ANGEWANDTE CHEMIE, Issue 41 2009
Katarzyna Gorska
Alles unter Kontrolle: Die Programmierbarkeit von Hybridisierungsvorgängen wurde zur Erzeugung einer kombinatorischen Bibliothek von Strukturen genutzt, die Topologien komplexer Kohlenhydrate imitieren, die mit einem Antikörper mit Breitbandwirkung gegen HIV wechselwirken. Bei dieser einfachen Methode werden mit Peptidnucleinsäuren markierte Oligosaccharide in kontrollierter Weise mit DNA-Templaten verknüpft (siehe Bild). [source]

Epitope of autoantibodies to N -methyl-d-aspartate receptor heteromers in paraneoplastic limbic encephalitis

ANNALS OF NEUROLOGY, Issue 1 2008
Yukitoshi Takahashi MD
No abstract is available for this article. [source]

C-Linked Disaccharide Analogue of the Thomsen,Friedenreich (T)-Epitope ,-O-Conjugated to L -Serine

CHEMISTRY - A EUROPEAN JOURNAL, Issue 12 2005
Loay Awad
Abstract Condensation of a silylated ,- D -galactopyranosylaldehyde (3) with isolevoglucosenone (4) in the presence of Et2AlI provided bicyclic enone 5. Subsequent addition of BnNHOMe gave adduct 6, which was converted into 4- O -acetyl-1,6-anhydro-3- C -[(1,R)-1,3,4,5,7-penta- O -acetyl-2,6-anhydro- D - glycero- L - manno -heptitol-1- C -yl]-2-azido-2,3-dideoxy-,- D - galacto -hexopyranose after liberation of the 2-amino group, its transformation into a 2-azido moiety, desilylation, and peracetylation. Ring-opening of the 1,6-anhydro galactopyranosyl unit and O -glycosidation with Fmoc-Ser-O- tBu afforded a 5:1 mixture of ,- and ,-galactosides. Treatment with CH3COSH gave pure N -[(9H -fluoren-9-ylmethoxy)carbonyl]-{4,6-di- O -acetyl-3- C -[(1,R)-2,6-anhydro 1,3,4,5,7-penta- O -acetyl- D - glycero - L - manno -heptitol-1- C -yl]-2-[(N- acetyl)amino]-2,3-dideoxy-,- D -galactopyranosyl}- l- serine tert -butyl ester (2), a protected form of a C-disaccharide analogue of the Thomsen,Friedenreich (or T) epitope (,- D -Galp -(1,3)-,- D -GalNAcp) ,-O-conjugated to L -serine. [source]

Spatially and temporally regulated expression of specific heparan sulfate epitopes in the developing mouse olfactory system

DEVELOPMENT GROWTH & DIFFERENTIATION, Issue 2 2010
Jun Takatoh
Heparan sulfate (HS) comprises a structurally diverse group of glycosaminoglycans present ubiquitously on cell surfaces and in the extracellular matrix. The spatially and temporally regulated expression of specific HS structures is essential for various developmental processes in the nervous system but their distributions in the mouse olfactory system have not been explored. Here, we examined the spatiotemporal distribution of particular HS species in the developing mouse olfactory system using three structure-specific monoclonal antibodies (HepSS-1, JM403 and NAH46). The major findings were as follows. (i) During olfactory bulb morphogenesis, the HepSS-1 epitope was strongly expressed in anterior telencephalic cells and coexpressed with fibroblast growth factor receptor 1. (ii) In early postnatal glomeruli, the JM403 epitope was expressed at different levels among individual glomeruli. The expression pattern and levels of the JM403 epitope were both associated with those of ephrin-A3. (iii) In the vomeronasal system, the JM403 epitope was expressed in all vomeronasal axons but became increasingly restricted to vomeronasal axons terminating in the anterior region of the accessory olfactory bulb by 3 weeks of age. Our results demonstrate that each HS epitope exhibits a unique expression pattern during the development of the mouse olfactory system. Thus, each HS epitope is closely associated with particular developmental processes of the olfactory system and might have a particular role in developmental events. [source]

Spatiotemporal distribution of heparan sulfate epitopes during myogenesis and synaptogenesis: A study in developing mouse intercostal muscle

DEVELOPMENTAL DYNAMICS, Issue 1 2002
Guido J. Jenniskens
Abstract Formation of a basal lamina (BL) ensheathing developing skeletal muscle cells is one of the earliest events in mammalian skeletal muscle myogenesis. BL-resident heparan sulfate proteoglycans have been implicated in various processes during myogenesis, including synaptic differentiation. However, attention has focused on the proteoglycan protein core, ignoring the glycosaminoglycan moiety mainly because of a lack of appropriate tools. Recently, we selected a panel of anti,heparan sulfate antibodies applied here to study the spatiotemporal distribution of specific heparan sulfate (HS) epitopes during myogenesis. In mouse intercostal muscle at embryonic day (E14), formation of acetylcholine receptor clusters at synaptic sites coincides with HS deposition. Although some HS epitopes show a general appearance throughout the BL, one epitope preferably clusters at synaptic sites but does so only from E16 onward. During elongation and maturation of primary myotubes, a process preceding secondary myotube development, significant changes in the HS epitope constitution of both synaptic and extrasynaptic BL were observed. As a whole, the data presented here strengthen previous observations on developmental regulation by BL components, and add to the putative roles of specific HS epitopes in myogenesis and synaptogenesis. © 2002 Wiley-Liss, Inc. [source]

