1.
Interleukin 15 levels in serum may predict a severe disease course in patients with early arthritis.
Source
Rheumatology Service, Hospital Universitario de La Princesa, IIS Princesa, Madrid, Spain.
Abstract
BACKGROUND:
Interleukin-15 (IL-15) is thought to be involved in the physiopathological mechanisms of RA and it can be detected in the serum and the synovial fluid of inflamed joints in patients with RA but not in patients with osteoarthritis or other inflammatory joint diseases. Therefore, the objective of this work is to analyse whether serum IL-15 (sIL-15) levels serve as a biomarker of disease severity in patients with early arthritis (EA).
METHODOLOGY AND RESULTS:
Data from 190 patients in an EA register were analysed (77.2% female; median age 53 years; 6-month median disease duration at entry). Clinical and treatment information was recorded systematically, especially the prescription of disease modifying anti-rheumatic drugs. Two multivariate longitudinal analyses were performed with different dependent variables: 1) DAS28 and 2) a variable reflecting intensive treatment. Both included sIL-15 as predictive variable and other variables associated with disease severity, including rheumatoid factor (RF) and anti-cyclic citrullinated peptide antibodies (ACPA). Of the 171 patients (638 visits analysed) completing the follow-up, 71% suffered rheumatoid arthritis and 29% were considered as undifferentiated arthritis. Elevated sIL-15 was detected in 29% of this population and this biomarker did not overlap extensively with RF or ACPA. High sIL-15 levels (β Coefficient [95% confidence interval]: 0.12 [0.06-0.18]; p<0.001) or ACPA (0.34 [0.01-0.67]; p = 0.044) were significantly and independently associated with a higher DAS28 during follow-up, after adjusting for confounding variables such as gender, age and treatment. In addition, those patients with elevated sIL-15 had a significantly higher risk of receiving intensive treatment (RR 1.78, 95% confidence interval 1.18-2.7; p = 0.007).
CONCLUSIONS:
Patients with EA displaying high baseline sIL-15 suffered a more severe disease and received more intensive treatment. Thus, sIL-15 may be a biomarker for patients that are candidates for early and more intensive treatment.
Sonic hedgehog maintains survival and growth of chronic myeloid leukemia progenitor cells through β-catenin signaling.
Source
Department of Hematology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China; Department of Hematology, the Affiliated Hospital of Medical College Qingdao University, Qingdao, China.
Abstract
Sonic hedgehog (Shh) signaling plays an important role in many human cancers and cancer stem cells. Here we investigate the activity and functional role of Shh signaling in chronic myeloid leukemia (CML) and leukemia progenitor cells. Differential activation of Shh signaling was found in about 50% CML-CP samples, 70% CML-AP samples and more than 80% CML-BP samples. Deregulated activation of Shh signaling was observed in CD34(+) and c-kit(+) leukemia progenitor cells. Stimulation of Shh signaling with exogenous Shh peptide induced expansion of CD34(+) and c-kit(+) progenitor cells (p<0.05), inversely, blocking the pathway with signal inhibitor induced cell apoptosis (p<0.05). Low level of Shh protein was observed in CML bone marrow stromal cells, besides, CD34(+) progenitor cells are less sensitive to exogenous Shh peptide and more sensitive to cyclopamine than CD34(-) cells (p<0.05), implying cell-autonomous activation of Shh signaling play a predominantly role in progenitor cells. Co-activation of Shh and β-catenin signaling was found in CD34(+) and c-kit(+) progenitor cells. Administration of Shh neutralizing antibody or Wnt3a neutralizing antibody in c-kit(+) progenitor cells induced cell apoptosis, however, Wnt3a peptide could salvage anti-Shh-induced cell apoptosis while Shh peptide failed to revert anti-Wnt3a-induced cell apoptosis. C-MYC, GLI1, BCL-2, P21 were also found to be downstream targets of Shh signaling, mediating apoptosis or G2/M cell cycle arrest of progenitor cells. Our results demonstrate that auto-activated Shh signaling provides survival and proliferative cues in CML progenitor cells through downstream β-catenin signaling, thus suggesting a novel therapeutic approach in CML.
Copyright © 2012 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.
Peptidyl-arginine deiminase: an additional marker of rheumatoid arthritis.
Source
Biotechnology Department, Heritage Institute of Technology, Chowbagha Road, Anandapur, Kolkata, India. psbasu.heritage@gmail.com
Abstract
BACKGROUND:
Antibodies against cyclic citrullinated peptide (anti-CCP) were thought to be more specific than rheumatoid factor (RF) for the diagnosis of rheumatoid arthritis (RA). The determination of anti-CCP in addition to RF could be helpful in the serological diagnosis and monitoring of patients with RA. Citrullination of proteins involves the enzymatic conversion of protein containing arginine residues to citrulline residues by the enzyme peptidylarginine deiminase (PAD). The present investigation was undertaken to estimate serum PAD enzyme activity in RA patients with a view to find its importance as a new diagnosis marker in a rheumatology clinic.
