RESUMEN
In recent years, nanoparticles based on cyclodextrins have been widely investigated, mainly for drug delivery. In this work, we synthesized nanoparticles with a hyaluronic acid backbone (11â kDa and 45â kDa) functionalized with γ-cyclodextrins. We tested sorafenib in the presence of the new hyaluronan-cyclodextrin conjugates in A2780 (ovarian cancer), SK-HeP-1 (adenocarcinoma) and MDA-MB-453 (breast cancer) cell lines. We found that hyaluronan-cyclodextrin conjugates improve the antiproliferative activity of sorafenib. Remarkably, the system based on the 11â kDa hyaluronan conjugate was the most effective and, in the MDA-MB-453â cell line, significantly reduced the IC50 value of sorafenib cells by about 75 %.
RESUMEN
Alzheimer's disease (AD) is the most common cause of dementia, characterized by a spectrum of symptoms associated with memory loss and cognitive decline with deleterious consequences in everyday life. The lack of specific drugs for the treatment and/or prevention of this pathology makes AD an ever-increasing economic and social emergency. Oligomeric species of amyloid-beta (Aß) are recognized as the primary cause responsible for synaptic dysfunction and neuronal degeneration, playing a crucial role in the onset of the pathology. Several studies have been focusing on the use of small molecules and peptides targeting oligomeric species to prevent Aß aggregation and toxicity. Among them, peptide fragments derived from the primary sequence of Aß have also been used to exploit any eventual recognition abilities toward the full-length Aß parent peptide. Here, we test the Aß8-20 fragment which contains the self-recognizing Lys-Leu-Val-Phe-Phe sequence and lacks Arg 5 and Asp 7 and the main part of the C-terminus, key points involved in the aggregation pathway and stabilization of the fibrillary structure of Aß. In particular, by combining chemical and biological techniques, we show that Aß8-20 does not undergo random coil to ß sheet conformational transition, does not form amyloid fibrils by itself, and is not toxic for neuronal cells. Moreover, we demonstrate that Aß8-20 mainly interacts with the 4-11 region of Aß1-42 and inhibits the formation of toxic oligomeric species and Aß fibrils. Finally, our data show that Aß8-20 protects neuron-like cells from Aß1-42 oligomer toxicity. We propose Aß8-20 as a promising drug candidate for the treatment of AD.
Asunto(s)
Enfermedad de Alzheimer , Fármacos Neuroprotectores , Humanos , Enfermedad de Alzheimer/metabolismo , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/uso terapéutico , Péptidos beta-Amiloides/metabolismo , Fragmentos de Péptidos/metabolismo , Amiloide/metabolismoRESUMEN
[This corrects the article DOI: 10.1021/acs.oprd.1c00368.].
RESUMEN
In the last two decades, the amyloid hypothesis, i.e., the abnormal accumulation of toxic Aß assemblies in the brain, has been considered the mainstream concept sustaining research in Alzheimer's Disease (AD). However, the course of cognitive decline and AD development better correlates with tau accumulation rather than amyloid peptide deposition. Moreover, all clinical trials of amyloid-targeting drug candidates have been unsuccessful, implicitly suggesting that the amyloid hypothesis needs significant amendments. Accumulating evidence supports the existence of a series of potentially dangerous relationships between Aß oligomeric species and tau protein in AD. However, the molecular determinants underlying pathogenic Aß/tau cross interactions are not fully understood. Here, we discuss the common features of Aß and tau molecules, with special emphasis on: (i) the critical role played by metal dyshomeostasis in promoting both Aß and tau aggregation and oxidative stress, in AD; (ii) the effects of lipid membranes on Aß and tau (co)-aggregation at the membrane interface; (iii) the potential of small peptide-based inhibitors of Aß and tau misfolding as therapeutic tools in AD. Although the molecular mechanism underlying the direct Aß/tau interaction remains largely unknown, the arguments discussed in this review may help reinforcing the current view of a synergistic Aß/tau molecular crosstalk in AD and stimulate further research to mechanism elucidation and next-generation AD therapeutics.
