-alpha. We have shown recently that IgG1 antimycobacterial antibodies play a role in augmenting TNF-alpha in purified protein derivative (PPD)-stimulated adherent cells from non-BCG-vaccinated donors. We now show that IgG1 antibodies can also augment TNF-alpha expression in stimulated adherent cells obtained from BCG-vaccinated donors and this augmentation is not linked to interleukin (IL)-10 secretion. In addition IgG1 antimycobacterial antibodies can reverse the effect of TNF-alpha blockers such as pentoxifylline and thalidomide. These studies therefore have clinical implications for anti-inflammatory drug treatments which are used increasingly to alleviate symptoms associated with chronic inflammation.

  17. Comparative analysis of B7-1 and B7-2 costimulatory ligands: expression and function

    PubMed Central

    1994-01-01

    Antigen-specific T cell activation requires the engagement of the T cell receptor (TCR) with antigen as well as the engagement of appropriate costimulatory molecules. The most extensively characterized pathway of costimulation has been that involving the interaction of CD28 and CTLA4 on the T cell with B7 (now termed B7-1) on antigen presenting cells. Recently, B7-2 a second costimulatory ligand for CTLA4, was described, demonstrating the potential complexity of costimulatory interactions. This report examines and compares the expression and function of B7-1 and B7-2. Overall these results indicate that (a) B7-1 and B7-2 can be expressed by multiple cell types, including B cells, T cells, macrophages, and dendritic cells, all of which are therefore candidate populations for delivering costimulatory signals mediated by these molecules; (b) stimulating B cells with either LPS or anti-IgD-dextran induced expression of both B7- 1 and B7-2, and peak expression of both costimulatory molecules occurred after 18-42 h of culture. Expression of B7-2 on these B cell populations was significantly higher than expression of B7-1 at all times assayed after stimulation; (c) blocking of B7-2 costimulatory activity inhibited TCR-dependent T cell proliferation and cytokine production, without affecting early consequences of TCR signaling such as induction of CD69 or interleukin 2 receptor alpha (IL-2R alpha); and (d) expression of B7-1 and of B7-2 can be regulated by a variety of stimuli. Moreover, expression of B7-1 and B7-2 can be independently regulated by the same stimulus, providing an additional complexity in the mechanisms available for regulating costimulation and hence immune response. PMID:7519245

  18. [Experimental study on TCRbeta idiotypic antigenic determinants DNA vaccine to induce anti-lymphoma antibodies].

    PubMed

    Zhang, Yeping; Zhu, Ping; Shi, Yongjin; Liu, Jihua; Pu, Dingfang; Cao, Xianghong; Zhu, Qiang; Wang, Yijia; Ma, Mingxin; Yu, Jiren

    2002-02-01

    To investigate the anti-human CEM lymphoma cell activities induced by TCR idiotypic DNA vaccine containing different antigen determinants in BALB/c mice. The specific rearranged gene fragment encoding TCRVbeta region of CEM cell line was obtained by RT-PCR technique. The PCR product was cloned into eukaryocytic expression vector pcDNA3, which was used as DNA vaccine and template for PCR amplifying different antigen determinant. Gene fragments encoding different antigen determinant were amplified and cloned into pcDNA3, separately. The experimental mice were immunized by intramuscular injection of the DNA vaccines. The specific anti-idiotype antibodies were detected by indirect immunofluorescence assay. TCRbetaV of CEM cell line contains five antigen determinants. Specific anti-idiotype antibody was detected in all of the six mice immunized with DNA vaccine containing all the five determinants (the highest titer was 1:480). Although the antibody could also be detected in four of the six mice immunized with DNA vaccine containing four of the five antigen determinants, the antibody titer was lower (the highest titer was 1:80). DNA vaccine containing two of the five determinants could not induce the specific antibody. The idiotypic DNA vaccine containing the whole TCRbetaV five antigen determinants could induce the specific anti-lymphoma idiotypic antibody in BALB/c mice.

  19. Interaction of anti-cancer drug-cisplatin with major proteinase inhibitor-alpha-2-macroglobulin: Biophysical and thermodynamic analysis.

    PubMed

    Zia, Mohammad Khalid; Siddiqui, Tooba; Ali, Syed Saqib; Ahsan, Haseeb; Khan, Fahim Halim

    2018-05-09

    Alpha-2-macroglobulin is a multifunctional, highly abundant, plasma protein which reacts with a wide variety of molecules and drugs including cisplatin. Cisplatin is commonly used anticancer drug widely used for treatment of testicular, bladder, ovarian, head and neck, lung and cervical cancers. This study is designed to examine the interaction of cisplatin with human alpha-2-macroglobulin through various biophysical techniques and drug binding through molecular modeling. Cisplatin alters the function of alpha-2-macroglobulin and the thiolesters are most likely the reactive sites for cisplatin. Our result suggests that cisplatin decreases the antiproteolytic potential and causes structural and functional change in human alpha-2-macroglobulin as evident by absorption and fluorescence spectroscopy. Change in secondary structure of alpha-2-macroglobulin was confirmed by CD and FTIR. Thermodynamics parameters such as entropy (ΔS), enthalpy (ΔH) and Gibb's free energy changes (ΔG) along with number of binding sites (N) of alpha-2-macroglobulin-cisplatin binding in solutions were determined by isothermal titration calorimetry (ITC). It was found that binding of cisplatin with alpha-2-macroglobulin was exothermic in nature. The interaction of drug with alpha-2-macroglobulin in the plasma could lead to structural alterations in the conformational status of alpha-2-macroglobulin resulting in its functional inactivation. Copyright © 2018 Elsevier B.V. All rights reserved.

  20. Mitogenic action of tumour necrosis factor-alpha and interleukin-8 on explants of human duodenal mucosa.

    PubMed

    Zachrisson, K; Neopikhanov, V; Wretlind, B; Uribe, A

    2001-08-07

    Our aim is to examine whether tumour necrosis factor-alpha (TNF-alpha) and interleukin affect the mitotic activity in explants of human duodenal mucosa and to estimate the release of cytokines from explants incubated with TNF-alpha. Biopsy specimens of normal duodenal mucosa were taken from 19 subjects that underwent upper endoscopy for investigation of dyspeptic symptoms or chronic gastrointestinal bleeding. The specimens were processed following guidelines for organ culture technique. Paired biopsy specimens from 12 subjects were cultured for 23 h to achieve steady state and thereafter the explants were incubated 25 h with 10(-13)-10(-9) M of TNF-alpha or IL-8. Mitoses were arrested in the metaphase by adding vincristine sulphate for the last three hours. The explants were then fixed and processed for microdissection. Fifteen crypts were microdissected and the total number of metaphases was determined using the whole crypt as reference volume. The number of metaphases per crypt was also estimated in explants incubated with 10(-10) M TNF-alpha in the presence of anti-IL-8 antibodies. Additional duodenal explants from seven subjects were incubated with 10(-10) M TNF-alpha for 25 h. Thereafter the release of IL-1-beta, IL-6, IL-8 and interferon gamma (IFN-gamma) into the culture medium was measured by enzyme immunoassay and expressed as pg/mg protein. TNF-alpha and IL-8 significantly increased the number of metaphases/crypts (P<0.0001). The addition of anti-IL-8 slightly reduced the number of metaphases/crypt compared to the values observed in the explants incubated with 10(-10) M TNF-alpha alone (P<0.0001). The number of metaphases/crypt in the explants incubated with 10(-10) M TNF-alpha in the presence of anti-IL-8 antibodies was, however, markedly and significantly higher than that of the controls (P<0.000). TNF-alpha induced the release of IL-8 (P<0.01) and IL-6 (P<0.05) from the duodenal explants. TNF-alpha and IL-8 are potent mitogens to human small intestinal

  1. Radioiodine Labeled Anti-MIF McAb: A Potential Agent for Inflammation Imaging

    PubMed Central

    Zhang, Chao; Hou, Gui-hua; Han, Jian-kui; Song, Jing; Liang, Ting

    2007-01-01

    Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine that may play a role in the pathogenesis of inflammation. Radiolabeled anti-MIF McAb can be used to detect in vivo inflammatory changes. The objective of this study was to investigate in vivo biology of radioiodinated anti-MIF McAb using the inflammation model mice. Anti-MIF McAb was radioiodinated with NaI125 by Iodogen method. Animal models were induced in the mice by intramuscular injection of S. aureus, E. coli, and turpentine oil. The biodistribution studies with radioiodinated anti-MIF McAb were performed on inflammation mice. The relationship between inflammatory lesions and anti-MIF McAb binding was investigated using the percent of injected dose per gram tissue (% ID/g) of tissue samples and whole-body autoradiography. The radioactivity of I125-anti-MIF McAb in the inflammatory tissue increased gradually for three inflammation models. The highest uptake was found in S. aureus group and the lowest was in E. coli group. The uptake in turpentine oil group was average. Whole-body autoradiography showed that all inflammation foci could be visualized clearly from 24 hours after injection, but 48 hours images were much clearer in accordance with the high T/NT ratio. These results demonstrate the ability of radioiodinated anti-MIF McAb to measure in vivo inflammatory events represented by high expression of MIF and suggests that radiolabeled anti-MIF McAb warrants further investigation as a potential inflammation-seeking agent for imaging to detect inflammatory disorders. PMID:18317509

  2. Immune monitoring and TCR sequencing of CD4 T cells in a long term responsive patient with metastasized pancreatic ductal carcinoma treated with individualized, neoepitope-derived multipeptide vaccines: a case report.

    PubMed

    Sonntag, Katja; Hashimoto, Hisayoshi; Eyrich, Matthias; Menzel, Moritz; Schubach, Max; Döcker, Dennis; Battke, Florian; Courage, Carolina; Lambertz, Helmut; Handgretinger, Rupert; Biskup, Saskia; Schilbach, Karin

    2018-02-06

    Cancer vaccines can effectively establish clinically relevant tumor immunity. Novel sequencing approaches rapidly identify the mutational fingerprint of tumors, thus allowing to generate personalized tumor vaccines within a few weeks from diagnosis. Here, we report the case of a 62-year-old patient receiving a four-peptide-vaccine targeting the two sole mutations of his pancreatic tumor, identified via exome sequencing. Vaccination started during chemotherapy in second complete remission and continued monthly thereafter. We tracked IFN-γ + T cell responses against vaccine peptides in peripheral blood after 12, 17 and 34 vaccinations by analyzing T-cell receptor (TCR) repertoire diversity and epitope-binding regions of peptide-reactive T-cell lines and clones. By restricting analysis to sorted IFN-γ-producing T cells we could assure epitope-specificity, functionality, and T H 1 polarization. A peptide-specific T-cell response against three of the four vaccine peptides could be detected sequentially. Molecular TCR analysis revealed a broad vaccine-reactive TCR repertoire with clones of discernible specificity. Four identical or convergent TCR sequences could be identified at more than one time-point, indicating timely persistence of vaccine-reactive T cells. One dominant TCR expressing a dual TCRVα chain could be found in three T-cell clones. The observed T-cell responses possibly contributed to clinical outcome: The patient is alive 6 years after initial diagnosis and in complete remission for 4 years now. Therapeutic vaccination with a neoantigen-derived four-peptide vaccine resulted in a diverse and long-lasting immune response against these targets which was associated with prolonged clinical remission. These data warrant confirmation in a larger proof-of concept clinical trial.

  3. Interferometer immunosensor based on porous silicon for determining alpha-fetoprotein

    NASA Astrophysics Data System (ADS)

    Lv, Xiaoyi; Jiang, Jing; Lv, Guodong; Mo, Jiaqing; Jia, Zhenhong

    2016-10-01

    An increased level of alpha-fetoprotein ( AFP) in the blood may be a sign of liver cancer. Porous silicon based optical microcavities structure is prepared as a label-free immunosensor platform for detecting AFP. After the antigen-antibody reaction, it is monitored that the red shift of the reflection spectrum of the immunosensor increases

  4. Labelling of histone H5 and its interaction with DNA. 1. Histone H5 labelling with fluorescein isothiocyanate.

    PubMed

    Favazza, M; Lerho, M; Houssier, C

    1990-06-01

    Histone H5 has been labelled with fluorescein isothiocyanate (FITC) with particular attention to the reaction conditions (pH, reaction time and input FITC/H5 molar ratio) and to the complete elimination of non-covalently bound dye. We preferred to use reaction conditions which yielded non-specific uniform labelling rather than specific alpha-NH2 terminal labelling, in order to obtain higher sensitivity in further studies dealing with the detection of perturbation at the binding sites of H5 on DNA. FITC-labelled H5 was further characterized by absorption and circular dichroism spectroscopy, and the fluorescein probe titrated in the 4-8 pH range. The structural integrity of H5 was found to be preserved after labelling. The positive electrostatic potential of the environment in which the FITC probe is embedded in the arginine/lysine-rich tails of H5 is believed to be responsible for the drop of pK of 1 unit found for H5-FITC as compared to free FITC. For the globular part of H5, the pK of covalently-bound FITC was only slightly lowered; this is a consequence of the much lower content in positively-charged amino-acid side chains in this region.

  5. Phthalocyanine tetrasulfonates affect the amyloid formation and cytotoxicity of alpha-synuclein.

    PubMed

    Lee, Eui-Nam; Cho, Hyun-Ju; Lee, Choong-Hwan; Lee, Daekyun; Chung, Kwang Chul; Paik, Seung R

    2004-03-30

    Alpha-synuclein is a pathological component of Parkinson's disease by constituting the filamentous component of Lewy bodies. Phthalocyanine (Pc) effects on the amyloidosis of alpha-synuclein have been examined. The copper complex of phthalocyanine tetrasulfonate (PcTS-Cu(2+)) caused the self-oligomerization of alpha-synuclein while Pc-Cu(2+) did not affect the protein, indicating that introduction of the sulfonate groups was critical for the selective protein interaction. The PcTS-Cu(2+) interaction with alpha-synuclein has occurred predominantly at the N-terminal region of the protein with a K(d) of 0.83 microM apart from the hydrophobic NAC (non-Abeta component of Alzheimer's disease amyloid) segment. Phthalocyanine tetrasulfonate (PcTS) lacking the intercalated copper ion also showed a considerable affinity toward alpha-synuclein with a K(d) of 3.12 microM, and its binding site, on the other hand, was located at the acidic C-terminus. These mutually exclusive interactions between PcTS and PcTS-Cu(2+) toward alpha-synuclein resulted in distinctive features on the kinetics of protein aggregation, morphologies of the final aggregates, and their in vitro cytotoxicities. The PcTS actually suppressed the fibrous amyloid formation of alpha-synuclein, but it produced the chopped-wood-looking protein aggregates. The aggregates showed rather low toxicity (9.5%) on human neuroblastoma cells (SH-SY5Y). In fact, the PcTS was shown to effectively rescue the cell death of alpha-synuclein overexpressing cells caused by the lactacystin treatment as a proteasome inhibitor. The anti-aggregative and anti-amyloidogenic properties of PcTS were also demonstrated with alcohol dehydrogenase, glutathione S-transferase, and amyloid beta/A4 protein under their aggregative conditions. The PcTS-Cu(2+), on the other hand, promoted the protein aggregation of alpha-synuclein, which gave rise to the fibrillar protein aggregates whose cytotoxicity became significant to 35.8%. Taken together, the

  6. Alterations in Intestinal Microbiota Lead to Production of Interleukin 17 by Intrahepatic γδ T-Cell Receptor-Positive Cells and Pathogenesis of Cholestatic Liver Disease.

    PubMed

    Tedesco, Dana; Thapa, Manoj; Chin, Chui Yoke; Ge, Yong; Gong, Minghao; Li, Jing; Gumber, Sanjeev; Speck, Patrick; Elrod, Elizabeth J; Burd, Eileen M; Kitchens, William H; Magliocca, Joseph F; Adams, Andrew B; Weiss, David S; Mohamadzadeh, Mansour; Grakoui, Arash

    2018-06-01

    Variants at the ABCB4 or MDR2 locus, which encodes a biliary transport protein, are associated with a spectrum of cholestatic liver diseases. Exacerbation of liver disease has been linked to increased hepatic levels of interleukin (IL) 17, yet the mechanisms of this increase are not understood. We studied mice with disruption of Mdr2 to determine how defects in liver and alteration in the microbiota contribute to production of IL17 by intrahepatic γδ T cells. We performed studies with Mdr2 -/- and littermate FVB/NJ (control) mice. IL17 was measured in serum samples by an enzyme-linked immunosorbent assay. Mice were injected with neutralizing antibodies against the γδ T-cell receptor (TCR; anti-γδ TCR) or mouse IL17A (anti-IL17A). Livers were collected and bacteria were identified in homogenates by culture procedures; TCRγδ + cells were isolated by flow cytometry. Fecal samples were collected from mice and analyzed by 16S ribosomal DNA sequencing. Cells were stimulated with antibodies or bacteria, and cytokine production was measured. We obtained tissues from 10 patients undergoing liver transplantation for primary sclerosing cholangitis or chronic hepatitis C virus infection. Tissues were analyzed for cytokine production by γδ TCR + cells. Mdr2 -/- mice had collagen deposition around hepatic bile ducts and periportal-bridging fibrosis with influx of inflammatory cells and increased serum levels of IL17 compared with control mice. Administration of anti-IL17A reduced hepatic fibrosis. Livers from Mdr2 -/- mice had increased numbers of IL17A + γδTCR + cells-particularly of IL17A + Vγ6Jγ1 γδ TCR + cells. Fecal samples from Mdr2 -/- mice were enriched in Lactobacillus, and liver tissues were enriched in Lactobacillus gasseri compared with control mice. Mdr2 -/- mice also had increased intestinal permeability. The γδ TCR + cells isolated from Mdr2 -/- livers produced IL17 in response to heat-killed L gasseri. Intraperitoneal injection of control mice

  7. Anti-inflammatory actions of clonidine, guanfacine and B-HT 920 against various inflammagen-induced acute paw oedema in rats.

    PubMed

    Kulkarni, S K; Mehta, A K; Kunchandy, J

    1986-02-01

    Clonidine (0.1-1.0 mg/kg, i.p.) exhibited anti-inflammatory activity in carrageenan-, formalin-, 5-HT- and histamine-induced paw oedema in rats. Similarly, other two alpha 2-adrenoceptor agonists, guanfacine and B-HT 920, also displayed an anti-inflammatory action in these models. The anti-inflammatory effect of all the three alpha 2-adrenoceptor agonists was reversed by yohimbine. However, prazosin failed to block the anti-inflammatory effect of clonidine. Intracerebroventricularly administered clonidine had a delayed onset of anti-inflammatory action, starting only from 60 min post carrageenan administration. This was in contrast to the systemically administered clonidine which was effective against both phases of carrageenan-induced oedema. On the other hand, irrespective of the route of administration, i.e. peripheral or central, guanfacine and B-HT 920 were effective against the early as well as against the delayed phases of the inflammatory reaction. The studies suggest that it is not the imidazoline moiety but the activation of alpha 2-adrenoceptors which is essential for the anti-inflammatory action of these agents.

  8. Interferon-alpha-induced changes in metallothionein expression in liver biopsies from patients with chronic hepatitis C.

    PubMed

    Nagamine, Takeaki; Suzuki, Keiji; Kondo, Toshihiko; Nakazato, Kyomi; Kakizaki, Satoru; Takagi, Hitoshi; Nakajima, Katuyuki

    2005-08-01

    An association between reactive oxygen species and liver damage has been postulated in the course of hepatitis C virus (HCV) infection. Metallothionein (MT), induced by HCV core protein and interferon (IFN), plays a role in scavenging free radicals. MT expression in liver biopsies obtained from 21 patients with chronic HCV infection before and after IFN-alpha therapy was investigated. Changes in Knodell histological activity index (HAI) scores, MT protein levels (immunohistochemistry), MT-I and MT-II messenger (m)RNA expression levels (in situ hybridization) and proliferating cell nuclear antigen (PCNA) labelling index were determined and compared in serial liver specimens. MT staining was clustered around the portal tracts with inflammatory cells and fibrosis. The pattern of MT protein before IFN-alpha therapy was similar in all patients, but was higher in IFN-sustained responders than in nonresponders after IFN-alpha therapy. HAI scores and PCNA labelling indexes were significantly reduced after IFN-alpha therapy. MT-II mRNA expression correlated positively with PCNA index before therapy and with HAI scores after therapy (P<0.05). No correlation was found between MT-I mRNA and HAI scores or PCNA index. The findings indicate that IFN-alpha-induced hepatic MT may participate in the therapeutic effects of IFN-alpha for HCV. In addition, MT-II mRNA expression may be involved in cell proliferation in the livers of patients with chronic HCV infection.

  9. Differential clinical efficacy of anti-CD4 monoclonal antibodies in rat adjuvant arthritis is paralleled by differential influence on NF-κB binding activity and TNF-α secretion of T cells

    PubMed Central

    Pohlers, Dirk; Schmidt-Weber, Carsten B; Franch, Angels; Kuhlmann, Jürgen; Bräuer, Rolf; Emmrich, Frank; Kinne, Raimund W

    2002-01-01

    The aim of this study was to analyze the differential effects of three anti-CD4 monoclonal antibodies (mAbs) (with distinct epitope specifities) in the treatment of rat adjuvant arthritis (AA) and on T-cell function and signal transduction. Rat AA was preventively treated by intraperitoneal injection of the anti-CD4 mAbs W3/25, OX35, and RIB5/2 (on days -1, 0, 3, and 6, i.e. 1 day before AA induction, on the day of induction [day 0], and thereafter). The effects on T-cell reactivity in vivo (delayed-type hypersensitivity), ex vivo (ConA-induced proliferation), and in vitro (mixed lymphocyte culture) were assessed. The in vitro effects of anti-CD4 preincubation on T-cell receptor (TCR)/CD3-induced cytokine production and signal transduction were also analyzed. While preventive treatment with OX35 and W3/25 significantly ameliorated AA from the onset, treatment with RIB5/2 even accelerated the onset of AA by approximately 2 days (day 10), and ameliorated the arthritis only in the late phase (day 27). Differential clinical effects at the onset of AA were paralleled by a differential influence of the mAbs on T-cell functions, i.e. in comparison with OX35 and W3/25, the 'accelerating' mAb RIB5/2 failed to increase the delayed-type hypersentivity (DTH) to Mycobacterium tuberculosis, increased the in vitro tumor necrosis factor (TNF)-α secretion, and more strongly induced NF-κB binding activity after anti-CD4 preincubation and subsequent TCR/CD3-stimulation. Depending on their epitope specificity, different anti-CD4 mAbs differentially influence individual proinflammatory functions of T cells. This fine regulation may explain the differential efficacy in the treatment of AA and may contribute to the understanding of such treatments in other immunopathologies. PMID:12010568

  10. Non-typhi Salmonella infection in patients with rheumatic diseases on TNF-alpha antagonist therapy.

    PubMed

    Peña-Sagredo, J L; Fariñas, M C; Perez-Zafrilla, B; Cruz-Valenciano, A; Crespo, M; Joven-Ibañez, B; Riera, E; Manero-Ruiz, F J; Chalmeta, I; Hernández, M V; Rodríguez-Gómez, M; Maíz, O; López, R; Cobo, T; Pita, J; Carmona, L; Gonzalez-Gay, M A

    2009-01-01

    The morbidity and mortality of patients with rheumatic diseases has improved considerably following the use of biologic therapies. However, an increase in the frequency of bacterial infections has been observed in patients receiving these drugs. In the present study we aimed to establish the incidence and clinical manifestations of non-typhi Salmonella infection in a large cohort of patients with rheumatic diseases undergoing TNF-alpha antagonist therapy due to severe rheumatic diseases refractory to conventional therapies. The rate of non-typhi Salmonella infection found in the Spanish Registry of Adverse Events of Biological Therapies in Rheumatic Diseases (BIOBADASER) was compared with that observed in a cohort of rheumatoid arthritis (RA) patients from the EMECAR (Morbidity and Clinical Expression of Rheumatoid Arthritis) Study, who were not treated with TNF-alpha antagonists. The rate found in the BIOBADASER registry was also compared with that available in a non-RA historic control cohort reported in a population from Huesca (Northern Spain). Seventeen cases of non-typhi Salmonella infection were observed in the series of patients exposed to anti-TNF-alpha therapies. The incidence rate of non-typhi Salmonella in BIOBADASER was 0.73 per 1000 patient-years (95% confidence interval [CI]: 0.45-1.17). The incidence rate in the EMECAR cohort was 0.44 per 1000 patient-years. The relative risk for non-typhi salmonellosis in RA patients exposed to TNF-alpha inhibitors compared to those not treated with biological therapies was 2.07 (95% CI: 0.27-15.73) (p=0.480) whereas the relative risk of non-typhi Salmonella infections in patients with rheumatic diseases undergoing TNF-alpha antagonist therapy compared with the non-RA Spanish control cohort was 0.63 (95% CI: 0.38-1.04) (p=0.07). Nine of the 17 patients with non-typhi salmonellosis presented a severe systemic infection. Incidence of non-typhi Salmonella infection is not increased significantly in rheumatic patients

  11. Tetraspanin CD151 regulates alpha6beta1 integrin adhesion strengthening

    NASA Technical Reports Server (NTRS)

    Lammerding, Jan; Kazarov, Alexander R.; Huang, Hayden; Lee, Richard T.; Hemler, Martin E.