METHODS:
The activity of the PAD enzyme was measured by spectrophotometric method at 530 nm in sera of control subjects and in patients of RA (Group I: RF negative and CCP positive: Group II: both RF and CCP positive) in terms of citrulline formation using benzoyl-arginine ethyl ester (BAEE) as substrate. Anti-CCP and RF were also estimated in two groups by enzyme immunoassay and immunoturbidimetry for comparison. Clinical variables (duration of morning stiffness, swollen and tender joint counts, patient's assessment of pain) and C-reactive protein were also evaluated.
RESULTS:
A marked increase in PAD enzyme activity (p < 0.001) was noted in RA patients in comparison to controls and the level diminished appreciably along with two known serological markers (anti-CCP and RF) after six months of disease modifying antirheumatic drug (DMARD) treatment. The Group II RA patients showed much higher enzyme activity than Group I RA patients. However, clinical variables did not differ significantly between the two Groups of RA patients.
CONCLUSIONS:
We conclude that determination of PAD enzyme activity may be used as an additional marker for monitoring disease progression and regression along with anti-CCP and RF in patients with RA. Moreover, this method is rapid, sensitive, and inexpensive and can be adopted in a laboratory having modest facilities.
- PMID:
- 22239037
- [PubMed - in process]
18F-Labeled N-(4-fluorobenzylidene)oxime-dimeric (ZHER2:477)2 .
Authors
Source
Molecular Imaging and Contrast Agent Database (MICAD) [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2004-2011.
2011 Nov 02 [updated 2012 Jan 04].
Excerpt
The 18F-labeled N-(4-fluorobenzylidene)oxime (FBO)-dimeric (ZHER2:477)2 conjugate, abbreviated as 18F-FBO-(ZHER2:477)2, is an affibody derivative synthesized by Cheng et al. for positron emission tomography (PET) of HER2-expressing tumors (1). Affibody molecules are a group of nonimmunogenic scaffold proteins that derive from the B-domain of staphylococcal surface protein A (2, 3). In the past several years, affibodies have drawn significant attention for developing imaging and therapeutic agents because of their unique features (3, 4). First, affibodies are small, with only 58 amino acid residues (~7 kDa) (3, 5). The small size allows affibodies to be generated with solid-phase peptidesynthesis and to be cleared quickly from kidneys. Second, affibodies have a high binding affinity and specificity to their targets. Their binding affinity can be further improved by generating multimeric constructs through the solvent-exposed termini of affibody Z-domain. The anti-HER2 monomeric affibody ZHER2:4 is an example that has a binding affinity of ~50 nM, but its dimeric form, (ZHER2:4)2, exhibits an improved binding affinity of up to ~3 nM in vitro (6). Third, affibodies lack cysteine residues and disulfide bridges in structure, and they fold rapidly. These features make it possible to chemically synthesize fully functional molecules and to introduce unique cysteine residues or chemical groups into affibodies for site-specific labeling. Several anti-HER2 affibody derivatives have been synthesized in this way. The imaging agent HPEM-His6-(ZHER2:4)2-Cys was generated by radiobrominating the dimeric (ZHER2:4)2 through the cysteine residues that were introduced to the C-terminus of (ZHER2:4)2 (7). Several affibody derivatives (e.g., 68Ga-DOTA-ZHER2:342-pep2, 111In-DOTA-ZHER2:342-pep2, 111In-benzyl-DOTA-ZHER2:342, and 111In-benzyl-DTPA-ZHER2:342) were synthesized by coupling a chelating agent with a specifically protected site group of the ZHER2:342peptide chain (3). Furthermore, affibody proteins can be selected and optimized with a strategy of sequence mutation and affinity maturation, and an example selected with this strategy is the anti-HER2 affibody ZHER2:342, which has an increased affinity of 50 nM (ZHER2:4, the first generation) to 22 pM (8). The investigators at Stanford University first tested the feasibility of the monomeric and dimeric forms of anti-HER2 affibody ZHER2:477 for molecular imaging. Both forms of the ZHER2:477 molecule were radiofluorinated with an 18F-labeled prosthetic group of 4-18F-fluorobenzaldehyde (18F-FBO-ZHER2:477 and 18F-FBO-(ZHER2:477)2, respectively) (1). The investigators have also coupled 64Cu to the affibody through DOTA, leading to the development of imaging agents 64Cu-DOTA- ZHER2:477 and 64Cu-DOTA-(ZHER2:477)2 (9). Interestingly, these studies showed that smaller affibody constructs performed better in vivo in terms of tumor uptake and clearance in spite of the lower affinity in vitro. The investigators then generated a class of small proteins consisting of two α-helix bundles of the 3-helix affibody by deleting the helix 3 because the binding domain localizes in the α-helices 1 and 2 bundles (5). One of these 2-helix proteins is MUT-DS, which has α-helices 1 and 2 bundles, with a disulfide bridge being formed between the two inserted homocysteines (10-12). MUT-DS showed a binding affinity to HER2 in the low-nM range. The radiolabeled MUT-DS derivatives exhibited favorable pharmacokinetics for both imaging and therapy of HER2-expressing tumors (refer to MUT-DS derived agents in MICAD). This series of chapters summarizes the data obtained with the ZHER2:477 derivatives, and this chapter presents the data obtained with 18F-FBO-(ZHER2:477)2 (1).
