Biomedical subjects

Constantinos Sakarellos

Publications and source records attributed to Constantinos Sakarellos.

24 records · Page 2Linked to original sources

PubMed2003

Idiotype-anti-idiotype circuit in non-autoimmune mice after immunization with the epitope and complementary epitope 289-308aa of La/SSB: implications for the maintenance and perpetuation of the anti-La/SSB response.

BACKGROUND: Antibodies to La/SSB are usually found in sera of patients with Sjogren's Syndrome (SS) and Systemic Lupus Erythematosus (SLE). Recent work from our laboratory (Mol Med 2002;8:293-305) revealed that an active idiotypic network involving antibodies to epitopes of La/SSB and their anti-idiotypes exist in human sera. The anti-idiotypic antibodies were detected using complementary peptides to B-cell epitopes of the autoantigen. The principle of the complementary peptides is based on the 'molecular recognition' theory. According to this theory, translation of two complementary RNA strands (coding and non-coding strand) into protein, generate a pair of peptides, which bind each other with specificity and high affinity. AIM: To investigate antibody production and T-cell responses in non-autoimmune-susceptible animal strains which were immunized with the epitope 289-308aa of La/SSB as well as its complementary epitope. MATERIALS AND METHODS: Balb/c mice were immunized with a peptide corresponding to epitope 289-308aa (pep) or its complementary (cpep) peptide (5 animals/group). The sera were tested for the presence of antibodies to pep and cpep as well as for epitope spreading to recombinant human La/SSB and a major B-cell epitope of La/SSB spanning the region 349-364aa. Another group of animals was sacrificed on day 10 and T-cell responses against pep and cpep were evaluated in cells from lymph nodes and spleen. RESULTS: Immunizations with either pep or cpep led to the appearance of antibodies against the immunogen peptide by day 31 which subsequently was followed by antibody production to its complementary peptide by day 55. In two out of five animals immunized with the epitope 289-308aa, a spreading of the immune response to epitope 349-364aa was observed. In the remaining three animals, negative for antibodies to pep349-364, a specific treatment of sera, using cpep349-364 revealed that anti-idiotypic antibodies masked antibodies to pep349-364. In all immunization experiments high T-cell proliferative responses to both pep and cpep peptides were detected. CONCLUSIONS: Complementary peptides to epitopes of La/SSB can be utilized as probes to study the development of an idiotypic-anti-idiotypic network towards the major autoantigen. The ability of pep and cpep peptides to induce both B-cell and T-cell responses may provide useful insights into understanding further the initiation and maintenance of autoimmune response and create new tools for therapeutic intervention.

Animals↗

PubMed2003

Linear epitopes of two different autoantigens-La/SSB and myelin basic protein--with a high degree of molecular similarity, cause different humoral immune responses.

The region 147-154aa of La/SSB presents 83% sequence similarity with the 139-146aa region of the human myelin basic protein (MBP). The aim of this study was to investigate the prevalence and significance of antibodies against both epitopes in sera from patients with systemic autoimmune diseases, and to compare the humoral responses produced after rabbit immunization. Peptides 147-154aa of La/SSB and 139-146aa of the MBP were attached on tetrameric sequential oligopeptide carriers and used for immunizations of New Zealand White rabbits. Antibodies to immunizing peptides, as well as to the peptides corresponding to other previously defined La/SSB epitopes (289-308aa, 349-364aa), to the intact human MBP (hMBP) and to the recombinant human La/SSB (rechLa) proteins, were identified using specific ELISA assays. Sera from 45 patients with Sjogren's syndrome (pSS), 49 with Systemic Lupus Erythematosus (SLE), and 18 with Rheumatoid Arthritis (RA) were tested against the two peptides and the hMBP. Twenty-two per cent of sera from pSS patients, 27% of SLE patients, and none from RA sera reacted with the La epitope; 27% from pSS sera, 22% of SLE sera, and 17% of RA sera gave a positive reaction against the MBP peptide. Finally, 19% of pSS, 30% of SLE, and 38% of RA sera reacted with the hMBP. Thirty-five days after immunization of rabbits with the La epitope, antibodies were produced against all three La/SSB peptides, the MBP peptide, and the hMBP and rechLa proteins. Rabbits immunized with the MBP peptide produced antibodies against the immunizing peptide and the mimicking peptide of La shortly after immunization, whilst antibodies against the other La epitopes and the two intact proteins were produced later. Inhibition experiments in rabbit sera with high reactivity against hMBP, using the MBP peptide as inhibitor, revealed that 80% of serum reactivity was abolished. In conclusion, a significant proportion of human autoimmune sera reacted with both La and MBP derived peptides, as well as with hMBP. La 147-154aa peptide, when used for animal immunizations, induces a fast epitope spreading involving both La and MBP. In contrast, the mimicking MBP epitope induces a delayed response against the other La epitopes. Thus, despite the fact that these peptides present molecular similarity, they induce different immune responses.