Asunto(s)
Enfermedad de Alzheimer , Amiloidosis , Enfermedad de Alzheimer/metabolismo , Amiloide , Péptidos beta-Amiloides/metabolismo , Proteínas Amiloidogénicas , Humanos , Iones , Lípidos/uso terapéutico , Metales , Proteínas tau/metabolismoRESUMEN
Somatostatin analogues are useful pharmaceuticals in peptide receptor radionuclide therapy. In previous studies, we analyzed a new bicyclic somatostatin analogue (BCS) in connection with Cu(II) ions. Two characteristic sites were present in the peptide chain: the receptor- and the metal-binding site. We have already shown that this ligand can form very stable imidazole complexes with the metal ion. In this work, our aim was to characterize the intramolecular interaction that occurs in the peptide molecule. Therefore, we analyzed the coordination abilities of two cyclic ligands, i.e., P1 only with the metal binding site and P2 with both sites, but without the disulfide bond. Furthermore, we used magnetic circular dichroism (MCD) spectroscopy to better understand the coordination process. We applied this method to analyze spectra of P1, P2, and BCS, which we have described previously. Additionally, we analyzed the MCD spectra of P3 ligand, which has only the receptor binding site in its structure. We have unequivocally shown that the presence of the Phe-Trp-Lys-Thr motif and the disulfide bond significantly increases the metal binding efficiency.
Asunto(s)
Complejos de Coordinación/química , Cobre , Somatostatina/análogos & derivados , Secuencia de Aminoácidos , Sitios de Unión , Complejos de Coordinación/síntesis química , Cobre/química , Cobre/metabolismo , Ligandos , Unión ProteicaRESUMEN
Diagnostics of Multiple Sclerosis (MS) are essentially based on the gold standard magnetic resonance imaging. Few alternative simple assays are available to follow up disease activity. Considering that the disease can remain elusive for years, identification of antibodies fluctuating in biological fluids as relevant biomarkers of immune response is a challenge. In previous studies, we reported that anti-N-glucosylated (N-Glc) peptide antibodies that can be easily detected in Solid-Phase Enzyme-Linked ImmunoSorbent Assays (SP-ELISA) on MS patients' sera preferentially recognize hyperglucosylated adhesin of non-typeable Haemophilus Influenzae. Since multivalency can be useful for diagnostic purposes to allow an efficient coating in ELISA, we report herein the development of a collection of Multiple N-glucosylated Peptide Epitopes (N-Glc MEPs) to detect anti-N-Glc antibodies in MS. To this aim, a series of N-Glc peptide antigens to be represented in the N-GlcMEPs were tested in competitive ELISA. We confirmed that the epitope recognized by antibodies shall contain at least 5-mer sequences including the fundamental N-Glc moiety. Using a 4-branched dendrimeric lysine scaffold, we selected the N-Glc MEP 24, carrying the minimal epitope Asn(Glc) anchored to a polyethylene glycol-based spacer (PEG) containing a 19-atoms chain, as an efficient multivalent probe to reveal specific and high affinity anti-N-Glc antibodies in MS.
RESUMEN
We report herein a novel ChemMatrix® Rink resin functionalised with two phenylboronate (PhB) moieties linked on the N-α and N-ε amino functions of a lysine residue to specifically capture deoxyfructosylated peptides, compared to differently glycosylated peptides in complex mixtures. The new PhB-Lys(PhB)-ChemMatrix® Rink resin allows for exploitation of the previously demonstrated ability of cis diols to form phenylboronic esters. The optimised capturing and cleavage procedure from the novel functionalised resin showed that only the peptides containing deoxyfructosyl-lysine moieties can be efficiently and specifically detected by HR-MS and MS/MS experiments. We also investigated the high-selective affinity to deoxyfructosylated peptides in an ad hoc mixture containing unique synthetic non-modified peptides and in the hydrolysates of human and bovine serum albumin as complex peptide mixtures. We demonstrated that the deoxyfructopyranosyl moiety on lysine residues is crucial in the capturing reaction. Therefore, the novel specifically-designed PhB-Lys(PhB)-ChemMatrix® Rink resin, which has the highest affinity to deoxyfructosylated peptides, is a candidate to quantitatively separate early glycation peptides from complex mixtures to investigate their role in diabetes complications in the clinics.