    2003-01-01

    The tetraspanin CD151 molecule associates specifically with laminin-binding integrins, including alpha6beta1. To probe strength of alpha6beta1-dependent adhesion to laminin-1, defined forces (0-1.5 nN) were applied to magnetic laminin-coated microbeads bound to NIH 3T3 cells. For NIH 3T3 cells bearing wild-type CD151, adhesion strengthening was observed, as bead detachment became more difficult over time. In contrast, mutant CD151 (with the C-terminal region replaced) showed impaired adhesion strengthening. Static cell adhesion to laminin-1, and detachment of beads coated with fibronectin or anti-alpha6 antibody were all unaffected by CD151 mutation. Hence, CD151 plays a key role in selectively strengthening alpha6beta1 integrin-mediated adhesion to laminin-1.

  12. Association of CD147 and Calcium Exporter PMCA4 Uncouples IL-2 Expression from Early TCR Signaling.

    PubMed

    Supper, Verena; Schiller, Herbert B; Paster, Wolfgang; Forster, Florian; Boulègue, Cyril; Mitulovic, Goran; Leksa, Vladimir; Ohradanova-Repic, Anna; Machacek, Christian; Schatzlmaier, Philipp; Zlabinger, Gerhard J; Stockinger, Hannes

    2016-02-01

    The Ig superfamily member CD147 is upregulated following T cell activation and was shown to serve as a negative regulator of T cell proliferation. Thus, Abs targeting CD147 are being tested as new treatment strategies for cancer and autoimmune diseases. How CD147 mediates immunosuppression and whether association with other coreceptor complexes is needed have remained unknown. In the current study, we show that silencing of CD147 in human T cells increases IL-2 production without affecting the TCR proximal signaling components. We mapped the immunosuppressive moieties of CD147 to its transmembrane domain and Ig-like domain II. Using affinity purification combined with mass spectrometry, we determined the domain specificity of CD147 interaction partners and identified the calcium exporter plasma membrane calcium ATPase isoform 4 (PMCA4) as the interaction partner of the immunosuppressive moieties of CD147. CD147 does not control the proper membrane localization of PMCA4, but PMCA4 is essential for the CD147-dependent inhibition of IL-2 expression via a calcium-independent mechanism. In summary, our data show that CD147 interacts via its immunomodulatory domains with PMCA4 to bypass TCR proximal signaling and inhibit IL-2 expression. Copyright © 2016 by The American Association of Immunologists, Inc.

  13. Inhibition of HIF-1{alpha} activity by BP-1 ameliorates adjuvant induced arthritis in rats

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Shankar, J.; Thippegowda, P.B., E-mail: btprabha@uic.edu; Kanum, S.A.

    Rheumatoid arthritis (RA) is a chronic inflammatory, angiogenic disease. Inflamed synovitis is a hallmark of RA which is hypoxic in nature. Vascular endothelial growth factor (VEGF), one of the key regulators of angiogenesis, is overexpressed in the pathogenesis of RA. VEGF expression is regulated by hypoxia-inducible factor-1{alpha} (HIF-1{alpha}), a master regulator of homeostasis which plays a pivotal role in hypoxia-induced angiogenesis. In this study we show that synthetic benzophenone analogue, 2-benzoyl-phenoxy acetamide (BP-1) can act as a novel anti-arthritic agent in an experimental adjuvant induced arthritis (AIA) rat model by targeting VEGF and HIF-1{alpha}. BP-1 administered hypoxic endothelial cells andmore » arthritic animals clearly showed down regulation of VEGF expression. Further, BP-1 inhibits nuclear translocation of HIF-1{alpha}, which in turn suppresses transcription of the VEGF gene. These results suggest a further possible clinical application of the BP-1 derivative as an anti-arthritic agent in association with conventional chemotherapeutic agents.« less

  14. Near-infrared emitting fluorescent nanocrystals-labeled natural killer cells as a platform technology for the optical imaging of immunotherapeutic cells-based cancer therapy

    NASA Astrophysics Data System (ADS)

    Taik Lim, Yong; Cho, Mi Young; Noh, Young-Woock; Chung, Jin Woong; Chung, Bong Hyun

    2009-11-01

    This study describes the development of near-infrared optical imaging technology for the monitoring of immunotherapeutic cell-based cancer therapy using natural killer (NK) cells labeled with fluorescent nanocrystals. Although NK cell-based immunotherapeutic strategies have drawn interest as potent preclinical or clinical methods of cancer therapy, there are few reports documenting the molecular imaging of NK cell-based cancer therapy, primarily due to the difficulty of labeling of NK cells with imaging probes. Human natural killer cells (NK92MI) were labeled with anti-human CD56 antibody-coated quantum dots (QD705) for fluorescence imaging. FACS analysis showed that the NK92MI cells labeled with anti-human CD56 antibody-coated QD705 have no effect on the cell viability. The effect of anti-human CD56 antibody-coated QD705 labeling on the NK92MI cell function was investigated by measuring interferon gamma (IFN- γ) production and cytolytic activity. Finally, the NK92MI cells labeled with anti-human CD56 antibody-coated QD705 showed a therapeutic effect similar to that of unlabeled NK92MI cells. Images of intratumorally injected NK92MI cells labeled with anti-human CD56 antibody-coated could be acquired using near-infrared optical imaging both in vivo and in vitro. This result demonstrates that the immunotherapeutic cells labeled with fluorescent nanocrystals can be a versatile platform for the effective tracking of injected therapeutic cells using optical imaging technology, which is very important in cell-based cancer therapies.

  15. Colloid labelled with radionuclide and method

    DOEpatents

    Atcher, Robert W.; Hines, John J.

    1990-01-01

    A ferric hydroxide colloid having an alpha-emitting radionuclide essentially on the outer surfaces and a method of forming same. The method includes oxidizing a ferrous hydroxide to ferric hydroxide in the presence of a preselected radionuclide to form a colloid having the radionuclide on the outer surface thereof, and thereafter washing the colloid, and suspending the washed colloid in a suitable solution. The labelled colloid is useful in cancer therapy and for the treatment of inflamed joints.

  16. Sensitization to the mammalian oligosaccharide galactose-alpha-1,3-galactose (alpha-gal): experience in a Flemish case series.

    PubMed

    Ebo, D G; Faber, M; Sabato, V; Leysen, J; Gadisseur, A; Bridts, C H; De Clerck, L S

    2013-01-01

    Recent observations have disclosed that the galactose-alpha (1,3)-galactose (alpha-gal) moiety of non-primate glycoproteins can constitute a target for meat allergy. To describe adults with allergic reactions to mammalian meat, dairy products and gelatin. To investigate whether patients could demonstrate sensitization to activated recombinant human coagulation factor VII ectapog alpha that is produced in baby hamster kidney cells. Ten adults with mammalian meat, dairy products and gelatin allergies were examined using quantification of specific IgE and/or skin prick test for red meat, milk, milk components, gelatin, cetuximab and eptacog alpha. Most patients demonstrate quite typical clinical histories and serological profiles, with anti-alpha-gal titers varying from less than 1% to over 25% of total serum IgE. All patients demonstrate negative sIgE for gelatin, except the patient with a genuine gelatin allergy. All patients also demonstrated a negative sIgE to recombinant milk components casein, lactalbumin and lactoglobulin. Specific IgE to eptacog was positive in 5 out of the 9 patients sensitized to alpha-gal and none of the 10 control individuals. This series confirms the importance of the alpha-gal carbohydrate moiety as a potential target for allergy to mammalian meat, dairy products and gelatin (oral, topical or parenteral) in a Flemish population of meat allergic adults. It also confirms in vitro tests to mammalian meat generally to be more reliable than mammalian meat skin tests, but that diagnosis can benefit from skin testing with cetuximab. Specific IgE to gelatin is far too insensitive to diagnose alphaa-gal related gelatin allergy. IgE binding studies indicate a potential risk of alpha-gal-containing human recombinant proteins produced in mammalians.

  17. Formatted anti-tumor necrosis factor alpha VHH proteins derived from camelids show superior potency and targeting to inflamed joints in a murine model of collagen-induced arthritis.

    PubMed

    Coppieters, Ken; Dreier, Torsten; Silence, Karen; de Haard, Hans; Lauwereys, Marc; Casteels, Peter; Beirnaert, Els; Jonckheere, Heidi; Van de Wiele, Christophe; Staelens, Ludovicus; Hostens, Jeroen; Revets, Hilde; Remaut, Erik; Elewaut, Dirk; Rottiers, Pieter

    2006-06-01

    The advent of tumor necrosis factor (TNF)-blocking drugs has provided rheumatologists with an effective, but highly expensive, treatment for the management of established rheumatoid arthritis (RA). Our aim was to explore preclinically the application of camelid anti-TNF VHH proteins, which are single-domain antigen binding (VHH) proteins homologous to human immunoglobulin V(H) domains, as TNF antagonists in a mouse model of RA. Llamas were immunized with human and mouse TNF, and antagonistic anti-TNF VHH proteins were isolated and cloned for bacterial production. The resulting anti-TNF VHH proteins were recombinantly linked to yield bivalent mouse and human TNF-specific molecules. To increase the serum half-life and targeting properties, an anti-serum albumin anti-TNF VHH domain was incorporated into the bivalent molecules. The TNF-neutralizing potential was analyzed in vitro. Mouse TNF-specific molecules were tested in a therapeutic protocol in murine collagen-induced arthritis (CIA). Disease progression was evaluated by clinical scoring and histologic evaluation. Targeting properties were evaluated by 99mTc labeling and gamma camera imaging. The bivalent molecules were up to 500 times more potent than the monovalent molecules. The antagonistic potency of the anti-human TNF VHH proteins exceeded even that of the anti-TNF antibodies infliximab and adalimumab that are used clinically in RA. Incorporation of binding affinity for albumin into the anti-TNF VHH protein significantly prolonged its serum half-life and promoted its targeting to inflamed joints in the murine CIA model of RA. This might explain the excellent therapeutic efficacy observed in vivo. These data suggest that because of the flexibility of their format, camelid anti-TNF VHH proteins can be converted into potent therapeutic agents that can be produced and purified cost-effectively.

  18. Estimation of free energy barriers in the cytoplasmic and mitochondrial aspartate aminotransferase reactions probed by hydrogen-exchange kinetics of C alpha-labeled amino acids with solvent

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Julin, D.A.; Wiesinger, H.; Toney, M.D.

    1989-05-02

    The existence of the postulated quinonoid intermediate in the cytoplasmic aspartate amino-transferase catalyzed transamination of aspartate to oxaloacetate was probed by determining the extent of transfer of tritium from the C alpha position of tritiated L-aspartate to pyridoxamine 5'-phosphate in single turnover experiments in which washout from the back-reaction was obviated by product trapping. The maximum amount of transferred tritium observed was 0.7%, consistent either with a mechanism in which a fraction of the net transamination reaction proceeds through a quinonoid intermediate or with a mechanism in which this intermediate is formed off the main reaction pathway. It is shownmore » that transfer of labeled hydrogen from the amino acid to cofactor cannot be used to differentiate a stepwise from a concerted transamination mechanism. The amount of tritium transferred is a function of the rate constant for torsional equilibration about the epsilon-amino group of Lys-258, the presumptive abstractor of the C alpha proton; the relative rate constants for hydrogen exchange with solvent versus cofactor protonation; and the tritium isotope effect on this ratio. The free energy barriers facing the covalent intermediate between aldimine and keto acid product (i.e., ketimine and possibly quinonoid) were evaluated relatively by comparing the rates of C alpha-hydrogen exchange in starting amino acid with the rates of keto acid formation. The value of theta (= kexge/kprod) was found to be 2.6 for the reaction of cytoplasmic isozyme with aspartate and ca. 0.5 for that of the mitochondrial form with glutamate.« less

  19. An Update on Plant Derived Anti-Androgens

    PubMed Central

    Grant, Paul; Ramasamy, Shamin

    2012-01-01

    Anti-androgens are an assorted group of drugs and compounds that reduce the levels or activity of androgen hormones within the human body. Disease states in which this is relevant include polycystic ovarian syndrome, hirsutism, acne, benign prostatic hyperplasia, and endocrine related cancers such as carcinoma of the prostate. We provide an overview and discussion of the use of anti-androgen medications in clinical practice and explore the increasing recognition of the benefits of plant-derived anti-androgens, for example, spearmint tea in the management of PCOS, for which some evidence about efficacy is beginning to emerge. Other agents covered include red reishi, which has been shown to reduce levels 5-alpha reductase, the enzyme that facilitates conversion of testosterone to dihydrotestosterone (DHT); licorice, which has phytoestrogen effects and reduces testosterone levels; Chinese peony, which promotes the aromatization of testosterone into estrogen; green tea, which contains epigallocatechins and also inhibits 5-alpha reductase, thereby reducing the conversion of normal testosterone into the more potent DHT; black cohosh, which has been shown to kill both androgenresponsive and non-responsive human prostate cancer cells; chaste tree, which has a reduces prolactin from the anterior pituitary; and saw palmetto extract, which is used as an anti-androgen although it shown no difference in comparison to placebo in clinical trials. PMID:23843810

  20. Influenza virus-specific TCR-transduced T cells as a model for adoptive immunotherapy

    PubMed Central

    Berdien, Belinda; Reinhard, Henrike; Meyer, Sabrina; Spöck, Stefanie; Kröger, Nicolaus; Atanackovic, Djordje; Fehse, Boris

    2013-01-01

    Adoptive transfer of T lymphocytes equipped with tumor-antigen specific T-cell receptors (TCRs) represents a promising strategy in cancer immunotherapy, but the approach remains technically demanding. Using influenza virus (Flu)-specific T-cell responses as a model system we compared different methods for the generation of T-cell clones and isolation of antigen-specific TCRs. Altogether, we generated 12 CD8+ T-cell clones reacting to the Flu matrix protein (Flu-M) and 6 CD4+ T-cell clones reacting to the Flu nucleoprotein (Flu-NP) from 4 healthy donors. IFN-γ-secretion-based enrichment of antigen-specific cells, optionally combined with tetramer staining, was the most efficient way for generating T-cell clones. In contrast, the commonly used limiting dilution approach was least efficient. TCR genes were isolated from T-cell clones and cloned into both a previously used gammaretroviral LTR-vector, MP91 and the novel lentiviral self-inactivating vector LeGO-MP that contains MP91-derived promotor and regulatory elements. To directly compare their functional efficiencies, we in parallel transduced T-cell lines and primary T cells with the two vectors encoding identical TCRs. Transduction efficiencies were approximately twice higher with the gammaretroviral vector. Secretion of high amounts of IFN-γ, IL-2 and TNF-α by transduced cells after exposure to the respective influenza target epitope proved efficient specificity transfer of the isolated TCRs to primary T-cells for both vectors, at the same time indicating superior functionality of MP91-transduced cells. In conclusion, we have developed optimized strategies to obtain and transfer antigen-specific TCRs as well as designed a novel lentiviral vector for TCR-gene transfer. Our data may help to improve adoptive T-cell therapies. PMID:23428899

  1. Goats' milk of defective alpha(s1)-casein genotype decreases intestinal and systemic sensitization to beta-lactoglobulin in guinea pigs.

    PubMed

    Bevilacqua, C; Martin, P; Candalh, C; Fauquant, J; Piot, M; Roucayrol, A M; Pilla, F; Heyman, M

    2001-05-01

    Contradictory results have been reported on the use of goats' milk in cows' milk allergy. In this study the hypothesis was tested, using a guinea pig model of cows' milk allergy, that these discrepancies could be due to the high genetic polymorphism of goats' milk proteins. Forty guinea pigs were fed over a 20 d period with pelleted diets containing one of the following: soyabean proteins (group S), cows' milk proteins (group CM), goats' milk proteins with high (group GM1) or low (group GM2) alpha(s1)-casein content. Parenteral sensitization to GM1 and GM2 proteins as also assessed. The sensitization was measured (1) by systemic IgG1 antibodies directed against bovine or caprine beta-lactoglobulin (beta-lg), alpha-lactalbumin (alpha-la) and whole caseins, and (2) by intestinal anaphylaxis measured in vitro in Ussing chambers, by the rise in short-circuit current (delta Isc) in response to milk proteins. Guinea pigs fed on CM and GM1 developed high titres (> 1500) of anti-beta-lg IgG1, with an important cross reactivity between goat and cow beta-lg. However, in guinea pigs fed on GM2, anti-goat beta-lg IgG1 antibodies were significantly decreased compared with GM1 guinea pigs (mean IgG1 titres were 546 and 2046 respectively), and the intestinal anaphylaxis was significantly decreased (3.5+/-4.5 microA/cm2) compared with that observed in GM1 guinea pigs (8.3+/-7.6 microA/cm2). Animals receiving GM1 or GM2 proteins via the parenteral route developed a marked sensitization. These results suggest that the discrepancies observed in the use of goats milk in cows' milk allergy could be due, at least in part, to the high genetic polymorphism of goats' milk proteins.

  2. Src homology 2-domain containing leukocyte-specific phosphoprotein of 76 kDa is mandatory for TCR-mediated inside-out signaling, but dispensable for CXCR4-mediated LFA-1 activation, adhesion, and migration of T cells.

    PubMed

    Horn, Jessica; Wang, Xiaoqian; Reichardt, Peter; Stradal, Theresia E; Warnecke, Nicole; Simeoni, Luca; Gunzer, Matthias; Yablonski, Deborah; Schraven, Burkhart; Kliche, Stefanie

    2009-11-01

    Engagement of the TCR or of chemokine receptors such as CXCR4 induces adhesion and migration of T cells via so-called inside-out signaling pathways. The molecular processes underlying inside-out signaling events are as yet not completely understood. In this study, we show that TCR- and CXCR4-mediated activation of integrins critically depends on the membrane recruitment of the adhesion- and degranulation-promoting adapter protein (ADAP)/Src kinase-associated phosphoprotein of 55 kDa (SKAP55)/Rap1-interacting adapter protein (RIAM)/Rap1 module. We further demonstrate that the Src homology 2 domain containing leukocyte-specific phosphoprotein of 76 kDa (SLP76) is crucial for TCR-mediated inside-out signaling and T cell/APC interaction. Besides facilitating membrane recruitment of ADAP, SKAP55, and RIAM, SLP76 regulates TCR-mediated inside-out signaling by controlling the activation of Rap1 as well as Rac-mediated actin polymerization. Surprisingly, however, SLP76 is not mandatory for CXCR4-mediated inside-out signaling. Indeed, both CXCR4-induced T cell adhesion and migration are not affected by loss of SLP76. Moreover, after CXCR4 stimulation, the ADAP/SKAP55/RIAM/Rap1 module is recruited to the plasma membrane independently of SLP76. Collectively, our data indicate a differential requirement for SLP76 in TCR- vs CXCR4-mediated inside-out signaling pathways regulating T cell adhesion and migration.

  3. Prostaglandin production by melanocytic cells and the effect of alpha-melanocyte stimulating hormone.

    PubMed

    Nicolaou, Anna; Estdale, Sian E; Tsatmali, Marina; Herrero, Daniel Pascual; Thody, Anthony J

    2004-07-16

    Prostaglandins are potent mediators of the inflammatory response and are also involved in cancer development. In this study, we show that human melanocytes and FM55 melanoma cells express cyclooxygenase-1 and -2 (COX-1 and -2) and thus have the capability to produce prostaglandins. The FM55 cells produced predominantly PGE2 and PGF2alpha, whereas the HaCaT keratinocyte cell line produced mainly PGE2. The anti-inflammatory peptide, alpha-melanocyte stimulating hormone (alpha-MSH), reduced prostaglandin production in FM55 and HaCaT cells and reversed the effect of the pro-inflammatory cytokine TNF-alpha in the former. These results indicate that melanocytes produce prostaglandins and that alpha-MSH, by inhibiting this response, may play an important role in regulating inflammatory responses in the skin.

  4. Endothelial monocyte activating polypeptide-II modulates endothelial cell responses by degrading hypoxia-inducible factor-1alpha through interaction with PSMA7, a component of the proteasome

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Tandle, Anita T.; Calvani, Maura; Uranchimeg, Badarch

    The majority of human tumors are angiogenesis dependent. Understanding the specific mechanisms that contribute to angiogenesis may offer the best approach to develop therapies to inhibit angiogenesis in cancer. Endothelial monocyte activating polypeptide-II (EMAP-II) is an anti-angiogenic cytokine with potent effects on endothelial cells (ECs). It inhibits EC proliferation and cord formation, and it suppresses primary and metastatic tumor growth in-vivo. However, very little is known about the molecular mechanisms behind the anti-angiogenic activity of EMAP-II. In the present study, we explored the molecular mechanism behind the anti-angiogenic activity exerted by this protein on ECs. Our results demonstrate that EMAP-IImore » binds to the cell surface {alpha}5{beta}1 integrin receptor. The cell surface binding of EMAP-II results in its internalization into the cytoplasmic compartment where it interacts with its cytoplasmic partner PSMA7, a component of the proteasome degradation pathway. This interaction increases hypoxia-inducible factor 1-alpha (HIF-1{alpha}) degradation under hypoxic conditions. The degradation results in the inhibition of HIF-1{alpha} mediated transcriptional activity as well as HIF-1{alpha} mediated angiogenic sprouting of ECs. HIF-1{alpha} plays a critical role in angiogenesis by activating a variety of angiogenic growth factors. Our results suggest that one of the major anti-angiogenic functions of EMAP-II is exerted through its inhibition of the HIF-1{alpha} activities.« less

  5. Anti-Hu antibodies activate enteric and sensory neurons

    PubMed Central

    Li, Qin; Michel, Klaus; Annahazi, Anita; Demir, Ihsan E.; Ceyhan, Güralp O.; Zeller, Florian; Komorowski, Lars; Stöcker, Winfried; Beyak, Michael J.; Grundy, David; Farrugia, Gianrico; De Giorgio, Roberto; Schemann, Michael

    2016-01-01

    IgG of type 1 anti-neuronal nuclear antibody (ANNA-1, anti-Hu) specificity is a serological marker of paraneoplastic neurological autoimmunity (including enteric/autonomic) usually related to small-cell lung carcinoma. We show here that IgG isolated from such sera and also affinity-purified anti-HuD label enteric neurons and cause an immediate spike discharge in enteric and visceral sensory neurons. Both labelling and activation of enteric neurons was prevented by preincubation with the HuD antigen. Activation of enteric neurons was inhibited by the nicotinic receptor antagonists hexamethonium and dihydro-β-erythroidine and reduced by the P2X antagonist pyridoxal phosphate-6-azo (benzene-2,4-disulfonic acid (PPADS) but not by the 5-HT3 antagonist tropisetron or the N-type Ca-channel blocker ω-Conotoxin GVIA. Ca++ imaging experiments confirmed activation of enteric neurons but not enteric glia. These findings demonstrate a direct excitatory action of ANNA-1, in particular anti-HuD, on visceral sensory and enteric neurons, which involves nicotinic and P2X receptors. The results provide evidence for a novel link between nerve activation and symptom generation in patients with antibody-mediated gut dysfunction. PMID:27905561

  6. A study of various synthetic routes to produce a halogen-labeled traction fluid

    NASA Technical Reports Server (NTRS)

    Jones, W. R., Jr.; Zimmer, H.

    1978-01-01

    Several synthetic routes were studied for the synthesis of the compound 1, 1, 3-trimethyl-1, 3-dicyclohexyl-2 chloropropane. This halogen-labeled fluid would be of use in the study of high traction lubricants under elastohydrodynamic lubrication conditions using infrared emission spectroscopy. The synthetic routes included: dimerization of alpha-methylstyrene, methanol addition to alpha-methylstyrene, a Wittig reaction, and an organometallic approach. Because of steric hindrance and competing reactions, none of these routes were successful.

  7. Gamma delta lymphocytes in endocrine autoimmunity: evidence of expansion in Graves' disease but not in type 1 diabetes.

