Avgi Mamalaki

Anatomy of the antigenic structure of a large membrane autoantigen, the muscle-type nicotinic acetylcholine receptor

Summary: The neuromuscular junction nicotinic acetylcholine receptor (AChR), a pentameric membrane glycoprotein, is the autoantigen involved in the autoimmune disease myasthenia gravis (MG). In animals immunized with intact AChR and in human MG, the anti‐AChR antibody response is polyclonal. However, a small extracellular region of the AChR a‐subunit, the main immunogenic region (MIR), seems to be a major target for anti‐AChR antibodies. A major loop containing overlapping epitopes for several anti‐MIR monoclonal antibodies (mAbs) lies within residues α67–76 at the extreme synaptic end of each a‐subunit; however, anti‐MIR mAbs are functionally and structurally quite heterogeneous. Anti‐MIR mAbs do not affect channel gating, but are very effective in the passive transfer of MG to animals; in contrast, their Fab or Fv fragments protect the AChR from the pathogenic effects of the intact antibodies. Antibodies against the cytoplas‐mic region of the AChR can be elicited by immunization with denatured AChR and the precise epitopes of many such mAbs have been identified; however, it is unlikely that such antibodies are present in significant amounts in human MG. Antibodies to other extracellular epitopes on all AChR subunits are present in both experimental and human MG; these include antibodies to the acetylcholine‐binding site which affect AChR function in various ways and also induce acute experimental MG. Finally, anti‐AChR antibodies cross‐reactive with noti‐AChR antigens exist, suggesting that MG may result from molecular mimicry. Despite extensive studies, many gaps remain in our understanding of the antigenic structure of the AChR, especially in relation to human MG. A thorough understanding of the antigenic structure of the AChR is required for an in‐depth understanding, and for possible specific immunotherapy, of MG.

Results of the first international round robin for the quantification of urinary and plasma hepcidin assays: need for standardization

This study indicates that hepcidin levels reported by various methods vary considerably but analytical variance is generally low and similar for all methods. See related perspective article on page 1631. The recently discovered iron regulatory peptide hormone hepcidin holds promise as a novel biomarker in iron metabolism disorders. To date, various mass spectrometry and immunochemical methods have been developed for its quantification in plasma and urine. Differences in methodology and analytical performance hinder the comparability of data. As a first step towards method harmonization, several hepcidin assays were compared. Worldwide eight laboratories participated in a urinary and plasma round robin in which hepcidin was analyzed. For both urine and plasma: (i) the absolute hepcidin concentrations differed widely between methods, (ii) the between-sample variation and the analytical variation of the methods are similar. Importantly, the analytical variation as percentage of the total variance is low for all methods, indicating their suitability to distinguish hepcidin levels of different samples. Spearman correlations between methods were generally high. The round robin results inform the scientific and medical community on the status and agreement of the current hepcidin methods. Ongoing initiatives should facilitate standardization by exchanging calibrators and representative samples.

Peptide HER2(776-788) represents a naturally processed broad MHC class II-restricted T cell epitope.

HER2/neu-derived peptides inducing MHC class II-restricted CD4+ T helper lymphocyte (Th) responses, although critical for tumour rejection, are not thoroughly characterized. Here, we report the generation and characterization of CD4+ T cell clones specifically recognizing a HER-2/neu-derived peptide (776–788) [designated HER2(776–788)]. Such clones yielded specific proliferative and cytokine [gamma-interferon(IFN)-γ] responses when challenged with autologous dendritic cells (DCs) loaded with HER2(776–788). By performing blocking studies with monoclonal antibodies (MAbs) and by using DCs from allogeneic donors sharing certain HLA-DR alleles, we found that HER2(776–788) is a promiscuous peptide presented, at least, by DRB5*0101, DRB1*0701 and DRB1*0405 alleles. One TCRVbeta6.7+ clone recognized the HLA-DRB5*0101+ FM3 melanoma cell line transfected with a full length HER-2/neu cDNA. Moreover, this clone recognized the HER-2/neu+ SKBR3 breast cancer cell line induced to express HLA-DR, thus demonstrating that HER2(776–788) represents a naturally processed and presented epitope. Our data demonstrate that helper peptide HER2(776–788) represents a promiscuous epitope binding to at least three HLA-DR alleles, thus offering a broad population coverage. The use of antigenic peptides presented by major histocompatibility complex (MHC) class II in addition to those presented by class I may improve the therapeutic efficacy of active immunization.

HER-2/neu-derived peptide 884-899 is expressed by human breast, colorectal and pancreatic adenocarcinomas and is recognized by in-vitro-induced specific CD4(+) T cell clones.