Asunto(s)
Ácidos Borónicos/química , Cromatografía de Afinidad/métodos , Fructosa/química , Péptidos/análisis , Péptidos/metabolismo , Biomarcadores/análisis , Biomarcadores/metabolismo , Glicosilación , Lisina/química , Péptidos/química , Prohibitinas , Albúmina Sérica Bovina/análisis , Albúmina Sérica Bovina/metabolismo , Albúmina Sérica Humana/análisis , Albúmina Sérica Humana/metabolismo , Espectrometría de Masas en TándemAsunto(s)
Péptidos Catiónicos Antimicrobianos/inmunología , Artritis Psoriásica/inmunología , Autoanticuerpos/inmunología , Inmunoglobulina G/inmunología , Adulto , Anciano , Estudios de Casos y Controles , Femenino , Humanos , Activación de Linfocitos/inmunología , Masculino , Persona de Mediana Edad , Psoriasis/inmunología , CatelicidinasRESUMEN
The design of molecules that mimic biologically relevant glycans is a significant goal for understanding important biological processes and may lead to new therapeutic and diagnostic agents. In this study we focused our attention on the trisaccharide human natural killer cell-1 (HNK-1), considered the antigenic determinant of myelin-associated glycoprotein and the target of clinically relevant auto-antibodies in autoimmune neurological disorders such as IgM monoclonal gammopathy and demyelinating polyneuropathy. We describe a structure-activity relationship study based on surface plasmon resonance binding affinities aimed at the optimization of a peptide that mimics the HNK-1 minimal epitope. We developed a cyclic heptapeptide that shows an affinity of 1.09×10-7 m for a commercial anti-HNK1 mouse monoclonal antibody. Detailed conformational analysis gave possible explanations for the good affinity displayed by this novel analogue, which was subsequently used as an immunological probe. However, preliminary screening indicates that patients' sera do not specifically recognize this peptide, showing that murine monoclonal antibodies cannot be used as a guide to select immunological probes for the detection of clinically relevant human auto-antibodies.
Asunto(s)
Antígenos CD57/química , Epítopos/química , Células Asesinas Naturales/química , Oligosacáridos/química , Oligosacáridos/inmunología , Péptidos/química , Péptidos/inmunología , Resonancia por Plasmón de Superficie , Animales , Anticuerpos Monoclonales/inmunología , Reacciones Antígeno-Anticuerpo , Antígenos CD57/inmunología , Epítopos/inmunología , Humanos , Células Asesinas Naturales/inmunología , Ratones , Conformación Proteica , Relación Estructura-ActividadRESUMEN
A través de un análisis estadístico tanto de los síntomas mentales registrados en 568 archivos clínicos resguardados en la base de datos de la Universidad Libre Internacional de Medicina Homeopática (Libera Università Internazionale di Medicina Omeopatica), como de los medicamentos que se prescribieron, pudo determinarse que la sycosis y la psora son los miasmas predominantes en los pacientes con enfermedad autoinmune. Esto significaría que, como han señalado algunos connotados médicos homeópatas, dicha afección no se debe a una perversión del sistema biológico (syphilis), sino a un estado hipo o híper alterado de una función biológica normal. Además, de lo anterior se deduce que las sustancias implicadas en la respuesta autoinmune (auto-anticuerpos) no serían proteínas "extrañas" o "pervertidas" en nuestro cuerpo, sino que son normales y se encuentran al servicio del sistema inmunológico, por ejemplo, para eliminar células viejas, reparar tejidos dañados y destruir malignidades.
Asunto(s)
Humanos , Enfermedades Autoinmunes , Anamnesis Homeopática , Medicamento Homeopático , MéxicoRESUMEN
INTRODUCTION: New low-cost strategies and enhancement of the already described methods to manufacture peptide molecules on an industrial scale are highly requested, particularly for peptides such as octreotide, which, along with goserelin and leuprolide, dominate the global peptide market. A number of patents related to the production of octreotide can be found, concerning both solution and solid-phase synthesis. Thus, there is a need to revise the existing synthetic approaches in order to organize them in a more comprehensible way. AREA COVERED: The octreotide patent landscape could help improvement of the methods for manufacturing of octreotide in industrial scale, leading to the appearance of innovative approaches. EXPERT OPINION: The pharmaceutical value of octreotide can be seen from its high market percentage among other peptide drugs. The complex chemical structure of octreotide represents the main challenge for its industrial production. Two synthetic steps are crucial in the preparation of octreotide: (i) threoninol attachment or on resin formation working in solid-phase and (ii) disulphide bond formation to achieve cyclic structure. Analysis of various patents filed to date allows us to see the trend in simplification of the synthetic approaches from the labor intensive syntheses in solution to the more versatile and rapid solid-phase methods.