Sections
18F-Labeled N-(4-fluorobenzylidene)oxime-monomeric ZHER2:477 .
Authors
Source
Molecular Imaging and Contrast Agent Database (MICAD) [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2004-2011.
2011 Nov 02 [updated 2012 Jan 04].
Excerpt
The 18F-labeled N-(4-fluorobenzylidene)oxime (FBO)-monomeric ZHER2:477 conjugate, abbreviated as 18F-FBO-ZHER2:477, is an affibody derivative synthesized by Cheng et al. for positron emission tomography (PET) of HER2-expressing tumors (1). Affibody molecules are a group of nonimmunogenic scaffold proteins that derive from the B-domain of staphylococcal surface protein A (2, 3). In the past several years, affibodies have drawn significant attention for developing imaging and therapeutic agents because of their unique features (3, 4). First, affibodies are small, with only 58 amino acid residues (~7 kDa) (3, 5). The small size allows affibodies to be generated with solid-phase peptidesynthesis and to be cleared quickly by the kidneys. Second, affibodies have a high binding affinity and specificity to their targets. Their binding affinity can be further improved by generating multimeric constructs through the solvent-exposed termini of affibody Z-domain. The anti-HER2 monomeric affibody ZHER2:4 is an example that has a binding affinity of ~50 nM, but its dimeric form, (ZHER2:4)2, exhibits an improved binding affinity of up to ~3 nM (6). Third, affibodies lack cysteine residues and disulfide bridges in structure, and they fold rapidly. These features make it possible to chemically synthesize fully functional molecules and to introduce unique cysteine residues or chemical groups into affibodies for site-specific labeling. Several anti-HER2 affibody derivatives have been synthesized in this way. The imaging agent HPEM-His6-(ZHER2:4)2-Cys was generated by radiobrominating the dimeric (ZHER2:4)2 through the cysteine residues that were introduced to the C-terminus of (ZHER2:4)2 (7). Several affibody derivatives (e.g., 68Ga-DOTA-ZHER2:342-pep2, 111In-DOTA-ZHER2:342-pep2, 111In-benzyl-DOTA-ZHER2:342, and 111In-benzyl-DTPA-ZHER2:342) were synthesized by coupling a chelating agent with a specifically protected site group of the ZHER2:342peptide chain (3). Furthermore, these small affibody proteins can be selected and optimized with a strategy of sequence mutation and affinity maturation, and an example selected with this strategy is the anti-HER2 affibody ZHER2:342, which has an increased affinity of 50 nM (ZHER2:4, the first generation) to 22 pM (8). The investigators at Stanford University first tested the feasibility of the monomeric and dimeric forms of anti-HER2 affibody ZHER2:477 for molecular imaging. Both forms of the ZHER2:477 molecule were radiofluorinated with an 18F-labeled prosthetic group of 4-18F-fluorobenzaldehyde (18F-FBO-ZHER2:477 and 18F-FBO-(ZHER2:477)2, respectively) (1). The investigators have also coupled 64Cu to the affibody through DOTA, leading to the development of imaging agents 64Cu-DOTA- ZHER2:477 and 64Cu-DOTA-(ZHER2:477)2 (9). Interestingly, these studies showed that smaller affibody constructs performed better in vivo in terms of tumor uptake and clearance in spite of the lower affinity in vitro. The investigators then generated a class of small proteins consisting of two α-helix bundles of the 3-helix affibody by deleting the helix 3 because the binding domain localizes in the α-helices 1 and 2 bundles (5). One of these 2-helix proteins is MUT-DS, which has α-helices 1 and 2 bundles, with a disulfide bridge being formed between the two inserted homocysteines (10-12). MUT-DS showed a binding affinity to HER2 in the low-nM range. The radiolabeled MUT-DS derivatives exhibited favorable pharmacokinetics for both imaging and therapy of HER2-expressing tumors (refer to MUT-DS derived agents in MICAD). This series of chapters summarizes the data obtained with the ZHER2:477 derivatives, and this chapter presents the data obtained with 18F-FBO-ZHER2:477 (1).
