RESUMO
Inflammatory pain associates with spinal glial activation and central sensitization. Systemic administration of IMT504, a non-CpG oligodeoxynucleotide originally designed as an immunomodulator, exerts remarkable anti-allodynic effects in rats with complete Freund´s adjuvant (CFA)-induced hindpaw inflammation. However, the anti-nociceptive mechanisms of IMT504 remain unknown. Here we evaluated whether IMT504 blocks inflammatory pain-like behavior by modulation of spinal glia and central sensitization. The study was performed in Sprague Dawley rats with intraplantar CFA, and a single lumbosacral intrathecal (i.t.) administration of IMT504 or vehicle was chosen to address if changes in glial activation and spinal sensitization relate to the pain-like behavior reducing effects of the ODN. Naïve rats were also included. Von Frey and Randall-Selitto tests, respectively, exposed significant reductions in allodynia and mechanical hypersensitivity, lasting at least 24 h after i.t. IMT504. Analysis of electromyographic responses to electrical stimulation of C fibers showed progressive reductions in wind-up responses. Accordingly, IMT504 significantly downregulated spinal glial activation, as shown by reductions in the protein expression of glial fibrillary acidic protein, CD11b/c, Toll-like receptor 4 (TLR4) and the phosphorylated p65 subunit of NFκB, evaluated by immunohistochemistry and western blot. In vitro experiments using early post-natal cortical glial cultures provided further support to in vivo data and demonstrated IMT504 internalization into microglia and astrocytes. Altogether, our study provides new evidence on the central mechanisms of anti-nociception by IMT504 upon intrathecal application, and further supports its value as a novel anti-inflammatory ODN with actions upon glial cells and the TLR4/NFκB pathway. Intrathecal administration of the non-CpG ODN IMT504 fully blocks CFA-induced mechanical allodynia and hypersensitivity, in association with reduced spinal sensitization. Administration of the ODN also results in downregulated gliosis and reduced TLR4-NF-κB pathway activation. IMT504 uptake into astrocytes and microglia support the concept of direct modulation of CFA-induced glial activation.
Assuntos
Sensibilização do Sistema Nervoso Central , Hiperalgesia , Animais , Hiperalgesia/tratamento farmacológico , Inflamação , Oligodesoxirribonucleotídeos , Dor , Ratos , Ratos Sprague-Dawley , Medula EspinalRESUMO
BACKGROUND: We have shown that oligodeoxynucleotide IMT504 improved blood glucose and islet beta-cell content in streptozotocin (STZ)-induced diabetic rats, inducing early expression of progenitor markers. Here we determined the effect of IMT504 on islet infiltration and on immunomodulatory proteins indoleamine 2,3-dioxygenase (IDO) and TNF-α-stimulated gene/protein 6 (TSG-6) in islets of STZ-diabetic rats, at the time of progenitor markers expression. METHODS: Male rats were i.p. injected with STZ [60 mg/kg body weight (BW)] or citrate buffer (control) (day 1). Starting on day 4, STZ animals were daily treated with saline (STZ-saline) or IMT504 (20 mg/kg BW/day s.c., STZ-IMT504) and killed after two consecutive decreases in blood glucose. Islet area and insulin expression, CD3 (T lymphocytes), CD68 (macrophages), IDO and TSG-6 immunostainings were determined. Islet infiltration was also evaluated by haematoxylin staining. RESULTS: STZ-induced diabetes in rats, with an important decrease in islet area was reversed by IMT504. Diabetes development did not involve islet infiltration, determined by haematoxylin and by the absence of significant T lymphocyte and macrophage presence. IMT504 did not induce changes in these parameters. IDO was not expressed in controls; the percentages of IDO-positive islets were very low and similar in STZ-saline and STZ-IMT504. Scarce TSG-6 was expressed in all groups, without significant differences. CONCLUSIONS: IMT504 improved insulin content but did not alter IDO or TSG-6 staining in islets of STZ-diabetic rats, suggesting that they do not participate in the IMT504-induced repair process. IMT504 did not per se modify leukocyte presence in islets of diabetic rats.