    PubMed Central

    Roura-Mir, I C; Alcalde, L; Vargas, F; Tolosa, E; Obiols, G; Foz, M; Jaraquemada, D; Pujol-Borrell, R

    1993-01-01

    Endocrine autoimmune disorders are mediated by T cell-dependent responses to organ-specific antigens, but the mechanisms initiating the process remain unknown. Lymphocytes which use the gamma delta heterodimer as T cell receptor (TCR) for antigen constitute a distinct subset of T cells whose function remains elusive. In order to investigate their possible involvement in endocrine autoimmunity we have determined the proportion of gamma delta T cells in the peripheral blood of 23 patients with type 1 (insulin-dependent) diabetes mellitus (type-1 DM) and 30 patients with autoimmune thyrotoxicosis (Graves' disease). T lymphocyte TCR expression was assessed by fluorescence-activated flow cytometry on peripheral blood mononuclear cells using MoAbs UCHT1 (CD3), TCR delta 1 (gamma delta TCR), WT31 and beta F1 (alpha beta TCR) and both the percentage of T cells expressing gamma delta and the ratio gamma delta/alpha beta were calculated. In the diabetic patients gamma delta cells were not significantly different from the control group (7.7 +/- 54% versus 8.0 +/- 5.5% of T cells, P NS). There was no relation between the proportion of gamma delta lymphocytes and the presence of islet cell antibodies (ICA) in the sera. The Graves' patients showed a tendency towards a higher proportion of gamma delta T lymphocytes than the controls (gamma delta/alpha beta ratios: 0.095 +/- 0.047 versus 0.063 +/- 0.022, P = 0.03). In 14 Graves' patients the number of gamma delta were measured in paired samples of peripheral and intrathyroidal lymphocytes, demonstrating an expansion of gamma delta within the thyroid glands (0.21 +/- 0.3 versus 0.095 +/- 0.047, P = 0.032). Immunohistochemical studies showed that gamma delta cells were scattered among the predominant alpha beta lymphocytes infiltrating the thyroid gland and that they account for 10% of intraepithelial lymphocytes. No relation was found between the increase of gamma delta lymphocytes and any clinical features. PMID:8485915

  8. Distinct T cell interactions with HLA class II tetramers characterize a spectrum of TCR affinities in the human antigen-specific T cell response.

    PubMed

    Reichstetter, S; Ettinger, R A; Liu, A W; Gebe, J A; Nepom, G T; Kwok, W W

    2000-12-15

    The polyclonal nature of T cells expanding in an ongoing immune response results in a range of disparate affinities and activation potential. Recently developed human class II tetramers provide a means to analyze this diversity by direct characterization of the trimolecular TCR-peptide-MHC interaction in live cells. Two HSV-2 VP16(369-379)-specific, DQA1*0102/DQB1*0602 (DQ0602)-restricted T cell clones were compared by means of T cell proliferation assay and HLA-DQ0602 tetramer staining. These two clones were obtained from the same subject, but show different TCR gene usage. Clone 48 was 10-fold more sensitive to VP16(369-379) peptide stimulation than clone 5 as assayed by proliferation assays, correlating with differences in MHC tetramer binding. Clone 48 gave positive staining with the DQ0602/VP16(369-379) tetramer at either 23 or 37 degrees C. Weak staining was also observed at 4 degrees C. Clone 5 showed weaker staining compared with clone 48 at 37 degrees C, and no staining was observed at 23 degrees C or on ice. Receptor internalization was not required for positive staining. Competitive binding indicates that the cell surface TCR of clone 48 has higher affinity for the DQ0602/VP16(369-379) complex than clone 5. The higher binding affinity of clone 48 for the peptide-MHC complex also correlates with a slower dissociation rate compared with clone 5.

  9. The coffee diterpene kahweol inhibits tumor necrosis factor-{alpha}-induced expression of cell adhesion molecules in human endothelial cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kim, Hyung Gyun; Kim, Ji Young; Hwang, Yong Pil

    2006-12-15

    Endothelial cells produce adhesion molecules after being stimulated with various inflammatory cytokines. These adhesion molecules play an important role in the development of atherogenesis. Recent studies have highlighted the chemoprotective and anti-inflammatory effects of kahweol, a coffee-specific diterpene. This study examined the effects of kahweol on the cytokine-induced monocyte/human endothelial cell interaction, which is a crucial early event in atherogenesis. Kahweol inhibited the adhesion of TNF{alpha}-induced monocytes to endothelial cells and suppressed the TNF{alpha}-induced protein and mRNA expression of the cell adhesion molecules, VCAM-1 and ICAM-1. Furthermore, kahweol inhibited the TNF{alpha}-induced JAK2-PI3K/Akt-NF-{kappa}B activation pathway in these cells. Overall, kahweol hasmore » anti-inflammatory and anti-atherosclerotic activities, which occurs partly by down-regulating the pathway that affects the expression and interaction of the cell adhesion molecules on endothelial cells.« less

  10. Thiazolidinediones inhibit REG I{alpha} gene transcription in gastrointestinal cancer cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Yamauchi, Akiyo; Laboratory of Molecular Genetics, Tohoku University Graduate School of Pharmaceutical Sciences, Sendai 980-8578; Department of Biochemistry, Nara Medical University, Kashihara 634-8521

    2009-02-13

    REG (Regenerating gene) I{alpha} protein functions as a growth factor for gastrointestinal cancer cells, and its mRNA expression is strongly associated with a poor prognosis in gastrointestinal cancer patients. We here demonstrated that PPAR{gamma}-agonist thiazolidinediones (TZDs) inhibited cell proliferation and REG I{alpha} protein/mRNA expression in gastrointestinal cancer cells. TZDs inhibited the REG I{alpha} gene promoter activity, via its cis-acting element which lacked PPAR response element and could not bind to PPAR{gamma}, in PPAR{gamma}-expressing gastrointestinal cancer cells. The inhibition was reversed by co-treatment with a specific PPAR{gamma}-antagonist GW9662. Although TZDs did not inhibit the REG I{alpha} gene promoter activity in PPAR{gamma}-non-expressingmore » cells, PPAR{gamma} overexpression in the cells recovered their inhibitory effect. Taken together, TZDs inhibit REG I{alpha} gene transcription through a PPAR{gamma}-dependent pathway. The TZD-induced REG I{alpha} mRNA reduction was abolished by cycloheximide, indicating the necessity of novel protein(s) synthesis. TZDs may therefore be a candidate for novel anti-cancer drugs for patients with gastrointestinal cancer expressing both REG I{alpha} and PPAR{gamma}.« less

  11. TCR and IL-7 Signaling Are Altered in the Absence of Functional GTPase of the Immune Associated Nucleotide Binding Protein 5 (GIMAP5)

    PubMed Central

    Chen, Xi-Lin; Serrano, Daniel; Ghobadi, Farnaz; Mayhue, Marian; Hoebe, Kasper; Ilangumaran, Subburaj; Ramanathan, Sheela

    2016-01-01

    GTPase of the immune associated nucleotide binding protein (GIMAP) family of proteins are expressed essentially in cells of the hematopoietic system. Mutation in the founding member of this gene family, Gimap5, results in the lymphopenic phenotype in Bio-Breeding diabetes prone rats. In mice, deletion of functional Gimap5 gene affects the survival and renewal of hematopoietic stem cells in addition to the defects observed in T cells. Here we show that T cells from OTII TCR-transgenic Gimap5sph/sph mice do not proliferate in response to its cognate antigen. Furthermore, T cells from Gimap5 mutant rats and mice show decreased phosphorylation of STAT5 following stimulation with IL-7. Our results suggest that functional Gimap5 is required for optimal signaling through TCR and IL-7R in T cells. PMID:27023180

  12. Comprehensive evaluation of the flavonol anti-oxidants, alpha-glycosyl isoquercitrin and isoquercitrin, for genotoxic potential.

    PubMed

    Hobbs, Cheryl A; Koyanagi, Mihoko; Swartz, Carol; Davis, Jeffrey; Kasamoto, Sawako; Maronpot, Robert; Recio, Leslie; Hayashi, Shim-Mo

    2018-03-01

    Quercetin and its glycosides possess potential benefits to human health. Several flavonols are available to consumers as dietary supplements, promoted as anti-oxidants; however, incorporation of natural quercetin glycosides into food and beverage products has been limited by poor miscibility in water. Enzymatic conjugation of multiple glucose moieties to isoquercitrin to produce alpha-glycosyl isoquercitrin (AGIQ) enhances solubility and bioavailability. AGIQ is used in Japan as a food additive and has been granted generally recognized as safe (GRAS) status. However, although substantial genotoxicity data exist for quercetin, there is very little available data for AGIQ and isoquercitrin. To support expanded global marketing of food products containing AGIQ, comprehensive testing of genotoxic potential of AGIQ and isoquercitrin was conducted according to current regulatory test guidelines. Both chemicals tested positive in bacterial reverse mutation assays, and exposure to isoquercitrin resulted in chromosomal aberrations in CHO-WBL cells. All other in vitro mammalian micronucleus and chromosomal aberration assays, micronucleus and comet assays in male and female B6C3F1 mice and Sprague Dawley rats, and Muta™ Mouse mutation assays evaluating multiple potential target tissues, were negative for both chemicals. These results supplement existing toxicity data to further support the safe use of AGIQ in food and beverage products. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

  13. Modulation of the immunogenicity of the Trypanosoma congolense cysteine protease, congopain, through complexation with alpha(2)-macroglobulin.

    PubMed

    Huson, Laura Elizabeth Joan; Authié, Edith; Boulangé, Alain Francçois; Goldring, James Phillip Dean; Coetzer, Theresa Helen Taillefer

    2009-01-01

    The protozoan parasite Trypanosoma congolense is the main causative agent of livestock trypanosomosis. Congopain, the major lysosomal cysteine proteinase of T. congolense, contributes to disease pathogenesis, and antibody-mediated inhibition of this enzyme may contribute to mechanisms of trypanotolerance. The potential of different adjuvants to facilitate the production of antibodies that would inhibit congopain activity was evaluated in the present study. Rabbits were immunised with the recombinant catalytic domain of congopain (C2), either without adjuvant, with Freund's adjuvant or complexed with bovine or rabbit alpha(2)-macroglobulin (alpha(2)M). The antibodies were assessed for inhibition of congopain activity. Rabbits immunised with C2 alone produced barely detectable anti-C2 antibody levels and these antibodies had no effect on recombinant C2 or native congopain activity. Rabbits immunised with C2 and Freund's adjuvant produced the highest levels of anti-C2 antibodies. These antibodies either inhibited C2 and native congopain activity to a small degree, or enhanced their activity, depending on time of production after initial immunisation. Rabbits receiving C2-alpha(2)M complexes produced moderate levels of anti-C2 antibodies and these antibodies consistently showed the best inhibition of C2 and native congopain activity of all the antibodies, with maximum inhibition of 65%. Results of this study suggest that antibodies inhibiting congopain activity could be raised in livestock with a congopain catalytic domain-alpha(2)M complex. This approach improves the effectiveness of the antigen as an anti-disease vaccine candidate for African trypanosomosis.

  14. Colloid labelled with radionuclide and method

    DOEpatents

    Atcher, R.W.; Hines, J.J.

    1990-11-13

    A ferric hydroxide colloid having an alpha-emitting radionuclide essentially on the outer surfaces and a method of forming same. The method includes oxidizing a ferrous hydroxide to ferric hydroxide in the presence of a preselected radionuclide to form a colloid having the radionuclide on the outer surface thereof, and thereafter washing the colloid, and suspending the washed colloid in a suitable solution. The labelled colloid is useful in cancer therapy and for the treatment of inflamed joints. No Drawings

  15. The T-cell antigen CD5 acts as a receptor and substrate for the protein-tyrosine kinase p56lck.

    PubMed Central

    Raab, M; Yamamoto, M; Rudd, C E

    1994-01-01

    CD5 is a T-cell-specific antigen which binds to the B-cell antigen CD72 and acts as a coreceptor in the stimulation of T-cell growth. CD5 associates with the T-cell receptor zeta chain (TcR zeta)/CD3 complex and is rapidly phosphosphorylated on tyrosine residues as a result of TcR zeta/CD3 ligation. However, despite this, the mechanism by which CD5 generates intracellular signals is unclear. In this study, we demonstrate that CD5 is coupled to the protein-tyrosine kinase p56lck and can act as a substrate for p56lck. Coexpression of CD5 with p56lck in the baculovirus expression system resulted in the phosphorylation of CD5 on tyrosine residues. Further, anti-CD5 and anti-p56lck coprecipitated each other in a variety of detergents, including Nonidet P-40 and Triton X-100. Anti-CD5 also precipitated the kinase from various T cells irrespective of the expression of TcR zeta/CD3 or CD4. No binding between p59fyn(T) and CD5 was detected in T cells. The binding of p56lck to CD5 induced a 10- to 15-fold increase in p56lck catalytic activity, as measured by in vitro kinase analysis. In vivo labelling with 32P(i) also showed a four- to fivefold increase in Y-394 occupancy in p56lck when associated with CD5. The use of glutathione S-transferase-Lck fusion proteins in precipitation analysis showed that the SH2 domain of p56lck could recognize CD5 as expressed in the baculovirus expression system. CD5 interaction with p56lck represents a novel variant of a receptor-kinase complex in which receptor can also serve as substrate. The CD5-p56lck interaction is likely to play roles in T-cell signalling and T-B collaboration. Images PMID:7513045

  16. Both α and β Subunits of Human Choriogonadotropin Photoaffinity Label the Hormone Receptor

    NASA Astrophysics Data System (ADS)

    Ji, Inhae; Ji, Tae H.

    1981-09-01

    It has been shown that a photoactivable derivative of human choriogonadotropin (hCG) labels the lutropin receptor on porcine granulosa cells [Ji, I. & Ji, T. H. (1980) Proc. Natl. Acad. Sci. USA 77, 7167-7170]. In an attempt to identify which of the hCG subunits labeled the receptor, three sets of different hCG derivatives were prepared. In the first set, hCG was coupled to the N-hydroxysuccinimide ester of 4-azidobenzoylglycine and radioiodinated. In the second set, only one of the subunits was radioiodinated, but both subunits were allowed to react with the reagent. In the third set, both the reagent and [125I]iodine were coupled to only one of the subunits. The binding activity of each hormone derivative was comparable to that of 125I-labeled hCG. After binding of these hormone derivatives to the granulosa cell surface, they were photolyzed. After solubilization, autoradiographs of sodium dodecyl sulfate/polyacrylamide gels of each sample revealed a number of labeled bands; the hCG derivatives containing 125I-labeled alpha subunit produced four bands (molecular weights 120,000 +/- 6,000, 96,000 +/- 5,000, 76,000 +/- 4,000, and 73,000 +/- 4,000) and those containing 125I-labeled beta subunit produced three bands (molecular weights 106,000 +/- 6,000, 88,000 +/- 5,000, and 83,000 +/- 4,000). Results were the same when the hormone-receptor complexes were solubilized in 0.5% Triton X-100 and then photolyzed or when the hormone was derivatized with a family of reagents having arms of various lengths. We conclude that both the alpha subunit and the beta subunit of hCG photoaffinity labeled certain membrane polypeptides and that these polypeptides are related to the hormone receptor.

  17. Specific tumor labeling enhanced by polyethylene glycol linkage of near infrared dyes conjugated to a chimeric anti-carcinoembryonic antigen antibody in a nude mouse model of human pancreatic cancer

    NASA Astrophysics Data System (ADS)

    Maawy, Ali A.; Hiroshima, Yukihiko; Zhang, Yong; Luiken, George A.; Hoffman, Robert M.; Bouvet, Michael

    2014-10-01

    Labeling of metastatic tumors can aid in their staging and resection of cancer. Near infrared (NIR) dyes have been used in the clinic for tumor labeling. However, there can be a nonspecific uptake of dye by the liver, lungs, and lymph nodes, which hinders detection of metastasis. In order to overcome these problems, we have used two NIR dyes (DyLight 650 and 750) conjugated to a chimeric anti-carcinoembryonic antigen antibody to evaluate how polyethylene glycol linkage (PEGylation) can improve specific tumor labeling in a nude mouse model of human pancreatic cancer. The conjugated PEGylated and non-PEGylated DyLight 650 and 750 dyes were injected intravenously into non-tumor-bearing nude mice. Serum samples were collected at various time points in order to determine serum concentrations and elimination kinetics. Conjugated PEGylated dyes had significantly higher serum dye concentrations than non-PEGylated dyes (p=0.005 for the 650 dyes and p<0.001 for the 750 dyes). Human pancreatic tumors subcutaneously implanted into nude mice were labeled with antibody-dye conjugates and serially imaged. Labeling with conjugated PEGylated dyes resulted in significantly brighter tumors compared to the non-PEGylated dyes (p<0.001 for the 650 dyes; p=0.01 for 750 dyes). PEGylation of the NIR dyes also decreased their accumulation in lymph nodes, liver, and lung. These results demonstrate enhanced selective tumor labeling by PEGylation of dyes conjugated to a tumor-specific antibody, suggesting their future clinical use in fluorescence-guided surgery.

  18. Incensole acetate, a novel anti-inflammatory compound isolated from Boswellia resin, inhibits nuclear factor-kappa B activation.

    PubMed

    Moussaieff, Arieh; Shohami, Esther; Kashman, Yoel; Fride, Ester; Schmitz, M Lienhard; Renner, Florian; Fiebich, Bernd L; Munoz, Eduardo; Ben-Neriah, Yinon; Mechoulam, Raphael

    2007-12-01

    Boswellia resin is a major anti-inflammatory agent in herbal medical tradition, as well as a common food supplement. Its anti-inflammatory activity has been attributed to boswellic acid and its derivatives. Here, we re-examined the anti-inflammatory effect of the resin, using inhibitor of nuclear factor-kappaB alpha (IkappaB alpha) degradation in tumor necrosis factor (TNF) alpha-stimulated HeLa cells for a bioassay-guided fractionation. We thus isolated two novel nuclear factor-kappaB (NF-kappaB) inhibitors from the resin, their structures elucidated as incensole acetate (IA) and its nonacetylated form, incensole (IN). IA inhibited TAK/TAB-mediated IkappaB kinase (IKK) activation loop phosphorylation, resulting in the inhibition of cytokine and lipopolysaccharide-mediated NF-kappaB activation. It had no effect on IKK activity in vitro, and it did not suppress IkappaB alpha phosphorylation in costimulated T-cells, indicating that the kinase inhibition is neither direct nor does it affect all NF-kappaB activation pathways. The inhibitory effect seems specific; IA did not interfere with TNFalpha-induced activation of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase. IA treatment had a robust anti-inflammatory effect in a mouse inflamed paw model. Cembrenoid diterpenoids, specifically IA and its derivatives, may thus constitute a potential novel group of NF-kappaB inhibitors, originating from an ancient anti-inflammatory herbal remedy.

  19. Progression of undiagnosed cutaneous lymphoma after anti-tumor necrosis factor-alpha therapy.

    PubMed

    Martinez-Escala, Maria Estela; Posligua, Alba L; Wickless, Heather; Rutherford, Audrey; Sable, Kimberly A; Rubio-Gonzalez, Belen; Zhou, Xiaolong A; Kaplan, Jason B; Pro, Barbara; Choi, Jaehyuk; Querfeld, Christiane; Rosen, Steven T; Guitart, Joan

    2018-06-01

    Cutaneous lymphoma diagnosed after anti-tumor necrosis factor-α therapy (anti-TNF-α) has been reported in the literature, yet a clear link between both events remains elusive. To review our experience with cutaneous lymphoma diagnosed during or after the use of anti-TNF-α therapies. This is a multicenter retrospective study and a literature review. A total of 22 cases, including 20 cutaneous T-cell lymphomas (CTCLs) and 2 cutaneous B-cell lymphomas, were identified. In the CTCL group, 75% of the patients received an anti-TNF-α agent for a presumed inflammatory skin condition. Mycosis fungoides and Sézary syndrome were the most common subtypes of CTCL diagnosed. Advanced disease (stage IIB to IVA) was commonly seen at time of diagnosis and required aggressive therapy, including stem cell transplant in 3 patients; 2 patients in whom cutaneous B-cell lymphomas was diagnosed had an indolent course. A total of 31 cases were gathered from a literature search. This is a retrospective study. Our findings suggest that the disease of most of the identified patients was misdiagnosed as psoriasis or eczema; therefore, a comprehensive morphologic and molecular review of skin biopsy specimens and peripheral blood samples should be considered before initiation of anti-TNF-α therapy in patients with poorly defined dermatitis or atypical presentations of psoriasis. Copyright © 2018 American Academy of Dermatology, Inc. Published by Elsevier Inc. All rights reserved.

  20. The Cish SH2 domain is essential for PLC-γ1 regulation in TCR stimulated CD8+ T cells.

    PubMed

    Guittard, Geoffrey; Dios-Esponera, Ana; Palmer, Douglas C; Akpan, Itoro; Barr, Valarie A; Manna, Asit; Restifo, Nicholas P; Samelson, Lawrence E

    2018-03-28

    Cish, participates within a multi-molecular E3 ubiquitin ligase complex, which ubiquitinates target proteins. It has an inhibitory effect on T cell activation mediated by PLC-γ1 regulation, and it functions as a potent checkpoint in CD8 + T cell tumor immunotherapy. To study the structural and functional relationships between Cish and PLC-γ1 during CD8 + T cell activation, we tested mutants of the Cish-SH2 (R107K) and D/BC (L222Q, C226Q) domains. We confirmed that Cish-SH2-specific binding was essential for PLC-γ1 ubiquitination and degradation. This domain was essential for the Cish-mediated inhibition of Ca 2+ release upon TCR stimulation. No effect on inhibition of cytokine release was observed with SH2 or D/BC mutants, although the absence of Cish led to an increased release of IFN-γ and TNF-α. Using imaging we showed that Cish was expressed mostly in the cytoplasm and we did not see any Cish clustering at the plasma membrane upon stimulation. We conclude that the Cish-SH2 domain is essential for PLC-γ1 regulation in TCR-stimulated CD8 + T cells.

  1. FDA preemption of drug and device labeling: who should decide what goes on a drug label?

    PubMed

    Valoir, Tamsen; Ghosh, Shubha

    2011-01-01

    The Supreme Court decided an issue that is critical to consumer health and safety last year. In April 2009, the Supreme Court held that extensive FDA regulation of drugs did not preempt a state law claim that an additional warning on the label was necessary to make the drug reasonably safe for use. Thus, states--and even courts and juries--are now free to cast their vote on what a drug label should say. This is in direct contrast to medical devices, where the federal statute regulating medical devices expressly provides that state regulations are preempted. This Article discusses basic preemption principles and drugs, and explores the policy ramifications of pro- and anti-preemption policy in the healthcare industry.

  2. Anti-CD22-chimeric antigen receptors targeting B-cell precursor acute lymphoblastic leukemia.

    PubMed

    Haso, Waleed; Lee, Daniel W; Shah, Nirali N; Stetler-Stevenson, Maryalice; Yuan, Constance M; Pastan, Ira H; Dimitrov, Dimiter S; Morgan, Richard A; FitzGerald, David J; Barrett, David M; Wayne, Alan S; Mackall, Crystal L; Orentas, Rimas J

    2013-02-14

    Immune targeting of B-cell malignancies using chimeric antigen receptors (CARs) is a promising new approach, but critical factors impacting CAR efficacy remain unclear. To test the suitability of targeting CD22 on precursor B-cell acute lymphoblastic leukemia (BCP-ALL), lymphoblasts from 111 patients with BCP-ALL were assayed for CD22 expression and all were found to be CD22-positive, with median CD22 expression levels of 3500 sites/cell. Three distinct binding domains targeting CD22 were fused to various TCR signaling domains ± an IgG heavy chain constant domain (CH2CH3) to create a series of vector constructs suitable to delineate optimal CAR configuration. CARs derived from the m971 anti-CD22 mAb, which targets a proximal CD22 epitope demonstrated superior antileukemic activity compared with those incorporating other binding domains, and addition of a 4-1BB signaling domain to CD28.CD3 constructs diminished potency, whereas increasing affinity of the anti-CD22 binding motif, and extending the CD22 binding domain away from the membrane via CH2CH3 had no effect. We conclude that second-generation m971 mAb-derived anti-CD22 CARs are promising novel therapeutics that should be tested in BCP-ALL.

  3. A Dual-Purpose Linker for Alpha Helix Stabilization and Imaging Agent Conjugation to Glucagon-Like Peptide-1 Receptor Ligands

    PubMed Central

    Zhang, Liang; Navaratna, Tejas; Liao, Jianshan; Thurber, Greg M.