Sections
Neutralizing Epitopes in the MPER of HIV-1 gp41 Are Influenced by the Transmembrane Domain and the Plasma Membrane.
Source
Dept. of Molecular Biology and Biochemistry.
Abstract
Failure to elicit broadly (b) neutralizing (Nt) antibodies (Abs) against the membrane proximal external region of HIV-1 gp41 (MPER), reflects the difficulty of mimicking its neutralization-competent structure (NCS). Here, we analyzed MPER antigenicity in the context of the plasma membrane, and identified a role for the gp41 transmembrane domain (TM) in exposing the epitopes of three bNt monoclonal (M)Abs (2F5, 4E10, Z13e1). We transiently expressed DNA constructs encoding gp41 ectodomain fragments fused to either the TM of the platelet-derived growth factor receptor (PDGFR), or the gp41 TM and cytoplasmic tail domain (CT). Constructs encoding the MPER tethered to the gp41 TM followed by a 27-residue CT fragment (MPER-TM1) produced optimal MAb binding. Critical binding residues for the three Nt MAbs were identified using a panel of 24 MPER-TM1 mutants bearing single amino-acid substitutions in the MPER; many were previously shown to affect MAb-mediated viral neutralization. Moreover, non-Nt mutants of MAbs 2F5 and 4E10 exhibited a reduction in binding to MPER-TM1, yet maintained binding to synthetic MPER peptides, indicating that MPER-TM1 better approximates the MPER NCS than peptides. Substitution of the gp41 TM and CT of MPER-TM1 with the PDGFR TM reduced binding by MAb 4E10, but not 2F5, indicating the gp41 TM plays a pivotal role in orienting the 4E10 epitope, and more globally, in affecting MPER exposure.
Evaluation of Biological Sample Preparation for Immunosignature Based Diagnostics.
Source
Center for Innovations in Medicine, The Biodesign Institute, Arizona State University, Tempe, AZ 85287-5901.
Abstract
To address the need for a universal system to assess health status, we previously described a method termed immunosignaturing which splays the entire humoral antibody repertoire across a peptide microarray. Two important issues relative to the potential broad use of immunosignatures are sample preparation and stability. In the present study we compared the immunosignatures developed from serum, plasma, saliva and antibodies eluted from blood dried onto filter paper. We found serum and plasma provide identical immunosignatures. Dried blood also correlated well with non-dried serum from the same individual. Immunosignatures derived from dried blood were capable of distinguishing naïve mice from those infected with influenza. Saliva was applied to the arrays and the IgA immunosignature correlated strongly with that from dried blood. Finally, we demonstrate that dried blood retains immunosignature information even when exposed to high temperature. This work expands the potential diagnostic uses for immunosignatures. These features suggest that different forms of archival samples can be used for diagnosis development and that in prospective studies samples can be easily procured.
A novel anti-MUC1 antibody against the MUC1 cytoplasmic tail domain: use in sensitive identification of poorly differentiated cells in adenocarcinoma of the stomach.
Source
Department of Human Pathology, Field of Oncology, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima, 890-8544, Japan, syoneza@m2.kufm.kagoshima-u.ac.jp.
Abstract
BACKGROUND:
Isolated cancer cells of non-solid type poorly differentiated adenocarcinoma (por2) or signet-ring cell carcinoma (sig) are frequently seen in scirrhous gastric cancers with a very poor prognosis. These cells are often scattered in granulation tissue or desmoplastic fibrotic tissue and tend to be overlooked in routine pathological examination. We aimed to raise a novel antibody that can identify the isolated cancer cells easily.
METHODS:
Because the MUC1 cytoplasmic tail domain (CTD) has many biological roles including tumor progression and cell adhesion disturbance and is expected to be expressed in isolated cancer cells, we raised a novel monoclonalantibody (MAb) MUC1-014E against an intracellular nonrepeating 19-amino-acid sequence (RYVPPSSTDRSPYEKVSAG: N-1217-1235-C) of the MUC1 CTD, using a synthetic peptide including the 7-amino-acid epitope (STDRSPY: N-1223-1229-C).
RESULTS:
In the immunohistochemical staining of 107 gastrectomy specimens including 48 por2 and 31 sig lesions, the MAb MUC1-014E showed high rates of positive staining (≥5% of carcinoma cells stained) for por2 (100%) and sig (97%), and of the highest intensity staining (4+, ≥75% of carcinoma cells stained) for por2 (100%) and sig (90%). In the 89 biopsy specimens including 82 por2 and 38 sig lesions, the MAb MUC1-014E showed high rates of positive staining for por2 (100%) and sig (100%) and of 4+ staining for por2 (87%) and sig (84%). All the rates were significantly higher than those with cytokeratins (AE1/AE3 or CAM5.2).