Assuntos
Diabetes Mellitus Experimental/imunologia , Ilhotas Pancreáticas/fisiologia , Oligodesoxirribonucleotídeos/farmacologia , Animais , Moléculas de Adesão Celular/metabolismo , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Ilhotas Pancreáticas/imunologia , Masculino , Ratos , Regeneração/efeitos dos fármacos , EstreptozocinaRESUMO
AIMS/HYPOTHESIS: IMT504 is an oligonucleotide that promotes tissue repair in bone injury and neuropathic pain models by stimulating progenitor cells. Here we evaluated the effect of IMT504 on the recovery of islet function in a streptozotocin (STZ)-induced model of diabetes in the rat. METHODS: Male Sprague-Dawley rats were injected with STZ (60 mg/kg, i.p., day 1) or citrate buffer (Control). Animals with glycaemia between 11 and 20 mmol/l on day 4 were injected with IMT504 (4 mg/animal in saline, s.c., STZ-IMT504) or with saline (STZ-Saline) for 10 days. Glycaemia and water and food intake were recorded for 33 days. Intraperitoneal glucose tolerance tests (IPGTTs) were performed on day 30. On day 35, overnight-fasted animals were killed and blood samples and pancreases collected for hormonal and histological studies. A second group of STZ-IMT504 rats was killed, together with Control and STZ-Saline rats, after two consecutive days of blood glucose decreases after the beginning of IMT504 treatment. Pancreases were collected and proliferating cell nuclear antigen (PCNA), nestin and neurogenin 3 (NGN3) detected by immunohistochemistry. RESULTS: IMT504 greatly improved blood glucose and food and water intakes in STZ-IMT504 rats by day 8, as well as IPGTTs on day 30. Significant increases in islet number and beta cell content were observed in STZ-IMT504 rats (day 33). Furthermore, after two to five IMT504 injections, blood glucose decreased, and an increase in pancreatic nestin (mainly in endothelial cells), PCNA and NGN3 production (in islets) was observed in STZ-IMT504 rats. CONCLUSIONS/INTERPRETATION: IMT504 induced a marked recovery of STZ-induced diabetes that correlated with early production of progenitor cell markers, such as nestin and NGN3.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Glicemia/metabolismo , Diabetes Mellitus Experimental/terapia , Células Secretoras de Insulina/metabolismo , Proteínas de Filamentos Intermediários/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Oligodesoxirribonucleotídeos/uso terapêutico , Análise de Variância , Animais , Contagem de Células , Diabetes Mellitus Experimental/metabolismo , Ingestão de Alimentos , Imuno-Histoquímica , Imunomodulação , Resistência à Insulina , Masculino , Nestina , Oligodesoxirribonucleotídeos/metabolismo , Pâncreas/metabolismo , Radioimunoensaio , Ratos , Ratos Sprague-Dawley , Células-Tronco , Resultado do TratamentoRESUMO
BACKGROUND: Although the treatment of asthma has been addressed in several guidelines, the management of the first acute wheezing episode in infants has not often been evaluated. We surveyed practicing pediatricians in Spain about the treatment they would provide in a simulated case. MATERIAL AND METHODS: A random sample of 3000 pediatricians and physicians who normally treated children was surveyed. The questionnaire inquired about how they would treat a first mild-to-moderate wheezing attack in a 5-month-old boy with a personal and family history of allergy. Pediatricians were asked about their professional background. RESULTS: A total of 2347 questionnaires were returned with useful data (78.2%). Most (90.4%) of the pediatricians would use a short-acting beta2 agonist (SABA) via a metered-dose inhaler with a spacer and a face mask or nebulizer. However, only 34.5% chose a SABA alone: 31.3% added an oral steroid and 27.6% added an inhaled corticosteroid (ICS). The factors associated with the use of ICS in the acute attack were: (1) lack of specific training in pediatrics (OR 1.45; 1.12-1.85) and (2) primary care health center setting (OR 1.31; 1.01-1.69) or rural setting (OR 1.28; 1.01-1.66). Forty-four percent did not recommend any follow-up treatment while 20.7% prescribed ICS as maintenance therapy. The factors related to this decision were the same as those described above. CONCLUSIONS: The management of a first wheezing episode seems to meet published guidelines among Spanish pediatricians with formal training in pediatrics and in those who work in a hospital setting or in urban areas.