    2016-01-01

    Peptides display many characteristics of efficient imaging agents such as rapid targeting, fast background clearance, and low non-specific cellular uptake. However, poor stability, low affinity, and loss of binding after labeling often preclude their use in vivo. Using the glucagon-like peptide-1 receptor (GLP-1R) ligands exendin and GLP-1 as a model system, we designed a novel alpha helix stabilizing linker to simultaneously address these limitations. The stabilized and labeled peptides showed an increase in helicity, improved protease resistance, negligible loss or an improvement in binding affinity, and excellent in vivo targeting. The ease of incorporating azidohomoalanine in peptides and efficient reaction with the dialkyne linker enables this technique to potentially be used as a general method for labeling alpha helices. This strategy should be useful for imaging beta cells in diabetes research and in developing and testing other peptide targeting agents. PMID:25594741

  4. A conjugate of camptothecin and a somatostatin analog against prostate cancer cell invasion via a possible signaling pathway involving PI3K/Akt, alphaVbeta3/alphaVbeta5 and MMP-2/-9.

    PubMed

    Sun, Li-Chun; Luo, Jing; Mackey, L Vienna; Fuselier, Joseph A; Coy, David H

    2007-02-08

    Camptothecin (CPT) was conjugated to the N-terminal of a somatostatin analog (SSA) directly via a carbamate group and a basic N-terminal linking motif, D-Lys-D-Tyr-Lys-D-Tyr-D-Lys. This new CPT-SSA conjugate termed JF-10-81 was evaluated as a receptor-specific delivery system for its anti-invasive and anti-angiogenic activities. It was found that, in addition to blocking migration and invasion of highly invasive prostate cancer PC-3 cells, this conjugate also inhibited in vitro capillary-like tube formation of endothelial cells and in vivo angiogenesis in C57B1/6N female mice. JF-10-81 was found to block PC-3 cell attachment to various extracellular matrix components, mainly to vitronectin, the ligand of cell surface receptors integrin alphaVbeta3 and alphaVbeta5. Additionally, JF-10-81 reduced expression of integrins alphaVbeta3 and alphaVbeta5 on PC-3 cell surfaces, without effects on beta1 or any alphabeta1 heterodimers. This conjugate also inactivated phosphorylation of protein kinase B (PKB/Akt), down-regulated the expression of latent matrix metalloproteinase (MMP) -2 and MMP-9, but had little effect on MMP-3/-10. Meanwhile, membrane type-1 matrix metalloproteinase (MT1-MMP) and the tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) were not detectable in PC-3 cells. alphaVbeta3/alphaVbeta5 and MMP-2/-9 are known to be highly expressed in many tumor cells and play an important role in tumor progression. Our results support that this conjugate could possibly inhibit prostate cancer PC-3 cell invasion through a signaling pathway involving PI3K/Akt, alphaVbeta3/alphaVbeta5 and MMP-2/-9, and this SSA could be used as an efficient vector to deliver CPT or other cytotoxic agents to target sites for cancer therapy.

  5. TCR-contacting residues orientation and HLA-DRβ* binding preference determine long-lasting protective immunity against malaria

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Alba, Martha P.; Suarez, Carlos F.; Universidad del Rosario, Bogotá D. C.

    Fully-protective, long-lasting, immunological (FPLLI) memory against Plasmodium falciparum malaria regarding immune protection-inducing protein structures (IMPIPS) vaccinated into monkeys previously challenged and re-challenged 60 days later with a lethal Aotus monkey-adapted P. falciparum strain was found to be associated with preferential high binding capacity to HLA-DRβ1* allelic molecules of the major histocompatibility class II (MHC-II), rather than HLA-DRβ3*, β4*, β5* alleles. Complete PPII{sub L} 3D structure, a longer distance (26.5 Å ± 1.5 Å) between residues perfectly fitting into HLA-DRβ1*PBR pockets 1 and 9, a gauche{sup −} rotamer orientation in p8 TCR-contacting polar residue and a larger volume of polar p2 residues was also found. Thismore » data, in association with previously-described p3 and p7 apolar residues having gauche{sup +} orientation to form a perfect MHC-II-peptide-TCR complex, determines the stereo-electronic and topochemical characteristics associated with FPLLI immunological memory. - Highlights: • Stereo-electronic and topochemical rules associated with FPLLI immunological memory. • Presence of very high long-lasting antibody titres against Plasmodium falciparum Spz. • Protective memory induction associated with a binding capacity to HLA-DRβ1*. • gauche{sup −} rotamer orientation in p8 polar residue is related to is related to immunological memory.« less

  6. Is there a benefit from the concomitant use of immunosupression with anti-TNF in Crohn's disease; heads or tails?

    PubMed

    Lakatos, Peter Laszlo

    2009-09-01

    Over the last some years the increasing knowledge on the pathogenesis of Crohn's disease led to the development of a number of biological agents targeting specific molecules involved in gut inflammation, first of all TNF-alpha and its receptors. Infliximab, adalimumab and certolizumab have been successful in inducing and maintaining remission in Crohn's disease at both short and long term. This was recently confirmed by a Cochrane meta-analysis and also open label extension follow-up and cohort studies. Emerging new data however indicate that combination therapy with infliximab-azathioprine appears to have added benefit in inducing steroid-free remission and mucosal healing than either infliximab or azathioprine alone in azathioprine-naïve patients with early disease. Similarly the combination of steroids induction and infliximab was efficacious in luminal Crohn's disease. In contrast, there seems to be no synergism between methotrexate and infliximab. It is also less clear whether it is beneficial to use short or long-term infliximab-azathioprine combination in patients who previously failed therapy with azathioprine. In contrast, combination may potentially be associated with increased risk for infection and cancer. In case control-studies, especially the combination of steroids and anti-TNF and older age increased the risk for infectious complications, while scattered case reports point to the potentially increased risk of a rare form of non-Hodgkin's lymphoma (Hepatosplenic T cell lymphoma) with the use of azathioprine-anti-TNF combination. The aim of this review is to summarize the benefits and risks for the use combination therapy with TNF-alpha inhibitors in the treatment of Crohn's disease.

  7. Activating STAT3 Alpha for Promoting Healing of Neurons

    NASA Technical Reports Server (NTRS)

    Conway, Greg

    2008-01-01

    A method of promoting healing of injured or diseased neurons involves pharmacological activation of the STAT3 alpha protein. Usually, injured or diseased neurons heal incompletely or not at all for two reasons: (1) they are susceptible to apoptosis (cell death); and (2) they fail to engage in axogenesis that is, they fail to re-extend their axons to their original targets (e.g., muscles or other neurons) because of insufficiency of compounds, denoted neurotrophic factors, needed to stimulate such extension. The present method (see figure) of treatment takes advantage of prior research findings to the effect that the STAT3 alpha protein has anti-apoptotic and pro-axogenic properties.

  8. Alpha lipoic acid in obstetrics and gynecology.

    PubMed

    Di Tucci, Chiara; Di Feliciantonio, Mara; Vena, Flaminia; Capone, Carmela; Schiavi, Michele Carlo; Pietrangeli, Daniela; Muzii, Ludovico; Benedetti Panici, Pierluigi

    2018-05-04

    Alpha-Lipoic acid (ALA) is a natural antioxidant synthetized by plants and animals, identified as a catalytic agent for oxidative decarboxylation of pyruvate and α-ketoglutarate. In this review, we analyzed the action of ALA in gynecology and obstetrics focusing in particular on neuropathic pain and antioxidant and anti-inflammatory action. A comprehensive literature search was performed in PubMed and Cochrane Library for retrieving articles in English language on the antioxidant and anti-inflammatory effects of ALA in gynecological and obstetrical conditions. ALA reduces oxidative stress and insulin resistance in women with polycystic ovary syndrome (PCOS). The association of N-acetyl cysteine (NAC), alpha-lipoic acid (ALA), and bromelain (Br) is used for prevention and treatment of endometriosis. In association with omega-3 polyunsaturated fatty acids (n-3 PUFAs) with amitriptyline is used for treatment of vestibulodynia/painful bladder syndrome (VBD/PBS). A promising area of research is ALA supplementation in patients with threatened miscarriage to improve the subchorionic hematoma resorption. Furthermore, ALA could be used in prevention of diabetic embryopathy and premature rupture of fetal membranes induced by inflamation. In conclusion, ALA can be safely used for treatment of neuropatic pain and as a dietary support during pregnancy.

  9. Cinnamon extract attenuates TNF-alpha-induced intestinal lipoprotein ApoB48 overproduction by regulating inflammatory, insulin, and lipoprotein pathways in enterocytes

    USDA-ARS?s Scientific Manuscript database

    We evaluated whether a water extract of cinnamon (CE = Cinnulin PF®) attenuates the dyslipidemia induced by TNF-alpha in Triton WR-1339-treated hamsters, and whether CE inhibited the over-secretion of apoB48-induced by TNF-alpha in enterocytes in a 35S-labelling study. In vivo, oral treatment with C...

  10. Combined effect of tumor necrosis factor (TNF)-alpha and heat shock protein (HSP)-70 in reducing apoptotic injury in hypoxia: a cell culture study.

    PubMed

    Goel, Gunjan; Guo, Miao; Ding, Jamie; Dornbos, David; Ali, Ahmer; Shenaq, Mohammed; Guthikonda, Murali; Ding, Yuchuan

    2010-10-15

    Studies have demonstrated neuroprotective effects of either TNF-alpha or HSP-70 in ischemia/reperfusion injury following exercise. However, the protective mechanisms involving combined effect of the two proteins, particularly in neuronal apoptosis, remain unclear. This study aims to elucidate the beneficial role of TNF-alpha and HSP-70 in the regulation of apoptotic proteins and ERK signaling in hypoxic injury. Cortical neurons from 20 Sprague-Dawley rat embryos were isolated and cultured in five groups with or without pretreatment with recombinant TNF-alpha, HSP-70 protein or both prior to hypoxic conditions: (1) control; (2) control/hypoxia; (3) TNF-alpha/hypoxia; (4) HSP-70/hypoxia and (5) TNF-alpha/HSP-70/hypoxia. Western blotting was used to detect pro- and anti-apoptotic proteins, including Bax, AIF, Bcl-xL, Bcl-2, and pERK1/2 protein. TNF-alpha and HSP-70 significantly (p<0.05) reduced the levels of pro-apoptotic proteins, Bax and AIF. Also, pretreatment of hypoxic brain tissue with TNF-alpha and HSP-70 significantly (p<0.05) enhanced the levels of anti-apoptotic protein, Bcl-xL. TNF-alpha and HSP-70 together increased Bcl-2 levels by 70%. Hypoxia caused a significant (p<0.05) increase in ERK1/2 phosphorylation levels by 224%. The most effective inhibition of ERK levels was obtained by the combined administration of TNF-alpha and HSP-70. This study suggested that TNF-alpha and HSP-70 together enhance the decrease in pro-apoptotic protein levels and the increase in anti-apoptotic protein levels in the event of neuronal hypoxia through ERK1/2 signal transduction. 2010. Published by Elsevier Ireland Ltd.

  11. The Validation of a Food Label Literacy Questionnaire for Elementary School Children

    ERIC Educational Resources Information Center

    Reynolds, Jesse S.; Treu, Judith A.; Njike, Valentine; Walker, Jennifer; Smith, Erica; Katz, Catherine S.; Katz, David L.

    2012-01-01

    Objective: To determine the reliability and validity of a 10-item questionnaire, the Food Label Literacy for Applied Nutrition Knowledge questionnaire. Methods: Participants were elementary school children exposed to a 90-minute school-based nutrition program. Reliability was assessed via Cronbach alpha and intraclass correlation coefficient…

  12. Electrochemiluminescence immunosensor for ultrasensitive detection of biomarker using Ru(bpy)(3)(2+)-encapsulated silica nanosphere labels.

    PubMed

    Qian, Jing; Zhou, Zhenxian; Cao, Xiaodong; Liu, Songqin

    2010-04-14

    Here, we describe a new approach for electrochemiluminescence (ECL) assay with Ru(bpy)(3)(2+)-encapsulated silica nanoparticle (SiO(2)@Ru) as labels. A water-in-oil (W/O) microemulsion method was employed for one-pot synthesis of SiO(2)@Ru nanoparticles. The as-synthesized SiO(2)@Ru nanoparticles have a narrow size distribution, which allows reproducible loading of Ru(bpy)(3)(2+) inside the silica shell and of alpha-fetoprotein antibody (anti-AFP), a model antibody, on the silica surface with glutaraldehyde as linkage. The silica shell effectively prevents leakage of Ru(bpy)(3)(2+) into the aqueous solution due to strong electrostatic interaction between the positively charged Ru(bpy)(3)(2+) and the negatively charged surface of silica. The porous structure of silica shell allowed the ion to move easily through the pore to exchange energy/electrons with the entrapped Ru(bpy)(3)(2+). The as-synthesized SiO(2)@Ru can be used as a label for ultrasensitive detection of biomarkers through a sandwiched immunoassay process. The calibration range of AFP concentration was 0.05-30 ng mL(-1) with linear relation from 0.05 to 20 ng mL(-1) and a detection limit of 0.035 ng mL(-1) at 3sigma. The resulting immunosensors possess high sensitivity and good analytical performance. Copyright 2010 Elsevier B.V. All rights reserved.

  13. Method of making colloid labeled with radionuclide

    DOEpatents

    Atcher, Robert W.; Hines, John J.

    1991-01-01

    A ferric hydroxide colloid having an alpha-emitting radionuclide essentially on the outer surfaces and a method of forming same. The method includes oxidizing a ferrous hydroxide to ferric hydroxide in the presence of a preselected radionuclide to form a colloid having the radionuclide on the outer surface thereof, and thereafter washing the colloid, and suspending the washed colloid in a suitable solution. The labelled colloid is useful in cancer therapy and for the treatment of inflamed joints.

  14. New Onset Autoimmune Hepatitis during Anti-Tumor Necrosis Factor-Alpha Treatment in Children.

    PubMed

    Ricciuto, Amanda; Kamath, Binita M; Walters, Thomas D; Frost, Karen; Carman, Nicholas; Church, Peter C; Ling, Simon C; Griffiths, Anne M

    2018-03-01

    To evaluate a large anti-tumor necrosis factor (TNF)-treated pediatric inflammatory bowel disease cohort for drug-induced liver injury (DILI) following presentation of an index case with suspected DILI with autoimmune features after infliximab exposure. To characterize the incidence, natural history, and risk factors for liver enzyme elevation with anti-TNF use. We reviewed the index case and performed a retrospective cohort study of 659 children receiving anti-TNF therapy between 2000 and 2015 at a tertiary pediatric inflammatory bowel disease center. Patients with alanine aminotransferase (ALT) ≥×2 the upper limit of normal were included. The incidence, evolution, and risk factors for liver injury were examined with univariate and multivariable proportional hazards regression. Causality was assessed using the Roussel-Uclaf Causality Assessment Method. The index case, a teenage girl with Crohn's disease, developed elevated liver enzymes and features of autoimmune hepatitis on liver biopsy 23 weeks after starting infliximab. The injury resolved entirely within 4 months of withdrawing infliximab without additional therapy. Overall, 7.7% of our cohort developed new ALT elevations while on anti-TNF. Most ALT elevations were mild and transient and attributable to alternate etiologies. No additional clear cases of autoimmune hepatitis were identified. Transient liver enzyme abnormalities are relatively common among anti-TNF-treated children. Anti-TNF-related DILI with autoimmune features is rare but must be recognized so that therapy can be stopped. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. Total alpha-globin gene cluster deletion has high frequency in Filipinos

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Hunt, J.A.; Haruyama, A.Z.; Chu, B.M.

    1994-09-01

    Most {alpha}-thalassemias [Thal] are due to large deletions. In Southeast Asians, the (--{sup SEA}) double {alpha}-globin gene deletion is common, 3 (--{sup Tot}) total {alpha}-globin cluster deletions are known: Filipino (--{sup Fil}), Thai (--{sup Thai}), and Chinese (--{sup Chin}). In a Hawaii Thal project, provisional diagnosis of {alpha}-Thal-1 heterozygotes was based on microcytosis, normal isoelectric focusing, and no iron deficiency. One in 10 unselected Filipinos was an {alpha}-Thal-1 heterozygote, 2/3 of these had a (--{sup Tot}) deletion: a {var_sigma}-cDNA probe consistently showed fainter intensity of the constant 5.5 kb {var_sigma}{sub 2} BamHI band, with no heterzygosity for {var_sigma}-globin region polymorphisms;more » {alpha}-cDNA or {var_sigma}-cDNA probes showed no BamHI or BglII bands diagnostic of the (--{sup SEA}) deletion; bands for the (-{alpha}) {alpha}-Thal-2 single {alpha}-globin deletions were only seen in Hb H cases. A reliable monoclonal anti-{var_sigma}-peptide antibody test for the (--{sup SEA}) deletion was always negative in (--{sup Tot}) samples. Southern digests with the Lo probe, a gift from D. Higgs of Oxford Univ., confirmed that 49 of 50 (--{sup Tot}) chromosomes in Filipinos were (--{sup Fil}). Of 20 {alpha}-Thal-1 hydrops born to Filipinos, 11 were (--{sup Fil}/--{sup SEA}) compound heterozygotes; 9 were (--{sup SEA}/--{sup SEA}) homozygotes, but none was a (--{sup Fil}/--{sup Fil}).« less

  16. Invited Commentary: Alpha-Gal Allergy: Tip of the Iceberg to a Pivotal Immune Response.

    PubMed

    Commins, Scott P

    2016-09-01

    The syndrome of delayed allergic reactions to the carbohydrate galactose-alpha-1,3-galactose ("alpha-gal") has become increasingly recognized in allergy and immunology clinics regionally throughout the southeastern USA. Due to the increasing awareness of this unique food allergy, cases have been identified in the northeastern and central USA as well as in Central and South America, Europe, Asia, Scandinavia, and Australia. Clinically, alpha-gal allergy is characterized by reactions to non-primate mammalian meat (e.g., beef, pork, lamb) that occur 3-6 h following exposure. The IgE response to alpha-gal is thought to develop after tick bites and can result in the loss of tolerance to foods that have been safely consumed for years. Although the initial description of alpha-gal allergy in 2009 was limited to red meat, this epitope is now identified in an expanded number of products, medications and foods-both labeled and unlabeled. Moreover, we are beginning to recognize that alpha-gal food allergy is the tip of the iceberg for this immune response.

  17. The chemokine CXCL12 generates costimulatory signals in T cells to enhance phosphorylation and clustering of the adaptor protein SLP-76.

    PubMed

    Smith, Xin; Schneider, Helga; Köhler, Karsten; Liu, Hebin; Lu, Yuning; Rudd, Christopher E

    2013-07-30

    The CXC chemokine CXCL12 mediates the chemoattraction of T cells and enhances the stimulation of T cells through the T cell receptor (TCR). The adaptor SLP-76 [Src homology 2 (SH2) domain-containing leukocyte protein of 76 kD] has two key tyrosine residues, Tyr(113) and Tyr(128), that mediate signaling downstream of the TCR. We investigated the effect of CXCL12 on SLP-76 phosphorylation and the TCR-dependent formation of SLP-76 microclusters. Although CXCL12 alone failed to induce SLP-76 cluster formation, it enhanced the number, stability, and phosphorylation of SLP-76 microclusters formed in response to stimulation of the TCR by an activating antibody against CD3, a component of the TCR complex. Addition of CXCL12 to anti-CD3-stimulated cells resulted in F-actin polymerization that stabilized SLP-76 microclusters in the cells' periphery at the interface with antibody-coated coverslips and increased the interaction between SLP-76 clusters and those containing ZAP-70, the TCR-associated kinase that phosphorylates SLP-76, as well as increased TCR-dependent gene expression. Costimulation with CXCL12 and anti-CD3 increased the extent of phosphorylation of SLP-76 at Tyr(113) and Tyr(128), but not that of other TCR-proximal components, and mutation of either one of these residues impaired the CXCL12-dependent effect on SLP-76 microcluster formation, F-actin polymerization, and TCR-dependent gene expression. The effects of CXCL12 on SLP-76 microcluster formation were dependent on the coupling of its receptor CXCR4 to G(i)-family G proteins (heterotrimeric guanine nucleotide-binding proteins). Thus, we identified a costimulatory mechanism by which CXCL12 and antigen converge at SLP-76 microcluster formation to enhance T cell responses.

  18. Cytochemical Labeling for Fungal and Host Components in Plant Tissues Inoculated with Fungal Wilt Pathogens

    NASA Astrophysics Data System (ADS)

    Ouellette, G. B.; Baayen, R. P.; Chamberland, H.; Simard, M.; Rioux, D.; Charest, P. M.

    2004-08-01

    Antibodies to detect pectin in present investigations attached to distinct fibrils in vessel lumina. In carnation infected with an isolate of Fusarium oxysporum f.sp., labeling of pathogen cells also occurred; in a resistant cultivar (cv.), it was coincident with proximate pectin fibrils and linked to altered fungal walls, which was the opposite in the susceptible cv., indicating that hindrance of pathogen ability to degrade pectin may be related to resistance. Labeling of the fungus in culture was nil, except in media containing pectin, showing that pectin is not native to the pathogen. Labeling of fungal walls for cellulose in elm (inoculated with Ophiostoma novo-ulmi) and carnation also occurred, linked to adsorbed host wall components. The chitin probe often attached to dispersed matter, in vessel lumina, traceable to irregularly labeled fungal cells and host wall degradation products. With an anti-horseradish peroxidase probe, host and fungal walls were equally labeled, and with a glucosidase, differences of labeling between these walls were observed, depending on pH of the test solution. Fungal extracellular matter and filamentous structures, present in fungal walls, predominantly in another elm isolate (Phaeotheca dimorphospora), did not label with any of the probes used. However, in cultures of this fungus, extracellular material labeled, even at a distance from the colony margin, with an anti-fimbriae probe.

  19. Type I interferon (IFN-alpha/beta) rescues B-lymphocytes from apoptosis via PI3Kdelta/Akt, Rho-A, NFkappaB and Bcl-2/Bcl(XL).

    PubMed

    Badr, Gamal; Saad, Heba; Waly, Hanan; Hassan, Khadega; Abdel-Tawab, Hanem; Alhazza, Ibrahim M; Ahmed, Emad A

    2010-01-01

    Although IFN-alpha was reported to promote the survival of peripheral B-lymphocytes via the PI3-kinase-Akt pathway, the triggered signalling pathways involved in the protection of B cell from apoptosis need to be clarified. Using flow cytometry and western blot analysis, we have found that type 1 IFNs (IFN-alpha/beta) protect human B cells in culture from spontaneous apoptosis and from apoptosis mediated by anti-CD95 agonist, in a dose- and time-dependant manner. IFN-alpha/beta-mediated anti-apoptotic effect on human B cells was totally abrogated by blockade of IFNR1 chain. Our data indicate that PI3Kdelta, Rho-A, NFkappaB and Bcl-2/Bcl(XL) are active downstream of IFN receptors and are the major effectors of IFN-alpha/beta-rescued B cells from apoptosis. Furthermore, immunohistochemical results show marked reduction in numbers of CD20 positive B cell in both spleen and Peyer's patches from mice treated with anti-IFNR1 blocking antibody compared with control group. Moreover, ultrastructural observations of these organs show an obvious increase in apoptotic cells from mice treated with anti-IFNR1 blocking antibody. Our results provide more details about the triggered signalling pathways and the phosphorylation cascade which are involved in the protection of B cell from apoptosis after treatment with IFN-alpha/beta. Copyright 2010 Elsevier Inc. All rights reserved.

  20. 15N-labeled glycine synthesis.

    PubMed

    Tavares, Claudinéia R O; Bendassolli, José A; Coelho, Fernando; Sant'ana Filho, Carlos R; Prestes, Clelber V

    2006-09-01

    This work describes a method for 15N-isotope-labeled glycine synthesis, as well as details about a recovery line for nitrogen residues. To that effect, amination of alpha-haloacids was performed, using carboxylic chloroacetic acid and labeled aqueous ammonia (15NH3). Special care was taken to avoid possible 15NH3 losses, since its production cost is high. In that respect, although the purchase cost of the 13N-labeled compound (radioactive) is lower, the stable tracer produced constitutes an important tool for N cycling studies in living organisms, also minimizing labor and environmental hazards, as well as time limitation problems in field studies. The tests were carried out with three replications, and variable 15NH3aq volumes in the reaction were used (50, 100, and 150 mL), in order to calibrate the best operational condition; glycine masses obtained were 1.7, 2, and 3.2 g, respectively. With the development of a system for 15NH3 recovery, it was possible to recover 71, 83, and 87% of the ammonia initially used in the synthesis. With the required adaptations, the same system was used to recover methanol, and 75% of the methanol initially used in the amino acid purification process were recovered.