Asunto(s)
Medicamentos Genéricos/síntesis química , Octreótido/síntesis química , Péptidos/síntesis química , Química Farmacéutica/métodos , Industria Farmacéutica/métodos , Medicamentos Genéricos/química , Humanos , Octreótido/química , Patentes como Asunto , Péptidos/químicaRESUMEN
The role of pathologic auto-antibodies against myelin oligodendrocyte glycoprotein (MOG) in multiple sclerosis is a highly controversial matter. As the use of animal models may enable to unravel the molecular mechanisms of the human disorder, numerous studies on multiple sclerosis are carried out using experimental autoimmune encephalomyelitis (EAE). In particular, the most extensively used EAE model is obtained by immunizing C57BL/6 mice with the immunodominant peptide MOG(35-55). In this scenario, we analyzed the anti-MOG antibody response in this model using the recombinant refolded extracellular domain of the protein, MOG(1-117). To assess the presence of a B-cell intramolecular epitope spreading mechanism, we tested also five synthetic peptides mapping the 1-117 sequence of MOG, including MOG(35-55). For this purpose, we cloned, expressed in Escherichia coli and on-column refolded MOG(1-117), and we applied an optimized microwave-assisted solid-phase synthetic strategy to obtain the designed peptide sequences. Subsequently, we set up a solid-phase immunoenzymatic assay testing both naïve and EAE mice sera and using MOG protein and peptides as antigenic probes. The results obtained disclose an intense IgG antibody response against both the recombinant protein and the immunizing peptide, while no response was observed against the other synthetic fragments, thus excluding the presence of an intramolecular epitope spreading mechanism. Furthermore, as the properly refolded recombinant probe is able to bind antibodies with greater efficiency compared with MOG(35-55), we hypothesize the presence of both linear and conformational epitopes on MOG(35-55) sequence.
Asunto(s)
Autoanticuerpos/química , Encefalomielitis Autoinmune Experimental/inmunología , Mapeo Epitopo , Epítopos/química , Glicoproteína Mielina-Oligodendrócito/química , Péptidos/síntesis química , Animales , Autoanticuerpos/biosíntesis , Linfocitos B/inmunología , Clonación Molecular , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/patología , Epítopos/inmunología , Escherichia coli/genética , Escherichia coli/metabolismo , Femenino , Expresión Génica , Humanos , Sueros Inmunes/química , Ratones , Ratones Endogámicos C57BL , Microondas , Modelos Moleculares , Esclerosis Múltiple/genética , Esclerosis Múltiple/inmunología , Esclerosis Múltiple/patología , Glicoproteína Mielina-Oligodendrócito/genética , Glicoproteína Mielina-Oligodendrócito/inmunología , Péptidos/inmunología , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Técnicas de Síntesis en Fase Sólida/métodosRESUMEN
The Nucleocapsid protein NCp7 (NC) is a nucleic acid chaperone responsible for essential steps of the HIV-1 life cycle and an attractive candidate for drug development. NC destabilizes nucleic acid structures and promotes the formation of annealed substrates for HIV-1 reverse transcription elongation. Short helical nucleic acid segments bordered by bulges and loops, such as the Trans-Activation Response element (TAR) of HIV-1 and its complementary sequence (cTAR), are nucleation elements for helix destabilization by NC and also preferred recognition sites for threading intercalators. Inspired by these observations, we have recently demonstrated that 2,6-disubstituted peptidyl-anthraquinone-conjugates inhibit the chaperone activities of recombinant NC in vitro, and that inhibition correlates with the stabilization of TAR and cTAR stem-loop structures. We describe here enhanced NC inhibitory activity by novel conjugates that exhibit longer peptidyl chains ending with a conserved N-terminal lysine. Their efficient inhibition of TAR/cTAR annealing mediated by NC originates from the combination of at least three different mechanisms, namely, their stabilizing effects on nucleic acids dynamics by threading intercalation, their ability to target TAR RNA substrate leading to a direct competition with the protein for the same binding sites on TAR, and, finally, their effective binding to the NC protein. Our results suggest that these molecules may represent the stepping-stone for the future development of NC-inhibitors capable of targeting the protein itself and its recognition site in RNA.