Assuntos
Administração de Caso/estatística & dados numéricos , Hipersensibilidade Imediata/tratamento farmacológico , Pediatria/estatística & dados numéricos , Padrões de Prática Médica/estatística & dados numéricos , Sons Respiratórios , Doença Aguda , Administração por Inalação , Administração Oral , Corticosteroides/administração & dosagem , Corticosteroides/uso terapêutico , Antagonistas de Receptores Adrenérgicos beta 2 , Antagonistas Adrenérgicos beta/administração & dosagem , Antagonistas Adrenérgicos beta/uso terapêutico , Antiasmáticos/administração & dosagem , Antiasmáticos/uso terapêutico , Asma/complicações , Asma/diagnóstico , Asma/tratamento farmacológico , Espasmo Brônquico/diagnóstico , Espasmo Brônquico/tratamento farmacológico , Espasmo Brônquico/etiologia , Pré-Escolar , Coleta de Dados , Quimioterapia Combinada , Fidelidade a Diretrizes , Humanos , Hipersensibilidade Imediata/diagnóstico , Hipersensibilidade Imediata/etiologia , Lactente , Espaçadores de Inalação , Máscaras , Inaladores Dosimetrados , Guias de Prática Clínica como Assunto , Recidiva , Espanha , Inquéritos e QuestionáriosRESUMO
Introducción: El sistema inmune no es capaz de reconocer los cambios en células transformadas por mutaciones en la leucemia linfática crónica de estirpe B (LLC-B). Su incubación con linfocitos autólogos no induce citotoxicidad. Puede deberse a la ausencia de linfocitos T citotóxicos o a la incapacidad de las células de LLC en expresar moléculas coestimulatorias (CD80, CD86), a pesar de tener expresión de HLA clase I y II. El ADN bacteriano y los ODNs que contienen motivos PyNTTrrGT, CpG y otros motivos, pueden activar a los monocitos, las células dendríticas y los linfocitos B. Aparte, algunos ODNs tienen efectos directos sobre las células LLC-B. Material y métodos: se incubaron células leucémicas provenientes de 20 pts. con LLC-B con 3 diferentes ODNs: a) IMT504, con motivos PyNTTrrGT b) 2006 con motivos CpG y c) ODN inactivo de control. Previo y posterior a la incubación se efectuaron determinaciones de CD80, CD86, CD40, MHC clase I y II, CD5, CD19 y CD20 por citometría de flujo. Además se determinó (por medio de la detección de fosfatidilserina por la unión ala Anexina V) la capacidad de inducir la apotosis de las células transformadas, y se estudió la morfología de las células en cultivo. Resultados: Se observó el aumento de la detección de fosfatidilserina por la unión ala Anexina V) la capacidad de inducir la apotosis de las células transformadas, y se estudió la morfología de las células en cultivo. Resultados: Se observó el aumento de la expresión de CD80, CD86 y CD40 en las células incubadas con IMT504 y 2006. Además las células LLC-B expresaron significativamente mayor cantidad de Anexina v. La morfología de las células en cultivo a largo plazo mostró las características de apoptosis. Los estudios efectuados con el ODN de control no mostraron ninguna de las características descritas. Conclusión: La incubación de ODN con motivos PyNTTTTGT (IMT504) y con motivos CpG (2006) induce en las células de LLC-B un fenotipo considerado de células presentadoras de antígenos, y además apoptosis. Perspectiva: En otros estudios preclínicos y clínicos los ODNs han demostrado muy baja toxicidad, lo que permitiria efectuar estudios de Fase I/II en LLC-B
Assuntos
Leucemia Linfocítica Crônica de Células B , OligonucleotídeosRESUMO
Introducción: El sistema inmune no es capaz de reconocer los cambios en células transformadas por mutaciones en la leucemia linfática crónica de estirpe B (LLC-B). Su incubación con linfocitos autólogos no induce citotoxicidad. Puede deberse a la ausencia de linfocitos T citotóxicos o a la incapacidad de las células de LLC en expresar moléculas coestimulatorias (CD80, CD86), a pesar de tener expresión de HLA clase I y II. El ADN bacteriano y los ODNs que contienen motivos PyNTTrrGT, CpG y otros motivos, pueden activar a los monocitos, las células dendríticas y los linfocitos B. Aparte, algunos ODNs tienen efectos directos sobre las células LLC-B. Material y métodos: se incubaron células leucémicas provenientes de 20 pts. con LLC-B con 3 diferentes ODNs: a) IMT504, con motivos PyNTTrrGT