GAD65 autoantibody epitopes in adult patients with latent autoimmune diabetes following GAD65 vaccination

DIABETIC MEDICINE, Issue 5 2007
L. M. Bekris
Abstract Aims Subcutaneous injection of recombinant human GAD65 (rhGAD65) in patients with latent autoimmune diabetes in adults (LADA) correlates with an increase in C-peptide levels. In this study we analysed the effect of rhGAD65 administration on the GAD65-specific autoimmune response. Methods Longitudinal serum samples obtained from LADA patients (n = 47) who received 4, 20, 100 or 500 µg alum-formulated rhGAD65 or placebo by subcutaneous injection twice (4 weeks apart) were analysed for their epitope recognition using GAD65-specific recombinant Fab and GAD65/67 fusion proteins. Results Overall, minor changes in the epitope pattern were observed using either approach. Only in the 500-µg dosage group was an increase in GAD65Ab level associated with a significant increase in the binding to a conformational epitope located at the middle part of GAD65. Conclusions Our data suggest that the apparent beneficial effects of 20 µg alum-formulated recombinant human GAD65 is not explained by changes in the GAD65Ab epitope pattern. [source]

LBY135, a novel anti-DR5 agonistic antibody induces tumor cell,specific cytotoxic activity in human colon tumor cell lines and xenografts,

DRUG DEVELOPMENT RESEARCH, Issue 2 2008
Jing Li
Abstract TRAIL (TNF-related apoptosis-inducing ligand) induces apoptosis on binding to DR4 and DR5 receptors on the surface of tumor cells. These receptors are of particular interest in the development of cancer therapeutics as they preferentially mediate tumor cell apoptosis. We have generated a chimeric anti-DR5 agonistic antibody, LBY135, from its murine parental antibody, LCR211, identified using hybridoma technology. Both LCR211 and LBY135 specifically bind to DR5 with nanomolar affinity, mimic TRAIL to induce cell death in tumor cells, and have little effect on non-transformed cells in vitro. The anti-DR5 antibody reduced viability in 45% of a panel of 40 human colon cancer cell lines with IC50 values of 20,nM or less. In vivo, using human colorectal tumor xenograft mouse models, LCR211 induced tumor regression and showed enhanced efficacy when combined with 5-FU. Both in vitro evaluation of ADCC (antibody-dependent cell-mediated cytotoxicity) and CDC (complement-dependent cytotoxicity), and in vivo studies using a non-functional DR5 specific antibody or SCID-Beige mice, suggested ADCC and CDC are unlikely to be the mechanism to ablate tumors in vivo. LBY135 and LCR211 appear to mediate cell death and tumor regression mainly through apoptosis, as demonstrated by the activation of caspase 3, caspase 8, M30, and TUNEL assay. In addition, the discovery of synergy between cross-linked LBY135 and TRAIL not only revealed the unique epitope of LBY135, but also demonstrated an additional mechanism of action for LBY135 in vivo. LBY135 demonstrates promise as a novel therapeutic for cancer treatment and is currently in Phase I clinical trials. Drug Dev Res 69: 69,82, 2008. © 2008 Wiley-Liss, Inc. [source]

Capillary electrophoresis of affinity complexes between subviral 80S particles of human rhinovirus and monoclonal antibody 2G2

ELECTROPHORESIS, Issue 13 2006
Leopold Kremser Dr.
Abstract Human rhinoviruses (HRVs), the main etiologic agents of the common cold, transform into subviral B- or 80S particles (they sediment at 80S upon sucrose density gradient centrifugation) during infection and, in,vitro, upon exposure to a temperature between 50 and 56°C. With respect to the native virion they lack the genomic RNA and the viral capsid protein VP4. 80S particles are unstable and easily disintegrate into their components, VP1, VP2, and VP3 in buffers containing SDS. However, this detergent was found to be a necessary constituent of the BGE for the analysis of these viruses and their complexes with receptors and antibodies by CE. We here demonstrate that dodecylpoly(ethyleneglycol ether) (D-PEG) a nonionic detergent, is suitable for analysis of subviral particles as it preserves their integrity, in contrast to SDS. Electrophoresis of the 80S particles in borate buffer (pH,8.3, 100,mM) containing 10,mM D-PEG resulted in a well-defined electrophoretic peak. The identity of the peak was confirmed, among other means, by complexation with mAb,2G2, which recognizes a structural epitope exclusively present on subviral particles but not on native virus. Upon incubation of the 80S particles with mAb,2G2 the peak disappeared, but a new peak, attributed to the antibody complex emerged. The separation system allowed following the time course of the transformation of intact HRV serotype,2 into 80S particles upon incubation at temperatures between 40 and 65°C. We also demonstrate that subviral particles derived from HRV2 labeled with the fluorescence dyes FITC or Cy3.5 were stable in the separation system containing D-PEG. Dye-modified particles were still recognized by mAb,2G2, suggesting that the exposed lysines that are derivatized by the reagent do not form part of the epitope of the antibody. [source]

Cross-reactivity of antibodies to actin- depolymerizing factor/cofilin family proteins and identification of the major epitope recognized by a mammalian actin-depolymerizing factor/cofilin antibody