CONCLUSIONS:
The MAb MUC1-014E is very useful for accurate detection of isolated cancer cells in scirrhous gastric cancers.
Anti-cytokine auto-vaccinations as tools for the analysis of cytokine function in vivo.
Source
Ludwig Institute for Cancer Research, Brussels Branch, Brussels, Belgium; Cellular Genetics Unit, Christian de Duve Institute of Cellular Pathology, Université Catholique de Louvain, Brussels, Belgium.
Abstract
Braking B cell tolerance to generate antibodies against autologous cytokines or chemokines offers an alternative to gene inactivation for functional analysis of these factors in vivo. It is clearly less potent than the genetic approach but offers the advantage of extreme flexibility. The basic principle is to enable a self-reactive B cell to attract T cell help by presenting foreign peptides, a process we called "deceptive" antigen presentation. We here review the different auto-vaccine procedures that are currently used and provide several examples of functional information acquired by this procedure or by mAbs derived from auto-vaccinated mice.
Copyright © 2011 Elsevier Ltd. All rights reserved.
Immunolocalization of sprouty-1 and sprouty-2 in developing rat lung.
Source
Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pa., USA.
Abstract
Objective: Sprouty, a common antagonist of fibroblast growth factor (FGF) and epidermal growth factor signaling, is a key player regulating tracheal branching and eye development in Drosophila. Four Sprouty homologs have been identified in vertebrates and all share a cysteine-rich region. However, the physiological function(s) of the individual Sprouty homologs is unknown. mRNA of Sprouty homologs is expressed during mouse lung development. In the present study, we investigated the immunolocalization of Sprouty proteins in rat lung at different stages of development. Methods: Rabbit antibodies were raised against peptides derived from rat Sprouty-1 and Sprouty-2 and were used in Western blot analysis to determine Sprouty distribution in subcellular fractions (pellets and supernatant centrifuged at 5,000 and 20,000 g) and bronchoalveolar lavage fluid (BAL) from adult rat lungs or used in immunohistochemistry. Results: Western blot analysis revealed a 30-kDa Sprouty-1 band and a 34-kDa Sprouty-2 band in the supernatant and pellet fractions centrifuged at 20,000 g. BAL contained a band of approximately 16 kDa with Sprouty-1 antibody derived from proteolytic fragmentation of Sprouty-1. In embryonic day (E) 14 and E16 lungs, Sprouty-1 and Sprouty-2 were expressed both in epithelial and peripheral mesenchymal cells. In adult rat lung, bronchiolar and alveolar type II epithelial cells showed staining for both Sprouty-1 and Sprouty-2. Sprouty-1 expression was also seen in alveolar type I epithelial cells. Conclusion: In light of the proximity of the distribution of Sprouty to that of FGF-10 (peripheral mesenchyme) and its receptor FGFR2IIIb (distal tubular epithelium) in lung development, and the finding that FGF-9, which is expressed in mesothelial cells, upregulates FGF-10, it appears that Sprouty expression in epithelial and mesenchymal cells during branching morphogenesis is closely related to signaling by FGF-9 and FGF-10.
Copyright © 2012 S. Karger AG, Basel.
Clinical Relevance of Anti-exenatide Antibodies: Safety, Efficacy, and Cross-reactivity with Long-term Treatment.
Source
Elcelyx Therapeutics, Inc., San Diego, CA Eli Lilly and Company, Indianapolis, IN, USA Diabetes Center, VU University Medical Center, Amsterdam, the Netherlands American Diabetes Association, Alexandria, VA, USA Amylin Pharmaceuticals, Inc., San Diego, CA.
Abstract
Aims Antibody formation to therapeutic peptides is common. This analysis characterizes the time-course and cross-reactivity of anti-exenatide antibodies and potential effects on efficacy and safety. Materials and Methods Data from ITT patients in 12 controlled (n=2225, 12-52wks) and 5 uncontrolled (n=1538, up to 3yrs) exenatide BID trials and 4 controlled (n=653, 24-30wks) exenatide once weekly (QW) trials with 1 uncontrolled period (n=128, 52wks) were analyzed. Results Mean titers peaked early (6-22wks), and subsequently declined. At 30wks, 36.7% of exenatide BID patients were antibody-positive; 31.7% exhibited low titers (≤125) and 5.0% had higher titers (≥625). Antibody incidence declined to 16.9% (1.4% higher titer) at 3yrs. Similarly, 56.8% of exenatide QW patients were antibody-positive (45.0% low/11.8% higher titer) at 24-30wks, declining to 45.4% positive (9.2% higher titer) at 52wks. Treatment-emergent anti-exenatide antibodies from a subset of patients tested did not cross-react with human GLP-1 or glucagon. Other than injection-site reactions, adverse event rates in antibody-positive and antibody-negative patients were similar. Efficacy was robust in both antibody-negative and antibody-positive patients (mean HbA(1c) : -1.0% and -0.9%, respectively, exenatide BID; -1.6% and -1.3% exenatide QW). No correlation between change in HbA(1c) and titer was observed for exenatide BID, although mean reductions were attenuated in the small subset of patients (5%) with higher titers. A significant correlation was observed for exenatide QW, with no difference between antibody negative and low-titer patients but an attenuated mean reduction in the subset of patients (12%) with higher titers. Conclusions Low titer anti-exenatide antibodies were common with exenatide treatment (32% exenatide BID, 45% exenatide QW patients), but had no apparent effect on efficacy. Higher titer antibodies were less common (5% exenatide BID, 12% exenatide QW) and within that titer group, increasing antibody titer was associated with reduced average efficacy that was statistically significant for exenatide QW. Other than injection-site reactions, anti-exenatide antibodies did not impact the safety of exenatide.