  1. Anti-CD22–chimeric antigen receptors targeting B-cell precursor acute lymphoblastic leukemia

    PubMed Central

    Haso, Waleed; Lee, Daniel W.; Shah, Nirali N.; Stetler-Stevenson, Maryalice; Yuan, Constance M.; Pastan, Ira H.; Dimitrov, Dimiter S.; Morgan, Richard A.; FitzGerald, David J.; Barrett, David M.; Wayne, Alan S.; Mackall, Crystal L.

    2013-01-01

    Immune targeting of B-cell malignancies using chimeric antigen receptors (CARs) is a promising new approach, but critical factors impacting CAR efficacy remain unclear. To test the suitability of targeting CD22 on precursor B-cell acute lymphoblastic leukemia (BCP-ALL), lymphoblasts from 111 patients with BCP-ALL were assayed for CD22 expression and all were found to be CD22-positive, with median CD22 expression levels of 3500 sites/cell. Three distinct binding domains targeting CD22 were fused to various TCR signaling domains ± an IgG heavy chain constant domain (CH2CH3) to create a series of vector constructs suitable to delineate optimal CAR configuration. CARs derived from the m971 anti-CD22 mAb, which targets a proximal CD22 epitope demonstrated superior antileukemic activity compared with those incorporating other binding domains, and addition of a 4-1BB signaling domain to CD28.CD3ζ constructs diminished potency, whereas increasing affinity of the anti-CD22 binding motif, and extending the CD22 binding domain away from the membrane via CH2CH3 had no effect. We conclude that second-generation m971 mAb-derived anti-CD22 CARs are promising novel therapeutics that should be tested in BCP-ALL. PMID:23243285

  2. Recombinant anti-tenascin antibody constructs

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    ZALUTSKY, MICHAEL R

    2006-08-29

    The general objective of this research is to combine genetically derived molecular constructs reactive with tenascin, with appropriate radionuclides and labeling methods in order to generate more effective diagnostic and therapeutic reagents for oncologic nuclear medicine. Tenascin, a polymorphic extracellular matrix glycoprotein, is of interest because of its high expression on glioma, melanoma, as well as prostate and breast carcinoma. Recently, we have also documented high levels of tenascin in lymphomas, particularly those of higher grade, making the potential clinical impact of tenascin-specific radiodiagnostics and therapeutics even greater. An essential feature of our work plan is the ability to exploitmore » our extensive clinical experience in order to design second-generation constructs with properties which could improve clinical efficacy. To date, we have treated over 150 brain tumor patients with 131I-labeled murine 81C6, an antibody which binds specifically to the alternatively spliced fibronectin type III repeats CD of the tenascin molecule. During the current grant period, we have made several observations which form the basis for our proposed specific aims. First, tissue distribution and catabolism experiments in animal models have demonstrated enhanced stability for a chimeric construct composed of murine variable regions and human IgG2 constant domains. Furthermore, pharmacokinetic studies in patients with 131I-labeled chimeric 81C6 have shown significantly longer retention in glioma tumor resection cavities compared with its murine parent. Second, we have initiated the first clinical trial of an endoradiotherapeutic labeled with the 7.2-hr -particle emitter 211At. Twelve glioma patients have received 211At-labeled chimeric 81C6 directly into their brain tumor resection cavity, and very encouraging results have been obtained. Now that the feasibility of human studies with 211At, has been demonstrated, the development and evaluation of anti

  3. Direct Measurement of T Cell Receptor Affinity and Sequence from Naïve Anti-Viral T Cells

    PubMed Central

    Zhang, Shuqi; Parker, Patricia; Ma, Keyue; He, Chenfeng; Shi, Qian; Cui, Zhonghao; Williams, Chad; Wendel, Ben S.; Meriwether, Amanda; Salazar, Mary A.; Jiang, Ning

    2016-01-01

    T cells recognize and kill a myriad of pathogen-infected or cancer cells using a diverse set of T cell receptors (TCR). The affinity of TCR to cognate antigen is of high interest in adoptive T cell transfer immunotherapy and antigen-specific T cell repertoire immune profiling because it is widely known to correlate with downstream T cell responses. Here, we introduce the in situ TCR affinity and sequence test (iTAST) for simultaneous measurement of TCR affinity and sequence from single primary CD8+ T cells in human blood. We demonstrate that the repertoire of primary antigen-specific T cells from pathogen inexperienced individuals has a surprisingly broad affinity range of 1000-fold composed of diverse TCR sequences. Within this range, samples from older individuals contained a reduced frequency of high affinity T cells compared to young individuals, demonstrating an age-related effect of T cell attrition that could cause holes in the repertoire. iTAST should enable the rapid selection of high affinity TCRs ex vivo for adoptive immunotherapy and measurement of T cell response for immune monitoring applications. PMID:27252176

  4. Measurement of the {sup 12}C({alpha},{gamma}){sup 16}O reaction at TRIAC

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Makii, H.; Miyatake, H.; Wakabayashi, Y.

    2012-11-12

    We have measured the {gamma}-ray angular distribution of the {sup 12}C({alpha},{gamma}){sup 16}O reaction at TRIAC (Tokai Radioactive Ion Accelerator Complex) to accurately determine the E1 and E2 cross sections. In this experiment, we used high efficiency anti-Compton NaI(T1) spectrometers to detect a {gamma}-ray from the reaction with large S/N ratio, intense pulsed {alpha}-beams to discriminate true event from background events due to neutrons from {sup 13}C({alpha},n){sup 16}O reaction with a time-of-flight (TOF) method. We succeeded in removing a background events due to neutrons and clearly detected {gamma}-ray from the {sup 12}C({alpha}{gamma}){sup 16}O reaction with high statistics.

  5. Anti-inflammatory and anti-arthritic activity of total flavonoids of the roots of Sophora flavescens.

    PubMed

    Jin, Jeong Ho; Kim, Ju Sun; Kang, Sam Sik; Son, Kun Ho; Chang, Hyun Wook; Kim, Hyun Pyo

    2010-02-17

    The roots of Sophora flavescens have long been used in Chinese medicine for the treatment of fever, inflammatory disorders, ulcers and skin burns. Sophora flavescens contains flavonoids and alkaloids. This study was conducted to develop a plant-based anti-inflammatory agent focused on chronic inflammatory disorders. To accomplish this, the alkaloid-free prenylated flavonoid-enriched fraction (PFS) of rhizomes of Sophora flavescens was prepared and its in vitro and in vivo anti-inflammatory activities were then evaluated for the first time. The inhibitory activity of PFS on PGE(2), NO, IL-6 and TNF-alpha production of lipopolysaccharide (LPS)-treated RAW 264.7 cells was measured. Additionally, adjuvant-induced arthritis in rats was used as an animal model of chronic inflammation to establish the in vivo anti-inflammatory effects of PFS. PFS inhibited cyclooxygenase-2 (COX-2)-catalyzed PGE(2) and inducible nitric oxide synthase (iNOS)-catalyzed NO production by lipopolysaccharide (LPS)-treated RAW 264.7 cells at 10-50 microg/ml, and these effects primarily occurred via COX-2 inhibition and iNOS down-regulation, respectively. PFS also inhibited IL-6 and TNF-alpha production. When tested against adjuvant-induced arthritis in rats (chronic inflammation), PFS strongly inhibited arthritic inflammation when administered orally at doses of 10-100mg/kg/day. In addition, PFS administered orally potently inhibited acetic acid-induced writhing in mice. Our results suggest that PFS inhibits chronic inflammatory response and the inhibition of proinflammatory molecules such as COX-2, iNOS and IL-6 may contribute, at least in part, to the anti-inflammatory activity in vivo. Overall, these results indicate that PFS from Sophora flavescens may have the potential for treatment of chronic inflammatory disorders such as rheumatoid arthritis. Copyright 2009 Elsevier Ireland Ltd. All rights reserved.

  6. alpha-Tocopheryl succinate promotes selective cell death induced by vitamin K3 in combination with ascorbate.

    PubMed

    Tomasetti, M; Strafella, E; Staffolani, S; Santarelli, L; Neuzil, J; Guerrieri, R

    2010-04-13

    A strategy to reduce the secondary effects of anti-cancer agents is to potentiate the therapeutic effect by their combination. A combination of vitamin K3 (VK3) and ascorbic acid (AA) exhibited an anti-cancer synergistic effect, associated with extracellular production of H(2)O(2) that promoted cell death. The redox-silent vitamin E analogue alpha-tocopheryl succinate (alpha-TOS) was used in combination with VK3 and AA to evaluate their effect on prostate cancer cells. Prostate cancer cells were sensitive to alpha-TOS and VK3 treatment, but resistant to AA upto 3.2 mM. When combined, a synergistic effect was found for VK3-AA, whereas alpha-TOS-VK3 and alpha-TOS-AA combination showed an antagonist and additive effect, respectively. However, sub-lethal doses of AA-VK3 combination combined with a sub-toxic dose of alpha-TOS showed to induce efficient cell death that resembles autoschizis. Associated with this cell demise, lipid peroxidation, DNA damage, cytoskeleton alteration, lysosomal-mitochondrial perturbation, and release of cytochrome c without caspase activation were observed. Inhibition of lysosomal proteases did not attenuate cell death induced by the combined agents. Furthermore, cell deaths by apoptosis and autoschizis were detected. These finding support the emerging idea that synergistic combinations of some agents can overcome toxicity and other side-effects associated with high doses of single drugs creating the opportunity for therapeutically relevant selectivity.

  7. Evaluation of pGL1-TNF-alpha therapy in combination with radiation

    NASA Technical Reports Server (NTRS)

    Li, J.; Andres, M. L.; Fodor, I.; Nelson, G. A.; Gridley, D. S.

    1998-01-01

    Long-term control of high-grade brain tumors is rarely achieved with current therapeutic regimens. In this study a new plasmid-based human tumor necrosis factor-alpha (TNF-alpha) expression vector was synthesized (pGL1-TNF-alpha) and evaluated together with radiation in the aggressive, rapidly growing C6 rat glioma model. pGL1-TNF-alpha was successfully transfected into C6 cells in vitro using a cationic polyamine method. Expression was detected up to 7 days and averaged 0.4 ng of TNF-alpha in the culture medium from 1x10(5) cells. The expressed protein was biologically functional, as evidenced by growth inhibition of L929, a TNF-alpha-susceptible cell line. Using fluorescence-labeled monoclonal antibodies and laser scanning cytometry, we confirmed that both the P55 and P75 receptors for TNF-alpha were present on the C6 cell membrane. However, the receptors were present at low density and P55 was expressed more than the P75 receptor. These findings were in contrast to results obtained with TNF-alpha-susceptible L929 cells. Tests in athymic mice showed that pGL1-TNF-alpha administered intratumorally 16-18 h before radiation (each modality given three times) significantly inhibited C6 tumor progression (P<0.05). This effect was more than additive, because pGL1-TNF-alpha alone did not slow tumor growth and radiation alone had little effect on tumor growth. These results indicate that pGL1-TNF-alpha has potential to augment the antitumor effects of radiation against a tumor type that is virtually incurable.

  8. Optimizing labelling conditions of 213Bi-DOTATATE for preclinical applications of peptide receptor targeted alpha therapy.

    PubMed

    Chan, Ho Sze; de Blois, Erik; Konijnenberg, Mark W; Morgenstern, Alfred; Bruchertseifer, Frank; Norenberg, Jeffrey P; Verzijlbergen, Fred J; de Jong, Marion; Breeman, Wouter A P

    2017-01-01

    213 Bismuth ( 213 Bi, T 1/2 = 45.6 min) is one of the most frequently used α-emitters in cancer research. High specific activity radioligands are required for peptide receptor radionuclide therapy. The use of generators containing less than 222 MBq 225 Ac (actinium), due to limited availability and the high cost to produce large-scale 225 Ac/ 213 Bi generators, might complicate in vitro and in vivo applications though.Here we present optimized labelling conditions of a DOTA-peptide with an 225 Ac/ 213 Bi generator (< 222 MBq) for preclinical applications using DOTA-Tyr 3 -octreotate (DOTATATE), a somatostatin analogue. The following labelling conditions of DOTATATE with 213 Bi were investigated; peptide mass was varied from 1.7 to 7.0 nmol, concentration of TRIS buffer from 0.15 mol.L -1 to 0.34 mol.L -1 , and ascorbic acid from 0 to 71 mmol.L -1 in 800 μL. All reactions were performed at 95 °C for 5 min. After incubation, DTPA (50 nmol) was added to stop the labelling reaction. Besides optimizing the labelling conditions, incorporation yield was determined by ITLC-SG and radiochemical purity (RCP) was monitored by RP-HPLC up to 120 min after labelling. Dosimetry studies in the reaction vial were performed using Monte Carlo and in vitro clonogenic assay was performed with a rat pancreatic tumour cell line, CA20948. At least 3.5 nmol DOTATATE was required to obtain incorporation ≥ 99 % with 100 MBq 213 Bi (at optimized pH conditions, pH 8.3 with 0.15 mol.L -1 TRIS) in a reaction volume of 800 μL. The cumulative absorbed dose in the reaction vial was 230 Gy/100 MBq in 30 min. A minimal final concentration of 0.9 mmol.L -1 ascorbic acid was required for ~100 MBq (t = 0) to minimize radiation damage of DOTATATE. The osmolarity was decreased to 0.45 Osmol/L.Under optimized labelling conditions, 213 Bi-DOTATATE remained stable up to 2 h after labelling, RCP was ≥ 85 %. In vitro showed a negative correlation between ascorbic acid

  9. Pig kidney transplantation in baboons: anti-Gal(alpha)1-3Gal IgM alone is associated with acute humoral xenograft rejection and disseminated intravascular coagulation.

    PubMed

    Bühler, L; Yamada, K; Kitamura, H; Alwayn, I P; Basker, M; Appel, J Z; Colvin, R B; White-Scharf, M E; Sachs, D H; Robson, S C; Awwad, M; Cooper, D K

    2001-12-15

    Kidneys harvested from miniature swine or pigs transgenic for human decay-accelerating factor (hDAF) were transplanted into baboons receiving an anti-CD154 monoclonal antibody (mAb) and either a whole body irradiation (WBI)- or cyclophosphamide (CPP)-based immunosuppressive regimen. Group 1 baboons (n=3) underwent induction therapy with WBI and thymic irradiation, pretransplantation antithymocyte globulin, and immunoadsorption of anti-Gal(alpha)1-3Gal (Gal) antibody (Ab). After transplantation of a miniature swine kidney, maintenance therapy comprised cobra venom factor, mycophenolate mofetil, and an anti-CD154 mAb (for 14-28 days). In group 2 (n=2), WBI was replaced by CPP in the induction protocol. Group 3 (n=3) animals received the group 2 regimen, but underwent transplantation with hDAF pig kidneys. Group 1 and 2 animals developed features of disseminated intravascular coagulation (DIC), with reductions of fibrinogen and platelets and increases of prothrombin time, partial thromboplastin time, and fibrin split products. Graft survival was for 6-13 days. Histology showed mild acute humoral xenograft rejection (AHXR) of the kidneys, but severe rejection of the ureters. Group 3 animals developed features of DIC in two of three cases during the fourth week, with AHXR in the third case. Graft survival was for 28 (n=1) or 29 (n=2) days. Histology of day 15 biopsy specimens showed minimal focal mononuclear cellular infiltrates, with predominantly CD3+ cells. By days 28 and 29, kidneys showed mild-to-moderate features of AHXR. In all groups, the humoral response was manifest by reappearance of anti-Gal IgM below baseline level, with no or low return of anti-Gal IgG. All excised kidneys showed IgM deposition, but no complement and no or minimal IgG deposition. No baboon showed a rebound of anti-Gal Ab immediately after excision of the graft, and anti-Gal Ab increased over pretransplantation levels only when anti-CD154 mAb was discontinued. DIC was observed with WBI- or

  10. The bean. alpha. -amylase inhibitor is encoded by a lectin gene

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Moreno, J.; Altabella, T.; Chrispeels, M.J.

    The common bean, Phaseolus vulgaris, contains an inhibitor of insect and mammalian {alpha}-amylases that does not inhibit plant {alpha}-amylase. This inhibitor functions as an anti-feedant or seed-defense protein. We purified this inhibitor by affinity chromatography and found that it consists of a series of glycoforms of two polypeptides (Mr 14,000-19,000). Partial amino acid sequencing was carried out, and the sequences obtained are identical with portions of the derived amino acid sequence of a lectin-like gene. This lectin gene encodes a polypeptide of MW 28,000, and the primary in vitro translation product identified by antibodies to the {alpha}-amylase inhibitor has themore » same size. Co- and posttranslational processing of this polypeptide results in glycosylated polypeptides of 14-19 kDa. Our interpretation of these results is that the bean lectins constitute a gene family that encodes diverse plant defense proteins, including phytohemagglutinin, arcelin and {alpha}-amylase inhibitor.« less

  11. Functional CD1d and/or NKT cell invariant chain transcript in horse, pig, African elephant and guinea pig, but not in ruminants.

    PubMed

    Looringh van Beeck, Frank A; Reinink, Peter; Hermsen, Roel; Zajonc, Dirk M; Laven, Marielle J; Fun, Axel; Troskie, Milana; Schoemaker, Nico J; Morar, Darshana; Lenstra, Johannes A; Vervelde, Lonneke; Rutten, Victor P M G; van Eden, Willem; Van Rhijn, Ildiko

    2009-04-01

    CD1d-restricted invariant natural killer T cells (NKT cells) have been well characterized in humans and mice, but it is unknown whether they are present in other species. Here we describe the invariant TCR alpha chain and the full length CD1d transcript of pig and horse. Molecular modeling predicts that porcine (po) invariant TCR alpha chain/poCD1d/alpha-GalCer and equine (eq) invariant TCR alpha chain/eqCD1d/alpha-GalCer form complexes that are highly homologous to the human complex. Since a prerequisite for the presence of NKT cells is the expression of CD1d protein, we performed searches for CD1D genes and CD1d transcripts in multiple species. Previously, cattle and guinea pig have been suggested to lack CD1D genes. The CD1D genes of European taurine cattle (Bos taurus) are known to be pseudogenes because of disrupting mutations in the start codon and in the donor splice site of the first intron. Here we show that the same mutations are found in six other ruminants: African buffalo, sheep, bushbuck, bongo, N'Dama cattle, and roe deer. In contrast, intact CD1d transcripts were found in guinea pig, African elephant, horse, rabbit, and pig. Despite the discovery of a highly homologous NKT/CD1d system in pig and horse, our data suggest that functional CD1D and CD1d-restricted NKT cells are not universally present in mammals.

  12. Effects of anti-tumour necrosis factor-alpha therapy on the quality of life in Crohn's disease.

    PubMed

    van Balkom, B P J; Schoon, E J; Stockbrügger, R W; Wolters, F L; van Hogezand, R A; van Deventer, S J H; Oldenburg, B; van Dullemen, H M; Russel, M G V M

    2002-06-01

    Infusion of anti-tumour necrosis factor-alpha appears to be highly effective in patients with Crohn's disease. To assess the effect of infliximab on the quality of life in patients with active or fistulizing disease, as measured by the inflammatory bowel disease questionnaire, and to examine the impact on its four dimensions. An observational study was conducted in 65 patients. An infusion of 5 mg/kg infliximab was given at week 0 in patients with active disease and at week 0, 2 and 6 in fistulizing disease. Changes from baseline in the total and dimensional inflammatory bowel disease questionnaire scores were calculated and compared between the patient groups. Potential predictors of change in the quality of life were identified. In the active disease group, at week 4, the mean total and dimensional inflammatory bowel disease questionnaire scores improved compared to baseline (P < 0.001). In the fistulizing group, at week 6, all scores changed from baseline (P < 0.05). Improvement in the total inflammatory bowel disease questionnaire score correlated well with the improvement of the Crohn's disease activity index. Systemic and social scores improved more than bowel and emotional scores. Inflammatory Crohn's disease and a young age at diagnosis were predictors for a better response to infliximab therapy. Infliximab therapy improves all dimensions of the quality of life in patients with Crohn's disease.

  13. In Vivo Defection of Thrombi with Indium-111-Labeled Platelets

    NASA Astrophysics Data System (ADS)

    Price, David C.; Lipton, Martin J.; Lusby, Robert J.; Engelstad, Barry L.; Stoney, Ronald J.; Prager, Robert J.; Hartmeyer, James A.; Holly, Anne S.

    1982-06-01

    The use of Indium-111-oxine labeled autologous platelets has been explored in a dog-catheter model, as well as in a variety of clinical disorders in man. Newly forming experimental thrombi in dogs label well during the first 45-90 minutes, then lose both label and thrombus mass in a manner consistent with fibrinolysis. Thrombus weight is linearly related to In-111 activity, so that in vivo scintigraphy will be a practical method to evaluate various thrombotic stimuli and anti-thrombotic interventions experimentally. Preformed thrombus, however, labels poorly and cannot be detected by imaging in this dog model. Initial clinical experience with a variety of arterial, venous and cardiac thrombotic states is reviewed, indicating some of the strengths and same of the potential weaknesses of this new scintigraphic technique.

  14. Diacylglycerol kinase α inactivation is an integral component of the costimulatory pathway that amplifies TCR signals.

    PubMed

    Arranz-Nicolás, Javier; Ogando, Jesús; Soutar, Denise; Arcos-Pérez, Raquel; Meraviglia-Crivelli, Daniel; Mañes, Santos; Mérida, Isabel; Ávila-Flores, Antonia

    2018-06-01

    The arsenal of cancer therapies has evolved to target T lymphocytes and restore their capacity to destroy tumor cells. T cells rely on diacylglycerol (DAG) to carry out their functions. DAG availability and signaling are regulated by the enzymes diacylglycerol kinase (DGK) α and ζ, whose excess function drives T cells into hyporesponsive states. Targeting DGKα is a promising strategy for coping with cancer; its blockade could reinstate T-cell attack on tumors while limiting tumor growth, due to positive DGKα functions in several oncogenic pathways. Here, we made a side-by-side comparison of the effects of commercial pharmacological DGK inhibitors on T-cell responses with those promoted by DGKα and DGKζ genetic deletion or silencing. We show the specificity for DGKα of DGK inhibitors I and II and the structurally similar compound ritanserin. Inhibitor treatment promoted Ras/ERK (extracellular signal-regulated kinase) signaling and AP-1 (Activator protein-1) transcription, facilitated DGKα membrane localization, reduced the requirement for costimulation, and cooperated with enhanced activation following DGKζ silencing/deletion. DGKiII and ritanserin had similar effects on TCR proximal signaling, but ritanserin counteracted long-term T-cell activation, an effect that was potentiated in DGKα -/- cells. In contrast with enhanced activation triggered by pharmacological inhibition, DGKα silencing/genetic deletion led to impaired Lck (lymphocyte-specific protein tyrosine kinase) activation and limited costimulation responses. Our results demonstrate that pharmacological inhibition of DGKα downstream of the TCR provides a gain-of-function effect that amplifies the DAG-dependent signaling cascade, an ability that could be exploited therapeutically to reinvigorate T cells to attack tumors.

  15. Time-resolved spectroscopy of dye-labeled photoactive yellow protein suggests a pathway of light-induced structural changes in the N-terminal cap.

    PubMed

    Hoersch, Daniel; Otto, Harald; Cusanovich, Michael A; Heyn, Maarten P

    2009-07-14

    The photoreceptor PYP responds to light activation with global conformational changes. These changes are mainly located in the N-terminal cap of the protein, which is approximately 20 A away from the chromophore binding pocket and separated from it by the central beta-sheet. The question of the propagation of the structural change across the central beta-sheet is of general interest for the superfamily of PAS domain proteins, for which PYP is the structural prototype. Here we measured the kinetics of the structural changes in the N-terminal cap by transient absorption spectroscopy on the ns to second timescale. For this purpose the cysteine mutants A5C and N13C were prepared and labeled with thiol reactive 5-iodoacetamidofluorescein (IAF). A5 is located close to the N-terminus, while N13 is part of helix alpha1 near the functionally important salt bridge E12-K110 between the N-terminal cap and the central anti-parallel beta-sheet. The absorption spectrum of the dye is sensitive to its environment, and serves as a sensor for conformational changes near the labeling site. In both labeled mutants light activation results in a transient red-shift of the fluorescein absorption spectrum. To correlate the conformational changes with the photocycle intermediates of the protein, we compared the kinetics of the transient absorption signal of the dye with that of the p-hydroxycinnamoyl chromophore. While the structural change near A5 is synchronized with the rise of the I(2) intermediate, which is formed in approximately 200 mus, the change near N13 is delayed and rises with the next intermediate I(2)', which forms in approximately 2 ms. This indicates that different parts of the N-terminal cap respond to light activation with different kinetics. For the signaling pathway of photoactive yellow protein we propose a model in which the structural signal propagates from the chromophore binding pocket across the central beta-sheet via the N-terminal region to helix alpha1

  16. Jellyfish mesogloea collagen. Characterization of molecules as alpha 1 alpha 2 alpha 3 heterotrimers.

    PubMed

    Miura, S; Kimura, S

    1985-12-05

    The mesogloea collagen of a primitive animal, the jellyfish Stomolophus nomurai, belonging to the class Scyphozoa in the Coelenterata, was studied with respect to its chain structure. Most of the mesogloea collagen was solubilized by limited digestion with pepsin and isolated by selective precipitation at 0.9 m NaCl in 0.5 M acetic acid. Upon denaturation, the pepsin-solubilized collagen produced three distinct alpha chains, alpha 1, alpha 2, and alpha 3, in comparable amounts which were separable by CM-cellulose chromatography. The nonidentity of these alpha chains was confirmed by amino acid and carbohydrate analyses and peptide mapping. Furthermore, the introduction of intramolecular cross-links into native molecules by formaldehyde yielded a large proportion of gamma 123 chain with chain structure alpha 1 alpha 2 alpha 3, as judged by chromatographic behavior and peptide maps. We concluded that mesogloea collagen is comprised of alpha 1 alpha 2 alpha 3 heterotrimers and is chemically like vertebrate Type V collagen. On the other hand, sea anemone mesogloea collagen from the class Anthozoa was previously reported to comprise (alpha)3 homotrimers (Katzman, R. L., and Kang, A. H. (1972) J. Biol. Chem. 247, 5486-5489). On the basis of these findings, we assume that alpha 1 alpha 2 alpha 3 heterotrimers arose in evolution with the divergence of Scyphozoa and Anthozoa.