Asunto(s)
Antraquinonas/farmacología , Duplicado del Terminal Largo de VIH , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/antagonistas & inhibidores , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismo , Antraquinonas/química , Antraquinonas/metabolismo , Sitios de Unión , Lisina/química , Ácidos Nucleicos/química , ARN Viral/metabolismo , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/químicaRESUMEN
The modulation of collagen turnover can be a relevant pharmacological target in the context of treating either pathological or pathophysiological conditions, such as collagen-related diseases and skin aging. Our recent work has focused on the search for short-chain peptides as lead compounds for further development of compounds that enhance the production of typeâ I collagen. In this study we selected and synthesized overlapping peptides of the C-terminal portion of serpinâ A1 (residues 393-418), the impact of which on collagen production has been reported previously, in order to identify shorter and still active fragments and to provide insight on the mechanisms involved. The biological activity of each fragment was evaluated with cultured normal human dermal fibroblasts, and changes in the amounts of collagen were monitored in collected culture media by a sandwich ELISA technique developed in house. Interestingly, we identified a decapeptide, termed SA1-III (Ac-MGKVVNPTQK-NH2 ), as a promising candidate for our purposes; it is able to induce a significant increase in typeâ I collagen levels in the culture medium of treated cells at micromolar concentrations.
Asunto(s)
Colágeno/metabolismo , alfa 1-Antitripsina/química , alfa 1-Antitripsina/farmacología , Células Cultivadas , Colágeno/biosíntesis , Fibroblastos/metabolismo , Humanos , Piel/metabolismo , alfa 1-Antitripsina/síntesis químicaRESUMEN
Primary biliary cirrhosis is an immune-mediated chronic liver disease whose diagnosis relies on the detection of serum antimitochondrial antibodies directed against a complex set of proteins, among which pyruvate dehydrogenase complex is considered the main autoantigen. We studied the immunological role of the lipoyl domain of this protein using synthetic lipoylated peptides, showing that the lipoyl chain chirality does not affect autoantibody recognition and, most importantly, confirming that both lipoylated and unlipoylated peptides are able to recognize specific autoantibodies in patients sera. In fact, 74% of patients sera recognize at least one of the tested peptides but very few positive sera recognized exclusively the lipoylated peptide, suggesting that the lipoamide moiety plays a marginal role within the autoreactive epitope. These results are supported by a conformational analysis showing that the lipoyl moiety of pyruvate dehydrogenase complex appears to be involved in hydrophobic interactions, which may limit its exposition and thus its contribution to the complex antigenic epitope. A preliminary analysis of the specificity of the two most active peptides indicates that they could be part of a panel of synthetic antigens collectively able to mimic in a simple immunoenzymatic assay the complex positivity pattern detected in immunofluorescence.
Asunto(s)
Epítopos Inmunodominantes/inmunología , Cirrosis Hepática Biliar/diagnóstico , Péptidos , Complejo Piruvato Deshidrogenasa/química , Complejo Piruvato Deshidrogenasa/inmunología , Antígenos/química , Ensayo de Inmunoadsorción Enzimática , Técnica del Anticuerpo Fluorescente , Humanos , Técnicas para Inmunoenzimas , Cirrosis Hepática Biliar/sangre , Cirrosis Hepática Biliar/enzimología , Conformación Molecular , Péptidos/síntesis química , Péptidos/química , Piruvato Deshidrogenasa (Lipoamida)/sangre , Piruvato Deshidrogenasa (Lipoamida)/química , Piruvato Deshidrogenasa (Lipoamida)/inmunología , Complejo Piruvato Deshidrogenasa/sangre , Relación Estructura-ActividadRESUMEN
Celiac disease (CD) patients usually present high levels of circulating IgA antibodies directed to different antigens, in particular tissue transglutaminase (tTG), gliadin (Glia), and endomysium. A series of synthetic peptide constructs containing cross-linked tTG and Glia deamidated peptides have been synthesized. Peptides were tested in enzyme-linked immunosorbent assays against celiac disease patients' sera versus normal blood donors, and their conformational features were evaluated by molecular modeling techniques. Four peptides were recognized as epitopes by autoantibodies (IgG class) circulating in CD patients' sera before gluten-free diet. The peptide II, containing Ac-tTG(553-564)-NH2 sequence cross-linked with deamidated Ac-α2-Glia(63-71)-NH2, was able to identify specific disease antibodies with a sensitivity of 50% and a specificity of 94.4%. Structural conformations of the linear fragments Ac-tTG(553-564)-NH2 and Ac-α2-Glia(63-71)-NH2 and the corresponding cross-linked peptide II were calculated by molecular modeling. Results showed that cross-linking is determinant to assume conformations, which are not accessible to the linear fragments.