Copyright © 2012 John Wiley & Sons A/S.
Circumscribing the Conformational Peptide Epitope Landscape.
Source
Department of Biochemistry and Molecular Biology, University of Bari, Via Orabona 4, 70126 Bari, Italy. d.kanduc@biologia.uniba.it.
Abstract
The development of vaccines for new and re-emerging pathologies and infections is based on the ability to define immunogenic epitopes. An immunogenic B-cell peptide epitope is a specific restricted antigen region that is capable of eliciting a humoral immune response and of combining with a specific site on antibodies. Using a number of experimental models and based on data from several literature reports, we identified low levels of sequence similarity to the host proteome as one of the main factors modulating the B-cell epitope repertoire in the humoral immune response. In point of fact, a low level of sequence identity to the host proteins is a common denominator unifying the composite, disparate assembly of linear peptide B-cell epitopes that has been experimentally validated and described in the literature. Here, we explore the proteomic similarity of conformational epitopes experimentally validated and described in published reports. Again, discontinuous epitopic structures formed by non-contiguous amino acid residues were found to define immunological peptide units with a low level of similarity to the host. The present meta-analysis adds further significance to the immunological low-similarity theory and its clinical implications. Potentially, low-similarity peptideepitopes pave the way for novel effective vaccines in cancer, autoimmunity, and infectious diseases.
- PMID:
- 22236129
- [PubMed - as supplied by publisher]
Epitope specificity of anti-HA2 antibodies induced in humans during influenza infection.
Source
Institute of Virology, Slovak Academy of Sciences, Bratislava, Slovak Republic Public Health Authority of the Slovak Republic, National Influenza Centre, Bratislava, Slovak Republic.
Abstract
Please cite this paper as: Stanekováet al. (2012) Epitope specificity of anti-HA2 antibodies induced in humans during influenza infection. Influenza and Other Respiratory Viruses DOI: 10.1111/j.1750-2659.2011.00328.x Background The conserved, fusion-active HA2 glycopolypeptide (HA2) subunit of influenza A hemagglutinin comprises four distinct antigenic sites. Monoclonal antibodies (MAbs) recognizing three of these sites are broadly cross-reactive and protective. Objectives This study aimed to establish whether antibodies specific to these three antigenic sites were elicited during a natural influenza infection or by vaccination of humans. Methods Forty-five paired acute and convalescent sera from individuals with a confirmed influenza A (subtype H3) infection were examined for the presence of HA2-specificantibodies. The fraction of antibodies specific to three particular antigenic sites (designated IIF4, FC12, and CF2 here) was investigated using competitive enzyme immunoassay. Results Increased levels of antibodies specific to an ectodomain of HA2 (EHA2: N-terminal residues 23-185 of HA2) were detected in 73% of tested convalescent sera (33/45), while an increased level of antibodies specific to the HA2 fusion peptide (N-terminal residues 1-38) was induced in just 15/45 individuals (33%). Competitive assays confirmed that antibodies specific to the IIF4 epitope (within HA2 residues 125-175) prevailed in 86% (13/15) over those specific to the other two epitopes during infection. However, only a negligible increase in HA2-specific antibodies was detectable following vaccination with a current subunit vaccine. Conclusions We observed that the antigenic site localized within N-terminal HA2 residues 125-175 was more immunogenic than that within residues 1-38 (HA2 fusion protein), although both are weak natural immunogens. We suggest that new anti-influenza vaccines should include HA2 (or specific epitopes localized within this glycopolypeptide) to enhance their cross-protective efficacy.
© 2012 Blackwell Publishing Ltd.
- PMID:
- 22236105
- [PubMed - as supplied by publisher]
CD40/APC-specific antibodies with three T-cell epitopes loaded in the constant domains induce CD4+ T-cell responses.