  17. Anti tumor necrosis factor - alpha adalimumab for complex regional pain syndrome type 1 (CRPS-I): a case series.

    PubMed

    Eisenberg, Elon; Sandler, Ifat; Treister, Roi; Suzan, Erica; Haddad, May

    2013-11-01

    Evidence suggests tumor necrosis factor-alpha (TNF-α) mediates, at least in part, symptoms and signs in complex regional pain syndrome (CRPS). Here, we present a case series of patients with CRPS type 1, in whom the response to the anti-TNF-α adalimumab was assessed. Ten patients with CRPS type 1 were recruited. Assessments were performed before treatment, at 1 week, and 1, 3, and 6 months following 3 biweekly subcutaneous injections (40 mg/0.8 mL) adalimumab (Humira(®) ) and included the followings: Pain intensity using a 0-10 cm visual analog scale; the Short Form of the McGill Pain Questionnaire; the Beck Depression Inventory; the SF-36 questionnaire and mechanical and thermal thresholds (Von frey hair and Thermal Sensory Analyzer, respectively). In addition to the description of individual patient responses, both intention to treat (ITT) and per-protocol (PP) analyses were performed for the entire group. Three subgroups of patients were identified (3 patients in each): "nonresponders", "partial responders", and "robust responders" in whom improvement in almost all parameters was noted. Both the ITT and PP analyses demonstrated only a trend toward improvement in mechanical pain thresholds following treatment (ITT χ² = 13.83, P = 0.008; PP χ² = 10.29, P = 0.036). These results suggest adalimumab, and possibly other anti-TNF-α, can be potentially useful in some (although not in all) patients with CRPS type 1. These preliminary results along with the growing body of evidence which points to the involvement of TNF-α in the pathogenesis of CRPS justify further studies in this area. © 2013 World Institute of Pain.

  18. Enzymatic preparation of. cap alpha. - and. beta. -deuterated or tritiated amino acids with l-methionine. gamma. -lyase

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Esaki, N.; Sawada, S.; Tanaka, H.

    L-Methionine ..gamma..-lyase catalyzes the exchange of ..cap alpha..- and ..beta..-hydrogens of L-methionine and S-methyl-L-cysteine with deuterium or tritium of solvents. The rate of ..cap alpha..-hydrogen exchange with deuterium was about 40 times faster than that of the elimination reactions. The deuterium and tritium were exchanged also with the ..cap alpha..- and ..beta..-hydrogens of the straight-chain amino acids which do not undergo the elimination: L-alanine, L-..cap alpha..-aminobutyrate, L-norvaline, and L-norleucine. No exchange occurs for the D-isomers, acidic L-amino acids, basic L-amino acids, and branched-chain L-amino acids, although ..cap alpha..-hydrogen of glycine, L-trypotophan, and L-phenylalanine is exchanged slowly. These enzymatic hydrogen-exchange reactionsmore » facilitate specific labeling of the L-amino acids with deuterium and tritium.« less

  19. TLR4 signaling in effector CD4+ T cells regulates TCR activation and experimental colitis in mice

    PubMed Central

    González-Navajas, José M.; Fine, Sean; Law, Jason; Datta, Sandip K.; Nguyen, Kim P.; Yu, Mandy; Corr, Maripat; Katakura, Kyoko; Eckman, Lars; Lee, Jongdae; Raz, Eyal

    2010-01-01

    TLRs sense various microbial products. Their function has been best characterized in DCs and macrophages, where they act as important mediators of innate immunity. TLR4 is also expressed on CD4+ T cells, but its physiological function on these cells remains unknown. Here, we have shown that TLR4 triggering on CD4+ T cells affects their phenotype and their ability to provoke intestinal inflammation. In a model of spontaneous colitis, Il10–/–Tlr4–/– mice displayed accelerated development of disease, with signs of overt colitis as early as 8 weeks of age, when compared with Il10–/– and Il10–/–Tlr9–/– mice, which did not develop colitis by 8 months. Similar results were obtained in a second model of colitis in which transfer of naive Il10–/–Tlr4–/– CD4+ T cells into Rag1–/– recipients sufficient for both IL-10 and TLR4 induced more aggressive colitis than the transfer of naive Il10–/– CD4+ T cells. Mechanistically, LPS stimulation of TLR4-bearing CD4+ T cells inhibited ERK1/2 activation upon subsequent TCR stimulation via the induction of MAPK phosphatase 3 (MKP-3). Our data therefore reveal a tonic inhibitory role for TLR4 signaling on subsequent TCR-dependent CD4+ T cell responses. PMID:20051628

  20. Generation of dendritic cells from positively selected CD14+ monocytes for anti-tumor immunotherapy.

    PubMed

    Curti, Antonio; Isidori, Alessandro; Ferri, Elisa; Terragna, Carolina; Neyroz, Paolo; Cellini, Claudia; Ratta, Marina; Baccarani, Michele; Lemoli, Roberto M

    2004-07-01

    Peripheral blood CD14+ monocytes from multiple myeloma (MM) patients can be induced to differentiate into fully functional, mature, CD83+ dendritic cells (DCs) which are highly efficient in priming autologous T lymphocytes in response to the patient-specific tumor idiotype (Id). We have recently scaled up our manufacturing protocol for application in a phase I-II clinical trial of anti-Id vaccination with DCs in MM patients. Elegible patients received a series of by-monthly immunizations consisting of three subcutaneous and two intravenous injections of Id-keyhole limpet hemocyanin (KLH)-pulsed DCs (5 x -, 10 x -, 50 x 10(6) cells and 10 x -, 50 x 10(6) cells, respectively). To generate DCs, monocytes were labeled with clinical grade anti-CD14 conjugates and positively selected by immunomagnetic separation. Cells were then cultured, according to Good Manufacturing Practice guidelines, in FCS-free medium in cell culture bags, and differentiated to DCs with GM-CSF plus IL-4 followed by TNF-alpha or, more recently, by a cocktail of IL-1beta, IL-6, TNF-alpha and prostaglandin-E2. Before maturation, Mo-DCs were pulsed with the autologous Id as whole protein or Id (VDJ)-derived HLA class I restricted peptides. Ten MM patients, who had been treated with two courses of high-dose chemotherapy with peripheral blood stem cell support, entered into the clinical study. CD14+ monocytes were enriched from 16.1+/-5.7% to 95.5+/-3.2% (recovery 67.9+/-15%, viability > 97%). After cell culture, phenotypic analysis showed that 89.6+/-6.6% of the cells were mature DCs. We obtained 2.89+/-1 x 10(8) DCs/leukapheresis which represented 24.5+/-9% of the initial number of CD14+ cells. Notably, the cytokine cocktail induced a significantly higher percentage and yield (31+/-10.9 of initial CD14+ cells) of DCs than TNF-alpha alone, secretion of larger amounts of IL-12, potent stimulatory activity on allogeneic and autologous T cells. Storage in liquid nitrogen did not modify the phenotype or

  1. [Antibacterial and anti-hemolysin activities of tea catechins and their structural relatives].

    PubMed

    Toda, M; Okubo, S; Ikigai, H; Shimamura, T

    1990-03-01

    Among catechins tested, (-)epigallocatechin (EGC), (-)epicatechin gallate (ECg), (-) epigallocatechin gallate (EGCg) inhibited the growth of Staphylococcus aureus, Vibrio cholerae O1 classical Inaba 569B and El Tor Inaba V86. S. aureus was more sensitive than V. cholerae O1 to these compounds. EGCg showed also a bactericidal activity against V. cholerae O1 569B. Pyrogallol showed a stronger antibacterial activity against S. aureus and V. cholerae O1 than tannic and gallic acid. Rutin or caffein had no effect on them. ECg and EGCg showed the most potent anti-hemolysin activity against S. aureus alpha-toxin, Vibrio parahaemolyticus thermostable direct hemolysin (Vp-TDH) and cholera hemolysin. Among catechin relatives, only tannic acid had a potent anti-hemolysin activity against alpha-toxin. These results suggest that the catechol and pyrogallol groups are responsible for the antibacterial and bactericidal activities, while the conformation of catechins might play an important role in the anti-hemolysin activity.

  2. Anti-thrombosis Repertoire of Blood-feeding Horsefly Salivary Glands*

    PubMed Central

    Ma, Dongying; Wang, Yipeng; Yang, Hailong; Wu, Jing; An, Shu; Gao, Li; Xu, Xueqing; Lai, Ren

    2009-01-01

    Blood-feeding arthropods rely heavily on the pharmacological properties of their saliva to get a blood meal and suppress immune reactions of hosts. Little information is available on antihemostatic substances in horsefly salivary glands although their saliva has been thought to contain wide range of physiologically active molecules. In traditional Eastern medicine, horseflies are used as anti-thrombosis material for hundreds of years. By proteomics coupling transcriptome analysis with pharmacological testing, several families of proteins or peptides, which exert mainly on anti-thrombosis functions, were identified and characterized from 60,000 pairs of salivary glands of the horsefly Tabanus yao Macquart (Diptera, Tabanidae). They are: (I) ten fibrin(ogen)olytic enzymes, which hydrolyze specially alpha chain of fibrin(ogen) and are the first family of fibrin(ogen)olytic enzymes purified and characterized from arthropods; (II) another fibrin(ogen)olytic enzyme, which hydrolyzes both alpha and beta chain of fibrin(ogen); (III) ten Arg-Gly-Asp-motif containing proteins acting as platelet aggregation inhibitors; (IV) five thrombin inhibitor peptides; (V) three vasodilator peptides; (VI) one apyrase acting as platelet aggregation inhibitor; (VII) one peroxidase with both platelet aggregation inhibitory and vasodilator activities. The first three families are belonging to antigen five proteins, which show obvious similarity with insect allergens. They are the first members of the antigen 5 family found in salivary glands of blood sucking arthropods to have anti-thromobosis function. The current results imply a possible evolution from allergens of blood-sucking insects to anti-thrombosis agents. The extreme diversity of horsefly anti-thrombosis components also reveals the anti-thrombosis molecular mechanisms of the traditional Eastern medicine insect material. PMID:19531497

  3. Expression of anti-tumor necrosis factor alpha (TNFα) single-chain variable fragment (scFv) in Spirodela punctata plants transformed with Agrobacterium tumefaciens.

    PubMed

    Balaji, Parthasarathy; Satheeshkumar, P K; Venkataraman, Krishnan; Vijayalakshmi, M A

    2016-05-01

    Therapeutic antibodies against tumor necrosis factor alpha (TNFα) have been considered effective for some of the autoimmune diseases such as rheumatoid arthritis, Crohn's diseases, and so on. But associated limitations of the current therapeutics in terms of cost, availability, and immunogenicity have necessitated the need for alternative candidates. Single-chain variable fragment (scFv) can negate the limitations tagged with the anti-TNFα therapeutics to a greater extent. In the present study, Spirodela punctata plants were transformed with anti-TNFα through in planta transformation using Agrobacterium tumefaciens strain, EHA105. Instead of cefotaxime, garlic extract (1 mg/mL) was used to remove the agrobacterial cells after cocultivation. To the best of our knowledge, this report shows for the first time the application of plant extracts in transgenic plant development. 95% of the plants survived screening under hygromycin. ScFv cDNA integration in the plant genomic DNA was confirmed at the molecular level by PCR. The transgenic protein expression was followed up to 10 months. Expression of scFv was confirmed by immunodot blot. Protein expression levels of up to 6.3% of total soluble protein were observed. β-Glucuronidase and green fluorescent protein expressions were also detected in the antibiotic resistant plants. The paper shows the generation of transgenic Spirodela punctuata plants through in planta transformation. © 2015 International Union of Biochemistry and Molecular Biology, Inc.

  4. GAS MOTION STUDY OF Ly{alpha} EMITTERS AT z {approx} 2 USING FUV AND OPTICAL SPECTRAL LINES {sup ,}

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Hashimoto, Takuya; Shimasaku, Kazuhiro; Nakajima, Kimihiko

    2013-03-01

    We present the results of Magellan/MMIRS and Keck/NIRSPEC spectroscopy for five Ly{alpha} emitters (LAEs) at z {approx_equal} 2.2 for which high-resolution FUV spectra from Magellan/MagE are available. We detect nebular emission lines including H{alpha} on the individual basis and low-ionization interstellar (LIS) absorption lines in a stacked FUV spectrum, and measure average offset velocities of the Ly{alpha} line, {Delta}v {sub Ly{alpha}}, and LIS absorption lines, {Delta}v {sub abs}, with respect to the systemic velocity defined by the nebular lines. For a sample of eight z {approx} 2-3 LAEs without active galactic nucleus from our study and the literature, we obtainmore » {Delta}v {sub Ly{alpha}} = 175 {+-} 35 km s{sup -1}, which is significantly smaller than that of Lyman-break Galaxies (LBGs), {Delta}v {sub Ly{alpha}} {approx_equal} 400 km s{sup -1}. The stacked FUV spectrum gives {Delta}v {sub abs} = -179 {+-} 73 km s{sup -1}, comparable to that of LBGs. These positive {Delta}v {sub Ly{alpha}} and negative {Delta}v {sub abs} suggest that LAEs also have outflows. In contrast to LBGs, however, the LAEs' {Delta}v {sub Ly{alpha}} is as small as |{Delta}v {sub abs}|, suggesting low neutral hydrogen column densities. Such a low column density with a small number of resonant scattering may cause the observed strong Ly{alpha} emission of LAEs. We find an anti-correlation between Ly{alpha} equivalent width (EW) and {Delta}v {sub Ly{alpha}} in a compilation of LAE and LBG samples. Although its physical origin is not clear, this anti-correlation result appears to challenge the hypothesis that a strong outflow, by means of a reduced number of resonant scattering, produces a large EW. If LAEs at z > 6 have similarly small {Delta}v {sub Ly{alpha}} values, constraints on the reionization history derived from the Ly{alpha} transmissivity may need to be revised.« less

  5. A human monoclonal anti-TNF alpha antibody (adalimumab) reduces airway inflammation and ameliorates lung histology in a murine model of acute asthma.

    PubMed

    Catal, F; Mete, E; Tayman, C; Topal, E; Albayrak, A; Sert, H

    2015-01-01

    A few experimental studies related to asthma have unveiled the beneficial effects of TNF alpha blocking agents on the airway histology, cytokine levels in bronchoalveolar lavage and bronchial hyper-responsiveness. In the current study, we aimed to assess the effect of adalimumab on the inflammation and histology of asthma in a murine model. Twelve-week-old BALB/c (H-2d/d) female rats (n=18) were allocated into three groups, including (group I) control (phosphate-buffered saline was implemented), (group II) asthma induced with OVA (n=6), and (group III) asthma induced with OVA+treated with adalimumab (n=6). Rats were executed on the 28th day of the study. The lung samples were fixed in 10% neutral buffered formalin. Lung parenchyma, alveolus, peribronchial and perivascular inflammation were assessed. Lung pathological scoring was performed. Severity of lung damage was found to be reduced significantly in the asthma induced with OVA+treated with adalimumab group. When compared with the untreated group, adalimumab significantly reduced the inflammatory cells around the bronchi and bronchioles, and reduced inflammation of the alveolar wall and alveolar wall thickness as well (median score=1, p=0.52). Peribronchial smooth muscle hypertrophy and oedema were significantly reduced after adalimumab administration. Adalimumab (a human monoclonal anti-TNF alpha antibody) therapy significantly reduced the severity of lung damage by decreasing cellular infiltration and improvement on the lung histology in a murine model of acute asthma. Copyright © 2013 SEICAP. Published by Elsevier Espana. All rights reserved.

  6. Tumor necrosis factor-alpha antagonists: differential clinical effects by different biotechnological molecules.

    PubMed

    Licastro, F; Chiappelli, M; Ianni, M; Porcellini, E

    2009-01-01

    Inhibitors of tumor necrosis factor-alpha have deeply changed the therapy of several inflammatory human diseases. For instance, clinical management of rheumatoid arthritis, psoriatic arthritis and ankylosing spondylitis have profoundly benefited after the introduction of new therapeutic tools, such as antagonist of TNF-alpha molecule. These drugs include etanercept, a soluble TNF-alpha receptor antagonist, three anti-TNF-alpha antibodies, adalimumab, infliximab, golimumab and certolizumab a humanized Fab fragment combined with polyethylene glycol. These compounds efficiently inhibit several TNF-alpha biological-mediated effects, however, they have also shown differential clinical efficacy in several trials from different autoimmune diseases. It is of clinical relevance that non-responders to one of these drugs often positively responded to another. Different mechanisms of action and diversity in pharmacokinetics of these three compounds may partially explain different clinical effects. However, partially diverse pathogenetic mechanisms in different diseases also contribute to differential therapeutic responses. Therefore, these apparently homogeneous agents can not be considered equivalent in their clinically efficacy. Differential therapeutic actions of these drugs may be advantageously used in clinical practice and further improve the great potential of individual TNF-alpha inhibitors.

  7. Liver alpha-amylase gene expression as an early obesity biomarker.

    PubMed

    Mojbafan, Marzieh; Afsartala, Zohreh; Amoli, Mahsa M; Mahmoudi, Mahdi; Yaghmaei, Parichehreh; Larijani, Bagher; Ebrahim-Habibi, Azadeh

    2017-04-01

    Obesity is a major health problem worldwide, for which preventive and therapeutic means are still needed. Alpha-amylase is a digestive enzyme whose inhibition has been targeted as a potential anti-obesity strategy. However, alpha-amylase gene expression has not been particularly attended to, and in contrast with pancreatic and salivary amylases, fewer studies have focused on liver alpha-amylase. The present study aimed at investigating the expression of alpha-amylase gene in obese and normal mice at RNA and protein level as well as acarbose effect on this gene expression in hepatocyte cell culture. Control and case groups were fed by normal mouse pellet and high-fat diet respectively, during 8 weeks. After this period, serum biochemical parameters including glucose, cholesterol, triglycerides, AST, ALT and alpha-amylase were assayed. Liver alpha-amylase gene was analyzed by real time PCR, and liver enzyme was assayed with Bernfeld and ELISA methods Hepatocyte cell culture derived from both group were also treated by acarbose and alpha-amylase activity and gene expression was analyzed by above mentioned methods. All biochemical factors showed an increase in obese mice, but the increase in ALT and AST were not statistically significant. Alpha-amylase levels were also increased in obese mice, both at RNA and protein level, while a decrease was seen in obese mice derived hepatocytes after acarbose treatment. Elevated liver alpha-amylase levels may be indicative of initial stages of obesity and the use of acarbose could be considered as a treatment of obesity which could be potentially effective at multiple levels. Copyright © 2016. Published by Elsevier Urban & Partner Sp. z o.o.

  8. Pathophysiology of disk-related low back pain and sciatica. II. Evidence supporting treatment with TNF-alpha antagonists.

    PubMed

    Mulleman, Denis; Mammou, Saloua; Griffoul, Isabelle; Watier, Hervé; Goupille, Philippe

    2006-05-01

    Strong evidence suggests that TNF-alpha may be among the chemical factors involved in disk-related sciatica. TNF-alpha is involved in the genesis of nerve pain in animal models and may promote pain-signal production from nerve roots previously subjected to mechanical deformation. In animal experiments, TNF-alpha has been identified in nucleus pulposus and Schwann cells. Local production of endogenous TNF-alpha may occur early in the pathogenic process. Exposure to exogenous TNF-alpha induces electrophysiological, histological, and behavioral changes similar to those seen after exposure to nucleus pulposus, and these changes are more severe when mechanical compression is applied concomitantly. TNF-alpha antagonists diminish or abolish abnormalities in animal models. Other cytokines may be involved also, as suggested by the potent inhibitory effects of compounds such as doxycycline. Two open-label studies in humans suggest dramatic efficacy of TNF-alpha antagonists in alleviating disk-related sciatica. In contrast, the results of the only controlled study available to date do not support a therapeutic effect of TNF-alpha antagonists. Thus, whether TNF-alpha antagonist therapy is warranted in patients with disk-related sciatica remains an open question, and further randomized controlled studies are needed.

  9. Antibody modified gold nano-mushroom arrays for rapid detection of alpha-fetoprotein.

    PubMed

    Li, Wanbo; Jiang, Xueqin; Xue, Jiancai; Zhou, Zhangkai; Zhou, Jianhua

    2015-06-15

    Localized surface plasmon resonance (LSPR) combined with immunoassay shows greatly potential in fast detection of tumor markers. In this paper, a highly sensitive LSPR substrate has been fabricated and modified for direct detection of alpha-fetoprotein (AFP). The biosensor was prepared by interference lithography, and modified by covalently immobilizing anti-AFP on the surface of gold nano-mushroom arrays (GNMA). The modification process was investigated by Vis-NIR reflectance spectra and cyclic voltammogram measurements. We revealed the optical properties of the modified GNMA by measuring the Vis-NIR reflectance spectra and simulating its electric intensity field distribution under light illumination. The GNMA substrate was highly sensitive, with a refractive index sensitivity of ~465 nm/RIU. The substrate can be applied to label-free detection of AFP, with the linear range and the limit of detection determined to be 20-200 ng/mL and 24 ng/mL (S/N=3), respectively. We also demonstrated its clinical application by directly detecting AFP in human serum samples. It is expected that our biosensor could be integrated on microfluidic chips for high-throughput detection in portable early diagnosis, post-operative and point-of-care (POC) in clinical applications. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. Alpha7 nicotinic receptors as novel therapeutic targets for inflammation-based diseases

    PubMed Central

    Bencherif, Merouane; Lippiello, Patrick M.; Lucas, Rudolf; Marrero, Mario B.

    2013-01-01

    In recent years the etiopathology of a number of debilitating diseases such as type 2 diabetes, arthritis, atherosclerosis, psoriasis, asthma, cystic fibrosis, sepsis, and ulcerative colitis has increasingly been linked to runaway cytokine-mediated inflammation. Cytokine-based therapeutic agents play a major role in the treatment of these diseases. However, the temporospatial changes in various cytokines are still poorly understood and attempts to date have focused on the inhibition of specific cytokines such as TNF-α. As an alternative approach, a number of preclinical studies have confirmed the therapeutic potential of targeting alpha7 nicotinic acetylcholine receptor-mediated anti-inflammatory effects through modulation of proinflammatory cytokines. This “cholinergic anti-inflammatory pathway” modulates the immune system through cholinergic mechanisms that act on alpha7 receptors expressed on macrophages and immune cells. If the preclinical findings translate into human efficacy this approach could potentially provide new therapies for treating a broad array of intractable diseases and conditions with inflammatory components. PMID:20953658

  11. Extensive alternative splicing and dual promoter usage generate Tcf-1 protein isoforms with differential transcription control properties.