Asunto(s)
Anticuerpos/sangre , Enfermedad Celíaca/sangre , Enfermedad Celíaca/inmunología , Epítopos/inmunología , Proteínas de Unión al GTP/inmunología , Gliadina/inmunología , Péptidos/síntesis química , Péptidos/inmunología , Transglutaminasas/inmunología , Adolescente , Adulto , Anticuerpos/inmunología , Reacciones Antígeno-Anticuerpo , Niño , Preescolar , Epítopos/química , Femenino , Proteínas de Unión al GTP/química , Gliadina/química , Humanos , Masculino , Modelos Moleculares , Péptidos/química , Proteína Glutamina Gamma Glutamiltransferasa 2 , Transglutaminasas/química , Adulto JovenRESUMEN
Rett syndrome (RTT), a neurodevelopmental disorder affecting exclusively (99%) female infants, is associated with loss-of-function mutations in the gene encoding methyl-CpG binding protein 2 (MECP2) and, more rarely, cyclin-dependent kinase-like 5 (CDKL5) and forkhead box protein G1 (FOXG1). In this study, we aimed to evaluate the function of the immune system by measuring serum immunoglobulins (IgG and IgM) in RTT patients (n = 53) and, by comparison, in age-matched children affected by non-RTT pervasive developmental disorders (non-RTT PDD) (n = 82) and healthy age-matched controls (n = 29). To determine immunoglobulins we used both a conventional agglutination assay and a novel ELISA based on antibody recognition by a surrogate antigen probe, CSF114(Glc), a synthetic N-glucosylated peptide. Both assays provided evidence for an increase in IgM titer, but not in IgG, in RTT patients relative to both healthy controls and non-RTT PDD patients. The significant difference in IgM titers between RTT patients and healthy subjects in the CSF114(Glc) assay (P = 0.001) suggests that this procedure specifically detects a fraction of IgM antibodies likely to be relevant for the RTT disease. These findings offer a new insight into the mechanism underlying the Rett disease as they unveil the possible involvement of the immune system in this pathology.
Asunto(s)
Sistema Inmunológico/inmunología , Inmunoglobulina G/inmunología , Inmunoglobulina M/inmunología , Síndrome de Rett/inmunología , Adolescente , Pruebas de Aglutinación/métodos , Niño , Preescolar , Ensayo de Inmunoadsorción Enzimática , Femenino , Factores de Transcripción Forkhead/genética , Glicopéptidos/inmunología , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Proteína 2 de Unión a Metil-CpG/genética , Mutación , Proteínas del Tejido Nervioso/genética , Proteínas Serina-Treonina Quinasas/genética , Síndrome de Rett/sangre , Síndrome de Rett/genética , Adulto JovenRESUMEN
Celiac disease (CD) is an autoimmune mediated disease with complex and multifactorial etiology. Gluten intake triggers a composite immune response involving T-cells and B-cells and leading to the secretion of autoantibodies if a genetic predisposition is present. Untreated CD patients show high levels of circulating autoantibodies directed to different auto-antigens present in the intestinal mucosa. The most important auto-antigen is the endomysial enzyme tissue transglutaminase (tTG). Both IgA and IgG antibody isotypes to tTG are known, but only the IgA antibodies demonstrate the highest disease specificity and thus are considered disease biomarkers. Because the pathogenicity and exact tTG binding properties of these autoantibodies are still unclear, the characterization of tTG antigenic domains is a crucial step in understanding CD onset and the autoimmune pathogenesis. Overlapping peptide libraries can be used for epitope mapping of selected protein portions to determine antigenic fragments contributing to the immunological activity and possibly develop innovative peptide-based tools with high specificity and sensitivity for CD. We performed an epitope mapping study to characterize putative linear auto-antigenic epitopes present in the tTG N-terminal portion (1-230). A library of 23 overlapping peptides spanning tTG(1-230) was generated by Fmoc/tBu solid-phase peptide synthesis and screened by immunoenzymatic assays employing patients' sera. The results indicate that four synthetic peptides, that is, Ac-tTG(1-15)-NH2 , Ac-tTG(41-55)-NH2 , Ac-tTG(51-65)-NH2 , and Ac-tTG(151-165)-NH2 , are recognized by IgA autoantibodies circulating in CD patients' sera. These results offer important insight on the nature of the antigen-antibody interaction.