Source
Department of Molecular Biosciences, Centre for Immune Regulation, University of Oslo, PO Box 1041, Blindern, NO-0316 Oslo, Norway.
Abstract
CD4(+) T lymphocytes play a central role in the orchestration and maintenance of the adaptive immune response. Targeting of antigen to antigen presenting cells (APCs) increases peptide loading of major histocompatibility complex (MHC) class II molecules and CD4(+) T-cell activation. APCs have been targeted by APC-specific recombinantantibodies (rAbs) with single T-cell epitopes integrated in the constant region of the heavy chain (C(H)). However, the strategy may be improved if several T-cell epitopes could be delivered simultaneously by one rAb. We here demonstrate that a single rAb can be loaded with multiple identical or different T-cell epitopes, integrated as loops between β-strands in C(H) domains. One epitope was inserted in C(H)1, while two were placed in C(H)2 of IgG. T-cell proliferation assays showed that all three peptides were excised from loops and presented on MHC class II to T-cells. Induction of T-cell activation by each epitope in the multi-peptide rAb was as good, or even better, than that elicited by corresponding single-peptide rAbs. Furthermore, following DNA vaccination of mice with plasmids that encode CD40-specific rAbs loaded with either one or three peptides, T-cell responses were induced. Thus, integration of multiple epitopes in C(H) region loops of APC-specific rAbs is feasible and may be utilized in design of multi-vaccines.
- PMID:
- 22233931
- [PubMed - as supplied by publisher]
Characterization of Insulin Degrading Enzyme and Other Amyloid-β Degrading Proteases in Human Serum: A Role in Alzheimer's Disease?
Source
Departments of Pharmacology and Experimental Therapeutics, Boston University Medical Campus, Boston, MA, USA Departments of Anesthesiology, The Second People's Hospital of Shenzhen, PR China.
Abstract
Sporadic Alzheimer's disease (AD) patients have low amyloid-β peptide (Aβ) clearance in the central nervous system. The peripheral Aβ clearance may also be important but its role in AD remains unclear. We aimed to study the Aβ degrading proteases including insulin degrading enzyme (IDE), angiotensin converting enzyme (ACE) and others in blood. Using the fluorogenic substrate V (a substrate of IDE and other metalloproteases), we showed that human serum degraded the substrate V, and the activity was inhibited by adding increasing dose of Aβ. The existence of IDE activity was demonstrated by the inhibition of insulin, amylin, or EDTA, and further confirmed by immunocapture of IDE using monoclonal antibodies. The involvement of ACE was indicated by the ability of the ACE inhibitor, lisinopril, to inhibit the substrate V degradation. To test the variations of substrate V degradation in humans, we used serum samples from a homebound elderly population with cognitive diagnoses. Compared with the elderly who had normal cognition, those with probable AD and amnestic mild cognitive impairment (amnestic MCI) had lower peptidase activities. Probable AD or amnestic MCI as an outcome remained negatively associated with serum substrate V degradation activity after adjusting for the confounders. The elderly with probable AD had lower serum substrate V degradation activity compared with those who had vascular dementia. The blood proteases mediating Aβ degradation may be important for the AD pathogenesis. More studies are needed to specify each Aβ degrading protease in blood as a useful biomarker and a possible treatment target for AD.
Quantitative peptidomics to measure neuropeptide levels in animal models relevant to psychiatric disorders.
Source
Dominick P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, Bronx, NY, USA.
Abstract
Neuropeptides play many important roles in cell-cell signaling and are involved in the control of anxiety, depression, pain, reward pathways, and many other processes that are relevant to psychiatric disorders. Mass spectrometry-based peptidomics techniques can identify the precise forms of peptides that are present in a given tissue. Utilizing this technique, peptides with any posttranslational modifications can be identified, and the exact sequence of the peptidescan be determined. Unlike radioimmunoassays, which are limited by specific antibodies and often cannot discriminate between different lengths of peptides from the same precursor, peptidomics reveals the precise sequence and allows for the identification of both known and novel peptides. The use of isotopic labels allows for quantitative peptidomics, which results in the ability to compare peptide levels between differently treated samples. These tags can be synthesized in five different isotopic forms, permitting multivariate analysis of up to five different groups of tissue extracts in a single liquid chromatography/mass spectrometry run; this is ideal for measuring changes in neuropeptides in animals subjected to drug treatments, or in comparing animal models of psychiatric disorders.
Development of molecular techniques for imaging and treatment of tumors.
Source
Department of Nuclear Medicine. University Hospital of Heidelberg, Heidelberg, Germany: 2 Clinical Cooperation Unit Nuclear Medicine, DKFZ and University of Heidelberg, Heidelberg, Germany: 3 Department of Radiopharmaceutical Chemistry, DKFZ Heidelberg, Heidelberg, Germany - uwe.haberkorn@med.uni-heidelberg.de.