Abstract. HER-2/neu peptides recognized in the context of HLA-DR molecules by CD4+ Th lymphocytes on antigen-presenting cells have been identified. In this report, we demonstrate for the first time that HER-2/neu helper epitopes are also expressed on the surface of metastatic breast, colorectal and pancreatic carcinomas. Peripheral blood mononuclear cells from an HLA-DR4 healthy donor were used to induce HER-2/neu peptide-specific CD4+ T cell clones by in vitro immunization with HER-2/neu peptide (884–899)-pulsed autologous dendritic cells (DCs). Strong proliferation and significant levels of IFN-γ were induced by the CD4+ T cell clones in response to specific stimulation with autologous DCs loaded with HER-2(884–899). Furthermore, these clones also recognized HER-2/neu+ tumor cell lines, and tumor cells from breast, colorectal and pancreatic adenocarcinomas induced to express HLA-DR4, but also the HLA-DR4+ melanoma cell line FM3 transfected to express HER-2/neu. The recognition of tumor cells was strongly inhibited by an anti-HLA-DR mAb. Taken altogether, we provide novel information for the role of HER-2(884–899) as a naturally processed epitope expressed by breast, colorectal and pancreatic carcinomas and the capacity of HER-2/neu protein to follow the endogenous class II processing pathway. Our results suggest that HER-2(884–899) might be attractive for broadly applicable vaccines and may prove useful for adoptive immunotherapy designed for breast, colorectal and pancreatic carcinomas.

Specific immunoadsorption of the autoantibodies from myasthenic patients using the extracellular domain of the human muscle acetylcholine receptor α-subunit. Development of an antigen-specific therapeutic strategy

Antibodies against the acetylcholine receptor (AChR) are the main pathogenic factor in myasthenia gravis (MG). Clinical improvement correlates well with a reduction in levels of circulating anti-AChR antibodies, and plasmapheresis is an efficient short-term MG treatment. The Sepharose-immobilized N-terminal extracellular domain of human muscle AChR alpha-subunit was used to immunoadsorb anti-AChR autoantibodies from 50 MG patients sera. The immunoadsorbents removed 60-94% of the anti-AChR antibodies in 10 sera and a mean of 35% from all samples combined. Immunoadsorption was fast, efficient, and the columns could be used repeatedly without any release or proteolysis of the polypeptide, suggesting the feasibility of antigen-specific MG immunoadsorption therapy.

Association of haptoglobin genotype and common cardiovascular risk factors with the amount of iron in atherosclerotic carotid plaques.

INTRODUCTION The aim of this study was to evaluate the iron burden of carotid atherosclerotic plaques removed from patients treated for carotid disease and find any relation with haptoglobin genotype and other common cardiovascular risk factors. METHODS Consecutive patients undergoing carotid endarterectomy were included in the study. All patients had high-grade carotid stenosis (>70%). The clinical characteristics and serum parameters of the study population were recorded and the haptoglobin genotype was determined. The presence of hemosiderin deposits in the plaques was identified using Perls stain on adjacent serial sections. RESULTS 70 specimens were processed for histologic examination: 27 plaques from diabetic patients (16 with the Hp 1-1 or 2-1 genotype and 11 with the Hp 2-2 genotype) and 43 plaques from non diabetic patients (20 with the Hp 1-1 or 2-1 genotype and 23 with the Hp 2-2 genotype). In plaques from diabetic patients the density of Perls iron stain was significantly higher in the Hp 2-2 group compared with that in the Hp 1-1 or 2-1 group (p = 0.008). The correlation and regression analysis of all possible clinical and laboratory predictors of intraplaque iron deposition showed that four factors were independently associated with intraplaque iron deposition: male gender, serum homocysteine, Hp 2-2 genotype and diabetes mellitus treatment. CONCLUSIONS Male diabetic patients with increased plasma levels of homocysteine and the Hp 2-2 genotype had higher carotid plaque iron deposition. Current evidence and pathophysiological considerations suggest that the increased intraplaque iron deposition may be associated with increased oxidative stress, affecting the stability of the carotid plaque.

High affinity single-chain Fv antibody fragments protecting the human nicotinic acetylcholine receptor

Univalent antibody fragments directed against the main immunogenic region (MIR) of the human acetylcholine receptor (AChR) are capable of protecting the AChR against loss induced by antibodies from myasthenia gravis (MG) patients. Our aim was to construct single-chain Fv (scFv) antibody fragments as a first step towards the production of therapeutic protecting molecules, from two high-affinity anti-MIR monoclonal antibodies (mAb 192 and mAb 195). During the construction of scFv192 fragment, two light chains co-secreted from the hybridoma mAb192 were identified. N-terminal amino acid and cDNA sequence analysis showed that one of the two light chains corresponded to the antigen binding molecule while the other originated from the non-secreting myeloma S194/5.XXO.BU.1 which was used in the production of the hybridoma. Functional scFv 192 and 195 fragments were constructed, expressed in Escherichia coli and affinity purified. The binding affinities of scFv192 and scFv195 (K(D) = 0.6 and 0.8 nM for human AChR) were two orders of magnitude higher than that of the earlier constructed scFv198. The scFv192 almost completely protected human AChR against binding of intact anti-MIR mAbs. Human AChR was also very efficiently protected (74-85%) by the scFv192 against binding of autoantibodies from MG sera with high anti-alpha subunit antibody fractions. These scFvs are good candidates for protection of MG patients after appropriate genetic modifications.