ELECTROPHORESIS, Issue 15 2004
Alisa E. Shaw
Abstract Members of the actin-depolymerizing factor (ADF)/cofilin family of proteins are expressed in all eukaryotic cells. In higher vertebrates, cells often express as many as three different ADF/cofilin genes and each of these proteins may be phosphorylated on serine 3, giving rise to up to six different species. Also, many avian, amphibian, and invertebrate systems have been useful in studying different aspects of ADF/cofilin function. Antibodies have been prepared against different members of the ADF/cofilin family, but no systematic examination of their cross-reactivity has been reported. Although ADF and cofilins within a single vertebrate species have about a 70% sequence homology, antibodies often differentiate between these proteins. Here, Western blotting was used with chemiluminescence substrates of different sensitivities to determine the relative immunoreactivities of different polyclonal rabbit antibodies and a mouse monoclonal antibody to purified ADF/cofilins from plants, protists, nematodes, insects, echinoderms, birds, and mammals. From immunocross-reactivities and sequence alignments, the principal epitope in mammalian ADF and cofilin-1 recognized by an antibody raised against avian ADF was identified. The specificity of an antibody to the phosphopeptide epitope of metazoan ADF/cofilins was confirmed by two-dimensional (2-D) immunoblot analysis. Futhermore, this bank of antibodies was used to identify by Western blotting a putative member of the ADF/cofilin family in the sea slug, Aplysia californica. [source]

Frequent occurrence of high affinity T cells against MELOE-1 makes this antigen an attractive target for melanoma immunotherapy

EUROPEAN JOURNAL OF IMMUNOLOGY, Issue 6 2010
Yann Godet
Abstract We recently showed that the infusion of tumor infiltrating lymphocytes specific for the MELOE-1 antigen was associated with a prolonged relapse-free survival for HLA-A2+ melanoma patients who received tumor infiltrating lymphocytes therapy. Here, we characterized the MELOE-1/A2-specific T-cell repertoire in healthy donors and melanoma patients to further support an immunotherapy targeting this epitope. Using tetramer enrichment followed by multicolor staining, we found that MELOE-1-specific T cells were present in the blood of healthy donors and patients at similar frequencies (around 1 in 1×105 CD8+ cells). These cells mainly displayed a naïve phenotype in 4/6 healthy donors and 3/6 patients, whereas high proportions of memory cells were observed in the remaining individuals of both groups. There was a recurrent usage of the V,12.1 chain for 17/18 MELOE-1-specific T-cell clones derived from healthy donors or patients, associated with diverse V, chains and V(D)J junctional sequences. All clones derived from melanoma patients (9/9) were reactive against the MELOE-136,44 peptide and against HLA-A2+ melanoma cell lines. This study documents the existence of a large TCR repertoire specific for the MELOE-1/A2 epitope and its capacity to give rise to antitumor CTL that supports the development of immunotherapies targeting this epitope. [source]

Altered effector functions of virus-specific and virus cross-reactive CD8+ T cells in mice immunized with related flaviviruses

EUROPEAN JOURNAL OF IMMUNOLOGY, Issue 5 2010
Derek W. Trobaugh
Abstract Memory cross-reactive CD8+ T-cell responses may induce protection or immunopathology upon secondary viral challenge. To elucidate the potential role of T cells in sequential flavivirus infection, we characterized cross-reactive CD4+ and CD8+ T-cell responses between attenuated and pathogenic Japanese encephalitis virus (JEV) and pathogenic West Nile virus (WNV). A previously reported WNV NS4b CD8+ T-cell epitope and its JEV variant elicited CD8+ T-cell responses in both JEV- and WNV-infected mice. The peptide variant homologous to the immunizing virus induced greater cytokine secretion and activated higher frequencies of epitope-specific CD8+ T cells. However, there was a virus-dependent, peptide variant-independent pattern of cytokine secretion; the IFN,+ -to-IFN,+TNF,+ CD8+ T-cell ratio was greater in JEV- than in WNV-infected mice. Despite similarities in viral burden for pathogenic WNV and JEV viruses, CD8+ T cells from pathogenic JEV-immunized mice exhibited functional and phenotypic profiles similar to those seen for the attenuated JEV strain. Patterns of killer cell lectin-like receptor G1 (KLRG1) and CD127 expression differed by virus type, with a rapid expansion and contraction of short-lived effector cells in JEV infection and persistence of high levels of short-lived effector cells in WNV infection. Such cross-reactive T-cell responses to primary infection may affect the outcomes of sequential flavivirus infections. [source]

Fine antigenic variation within H5N1 influenza virus hemagglutinin's antigenic sites defined by yeast cell surface display

EUROPEAN JOURNAL OF IMMUNOLOGY, Issue 12 2009
Jian Li
Abstract Fifteen strains of mAb specific for HA of the A/Hong Kong/482/97 (H5N1) influenza virus were generated. The HA antigenic sites of the human A/Hong Kong/482/97 (H5N1) influenza virus were defined by using yeast cell surface-displaying system and anti-H5 HA mAb. Evolution analysis of H5 HA identified residues that exhibit diversifying selection in the antigenic sites and demonstrated surprising differences between residue variation of H5 HA and H3 HA. A conserved neutralizing epitope in the H5 HA protein recognized by mAb H5M9 was found using viruses isolated from 1997,2006. Seven single amino acid substitutions were introduced into the HA antigenic sites, respectively, and the alteration of antigenicity was assessed. The structure obtained by homology-modeling and molecular dynamic methods showed that a subtle substitution at residue 124 propagates throughout its nearby loop (152,159). We discuss how the structural changes caused by point mutation might explain the altered antigenicity of the HA protein. The results demonstrate the existence of immunodominant positions in the H5 HA protein, alteration of these residues might improve the immunogenicity of vaccine strains. [source]