Abstract
With the advances in molecular biology and biochemistry new imaging and treatment modalities based on the biological properties of tissues have been developed. In oncology, the major progress has been achieved using peptide andantibody targeting vectors. Besides the identification of new target structures, progress in molecular biology also made new techniques for the development of new biomolecules. This relies on the identification of lead compounds and on the screening of various derivatives of these compounds one at a time. The principle of high-troughput methods for the identification of novel high affinity binders is to generate a vast library of possible variants of the molecule of interest and screen the population for the few variants that show the property of interest. The attracting feature of the concept arises from the huge number of candidate molecules that can be used for further evaluation. After the characterization of the structure-function relationships for the lead compounds found in this process further improvement by rational design of analogs can be performed.
Practical theoretic guidance for the design of tumor-targeting agents.
Source
Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA; Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
Abstract
Theoretical analyses of targeting agent pharmacokinetics provides specific guidance with respect to desirable design objectives such as agent size, affinity, and target antigen. These analyses suggest that IgG-sized macromolecular constructs exhibit the most favorable balance between systemic clearance and vascular extravasation, resulting in maximal tumor uptake. Quantitative predictions of the effects of dose and binding affinity on tumor uptake and penetration are also provided. The single bolus dose required for saturation of xenografted tumors in mice can be predicted from knowledge of antigen expression level and metabolic half-life. The role of high binding affinity in tumor uptake can be summarized as: essential for small peptides, less important for antibodies, and negligible for nanoparticles.
Copyright © 2012 Elsevier Inc. All rights reserved.
- PMID:
- 22230572
- [PubMed - in process]
T cell receptor engineering.
Abstract
T lymphocytes express on their surface a heterodimeric αβ receptor, called the T cell receptor (TCR), which recognizes foreign antigens. Unlike antibodies, the recognition requires both an antigenic peptide epitope and a protein encoded by the major histocompatibility complex (MHC). In contrast to conventional antibody-directed target antigens, antigens recognized by the TCR can include the entire array of potential intracellular proteins, which are processed and delivered to the cell surface as a peptide/MHC complex. In the past 10 years, there have been significant efforts to engineer TCRs in various formats, which would allow improved recognition and destruction of virus-infected cells or cancer. The proposed therapeutic approaches involve either the use of engineered, high-affinity TCRs in soluble forms, analogous toantibody-directed therapies, or the use of engineered TCRs whose genes are reintroduced into autologous T cells and transferred back into patients (T cell adoptive therapies). This chapter describes three methods associated with the engineering of TCRs for these therapeutic purposes: (1) use of a yeast display system to engineer higher affinity single-chain VαVβ TCRs, called scTv; (2) use of a T cell display system to engineer higher affinity full-length TCRs; and (3) expression, purification, and characterization of soluble TCRs in an Escherichia coli system.
Copyright © 2012 Elsevier Inc. All rights reserved.
- PMID:
- 22230570
- [PubMed - in process]
Association of ferritin autoantibodies with giant cell arteritis/polymyalgia rheumatica.
Source
1Department of Immunology and Rheumatology, Medical University, Hannover, Germany.
Abstract
OBJECTIVES:
Polymyalgia rheumatica (PMR) and giant cell arteritis (GCA) are relatively common inflammatory disorders. Establishing the diagnosis however may be difficult, since so far no specific biomarkers of the disorders are available.
METHODS:
As a screening procedure, the authors used protein arrays for the detection of new autoantigens in GCA and PMR. The results of the protein array were confirmed by different ELISAs detecting IgG antibodies against the human ferritin heavy chain, N-terminal 27 amino acids of the human ferritin heavy chain or the homologous peptide of Staphylococcus epidermidis. Sera of patients with only GCA (n=64), only PMR (n=47) and both PMR and GCA (n=31) were used.
RESULTS:
In the ELISA using the human ferritin peptide, the sensitivity of IgG antibodies against ferritin was 92% in 36 GCA and/or PMR patients before initiation of treatment, 22/32 (69%) in patients with disease flares and 64/117 (55%) in the total cohort including treated and inactive patients. In controls, the false positive rate was 11/38 (29%) in systemic lupus erythematosus, 1/36 (3%) in rheumatoid arthritis, 0/31 (0%) in late onset rheumatoid arthritis, 3/46 (6.5%) in B-non-Hodgkin's lymphoma and 1/100 (1%) in blood donors. In the ELISA using the ferritin peptide of S epidermidis, 89% of 27 patients with untreated GCA and PMR were positive.
CONCLUSION:
Antibodies against the ferritin peptide were present in up to 92% of untreated, active GCA and PMR patients. They can be useful as a diagnostic marker of PMR and GCA.
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