Animals↗

PubMed2003

Mapping the binding domains of the alpha(IIb) subunit. A study performed on the activated form of the platelet integrin alpha(IIb)beta(3).

alpha(IIb)beta(3), a member of the integrin family of adhesive protein receptors, is the most abundant glycoprotein on platelet plasma-membranes and binds to adhesive proteins via the recognition of short amino acid sequences, for example the ubiquitous RGD motif. However, elucidation of the ligand-binding domains of the receptor remains controversial, mainly owing to the fact that integrins are conformationally labile during purification and storage. In this study, a detailed mapping of the extracellular region of the alpha(IIb) subunit is presented, using overlapping 20-peptides, in order to identify the binding sites of alpha(IIb) potentially involved in the platelet-aggregation event. Regions alpha(IIb) 313-332, alpha(IIb) 265-284 and alpha(IIb) 57-64 of alpha(IIb)beta(3) were identified as putative fibrinogen-binding domains because the corresponding peptides inhibited platelet aggregation and antagonized fibrinogen association, possibly by interacting with this ligand. The latter is further supported by the finding that the above peptides did not interfere with the binding of PAC-1 to the activated form of alpha(IIb)beta(3). Furthermore, alpha(IIb) 313-332 was found to bind to fibrinogen in a solid-phase binding assay. It should be emphasized that all the experiments in this study were carried out on activated platelets and consequently on the activated form of this integrin receptor. We hypothesize that RAD and RAE adhesive motifs, encompassed in alpha(IIb) 313-332, 265-284 and 57-64, are capable of recognizing complementary domains of fibrinogen, thus inhibiting the binding of this ligand to platelets.

Adenosine Diphosphate↗

PubMed2002

Unmasking the anti-La/SSB response in sera from patients with Sjogren's syndrome by specific blocking of anti-idiotypic antibodies to La/SSB antigenic determinants.

BACKGROUND: Autoantigen La/SSB is molecular target of humoral autoimmunity in patients with primary Sjogren's Syndrome (pSS) and systemic lupus erythematosus (SLE). In this study, we investigated the existence and possible influence of anti-idiotypic response to anti-La/SSB antibodies. MATERIALS AND METHODS: Synthetic peptide analogs (pep) of the major antigenic determinants of La/SSB (289-308 aa and 349-364 aa) were prepared. Based on "molecular recognition" theory, complementary peptides (cpep), derived by anti-parallel readings of the noncoding strand of La/SSB DNA encoding for its antigenic determinants, were constructed. Sera from 150 patients with anti-La/SSB antibodies, 30 patients without anti-La/SSB antibodies, and 42 normal individuals were tested against all four peptides. F(ab')(2) fragments from anti-peptide IgG were prepared and F(ab')(2) - IgG interactions were evaluated using a specific anti-idiotypic ELISA. RESULTS: All four peptides were recognized by anti-La positive sera (83% and 51% for pep and cpep 349-364 and 51% and 28% for pep and cpep289-308, respectively). Anti-cpep F(ab')(2 )bound to a common idiotype (Id) located within or spatially close to the antigen combining site of anti La/SSB (anti-pep) antibodies. Homologous and cross-inhibition experiments further confirmed this relation. The anti-idiotypic antibodies inhibited the anti-La/SSB antibody binding to recombinant La/SSB by 91%. To overcome the anti-idiotypic interference in anti-La/SSB detection, a specific assay was developed. Sera were heated for dissociation of Id-anti-Id complexes, anti-Id antibodies blocked with cpep, and anti-La/SSB reactivity was recovered. Application of this method to anti-Ro positive-anti-La/SSB "negative" sera showed that all anti-Ro/SSA positive autoimmune sera also possess anti-La/SSB antibodies. This reaction was not observed in 14 anti-Ro negative- anti-Sm/RNP positive sera from patients with SLE. CONCLUSIONS: Autoimmune sera from patients with pSS and SLE contain anti-idiotypic antibodies targeting a common anti-La/SSB idiotype. These antibodies can be detected using complementary peptides of La/SSB epitopes. The antiidiotypic antibodies mask the anti-La/SSB response. Hidden anti-La/SSB antibodies can be released and detected using complementary epitope analogs.