Getting to the crux of the matter: IL-23 and Th17 cell accumulation in the CNS

EUROPEAN JOURNAL OF IMMUNOLOGY, Issue 7 2009
Benjamin M. Segal
Abstract IL-23 plays a critical role in EAE induced by the active immunization of C57BL/6 mice with an immunodominant epitope of myelin oligodendrocyte glycoprotein (MOG35,55). It was initially assumed that the pathogenic effects of IL-23 were directly related to the generation, expansion and/or stabilization of autoreactive CD4+ Th17 cells. However, a number of recent studies have uncovered discrepancies between the requirement for IL-23, as opposed to Th17 cells or their products (IL-17A, IL-17F and IL-22), in the development of EAE. In this issue of the European Journal of Immunology, it is demonstrated that impairment of IL-23 signaling does not impede the expansion of myelin-specific CD4+ T cells in peripheral lymphoid tissues but inhibits their accumulation in the CNS. This paper contributes to a growing body of data that implicates IL-23 in the acquisition of CNS homing properties by autoreactive effector cells. [source]

In vivo application of mAb directed against the ,, TCR does not deplete but generates "invisible" ,, T cells

EUROPEAN JOURNAL OF IMMUNOLOGY, Issue 2 2009
Christian Koenecke
Abstract mAb targeting the ,, TCR have been used for ,, T-cell depletion with varying success. Although the depletion-capacity of the anti-,, TCR mAb clone GL3 has been disputed repeatedly, many groups continue to use ,, T-cell depletion protocols involving the mAb clone UC7-13D5 and find significant biological effects. We show here that treatment with both GL3 and UC7-13D5 antibodies does not deplete ,, T cells in vivo, but rather leads to TCR internalization and thereby generates "invisible" ,, T cells. We addressed this issue using anti-,, TCR mAb injections into WT mice as well as into reporter TCR delta locus-histone 2B enhanced GFP knock-in mice, in which ,, T cells can be detected based on an intrinsic green fluorescence. Importantly, the use of TCR delta locus-histone 2B enhanced GFP mice provided here for the first time direct evidence that the "depleted" ,, T cells were actually still present. Our results show further that GL3 and UC7-13D5 mAb are in part cross-competing for the same epitope. Assessed by activation markers, we observed in vitro and in vivo activation of ,, T cells through mAb. We conclude that ,, T-cell depletion experiments must be evaluated with caution and discuss the implications for future studies on the physiological functions of ,, T cells. [source]

Availability of autoantigenic epitopes controls phenotype, severity, and penetrance in TCR Tg autoimmune gastritis

EUROPEAN JOURNAL OF IMMUNOLOGY, Issue 12 2008
Ditza Levin
Abstract We examined TCR:MHC/peptide interactions and in vivo epitope availability to explore the Th1- or Th2-like phenotype of autoimmune disease in two TCR Tg mouse models of autoimmune gastritis (AIG). The TCR of strains A23 and A51 recognize distinct IAd -restricted peptides from the gastric parietal cell H/K-ATPase. Both peptides form extremely stable MHC/peptide (MHC/p) complexes. All A23 animals develop a Th1-like aggressive, inflammatory AIG early in life, while A51 mice develop indolent Th2-like AIG at 6,8,wk with incomplete penetrance. A51 T cells were more sensitive than A23 to low doses of soluble antigen and to MHC/p complexes. Staining with IAd/peptide tetramers was only detectable on previously activated T cells from A51. Thus, despite inducing a milder AIG, the A51 TCR displays a higher avidity for its cognate IAd/peptide. Nonetheless, in vivo proliferation of adoptively transferred A51 CFSE-labeled T cells in the gastric lymph node was relatively poor compared with A23 T cells. Also, DC from WT gastric lymph node, presenting processed antigen available in vivo, stimulated proliferation of A23 T cells better than A51. Thus, the autoimmune potential of these TCR in their respective Tg lines is strongly influenced by the availability of the peptide epitope, rather than by differential avidity for their respective MHC/p complexes. [source]

Structural requirements for initiation of cross-reactivity and CNS autoimmunity with a PLP139,151 mimic peptide derived from murine hepatitis virus

EUROPEAN JOURNAL OF IMMUNOLOGY, Issue 10 2006
Ludovic Croxford
Abstract MS is an autoimmune CNS demyelinating disease in which infection appears to be an important pathogenic factor. Molecular mimicry, the cross-activation of autoreactive T cells by mimic peptides from infectious agents, is a possible explanation for infection-induced autoimmunity. Infection of mice with a non-pathogenic strain of Theiler's murine encephalomyelitis virus (TMEV) engineered to express an epitope from Haemophilus influenzae (HI) sharing 6/13 amino acids with the dominant proteolipid protein (PLP) epitope, PLP139,151, can induce CNS autoimmune disease. Here we demonstrate that another PLP139,151 mimic sequence derived from murine hepatitis virus (MHV) which shares only 3/13 amino acids with PLP139,151 can also induce CNS autoimmune disease, but only when delivered by genetically engineered TMEV, not by immunization with the MHV peptide. Further, we demonstrate the importance of proline at the secondary MHC class,II contact residue for effective cross-reactivity, as addition of this amino acid to the native MHV sequence increases its ability to cross-activate PLP139,151 -specific autoreactive T cells, while substitution of proline in the HI mimic peptide has the opposite effect. This study describes a structural requirement for potential PLP139,151 mimic peptides, and provides further evidence for infection-induced molecular mimicry in the pathogenesis of autoimmune disease. [source]