Asunto(s)
Autoantígenos/inmunología , Enfermedades Autoinmunes/inmunología , Enfermedad Celíaca/inmunología , Mapeo Epitopo , Epítopos/inmunología , Proteínas de Unión al GTP/inmunología , Inmunoglobulina A/inmunología , Biblioteca de Péptidos , Transglutaminasas/inmunología , Autoantígenos/química , Epítopos/química , Femenino , Proteínas de Unión al GTP/química , Humanos , Inmunoglobulina A/química , Masculino , Proteína Glutamina Gamma Glutamiltransferasa 2 , Transglutaminasas/químicaRESUMEN
The chemical analysis of egg-based wall paintings-the mezzo fresco technique-is an interesting topic in the characterisation of organic binders. A revised procedure for a dot-enzyme-linked immunosorbent assay (dot-ELISA) able to detect protein components of egg-based wall paintings is reported. In the new dot-ELISA procedure we succeeded in maximizing the staining colour by adjusting the temperature during the staining reaction. Quantification of the colour intensity by visible reflectance spectroscopy resulted in a straight line plot of protein concentration against reflectance in the wavelength range 380-780 nm. The modified dot-ELISA procedure is proposed as a semi-quantitative analytical method for characterisation of protein binders in egg-based paintings. To evaluate its performance, the method was first applied to standard samples (ovalbumin, whole egg, egg white), then to model specimens, and finally to real samples (Giotto's wall paintings). Moreover, amino acid analysis performed by innovative ultra-performance liquid chromatography was applied both to standards and to model samples and the results were compared with those from the dot-ELISA tests. In particular, after protein hydrolysis (24 h, 114 °C, 6 mol L(-1) HCl) of the samples, amino acid derivatization by use of 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate enabled reproducible analysis of amino acids. This UPLC amino acid analysis was rapid and reproducible and was applied for the first time to egg-based paintings. Because the painting technique involved the use of egg-based tempera on fresh lime-based mortar, the study enabled investigation of the effect of the alkaline environment on egg-protein detection by both methods.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Proteínas del Huevo/química , Ensayo de Inmunoadsorción Enzimática/métodos , Pintura/análisis , Aminoácidos/análisis , Huevos/análisis , PinturasRESUMEN
BACKGROUND: The feline leukaemia virus (FeLV) is a gammaretrovirus commonly affecting cats. Infection with this virus often leads to fatal outcomes and, so far, no cure is available for this disease. Synthetic peptides with structures mimicking the transmembrane protein of the viral surface proteins hold the potential to effectively interfere with viral entry by hampering the fusion of viral and host cell membranes and constitute a novel approach for the treatment of infections with retroviruses. We identified and synthetically produced 11 FeLV peptides and evaluated their potential to block FeLV infection in vitro. METHODS: Cell cultures were exposed to FeLV subgroup A prior to the addition of the peptides. The inhibitory effect of the peptides was assessed by measuring FeLV gag protein in the supernatant of peptide versus mock-treated cell cultures using an ELISA. RESULTS: A peptide (EPK364) of 37 amino acids in length, with sequence homology to the HIV fusion inhibitor T-20, significantly suppressed viral replication by 88%, whereas no effects were found for shorter peptides. Two structurally modified variants of EPK364 also inhibited viral replication by up to 58% (EPK397) and 27% (EPK398). CONCLUSIONS: Our data support the identification of synthetic FeLV peptides that have the potential for a curative short-term therapy of viraemic cats. In addition, these peptides might become an important tool in xenotransplantation, where endogenous gammaretroviruses of the donor species might be able to infect the host.