    PubMed Central

    Van de Wetering, M; Castrop, J; Korinek, V; Clevers, H

    1996-01-01

    Previously, we reported the isolation of cDNA clones representing four alternative splice forms of TCF-1, a T-cell-specific transcription factor. In the present study, Western blotting (immunoblotting) yielded a multitude of TCF-1 proteins ranging from 25-55 kDa, a pattern not simply explained from the known splice alternatives. Subsequent cDNA cloning, PCR amplification, and analysis by rapid amplification of 5' cDNA ends revealed (i) the presence of an alternative upstream promoter, which extended the known N terminus by 116 amino acids, (ii) the presence of four alternative exons, and (iii) the existence of a second reading frame in the last exon encoding an extended C terminus. Inclusion of the extended N terminus into the originally reported protein resulted in a striking similarity to the lymphoid factor Lef-1. Several of the TCF-1 isoforms, although less potent, mimicked Lef-1 in transactivating transcription through the T-cell receptor alpha-chain (TCR-alpha) enhancer. These data provide a molecular basis for the complexity of the expressed TCF-1 proteins and establish the existence of functional differences between these isoforms. Furthermore, the functional redundancy between Tcf-1 and Lef-1 explains the apparently normal TCR-alpha expression in single Tcf-1 or Lef-1 knockout mice despite the firm in vitro evidence for the importance of the Tcf/Lef site in the TCR-alpha enhancer. PMID:8622675

  12. Progesterone's 5 alpha-reduced metabolite, 3 alpha,5 alpha-THP, mediates lateral displacement of hamsters.

    PubMed

    Frye, Cheryl A; Rhodes, Madeline E

    2005-03-15

    5 alpha-Pregnan-3 alpha-ol-20-one (3 alpha,5 alpha-THP), progesterone (P4)'s 5 alpha-reduced, 3 alpha-hydroxysteroid oxidoreduced product, facilitates lordosis of rodents in part via agonist-like actions at GABA(A)/benzodiazepine receptor complexes in the ventral tegmental area (VTA). Whether 3 alpha,5 alpha-THP influences another reproductively-relevant behavior, lateral displacement, of hamsters was investigated. Lateral displacement is the movement that female hamsters make with their perineum towards male-like tactile stimulation. This behavior facilitates, and is essential for, successful mating. Hamsters in behavioral estrus had greater lateral displacement responses when endogenous progestin levels were elevated compared to when progestin levels were lower. Administration of P4, a prohormone for 3 alpha,5 alpha-THP, dose-dependently (500 > 200 > 100, 50, or 0 microg) enhanced lateral displacement of ovariectomized hamsters that had been primed with SC estradiol benzoate (5 or 10 microg). Inhibiting P4's metabolism to 3 alpha,5 alpha-THP by co-administering finasteride, a 5 alpha-reductase inhibitor, or indomethacin, a 3 alpha-hydroxysteroid oxidoreductase inhibitor, either systemically or to the VTA, significantly decreased lateral displacement and midbrain progestin levels of naturally receptive or hormone-primed hamsters compared to controls. These data suggest that lateral displacement is progestin-sensitive and requires the formation of 3 alpha,5 alpha-THP in the midbrain VTA.

  13. Anti-fibrotic effects of thalidomide on hepatic stellate cells and dimethylnitrosamine-intoxicated rats.

    PubMed

    Chong, Lee-Won; Hsu, Yi-Chao; Chiu, Yung-Tsung; Yang, Kuo-Ching; Huang, Yi-Tsau

    2006-05-01

    Tumor necrosis factor-alpha (TNF-alpha) plays a central role in cellular necrosis, apoptosis, organ failure, tissue damage, inflammation and fibrosis. These processes, occurring in liver injury, may lead to cirrhosis. Thalidomide, alpha-N-phthalidoglutarimide, (C(13)H(10)N(2))(4), has been shown to have immunomodulatory and anti-inflammatory properties, possibly mediated through its anti-TNF-alpha effect. In this study, we investigated the in vitro and in vivo effects of thalidomide on hepatic fibrosis. A cell line of rat hepatic stellate cells (HSC-T6) was stimulated with transforming growth factor-beta1 (TGF-beta1) or TNF-alpha. The inhibitory effects of thalidomide on the NFkappaB signaling cascade and fibrosis markers including alpha-smooth muscle actin (alpha-SMA) and collagen, were assessed. An in vivo therapeutic study was conducted in dimethylnitrosamine (DMN)-treated rats, which were randomly assigned to 1 of 4 groups: vehicle (0.7% carboxyl methyl cellulose, CMC), thalidomide (40 mg/kg), thalidomide (200 mg/kg), or silymarin (50 mg/kg), each given by gavage twice daily for 3 weeks starting after 1 week of DMN administration. Thalidomide (100-800 nM) concentration-dependently inhibited NFkappaB transcriptional activity induced by TNF-alpha, including IKKalpha expression and IkappaBalpha phosphorylation in HSC-T6 cells. In addition, thalidomide also suppressed TGF-beta1-induced alpha-SMA expression and collagen deposition in HSC-T6 cells. Fibrosis scores of livers from DMN-treated rats receiving high dose of thalidomide (0.89 +/- 0.20) were significantly reduced in comparison with those of DMN-treated rats receiving vehicle (1.56 +/- 0.18). Hepatic collagen contents of DMN rats were also significantly reduced by either thalidomide or silymarin treatment. Immunohistochemical double staining results showed that alpha-SMA- and NFkappaB-positive cells were decreased in the livers from DMN rats receiving either thalidomide or silymarin treatment. In addition

  14. Anti-polymorphonuclear neutrophil antibodies in patients with leukopenia or neutropenia.

    PubMed

    Riera, N E; Rosso Saltó, M; Galán, V; Canalejo, K; Khoury, M; Aixalá, M; Kantor, G L; Vermeulen, M; Bengió, R; De Bracco, M M E

    2010-02-01

    Immune humoral neutropenia (Np) could be the consequence of anti-polymorphonuclear neutrophil (PMN) antibodies, circulating immune complexes (CIC) and/or antibodies against myeloid precursors. Granulocyte immunofluorescence test (GIFT) and a leukoagglutination technique (LAGT) assays are recommended for its diagnosis. Fifty adult patients with secondary Np were screened for anti-PMN. GIFT by flow cytometry from viable PMN and LAGT were employed. In addition, CIC levels, low expression of CD16(b) (CD16 (b)(low)), PMN phenotype and sera tumor necrosis factor-alpha (TNF-alpha) were also evaluated. Direct IgG-PMN binding (dir-GIFT) was positive in 16% of the patients. Antibodies against autologous PMN were detected in 32% of the samples by indirect (ind)-GIFT and demonstrated in 70% of the sera by both ind-GIFT and/or LAGT. Predominance of human neutrophil alloantigen (HNA)-1b and HNA-2 expression was confirmed. CD16(b)(low) was detected in 16% of the patient's PMN and TNF-alpha in 68% of sera patients. Our results suggest that diagnosis of immune Np in the laboratory may be improved by focusing on patient's PMN together with the assessment of cellular markers.

  15. NBBA, a synthetic small molecule, inhibits TNF-{alpha}-induced angiogenesis by suppressing the NF-{kappa}B signaling pathway

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kim, Nam Hee; Jung, Hye Jin; Shibasaki, Futoshi

    2010-01-15

    Nuclear factor-{kappa}B (NF-{kappa}B) is a crucial transcription factor that contributes to cancer development by regulating a number of genes involved in angiogenesis and tumorigenesis. Here, we describe (Z)-N-(3-(7-nitro-3-oxobenzo[d][1,2]selenazol-2(3H)-yl)benzylidene) propan-2-amine oxide (NBBA) as a new anti-angiogenic small molecule that targets NF-{kappa}B activity. NBBA showed stronger growth inhibition on human umbilical vein endothelial cells (HUVECs) than on the cancer cell lines we tested. Moreover, NBBA inhibited tumor necrosis factor-alpha (TNF-{alpha})-induced tube formation and invasion of HUVECs. In addition, NBBA suppressed the neovascularization of chorioallantonic membrane from growing chick embryos in vivo. To address the mode of action of the compound, the effectmore » of NBBA on TNF-{alpha}-induced NF-{kappa}B transcription activity was investigated. NBBA suppressed TNF-{alpha}-induced c-Jun N-terminal kinase phosphorylation, which resulted in suppression of transcription of NF-{kappa}B and its target genes, including interleukin-8, interleukin-1{alpha}, and epidermal growth factor. Collectively, these results demonstrated that NBBA is a new anti-angiogenic small molecule that targets the NF-{kappa}B signaling pathway.« less

  16. PET Imaging of Abdominal Aortic Aneurysm with 64Cu-Labeled Anti-CD105 Antibody Fab Fragment.

    PubMed

    Shi, Sixiang; Orbay, Hakan; Yang, Yunan; Graves, Stephen A; Nayak, Tapas R; Hong, Hao; Hernandez, Reinier; Luo, Haiming; Goel, Shreya; Theuer, Charles P; Nickles, Robert J; Cai, Weibo

    2015-06-01

    The critical challenge in abdominal aortic aneurysm (AAA) research is the accurate diagnosis and assessment of AAA progression. Angiogenesis is a pathologic hallmark of AAA, and CD105 is highly expressed on newly formed vessels. Our goal was to use (64)Cu-labeled anti-CD105 antibody Fab fragment for noninvasive assessment of angiogenesis in the aortic wall in a murine model of AAA. Fab fragment of TRC105, a mAb that specifically binds to CD105, was generated by enzymatic papain digestion and conjugated to NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid) for (64)Cu labeling. The binding affinity/specificity of NOTA-TRC105-Fab was evaluated by flow cytometry and various ex vivo studies. BALB/c mice were anesthetized and treated with calcium phosphate to induce AAA and underwent weekly PET scans using (64)Cu-NOTA-TRC105-Fab. Biodistribution and autoradiography studies were also performed to confirm the accuracy of PET results. NOTA-TRC105-Fab exhibited high purity and specifically bound to CD105 in vitro. Uptake of (64)Cu-NOTA-TRC105-Fab increased from a control level of 3.4 ± 0.1 to 9.5 ± 0.4 percentage injected dose per gram (%ID/g) at 6 h after injection on day 5 and decreased to 7.2 ± 1.4 %ID/g on day 12, which correlated well with biodistribution and autoradiography studies (i.e., much higher tracer uptake in AAA than normal aorta). Of note, enhanced AAA contrast was achieved, due to the minimal background in the abdominal area of mice. Degradation of elastic fibers and highly expressed CD105 were observed in ex vivo studies. (64)Cu-NOTA-TRC105-Fab cleared rapidly through the kidneys, which enabled noninvasive PET imaging of the aorta with enhanced contrast and showed increased angiogenesis (CD105 expression) during AAA. (64)Cu-NOTA-TRC105-Fab PET may potentially be used for future diagnosis and prognosis of AAA. © 2015 by the Society of Nuclear Medicine and Molecular Imaging, Inc.

  17. Kinetic studies of the inhibition of a human liver 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase isozyme by bile acids and anti-inflammatory drugs.

    PubMed

    Miyabe, Y; Amano, T; Deyashiki, Y; Hara, A; Tsukada, F

    1995-01-01

    We have investigated the steady-state kinetics for a cytosolic 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase isozyme of human liver and its inhibition by several bile acids and anti-inflammatory drugs such as indomethacin, flufemanic acid and naproxen. Initial velocity and product inhibition studies performed in the NADP(+)-linked (S)-1-indanol oxidation at pH 7.4 were consistent with a sequential ordered mechanism in which NADP+ binds first and leaves last. The bile acids and drugs, competitive inhibitors with respect to the alcohol substrate, exhibited uncompetitive inhibition with respect to the coenzyme, with Ki values less than 1 microM, whereas indomethacin exhibited noncompetitive inhibition (Ki < 24 microM). The kinetics of the inhibition by a mixture of the two inhibitors suggests that bile acids and drugs, except indomethacin, bind to overlapping sites at the active center of the enzyme-coenzyme binary complex.

  18. [In vitro anti-inflammatory and free radical scavenging activities of flavans from Ilex centrochinensis].

    PubMed

    Li, Lu-jun; Yu, Li-juan; Li, Yan-ci; Liu, Meng-yuan; Wu, Zheng-zhi

    2015-04-01

    This study was carried out to evaluate the anti-inflammatory and free radical scavenging activities of flavans from flex centrochinensis S. Y. Hu in vitro and their structure-activity relationship. LPS-stimulated RAW 264.7 macrophage was used as inflammatory model. MTT assay for cell availability, Griess reaction for nitric oxide (NO) production, the content of TNF-alpha, IL-1beta, IL-6 and PGE, were detected with ELISA kits; DPPH, superoxide anion and hydroxyl free radicals scavenging activities were also investigated. According to the result, all flavans tested exhibited anti-inflammatory effect in different levels. Among them, compounds 1, 3, 4 and 6 showed potent anti-inflammatory effect through the inhibition of NO, TNF-alpha, IL-lp and IL-6, of which 1 was the most effective inhibitor, however, 2 and 5 were relatively weak or inactive. The order of free radical scavenging activities was similar to that of anti-inflammatory activities. Therefore, these results suggest that 3, 4 and 6, especially of 1, were,in part responsible for the anti-inflammatory and free radical scavenging activity of Ilex centrochinensis. Hydroxyl group at 4'-position of B-ring plays an important role in the anti-inflammatory and free radical scavenging capacities.

  19. Unlike PPAR{gamma}, PPAR{alpha} or PPAR{beta}/{delta} activation does not promote human monocyte differentiation toward alternative macrophages

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Bouhlel, Mohamed Amine; Inserm U545, F-59000 Lille; UDSL, F-59000 Lille

    2009-08-28

    Macrophages adapt their response to micro-environmental signals. While Th1 cytokines promote pro-inflammatory M1 macrophages, Th2 cytokines promote an 'alternative' anti-inflammatory M2 macrophage phenotype. Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors expressed in macrophages where they control the inflammatory response. It has been shown that PPAR{gamma} promotes the differentiation of monocytes into anti-inflammatory M2 macrophages in humans and mice, while a role for PPAR{beta}/{delta} in this process has been reported only in mice and no data are available for PPAR{alpha}. Here, we show that in contrast to PPAR{gamma}, expression of PPAR{alpha} and PPAR{beta}/{delta} overall does not correlate with the expressionmore » of M2 markers in human atherosclerotic lesions, whereas a positive correlation with genes of lipid metabolism exists. Moreover, unlike PPAR{gamma}, PPAR{alpha} or PPAR{beta}/{delta} activation does not influence human monocyte differentiation into M2 macrophages in vitro. Thus, PPAR{alpha} and PPAR{beta}/{delta} do not appear to modulate the alternative differentiation of human macrophages.« less

  20. BCR CDR3 length distributions differ between blood and spleen and between old and young patients, and TCR distributions can be used to detect myelodysplastic syndrome

    NASA Astrophysics Data System (ADS)

    Pickman, Yishai; Dunn-Walters, Deborah; Mehr, Ramit

    2013-10-01

    Complementarity-determining region 3 (CDR3) is the most hyper-variable region in B cell receptor (BCR) and T cell receptor (TCR) genes, and the most critical structure in antigen recognition and thereby in determining the fates of developing and responding lymphocytes. There are millions of different TCR Vβ chain or BCR heavy chain CDR3 sequences in human blood. Even now, when high-throughput sequencing becomes widely used, CDR3 length distributions (also called spectratypes) are still a much quicker and cheaper method of assessing repertoire diversity. However, distribution complexity and the large amount of information per sample (e.g. 32 distributions of the TCRα chain, and 24 of TCRβ) calls for the use of machine learning tools for full exploration. We have examined the ability of supervised machine learning, which uses computational models to find hidden patterns in predefined biological groups, to analyze CDR3 length distributions from various sources, and distinguish between experimental groups. We found that (a) splenic BCR CDR3 length distributions are characterized by low standard deviations and few local maxima, compared to peripheral blood distributions; (b) healthy elderly people's BCR CDR3 length distributions can be distinguished from those of the young; and (c) a machine learning model based on TCR CDR3 distribution features can detect myelodysplastic syndrome with approximately 93% accuracy. Overall, we demonstrate that using supervised machine learning methods can contribute to our understanding of lymphocyte repertoire diversity.

  1. Synthesis of methyl 3-O-alpha-D-mannopyranosyl-alpha-D-talopyranoside and methyl 3-O-alpha-D-talopyranosyl-alpha-D-talopyranoside.

    PubMed

    Dubey, R; Jain, R K; Abbas, S A; Matta, K L

    1987-08-01

    Methyl 2-O-benzyl-3-O-(2,3,4,6-tetra-O-acetyl-alpha-D-mannopyranosyl)-alpha- D-mannopyranoside (4) and methyl 2-O-benzyl-3-O-alpha-D-mannopyranosyl-alpha-D-mannopyranoside (6) were prepared from a common intermediate, namely, methyl 2-O-benzyl-4,6-O-benzylidene-3-O-(2,3,4,6-tetra-O-acetyl-alpha-D- mannopyranosyl)-alpha-D-mannopyranoside. On treatment with tert-butylchlorodiphenylsilane, in N,N-dimethylformamide in the presence of imidazole, 4 and 6 afforded methyl 2-O-benzyl-6-O-tert-butyldiphenylsilyl-3-O-(2,3,4,6-tetra-O-acetyl -alpha-D- mannopyranosyl)-alpha-D-mannopyranoside (7), and methyl 2-O-benzyl-6-O-tert-butyldiphenylsilyl-3-O-(6-O-tert- butyldiphenylsilyl-alpha-D-mannopyranosyl)-alpha-D-mannopyranoside (8), respectively. Compound 8 was converted into its 2,3-O-isopropylidene derivative (9), and oxidation of 7 and 9 with pyridinium chlorochromate, and reduction of the resulting carbonyl intermediates gave methyl 2-O-benzyl-6-O-tert-butyldiphenylsilyl-3-O-(2,3,4,6-tetra-O-acetyl -alpha-D- mannopyranosyl)-alpha-D-talopyranoside and methyl 2-O-benzyl-6-O-tert-butyldiphenylsilyl-3-O-(6-O-tert-butyldiphe nylsilyl- 2,3-O-isopropylidene-alpha-D-talopyranosyl)-alpha-D-talopyranoside , respectively. Removal of the protecting groups furnished the title disaccharides.

  2. Curcumin inhibits interferon-{alpha} induced NF-{kappa}B and COX-2 in human A549 non-small cell lung cancer cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lee, Jeeyun; Im, Young-Hyuck; Jung, Hae Hyun

    2005-08-26

    The A549 cells, non-small cell lung cancer cell line from human, were resistant to interferon (IFN)-{alpha} treatment. The IFN-{alpha}-treated A549 cells showed increase in protein expression levels of NF-{kappa}B and COX-2. IFN-{alpha} induced NF-{kappa}B binding activity within 30 min and this increased binding activity was markedly suppressed with inclusion of curcumin. Curcumin also inhibited IFN-{alpha}-induced COX-2 expression in A549 cells. Within 10 min, IFN-{alpha} rapidly induced the binding activity of a {gamma}-{sup 32}P-labeled consensus GAS oligonucleotide probe, which was profoundly reversed by curcumin. Taken together, IFN-{alpha}-induced activations of NF-{kappa}B and COX-2 were inhibited by the addition of curcumin in A549more » cells.« less

  3. Tumor necrosis factor alpha converting enzyme: an encouraging target for various inflammatory disorders.

    PubMed

    Bahia, Malkeet S; Silakari, Om

    2010-05-01

    Tumor necrosis factor alpha is one of the most common pro-inflammatory cytokines responsible for various inflammatory disorders. It plays an important role in the origin and progression of rheumatoid arthritis and also in other autoimmune disease conditions. Some anti-tumor necrosis factor alpha antibodies like Enbrel, Humira and Remicade have been successfully used in these disease conditions as antagonists of tumor necrosis factor alpha. Inhibition of generation of active form of tumor necrosis factor alpha is a promising therapy for various inflammatory disorders. Therefore, the inhibition of an enzyme (tumor necrosis factor alpha converting enzyme), which is responsible for processing inactive form of tumor necrosis factor alpha into its active soluble form, is an encouraging target. Many tumor necrosis factor alpha converting enzyme inhibitors have been the candidates of clinical trials but none of them have reached in to the market because of their broad spectrum inhibitory activity for other matrix metalloproteases. Selectivity of tumor necrosis factor alpha converting enzyme inhibition over matrix metalloproteases is of utmost importance. If selectivity is achieved successfully, side-effects can be over-ruled and this approach may become a novel therapy for treatment of rheumatoid arthritis and other inflammatory disorders. This cytokine not only plays a pivotal role in inflammatory conditions but also in some cancerous conditions. Thus, successful targeting of tumor necrosis factor alpha converting enzyme may result in multifunctional therapy.

  4. In vitro cell studies of technetium-99m labeled RGD-HYNIC peptide, a comparison of tricine and EDDA as co-ligands.

    PubMed

    Su, Zi-Fen; He, Jiang; Rusckowski, Mary; Hnatowich, Donald J

    2003-02-01

    The level of alpha(V)beta(3) integrins on endothelial cells is elevated in angiogenesis. The high binding specificity to alpha(V)beta(3) integrins of peptides containing Arg-Gly-Asp (RGD) residues suggests that the radiolabeled RGD peptides may be useful as tumor specific imaging agents. In this research, cyclised peptides containing Arg-Gly-Asp (RGD) and Arg-Gly-Glu (RGE, as control) residues were conjugated with HYNIC and labeled with (99m)Tc. The goal was to evaluate the influence of co-ligand, either tricine or ethylenediamine-N,N'-diacetic acid (EDDA) on protein and integrin binding and on cellular uptake in culture. The n-octanol/water partition coefficient, binding to bovine serum albumin (BSA) and human umbilical vein endothelial (HUVE) cells, and cell lysate distributions of the radiolabeled peptides were evaluated. The co-ligands had a significant effect on the labeling efficiency of the HYNIC conjugates and on certain properties of the (99m)Tc complexes. The labeling efficiency with tricine was 10 fold higher and BSA binding was over 8 fold greater compared to EDDA. Both RGD labels showed higher (6 to 28 fold) binding to HUVE cells than that of the RGE labels, indicating binding specificity. After cell-lysis, only a small percentage of the total RGD label that accumulated in the cells was found bound to cellular proteins (9% of RGD/tricine and 5% of RGD/EDDA), implying that over 90% of the radiolabeled peptides were internalized for both radiolabeled RGDs. The number of the RGD molecules bound to proteins was estimated to be approximately three per cell, suggesting that only a small number of alpha(V)beta(3) integrin proteins are expressed on the cells. Apart from the differences in radiolabeling, the only important effect of substituting EDDA for tricine as co-ligand on the HYNIC-peptides was the lower degree of serum protein binding. In spite of the lower serum protein binding potential, in vivo tumor accumulation of the RGD/EDDA may not be improved

  5. A retrospective analysis of the potential impact of IgG antibodies to agalsidase beta on efficacy during enzyme replacement therapy for Fabry disease.

    PubMed

    Bénichou, Bernard; Goyal, Sunita; Sung, Crystal; Norfleet, Andrea M; O'Brien, Fanny

    2009-01-01

    Fabry disease results from a genetic deficiency of alpha-galactosidase A (alpha GAL) and the impaired catabolism of globotriasoylceramide (GL-3) and other glycosphingolipid substrates, which then accumulate pathogenically within most cells. Enzyme replacement therapy (ERT) with agalsidase beta (Fabrazyme), one of two available forms of recombinant human alpha GAL, involves regular intravenous infusions of the therapeutic protein. Immunoglobulin G (IgG) antibodies to recombinant alpha GAL develop in the majority of patients upon repeated infusion. To explore whether anti-alpha GAL IgG interferes with therapeutic efficacy, retrospective analyses were conducted using data obtained from a total of 134 adult male and female patients with Fabry disease who were treated with agalsidase beta at 1mg/kg every 2 weeks for up to 5 years during placebo-controlled trials and the corresponding open-label extension studies. The analyses did not reveal a correlation between anti-alpha GAL IgG titers and the onset of clinical events or the rate of change in estimated GFR during treatment, and no statistically significant association was found between anti-alpha GAL IgG titers and abnormal elevations in plasma GL-3 during treatment. However, a statistically significant association was found between anti-alpha GAL IgG titers and observation of some GL-3 deposition in the dermal capillary endothelial cells of skin during treatment, suggesting that GL-3 clearance may be partially impaired in some patients with high antibody titers. Determination of the long-term impact of circulating anti-alpha GAL IgG antibodies on clinical outcomes will require continued monitoring, and serology testing is recommended as part of the routine care of Fabry disease patients during ERT.

  6. Interleukin-10 to tumor necrosis factor-alpha ratio is a predictive biomarker in nonalcoholic fatty liver disease: interleukin-10 to tumor necrosis factor-alpha ratio in steatohepatitis.