Breakpoints in immunoregulation required for Th1 cells to induce diabetes

EUROPEAN JOURNAL OF IMMUNOLOGY, Issue 9 2006
Margaret Neighbors
Abstract We describe a novel TCR-transgenic mouse line, TCR7, where MHC class,II-restricted, CD4+ T cells are specific for the subdominant H-2b epitope (HEL74,88) of hen egg lysozyme (HEL), and displayed an increased frequency in the thymus and in peripheral lymphoid compartments over that seen in non-transgenic littermate controls. CD4+ T cells responded vigorously to HEL or HEL74,88 epitope presented on APC and could develop into Th1 or Th2 cells under appropriate conditions. Adoptive transfer of TCR7 Ly5.1 T cells into Ly5.2 rat insulin promoter (RIP)-HEL transgenic recipient hosts did not lead to expansion of these cells or result in islet infiltration, although these TCR7 cells could expand upon transfer into mice expressing high levels of HEL in the serum. Islet cell infiltration only occurred when the TCR7 cells had been polarized to either a Th1 or Th2 phenotype prior to transfer, which led to insulitis. Progression from insulitis to autoimmune diabetes only occurred in these recipients when Th1 but not Th2 TCR7 cells were transferred and CTLA-4 signaling was simultaneously blocked. These findings show that regulatory pathways such as CTLA-4 can hold in check already differentiated autoreactive effector Th1 cells, to inhibit the transition from tolerance to autoimmune diabetes. See accompanying commentary at http://dx.doi.org/10.1002/eji.200636591 [source]

Induction of central T cell tolerance: Recombinant antibodies deliver peptides for deletion of antigen-specific CD4+8+ thymocytes

EUROPEAN JOURNAL OF IMMUNOLOGY, Issue 11 2005
Karoline, Western Schjetne
Abstract In order to prevent or ameliorate autoimmune disease, it would be desirable to induce central tolerance to peripheral self-antigens. We have investigated whether recombinant antibodies (Ab) that deliver T cell epitopes to antigen-presenting cells (APC) in the thymus can be used to induce thymocyte deletion. Troybodies are recombinant Ab with V regions specific for APC surface molecules that have T cell epitopes genetically introduced in their C domains. When MHC class II-specific Troybodies with the ,2315 T cell epitope were injected into ,2315 -specific TCR transgenic mice, a profound deletion of CD4+8+ thymocytes was observed. MHC class II-specific Troybodies were 10,100-fold more efficient than non-targeting peptide Ab, and 500-fold more efficient than synthetic peptide at inducing deletion. Similar findings were observed when MHC class II-specific Troybodies with the OVA323,339 T cell epitope were injected into OVA-specific TCR transgenic mice. Although deletion was transient after a single injection, newborn mice repeatedly injected with MHC class II-specific Troybodies for 4,weeks, had reduced antigen-specific T cells in peripheral lymphoid tissues and reduced T cell responses. These experiments suggest that Troybodies constructed to target specifically thymic APC could be useful tools for induction and maintenance of central T cell tolerance in autoimmune diseases. [source]

Isolation and characterization of human anti-acetylcholine receptor monoclonal antibodies from transgenic mice expressing human immunoglobulin loci

EUROPEAN JOURNAL OF IMMUNOLOGY, Issue 6 2005
Evdokia Protopapadakis
Abstract The isolation of human antibodies against muscle acetylcholine receptor (AChR), the autoantigen involved in myasthenia gravis (MG), is important for the development of therapeutically useful reagents. Monovalent antibody fragments from monoclonal antibodies against the main immunogenic region (MIR) of AChR protect the receptor from the destructive activity of MG autoantibodies. Human anti-AChR ,-subunit antibody fragments with therapeutic potential have been isolated using phage display antibody libraries. An alternative approach for obtaining human mAb has been provided by the development of humanized mice. In this report, we show that immunization of transgenic mouse strains with the extracellular domain of the human AChR ,-subunit results in antibody responses and isolation of hybridomas producing human mAb. Four specific IgM mAb were isolated and analyzed. mAb170 recognized the native receptor the best and was capable of inducing AChR antigenic modulation, suggesting its specificity for a pathogenic epitope. Moreover, the recombinant antigen-binding (Fab) fragment of this mAb competed with an anti-MIR mAb, revealing that its antigenic determinant lies in or near the MIR. Finally, Fab170 was able to compete with MG autoantibodies and protect the AChR against antigenic modulation induced by MG sera. This approach will be useful for isolating additional mAb with therapeutic potential against the other AChR subunits. [source]

High epitope density in a single protein molecule significantly enhances antigenicity as well as immunogenicity: a novel strategy for modern vaccine development and a preliminary investigation about B,cell discrimination of monomeric proteins