Amino Acid Sequence↗

PubMedDate not supplied

Synthesis and comparison of antibody recognition of conjugates containing herpes simplex virus type 1 glycoprotein D epitope VII.

Synthetic oligopeptides comprising linear or continuous topographic B-cell epitope sequences of proteins might be considered as specific and small size antigens. It has been demonstrated that the strength and specificity of antibody binding could be altered by conjugation to macromolecules or by modification in the flanking regions. However, no systematic studies have been reported to describe the effect of different carrier macromolecules in epitope conjugates. To this end, the influence of carrier structure and topology on antibody recognition of attached epitope has been studied by comparing the antibody binding properties of a new set of conjugates with tetratuftsin analogue (H-[Thr-Lys-Pro-Lys-Gly](4)-NH(2), T20) sequential oligopeptide carrier (SOC(n)), branched chain polypeptide, poly[Lys(Ser(i)-DL-Ala(m))] (SAK), multiple antigenic peptide (MAP), and keyhole limpet hemocyanine (KLH). In these novel constructs, peptide (9)LKNleADPNRFRGKDL(22) ([Nle(11)]-9-22) representing an immunodominant B cell epitope of herpes simplex virus type 1 glycoprotein D (HSV-1 gD) was conjugated to polypeptides through a thioether or amide bond. Here we report on the preparation of sequential and polymeric polypeptides possessing chloroacetyl groups in multiple copies at the alpha- and/or epsilon-amino group of the polypeptides and its use for the conjugation of epitope peptides possessing Cys at C-terminal position. We have performed binding studies (direct and competitive ELISA) with monoclonal antibody (Mab) A16, recognizing the HSV gD-related epitope, [Nle(11)]-9-22, and conjugates containing identical and uniformly oriented epitope peptide in multiple copies attached to five different macromolecules as carrier. Data suggest that the chemical nature of the carrier and the degree of substitution have marked influence on the strength of antibody binding.

Amino Acid Sequence↗

PubMedDate not supplied

A peptide carrier with a built-in vaccine adjuvant: construction of immunogenic conjugates.

A multifunctional carrier combining B/T cell epitopes (i), a built-in vaccine adjuvant (ii), and a universal T cell epitope (iii) for the construction of potent and specific immunogenic conjugates is presented. The IL-1beta(163-171) fragment known to reproduce the immunostimulatory and adjuvant effects of the whole IL-1beta without possessing any of the pro-inflammatory properties of IL-1beta was covalently anchored to the N-terminus of the Sequential Oligopeptide Carrier, SOC(n), formed by the repeating tripeptide unit Lys-Aib-Gly. A promiscuous T cell epitope derived from the tetanus toxin, TT(593-599), was also positioned in the carboxy terminus of SOC(n) as a universal immunogen to provide broad immunogenicity. Selected B/T cell epitopes from the Sm and La/SSB autoantigens, against which is directed the humoral autoimmunity in patients with systemic lupus erythematosus and Sjögren's Syndrome, respectively, were coupled to the Lys-N(epsilon)H2 groups of the carrier, and the formulated constructs were administered in animals following the conventional immunization protocol of complete/incomplete Freund's adjuvant. The induced immune responses were compared with that produced when the Sm- and La/SSB-reconstituted immunogenic conjugates were injected alone. High titers of specific antibodies recognizing the priming construct, as well as the cognate autoantigen, were obtained when administered alone without the assistance of Freund's adjuvant. It is concluded that our approach provides the conceptual and experimental framework for the development of multifunctional immunogenic conjugates eliciting enhanced, specific, and prolonged humoral response for usage as human vaccine candidates.

Adjuvants, Immunologic↗