b) 2006 con motivos CpG y c) ODN inactivo de control. Previo y posterior a la incubación se efectuaron determinaciones de CD80, CD86, CD40, MHC clase I y II, CD5, CD19 y CD20 por citometría de flujo. Además se determinó (por medio de la detección de fosfatidilserina por la unión ala Anexina V) la capacidad de inducir la apotosis de las células transformadas, y se estudió la morfología de las células en cultivo. Resultados: Se observó el aumento de la detección de fosfatidilserina por la unión ala Anexina V) la capacidad de inducir la apotosis de las células transformadas, y se estudió la morfología de las células en cultivo. Resultados: Se observó el aumento de la expresión de CD80, CD86 y CD40 en las células incubadas con IMT504 y 2006. Además las células LLC-B expresaron significativamente mayor cantidad de Anexina v. La morfología de las células en cultivo a largo plazo mostró las características de apoptosis. Los estudios efectuados con el ODN de control no mostraron ninguna de las características descritas. Conclusión: La incubación de ODN con motivos PyNTTTTGT (IMT504) y con motivos CpG (2006) induce en las células de LLC-B un fenotipo considerado de células presentadoras de antígenos, y además apoptosis. Perspectiva: En otros estudios preclínicos y clínicos los ODNs han demostrado muy baja toxicidad, lo que permitiria efectuar estudios de Fase I/II en LLC-B(AU)
Assuntos
Oligonucleotídeos , Leucemia Linfocítica Crônica de Células BRESUMO
Molecular variants of GnRH were characterized by reverse-phase, high-performance liquid chromatography from brain extracts of fish in three different orders: Synbranchiformes (swamp eel [Synbranchus marmoratus]), Cyprinidontiformes (platyfish [Xiphophorus maculatus] and green swordtail [X. helleri]), and Atheriniformes (Patagonia pejerrey [Odontesthes hatchery]). Also, pituitary gland extracts from the pejerrey O. bonariensis (Atheriniformes) were characterized. Eluted fractions were tested in radioimmunoassays with antisera specific to GnRH, including both antisera that detected only one form of GnRH and those that detected several forms. The results show that brain extracts obtained from all species contained the same three molecular forms of GnRH, which were immunologically and chromatographically undistinguishable from chicken GnRH-II, pejerrey GnRH (pjGnRH), and salmon GnRH. This study supports the hypothesis that expression of these three forms is common in different fish orders and that pjGnRH is the main regulator of pituitary function in these fish.
Assuntos
Expressão Gênica , Hormônio Liberador de Gonadotropina/genética , Smegmamorpha/metabolismo , Animais , Química Encefálica , Cromatografia Líquida de Alta Pressão , Ciprinodontiformes/metabolismo , Feminino , Hormônio Liberador de Gonadotropina/isolamento & purificação , Masculino , Hipófise/química , Extratos de Tecidos/químicaRESUMO
Rat and hamster brain tissues were used to investigate the possible existence of a follicle stimulating hormone (FSH)-releasing factor with similar characteristics to the lamprey gonadotropin-releasing hormone III (lGnRH-III) form proposed in previous reports. The present studies involved isolation and purification of the molecule by high-performance liquid chromatography (HPLC), identification by radioimmunoassay, sequence analysis by automated Edman degradation, mass spectrometry and examination of biological activity. Hypothalamic extracts from both species contained an HPLC fraction that was immunoreactive to GnRH and coeluted with lGnRH-III and 9-hydroxyproline mGnRH ([Hyp(9)]GnRH). Determination of primary structure from purified total brain material demonstrated that the isolated molecule was [Hyp(9)]GnRH. This is the first report showing the presence of the posttranslationally modified form already known as [Hyp(9)]GnRH by primary sequence analysis. The biological activity of distinct GnRH peptides was also tested in vitro for gonadotropin release using rat pituitary primary cell cultures. The results showed that [Hyp(9)]GnRH stimulated both luteinizing hormone and FSH release, as already reported, whereas lGnRH-III had no action on the secretion of either gonadotropin.