EUROPEAN JOURNAL OF IMMUNOLOGY, Issue 2 2005
Wanli Liu
Abstract Although early studies have shown a close correlation between epitope density and epitope-specific humoral immune responses, few attempts have been made to quantitatively compare the antigenic and immunogenic differences between protein molecules bearing low or high degrees of epitope density, nor have studies quantitatively investigated the mechanism of B,cell discrimination of monomeric antigens. In this study, we prepared glutathione S-transferase (GST) fusion proteins bearing various copies of the M2e epitope from the influenza virus M2,protein [GST-(M2e)8, GST-(M2e)4 and GST-(M2e)1], which were used to detect and compare the real-time kinetic binding with M2e-specific mAb by surface plasma resonance. Our data show clearly that fusion proteins bearing higher M2e epitope density resulted in higher average avidity for M2e-specific mAb. Furthermore, it was observed that fusion proteins bearing high M2e epitope density could induce polyclonal antibodies (pAb) with enhanced an average affinity constant (KA) for M2e epitope peptide compared to fusion proteins bearing low epitope density. The average KA of pAb induced by GST-(M2e)8 (3.08 × 108,M,1 or 9.96 × 108,M,1) was up to two orders of magnitude greater than the average KA of pAb induced by GST-(M2e)1 (2.00 × 106,M,1 or 3.43 × 106,M,1). Thus, the data presented here demonstrate that high epitope density in a single protein molecule significantly enhances antigenicity and immunogenicity. These findings enrich our knowledge of how epitope density might relate to the recognition, activation and antibody production processes of epitope-specific immature B,cells. [source]

Specificity, magnitude, and kinetics of MOG-specific CD8+ T,cell responses during experimental autoimmune encephalomyelitis

EUROPEAN JOURNAL OF IMMUNOLOGY, Issue 1 2005
Mandy
Abstract Experimental autoimmune encephalomyelitis (EAE) has traditionally been thought to be almost exclusively mediated by CD4+ effector T,cells. Here, we provide evidence for the existence of mouse CD8+ T,cells that are specific for an epitope of the myelin oligodendrocyte glycoprotein (MOG). Using a panel of truncated MOG peptides, we have identified the minimal epitope recognized by these T,cells as MOG,37,46. This peptide, while possessing relatively low affinity for H-2Db, efficiently stimulates IFN-, production from MOG-specific CD8+ T,cell lines in vitro and induces EAE in vivo. To further characterize the magnitude and kinetics of expansion of the MOG-specific CD8+ T,cell population in vivo, we used MOG,37,50/H-2Db MHC tetramers to visualize MOG-specific CD8+ effectors in the peripheral lymphoid organs and central nervous system during the course of EAE induction and progression. Our results identify MOG-specific CD8+ T,cells in the central nervous system prior to and after the onset of disease, suggesting that CD8+ T,cells are a possible target for therapeutic intervention during EAE. [source]

Cross-presentation of a human tumor antigen delivered to dendritic cells by HSV VP22-mediated protein translocation

EUROPEAN JOURNAL OF IMMUNOLOGY, Issue 10 2004
Arvind Chhabra
Abstract Dendritic cells (DC) capture antigens from apoptotic and/or necrotic tumor cells and cross-present them to T,cells, and various ways of delivering tumor antigens to DC in vitro and in vivo are being pursued. Since fusions of antigenic proteins with the HSV integument protein VP22 are capable of intercellular trafficking, this approach has been exploited for delivery of antigens to antigen-presenting cells. Adenoviral vectors were used to express the tumor-associated-but-self-antigen MART-1 fused to HSV VP22 in MART-1-negative A375 melanoma cells and in DC. When expressed in A375 cells and allowed to spread to DC across a transwell barrier, the VP22-MART-1 fusion protein localized to both early and late endosomal structures of the DC. The DC loaded with the VP22-MART-1 fusion by intercellular trafficking efficiently presented the MART-127,35 epitope to MART-127,35 -specific CTL. Furthermore, transloaded DC were capable of expanding the population of MART-127,35 -specific CTL. Thus, a tumor antigen acquired by intercellular trafficking can be cross-presented by DC. This experimental approach should therefore be useful not only for studying the mechanism of cross-presentation but also for vaccine development. [source]

Dissecting cytotoxic T,cell responses towards the NY-ESO-1 protein by peptide/MHC-specific antibody fragments

EUROPEAN JOURNAL OF IMMUNOLOGY, Issue 10 2004
Gerhard Held
Abstract NY-ESO-1 is a germ cell antigen aberrantly expressed by different tumor types that elicits strong humoral and cellular immune responses, representing one of the most promising candidates for vaccination of cancer patients. A detailed analysis of CD8+ T,cells generated in vaccine trials using NY-ESO-1-derived peptides (157,165 and 157,167) revealed that the dominant immune response was directed against a cryptic epitope (159,167) diverting the immune response from tumor recognition. Only CTL reactivity to the NY-ESO-1157,165 peptide appeared to be capable of lysing NY-ESO-1/HLA-A0201-expressing tumor cells. To study the process of NY-ESO-1 peptide presentation by tumor cells in more detail we generated a high-affinity (KD=60,nM) antibody fragment that specifically recognizes the NY-ESO-1157,165 peptide/HLA-A0201 complex. Peptide variants such as the NY-ESO-1157,167 peptide or the cryptic NY-ESO-1159,167 peptide were not recognized. The antibody fragment blocked in a dose-dependent fashion the recognition of NY-ESO-1/HLA-A2-positive tumor cells by NY-ESO-1157,165 peptide-specific CD8+ T,cells. This antibody fragment is a novel reagent that binds with TCR-like specificity to the NY-ESO-1157,165/HLA-A2 complex thus distinguishing between CTL responses against immunological meaningful or cryptic NY-ESO-1-derived peptides. It may therefore become a useful monitoring tool for the development of NY-ESO-1-based cancer vaccines. [source]