    PubMed

    Hashem, Reem M; Mahmoud, Mona F; El-Moselhy, Mohamed A; Soliman, Hala M

    2008-10-01

    Fatty liver disease is commonly associated with diabetes mellitus (DM). Insulin resistance (IR) as an investigative biomarker is only concerned with fatty liver that results from DM type 2 associated with metabolic syndrome. Irrespective of IR, DM is generally characterized by overproduction of the proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha), whereas action of the latter is modulated by the anti-inflammatory cytokine interleukin-10 (IL-10). The aim of this study was to investigate the efficacy of using TNF-alpha alone or IL-10/TNF-alpha ratio compared to IR, as a promising biomarker for fatty liver assessment in DM. Furthermore, we hypothesized that using garlic as an immunomodulator may decrease TNF-alpha and increase IL-10 production to improve steatohepatitis. DM was induced metabolically by a high-fat diet to bring about IR, or chemically by alloxan, producing insulin deficiency, in male albino rats. Garlic powder was supplemented (15 mg/kg per day) for 3 weeks. Fatty liver was depicted histologically and biochemically (aspartic aminotransferase, alanine aminotransferase, HOMA-IR, TNF-alpha, IL-10, IL-10/TNF-alpha ratio). We found that, in contrast to obese rats, garlic decreased IL-10/TNF-alpha ratio, despite decreasing TNF-alpha in alloxan diabetic rats in agreement with the histology, which revealed more prominent improvement in the obese group. Moreover, the effect of garlic was not linked to improvement of IR in obese rats. We conclude that IL-10/TNF-alpha ratio may be considered as a convenient biomarker for investigation of fatty liver of different grades, apart from being associated with IR, and immunomodulation of this ratio in favor of increasing it may exert significant improvement.

  7. Ovarian response markers lead to appropriate and effective use of corifollitropin alpha in assisted reproduction.

    PubMed

    La Marca, Antonio; D'Ippolito, Giovanni

    2014-02-01

    Corifollitropin alpha is a highly effective gonadotrophin, which maintains multifollicular growth for a week. The advantages of its administration include ease of use of the drug, making the treatment more patient friendly, resulting in a lower level of distress for the patient. At the same time, the pregnancy rate resulting from its use in IVF/intracytoplasmic sperm injection cycles is similar to that found when daily recombinant FSH is administered. The ovarian response to corifollitropin alpha is dependent on clinically established predictors such as baseline FSH, antral follicle count (AFC) and age. There is a general trend towards a higher ovarian response with an increasing AFC and the number of oocytes per attempt decreased with increasing baseline FSH and age. Even if the risk of ovarian hyperstimulation syndrome following corifollitropin alpha is very similar to the rate reported in literature for young women undergoing IVF, the risk of overstimulation may be reduced by avoiding maximal ovarian stimulation in women anticipated to be hyperresponders. High basal anti-Müllerian hormone and/or AFC can identify women with enhanced functional ovarian reserve at risk of overstimulation, and the risk is even higher if maximally stimulated with corifollitropin alpha or high dose of daily recombinant FSH. Corifollitropin alpha is a highly effective gonadotrophin which maintains multifollicular growth for a week. The ovarian response to corifollitropin was demonstrated to be dependent on clinically established predictors such as baseline FSH, antral follicle count (AFC) and age. There was a general trend toward a higher ovarian response with an increasing AFC and the mean number of oocytes per attempt decreased with increasing baseline FSH and age. Even if the risk of ovarian hyperstimulation syndrome (OHSS) following corifollitropin alpha is very similar to the rate of OHSS reported in literature for young women undergoing IVF, the risk of overstimulation may be

  8. Evaluation of IgY capture ELISA for sensitive detection of alpha hemolysin of Staphylococcus aureus without staphylococcal protein A interference.

    PubMed

    Reddy, Prakash Kudumala; Shekar, Aravind; Kingston, Joseph Jeyabalaji; Sripathy, Murali Harishchandra; Batra, Harshvardhan

    2013-05-31

    Staphylococcal protein A (Spa) secreted by all Staphylococcus aureus strains is the major hindrance in development of specific immunoassays for detecting S. aureus antigens, because of its characteristic feature of binding to Fc region of most mammalian immunoglobulins and also to Fab region of certain classes of mammalian immunoglobulins. Immunoglobulin Y (IgY) is the avian equivalent of mammalian IgG which does not have any affinity to Spa. In the present study we report that using chicken egg yolk IgY over mammalian IgG as capture antibody prevents both soluble and surface bound protein A from causing false positives quantified by chicken anti-protein A antibodies. This was demonstrated by development of sandwich ELISA for detection of alpha hemolysin toxin from culture supernatants of S. aureus strains with anti alpha hemolysin IgY as capture and rabbit anti alpha hemolysin IgG as revealing antibody. This indirect sandwich ELISA was evaluated onto a large number of S. aureus isolates recovered from clinical sources for alpha hemolysin secretion. Results of sandwich ELISA were compared with PCR and Western blot analysis. The immunoassay is highly specific and has high sensitivity of detecting less than 1 ng/ml. This procedure is highly effective in eliminating Spa interference and can be extended to detection of other important superantigen toxins of S. aureus. Copyright © 2013 Elsevier B.V. All rights reserved.

  9. Evidence for a prolonged role of alpha 4 integrin throughout active experimental allergic encephalomyelitis.

    PubMed

    Keszthelyi, E; Karlik, S; Hyduk, S; Rice, G P; Gordon, G; Yednock, T; Horner, H

    1996-10-01

    The leukocyte integrin receptor, alpha 4 beta 1, and its endothelial cell ligand, vascular cell adhesion molecule 1, appear to be of critical importance in the leukocyte trafficking that accompanies CNS damage in experimental allergic encephalomyelitis (EAE). In this study, the persistence of the role for alpha 4 beta 1/VCAM-1 in EAE was established by observing antibody-mediated disease reversal up to 1 month following disease onset. Limited treatment with a monoclonal antibody against alpha 4 integrin, GG5/3, resulted in a significant decrease in both clinical and histopathologic signs. This was not observed in isotype control experiments. In the latter phase of progressive disease, widespread demyelination occurred in the animals that did not respond to 6 days of anti-alpha 4 treatment. These results demonstrate an essential role for alpha 4 beta 1 interactions throughout active EAE and illustrate the difference between reversible clinical deficits caused by edema and irreversible deficits associated with demyelination.

  10. Immunolocalization of thymosin alpha 1, thymopoietin and thymulin in mouse thymic epithelial cells at different stages of culture: a light and electron microscopic study.

    PubMed Central

    Fabien, N; Auger, C; Monier, J C

    1988-01-01

    The secretory evolution of the thymic hormones (thymulin, thymosin alpha 1 and thymopoietin) in cultured thymic reticuloepithelial cells (TREC) was studied by immunocytochemical techniques using monoclonal anti-thymulin or anti-thymosin alpha 1 and polyclonal anti-thymopoietin antibodies (Ab). The culture of TREC was performed with a medium where L-valine was replaced by D-valine, thus ensuring rapid and selective development of these cells. The number of thymulin, thymosin alpha 1 or thymopoietin-containing cells increased progressively from Day 6 to Day 12 of the culture. The localization of the three thymic hormones within the TREC also varied according to the age of the culture. By light microscopy the staining of the three hormones was localized in some cytoplasmic granules at the beginning of the culture and at Day 90, while at Day 12 it was throughout the cytoplasm. In electron microscopy these localizations corresponded respectively to vacuoles of different sizes and to cytosol. All these results show that the synthesis and excretion of thymulin, thymosin alpha 1 and thymopoietin evolve during the development of TREC in culture. Images Figure 1 Figure 2 PMID:3284819

  11. Synthesis of methyl 2-O-alpha-D-mannopyranosyl-alpha-D-talopyranoside and methyl 2-O-alpha-D-talopyranosyl-alpha-D-talopyranoside.

    PubMed

    Jain, R K; Dubey, R; Abbas, S A; Matta, K L

    1987-03-15

    Treatment of methyl 3-O-benzyl-2-O-(2,3,4,6-tetra-O-acetyl-alpha-D-mannopyranosyl)-alpha-D- mannopyranoside (1) with tert-butyldiphenylsilyl chloride in N,N-dimethylformamide afforded methyl 3-O-benzyl-6-O-tert-butyldiphenylsilyl-2-O-(2,3,4,6-tetra-O-acetyl -alpha-D- mannopyranosyl)-alpha-D-mannopyranoside (2). Oxidation of 2 with pyridinium chlorochromate, followed by reduction of the carbonyl group, and subsequent O-deacetylation afforded methyl 3-O-benzyl-6-O-tert-butyldiphenylsilyl-2-O-alpha-D-mannopyranosyl- alpha-D- talopyranoside (5). Cleavage of the tert-butyldiphenylsilyl group of 5 with tetrabutylammonium fluoride in oxolane, followed by hydrogenolysis, gave methyl 2-O-alpha-D-mannopyranosyl-alpha-D-talopyranoside (7). O-Deacetylation of 1 gave methyl 3-O-benzyl-2-O-alpha-D-mannopyranosyl-alpha-D-mannopyranoside (8). Treatment of 8 with tert-butyldiphenylsilyl chloride afforded a 6,6'-disilyl derivative, which was converted into a 2',3'-O-isopropylidene derivative, and then further oxidized with pyridinium chlorochromate. The resulting diketone was reduced and removal of the protecting groups gave methyl 2-O-alpha-D-talopyranosyl-alpha-D-talopyranoside (15). The structures of both 7 and 15 were established by 13C-n.m.r. spectroscopy.

  12. Development of an RNA-based kit for easy generation of TCR-engineered lymphocytes to control T-cell assay performance.

    PubMed

    Bidmon, Nicole; Kind, Sonja; Welters, Marij J P; Joseph-Pietras, Deborah; Laske, Karoline; Maurer, Dominik; Hadrup, Sine Reker; Schreibelt, Gerty; Rae, Richard; Sahin, Ugur; Gouttefangeas, Cécile; Britten, Cedrik M; van der Burg, Sjoerd H

    2018-07-01

    Cell-based assays to monitor antigen-specific T-cell responses are characterized by their high complexity and should be conducted under controlled conditions to lower multiple possible sources of assay variation. However, the lack of standard reagents makes it difficult to directly compare results generated in one lab over time and across institutions. Therefore TCR-engineered reference samples (TERS) that contain a defined number of antigen-specific T cells and continuously deliver stable results are urgently needed. We successfully established a simple and robust TERS technology that constitutes a useful tool to overcome this issue for commonly used T-cell immuno-assays. To enable users to generate large-scale TERS, on-site using the most commonly used electroporation (EP) devices, an RNA-based kit approach, providing stable TCR mRNA and an optimized manufacturing protocol were established. In preparation for the release of this immuno-control kit, we established optimal EP conditions on six devices and initiated an extended RNA stability study. Furthermore, we coordinated on-site production of TERS with 4 participants. Finally, a proficiency panel was organized to test the unsupervised production of TERS at different laboratories using the kit approach. The results obtained show the feasibility and robustness of the kit approach for versatile in-house production of cellular control samples. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

  13. Improved labelling of DTPA- and DOTA-conjugated peptides and antibodies with 111In in HEPES and MES buffer.

    PubMed

    Brom, Maarten; Joosten, Lieke; Oyen, Wim Jg; Gotthardt, Martin; Boerman, Otto C

    2012-01-27

    In single photon emission computed tomography [SPECT], high specific activity of 111In-labelled tracers will allow administration of low amounts of tracer to prevent receptor saturation and/or side effects. To increase the specific activity, we studied the effect of the buffer used during the labelling procedure: NaAc, NH4Ac, HEPES and MES buffer. The effect of the ageing of the 111InCl3 stock and cadmium contamination, the decay product of 111In, was also examined in these buffers. Escalating amounts of 111InCl3 were added to 1 μg of the diethylene triamine pentaacetic acid [DTPA]- and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid [DOTA]-conjugated compounds (exendin-3, octreotide and anti-carbonic anhydrase IX [CAIX] antibody). Five volumes of 2-(N-morpholino)ethanesulfonic acid [MES], 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid [HEPES], NH4Ac or NaAc (0.1 M, pH 5.5) were added. After 20 min at 20°C (DTPA-conjugated compounds), at 95°C (DOTA-exendin-3 and DOTA-octreotide) or at 45°C (DOTA-anti-CAIX antibody), the labelling efficiency was determined by instant thin layer chromatography. The effect of the ageing of the 111InCl3 stock on the labelling efficiency of DTPA-exendin-3 as well as the effect of increasing concentrations of Cd2+ (the decay product of 111In) were also examined. Specific activities obtained for DTPA-octreotide and DOTA-anti-CAIX antibody were five times higher in MES and HEPES buffer. Radiolabelling of DTPA-exendin-3, DOTA-exendin-3 and DTPA-anti-CAIX antibody in MES and HEPES buffer resulted in twofold higher specific activities than that in NaAc and NH4Ac. Labelling of DTPA-exendin-3 decreased with 66% and 73% for NaAc and NH4Ac, respectively, at day 11 after the production date of 111InCl3, while for MES and HEPES, the maximal decrease in the specific activity was 10% and 4% at day 11, respectively. The presence of 1 pM Cd2+ in the labelling mixture of DTPA-exendin-3 in NaAc and NH4Ac markedly reduced the labelling

  14. Reaction process of {alpha} {yields} {gamma} massive transformation in Ti-rich TiAl alloy

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kumagai, T.; Abe, E.; Nakamura, M.

    1995-08-01

    Reaction sequence of the massive transformation from the high-temperature {alpha}-Ti phase to the {gamma}-TiAl phase ({gamma}{sub m}) in a Ti-48at.% Al alloy has been examined in terms of optical and transmission electron microscopes. Both transformed and untransformed regions were macroscopically observed in the sample quenched from the high-temperature {alpha} phase field, when the sample was held there for a extended period of time prior to quenching. The transformed region consists of randomly oriented fine {gamma} single phase grains, in which many thermal anti-phase domains (TAPDs), together with a number of stacking faults were observed. In contrast, the untransformed region comprisesmore » extremely fine lamellae of the {gamma} and {alpha}{sub 2}-Ti{sub 3}Al phases, and the {gamma} plates were found to run through the TAPDs caused by {alpha} {yields} {alpha}{sub 2} ordering. Subsequent aging at 1,273 K causes the microstructure change in the untransformed region from {alpha}{sub 2}/{gamma} lamellae to {gamma}/{gamma} lamellae spontaneously and expands the {gamma}{sub m} region. These observations suggest that the {alpha} {yields} {gamma}{sub m} transformation proceeds through formation of fine {gamma} plates.« less

  15. Synthesis of 3 alpha, 5 alpha-tetrahydroaldosterone.

    PubMed

    Harnik, M; Kashman, Y; Morris, D J

    1984-06-01

    3 alpha, 5 alpha-Tetrahydroaldosterone (12a), a metabolite of aldosterone, has been synthesized from the lactone 2a or, preferably 11 beta, 21-dihydroxy-5-pregnene-3,20-dione-18-oic acid 3,20-di-(ethylene glycol)-ketal (18----11) lactone 21-acetate (6b), via 11 beta, 21-dihydroxy-5 alpha-pregnane-3,20-dione-18-oic acid 3,20-di-(ethylene glycol) ketal (18----11) lactone 21-acetate (4b). Selective hydrolysis of the latter at C-3 furnished the monoketal 5 which, on reduction with potassium tri-sec-butylborohydride, yielded predominantly 3 alpha, 11 beta, 21-trihydroxy-5 alpha-pregnan-20-one-18-oic acid 20-(ethylene glycol)-ketal (18----11) lactone (8a) and its acetate 8b. Further reduction with diisobutylaluminum hydride afforded 3 alpha, 5 alpha-tetrahydroaldosterone-20-ketal (10a), which was directly hydrolyzed to 12a with dilute acid in tetrahydrofuran-dioxan. Periodate oxidation led to the gamma-etiolactone 14a, which was then further converted into 5 alpha-dihydroaldosterone-gamma-etiolactone (14c).

  16. Inhibition of B cell proliferation by antisense DNA to both alpha and beta forms of Fc epsilon R II.

    PubMed

    Bhatti, L; Behle, K; Stevens, R H

    1992-10-01

    Epstein-Barr Virus (EBV) infection activates B lymphocyte proliferation through partially understood mechanisms, resulting in phenotypic changes, including the appearance of new antigens. One such antigen is Fc epsilon R II/CD-23 which may be relevant for B cell proliferation. We have used anti-sense oligonucleotides to study the importance of the two forms of this molecule for proliferation in the EBV-transformed, Fc epsilon R II +ve lymphoblastoid B cell line, RPMI 8866. Anti-sense oligodeoxynucleotides were generated to the two forms of Fc epsilon R II; Fc epsilon R IIa (alpha) and IIb (beta) which differ only in their intracytoplasmic domains. Addition of increasing concentrations of anti-sense oligonucleotides, ranging from 1 to 30 microM, significantly decreased cellular proliferation as measured by the incorporation of [3H]thymidine (inhibition range 8-88%). Optimum inhibition of cellular proliferation was apparent at 15 microM concentration of both anti-sense Fc epsilon R IIa and IIb (Fc epsilon R IIa, mean +/- SE = 75 +/- 7% inhibition, p less than 0.001; Fc epsilon R IIb, mean +/- SE = 71 +/- 7% inhibition, p less than 0.001). Anti-sense oligonucleotides complementary to the common part of Fc epsilon R II resulted in a similar inhibition of proliferation. Sense oligonucleotides did not induce significant inhibition. Preincubation of sense and anti-sense oligonucleotides resulted in an abrogation of proliferation inhibition. Moreover, none of these oligonucleotides had any effect on a Fc epsilon R II -ve cell line. Incubation with both anti-sense IIa and IIb resulted in additive, but not synergistic inhibition of proliferation. Addition of soluble Fc epsilon R II did not reverse inhibition of proliferation, suggesting that membrane-bound or intracellular rather than soluble Fc epsilon R II was important for the induced proliferation. Analysis of cell surface expression for Fc epsilon II indicated that while there was a pronounced effect on cell number

  17. Effect of alpha-lipoic acid on endometrial implants in an experimental rat model.

    PubMed

    Pınar, Neslihan; Soylu Karapınar, Oya; Özcan, Oğuzhan; Özgür, Tümay; Bayraktar, Suphi

    2017-10-01

    To investigate the antioxidant and anti-inflammatory effects of alpha-lipoic acid (ALA) in the treatment of endometriosis in an experimental rat model by evaluating biochemical and histopathologic parameters. Experimental endometriosis was induced by the peritoneal implantation of autologous endometrial tissue. The rats were randomly divided into two groups with eight rats each. Group I was intraperitoneally administered ALA 100 mg/kg/day for 14 days. Group II was intraperitoneally administered saline solution at the same dosage and over the same period. Endometrial implant volume was measured in both groups both pre- and post-treatment. Tumor necrosis factor alpha (TNF-α) was measured in peritoneal fluid. Total antioxidant status (TAS), total oxidant status (TOS), and oxidative stress index (OSI) were assessed in serum. The implants were histopathologically evaluated. In the ALA group, the serum TOS and OSI levels, the endometrial implant volumes, the TNF-α levels in serum and peritoneal fluid, and the histopathologic scores were significantly lower compared to the control group (P < 0.05). Alpha-lipoic acid may have a therapeutic potential in the treatment of endometriosis due to its antioxidant and anti-inflammatory effects. © 2017 Société Française de Pharmacologie et de Thérapeutique.

  18. Mechanisms of stimulation of interleukin-1 beta and tumor necrosis factor-alpha by Mycobacterium tuberculosis components.

    PubMed Central

    Zhang, Y; Doerfler, M; Lee, T C; Guillemin, B; Rom, W N

    1993-01-01

    The granulomatous immune response in tuberculosis is characterized by delayed hypersensitivity and is mediated by various cytokines released by the stimulated mononuclear phagocytes, including tumor necrosis factor-alpha (TNF alpha) and IL-1 beta. We have demonstrated that Mycobacterium tuberculosis cell wall component lipoarabinomannan (LAM), mycobacterial heat shock protein-65 kD, and M. tuberculosis culture filtrate, devoid of LPS as assessed by the Amebocyte Lysate assay, stimulate the production of TNF alpha and IL-1 beta proteins and mRNA from mononuclear phagocytes (THP-1 cells). The effect of LAM on the release of these cytokines was specific, as only LAM stimulation was inhibited by anti-LAM monoclonal antibody. Interestingly, we found that LAM and Gram-negative bacterial cell wall-associated endotoxin LPS may share a similar mechanism in their stimulatory action as demonstrated by inhibition of TNF alpha and IL-1 beta release by monoclonal antibodies to CD14. Anti-CD14 monoclonal antibody MY4 inhibited both TNF alpha and IL-1 beta release with LAM and LPS but no effect was observed with other mycobacterial proteins. An isotype antibody control did not inhibit release of cytokines under the same experimental conditions. M. tuberculosis and its components upregulated IL-1 beta and TNF alpha mRNAs in THP-1 cells. Nuclear run-on assay for IL-1 beta demonstrated that LAM increased the transcription rate. The induction of IL-1 beta was regulated at the transcriptional level, in which these stimuli acted through cis-acting element(s) on the 5' flanking region of the IL-1 beta genomic DNA. M. tuberculosis cell wall component LAM acts similarly to LPS in activating mononuclear phagocyte cytokine TNF alpha and IL-1 beta release through CD14 and synthesis at the transcriptional level; both cytokines are key participants in the host immune response to tuberculosis. Images PMID:7683696

  19. Interferon-alpha and interferon-gamma modulate Fas-mediated apoptosis in mitomycin-C-resistant human Tenon's fibroblasts.

    PubMed

    Wang, Xiao Yang; Crowston, Jonathan G; White, Andrew J R; Zoellner, Hans; Healey, Paul R

    2014-08-01

    The aim of the study was to investigate, using a native mitomycin-C-resistant human Tenon's fibroblast cell line, the possibility that interferon-alpha and gamma could be used with Fas agonists as an alternative anti-fibrotic strategy to mitomycin-C in trabeculectomy. A clinically resistant and in vitro verified mitomycin-C-resistant human Tenon's fibroblast cell line was pretreated with interferon-alpha and interferon-gamma for 48 h before stimulation with an agonistic Fas antibody (CH11) for 2 days to induce cell death. Cell death assays were undertaken. Changes in apoptosis-related proteins were determined by flow cytometry and Western blot. Pretreatment with interferon-alpha or interferon-gamma for 48 h increased Fas, Fas-associated protein with death domain and caspase-8 expression. Protein expression was further increased by combined exposure to interferon-alpha and gamma. Pretreatment with cytokines had no effect on Fas-L and Bcl-2. Interferon-alpha alone did not change the rate of induced cell death. A combination of interferon-alpha and gamma synergistically increased the sensitivity of mitomycin-C-resistant human Tenon's fibroblast cell line to induced cell death. An antagonistic anti-Fas antibody (ZB4) completely blocked induced cell death. Broad caspase inhibitors specific for caspases-8 and -3 reduced induced deaths in interferon pretreated mitomycin-C-resistant human Tenon's fibroblast cell line in a dose-dependent manner. Interferon-alpha and interferon-gamma render mitomycin-C-resistant human Tenon's fibroblast cell line sensitive to Fas-mediated apoptosis. The mechanism involves increased death-inducing signalling complex formation by upregulation of Fas, Fas-associated protein with death domain and caspase-8 expression. © 2013 Royal Australian and New Zealand College of Ophthalmologists.

  20. A novel electrochemical immunosensor using β-cyclodextrins functionalized silver supported adamantine-modified glucose oxidase as labels for ultrasensitive detection of alpha-fetoprotein.

    PubMed

    Gao, Jian; Ma, Hongmin; Lv, Xiaohui; Yan, Tao; Li, Na; Cao, Wei; Wei, Qin

    2015-09-17

    In this work, a novel sandwich-type electrochemical immunosensor based on host-guest interaction was fabricated for the detection of alpha-fetoprotein (AFP). Due to the large specific surface area of multiwalled carbon nanotubes and the unique supramolecular recognition ability of β-cyclodextrins, ferrocenecarboxylic acid (Fc) was incorporated into this sensor platform by host-guest interaction to generate an electrochemical signal. And β-cyclodextrins functionalized silver supported adamantine-modified glucose oxidase (GOD-CD-Ag), was used as a label to improve the analytical performance of the immunosensor by the dual amplification strategy. The obtained GOD-CD-Ag conjugates could convert glucose into gluconic acid with the formation of hydrogen peroxide (H2O2). And then silver nanoparticles could in situ catalyze the reduction of the generated H2O2, dramatically improving the oxidation reaction of Fc. The developed immunosensor shows a wide linear calibration range from 0.001 to 5.0 ng/mL with a low detection limit (0.2 pg/mL) for the detection of AFP. The method, with ideal reproducibility and selectivity, has a wide application prospect in clinical research. Copyright © 2015 Elsevier B.V. All rights reserved.