Assuntos
Encéfalo/metabolismo , Hormônio Liberador de Gonadotropina/química , Hormônio Liberador de Gonadotropina/farmacologia , Sequência de Aminoácidos/genética , Animais , Cricetinae , Feminino , Hormônio Foliculoestimulante/metabolismo , Hormônio Liberador de Gonadotropina/análogos & derivados , Hormônio Liberador de Gonadotropina/genética , Hormônio Liberador de Gonadotropina/isolamento & purificação , Hidroxiprolina/análogos & derivados , Hidroxiprolina/farmacologia , Hipotálamo/metabolismo , Hormônio Luteinizante/metabolismo , Masculino , Espectrometria de Massas , Mesocricetus , Hipófise/citologia , Hipófise/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/isolamento & purificação , Isoformas de Proteínas/farmacologia , Ratos , Relação Estrutura-AtividadeRESUMO
The neuropeptide GnRH is the major regulator of reproduction in vertebrates acting as a first signal from the hypothalamus to pituitary gonadotropes. Three GnRH molecular variants were detected in the brain of a fish, pejerrey (Odontesthes bonariensis), using chromatographic and immunological methods. The present study shows that one form is identical to chicken GnRH-II (sequence analysis and mass spectrometry) and the second one is immunologically and chromatographically similar to salmon GnRH. The third form was proven to be a novel form of GnRH by isolating the peptide from the brain and determining its primary structure by chemical sequencing and mass spectrometry. The sequence of the novel pejerrey GnRH is pGlu-His-Trp-Ser-Phe-Gly-Leu-Ser-Pro-Gly-NH(2), which is different from the known forms of the vertebrate and protochordate GnRH family. The new form of GnRH is biologically active in releasing gonadotropin and GH from pituitary cells in an in vitro assay.
Assuntos
Química Encefálica , Peixes/metabolismo , Hormônio Liberador de Gonadotropina/química , Aminoácidos/análise , Animais , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Hormônio Liberador de Gonadotropina/síntese química , Hormônio Liberador de Gonadotropina/farmacologia , Peptídeos/síntese química , Peptídeos/química , Peptídeos/isolamento & purificação , Hipófise/efeitos dos fármacos , Hipófise/metabolismo , Radioimunoensaio , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
In reptiles as in other vertebrates, multiple forms of gonadotropin-releasing hormone (GnRH) within a single brain have been identified. In this group the following GnRH molecular variants have been characterized either by direct or indirect methods: chicken GnRH I (cGnRH-I), chicken GnRH II (cGnRH-II), salmon GnRH (sGnRH) and several unidentified GnRH-like forms. In the present study GnRH variants were investigated in brain extracts of the lizard Tupinambis teguixin (= T. merinae) by combining high-performance liquid chromatography (RP-HPLC) followed by radioimmunoassays (RIA). Two peaks showing GnRH immunoreactivity with the elution position of synthetic mammalian GnRH (mGnRH) and cGnRH-II were detected. Both peaks were further analyzed with different radioimmunoassay systems specific for mGnRH, cGnRH-I, and cGnRH-II. Pooled fractions corresponding to the first eluting peak showed no crossreactivity when analyzed with a cGnRH-I specific assay and logit-log displacement curves were not significantly different from those of synthetic mGnRH with homologous RIA systems. The second peak showed immunological characteristics of cGnRH-II when analyzed with a specific antiserum. The first ir-GnRH peak was selected for further RP-HPLC purification showing similar chromatographic behavior as mGnRH synthetic standard. We demonstrated the absence of cGnRH-I in this lizard using well-characterized antisera.
Assuntos
Química Encefálica , Variação Genética , Hormônio Liberador de Gonadotropina/análogos & derivados , Hormônio Liberador de Gonadotropina/análise , Hormônio Liberador de Gonadotropina/genética , Lagartos/metabolismo , Mamíferos/metabolismo , Animais , Cromatografia Líquida de Alta Pressão , Feminino , Masculino , Ácido Pirrolidonocarboxílico/análogos & derivados , RadioimunoensaioRESUMO
1. In a previous paper we reported evidence for the presence of mGnRH- and sGnRH-like peptides in the preoptic-hypothalamic region of the capybara Hydrochaeris hydrochaeris (Montaner et al., 1998). In that study, the presence of a cGnRH-II like molecule in olfactory bulb extracts was suggested. 2. The capybara, the largest living rodent in the world, belongs to the order Hystricomorpha, which is considered to be one of the oldest groups of rodents. Some authors consider that this group is the ancestor of all remaining rodents. 3. In this study we have characterized GnRH molecular variants found in extracts from the olfactory bulbs and the mesencephalic region of capybara. These regions represent the two GnRH neuronal systems: the terminal nerve-septopreoptic and the midbrain systems. 4. An indirect method combining reverse-phase high-performance liquid chromatography (RP-HPLC) and radioimmunoassay (RIA) was used to characterize GnRH variants. The analysis of both extracts with two different RIA systems revealed three immunoreactive GnRH peaks, coeluting with mGnRH, cIIGnRH, and sGnRH synthetic standards. These results were additionally supported by serial dilution studies with specific antisera. 5. To our knowledge this the first report on the presence of three GnRH variants in the brain of an eutherian mammal. These results suggest that, similarly to other vertebrates, the expression of multiple GnRH variants may also be a common pattern in mammals.