Bet,v,1, the major birch pollen allergen, initiates sensitization to Api,g,1, the major allergen in celery: evidence at the T,cell level

EUROPEAN JOURNAL OF IMMUNOLOGY, Issue 12 2003
Barbara Bohle
Abstract Due to IgE cross-reactivity, birch pollen-allergic individuals frequently develop type,I hypersensitivity reactions to celery tuber. We evaluated the T,cell response to the major allergen in celeriac, Api,g,1, and the cellular cross-reactivity with its homologous major allergen in birch pollen, Bet,v,1. Api,g,1-specific T,cell lines (TCL) and clones (TCC) were established from peripheralblood mononuclear cells of allergic patients. Epitope mapping of Api,g,1 with overlapping Api,g,1-derived peptides revealed one dominant T,cell-activating region, Api,g,1109,126. TCL and TCC generated with Api,g,1 cross-reacted with the birch pollen allergen and, although initially stimulated with the food allergen, cellular responses to Bet,v,1 were stronger than to Api,g,1. Epitopemapping with Bet,v,1-derived peptides revealed that T,cells specific for several distinct epitopes distributed over the complete Bet,v,1 molecule could be activated by Api,g,1. Bet,v,1109,126 was identified as the most important T,cell epitope for cross-reactivity with Api,g,1. This epitope shares 72% amino acid sequence similarity with the major T,cell-activating region of the food allergen, Api,g,1109,126. Our data provide evidence that humoral as well as cellular reactivity to the major celery allergen is predominantly based on cross-reactivity with the major birch pollen allergen. The activation of Bet,v,1-specific Th2 cells by Api,g,1, in particular outside the pollen season, may have consequences for birch pollen-allergic individuals. [source]

The cytoplasmic tail of invariant chain modulates antigen processing and presentation

EUROPEAN JOURNAL OF IMMUNOLOGY, Issue 2 2003

Abstract The MHC class II-associated invariant chain (Ii) has several important functions in antigen presentation. In this study, we have examined the effect of Iip33 expression on endocytic transport and antigen presentation. We find that degradation of both endocytosed antigen and Ii itself is delayed in cells expressing high levels of Ii, whereas a mutant Ii with an altered charge distributionin the cytoplasmic tail was unable to exert this effect. Furthermore, the Ii mutant did not enhance the presentation of an Ii-dependent MHC class II-restricted epitope to the same extent as the wild type. In a parallel study, we investigated the effect of charge in the cytoplasmic tail of Ii. We find that due to exposed negative charges, it promotes endosome fusion events, and we suggest thatthis causes endosomal retention (Nordeng et al., Mol. Biol. Cell 2002). Together, the data reveal an additional property of the Iip33 cytoplasmic tail that contributes to the modulation of antigen processing and presentation. [source]

Is my antibody-staining specific?

EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 12 2008
How to deal with pitfalls of immunohistochemistry
Abstract Immunohistochemistry is a sensitive and versatile method widely used to investigate the cyto- and chemoarchitecture of the brain. It is based on the high affinity and selectivity of antibodies for a single epitope. However, it is now recognized that the specificity of antibodies needs to be tested in control experiments to avoid false-positive results due to non-specific binding to tissue components or recognition of epitopes shared by several molecules. This ,Technical Spotlight' discusses other pitfalls, which are often overlooked, although they can strongly influence the outcome of immunohistochemical experiments. It also recapitulates the minimal set of information that should be provided in scientific publications to allow proper evaluation and replication of immunohistochemical experiments. In particular, tissue fixation and processing can have a strong impact on antigenicity by producing conformational changes to the epitopes, limiting their accessibility (epitope masking) or generating high non-specific background. These effects are illustrated for an immunoperoxidase staining experiment with three antibodies differing in susceptibility to fixation, using tissue from mice processed under identical conditions, except for slight variations in tissue fixation. In these examples, specific immunostaining can be abolished depending on fixation strength, or detected only after prolonged postfixation. As a consequence, antibody characterization in immunohistochemistry should include their susceptibility towards fixation and determination of the optimal conditions for their use. [source]

Postnatal maturation of Na+, K+, 2Cl, cotransporter expression and inhibitory synaptogenesis in the rat hippocampus: an immunocytochemical analysis

EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 2 2002
Serge Marty
Abstract GABA, a major inhibitory neurotransmitter, depolarizes hippocampal pyramidal neurons during the first postnatal week. These depolarizations result from an efflux of Cl, through GABAA -gated anion channels. The outward Cl, gradient that provides the driving force for Cl, efflux might be generated and maintained by the Na+, K+, 2Cl, cotransporter (NKCC) that keeps intracellular Cl, concentration above electrochemical equilibrium. The developmental pattern of expression of the cotransporter in the hippocampus is not known. We studied the postnatal distribution pattern of NKCC in the hippocampus using a monoclonal antibody (T4) against a conserved epitope in the C-terminus of the cotransporter molecule. We also examined the temporal relationships between the developmental pattern of NKCC expression and the formation of perisomatic GABAergic synapses. This study was aimed at determining, with antivesicular inhibitory amino acid transporter (VIAAT) antibodies, whether perisomatic GABAergic synapses are formed preferentially at the time when GABA is depolarizing. During the first postnatal week, NKCC immunolabelling was restricted to cell bodies in the pyramidal cell layer and in the strata oriens and radiatum. In contrast, at postnatal day 21 (P21) and in adult animals little or no labelling occurred in cell bodies; instead, a prominent dendritic labelling appeared in both pyramidal and nonpyramidal neurons. The ultrastructural immunogold study in P21 rat hippocampi corroborated the light-microscopy results. In addition, this study revealed that a portion of the silver-intensified colloidal gold particles were located on neuronal plasmalemma, as expected for a functional cotransporter. The formation of inhibitory synapses on perikarya of the pyramidal cell layer was a late process. The density of VIAAT-immunoreactive puncta in the stratum pyramidale at P21 reached four times the P7 value in CA3, and six times the P7 value in CA1. Electron microscopy revealed that the number of synapses per neuronal perikaryal profile in the stratum pyramidale of the CA3 area at P21 was three times higher than at P7, even if a concomitant 20% increase in the area of these neuronal perikaryal profiles occurred. It is concluded that, in hippocampal pyramidal cells, there is a developmental shift in the NKCC localization from a predominantly somatic to a predominantly dendritic location. The presence of NKCC during the first postnatal week is consistent with the hypothesis that this transporter might be involved in the depolarizing effects of GABA. The depolarizing effects of GABA may not be required for the establishment of the majority of GABAergic synapses in the stratum pyramidale, because their number increases after the first postnatal week, when GABA action becomes hyperpolarizing. [source]

Oligodendroglial tau filament formation in transgenic mice expressing G272V tau

EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 11 2001
Jürgen Götz
Abstract Genetic evidence indicates that several mutations in tau, including G272V, are linked to frontotemporal dementia with parkinsonism. We expressed this mutation in mouse brains by combining a prion protein promoter-driven expression system with an autoregulatory transactivator loop that resulted in high expression of human G272V tau in neurons and in oligodendrocytes. We show that G272V tau can form filaments in murine oligodendrocytes. Electron microscopy established that the filaments were either straight or had a twisted structure; these were 17,20 nm wide and had a periodicity of ,,75 nm. Filament formation was associated with tau phosphorylation at distinct sites, including the AT8 epitope 202/205 in vivo. Immunogold electron microscopy of sarcosyl-extracted spinal cords from G272V transgenic mice using phosphorylation-dependent antibodies AT8 or AT100 identified several sparsely gold-labelled 6-nm filaments. In the spinal cord, fibrillary inclusions were also identified by thioflavin-S fluorescent microscopy in oligodendrocytes and motor neurons. These results establish that expression of the G272V mutation in mice causes oligodendroglial fibrillary lesions that are similar to those seen in human tauopathies. [source]

Detection of pemphigus desmoglein 1 and desmoglein 3 autoantibodies and pemphigoid BP180 autoantibodies in saliva and comparison with serum values

EUROPEAN JOURNAL OF ORAL SCIENCES, Issue 5 2006
Dimitrios Andreadis
Although there is much literature on the detection of pemphigus and pemphigoid autoantibodies by enzyme-linked immunosorbent assay (ELISA) in serum, nothing is known about their presence in saliva. The aim of this study was to evaluate the salivary levels of these autoantibodies in pemphigus and pemphigoid patients. Autoantibodies against desmoglein3, desmoglein1, and BP180 were assayed, by ELISA, in serum and saliva samples of patients and healthy controls. The titres of autoantibodies against Dsg1/3 found in both serum and saliva of pemphigus patients showed a statistically significant correlation, suggesting that saliva may be a useful biological material for diagnostic purposes, in monitoring disease activity, as well as for the early detection of relapses. By contrast, the titres of autoantibodies against BP180 in the serum and saliva of bullous pemphigoid patients were not statistically related, and further study of the usefulness of the BP180 ELISA for saliva in this disease is needed. In addition, based on our results, the BP180 ELISA with a recombinant NC16a epitope failed to detect the autoantibodies against BP180 in the serum and saliva of mucous membrane pemphigoid patients. [source]

Fucosyltransferase VII-positive, skin-homing T cells in the blood and skin lesions of atopic dermatitis patients

EXPERIMENTAL DERMATOLOGY, Issue 3 2008
Yoshiko Mizukawa
Abstract: Patients with atopic dermatitis (AD) have an abnormally increased frequency of cutaneous lymphocyte antigen (CLA)+ Th2 cells responsible for local inflammation; however, this is paradoxical, given the well-recognized defective capacity of Th2 cells to migrate to the skin sites of inflammation. These discrepant observations would stem from the ambiguity of CLA+ T cells, because CLA does not represent the epitope required for binding to E-selectin but the epitope generated by fucosyltransferase VII (Fuc-TVII) and because skin-homing T cells are composed of three distinct subpopulations; Fuc-TVII+ E-selectin ligand (ESL)+ CLA,, Fuc-TVII+ ESL+ CLA+ and Fuc-TVII, ESL, CLA+ cells. We therefore asked which subpopulations of skin-homing Th2 cells could be increased in the blood and skin lesions of AD. We analysed the frequencies of the three subpopulations in purified CD4+ peripheral blood T cells from AD patients and healthy controls by immunohistochemistry and flow cytometry. The Fuc-TVII+ CLA+ or CLA+ ESL+ CCR4+ cells were dramatically increased in frequency not only in the blood but also in the skin lesions of AD patients and this increase was related to the severity of the clinical symptoms. Our data indicate the clinical importance of identifying skin-homing T cells with the potent capacity to migrate into the skin by analysing their Fuc-TVII expression and E-selectin binding ability in patients with AD. [source]