Construction and characterization of a humanized single chain Fv antibody fragment against the main immunogenic region of the acetylcholine receptor.

The single chain Fv fragment of mAb198 (scFv198) directed against the main immunogenic region (MIR) of the nicotinic acetylcholine receptor (AChR), can efficiently protect the AChR in muscle cell cultures against the destructive activity of human myasthenic autoantibodies. Humanization of the scFv198 antibody fragment should prove useful for therapeutic application by reducing its immunogenicity. Framework sequences from human immunoglobulins homologous to the rat scFv198 sequences were selected and a totally synthetic humanized scFv198 antibody fragment was constructed in vitro. Humanized VH and VL domains were synthesized using two overlapping sets of 225 bases long oligonucleotides overlap extension and polymerase chain reaction (PCR), then assembled into a full-length gene by overlap extension of single-stranded DNA (ssDNA) fragments and PCR. The initial humanized antibody fragment had a very low affinity for the AChR. Molecular modeling was then performed and four residues from the framework regions (FR) of the humanized VH domain were selected to be replaced by the corresponding amino acid from the rat sequence. Three mutants were constructed by overlap extension, using PCR. The humanized variant containing replacements at VH residues 27, 29, 30 and 71 showed very good recovery of AChR binding activity; its binding affinities for Torpedo or human AChR (K(D): 8.5 or 323 nM, respectively) being only four times lower than those of the parental scFv198 (K(D): 2 or 80 nM, respectively). This variant was able to protect the human AChR against the binding of anti-MIR mAb and anti-alpha autoantibodies from a myasthenic patient. It was also able to protect AChR against antigenic modulation induced by the anti-MIR mAb198.

Expression of Endopeptidase-24.11 (Common Acute Lymphoblastic Leukaemia Antigen CDI0) in the Sciatic Nerve of the Adult Rat After Lesion and During Regeneration

Endopeptidase‐24.11, which is identical with the common acute lymphoblastic leukaemia antigen CD1O (CALLA), is a cell surface Zn2+ metalloprotease that regulates peptide‐induced responses in different tissues, including the nervous and immune systems. In the peripheral nervous system, high levels of the enzyme are present in all neonatal and early postnatal Schwann cells, while as myelination proceeds it is gradually suppressed in the majority of cells that form myelin but retained in non‐myelin‐forming cells in the adult animal. In the present study we have investigated the effects of transection, crush and regeneration of the adult rat sciatic nerve on the expression of the endopeptidase by Schwann cells in situ. Endopeptidase‐24.11 was monitored by immunocytochemistry using the monoclonal anti‐endopeptidase antibody 23811. For comparison, a parallel study was carried out with a monoclonal antibody directed against the rat nerve growth factor receptor. We found that (i) all Schwann cells of the distal segment re‐expressed endopeptidase‐24.11 as early as 4 days after axotomy, the level of immunostaining reaching a maximum after 2 weeks, (ii) axonal regeneration repressed Schwann cell expression of endopeptidase‐24.11, and (iii) the induction of the nerve growth factor receptor followed a similar pattern to that of endopeptidase‐24.11 in the transected and crushed nerve. Enzymatic amplification of endopeptidase‐24.11 cDNA from normal and axotomized adult rat sciatic nerve confirmed the expression of endopeptidase‐24.11 in these tissues. Our results show that the expression of endopeptidase‐24.11 in Schwann cells, as is the case with the nerve growth factor receptor, is induced by the loss of the normal axon‐Schwann cell contact. The significant increase in the expression of endopeptidase‐24.11 by Schwann cells after axonal damage suggests that the enzyme could play a role in axonal regeneration.

Nicotinic acetylcholine receptor: Structure, function and main immunogenic region

The nicotinic acetylcholine receptor (AChR) family is a member of the ligand-gated ion-channel gene superfamily, which includes the y-aminobutyric acid (GABA) and glycine receptors (Barnard et al., 1987; Stroud et al., 1990). It contains receptors from the post-synaptic membranes of vertebrate neuromuscular junctions, from the electric organs of electric fish (Torpedo and Electrophorus) and from the central nervous systems of vertebrates and insects. These receptors have several structural and functional features in common, but also have many differences (Lucas and Bencherif, 1992). The muscle-type AChR (i.e. that of the neuromuscular junction and fish electric organs) is the best-known member of the AChR family. It is an integral pentameric membrane protein (Popot and Changeux, 1984) formed from four types of subunits in the stoichiometry @yS or OL