Assuntos
Hormônio Liberador de Gonadotropina/análogos & derivados , Hormônio Liberador de Gonadotropina/análise , Mesencéfalo/química , Bulbo Olfatório/química , Animais , Evolução Biológica , Feminino , Masculino , RoedoresRESUMO
The molecular variants of Gonadotropin releasing hormone (GnRH) in brain extracts of the eutherian mammal Hydrochaeris hydrochaeris (Mammalia, Rodentia) were characterized. An indirect method combining reverse-phase high-performance liquid chromatography (RP-HPLC) and radioimmunoassay (RIA) with different antisera was used. Two different forebrain regions (olfactory bulbs and preoptic-hypothalamic region) were analyzed. Characterization of RP-HPLC fractions from preoptic-hypothalamic extracts with three different RIA systems revealed two immunoreactive GnRH (ir-GnRH) peaks coeluting with mammalian GnRH (mGnRH) and salmon GnRH (sGnRH) synthetic standards. These results were additionally supported by serial dilution studies with specific antisera. Similar results were obtained from olfactory bulb extracts with the same methodology. However, a third ir-GnRH peak in a similar position to that of chicken GnRH II (cIIGnRH) synthetic standard was revealed. As far as we know, this is the first report showing chromatographic and immunological evidences for the presence of a second GnRH variant in the forebrain of an eutherian mammal.
Assuntos
Hormônio Liberador de Gonadotropina/imunologia , Prosencéfalo/química , Roedores/fisiologia , Animais , Cromatografia Líquida de Alta Pressão/métodos , Feminino , Hormônio Liberador de Gonadotropina/química , Hormônio Liberador de Gonadotropina/metabolismo , Hipotálamo/química , Hipotálamo/metabolismo , Masculino , Mamíferos/fisiologia , Bulbo Olfatório/química , Bulbo Olfatório/metabolismo , Área Pré-Óptica/química , Área Pré-Óptica/metabolismo , Prosencéfalo/metabolismo , RadioimunoensaioRESUMO
Five peptides derived from human immuno deficiency virus (HIV-1) gp41 transmembrane protein have been synthesized: M9 (610-618), M12 (598-609), M15 (600-614), M21 (584-604) and M23 (587-609). These sequences partially overlap in the region vicinal to the immunodominant epitope CSGKLIC, between two cysteine residues 603-609 and three of them (M12, M15 and M23) include this complete heptapeptide. M23, the longer peptide, includes an hydrophilic chain in addition to the heptapeptide loop. The purpose of this work was to determine the influence of contiguous chains to the heptapeptide loop on antibody recognition in fluid and solid phases, and dissociation constants (KD) of each sequence with human anti-HIV-1 antibodies. Two peptides, M13 and M23, overlapped on this loop, were found to be more reactive. Antigen-antibody dissociation constants were determined for both peptides by competition enzyme-linked immunosorbent assay, using each peptide alternatively as the solid phase-immobilized antigen. In addition to the influence of solid-phase antigen on calculated dissociation constants (a phenomenon described by Seligman, 1994), the inhibitory effect of M15 in liquid phase on antibody binding to solid phase M23 was higher than exerted by M23 in solution over antibody binding to M15 on solid phase. On the basis of peptide sequence and predicted antigenicity, this behavior appeared to be contradictory. It is assured that the possible origin of this phenomenon is due to unfavorable conformation of the longer peptide. Even though synthetic peptides mimic mainly sequential epitopes, conformational preferences in fluid or solid phase play an important role in epitope functionality. In particular, addition of residues to known immunodominant sequences may not always amplify antibody recognition if conformation provokes steric hindrance in the native epitope.